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Doctoral Thesis

Root-secreted phosphomonoesterases mobilizing phosphorusfrom the rhizosphereA molecular physiological study in Solanum tuberosum

Author(s): Zimmermann, Philip

Publication Date: 2003

Permanent Link: https://doi.org/10.3929/ethz-a-004583500

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Diss. ETH Nr. 15027

Root-secreted phosphomonoesterases

mobilizing phosphorus from the rhizosphere

A molecular physiological study in Solanum tuberosum

A dissertation submitted to the

SWISS FEDERAL INSTITUTE OF TECHNOLOGY ZURICH

for the degree of

DOCTOR OF SCIENCES

presented by

PHILIP ZIMMERMANN

Dipl. Ing. Agronom, ETH Zurich

born January 31st, 1974

from Charmoille, JU

accepted on the recommendation of

Prof. Dr. Emmanuel Frossard, examiner

Prof. Dr. Nikolaus Amrhein, co-examiner

Dr. Marcel Bücher, co-examiner

Dr. Markus Wyss, co-examiner

2003

Dedication

To Daman's, for her love and support.

Table of contents I

Table of contents

Table of contents /

List of abbreviations ///

Abstract V

Résumé VI

Zusammenfassung VII

11ntroduction 1

1.1 Recent developments in agricultural crop production 1

1.2 How can plant biotechnology contribute? 2

2 Literature review 3

2.1 Phosphate availability and the plant's responses to P-deficiency 3

2.1.1 Phosphorus cycle in an agro-ecosystem 3

2.1.2 Plant responses to P-deficiency: the lupin model 4

2.1.3 Root hairs and the P-deficiency response 5

2.2 Organic phosphates and phosphatases 7

2.2.1 Organic phosphates in soils 7

2.2.2 Phosphatases in the rhizosphere 7

2.2.3 Phosphatases in the plant: the case of purple acid phosphatases 10

2.3 Inositol phosphates and phytases 16

2.3.1 Inositol phosphates 16

2.3.2 Phytases 23

3 Objectives of dissertation research 30

4 Materials & Methods 31

4.1 Materials and chemicals 31

4.2 Methods 35

4.2.1 Molecular biology 35

4.2.2 Physiological and biochemical measurements 37

4.2.3 Plant growth conditions and tissue harvest 41

4.2.4 Computer analyses 45

5 Purple acid phosphatases from potato 47

5.1 Introduction 47

5.2 Results 48

Secreted Phosphomonoesterase activity of potato roots 48

Cloning of StPAPI, StPAP2 and StPAP3 49

Protein sequence analysis 49

Tissue-specific expression of StPAPI, StPAP2 and StPAP3 53

Induction of expression after P deprivation 53

Effect of mycorrhizal symbiosis 53

Regulatory aspects of expression of StPAPI, 2 and 3 54

5.3 Discussion 56

6 Expression of a consensus phytase in potato root hairs 59

Il

6.1 Introduction 59

6.2 Results 61

Potato root hair growth in P-deficient conditions 61

The root hair-specific promoter LeExtl. 1 61

Generation of transgenic potato lines expressing a consensus phytase 62

The consensus phytase properties are maintained in transgenic plants 63

The PHY protein is secreted from the roots 64

Kinetics of phytic acid degradation by root exudates 64

Phenotype of PHY plants 66

6.3 Discussion 68

7 General conclusions and outlook 71

7.1 Potato purple acid phosphatases 71

7.2 P mobilization from phytate in transgenic plants secreting phytase 76

7.3 Designing plants more effectively mobilizing P 77

7.4 Phosphatases, phytases and beyond 80

8 References 83

9 Appendix 97

Appendix 1. DNA and amino acid sequences of StPAPI 98

Appendix 2. DNA and amino acid sequences of StPAP2 99

Appendix 3. DNA and amino acid sequences of StPAP3 100

Appendix 4. DNA and amino acid sequences of the SP/PHY chimeric gene 101

Appendix 5. Analysis of the signal sequence of SP/PHY 102

10 Acknowledgements 103

11 Curriculum vitae 105

List of abbreviations III

List of abbreviations

AMF arbuscular mycorrhizal fungus / fungiAmpr ampicillin resistent

ANOVA analysis of variance

APase acid phosphataseBLAST basic local alignment search tool

bp basepair(s)BSA bovine serum albumin

CaMV cauliflower mosaic virus

cDNA complementary DNA

CIP (CIAP) calf intestinal (alkaline) phosphataseddH20 double distilled water

dH20 deionized water

DNase deoxyribonucleaseDTT dithiothreitol

DW dry weightDMF dimethylformamidedNTP deoxynucleoside triphosphateEDTA sodium ethylenediaminetetraacetateEST expressed sequence tagEtOH ethanol

GFP green fluorescent proteinGFP buffer glyoxal / formamide / phosphate buffer

GPI glycosylphosphatidylinositolGUS ß-glucuronidaseH PLC high performance liquid chromatographyICP inductively coupled plasmaKanr kanamycin resistent

kbp kilobasepair(s)M ES 2-morpholinoethanesulfonic acid monohydratemRNA messenger RNA

MS medium Murrashige and Skoog medium

NaAc sodium acetate

OD optical densityORF open reading frame

PCR polymerase chain reaction

P phosphorusPAP purple acid phosphatasePase phosphatasePi orthophosphate (inorganic phosphate)PDEase phosphodiesterasePMEase Phosphomonoesterase

pNPP p-nitrophenyl phosphatePo organic phosphateRACE rapid amplification of cDNA ends

RNase ribonuclease

RT-PCR reverse transcription PCR

TIGR The Institute for Genomic Research

X-Gluc 5-bromo-4-chloro-3-indolyl-beta-D-glucuronicacid

Abstract V

Abstract

Phosphorus (P) is one of the most limiting plant nutrients in crop production worldwide.

Owing to the strong reactivity of phosphates with soil minerals, P is largely unavailable to

plants. Furthermore, P is exported from the field in the harvested products. The addition of P

fertilizers to sustain crop production is thus required. However, not only are natural resources

of P for the production of fertilizers limited and non-renewable, but also, the excessive use of

P in high-input agricultural systems has resulted in environmental pollution. Low-input

systems, in contrast, use low amounts of P fertilizers and crop production depends on the

amount of available P in the soil, on the efficiency of their cropping system and on the plant

genotypes used.

This thesis deals with increasing the ability of crop plants to mobilize soil P to improve crop

production. The specific objective was to study, on the one hand, the plant's natural

responses to P deficiency, and, on the other hand, to engineer a crop plant for improved

mobilization of P from soils. Between 30 and 65% of P in soils is in organic form and largely

unavailable for plant uptake. This work therefore focused specifically on the study of

phosphatases secreted from plant roots and on engineering the secretion of phytase from

roots by expressing a phytase gene in root hairs of potato.

Three cDNAs encoding polypeptides belonging to the family of purple acid phosphatases

(PAP) were isolated from Solanum tuberosum, and the expression of the corresponding

genes was characterized. StPAPI was shown to be expressed in roots, stems, leaves,

flowers and stolons, and did not respond to P deprivation. Both StPAP2 and StPAP3 were

induced by P starvation and expressed mainly in roots, with StPAP3 being additionally

expressed in stem. Based on sequence analysis, all three PAPs are predicted to be

secretory proteins. The precise function of these genes could not be elucidated within the

frame of this work. However, the data obtained suggests that StPAP2 and StPAP3 contribute

to the phosphatase activity in potato root exudates and therefore presumably function in the

mobilization of P from organic P sources in the rhizosphere.

The expression of a synthetic phytase in root hairs of potato resulted in a more than 50-fold

increased phytase activity in root exudates. The recombinant phytase showed high thermo¬

stability and was able to degrade extraradical phytic acid to lower inositol phosphates. In a

soil-quartz substrate to which phytate had been added, transgenic plants secreting the

synthetic phytase exhibited a 40% higher P concentration in the leaves, were on average

20% taller, but in our experimental conditions did not have a significantly higher biomass

production. From the data obtained we cannot conclude whether phytate availability in soils

or phytase activity in the rhizosphere is the limiting step in the mobilization of P from soil

phytate for plant uptake. These issues are discussed in the light of our observations.

Résumé VI

Résumé

Le phosphore (P) est un des éléments nutritifs végétaux les plus limitants pour la production

agricole mondiale. En raison de la forte adsorption des phosphates aux éléments minéraux

du sol, le P est largement indisponible pour le prélèvement par les plantes. Par conséquent,

la fertilisation en phosphates est essentielle pour une production agricole soutenue. Non

seulement les réserves en P naturel sont limitées, mais l'utilisation excessive de P dans les

systèmes de production intensive cause des problèmes de pollution environnementale. Par

contre, les systèmes de production extensifs n'ont souvent pas les moyens de se procurer

les fertilisants P nécessaires, ce qui les rend davantage dépendants de la capacité de leurs

cultures à prélever le P présent dans leurs sols.

Le concept de cette thèse est d'augmenter la capacité des plantes à mobiliser le P du sol

afin d'améliorer la production végétale. L'objectif spécifique est, d'une part, d'étudier les

réponses naturelles de la plante à une carence en P, et d'autre part, de créer une plante

transgénique capable de mobiliser davantage de P du sol. 30-65% du P dans le sol est sous

forme organique (Po), dont une partie, telle les inositols phosphates (phytates), n'est pas

disponible pour le prélèvement par les plantes. Le travail présenté a donc porté surtout sur

l'étude de phosphatases sécrétées par les racines et sur l'augmentation de la sécrétion de

phytase par les racines en exprimant un gène de phytase dans le poil absorbant des racines

de pommes de terre.

Trois gènes de pomme de terre codant pour des polypeptides appartenant à la famille des

phosphatases acides pourpres (PAP) furent isolés et characterises au niveau de leur

expression dans la plante. Il s'avère que StPAPI est exprimée dans les racines, feuilles,

fleurs et stolons, et qu'elle ne réagit pas à l'apport de P. StPAP2 et StPAP3, par contre, sont

exprimées en conditions de carence en P principalement dans les racines (StPAP2 et 3) et

dans la tige (StPAPS). L'analyse des séquences d'acides aminés suggère que les trois

gènes contiennent un signal de sécrétion, et que StPAP2 pourrait avoir un signal d'ancrage

dans la membrane par GPI. La fonction précise des ces gènes ne fut pas possible dans le

cadre du travail présenté. Néanmoins, les données obtenues permettent de supposer que

StPAP2 et StPAP3 jouent un rôle dans l'activité phosphatasique des sécrétions racinaires et

par conséquent dans la mobilisation de P du P organique dans la rhizosphere.

L'expression d'une phytase synthétique dans le poil absorbant des racines de pomme de

terre produisit une activité de phytase plus de 50 fois plus élevée dans les exsudats

racinaires. La phytase synthétique montra une grande thermo-stabilité et fut capable de

dégrader l'inositol hexa/c/'sphosphate en formes réduites d'inositols phosphates. Dans un

substrat composé de sol et de quartz contenant de la phytate, les plantes transgéniques

sécrétant une phytase artificielle purent prélever davantage de P, étaient 20% plus grandes,

mais dans nos conditions expérimentales n'accumulèrent pas davantage de biomasse. Sur

la base de ces données, il n'est pas possible de déterminer si la mobilisation de P des

phytates d'un sol naturel est limitée par la disponibilité des phytates ou par la quantité de

l'enzyme de phytase. Néanmoins, certains aspects sont discutés sur la base des résultats

obtenus.

Zusammenfassung VII

Zusammenfassung

Phosphor (P) ist weltweit einer der am meisten limitierenden essentiellen Pflanzennährstoffe

in der landwirtschaftlichen Pflanzenproduktion. Aufgrund des starken Bindungsvermögens

von Phosphaten an Bodenelemente ist er nur in geringen Mengen für die Pflanzenaufnahme

verfügbar. Demzufolge ist eine P Düngung zur Aufrechterhaltung der Pflanzenproduktion

unentbehrlich. Die natürlichen Ressourcen zur Herstellung von P Dünger sind aber begrenzt.

Zudem hat der übermässige Gebrauch dieser Ressourcen in den intensiven

landwirtschaftlichen Systemen zu Umweltbelastungen beigetragen. In vielen anderen

Gebieten werden hingegen nur sehr wenige P Dünger verwendet, und diese Gebiete sind

daher stärker vom P Gehalt ihrer Böden, des Anbausystems, sowie der verwendeten

Genotypen der Kulturpflanzen abhängig.

Die vorgelegte Arbeit basiert auf dem Konzept einer Vergrösserung der Fähigkeit

landwirtschaftlich nutzbarer Pflanzen, Boden P zu mobilisieren, und damit zur Verbesserung

der Produktion und somit zur Linderung der Abhängigkeit von externen P Quellen

beizutragen. Das Ziel war einerseits, die natürlichen biologischen Reaktionen von Pflanzen

auf P Mangel zu studieren, und andererseits, eine Pflanze mit erhöhter P-

Mobilisierungsfähigkeit durch gentechnische Methoden zu erzeugen. Da der Boden 30-65%

des P in organischer Form enthält, wovon ein wesentlicher Teil in Form von Phytat nicht

pflanzenverfügbar ist, wurde diese Arbeit auf die Untersuchung der von Wurzeln

ausgeschiedenen Phosphatasen sowie auf die Erzeugung transgener Pflanzen mit erhöhter

Sekretion von Phytase in den Wurzeln ausgerichtet.

Drei cDNAs aus Kartoffel, die für Polypeptide kodieren, die der Familie der purpuren sauren

Phosphatasen (PAP) angehören, wurden isoliert und auf Expressionsebene charakterisiert.

StPAPI wird in Wurzeln, Stengeln, Blättern, Blüten sowie Stolonen exprimiert und reagierte

nicht auf Änderungen der P Konzentration im Nährmedium. StPAP2 und StPAP3 wurden

hingegen unter P Mangel stark induziert und waren vor allem in den Wurzeln exprimiert.

StPAP3 war zusätzlich noch im Stengel stark exprimiert. Die Analyse der

Aminosäuresequenzen ergab, dass alle drei PAPs eine mögliche Sekretionssignalsequenz

aufweisen und dass StPAP2 eine Signalsequenz für GPI Verankerung in der Membran

haben könnte. Die genaue Funktion dieser drei Gene konnte im Rahmen dieser Arbeit nicht

bestimmt werden, doch die erhaltenen Resultate lassen auf eine mögliche Funktion von

StPAP2 und StPAP3 in der P-Mobilisierung von organischem P in der Rhizosphäre

schliessen.

Die Expression einer synthetischen Phytase in Wurzelhaaren der Kartoffel ergab eine mehr

als 50-fache Erhöhung der Phytaseaktivität in den Wurzelexudaten. Die rekombinante

Phytase zeigte eine ausgesprochen hohe Hitzestabilität und konnte Phytinsäure

degradieren. Die Phytase sezernierenden transgenen Pflanzen, die auf einem Bodensubstrat

mit Phytatzusatz angezogen wurden, hatten 40% mehr P in den Blättern als Wldtyp

Pflanzen. Der Spross war 20% höher als beim Wldtyp, aber die gesamte Biomasse war in

diesem Experiment statistisch nicht signifikant unterschiedlich. Diesen Daten kann nicht

entnommen werden, ob in einem natürlichen Boden die Verfügbarkeit des Phytats oder die

Menge an Phytase für die Mobilisierung von P aus Phytat limitierend wirken. Abschliessend

werden mögliche Hypothesen diskutiert.

1 Introduction 1

1I Introduction

1.1 Recent developments in agricultural crop production

The last century has experienced an unprecedented evolution in world agriculture. Up to the

1950's, increases in agricultural production were largely achieved by an expansion of the

area under cultivation, by the use of chemical fertilizers, and also by the more recent use of

chemicals for crop protection. Since then, significant increases have also been obtained in

many regions of the world by crop breeding in combination with the use of fertilizers and

irrigation. Nevertheless, despite significant increases in crop production, food security

remains a major concern.

To improve food security means to enhance both the access to and the availability of food

(Runge-Metzger, 1995). A major factor in improving food availability in many countries is the

capability of increasing local production of foods. Increased local production can be achieved

by increased input and/or improved agricultural techniques and crop varieties. In the case of

phosphorus (P), which is the most limiting nutrient in crop production worldwide, increased

input is logistically and technically difficult to achieve for many regions. In addition, limited P

reserves and the inefficient use of P bear the potential for a future phosphate crisis in

agriculture (Abelson, 1999). In this context, the use of alternative cropping systems and the

breeding of plants adapted to low nutrient soils can significantly contribute to increased local

production of foods. Although the use of external fertilizer inputs remains indispensible to

compensate losses by crop removal from fields, improved crop varieties may increase the

efficiency of utilization of nutrients from soils and from external inputs. In order to achieve this

goal, a deeper understanding of the processes involved in nutrient cycling, mobilization, and

uptake by crop plants is needed.

Although much research has been done to improve agricultural systems in regions where P

is limiting, the basic processes governing the movement of P in agro-ecosystems, fixation of

P in soils, mobilization by plants and uptake by the roots are still not fully understood. Recent

advances in describing these systems (Oberson et al., 1999; Raghothama, 1999; Frossard et

al., 2000b; Bucher et al., 2001) raise new possibilities in developing both efficient

management systems and crops more efficient in P mobilization and uptake. These

improvements would result in increased productivity in those regions where P fertilizers are a

rare good. Recent developments in root research, including molecular biological approaches,

set the stage for new technologies that can be used to improve crops. In fact, plant

biotechnology is expected to make a major contribution to the development of plants efficient

in P acquisition (Hell and Hillebrand, 2001).

1.1 Recent developments in agricultural crop production

1 Introduction 2

1.2 How can plant biotechnology contribute?

The application of biotechnology as a tool for increasing food security by crop improvement

has been discussed at length (Ortiz, 1998; Conway and Toenniessen, 1999; Herrera-

Estrella, 1999; Kishore and Shewmaker, 1999; Serageldin, 1999). Advances in plant

breeding and in the understanding of basic biological processes have allowed new

biotechnologies to develop. Initially, products of crop biotechnology embraced mainly

resistance to herbicides and to pathogens, and were commercialized primarily in

industrialized countries such as the United States and Canada. New developments in

research, however, have revealed a significant potential for crop improvements for

developing countries. Some examples of such achievements include the modification of seed

contents for improvement of the nutritional value of crops (Ye et al., 2000; Lucca et al.,

2002), resistance to pathogens (Clausen et al., 2000), and tolerance to drought, salt and

freezing (Kasuga et al., 1999; Zhang and Blumwald, 2001). Recent advances have shown

possibilities in improving the ability of plants to mobilize nutrients from soils, such as Fe and

P (Samuelsen et al., 1998; Koyama et al., 2000; Lopez-Bucio et al., 2000; Delhaize et al.,

2001; Richardson et al., 2001a; Takahashi et al., 2001a). In parallel to the introduction of

new traits in transgenic crops by expression of transgenes, advances in plant functional

genomics are leading to a better understanding of the metabolic pathways governing plant

responses to stress. Ultimately, one can expect crop biotechnology to evolve from a

straightforward, simple approach to more complex and comprehensive strategies in

combination with plant breeding to engineer crops better adapted to their environment. It is

thus important both to assess the expression of transgenes on plant productivity as well as to

understand the function of endogenous genes, to identify target genes for transgene

expression, and, furthermore, to identify genetic loci of interest by marker-assisted breeding.

In addition, gene knock-out mutants and plants expressing foreign genes can serve as model

plants to other sciences such as soil sciences, phytopathology and entomology, which, in

turn, would yield new knowledge resulting in improved crop management.

This work embraces all three aspects, namely, the characterisation of genes involved in the

potato plant's response to P deficiency, the expression of a foreign phytase gene to improve

P acquisition, and, finally, the possibility to use recombinant technology as a tool to modify

the rhizosphere by targeted expression of genes in root hairs.

1.2 How can plant biotechnology contribute?


2^h Literature review

2.1 Phosphate availability and the plant's responses to P-deficiency

2.1.1 Phosphorus cycle in an agro-ecosystem

Sibbesen and Runge-Metzger (1995) proposed a model of P cycling in the agro-ecosystem

composed of three main compartments: soils, crops and animals (Figure 2.1).

"

Milk, eggs and

animals for slaughter

Surface runoff, wind erosion (Leaching)

Figure 2.1 P-cycling in agroecosystems. Modified from Sibbesen and Runge-Metzger (1995).

In intensive agricultural systems, large quantities of P are transferred from one compartment

to another. Nevertheless, P is one of the most limiting essential plant nutrients in agricultural

production worldwide. The availability of P in soils to plants remains limited owing to the



strong reactivity of phosphates with the soil matrix (Holford, 1997). In fact, many soils contain

pools of organic and inorganic P that would be large enough for sustained agricultural crop

production, but only a reduced fraction of P is available to the roots (Marschner, 1995;

Frossard et al., 2000a). Up to 30 million tons of non-renewable phosphate fertilizers are

applied each year worldwide (Prud'homme, 2001), of which only a small fraction is actually

utilized by the crops (Gallet et al., 2003). In low-input systems, where P fertilization is scarce,

crops are regularly removed from the surfaces, and little or no P is returned back to the soil,

resulting in soil mining and poor crop production. To cope with these problems, new

management systems and the development of crops more efficient in P mobilization from

soils have been and are being pursued. To better target research for crop improvement, it is

a prerequisite to understand which mechanisms naturally occurring in plants allow them to

cope with P deficiency stress. For this purpose, model plants such as white lupin (Lupinus

albus, L.) have been used. White lupin, a non-mycorrhizal plant, is an interesting case

because it has a very high capacity to mobilize P from low-P soils. Another model plant

would be Arabidopsis, also non-mycorrhizal, which has the advantage of a fully sequenced

genome and therefore can easily be studied via functional genomics. Lotus and Medicago

are also well described plants, having the advantage of being mycorrhizal.

2.1.2 Plant responses to P-deficiency: the lupin model

Plants react to low levels of available P in the rhizosphere by activating a large number of

morphological and physiological responses. The P-efficient model plant lupin (Lupinus albus,

L.) has been extensively studied and has been shown to initiate various strategies to acquire

P from soils (Figure 2.2).

Some of these responses appear also more generally in higher plants and include the

secretion of increased amounts of organic acids and phosphatases (Ozawa et al., 1995;

Johnson et al., 1996; Ascencio, 1997; Gilbert et al., 1999; Neumann et al., 2000; Gaume et

al., 2001), modification of root architecture (Johnson et al., 1996; Wlliamson et al., 2001),

induction of H+-ATPases and subsequent secretion of protons (Yan et al., 2002), activation of

high-affinity P transporters (Smith et al., 1997; Daram et al., 1998; Liu et al., 1998),

production of anthocyanins (Ticconi et al., 2001; Bloor and Abrahams, 2002), and activation

of P-scavenging enzymes (Plaxton, 1998; Neumann et al., 1999). Some species possess

other adaptive features like increased seed size (Milberg and Lamont, 1997) and slower

growth.

The multiplicity of P-starvation responses occurring simultaneously in lupin, the intensity of

these responses, and their colocalisation in root clusters (proteoid roots) suggest that the

effectiveness of phosphate mobilization from the soil may depend on the joint activation of

these different strategies and on the respective concentrations of secreted products involved.

However, it is known from other (non-proteoid) plant species, e.g. Arabidopsis thaliana and

Zea mays, that the development of root hairs, as well as the secretion of acid phosphatases

and organic acids, is higher in P-efficient varieties than in their inefficient counterpart

genotypes (Narang et al., 2000; Gaume et al., 2001). In other words, although these



strategies considered alone may not be as effective as in combination, they seem to play a

role in the P mobilization from soils. Of particular interest in this work are the increased

growth of root hairs (see below) and the secretion of phosphatases from plant roots (see

chapter 2.2.2).

Activation of P

scavenging enzymes

Modification of

redox-potential

Induction of

high-affinityP transporters

"^

Secretion of phenolics

Production of anthocyanins

Secretion of organic acids

Secretion of phosphatases

H+-release or uptake

Secretion of

chelating compounds

Figure 2.2 Morphological, physiological and molecular responses of white lupin to P deficiency

2.1.3 Root hairs and the P-deficiency response

Root hairs are tubular-shaped tip-growing root epidermal (rhizodermal) cells which extend

into the ambient soil. Root hair length and density vary between plant species. Whereas

certain species produce almost no root hairs, others such as maize, alfalfa and ryegrass

produce several hundred hairs per mm2 surface area of the root cylinder (Jungk, 2001). Root

hairs generate an extended area of contact between the soil and the root, resulting in the

mobilization and uptake of nutrients from larger soil volumes. Comparable to the thin hyphae

of symbiotic mycorrhizal fungi, root hairs are particularly efficient in mining the soil for

scarcely abundant nutrients such as P. In fact, zones around roots have been shown to

become increasingly depleted in P, largely due to the action of root hairs, resulting in P-

depletion zones up to a few mm distance from the root surface (Fusseder and Kraus, 1986;

see Figure 2.3).



Since root hairs represent the major root surface area (up to 90% of the total root surface

area; Itoh and Barber, 1983; Bates and Lynch, 1996), genes involved in P mobilization and

uptake are predominantly expressed in this tissue (Bucher et al., 2001). In fact, several

genes encoding high-affinity Pi transporters have been cloned and shown to be

predominantly expressed at two root interfaces, i.e. in rhizodermal cells including root hairs,

and in root zones below the rhizodermis colonized by arbuscular-mycorrhizal fungi (Daram et

al., 1998; Liu et al., 1998; Muchhal and Raghothama, 1999; Rausch et al., 2001; Harrison et

al., 2002; Karthikeyan et al., 2002; Paszkowski et al., 2002; Smith and Barker, 2002). In

response to P deprivation, increased root hair length was measured, for example, in

Arabidopsis thaliana (Bates and Lynch, 1996), barley (Gahoonia and Nielsen, 1997), and

wheat (Gahoonia et al., 1997). The role of root hairs in P uptake has been demonstrated

repeatedly, for instance in six different species (Itoh and Barber, 1983), in wheat and barley

cultivars (Gahoonia et al., 1997), and by using root hair mutants of Arabidopsis (Bates and

Lynch, 2000). Bailey et al. (2002) reported that root hairs also promote anchorage against

uprooting forces.

Figure 2.3 3-dimensional model of a P-depletion zone around roots A = root cylinder, B = root hair cylinder,C = maximal depletion zone Source Fusseder and Kraus (1986)

While, in an initial phase, plants tend to increase their root surface area under P deficiency

stress (Mollier and Pellerin, 1999), they generally also secrete various compounds. The

secretion of high molecular weight compounds such as phosphatases may play an important

role in the mobilization of organic P sources and has been widely mentioned in the literature.

This topic will be dealt with in the next chapter.



2.2 Organic phosphates and phosphatases

2.2.1 Organic phosphates in soils

During soil development, organic P (Po) accumulates in the upper soil horizon (Syers and

Curtin, 1989) and represents between 30% and 65% of the total P in soils (Harrison, 1987).

Due to the dynamic nature of Po in soils, the composition and turnover of these fractions are

difficult to assess. Soil Po occurs as monoesters ((RO)P03H2) and diesters ((RO)(R'0)P02H)

and their chemical diversity resides in the organic moieties (R and R'). Although the structure

of numerous forms of Po in soils remains unknown, the use of 31P-NMR techniques has

given new insights into soil Po forms (Hawkes et al., 1984; Condron et al., 1990). The most

prevalent forms of Po include inositol phosphates, sugar phosphates, nucleic acids and

phospholipids. Of particular relevance for our work are inositol phosphates, which represent

up to 50% of the organic P in soils (Dalai and Hallsworth, 1977; Anderson, 1980). For a more

detailed analysis of inositol phosphates and phytases, see chapter 2.3 in this thesis.

The microbial activity in soils significantly contributes to the transformation processes

involved in P cycling (Stewart and Tiessen, 1987; Oberson et al., 1996; Rodriguez and

Fraga, 1999). However, gamma-sterilized soils also actively degrade Po because of

biochemical P mineralisation due to the remaining activity of phosphohydrolases (Burns,

1982). In fact, enzymes can be stabilized in soils by sorption onto soil compounds such as

clay minerals while maintaining their activity (Leprince and Quiquampoix, 1996; Pant and

Warman, 2000).

Although several of the mechanisms involved in the cycling and transformation processes of

organic phosphates are not fully understood, there is increasing evidence that these forms of

phosphate may play a role in plant nutrition (Stewart and Tiessen, 1987; Magid et al., 1996;

Rodriguez and Fraga, 1999; Oehl et al., 2001; Oehl et al., 2002). In fact, a number of authors

have shown that phosphatases generally have low substrate specificity and thus are

probably able to hydrolyze P from a large array of organic P compounds from soils.

2.2.2 Phosphatases in the rhizosphere

Terminology

The name "phosphatase" has generally been attributed to any enzyme that can hydrolyze

phosphate esters and anhydrides. This group includes for example phosphoprotein

phosphatases, phosphodiesterases, diadenosine tetraphosphatases, exonucleases, 5'-

nucleotidases, phytases, alkaline and acid phosphatases, respectively, and other types of

phosphomonoesterases. In rhizosphere research, this term more generally refers to proteins

from an unknown composition of enzymes able to cleave chromogenic substrates such as p-

nitrophenylphosphate, which has been the method of choice for measuring soil phosphatase

activity since 1969 (Tabatabai and Bremner, 1969). Phosphate ester hydrolysing activities

would more correctly be indicated by the substrate used, for example p-nitrophenyl



phosphatase activity. However, for practical reasons, the use of the general term

"phosphatase" has been maintained in the scientific vocabulary. Some more recent

publications refer to the more restrictive term "Phosphomonoesterase" (PMEase), "acid

Phosphomonoesterase" (AcPMEase), "alkaline Phosphomonoesterase" (alkPMEase) or

"phytase" to account for the substrate or pH range, respectively, used for activity

measurements.

Microbially-secreted phosphatases

Many soil microorganisms studied to date show a propensity to produce and secrete

phosphatases. Some fungi, including ecto-mycorrhizal fungi, are well known for their

secretion of PM Eases (e.g., Greaves and Webley, 1965; Antibus et al., 1992; Yadav and

Yadav, 1996; Leake, 2002). PMEase secretion has also been described for microorganisms

such as Bacillus (Kerovuo et al., 1998; Idriss et al., 2002), Pseudomonas (Rodriguez and

Fraga, 1999; Home et al., 2002), Rhizobium (Rodriguez and Fraga, 1999), Pénicillium

(Reyes et al., 2002), various Enterobacteriaceae, and Paecilomyces (Silva and Vidor, 2001).

Root-secreted phosphatases

Most plants respond to P deficiency by producing increased amounts of acid phosphatases

in roots. As mentioned in chapter 2.1.2, the model plant lupin has been extensively studied in

this respect (Adams and Pate, 1992; Ozawa et al., 1995; Li and Tadano, 1996; Li et al.,

1997a; Olczak et al., 1997; Gilbert et al., 1999; Neumann et al., 1999; Miller et al., 2001), but

increased acid Phosphomonoesterase activities in root protein extracts or root exudates have

also been reported for a number of other species including rice (Chen et al., 1992), barley

(Asmar et al., 1995), wheat (Szabo-Nagy and Erdei, 1995), maize (Gaume et al., 2001), as

well as gray birch and red maple (Antibus et al., 1997), tomato (Kaya et al., 2000), and a

variety of other plants (Tadano and Sakai, 1991; Dinkelaker and Marschner, 1992; Ascencio,

1997; Hayes et al., 1999). Several authors have demonstrated that plants do not only secrete

PMEases, but a more general group of P-cleaving enzymes including, additionally,

phosphodiesterases (PDases; Asmar and Gissel-Nielsen, 1997; Abel et al., 2000; Chen et

al., 2002) and ribonucleases (RNases; Nürnberger et al., 1990; Löffler et al., 1992; Bosse

and Koeck, 1998; Bariola et al., 1999; Abel et al., 2000). In terms of Pi release from organic

sources, PMEases are thought to have a major function compared to other

phosphohydrolases due to their broad substrate specificity and to the significant proportion of

monoester P forms in soils (Condron et al., 1990).

The level of PMEase activity depends on different physiological factors such as root age and

morphology, the P-status, the supply in other nutrients, genotype, and various forms of stress

(Grierson and Comerford, 2000; Chen et al., 2002). The secretion of PMEase from roots

does not result from a higher leakiness of cell membranes due to P deficiency, but from an

active export of enzymes via the cellular secretory pathway (Gaume et al., 2001; Yadav and

Tarafdar, 2001). This finding is further corroborated by the discovery of several phosphatase

genes which are specifically expressed in roots under phosphate starvation. Most of these



phosphatases contain a signal sequence for secretion via the endoplasmatic reticulum (Deng

et al., 1998; del Pozo et al., 1999; Haran et al., 2000; Wasaki et al., 2000; Baldwin et al.,

2001; Miller et al., 2001; Li et al., 2002; see also chapter 2.2.3). To sum up, many plants

respond to P deficiency by secreting increased amounts of phosphatases from the roots. It is

generally held that the function of these phosphatases is to mobilize P from organic sources

in the rhizosphere, thereby increasing the P availability in that soil compartment.

Phosphatases and P availability in the rhizosphere

Phosphatase activity can be detected in virtually all cultivated soils. The orthophosphate

present in different Po forms in soils (see chapter 2.2.1) can theoretically be released by at

least one group of phosphatases present in soils. Owing to the relatively high Po

concentration in soils, and to the presence of root-secreted phosphatases together with

fungal phosphatases and bacterial alkaline and acid phosphatases, they are believed to be

potentially important sources of P for plants. The ability of phosphatases to mobilize P from

soil organic sources has been shown in a number of cases (Tarafdar and Claassen, 1988;

Helal, 1990; Adams and Pate, 1992; Fox and Comerford, 1992). Others (Thompson and

Black, 1970) have reported, however, that the addition of phosphatases to the soil did not

decrease its organic P content. The quantitative contribution of plant-secreted phosphatases

to plant nutrition and the contribution of individual substrates to the enzymatic release of P

are thus not clear (Häussling and Marschner, 1989; Jungk, 1996). Tarafdar and Jungk (1987)

measured the PMEase activities in soil around plant roots in relation to the depletion of

different P fractions in the rhizosphere and showed that there is a strong correlation between

both factors. A similar study was performed by Chen et al. (2002) in a pot experiment with

ryegrass and Pinus radiata. The results obtained revealed that pine roots occasioned a

larger depletion zone of Po than ryegrass (Figure 2.4). This depletion correlated with greater

concentrations of water-soluble organic carbon, higher microbial biomass and increased

alkaline and acid phosphatase and PDase activities.

A B

0 2 4 6 8 1Ü 12 14

Distance from root surface (mm)

'J Sil85

6.

2 x

1300

1200

1100

1000

900

800

—9

Q 2 4 6 8 10 12 14

Distance from root surface (nun)

Figure 2.4 (A) Sodium hydroxide extractable organic P (NPo) content, and (B) acid PMEase

activities in an Orthic Brown Soil (Dystrochrept) at different distances from roots of Pinus radiata D

Don (T) and Lohum perenne L (o) As a control soil only (•) Figures from Chen et al (2002)



Asmar et al. (1995) reported that the depletion of bicarbonate-extractable Po by barley

(Hordeum vulgare, L.) roots was positively correlated with phosphatase activity in the

rhizosphere. Häussling and Marschner (1989) found that readily hydrolysable Po in the

rhizosphere of 80 year-old Norway spruce (Picea abies, (L) Karst.) was accompanied by a

higher phosphatase activity. Recently, Bhadraray et al. (2002) compared phosphatase

activities in rhizosphere soil to plant biomass production and P uptake in rice varieties and

found that among the enzymatic activities that were compared, alkaline PMEase activity was

found to be most significantly correlated to plant biomass and P uptake. Whether there was a

causal relationship between alkaline PMEase activity and biomass production is not known.

The contribution of phosphatases from microbial origin to the hydrolysis of Po in soils is

unclear. While ecto-mycorrhizal fungi are known to secrete phytase and are thought to be

able to mobilize P from phytate (see chapter 2.3.1), the quantitative contribution of

extracellular phosphatases from AM fungi to Po hydrolysis is thought to be insignificant

(Joner et al., 2000).

Despite this wealth of evidence, other authors have suggested that increased phosphatase

activities in the rhizosphere were in some cases rather a natural plant response to P- or other

nutrient starvation stresses and to an increase in root density, and that there was no causal

relationship between phosphatase secretion and mobilization of P from Po (Hedley et al.,

1983; Furlani et al., 1984). Boreo and Thien (1979) equally found that the increased levels of

phosphatase activity in the rhizosphere were not related to the mineralization of organic P.

In light of these controversies, new methods must be developed to provide new insight into

the effective roles of phosphatases in soils. The study of root-secreted phosphatases, and

more precisely, the identification of genes responsible for phosphatase synthesis, is one

approach that may help answer some of these questions. The next chapter will deal with a

particular family of plant phosphatases, some members of which have been shown to be

synthesized in increased amounts in roots in response to P deficiency.

2.2.3 Phosphatases in the plant: the case of purple acid phosphatases

Biochemical characteristics

Purple acid phosphatases (PAPs) comprise a family of metal-containing glycoproteins that

catalyse the hydrolysis of a wide range of phosphate esters and anhydrides. Members of this

group have been identified in plants, animals, fungi and bacteria (Oddie et al., 2000; Schenk

et al., 2000a). Their active sites exhibit a binuclear, redox-active Fe(lll)-M(ll)/M(lll) center,

where M(ll) is either Fe, Zn or Mn, and M(lll) is Fe (Doi et al., 1988; Vincent and Averill,

1990; Barford et al., 1998; Schenk et al., 2001). The characteristic purple colour is due to a

tyrosine-M(lll) charge-transfer transition (Klabunde and Krebs, 1997). PAPs are also known

as tartrate-resistant acid phosphatases (TRAPs) due to their insensitivity to inhibition by

tartrate.

In animals, PAPs appear to form a homogeneous group of 35 kDa monomeric proteins

containing an antiferromagnetically coupled binuclear Fe(lll)-Fe(ll)/Fe(lll) center. Plant PAPs

appear to be more diverse than animal forms, both in size and in the nature of the second



metal involved in the active site. Two families of different molecular size can be

distinguished. The low-molecular weight (LMW) PAPs (-35 kDa) occur both as monomers

and as dimers derived from disulphide-linked monomer fragments. High-molecular weight

(HMW) PAPs (-55 kDa) are unique to plants and form homodimers through disulfide bonds.

In Arabidopsis thaliana, several genes from each family have been identified based on

sequence similarity (Li et al., 2002). Members of the HMW PAP family possess an extended

N terminus without catalytic function, and their C terminus has sequence similarity with the

LMW family of PAPs. Five conserved motifs containing seven residues involved in metal

binding are conserved throughout all known plant, animal and cyanobacterial PAPs. The

binuclear catalytic center of plant PAPs has been shown to possess both Fe(lll)-Zn(ll) as well

as Fe(lll)-Mn(ll) complexes (Beck et al., 1986; Schenk et al., 2001). The replacement of

Zn(ll) by Fe(ll) in a plant PAP yielded an enzyme with full activity and spectral properties

similar to animal PAPs (Beck et al., 1988). As far as their substrate specificities are

concerned, plant PAPs have not been well characterized. They are generally thought to be

non-specific acid phosphomonoesterases (Li and Tadano, 1996).

Purple acid phosphatases in plants

Since the first purification and isolation of a human PAP protein in 1971, a number of PAP

proteins have been characterized in the literature. The majority of these PAPs were

discovered in the context of biomedical research, mainly in beef and rat spleen (Davis et al.,

1981; Davis and Averill, 1982; Averill et al., 1983; Hara et al., 1984) and in the uterine

secretion of sows (Murray et al., 1972). Owing to their iron content and their suspected role

in iron transport, the latter proteins were often called uteroferrins. These proteins all belong

to the family of low molecular weight PAPs. In plants, PAPs were first discovered in sweet

potato (Uehara et al., 1974a; Uehara et al., 1974b) and have since been isolated from other

plant species, including spinach leaves (Fujimoto et al., 1977), red kidney beans (Beck et al.,

1986), sweet potato tubers (Hefler and Averill, 1987), soybean suspension cultures

(Lebansky et al., 1993) and yellow lupin seeds (Olczak et al., 1997). A more detailed

characterisation of a plant PAP was undertaken with a purified PAP (initially named PvPAPI ;

in this work named PvPAPI; see chapter 7.1) from red kidney bean (Beck et al., 1986). This

protein was later characterized with respect to its secondary structure and amino acid and

metal composition (Cashikar and Rao, 1995). PvPAPI was found to be a dimeric

glycoprotein with a molecular mass of 110 kDa, thus belonging to a new class of high

molecular weight PAPs. Its primary structure and the structure of its oligosaccharides were

also reported (Klabunde et al., 1994; Stahl et al., 1994). The first crystal structure of a plant

PAP was published shortly thereafter (Strater et al., 1995) and led to a better understanding

of the structure and functioning of the active site.

The characterisation of plant genes encoding PAPs has only been reported recently.

Nakazato et al. (1997) reported the cloning of a cDNA encoding a putative

glycosylphosphatidylinositol (GPI) anchored phosphatase from Spirodela oligorrhiza. One

year later, it was reported that this phosphatase is a member of the PAP family of

phosphatases (Nakazato et al., 1998) and it was recently shown by immunohistochemical

staining to be preferentially distributed in the cell walls of the outermost cortical cells but not



at the epidermis. Furthermore, PAP was released by digestion of the cell wall fraction with

cellulases, indicating that in fact it is GPI anchored in the plasma membrane and positioned

to the cell wall (Figure 2.5), thus possibly having a role in acquiring Pi from Po close to the

cell surface. Since then, a moderate number of cDNA clones encoding PAPs have been

described. Durmus (1999) published the sequences of cDNA fragments of two PAP

isozymes of sweet potato. The encoded amino acid sequences of these two isozymes

showed a similarity of 72-77% not only to each other, but also to the primary structure of the

purple acid phosphatase from red kidney bean (PvPAPI). The biochemical and biophysical

characterisation of three PAPs from soybean and sweet potato revealed that they had >66%

sequence identity with the previously characterized PvPAPI, and all of the metal ligands

were conserved (Schenk et al., 1999). Moreover, the metals involved in the active site were,

in contrast to the characteristic Fe-Fe pattern found previously in mammals, Fe-Mn in the

sweet potato enzyme and Fe-Zn in soybean. This report was the first to unambiguously

demonstrate the involvement of Mn in the active site of an enzyme.

Figure 2.5 Simplified model of the addition of the GPI lipid to the protein by a transamidation

reaction mechanism The process takes place within the lumen of the endoplasmatic reticulum

(Model redrawn from diverse sources)

A more specific gene expression and functional analysis, respectively, of a plant PAP was

reported for AtACP5 (AtPAPU), a gene induced by P starvation and by some other types of

P mobilizing/oxidative stresses in Arabidopsis (del Pozo et al., 1999). This gene encodes a

secretory protein homologous to the mammalian LMW PAPs. Promoter-GUS analysis

revealed transcription activation in older leaves and in roots upon P deprivation. Treatment

with abscisic acid (ABA) and hydrogen peroxide (H202) also induced gene expression,

mainly in leaves. Wasaki et al. (2000) reported the cloning and characterisation of two HMW

PAPs from white lupin (LaPAPI and LaPAP2). LaPAP2 is expressed in roots under P



starvation and contains a predicted secretory signal sequence. Both proteins were suspected

to play a role in P mobilization from organic P sources in the rhizosphere. Miller et al. (2001)

reported the cloning of another HMW PAP from white lupin which is preferentially expressed

in proteoid roots under P starvation. The promoter sequence revealed a 50 bp region

showing 72% identity to the promoter of AtPAPU. The cloning of a novel phytase from

soybean with sequence similarity to PAPs was reported by Hegeman and Grabau (2001).

This enzyme exhibited a high affinity for phytic acid and had low pH optimum, indicating that

it could be a vacuolar protein. Very recently, Li et al. (2002) published a list of 29 putative

PAPs of Arabidopsis thaliana identified in several sequence databases. Semi-quantitative

RT-PCR of fragments from 7 genes done on RNA extracted from suspension cell cultures

showed differential transcriptional responses to P deprivation. Only one PAP clone was

shown to be strongly inducible by P starvation (AtPAPU), a second appeared to be

moderately induced (AtPAP12), while the remaining clones more or less did not respond to P

nutrient stress (AtPAPl, AtPAPS, AtPAP9, AtPAPIO and AtPAP13). None of the 29 genes

was characterized on a whole plant level, except for one previously reported cDNA clone

(AtPAPU; del Pozo etal., 1999).

Possible functions of PAPs

The biological roles of PAPs are still unclear. Mammalian PAPs have been implicated in iron

transport (Nuttleman and Roberts, 1990) and in bone resorption because of the high level of

expression of such a gene in bone-resorbing osteoclasts (Hayman and Cox, 1994). Plant

PAPs are thought to play a role in P mobilization or scavenging. In fact, five plant PAPs have

been implicated in the P-starvation responses of Arabidopsis thaliana, Lupinus albus and

Spirodela oligorrhiza (Nakazato et al., 1998; del Pozo et al., 1999; Haran et al., 2000). In

microorganisms, PAPs are found only in a restricted number of organisms (myco- and

cyanobacteria). Schenk et al. (2000a) suggested that PAPs may have a function in survival

of eukaryotic parasites in their hosts by inhibiting the respiratory burst of their host and

removing reactive oxygen species in a Fenton-type reaction (Sibille et al., 1987). The

localisation of GPI-anchored PAPs at the surface of the cell may indicate that some of them

could play a role in defense or in parasite recognition by signal transduction through the

plasma membrane (Hiscox et al., 2002). PAPs have also been suggested to have

intracellular metabolic functions. The functional analysis of plant PAPs using mutants has not

yet been described.

In summary, few PAP genes have so far been isolated from plants and their functions in

plant metabolism remain to be elucidated. An interesting observation is that there are a

number of Arabidopsis mutants that are either defective in P starvation-induced phosphatase

secretion, or which secrete phosphatase constitutively (Trull et al., 1998). In the latter case,

given the high number of PAP genes identified in Arabidopsis, this finding could indicate that

in this mutant either one of the phosphatase genes is overexpressed, or that several

secretory phosphatase genes are regulated via a common signaling transduction pathway.



Transcriptional regulation of plant phosphatases

In many respects, the molecular genetics of the yeast Saccharomyces cerevisiae and of

higher plants are relatively well conserved. This may hold true for some aspects regarding

the P-starvation responses in yeast. As in other microorganisms, Saccharomyces cerevisiae

responds to low levels of P in its environment by activating the transcription of a number of

genes involved in Pi scavenging. These genes are clustered in what is called the PHO

regulon, comprising three genes coding for secretory phosphatases (PH05, PHO10 and

PH011), a vacuolar alkaline phosphatase (PH08), and a high-affinity Pi transporter (PH084)

(Yoshida et al., 1989). The expression of these genes is often coordinated in some sort of

"collective emergency response" to Pi deficiency. Although the induction is very strong for

PH05 (more than 500-fold increase in level of transcript) and PH084 (60-fold), it is weaker in

the case of the alkaline phosphatase (PH08). This differential regulation is in accordance

with their putative functions as Pi scavenger, transporter and storage management proteins,

respectively. In fact, the mobilization of Pi from Po is probably the most limiting factor in case

of P-limitation. The PHO regulon components are transcriptionally regulated by at least five

other proteins: PH081 (a cyclin-dependent kinase (CDK) inhibitor), PHO80 (a cyclin),

PH085 (a CDK), PH02 and PH04 (both transcription factors; Lenburg and O'Shea, 1996;

Oshimaetal., 1996).

It is hypothesized that plants may possess a regulatory network homologous to the yeast

PHO regulon (Wykoff et al., 1999; Rubio et al., 2001; Tomscha, 2001). The first argument

supporting this hypothesis is that there are a number of Arabidopsis mutants defective in

multiple aspects of P deficiency symptoms. One example is the pho3 mutant, which exhibits

a number of characteristics normally associated with low-P stress and does not seem to be

able to respond to low internal P levels (Zakhleniuk et al., 2001). Characteristics of this

mutant include a lack of increase in acid phosphatase activity in response to P deprivation,

reduced accumulation of P in roots and shoots when supplied with sufficient amounts of P in

growth media, delayed flowering, less shoot biomass when grown in soil, accumulation of

starch, lower chlorophyll content when grown in P-sufficient agar media, low fertility, and

anthocyanin accumulation. This multiplicity of deficiency in typical P-starvation responses

indicates that the pho3 mutant may lack a regulatory component of a putative PHO regulon

homolog (Zakhleniuk et al., 2001).

The second argument for the existence of a plant PHO regulon is based on evolutionary

considerations. The PHO regulon is relatively well conserved throughout different organisms,

from prokaryotes up to fungi, such as yeast and Neurospora crassa. The latter controls

expression of phosphatases and Pi transporters with a system similar to the yeast PHO

regulon, in addition to controlling the production of vacuolar and secreted ribonucleases.

Recently, mutants of the unicellular green alga Chlamydomonas reinhardtii defective in a

number of specific Pi starvation responses were identified (psr, phosphorus starvation

response; Shimogawara et al., 1999). psrl is a single recessive mutation that results in the

inability to activate transcription of secreted phosphatases and high-affinity Pi transporters in

response to P-stress. psr2 is a single dominant mutation resulting in constitutively high

expression of secretory phosphatase genes in P-sufficient conditions (Shimogawara et al.,

1999). The cloning of the Psr1 gene responsible for the psrl mutation revealed that it has



sequence characteristics associated with transcriptional activators. However, the Psrl

protein has no sequence homology to yeast PHO regulon proteins. A similar gene (PHR1)

was found in Arabidopsis (Rubio et al., 2001). In contrast to PH04 in yeast, both Psrl and

PHR1 were shown to be permanently detectable in the nucleus, indicating that

photosynthetic organsims possess a different, and probably more complex, gene regulatory

system responding to P-deficiency. However, there appear to be some common pathways

between the yeast PHO regulon and a putative plant PHO regulon in that some genes

induced by low cellular P-concentrations contain a phosphate response domain within their

promoter which has homology to the PH04 binding site (Karthikeyan et al., 2002; Rausch

and Bucher, 2002). In view of the large number of phosphatase genes found in the

Arabidopsis genome, and the fact that there exist both phosphatase underproducing (pup)

and constitutive phosphatase secretion (cps) mutants (Trull et al., 1998), it is possible that

many, if not most, plant secreted phosphatases are transcriptionally regulated via a common

signal transduction pathway.



2.3 Inositol phosphates and phytases

2.3.1 Inositol phosphates

Nomenclature of inositol phosphates

Inositol phosphates are a group of phosphate esters of hexahydroxycyclohexane (inositol). A

number of stereoisomers exist, including myo-, neo-, scyllo-, and D-c/7/ro-inositol phosphates

(Figure 2.6 (A)). The number of phosphate groups may vary between one and six, indicated

by the prefixes mono, bis, tris, tetra/c/'s, penta/c/'s and hexa/c/'s (IUPAC, 1971). The positions of

the phosphate groups are given by the position number of the carbon in the inositol ring to

which they are attached. For example, lns(1,2,3,5,6)P5 is a D-myo-inositol

penta/c/'sphosphate, i.e. with no phosphate moiety at carbon position 4. By convention, the

abbreviation "Ins" always refers to myo-inositol with the numbering of the ID-configuration.

The most common form of inositol phosphates in nature is myo-lnsP6 (Figure 2.6 (B)) and is

referred to by a number of trivial names. "Phytic acid" refers to the free acid form, while

"phytate" is the salt of phytic acid. A more ancient name, "phytin", refers specifically to the

Ca-Mg salt of phytic acid, which is the dominant form of inositol phosphate in cereal grains

(Wheeler and Ferrel, 1971).

myo - inositol neo - inositol scyllo - inositol 1 D-chiro - inositol

B

H203P0^5

H203PO

lnsP6 (l-D-myo-inositol hexa/a'sphosphate)

Figure 2.6 (A) Some stereoisomers of inositols (B) lnsP6 (1D-myo-mositol hexa/asphosphate, or

"phytic acid") in the energetically most favorable configuration



Chemistry of inositol phosphates

Phytic acid has 12 ionizable protons. Correspondingly, 12 pKa values will define the charge

of lnsP6 at a given pH (Figure 2.7). Due to their high anionic charge, inositol phosphates act

as strong ligands. Their cation complexing properties have been extensively studied in the

medical and biological fields (Lutrell, 1992; Martin, 1995). The affinity of lnsP6 for polyvalent

cations varies with pH. At low pH, phytic acid binds metal ions with the following affinity:

Cu(ll) » Zn(ll) = Cd(ll) > Mn(ll) > Mg(ll) > Co(ll) > Ni(ll) (Turner et al., 2002). At pH 6-8,

insoluble complexes are formed with ion:lnsP6 ratios of 6:1 for Co, Ni and Cu, while Zn and

Ca show ion:lnsP6 binding ratios of 3.5 and 4.8, respectively (Martin and Evans, 1987). The

high potential charge density of phytic acid results in its strong adsorption to different

materials. For example, phytate binds strongly to iron oxides and competes with inorganic

phosphate for binding sites. The addition of Ca2+ increases the adsorption of lnsP6 to these

surfaces, and the chemical reduction of iron oxides results in insoluble Fe-lnsP6 (4:1)

precipitates (Bowman et al., 1967; De Groot and Golterman, 1993).

pK2 pKj pK*

1.5 1.5 1.7

pK,; pKz pK#

2.1 5.7 6.9

pK« pK|j pK,2

10.0 10.0 12.0

pH

Figure 2.7 pKa data and estimated net charge on myo-lnsP6 over a range of pH values

(adapted from Costello et al, 1976)

Phytic acid

Phytic acid is thought to have been discovered as early as 1872 by Wlhelm Pfeffer, who

showed that subcellular particles in wheat endosperm contained a calcium/magnesium salt

of organic phosphate (cited in: Cosgrove, 1980). Two structures were proposed for phytic

acid: in 1908, Neumann proposed a structure based on three cyclic pyrophosphate moieties,

while Anderson proposed a structure based on esterification of six hydroxyl groups of myo¬

inositol by orthophosphate moieties (cited in: Cosgrove, 1980). The final proof for the

structure of phytic acid was given by Johnson and Tate (1969b) using NMR-spectroscopy,

confirming the second hypothesis.

pK,

1.1

pKs

2.1

pK»

7.6



Phytic acid and inositol phosphates in plants

Phytic acid accumulates mainly in seeds and pollen, but is also found in tubers and fruits,

and is a rare component in leaves (Plaami and Kumpalainen, 1997). The synthesis of phytic

acid occurs through different pathways, but basically starts with the synthesis of myo-inositol

from D-glucose-6-phosphate. This is the first committed step in the production of inositol-

containing compounds. D-glucose-6-phosphate is initially transformed to 1L-myo-inositol-1-

phosphate (lns(1)P) by the enzyme myo-inositol-1-phosphate synthase (INPS; Nelson et al.,

1998) and then dephosphorylated to myo-inositol by the myo-inositol-monophosphatase

(IMP) enzyme (Gillaspy et al., 1995). Yoshida et al. (1999) characterized a cDNA encoding

an INPS homolog from rice (pRINOI), and showed that corresponding transcripts

accumulated in embryos, while they were absent from other plant tissues. In situ

hybridisation analysis revealed that pRINOI transcripts temporally and spatially colocalized

with the accumulation of lnsP6-containing globoids in the scutellum and aleuron layer. An

alternative pathway includes the formation of D-myo-inositol-3-P from D-glucose-6-P,

resulting in the synthesis of a number of inositol phosphate compounds and concomitant

accumulation of lnsP6 as the final product. The first complete description of the synthetic

sequence of lnsP6 in the plant kingdom was suggested by Brearley and Hanke (1996). In

Spirodela oligorrhiza, the synthesis pathway leading to lnsP6 proceeded through lns(3)P,

lns(3,4)P2, lns(3,4,6)P3, lns(3,4,5,6)P4, lns(1,3,4,5,6)P5 to lnsP6. This pathway differed in

the sequence and forms of inositol phosphates from that reported in the slime mold

Dictyostelium discoideum (Van der Kaay et al., 1995).

lnsP6 has a number of functions in plant metabolism, such as providing a P and mineral

storage form (Raboy, 1997), a pool in inositol phosphate pathways, as a second messenger

ligand (Sasakawa et al., 1995), complexation of multivalent cations and thereby regulation of

the levels of inorganic ions (Cosgrove, 1980; Loewus and Murthy, 2000), and DNA double

strand break repair (Hanakahi et al., 2000). More recently, lower phosphate esters of myo¬

inositol have been shown to have several functions in cell metabolism and in cellular

signalling in mammalian (Streb et al., 1983; Taylor, 1998) and plant cells, respectively

(DeWald et al., 2001; Takahashi et al., 2001b; Mueller-Roeber and Pical, 2002). The inositol

phosphate-mediated signal transduction pathways in plants are related to phospholipid

metabolism and Ca2+ homeostasis, and ultimately to the response of plants to hyperosmotic

stress (DeWald et al., 2001; Takahashi et al., 2001b). Both in plants and animals, subtypes

of inositol trisphosphate (lnsP3) receptors are involved in forming intracellular Ca2+ channels

that are regulated both by lnsP3 and Ca2+. These channels release Ca2+ from intracellular

stores, thus modifying the Ca2+ concentration in different compartments and leading to the

activation of precise cellular functions (Lutrell, 1992; Dasgupta et al., 1996). The

physiological significance of these channels is not always clear, but they could take part in

rapid reaction cascades induced by external stimuli. Inositol phosphates also play an

important role in membranes by the formation of inositol-containing lipids such as

phosphatidyl-inositol phosphates (mainly Ptdlns(4)P and Ptdlns(4,5)P2; for a review see

Mueller-Roeber and Pical, 2002). Inositol is further important by serving as a potential signal

promoting Na+ uptake (Nelson et al., 1999). Worth mentioning is also that lnsP3 and



phospholipids have been shown to be involved in the short- and long-term responses to

gravistimulation (Perera etal., 1999).

The dephosphorylation of phytic acid to lower inositol phosphate intermediates is mediated

by phytase (see chapter 2.3.2). In plants, phytases are found mainly in germinating seedlings

and serve to mobilize Pi from the phytate stored in the seed (Hegeman and Grabau, 2001).

Since inositol phosphates exhibit such a large array of biological functions, the question

arises as to whether and how they might be affected by the presence of phytase in the

cytosol. The study of the intra- and extracellular location of acid phosphatases, alkaline

phosphatases and phytase in six different fungi revealed that only 25% of the total acid

phosphatase and 22% of the total alkaline phosphatase were secreted, while more than 97%

of the total phytase was released into the extracellular space (Tarafdar et al., 2002).

Although few plant phytases have been cloned to date, those that have been characterized

frequently were found to possess signal sequences for targeting to the endoplasmatic

reticulum (Maugenest et al., 1997; Hegeman and Grabau, 2001). It is possible that

compartmentation or secretion of phytase proteins avoids their interactions with inositol

phosphates in the cytosol.

Interestingly, no particular phenotypic changes have been reported for Arabidopsis plants

constitutively expressing an Aspergillus niger phytase gene without secretory signal

sequence (Richardson et al., 2001a). Brinch-Pedersen et al. (2000) observed that wheat

transformants with constitutive heterologous expression of an Aspergillus phytase gene

without secretory signal sequence exhibited a highly complex integration pattern of the

transgene as compared to the lines transformed with the phytase gene including a signal

sequence. In addition, phytase activity levels were eight times lower in these lines. These

findings may indicate that cytosolic accumulation of phytase may have an impact on cellular

metabolism affecting normal growth. In contrast, no anomalies were noted for soybean

(Denbow et al., 1998), canola (Zhang et al., 2000b) or Arabidopsis (Richardson et al., 2001a)

expressing a secretory Aspergillus phytase.

In summary, inositol phosphates have been described to exhibit numerous functions in plant

metabolism. Quantitatively, myo-lnsP6 is the most prevalent form of inositol phosphates in

the plant kingdom. Its accumulation in seeds is of particular ecological and pedological

relevance, since seeds are easily transported throughout the terrestrial ecosystem by

animals and natural processes like wind, resulting in a widespread occurrence of lnsP6 in the

terrestrial ecosystem and ultimately in soils.

The inositol phosphate cycle in the terrestrial ecosystem

Inositol phosphates are synthesized in plants as a storage form of P and represent between

50% and 86% of total P in seeds (Lolas et al., 1976), almost exclusively as the myo-

stereoisomer of lnsP6. Kasim and Edwards (1998) reported that myo-lnsP6 often comprises

100% of the total inositol phosphates detectable in most cereals, legumes and oil seeds.

Phytate is generally degraded during germination (Centeno et al., 2001) or follows different

pathways down to the soil either via decomposition of ungerminated seeds, or through the

digestive tract of animals, where, particularly in the case of monogastric animals, it remains



undegraded and is excreted (Poulsen, 2000). Manure and sewage sludge are thus

potentially important sources of phytate in intensive agro-ecosystems, containing large

amounts of orthophosphate monoesters (Turner et al., 2002).

Although the soil inputs from plant and animal wastes are principally in form of myo-lnsP6, all

possible forms of inositol-P have been detected in soils (Minear et al., 1988) including other

types of stereoisomers generally not found elsewhere in nature. These are present mostly in

the order myo > scyllo > D-chiro > neo, with myo-lnsP6 representing up to 90% of total

lnsP6. Scy//o-lnsP6 (up to 20-50%) and D-c/7/'ro-lnsP6 (up to 10%) are the other forms

present in significant amounts (McKercher and Anderson, 1968).

1000-j

900 -

4p 800 -

q. 700 -

a 6oo -

û- 500 -

£ 400 -

| 300 -

200 -

100 -

0 -

Figure 2.8 Sorption of organic phosphates on a clayey soil

(Adapted from McKercher and Anderson, 1989)

A major proportion (60-90% in alkaline soil extracts) of identifiable organic P exists as

monoester-P (Condron et al., 1990; CadeMenun and Preston, 1996). Since inositol

phosphates often comprise more than 60% of the soil orthophosphate monoester-P fraction

(Dalai and Hallsworth, 1977; Harrison, 1987), they represent a major component of the

organic P fraction. However, the total amount of phytate and its contribution to the organic P

vary greatly between soils (Turner et al., 2002). The composition of organic P inputs to the

soil from fresh plant material and animal wastes does not reflect the proportions of different

organic P forms found in soils (Magid et al., 1996). In fact, inositol-P accumulates in soils,

while other organic P sources, in particular diester-P forms, are rapidly degraded. This may

be explained by a differential stabilization of organic P compounds in the soil. Since

adsorption of organic P compounds is principally determined by the number of phosphate

groups, sugar phosphates and phosphodiesters are only weakly adsorbed and remain prone

to degradation by soil enzymes, while lnsP6 strongly interacts with soil particles, resulting in

preferential stabilization, preventing hydrolysis by phytases and phosphatases (Greaves et

10 20 30 40 50 60

P in final solution (ug P ml1)

70



al., 1963; McKercher and Anderson, 1989; Celi et al., 2001; see figure 2.8). The

concentrations of inositol-phosphates in soils thus depend on the interplay between

enzymatic activity and adsorption processes. The latter are controlled by a number of factors

including Po content, total P content, pH, total organic carbon, and total nitrogen (Turner et

al., 2002). These factors, finally, will affect the biological availability of lnsP6 in soils.

The accumulation of inositol phosphates in soils suggests that they are relatively unavailable

for biological uptake. In fact, although a number of plants have been shown to secrete

phytases (Li et al., 1997c; for phytases, see also chapter 2.3.2), and despite the high levels

of phytase activity detected in many microorganisms (Chen, 1998; Richardson and Hayes,

2000; Tarafdar et al., 2002), few plants have been shown to possess the ability to mobilize P

from phytate in P-limited environments. There is evidence for P-uptake from lnsP6 in a Carex

species (Corona et al., 1996) and in the arctic tundra plant Eriophorum vaginatum (Kroehler

and Linkins, 1991). Adams and Pate (1992) reported that in sand culture, phytate was at

least equal to KH2P04 as a source of P for the growth of lupins, but a much poorer source in

soils, where RNA and glycerophosphates were more readily taken up by lupin plants. They

concluded that the difference in availability of different P sources is related to their solubility

in soils rather then their susceptibility to degradation by phosphatases and phytases.

Similarly, Jackman and Black (1952) showed that increasing the phytase activity of soils did

not increase the level of extractable Pi. The addition of soluble phytate to the soil, however,

increased the level of soil extractable Pi. These findings indicate that the availability of lnsP6

is a crucial factor influencing its degradation by phytase in soils. It appears from the work of

Martin and Cartwright (1971) that the P fixing capacity of a soil strongly affects the biological

availability of lnsP6. In fact, lnsP6 added to soils was more available to plants in soils with

low P-adsorbing capacity than in strongly P-adsorbing soils. On the other hand, a number of

authors have pointed out that in some cases, the utilization of lnsP6 is clearly limited by the

quantity of phytase present in the soil solution, both of plant and microbial origin (Findenegg

and Nelemans, 1993; Hayes et al., 2000b).

The contribution of microorganisms to the mobilization of P from soil phytates has been

demonstrated by Richardson et al. (2001b), Greenwood and Lewis (1977) and Kim et al.

(2002). However, it is not clear which mechanisms determine the availability of lnsP6 as a

substrate for microbial phytase enzymes in the soil solution. In fact, plant growth on a soil

inoculated with a high phytase-secreting Pseudomonas species was lower than when grown

in the same soil to which a species-rich mix of soil microorganisms had been added

(Richardson et al., 2001b). This finding confirms the work by Greaves and Webley (1965)

who reported that, although 30-50% of soil bacterial isolates were able to secrete phytases,

their ability to access lnsP6 in the presence of soil was extremely limited. Ectomycorrhizal

fungi may play an important role in P-mobilization from soil phytate. Leake (2002) reported

that phytase activity was detected in all 20 species of ectomycorrhizal fungi so far tested.

However, the role of the ectomycorrhizal associations in improving the P-acquisition

efficiency of plants from lnsP6 sources is not clear. Antibus et al. (1992) showed that the

mycelium of four basidiomycete species could hydrolyse and take up 32P from radio-labelled,

insoluble lnsP6. Monoester-P forms, of which phytate is the major component, were reduced

in coniferous plantations grown on pasture soils as compared to the pasture control and



these activities were attributed to ectomycorrhizal fungi (Antibus et al., 1992). For AM fungi,

no such information could be found in the literature.

myo-inositol hexa/t/sphosphate

Figure 2.9 Model configuration of the myoinositol hexa/asphosphate-Goethite complexModified from Ognalaga et al (1994)

The current knowledge of the complex interplay of bacterial, fungal and root activities in the

rhizosphere with respect to P-mobilization from lnsP6 is still very limited. Whether, and to

which extent, substrate availability or enzyme activity, respectively, govern these processes

and how these processes are influenced by the different soil factors still have to be clarified.

Root-induced changes in the rhizosphere, such as secretion of organic acids or protons, may

significantly modify the adsorption/desorption properties of lnsP6, creating new opportunities

for enzymes to access the substrate. As a matter of fact, protons and organic acids such as

citric acid have been shown to play an important role in the solubilization of inorganic P

minerals and phosphate rock in a number of cases (Hedley et al., 1982; Hinsinger, 2001).

Hayes et al. (2000a) showed that only a small fraction of soil Po extracted by water and

NaHC03 was hydrolyzable by phytase, while the addition of citric acid resulted in the

solubilization of a greater amount of enzyme-labile Po. One may also ask the question as to

whether phytosiderophores and other chelating molecules can induce desorption of lnsP6.

Although there is no direct evidence for this hypothesis in the literature, some data suggests

that they could play a role in P mobilization (Shen et al., 2001). The lowering of soil pH by

active secretion of H+ from the roots, in contrast, is rather expected to decrease the solubility

of lnsP6 owing to precipitation reactions with Fe and Al-oxides (Jackman and Black, 1951).

Furthermore, the binding of lnsP6 to clays is dependent on the presence and type of cations

present in the soil solution and on soil pH (Celi et al., 2001). In fact, P sorption was more

strongly increased by addition of Ca2+ than of K+ to the solution, especially at higher pH

values. Ognalaga et al. (1994) showed that lnsP6 was adsorbed by ligand exchange to the



surface hydroxyls of Goethite and proposed a tentative configuration for the

organophosphate-Goethite complex (Figure 2.9).

Further research is thus required to better characterize and understand the processes

revolving around inositol phosphates in the soil and their availability for plant nutrition.

2.3.2 Phytases

A first reference to phytase in the literature dates from 1907 (Suzuki et al., 1907) and

describes phytase as an enzyme capable of lowering phytic acid content in rice-bran. This

first report opened the way to a large number of industrial applications for this enzyme,

particularly in animal nutrition. In fact, phytic acid is the major storage form of P in plant

seeds, as 60-90% of all organically bound P is found in this form (Plaami and Kumpalainen,

1997). In foods and feeds, phytic acid reduces the availability of inositol, phosphorus, and

essential minerals by forming non-assimilable salt complexes.

3-phytase

(EC 3.1.3.8)H203PO

H203PO

H203PO

OP03H2

OP03H2

H203PO

H203PO

H203PO

OP03H2

1 OP03H2H203PO

1 -D-myo-inositol hexatosphosphate

6-phytase

(EC 3.1.3.26)

1-D-myo-mositol 1,2,4,5,6-penta/flsphosphate

H203PO

H,0,PO

H203PO

OP03H2

3 OP03H2

1-L-myo-mositol 1,2,3,4,5-pentatosphosphate

(identical to 1-D-myo-mositol 1,2,3,5,6-pentatosphosphate)

Figure 2.10 Dephosphorylation of myo-lnsP6 by 3-phytase and 6-phytase Carbon position 4 in the

D-configuration corresponds to position 6 in the L-configurationModified from Dvorakova (1998)



Classification and properties of phytases

Phytase (myo-inositol hexa/c/'sphosphate phosphohydrolase) belongs to a group of

phosphoric monoester hydrolases catalyzing the hydrolysis of myo-inositol hexa/c/'sphosphate

to inorganic phosphate and lower phosphoric esters of myo-inositol. One classification

distinguishes between purple acid phosphatase, alkaline phosphatase, high- and low-

molecular-weight acid phosphatase and protein phosphatase (Vincent et al., 1992). Phytases

belong to a subfamily of the high-molecular-weight histidine acid phosphatases. Based on

the first phosphate group attacked by the enzyme, two types of phytases are distinguished

by the Enzyme Nomenclature Committee of the International Union of Biochemistry. 3-

phytase and 6-phytase. 3-Phytases are mainly associated with microorganisms, while 6-

phytases are found predominantly in plants (Cosgrove, 1969; Johnson and Tate, 1969a;

Cosgrove, 1970b; see Figure 2.10).

However, other dephosphorylation patterns have been described in the literature. For

example, Nakano et al. (2000) established by nuclear magnetic resonance spectrometry that

the dephosphorylation of lnsP6 by phytases from wheat bran followed two alternative

pathways (see Figure 2.11). Greiner et al. (2002) confirmed one of the two

dephosphorylation pathways for phytases from legume seeds using high performance ion

chromatography (HPIC) analysis and kinetic studies. The dephosphorylation of lnsP6 by a

phytase from Paramecium sp., however, appeared to occur in the sequence 6/5/4/1 (Van der

Kaay and Van Haastert, 1995).

Many naturally occurring phytases are thermostable up to 70 °C (Wyss et al., 1998; Wyss et

al., 1999b; Lehmann et al., 2000b; Simon and Igbasan, 2002). With respect to their pH

activity profiles, fungal phytases typically exhibit enzymatic activity within the range pH 2.5 to

pH 8.0 (Wyss et al., 1999a; Simon and Igbasan, 2002). The proteolytic stability of phytases

varies widely and strongly depends on the type of protease. For example, Aspergillus niger

phytase was found to be more resistant against pepsin than wheat phytase (Phillippy, 1999),

and E.coli AppA phytase exhibited even higher stability than A. niger phytase in the presence

of the same protease (Rodriguez et al., 1999; Golovan et al., 2000). A Bacillus phytase was

found to be extremely resistant to proteolytic degradation by papain, pancreatin and trypsin,

but appeared to be highly susceptible to pepsin (Kerovuo et al., 2000).

Plant phytases have not been characterised as extensively as microbial phytases. Of

importance to this work are root-secreted and soil-borne phytases.



Ins Pe

I

Ins (1,2,5,6) P4 Ins (1,2,3,5,6) P5 Ins (1,2,3,6) P4

I I

/ Ins (1,2,3) P3

Figure 2.11 Proposed dephosphorylation pathway of phytic acid by phytases from wheat bran

(Nakano et al, 2000)



Root-secreted and soil microbial phytases

Early in the 1950's, soil scientists started to investigate the role of the soil microbial

community in the dephosphorylation of soil-bound lnsP6. Jackman and Black (1952)

reported the presence of phytase activity in soils and correlated it to the occurrence of

microorganisms. In the same year, Courtois and Manet (1952) showed that some bacilli are

efficient in mineralizing inositol phosphates. Later on, Greaves et al. (1963) demonstrated

that a significant proportion of soil and root-surface microorganisms secreted enzymes

capable of P-hydrolysis from Na-phytate. Saxena (1964) published a first detailed study of

the role of plants in the overall inositol-phosphate mineralizing process, both in the presence

and absence of microorganisms. Since that time, a large number of papers have been

published showing that although some plants do secrete certain amounts of phytases, the

importance of these enzymes for the release of Pi from lnsP6 remains unclear.

More recently, Li et al. (1997c) measured phytase activity in root exudates of several plant

species under P deficiency, while Asmar (1997) reported different levels of phytase secretion

from barley roots between different genotypes. A number of authors reported on phytase

activity in crude protein extracts from roots (Li et al., 1997b; Hayes et al., 1999). The

relevance of phytase secretion in terms of mobilization of P from soil-bound phytate was not

assessed in either of these experiments. It is generally assumed that plants have low ability

to acquire P from phytate. In fact, although some authors have published that phytate could

be used as a P source by plants (Islam et al., 1979; Tarafdar and Claassen, 1988), others

have shown that this ability is, at most, restricted (Findenegg and Nelemans, 1993;

Richardson et al., 2000). Richardson et al. (2000) proved in experiments conducted in sterile

conditions that wheat plants were able to take up P from a variety of organic monoester-P

compounds but were largely unable to use phytate as a source of P. At the same time,

Hayes et al. (2000a) examined different soil Po substances as substrates for hydrolysis by

acid phosphatases and phytases. Phytases with narrow substrate specificity and high

specificity against phytate could hydrolyse only a limited proportion of Po extracted both with

NaHC03 and citric acid, while phosphomonoesterases with broader substrate specificity

were able to hydrolyze up to 79% of extracted Po. It thus appears that plants synthesize and

secrete phytases in modest amounts under P-starvation conditions, and that these phytases

are unlikely to play a significant role in the hydrolysis of soil phytate. On the other hand, soil

microorganisms, which are known to secrete large amounts of phytase, may have a function

in this respect. In fact, Richardson et al. (2001b) demonstrated that plant growth was

significantly improved in the presence of soil microorganisms effective in phytase secretion.

However, the response to inoculation was dependent on the source of microorganisms and

the growth medium. Cultured populations of soil microorganisms were more effective in

releasing P from lnsP6 than inoculation with a single isolate of Pseudomonas sp. selected for

its efficiency in P hydrolysis from phytate. The ability of phytases to mobilize P from phytates

in soils thus seems to be additionally related to other factors that may act in changing

substrate availability and affinity, as well as adsorption of released P.



Industrial applications of phytase

The use of phytase in animal feeding began in the 1960's, as one became aware that

monogastric animals were unable to digest phytic acid, resulting in a reduced uptake of P

(Nelson, 1967). The treatment of soybean meals with a mold phytase improved the uptake of

P from this diet (Nelson et al., 1968). The search for microorganisms producing phytases

resulted in the identification of a number of species particularly efficient in phytase synthesis.

Shieh and Ware (1968) tested over 2000 microorganisms isolated from soil and could isolate

only 30 secreting significant amounts of phytase. All phytase producing strains were

filamentous fungi, of which the genus Aspergillus accounted for 28, the other two belonging

to the genera Pénicillium and Mucor. Since then, a number of other organisms have been

shown to produce phytase, including Escherichia coli (Greiner et al., 1993), Pseudomonas

sp. (Cosgrove, 1970a; Richardson and Hadobas, 1997), Klebsiella terrigena (Greiner et al.,

1997), Bacillus subtilis (Kerovuo et al., 1998; Kim et al., 1998), anaerobic ruminai bacteria

(Yanke et al., 1998), Talaromyces thermophilus (Pasamontes et al., 1997b), and a few other

fungal strains (Wyss et al., 1998; Igbasan et al., 2000; Lassen et al., 2001).

The use of phytase as a feed additive is now widely spread and has been shown to improve

the availability of P in pigs (Jongbloed et al., 1992; Cromwell et al., 1993; Lei et al., 1993;

Simoes Nunes and Guggenbuhl, 1998) and broilers (Simons et al., 1990; Perney et al., 1993;

Broz et al., 1994; Denbow et al., 1995). As phytic acid is known as an anti-nutrient, especially

in the case of Fe, Ca, Zn, Cu, Mn (Cheryan, 1980; Simpson and Wise, 1990; Lonnerdal,

2002), the addition of phytase to diets has been shown to improve uptake of these elements

(Lei et al., 1993; Rimbach et al., 1995; Sebastian et al., 1996; Stahl et al., 1999). More

recently, transgenic mice and pigs expressing an E. coli phytase in their salivary glands were

able to use phytate as a source of P, resulting in a decreased excretion of P by up to 70%

(Golovan et al., 2001a; Golovan et al., 2001b). The development of transgenic plants

expressing fungal phytase genes for human and animal nutrition has also been pursued

(Brinch-Pedersen et al., 2000; Lucca et al., 2001; Brinch-Pedersen et al., 2002; Lopez et al.,

2002; Lucca et al., 2002; Zimmermann and Hurrell, 2002).

An alternative use of phytase has resulted from an increasing need of preparations of various

inositol phosphates and derivatives. The latter can be used as enzyme stabilizers (Siren,

1986), substrates for metabolic investigation, enzyme inhibitors, and as chiral building blocks

(Laumen and Ghisalba, 1994).

To be more effective in the digestive tract of animals and humans, and to resist the high

temperatures occurring during the pelleting process of feeds (60-90 °C; Simon and Igbasan,

2002), phytases need to exhibit particular properties like high thermo- and protease stability,

as well as low pH optimum. For this reason, the development of new, engineered phytases

has become a high priority. The next paragraph will summarize attempts to improve phytases

for applications in animal feeding, resulting in the development of so-called consensus

phytases, of which one has been used in the frame of this thesis.



Development of the consensus phytases at Roche Vitamins Ltd.

The development of the so-called consensus phytase proteins was preceded by the study of

fungal phytases (Pasamontes et al., 1997a). A genomic library of Aspergillus fumigatus was

screened for the phytase gene using a PCR fragment amplified with degenerate primers

derived from conserved sequences of A. niger phytase and other histidine acid

phosphatases. The purified A. fumigatus phytase recovered after heat-inactivation up to 100

°C with only a loss of 10% of its activity.

During the same period, the thermostability of three histidine acid phosphatases, i.e. A.

fumigatus phytase, A. niger phytase, and A. niger optimum pH 2.5 acid phosphatase, were

assessed. Of these, A. fumigatus phytase revealed the highest temperature stability

(refolding after exposure to >90 °C; Wyss et al., 1998).

Wyss et al. (1999b) overexpressed six different wild-type fungal phytase genes in either

filamentous fungi or yeast and purified the recombinant proteins. All proteins examined were

monomeric. Compared to Escherichia coli phytase, which is not glycosylated, all fungal

phytases exhibited highly variable patterns of glycosylation. The different extents of

glycosylation of single phytases expressed in different organisms did not affect their activity,

thermostability or refolding properties. Expressed in A. niger, the phytases were weakly

susceptible to proteolysis, and site-directed mutagenesis at the cleavage sites significantly

increased the resistance of the proteins towards proteolysis. It was concluded that

engineering of surface loops may increase the stability of the phytase protein in the digestive

tract of animals.

The biochemical characterisation of wild-type phytases was extended by characterisation of

the catalytic properties of the enzymes. At high enzyme concentrations, all of the considered

phytases were able to release five phosphate groups from phytic acid. The final product was

generally myo-inositol 2-monophosphate. A combination of phytases was able to release all

six phosphates. For some enzymes, the final products that accumulated were myo-inositol

tris- and bisphosphates, respectively. The specificity of activity towards phytic acid was high

in A. niger, A. terreus, and E. coli phytases, respectively, whereas A. fumigatus, Emericella

nidulans, and Myceliophthora thermophila phytases were able to hydrolyse a large spectrum

of phosphate compounds (Wyss et al., 1999a).

The study of these different enzymes led to the development of a synthetic, thermostable

phytase based on protein sequence comparison (Lehmann et al., 2000b; Lehmann et al.,

2000a). Although A. niger phytase is being successfully used in animal feeding, where it is

either added to feed pellets before or after the pelleting process, the need to develop more

themostable phytases remains. In fact, feed pelleting reaches temperatures between 60 and

90 °C, partially denaturing phytases added prior to pelleting. To improve thermostability, a

consensus enzyme was constructed using primary protein sequence comparisons of 13

fungal phytases. While the enzyme exhibited similar catalytic properties as each of its

"parents", there was a 15-22 °C increase in the unfolding temperature. The crystal structure

of the purified consensus phytase was determined and compared with known structures

(Lehmann et al., 2000b). Most consensus amino acids contributed to the stability of the

protein. Some consensus residues, however, predicted by structural comparisons to



destabilize the protein, in fact increased the unfolding temperature of the protein. On the

whole, protein sequence conservation and stability of proteins were correlated, confirming

the consensus concept for protein engineering.

In addition to the requirements in terms of thermostability, phytases used for animal feeding

should be active in the low pH range. For this purpose, A. fumigatus and consensus

phytases were engineered to exhibit high activity at low pH (Tomschy et al., 2002). In a first

attempt, glycinamidylation of the surface carboxy groups lowered the pH optimum but also

reduced the phytase activity by 70%. In a second strategy, active site amino acid residues

considered to be correlated to a low pH activity profile in other fungal phytases were

identified by sequence alignments and by three-dimensional structure analyses. Site-directed

mutagenesis of a number of residues in A. fumigatus wild-type phytase gave rise to a second

pH optimum at pH 2.8-3.4 (the first being at pH 6.0), while a single mutation in the consensus

phytase backbone could decrease the pH optimum with phytic acid as a substrate by

approximately 0.5-1 unit.

Recently, six new phytase sequences (additionally to the 13 previously used) were taken into

account for designing a new, more thermostable consensus phytase sequence (Lehmann et

al., 2002). Thirty eight amino-acid residues from the first consensus sequence were replaced

by newly ranked conserved residues at the respective positions in either one or both of the

new consensus phytases-10 and -11. It was observed that mutations could yield both

stabilizing and destabilizing effects. Single mutations always resulted in changes in unfolding

temperatures of smaller than 3 °C. The introduction of all stabilizing amino acid residues in a

multiple mutant of consensus phytase-1 resulted in an increase of the unfolding temperature

from 78 °C to 88.5 °C. The back-mutation of the four destabilizing amino acids, as well as the

introduction of a stabilizing residue in consensus phytase-10 further increased the unfolding

temperature from 85.4 °C to 90.4 °C. The improvements observed were the result of the

exchange of a specific combination of individual mutations rather than that of dominating

mutations alone, or the replacement of all amino acid residues considered with their

respective conserved residues.


3 Objectives and hypothesis 30

3\J Objectives of dissertation research

Plants respond to P deficiency by secreting higher amounts of phosphatases from the roots.

The first objective was to identify and study the expression of phosphatase genes in potato to

further the knowledge on the regulation of these genes and possible functions associated

with them.

• The first hypothesis is that there are genes encoding secretory phosphatases in

potato that are expressed in roots under P starvation.

• The second hypothesis is that these genes may be transcriptionally regulated via a

common signal transduction pathway.

Based on the fact that plants have a low ability to take up P from phytate in the soil, another

objective was to test whether the overexpression of a synthetic, secretory consensus

phytase gene in the root hairs could improve the Pi-acquisition efficiency of potato.

• The first hypothesis is that the expression of a synthetic secretory phytase in root

hairs will result in an increased phytase activity in the root exudates.

• The second hypothesis is that under conditions where lnsP6 is present in the soil

solution, plants secreting a phytase protein will possess an advantage over wild-type

plants in terms of P acquisition from lnsP6, both under sterile and non-sterile

conditions.

4 Materials and methods 31

4 Materials & Methods

4.1 Materials and chemicals

Bacterial strains

Escherichia coli DH5a (GIBCO, Basel, Switzerland)

Agrobacterium tumefaciens C58C1 pGV2260, Rif, Ampr (Deblaere et al., 1985)

Plasmid DNA

pBluescript SK (+/-) Ampr (Stratagene Europe, Amsterdam, Netherlands)

pBin19 Kanr (Bevan, 1984)

pCRScript Ampr (Stratagene Europe, Amsterdam, Netherlands)

pUC18 Ampr (Stratagene Europe, Amsterdam, Netherlands)

Enzymes

Restriction enzymes (MBI Fermentas GmbH, Nunningen, Switzerland)

T4 DNA ligase (MBI Fermentas GmbH, Nunningen, Switzerland)

Taq polymerase (MBI Fermentas GmbH, Nunningen, Switzerland)

Pfu polymerase (Stratagene Europe, Amsterdam, Netherlands)

SuperScript II, moloney murine leukemia virus reverse transcriptase (GIBCO,

Basel, Switzerland)

Mung Bean Nuclease (New England Biolabs)



Oligonucleotides

Oligonucleotides were synthesized at Microsynth GmbH (Balgach, Switzerland) in the

desalted grade at a scale of 40 nmol.

Kits

• QIAprep Spin Plasmid Kit (Qiagen, Basel, Switzerland)

• DNA Extraction Kit (MBI Fermentas GmbH, Nunningen, Switzerland)• Nucléon Phytopure Plant DNA Extraction Kit (Amersham Biosciences, Dübendorf,

Switzerland)• Prime-It II Kit (Stratagene Europe, Amsterdam, Netherlands)

• Megaprime DNA Labelling System (Amersham Biosciences, Dübendorf, Switzerland)• ABI PRISM Dye Terminator Cycle Sequencing Ready Reaction-Kit (Perkin Elmer,

Boston, USA)• HighPure PCR Product Purification-Kit (Roche Molecular Biochemicals, Mannheim,

Germany)

Radiochemicals

(a-32P) dCTP (Hartmann Analytics, Braunschweig, Germany)

KH232P04 (ICN Biomedicals, Eschwege, Germany)

General chemicals and materials

Agar, granulated

Antibiotics (kanamycin, ampicillin,

gentamycin, rifampicin)

a-naphthyl phosphate

ß-naphthyl phosphate

Biomax MS film

BSA (fraction V)

Daichin agar

Fast Black K

Fast Blue BB

Fast Red TR

Glyoxal 40%; 7.1 M Ultrapure

Difco (New Jersey, USA)

Sigma (Fluka, Buchs, Switzerland)

Fluka (Buchs, Switzerland)


Kodak (product ordering via Integra Biosciences,

Eschenbach, Switzerland)

Serva (Catalysis, Wallisellen, Switzerland)

Brunschwig Chemie (Basel, Switzerland)



Serva (Heidelberg, Germany)

Clontech (Allschwil, Switzerland)



Hybond NX membrane

Meat peptone

MicrospinTM S-400 HR columns

MS salts

Nitro-cellulose membranes

Phytic acid (from corn and rice)

Protease inhibitor cocktail

Timenten

Tryptone, yeast extract

X-Gluc

Zeatin riboside

Amersham Biosciences (Dübendorf, Switzerland)

Life Technologies Inc. (Basel, Switzerland)

Amersham Biosciences (Dübendorf, Switzerland)


Schleicher & Schuell (Dassel, Germany)



Smith Kline Beecham Pharmaceuticals (Thoerishaus,

Switzerland)

Difco (New Jersey, USA)

Biosynth AG (Staad, Switzerland)


All other chemicals were purchased from Fluka Chemie AG (Buchs, Switzerland)

Plants

Solanum tuberosum L. var. Désirée

Materials for plant growth

Sterile culture 2MS medium (Murrashige and Skoog, 1962), containing:- 4.3 g/l MS salts (Sigma, without vitamins)- 5 ml/l MS vitamins (see below)- 20 g/l sucrose

- 8 g/l agar (Difco)

medium set to pH 5.8 with 0.1 M KOH and autoclaved

MS vitamins:

0.1 g/l nicotinic acid

0.1 g/l pyridoxine HCl

0.02 g/l thiamine HCl

0.4 g/l glycine20 g/l myo-inositol

solution was dissolved, filtered through a 0.2 urn sterile filter

and stored at -20 °C

Aeroponics An aeroponic system (see figure 4.1) was developed in

collaboration with AIRWATECH (Bern, Switzerland).



Plexiglasshield

Temperatureand humidity

sensor

Temperaturesensor for

'

root chamber

Root growthchamber

Electronic

control unit

Air-flow

propeller

Nutrient

solution

reservoir

Water

pump

Alternative

outlet

Atomizing disc

Control valve

UV sterilizing lamp

Cooling unit

Figure 4.1 Schematic representation and photography of the aeroponic system used for the

controlled growth of plants and root systems.

Pot growth substrate Contained the following substrates:

• 10% loess subsoil from Frick, Switzerland, containing

550 mg total P / kg (obtained from Dr. Paul Mäder,

FIBL, Frick, Switzerland)

• 85% quartz sand 0.7-1.2 mm diameter

• 5% peat (Type P; Einheitserdewerk, Sinntal-Jossa,

Germany) containing 100 mg total P per kg dry matter.

Plant-available P in this substrate was determined to be 8 mg

Pi / kg dry soil by extraction with NaHC03 (Olsen et al., 1954).



4.2 Methods

4.2.1 Molecular biology

DNA recombinant methods

All standard DNA manipulation methods were done basically according to Sambrook et al.

(1989). Enzymes and kits used are described above.

DNA cloning of StPAPI, StPAP2, and StPAP3

The cDNA clone of StPAPI was isolated by phage screening of a cDNA library generated

from RNA originating from P-starved potato roots (Leggewie et al., 1997) using a sequence

fragment from the cDNA of AtPAPU (del Pozo et al., 1999) as a probe. A protein sequence

based BLAST search in the TIGR potato sequence database (http://www.tigr.org/tdb/tgi/stgi/)

revealed the existence of two further PAP expressed sequence tag (EST) clones. The clones

EST393242 and EST519948 were ordered at ResGen, Invitrogen Corporation (Carlsbad,

California, USA). The 5'-end sequence of EST393242 was lacking and cloned by the rapid

amplification of 5' cDNA ends method (5'-RACE; see below). The full length clones

corresponding to EST393242 and EST519948 were subsequently named StPAP2 and

StPAP3, respectively.

5'-RACE and PCR amplification

The missing 5'-end of the StPAP2 sequence was cloned using a modified protocol of a 5'-

RACE method described by Frohman et al. (1988).

First, a reverse transcription-PCR (RT-PCR) was performed on RNA of P starved potato

roots with primer A (5'-ttggtagctcttcctcaagcc-3'):

1 ul total RNA (2 ug/ul)

4 ul of 5x first strand buffer

1 ul of 10mM dNTP mix

1 ul of 10 uM primer A

2 ulofO.1 mM DTT

H20 to 18 ul total volume

This reaction was heated for 5 minutes at 65 °C and immediately cooled on ice. After

addition of 2 ul of reverse transcriptase, samples were incubated at 42 °C for two hours. The

resulting cDNA-RNA duplex was subsequently purified using the StrataPrep PCR purification

kit (Stratagene Europe, Amsterdam, Netherlands).

Second, an adaptor was created by heating to 95 °C and slow cooling to room temperature

of both complementary sequences 5'-gaccacgcgtatcgatgtcgacttttttttttttttttv-3' and 5'-

4.2 Methods


gtcgacatcgatacgcgtggtc-3' (Primer B). The adaptor was ligated to the cDNA-RNA duplex

strands by incubation of the following mixture overnight at 16 °C:

36 ul cDNA-RNA duplex

4 ul adaptor (20 pmol/ ul)

5 ul 10x ligation buffer (Fermentas)

5 ul T4 DNA ligase HC (Fermentas)

A first PCR was performed using primers A and B and 3 ul of the cDNA-mRNA duplex -

anchor ligation solution. Two sequential nested PCR amplifications were performed with

primer B in combination with primers C (5'-gtattgaggagtgtatttgccatatgc-3') and D (5'-

ctcgcttgattgaataccaaagtgg-3').

DNA cloning of the consensus phytase construct

A gene expression cassette (named LeExt1.1:.SP/PHY-OCS, see figure 4.2) was

constructed using the root hair-specific promoter LeExtl.1 from tomato (Bucher et al., 2002),

a secretory signal peptide sequence (SP) from ß-1,3 glucanase from barley (Leah et al.,

1991) and a gene encoding a thermostable consensus phytase (PHY) from Roche Vitamins

Ltd, Basel (Lehmann et al., 2000b). The PHY gene was synthesized using the maize codon

usage and was based on Consensus-Phytase-1 with four point mutations (Q24T, E32A,

R265I, G378A, with reference to the start methionine of the PHY gene). This numbering is

shifted by 3 amino acids as compared to the standard numbering used by Roche Vitamins

Ltd. In their system, the mutations would be numbered Q27T, E35A, R268I and G381A.

pBin19-LeExt1.1-SP/PHY-OCS

[Sal l/Ncol] Kpnl

Xbal I Smal

Neo I Smal

Smal'

Xbal '

Smal Pstl

Figure 4.2 Genetic construct used for the root hair-targeted

expression of a secretory phytase in potato roots

The LeExtl.1 promoter was cloned from a pBin19 vector (Bucher et al., 2002) into

pBluescript KS (-) (Stratagene, Amsterdam, The Netherlands) containing the gene terminator

sequence from octopine synthase (OCS). An additional ATG site at the 3' end of LeExtl.1

was removed using Mung Bean Nuclease (New England Biolabs). Suitable restriction sites

were introduced into the signal sequence and the phytase gene via PCR according to

standard procedures (Sambrook et al, 1989), and both sequences were fused in frame and

cloned downstream of LeExtl. 1. Between the predicted cleavage site of SP and the start

methionine of PHY there are four additional amino acids (IGVS), and the first amino acid

4.2 Methods


after the start methionine of PHY was mutated from S to G (see appendix 4). This construct

was sequenced and inserted into the binary vector Bin19 for plant transformation.

Sequencing

DNA sequencing was performed on DNA extracted from E. coli cultures with the QIAprep

Spin Plasmid Kit (Qiagen, Basel, Switzerland) and purified by NaAc-EtOH precipitation

(Sambrook et al., 1989). The sequencing PCR reaction was done with the ABI PRISM Dye

Terminator Cycle Sequencing Ready Reaction-Kit (Perkin Elmer, Boston, USA) and samples

were sent to Microsynth (Balgach, Switzerland) for sequence analysis.

RNA isolation and gene expression analysis

RNA extraction from potato (Verwoerd et al., 1995) and RNA gel blot analysis were

performed as previously described (Bûcher et al., 1997). Radioactively labelled cDNA

fragments of roughly 600-900 bp were used (PHY: BamHI fragment at 3'-end; StPAPI: Ncol-

Ncol fragment; StPAP2: Xhol-Ncol fragment including 3' non-translated region; StPAP3:

Ncol-Smal fragment). Hybridisation was performed with 5 X SSC, 5 X Denhardt's and 0.5%

SDS (w/v) at 65 °C with a final washing using 0.1 X SSC and 0.1% SDS at 55 °C.

4.2.2 Physiological and biochemical measurements

ß-Glucuronidase (GUS) assay

Plant roots from either root tip cultures, hairy root cultures, or from aeroponically grown

plants were stained for ß-glucuronidase activity by vacuum-infiltration for 2x1 min in GUS-

staining solution and incubation at 37 °C for 30 min to 4 h.

GUS staining solution:

0.1 g X-Gluc dissolved in DMF

1ml 10% Triton X-100

5 ml 1 M sodium phosphate buffer, pH 7.2

mixed in a final volume of 100 ml and stored in aliquots at -20 °C

Microscopy

Root hair length was measured by digital analysis of root photographs obtained from the

stereo-microscope (Olympus SZX12, Olympus Optical Co., Tokyo, Japan) using SIS image

analysis software (Soft Imaging System, Munster, Germany).

Tissue sections were photographed at 50x to 200x magnification under a compound

microscope Olympus Provis AX70 (Olympus Optical Co., Tokyo, Japan).

4.2 Methods


Protein extraction from roots and determination of protein concentration

Crude protein extracts from roots were prepared as described by Aarts et al. (1991) using an

extraction buffer containing 100 mM Tris-acetate at pH 7.9, 100 mM potassium-acetate, 10%

(v/v) glycerol, 2 mM EDTA, 0.1 mM PMSF, 5 mM DTT and 250 mM sodium-ascorbate. Three

ml extraction buffer were used for protein extraction from 1 g root fresh weight. Protein

concentration in root extracts was measured basically according to Bradford (1976) relative

to standard solutions of bovine serum albumin (BSA). One volume of protein extract (10-20

ul, usually around 20 ug) was diluted into 39 volumes of water and 10 volumes of Bradford

reagent (BioRad Laboratories, Inc.). After 10 min incubation, optical density was measured

spectrophotometrically by absorbance at 595 nm.

Enzymatic activities in crude protein extracts

Total Phosphomonoesterase (PMEase) activities were determined by incubating 20 ug crude

protein extract in 25 mM MES buffer at pH 5.5 containing 10 mM p-nitrophenyl phosphate

(pNPP), 5 mM cysteine and 1 mM EDTA for 20 min at 37 °C. The reaction was stopped by

addition of a half sample volume of 0.5 M NaOH. The activity was calculated from the

production of p-nitrophenol as determined by spectrophotometry at 410 nm relative to

standard solutions. For phytase activity measurements, crude protein extracts were diluted to

a final protein concentration of 0.1 ug/ul. Two ug of protein extract was mixed with 49

volumes of 25 mM MES buffer at pH 5.5 containing 2 mM lnsP6 (Sigma, Cat. No. P8810),

1 mM EDTA, and 5 mM cysteine. Phytase activity was calculated from the release of

phosphate (Pi) as determined spectrophotometrically at 610 nm relative to standard solutions

using the malachite green method (Ohno and Zibilske, 1991) and after subtraction of

concentrations of contaminant Pi in reaction solutions and protein extract. Enzyme activities

were calculated as mU per unit protein content, where 1 unit (U) releases 1 umol Pi / min

under the conditions described above.

Collection of root exudates and measurement of enzymatic activities

To determine enzymatic activities in root exudates, rooted potato cuttings were grown

aeroponically for two weeks, carefully removed from the aeroponic system and rinsed three

times in deionized water. Roots were then incubated in a buffer (exudation solution)

containing 5 mM maleate buffer, pH 5.5, 2% sucrose, 2 mM CaCI2 and 0.01% (v/v) protease

inhibitor cocktail (SIGMA, Cat. No. P9599). Plants were illuminated with cool white

fluorescent tubes with a mean photon flux density of 110 umol m"2 s"1 at canopy level.

Exudates were collected 90 min after start of incubation and assayed for PMEase and

phytase activity. For PMEase activity, exudation solution was mixed with an equal volume of

100 mM MES buffer at pH 5.5 containing 10 mM pNPP, 5 mM cysteine and 1 mM EDTA, and

incubated for 60 min at 37 °C. The reaction was stopped by addition of half a volume of 0.5

M NaOH and the activity calculated as descibed above. For phytase activity measurements,

exudation solution was mixed with 250 mM MES buffer (9:1, v/v), pH 5.5, containing 5 mM

lnsP6 (Sigma P8810), 1 mM EDTA, 5 mM cysteine, and incubated for 60 min at 37 °C. The

reaction was stopped with half a volume of 15% trichloroacetic acid (TCA). Phytase activity

4.2 Methods


was calculated from the release of inorganic phosphate as described above (Ohno and

Zibilske, 1991). Enzyme activities were calculated as mU per unit root fresh weight or per

root tip, where 1 unit (U) releases 1 umol Pi / min under the conditions described above.

Visual staining for Phosphomonoesterase activity

Visual staining for PMEase activity on roots was adapted from the description given by

Dinkelaker and Marschner (1992). The staining solution was a 50 mM Tri-Sodium-Citrate

(TSC) buffer adjusted to pH 5.5, containing 37.5 mM a-naphthyl phosphate and 2.7 mM Fast

Red TR. To obtain darker colors, Blue B was added at equal amounts (w/w) to Fast Red TR

to the staining solution.

Native Polyacrylamide Gel Electrophoresis (PAGE) of a thermostable phytase

Native PAGE was performed basically as described (Bucher et al., 1994). Eight ul of loading

buffer was mixed to an aliquot of protein extract containing 25 ug of total protein. Because

the recombinant phytase was thermotolerant, but endogenous phosphatases were not,

samples were heated for 10 minutes at 70 °C prior to loading to the gel. Gel electrophoresis

was performed during 2 h in the BioRad Mini-Protean II apparatus cooled on ice and set to

150 V and 80 mA (for two gels). The gel was stained for PMEase activity during 2 h in a 50

mM TSC buffer containing 0.5 mg/ml Fast Black K and 0.3 mg/ml ß-naphthyl phosphate.

HPLC analysis of the time course of accumulation of phytic acid degradation

intermediates

23000

18000

=; 13000

TO

c

•2> 8000CO

3000

-2000

Time [min]

Figure 4.3 Example of HPLC analysis of inositol phosphates using the method described by Egh (2001)

Roots of plants grown aeroponically for two weeks were washed in deionized water and

incubated in 50 ml of 5 mM maleate buffer, pH 5.5, containing 2 mM CaCI2, 0.01% protease

inhibitor cocktail (Sigma, P9599) and 2 mM lnsP6 from rice (Sigma, cat. no. P3168). Every

hour after beginning of incubation, 200 ul and 20 ul samples were collected for PMEase and

4.2 Methods

lnsP3 lnsP5

lnsP4

lnsP6

123456789


phytase activity measurements, respectively. In addition, 1 ml was sampled every hour for

HPLC analysis, set on ice, and phytase activity stopped by addition of 0.5 ml 15% TCA.

Samples were centrifuged to remove cell debris and 1.45 ml of the supernatant was adjusted

to pH 2-3 by addition of 1.4 ml 0.5 M KOH, purified through an anion exchange resin and

used for HPLC analysis basically as described (Egli, 2001). Peaks were obtained for lnsP6,

lnsP5, lnsP4 and lnsP3. lnsP2, InsPI and Ins could not be resolved from background noise

(Figure 4.3). Because the sum of InsP forms was relatively constant (+/- 6%), values for the

sum of lnsP2, InsPI and Ins were calculated as the difference from the total quantity of initial

lnsP6 to the sum of other forms present in solution.

Concentrations of total P, Ca, Cu, Fe, Mg, Mn, Na and Zn in plant tissue

Leaf, root and tuber samples (approx. 1 g) were harvested and plant tissue dry weight was

measured after drying at 80 °C for 36 h (leaves, roots) or 72 h (peeled tuber slices). Dried

samples were incinerated at 550 °C for 8 h. The ash was solubilized with 2 ml of 6.0 M HCl

(20% vol.), shortly heated to 100 °C, filtered through Whatman No. 40 ashless filter papers

and diluted to 50 ml with double distilled water (see figure 4.4).

Figure 4.4 Schematic representation of the extraction of total P from ashes from plant tissue

Phosphate concentration in the extracts was measured by the malachite green method

(Ohno and Zibilske, 1991). Additionally, the concentrations of P and other elements (Ca, Cu,

Fe, Mg, Mn, Na and Zn) were measured in these extracts using ICP emission-spectroscopy

(Varian Liberty 220 equipped with an ultrasonic nebulizer CETAC U-5000 AT+, Varian Inc.,

Palo Alto, CA, USA).

4.2 Methods


Measurement of soluble Pi in leaves

To measure soluble Pi content in plant tissue, leaf samples (approx. 1 g) were harvested and

an extract was prepared as described (Hurry et al., 2000). Pi measurements in the extract

were done using the malachite green method (Ohno and Zibilske, 1991).

Plant growth parameters

Total root and shoot dry weight were measured after drying in an oven for 72 h at 80 °C.

Total leaf area was measured using a portable leaf area meter (LI-COR, LI-3000A combined

with the LI-3050A Transparent Belt Conveyer Accessory).

4.2.3 Plant growth conditions and tissue harvest

Sterile culture

Wild-type and transgenic potato plants (Solanum tuberosum var. Désirée) were propagated

in-vitro under sterile conditions in glass pots containing 100 ml of 2MS medium (Murrashige

and Skoog, 1962) supplied with various sources of P and set at pH 5.8. Plants were grown at

22 °C with a 16 h/8 h light/dark cycle.

Preparing plants for culture in the greenhouse

For experiments in the greenhouse, plants were first grown in quartz or under aeroponic

conditions supplied with half-strength Hoagland's solution. After excision of the primary

shoot, lateral shoots were allowed to grow for 1-2 weeks. Lateral shoots (3-5 cm long) were

excised and transferred to stonewool supplied with deionized water for 1 week and with %

Hoagland solution for another week. Rooted potato cuttings were either transferred to an

aeroponic system (Figure 4.5) or to solid substrates.

Figure 4.5 Use of lateral shoots from potato plants for rooting and growth under aeroponic conditions.

4.2 Methods


Aeroponics

A spinning-disc operated aeroponic system (see Figure 4.1) was supplied with half-strength

Hoagland's solution at pH 5.5 and cooled using an external cooling facility set at 5 °C. The

temperature in the root growth chamber varied between 18 °C and 20 °C. Roots were

sprayed for 1 minute every 4 minutes (or 1 minute every 2-3 minutes in summer). Plant

shoots were exposed to daylight or to a minimum photon flux density of 100 umol m"2 s"1 at

canopy level with incandescent light (Philips sodium bulbs, HPL-N 400 W) and to

temperatures between 22 °C and 25 °C.

Harvest of root hairs

Roots of aeroponically-grown potato plants were removed from the aeroponic system and

immediately frozen in liquid nitrogen. After gently breaking larger root pieces with a plastic

pipette, the liquid nitrogen containing root pieces was mechanically stirred with a glass rod

for 15-20 minutes. Every 5 minutes, the liquid nitrogen was filtered through a 200 urn mesh

and new liquid nitrogen added to the roots. The filtering procedure was continued until

sufficient root hairs were harvested. The quality of the harvested tissue was verified under

the microscope (Figure 4.6).

Figure 4.6 (A) Harvest of root hairs from plant roots grown aeroponically using liquid nitrogen and a

200 urn mesh filter. (B) Photograph of root hairs harvested with this method; bar size is 50 urn.

Nutrient starvation experiments

For nutrient starvation analysis, ten plants were grown under aeroponic conditions with half-

strength Hoagland's solution until the root system reached an average length of 20 cm. To

start starvation, the aeroponic system was rinsed with deionized water and the nutrient

4.2 Methods


solution replaced with half-strength Hoagland without P, where (NH4)H2P04 was replaced by

equimolar amounts of NH4CI. Plants were starved for P for eight days and resupplied with P

from day 8 to day 16. Approximately 1-2 g of root material was harvested at each time point

for RNA extraction and Northern blot analysis.

Measurement of plant-available P in substrates

To obtain an estimate of soil P available for plant uptake, P was extracted from substrates

using sodium bicarbonate (NaHC03), which decreases the ionic activity of Ca2+ by

precipitation of CaC03, thus increasing P solubility (Olsen et al., 1954). P was extracted

during 30 min by incubation in a 0.5 M NaHC03 solution at pH 8.5, with a soil-solution ratio of

1 g : 20 ml at ambient temperature. After filtration of the extracts (Sartorius, mesh 0.2 urn), P

concentration was determined using malachite green colorimetry (Ohno and Zibilske, 1991).

Mycorrhization of roots

To test the effect of mycorrhization on gene expression of StPAPI, StPAP2 and StPAP3,

mycorrhized and non-mycorrhized roots were obtained as described by Rausch et al. (2001)

and RNA was extracted from these roots.

Split-root experiment

A split-root experiment in the aeroponic system was devised to study possible gene

regulatory effects of leaves and P status on PAP expression (Figure 4.7). Root systems of

four plants per treatment were split into two equal parts and sprayed with half-strength

Hoagland's solution without P for seven days. Subsequently, in treatments (a) and (b) one

half of the root system was supplied with nutrient solution containing P, while in treatment (c),

both halves remained starved as a negative control. In (a) and (c), plants were not defoliated,

whereas in treatment (b) all leaves were removed except for the three youngest visible

leaves at the shoot tip to test whether PAP expression in roots is regulated by a signal

originating from source leaves. At times 0 (just before resupply), 4 h and 24 h after P

resupply, 3 root tips (approximately 3 cm long) were harvested from each part of the root

systems and the PMEase activity of secreted phosphatases was subsequently assessed. An

average value was calculated for each part of the root systems, and the average for each

treatment was calculated from the resulting four values. Approximately 1 g of root material

was harvested in both parts of root systems for each treatment at time 0, 4 h and 24 h for

RNA extraction and gene expression analysis. In addition, one to two leaves per plant were

harvested at the three time points for the measurement of soluble Pi.

4.2 Methods


Figure 4.7 Aerial view of a modified setup of three aeroponic systems (1, 2 and 3) for use in a tri-

chamber split-root design. The root systems of four plants per treatment (a, b, c) were split into two

and each half exposed to a different growth chamber supplied from a separate aeroponic device.

Pot experiments

A substrate was established containing 85% quartz, 10% loess subsoil from Frick and 5% of

a peat-derived substrate (Typ P). Rooted potato cuttings were transferred to this substrate

mixture (4 kg per pot) and were irrigated with deionized water and supplemented twice a

week with 300 ml of half-strength Hoagland either without P, or with 100 uM Na-lnsP6 (myo¬

inositol hexa/c/'sphosphate dodecasodium salt; SIGMA, Cat. No. P8810; Mr = 932 g/mol).

Each plant was thus supplemented with 56 mg Na-lnsP6 per week, corresponding to

approximately 35 mg Pi or 11 mg P. Eight plants per treatment were cultivated in a

randomized complete block design in a conventional greenhouse under daylight,

supplemented by incandescent light from Philips sodium bulbs (HPL-N 400 W) with a

maximum of 120 umol s"1m"2 at canopy level. Relative ambient humidities varied between 40-

80% and temperatures were between 22-28 °C during the day and 18-22 °C during the night.

The experiment was carried out over a period of five weeks. Element concentrations in

leaves were measured in week three, while biomass production and leaf area were

measured at the end of the experiment.

4.2 Methods


4.2.4 Computer analyses

Statistics

All numerical data was analysed by one-way ANOVA using SYSTAT 10.0 (SYSTAT

Software Inc., CA, USA). LSD-based F tests were performed at a 5% and 1% significance

level (P<0.05 and P<0.01, respectively) to identify significant differences between treatment

means.

Restriction site analysis

Restriction site analysis was performed online at:

• Webcutter (http://www.firstmarket.com/cutter/cut2.html) and at

• N EBcutter (httpJ/tools. neb. com/NEBcutter/index.php3)

BLAST

Searches for similar sequences in nucleotide and protein databases were performed using

BLAST (Altschul et al., 1990):

• NCBI, National Center for Biotechnology Information

(httpJ/www. ncbi. nlm. nih. gov/blast/)

• TIGR, The Institute for Genomic Research (http://www.tigr.org/tdb/tgi/)

Sequence alignments

Sequence alignments were performed at various sites:

• Multalin (http://prodes.toulouse.inra.fr/multalin/multalin.htmf)

• ClustalW (Thompson et al., 1994) at EBI, European Bioinformatics Institute

(http://www. ebi.ac. uk/clustalwl)

• Other sources listed in

httpJ/www. techfak. uni-bielefeld. de/bcd/Curric/MulAli/welcome. html

Phylogeny analysis

Phylogenetic relationships were inferred using web-applications of the PHYLIP Program

Package using the neighbour joining algorithm (Felsenstein, 1993) at http://bioweb.pasteur.fr/

4.2 Methods


Signal sequence prediction analysis

Signal sequence prediction servers are available at:

• CBS, Center for Biological Sequence Analysis

(http://www. cbs. dtu. dk/services/SignalP-2.0/)

• Leeds University (http://bioinformatics.leeds.ac.uk/prot_analysis/Signal.html)

GPI-anchoring signal prediction analysis

The analysis for prediction of GPI-anchoring sites was performed at:

• http://mendel. imp. univie. ac. at/sat/gpi/gpi_server. html

• http://129.194.185.165/dgpi/index_en.html

4.2 Methods

5 Potato purple acid phosphatases 47

5\tw Purple acid phosphatases from potato

*

5.1 Introduction

The secretion of acid phosphatases from plant roots in response to P deficiency is thought to

play a major role in the mobilization of phosphate (Pi) from organic P sources in the

rhizosphere and in P scavenging (Duff et al., 1994). Although there are numerous reports

describing increased phosphatase activities in plant root exudates under P starvation, few

genes encoding secretory phosphatases which are expressed in roots have been isolated so

far (Deng et al., 1998; Nakazato et al., 1998; del Pozo et al., 1999; Haran et al., 2000;

Wasaki et al., 2000; Miller et al., 2001). Those isolated belong to different gene families, of

which the purple acid phosphatase (PAP) family is of particular interest due to the large

number of its members and therefore the possible diversity of functions.

Purple acid phosphatases comprise a family of metal-containing glycoproteins that catalyse

the hydrolysis of a wide range of phosphate esters and anhydrides. Members of this group

have been identified in plants, animals and fungi (Oddie et al., 2000; Schenk et al., 2000a).

In plants, two families of different molecular weight have been identified (Nakazato et al.,

1998; Schenketal., 2000b).

The functions of PAPs in plants are still unclear. Five plant PAPs have been implied in the P-

starvation responses of Arabidopsis thaliana, Lupinus albus and Spirodela oligorrhiza

(Nakazato et al., 1998; del Pozo et al., 1999; Wasaki et al., 2000; Miller et al., 2001). AtACP5

(recently renamed AtPAPU; Li et al., 2002) was shown to be additionally involved in

oxidative stress by exposure to H202. The high number of PAP genes found in plants and the

structural and biochemical diversity of PAPs may reveal a multiplicity of functions originating

from this gene family. At the same time, functional investigations may be hindered because

of genetic and functional redundancies (Li et al., 2002).

We report the isolation and characterisation of three novel PAP genes from potato and their

pattern of expression in different tissues. The primary structure of the encoded PAP proteins

gives indications to their possible roles in plant metabolism. In a split-root experiment, we

examine some regulatory aspects related to the expression of these three genes in potato

roots.

*

Major parts of this chapter were submitted for peer-reviewed publication with the title "Differential regulation of three purpleacid phosphatases from potato", by Philip Zimmermann, Babette Regierer, Jens Kossmann, Emmanuel Frossard, Nikolaus

Amrhein and Marcel Bucher

5.1 Introduction


5.2 Results

Secreted Phosphomonoesterase activity of potato roots

The activity of PMEase secreted from sterile potato roots grown in P-sufficient and P-

deficient conditions was visualized on agar by the intensity of precipitated red a-Naphthol-

Fast Red complex (Dinkelaker and Marschner, 1992). Potato roots responded to P deficiency

by an increased activity of secreted PMEase all along the root (Figure 5.1 (A)). Exudates

from root tips showed higher activities, both in P-sufficient and P-deficient conditions, than

the older parts of the roots. The induction of PMEases appeared to be more pronounced in

the root tips than in other root zones.

B

460 aa 330 aa

-< 1 1

1 23 4

1

5 67

stPAPi mm i I 1 1

stPAP2 warn i 1 1 1 11

StPAPS WÊÊKÊ 1 I I i 1

| Metal coordinating amino acid residues

M Predicted secretion signal sequence

Predicted GPI-anchoring signal sequence

Figure 5.1 (A) Potato roots stained for activity of secreted phosphomonoesterases under P-

sufficient (+P) and P-deficient (-P) conditions. (B) Primary structures of three PAPs from potato with

lengths of 328 (StPAPI), >448 (StPAP2) and 477 (StPAP3) amino acids. All three sequences have

a predicted secretory signal sequence. StPAP2 is predicted to possess a sequence encoding a

GPI-anchoring signal. Seven metal coordinating amino acid residues characteristic for PAPs are

indicated. Sequences are aligned according to the first conserved residue.

5.2 Results


Cloning of StPAPI, StPAP2 and StPAP3

StPAPI is 984 bp long and contains an open reading frame encoding a 328-amino acid

polypeptide with a molecular mass of 34.8 kDa. This polypeptide has sequence homology to

the family of PAP proteins with low molecular weight (LMW; -35 kDa). Members of the LMW

family are found in mammals, plants, fungi and cyanobacteria (Schenk et al., 2000a) and are

relatively well conserved in sequence (Figure 5.2).

A sequence similarity search through the TIGR potato expressed sequence tag (EST)

database (http://www.tigr.org/) revealed the existence of at least two more potato PAPs

expressed in roots (EST393242 and EST519948), of which the cDNAs are referred to here

as StPAP2 and StPAP3, respectively. StPAP2 is -1390 bp long and contains an open

reading frame encoding a - 462 amino acid polypeptide, whereas StPAP3 has 1431 bp

encoding 477 amino acids. The protein sequences derived from both genes show homology

to the second family of PAP proteins with higher molecular weight (HMW; - 55 kDa). StPAP2

exhibits a high degree of sequence identity (58%) to StPAP3, while both have a low degree

of identity to the LMW protein StPAPI The mature proteins of StPAPI, StPAP2 and StPAP3

(after cleavage of the N-terminal signal peptide sequence) have 304, 441 and 456 amino

acids, respectively. (At the time of writing this thesis, the N-terminal sequence of StPAP2

was still lacking approximately 10-15 amino acids (see Figure 5.3)).

Protein sequence analysis

Protein sequence analysis revealed that the three potato PAPs show structural features

typical for PAPs. In fact the analysis of the N-terminal sequences of the three potato PAP

amino acid sequences using the signal IP program (Nielsen et al., 1997) indicated that all

three proteins contain a predicted secretory signal sequence (Figure 5.1 (B)). This structure

is common to many previously described PAPs (Hegeman and Grabau, 2001; Li et al.,

2002). Five conserved motifs containing the seven described residues involved in metal

binding are found throughout all compared sequences (Figures 5.2 and 5.3). GPI-prediction

analysis using the algorithm described by Eisenhaber et al. (1999) indicated that the C-

terminal end of StPAP2 may contain a GPI-modification signal, which was not detected in the

other two PAPs.

Protein sequence alignments between StPAPI, 2 and 3 and other plant, animal and

cyanobacterial PAPs show a high degree of conservation throughout all kingdoms (Figures

5.2 and 5.3). A proposed phylogenetic tree based on the neighbour-joining method (PHYLIP

Program v. 3.5) reveals two distinct families of PAPs in plants, with StPAP2 and StPAP3

belonging to the HMW PAPs (-55 kDa) and StPAPI belonging to the LMW PAPs (-35 kDa),

the latter group also comprising families of PAP proteins from mammals and cyanobacteria

(Figure 5.4).

5.2 Results


AtPAP17 1 MNSGRRSLMSATASLSLLLCIFTTFVWSNGELQRFIEPAKSDGS|SE|JVAAF6 0315 1 MAVYSGISMVLCLWVGWFGVCLASAIVELPTFHHPTKGDGSJSfIvStPAPI 1 KYMASMKILNIFISFLLLLLFPAAMAELHRLEHPVNTDGslsEjlvAAF60316 1 MGTQRSKPSCTIVAIFLAFCCFVSSSKAKLESLQHAPKADGsjsFjjVAAF60317 1 MAGLG¥WLAFIGVCFLNVSALLQRLEHPVKADGs|jsL|V

AAL34 937 1 MAVALALLAAMSALSSCTSPATAELTRHEHPVAAGApIrlIv

P1368 6 1 MDMWTALLILQALLLPSLADGATPaIreÏaNP 485726 1 MNLKRRQFLFLSSLSAVGTGLLAWKFAHKYYQSSDLAIASPPKKDLlIreÏs

AtPAP17

AAF60315

StPAPI

AAF60316

AAF60317

AAL34937

P13686

NP 485726

-RRBSFNQS LVAYŒ

-RKjDYNQS QVAFQ

-RF*TFNQS---QVAQQ|-RKjAYNQS LVAFQ

-RKjTYNQS EVSAQ

-rkIgynqt—rvaeq!

gvpn|pfhtaremanake|artvqil&t[argqy—avara|||tlyhkqnpy

GKIGEKIDL

GEIGDQLAI

GIIGEKLNI

GVIGEKLDV

GRVGAKLNI

GKVAEETEI

LFSEHDPN

LTGEHDD,

LTGVDDP,

LTGVFDPS

LSGVDDP

LAGVDDM,

vqdindk:

EIEKVNA

ÏEQS

|TES

EES

EES

ELS

HDS

jERP|fcg

AtPAP17

AAF60315

StPAPI

AAF60316

AAF60317

AAL34937

P13686

NP 485726

SYTAPSLQKQ-fi--Ytaeslqkq|ysvl|

loi tnIytapslqkn-IynvlI

106

103 SNiYTAPSLQKQjYSVLl

ytapslqkk-IynvlIytakslqkq-Iysvlytaqslhkp-1ylvl|fsdrslrkvpIyvl.

lkqg vkj§qacl|

jLSSVLREIDSRWICLRSl|lsshlrkldsrwpclrs||lspilkqkdnrwicmrs|jlSHVLRYRDNRWVCFRsi|LNTILQKIDPRWICQRs|jlDPALRKIDSRFICMRsiIIA—YSKISKRWNFPSP1

GDPQVRYPGFNMNGRRYI

WDAELVEMFF

1WNTETVDLFF

'IVNTDVAEFFF

(TLNSENVDFFF:IVDTEIAEFFF

:IVSAGIVDFFF

[YRLHFKIPQTNlITFRRDRVQFFA

AtPAP17

AAF60315

StPAPI

AAF60316

AAF60317

AAL34937

P13686

NP 485726

165 DTTPFVKEYYT

162 DTTPFVEEYFN

160 DTTPFQDMYFT

162 DTTPYVDKYFI

154 DTTPFVDKYFL

157 DTTPFQLQYWT

14 9 SVAIFMLDTVT

160 DTN

EADGHSYDWRAVPSRNSYVK-AL

SPE-HVYDWRGVFPQQTYTK-NV

TPKDHTYDWRNVMPRKDYLS-QV

EDKGHNYDWRGILPRKRYTS-NL

KPKDHTYDWTGVLPRDKYLS-KL

DPGEDHYDWRGVAPRDAYIA-NL

LCGNSDDFLSQQPERPRLTART

SNADWQN

lEVSjKSSKA:IeyaImkstt:

Ire s sa:

|rqstat|jkdsta:ikkstati

AAARE

QÜIekeIsssnap|

AtPAP17

AAF60315

StPAPI

AAF60316

AAF60317

AAL34937

P13686

NP 485726

224 IGHH

22 0 AGHH

219 AGHH

221 IGHH

213 IGHH

216 VSAH

209 IAEH

197 SGVY

dtkelneell

dtkelverll

sseelgvhil

dtqellihfl

dtqelirhll

dtqellelll|pthclvkql

SNQAFIKTFTl

CLQHMSDEDSPIC

SLEHISDDESPIC

CLEHISSSDSPLC

CLEHISSLDSSVC

CLEHISSTSSQI

CLEHISSRNSPI

NLQYLQDENG-Vi

SYERTRAIDG-TTl

SKAWRGDI

SKAWRGDV

SKSWRGDM

SKAWRGDT

SKAWKGDH

SKAWRGIF

INFMDPSKR—AGNR

AtPAP17 284 NPVTINPKLLKFYYDGQ'

AAF60315 280 TMDRKGVSFFYDGQ'

StPAPI 27 9 N—WWNPKEMKFYYDGQ'

AAF60316 281 K—QSEGDEMKFYYDGQ'

AAF60317 273 L—IKMGKMGQRFTMMD'

AAL34 937 27 6 Q QNEDKLQFFYDGQ'

P13686 268 HQRKVPNGYLRFHYGTEDS

NP 485726 252 P VGRSKWTEYSTSD—

LGG

MSARFTHSDAEIVFYDVFGEILHKWVTSKQLLHSSV-

iMSVQLVETDIGIVFYGC

iMAMQITQTQVWIQFFDIFGNILHKWSAS-KNLVSIM-

(MSVHISQTQLRISFFDVFGNAIHKWNTC-KFDSSDM-

ILQVWRFKKSIPKLFIMIFLAKFCKLLICPRGYVMCMP

1LSLELSENRARFAFYDVFGEALYHWSFSKANLQKVQS

iAYVEISSKEMTVTYIEASGKSLFKTRLPRRARP

LSlATYEVYPDRIELNAIATNNRIFDRGIIRRVEVSGV-

AtPAP17

AAF60315

StPAPI

AAF60316

AAF60317

AAL34937

P13686

NP 485726

327 YNSLI—

32 9 SASVTEE

Figure 5.2 Multiple sequence alignment of LMW PAP ammo acid sequences from potato (StPAPI),

soybean (AAF60316), Arabidopsis (AtPAP17), sweet potato (AAF60315), red kidney bean

(AAF60317), rice (AAL34937), human (P13686) and Nostoc sp (NP_485726) Conserved ammo

acid residues involved in metal binding are indicated with asterisks Ammo acids conserved

throughout all sequences aligned are shaded in black, while those conserved to a lower degree but

having similar biophysical properties are shaded in grey Predicted signal peptides are underlined

5.2 Results


SGPTSGEVTSSStPAP2 1

IbPAP2 1 MGASRTGCYLLAWLAAVMNAAIAGITSS

GmPAPl 1 —MGWEGLLALALVLSACVMCNGGSSSP

PvPAPI 1 —MGWKGLLALALVLNWWSNGGKSSN

LaPAP2 1 MGYSSFVAIALLMSWWCNGGKTSTI

StPAP3 1 MLLHIFFLLSLFLTFIDNGSAGITS

StPAP2

IbPAP2

GmPAPl

PvPAPI

LaPAP2

StPAP3

HVE WSEKSKLKNKAN

WSENSQHKKVAR

WSENSDKKKIAE

WSEKNGRKRIAK

WSDSSLQNFTAE

GLSEGKYDVTVE

k|tt In T

n|rt T| In T

iJIvt R1J In S

kJIst Rjl In S

EjjjjFT Tl In T

t|nn T|«Be

StPAP2

IbPAP2

GmPAPl

PvPAPI

LaPAP2

StPAP3

StPAP2

IbPAP2

GmPAPl

PvPAPI

LaPAP2

StPAP3

StPAP2

IbPAP2

GmPAPl

PvPAPI

LaPAP2

StPAP3

StPAP2

IbPAP2

GmPAPl

PvPAPI

LaPAP2

StPAP3

StPAP2

IbPAP2

GmPAPl

PvPAPI

LaPAP2

StPAP3

StPAP2

IbPAP2

GmPAPl

PvPAPI

LaPAP2

StPAP3

81 LP Mil TE puÉMifl s

99 LPm TE ünSi s

97 LP

8_\

TE Sural s

7 0 LR SE liiflÉSÉl s

95 LP

95 FK

SE

EK si

Ie|YA|EA|WW

niyS ByaIea

m ^nn|tt

kiwvnIrvIhp jdestaaks—

smwvs|rfIhp Iddstttkl—slwsf|ry|hp Iddstahvsh-svwffBrhIyp IDDSTklwlf|ry|np Indstihip—sftlhBqybgsglrrrklnknhInsviserpfs

StPAP2

IbPAP2

GmPAPl

PvPAPI

LaPAP2

StPAP3 475 ARL

Figure 5.3 Multiple sequence alignment of HMW PAP ammo acid sequences from potato (StPAP2and StPAP3), lupin (LaPAP2), sweet potato (lbPAP2), soybean (GmPAPl) and red kidney bean

(PvPAPI) Conserved ammo acid residues involved in metal binding are indicated with asterisks

Ammo acids conserved throughout all sequences aligned are shaded in black, while those

conserved to a lower degree but having similar biophysical properties are shaded in grey Predicted

signal peptides are underlined

5.2 Results


Plants

AtPAP12

lbPAP3

IbPAPI

AtPAPIO

StPAP2 |bPAP2N

PvPAPI.

GmPAPl~

LaPAP2

AtPAPU A1PAP25

I,

StPAP3

AtPAP5

1000

AtPAP20

AtPAP22

11000/

GmPhy

AtPAP15

AtPAP13

LaPAPI -

SoPAPI

974

1000/945 S60

HMW

1000

^1000728

_

StPAPI

/lOOO

1000

::;^~~~"~" AAF60316

NNs/*^^- AtPAP3

\ \\. AtPAPS

/999\ \. AAF60315

B27035 s^j4 / ,8111\ AtPAP17

pnme'V AVAA

\ AAF60317

P09889

AF292105AAL16925 / L16924 AAL34937

/ AAL16926

! |NP_485726

i I

I

Mammals Plants

LMW

Cyanobacteria

Figure 5.4 Phylogenese relationships between proteins identified as PAPs from mammalian,

cyanobacterial and plant origin based on CLUSTALW protein alignment and the neighbour joining

method in the PHYLIP program 1000 bootstrap replicates were used to generate the consensus

tree Bootstrap values are indicated for major branches One can distinguish between high

molecular-weight (HMW) and low molecular-weight (LMW) proteins, respectively The three potatoPAPs reported in this work are shaded Plant proteins mentioned in the literature are indicated with

their respective names, others are given by accession number Mammalian PAPs shown are from

Bos taurus (B27035), Homo sapiens (P13686), Mus scrofa (AF292105) and Sus scrofa (P09889)

Cyanobacterial PAPs are from Aphanizomenon sp (AAL16924), Aphanizomenon baltica

(AAL16926), Nodulana spumigena (AAL16925) and Nostoc sp (NP_485726) PAPs from plant

origin are from Arabidopsis thaliana (AtPAP3-AtPAP25, see also Li et al, 2002), Glycine max

(GmPAPl (AF200824), GmPhy (AAK49438), and AAF60316), Ipomoea batatas (IbPAPI

(AAF19821), lbPAP2 (AAF19822), lbPAP3 (CAA07280), and AAF60315), Lupinus albus (LaPAPI

(AB023385) and LaPAP2 (AB037887)), Phaseolus vulgaris (PvPAPI (S51031) and AAF60317),

Oryza sativa (AAL34937), Solanum tuberosum (StPAPI, StPAP2, StPAP3), and Spirodela

oligorrhiza (SoPAPI (AB039746))

5.2 Results


Tissue-specific expression of StPAPI, StPAP2 and StPAP3

To determine the expression patterns of the three PAP genes in different plant organs, we

extracted RNA from potato plants grown in an aeroponic system either containing or lacking

Pi. Root tissue was harvested from root tips (distal 2 mm), root hair elongation zone

(encompassing the zone from 5 to 10 mm distance from the root tip) and root hair zone (at

least 30 mm distant from the root tip), respectively. Each PAP had a specific pattern of

expression distinct from those of the other two, as determined by RNA gel blot analysis.

StPAPI was strongly expressed in roots and stem, but also was moderately expressed in

young leaves (Figure 5.5 (A)) and at intermediate levels in stolons and flowers (data not

shown). This gene was not responsive to P starvation. StPAP2, in contrast, responded

strongly to P deficiency stress and was expressed highly in roots and less in leaves and

stem. Similar to StPAP2, StPAP3 was P starvation-inducible, but showed highest expression

in the stem, intermediate levels of expression in roots and moderate levels of expression in

leaves (Figure 5.5 (A)).

Secreted phosphatases are presumed to function in P mobilization from organic P sources in

the rhizosphere. Since all genes were expressed in roots, we analyzed gene expression

patterns more precisely in different root fractions to get an indication of the precise sites of

expression and possible functions of the encoded proteins in the roots. Root hairs and

stripped roots, as well as root tip, elongation zone and root hair zone tissues, respectively,

were isolated from aeroponically-grown plants in P-deficient conditions. RNA gel blot

analysis showed that StPAPI was expressed both in root hairs and in whole roots, while

transcripts of StPAP2 and StPAP3 were almost absent from root hairs (Figure 5.5 (B)). The

latter two showed higher levels of expression in the root tips than in the elongation and root

hair zone, respectively. StPAPI transcripts, on the other hand, were more abundant in the

root hair zone and elongation zone, where root hair development is initiated, but were almost

absent at the root tip (Figure 5.5 (B)).

Induction of expression after P deprivation

Aeroponically grown roots were harvested at different time points after initiation of P-deficient

conditions and resupply of P. Expression analysis of RNA extracted from these tissues

revealed that StPAP2 was the most strongly P starvation-inducible PAP gene, with intensity

of induction comparable to the P starvation marker gene StPT2, which encodes a high-

affinity phosphate transporter (Leggewie et al., 1997; Figure 5.5 (D)). StPAPI expression

was not induced under P deficiency, while StPAP3 expression was moderately activated by

low-P conditions in comparison to its homolog StPAP2.

Effect of mycorrhizal symbiosis

To test whether mycorrhizal infection and symbiosis would affect the abundance of PAP

transcripts in roots. RNA was extracted from plants grown with and without mycorrhizal

association under both P-sufficient and P-deficient conditions (Rausch et al., 2001). For

StPAPI, StPAP2 and StPAP3, no effect of mycorrhizal colonisation was observed on the

5.2 Results


level of gene expression in root tissues (Figure 5.5 (C)), while transcripts of the mycorrhiza-

specific Pi transporter StPT3 were absent in non-mycorrhized roots but highly abundant in

infected roots (Rausch et al., 2001). (Roots from the sames plants were used for the

experiments presented in Rausch et al. (2001) and for the experiment described here).

A

StPAPI

StPAP2

StPAP3

rRNA

rRNA

o

ooDC

OO

oo a

LU

ooa.

£

a.

E

Figure 5.5 Transcript levels of StPAPI, StPAP2 and StPAP3. (A) Gene expression in young

leaves, old leaves, stem and roots both under P-sufficient (+P) and P-deficient (-P) conditions. (B)Gene expression in different root tissues under P-deficient conditions. (C) Effect of mycorrhizationon the expression of StPAPI, StPAP2 and StPAP3 in roots with (+myc) and without (-myc)

mycorrhizas, both under P-sufficient (+P) and deficient (-P) conditions. (D) Analysis of PAP gene

expression in potato roots at different time points after transfer to P-deficient growth conditions. At

day 8, plants were resupplied with P until day 16. The high-affinity P transporter StPT2 is used as a

P starvation inducible marker gene.

Regulatory aspects of expression of StPAPI, 2 and 3

A split-root experiment in an aeroponic system (see chapter 4.1) was carried out to identify

conditions involved in the regulation of phosphatase expression in roots. Plants were initially

starved for P for 7 days and resupplied with P for the remaining period of investigation. The

addition of high levels of Pi to one half of the plant roots resulted in a decreased activity of

secreted PMEase for both halves of the separated roots (Figure 5.6 (A)). This reduction

started to be measurable within four hours after application of Pi to one half of the root

5.2 Results


system and was statistically significantly different (P<0.05) 24 h after resupply of Pi. In plants

from which older leaves had been removed (treatment b) just after beginning of resupply (t =

0), the decrease in PMEase activity was not statistically significant (P<0.05) but showed a

trend towards lower values after 24 h (Figure 5.6 (B)). In plants continuously supplied with

nutrient solution without P, in contrast, the PMEase activity remained constant in both parts

of the root system (Figure 5.6 (C)). In all three treatments, there was no significant difference

between both parts of the root system. Total soluble inorganic P (Pi) in leaves remained

constant in all three treatments within the experimental period (Figure 5.6 (D)).

D

ct! 300 -

£

0 4 24

time (h)

*- 0.0.0-0.0.0.0.0.0.+ i

00©000'Bt'*t'Bt

(UrUJ2.QUU(?'fl!.2

stpAP2 • it 11 m • Äfc m mÛ. û. Û- Û. û. û.

£ û. Û. + +"*" ' ' -* Tt Tf ** Tl" Tt

CU fU -O -Q

StPAP2 Ü

Time (h)

Figure 5.6 Phosphomonoesterase (PMEase) activities in exudates of root tips, and gene

expression StPAP2 in P-starved roots after P resupply in a split-root setup. Half of the root

systems remained in P-deficient conditions (-P), while the other half was sprayed with a nutrient

mist either with (+P) or without (-P) phosphate. (A-C) PMEase activity of exudates from root tipsfrom both halves of the root systems of plants possessing all leaves (A and C) or stripped of the

fully developed leaves (B). In (A) and (B), half of the root systems was resupplied with P after time

0 (grey columns), while the other halves remained under P starvation conditions (black columns). In

C, both halves of the root system remained in P-deficient conditions as a negative control. (D)Soluble Pi content in leaves of plants from treatments A-C. (E) Gene expression of StPAP2 in roots

of plants grown in the conditions as described above.

5.2 Results


Gene expression analysis of StPAP2 in roots of both parts of the root systems in the

treatments A to C (see also Figure 4.7) did not correlate with the levels of secreted PMEase

measured in root tips from these same parts (Figure 5.6). It appears that there are

irregularities in gene expression throughout the experiment. In fact, there is no consistent

relationship between P nutrition and level of gene expression, except perhaps for treatment

C, the roots of which were continuously lacking P in the nutrient solution, and from which the

levels of StPAP2 transcripts were generally higher. These results could be attributed to the

sampling of the roots, or to unknown factors influencing the expression of StPAP2 in roots.

Since transcripts of StPAP2 are more abundant in root tips than in other root parts (Figure

5.5), the proportion of root tips harvested can be expected to influence the level of gene

expression in harvested root tissue and may yield an explanation to the inconsistencies

observed. The measurements of secreted PMEase activity, in contrast, were performed with

root tips of similar length and age, which was not possible for RNA extraction, for which

larger amounts of root tissue were required.

5.3 Discussion

The primary structures of StPAPI, StPAP2 and StPAP3 reveal the presence of at least three

distinct types of PAPs in potato (Figure 5.1). StPAPI is a LMW protein homologous to

mammalian and cyanobacterial PAPs, while StPAP2 and StPAP3 are HMW types unique to

plants. StPAP2 may have a predicted putative GPI anchoring signal for anchoring of the

protein in the plasma membrane (Figure 5.1). Plant homologs to StPAPI were found in

Ipomoea batatas (AAF60315), Glycine max (AAF60316) Arabidopsis thaliana (AtPAP8 and

AtPAP17), Phaseolus vulgaris (AAF60317) and Oryza sativa (AAL34937). The

characterisation of the expression of a LMW PAP at the transcript level in plants has been

reported only in Arabidopsis (del Pozo et al., 1999). This gene was found to be expressed in

roots and shoots under P-deficient conditions, but not in the presence of P. Furthermore,

promoter-GUS fusions showed activation of the GUS gene under other types of stresses,

such as oxidative stress under hydrogen peroxide (del Pozo et al., 1999). GUS staining was

not found in the stem, in contrast to expression of StPAPI in potato plants. StPAPI is

furthermore expressed both in P-deficient and P-sufficient conditions and appears to be

expressed in most tissues analyzed. One can therefore expect it to have a more general role

in plant metabolism, probably not related to the plant's P-starvation response.

HMW plant PAPs have been characterized in Spirodela oligorrhiza (Nakazato et al., 1998;

Nishikoori et al., 2001), white lupin (Wasaki et al., 2000; Miller et al., 2001) and soybean

(Hegeman et al., 2001). With the exception of a PAP with homology to phytases that was

induced in cotyledons during germination of soybean seedlings (Hegeman et al., 2001), all

other PAPs appeared to be regulated by P-starvation. In the present work, transcripts of both

StPAP2 and StPAP3 started to accumulate within a few hours after transfer of the plants to

medium without P (Figure 5.5 (D)). Infection by mycorrhizal fungi did not affect gene

expression of StPAP2 and StPAP3 in roots under P-deficient conditions (Figure 5.5 (C)).

Another type of phosphatases has been shown to be induced by pathogen attack in potato

5.2 Discussion


leaves infected with Pseudomonas syringae pv. maculicola and Phytophthora infestans

(Petters et al., 2002), but so far no plant phosphatase gene could be associated with

mycorrhizal infection.

All reported HMW plant PAPs contained a predicted signal sequence for secretion. The

functions of these genes were thus assumed to be related to P nutrition and P remobilization.

The regulation of these genes has been poorly investigated. Whether gene expression is

controlled locally upon environmental stimuli, or systemically is not known. The measurement

of phosphatase activity of root tips from two parts of the roots of potato plants that were P-

deficient revealed that resupply of P to one part of the root system affected secreted

phosphatase activity in both parts (Figure 5.6 (A)). In fact, both parts of the root system of the

plants reduced their excretion of phosphatase to a similar extent, while plants that were

continuously starved maintained the level of phosphatase secretion in both parts of the roots

(Figure 5.6 (B)). These results would indicate that there is a systemic signal regulating gene

expression of secreted phosphatases in potato. To test whether this signal is Pi, Pi

concentrations in leaves were measured. In all treatments there was no significant

difference, at any time point of this experiment, between plants supplied with P in part of their

root systems and plants that were maintained in P starvation conditions. Stripping the shoot

of all leaves except for the three youngest visible leaves resulted in a delayed reduction in

phosphatase activity. From this experiment alone, it is not possible to conclude whether Pi

concentrations in leaves are in fact involved in the sigaling cascade controlling gene

expression of PAPs in roots. However, on can hypothesize that post-transcriptional events

may control the level of PAP protein concentration and activity in the cells.

5.2 Discussion


5.2 Discussion

6 Consensus phytase 59

\# Expression of a consensus phytase in

potato root hairs *

6.1 Introduction

Plants are sessile organisms, and their survival and productivity depend largely on their

ability to cope with the local environmental conditions. To tolerate stress originating in the

rhizosphere, plants must exhibit a specific subset of genetically controlled mechanisms of

which many are targeted to the root-soil interface. The knowledge of such mechanisms has

provided new opportunities for the breeding or genetic engineering of crop plants more

tolerant to rhizosphere stress. To date, a limited number of transgenic plants have been

developed that show a potential for improved tolerance to rhizosphere-related stress

(Samuelsen et al., 1998; Kasuga et al., 1999; Richardson et al., 2001a; Takahashi et al.,

2001a; Zhang and Blumwald, 2001). The model plants thus developed expressed the

transgenes either constitutively or simultaneously in several plant parts, under the direction

of the constitutive cauliflower mosaic virus (CaMV35S) or other promoters not specifically

driving expression in roots, respectively.

The constitutive expression of certain transgenes may result in metabolic disorders and

growth retardation, as was recently illustrated by the constitutive expression of a stress-

inducible transcription factor (Kasuga et al., 1999). In contrast, the expression of this gene

under the control of its own promoter had no detrimental consequences. Therefore, the

analysis of tissue-specific gene expression and the availability of suitable promoters is a

prerequisite for the successful application of molecular-genetic tools towards modification of

root properties (Atkinson et al., 1995; Bücher, 2002). Expression of endogenous or

heterologous genes can then be targeted in a precise spatial and temporal manner to alter

root characteristics, avoiding undesired side-effects.

Root hairs have a large surface area which is in direct contact with the soil environment.

They make up between 70% and 90% of the total root surface area (Bates and Lynch, 1996).

Root hairs are tubular extensions of root epidermal cells extending from the root surface by

tip growth. This cell type has been shown to play a dominant role in a number of root

*

Major parts of this chapter were submitted for peer-reviewed publication with the title "Engineenng the root-soil interface via

targeted expression of a synthetic phytase gene in tnchoblasts", by Philip Zimmermann, Gerardo Zardi, Martin Lehmann,

Christophe Zeder, Emmanuel Frossard, Nikolaus Amrhein and Marcel Bücher

6.1 Introduction


functions. For example, membrane proteins responsible for nutrient uptake from the soil

solution have often been localized to the root epidermal layer, including root hairs. Nitrate

and ammonium transporter genes were reported to be expressed in root hairs (Lauter et al.,

1996; von Wren et al., 2000). Phosphate, sulfate and ammonium transporters have also

been localized to the external root layers, including root hairs (Daram et al., 1998; Hartje et

al., 2000; Takahashi et al., 2000; Yoshimoto et al., 2002). Two genes related to iron

mobilization and uptake were predominantly expressed in the external root cell layers (Vert

et al., 2002; Waters et al., 2002). Increases in root hair length and density in response to Fe

and P deficiency have been reported (Bates and Lynch, 1996; Ma et al., 2001; Schmidt and

Schikora, 2001), and the study of root hairless mutants revealed an important role of root

hairs in nutrient uptake from the soil solution (Bates and Lynch, 2000, 2001). In addition, root

hairs are instrumental in the establishment of the Rhizobium symbiosis in legumes (Kalsi and

Etzler, 2000; Cullimore et al., 2001; Wubben et al., 2001) as well as in the anchorage of the

plants in the soil (Bailey et al., 2002). Promoters directing root hair-specific expression in

crop plants are therefore of advantage in engineering new traits in biotic and abiotic stress

tolerance.

Here, we report on the use of a synthetic phytase gene and its tissue-specific expression to

engineer plants able to modify the rhizosphere for improved plant nutrition. We show that the

targeted secretion of the synthetic phytase exhibiting higher stability to thermal inactivation

and protease degradation (Wyss et al., 1999b; Lehmann et al., 2000a) in root hairs of potato

induces changes in the rhizosphere which result in higher P mobilization from substrates

containing phytate.

6.1 Introduction


6.2 Results

Potato root hair growth in P-deficient conditions

The number, density and elongation of root hairs increase in response to nutrient stress in a

number of species (Bates and Lynch, 1996; Gahoonia et al., 1997; Gilroy and Jones, 2000;

Jungk, 2001). Root hairs are sites of phosphate transporter (Daram et al., 1998) and H+-

ATPase (Moriau, 1999) activity, respectively, both of which are necessary for P uptake from

the soil solution. To investigate how potato root hairs respond to P starvation, we measured

root hair length and the expression of the high-affinity phosphate transporter gene StPT2

(Leggewie et al., 1997) in root hairs of aeroponically grown potato plants both in P-deficient

and P-sufficient conditions. After 4 days of P deprivation, average root hair length of newly

grown root hairs increased by approximately 40%, from an average of 390 to 570 urn, while it

decreased to control levels after resupplying the roots with P for four days (Figure 6.1 (B)).

Under low-P conditions, the variability of root hair length was high, varying from 400 to 850

urn for 90% of the root hairs (Figure 6.1 (A)). Roots supplied with sufficient P, however,

developed root hairs with a more homogeneous hair length distribution, 90% of root hairs

having lengths between 300 and 550 urn. Due to the high density of hairs, it was not possible

to measure root hair density, which has been reported to increase in response to P starvation

in other plant species (Jungk, 2001). It is safe to assume, however, that the root surface area

increased (as a minimal estimate) in proportion to root hair length, i.e. 40%, under P

deficiency.

The expression of StPT2 in root hairs was induced within 6 hours of P deprivation and

reached high levels within 4 days, while little or no StPT2 transcripts were detected in the

control plants supplied with P (Figure 6.1 (C)). The data confirmed the role of potato root

hairs in increasing both root surface area and phosphate uptake. Root hairs are thus suitable

target cells for the expression of transgenes involved in P mobilization and uptake.

The root hair-specific promoter LeExt1.1

The root hair-specific promoter LeExt1.1 of tomato (Bucher et al., 1997; Bucher et al., 2002)

was used to direct ß-glucuronidase (GUS) reporter gene expression in potato. As in tomato,

expression was root hair-specific and was additionally found in dry pollen, growing pollen

tubes and occasionally in vascular tissues of potato tubers (Figure 6.1, (D) and (E)). GUS

activity was observed in root hairs, but not in root tips, of transformed hairy roots and of

potato plants grown either in tissue culture, in an aeroponic system, or in the soil (data not

shown).

6.2 Results


Root hair length (pm)

Figure 6.1 Root hair growth and gene expression analysis. (A) Distribution of root hair lengths of

roots grown under high and low phosphorus (P) conditions. Insets show root segments of

aeroponically grown potato roots at high and low P. (B) Average root hair length of roots grown

permanently in P-sufficient conditions (black columns), and under 4 days of P starvation and after

four days of resupply, respectively (grey columns). (C) Expression of the high-affinity phosphate

transporter gene StPT2 in root hairs after 6 h, 24 h and 4 days of P deprivation (-P) and under P-

sufficient conditions (+P) during the same period. (D and E) Expression analysis of the GUS gene

under the control of the root hair-specific promoter LeExt1.1 in whole plants (D) and in longitudinalroot section, dry pollen, germinating pollen, root tip, transverse section, and potato tuber slice (E,from left to right and top to bottom, respectively).

Generation of transgenic potato lines expressing a consensus phytase

To test the suitability of root hairs to achieve a modification of the rhizosphere, we

engineered root hairs to secrete the enzyme phytase. To this end, a gene expression

cassette was constructed (Figure 6.2 (A)) using the LeExt1.1 promoter (Bucher et al., 1997;

Bucher et al., 2002) and a consensus phytase gene (PHY; Lehmann et al., 2000b) fused to

the barley ß-glucanase signal peptide (Leah et al., 1991; Figure 6.2 (A)). Transgenic potato

lines obtained after leaf-disc mediated genetic transformation using Agrobacterium

tumefaciens were tested for expression of the PHY gene by RNA gel blot analysis. A number

of lines with high (lines 120 and 124) or intermediate (lines 127 and 129) levels of PHY

transcripts were used for further analysis (Figure 6.2 (B)). The transcript levels of PHY

increased moderately in aeroponically grown roots under P deficiency as compared to plants

6.2 Results


supplied with control levels of P. This may reflect the higher metabolic activity of root hairs

when plants are exposed to P nutrient deprivation stress.

Figure 6.2 (A) T-DNA fragment used for plant transformation containing the root hair-specific

promoter (LeExt1.1), an ER targeting signal sequence (SP), the consensus phytase gene (PHY)and the octopine synthase terminator sequence (OCS). (B) Expression of the PHY gene in different

transgenic lines and in response to P deprivation (from day 0 to day 8) and after resupply of P (from

day 8 to day 16), with the Pi transporter StPT2 as a P-starvation marker gene. (C) PMEase activity

staining of heat-treated roots of wild-type (left) and transgenic line PHY129 (right) using a-naphthyl

phosphate and Fast-Red TR / Blue B reagents. (D) Phosphomonoesterase (PMEase) activity

staining of a native Polyacrylamide gel of heat-denatured crude protein extracts from different

transformed lines, as well as of authentic purified phytase, using the same reagents as in (C).

The consensus phytase properties are maintained in transgenic plants

As the consensus phytase is thermotolerant (Lehmann et al., 2000b), visualisation of the

activity of the recombinant PHY protein was achieved using standard staining methods for

Phosphomonoesterase (PMEase) activity after heating the potato roots to 90 °C for 5

minutes to inactivate endogenous root PMEases. Roots from transgenic plants (PHY129)

had high PMEase activity, while roots of untransformed plants had no PMEase activity under

these conditions (Fig. 6.2 (C)). In order to verify that the PMEase activity observed was

related to the phytase protein, heat-inactivated crude protein extracts were subjected to

PAGE under non-denaturing conditions and stained for PMEase activity. Only a single band

migrating in the gel like authentic consensus phytase was found in each of the transgenic

lines, while no activity was detected in the extracts of control plants (Fig. 6.2 (D)).

In non-denaturated crude protein extracts from roots, total PMEase activity was 30% higher

in PHY124 as compared to wild-type (Figure 6.3 (A)). The pH activity profiles for total

PMEase were similar for both PHY124 and wild-type, with an optimum at pH 5.5 (data not

shown).

Specific phytase activity was measured as Pi released from phytate added to the crude

protein extracts. The phytase activity of crude protein extracts was more than 4 times higher

in PHY124 than in wild-type plants (Fig 6.3 (B)). This activity results from both cellular

phytase and secreted phytase bound to the cell walls of root hairs.

6.2 Results


B

400 200

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Figure 6.3 Specific enzymatic activities in crude protein extracts and exudates from roots. PMEase

(A) and phytase (B) activities in crude protein extracts from roots of wild-type (WT) and transgenic

(PHY124) lines, respectively. PMEase (C) and phytase (D) activities in root exudates of WT and

PHY124, respectively. (E) pH activity profile of phytase activity in root exudates from wild-type (o)

andPHY124(«).

The PHY protein is secreted from the roots

To be able to hydrolyze P from phytates in the soil, the PHY protein must be secreted from

the root hairs into the rhizosphere. To verify this, plants were grown in an aeroponic system,

in which root hair growth is not impaired and roots are not injured upon removal, and were

subsequently incubated in a buffer for collection of exudates. Total PMEase activity in root

exudates of PHY124 plants was 2 to 3 times higher than in controls (Figure 6.3 (C)). The

level of specific phytase activity secreted by wild-type plants was hardly above background,

whereas high levels of activity were measured in exudates collected from line PHY124

(Figure 6.3 (D)). The pH optimum of the root-secreted phytase activity was around pH 6.0

(Figure 6.3 (E)), which is similar to the pH activity profile as determined for recombinant

consensus phytases expressed in yeast (Lehmann et al., 2000b).

Kinetics of phytic acid degradation by root exudates

Fungal and consensus phytases hydrolyze myo-inositol hexa/c/'sphosphate (lnsP6) to lower

inositol phosphates following a particular degradation sequence (Wyss et al., 1999a). We

therefore determined the kinetics and intermediates of phytic acid degradation by HPLC of

potato root exudates. The roots of transgenic and wild-type plants, grown aeroponically

under low P conditions, were incubated for 16 hours in a buffer containing 2 mM lnsP6 as the

6.2 Results


sole P source. Samples were collected every hour to determine myo-inositol phosphates.

PMEase and phytase activities were monitored during the entire period (Figure 6.4 (A) and

(B)). The PMEase activity followed a sigmoidal pattern for both PHY124 and wild-type, with

an initial lag phase and a saturation of product accumulation after 6 h (Figure 6.4.(A)).

Phytase activity, measured as Pi released to the incubation solution, was very low for control

plants, while Pi accumulated rapidly in exudates of PHY124 after an initial lag phase of 6 h,

the rate levelling off after 16 h of incubation (Fig 6.4 (B)). HPLC analysis of inositol

phosphates revealed that exudates from wild-type roots were unable to degrade lnsP6

(Figure 6.4 (C)), while PHY124 exudates rapidly degraded lnsP6 with concomitant

accumulation of lnsP2, InsPI and InsP (Figure 6.4 (D)). lnsP5 and lnsP4 were observed as

intermediates. The calculation of the rates of degradation of intermediates revealed that

lnsP4 and lnsP5 were more rapidly degraded than lnsP6 and lnsP3 (data not shown). These

findings confirm the kinetics of phytic acid degradation observed in fungal and bacterial

phytases (Wyss et al., 1999a) and furthermore provide proof that the root-secreted

recombinant phytase is able to degrade phytic acid present in soluble form in a liquid

medium.

A B

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80

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0 2 4 6 8 10 12 14 16 18

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Figure 6.4 Phosphomonoesterase activity in, and kinetics of phytic acid degradation by root

exudates of wild-type (WT) and transgenic (PHY124) plants (A) Phosphomonoesterase activity in

root exudates of PHY124 (o) and WT (•) plants (B) Release of Pi from phytic acid by root exudates

from PHY124 (o) and WT (•) (C and D) Intermediates of phytic acid degradation by root exudates

of WT and PHY124 plants, respectively Percent values indicate percent of total InsP forms presentin solution, (•) lnsP6, (o) lnsP5, (T) lnsP4, (V) lnsP3, () lnsP2 + InsPI + Ins

6.2 Results


Phenotype of PHY plants

Having established that the transgenic plants excrete enzymatically active consensus

phytase under aeroponic conditions, we next tested whether plants can utilize phytic acid as

a P source under various growth conditions. Potato cuttings grown in sterile conditions were

starved for P during two growth cycles prior to transfer to agar containing either no P (-Pi), Pi

as Na2HP04 (+Pi) or as Na-lnsP6 (+lnsP6). No significant differences in growth were

measured between PHY124 and wild-type plants, respectively, in the treatments -Pi and +Pi.

However, in the +lnsP6 treatment, PHY124 produced a 30% higher biomass than wild-type

(data not shown).

Since PHY124 plants were obviously able to utilize lnsP6 as a source of P in sterile

conditions, in contrast to control plants, we tested whether this effect could be observed

under non-sterile conditions in an artificial substrate containing quartz (85%), loess (10%)

and peat (5%). Ten day-old rooted potato cuttings were transferred to this substrate supplied

with half strength Hoagland solution containing 100 uM lnsP6 as sole source of P. After ten

days of growth, first differences were visible in plant height, and these differences were

observed throughout the experiment (i.e. during 4 weeks). Three week-old PHY124 plants

were up to 20% taller than wild-type (Figure 6.5 (B)). Leaf shape of some leaves was altered

in the transgenic plants, with a predominant growth of the terminal leaflet and reduced

growth of the lateral leaflets (Figure 6.5 (C)). The ratio of leaf area to shoot dry weight was

significantly higher in the wild-type as compared to PHY124 (P<0.05), concomitant with a

higher root-shoot ratio in the wild-type plants (Figure 6.5 (A)). This finding may reflect the P

status of the plant. In fact, PHY124 had a 40% higher total P concentration in leaves

(P<0.01) as compared to wild-type. As plants of both genotypes had similar dry matter

production, the total P mobilization and uptake from the soil substrate was 40% higher in line

PHY124 than in wild-type. With respect to micronutrients, no statistically significant increases

in Fe, Ca, Zn and Mn were measured (P<0.01; Figure 6.5 (A)). In a parallel experiment,

however, PHY124 plants exhibited significantly higher concentrations not only of P, but also

of Fe and Zn in the tubers when grown in substrates supplemented with Na-lnsP6 (data not

shown). Both results confirm that transgenic plants secreting phytase via their root hairs are

able to take up additional P from an unsterile, P-sorbing substrate supplemented with phytic

acid in soluble form in the nutrient solution. Moreover, phytase-mediated P-mobilization from

phytate may interfere with the acquisition of some micronutrient elements.

6.2 Results


A

1) DW shoot DWroot DW total Root/shoot Leaf area/

(g) (g) (g) Ratio (%) DW shoot

(cm2 / g)

PHY124 8 63 ±0 16 161 ±0 15 10 24 ±0 23 18 7 68 4 ± 1 9 *

WT 8 44 ±0 20 178 ±0 11 10 22 ±0 28 211 75 5 ± 1 6

2)a P Ca Fe Zn Mn

PHY124 4076 ±276** 8390 ± 646 103±70 32 2 ± 2 4 36 6 ± 1 6

WT 2886 ±242 8918 ±663 99 ± 4 5 27 1 ± 1 9 38 8 ± 1 5

aAN values are given m mg/kg dry weight

**

Significant at P < 0 01*

Significant at P < 0 05 n=8

PHY 124 WT

Figure 6.5 Growth response of transgenic (PHY124) and wild-type (WT) plants in a soil substrate

(A) Growth parameters and total nutrient concentrations in leaves of PHY124 and WT (B)

Phenotype of PHY124 and WT (C) Shape of some leaves from PHY124 and WT plants

6 2 Results


6.3 Discussion

To test the approach of biochemically modifying the root-soil interface via root hair-mediated

secretion of recombinant proteins, we chose to express in root hairs of potato a synthetic

gene encoding a secretory phytase engineered for high heat and proteolytic stability.

Previously, the constitutive expression of an Aspergillus niger phytase gene in all organs of

transgenic Arabidopsis thaliana plants and the subsequent secretion of the recombinant

protein were shown to improve the ability of the plants to mobilize P from soluble phytate in

sterile agar culture (Richardson et al., 2001a). In our case, root hair-specific secretion of the

engineered phytase was shown to be sufficient to improve the ability of the transgenic plants

to acquire P from phytate both in sterile agar and in an unsterile substrate (Figure 6.5).

The increased length of root hairs and the expression of the high-affinity phosphate

transporter gene StPT2 in root hairs under low P conditions (Figure 6.1 (A) to (C)) confirmed

that a root hair-specific promoter is suitable for the expression of genes involved in P

mobilization and uptake. Although the total root surface area was measured to increase up to

40% in our experimental conditions for potato, other reports show that increases in root hair

length in response to nutrient starvation in other species can result in an increased total root

surface area by up to 340% (Gahoonia et al., 1997).

LeExt1.1 promoter-GUS analysis revealed that expression in potato roots is limited to the

root hair cells and is absent at the root tip and in the root cylinder (Figure 6.1 (D) and (E);

Bûcher et al., 2002). We thus chose LeExt1.1 as a root hair-specific promoter to express the

PHY gene.

The rhizosphere represents a harsh environment for enzymes due to high microbial activities

and numerous soil chemical processes. For this reason, the secretion of a highly stable

phytase from roots could confer an advantage over soil microbial phytases and

phosphatases. Assessment of PMEase activity of heat-treated roots and in crude protein

extracts revealed that none of the endogenous PMEases matched the level of thermostability

exhibited by the recombinant protein (Figure 6.2 (C) and (D)). Secretion experiments and

HPLC analysis demonstrated that root hairs can produce and secrete sufficient amounts of

active PHY protein within a few hours to hydrolyse lnsP6 in the medium (Figure 6.4 (D)). The

phytase activities measured were comparable to those obtained by Richardson et al. (2001a)

after constitutive expression of a fungal phytase gene in Arabidopsis. The improved P uptake

in an unsterile, artificial soil substrate by transgenic plants as compared to wild-type (Figure

6.5) appears to indicate that the phytase activity of the recombinant protein is in excess of

the activities of phytases produced by soil microorganisms at the root-soil interface. Apart

from the amounts of PHY protein secreted by the transgenic lines, the higher level of P

mobilization could also be related to the particular stability of the PHY protein.

Exudates from PHY124 plants were able to degrade lnsP6 at rates more than 100 times

higher than exudates from wild-type plants (Figure 6.4). One would therefore expect the

PHY124 plants to be able to take up substantially more P than wild-type plants from a low-P

substrate supplied with phytic acid. In the pot experiment reported here, however, biomass

6.3 Discussion


production was not higher in PHY124 than in wild-type plants, while P concentration in

leaves was 40% higher. Moreover, P concentrations in the leaves of wild-type plants were in

a near to normal range (3.8 g / kg DW). One can therefore conclude that, either the wild-type

plants could have access to P released from phytate (by root-secreted or microbial

enzymes), or there may have been other sources of P in the growth substrate or in the

irrigation water allowing wild-type plants to produce similar biomass as PHY124 plants. To

address this issue, the possible sources of P for plant nutrition in that experiment were

calculated and are discussed.

First, in a prelimary experiment to determine phytic acid intermediates by HPLC of potato

root exudates (see also chapter 4.2.2), it was observed that the phytate (P8810 from Sigma,

Fluka, Buchs, Switzerland) added to the soil substrate was composed of 69% of lnsP6, 25%

of lnsP5 and 6% of lnsP4 (data not shown). It is thought that extracellular enzymes other

than phytase can dephosphorylate lower forms of inositol phosphates such as lnsP5, lnsP4

and lnsP3, and that therefore wild-type plants may have been able to hydrolyse P from lower

forms of inositol phosphates. This hypothesis cannot be confirmed by the data. In fact, after

24 h of incubation, the proportions in the buffer containing roots of wild-type potato plants

changed to 67%, 24% and 9%, with respect to lnsP6, lnsP5 and lnsP4, respectively. It

therefore appears that inositol phosphates, including lnsP6, were partially degraded in

solution in the presence of roots of wild-type plants, but the rates of hydrolysis were more

than 100 times lower than those observed in the presence of roots from PHY plants (data not

shown).

Second, the source of phytate used (P8810) contained Pi as a contaminant at 0.1% (w/w).

The total Pi thus added to each pot throughout the whole experiment was approximately 0.28

mg. Based on the measurements of Pi content in leaves and on biomass production, one can

estimate the total Pi uptake throughout the whole experiment to be approximately 29 (wild-

type) to 41 (PHY124) mg per plant, which is two orders of magnitude higher than the

contaminant Pi in the phytate added. Therefore, the level of Pi contaminant also cannot

explain the relatively high amounts of Pi taken up by wild-type plants in this substrate.

Third, the amount of soluble Pi supplied to each pot by the irrigation water (deionized)

throughout the whole period of growth was calculated to be 0.5 mg. This source of Pi also

cannot account for the relatively large amounts of P taken up by wild-type plants.

Fourth, the soil substrate may have had sufficient available P to sustain normal plant growth.

In fact, extraction of Pi from the soil substrate using the sodium bicarbonate method (Olsen

et al., 1954) indicated that 8 ± 0.6 mg P / kg soil were available for plant uptake (without the

added phytate). Each pot culture thus contained 32 ± 2.4 mg available Pi, which is

approximately equivalent to that taken up by wild-type plants. However, knowing that Pi has

a very low mobility in soils (see chapter 2.2), and that the roots did not have access to the

entire soil volume within the five weeks of the experiment, it is safe to assume that the total P

uptake from soil available P was lower than 32 mg Pi per plant. In the best case, this value

could account for the Pi taken up by wild-type plants. However, it can not be excluded that

wild-type plants could take up a fraction of P released from phytate. Since HPLC analysis of

phytic acid degradation by root exudates shows that exudates from wild-type roots degrade

6.3 Discussion


inositol phosphates at a very low rate (not sufficient to hydrolyse significant amounts of

phytate within the five weeks of the experiment), one must conclude that a possible P

release from phytate is the result of processes other than enzyme activity of plant root

exudates.

In contrast, plants of PHY124 took up approximately 11 mg Pi per plant more than wild-type

plants (41 mg (PHY124) - 29 mg (wild-type)) and approximately 9 mg Pi more than the

estimated available P from the soil substrate alone. This difference may therefore be

attributed to the P-hydrolysis from phytate by root-secreted recombinant phytase. The total

amount of hydrolysable Pi in form of phytate that was added to each pot culture throughout

the experiment was 175 mg. Assuming that 10 mg Pi was released from phytate and taken

up by PHY124 plants, the recovery rate of P from phytate under the culture conditions

described is near to 6%, which is higher than the rates of 1.5% calculated for phytate added

to natural soils (Findenegg and Nelemans, 1993). In summary, the soil substrate used had

relatively high levels of available P, thus not allowing a strong effect of phytase secretion

from roots on the total P mobilization and uptake from the substrate containing phytate, and

thereby on improving plant growth of transgenic plants, as compared to wild-type plants.

Nevertheless, the above estimates allow concluding that approximately 25% of the total P

taken up by the PHY124 plants resulted from P hydrolysis from phytate. This remains to be

proven, for example by using radioactively labelled phytic acid as a source of P.

6.3 Discussion

7 Conclusions and outlook 71

ff General conclusions and outlook

The results on the identification, cloning, and analysis of three purple acid phosphatase

(PAP) genes from potato will be discussed first. Particular attention will be given to link these

findings to the current body of knowledge about plant PAPs. The second part of the

discussion will relate the results obtained with the expression of a synthetic phytase in potato

root hairs to other work in this field. Finally, a comprehensive and conceptual overview of the

role of phosphatases and phytases in soils and on how the current knowledge and the

presently available tools may change our perspectives in rhizosphere research will be given.

7.1 Potato purple acid phosphatases

Analysis of StPAPI, StPAP2 and StPAP3

In recent years, several names have been proposed for individual plant PAP genes and

proteins in different publications. To clarify this unsatisfactory state, a comparison of amino

acid sequences of known plant PAPs was performed to identify unique sequences and a

systematic list of names was created (see table 7.1). In the left column are the names which

are used in this report.

Early reports on plant PAPs were based on the proteins purified from red kidney bean and

from tubers of sweet potato, and on the characterisation of their biochemical and biophysical

properties (see table 7.1). The elucidation of the crystal structure of red kidney bean PAP

(PvPAPI) confirmed previous proposals on the structure of the protein and the mechanism of

phosphate ester hydrolysis. In more recent times, cDNA clones have been isolated and gene

expression data, together with protein characterisation and localisation, have yielded

important information on the biological functions of these proteins (see table 7.2), but in no

case could the respective biological function be fully demonstrated.

However, some authors have deduced possible biological functions from their results. Using

histoenzymological methods, Cashikar et al. (1997) showed that PvPAPI was localized

exclusively in the cell walls of the peripheral two to three rows of cells in the cotyledons.

Additionally, in vitro experiments showed that pectin, a major component of the cell wall,

altered the kinetic properties of PvPAPI. Based on these two findings, they suggested that

PvPAPI may have a role in mobilizing organic phosphates during seed germination. In

Arabidopsis, transcripts of AtPAPU (=AtACP5) were shown to be localized both in leaves

and in roots, and gene expression to be regulated by a large number of environmental

7.1 Purple acid phosphatases


factors. It was therefore thought to be controlled via several signal transduction pathways

(del Pozo et al., 1999). Individual functions for this protein were not suggested. However, a

general role in recycling of phosphate from the plant's phosphate ester pool was proposed.

The first account of a PAP-like protein to have a relatively clear metabolic function was

recently reported for a soybean PAP exhibiting phytase activity (Hegeman and Grabau,

2001). In contrast to PvPAPI, which does not have any phytase activity, this protein, named

GmPhy, had a high affinity for phytic acid, while it showed a 340-fold and 540-fold lower

affinity for ATP and pNPP, respectively, than PvPAPI Since the GmPhy protein was highly

concentrated in cotyledons of germinating soybean, its role in the mobilisation of P from seed

phytate was suggested.

Name in

this

report:

Authors

A1 A2 A3 A4 A5 A6 A7 A8

IbPAPI IbPAPI SP-PAP1 -

lbPAP2 lbPAP2 SP-PAP2 SpPAPI

lbPAP3 lbPAP3 SP-PAP3 SpPAP2

SoPAPI Sp1

Spirodela

oligorrhizaPAP

LaPAPI La1La1,

LASAP1

LaPAP2 LASAP2

GmPAPl GmPAPl SB-PAP

GmPhy GmPhy

AtPAP17 AtACP5 StPAPI 7

PvPAPI PvPAPIKB-PAP,

Pvu

LaAPaseLupinAPase

Authors

A1 Hegeman et al (2001)A2 Schenketal (1999,2000b)A3 Durmusetal (1999)A4 Nakazato et al (1997, 1998) and Nishikoon et al (2001)A5 Wasaki et al (2000)A6 del Pozo et al (1999)A7 Li et al (2002)A8 Miller et al (2001)

Corresponding plants

IbPAPI, 2 and 3

SoPAPI

LaPAPI and 2, and LaAPase

GmPAPl and GmPhyAtPAP17

PvPAPI

sweet potato (Ipomoea batatas)duckweed (Spirodela oligorrhiza)white lupin (Lupinus albus)

soybean (Glycine max)

Arabidopsis thaliana

red kidney bean (Phaseolus vulgaris)

Table 7.1 Names given to recently published plant PAPs The first occurrence in the literature is

given in bold The left column indicates names that are used in this report Authors and plants

corresponding to the genes mentioned are given below the table



In the current work, three cDNA clones encoding phosphatases belonging to the PAP family

have been cloned and characterized. StPAPI is homologous to the LMW PAPs also found in

mammals and cyanobacteria. StPAP2 and StPAP3 are homologous to the HMW PAPs, a

family of proteins exclusively found in plants to date, of which some have been extensively

characterized on a biochemical and biophysical level (Beck et al., 1986; Strater et al., 1995;

Klabunde et al., 1996). Based on amino acid sequence analysis, StPAPI, as well as StPAP2

and StPAP3, have a predicted signal sequence for secretion. As typically for signal peptides,

their sequences are not conserved, in contrast to the sequences encoding the mature

proteins, which are highly conserved.

Biological analysis Protein characterisation

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IbPAP? / 1

PvPAPI / Fe-Zn 2

PvPAPI / Fe-"? 3

PvPAPI / • / • Fe-Zn 4

PvPAPI / • / / • • / Fe-Zn 5

PvPAPI / • / / • • / Fe-Zn 6

PvPAPI / • / • • 7

SoPAPI / / / / / Fe-Mn 8-10

IbPAPI / / • / Fe-Mn 11

lbPAP2 / / • / Fe-Mn 11, 12

lbPAP3 / / • / Fe-Mn 11, 12

AtPAP17 / / / / • / / / / 13

LaPAPI / / / 14

LaPAP2 / / / / 15

GmPAPl / / / / Fe-Zn 11

GmPhy / / / • / / • / / 16

LaAPase / 17

LePAPI / / / 18

NtPAP? / / 19

StPAPI / / / This work

StPAP2 / / / This work

StPAP3 / / / This work

Table 7.2 Selected publications on plant PAPs since 1974 (1) (Uehara et al ) (1974 a, b), (2) Beck

et al (1986), (3) Hefler and Averill (1987), (4) Cashikar et al (1995), (5) Strater et al (1995), (6)Klabunde et al (1996), (7) Cashikar et al (1997), (8) Nakazato et al (1997), (9) Nakazato et al

(1998), (10) Nishikoon et al (2001), (11) LeBansky et al (1991), (12) Schenk et al (1999), (13)Durmus et al (1999), (14) del Pozo et al (1999), (15) Wasaki et al (2000), (16) Hegeman et al

(2001), (17) Miller et al (2002), (18) Varadarajan et al (2002), (19) Kaida and Kaneko (2002)



The search for particular sequence motifs revealed, in addition, that StPAP2 may contain a

GPI anchoring signal (Figure 5.1 (B)). Since GPI anchors do not have a very strict structure

allowing high probability predictions, this information must be taken with caution. In fact, the

algorithm described by Eisenhaber et al. (1999) could not unanimously detect a GPI anchor

in the C-terminal sequence of the Spirodela oligorrhiza PAP, in contrast to experimental data

that confirm this hypothesis. Reciprocally, a positive prediction may not necessarily prove the

presence of a GPI anchor.

Although all three potato PAPs are preferentially expressed in roots and stems based on

RNA gel blot analysis, some level of expression is also found in leaves, and for StPAPI

additionally in stolons, growing sprouts and flowers, but not in tubers (see Figure 5.4).

StPAPI was not induced by P starvation, in contrast to StPAP2 and StPAP3. Due to the fact

that StPAPI is expressed in most tissues tested and, furthermore, that it is not responsive to

P starvation stress, it may have a rather constitutive function, probably in the cell walls, e.g.

by interacting with ß-glucans or pectins. In fact, Kaida and Kaneko (2002) found that

overexpression of a PAP in yeast enhanced the deposition of ß-glucan at the surface of

protoplasts.

StPAP2 and StPAP3, being responsive to P starvation and localized mainly in roots, can be

expected to play a role in the remobilization of organic P, such as ATP, which is present in

the extracellular matrix of multicellular organisms and in the extracellular fluid of unicellular

organisms (Thomas et al., 1999), from the apoplast or at the soil-root interface. StPAP2 may

additionally have other functions in conjunction with its putative localisation at the external

surface of the cell membranes, possibly via interactions with cell wall polymers, or by taking

part in the activation or inactivation of other GPI-anchored proteins clustered in membrane

microdomains ("rafts"). Such cell surface raft microdomains composed of GPI-anchored

proteins and lipids have been shown to be enriched in a number of signal transduction

components in animal cells (Horejsi et al., 1999).

The results presented here do not include any precise localisation studies, such as by in situ

RNA hybridisation or immunolocalisation. Furthermore, the cloning of RNA interference

constructs to trace protein function via knock-out could not be terminated. Therefore, the

precise function of the three potato PAPs analysed in this work cannot be presented. Further

research is thus needed to give a conclusive answer to the question of the biological roles of

these genes.

Potato PAPs: outlook

Table 7.2 reveals that the biochemical and biophysical characterisation of PAPs is well

advanced, while biological data are scarce. Few authors have reported gene expression

data, protein localisation, protein purification and substrate hydrolysis kinetics, effect of

inhibitors, and promoter-reporter gene studies. Only recently, the use of knock-out mutants

has been taken into consideration for Arabidopsis (Li et al., 2002). Functional analysis of

these genes must be pursued with priority.

Probably the most promising approach for potato, for which single gene knock-out mutants

are not yet descibed, is the establishment of transgenic lines in which gene expression of



individual or several PAPs, respectively, is down-regulated by RNA interference (Smith et al.,

2000). The study of PAPs may also be accelerated by the concomitant analysis of

Arabidopsis PAPs, for which the sequences of 29 PAP homologs are available (Li et al.,

2002) and where gene knock-out mutants are available as well. A search of the Syngenta

Arabidopsis Insertion Library (SAIL) using an Arabidopsis sequence homolog to StPAPI

revealed the existence of at least three putative knock-outs. The analysis of repressed

transgenic lines or knock-out mutants is a complex task due to the redundancy of PAPs in

plants and the likelihood that they may substitute for each other. Furthermore, the use of

antibodies for immunolocalisation and Western blot analyses could result in cross-reactions,

as illustrated by the cross-reaction of an anti-/\raö/'cfops/s PAP antibody with the S.

oligorrhiza PAP (Nishikoori et al., 2001).

A complementary approach that would assist in interpreting genetic results could be the

study of protein substrate hydrolysis specificity and protein-protein binding using protein

microarray technology (Templin et al., 2002). This technique would allow screening of

hundreds or thousands of substrates and known proteins in a relatively short time, leading to

more targeted biological experiments which may allow defining precise functions to the PAP

proteins tested.

Another interesting aspect of PAPs is their regulation. In fact, despite the large number of

PAPs, there are Arabidopsis mutants that have lost their P-starvation response for

phosphatase secretion (see chapter 2.2.3). As many PAPs contain signal sequences for

secretion, this could indicate that there is a common signal transduction pathway controlling

several secretory phosphatase genes. The elucidation of these control mechanisms would

be a big step towards understanding the plants' responses to P deficiency stress.

Comparative PAP promoter sequence analysis would allow the identification of conserved

response elements. Gel-shift and deoxyribonuclease-l footprinting assays would then allow

to identify putative transcription factors and their corresponding DNA binding sites. One such

response domain has already been shown to bind homeodomain leucine zipper proteins in

soybean, suggesting a role for these transcription factors in P-modulated gene expression

(Tangetal., 2001).

Not to be ignored are possible functions of PAPs associated with plant-pathogen

interactions. In fact, a potato phosphatase cDNA clone has recently been identified. The

corresponding transcript was upregulated in leaf tissues infected with the fungal pathogen

Phytophthora infestans and the bacterium Pseudomonas syringae pv. Maculicola (Petters et

al., 2002). Similarly, the transcript levels of a phosphatase gene in bean was correlated to

disease resistence, but not to wounding (Jakobek and Lindgren, 2002). In our case,

arbuscular mycorrhizal infection of roots did not affect the transcript levels of StPAP2 and

StPAP3. However, it has been reported that mycorrhizal infection may affect phosphatase

secretion (McArthur and Knowles, 1993). Since P is thought to be transported through the

mycelium of certain species mainly as polyphosphates, but also as Pi and Po (Boddington

and Dodd, 1999; Ezawa et al., 2002), polyphosphates and Po must be hydrolysed for release

through the fungal membrane and uptake by the plant cell. Whether organic forms of P are

also released by the fungus and require the activity of a plant cell membrane-bound

phosphatase for hydrolysis and subsequent uptake into the plant is not known. The GPI-



anchoring machinery would provide the means for polar or targeted accumulation of surface

proteins (Chatterjee and Mayor, 2001) within the peri-arbuscular membrane. By analogy to

PAP activation in response to pathogen attack, one can thus suggest a role of PAPs in the

events leading to mycorrhizal symbiosis.

7.2 P mobilization from phytate in transgenic plants secretingphytase

It has long been debated whether the availability of phytate in soils is limited by its solubility

or by the quantity of phytase present in the soil solution. There are still conflicting views in

the literature about this topic. For example, in the 1950's, Jackman and Black (1952) found

that the hydrolysis of soil phytate was strongly controlled by its solubility. Flaig et al. (1960),

in contrast, stated that the rate of phytate hydrolysis was limiting the rate of P uptake from

phytate by the plants. Since that time, depending on the conditions chosen, it was either

reported that the activity of root-secreted or microbial phytase was sufficient for mobilizing P

from phytate (Tarafdar and Claassen, 1988; Adams and Pate, 1992; Findenegg and

Nelemans, 1993; Hayes et al., 2000b) or that the availability of phytate for enzymatic

degradation was a function of its solubility in the soil (Martin and Cartwright, 1971;

McKercher and Anderson, 1989; see also figure 2.8). Some experiments were done in sterile

conditions (Hayes et al., 2000b; Richardson et al., 2000), while most others were performed

in non-sterile greenhouse pot experiments. Results obtained with plants grown in sand

culture appear to support the second hypothesis, while experiments in pot cultures

containing bulk soil emphasized the availability of phytate as the bottleneck for the release of

P from phytate. One can assume that all authors were right about their conclusions, as far as

their own experimental conditions were concerned, but with the limitation that the

conclusions are valid only to a restricted number of other conditions. Moreover, considering

the complexity of the rhizosphere, it is not surprising to be confronted with divergent results.

The role of soil microorganisms, including ecto-mycorrhizal fungi, in mobilizing phytate from

soils has also been demonstrated (Antibus et al., 1992, 1997; Richardson et al., 2001b;

Tarafdar et al., 2001).

In the present work, transgenic plants secreting a synthetic phytase from their roots were

able to degrade phytic acid in a buffer solution. One would therefore expect transgenic plants

grown in a medium containing soluble phytate to have a higher P mobilization capacity and

thus higher P uptake than wild-type plants. In fact, in the experiments described here,

transgenic plants grown in sterile culture or in a quartz sand mixture, to which soluble phytate

had been added, grew better than wild-type plants, in terms of biomass production (in sterile

culture; data not shown) and of total P content in the leaves (Figure 6.5). Up to this point, the

results do not contradict those of Richardson et al. (2001a), obtained by constitutively

overexpressing a fungal phytase in Arabidopsis, or other data from experiments in which

plant growth was assessed after phytase addition to sandy soil substrates or agar medium

(Findenegg and Nelemans, 1993; Richardson and Hayes, 2000). In a preliminary experiment

using a soil from the Federal Research Station Reckenholz (Zurich, Switzerland), however,

no significant difference in total P concentrations in leaves was measured between

7.2 Plants secreting phytase


transgenic and wild-type plants (data not shown). Given the high rates of phytate hydrolysis

measured when incubating plants in a solution containing phytate (see figure 6.4), one

should assume that the phytase activity in exudates is not limiting. Knowing that soils

generally contain phytate concentrations in the range of 50 - 400 mg / kg soil, representing

10-35% of the total P in soils, the potential P that could be released from phytate is in the

same order of magnitude. The calculated total P uptake by the plants in this pot experiment

(~ [20 g plant DW]*

[2000 mg P / kg DW]) is around 40 mg, which is far below the amounts

which could theoretically be taken up by the plants if all the phytate was degraded and the P

was available for plant uptake. The results of this experiment using a natural soil thus

suggest that the availability of phytate for enzymatic hydrolysis is limiting the P mobilization

from phytate by the enzyme in the conditions used, or that, alternatively, the enzymatic

cleavage takes place to a certain extent, but the Pi released is rapidly fixed to the soil and

only part of it remains available for plant uptake. Findenegg and Nelemans (1993) showed

that commercial phytase added to the growth substrates of plants had an effect on plant

growth only in those substrates to which soluble phytate had been added prior to the

experiment. Added phytase did not stimulate P-uptake and growth when no phytate was

added to the soil. The addition of very high amounts of phytase (10'000 U / kg soil, which is

three orders of magnitude higher than soil phytase activity) did not result in increased P-

uptake from three soils tested, but in one soil it resulted in a more than two-fold higher

uptake of P by the plants. However, in the latter case, it was not known whether the

increased uptake resulted from the activity of the phytase or from the P contamination (153

umol P per pot) in the solution of phytase added. Since the recovery rate of P in this soil was

much higher than that which is usually measured for P (69% as compared to 10%), the

authors concluded that the increased P uptake resulted from an increase in the hydrolysis

rate of soil phytin.

The results from the present work are not sufficient to confirm one or the other of the two

hypotheses, but in any case do not seem to contradict the general contention that the soil

solubility of phytate determines its availability for plant uptake. Should this be the case, the

effectiveness of a recombinant phytase secreted from plant roots would certainly be

increased if the plant had the additional capacity to increase the availability of Po and phytate

in the soil. The next chapter will discuss possibilities to genetically engineer plants towards

that end.

7.3 Designing plants more effectively mobilizing P

Based on our current knowledge and experience, and on our understanding of plant P

metabolism, it is time to think about developing a "super-mobilizing-transporting-recycling"

crop plant, or more simply, a "lupinized" plant. This idea is not new, but first positive

experiences in this field are encouraging to the development of such a crop. Figure 7.1

shows a model of such a plant, as well as possible requirements to achieve increased P

mobilization efficiency, uptake and recycling.

7.2 Plants secreting phytase


P retranslocation

P sensing & signaling

phytosiderophores

Proteoid roots

Enzymes• phytase• RNase

• alkaline phosphatase• acid phosphatase• apyrase

Figure 7.1 Model of a "lupmized" plant, with addition of mycorrhizal association and elements in P

sensing and signalling, P retranslocation within the plant and P metabolism

The expression of a synthetic phytase in root hairs of potato resulted in an increased

mobilization of P from phytate added to the growth substrate. It appears, however, from the

data obtained, that the effect may be lower in a natural soil, where phytic acid is not soluble,

but bound to the soil matrix. The particularly high adsorption of phytic acid to different soils

has been demonstrated previously (McKercher and Anderson, 1989). Some soils, especially

those with high organic matter content, exhibited lower adsorption profiles, suggesting that

organic matter, and by extension the generally increased biological activity, may affect the

adsorption of phytic acid to soil minerals (McKercher and Anderson, 1989).

It is known that plants can significantly modify the organic content and biological activity of

soils by secreting organic compounds and therefore encouraging the development of the

rhizosphere microflora (Zhang et al., 2000a; Nardi et al., 2002). Furthermore, lupin plants are

particularly effective in secreting organic acids in root clusters, thereby concentrating

exudates in a relatively small volume (Neumann and Martinoia, 2002). The concomitant

secretion of phosphatases, organic acids and protons in root clusters results in the

establishment of miniature "P mining factories". It is believed that organic acids such as



citrate play a role in making sources of organic P more effectively available to enzymatic

degradation. To be effective, this process is expected to require a minimum concentration of

organic acids, together with sufficiently high phosphatase and phytase activities. This could

explain why lupin is more effective in P mobilization than other plants equally secreting both

phosphatases and organic acids under P deprivation. By analogy, one would expect

transgenic plants secreting a fungal phytase in high amounts to be more effective in P uptake

if they were additionally engineered for increased organic acid secretion. The latter has been

shown not to be simply done by the expression of a bacterial citrate synthase in the cytosol

(Delhaize et al., 2001). The expression of a mitochondrial citrate synthase gene, however,

was shown to improve the P acquisition efficiency of Arabidopsis plants (Koyama et al.,

2000). One can speculate whether in this case the effect was also a function of the chosen

growth condition. In fact, it is known that aluminum ions induce the secretion of citric and

malic acids by activating a citrate-permeable anion channel (Kollmeier et al., 2001; Zhang et

al., 2001). The soil used in the experiment reported by Koyama et al. (2000) was rich in Al-

phosphates, which could explain that in this soil an effect of increased citrate production and

subsequent secretion could be measured. A more robust and reproducible strategy may be

to additionally express genes encoding di- and tri-carboxylate carriers directed to the

mitochondrial and cell membranes. Such carriers have been identified in Arabidopsis

mitochondria (Picault et al., 2002). Even artificial membrane channels are currently being

designed, including switches (Bayley, 1999), which could be used for increased secretion of

citric and malic acids.

Other chelating agents like phytosiderophores could play a role in rendering organic P

available for enzymatic hydrolysis. The study of the effects of exudates from roots of

elephantgrass (Pennise clundestinum L., cv. Nayier 62), which is very efficient in utilizing P in

acid soils, revealed that rhizosphere acidification and phosphatase activity could not account

for this efficiency. Exudates contained high amounts of pentanedioic acid, a

phytosiderophore, in response to P deficiency, and the presence of pentanedioic acid

correlated with improved P uptake (Shen et al., 2001). Transgenic plants secreting higher

amounts of phytosiderophores have been obtained and had an increased capacity to take up

Fe (Takahashi et al., 2001a). It is not known whether these plants exhibited higher capacities

to mobilize and take up P.

In lupin, increased proton secretion was observed in root clusters under P-deficient

conditions (Yan et al., 2002). It was suggested that the activation of H+-ATPase is

instrumental in the acidification of the rhizosphere by active proteoid roots. Whether

acidification is essential for P mobilization is not clear. The overexpression of a H+-ATPase in

plant roots could thus mimic what is observed in cluster roots.

Introducing mycorrhization capability to plants like lupin or Arabidopsis may sound like a

fancy idea. However, since the control mechanisms allowing mycorrhization by different fungi

are not yet well understood, the assumption can be made that non-mycorrhizal plants may

have lost the ability to be mycorrhizal during evolution, and that this loss-of-function could be

related to a limited number of genes, possibly even a single gene. Reactivation or

reintroduction of such a gene in non-mycorrhizal plants may provide the means to allow fungi

to enter mutualistic association with its host.



The modification of retranslocation of P throughout the plant, altering of P sensing patterns,

and changing metabolic pathways to less expensive ones in terms of P are all theoretically

possible ways to improve the P utilisation efficiency of crops. In the last resort, the dream

plant of a plant P nutritionist would embrace increased efficiency of P mobilization from

several P sources in the soil, more efficient transport and allocation, and higher P use

efficiency within plant metabolism.

7.4 Phosphatases, phytases and beyond

Taking into account the large number of publications dealing with phosphatase secretion

from plant roots and phosphatase activities in the soil, one would expect the current

knowledge to be large enough to yield a clear concept in terms of the contribution of

phosphatases to plant nutrition. Unfortunately, throughout the years, new data have either

confirmed or questioned previous findings in new experimental setups, but have not

necessarily shed new light in understanding the processes involved (Figure 7.2). This lack of

significant progress is certainly not due to uncreative research strategies, but much more to

the lack of adequate tools for rhizosphere investigation, and undoubtedly also to the

complexity and variability of the rhizosphere. In fact, results obtained in a particular soil

condition may not hold true for another closely related soil, not to mention other, unrelated

soil types. The reductionist approach, where individual processes were studied in tightly

controlled and simplified systems, resulted in interesting discoveries, but often failed at the

integration step into the natural, complex system. Obviously, there is a conceptual black hole

in rhizosphere research.

Figure 7.2 Current concept of the role of phosphatases in the rhizosphere phosphatases are

secreted into the rhizosphere and interact with scarcely available sources of organic P (P-org ) The

soil matrix and colloids exert electrostatic interactions with enzymes, resulting in immobilisation on

mineral surfaces (not shown) Organic P is located either in soil solution or in complex soil-humic

complexes The uptake of Pi occurs via P transporters in conjunction with the activity of proton-

secreting pumps (H+-ATPases) The rhizoplane contains scores of living organisms that also

release phosphatases, phytases and other hydrolases



Recently developed technologies in enzymology, soil sciences and root biology may help to

fill the gap between basic process understanding in plants and in soil, and in the integration

of both disciplines in a more complex (or natural) system. For example, the use of the

APIZYME technology (BioMérieux, Lyon, France), which allows to screen for at least 20

enzymatic reactions, would rapidly give a profile of enzymatic activities in a given system.

Using this method, one could then modify single parameters in the system and check the

profile for modifications. This would be a kind of soil enzyme microarray allowing the rapid

detection of changes in individual enzymatic activities. A complex system cannot be

understood only by reduction to its single elements, but also requires the collection of large

databases of information that give some sort of fingerprint profiles for each studied system.

Such fingerprint profiles would cover the fields of enzymology, microbial species diversity,

mineralogy, perhaps soil organic P by using P-NMR studies in rhizo, and of course plant

gene expression profiling. The computing of these different profiles into a single database,

and the comparison of databsets from strictly controlled experiments by statistical analysis

can be expected to yield valuable information on processes in the rhizosphere. Some of

these profiles can already be achieved. As mentioned, enzyme-substrate arrays are already

available on the market (Templin et al., 2002). Species diversity can be assessed for

example by molecular identification of the ITS2 region of rDNA in microorganisms (Jansa et

al., 2002) or by using other standard tests for bacterial identification, such as API strips from

BioMérieux. A rapid mineralogical profile may be more difficult to achieve, but is certainly

possible. The profiling approach could be extended to other parameters, but this would go

beyond the scope of this analysis.

Figure 7.3 An imaginable concept of rhizosphere research



The combination of such data with molecular biological tools to modifiy root properties, as

well as the study of genes having a function in the root-soil interface can be expected to

open new fields in rhizosphere research. For example, the expression of a synthetic phytase

in root hairs of potato allows the setup of novel experimental systems for the study of lnsP6

availability and mobilization in soils. The wild-type and transgenic plants differ in a single but

important trait, allowing the study of mechanisms involved in P mobilization and uptake in

better controlled systems. This is but one example. One can further imagine the development

of transgenic plants secreting a large number of substances that would affect biological

activity, biochemistry, and chemistry of soils, including toxins, enzyme inhibitors, allelopathic

substances, or maybe even sugars. After engineering plants with improved nutrient uptake

capabilities, better resistance to pathogens, and higher quality, one could think of

engineering the rhizosphere to increase the quality of soils, not only by phytoremediation, but

much more for increasing the biological activity, by improving the quality of life of those little

beings which we do not see, but who do more for us than we generally assume.


8 References 83

O^J References

Aarts, J.M., Hontelez, J.G., Fischer, P., Verkerk, R., van Kämmen, A., and Zabel, P. (1991) Acid

phosphatase-1, a tightly linked molecular marker for root-knot nematode resistance in tomato from

protein to gene, using PCR and degenerate primers containing deoxymosme Plant Mol Biol 16, 647-

661

Abel, S., Nürnberger, T., Ahnert, V., Krauss, G.J., and Glund, K. (2000) Induction of an extracellular cyclicnucleotide phosphodiesterase as an accessory nbonucleolytic activity during phosphate starvation of

cultured tomato cells Plant Physiol 122, 543-552

Abelson, P.H. (1999) A Potential Phosphate Crisis Science 283, 2015-2016

Adams, M.A., and Pate, J.S. (1992) Availability of organic and inorganic forms of phosphorus to lupins (Lupinus

spp) Plant Soil 145, 107-113

Altschul, S., Gish, W., Miller, W., Meyers, E., and Lipman, D. (1990) Basic local alignment search tool J Mol

Biol 215, 403-410

Anderson, G. (1980) Assessing organic phosphorus in soils In The Role of Phosphorus m Agriculture, F

Khasawneh, E Sample, and E Kamprath, eds (Madison, USA American Society of Agronomy), pp

411-432

Antibus, R.K., Sinsabaugh, R.L., and Linkins, A.E. (1992) Phosphatase activities and phosphorus uptake from

inositol phosphate by ectomycorrhizal fungi Can J Bot 70, 794-801

Antibus, R.K., Bower, D., and Dighton, J. (1997) Root surface phosphatase activities and uptake of 32P-labelled inositol phosphate in field-collected gray birch and red maple roots Mycorrhiza 7, 39-46

Ascencio, J. (1997) Root secreted acid phosphatase kinetics as a physiological marker for phosphorus

deficiency J Plant Nutr 20, 9-26

Asmar, F. (1997) Variation in activity of root extracellular phytase between genotypes of barley Plant Soil 195,61-64

Asmar, F., and Gissel-Nielsen, G. (1997) Extracellular phosphomono- and phosphodiesterase associated with

and released by the roots of barley genotypes a non-destructive method for the measurement of the

extracellular enzymes of roots Biol Fertil Soils 25, 117-122

Asmar, F., Singh, R., Gahoonia, and Nielsen, N.E. (1995) Barley genotypes differ in activity of soluble

extracellular phosphatase and depletion of organic phosphorus in the rhizosphere soil Plant Soil 172,117-122

Atkinson, H., Urwin, P., Hansen, E., and McPherson, M. (1995) Designs for engineered resistance to root-

parasitic nematodes Trends Biotechnol 13, 369-374

Averill, B.A., Burman, S., Davis, J.C., and Kauzlarich, S.M. (1983) The purple acid-phosphatase from beef

spleen - a novel iron enzyme Abstr Pap Am Chem Soc 186, 298

Bailey, P.H., Currey, J.D., and Fitter, A.H. (2002) The role of root system architecture and root hairs in

promoting anchorage against uprooting forces in Allium cepa and root mutants of Arabidopsis thaliana J

Exp Bot 53, 333-340

Baldwin, J.C., Karthikeyan, A.S., and Raghothama, K.G. (2001) LEPS2, a phosphorus starvation-induced

novel acid phosphatase from tomato Plant Physiol 125, 728-737

Barford, D., Das, A.K., and Egloff, M.P. (1998) The structure and mechanism of protein phosphatases insightsinto catalysis and regulation Annu Rev Biophys Biomol Struct 27, 133-164

Bariola, P.A., Macintosh, G., and Green, P. (1999) Regulation of S-hke nbonuclease levels in ArabidopsisAntisense inhibition of RNS1 or RNS2 elevates anthocyanm accumulation Plant Physiol 119, 331-342

Bates, T.R., and Lynch, J.P. (1996) Stimulation of root hair elongation in Arabidopsis thaliana by low

phosphorus availability Plant Cell Environ 19, 529-538

Bates, T.R., and Lynch, J.P. (2000) Plant growth and phosphorus accumulation of wild type and two root hair

mutants of Arabidopsis thaliana (Brassicaceae) Am J Bot 87, 958-963

Bates, T.R., and Lynch, J.P. (2001) Root hairs confer a competitive advantage under low phosphorus

availability Plant Soil 236, 243-250

Bayley, H. (1999) Designed membrane channels and pores CurrOpm Biotechnol 10, 94-103

Beck, J., McConaghie, L., Summors, A., Arnold, W., de Jersey, J., and Zerner, B. (1986) Properties of a

purple acid phosphatase from red kidney bean a zinc-iron metalloenzyme Biochim Biophys Acta 869,61-68

Bevan, M. (1984) Binary Agrobacterium vectors for plant transformation Nucleic Acids Res 12, 8711-8721

8 References 84

Bhadraray, S., Purakayastha, T.J., Chhonkar, P.K., and Verma, V. (2002) Phosphorus mobilization in hybridrice rhizosphere compared to high yielding varieties under integrated nutrient management Biol Fertil

Soils 35, 73-78

Bloor, S.J., and Abrahams, S. (2002) The structure of the major anthocyanm in Arabidopsis thaliana

Phytochemistry 59, 343-346

Boddington, C, and Dodd, J. (1999) Evidence that differences in phosphate metabolism in mycorrhizas formed

by species of Glomus and Gigaspora might be related to their life-cycle strategies New Phytol 142, 531-

538

Boreo, G., and Thien, S. (1979) Phosphatase activity and phosphorus availability in the rhizosphere In The soil-

root interface, J Harley and R Scott Russell, eds (London Academic Press), pp 231-242

Bosse, D., and Koeck, M. (1998) Influence of phosphate starvation on phosphohydrolases during developmentof tomato seedlings Plant Cell Environ 21, 325-332

Bowman, B., Thomas, R., and Elrick, D. (1967) The movement of phytic acid in soil cores Soil Sei Soc Am

Proc 31, 477-481

Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein

utilizing the principle of protein-dye binding Anal Biochem 72, 248-254

Brearley, CA., and Hanke, D.E. (1996) Metabolic evidence for the order of addition of individual phosphateesters in the myoinositol moiety of inositol hexa/c/sphosphate in the duckweed Spirodela polyrhiza L

Biochem J 314 ( Pt 1), 227-233

Brinch-Pedersen, H., Sorensen, L.D., and Holm, P.B. (2002) Engineering crop plants getting a handle on

phosphate Trends Plant Sei 7, 118-125

Brinch-Pedersen, H., Olesen, A., Rasmussen, S.K., and Holm, R.B. (2000) Generation of transgenic wheat

(Tnticum aestivum L ) for constitutive accumulation of an Aspergillus phytase Mol Breeding 6, 195-206

Broz, J., Oldale, P., Perrin-Voltz, A.H., Rychen, G., Schulze, J., and Nunes, CS. (1994) Effects of

supplemental phytase on performance and phosphorus utilisation in broiler chickens fed a low

phosphorus diet without addition of inorganic phosphates Br Poult Sei 35, 273-280

Bûcher, M. (2002) Molecular root bioengineering In Plant Roots the Hidden Half, Y Waisel, ed (

Bûcher, M., Brandie, R., and Kuhlemeier, C. (1994) Ethanohc fermentation in transgenic tobacco expressing

Zymomonas mobilis pyruvate decarboxylase EMBO J 13, 2755-2763

Bûcher, M., Rausch, C, and Daram, P. (2001) Molecular and biochemical mechanisms of phosphorus uptakeinto plants J Plant Nutr Soil Sc 164, 209-217

Bucher, M., Schroeer, B., Willmitzer, L., and Riesmeier, J.W. (1997) Two genes encoding extensin-hke

proteins are predominantly expressed in tomato root hair cells Plant Mol Biol 35, 497-508

Bûcher, M., Brunner, S., Zimmermann, P., Zardi, G.I., Amrhein, N., Willmitzer, L., and Riesmeier, J.W.

(2002) The expression of an extensm-hke protein correlates with cellular tip growth in tomato Plant

Physiol 128,911-923

Burns, R. (1982) Enzyme activity in soil location and possible role in microbial ecology Soil Biol Biochem 12,423-427

CadeMenun, B.J., and Preston, CM. (1996) A comparison of soil extraction procedures for P-31 NMR

spectroscopy Soil Sei 161, 770-785

Cashikar, A., and Rao, M. (1995) Unique Structural Features of Red Kidney Bean Purple Acid- PhosphataseIndian Journal of Biochemistry & Biophysics 32, 130-136

Cashikar, A.G., Kumaresan, R., and Rao, N.M. (1997) Biochemical characterization and subcellular localization

of the red kidney bean purple acid phosphatase Plant Physiol 114, 907-915

Celi, L., Presta, M., Ajmore-Marsan, F., and Barberis, E. (2001) Effects of pH and electrolytes on inositol

hexaphosphate interaction with Goethite Soil Sei Soc Am J 65, 753-760

Centeno, C, Viveros, A., Brenes, A., Canales, R., Lozano, A., and de la Cuadra, C. (2001) Effect of several

germination conditions on total P, phytate P, phytase, and acid phosphatase activities and inositol

phosphate esters in rye and barley J Agnc Food Chem 49, 3208-3215

Chatterjee, S., and Mayor, S. (2001) The GPI-anchor and protein sorting Cell Mol Life Sei 58,1969-1987

Chen, CR., Condron, L.M., Davis, M.R., and Sherlock, R.R. (2002) Phosphorus dynamics in the rhizosphereof perennial ryegrass (Lolium perenne L ) and radiata pine (Pinus radiata D Don ) Soil Biol Biochem 34,487-499

Chen, J.C (1998) Novel screening method for extracellular phytase-producmg microorganisms Biotechnol Tech

12, 759-761

Chen, W.S., Huang, Y.F., and Chen, Y.R. (1992) Localization of acid phosphatase in root cap of rice plant Bot

Bull Acad Smica 33, 233-239

Cheryan, M. (1980) Phytic acid interactions in food systems Crit Rev Food Sei Nutr 13, 297-335

Clausen, M., Krauter, R., Schachermayr, G., Potrykus, I., and Sautter, C. (2000) Antifungal activity of a virallyencoded gene in transgenic wheat Nat Biotechnol 18, 446-449

Condron, L.M., Frossard, E., Tiessen, H., Newman, R.H., and Stewart, J.W.B. (1990) Chemical Nature of

Organic Phosphorus in Cultivated and Uncultivated Soils under Different Environmental-Conditions J

Soil Sei 41, 41-50

Conway, G., and Toenniessen, G. (1999) Feeding the world in the twenty-first century Nature 402, C55-C58

Corona, M., Van der Klundert, I., and Verhoeven, J. (1996) Availability of organic and inorganic phosphorus

compounds as phosphorus sources for Carex species New Phytol 133, 225-331

8 References 85

Cosgrove, D.J. (1969) Ion-exchange chromatography of inositol polyphosphates Annals of the New York

Academy of Sciences 165, 677-&

Cosgrove, D.J. (1970) Inositol phosphate phosphatases of microbiological origin - inositol phosphateintermediates in dephosphorylation of hexaphosphates of myoinositol, scy//o-mositol, and D-chiro-

mositol by a bacterial (Pseudomonas-Sp) phytase Aust J Biol Sei 23, 1207

Cosgrove, D.J. (1980) Inositol phosphates Their chemistry, biochemistry and physiology (Amsterdam

Elsevier)Costello, A., Gonek, T., and Myers, T. (1976) 31P nuclear magnetic resonance-pH titrations of myoinositol

hexaphosphate Carbohydr Res 46, 159-171

Courtois, J.E., and Manet, L. (1952) The very acid phytase and glycero-phosphatase of colon bacilli In CongrInter Biochem

,Résumés Communs, 2 (Pans)

Cromwell, G.L., Stahly, T.S., Coffey, R.D., Monegue, H.J., and Randolph, J.H. (1993) Efficacy of phytase in

improving the bioavailability of phosphorus in soybean meal and corn-soybean meal diets for pigs J

AnimSci71, 1831-1840

Cullimore, J.V., Ranjeva, R., and Bono, J.J. (2001) Perception of lipo-chitoohgosaccharidic Nod factors in

legumes Trends Plant Sei 6, 24-30

Dalai, R.C, and Hallsworth, E.G. (1977) Measurement of isotopic exchangeable soil phosphorus and

interrelationship among parameters of quantity, intensity, and capacity factors Soil Sei Soc Am J 41, 81-

86

Daram, P., Brunner, S., Persson, B.L., Amrhein, N., and Bûcher, M. (1998) Functional analysis and cell-

specific expression of a phosphate transporter from tomato Planta 206

Dasgupta, S., Dasgupta, D., Sen, M., Biswas, S., and Biswas, B.B. (1996) Interaction of myo-

mositoltnsphosphate-phytase complex with the receptor for intercellular Ca2+ mobilization in plants

Biochemistry 35, 4994-5001

Davis, J.C, and Averill, B.A. (1982) Evidence for a Spin-Coupled Binuclear Iron Unit at the Active-Site of the

Purple Acid-Phosphatase from Beef Spleen Proc Natl Acad Sei U S A 79, 4623-4627

Davis, J.C, Lin, S.S., and Averill, B.A. (1981) Kinetics and Optical Spectroscopic Studies on the Purple Acid-

Phosphatase from Beef Spleen Biochemistry 20, 4062-4067

De Groot, C, and Golterman, H. (1993) On the presence of organic phosphate in some Camargue sediments

evidence for the importance of phytate Hydrobiologia 252, 117-126

Deblaere, R., Bytebier, B., Dégrevé, H., Deboeck, F., Schell, J., Vanmontagu, M., and Leemans, J. (1985)Efficient octopme Ti plasmid-denved vectors for agrobactenum-mediated gene-transfer to plants Nucleic

Acids Res 13, 4777-4788

del Pozo, J.C, Allona, I., Rubio, V., Leyva, A., de la Pena, A., Aragoncillo, C, and Paz-Ares, J. (1999) A

type 5 acid phosphatase gene from Arabidopsis thaliana is induced by phosphate starvation and bysome other types of phosphate mobihsing/oxidative stress conditions Plant J 19, 579-589

Delhaize, E., Hebb, D.M., and Ryan, P.R. (2001) Expression of a Pseudomonas aeruginosa citrate synthase

gene in tobacco is not associated with either enhanced citrate accumulation or efflux Plant Physiol 125,2059-2067

Denbow, D.M., Ravindran, V., Kornegay, E.T., Yi, Z., and Hulet, R.M. (1995) Improving phosphorus availabilityin soybean meal for broilers by supplemental phytase Poult Sei 74, 1831-1842

Denbow, D.M., Grabau, E.A., Lacy, G.H., Kornegay, E.T., Russell, D.R., and Umbeck, P.F. (1998) Soybeanstransformed with a fungal phytase gene improve phosphorus availability for broilers Poult Sei 77, 878-

881

Deng, S., Summers, M.L., Khan, M.L., and McDermott, T.R. (1998) Cloning and characterization of a

Rhizobium mehloti nonspecific acid phosphatase Arch Microbiol 170, 18-26

DeWald, D.B., Torabinejad, J., Jones, CA., Shope, J.C, Cangelosi, A.R., Thompson, J.E., Prestwich, G.D.,and Hama, H. (2001) Rapid accumulation of phosphatidylmositol 4,5-bisphosphate and inositol 1,4,5-

tnsphosphate correlates with calcium mobilization in salt-stressed Arabidopsis Plant Physiol 126, 759-

769

Dinkelaker, B., and Marschner, H. (1992) In vivo demonstration of acid phosphatase activity in the rhizosphereof soil-grown plants Plant Soil 144, 199-205

Doi, K., McCracken, J., Peisach, J., and Aisen, P. (1988) The binding of molybdate to uterofernn Hyperfmeinteractions of the binuclear center with 95Mo, 1H, and 2H J Biol Chem 263, 5757-5763

Duff, S.M.G., Sarath, G., and Plaxton, W.C (1994) The role of acid phosphatases in plant phosphorusmetabolism Physiol Plantarum 90, 791-800

Durmus, A., Eicken, C, Spener, F., and Krebs, B. (1999) Cloning and comparative protein modeling of two

purple acid phosphatase isozymes from sweet potatoes (Ipomoea batatas) Biochim Biophys Acta 1434,202-209

Dvorakova, J. (1998) Phytase sources, preparation and exploitation Folia Microbiol 43, 323-338

Egli, I. (2001) Traditional food processing methods to increase mineral bioavailability from cereal and legumebased weaning foods (Zurich ETH, Naturwissenschaften Diss Nr 13980)

Eisenhaber, B., Bork, P., and Eisenhaber, F. (1999) Prediction of potential GPI-modification sites in proprotem

sequences J Mol Biol 292, 741-758

Ezawa, T., Smith, S.E., and Smith, F.A. (2002) P metabolism and transport in AM fungi Plant Soil 244, 221-

230

8 References 86

Felsenstein, J. (1993) PHYLIP (phylogeny inference package) version 3 5 A computer program distributed bythe author (Department of Genetics, University of Washington, Seattle, Washington)

Findenegg, G.R., and Nelemans, J.A. (1993) The effect of phytase on the availability of P from myoinositol

hexaphosphate (phytate) for maize roots Plant Soil 154, 189-196

Flaig, W., Schmid, W., Wagner, E., and Keppel, H. (1960) Die Aufnahme von Phosphor aus Inositol

hexaphosphat Landwirtsch Forsch Sonderh 14, 43-48

Fox, T.R., and Comerford, N.B. (1992) Rhizosphere phosphatase-activity and phosphatase hydrolyzable

organic phosphorus in two forested spodosols Soil Biol Biochem 24, 579-583

Frohman, M.A., Dush, M.K., and Martin, G.R. (1988) Rapid production of full-length cDNAsfrom rare

transcripts amplification using a single gene-specific oligonucleotide primer Proc Natl Acad Sei U S A

85, 8998-9002

Frossard, E., Condron, L.M., Oberson, A., Sinaj, S., and Fardeau, J.C. (2000a) Processes governing

phosphorus availability in temperate soils J Environ Qual 29, 15-23

Fujimoto, S., Nakagawa, T., Ishimitsu, S., and Ohara, A. (1977) Purification and Some Properties of Violet-

Colored Acid-Phosphatase from Spinach Leaves Chem Pharm Bull 25,1459-1462

Furlani, M., Clark, R., Maranville, J., and Ross, W. (1984) Root phosphatase activity of sorghum genotypes

grown with organic and inorganic sources of phosphorus J Plant Nutr 7, 1583-1595

Fusseder, A., and Kraus, M. (1986) Individual root competition and exploitation of the hmmobile macronutnents

in the root space of maize Flora 178, 11-18

Gahoonia, T.S., and Nielsen, N.E. (1997) Variation in root hairs of barley cultivars doubled soil phosphorus

uptake Euphytica 98, 177-182

Gahoonia, T.S., Care, D., and Nielsen, N.E. (1997) Root hairs and phosphorus acquisition of wheat and barleycultivars Plant Soil 191, 181-188

Gallet, A., Flisch, R., Ryser, J.-P., Nösberger, J., Frossard, E., and Sinaj, S. (2003) Uptake of residual

phosphate and freshly applied diammonium phosphate by Lolium perenne and Trifolium repens in

press

Gaume, A., Machler, F., De Leon, C, Narro, L., and Frossard, E. (2001) Low-P tolerance by maize (Zea maysL ) genotypes Significance of root growth, and organic acids and acid phosphatase root exudation Plant

Soil 228, 253-264

Gilbert, G.A., Knight, J.D., Vance, C.P., and Allan, D.L. (1999) Acid phospatase activity in phosphorus-deficient white lupin roots Plant Cell Environ 22, 801-810

Gillaspy, G.E., Keddie, J.S., Oda, K., and Gruissem, W. (1995) Plant inositol monophosphatase is a hthium-

sensitive enzyme encoded by multigene family Plant Cell 7, 2175-2182

Gilroy, S., and Jones, D.L. (2000) Through form to function root hair development and nutrient uptake Trends

Plant Sei 5, 56-60

Golovan, S., Wang, G., Zhang, J., and Forsberg, C.W. (2000) Characterization and overproduction of the

Escherichia coli appA encoded afunctional enzyme that exhibits both phytase and acid phosphataseactivities Can J Microbiol 46, 59-71

Golovan, S.P., Hayes, M.A., Phillips, J.P., and Forsberg, C.W. (2001a) Transgenic mice expressing bacterial

phytase as a model for phosphorus pollution control Nat Biotechnol 19, 429-433

Golovan, S.P., Meidinger, R.G., Ajakaiye, A., Cottrill, M., Wiederkehr, M.Z., Barney, D.J., Plante, C, Pollard,

J.W., Fan, M.Z., Hayes, M.A., Laursen, J., Hjorth, J.P., Hacker, R.R., Phillips, J.P., and Forsberg,C.W. (2001b) Pigs expressing salivary phytase produce low-phosphorus manure Nat Biotechnol 19,741-745

Greaves, S.R., and Webley, D.M. (1965) A study of the breakdown of organic phosphates by micro-organisms

from the root region of certain pasture grasses J Appl Bactenol 28, 454-465

Greaves, S.R., Anderson, G., and Webley, D.M. (1963) A rapid method of determining the phytase activity of

soil micro-organisms Nature 200, 1231-1232

Greenwood, A.J., and Lewis, D.H. (1977) Phosphatases and Utilization of Inositol Hexaphosphate by Soil

Yeasts of Genus Cryptococcus Soil Biol Biochem 9, 161-166

Greiner, R., Konietzny, U., and Jany, K.D. (1993) Purification and characterization of two phytases from

Escherichia coli Arch Biochem Biophys 303, 107-113

Greiner, R., Haller, E., Konietzny, U., and Jany, K.D. (1997) Purification and characterization of a phytase from

Klebsiella terngena Arch Biochem Biophys 341, 201-206

Greiner, R., Larsson Alminger, M., Carlsson, N.G., Muzquiz, M., Burbano, C, Cuadrado, C, Pedrosa, M.M.,and Goyoaga, C. (2002) Pathway of dephosphorylation of myoinositol hexa/asphosphate by phytasesof legume seeds J Agnc Food Chem 50, 6865-6870

Grierson, P.F., and Comerford, N.B. (2000) Non-destructive measurement of acid phosphatase activity in the

rhizosphere using nitrocellulose membranes and image analysis Plant Soil 218, 49-57

Hanakahi, L.A., Bartlet-Jones, M., Chappell, C, Pappin, D., and West, S.C (2000) Binding of inositol

phosphate to DNA-PK and stimulation of double-strand break repair Cell 102, 721-729

Hara, A., Sawada, H., Kato, T., Nakayama, T., Yamamoto, H., and Matsumoto, Y. (1984) Purification and

Characterization of a Purple Acid-Phosphatase from Rat Spleen J Biochem (Tokyo) 95, 67-74

Haran, S., Logendra, S., Seskar, M., Bratanova, M., and Raskin, I. (2000) Characterization of Arabidopsis acid

phosphatase promoter and regulation of acid phosphatase expression Plant Physiol 124, 615-626

Harrison, A.F. (1987) Soil Organic Phosphorus A Review of World Literature (CAB International, Oxford)

8 References 87

Harrison, M.J., Dewbre, G.R., and Liu, J. (2002) A Phosphate Transporter from Medicago truncatula Involved

in the Acquisition of Phosphate Released by Arbuscular Mycorrhizal Fungi Plant Cell 14, 2413-2429

Hartje, S., Zimmermann, S., Klonus, D., and Mueller-Roeber, B. (2000) Functional characterisation of LKT1, a

K+ uptake channel from tomato root hairs, and comparison with the closely related potato inwardly

rectifying K+ channel SKT1 after expression in Xenopus oocytes Planta 210, 723-731

Häussling, M., and Marschner, H. (1989) Organic and inorganic soil phosphates and acid phosphatase activityin the rhizosphere of 80-year-old Norway spruce (Picea abies (L) Karst ) trees Biol Fertil Soils 8, 128-

133

Hawkes, G., Powlson, D., Randall, E., and Tate, K. (1984) A31P nuclear magnetic resonance study of the

phosphorus species in alkali extracts of soils from long-term field experiments J Soil Sei 35, 35-45

Hayes, J.E., Richardson, A.E., and Simpson, R.J. (1999) Phytase and acid phosphatase activities in extracts

from roots of temperate pasture grass and legume seedlings Aust J Plant Physiol 26, 801-809

Hayes, J.E., Richardson, A.E., and Simpson, R.J. (2000a) Components of organic phosphorus in soil extracts

that are hydrolysed by phytase and acid phosphatase Biol Fertil Soils 32, 279-286

Hayes, J.E., Simpson, R.J., and Richardson, A.E. (2000b) The growth and phosphorus utilisation of plants in

sterile media when supplied with inositol hexaphosphate, glucose 1-phosphate or inorganic phosphatePlant Soil 220, 165-174

Hayman, A.R., and Cox, T.M. (1994) Purple acid-phosphatase of the human macrophage and osteoclast-

characterization, molecular-properties, and crystallization of the recombinant di-iron-oxo protein secreted

by Baculovirus- infected insect cells J Biol Chem 269, 1294-1300

Hedley, M., Nye, P., and White, R. (1982) Plant-induced changes in the rhizosphere or rape (Brassica napus

var Emerald) seedlings II Origin of the pH change New Phytol 91, 31-44

Hedley, M., Nye, P., and White, R. (1983) Plant-induced changes in the rhizosphere of rape (Brassica napus

Var Emerald) seedlings IV The effect of the rhizosphere phosphorus status on the pH, phosphatase

activity and depletion of soil phosphorus fractions in the rhizosphere and on the cation-anion balance in

the plants New Phytol 95, 69-82

Hefler, S.K., and Averill, B.A. (1987) The "manganese(lll)-containing" purple acid phosphatase from sweet

potatoes is an iron enzyme Biochem Biophys Res Commun 146, 1173-1177

Hegeman, CE., and Grabau, E.A. (2001) A novel phytase with sequence similarity to purple acid phosphatasesis expressed in cotyledons of germinating soybean seedlings Plant Physiol 126, 1598-1608

Hegeman, CE., Good, L.L., and Grabau, E.A. (2001) Expression of D-myoinositol-3-phosphate synthase in

soybean Implications for phytic acid biosynthesis Plant Physiol 125, 1941-1948

Helal, H.M. (1990) Varietal differences in root phosphatase activity as related to the utilization of organic

phosphates Plant Soil 123, 1429-1436

Hell, R., and Hillebrand, H. (2001) Plant concepts for mineral acquisition and allocation Curr Opm Biotechnol

12, 161-168

Herrera-Estrella, L. (1999) Transgenic plants for tropical regions some considerations about their developmentand their transfer to the small farmer Proc Natl Acad Sei U S A 96, 5978-5981

Hinsinger, P. (2001) Bioavailability of soil inorganic P in the rhizosphere as affected by root-induced chemical

changes a review Plant Soil 237, 173-195

Hiscox, S., Hallett, M.B., Morgan, B.P., and van den Berg, CW. (2002) GPI-anchored GFP signals Ca2+ but is

homogeneously distributed on the cell surface Biochem Biophys Res Commun 293, 714-721

Holford, I. (1997) Soil phosphorus, its measurements and its uptake by plants Aust J Soil Res 35, 227-239

Horejsi, V., Drbal, K., Cebecauer, M., Cerny, J., Brdicka, T., Angelisova, P., and Stockinger, H. (1999) GPI-

microdomains a role in signalling via immunoreceptors Immunol Today 20, 356-361

Home, I., Harcourt, R.L., Sutherland, T.D., Russell, R.J., and Oakeshott, J.G. (2002) Isolation of a

Pseudomonas monteilli strain with a novel phosphotnesterase FEMS Microbiol Lett 206, 51-55

Hurry, V., Strand, A., Furbank, R., and Stitt, M. (2000) The role of inorganic phosphate in the development of

freezing tolerance and the acclimatization of photosynthesis to low temperature is revealed by the phomutants of Arabidopsis thaliana Plant J 24, 383-396

Idriss, E.E., Makarewicz, O., Farouk, A., Rosner, K., Greiner, R., Bochow, H., Richter, T., and Borriss, R.

(2002) Extracellular phytase activity of Bacillus amyloliquefaciens FZB45 contributes to its plant-growth-

promoting effect Microbiology-(UK) 148, 2097-2109

Igbasan, F.A., Manner, K., Miksch, G., Borriss, R., Farouk, A., and Simon, O. (2000) Comparative studies on

the in vitro properties of phytases from various microbial origins Arch Tierernahr 53, 353-373

Islam, A., Mandai, R., and Osman, K.T. (1979) Direct absorption of organic phosphate by rice and jute plantsPlant Soil 53, 49-54

Itoh, S., and Barber, S.A. (1983) Phosphorus uptake by 6 plant species as related to root hairs Agron J 75,457-461

IUPAC (1971) IUPAC commission on the nomenclature of organic chemistry (CNOC) and IUPAC-iub

commission on biochemical nomenclature (CBN) Tentative rules for carbohydrate nomenclature Eur J

Biochem 21, 455-477

Jackman, R.H., and Black, CA. (1951) Solubility of iron, aluminium, calcium and magnesium inositol

phosphates at different pH values Soil Sei 72, 179-186

Jackman, R.H., and Black, CA. (1952) Hydrolysis of phytate phosphorus in soils Soil Sei 73, 167-171

Jakobek, J.L., and Lindgren, P.B. (2002) Expression of a bean acid phosphatase cDNA is correlated with

disease resistance J Exp Bot 53, 387-389

8 References 88

Jansa, J., Mozafar, A., Anken, T., Ruh, R., Sanders, LR., and Frossard, E. (2002) Diversity and structure of

AMF communities as affected by tillage in a temperate soil Mycorrhiza 12, 225-234

Johnson, J.F., Vance, C.P., and Allan, D.L. (1996) Phosphorus deficiency in Lupinus albus Altered lateral root

development and enhanced expression of phosphoenolpyruvate carboxylase Plant Physiol 112, 31-41

Johnson, L.F., and Tate, M.E. (1969a) Structure of myoinositol pentaphosphates Annals of the New York

Academy of Sciences 165, 526-&

Johnson, L.F., and Tate, M.E. (1969b) Structure of "phytic acids" Can J Chem 47,63-73

Joner, E.J., van Aarle, I.M., and Vosatka, M. (2000) Phosphatase activity of extra-radical arbuscular

mycorrhizal hyphae A review Plant Soil 226, 199-210

Jongbloed, A.W., Mroz, Z., and Kemme, P.A. (1992) The effect of supplementary Aspergillus niger phytase in

diets for pigs on concentration and apparent digestibility of dry matter, total phosphorus, and phytic acid

in different sections of the alimentary tract JAnimSci70, 1159-1168

Jungk, A. (1996) Dynamics of nutrient movement at the soil-root interface In Plant Roots Tthe Hidden Half, Y

Waisel, A Eschel, and U Kafkafi, eds (New York Marcel Dekker), pp 581-605

Jungk, A. (2001) Root hairs and the acquisition of plant nutrients from soil J Plant Nutr Soil Sc 164, 121-129

Kaida, R., and Kaneko, T. (2002) Overexpression of purple acid phosphatase enhances accumulation of beta-

gulcan on surface of protoplasts during cell wall regeneration Plant Cell Physiol 43, S139-S139

Kalsi, G., and Etzler, M.E. (2000) Localization of a nod factor-binding protein in legume roots and factors

influencing its distribution and expression Plant Physiol 124, 1039-1048

Karthikeyan, A.S., Varadarajan, D.K., Mukatira, U.T., D'Urzo, M.P., Damsz, B., and Raghothama, K.G.

(2002) Regulated expression of Arabidopsis phosphate transporters Plant Physiol 130, 221-233

Kasim, A., and Edwards, H. (1998) The analysis for inositol phosphate forms in feed ingredients J Sei Food

Agnc76, 1-9

Kasuga, M., Liu, Q., Miura, S., Yamaguchi-Shinozaki, K., and Shinozaki, K. (1999) Improving plant drought,

salt, and freezing tolerance by gene transfer of a single stress-mducible transcription factor Nat

Biotechnol 17, 287-291

Kaya, C, Higgs, D., and Burton, A. (2000) Plant growth, phosphorus nutrition, and acid phosphatase enzyme

activity in three tomato cultivars grown hydroponically at different zinc concentrations J Plant Nutr 23,569-579

Kerovuo, J., Lappalainen, I., and Reinikainen, T. (2000) The metal dependence of Bacillus subtilis phytaseBiochem Biophys Res Commun 268, 365-369

Kerovuo, J., Lauraeus, M., Nurminen, P., Kalkkinen, N., and Apajalahti, J. (1998) Isolation, characterization,molecular gene cloning, and sequencing of a novel phytase from Bacillus subtilis Appl Environ Microbiol

64, 2079-2085

Kim, Y.H., Gwon, M.N., Yang, S.Y., Park, T.K., Kim, CG., Kim, C.W., and Song, M.D. (2002) Isolation of

phytase-producmg Pseudomonas sp and optimization of its phytase production J Microbiol Biotechnol

12, 279-285

Kim, Y.O., Lee, J.K., Kim, H.K., Yu, J.H., and Oh, T.K. (1998) Cloning of the thermostable phytase gene (phy)from Bacillus sp DS11 and its overexpression in Escherichia coli FEMS Microbiol Lett 162, 185-191

Kishore, G.M., and Shewmaker, C. (1999) Biotechnology enhancing human nutrition in developing and

developed worlds Proc Natl Acad Sei U S A 96, 5968-5972

Klabunde, T., and Krebs, B. (1997) The dimetal center in purple acid phosphatases Struct Bonding 89, 177-

198

Klabunde, T., Strater, N., Fröhlich, R., Witzel, H., and Krebs, B. (1996) Mechanism of Fe(lll)-Zn(ll) purple acid

phosphatase based on crystal structures J Mol Biol 259, 737-748

Klabunde, T., Stahl, B., Suerbaum, H., Hahner, S., Karas, M., Hillenkamp, F., Krebs, B., and Witzel, H.

(1994) The amino-acid-sequence of the red kidney bean Fe(ln)-Zn(li) purple acid-phosphatase -

determination of the ammo-acid- sequence by a combination of matrix-assisted laser-desorptionionization mass-spectrometry and automated Edman sequencing Eur J Biochem 226, 369-375

Kollmeier, M., Dietrich, P., Bauer, CS., Horst, W.J., and Hedrich, R. (2001) Aluminum activates a citrate-

permeable anion channel in the aluminum-sensitive zone of the maize root apex A comparison between

an aluminum- sensitive and an aluminum-resistant cultivar Plant Physiol 126, 397-410

Koyama, H., Kawamura, A., Kihara, T., Hara, T., Takita, E., and Shibata, D. (2000) Overexpression of

mitochondrial citrate synthase in Arabidopsis thaliana improved growth on a phosphorus-limited soil

Plant Cell Physiol 41, 1030-1037

Kroehler, C.J., and Linkins, A.E. (1991) The Absorption of Inorganic-Phosphate from 32P-Labeled Inositol

Hexaphosphate by Enophorum-Vaginatum Oecologia 85, 424-428

Lassen, S.F., Breinholt, J., Ostergaard, P.R., Brugger, R., Bischoff, A., Wyss, M., and Fuglsang, C.C.

(2001) Expression, gene cloning, and characterization of five novel phytases from four basidiomycete

fungi Peniophora lycn, Agrocybe pediades, a Cenpona sp ,and Trametes pubescens Appl Environ

Microbiol 67, 4701-4707

Laumen, K., and Ghisalba, O. (1994) Preparative-scale chemo-enzymatic synthesis of optically pure D-myo-mositol 1-phosphate Biosci Biotechnol Biochem 58, 2046-2049

Lauter, F.R., Ninnemann, O., Bûcher, M., Riesmeier, J.W., and Frommer, W.B. (1996) Preferential

expression of an ammonium transporter and of two putative nitrate transporters in root hairs of tomato

Proc Natl Acad Sei U S A 93, 8139-8144

8 References 89

Leah, R., Tommerup, H., Svendsen, I., and Mundy, J. (1991) Biochemical and molecular characterization of

three barley seed proteins with antifungal properties J Biol Chem 266, 1564-1573

Leake, J. (2002) Organic phosphorus utilisation by mycorrhizal plants and fungi how much do we really know"?

In Phosphatases in the environment, B Whitton and I Hernandez, eds (Dordrecht, The Netherlands

Kluwer Academic), pp in press, cited by Turner et al (2002)Lebansky, B.R., McKnight, T.D., and Griffing, L.R. (1991) Purification and characterization of a secreted

purple phosphatase from soybean suspension cultures Plant Physiol 99, 391-395

Lebansky, B.R., McKnight, T.D., and Griffing, L.R. (1993) Purification of a purple acid-phosphatase secreted

by soybean suspension-cultures and identification of intracellular forms Plant Physiol 102, 58-58

Leggewie, G., Willmitzer, L., and Riesmeier, J.W. (1997) Two cDNAs from potato are able to complement a

phosphate uptake-deficient yeast mutant identification of phosphate transporters from higher plantsPlant Cell 9, 381-392

Lehmann, M., Pasamontes, L., Lassen, S.F., and Wyss, M. (2000a) The consensus concept for thermostability

engineering of proteins Biochim Biophys Acta 1543, 408-415

Lehmann, M., Kostrewa, D., Wyss, M., Brugger, R., D'Arcy, A., Pasamontes, L., and van Loon, A.P. (2000b)From DNA sequence to improved functionality using protein sequence comparisons to rapidly design a

thermostable consensus phytase Protein Eng 13, 49-57

Lehmann, M., Loch, C, Middendorf, A., Studer, D., Lassen, S.F., Pasamontes, L., van Loon, A.P., and

Wyss, M. (2002) The consensus concept for thermostability engineering of proteins further proof of

concept Protein Eng 15, 403-411

Lei, X.G., Ku, P.K., Miller, E.R., Yokoyama, M.T., and Ullrey, D.E. (1993) Supplementing corn-soybean meal

diets with microbial phytase maximizes phytate phosphorus utilization by weanling pigs J Anim Sei 71,3368-3375

Lenburg, M.E., and O'Shea, E.K. (1996) Signaling phosphate starvation Trends Biochem Sei 21, 383-387

Leprince, F., and Quiquampoix, H. (1996) Extracellular enzyme activity in soil effect of pH and ionic strengthon the interaction with montmorillonite or two acid phosphatases secreted by the ectomycorrhizal fungusHebeloma cylindrosporum Eur J Soil Sei 47, 511-522

Li, D., Zhu, H., Liu, K., Liu, X., Leggewie, G., Udvardi, M., and Wang, D. (2002) Purple acid phosphatases of

Arabidopsis thaliana Comparative analysis and differential regulation by phosphate deprivation J Biol

Chem 277, 72-81

Li, M., and Tadano, T. (1996) Comparison of characteristics of acid phosphatases secreted from roots of lupinand tomato Soil Sei Plant Nutr 42, 753-763

Li, M., Shinano, T., and Tadano, T. (1997a) Distribution of exudates of lupin roots in the rhizosphere under

phosphorus deficient conditions Soil Sei Plant Nutr 43, 237-245

Li, M., Osaki, M., Honma, M., and Tadano, T. (1997b) Purification and characterisation of phytase induced in

tomato roots under phosphorus-deficient conditions Soil Sei Plant Nutr 43, 179-190

Li, M., Osaki, M., Rao, I.M., and Tadano, T. (1997c) Secretion of phytase from roots of several plant species

under phosphorus-deficient conditions Plant Soil 195, 161-169

Liu, C, Muchhal, U.S., Uthappa, M., Kononowicz, A.K., and Raghothama, K.G. (1998) Tomato phosphate

transporter genes are differentially regulated in plant tissues by phosphorus Plant Physiol 116, 91-99

Loewus, F.A., and Murthy, P.P.N. (2000) myoinositol metabolism of plants Plant Sei 150, 1-19

Löffler, A., Abel, S., Jost, W., Beintema, J.J., and Glund, K. (1992) Phosphate-regulated induction of

intracellular nbonucleases in cultured tomato (Lycopersicon esculentum) cells Plant Physiol 98, 1472-

1478

Lolas, G., Palamidis, N., and Markakis, P. (1976) The phytic-acid total phosphorus relationship in barley, oats,

soybeans and wheat Cereal Chem 53, 867-870

Lonnerdal, B. (2002) Phytic acid-trace element (Zn, Cu, Mn) interactions Int J Food Sei Technol 37, 749-758

Lopez, H.W., Leenhardt, F., Coudray, C, and Remesy, C. (2002) Minerals and phytic acid interactions is it a

real problem for human nutrition'? Int J Food Sei Technol 37, 727-739

Lopez-Bucio, J., Martinez de la Vega, O., Guevara-Garcia, A., and Herrera-Estrella, L. (2000) Enhanced

phosphorus uptake in transgenic tobacco plants that overproduce citrate Nature Biotechnology 18, 450-

453

Lucca, P., Hurrell, R., and Potrykus, I. (2001) Genetic engineering approaches to improve the bioavailabilityand the level of iron in rice grains Theor Appl Genet 102, 392-397

Lucca, P., Hurrell, R., and Potrykus, I. (2002) Fighting iron deficiency anemia with iron-rich rice J Am Coll Nutr

21, 184S-190S

Lutrell, B. (1992) The biological relevance of the binding of calcium ions by inositol phosphates J Biol Chem

268, 1521-1524

Ma, Z., Bielenberg, D.G., Brown, K.M., and Lynch, J.P. (2001) Regulation of root hair density by phosphorus

availability in Arabidopsis thaliana Plant Cell Environ 24, 459-467

Magid, J., Tiessen, H., and Condron, L.M. (1996) Dynamics of organic phosphorus in soils under natural and

agricultural ecosystems In Humic substances in terrestrial ecosystems, A Piccolo, ed (Oxford Elsevier

Science), pp 429-466

Makarov, M.I., Haumaier, L., and Zech, W. (2002) Nature of soil organic phosphorus an assessment of peak

assignments in the diester region of P-31 NMR spectra Soil Biol Biochem 34, 1467-1477

Marschner, H. (1995) Mineral nutrition of higher plants (London Academic Press)

8 References 90

Martin, C. (1995) Reaction of the co-ordinate complexes of inositol hexaphosphate with first row transition series

cations and Cd(ll) with calf intestinal alkaline phosphatase J Inorg Biochem 58, 89-107

Martin, C, and Evans, W. (1987) Phytic acid divalent cation interactions V Titrimetric, calorimetric, and bindingstudies with cobalt(ll), nickel(il) and their comparison with other metal ions J Inorg Biochem 30, 101-

119

Martin, J., and Cartwright, B. (1971) The comparative plant availability of 32P myo-inositol hexa/asphosphateand KH232P04 added to soils Commun Soil Sei Plant Anal 2,375-381

Maugenest, S., Martinez, I., and Lescure, A.M. (1997) Cloning and characterization of a cDNA encoding a

maize seedling phytase Biochem J 322, 511-517

McArthur, D., and Knowles, N.R. (1993) Influence of Vesicular-Arbuscular Mycorrhizal Fungi on the Responseof Potato to Phosphorus Deficiency Plant Physiol 101, 147-160

McKercher, R., and Anderson, G. (1968) Characterization of inositol penta- and hexaphosphate fractions of a

number of Canadian and Scottish soils J Soil Sei 19, 302

McKercher, R., and Anderson, G. (1989) Organic phosphate sorption by neutral and basic soils Commun Soil

Sei Plant Anal 20,723-732

Milberg, P., and Lamont, B.B. (1997) Seed/cotyledon size and nutrient content play a major role in early

performance of species on nutrient-poor soils New Phytol 137, 665-672

Miller, S.S., Liu, J., Allan, D.L., Menzhuber, C.J., Fedorova, M., and Vance, C.P. (2001) Molecular control of

acid phosphatase secretion into the rhizosphere of proteoid roots from phosphorus-stressed white lupinPlant Physiol 127, 594-606

Minear, R.A., Segars, J.E., Elwood, J.W., and Mulholland, P.J. (1988) Separation of inositol phosphates by

high-performance ion exchange chromatography Analyst 113, 645-649

Mollier, A., and Pellerin, S. (1999) Maize root system growth and development as influenced by phosphorus

deficiency J Exp Bot 50, 487-497

Moriau, L. (1999) Expression analysis of two gene subfamilies encoding the plasma membrane H+-ATPase in

Nicotiana plumbaginifoha reveals the major transport functions of this enzyme Plant J 19, 31-41

Muchhal, U.S., and Raghothama, K.G. (1999) Transcriptional regulation of plant phosphate transporters Proc

Natl Acad Sei U S A 96, 5868-5872

Mueller-Roeber, B., and Pical, C. (2002) Inositol phospholipid metabolism in Arabidopsis Characterized and

putative isoforms of inositol phospholipid kinase and phosphomositide-specific phosphohpase C Plant

Physiol 130, 22-46

Murrashige, T., and Skoog, F. (1962) A revised medium for rapid growth and bioassays with tobacco tissue

culture Physiol Plantarum 15, 473-497

Murray, F.A., Jr., Bazer, F.W., Wallace, H.D., and Warnick, A.C. (1972) Quantitative and qualitative variation in

the secretion of protein by the porcine uterus during the estrous cycle Biol Reprod 7, 314-320

Nakano, T., Joh, T., Narita, K., and Hayakawa, T. (2000) The pathway of dephosphorylation of myoinositol

hexa/asphosphate by phytases from wheat bran of Tnticum aestivum L cv Nounn#61 Biosci

Biotechnol Biochem 64, 995-1003

Nakazato, H., Okamoto, T., Nishikoori, M., Washio, K., Morita, N., Haraguchi, K., Thompson, G.A., Jr., and

Okuyama, H. (1997) Characterisation and cDNA cloning of the GPI-anchored phosphatase from

Spirodela oligorrhiza In Plant nutrition - For sustainable food production and environment, T Ando, K

Fujita, T Mae, H Matsumoto, S Mon, and J Sekiya, eds (Dordrecht, The Netherlands Kluwer

Academic Publishers), pp 229-230

Nakazato, H., Okamoto, T., Nishikoori, M., Washio, K., Morita, N., Haraguchi, K., Thompson, G.A., Jr., and

Okuyama, H. (1998) The glycosylphosphatidylmositol-anchored phosphatase from Spirodela oligorrhizais a purple acid phosphatase Plant Physiol 118, 1015-1020

Narang, R.A., Bruene, A., and Altmann, T. (2000) Analysis of phosphate acquisition efficiency in different

Arabidopsis accessions Plant Physiol 124, 1786-1799

Nardi, S., Sessi, E., Pizzeghello, D., Sturaro, A., Relia, R., and Parvoli, G. (2002) Biological activity of soil

organic matter mobilized by root exudates Chemosphere 46, 1075-1081

Nelson, D.E., Rammesmayer, G., and Bohnert, H.J. (1998) Regulation of cell-specific inositol metabolism and

transport in plant salinity tolerance Plant Cell 10, 753-764

Nelson, D.E., Koukoumanos, M., and Bohnert, H.J. (1999) Myoinositol-dependent sodium uptake in ice plantPlant Physiol 119, 165-172

Nelson, T.S. (1967) The utilization of phytate phosphorus by poultry-a review Poult Sei 46, 862-871

Nelson, T.S., Shieh, T.R., Wodzinski, R.J., and Ware, J.H. (1968) The availability of phytate phosphorus in

soybean meal before and after treatment with a mold phytase Poult Sei 47, 1842-1848

Neumann, G., and Martinoia, E. (2002) Cluster roots-an underground adaptation for survival in extreme

environments Trends Plant Sei 7, 162-167

Neumann, G., Massonneau, A., Martinoia, E., and Romheld, V. (1999) Physiological adaptations to

phosphorus deficiency during proteoid root development in white lupin Planta 208, 373-382

Neumann, G., Massonneau, A., Langlade, N., Dinkelaker, B., Hengeler, C, Romheld, V., and Martinoia, E.

(2000) Physiological aspects of cluster root function and development in phosphorus-deficient white

lupin (Lupinus albus L ) Ann Bot-London 85, 909-919

Nielsen, H., Engelbrecht, J., Brunak, S., and von Heijne, G. (1997) Identification of prokaryotic and eukaryotic

signal peptides and prediction of their cleavage sites Protein Eng 10, 1-6

8 References 91

Nishikoori, M., Washio, K., Hase, A., Morita, N., and Okuyama, H. (2001) Cloning and characterization of

cDNA of the GPI-anchored purple acid phosphatase and its root tissue distribution in Spirodela

oligorrhiza Physiol Plantarum 113, 241-248

Nürnberger, T., Abel, S., Jost, W., and Glund, K. (1990) Induction of an extracellular ribonuclease m cultured

tomato cells upon phosphate starvation Plant Physiol 92, 970-976

Nuttleman, P.R., and Roberts, R.M. (1990) Transfer of Iron from Uterofernn (Purple Acid-Phosphatase) to

Transferrin Related to Acid-Phosphatase-Activity J Biol Chem 265, 12192-12199

Oberson, A., Besson, J.M., Maire, N., and Sticher, H. (1996) Microbiological processes in soil organic

phosphorus transformations in conventional and biological cropping systems Biol Fertil Soils 21, 138-

148

Oberson, A., Friesen, D.K., Tiessen, H., Morel, C, and Stahel, W. (1999) Phosphorus status and cycling in

native savanna and improved pastures on an acid low-P Colombian Oxisol Nutrient Cycling in

Agroecosystems 55, 77-88

Oddie, G.W., Schenk, G., Angel, N.Z., Walsh, N., Guddat, L.W., de Jersey, J., Cassady, A.I., Hamilton, S.E.,and Hume, D.A. (2000) Structure, function, and regulation of tartrate-resistant acid phosphatase Bone

27, 575-584

Oehl, F., Oberson, A., Probst, M., Fliessbach, A., Roth, H.R., and Frossard, E. (2001) Kinetics of microbial

phosphorus uptake in cultivated soils Biol Fertil Soils 34, 31-41

Oehl, F., Oberson, A., Tagmann, H.U., Besson, J.M., Dubois, D., Mader, P., Roth, H.R., and Frossard, E.

(2002) Phosphorus budget and phosphorus availability in soils under organic and conventional farmingNutr Cycl Agroecosys 62, 25-35

Ognalaga, M., Frossard, E., and Thomas, F. (1994) Glucose-1-phosphate and myoinositol hexaphosphate

adsorption mechanisms on Geothite Soil Sei Soc Am J 58, 332-337

Ohno, T., and Zibilske, L.M. (1991) Determination of low concentrations of phosphorus in soil extracts using

malachite green Soil Sei Soc Am J 55, 892-895

Olczak, M., Watorek, W., and Morawiecka, B. (1997) Purification and charactenzhation of acid phosphatasefrom yellow lupin (Lupinus luteus) seeds Biochim Biophys Acta 1341, 14-25

Olsen, S., Cole, C, Watanabe, A., and Dean, L. (1954) Estimation of available phosphorus in soils byextraction with sodium bicarbonate USDA, 171-188

Ortiz, R. (1998) Critical role of plant biotechnology for the genetic improvement of food crops perspectives for

the next millenium El J Biotechnol 1, 1-8

Oshima, Y., Ogawa, N., and Harashima, S. (1996) Regulation of phosphatase synthesis in Saccharomycescerevisiae-a review Gene 179, 171-177

Ozawa, K., Osaki, M., Matsui, H., Honma, M., and Tadano, T. (1995) Purification and properties of acid

phosphatase secreted from lupin roots under phosphorus-deficiency conditions Soil Sei Plant Nutr 41,461-469

Pant, H.K., and Warman, P.R. (2000) Enzymatic hydrolysis of soil organic phosphorus by immobilized

phosphatases Biol Fertil Soils 30, 306-311

Pant, H.K., Warman, P.R., and Nowak, J. (1999) Identification of soil organic phosphorus by P-31 nuclear

magnetic resonance spectroscopy Communications in Soil Science and Plant Analysis 30, 757-772

Pasamontes, L., Haiker, M., Wyss, M., Tessier, M., and van Loon, A.P. (1997a) Gene cloning, purification,and characterization of a heat-stable phytase from the fungus Aspergillus fumigatus Appl Environ

Microbiol 63, 1696-1700

Pasamontes, L., Haiker, M., Henriquez-Huecas, M., Mitchell, D.B., and van Loon, A.P. (1997b) Cloning of

the phytases from Emencella nidulans and the thermophilic fungus Talaromyces thermophilus Biochim

Biophys Acta 1353, 217-223

Paszkowski, U., Kroken, S., Roux, C, and Briggs, S.P. (2002) Rice phosphate transporters include an

evolutionary divergent gene specifically activated in arbuscular mycorrhizal symbiosis Proc Natl Acad

Sei USA99, 13324-13329

Perera, I.Y., Heilmann, I., and Boss, W.F. (1999) Transient and sustained increases in inositol 1,4,5-

tnsphosphate precede the differential growth response in gravistimulated maize pulvmi Proc Natl Acad

Sei U S A 96, 5838-5843

Perney, K.M., Cantor, A.H., Straw, M.L., and Herkelman, K.L. (1993) The effect of dietary phytase on growth

performance and phosphorus utilization of broiler chicks Poult Sei 72, 2106-2114

Petters, J., Gobel, C, Scheel, D., and Rosahl, S. (2002) A pathogen-responsive cDNAfrom potato encodes a

protein with homology to a phosphate starvation-induced phosphatase Plant Cell Physiol 43, 1049-

1053

Phillippy, B.Q. (1999) Susceptibility of wheat and Aspergillus niger phytases to mactivation by gastrointestinal

enzymes J Agric Food Chem 47, 1385-1388

Picault, N., Palmieri, L., Pisano, I., Hodges, M., and Palmieri, F. (2002) Identification of a novel transporter for

dicarboxylates and tricarboxylat.es in plant mitochondria Bacterial expression, reconstitution, functional

characterization, and tissue distribution J Biol Chem 277, 24204-24211

Plaami, S., and Kumpalainen, J. (1997) Inositol phosphate content of some cereal-based foods Journal of

Food Composition and Analysis 8, 324-335

Plaxton, W.C. (1998) Metabolic aspects of phosphate starvation in plants In Phosphorus in Plant Biology

Regulatory Roles in Molecular, Cellular, Organismic, and Ecosystem Processes,J P Lynch and J

Deikman, eds (ASPP), pp 229-241

8 References 92

Poulsen, H. (2000) Phosphorus utilization and excretion in pig production J Environ Qual 29, 24-27

Prud'homme, M. (2001) Summary report global agricultural situation and fertilizer consumption in 2000 and

2001 (Pans International Fertilizer Industry Association (IFA))

Raboy, V. (1997) Accumulation and storage of phosphate and minerals In Cellular and molecular biology of

plant seed development, B Larkms and I Vasil, eds (Dordrecht Kluwer Academic Publishers), pp 441-

477

Raghothama, K.G. (1999) Phosphate acquisition Annu Rev Plant Physiol Plant Mol Biol 50,665-693

Rausch, C, and Bucher, M. (2002) Molecular mechanisms of phosphate transport in plants Planta 216, 23-37

Rausch, C, Daram, P., Brunner, S., Jansa, J., Laloi, M., Leggewie, G., Amrhein, N., and Bucher, M. (2001)A phosphate transporter expressed in arbuscule-contammg cells in potato Nature 414, 462-470

Reyes, I., Bernier, L., and Antoun, H. (2002) Rock phosphate solubilization and colonization of maize

rhizosphere by wild and genetically modified strains of Pénicillium rugulosum Microb Ecol 44, 39-48

Richardson, A.E., and Hadobas, P.A. (1997) Soil isolates of Pseudomonas spp that utilize inositol phosphatesCanadian Journal of Microbiology 43, 509-516

Richardson, A.E., and Hayes, J.E. (2000) Microbial phytase improves the ability of plants to acquire

phosphorus from phytate In International Symposium on Phosphorus in the Soil-Plant Continuum

(Beijing, P R China)

Richardson, A.E., Hadobas, P.A., and Hayes, J.E. (2000) Acid Phosphomonoesterase and phytase activities of

wheat (Tnticum aestivum L ) roots and utilization of organic phosphorus substrates by seedlings grown

in sterile culture Plant Cell Environ 23, 397-405

Richardson, A.E., Hadobas, P.A., and Hayes, J.E. (2001a) Extracellular secretion of Aspergillus phytase from

Arabidopsis roots enables plants to obtain phosphorus from phytate Plant J 25, 641-649

Richardson, A.E., Hadobas, P.A., Hayes, J.E., O'Hara, C.P., and Simpson, R.J. (2001b) Utilization of

phosphorus by pasture plants supplied with myoinositol hexaphosphate is enhanced by the presence of

soil micro-organisms Plant Soil 229, 47-56

Rimbach, G., Pallauf, J., Brandt, K., and Most, E. (1995) Effect of phytic acid and microbial phytase on Cd

accumulation, Zn status, and apparent absorption of Ca, P, Mg, Fe, Zn, Cu, and Mn in growing rats Ann

Nutr Metab 39, 361-370

Rodriguez, E., Porres, J.M., Han, Y., and Lei, X.G. (1999) Different sensitivity of recombinant Aspergillus niger

phytase (r-PhyA) and Escherichia coli pH 2 5 acid phosphatase (r-AppA) to trypsin and pepsin in vitro

Arch Biochem Biophys 365, 262-267

Rodriguez, H., and Fraga, R. (1999) Phosphate solubihzmg bacteria and their role in plant growth promotionBiotechnol Adv 17,319-339

Rubio, V., Linhares, F., Solano, R., Martin, A.C., Iglesias, J., Leyva, A., and Paz-Ares, J. (2001) A conserved

MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in

unicellular algae Genes Dev 15, 2122-2133

Runge-Metzger, A. (1995) Closing the cycle obstacles to efficient P management for improved global food

security In Phosphorus in the global environment, H Tiessen, ed (New York Wley), pp 27-42

Sambrook, J., Frisch, E., and Maniatis, T. (1989) Molecular cloning a laboratory manual, 2nd edition (Cold

Spring Harbor, NY Cold Spring Harbor Laboratory Press)

Samuelsen, A.I., Martin, R.C, Mok, D.W., and Mok, M.C (1998) Expression of the yeast FRE genes in

transgenic tobacco Plant Physiol 118, 51-58

Sasakawa, N., Sharif, M., and Hanley, M.R. (1995) Metabolism and biological activities of inositol

penta/asphosphate and inositol hexa/asphosphate Biochem Pharmacol 50, 137-146

Saxena, S.N. (1964) Phytase activity of plant roots J Exp Bot 15, 654-658

Schenk, G., Korsinczky, M.L., Hume, D.A., Hamilton, S., and DeJersey, J. (2000a) Purple acid phosphatasesfrom bacteria similarities to mammalian and plant enzymes Gene 255, 419-424

Schenk, G., Guddat, L.W., Ge, Y., Carrington, L.E., Hume, D.A., Hamilton, S., and de Jersey, J. (2000b)Identification of mammalian-like purple acid phosphatases in a wide range of plants Gene 250, 117-125

Schenk, G., Ge, Y., Carrington, L.E., Wynne, C, Searle, I., Caroll, B., Hamilton, S., and de Jersey, J. (1999)Binuclear metal centers in plant purple acid phosphatases Fe-Mn in sweet potato and Fe-Zn in

Soybean Arch Biochem Biophys 370, 183-189

Schenk, G., Boutchard, CL., Carrington, L.E., Noble, C.J., Moubaraki, B., Murray, K.S., de Jersey, J.,

Hanson, G.R., and Hamilton, S. (2001) A purple acid phosphatase from sweet potato contains an

antiferromagnetically coupled binuclear Fe-Mn center J Biol Chem 276, 19084-19088

Schmidt, W., and Schikora, A. (2001) Different pathways are involved in phosphate and iron stress- induced

alterations of root epidermal cell development Plant Physiol 125, 2078-2084

Sebastian, S., Touchburn, S.P., Chavez, E.R., and Lague, P.C. (1996) The effects of supplemental microbial

phytase on the performance and utilization of dietary calcium, phosphorus, copper, and zinc in broiler

chickens fed corn-soybean diets Poult Sei 75, 729-736

Serageldin, I. (1999) Biotechnology and food security in the 21st century Science 285, 387-389

Shen, H., Wang, X.C, Shi, W.M., Cao, Z.H., and Yan, X.L (2001) Isolation and identification of specific root

exudates in elephantgrass in response to mobilization of iron- and aluminum-phosphates J Plant Nutr

24, 1117-1130

Shieh, T.R., and Ware, J.H. (1968) Survey of microorganism for the production of extracellular phytase ApplMicrobiol 16, 1348-1351

8 References 93

Shimogawara, K., Wykoff, D.D., Usuda, H., and Grossman, A.R. (1999) Chlamydomonas reinhardtn mutants

abnormal in their responses to phosphorus deprivation Plant Physiol 120, 685-694

Sibbesen, E., and Runge-Metzger, A. (1995) Phosphorus balance in European agriculture - status and policy

options In Phosphorus in the Global Environment, H Tiessen, ed (New York Wley), pp 43-57

Silva, G.N., and Vidor, C. (2001) Phosphate solubihzmg activity of microorganisms in the presence of nitrogen,

iron, calcium and potassium Pesqui Agropecu Bras 36, 1495-1508

Simoes Nunes, C, and Guggenbuhl, P. (1998) Effects of Aspergillus fumigatus phytase on phosphorus

digestibility, phosphorus excretion, bone strength and performance in pigs Reprod Nutr Dev 38, 429-

440

Simon, O., and Igbasan, F. (2002) In vitro properties of phytases from various microbial origins Int J Food Sei

Technol 37, 813-822

Simons, P.C., Versteegh, H.A., Jongbloed, A.W., Kemme, P.A., Slump, P., Bos, K.D., Wolters, M.G.,

Beudeker, R.F., and Verschoor, G.J. (1990) Improvement of phosphorus availability by microbial

phytase in broilers and pigs Br J Nutr 64, 525-540

Simpson, C.J., and Wise, A. (1990) Binding of zinc and calcium to inositol phosphates (phytate) in vitro Br J

Nutr 64, 225-232

Siren, M. (1986) Stabilized pharmaceutical and biological material composition In Pat SE 003 165

Smith, F.W., Ealing, P.M., Dong, B., and Delhaize, E. (1997) The cloning of two Arabidopsis genes belongingto a phosphate transporter family Plant J 11, 83-92

Smith, N., Surinder, P., Wang, M., Stoutjesdijk, P., Green, A., and Waterhouse, P. (2000) Total silencing by

mtron-sphced hairpin RNAs Nature 407, 319-320

Smith, S.E., and Barker, S.J. (2002) Plant phosphate transporter genes help harness the nutritional benefits of

arbuscular mycorrhizal symbiosis Trends Plant Sei 7, 189-190

Stahl, B., Klabunde, T., Witzel, H., Krebs, B., Steup, M., Karas, M., and Hillenkamp, F. (1994) The

oligosaccharides of the Fe(hi)-Zn(h) purple acid- phosphatase of the red kidney bean - determination of

the structure by a combination of matrix-assisted laser-desorption ionization mass-spectrometry and

selective enzymatic degradation Eur J Biochem 220, 321-330

Stahl, C.H., Han, Y.M., Roneker, K.R., House, W.A., and Lei, X.G. (1999) Phytase improves iron bioavailabilityfor hemoglobin synthesis in young pigs J Anim Sei 77, 2135-2142

Stewart, J.W.B., and Tiessen, H. (1987) Dynamics of soil organic phosphorus Biogeochemistry 4, 41-60

Strater, N., Klabunde, T., Tucker, P., Witzel, H., and Krebs, B. (1995) Crystal structure of a purple acid-

phosphatase containing a dmuclear Fe(lll)-Zn(ll) active-site Science 268, 1489-1492

Streb, H., Irvine, R.F., Berridge, M.J., and Schulz, I. (1983) Release of Ca2+ from a nonmitochondrial

intracellular store in pancreatic acinar cells by mositol-1,4,5-tnsphosphate Nature 306, 67-69

Suzuki, U., Yoshimura, K., and Takaishi, M. (1907) Cited in Inositol phosphates their chemistry, biochemistryand physiology, D J Cosgrove, ed (Amsterdam Elsevier)

Syers, J., and Curtin, D. (1989) Inorganic reactions controlling phosphorus cycling In Phosphorus cycles in

terrestrial and aquatic ecosystems, H Tiessen, ed.pp 17-29

Szabo-Nagy, A., and Erdei, L. (1995) Phosphatase induction under stress conditions in wheat In Structure and

function of roots, R e a Baluska, ed (Kluwer Academic Publishers), pp 163-167

Tabatabai, M.A., and Bremner, J.M. (1969) Use of p-nitrophenyl phosphate for assay of soil phosphatase

activity Soil Biol Biochem 1, 301-307

Tadano, T., and Sakai, H. (1991) Secretion of acid phosphatase by the roots of several crop species under

phosphorus-deficient conditions Soil Sei Plant Nutr 37, 129-140

Takahashi, H., Watanabe-Takahashi, A., Smith, F.W., Blake-Kalff, M., Hawkesford, M.J., and Saito, K.

(2000) The roles of three functional sulphate transporters involved in uptake and translocation of

sulphate in Arabidopsis thaliana Plant J 23, 171-182

Takahashi, M., Nakanishi, H., Kawasaki, S., Nishizawa, N.K., and Mori, S. (2001a) Enhanced tolerance of rice

to low iron availability in alkaline soils using barley nicotianamine aminotransferase genes Nat

Biotechnol 19, 466-469

Takahashi, S., Katagiri, T., Hirayama, T., Yamaguchi-Shinozaki, K., and Shinozaki, K. (2001b) Hyperosmoticstress induces a rapid and transient increase in inositol 1,4,5-tnsphosphate independent of abscisic acid

in Arabidopsis cell culture Plant Cell Physiol 42, 214-222

Tang, Z., Sadka, A., Morishige, D.T., and Mullet, J.E. (2001) Homeodomam leucine zipper proteins bind to the

phosphate response domain of the soybean vspb tripartite promoter Plant Physiol 125, 797-809

Tarafdar, J.C, and Jungk, A. (1987) Phosphatase activity in the rhizosphere and its relation to the depletion of

soil organic phosphorus Biol Fertil Soils 3, 199-204

Tarafdar, J.C, and Ciaassen, N. (1988) Organic phosphorus compounds as a phosphorus source for higher

plants through the activity of phosphatases produced by plant roots and microorganisms Biol Fertil Soils

5, 308-312

Tarafdar, J.C, Yadav, K.D., and Meena, S. (2001) Comparative efficiency of acid phosphatase originated from

plant and fungal sources J Plant Nutr Soil Sc 164, 279-282

Tarafdar, J.C, Yadav, R.S., and Niwas, R. (2002) Relative efficiency of fungal intra- and extracellular

phosphatases and phytase J Plant Nutr Soil Sei-Z Pflanzenernahr Bodenkd 165,17-19

Taylor, CW. (1998) Inositol tnsphosphate receptors Ca2+-modulated intracellular Ca2+ channels Biochim

Biophys Acta 1436, 19-33

8 References 94

Templin, M., Stoll, D., Schrenk, M., Traub, P., Vöhringer, C, and Joos, T. (2002) Protein microarray

technology Drug Discovery Today 7, 815-821

Thomas, C, Sun, Y., Naus, K., Lloyd, A., and Roux, S. (1999) Apyrase functions in plant phosphate nutrition

and mobilizes phosphate from extracellular ATP Plant Physiol 119, 543-552

Thompson, E., and Black, CA. (1970) Changes in extractable phosphorus in soil in the presence and absence

of plants, III Phosphatase effect Plant Soil 32, 335-348

Thompson, J., Higgins, G., and Gibson, T. (1994) CLUSTALW increasing the sensitivity of progressive

multiple sequence alignment through sequence weighting, position-specific gap penalties and weightmatrix choice Nucleic Acids Res 22, 4673-4680

Ticconi, CA., Delatorre, CA., and Abel, S. (2001) Attenuation of phosphate starvation responses by phosphitein Arabidopsis Plant Physiol 127, 963-972

Tomscha, J. (2001) Phosphatase secretion mutants in Arabidopsis thaliana (Thesis, Pennsylvania State

University), pp 102

Tomschy, A., Brugger, R., Lehmann, M., Svendsen, A., Vogel, K., Kostrewa, D., Lassen, S.F., Burger, D.,

Kronenberger, A., van Loon, A.P., Pasamontes, L., and Wyss, M. (2002) Engineering of phytase for

improved activity at low pH Appl Environ Microbiol 68, 1907-1913

Trull, M.C., Tomscha, J., and Deikman, J. (1998) Arabidopsis thaliana mutants defective in the phosphorus-starvation response In Phosphorus in Plant Biology Regulatory Roles in Molecular, Cellular,

Organismic, and Ecosystem Processes,J P Lynch and J Deikman, eds (ASPP), pp 281-289

Turner, B.L., Paphazy, M.J., Haygarth, P.M., and McKelvie, I.D. (2002) Inositol phosphates in the

environment Philos Trans R Soc Lond Ser B-Biol Sei 357, 449-469

Uehara, K., Fujimoto, S., and Taniguchi, T. (1974a) Studies on violet-colored acid phosphatase of sweet

potato I Purification and some physical properties J Biochem (Tokyo) 75, 627-638

Uehara, K., Fujimoto, S., Taniguchi, T., and Nakai, K. (1974b) Studies on violet-colored acid phosphatase of

sweet potato II Enzymatic properties and ammo acid composition J Biochem (Tokyo) 75, 639-649

Van der Kaay, J., and Van Haastert, P.J. (1995) Stereospecificity of inositol hexa/asphosphate

dephosphorylation by Paramecium phytase Biochem J 312 ( Pt 3), 907-910

Van der Kaay, J., Wesseling, J., and Van Haastert, P.J. (1995) Nucleus-associated phosphorylation of

lns(1,4,5)P3to lnsP6 in Dictyostelium Biochem J 312 ( Pt3), 911-917

Varadarajan, D.K., Karthikeyan, A.S., Matilda, P.D., and Raghothama, K.G. (2002) Phosphite, an analog of

phosphate, suppresses the coordinated expression of genes under phosphate starvation Plant Physiol

129, 1232-1240

Vert, G., Grotz, N., Dedaldechamp, F., Gaymard, F., Guerinot, M.L., Briat, J.F., and Curie, C. (2002) IRT1,an Arabidopsis transporter essential for iron uptake from the soil and for plant growth Plant Cell 14,1223-1233

Verwoerd, T.C, van Paridon, P.A., van Ooyen, A.J., van Lent, J.W., Hoekema, A., and Pen, J. (1995) Stable

accumulation of Aspergillus niger phytase in transgenic tobacco leaves Plant Physiol 109, 1199-1205

Vincent, J.B., and Averill, B.A. (1990) An enzyme with a double identity purple acid phosphatase and tartrate-

resistant acid phosphatase Faseb J 4, 3009-3014

Vincent, J.B., Crowder, M., and Averill, B.A. (1992) Hydrolysis of phosphate monoesters a biological problemwith multiple chemical solutions Trends Biochem Sei 17, 105-110

von Wiren, N., Lauter, F.R., Ninnemann, O., Gillissen, B., Walch-Liu, P., Engels, C, Jost, W., and Frommer,W.B. (2000) Differential regulation of three functional ammonium transporter genes by nitrogen in root

hairs and by light in leaves of tomato Plant J 21, 167-175

Wasaki, J., Omura, M., Ando, M., Dateki, H., Shinano, T., Osaki, M., Ito, H., Matsui, H., and Tadano, T.

(2000) Molecular cloning and root specific expression of secretory acid phosphatase from phosphatedeficient lupin (Lupinus albus L ) Soil Sei Plant Nutr 46, 427-437

Waters, B.M., Blevins, D.G., and Eide, D.J. (2002) Characterization of FR01, a pea fernc-chelate reductase

involved in root iron acquisition Plant Physiol 129, 85-94

Wheeler, E., and Ferrel, R. (1971 ) A method for phytic acid determination in wheat and wheat fraction Cereal

Chem 48, 312-320

Williamson, L.C, Ribrioux, S.P., Fitter, A.H., and Leyser, H.M. (2001) Phosphate availability regulates root

system architecture in Arabidopsis Plant Physiol 126, 875-882

Wubben, M.J.E., Su, H., Rodermel, S.R., and Baum, T.J. (2001) Susceptibility to the sugar beet cyst nematode

is modulated by ethylene signal transduction in Arabidopsis thaliana Molecular Plant-Microbe

Interactions 14, 1206-1212

Wykoff, D.D., Grossman, A.R., Weeks, D.P., Usuda, H., and Shimogawara, K. (1999) Psrl, a nuclear

localized protein that regulates phosphorus metabolism in Chlamydomonas Proc Natl Acad Sei U S A

96, 15336-15341

Wyss, M., Pasamontes, L., Remy, R., Köhler, J., Kusznir, E., Gadient, M., Muller, F., and van Loon, A.

(1998) Comparison of the thermostability properties of three acid phosphatases from molds Aspergillus

fumigatus phytase, A niger phytase, and A niger PH 2 5 acid phosphatase Appl Environ Microbiol 64,4446-4451

Wyss, M., Brugger, R., Kronenberger, A., Remy, R., Fimbel, R., Oesterhelt, G., Lehmann, M., and van Loon,A.P. (1999a) Biochemical characterization of fungal phytases (myoinositol hexa/asphosphate

phosphohydrolases) catalytic properties Appl Environ Microbiol 65, 367-373

8 References 95

Wyss, M., Pasamontes, L., Friedlein, A., Remy, R., Tessier, M., Kronenberger, A., Middendorf, A.,

Lehmann, M., Schnoebelen, L., Rothlisberger, U., Kusznir, E., Wahl, G., Muller, F., Lahm, H.W.,

Vogel, K., and van Loon, A.P. (1999b) Biophysical characterization of fungal phytases (myoinositol

hexa/asphosphate phosphohydrolases) molecular size, glycosylation pattern, and engineering of

proteolytic resistance Appl Environ Microbiol 65, 359-366

Yadav, R., and Yadav, K.D. (1996) An extracellular phosphatase from Aspergillus fumigatus Indian J Exp Biol

34, 1257-1260

Yadav, R.S., and Tarafdar, J.C. (2001) Influence of organic and inorganic phosphorus supply on the maximum

secretion of acid phosphatase by plants Biol Fertil Soils 34, 140-143

Yan, F., Zhu, Y.Y., Müller, C, Zorb, C, and Schubert, S. (2002) Adaptation of H+-pumping and plasmamembrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency Plant Physiol

129, 50-63

Yanke, L.J., Bae, H.D., Selinger, L.B., and Cheng, K.J. (1998) Phytase activity of anaerobic ruminai bacteria

Microbiology 144 ( Pt 6), 1565-1573

Ye, X., Al-Babili, S., Kloti, A., Zhang, J., Lucca, P., Beyer, P., and Potrykus, I. (2000) Engineering the

provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) rice endosperm Science 287,303-305

Yoshida, K., Ogawa, N., and Oshima, Y. (1989) Function of the PHO regulatory genes for repressive acid

phosphatase synthesis in Saccharomyces cerevisiae Mol Gen Genet 217, 40-46

Yoshida, K.T., Wada, T., Koyama, H., Mizobuchi-Fukuoka, R., and Naito, S. (1999) Temporal and spatial

patterns of accumulation of the transcript of Myoinositol-1 -phosphate synthase and phytin-contaming

particles during seed development in rice Plant Physiol 119, 65-72

Yoshimoto, N., Takahashi, H., Smith, F.W., Yamaya, T., and Saito, K. (2002) Two distinct high-affinity sulfate

transporters with different mducibihties mediate uptake of sulfate in Arabidopsis roots Plant J 29, 465-

473

Zakhleniuk, O.V., Raines, CA., and Lloyd, J.C. (2001) pho3 a phosphorus-deficient mutant of Arabidopsisthaliana (L ) Heynh Planta 212, 529-534

Zhang, B., Zhang, P., and Chen, X. (2000a) Factors affecting colonization of introduced microorganisms on

plant roots Ymg Yong Sheng Tai Xue Bao 11, 951-953

Zhang, H.X., and Blumwald, E. (2001) Transgenic salt-tolerant tomato plants accumulate salt in foliage but not

in fruit Nat Biotechnol 19, 765-768

Zhang, W.H., Ryan, P.R., and Tyerman, S.D. (2001) Malate-permeable channels and cation channels activated

by aluminum in the apical cells of wheat roots Plant Physiol 125, 1459-1472

Zhang, Z.B., Kornegay, E.T., Radcliffe, J.S., Wilson, J.H., and Veit, H.P. (2000b) Comparison of phytase from

genetically engineered Aspergillus and canola in weanling pig diets J Anim Sei 78, 2868-2878

Zimmermann, M.B., and Hurrell, R.F. (2002) Improving iron, zinc and vitamin A nutrition through plant

biotechnology Curr Opm Biotechnol 13, 142-145

8 References 96

9 Appendix 97

g%J Appendix

Appendix 1. DNA and amino acid sequences of StPAPI

Appendix 2. DNA and amino acid sequences of StPAP2


Appendix 4. DNA and amino acid sequences of the SP/PHY chimeric gene

Appendix 5. Analysis of the signal sequence of SP/PHY

9 Appendix 98

Appendix 1. DNA and amino acid sequences of StPAPI

1 MASMKILNIFI

1 TCGGCACGAGTTCTTNTCTAAAAATATATGGCTTCCATGAAAATACTCAACATTTTCATT

12 SFLLLLLFPAAMAELHRLEH

61 AGTTTCTTGTTGTTGTTACTATTTCCGGCAGCCATGGCTGAGCTCCACCGGTTAGAACAT

32 PVNTDGSISFLVVG WGRRG

121 CCGGTGAACACCGACGGCTCGATTAGTTTTTTGGTCGTCGGAGATTGGGGAAGAAGAGGA

52 TFNQSQVAQQMGIIGEKLNI

181 ACCTTTAACCAATCTCAAGTTGCTCAACAAATGGGAATAATTGGAGAGAAATTAAACATA

72 DFVVSTG NF DDGLTGVDD

2 41 GATTTTGTTGTATCAACTGGAGACAATTTCTATGATGATGGATTGACTGGTGTGGATGAT

92 PAFEESFTNVYTAPSLQKNW

301 CCTGCCTTTGAGGAATCTTTTACCAATGTCTACACAGCTCCAAGCTTACAAAAAAATTGG

112 YNVLG HDYRGDALAQLSPI

361 TATAACGTTTTGGGGAACCATGACTACAGAGGTGATGCTTTAGCACAATTAAGTCCTATT

132 LKQKDNRWICMRSYIVNTDV

4 21 CTTAAGCAAAAGGATAACAGATGGATT TGTATGAGGTCT TATAT TGT TAATACAGATGTG

152 AEFFFVDTTPFQDMYFTTPK

4 81 GCAGAATTTTTCTTTGTAGATACAACTCCTTTTCAAGATATGTATTTCACAACTCCTAAA

172 DHTYDWRNVMPRKDYLSQVL

541 GATCATACTTATGATTGGAGAAATGTTATGCCTCGAAAAGATTATCTTTCCCAAGTTTTG

192 KDLDSALRESSAKWKIVVG

601 AAGGATT TGGACTCAGCATTAAGGGAATCAAGTGCAAAATGGAAAATAGTAGTTGGTCAC

212 HTIKSAGHHGSSEELGVHIL

661 CACACCATTAAAAGTGCTGGACACCATGGTAGCTCTGAGGAGCTTGGAGTCCACATTCTT

232 PILQANNVDFYLNG D CLE

7 21 CCCATATTACAGGCAAACAATGTTGACTTTTACCTAAATGGGCATGACCATTGCTTGGAG

252 HISSSDSPLQFLTSGGGSKS

7 81 CATATCAGCAGT TCAGATAGTCCACTACAATT T T TGACAAGTGGTGGGGGTTCAAAATCA

272 WRGDMNWWNPKEMKFYYDGQ

8 41 TGGAGGGGTGATATGAATTGGTGGAATCCAAAGGAAATGAAATTTTATTATGATGGACAA

292 GFMAMQITQTQVWIQFFDI F

9 01 GGATTTATGGCTATGCAAATTACTCAAACACAAGTTTGGATACAATTTTTTGACATTTTT

312 GNILHKWSASKNLVSIM-

961 GGAAACATTTTGCATAAATGGAGTGCATCAAAAAACCTTGTTTCCATTATGTAAACAACT

1021 CAAAATAAAAAAAAATGTTGAACAAAAAATAGCCAAAAAGAAATTATGGATT TAATT TGT

1081 TTCTGCTAATTAGCAGTTAAATATTATCCTAATCATTGATGTAATTGTATCCAATATGTT

1141 CTATTGAAATATTTATTTATGTTAAATCTGAGTTTATTTGCAGTAAAAAAAAAAAAAAAA

12 01 AAAAAACTCGAGACTAGTTCTCTCCTNCGTGCCGAATTGCGGCCGCGAATTCCTGCAGCC

9 Appendix 99


1 SGPTSGEVTSSFVRKIEKTIDMPLDSDVFR

1 TCCGGCCCAACTTCCGGAGAAGTCACCAGTAGTTTTGTTAGGAAAATTGAGAAGACAATTGATATGCCTCTGGATAGTGATGTCTTCCGT

31 VPPGYNAPQQVHITQGDHVGKAVIVSWVTM

91 GTTCCTCCTGGATATAATGCGCCTCAACAGGTTCATATAACACAAGGAGATCATGTGGGAAAGGCGGTAATTGTTTCATGGGTGACTATG

61 DEPGSSTVVYWSEKSKLKNKANGKVTTYKF

181 GATGAACCTGGTTCAAGTACAGTAGTATACTGGAGTGAGAAAAGCAAGCTAAAGAATAAGGCAAATGGAAAAGTTACTACCTATAAGTTT

91 YNYTSGYIHHCNIKNLKFDTKYYYKIGIGH

271 TATAACTATACATCTGGTTACATCCACCACTGCAATATCAAAAATTTGAAGTTCGATACCAAATACTACTATAAGATTGGGATTGGACAC

121 VARTFWFTTPPEAGPDVPYTFGLIGDLGQS

361 GTGGCACGAACCTTCTGGTTCACAACCCCTCCAGAAGCCGGCCCTGATGTACCCTATACATTTGGTCTTATAGGGGATCTTGGTCAGAGT

151 FDSNKTLTHYELNPIKGQAVSFVGDISYAD

4 51 TTCGATTCAAACAAGACACTCACACATTATGAATTAAATCCAATTAAGGGGCAAGCAGTGTCGTTCGTAGGCGACATATCTTACGCAGAT

181 NYPNHDKKRWDTWGRFAERSTAYQPWIWTA

541 AACTACCCAAATCATGACAAAAAAAGATGGGACACATGGGGAAGGTTTGCAGAGAGAAGTACTGCTTATCAACCTTGGATTTGGACAGCA

211 GNHEIDFAPEIGETKPFKPYTHRYHVPFRA

631 GGAAACCATGAGATAGATTTTGCTCCTGAAATTGGGGAAACAAAACCATTCAAGCCCTACACTCATCGGTATCATGTCCCATTCAGAGCA

241 SDSTSPLWYSIKRASAYIIVLSSYSAYGKY

7 21 TCAGACAGCACATCTCCACTTTGGTATTCAATCAAGCGAGCTTCAGCGTATATCATAGTTTTATCCTCATATTCAGCATATGGCAAATAC

271 TPQYKWLEEELPKVNRTETPWLIVLVHSPW

811 ACTCCTCAATACAAGTGGCTTGAGGAAGAGCTACCAAAGGTTAACAGGACTGAGACTCCGTGGCTGATTGTTCTAGTACATTCGCCATGG

301 YNSYNYHYMEGETMRVMYE PWFVQYKVNMV

9 01 TATAACAGCTACAACTATCACTATATGGAAGGGGAAACCATGAGAGTAATGTATGAACCATGGTTTGTACAGTACAAAGTGAATATGGTT

331 FAGHVHAYERTERI SNVAYNVVNGECS PI K

9 91 TTTGCAGGTCATGTTCATGCTTATGAACGAACGGAACGGATTTCTAATGTGGCCTACAACGTTGTCAATGGAGAATGCAGTCCTATTAAA

361 DQSAPIYVTIGDGGNLEGLATNMTEPQPAY

1081 GATCAGTCTGCTCCAATTTATGTAACAATCGGTGATGGAGGAAATCTTGAAGGCCTAGCCACCAACATGACAGAGCCACAACCAGCTTAC

391 SAFREASFGHATLAIKNRTHAYYSWHRNQD

1171 TCTGCATTCCGCGAGGCTAGTTTTGGACATGCCACTCTTGCCATCAAGAATAGAACTCATGCTTATTATAGTTGGCATCGTAATCAAGAT

421 GYAVEADKIWVNNRVWHPVDESTAAKS-

12 61 GGATATGCTGTGGAAGCTGATAAAATATGGGTTAACAACCGAGTTTGGCATCCAGTTGATGAGTCCACAGCAGCCAAATCATGATGATAT

1351 ACACGAAATTTCATCTATCTTTTCTTTCCTTTTCCTCAGTAACATTGTGCACTTGTTGATGAATAAACGTTTCATTATTTCAAGCTCTTG

14 41 CTGCCTCATAATTTGTTAAACGTCCATTTGGGACATGGCAGAAGAGTCATTGTGTGGTAAACGATAAAAACGTCGTAAAAGAAAATCGAA

1531 GGACATACATTTGTTCATATTACTTATTTATCCAAATTATAATTCTAATCATTAAAAAAAAAAAAAAAAAACTCGAGGGGGGGCCCGGTA

1621 CCCA

9 Appendix 100


1 MLLHIFFLLSLFLTFIDNGSAGIT

3 GTTAGTGGAGAGGAGACAATGTTGCTTCATATCTTCTTTTTGTTATCTCTCTTTTTGACATTTATAGACAATGGGAGTGCTGGTATAACA

31 SAFIRTQFPSVDIPLENEVLSVPNGYNAPQ

93 AGTGCATTCATTCGAACTCAGTTTCCGTCTGTTGATATTCCCCTTGAAAATGAAGTACTTTCAGTTCCAAATGGTTATAACGCTCCACAG

61 QVHITQGDYDGEAVIISWVTADEPGSSEVR

183 CAAGTGCATATTACACAAGGTGACTATGATGGGGAAGCTGTCATTATCTCATGGGTAACTGCTGATGAACCAGGGTCTAGCGAAGTGCGA

91 YGLSEGKYDVTVEGTLNNYTFYKYESGYIH

27 3 TATGGCTTATCTGAAGGGAAATATGATGTTACTGTTGAAGGGACTCTAAATAACTACACATTCTACAAGTACGAGTCTGGTTACATACAT

121 QCLVTGLQYDTKYYYEIGKGDSARKFWFET

363 CAGTGCCTTGTAACTGGCCTTCAGTATGACACAAAGTACTACTATGAAATTGGAAAAGGAGATTCTGCACGGAAGTTTTGGTTTGAAACT

151 PPKVDPDASYKFGIIGDLGQTYNSLSTLQH

4 53 CCTCCAAAAGTTGATCCAGATGCTTCTTACAAATTTGGCATCATAGGTGACCTTGGTCAAACATATAATTCTCTTTCAACTCTTCAGCAT

181 YMASGAKSVLFVGDLSYADRYQYNDVGVRW

54 3 TATATGGCTAGTGGAGCAAAGAGTGTCTTGTTTGTTGGAGACCTCTCTTATGCTGACAGATATCAGTATAATGATGTTGGAGTCCGTTGG

211 DTFGRLVEQSTAYQPWIWSAGNHEIEYFPS

633 GATACATTTGGCCGCCTAGTTGAACAAAGTACAGCATACCAGCCATGGATTTGGTCTGCTGGGAATCATGAGATAGAGTACTTTCCATCT

241 MGEEVPFRSFLSRYPTPYRASKSSNPLWYA

7 23 ATGGGGGAAGAAGTTCCATTCAGATCGTTTCTATCTAGATACCCCACACCTTATCGAGCTTCAAAAAGCAGTAATCCCCTTTGGTATGCC

271 IRRASAHIIVLSSYSPFVKYTPQWHWLKQE

813 ATCAGAAGGGCATCTGCTCACATAATTGTCCTATCAAGCTATTCCCCTTTTGTAAAATATACACCTCAATGGCATTGGCTGAAACAGGAA

301 FKKVNREKTPWLIVLMHVPIYNSNEAHFME

903 TTTAAAAAGGTGAACAGAGAGAAAACTCCTTGGCTTATAGTCCTTATGCATGTTCCTATCTACAACAGTAATGAAGCCCATTTCATGGAA

331 GESMRSAYERWFVKYKVDVI FAGHVHAYER

9 93 GGGGAAAGCATGAGATCCGCCTACGAAAGATGGTTTGTCAAATACAAAGTCGATGTGATCTTTGCTGGCCACGTCCATGCTTATGAAAGA

361 SYRISNIHYNVSGGDAYPVPDKAAPIYITV

1083 TCATATCGCATATCTAATATACACTACAATGTCTCGGGTGGTGATGCTTATCCCGTACCAGATAAGGCAGCTCCTATTTACATAACTGTT

391 GDGGNSEGLASRFRDPQPEYSAFREASYGH

117 3 GGTGATGGAGGAAATTCAGAAGGTCTTGCTTCAAGATTTAGAGATCCCCAGCCAGAATATTCTGCTTTCCGTGAAGCCAGCTATGGTCAT

421 STLDIKNRTHAIYHWNRNDDGNNITTDSFT

12 63 TCCACTCTAGATATCAAGAATAGAACACATGCTATCTACCACTGGAATCGAAATGATGATGGAAATAACATTACAACTGACTCATTTACA

451 LHNQYWGSGLRRRKLNKNHLNSVISERPFS

1353 TTGCACAACCAGTATTGGGGAAGTGGTCTTCGCAGGAGAAAGTTGAACAAGAATCATCTAAACTCTGTCATTTCCGAAAGGCCCTTCTCT

481 A R L -

14 4 3 GCGCGACTCTGAGACACAGTTTCTCAAACATGTTAGTCAAACTATGGGTAATTTTATGCCATGATCCTAGTATGTAGTTATATTATAAAA

1533 TCTATCTACTTTTGTTGGAGAGAGTGGATCAAGCTATTTTCCCAGTGTATTGGTTCATGTAAAATAAGGATTTGTGTTGTTTATATGACA

162 3 GCATTATGGAAAGTATAGCTCTTGTAAATTTGAAATAGCTACTTCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA

1713 AAAAAAAAAAAAAAAAAAAAA

9 Appendix 101

Appendix 4. DNA and amino acid sequences of the SP/PHY chimeric gene

GGATCCATGGCTAGAAAAGATGTTGCCTCCATGTTTGCAGTTGCTCTCTTCATTGGAGCA

21 f*'*k*'rV'l'**f'i *"*'* '••'">l<Äx%^^^^B S | G H S C D

61 TTCGCTGCTGTTCCTACGAGTGTGCAGTCCATCGGCGTATCCATGGGCCACTCCTGCGAC

41 TVDGGYQCFPEISHLWGTYS

121 ACCGTGGACGGCGGCTACCAGTGCTTCCCGGAGATCTCCCACCTCTGGGGCACCTACTCC

61 PYFSLADESAISPDVPDDCR

181 CCGTACTTCTCCCTCGCCGACGAGTCCGCCATCTCCCCGGACGTGCCGGACGACTGCCGC

81 VTFVQVLSRHGARYPTSSKS

2 41 GTGACCTTCGTGCAGGTGCTCTCCCGCCACGGCGCCCGCTACCCGACCTCCTCCAAGTCC

101 KAYSALIEAIQKNATAFKGK

3 01 AAGGCCTACTCCGCCCTCATCGAGGCCATCCAGAAGAACGCCACCGCCTTCAAGGGCAAG

121 YAFLKTYNYTLGADDLTPFG

3 61 TACGCCTTCCTCAAGACCTACAACTACACCCTCGGCGCCGACGACCTCACCCCGTTCGGC

141 ENQMVNSGIKFYRRYKALAR

421 GAGAACCAGATGGTGAACTCCGGCATCAAGTTCTACCGCCGCTACAAGGCCCTCGCCCGC

161 KIVPFIRASGSDRVIASAEK

4 81 AAGATCGTGCCGTTCATCCGCGCCTCCGGCTCCGACCGCGTGATCGCCTCCGCCGAGAAG

181 FIEGFQSAKLADPGSQPHQA

541 TTCATCGAGGGCTTCCAGTCCGCCAAGCTCGCCGACCCGGGCTCCCAGCCGCACCAGGCC

201 SPVIDVIIPEGSGYNNTLDH

6 01 TCCCCGGTGATCGACGTGATCATCCCGGAGGGCTCCGGCTACAACAACACCCTCGACCAC

221 GTCTAFEDSELGDDVEANFT

6 61 GGCACCTGCACCGCCTTCGAGGACTCCGAACTCGGCGACGACGTGGAGGCCAACTTCACC

241 ALFAPAIRARLEADLPGVTL

721 GCCCTCTTCGCCCCGGCCATCCGCGCCCGCCTCGAGGCCGACCTCCCGGGCGTGACCCTC

261 TDEDVVYLMDMCPFETVART

7 81 ACCGACGAGGACGTGGTGTACCTCATGGACATGTGCCCGTTCGAGACCGTGGCCCGCACC

281 SDATELSPFCALFTHDEWIQ

8 41 TCCGACGCCACCGAACTCTCCCCGTTCTGCGCCCTCTTCACCCACGACGAGTGGATCCAG

lii:

Start methionines of

ß-1,3 glucanase signal

sequence and of the

modified sequence of

Consensus-phytase

(see chapter 4.2)

Signal peptide (SP)for secretion

sequence followingSP in ß-1,3 glucanase

Bold: mutations in AA

sequence:

• I of ß-1,3 glucanase

changed to S

• S of consensus

phytase changed to G

Arrow: cleavage site of

signal peptide

301

901

YDYLQSLGKYYGYGAGNPLG

TACGACTACCTCCAGTCCCTCGGCAAGTACTACGGCTACGGCGCCGGCAACCCGCTCGGC

321

961

PAQGVGFANELIARLTRS PV

CCGGCCCAGGGCGTGGGCTTCGCCAACGAACTCATCGCCCGCCTCACCCGCTCCCCGGTG

341

1021

QDHTSTNHTLDSNPATFPLN

CAGGACCACACCTCCACCAACCACACCCTCGACTCCAACCCGGCCACCTTCCCGCTCAAC

361

1081

ATLYADFSHDNSMISIFFAL

GCCACCCTCTACGCCGACTTCTCCCACGACAACTCCATGATCTCCATCTTCTTCGCCCTC

381

1141

GLYNGTAPLSTTSVESIEET

GGCCTCTACAACGGCACCGCCCCGCTCTCCACCACCTCCGTGGAGTCCATCGAGGAGACC

401

1201

DGYSASWTVPFAARAYVEMM

GACGGCTACTCCGCCTCCTGGACCGTGCCGTTCGCCGCCCGCGCCTACGTGGAGATGATG

421

1261

QCQAEKEPLVRVLVNDRVVP

CAGTGCCAGGCCGAGAAGGAGCCGCTCGTGCGCGTGCTCGTGAACGACCGCGTGGTGCCG

441

1321

LHGCAVDKLGRCKRDDFVEG

CTCCACGGCTGCGCCGTGGACAAGCTCGGCCGCTGCAAGCGCGACGACTTCGTGGAGGGC

461

1381

LSFARSGGNWAECFA-LIKR

CTCTCCTTCGCCCGCTCCGGCGGCAACTGGGCCGAGTGCTTCGCCTGATTAATTAAGAGA

9 Appendix 102

Appendix 5. Analysis of the signal sequence of SP/PHY

l 0

0 8

0 6

0 4

l!2

OiJ

-

X**i

& St.Ort —

> < ut

h

h k >'

-' \ ; t

-

' *.. f '< 1

»

yi

i

i

t4'•

r>

;/"

t-*"\"*

V\ i / K, f*' l

l ! i *r "--"'^tv-.-t--^x-;"' ,/•....

G3*ttR<r»aVFAVALF1 &6FMVFTS/3S19/3XH903TCB3MCFPE 1SHWGTYSPVFSLAEESA

l l l i i l i

20 30 40 50 60Positon

Change from polar to apolar

Polarity

Charge

AA sequence

apppaapaaaaaaaaaaaaaaappappaaaapaa

0++-000000000000000000000000000000

IGVCYGV

, cleavagesite

MARKDVASMFAVALFIGAFAAWTSJvja^

h

The general model describes three domains: a positively charged, polar domain (n), a neutral

and hydrophobic core (h) and a neutral, polar domain close to the cleavage site (c). The

algorithm for signal sequence prediction has the following requirements:

1. apolar (hydrophobic), uncharged core surrounded by two polar (hydrophilic) sequences.

2. Clear change from polar (signal sequence) to apolar (mature protein) at cleavage site

3. Last amino acid usually S, third last usually V.

The SP/PHY construct fulfils all the theoretical requirements and was tested positive in all

four tests performed by the SignallP prediction server

(http://www. cbs. dtu. dk/services/SignalP-2.0/).

10 Acknowledgements 103

\ß Acknowledgements

I wish to thank:

Dr. Marcel Bücher for giving me a good start in molecular biology and his continuous support

throughout the project. The creative discussions, the freedom given me to try also personalideas, and the help during dissertation writing are particularly appreciated.

Prof. Dr. Emmanuel Frossard for providing me the opportunity to pursue a PhD in his group

and giving me a good and agréable place to work. Thanks also for the advices, suggestions,and conceptual ideas during thesis writing.

Prof. Dr. Nikolaus Amrhein for his support and for the good questions raised duringseminars, resulting in improved experiments. Thank you for the great contribution in

language style and scientific precision of content to this thesis.

Dr. Markus Wyss for providing us with the consensus phytase gene and protein samples, the

good scientific discussions during the early processes of patenting, and finally for his detailed

corrections and critical review of the phytase manuscript and of the whole thesis. The goodcollaboration with Roche Vitamins Ltd is greatly appreciated. Thanks also to Martin Lehmann

for investing time and energy to synthesize a consensus phytase with maize codon usage.

My colleagues from the Bucher lab, Gerardo Zardi, Christine Rausch, Réka Nagy, Pierre

Daram, Silvia Brunner, Volodya Karandashov, Sarah Wegmüller, and Cyril Steiner for the

good atmosphere in the lab and their help in learning new methods in plant molecular

biology.

My colleagues from the "Frossard Group" for the good times we spent together, in the office

and elsewhere. Special thanks to Theres Rösch and Thomas Flura for their help in ICP

analysis, and to Christiane Gujan for readily organizing the administrative parts of the project.Special thanks also to Jan Jansa, who, next to being a good scientific collaborator, alwaysremained a dynamic and interesting friend.

Christophe Zeder and Prof. Dr. Richard Hurrell from the group of Human Nutrition, ETH

Zurich, for their great help in HPLC analysis.

My wife Damaris, who always stood by my side, ever trying to help and understand; her

constant support and encouragements, throughout the whole period of the thesis and despite

my frequent absences from home, were greatly appreciated.

Both our families Zimmermann and Oertle, who always provided me with optimism and

happiness by their cheerful receiving us at their homes.

The financial support of the Swiss Federal Institute of Technology (ETH) Zurich, and of the

Hochstrasser Stiftung for a three months prolongation.

1

11 Curriculum vitae 105

11 Curriculum vitae

Name:

Date of birth

Citizenship

Philip Zimmermann

31st January 1974

Swiss, from Charmoille, JU

07.1999-today

12.1998-06.1999

10.1998- 12.1998

04.1998-09.1998

10.1992-04.1998

07.1996- 11.1996

1989- 1992

1986- 1989

1984- 1986

1980- 1984

31st January 1974

PhD thesis at the ETH Zurich. Project as a collaboration

between the groups of Plant Nutrition (Prof. Dr. E. Frossard)and Plant Biochemistry and Physiology (Prof. Dr. N. Amrhein)

Training in molecular biology methods in the lab of Dr. Marcel

Bucher, ETH Zurich, Group of Prof. Dr. N. Amrhein.

Training course at the SRVA (Service Romand de Vulgarisation

Agricole) in Lausanne

Diploma thesis at the Texas A&M University in Lubbock, TX, in

the group of Soil Physics (Prof. Dr. R.J. Lascano). Awarded

with the ETH medal.

Studies as agronomist in the Department of Food Science and

Agriculture, ETH Zurich

Training course in the Ramat Negev Agro-Research Station,Israel.

Lycée Cantonal, Porrentruy, Type C, June 1992

Ecole secondaire, Porrentruy, Switzerland

Schweizer Sekundärschule, Papua New Guinea

Primary School, Papua New Guinea

Born in Kassam, Eastern Highlands Province,

Papua New Guinea

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