the role of rab gtpases in cell wall metabolism

Journal of Experimental Botany, Vol. 59, No. 15, pp. 4061–4074, 2008

doi:10.1093/jxb/ern255 Advance Access publication 22 October, 2008

REVIEW ARTICLE

The role of Rab GTPases in cell wall metabolism

Grantley Lycett*

Plant and Crop Sciences Division, School of Biosciences, University of Nottingham, Sutton Bonington Campus,Near Loughborough, LE12 5RD, UK

Received 28 July 2008; Revised 15 September 2008; Accepted 17 September 2008

Abstract

The synthesis and modification of the cell wall must

involve the production of new cell wall polymers and

enzymes. Their targeted secretion to the apoplast is

one of many potential control points. Since Rab

GTPases have been strongly implicated in the regula-

tion of vesicle trafficking, a review of their involvement

in cell wall metabolism should throw light on this

possibility. Cell wall polymer biosynthesis occurs

mainly in the Golgi apparatus, except for cellulose and

callose, which are made at the plasma membrane by

an enzyme complex that cycles through the endomem-

brane system and which may be regulated by this

cycling. Several systems, including the growth of root

hairs and pollen tubes, cell wall softening in fruit, and

the development of root nodules, are now being

dissected. In these systems, secretion of wall poly-

mers and modifying enzymes has been documented,

and Rab GTPases are highly expressed. Reverse

genetic experiments have been used to interfere with

these GTPases and this is revealing their importance

in regulation of trafficking to the wall. The role of the

RabA (or Rab11) GTPases is particularly exciting in

this respect.

Key words: Cell wall, fruit softening, pollen tube, Rab GTPase,

root hair, root nodule, trafficking, vesicle.

Introduction

Control of the process of trafficking has been implicated inthe control of a wide range of cellular and physiologicalprocesses, including gravitropism, polar auxin transport,autophagy, cytokinesis, abscisic acid (ABA) and stressresponses, and pathogen resistance (Surpin and Raikhel,2004), and cell wall metabolism is clearly another

important example. The vitally important process of howthe cell wall is synthesized and modified has been difficultto study at the molecular level because it occurs partly inthe apoplast. Attempts to study cell wall modification byinhibiting single enzymes have met with limited success(Brummell, 2006) and it is clear that this is partly becausethe process needs to be highly coordinated. The elementsinvolved are produced in different parts of the cell:cellulose at the plasma membrane (PM), other polysacchar-ides in the Golgi apparatus, and the enzymes responsiblefor both synthesis and modification on the endoplasmicreticulum (ER). Coordination of the delivery of theseelements to the apoplast at the correct time must, therefore,involve trafficking through the endomembrane system.Studies involving reverse genetic approaches, mostly,

but not exclusively, by blocking the action of RabGTPases, have now reached an exciting stage where theresults from several systems are revealing their centralimportance in cell wall metabolism, both in highlypolarized cells such as root hairs and pollen tubes and inprocesses such as fruit ripening. This review sets out toexamine the processes by which materials destined for thecell wall reach their destination and the extent to whichreverse genetic approaches, supplemented by other evi-dence, have established the central importance of RabGTPases in regulating these processes.

Rab GTPases help to regulate different vesicletrafficking pathways

Most macromolecules that are secreted from the cell aretransported by vesicles through the endomembrane system.Polysaccharides are synthesized at the cell membrane or inthe Golgi apparatus (see later section) and secreted proteinsare usually synthesized on the ER and transported via theGolgi apparatus and the trans-Golgi network (TGN) ontheir way to the PM (Hanton and Brandizzi, 2006; Fig. 1).

* To whom correspondence should be addressed. E-mail: [email protected]

ª The Author [2008]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.For Permissions, please e-mail: [email protected]

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Within this trafficking system, transport is by vesiclesthat bud from one organelle and fuse with another, andthese may move in an anterograde or retrograde di-rection. Fusion is promoted by complementary SNAREs(soluble N-ethylmaleimide-sensitive factor attachmentproteins), the v-SNAREs on the vesicle membrane andthe t-SNAREs on the target membrane, which are alsooften referred to as syntaxins or SYP proteins. The role ofSNAREs in plants has been reviewed (Sutter et al., 2006).However, it is thought that Rab GTPases are oneregulatory factor in the fusion of the correct vesicle withthe correct target membrane, and in mammalian systemsthere are 41 subclasses of these, each of which regulatesdocking of different vesicles with their target membranes(Zerial and McBride, 2001). In Arabidopsis thaliana, the57 Rab GTPases have close homology to only eight of thetypes found in mammals (Pereira-Leal and Seabra, 2001;Rutherford and Moore, 2002; Vernoud et al., 2003), andthe same eight groups are also found in other plants(Zhang et al., 2007; Abbal et al., 2008; Table 2) thoughthe total number of Rabs in grapevine was about half thatin Arabidopsis. However, these eight have many moremembers than the corresponding mammalian groups andthey have been renamed RabA, RabB, RabC, RabD,RabE, RabF, RabG, and RabH, and further divided intosubclades (Pereira-Leal and Seabra, 2001; Rutherford andMoore, 2002; Vernoud et al., 2003; Table 1). Mostnotably, the two mammalian Rab11 GTPases correspond

to the Arabidopsis RabA clade, which has 26 members,divided into six subgroups (RabA1 – RabA6). This hasled to the hypothesis that these subgroups may haveevolved different functions (Rutherford and Moore, 2002),which means that we cannot be sure of the functions ofthese types in plants, though the evidence so far suggeststhat some groups have largely similar functions to theirmammalian counterparts (Vernoud et al., 2003: Molendijket al., 2004).Different Rab GTPases regulate trafficking between

different membrane compartments, and assumptions basedupon homology to mammalian Rabs are now beingsupplemented by evidence from plant systems so that,with the exception of RabC (Rab18) for which evidence isstill scarce, it is possible to predict which ones may beinvolved in regulating transport to and from the cell wall(Table 1, Fig. 1). RabF (corresponding to Rab5) andRabG (Rab7) proteins are expected to be associated withendocytosis, and evidence suggests that RabG is indeedinvolved in internalization of dye into the vacuole (Mazelet al., 2004), though this is a process that would involvetrafficking steps through several compartments. Similarly,different RabFs may be involved in either endocytosis orvacuolar trafficking (Ueda et al., 2001; Grebe et al., 2003;Sohn et al., 2003; Bolte et al., 2004; Kotzer et al., 2004;Lee et al., 2004). RabE is related to the Golgi to PM-associated Rabs of mammals such as Rab8 and seems tohave the same role in plants (Zheng et al., 2005). RabD

Fig. 1. Simplified schematic representation of the trafficking pathways to and from the cell wall. Pathways are shown as arrows with probablecargoes indicated. The Rab GTPases probably involved at each step are indicated in parentheses. Pathways to and from the vacuole have beenomitted for simplicity. TGN/EE refers to the trans-Golgi network and early endosomal compartments which now seem to partially overlap in plantcells and possibly function in both endocytosis and exocytosis.

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(Rab1) and RabB (Rab2) are expected to be involved inER to Golgi transport, and plant studies so far confirm this(Batoko et al., 2000; Cheung et al., 2002). RABH (Rab6)will complement its yeast homologue, ypt6, which isimplicated in retrograde Golgi to ER transport (Bednareket al., 1994). The role of the RabA (Rab11) GTPases hasbeen the most difficult to assign. The mammalian Rab11GTPases are traditionally associated with recycling (Zerialand McBride, 2001) though there are also reportsindicating that they can be involved in exocytosis fromthe TGN (Chen et al., 1998). Evidence from plants issuggesting that at least some groups are associated withtransport to the cell membrane (Ueda et al., 1996; Luet al., 2001; Inaba et al., 2002; de Graaf et al., 2005;Chow et al., 2008; Rehman et al., 2008). As discussed indetail later, one of the exciting developments is that thisclass may be vital for trafficking to the cell wall.

Cell wall synthesis and modification

Before considering how trafficking to the cell wall may beregulated, it is necessary to consider the components ofthe cell wall itself and how they are made. These will bediscussed only briefly, and readers interested in a detailedtreatment of the structure and experimental evidenceshould consult recent reviews (O’Neill and York, 2003;Brummell, 2006). There are three main groups ofpolysaccharides in the plant cell wall. Cellulose (b-1,4-D-glucan) chains are grouped into crystalline microfibrilsembedded in a matrix of other polysaccharides. Thesecond class of polysaccharides, the matrix glycans orhemicelluloses, comprises four main types: xyloglucans,gluconoarabinoxylans, glucomannans, and mixed linkageglucans (MLGs). The third class of wall polysaccharides

are the pectins, which again fall into four main types:homogalacturonans, xylogalacturonans, rhamnogalactur-onan I and rhamnogalacturonan II. The synthesis of thesepolysaccharides by membrane-associated enzymes hasagain been extensively reviewed (Doblin et al., 2003;Lerouxel et al., 2006; Scheller et al., 2007). Cellulose issynthesized by hexameric rosette complexes in the PM.The catalytic subunits are encoded by the CESA genes,but other associated proteins also seem to be needed. Thepioneering study of Northcote and Pickett-Heaps (1966)employed [3H]glucose to show that pectic polysaccharidesmove through the Golgi from cis cisternae to transcisternae, then through the TGN and Golgi-derivedvesicles to the primary cell wall. Since then, antibodieshave been used to show localization of xyloglucans andpectin in the Golgi cisternae and the TGN, and isolatedGolgi preparations have been shown to incorporatelabelled sugars into MLGs, xyloglucan, gluconoarabinox-ylan, glucomannan, and galactomannan (for references,see Doblin et al., 2003). There is, however, some doubtabout the site of synthesis of MLGs because two recentimmunolabelling studies have indicated that these aresynthesized at the PM (Philippe et al., 2006; Wilson et al.,2006). Glycan synthases, which make the backbones, andglycosyl transferases, which add side chains, have alsobeen found in the Golgi bodies (Doblin et al., 2003;Scheller et al., 2007). Glycan synthases are type IIImembrane proteins with multiple membrane-spanningregions and the glycosyl transferases are type II mem-brane proteins. Therefore, it is clear that all of theseenzymes must be correctly targeted and trafficked to thecorrect membrane (Fig. 1). The Golgi-synthesized poly-saccharides too must be correctly transported to theapoplast, so that vesicle trafficking must play an importantrole in cell wall synthesis.

Table 1. Nomenclature and functions of different Rab groups

New Arabidopsisnames

Equivalentmammalian groups

Function based on mammalian studies Probable function in plantsa

RabA Rab11 Recycling through endosomesand Golgi

Several subgroups in plants sofunctions possibly diverse. Associatedwith Golgi vesicles and TGN/endosomes.Golgi/PM trafficking? Also involved in signalling?

RabB Rab2 cis-Golgi. ER/Golgi trafficking ER/Golgi trafficking?RabC Rab18 Lack of evidence Lack of evidenceRabD Rab1 ER/Golgi trafficking ER/Golgi trafficking?RabE Rab8/10/12 Constitutive exocytosis, especially

in polarized cells. TGN/PMGolgi/PM?

RabF Rab5/22 Early exocytic pathway. Earlyendosome fusion

PM/endosome and/or PVC/vacuole. Osmotic stress

RabG Rab7 Late endocytic pathwayIntraendosomal andendosome/lysosome transport

PM/vacuole? Osmotic stress

RabH ab6 Retrograde intra-Golgi andGolgi/ER transport

Stress responses. PVC/late endosome/vacuole trafficking

a See text for discussion and references.PVC, pre-vacuolar compartment.

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Table 2. Fruit-related expression profiles of Rab genes in the Dfi tomato gene indexa

Accessionnumber

Type of Rab Expression in different stages of fruit development

Ovary Developing/immaturegreen fruit

Maturingfruit

Maturegreen fruit

Breakerfruit

Ripeningpericarp

Red ripefruit

Microtomfruit

All tissues andtreatments

TC170014b Rab1/RabD 3 1 12TC170107c Rab1/RabD 8 1 1 1 2 35TC170420d Rab1/RabD 2 2 22TC172978 Rab1/RabD 4 7TC175258 Rab1/RabD 1 2 2 5 29TC175806 Rab1/RabD 2 2 6 1 40TC178895 Rab1/RabD 1 1 1 7TC191282 Rab1/RabD 1 7TC170492 Rab2/RabB 1 3 1 5 17TC175074 Rab2/RabB 1 1 7TC178011 Rab2/RabB 2 6TC187092 Rab2/RabB 1 5TC172108 Rab5/RabF 2 14TC177152 Rab5/RabF 1 1 1 7TC182542 Rab5/RabF 2 6TC176115 Rab5/RabF 2 1 3 1 16TC177750 Rab6/RabH 1 1 1 5TC183750 Rab6/RabH 1 1 3TC184604 Rab6/RabH 3 3 1 2 27TC187308 Rab6/RabH 1 2 4TC187358 Rab6/RabH 1 2TC171871 Rab7/RabG 6TC172410 Rab7/RabG 1 4 1 19TC174722 Rab7/RabG 1 5TC177559 Rab7/RabG 3TC177740 Rab7/RabG 1 5TC181072 Rab7/RabG 2 8 2 1 2 31TC170347 Rab8/RabE 1 2 1 1 13TC170684e Rab8/RabE 4 1 1 12TC171031 Rab8/RabE 4 4 1 4 22TC173103 Rab8/RabE 8TC174020 Rab8/RabE 2 1 1 11TC175489 Rab8/RabE 5TC186042 Rab8/RabE 5TC188589 Rab8/RabE 1 2TC190150 Rab8/RabE 2TC171570 Rab11/RabA 8TC172616 Rab11/RabA 3 1 10TC173506 Rab11/RabA 3 1 7TC173594 Rab11/RabA 6TC174729 Rab11/RabA 6TC176217f Rab11/RabA 4 1 9TC176819 Rab11/RabA 2 1 7TC177424 Rab11/RabA 1 3TC178173 Rab11/RabA 1 1 9TC180101 Rab11/RabA 3TC180423 Rab11/RabA 2 11TC180587 Rab11/RabA 1 1 1 8TC180845 Rab11/RabA 1 2 4TC181854 Rab11/RabA 2 1 9TC182133 Rab11/RabA 2 8TC182649 Rab11/RabA 2 1 7TC183847 Rab11/RabA 1 7TC185090 Rab11/RabA 7TC183933 Rab18/RabC 1 1 4

Classification of Rab GTPases is according to Rutherford and Moore (2002).a http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb¼tomato June 2008.b Rab1C of Loraine et al. (1996) (see text).c Rab1A of Loraine et al. (1996) (see text).d Rab1B of Loraine et al. (1996) (see text).e Rab8 of Zegzouti et al. (1999) (see text).f Rab11a of Lu et al. (2001) (see text).

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Recent data throw some light on the potential importanceof vesicle trafficking in regulating cellulose synthesis.Certain enzymes are recycled within the cell, and this maybe a means of regulating synthesis. By employing a freeze-fracture technique on cultured Zinnia elegans cells, Haiglerand Brown (1986) have shown that cellulose synthase(CESA) rosettes are present in the Golgi and Golgi-derivedvesicles throughout cell wall deposition, suggesting that thesystem is dynamic and new rosettes need to be constantlyinserted into the wall. Dunkley et al. (2006) have alsolocalized CESA to the Golgi by a membrane fractionationtechnique. CESA proteins in young developing xylem cellsare localized intracellularly, whereas in older cells that aresynthesizing cellulose more actively they co-localize withbands of microtubules at the cell surface that mark sites ofcell wall deposition. The intracellular pattern is consistentwith some or all being in the ER (Gardiner et al., 2003). Inthe absence of one of the three subunits, the proteins appearto stay in the ER, indicating that a complex must beassembled before it can be transported to the site ofsynthesis. Disruption of the microtubules causes theproteins to redistribute intracellularly.Yellow fluorescent protein (YFP) fusions have been

used to visualize CESA proteins in the Golgi and instructures that may be secretory vesicles. In the PM, thefusion proteins assemble into structures, assumed to berosettes, that move along microtubules. Inhibition ofcellulose synthesis by isoxaben causes the YFP fluores-cence at the PM to disappear (Paredez et al., 2006). It ispossible that these are recycled to the Golgi, eithercontinuously or to be stored. Green fluorescent protein(GFP)–CESA is found primarily in the PM in slowlygrowing hypocotyl cells but has a more intracellulardistribution in larger cells further down the hypocotyls(Johansen et al., 2006). KORRIGAN, a b-1,4-glucanaseassociated with the cellulose synthetic rosettes andnecessary for their action, also appears to cycle throughdifferent compartments depending upon the age of the celland its growth rate (Robert et al., 2005). Therefore,cellulose synthesis may be regulated not only by thesynthesis of CESA proteins but also by their redistributionaway from the PM, and perhaps even by their subsequentrecycling back to the PM.Pectin polysaccharides themselves also seem to be

recycled in dividing cells. Antibodies have been raisedagainst cross-linked rhamnogalacturonan–borate com-plexes that are believed to form only after delivery to thecell wall. These have revealed that these pectic poly-saccharide complexes may be recycled back into the celland may then be inserted into the growing cell plate(Baluska et al., 2002, 2005; Dhonukshe et al., 2006).The wall also contains structural proteins, which may act

to stiffen the wall by peroxidase-mediated cross-linking(Passardi et al., 2004). Recent evidence confirms theirimportance because modifying levels of hydroxyproline-rich

glycoproteins in the wall can change stem thickness(Roberts and Shirsat, 2006). Extensins are induced byelicitors and by pathogen attack (Bowles, 1990), and,recently, Wei and Shirsat (2006) have shown that transgenicplants with increased levels of these proteins have reducedpathogen invasiveness. As expected for secreted proteins,these all have N-terminal signal sequences and glycosyla-tion has been shown to occur in the Golgi (Johnson et al.,2003).Therefore, it is clear that both polysaccharides and

proteins must be transported to, and sometimes from, thecell surface in a regulated manner. It is also clear thatthis transport must be via the Golgi apparatus througha classical vesicle trafficking route (Fig. 1). However, theremust be more than one route and more than one control.Homann and Tester (1997) showed that exocytosis inbarley aleurone protoplasts could be by a Ca2+-independentpathway or by a Ca2+-dependent one requiring GTP-binding protein. This indicates that control may be byclassical signalling molecules and by GTPases. Furtherevidence for differentially regulated pathways comes fromthe finding that one of two SNARES involved in traffickingto the PM is implicated in ABA-responsive secretion(Leyman et al., 1999, 2000). New tools for studying theinvolvement of different SNAREs in different pathways(Tyrrell et al., 2007) offer the probability of great strides inthis exciting area. Different trafficking routes may alsocarry different constituents of the cell wall. In pea seed-lings, a low concentration of brefeldin A was shown toinhibit incorporation of matrix polysaccharides and cellu-lose, but only slightly inhibited incorporation of labelledamino acids into PM and wall proteins (Lanubile et al.,1997). Blocking vesicle docking by blocking the actionof an ABA-inducible syntaxin in tobacco leaf protoplastsdid not inhibit polysaccharide incorporation into the walland actually slightly increased cellulose incorporation(Leucci et al., 2007). However, the transport of secretedproteins was reduced by half. Therefore, there seem to beparallel routes to the apoplast that may be controlledindependently and by different mechanisms, and these maycarry different cargoes.Following cell wall synthesis, there are numerous

circumstances in which the wall should not remain rigid.One of these is cell expansion, during which it isnecessary for some of the bonds between primary wallpolymers to be selectively loosened and reformed ina controlled manner to allow it to stretch (Cosgrove,2003). Cell wall modification is also integral to fruitripening, and there are many developmental processeswhere cells need to separate, and these include growth ofpollen tubes through the style, abscission of leaves flowersand fruit, dehiscence of pods and anthers, emergence oflateral roots through the cortex and radicles through theendosperm, formation of leaf mesophyll, aerenchyma,hydathodes, laticifers, stomata, and tracheary elements,




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and the shedding of root caps (Roberts et al., 2002). All ofthese processes involve hydrolytic enzymes and other keyproteins such as expansins.With the advent of genomics, many families of

glycoside hydrolases have been identified and these areinvolved not only in cell wall polysaccharide synthesisand degradation during cell separation, but also incellulose synthesis, cell division, storage reserve mobili-zation, defence, symbiosis, secondary metabolism, andsignalling (Minic, 2008). It would normally be expectedthat proteins bound for the cell wall would be transportedvia vesicles, but this has been very little studied. Recentanalyses of apoplastic or cell wall protein fractions haveidentified many glycoside hydrolases, and the majority ofthese were predicted to have signal peptides (Chivasaet al., 2002; Watson et al., 2004; Boudart et al., 2005;Kwon et al., 2005; Bayer et al., 2006; Zhu et al., 2006),which would indicate that they entered the endomembranesystem during synthesis. Therefore, it would be expectedthat they would be trafficked via vesicles and thereforemight potentially be regulated in part by Rab GTPases.Alteration of cell wall metabolism by wounding is an

interesting specialized case where recent work has shedsome light. The response to wounding is mediated bya number of local and systemic signals including nitricoxide, systemin, jasmonates, ABA, ethylene, and cellwall-derived oligosaccharides (Leon et al., 2001; Pariset al., 2006). Whereas some signalling oligosaccharidesmay be produced mechanically, one particular wound-inducible polygalacturonase (PG) from tomato leaves maybe responsible for the production of oligogalacturonidesignalling molecules (Bergey et al., 1999). Callosesynthesis is a major response to wounding and, followingwounding, b-1,3-glucan synthase appears at sites ofcallose synthesis on the PM. The silencing of callosesynthase genes prevents wound-induced callose synthesis(Jacobs et al., 2003; Nakashima et al., 2003). A widerange of other glycan synthases and transferases, as wellas expansins and structural wall proteins, are also inducedby wounding (Dimmer et al., 2004; Guzzardi et al., 2004;Park et al., 2006a; Reilly et al., 2006), though we cannotbe sure that all of these are directly involved in woundrepair. In fruit, the situation is rather different because innormal fruit ripening, cell wall hydrolases are produced inresponse to ethylene (see next section). However, wound-ing of tomato fruit has been shown to halt production ofsome of these (Chung et al., 2006).Rab GTPases presumably must be involved in traffick-

ing of wound-induced cell wall metabolic enzymes, butthis has been little studied. In addition to inducing theproduction of Rab GTPases in ripening fruit (see nextsection), ethylene induces Rab11 and Rab8 in peahypocotyls and Arabidopsis leaves (Moshkov et al.,2003a, b). However, a direct connection between RabGTPases and wound repair must await further study.

Selected systems

There are several systems where the key role of vesicletrafficking in synthesis and modification of cell walls hasbeen well studied. Some of the evidence is indirect;typically this involves a correlation between the synthesisof key trafficking elements such as Rab GTPases with theprocess of wall growth and modification. More excitingly,in the last few years, evidence has emerged from reversegenetic approaches to suggest that these processes may becontrolled at the level of trafficking. Three of these areconsidered here.

Fruit

One of several obvious changes that occur during fruitripening is softening of the fruit. This is caused by changesin the cell wall brought about by the synthesis and secretionof enzymes and also due to synthesis of new cell wallmaterials (for a review, see Brummell, 2006). However,this process is extremely complex, as shown by attempts toreduce fruit softening by the use of antisense transgenes toinhibit the action of single enzymes. In tomato fruit, the useof gene silencing to inhibit the synthesis of PG reduced PGlevels to as little as 1% and inhibited pectin depolymeriza-tion but had only very small effects upon fruit firmness(Sheehy et al., 1988; Smith et al., 1988, 1990; Krameret al., 1992; Langley et al., 1994). Similarly, inhibition oroverexpression of pectinesterase (PE; Tieman et al., 1992;Hall et al., 1993), b-glucanase (Lashbrook et al., 1998;Brummell et al., 1999a), and xyloglucan endotransglycosy-lase (de Silva et al., 1994) had little or no effect upon fruitsoftening, though suppression of galactanase gene expres-sion reduced softening by 40% (Smith et al., 2002) andinhibition of the ripening-specific expansin reduced soften-ing by up to 20% and increased the degree of pectinpolymerization (Brummell et al., 1999b). Therefore, mostof these attempts have met with limited success, anda recent study has even shown that inhibition of PE intomato causes cell walls to soften more quickly (Phanet al., 2007). Recent work on pollen tubes has also shownthat regulation of cell wall stiffness by PE and PE inhibitorsis complex (Rockel et al., 2008; see later section).Therefore, it is clear that this process of wall disassem-

bly is highly coordinated and requires many enzymesacting in concert. The prospects for effecting biotechno-logical improvements in post-harvest quality of fruit arebetter using approaches that inhibit more than one enzymesimultaneously. When plants silenced for PG and thosesilenced for expansin were crossed, the effects weresynergistic and the fruits were firmer than in either singleline (Powell et al., 2003) and gave more viscous paste(Kalamaki et al., 2003). Inhibition of ethene biosynthesis,which would in turn inhibit the synthesis of a rangeof ethene-inducible enzymes, did cause dramaticallyfirmer fruit (Hamilton et al., 1990) though, unfortunately,

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inhibition of ethene production can also affect colour andflavour development. The inhibition of vesicle traffickingto the apoplast offers the potential of inhibiting many cellwall hydrolases at once and also the deposition of newpolymers. This could lead to new biotechnologicalapplications in post-harvest technology.Elements of the secretory machinery, notably Rab

GTPases, have been shown to be expressed preferentiallyin fruit (Loraine et al., 1996; Zainal et al., 1996; Zegzoutiet al., 1999; Lu et al., 2001; Park et al., 2006b; Abbalet al., 2008), often in a ripening-related and ethene-regulated manner. Three Rab1 proteins from tomato areexpressed strongly in fruit (Loraine et al., 1996). One ofthese (LeRab1C) is expressed most strongly in expandinggreen fruit, and the other two (LeRab1A and LeRab1B)more strongly in ripe fruit. However, the protein productsof these two genes may not be totally redundant in theirfunctions because prenylation at the C-terminus isimportant for the function of Rab GTPases, and these twodiffered in their ability to interact with geranylgeranyltransferases from yeast, mammalian cells, and plant cells.Thus, though they are all the same class of protein andwould, therefore, be expected to regulate ER–Golgitransport and they are all expressed in fruit, there may besubtle differences in their roles. Rab8 and Rab11 geneshave also been found to be preferentially expressed intomato fruit (Zegzouti et al., 1999; Lu et al., 2001) and,subsequently, the same classes have been found in applefruit (Park et al., 2006b). Examination of the tomatoTIGR database (Table 2) shows that many of the Rabexpressed sequence tags (ESTs) in the database have beenrecovered from carpel or fruit pericarp tissues at variousstages of development.It has been suggested that Rab GTPases enable or

regulate secretion of the enzymes that modify the cell wallto bring about fruit softening (Loraine et al., 1996; Zainalet al., 1996). This has been investigated for the Rab11class, first identified in ethene-treated mango fruit meso-carp (Zainal et al., 1996). A tomato orthologue (LeR-ab11a) was silenced by transformation with antisenseconstructs. The transgenic tomato plants had fruit thatremained firm for a very long period, and it was shownthat the levels of extractable PG and PE were reduced inthese fruit (Lu et al., 2001).The Rab11 antisense tomato plants also showed

physiological and developmental differences, includingaltered floral organ identity, highly branched inflorescen-ces, rumpled leaves, ectopic shoots from leaves, de-terminate growth, reduced apical dominance, and alteredethene production. These effects are reminiscent of de-velopmental abnormalities and altered hormone levelsseen in studies where Rab11 GTPases have been overex-pressed (Kamada et al., 1992; Sano et al., 1994; Aspuriaet al., 1995) and are not likely to be due to alteredtrafficking to the wall but instead it has been argued (Lu

et al., 2001) that these may be the result of misdirection ofhomeobox proteins, hormone carriers, and/or hormonereceptors. However, there was not a general failure oftrafficking in LeRab11a antisense tomatoes because,although a constitutive cauliflower mosaic virus (CaMV)35S promoter was used to drive the antisense construct,the plants grew at the normal rate, and leaves and fruitreached the usual size. Again, this indicates that there issome subtlety to the regulation of secretion to the cellwall. In a more recent study using tobacco leaf proto-plasts, LeRab11a–GFP fusions were shown to be associ-ated with the TGN, and dominant-negative mutantversions of the Rab gene also inhibited exocytosis ofsecreted GFP (Rehman et al., 2008). Therefore, tomatoRab11a does seem to be involved in trafficking from theGolgi to the PM, though the distinctiveness of theendosome and the TGN has recently been questioned(Dettmer et al., 2006; Lam et al., 2007) and the exactroute by which material is trafficked through post-Golgiendomembrane compartments is unclear. Both the studyby Rehman et al. (2008) and a recent study by Chow et al.(2008) on Arabidopsis root tips indicate that there may beseveral partially overlapping routes that are regulated, inpart, by several classes of RabA GTPases. Chow et al.(2008) showed that fluorescently labelled AtRabA2 andAtRabA3 both label the cell plate, and dominant-negativemutants of these Rabs show disruption of cell divisionpatterns indicating that these are involved in trafficking tothe newly forming cell plate in mitosis. One dominantnegative also targets the cell periphery in interphase cells,so there might be an involvement in Golgi to PMtrafficking too. The fluorescently labelled AtRabA2 andAtRabA3 completely co-localized with an endocyticmarker FM4-64 and partially co-localized with a TGNmarker VHA-a1. Conversely, Rehman et al. (2008)showed that the GFP fusions of LeRab11a (an AtRabA1homologue) co-localized completely with the TGN markerVenus-Syp61 and only partially with FM4-64. Whethereither of these overlapping post-Golgi compartmentscorresponds to classic TGN or classic early endosomes isunclear, though it is interesting that material trafficked tothe cell plate appears to come partly from endocytosis ofmaterial previously incorporated into the cell wall(Baluska et al., 2002, 2005; Dhonukshe et al., 2006).Clearly, elucidation of the relationships between thesecompartments requires further work, especially as the twomain studies reported here are from different systems.Rehman et al. (2008) went on to show that inhibiting

the syntaxin SYP121 pathway as well as Rab11a causeda greater inhibition of trafficking than just the LeRab11adominant-negative mutant alone, whereas inhibition ofSYP122 did not. This indicates that SYP122 and Rab11aare probably involved in the same pathway, but SYP121,which has been implicated in ABA-responsive secretion(Leyman et al., 1999, 2000), may be involved in




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a different pathway. This may shed some light on one wayin which subtlety of regulation of trafficking to the cellwall may be generated.

Root nodules

The formation of root nodules is a process that involvesboth growth and breakdown of the cell wall at severalstages. Formation of the nodule involves cell division andcell expansion, and take up of the symbiotic bacteriainvolves the formation of a novel tip-growing structure,the infection thread. Loosening of cell walls will benecessary as bacteria enter the root hair, as the infectionthread grows through cortical cells and as the bacteria arereleased from the infection thread.A PG and a PE have been found in Sinorhizobium

meliloti–Medicago symbiotic tissues. In both cases theseare encoded by genes that are distinct from, but veryclosely related to, those expressed in pollen (Munoz et al.,1998; Rodriguez-Llorente et al., 2004). The PG is alsolocalized at the tips of uninoculated root hairs (Rodriguez-Llorente et al., 2003b), and the authors proposed that thesegenes have evolved from those that support tip growth ofpollen to support tip growth of the infection thread.Promoter–b-glucosidase (GUS) fusion studies from thesame laboratory (Munoz et al., 1998; Rodriguez-Llorenteet al., 2003a) have shown that the PG gene is expressed innodule primordia, young nodules, and the invasion zone ofmature nodules. Because the gene is also expressed inlateral root primordia, it is clearly not confined to a role ininfection thread growth and may be secreted to loosen cellwalls of normal growing cells within the nodule.More direct investigations of control of secretion in root

nodules have centred on the role of Rab GTPases. Cheonet al. (1993) identified a Rab7 GTPase and a Rab1 GTPasein the nodules of soybean, and later Borg et al. (1997) useddegenerate probes to isolate a total of 29 Rab cDNAs from3-week-old nodules of Lotus japonicus. These includedseven copies of the ER–Golgi implicated Rab1, and fiveRab8 and 10 Rab11 cDNAs, each of which seems to beimplicated in secretion from the Golgi/TGN to the apoplast(Rutherford and Moore, 2002; Vernoud et al., 2003).Whilst many of these were not established as being specificto root nodules, Schiene et al. (2004) showed, usingwestern blots, that Medicago sativa Rab11f is specific tonodules. One Rab11 has recently been implicated innodulation by specific rhizobia (Meschini et al., 2008).In a reverse genetic approach to test the importance of

Rab1 and Rab7 in nodulation, Cheon et al. (1993)transformed antisense constructs under the control ofa nodule-specific leghaemoglobin promoter into hairyroots. Both antisense Rab genes caused smaller noduleswith reduced nitrogenase activity. The Rab7-suppressednodules showed a perinuclear accumulation of late endo-somes and multivesicular bodies. The Rab1 antisensenodules had fewer bacteroids per cell, release of bacte-

roids into the central vacuole, and reduced expansion ofinfected cells. Since this was a very early study, and thefirst that I am aware of to show an effect on cell expansiondue to inhibition of an element of the secretory machinery,it would be interesting to see further investigations of thisphenomenon in root nodules.

Apical growth systems

Two of the best studied experimental systems involvingsecretion to the cell wall are the tip growing cells: pollentubes and root hairs. These structures grow at a prodigiousrate and traffic large quantities of new material to thegrowing tip (for recent reviews, see Samaj et al., 2006;Campanoni and Blatt, 2007). Caution must be exercised ingeneralizing from these systems because they are veryspecialized and growth of the cell is highly polarized.Also, most other systems studied have involved traffickingof enzymes that modify cell walls, rather than new wallmaterial.However, there seem to be some similarities between

these systems and fruit ripening. Studies involving genesilencing to inhibit elements of the trafficking machineryhave shown that similar Rab GTPases are implicated (seebelow). In pollen tubes, there is also evidence fortrafficking of cell wall-modifying proteins, including PE(Bosch et al., 2005) and b-expansin (Cosgrove, 2000).Jiang et al. (2005) found that the VANGUARD1 geneencodes a PE expressed in pollen tubes, and the vgd1mutant has reduced fertility because of impaired growththrough the style tissue. They propose that this PE issecreted out of the pollen tube tip and modifies the wallsof the stylar cells to facilitate the growth of the tubethrough the style. This is supported by evidence thatexternally applied pectinase and PE could reduce wallstiffness and promote tip growth in Solanum chacoensepollen tubes (Parre and Geitmann, 2005). When anantisense transgene was used to silence expression ofa pollen-specific PE in tobacco, genetic segregation ratioswere perturbed and growth of the pollen tube through thestyle was inhibited whereas growth in vitro was un-affected (Bosch and Hepler, 2006). In this case, theauthors argued that the PE is more important in signallingthan in modification of the style cell walls but, in eithercase, the enzyme would need to be secreted through thecell membrane. PE is not the only protein secreted in thisway. Maize pollen (Cosgrove et al., 1997) and the pollenof other grasses (Cosgrove, 2000) secretes b-expansin.Because the maize expansin is secreted in such largequantities, is very soluble, and was shown to loosen thecell walls of grasses, it seems probable that the expansinfunctions to loosen the cell walls of the stylar tissue toallow the pollen tube to penetrate more readily. In-terestingly, the pollen tubes of dicotyledonous plants donot secrete this type of expansin and dicot cell walls arenot susceptible to it (Cosgrove et al., 1997).

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Trafficking during the growth of the cell wall has alsobeen extensively studied in both pollen and root hairs.Inhibition of vesicle formation by brefeldin treatment ofpollen caused reduced pollen germination, retardedpollen tube growth, abnormal pollen shape, and alteredcell wall composition and structure (Wang et al., 2005).In particular, the protein and polysaccharide content ofthe wall, especially the pectin content, was reduced at thetube apex. In this study, brefeldin treatment inhibitedexocytosis but, more unexpectedly, stimulated endocyto-sis. This led Wang et al. (2005) to note that cross-linkedpectin has been shown to be recycled in root meriste-matic cells (Baluska et al., 2002, 2005). Perhaps,therefore, such recycling is important in maintenance ofthe plasticity of the apical wall, and increased endocyto-sis could account for the reduced pectin content of thewall. This idea is supported by the observation that inlatrunculin-treated root hairs, the inhibition of actin-driven movement of endosomes was coincident withcessation of tip growth (Voight et al., 2005). Theseauthors proposed that the prevention of pectin recyclingmight also be the cause of growth cessation in thissystem. Exciting recent data show that several differentPE isoforms and several PE inhibitors are produced inArabidopsis pollen tubes. Some of these accumulatedifferentially at the tip and the flanks of the tube and mayregulate the extensibility of the wall by changing theesterification of the pectins. This differential accumula-tion appears to involve not only exocytosis of newproteins but also endocytosis of PE inhibitor from theflanks of the tube (Rockel et al., 2008). Thereforeregulation of vesicle trafficking is crucial to maintainingappropriate cell wall extensibility.The roles of elements of the trafficking machinery and

particularly Rab GTPases have also been investigated inthese tip growing systems. AtRabF1 and AtRabF2a wereshown to associate with endosomes in root hairs andother tissues (Voight et al., 2005), and the ER–Golgi-associated AtRabB1c (Rab2B) was shown to have a highlevel of expression in pollen (Moore et al., 1997). Atobacco gene of the same class, NtRab2, is highlyexpressed in pollen and also expressed in root hairs andother tissues that secrete enzymes or other material tothe cell wall: young seedlings, pistils, and germinatingseeds (Cheung et al., 2002). A dominant-negativemutant of this gene blocked ER–Golgi trafficking andprevented delivery of membrane proteins and invertaseto the cell surface. This same mutant inhibited pollentube growth, strongly implicating this Rab GTPase incontrol of trafficking of new cell wall material to thegrowing tip.Once again, the Rab11/RabA class of GTPases has

been best characterized. MtRab11G and AtRabA4b havebeen shown to be expressed in root hairs of Medicagoand Arabidopsis (Covitz et al., 1998; Preuss et al.,

2004). AtRabA4b was shown to localize to the tips ofroot hairs only when actively growing, but did not co-localize with either Golgi or the TGN. AtRabA5c(previously called Ara-4) is expressed in pollen tubesand was shown to co-localize with the Golgi cisternae,the TGN, and Golgi-derived vesicles (Ueda et al., 1996).NtRab11b localizes to the cone-shaped region, almosttotally occupied by vesicles, at the tip of tobacco pollentubes. This GTPase seems to be a key element incorrectly delivering material to the growing cell wallbecause a dominant-negative mutant, when overex-pressed in pollen, caused a reduced growth rate of thepollen tube and reduced fertility (de Graaf et al., 2005).It also plays some role in orienting growth because thesame mutant induced a wandering deformation in thepollen tubes. These phenotypic effects were shown to bemediated by the inhibition of exocytic and endocyticvesicle targeting to the cone region and reduced deliveryof cell wall proteins to the apoplast. T-DNA knockoutsof several Arabidopsis Rab11/RabA GTPases gave nocomparable phenotype (de Graaf et al., 2005), and it ispossible that this reflects functional redundancy becauseat least 10 RabA genes are expressed in pollen at roughlysimilar levels (Pina et al., 2005). Rab8 is also classicallyimplicated in Golgi–PM transport, but its involvement inthese systems remains to be determined.How are Rab GTPases involved in growth orientation?

This may be directly, through interaction with the exocyst,but Ca2+ signalling, which is known to play a role inorientation of growth, may also be involved. In a yeasttwo-hybrid system, AtRabA4b was shown to interactspecifically with the phosphatidylinositol-4-OH kinase,PI-4Kb1, but not other classes of PI-4Ks, whereas thedominant-negative mutant form of RabA4b would notinteract (Preuss et al., 2006). AtRabA4b and PI-4Kb1 were also shown to co-localize in root hairs. In thesame system, PI-4Kb1 interacted specifically with thecalcineurin-like Ca2+ sensor, AtCBL1, but not AtCBL3 orAtCBL5. PI-4Kb1/PI-4Kb2 T-DNA double insertionmutants had abnormal root hairs, and treatment with theionophore A23187 to disrupt the Ca2+ gradient in the roothair caused dispersion of RabA4b away from the tip(Preuss et al., 2006). A further study (Thole et al., 2008)has shown that ROOT HAIR DEFECTIVE4 encodesa phosphatidylinositol-4-phosphate phosphatase that co-localizes with RabA4b and may also help to regulatephosphatidylinositol-4-phosphate levels in the root hair tip.Therefore, it is clear that Ca2+ signals are directly linked toRab action.Although these tip growth systems are difficult to

dissect because inhibition of related factors such asendocytosis and signalling by Rop GTPases also affectsgrowth (see Cole and Fowler, 2006; Samaj et al., 2006;Campanoni and Blatt, 2007), there does seem to beevidence for the importance of control of secretion to the




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cell wall in these tip growing systems and a possibleregulatory role for Rab GTPases.

Concluding remarks

It is now clear that vesicle trafficking is necessary for cellwall synthesis in several ways. The enzymes thatsynthesize the polysaccharides need to be trafficked to thecell wall, and it seems that the retrograde trafficking ofcellulose synthase to storage compartments may be animportant regulatory step. In addition, the polysaccharidesthemselves need to be trafficked to the wall and alsorecycled so that they can be re-used when rapid cell wallsynthesis is required. Blocking of trafficking to the apicesof pollen tubes and root tips causes inhibition of tipgrowth. Cell wall hydrolases and other wall-modifyingenzymes also need to be trafficked to the cell wall, andinhibition of vesicle trafficking in fruit causes a reductionin enzyme levels in the walls and a reduction of softening.Rab GTPases have long been considered key players in

the regulation of vesicle trafficking in animal systems andtheir roles are now being evaluated in plants. Based onanimal models, the Rab11/RabA class of GTPase wasthought to be involved in recycling to endosomes (Zerialand McBride, 2001). However, the Rab11 clade inArabidopsis has 26 members compared with the two inmammals, and this has led to speculation that they havediversified structurally and functionally (Rutherford andMoore, 2002). Based upon the effect of inhibiting these,several of them now seem to be implicated in traffickingto the cell wall. First of all, a tomato RabA1 homologue isrequired for trafficking to the wall in whole plants (Luet al., 2001) and has recently been shown to be involvedin Golgi–PM transport in transient expression studies(Rehman et al., 2008). Also, recently, a study of twoother RabA clades, RabA2 and RabA3, in Arabidopsisroot tips has shown that they too are involved intrafficking to the PM but in this case also to thephragmoplast (Chow et al., 2008). A tobacco RabAhomologue has also been shown to be required fordelivery of vesicles to the PM in pollen tubes (de Graafet al., 2005). Therefore, several, but not all, members ofthe Rab11/RabA clade are now firmly established ashaving a major role in secretion to the cell wall in plantsand probably play a role in regulating growth andmodification of the wall and production of new wallduring cytokinesis.Although there are systems where interference with

vesicle trafficking has given rise to altered growthcharacterisitics (McElver et al., 2000), in several systemsdescribed here, where natural mutants or mutants gener-ated by gene silencing with a constitutive promoter haveinterfered with production or delivery of PE (Jiang et al.,2005) or a range of cell wall-modifying enzymes (Lu

et al., 2001) and have produced specific phenotypes inspecific organs, the overall size and rate of growth of theplants have been normal. Many of these genes aredifferentially regulated and belong to gene families so thismight potentially explain the phenomenon. However,several Rab GTPases have been shown to be expressed ina range of actively growing or highly secretory tissues (Luet al., 2001; Cheung et al., 2002) and inhibition ofLeRab11a did not inhibit overall growth (Lu et al., 2001).It is also possible that different proteins and polysacchar-ides destined for the cell wall are delivered by differentvesicles, and support for this has recently emerged (Leucciet al., 2007; Rehman et al., 2008).Trafficking is important for the location of hormone

receptors and other elements of the signalling machineryand for localization of ion channels. Interference withthese processes would be expected to have effects uponcell wall metabolism, and this is probably important in tipgrowing systems (see Cole and Fowler, 2006; Samajet al., 2006; Campanoni and Blatt, 2007) and has alsobeen observed in plants with a silenced tomato fruit RabGTPase (Lu et al., 2001). Therefore, future studies mustaim to distinguish between direct control of trafficking tothe cell wall and more global effects due to altered signaltransduction. However, despite this reservation, it seemsclear that trafficking pathways, and Rab GTPases inparticular, must play a role in controlling cell wallsynthesis and modification.

Acknowledgements

The author would like to thank Professor Greg Tucker and Dr TimRobbins for making helpful suggestions during the production ofthe manuscript.

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the role of rab gtpases in cell wall metabolism

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