starch biosynthesis, its regulation and biotechnological

Biotechnology Advances xxx (2013) xxx–xxx

JBA-06696; No of Pages 20

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Biotechnology Advances

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Starch biosynthesis, its regulation and biotechnological approaches to improvecrop yields

Abdellatif Bahaji a,1, Jun Li a,1, Ángela María Sánchez-López a, Edurne Baroja-Fernández a,Francisco José Muñoz a, Miroslav Ovecka a,b, Goizeder Almagro a, Manuel Montero a, Ignacio Ezquer a,Ed Etxeberria c, Javier Pozueta-Romero a,⁎a Instituto de Agrobiotecnología (CSIC/UPNA/Gobierno de Navarra), Mutiloako etorbidea z/g, 31192 Mutiloabeti, Nafarroa, Spainb Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Cell Biology, Faculty of Science, Palacky University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republicc University of Florida, Institute of Food and Agricultural Sciences, Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850-2299, USA

Abbreviations: ADPG, ADPglucose; AGP, ADPG pyrophglucose-1-phosphate; G6P, glucose-6-phosphate; GBSS, gBT1; MCF, mitochondrial carrier family; MVs, microbial vocess; NTRC, NADP-thioredoxin reductase C; OPPP, oxidatphosphoglucoseisomerase; pPGM, plastidial phosphoglusynthase; SS, starch synthase; T6P, trehalose-6-phosphate⁎ Corresponding author. Tel.: +34 948168009; fax: +3

E-mail address: [email protected] (J. Pozueta1 These authors contributed equally to this work.

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Keywords:ADPglucoseCalvin–Benson cycleCarbohydrate metabolismGenetic engineeringMicrobial volatilesMIVOISAPPlant–microbe interactionEndocytosisStarch futile cyclingSucrose synthase

Structurally composed of the glucose homopolymers amylose and amylopectin, starch is themain storage carbo-hydrate in vascular plants, and is synthesized in the plastids of both photosynthetic and non-photosynthetic cells.Its abundance as a naturally occurring organic compound is surpassed only by cellulose, and represents both acornerstone for human and animal nutrition and a feedstock formany non-food industrial applications includingproduction of adhesives, biodegradable materials, and first-generation bioethanol. This review provides an up-date on the different proposed pathways of starch biosynthesis occurring in both autotrophic and heterotrophicorgans, and provides emerging information about the networks regulating them and their interactions with theenvironment. Special emphasis is given to recent findings showing that volatile compounds emitted bymicroor-ganisms promote both growth and the accumulation of exceptionally high levels of starch in mono- and dicoty-ledonous plants. We also review how plant biotechnologists have attempted to use basic knowledge on starchmetabolism for the rational design of genetic engineering traits aimed at increasing starch in annual crop species.Finally we present some potential biotechnological strategies for enhancing starch content.

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Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Proposed starch biosynthetic pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.1. Starch biosynthesis in leaves . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.1. A classic model of starch biosynthesis according to which the starch biosynthetic pathway is linked to the Calvin–Benson cycle bymeans of

plastidial phosphoglucose isomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.1.2. Models of starch biosynthesis according to which the starch biosynthetic pathway is not linked to the Calvin–Benson cycle by

means of plastidial phosphoglucose isomerase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2. Starch biosynthesis in heterotrophic organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

2.2.1. Mechanisms of sucrose unloading in heterotrophic organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02.2.2. Proposed mechanisms of sucrose–starch conversion in heterotrophic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

osphorylase; AGPP, ADPG pyrophosphatase; A/N-inv, alkaline/neutral invertase; BT1, Brittle 1; F6P, fructose-6-phosphate; G1P,ranule bound starch synthase; GPT, glucose-6-phosphate translocator; GWD, glucan, water dikinase; HvBT1, Hordeum vulgarelatiles; NPP, nucleotide pyrophosphatase/phosphodiesterase; MIVOISAP, MIcrobial VOlatiles Induced Starch Accumulation Pro-ive pentose phosphate pathway; Pi, orthophosphate; 3PGA, 3-phosphoglycerate; pHK, plastidial hexokinase; pPGI, plastidialcomutase; PPi, inorganic pyrophosphate; pSP, plastidial starch phosphorylase; RSR1, Rice Starch Regulator1; SuSy, sucrose; Trx, thioredoxin; UDPG, UDPglucose; UGP, UDPG pyrophosphorylase; WT, wild type; ZmBT1, Zea mays BT1.4 948232191.-Romero).

ghts reserved.

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2 A. Bahaji et al. / Biotechnology Advances xxx (2013) xxx–xxx

3. Microbial volatiles promote both growth and accumulation of exceptionally high levels of starch in mono- and dicotyledonous plants . . . . . . . . 04. Biotechnological approaches for increasing starch content in heterotrophic organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4.1. Enhancement of AGP activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.2. Increasing supply of starch precursors to the amyloplast . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.3. Increasing supply of sucrose to heterotrophic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.4. Enhancement of sucrose breakdown within the heterotrophic cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.5. Enhancement of expression of starch synthase class IV . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.6. Blocking starch breakdown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.7. Altering the expression of global regulators . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.8. Enhancing trehalose-6-phosphate content . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.9. Potential strategies for increasing starch content in heterotrophic organs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

4.9.1. Over-expression of BT1 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.9.2. Blocking the activity of ADPG breakdown enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.9.3. Ectopic expression of SuSy in the plastid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04.9.4. Blocking the synthesis of storage proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

5. Conclusions and main challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction

Starch is the main storage carbohydrate in vascular plants. Its abun-dance as a naturally occurring compound of living terrestrial biomass issurpassed only by cellulose, and represents the primary source of calo-ries in the human diet. Because starch is the principal constituent ofthe harvestable organ of many agronomic plants, its synthesis and accu-mulation also influences crop yields.

Synthesized in the plastids of both photosynthetic and non-photosynthetic cells, starch is an insoluble polyglucan produced bystarch synthase (SS) using ADP-glucose (ADPG) as the sugar donor mol-ecule. Starchhas twomajor components, amylopectin and amylose, bothof which are polymers of α-D-glucose units. Amylose is a linear polymerof up to several thousand glucose residues, whereas amylopectin is alarger polymer regularly branched with α-1,6-branch points. Thesetwo molecules are assembled together to form a semi-crystalline starchgranule. The exact proportions of these molecules and the size andshape of the granule vary between species and between organs of thesame plant. The diversity of both composition and physical parametersof starches from different botanical sources gives rise to their diverseprocessing properties and applications in both non-food sectors such assizing agents in textile and paper industry, adhesive gums, and biode-gradable materials, and in food industries, particularly in bakery, thick-ening, confectionary and emulsification (Burrell, 2003; Mooney, 2009;Slattery et al., 2000). Starch is also used as a feedstock for first generationbioethanol production (Goldemberg, 2007). From an industrial perspec-tive, the utilization of starch as a cheap and renewable polymer and cleanenergy source is becoming increasingly attractive as a consequence of so-cial concerns about industrial wastes generated from fossil fuels.

In general, the harvested parts of our staple crop plants are hetero-trophic starch-storing organs. Worldwide annual starch production ofcereal seeds (rice, maize, wheat, barley, etc.) is approximately2 billion tons, whereas worldwide annual starch production of roots(e.g. cassava and taro) and tubers (e.g. potato and sweet potato) ex-ceeds 700 million tons (Food and Agriculture Organization of the Unit-ed Nations, http://faostat.fao.org), much of which is destined to non-food purposes. In 2012 for instance, 75 million tons of starch wereextracted and used in many industrial applications (http://www.starch.dk/). The increasing demand of starch from non-food industries,the continuing rapid increase in the world's population, predicted toreach 9 billion by 2050, and the growing loss of arable land to urbaniza-tion have spurred on efforts to synthesize plants with higher starchcontent. Therefore, a thorough understanding of the mechanisms in-volved in starch metabolism and regulation is critically important forthe rational design of experimental traits aimed at improving yields inagriculture, and producing more and better polymers that fit both

Please cite this article as: Bahaji A, et al, Starch biosynthesis, its regulationAdv (2013), http://dx.doi.org/10.1016/j.biotechadv.2013.06.006

industrial needs and social demands. However, while much is knownabout starch metabolism, there are still major gaps in our knowledgethat prevent breeders and biotechnologists from advancing in their at-tempts to increase starch content in a predictable way (Chen et al.,2012).

In this paper, we first review recent advances in the biochemistry ofstarch biosynthesis and its regulation, and provide an update on differentproposed pathways of starch biosynthesis occurring in both autotrophicand heterotrophic organs. Special emphasis is given to recent findingsshowing that volatile compounds emitted by microorganisms promoteboth growth and the accumulation of exceptionally high levels of starch.Finally, we assess the progress in molecular strategies for increasingstarch in annual crop species by genetic engineering approaches.

2. Proposed starch biosynthetic pathways

Starch is found in the plastids of photosynthetic and non-photosynthetic tissues. Mature chloroplasts occurring in photosynthet-ically active cells possess the capacity of providing energy (ATP) andfixed carbon for the synthesis of starch during illumination. By contrast,production of long-term storage of starch taking place in amyloplasts ofreserve organs such as tubers, roots and seed endosperms dependsupon the incoming supply of carbon precursors and energy from thecytosol. This difference between themetabolic capacities of chloroplastsand amyloplasts has led to the generally accepted view that the path-way(s) involved in starch production are different in photosyntheticand non-photosynthetic cells.

2.1. Starch biosynthesis in leaves

In leaves, a portion of the photosynthetically fixed carbon is retainedwithin the chloroplasts during the day to synthesize starch, which isthen remobilized during the subsequent night to support non-photosynthetic metabolism and growth by continued export of carbonto the rest of the plant. Due to the diurnal rise and fall cycle of its levels,foliar starch is termed “transitory starch”.

Transitory starch is a major determinant of plant growth (Sulpiceet al., 2009), and its metabolism is regulated to avoid a shortfall of car-bon at the end of the dark period. It is typically degraded in a near-linearmanner during darkness, with only a small amount remaining at theend of the night. When changes in the day length occur that leads to atemporary period of carbon starvation, the carbon budget is rebalancedby increasing the rate of starch synthesis, decreasing the rate of starchbreakdown and, consequently, decreasing the rate of growth duringdarkness. The importance of starch turnover in plant growth and

and biotechnological approaches to improve crop yields, Biotechnol

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biomass production is demonstrated by studies of mutants that are de-fective in starch synthesis and mobilization. These mutants grow at thesame rate as wild-type (WT) plants under continuous light or duringvery long days, but show a large inhibition of growth in short days(Caspar et al., 1985; Gibon et al., 2004; Lin et al., 1988), which is dueto the transient depletion of carbon during the night.

Leaf starch mainly accumulates in the photosynthetic palisade andspongy mesophyll cells, although it can also be synthesized in epider-mal cells, stomatal guard cells and bundle sheath cells surroundingthe vasculature (Tsai et al., 2009). Various mechanisms have been pro-posed to describe starch synthesis and degradation in leaves. Given thatstarch breakdown has been thoroughly discussed in recent reviews(Streb and Zeeman, 2012), in the following lines we will focus on de-scribing, analyzing and comparing the proposed mechanisms of starchsynthesis in leaves.

2.1.1. A classic model of starch biosynthesis according to which the starchbiosynthetic pathway is linked to the Calvin–Benson cycle by means ofplastidial phosphoglucose isomerase

It is widely assumed that the whole starch biosynthetic process re-sides exclusively in the chloroplast and segregated from the sucrose bio-synthetic process that takes place in the cytosol (Neuhaus et al., 2005;Stitt and Zeeman, 2012; Streb et al., 2009). According to this classicalview of starch biosynthesis (which is schematically illustrated inFig. 1), starch is considered the end-product of a metabolic pathwaythat is linked to the Calvin–Benson cycle by means of the plastidialphosphoglucose isomerase (pPGI). This enzyme catalyzes the conver-sion of fructose-6-phosphate (F6P) from the Calvin–Benson cycle intoglucose-6-phosphate (G6P), which is then converted into glucose-1-phosphate (G1P) by the plastidial phosphoglucomutase (pPGM).ADPG pyrophosphorylase (AGP) then converts G1P and ATP into inor-ganic pyrophosphate (PPi) and ADPG necessary for starch biosynthesis.These three enzymatic steps are reversible, but the last step is rendered

Chloroplast

4’

5’

CO2

G6P

G1P

ADPG

Starch 910

Triose-P

FBP

1’Calvin-Benson

cycle

F6P

2’

Fig. 1. Classic interpretation of the mechanisms of starch and sucrose syntheses in leaves. The e1,6-bisphosphatase; 3, PPi: fructose-6-P 1-phosphotransferase; 4, 4′, PGI; 5, 5′, PGM; 6, UGP;According to this view, the starch biosynthetic process resides exclusively in the chloroplast an


irreversible upon hydrolytic breakdown of PPi by plastidial alkalinepyrophosphatase.

pPGI is strongly inhibited by light (Heuer et al., 1982) and byCalvin–Benson cycle intermediates such as erythrose-4-P and 3-phosphoglycerate (3PGA) (the latter being an indicator of photosyn-thetic carbon assimilation) that accumulate during the day at stro-mal concentrations higher than the Ki values for pPGI (Backhausenet al., 1997; Dietz, 1985; Kelly and Latzko, 1980; Zhou and Cheng,2008). Although both these characteristics of pPGI, and the low stro-mal G6P/F6P ratio occurring during illumination (Dietz, 1985) wouldindicate that this enzyme is inactive during illumination (and thusnot involved in transitory starch biosynthesis), genetic evidenceshowing that transitory starch biosynthesis occurs solely by thepPGI pathway has been obtained from characterization of starch-deficient pPGI mutants from various species (Jones et al., 1986;Kruckeberg et al., 1989; Kunz et al., 2010; Niewiadomski et al.,2005; Yu et al., 2000).

The classic view of leaf starch biosynthesis illustrated in Fig. 1 alsoimplies that AGP is the sole source of ADPG, and functions as themajor regulatory step in the starch biosynthetic process (Neuhauset al., 2005; Stitt and Zeeman, 2012; Streb and Zeeman, 2012; Strebet al., 2009). This enzyme is a heterotetramer comprising two types ofhomologous but distinct subunits, the small (APS) and the large (APL)subunits (Crevillén et al., 2003, 2005). In Arabidopsis, six genes encodeproteins with homology to AGP. Two of these genes code for small sub-units (APS1 and APS2, the latter being in a process of pseudogenization)and four (APL1–APL4) encode large subunits. Whereas APS1, APL1 andAPL2 are catalytically active, APL3 and APL4 have lost their catalyticproperties during evolution (Ventriglia et al., 2008). In Arabidopsis, thelarge subunits are highly unstable in the absence of small subunits(Wang et al., 1998). Therefore,APS1T-DNAnullmutants contain neitherthe large nor the small subunit proteins, which result in a total lack ofAGP activity (Bahaji et al., 2011a; Ventriglia et al., 2008).

Sucrose-P

Sucrose

Cytosol

1

2

4

5

67

8

3

Triose-P

FBP

F6P

G6P

G1P

UDPG

nzymes are numbered as follows: 1, 1′, fructose-1,6-bisphosphate aldolase; 2, 2′, fructose7, sucrose-phosphate-synthase; 8, sucrose phosphate phosphatase; 9, AGP; and 10, SS.d is segregated from the sucrose biosynthetic pathway that takes place in the cytosol.


image of Fig.�1



AGP is allosterically activated by 3PGA, and inhibited by Pi(Kleczkowski, 1999). This propertymakes the production of ADPGhigh-ly sensitive to changes in photoassimilate (e.g. 3PGA) availability in thechloroplast, and helps to coordinate photosynthetic CO2 fixation withstarch synthesis in leaves. AGP activity is also subjected to APS redoxregulation (Hendriks et al., 2003; Kolbe et al., 2005; Li et al., 2012;Michalska et al., 2009). Under oxidizing conditions the twoAPS subunitsare covalently linked via an intermolecular disulfide bridge, thusforming a stable dimer within the heterotetrameric AGP enzyme (Fuet al., 1998; Hendriks et al., 2003). Conversely, under reductive condi-tions APSmonomerization is accompanied by activation of AGP activity.APS dimerization markedly decreases the activity of AGP and alters itskinetic properties, making it less sensitive to activation by 3PGA and in-creasing its Km for ATP (Hendriks et al., 2003). In turn, APSmonomerization activates AGP, which is then sensitive to fine controlthrough allosteric activation by 3PGA.

By examining the correlation between APS redox status, AGP activityand starch levels in leaves, different works have provided evidence thattransitory starch accumulation is finely regulated by APS post-translational redox modification in response to environmental inputssuch as light, sugars and biotic stress (Gibon et al., 2004; Hendrikset al., 2003; Kolbe et al., 2005; J. Li et al., 2011). However, using aps1Arabidopsis plants ectopically expressing a redox-insensitive, mutatedAPS1 form, Li et al. (2012) andHädrich et al. (2012) have independentlyshown that the rates of transitory starch accumulation in these plantsare comparable to that of WT leaves. In addition, Li et al. (2012) report-ed WT rates of starch accumulation in aps1 plants expressing in thechloroplast a redox-insensitive AGP from Escherichia coli, and concludedthat post-translational redoxmodification of APS1 in response to light isnot a major determinant of fine regulation of transitory starch accumu-lation in Arabidopsis leaves.

Plastidial NADP-thioredoxin reductase C (NTRC) plays an im-portant role in protecting plants against oxidative stress (Pulidoet al., 2010; Serrato et al., 2004). Serrato et al. (2004) and Michalskaet al. (2009) reported that Arabidopsis ntrc mutants grown at180 μmol photons s−1 m−2 are small and their leaves accumulatelow levels of starch when compared with WT leaves. Furthermore,Michalska et al. (2009) reported that ntrc leaves exhibit a decrease inthe extent of APS1 activation (reduction) by light. These authorsthus concluded that NTRC is a major determinant of both light-dependent APS1 redox status and transitory starch accumulation.However, Li et al. (2012) have recently found that the size ofArabidopsis ntrc mutants is similar to that of WT plants whencultured at 90 μmol photons s−1 m−2. Under these conditions, APS1redox status and starch accumulation rates in leaves of ntrc mutantsare similar to those of WT leaves, strongly indicating that NTRC playsa minor role, if any, in the control of both light-dependent APS1 redoxstatus and transitory starch accumulation in Arabidopsis cultured undernon-stressing conditions.

In vitro assays using heterologously expressed potato and pea AGPhave shown that APS can be activated by plastidial thioredoxins (Trxs) fand m (Ballicora et al., 2000; Geigenberger et al., 2005), indicating thatthese proteins could act as major determinants of APS redox status andof fine regulation of transitory starch accumulation. However, Li et al.(2012) found no differences in the rate of starch accumulation betweenWT leaves and leaves of homozygous trxf1, trxf2, trxm2 and trxm3 mu-tants. Furthermore, Thormählen et al. (2013) reported that starch levelsin trxf1 leaves were only slightly reduced when compared with WTleaves. In addition, Sanz-Barrio et al. (2013) have shown that APS redoxstatus in high-starch tobacco leaves overexpressing Trxf and Trxm genesfrom the plastid genome is similar to that of WT leaves. The overalldata thus indicate that, individually, plastidial Trxs are notmajor determi-nants of APS1 redox status-controlled transitory starch content.

Genetic evidence showing that transitory starch biosynthesis occurssolely by the AGP pathway has been obtained from the characterizationof leaves with reduced pPGM and AGP activities. For example, leaves of


APS1 and pPGM antisensedpotato plants accumulate low levels of starchwhen compared with WT leaves (Lytovchenko et al., 2002; Müller-Röber et al., 1992; Muñoz et al., 2005). Furthermore, Arabidopsis pgmnull mutants (Caspar et al., 1985; Kofler et al., 2000), adg1-1 mutantswith less than 3% of the WT AGP activity (Lin et al., 1988; Wanget al., 1998), and APS1 T-DNA null mutants completely lacking AGPactivity (Bahaji et al., 2011a; Ventriglia et al., 2008) accumulateless than 2% of the WT starch. Further evidence supporting thatAGP plays a crucial role in starch biosynthesis in leaves comes fromalkaline pyrophosphatase-silenced plants of Nicotiana benthamiana,since their leaves accumulate ca. 60% of the WT starch (George et al.,2010).

2.1.2. Models of starch biosynthesis according to which the starchbiosynthetic pathway is not linked to the Calvin–Benson cycle by meansof plastidial phosphoglucose isomerase

A vast amount of biochemical and genetic data appear to support thestarch biosynthetic pathway illustrated in Fig. 1 according to which (a)the whole starch biosynthetic process resides exclusively in the chloro-plast, (b) pPGI links the Calvin–Benson cycle with starch biosynthesis,and (c) AGP is the sole source of ADPG linked to starch biosynthesis.However, in recent years an increasing volume of evidence has beenprovided that supports the occurrence of additional/alternative starchbiosynthetic pathways involving the cytosolic and plastidial compart-ments wherein the supply of ADPG is not directly linked to theCalvin–Benson cycle by means of pPGI. Thus, Fettke et al. (2011)found that the envelope membranes of chloroplasts in mesophyll cellspossess a yet to be identified G1P transport machinery enabling the in-corporation of cytosolic G1P into the stroma, which is subsequentlyconverted into starch by the stepwise AGP and SS reactions. Accordingto these authors, such mechanism would only allow the accumulationof 1% of the WT starch, and would explain the occurrence of sometrace amounts of starch in the pPGM mutants (Caspar et al., 1985;Muñoz et al., 2005; Streb et al., 2009).

Kunz et al. (2010) found that leaves of the pgi1-2 null mutant im-paired in pPGI activity accumulate 10% of the WT starch content,which is restricted to bundle sheath cells adjacent to the mesophylland stomatal guard cells. This mutant exhibits high G6P transport activ-ity as a consequence of the elevated expression of GPT2 (Kunz et al.,2010), a gene that codes for a G6P/Pi translocator (GPT) mainly operat-ing in heterotrophic tissueswhere the imported G6P can be used for thesynthesis of starch and fatty acids or to drive the oxidative pentosephosphate pathway (Bowsher et al., 2007; Kammerer et al., 1998;Kang and Rawsthorne, 1996; L. Zhang et al., 2008). According to Kunzet al. (2010) the unexpected high levels of starch occurring in leavesof pPGI mutants can be ascribed to high GPT2-mediated incorporationof G6P into the chloroplast of bundle sheath cells adjacent to the meso-phyll and stomatal guard cells, where this hexose-phosphate can bethen converted to starch bymeans of pPGM, AGP and SS as schematical-ly illustrated in Fig. 2. This interpretation, however, is hardly reconcil-able with previous studies carried out by the same group showingthat the starch-deficient phenotype of pPGI mutants can be totallyreverted to WT starch phenotype by the ectopic expression of GPT2(Niewiadomski et al., 2005), and with the fact that leaves of the pgi1-2/gpt2 double mutant accumulate as much as 60% of the starch occur-ring in pgi1-2 leaves (Kunz et al., 2010). Furthermore, considering that(a) most of leaf starch accumulates in the mesophyll cells, (b) micro-scopic analyses revealed that stomatal guard cells and bundle sheathcells adjacent to the mesophyll of pPGI mutants accumulate nearlyWT starch content (Kunz et al., 2010; Tsai et al., 2009), and (c) guardcells of WT leaves possess a G6P transport machinery (Overlach et al.,1993), it is difficult to understand how guard cells and bundle sheathcells adjacent to the mesophyll of pPGI mutants can accumulate asmuch as 10% of the WT starch as a consequence of high GPT2 activity.Needless to say, further investigations will be necessary to understandhow and where pgimutants accumulate ca. 10% of the WT starch.



Triose-P

Chloroplast

5’G6P

G1P

ADPG

Starch

Cytosol

1

2

4

3

910

Triose-P

FBP

F6P

G6PGPT

CO2

BensonCalvin-

cycle

Fig. 2. Suggestedmodel of starch biosynthesis in bundle sheath cells adjacent to themesophyll and stomatal guard cells of pgi-2 Arabidopsismutants (fromKunz et al., 2010). Numbering ofenzyme activities is the same as in Fig. 1.


Gerrits et al. (2001) have provided evidence that a sizable pool ofsucrose in the plant cell has a plastidial localization. Consistent withthe occurrence of a plastidial pool of sucrose, Murayama and Handa(2007) and Vargas et al. (2008) reported the presence of a functionalalkaline/neutral invertase (A/N-inv) inside plastids. Noteworthy,Vargas et al. (2008) reported that leaves of Arabidopsismutants impairedin A/N-inv accumulate ca. 70% of the WT starch content. The same au-thors hypothesized that chloroplastic sucrose plays a sensing role in amechanism of control of carbon partitioning and sucrose/starch ratiowherein glucose and fructose generated by A/N-inv are sensed throughplastidial hexokinase (pHK) (Giese et al., 2005; Olsson et al., 2003). How-ever, a possibility cannot be ruled out that, as schematically illustrated inFig. 3, plastidial sucrose acts as precursor for starch biosynthesis, itsA/N-inv breakdown products being converted into starch by meansof pHK, pPGM and AGP.

Sufficient evidence exists to support the view that a sizable pool ofADPG in leaves accumulates in the cytosol of photosynthetically compe-tent cells. This hypothesis is compatible with the occurrence of cytosolicADPGmetabolizing enzymes such as ADPG phosphorylase (McCoy et al.,2006), glucan synthases (Tacke et al., 1991), sucrose synthase (SuSy) andADPG hydrolases (Olejnik and Kraszewska, 2005; Rodríguez-López et al.,2000), and with the occurrence in the chloroplast envelope membranesof yet to be molecularly identified ADPG transport machineries whoseactivities can account for rates of starch accumulation occurring in leaves(Pozueta-Romero et al., 1991a). Genetic evidence supporting the occur-rence of important ADPG source(s), other than AGP, has been obtainedfrom different sources. First, leaves of transgenic potato and Arabidopsisplants ectopically expressing in the cytosol a bacterial ADPG hydrolase(Moreno-Bruna et al., 2001) accumulate lower ADPG levels than WTleaves, as determined by HPLC analyses (Bahaji et al., 2011a; Baroja-Fernández et al., 2004). Second, ADPG content in the leaves of AGP andpPGM mutants is comparable to that of WT leaves, as confirmed byboth HPLC (Bahaji et al., 2011a; Muñoz et al., 2005) and HPLC:MS


analyses (Baroja-Fernández et al., 2013). Third, HPLC:MS analyses ofthe triple ss3/ss4/aps1mutant impaired in AGP and SS class III and IV re-vealed that leaves from this mutant accumulate ca. 40-fold more ADPGthan WT leaves (Ragel, 2012).

SuSy catalyzes the reversible conversion of sucrose and NDP intofructose and the correspondingNDP-glucose,whereN stands for uridine,adenosine, guanosine, cytidine, thymidine or inosine. Although UDP isgenerally considered to be the preferred nucleoside diphosphate forSuSy, ADP also serves as an effective substrate to produce ADPG(Baroja-Fernández et al., 2003, 2012; Cumino et al., 2007; Delmer,1972; Nakai et al., 1998; Porchia et al., 1999; Silvius and Snyder, 1979;Zervosen et al., 1998). SuSy is highly regulated at both transcriptionaland post-transcriptional levels (Asano et al., 2002; Ciereszko andKlezkowski, 2002; Déjardin et al., 1999; Fu and Park, 1995; Hardinet al., 2004; Koch et al., 1992; Pontis et al., 1981; Purcell et al., 1998; fora review see Kleczkowski et al., 2010). Although this sucrolytic enzymeis very active in reserve organs, it also expresses in the mesophyll cellsof source leaves (Fu et al., 1995; Wang et al., 1999), its activity in potatoand Arabidopsis leaves greatly exceeding the minimum needed to sup-port normal rate of starch accumulation during illumination (Baroja-Fernández et al., 2012; Muñoz et al., 2005). Accordingly, a metabolicmodel of transitory starch biosynthesis has been proposed wherein (a)both sucrose and starch metabolic pathways are tightly interconnectedby means of cytosolic ADPG producing enzymes such as SuSy (actingwhen cytosolic sucrose transiently accumulates during illumina-tion), and by the action of an ADPG translocator located at the chlo-roplast envelope membranes, and (b) both AGP and pPGM play animportant role in the scavenging of glucose units derived from starchbreakdown occurring during starch biosynthesis and during the bio-genesis of the starch granule (Bahaji et al., 2011a; Baroja-Fernándezet al., 2005; Muñoz et al., 2005, 2006) (Fig. 4). Thus, the net rate ofstarch accumulation is determined by the balance between therates of SuSy-mediated ADPG synthesis in the cytosol, import of


image of Fig.�2


Triose-P

Chloroplast

CO2

ADPGStarch

Cytosol

1-8

Triose-P

G6P

11

BensonCalvin-

cycle

Glucose

Sucrose

G1P

Sucrose

Fructose

12

5’910

F6P4’

12

Fig. 3. Suggestedmodel of starch biosynthesiswhere sucrose entering the plastid is broken downbyA/N-inv to produce fructose and glucose, the latter being converted toG6P bymeans ofpHK. G6P is subsequently metabolized to starch by the stepwise reactions of pPGM, AGP and SS. Enzyme activities 1–10 and 4′ and 5′ are the same as in legend of Fig. 1. Other enzymesinvolved are numbered as follows: 11, A/N-inv; 12, pHK.


cytosolic ADPG to the chloroplast, starch synthesis, starch break-down and by the efficiency with which starch breakdown productscan be recycled back to starch via the coupled reactions of pPGMand AGP. Accordingly, this view predicts that the recovery towardstarch biosynthesis of the glucose units derived from the starchbreakdown will be deficient in pPGM and AGP mutants, resulting ina parallel decline of starch accumulation.

The occurrence of a substrate or “futile” cycle as a result of the simul-taneous synthesis and breakdown of starch in leaves is not surprisingsince pulse-chase and starch-preloading experiments using isolatedchloroplasts (Fox and Geiger, 1984; Stitt and Heldt, 1981), intact leaves(Scott and Kruger, 1995; Walters et al., 2004), or cultured photosyn-thetic cells (Lozovaya et al., 1996) have shown that the illuminatedchloroplasts can synthesize and mobilize starch simultaneously. Fur-thermore, enzymes involved in starch breakdown such as SEX1 andBAM1 are redox-activated during the light under different environmen-tal conditions (Mikkelsen et al., 2005; Sparla et al., 2006; Valerio et al.,2010). Futile cycles lead to a net consumption of ATP, which in some in-stances can be estimated at about 60% of the total ATP produced by thecell (Alonso et al., 2005; Hill and ap Rees, 1994). Although these cyclesappear to waste energy without any apparent physiological reason,they form part of the organization of the plant central metabolism,and contribute to its flexibility (Rontein et al., 2002). As presentedabove, starch acts as a major integrator in the plant growth that accu-mulates to cope with temporary starvation imposed by the environ-ment (Sulpice et al., 2009). It is thus conceivable that highly regulatedstarch futile cycling may entail advantages such as sensitive regulationand rapid metabolic channeling toward various pathways in responseto physiological and biochemical needs of the plant. Therefore, due tothe importance of starch in the overall plant metabolism and growth,it is tempting to speculate that both redundancy of ADPG sources andstarch futile cycling have been selected during plant evolution to


warrant starch production and rapid connection of starch metabolismwith other metabolic pathways.

The postulated starch biosynthetic pathways involving the plastidialand cytosolic compartments are consistent with still enigmatic resultsobtained from experiments carried out more than 50 years ago showingthat 14C in the glucose moiety of sucrose, starch, UDP-glucose andhexose-Ps is asymmetrically distributed in green leaves exposed to14CO2 for a short period of time (Gibbs and Kandler, 1957; Havir andGibbs, 1963; Kandler and Gibbs, 1956). Unlike the “classic” view on tran-sitory starch biosynthesis (Fig. 1) predicting that green leaves exposed to14CO2 for a short period of time should synthesize starch with 14C sym-metrically distributed in the glucose moiety, the proposed mechanismsof starch synthesis illustrated in Figs. 2–4 provide a possible explanationfor solving the above enigma. As shown in Fig. 4, triose-Ps produced inthe Calvin–Benson cycle are exported to the cytosol where they can bechanneled into the oxidative pentose-P pathway (OPPP), thereby leadingto a randomization of the carbons giving rise to the asymmetric 14C dis-tribution observed in hexose-Ps, nucleotide-sugars and sucrose upon ex-posure to 14CO2 for a short period of time. Cytosolic G1P, G6P, sucroseand/or ADPG will be then transported into the chloroplast and utilizedas precursors for the synthesis of starch molecules possessing glucosemolecules with asymmetric 14C distribution.

Genetic evidence consistentwith the occurrence of a link between su-crose and transitory starch metabolism can be obtained from sucrose-phosphate-synthase and cytosolic PGM deficient plants (Fernie et al.,2002; Strand et al., 2000), since their leaves contain low levels of both su-crose and starch as compared with WT leaves. Further genetic evidencecan be obtained from sedoheptulose-1,7-bisphosphate overexpressingplants (Lefebvre et al., 2005; Miyagawa et al., 2001), since their leavesare characterized by having high levels of both sucrose and starch whencompared with WT leaves. Finally, genetic evidence indicating that leafsucrose and starch metabolic pathways are linked by SuSy has been


image of Fig.�3


Triose-P

Chloroplast

9

CO2

G1P

ADPGStarch

ADP

Cytosol

1-81’- 4’

5’

10

Triose-P

Sucrose

ADPG

16

G6P

15

BensonCalvin-

cycle

?

Glucose12

UDPG

14

SulfolipidsFatty acids,OPPP, etc.

13

Fructose

GPT

OPPP

Hexose-Ps

Fig. 4. Suggested interpretation of themechanism of starch biosynthesis in leaves according towhich (a) ADPG is produced in the cytosol and transported to the chloroplast by the actionof a yet to be identified ADPG translocator, (b) AGP and pPGM play an important role in the scavenging of glucose units derived from starch breakdown, and (c) starch metabolism isconnectedwith othermetabolic pathways through themetabolites generated by the starch futile cycle. Enzyme activities 1–10 and 1′–5′ are the same as in legend of Fig. 1. Other enzymesinvolved are numbered as follows: 12, pHK; 13, plastidial UGP (Okazaki et al., 2009); 14, plastidial AGPP; 15, pSP (Zeeman et al., 2004); and 16, SuSy. Starch to glucose conversion wouldinvolve the coordinated actions of amylases, isoamylases and disproportionating enzymes (Asatsuma et al., 2005; Fulton et al., 2008; Kaplan and Guy, 2005). Note that starch futile cycling(indicated with red arrows) may entail advantages such as rapid metabolic channeling toward various pathways (such as fatty acid (Periappuram et al., 2000), OPPP under stress condi-tions (Zeeman et al., 2004) and sulfolipid biosynthesis (Okazaki et al., 2009)) in response to physiological and biochemical needs. This view predicts that the recovery toward starch bio-synthesis of the glucose units derived from the starch breakdown will be deficient in pPGM and AGP mutants, resulting in a parallel decline of starch accumulation and enhancement ofsoluble sugars content since starch breakdown derived products (especially glucose and G6P)will leak out the chloroplast through very active glucose translocator (Cho et al., 2011) andGPT2 (which is highly expressed in pgi, pgm and agpmutants, Kunz et al., 2010). (For interpretation of the references to color in thisfigure legend, the reader is referred to theweb versionof this article.)


obtained from SuSy-overexpressing potato plants whose leaves accumu-late higher ADPG and starch contents than WT leaves (Muñoz et al.,2005). Further endeavors based on the characterization ofmutants totallylacking SuSy will be necessary to confirm (or refute) the involvement ofSuSy in starch biosynthesis in leaves.

2.2. Starch biosynthesis in heterotrophic organs

Sucrose produced in leaves is imported by the heterotrophic organsand used as carbon source for energy production and starch synthesis inthe amyloplast. As in the case of leaves, various mechanisms have beenproposed to describe starch biosynthesis in the heterotrophic organs,which together with the mechanisms of sucrose unloading, will bepresented and discussed below.

2.2.1. Mechanisms of sucrose unloading in heterotrophic organsUnloading of sucrose arriving from aerial parts of the plant into sink

organs occurs either symplastically (moving via plasmodesmata) orapoplastically. The route depends on the species, organ or tissue. Inthe apoplastic delivery of sucrose to rapidly growing sinks, apoplasticsucrose that has been released into the extracellular space can be splitby cell wall-bound invertases into glucose and fructose for subsequentuptake into the cell by hexose transporters. Alternatively, sucrose canbe taken up by plasma membrane-bound sink specific sucrose trans-porters of non-dividing storage sinks (Sauer, 2007). Sucrose is thentransported via yet-to-be-identified tonoplast sucrose transporters tothe vacuole, which serves as temporary reservoir. Apoplastic sucrosecan also be taken up by a sucrose-induced endocytic process, andtransported to the central vacuole of heterotrophic cells (Etxeberriaet al., 2005a, 2005b; for a review see Etxeberria et al., 2012). Endocytic


uptake of extracellular sucrose and subsequent transport to the vacuoleis not in conflict with transport through membrane-bound carriersgiven that cell homeostasis can be bettermaintained if bothmechanismsoperate in parallel (Etxeberria et al., 2005a). Consistent with this view,Baroja-Fernández et al. (2006) reported that carrier-mediated sucroseimport in starved cells of sycamore (Acer pseudoplatanus L.) plays an im-portant role in the overall sucrose–starch conversion process during theinitial period of sucrose unloading, whereas endocytosismay play an im-portant role in transporting the bulk of sucrose to the vacuole and itssubsequent conversion into starch after prolonged period of sucroseunloading.

2.2.2. Proposed mechanisms of sucrose–starch conversion in heterotrophiccells

Sucrose entering the heterotrophic cell by any of the mechanismsdescribed above must be metabolized to molecules that can betransported to the amyloplast for subsequent conversion into starch.Because both chloroplasts and amyloplasts are ontogenically related,and because chloroplasts possess a very active triose-P translocatorconnecting the plastidial and cytosolic compartments, it was originallyassumed that cytosolic triose-Ps entering the amyloplast by means ofa triose-P translocator act as precursors for starch biosynthesis (Boyer,1985). However, NMR spectroscopy experiments of starch biogenesisin wheat and maize grains using 13C NMR (Hatzfeld and Stitt, 1990;Keeling et al., 1988) demonstrated that a C-6 molecule, not a triose-P(C-3), is the precursor of starch biosynthesis in amyloplasts. Further in-vestigations of the enzyme capacities of amyloplasts (Entwistle and apRees, 1988; Frehner et al., 1990) supported the view that C-6moleculesentering amyloplasts are the precursors of starch biosynthesis.Depending on the nature of the C-6 molecule entering the amyloplast


image of Fig.�4



(G6P or ADPG), and depending on the enzymes involved in ADPGsynthesis, various mechanisms have been proposed to explain thesucrose–starch conversion process in heterotrophic cells.

2.2.2.1. Models of sucrose–starch conversion in heterotrophic cellsaccording to which AGP is the sole source of ADPG linked to starchbiosynthesis. It is widely assumed that AGP is the sole source of ADPGin heterotrophic organs of mono- and dicotyledonous plants. Maximalin vitro AGP activity greatly exceeds the minimum required to supportthe normal rate of starch accumulation in starch storing organs(Denyer et al., 1995; Li et al., 2013;Weber et al., 2000). This would indi-cate that AGP is not a rate-limiting step in the sucrose–starch conver-sion process in many heterotrophic organs. In fact, the flux controlcoefficient of AGP has been estimated to be as low as 0.08 in some het-erotrophic organs (Denyer et al., 1995; Rolletschek et al., 2002; Weberet al., 2000). As presented above, AGP is a highly regulated enzyme.However, whereas evidence has been provided that starch syn-thesis is regulated by post-translational redox modification of AGPand 3PGA/Pi balance in potato tubers (Tiessen et al., 2003), AGP in devel-oping barley, wheat, pea and bean seeds is insensitive to 3PGA and Pi

Amyloplast

ADPGStarch

G6P

G1PADPADPATP

ATP

PPi

G65

9

10

ATP

GPT

Amyloplast

AADPG10

Starch

A

B

Fig. 5. Classic interpretation of themechanisms of sucrose–starch conversion in (A) heterotrophthe sole source of ADPG linked to starch biosynthesis. Note that in heterotrophic cells of dicotylstrate compound entering the amyloplast for subsequent conversion into starch is G6P, and (cG1P pools are likely connected bymeans of a yet to be identified G1P translocator occurring inhas a cytosolic localization, and (b) the cytosolic carbon substrate compound entering the amylosame as in legends of Figs. 1–4.


regulation (Gómez-Casati and Iglesias, 2002; Hylton and Smith, 1992;Kleczkowski et al., 1993; Weber et al., 1995).

In heterotrophic organs of dicotyledonous plants, sucrose entering thecytosolic compartment of the heterotrophic cell is broken down by SuSyto produce fructose and UDPglucose (UDPG), the latter being convertedto G1P and PPi by UDPG pyrophosphorylase (UGP). G1P is subsequentlymetabolized to G6P by means of the cytosolic phosphoglucomutase.Cytosolic G6P then enters the amyloplast, where it is converted to starchby the sequential activities of pPGM, AGP and SS (Fig. 5A). Unlike chloro-plasts, amyloplasts are unable to photosynthetically generate ATP andtherefore, cytosolic ATP must enter the amyloplast to produce ADPG bymeans of AGP. The presence of an ATP/ADP translocator in the envelopemembranes of amyloplasts has been firmly established by different labo-ratories (Pozueta-Romero et al., 1991b; Tjaden et al., 1998), and evidenceabout its relevance in the starch biosynthetic process has been providedby Tjaden et al. (1998) and Geigenberger et al. (2001) who reportedthat potato tuberswithdecreasedplastidic ATP/ADP transporter activitiesexhibit reduced starch content.

Genetic evidence demonstrating the importance of SuSy in thesucrose–starch conversion process in heterotrophic organs of

Cytosol

Sucrose

P G1P UDPG

UTP PPi

UDP

Fructose5 6

16

SucroseCarriers/Endocytosis

Cytosol

Sucrose

G1P UDPG

UTP PPi

UDP

Fructose6

16

DPG

PPi ATP

9


ic cells of dicotyledonous plants and (B) cereal endosperm cells according towhich AGP isedonous plants (a) AGP is exclusively localized in the plastid, (b) the cytosolic carbon sub-) cytosolic UGP is involved in the sucrose–G6P conversion process. Plastidial and cytosolicamyloplasts (Fettke et al., 2010). Note also that in cereal endosperms cells (a) most of AGPplast for subsequent conversion into starch is ADPG. Numbering of enzyme activities is the


image of Fig.�5



dicotyledonous plants comes from the reduced levels of starch in potatotubers and carrot roots exhibiting low SuSy activity (Tang and Sturm,1999; Zrenner et al., 1995). In addition, genetic evidence showing thatstarch biosynthesis in heterotrophic organs involves the incorporationof G6P and its subsequent conversion into ADPG by means of pPGMand AGP has been obtained from the characterization of Vicianarbonensis embryos expressing antisense GPT (Rolletschek et al.,2007) (these embryos, however, expressed lower levels of starch-related genes such as AGP and SuSy encoding genes), geneticallyengineered pea seeds and potato tubers with reduced pPGM(Harrison et al., 1998; Tauberger et al., 2000) and from V. narbonensisseeds and potato tubers with reduced AGP activity (Müller-Röberet al., 1992; Weber et al., 2000). These organs all exhibit reduced starchcontent when compared with their corresponding WT organs.

Using disks fromWT and pPGM antisensed potato tubers incubatedwith radiolabeled G1P, Fettke et al. (2010) reported that G1P can be ef-ficiently taken up by potato tuber parenchyma cells and converted tostarch. Moreover, these authors reported that exogenously addedG1P-dependent starch synthesis was diminished in tuber disks of pota-to plants with low plastidial starch phosphorylase (pSP) activity. Fettkeet al. (2010) thus proposed that G1P can be taken up by amyloplast by ayet to be identified transporter to be subsequently used as substrate forpSP- and/or AGP-mediated starch biosynthesis. However, although thishypothesis can explain the results obtained using potato tuber disks, itconflicts with previous reports using pSP antisensed potato tubersshowing that, in planta, the lack of pSP did not exert any effect on starchaccumulation (Sonnewald et al., 1995). Furthermore, this hypothesis ishardly reconcilablewith the strong reduction of starch content in tubersof pPGM antisensed potato tubers, indicating that plastidial G6P islinked to starch biosynthesis (Tauberger et al., 2000).

Unlike dicotyledonous plants where AGP is exclusively localized inthe plastidial compartment, most of AGP in cereal endosperm cells hasa cytosolic localization (Beckles et al., 2001; Denyer et al., 1996;Johnson et al., 2003; Kleczkowski, 1996; ThornbjØrnsen et al., 1996a,1996b; Villand and Kleczkowski, 1994). Consistently, most of ADPG hasa cytosolic localization in cereal endosperm cells (Liu and Shannon,1981; Tiessen et al., 2012). Import studies using amyloplasts isolatedfrom maize and barley endosperms of WT plants and starch-deficient/ADPG-excess maize Zmbt1 and barley Hvbt1 mutants provided geneticevidence that Zea mays brittle1 (BT1) (ZmBT1) and Hordeum vulgareBT1 (HvBT1) are membrane proteins that can incorporate cytosolicADPG into the amyloplast (Liu et al., 1992; Möhlmann et al., 1997;Patron et al., 2004; Shannon et al., 1996, 1998). Furthermore, cell frac-tionation studies revealed that most of ADPG accumulating in “high-ADPG” developing endosperms of the Riso 13 Hvbt1 mutant has a cyto-solic localization (Tiessen et al., 2012). The overall data thus indicatethat, in cereal endosperm cells, BT1 proteins facilitate the transfer ofcytosolic ADPG into the amyloplast for starch biosynthesis and arerate-limiting steps in this process. Based on this evidence, a starch bio-synthesis model has been proposed for cereal endosperms wherein thestepwise reactions of SuSy, UGP and AGP take place in the cytosol to con-vert sucrose into ADPG, which enters the amyloplast by means of BT1 tobe utilized as glucosyl donor for starch biosynthesis (Fig. 5B) (Denyeret al., 1996; Villand and Kleczkowski, 1994). Genetic evidence demon-strating the importance of SuSy in the sucrose–starch conversion processin cereal endosperms comes from QTL analyses in maize endosperms(Thévenot et al., 2005) and from the reduced levels of starch in maizeseeds exhibiting low SuSy activity (Chourey andNelson, 1976). Evidenceshowing the importance of AGP in starch biosynthesis in cereal endo-sperms comes from AGP mutants exhibiting reduced starch content(Johnson et al., 2003; Tsai and Nelson, 1966).

2.2.2.2. Models of sucrose–starch conversion in heterotrophic cellsaccording to which both SuSy and AGP are involved in the synthesis ofADPG linked to starch biosynthesis. Sufficient evidence exists to supportthe view that heterotrophic organs possess in the cytosol important


sources, other than AGP, of ADPG linked to starch biosynthesis. This hy-pothesis is compatible with the occurrence in the amyloplast envelopemembranes of ADPG transporters (Naeem et al., 1997; Pozueta-Romero et al., 1991b; Shannon et al., 1998), and with the occurrencein heterotrophic organs of cytosolic ADPG metabolizing enzymes suchas ADPG phosphorylase (Dankert et al., 1964; McCoy et al., 2006;Murata, 1977) and SuSy. As presented in Section 2.1.2, SuSy is a highlyregulated enzyme that can produce ADPG from sucrose and ADP. Itplays a predominant role in determining both sink strength andphloem loading, and in the entry of carbon into metabolism innonphotosynthetic cells.Maximum in vitro ADPG producing SuSy activ-ity is particularly high in starch storing organs, being comparable to thatof AGP in potato tubers (Baroja-Fernández et al., 2009) and 3–4 foldhigher than that of AGP in developing barley and maize endosperms(Baroja-Fernández et al., 2003; Li et al., 2013). Furthermore, maximalADPG producing SuSy activity in heterotrophic organs such as develop-ingmaize endosperm greatly exceeds theminimum required to supportthe normal rate of starch accumulation in this organ (Li et al., 2013),which would indicate that ADPG producing SuSy activity is not a ratelimiting step in the sucrose–starch conversion process in heterotrophicorgans.

Essentially in line with investigations describing the occurrence ofsimultaneous synthesis and breakdown of glycogen in heterotrophicbacteria and animals (Bollen et al., 1998; David et al., 1990; Gaudetet al., 1992; Guedon et al., 2000; Massillon et al., 1995; Montero et al.,2011), pulse chase as well as starch pre-loading experiments using iso-lated amyloplasts and heterotrophic organs have provided evidenceabout the occurrence of simultaneous synthesis and breakdown ofstarch in non-photosynthetic cells of plants under conditions of activestarch accumulation (Neuhaus et al., 1995; Pozueta-Romero andAkazawa, 1993; Sweetlove et al., 1996). The occurrence of starch futilecycling in heterotrophic organs has been further fortified by recentstudies showing that global regulators of storage substance accumula-tion such as FLOURY ENDOSPERM2 (FLO2) positively co-regulate theexpression of starch synthesis and breakdown genes during starchaccumulation in developing rice seeds (She et al., 2010) (seeSection 4.7), and by the fact that down-regulation of starch breakdownfunctions in genetically engineered wheat and rice plants results inincreased grain yield (Hakata et al., 2012; Ral et al., 2012) (seeSection 4.6). Furthermore, starch breakdown enzymatic activities arehigh in heterotrophic organs (Li et al., 2013). Consistently, “alterna-tive/additional” models for the sucrose–starch conversion process inheterotrophic organs of dicotyledonous and monocotyledonous plantshave been proposed according to which (a) cytosolic enzymes such asSuSy catalyze directly the de novo production from sucrose of ADPG,which is subsequently imported into the amyloplast by the action ofan ADPG translocator, and (b) pPGM and AGP play an important rolein the scavenging of the glucose units derived from the starch break-down (Fig. 6). Accordingly, these models preview that the net rate ofstarch accumulation in heterotrophic cells is determined by the balancebetween the rates of ADPG synthesis in the cytosol, import of cytosolicADPG to the amyloplast, starch synthesis and starch breakdown, andby the efficiencywithwhich starch breakdownproducts can be recycledback to starch via the coupled reactions of pPGM and AGP. Also, thesemodels predict that the recovery toward starch biosynthesis of starchbreakdown products will be deficient in pPGM and AGP mutants,resulting in a parallel decline of starch accumulation.

The occurrence of the sucrose–starch conversion pathway illustrat-ed in Fig. 6A implies that neither UDPG produced by SuSy, nor cytosolichexose-Ps derived from the action of UGP on UDPG, are involved instarch biosynthesis in heterotrophic organs of dicotyledonous plants.This view is consistent with the unexpected WT starch content pheno-type of UGP antisensed potato tubers (Zrenner et al., 1993), and withtheWT G6P and G1P contents of starch-deficient SuSy antisensed pota-to tubers (Baroja-Fernández et al., 2003). Also, this view is coherentwith the unexpected starch-deficient phenotype of transgenic potato



A

B

Amyloplast

ADPG

Cytosol

G1P

Sucrose

ADPFructose

5

9

16

G6P

ADPGStarch

Glucose

12

15

SucroseCarriers/Endocytosis10

GPT

Amyloplast

ADPG

Cytosol

G1P

Sucrose

G1P

ADPFructose

9

16ADPG

UDPG

PPiUTP

6

UDP

Fructose16

ATP

PPi9

ATP

PPi

15

Starch

G6P

Glucose

125


10

GPT

Fig. 6. Suggested interpretation of themechanisms of sucrose–starch conversion in (A) heterotrophic cells of dicotyledonous plants and (B) cereal endosperms according towhich (a) bothSuSy and AGP are involved in the synthesis of ADPG linked to starch biosynthesis, (b) the cytosolic carbon substrate compound entering the amyloplast for subsequent conversion intostarch is ADPG, (c) pPGM and AGP are involved in the scavenging of starch breakdown products, and (d) the rate of starch accumulation would be the result of the net balance betweenstarch synthesis and breakdown. Note that in heterotrophic cells of dicotyledonous plants neither cytosolic hexose-phosphates, nor cytosolic UGP is involved in the sucrose–starch con-version process. Note also that in cereal endosperm cells SuSy catalyzes the direct conversion of sucrose into both ADPG and UDPG, the latter being converted to ADPG in the cytosol by thestepwise reactions of UGP andAGP. This viewpredicts that in pPGMand AGPmutants the recovery toward starch biosynthesis of the glucose units derived from the starch breakdownwillbe deficient, resulting in a parallel decline of starch accumulation since starch breakdown products (especially glucose and G6P) will leak out the amyloplast. Numbering of enzyme ac-tivities is the same as in legends of Figs. 1–4.


tubers heterologously expressing in the cytosol either yeast invertaseplus glucokinase (Trethewey et al., 1998) or bacterial sucrose phos-phorylase (Trethewey et al., 2001), since the high sucrolytic activityoccurring in these tubers leads to drastic reduction of cytosolic sucroselevels, thus limiting ADPG-producing SuSy activity.

Genetic evidences demonstrating the significance of ADPG-producing SuSy in the sucrose–starch conversion process in heterotro-phic cells of both mono- and dicotyledonous plants are the same asthose presented in Section 2.2.2.1.

3. Microbial volatiles promote both growth and accumulation ofexceptionally high levels of starch in mono- anddicotyledonous plants

Microbes synthesize and emit many volatile compounds. Volatileemissions from rhizobacterial isolates of Bacillus spp. promote growthin Arabidopsis plants by facilitating nutrient uptake, photosynthesis anddefense responses, and by decreasing glucose sensing and abscisic acidlevels (Ryu et al., 2003, 2004; H. Zhang et al., 2008, 2009). In contrast,volatiles from Pseudomonas spp., Serratia spp. and Stenotrophomonas


spp., and from some fungal species inhibit growth in Arabidopsis plants(Splivallo et al., 2007; Tarkka and Piechulla, 2007; Vespermann et al.,2007). Given the lack of knowledge on howmicrobial volatiles (MVs) af-fect reprogramming of carbohydrate metabolism in plants, Ezquer et al.(2010a) explored the effect on starch metabolism of volatiles releasedfrom different microbial species ranging from Gram-negative andGram-positive bacteria to different fungi. Toward this end, the authorsmeasured the starch and soluble sugar contents in leaves of plantscultured in the presence or in the absence of adjacentmicrobial cultures.Noteworthy, Ezquer et al. (2010a) reported that, in the absence of phys-ical contact between plant and microbe, all microbial species tested(including plant pathogens and microbes that normally do not interactwith plants) emitted volatiles that promoted enhancement of the intra-cellular ADPG levels and a rapid accumulation of exceptionally highlevels of starch in leaves of bothmono- and dicotyledonous plants. Levelsof starch reached byMV-treated leaves were comparable to those occur-ring in heterotrophic organs such as tubers and cereal seeds. This phe-nomenon, designated as MIVOISAP (for MIcrobial VOlatiles InducedStarch Accumulation Process), was also accompanied by strong promo-tion of growth. Therefore, MIVOISAP cannot be ascribed to utilization


image of Fig.�6



of surplus photosynthates and/or ATP for starch biosynthesis that wouldoccur under growth arrest conditions.

Transcriptome, metabolite content and enzyme activity analyses ofpotato leaves exposed to volatiles emitted by the plant pathogenAlternaria alternata revealed that starch over-accumulation was accom-panied by enhanced 3PGA/Pi ratio and up-regulation of SuSy, acid in-vertase inhibitors, SSIII and SSIV, starch branching enzyme, GPT2, andsynthesis of phosphatidylinositol-phosphates implicated in processessuch as endocytosis and vesicle traffic (Ezquer et al., 2010a).MIVOISAP was also accompanied by down-regulation of acid invertase,plastidial Trxs, plastidial β-amylase and pSP, and proteins involved inthe conversion of plastidial triose-phosphates into cytosolic G6P. Nochanges were found neither in the expression levels of AGP encodinggenes nor in the redox status of APS1. Furthermore AGP-antisensed po-tato leaves accumulated exceptionally high levels of starch in the pres-ence ofMVs. The overall data thus indicated that potatoMIVOISAP is theconsequence of transcriptionally and post-transcriptionally regulatedmetabolic reprogramming involving (a) up-regulation of ADPG-producing SuSy, SSIII and SSIV, and proteins involved in the endocyticuptake and traffic of sucrose, and (b) down-regulation of acid invertaseand starch breakdown enzymes. During potatoMIVOISAP there also oc-curs a down-regulation of ATP-consuming functions involved in inter-nal amino acid provision such as proteases and enzymes involved inde novo synthesis of amino acids. Ezquer et al. (2010a, 2010b) thus hy-pothesized that, under conditions of limited ATP-consuming proteinbreakdown occurring during exposure toMVs, the surplus ATP and car-bon are diverted from protein metabolism to starch biosynthesis.

Time-course analyses of starch and maltose contents in illuminatedArabidopsis leaves of plants exposed to volatiles emitted by A. alternatarevealed that both starch synthesis and β-amylase-dependent starchdegradation were enhanced upon MV treatment, although the finalbalance was positive for synthesis over breakdown (J. Li et al., 2011).The increase of starch content in illuminated leaves of MV-treated hy1/cry1, hy1/cry2 and hy1/cry1/cry2 Arabidopsis mutants was many-foldlower than that of WT leaves, indicating that Arabidopsis MIVOISAP issubjected to photoreceptor-mediated control. Transcriptomic analysesof Arabidopsis leaves exposed to A. alternata volatiles revealed changesin the expression of genes involved in multiple processes. However, un-like potato, no changes could be observed in the expression of starch re-lated genes (J. Li et al., 2011). Also, unlike potato plants, ArabidopsisMIVOISAPwas accompanied by 2-3-fold increase of the levels of reduced(active) form of APS1. Using different Arabidopsis knockout mutants J. Liet al. (2011) observed that the magnitude of the MV-induced starch ac-cumulation was low in mutants impaired in SSIII, SSIV and NTRC. UnlikeWT leaves, no changes in the redox status of AGP occurred in ntrcmutants, providing evidence that MV-promoted monomerization(activation) of AGP is mediated by NTRC. The overall data thus stronglyindicated that Arabidopsis MIVOISAP involves a photocontrolled, tran-scriptionally and post-transcriptionally regulated networkwherein pho-toreceptors and post-translational changes (e.g. changes in the redoxstatus) of plastidial enzyme(s) such as AGP, SSIII and SSIV play importantroles. Consistent with the possible involvement of changes in redox sta-tus of SSs in MIVOISAP, Glaring et al. (2012) have recently reported thatSSI and SSIII are reductively activated enzymes.

4. Biotechnological approaches for increasing starch content inheterotrophic organs

Despite themonumental progressmade inunderstanding the geneticand biochemical mechanisms of starch biosynthesis in plants, up to thelate 2000s relatively little hadbeen achieved in terms of increasing starchcontent and yield using biotechnological approaches, particularly in het-erotrophic organs of cereal crops (Smith, 2008). However, within the last5 years, a series of major developments has produced significant resultsthat indicate the likelihood of enhancing starch production. We nextassess the progress made in molecular strategies, both successful and


unsuccessful, for improving starch accumulation in heterotrophic organsof plants of agronomic interest. Finally we will present some potentialstrategies for enhancing starch content.

4.1. Enhancement of AGP activity

Most attempts to increase starch accumulation have focused on en-hancing AGP activity. The focus on AGP arose originally from the beliefthat this enzymatic reaction catalyzes a “rate-limiting step” in the pro-cess of starch synthesis and thus, increases in its activitywould concom-itantly result in enhanced starch accumulation.

One strategy for enhancing AGP activity in plants consisted of heter-ologously expressing glgC16 (a mutant variant of the E. coli glgC gene)that encodes an allosterically insensitive AGP form. Recent studies,however, have shown that the allosterically sensitive glgC equallyserves the same purpose, since ectopic glgC expression restores WTstarch content in aps1 Arabidopsis mutants (Li et al., 2012). First at-tempts to increase AGP activity in plants were made in potatoesthrough expression of glgC16 under the control of the constitutive 35Spromoter. Using this approach, Stark et al. (1992) reported that whenthe product of glgC16 was targeted to plastids, tubers from the trans-genic lines accumulated more starch than control tubers, althoughthere was no correlation between starch levels and AGP activity in dif-ferent lines. Subsequent attempts to reproduce these results on a differ-ent cultivar of potato expressing glgC16 under the control of a tuber-specific promoter did not render tubers with increased levels of starchand ADPG, although they showed increased levels of AGP activity(Sweetlove et al., 1996). Ectopic expression of allosterically insensitiveE. coli AGP has also been used in other crops such as rice (Nagai et al.,2009; Sakulsingharoj et al., 2004), maize (Wang et al., 2007) or cassava(Ihemere et al., 2006). In these cases enhanced AGP activity in seedsresulted in enhanced biomass, but there was no increase in the starchcontent on a fresh weight basis.

Other attempts at achieving increased starch content in cereal endo-sperms have used modified variants of the AGP large subunit, whichcontributes little to the catalytic activity of the enzyme but is importantin determining its regulatory properties. Giroux et al. (1996) obtainedmaize lines expressing Sh2r6hs (a mutated variant of the AGP largesubunit gene), which encodes an enzyme that is allosterically insensi-tive. The starch content of these lines was higher than in control lines;however there was no increase in starch as a percentage of seed weightand, instead, these lines had significantly heavier seeds (Giroux et al.,1996). Ectopic expression of Sh2r6hs has been conducted in maize,rice and wheat (Meyer et al., 2004; Smidansky et al., 2002, 2003). Inthese cases enhanced AGP activity in seeds resulted in increased indi-vidual seed weight and seed yield per plant.

Most recently N. Li et al. (2011) have shown that ectopic expressionof either the BT2 gene coding for the small subunit maize AGP, or SH2gene coding for the maize AGP large subunit, results in enhanced seedweight and starch content.

4.2. Increasing supply of starch precursors to the amyloplast

As indicated above, synthesis of storage starch in the amyloplast re-quires the incorporation of ATP and carbon skeletons from the cytosol.Tjaden et al. (1998) and Geigenberger et al. (2001) provided evidencethat increasing the supply of ATP to the plastid in potato tubers couldbe a strategy to enhance tuber starch content, since transgenic potatotubers ectopically expressing a plastidic ATP/ADP transporter fromArabidopsis under the control of the cauliflower mosaic virus 35S pro-moter accumulated 2-fold more ADPG and 16%–36% more starch pergram fresh weight than control tubers. Although these results wouldsuggest that starch content is very sensitive to changes in the transportmachinery that supplies ATP, more recent studies using transgenic po-tato plants ectopically expressing the Arabidopsis ATP/ADP transporterunder the control of a tuber specific promoter did not result in increased




levels of starch (L. Zhang et al., 2008). Furthermore, these studiesshowed that increasingG6P import to the amyloplast by ectopic expres-sion of a pea GPT had no effect on tuber starch content or tuber yield. Bycontrast, double transformants simultaneously expressing the pea GPTand the Arabidopsis ATP/ADP transporter exhibited an enhanced tuberyield and starch content, the overall data indicating that both ATP/ADPand GPT share control of the starch content and tuber yield, most prob-ably by co-limiting substrate (ATP and hexose-P) supply to AGP.

Indirect evidence that increasing ATP supply to the amyloplast is astrategy for increasing starch content comes from the studies carriedout by Regierer et al. (2002) who reported that constitutivedownregulation of expression of plastidial adenylate kinase, an enzymethat catalyzes the inter-conversion of ATPwith AMP and ADP, increasedthe ADPG and starch contents of potato tubers (see also patent WO 02/097101 A1). These changes were accompanied by increases in the totaladenylate pools (includingATP), although not by changes in the adenyl-ate energy charge or by changes in developmental phenotype. Regiereret al. (2002) thus hypothesized that adenylate kinase acts in the ATP-consuming direction, competing for the same ATP pool with AGP. Theincrease in starch content (2 fold) is the largest so far reported for anytargeted manipulation of starch synthesis.

4.3. Increasing supply of sucrose to heterotrophic cells

The occurrence of high levels of sucrose transporters in heterotrophicorgans where apoplastic sucrose unloading occurs (e.g. developing rice,maize, barley, faba bean, and wheat seeds) has led to the conclusionthat sucrose transporters may play a role in sucrose uptake into endo-sperm cells for the synthesis of storage compounds during grain filling(Aoki et al., 1999, 2002, 2003; Harrington et al., 1997; Hirose et al.,1997; Sivitz et al., 2005;Weschke et al., 2000). Supporting this hypothe-sis, Scofield et al. (2002) reported that antisense inhibition of the rice su-crose transporter OsSUT1 leads to impaired grain filling, the final grainweight being reduced. Toward the aim of evaluating the potential of im-proved sucrose uptake capacity on wheat grain quality and yieldWeichert et al. (2010) produced transgenic wheat plants ectopically ex-pressing the barley sucrose transporter HvSUT1 under the control of anendosperm-specific promoter. The authors reported that, although pro-tein content in transgenic grains was higher than in WT grains, starchconcentration was unchanged.

In very early stages of potato tuber development, i.e. during the elon-gation phase of stolon growth, apoplasmic sucrose unloading predomi-nates over symplastic unloading. Subsequent tuberization processinvolves a switch from apoplastic to symplastic phloem unloading(Viola et al., 2001). Although this would apparently imply that sucrosetransporters play a minor role in sucrose unloading in developing tubersand subsequent uptake of sucrose in parenchymatic cells, Kühn et al.(2003) reported that reduction of StSUT1 expression in antisensed potatoplants leads to reduced yield during early stages of tuber development,indicating that in this phase StSUT1 plays an important role for sugartransport. To evaluatewhether it is possible to improve potato plant per-formance by overexpressing a sucrose transporter Leggewie et al. (2003)produced transgenic potato plants heterologously expressing a spinachsucrose transporter under the control of the 35S promoter. These plantsproduced tubers with WT levels of starch content and yield. Thus, theoverall data obtained using both mono- and dicotyledonous cropswould indicate that up-regulation of sucrose transport is not a satisfacto-ry strategy for increasing starch content in heterotrophic organs.

4.4. Enhancement of sucrose breakdown within the heterotrophic cell

Inspired on the widely accepted view that cytosolic hexose-Ps areprecursors for starch biosynthesis in heterotrophic organs of dicotyle-donous plants (see Fig. 5A), attempts to direct more carbon into starchproduction in potato tubers consisted of heterologously expressing inthe cytosol enzymes such as yeast invertase plus bacterial glucokinase,


bacterial sucrose phosphorylase or fructokinase, that facilitate the con-version of sucrose or sucrose breakdown products (i.e. glucose and fruc-tose) into cytosolic hexose-Ps. However, contrary to expectations,transgenic tubers expressing in the cytosol bacterial sucrose phosphor-ylase, fructokinase or yeast invertase plus bacterial glucokinase allexhibited reduced starch content (Davies et al., 2005; Trethewey et al.,1998, 2001). Despite extensive research, the mechanisms leading tothese unexpected results remain obscure (see Section 2.2.2.2).

More successful attempts to promote sucrose–starch conversionhave centered on enhancing SuSy activity. The focus on SuSy arosefrom the belief that this sucrolytic enzyme is a major determinant ofsink strength and starch accumulation in both mono- and dicotyledon-ous plants. First attempts to increase SuSy activity were conducted inpotatoes through expression of StSUS4under the control of the constitu-tive 35S promoter. Baroja-Fernández et al. (2009) reported that UDPG,ADPG and starch contents in SuSy overexpressing tubers were higherthan in WT tubers. Furthermore, starch yield per plant was shown tobe higher in SuSy-overexpressing plants than in WT plants, underboth greenhouse and open field conditions (Baroja-Fernández et al.,2009). “High starch” SuSy-overexpressing tubers exhibited a trend-wise increase of AGP activity. Furthermore, they exhibited reducedacid invertase activity, which together with previous studies showingthat down-regulation of SuSy is accompanied by a concomitant reduc-tion of starch levels and enhancement of acid invertase activity(Zrenner et al., 1995) indicates that (a) SuSy, but not acid invertase, isinvolved in the sucrose–starch conversion process, (b) SuSy and acid in-vertase compete for the same substrate (sucrose), and (c) the balancebetween SuSy- and acid invertase-mediated sucrolytic pathways is amajor determinant of starch accumulation in potato tubers. Interesting-ly enough SuSy overexpressing tubers exhibited other characteristics ofagronomic interest such as substantial increase in antioxidant activityand resistance to enzymatic browning when compared to control tu-bers. Protection against browning was ascribed to the involvement ofSuSy in the production of UDPG necessary for glycosylation and stabili-zation of polyphenols (patent WO 2010/055186 A1).

Most recently Li et al. (2013) produced transgenicmaize plants ectop-ically expressing StSUS4under the control of the constitutiveUBI promot-er. The five independent transgenic lines characterized produced seedsexhibiting higher SuSy activity, higher ADPG content and ca. 10% morestarch than WT seeds at the mature stage. Furthermore, StSUS4 express-ing seeds contained a higher amylose/amylopectin balance that WTseeds. SuSy (and AGP) activity in WT seeds greatly exceeded theminimum required to support normal rate of starch accumulation in de-veloping maize endosperms (Li et al., 2013). Consequently, SuSy over-expression should not lead to enhancement of starch content. To explainthe observed positive effect of SuSy over-expression on starch content,the authors argued that the rate of starch accumulation is the result ofthe balance between SuSy–AGP–ADPG transporter-SS-mediated starchsynthesis, and amylase- and/or SP-mediated starch breakdown. There-fore, up-regulation of SuSy results in a concomitant increase of thebalance between starch synthesis and breakdown, thereby leading to en-hancement of starch accumulation. The enhancement of ADPG content inSuSy over-expressing lines (also occurring in AGP over-expressing riceendosperms, Nagai et al., 2009)would indicate that one ormore reactionsconstrain carbonflux from cytosolic ADPG into starch. Li et al. (2013) thushypothesized that the transport of ADPG is perhaps the more importantlimiting factor in starch synthesis in SuSy over-expressing maizeendosperms.

The overall data thus indicate that enhancement of SuSy activityrepresents a useful strategy for increasing starch accumulation andyield in heterotrophic organs of both mono- and dicotyledonous crops.

4.5. Enhancement of expression of starch synthase class IV

Five distinct classes of SSs are known in plants: granule-bound SS(GBSS), which is responsible for the synthesis of amylose, and soluble




SS classes I, II, III, and IV (SSI, SSII, SSIII, and SSIV, respectively),which areresponsible for the synthesis of amylopectin. Using developing wheatendosperms exposed to SS inactivating temperatures, Keeling et al.(1993) reported that SSs have a starch biosynthesis flux control coeffi-cient approaching to unity. This and the fact thatmaximum catalytic ac-tivity of SS is close to metabolic flux through starch biosynthesis inseveral starch storing organs would indicate that SS is a major site ofregulation of starch synthesis, and therefore, a good target for theproduction of genetically engineered “high-starch” plants. However,ectopic expression of SS genes has hardly ever been used to increasethe accumulation of starch. This is mainly due to the presence of differ-ent classes of SSs in plants interactingwith each other in a protein com-plex and displaying a specific role in the synthesis of the finalarchitecture of the starch granule (Hennen-Bierwagen et al., 2008).Consequently, changes in the expression of a single SS may lead to pro-found and unpredictable effects not only on starch structure, but also onstarch content, revealing that SS isoforms have specific but inter-dependent roles in starch polymer synthesis. This problem is wellexemplified by the results obtained using transgenic potato plants ex-pressing the E. coli glycogen synthase (an enzyme that catalyzes thesame reaction as SS) whose tubers accumulated less starch thanWT tu-bers. Furthermore, the starch from the transgenic tubers had alteredstructural properties (Shewmaker et al., 1994).

Roldán et al. (2007) reported that SSIV T-DNA mutants accumulateonly one large starch granule in Arabidopsis chloroplasts. In addition,Szydlowski et al. (2009) reported that Arabidopsis SSIII/SSIV double T-DNA mutants accumulate very reduced starch levels, the overall dataindicating that SSIV plays a key role both in the starch granule initiationprocess and in starch accumulation, beingmandatory to render the regu-lar number of starch granule found inWT plants. That elimination of SSIVresults in both reduction of number of starch granules and starch contentin Arabidopsis leaves suggested that the number of starch granules couldrepresent a regulatory step in the control of starch accumulation(D'Hulst and Mérida, 2010). Confirming this hypothesis Gámez-Arjonaet al. (2011) reported that SSIV overexpression results in enhanced levelsof transitory starch inArabidopsis leaves. Noteworthy, high levels of starchaccumulated in the end of the day were completely mobilized during thenight in SSIV overexpressing plants, allowing them to grow at a higherrate thanWTplants. Gámez-Arjona et al. (2011) also reported that ectop-ic expression of SSIV results in increased levels of reserve starch and yieldin tubers of transgenic potato plants cultured both under greenhouse andopen field conditions. Interestingly enough, SSIV overexpression wasaccompanied by pleiotropic increase in AGP and SuSy activities, reductionof acid invertase activity and enhancement of ADPG content. Gámez-Arjona et al. (2011) thus hypothesized that enhanced starch accumula-tion in SSIV overexpressing tubers would be the result of enhancedSSIV, AGP and SuSy activities and reduced acid invertase. Further investi-gationswill be necessary to determinewhether ectopic expressionof SSIVrepresents a useful strategy for increasing starch content and yield inother crops of agronomic interest such as cereal crops.

4.6. Blocking starch breakdown

As discussed above (see Section 2.2.2.2), substantial evidence hasbeen provided showing the occurrence of simultaneous synthesis andbreakdown of starch in some starch accumulating heterotrophic cells.It is thus highly conceivable that starch accumulated by heterotrophicorgans with active starch turnover is the result of the net balance be-tween starch synthesis and breakdown (Baroja-Fernández et al., 2003,2009; Li et al., 2013; Muñoz et al., 2006). Consequently, down-regulation of starch breakdown functions should be a viable means ofincreasing starch content in heterotrophic organs.

It is generally accepted that α-amylase plays a central role in endo-sperm starch degradation. This activity is high in developing seed endo-sperms during the process of starch accumulation (Hakata et al., 2012;Li et al., 2013). On the other hand, genes encoding glucan, water-


dikinases (GWD) involved in starch phosphorylation required for amy-lase mediated starch breakdown are expressed in starch storing organs.Supporting the above hypothesis that reducing starch breakdownwould be a viable means of increasing starch content in storage organs(see above), Ral et al. (2012) recently reported that RNAi-mediateddown-regulation of GWD in wheat endosperm under the control of anendosperm-specific promoter resulted in an increase in grain yield de-termined by increases in seed weight, tiller number, spikelets perhead and seed number per spike (see also patent WO 2009/067751A1). These changeswere not accompanied by changes in the percentageof starch per total dried weight, which is a phenomenon comparable tothat occurring in AGP overexpressing plants (see above). The work ofRal et al. (2012) provided a promising mechanism for enhancing bio-mass and grain yield in wheat, with potential application to othercrops. Further evidence supporting that down-regulation of starch deg-radation may contribute to enhance starch content in developing seedshas been provided by Hakata et al. (2012), who reported that suppres-sion of α-amylase genes improves the quality of rice grain when plantsare cultured under conditions of high temperature.

4.7. Altering the expression of global regulators

SnRK1 (for Sucrose non-fermenting-1-related kinase-1) is a serine/threonine protein kinase that forms a complex with sucrose non-fermenting (SNF1) and AMP-activated protein kinase (AMPK). It actsas a central integrator of transcription networks in plant stress and en-ergy signaling, and as such, plays a role in regulating metabolism in re-sponse to carbon availability (Baena-González and Sheen, 2008; Baena-González et al., 2007).

Upon sensing the energy deficit associated with stress or nutrientdeprivation, SnRK1 triggers extensive transcriptional changes that con-tribute to restoring homeostasis. In general, SnRK1 activates genes in-volved in nutrient remobilization and represses those involved inbiosynthetic processes and storage, thus providing alternative sourcesof energy through catabolism of amino acids and starch during nutrientstarvation. Thus, SnRK1 positively regulates the expression of starchbreakdown genes, since antisense expression of SnRK1 reduces α-amylase gene expression in cultured wheat and rice embryos duringsugar starvation (Laurie et al., 2003; Lu et al., 2007). Intriguingly, al-though the attributed role of SnRK1 is to repress functions involved inbiosynthetic processes and storage, SnRK1 has been shown to be neces-sary for starch synthesis, since (a) it is required for SuSy gene expressionin potato tubers (Debast et al., 2011; Purcell et al., 1998), and (b)McKibbin et al. (2006) reported that tubers of SnRK1 overexpressingpo-tato plants accumulate up to 30% more starch than control tubers. Asdiscussed by McKibbin et al. (2006), starch content enhancementresulting from SnRK1 overexpression can be ascribed to up-regulationof SuSy and AGP activities occurring in the SnRK1 overexpressinglines. Although more research using different crop species is necessaryto investigate the role of SnRK1 in the control of starch metabolism,the work of McKibbin et al. (2006) provided evidence that SnRK1overexpression represents a useful strategy for increasing starch accu-mulation and yield in heterotrophic organs.

Genome-wide co-expression analyses conducted to identify candi-date regulators for starch biosynthesis in rice revealed that Rice StarchRegulator1 (RSR1), an APETALA2/ethylene-responsive element bindingprotein family transcription factor, negatively co-regulates the expres-sion of many starch synthesis genes including those coding for AGP,SSs, GBSS, branching enzymes and starch debranching enzymes (Fuand Xue, 2010). RSR1 highly expresses in rice organs and tissues withreduced starch content such as roots, seedlings and leaves, and ex-presses tomuch lesser extent in developing seed endosperms. Althoughstarch content and amylose/amylopectin balance in RSR1 over-expressing seeds were shown to be comparable to those of WT seeds,seeds of rsr1 mutants displayed increased amylose content as a conse-quence of up-regulation of GBSS expression (Fu and Xue, 2010), had




WT starch content and were larger than WT seeds, a typical phenotypeof AGP over-expression (see above). It thus appears that down-regulation of RSR1 is a good strategy for increasing starch yield in rice.

She et al. (2010) identified FLO2 as a genehighly expressing in devel-oping rice seeds coincident with starch accumulation whose mutationresults in both reduced rice grain size and reduced amylose/amylopectinbalance. The same authors reported that FLO2 positively co-regulates theexpression of genes coding for many starch synthesis enzymes such asAGP (AGPL1–4, AGPS1, AGPS2a and AGPS2b), SuSy (SuSy1 and SuSy2),SSs (SSIIb, SSIIIb, SSIVa), GBSS and branching enzymes (BE1 and BEIIb),and enzymes involved in starch breakdown such as α-amylase(Amy3C, Amy3D and Amy3E), isoamylases (ISA1 and ISA2) andpullulanase. Importantly, FLO2 overexpressing rice lines produce largerand heavier grains compared with WT plants, providing evidence thatFLO2 overexpression is a good strategy for increasing starch contentand yield in rice (She et al., 2010). Noteworthy, FLO2 belongs to a familyof rice genes whose orthologs widely exist in plants. Provided the func-tion of FLO2 orthologs encoded proteins is similar to that of FLO2, it islikely that up-regulation of FLO2 orthologs in other plant speciesmay re-sult in enhanced starch content and yield. Further researchwill be neces-sary to confirm or refute this hypothesis.

4.8. Enhancing trehalose-6-phosphate content

Trehalose-6-phosphate (T6P) is a sugar signal of emerging signifi-cance that has been suggested to regulate starch biosynthesis via Trx-mediatedposttranslational redox activation of AGP in response to sucrosein leaves, reporting onmetabolite status between cytosol and chloroplast(Kolbe et al., 2005; Lunn et al., 2006; Schluepmann et al., 2003).

Leaves of transgenic plants with enhanced T6P have increased redoxactivation of AGP and increased starch levels, whereas leaves of plantswith reduced T6P show the opposite effect (Kolbe et al., 2005). The oc-currence of a positive correlation between intracellular T6P levels,redox AGP activation and rates of starch synthesis in Arabidopsis (Kolbeet al., 2005; Lunn et al., 2006), and the promotion of SnRK1-dependentredox activation of AGP by trehalose or sucrose feeding to potato tuberdisks (Tiessen et al., 2003) led to the hypothesis that T6P is a signal of su-crose status that promotes starch accumulation in a SnRK1-mediatedprocess involving redox AGP activation (Geigenberger et al., 2005;Tiessen et al., 2003). However, this proposal conflicts with recent find-ings showing that T6P is a strong inhibitor of SnRK1 (Martínez-Barajaset al., 2011; Y. Zhang et al., 2009) and with the fact that genes normallyinduced by SnRK1 are repressed by T6P and vice-versa (Y. Zhang et al.,2009). To investigate the role of T6P signaling in starch biosynthesis inheterotrophic organs Debast et al. (2011) generated transgenic potatoplants with high levels of T6P as a consequence of the heterologous ex-pression of E. coli OtsA (which encodes a T6P synthase). They also charac-terized transgenic potato plants with low levels of T6P as a consequenceof the ectopic expression of E. coli OtsB (which encodes a T6P phospha-tase). These authors reported that tubers from transgenic lines with ele-vated T6P levels displayed reduced starch content. On the other hand,tubers from lines with reduced T6P accumulated WT starch content,although they showed a strongly reduced yield as a consequence ofdown-regulation of cell proliferation and growth. Transcriptional profil-ing of “lowT6P” tubers revealed that SnRK1 target genes are strongly up-regulated, which confirms the inhibitory effect of T6P on SnRK1 activity.AlthoughDebast et al. (2011) concluded that T6P plays an important rolefor potato tuber growth, their results indicated that altering T6P intracel-lular levels is not a good strategy for increasing starch content in hetero-trophic organs of plants.

4.9. Potential strategies for increasing starch content in heterotrophic organs

4.9.1. Over-expression of BT1As pointed out in Section 2.2.2.1, data on ADPG content in seeds of

Zmbt1 maize and Hvbt1 barley mutants, and on ADPG transport


capacities of amyloplasts isolated from Zmbt1 andHvbt1 seeds indicatedthat, in cereal endosperms, BT1 proteins facilitate the transfer ofcytosolic ADPG into the amyloplast for starch biosynthesis and arerate-limiting steps in this process. This has led to the proposal that up-regulation of BT1 functionwould be a strategy for increasing starch con-tent in cereal seeds (Chen et al., 2012; Slattery et al., 2000).

BT1 proteins belong to the mitochondrial carrier family (MCF) ofproteins that are involved in the transport of metabolites across themi-tochondrial membrane (Haferkamp, 2007; Millar and Heazlewood,2003). AlthoughMCFproteins are all presumed to be targeted to themi-tochondrial inner membrane (Millar and Heazlewood, 2003), some ofthem occur in other subcellular compartments. Kirchberger et al.(2007, 2008) for instance provided evidence that BT1 is exclusivelylocalized to the inner plastidial envelope membranes of plastids. How-ever, more recent studies have shown that BT1 proteins are duallytargeted to mitochondria and plastids (Bahaji et al., 2011c).

Kirchberger et al. (2008) reported that homozygous Atbt1Arabidopsis mutants displayed an aberrant growth and sterility pheno-type, which was ascribed to impairments in export of newly synthe-sized adenylates from plastids to the cytosol. However, Bahaji et al.(2011b) have recently shown that the aberrant growth and sterilityphenotype of Atbt1mutants could be reverted toWT phenotype by spe-cific delivery of AtBT1 to mitochondria, indicating that AtBT1 may playcrucial roles, other than transport of newly synthesized adenylatesfromplastids to the cytosol, that are connected tomitochondrialmetab-olism. Taking into account that (a) similar to AtBT1, ZmBT1 is duallytargeted to plastids and mitochondria, (b) similar to homozygousAtbt1 Arabidopsismutants, homozygous Zmbt1mutant seeds germinatepoorly and produce plants with low vigor (Mangelsdorf, 1926), (c) mi-tochondria do not synthesize starch, and (d) there are many “lowstarch” maize mutant seeds that germinate and produce plants thatgrow normally, Bahaji et al. (2011a) hypothesized that (a) the aberrantgrowth and sterility phenotype of homozygous Zmbt1 mutants wouldnot be ascribed to starch deficiency and/or to reduced ADPG transportactivity in endosperm amyloplasts, and (b) similar to AtBT1, ZmBT1would be involved in yet to be identified processes occurring in mito-chondria, other than ADPG transport, that are important for normalgrowth and fertility that indirectly influence starch accumulation. Need-less to say further studies are necessary to confirm or refute the latterhypothesis and to know whether up-regulation of BT1 constitutes avaluable strategy for increasing starch content and yields.

4.9.2. Blocking the activity of ADPG breakdown enzymesAn alternative approach to enhancing starch content in crops would

be to reduce the rate of ADPG degradation. Plants possess a widely dis-tributed enzymatic activity, designated as ADPG pyrophosphatase(AGPP), that catalyzes the hydrolytic breakdown of ADPG to G1P andAMP (Rodríguez-López et al., 2000). Two different protein entities areresponsible for this activity: “Nudix” hydrolases and nucleotidepyrophosphatase/phosphodiesterases (NPP). Nudix hydrolases havebeen suggested to act as “housecleaning” enzymes that prevent accu-mulation of reactive nucleoside diphosphate derivates, cell signalingmolecules or metabolic intermediates by diverting them to metabolicpathways in response to biochemical and physiological needs(McLennan, 2006). AGPPs belonging to this family have been foundboth in the plastidial and cytosolic compartments (Muñoz et al., 2008;Olejnik and Kraszewska, 2005). Up-regulation of the plastidial AGPPNudix hydrolase in transgenic potato plants results in reduced levelsof starch content in both leaves and tubers, indicating that this enzymehas access to the pool of ADPG linked to the biosynthesis of both transi-tory and reserve starch in leaves and tubers, respectively (Muñoz et al.,2008). The control of the intracellular levels of ADPG linked to starchproduction likely represents an important, though still poorly investi-gated point of control of starch metabolism. Continued molecular ge-netic analysis will be needed to determine whether AGPPs play acrucial role in preventingmetabolic flux toward starch in heterotrophic




organs of plants, and whether down-regulation of the AGPP functionconstitutes a good strategy for enhancing starch content and yield inmajor crops.

4.9.3. Ectopic expression of SuSy in the plastidAs presented in Section 2.1.2., a sizable pool of sucrose has a plastid-

ial localization (Gerrits et al., 2001; see Section 2.1.2). SuSys fromcyanobacteria are highly specific for ADP and have high affinity forthis nucleotide (Curatti et al., 2000; Figueroa et al., 2013; Porchia et al.,1999). Thus, a more radical possibility for increasing the starch contentof crop plants would be to express SuSy (especially from cyanobacterialorigin) in the plastid to produce ADPG.

4.9.4. Blocking the synthesis of storage proteinsDuring development of some heterotrophic organs such as tubers

and seed endosperms, cell division is followed by cell elongation, differ-entiation and storage.When cell division ceases, a sizable pool of carbonand ATP is mainly utilized to produce massive amount of storage starchand proteins. Wemust emphasize that a similar phenomenon has beenobserved in bacterial cultures entering the stationary phase, where ATPand carbon are diverted from processes required for cell division andgrowth to glycogen production (Montero et al., 2009; Rahimpouret al., 2013).

Storage protein production is expensive in terms of carbon and ATPcosts and it is highly likely that both storage protein and starch biosyn-thetic processes compete for the same ATP and carbon pools. Therefore,blocking storage protein synthesis during the latter phase of develop-ment could be a potential strategy for increasing starch content in het-erotrophic organs. One way to do it would consist in altering theexpression of factors globally controlling the production of storage pro-teins. This, however, is difficult since it has been reported that the ex-pression of genes involved in storage starch and protein synthesis ishighly coordinated by the same factors (Giroux et al., 1994; She et al.,2010). One alternative would consist in moderately down-regulatingthe expression of genes directly or indirectly involved in the synthesisof amino acids by using promoters that are specifically expressed duringthe latter part of the heterotrophic organ development. In this respectwe must emphasize that accumulation of exceptionally high levels ofstarch in leaves of plants exposed to MVs was accompanied by reduc-tion of the levels of amino acids as a consequence of down-regulationof genes directly or indirectly involved in amino acid biosynthesis (seeSection 3, Ezquer et al., 2010a).

5. Conclusions and main challenges

The discrepancies between the different proposed models ofstarch biosynthesis in leaves (illustrated in Figs. 1–4) and heterotro-phic organs (Figs. 5 and 6), and the unexpected failures of some mo-lecular strategies aimed at improving storage starch content inplants denotes that our knowledge of the starch biosynthetic processis still at a rudimentary level. Better understanding of the regulationof starch synthesis will require additional research, either at the bio-chemical, cell biological and molecular levels, which in turn willallow plant breeders and biotechnologists to advance in theirattempts to increase starch in a predictable way. Additional knowl-edge on resource allocation within the plant, sucrose uptake in het-erotrophic cells, sources of ADPG, mechanisms of ADPG transportin plastids, interaction of the plant with both biotic and abiotic envi-ronmental factors, and metabolic interaction of the plastidial com-partment with other cellular compartments will be crucial. In thislast respect we must emphasize that recent studies have demon-strated the occurrence of strong, but unexplored metabolic links be-tween plastids and endomembrane organelles. Thus, Nanjo et al.(2006) and Kitajima et al. (2009) have shown that α-amylase andNPPs traffic between endoplasmic reticulum-Golgi and the plastidialcompartment. Furthermore, Wang et al. (2013) have recently shown


that autophagy contributes to starch degradation in N. benthamianaleaves, cytosolic starch granule-like structures being sequesteredby autophagic bodies and delivered to vacuoles for their subsequentbreakdown. Needless to say, further studies will be necessary toevaluate the relevance of such links in the overall starch metabolism.Although the study of endocytosis in higher plants remained dor-mant because of the notion that this mechanism would not workagainst the high turgor pressure in plant cells, mounting evidencehas been compiled during the last years showing that endocytosisis essential for many processes in plants (Müller et al., 2007; Samajet al., 2005), including carbohydrate metabolism. We now knowthat in heterotrophic organs where apoplastic sucrose unloading oc-curs endocytosis may play an important role in transporting the bulkof sucrose from the apoplast to the vacuole and its subsequent con-version into starch (see Section 2.2.1). Thus, further investigationswill be necessary to understand the molecular and signaling mecha-nisms involved in this process.

Starch biosynthesis is a highly regulated process, both at transcrip-tional and post-transcriptional level that is interconnected with a widevariety of cellular processes and metabolic pathways. Its regulation in-volves a complex and an as yet not well defined assemblage of factorsthat are adjusted to the physiological status of the cell. However, todate most studies on genetic regulation of starch metabolism have fo-cused on the effects of single genes on starch biosynthesis, and analysesof the effect of global regulatory factors on starchmetabolism, aswell assystematic studies of the regulatory mechanisms of starch biosynthesisare still scanty. Evidence has been provided that, similar to the bacterialsystem where glycogen synthesis and breakdown genes are co-expressed and co-regulated with genes involved in diverse biologicalprocesses (Montero et al., 2011), starch metabolism genes are co-expressed and coordinated with other functions such as amino acidand fatty acid biosynthesis (Fu and Xue, 2010; Giroux et al., 1994; Sheet al., 2010; Tsai et al., 2009). Systematic analyses-based characteriza-tion of the interactome of starchmetabolism-related genes and proteinswith genes and proteins involved in other biological processes, and theidentification of global factors controlling the expression of core starchmetabolism functions will be crucially important to rationally designmolecular strategies aimed at enhancing storage starch content in het-erotrophic organs of main crops. In this respect, we must emphasizethat genome-wide co-expression analyses, which are based on the as-sumption that genes with similar expression patterns are more likelyto be functionally related, have proven to be a powerful tool to identifyregulatory factors in transcriptional networks regulating starch metab-olism in crop species such as rice and potato (Ferreira et al., 2010; Fuand Xue, 2010). Similar type of comparative transcriptome and prote-ome analyses between different plant organs of WT plants andmutantswith altered starch content should be conducted using a wide range ofspecies of agronomic interest to identifymajor determinants and globalregulators of starch biosynthesis.

MV-promoted accumulation of exceptionally high levels of starch isa recently discovered mechanism for the elicitation of plant carbohy-drate metabolism by microbes that still needs to be investigated inmore detail before it can be fully understood and used as a source ofideas and tools for strategies to complement breeding and biotechnolo-gy programs aimed at improving crop yields. At short run, further effortsand research will be necessary to identify the volatiles and sensingmechanisms involved in MIVOISAP.

Acknowledgments

This research was partially supported by the grants BIO2010-18239from the Comisión Interministerial de Ciencia y Tecnología and FondoEuropeo de Desarrollo Regional (Spain) and IIM010491.RI1 from theGovernment of Navarra, and by Iden Biotechnology. M.O. was partlysupported by grant N. ED0007/01/01 Centre of the Region Haná for Bio-technological and Agricultural Research.




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starch biosynthesis, its regulation and biotechnological

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