12

Plant cell expansion: scaling the wall Fr6deric Nicol and Herman H6fte*

The regulation of plant cell size and shape is poorly understood at the molecular level. Recently, two loci required for normal cell expansion in Arabidopsis were cloned. They both encode enzymes involved in the construction of the cell wall. These studies are the first promising examples of the use of Arabidopsis molecular genetics for the study of wall synthesis and assembly during plant cell elongation.

Addresses INRA, Laboratoire de Biologie Ceilulaire, Route de St-Cyr, 78026 Versailles Cedex, France *e-maih hofte@versailles.inra.fr

Current Opinion in Plant Biology 1998, 1:12-17

http://biomednet.com/elecref/1369526600100012

© Current Biology Ltd ISSN 1369-5266

Abbreviations EGase endo-1,4-~-d-glucanase EST expressed sequence tag DCB dichlorobenzonitrile TC terminal complex

Introduction Cell expansion plays a crucial role in plant development. The shape of plant organs and the function of the tissues are to a large extent dependent on the regulation of the size and the shape of the constituent cells. Cell size itself may also act as a signal to trigger cell division [1,2] or even differentiation [3].

Our current understanding of the melecular basis of cell enlargement has been extensively covered in recent reviews [4-6] and some key features are summariscd in Figure 1. Briefly, the rigidity of a plant cell is the result of the osmotically induced turgor pressure which can be built up by virtue of the cell wall strength. Since, in a growing cell, the turgor pressure generally remains constant, cell enlargement must be the result of the controlled expansion of the primary wall. The growing plant cell wall is a polymeric structure, consisting of crystalline cellulose microfibrils coated with hemicellulose chains which are embedded in a highly hydrated pectin matrix with small amounts of protein. The direction of cell expansion is thought to be controlled by the orientation of the cellulose microfibrils. In growing cells these microfibrils are laid down transversely to the axis of elongation, thus forming a spring-like structure reinforcing the cell laterally and favouring longitudinal expansion. Control of the orientation of cellulose has been attributed to the cortical microtubules, which are normally aligned in the same orientation as the cellulose microfibrils and may guide the cellulose synthase complexes [7]. Consistent with this model, anisotropic growth is abolished upon chemical

inhibition of cellulose synthesis or microtubule polymeri- sation [8]. Hemicelluloses and pectins are synthesised in the Golgi apparatus and transported in vesicles to the cell surface. Upon secretion into the apoplast, hemicelluloses bind tightly to cellulose and are thought to link the microfibrils in a stress-resistant network. Cell expansion requires the constant rearrangement of the bonds within this network, to allow the movement of wall polymers and the incorporation of new wall material into the existing architecture. According to the acid growth theory, the cell wall pH plays a coordinating role in this process [9]. Activation of the H+ ATPase under influence of auxin, and maybe other growth factors, lowers the wall pH. This, in turn, stimulates the highly pH-dependent activity of three classes of wall proteins potentially involved in rearranging wall polymers: expansins, xyloglucan endotransglycosy- lases and endo-l,4-~-D-glucanases (Figure 1). Increasing the pH would arrest cell expansion by inhibiting the activities of these proteins and by inhibiting the activation of enzymes thought to be involved in wall cross-linking. The role of polysaccharide biosynthesis and vesicular transport in the control of cell expansion is not understood [10]. Also, our knowledge of how growth factors, light, mechanical stimuli, nutrition and cell-cell interactions influence the extent and direction of cell enlargement remains very rudimentary.

The study of Arabidopsis molecular genetics now sheds new light on this research area: we will devote the majority of this review to new insights obtained from two cell wall mutants that encode a cellulose synthase subunit and an unusual endo 1,4-[3-glucanase. We will then go on to discuss other cell expansion mutants with potential wall defects and the importance of cellulose synthase-like genes.

A cel lulose synthase mutant Cellulose is composed of unbranched polymers of 1,4- 13-D-linked glucose residues that form crystalline arrays of many parallel chains which make up microfibrils [11]. All available evidence suggests that cellulose is synthesised at the plasma membrane, presumably by a multisubunit protein complex, the so-called terminal complex (TC), which can be visualised as hexameric rosettes on freeze-fractured membranes [12]. Despite much effort, plant cellulose synthase has so far resisted purification, in part due to its low in vitro activity [13]. A major step forward was the identification of a family of candidate genes for the catalytic subunit of cellulose synthase through expressed sequence tag (EST) sequencing [14]. (For recent reviews on cellulose synthesis, see [15,16]). During the past year a further breakthrough was reported with the positional cloning of

Plant cell expansion Nicol and H6fte 13

Figure 1

Z~ Elongation

Plasma membrane

• Membrane-bound endo-1,4-~-D-glucanase

l l ~ Soluble endo-1,4-~-glucanase

Xyloglucan endotransglycosylase

Expansin

Cellulose synthase complex

@ Lateral view ~ Top view

I ~ H+/ATPase

Secretory vesicle

Cellulose microfibril

Xyloglucans

Current Opinion in Plant Biology

Schematic representation of some of the cellular processes thought to be involved in cell expansion in an elongating dicotyledonous cell, adapte d from Cosgrove [10]. Cellulose microfibrils are thought to be synthesised by plasma membrane-bound hexameric cellulose synthase complexes. All other cell wall polysaccharides are synthesised in the Golgi apparatus and transported in vesicles to the cell surface. Xyloglucans are tightly bound to microfibrils by hydrogen bonds and form cross-links between them, thus constituting a load-bearing network. Only a small number of xyloglucan chains are represented for simplicity. In elongating cells, the microfibrils are deposited perpendicular to the axis of elongation, forming a spring-like structure that reinforces lateral walls and favours longitudinal growth. Expansins are thought to act at the xyloglucan-cellulose interface and promote movement of the polymers; endo-l,4-~-D-glucanases cleave xyloglucans, whereas xyloglucan endotransglycosylases cleave xyloglucan and join the newly generated reducing end to other xyloglucan chains represented by an arrow in the figure. The increase in wall surface then leads to the uptake of water and solutes, which results in an increase in volume. Cell growth arrests as soon as the wall pH increases, for instance through inhibition of the proton ATPase, which would inhibit cell wall loosening proteins, and activate a set of enzymes potentially involved in cross-linking the wall, for example pectin-methyl esterases (PMEs) or peroxidases.

a member of this gene family from a cellulose-deficient mutant in Arabidopsis, as discussed below.

Williamson and colleagues [17] carried out a screen for temperature-sensitive mutants that showed exaggerated radial expansion of root cells at the restrictive tem- perature (31°C), reminiscent of the phenotype obtained after treatment with the cellulose synthesis inhibitor dichlorobenzonitrile (DCB). Mutants identifying four Root

Swelling (RSW) loci indeed show a significant reduction of the amount of crystalline cellulose found in cell walls when grown at 31°C. Recently, the positional cloning of one of the loci, RSW1, was carried out (unpublished data). RSW1 encodes a 122 kDa protein, with a low but significant sequence similarity (about 30% amino acid identity) to the catalytic subunit of bacterial cellulose synthases [18-20]. RSW1 is also highly similar to the products of two cDNAs (CelA1 and CelA2) that had been previously identified

14 Growth and development

through random EST sequencing of a cDNA library from maturing cotton fibres [14]. In addition to the sequence similarity, two other observations suggest that CELA1 indeed encodes a catalytic subunit of cellulose synthase. First, the mRNA levels of CELA1 were strongly induced in fibre cells at the onset of massive cellulose synthesis during secondary wall formation, and second, a fragment of CELA1 expressed in Escherichia coli specifically bound UDP-glucose, the substrate for cellulose synthase, in a magnesium-dependent way.

What can we learn from the rswl mutant phenotype? Mutant plants grown at 31°C show increased radial expan- sion in all cell types investigated, including tip-growing cells such as trichomes and root hairs ([17] and personal communication). RSW1, therefore, seems to be required for primary wall synthesis in all cells. It is not clear to what extent secondary wall formation is affected as well. Shifting the temperature from 18°C to 31°C causes the disappearance of the hexameric rosettes in the plasma membrane as shown by freeze etching. At the same time, over 50% reduction in the production of crystalline cellulose can be observed. Unexpectedly, a 1,4-13-1inked glucan that behaves in all aspects as a noncrystalline polymer accumulates. Indeed, this glucan, in contrast to crystalline cellulose, co-extracts with the wall pectins in ammonium oxalate, and is susceptible, to hydrolysis by trifluoroacetic acid, or an endo-cellutase/1,4-13-glucosidasc mixture. The simplest interpretation of these results is that the mutation prevents the assembly of glucan chains into microfibrils but does not inhibit g!ucan synthesis per se. At the restrictive temperature the mutant synthase complexes would disassemble to monomers. The monomers of the catalytic subunit would still be catalytically active, but the synthesis of 1,4-13-glucan chains at dispersed sites would prevent microfibril assembly.

In summary, the mutant phenotype of Arabidopsis rswl together with the observations on cotton Ce/A1 now strongly support the idea that CELA1 and the closely related protein RSWl are both bonafide catalytic subunits of cellulose synthase.

What can we learn from the RSW1 sequence? The hypothetical structure of RSW1 is shown in Figure 2. The protein has two amino-terminal and six carboxy-terminal predicted membrane spanning domains, with a large cen- tral domain which is presumably cytosolic. Experiments with epitope-tagged cotton CELA1 expressed in yeast provided evidence for the cytosolic orientation of the amino terminus and the extracellular orientation of the last loop (D Delmer, personal communication). Within the central domain one can distinguish four regions conserved in all processive glycosyl transferases in plants and bacteria; these regions contain three conserved aspartic acid residues and a QXXRW motif (single-letter code for amino acids) which are thought to be critical for catalysis and UDP-glucose binding [11]. The cysteine-rich

amino terminus has two predicted zinc finger domains forming a LIM-like motif: a structural zinc-binding motif potentially involved in protein-protein interactions, found in animals and plants [21]. The amino terminal domain of CELA1 produced in E. coli as a glutathione-S-transferase fusion protein effectively binds zinc with a protein:Zn stoichiometry of 1:2 (D Delmer, personal communication). This domain shows sequence similarity to two bZIP transcription factors from soybean (unpublished data) and is most likely involved in protein-protein interactions. Having identified RSW1 and related genes, the rapid isolation of the other subunits of the cellulose synthase complex using yeast two-hybrid or biochemical techniques can be expected. Also the regulation of cellulose synthesis, crystallisation and orientation during cell enlargement and secondary wall formation can now be addressed more easily.

An unusual endo- l , 4 -~ -D-g lucanase mutant The recessive mutation korrigan (kor) was identified in a screen for short hypocotyl mutants of Arabidopsis [22]. As with rswl, kor cells show reduced elongation and increased radial cell expansion. The mutation affects all cell types examined, except for the tip-growing root hairs, trichomes and pollen tubes. Judged from analysis of sections of fixed material; mutant primary cell walls are thicker and have a highly undulated surface. In contrast to wild-type walls, no layered structure of cellulose microfibrils can be distinguished in kor walls, and aggregates of polysaccharides containing cellulosic material accumulate at the cytoplasmic side of the cell wall ([22] and our unpublished data).

The T-DNA-tagged Kor gene was cloned and encodes a member of the endo-l,4-13-D-glucanase (EGase) family ([22] and our unpublished data). EGases form an ancient class of enzymes present in plants, bacteria, fungi and animals that hydrolyse 1,4-13 linkages adjacent to nonsubstituted glucose residues. In contrast to bacterial and fungal EGases which can degrade crystalline cellulose, plant EGases lack a cellulose binding domain and only degrade noncrystalline 1,4-13-glucans in vitro. The exact in vivo substrate is not known, but it is thought to be xyloglucan [23]. Plant EGases are encoded by large gene families: Arabidopsis expresses at least ten different EGase genes ([22] and our unpublished data). The expression of plant EGase genes is tightly regulated and the expression patterns suggest a role for these enzymes in plant development. The expression of one group of genes is ethylene-inducible and correlated with massive wall degradation during fruit ripening and leaf abscission [23]. The expression of a second class of EGase genes appears to be correlated with the more subtle hydrolytic processes thought to take place during the rearrangement of polysaccharides in growing cells [23-25]. The hypothetical mode of action of EGases in cell expansion could be either to promote wall loosening, thereby acting cooperatively with expansins [5], or to

Figure 2

Cell wall

Cytosol

0

!l F

Plant cell expansion Nicol and H~Sfte 15

,~ Zinc finger domain DDD, QVLRW Conserved motif and residues of processive I~-glycosyl transferases @ Plant conserved region (PCR) • Highly variable region (HVR)

Current Opinion in Plant Biology

Predicted membrane topology and other structural features of RSWl. The protein has two amino-terminal and six carboxy-terminal predicted membrane-spanning domair;s. In the centre, a large, most likely cytosolic, domain can be distinguished containing four regions conserved for all processive glycosyl transferases in plants and bacteria, and which are predicted to be critical for UDP-glucose binding and catalysis in bacterial CelA. The three critical aspartic acid (D) residues and the O.XXRW motif in these regions are indicated. Also in the central domain, two plant-specific regions of unknown function can be distinguished, a plant conserved region (PCR) and a highly variable region (HVR). The cytosolic orientation of the amino terminus and the extracellular orientation of the last loop have been experimentally confirmed for the cotton CELA1 (D Delmer, personal communication). The amino terminus contains two zinc-finger domains, which are most likely involved in protein-protein interactions,

contribute to the incorporation of new microfibrils in the growing wall. The mRNA induction kinetics of an auxin-inducible isoform, CEL7, expressed in the tomato hypocotyl, indicates that the increased expression is not required for the rapid growth response to auxin, and suggests rather that the enzyme has a role in sustained growth [26].

In contrast to other plant EGases, the KOR sequence predicts an integral membrane protein with a short amino terminus in the cytosol and an external catalytic domain (type II topology). Free flow electrophoresis analysis lo- calised the protein mainly in plasma-membrane-enriched fractions, with a minor portion in internal membranes that were enriched for a tonoplast marker. CEL3, a

tomato EGase highly similar to KOR, was also localised in plasma-membrane-enriched membrane fractions and in a membrane fraction comigrating with Golgi markers in a linear sucrose gradient [27°].

KOR mRNAs are present in all plant tissues, and in germinating seedlings appear concomitantly with the onset of hypocotyl elongation. For the tomato homologue, CEL3, mRNA levels are not influenced by external addition of auxin, ethylene or brassinosteroids [27°]. We can only speculate on the significance of the membrane location. A membrane-bound enzyme may be more easily targeted either to the inside of the wall or to specific wall domains (for instance, to the longitudinal walls of elongating cells). Alternatively, a membrane location may

16 Growth and development

allow the abundance of the enzyme in the cell wall to be precisely controlled, for instance through endocytosis. Also, a membrane anchor may allow the targeting of a fraction of the protein to the Golgi apparatus where the enzyme might have a role in the priming of the synthesis of xyloglucans, the polysaccharides that bind to cellulose and form the major load bearing network of the primary wall [27°]. Finally, a membrane-associated EGase may in fact be a part of the cellulose synthase complex and play a role in the synthesis of cellulose. Indeed, the only other known example of a membrane-bound EGase was reported for Agrobacterium tumefaciens, where the gene (CelC) is part of the cellulose synthase operon [20,28]. In Agrobacterium CelC is essential for cellulose synthesis as shown by transposon mutagenesis, and the authors provide evidence for a role of this protein in the polymerisation of lipid-linked oligosaccharides to cellulose. The existence of lipid-linked intermediates for cellulose synthesis in bacteria and certainly for plants remains controversial but so far has not been ruled out [15]. The unordered accumulation of cellulosic material in kor mutant walls is not incompatible with a role for this enzyme in an as yet unidentified step in the biosynthesis or crystallisation of cellulose.

In summary the results show a crucial role for a membrane- bound EGase in cell elongation and normal wall assembly, and raise new interesting questions as to its function that can now be experimentally addressed.

More cell expansion mutants A number of increased radial cell expansion mutants presenting phenotypes very similar to that of rswl and kor have been described. Recessive mutants identifying three other root-swelling loci (RSW2, RSW3, RSW5) show, like rswl, a temperature-sensitive reduction in cellulose accumulation (R Williamson, personal communication). Other mutants of this class were described as conditional root expansion (CORE) mutants [29]. These mutants identify six loci (POMPOM1 [POM1], POMPOM2 [POM2], COBRA [COB], LION'S TAlL [LIT], QUILL [QUI] and CUDGE [CUD]); lit and qui mutants are respectively allelic to rsw2 (R Williamson, personal communication) and procustel (prcl) [30]; H H6fte and P Benfey, unpublished data). The cloning of at least some of these loci is expected ultimately to identify other structural proteins of the cellulose synthase complex or regulators of cellulose synthesis or deposition. The recessive mutant prc (qui) shows, in addition to its root phenotype, an increased and uncoordinated radial expansion in hypocotyls, but only in dark-grown seedlings. Light, through the action of phytochrome, completely rescues the hypocotyl-growth defect [30]. PRC1 may therefore represent a link between phytochrome control of cell elongation and cell wall assembly. Several other mutants show defects in cellulose synthesis, but specifically during secondary wall formation [31,32], indicating that this process requires specialised

sets of genes not required in growing cells. Other mutants with altered wall polysaccharide composition were described by Reiter and colleagues [33-35] and reviewed elsewhere [36].

Gene-families encoding cellulose synthases or related enzymes Inspection of sequence databases shows that RSW1, CelA1 and CelA2 are members of a large gene family present in dicots and monocots. So far, at least seventeen related genes have been identified among ESTs and genomic sequences from Arabidopsis [37] (S Cutler, personal communication). In view of this high apparent redundancy, it is surprising that a mutation in the single gene RSW1 causes such a strong phenotype in all cell types.

In addition, a class of more distantly related genes, the cellulose-synthase-like, or Csl, genes, represented by at least nine genes in Arabidopsis, can be distinguished (S Cutler, personal communication). The Csl products contain seven or eight predicted membrane-spanning domains, and also contain the three conserved aspartic acid residues and the QXXRW motif, characteristic of processive glycosyl transferases [11]. The overall sequence similarity with CELA is low, for instance, CSL1 shows about 25% amino acid identity with CELA. CelA products are in fact more related to their bacterial homologues than to Csl, suggesting that their divergence preceded the appearance of eukaryotes in evolution. It is conceivable that CSLs are processive glycosyl transferases involved in the biosynthesis of other 1,4-~- or 1,3-[~-linked polysac- charides in plants [37] and may represent a subset of the estimated several hundred enzymes that are required to produce the complete set of wall polysaccharides [38]. The use of reverse genetics is expected to clarify the role of these different family members in wall biosynthesis.

Conclusions With the help of Arabidopsis molecular genetics, a corner of the veil covering the cellular processes involved in polysac- charide biosynthesis and assembly in the growing primary wall has been lifted. The temperature-sensitive rswl mutants identify beyond reasonable doubt the catalytic subunit of the cellulose synthase complex. The cloning of the gene encoding a plasma membrane-associated EGase essential for cell elongation and normal wall assembly has added an intriguing new element to the cell elongation machinery, with a role for the EGase either in the rearrangement of wall polymers or in the synthesis or crystallisation of cellulose. These findings will greatly accelerate the identification of other proteins of the cellulose synthase complex and the molecular dissection of cellulose synthesis. The functional study of these proteins and other CELA family members, together with the cloning of the genes identified by other radial cell expansion mutants, may revolutionise our understanding of cell-wall-associated processes involved in the expansion of plant cells.

Plant cell expansion Nicol and H6fte 1 ?

Acknowledgements The authors wish to thank Richard Williamson and Tony Arioli, Debby Delmer, Sean Cutler, Marie-Therbs Hauser and Philip Benfey for allowing us to cite their unpublished results. We apologise to those colleagues working in the field whose work could not be mentioned due to space limitations, and thank Heather McKann, Ageeth Van Tuinen and Rachel Cowling for critical reading of the manuscript. Our research was in part financed by a grant, ACC-SV number 9501006 from the Minist6re de la Recherche et de la Technologie to Herman Hbfte.

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