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Plants depend on various cell surface receptors to integrate extracellular signals with developmental programs. One of the best-studied receptors is BRASSINOSTEROID INSENSITIVE 1 (BRI1) in Arabidopsis (Arabidopsis thaliana). Upon binding of its hormone ligands, BRI1 forms a complex with a shape-complementary coreceptor and initiates a signal transduction cascade, which leads to a variety of responses. At the macroscopic level, brassinosteroid (BR) biosynthetic and receptor mutants have similar growth defects, which initially led to the assumption that the signaling pathways were largely linear. However, recent evidence suggests that BR signaling is interconnected with several other pathways through various mechanisms. We recently described that feedback from the cell wall is integrated at the level of the receptor complex through interaction with RECEPTOR-LIKE PROTEIN 44 (RLP44). Moreover, BRI1 is required for another function of RLP44: the control of procambial cell fate. Here, we report a BRI1 mutant, bri1(cnu4), which differentially affects canonical BR signaling and RLP44 function in the vasculature. Although BR signaling is only mildly impaired, bri1(cnu4) mutants show ectopic xylem in place of procambium. Mechanistically, this is explained by an increased association between RLP44 and the mutated BRI1 protein, which prevents the former from acting in vascular cell fate maintenance. Consistent with this, the mild BR response phenotype of bri1(cnu4) is a recessive trait, whereas the RLP44-mediated xylem phenotype is semidominant. Our results highlight the complexity of plant plasma membrane receptor function and provide a tool to dissect BR signaling-related roles of BRI1 from its noncanonical functions.

The plant steroid hormones brassinosteroids (BRs) regulate growth in part through altering the properties of the cell wall, the extracellular matrix of plant cells. Conversely, cell wall signalling connects the state of cell wall homeostasis to the BR receptor complex and modulates BR activity. Here we report that both pectin-triggered cell wall signalling and impaired BR signalling result in altered cell wall orientation in the Arabidopsis root meristem. BR-induced defects in the orientation of newly placed walls are associated with aberrant localization of the cortical division zone but with normal specification of its positioning. Tissue- specific perturbations of BR signalling revealed that the cellular malfunction is unrelated to previously described whole organ growth defects. Thus, tissue type separates the pleiotropic effects of cell wall/BR signals and highlights their importance during cell wall placement.

The cells of all organisms are sugarcoated with polysaccharides, glycoproteins, and glycolipids. In multicellular organisms these polymers not only have a structural role in the organization of tissues and organs, but also have signaling activities. In the animal extracellular matrix for instance, the negatively charged hyaluronic acid (HA) polymers contribute to morphogenesis through mechanosensing, whereas HA fragments that are released from the polymer under certain conditions trigger inflammatory responses and angiogenesis (1). In plants, pectins are the major charged polysaccharides in the cell wall and thus can be considered the equivalent of HA. In addition, pectin-derived oligosaccharides released from the host cell wall during pathogen infection have been shown to trigger immune responses (2⇓–4). Gallego-Giraldo et al. (5) in PNAS now provide evidence for a role of pectin oligosaccharide signaling also in the response of plant cells to cell wall perturbation. The study is part of a larger effort of Gallego-Giraldo et al. (5) to tailor plant biomass for improved digestibility (for animal feed) or conversion into fermentable sugars (saccharification, to produce second-generation biofuels and chemicals) (6). Plants, in contrast to animals, contain lignins in their extracellular matrix, besides polysaccharides and glycoproteins. Lignins consist of oxidatively cross-linked phenolic molecules, which form a hydrophobic network attached to the polysaccharides. Previous studies by Gallego-Giraldo et al. and others have shown that lignins are a major obstacle for biomass digestibility/saccharification, mainly because they limit the access of cell … [↵][1]1To whom correspondence may be addressed. Email: hermanus.hofte{at}inra.fr. [1]: #xref-corresp-1-1

Growth of etiolated Arabidopsis hypocotyls is biphasic. During the first phase, cells elongate slowly and synchronously. At 48 h after imbibition, cells at the hypocotyl base accelerate their growth. Subsequently, this rapid elongation propagates through the hypocotyl from base to top. It is largely unclear what regulates the switch from slow to fast elongation. Reverse genetics-based screening for hypocotyl phenotypes identified three independent mutant lines of At1g70990, a short extensin (EXT) family protein that we named EXT33, with shorter etiolated hypocotyls during the slow elongation phase. However, at 72 h after imbibition, these dark-grown mutant hypocotyls start to elongate faster than the wild type (WT). As a result, fully mature 8-day-old dark-grown hypocotyls were significantly longer than WTs. Mutant roots showed no growth phenotype. In line with these results, analysis of native promoter-driven transcriptional fusion lines revealed that, in dark-grown hypocotyls, expression occurred in the epidermis and cortex and that it was strongest in the growing part. Confocal and spinning disk microscopy on C-terminal protein-GFP fusion lines localized the EXT33-protein to the ER and cell wall. Fourier-transform infrared microspectroscopy identified subtle changes in cell wall composition between WT and the mutant, reflecting altered cell wall biomechanics measured by constant load extensometry. Our results indicate that the EXT33 short EXT family protein is required during the first phase of dark-grown hypocotyl elongation and that it regulates the moment and extent of the growth acceleration by modulating cell wall extensibility.

International audience; Despite an ever-increasing interest for the use of pectin-derived oligogalacturonides (OGs) as biological control agents in agriculture, very little information exists-mainly for technical reasons-on the nature and activity of the OGs that accumulate during pathogen infection. Here we developed a sensitive OG profiling method, which revealed unsuspected features of the OGs generated during infection of Arabidopsis thaliana with the fungus Botrytis cinerea. Indeed, in contrast to previous reports, most OGs were acetyl-and methylesterified, and 80% of them were produced by fungal pectin lyases, not by polygalacturonases. Polygalacturonase products did not accumulate as larger size OGs but were converted into oxidized GalA dimers. Finally, the comparison of the OGs and transcriptomes of leaves infected with B. cinerea mutants with reduced pectinolytic activity but with decreased or increased virulence, respectively, identified candidate OG elicitors. In conclusion, OG analysis provides insights into the enzymatic arms race between plant and pathogen and facilitates the identification of defense elicitors. oligogalacturonides | plant-pathogen interaction | Arabidopsis thaliana | Botrytis cinerea | pectin lyase T he cell wall forms the first line of defense in the interaction of plants with their microbial environment. Cell walls consist of complex polysaccharide networks, which combine multiple functional properties such as strength (to resist turgor pressure), ex-tensibility (to allow growth), as well as protection against microbial attack. Such protection involves so called "basal immunity," which is triggered upon recognition of pathogen-associated molecular patterns but also of plant-derived molecules associated with infection, referred to as damage-associated molecular patterns (DAMPs). The latter include cell wall-derived oligosaccharides with elicitor activity (also called oligosaccharins) (1) that are produced during infection with microbes and, in particular, necrotrophic bacteria and fungi, which feed on cell walls and have large arsenals of cell wall-degrading enzymes (2). A major source of cell wall-derived DAMPs are pectins. The pectic polymer, homogalacturonan (HG) for example, represents in Arabidopsis 20% of the wall of growing cells. HG is a linear polymer of α-1-4-linked galacturonic acid (GalA) residues secreted in a methylesterified (on C6) and often acetylesterified (on C2 and/or C3) form (3), which can be enzymatically deacetylesterified by pectin acetylesterases and demethylesterified by pectin methyl-esterases (PMEs) in muro (4). Demethylesterification exposes the polymer to degradation by polygalacturonases (PGs) and pectate lyases (PLs, that cleave unmethylesterified HG by β-elimination, thus generating an unsaturated bond at the nonreducing end), which can be of plant or fungal origin. HG demethylesterification plays a key role in the control of cell wall rheology underlying plant growth (5). In addition, HG turnover generates oligogalacturonides (OGs)

A major challenge in plant systems biology is the development of robust, predictive multiscale models for organ growth. In this context it is important to bridge the gap between the, rather well-documented molecular scale and the organ scale by providing quantitative methods to study within-organ growth patterns. Here, we describe a simple method for the analysis of the evolution of growth patterns within rod-shaped organs that does not require adding markers at the organ surface. The method allows for the simultaneous analysis of root and hypocotyl growth, provides spatio-temporal information on curvature, growth anisotropy and relative elemental growth rate and can cope with complex organ movements. We demonstrate the performance of the method by documenting previously unsuspected complex growth patterns within the growing hypocotyl of the model species Arabidopsis thaliana during normal growth, after treatment with a growth-inhibiting drug or in a mechano-sensing mutant. The method is freely available as an intuitive and user-friendly Matlab application called KymoRod.

International audience; Somatic polyploidy caused by endoreplication is observed in arthropods, molluscs, and vertebrates but is especially prominent in higher plants, where it has been postulated to be essential for cell growth and fate maintenance. However, a comprehensive understanding of the physiological significance of plant endopolyploidy has remained elusive. Here, we modeled and experimentally verified a high-resolution DNA endoploidy map of the developing Arabidopsis thaliana root, revealing a remarkable spatiotemporal control of DNA endoploidy levels across tissues. Fitting of a simplified model to publicly available data sets profiling root gene expression under various environmental stress conditions suggested that this root endoploidy patterning may be stress-responsive. Furthermore, cellular and transcriptomic analyses revealed that inhibition of endoreplication onset alters the nuclear-to-cellular volume ratio and the expression of cell wall-modifying genes, in correlation with the appearance of cell structural changes. Our data indicate that endopolyploidy might serve to coordinate cell expansion with structural stability and that spatiotemporal endoreplication pattern changes may buffer for stress conditions, which may explain the widespread occurrence of the endocycle in plant species growing in extreme or variable environments.

While Brachypodium distachyon (Brachypodium) is an emerging model for grasses, no expression atlas or gene coexpression network is available. Such tools are of high importance to provide insights into the function of Brachypodium genes. We present a detailed Brachypodium expression atlas, capturing gene expression in its major organs at different developmental stages. The data were integrated into a large-scale coexpression database ( www.gene2function.de), enabling identification of duplicated pathways and conserved processes across 10 plant species, thus allowing genome-wide inference of gene function. We highlight the importance of the atlas and the platform through the identification of duplicated cell wall modules, and show that a lignin biosynthesis module is conserved across angiosperms. We identified and functionally characterised a putative ferulate 5-hydroxylase gene through overexpression of it in Brachypodium, which resulted in an increase in lignin syringyl units and reduced lignin content of mature stems, and led to improved saccharification of the stem biomass. Our Brachypodium expression atlas thus provides a powerful resource to reveal functionally related genes, which may advance our understanding of important biological processes in grasses.

Antagonistic growth effects of two T-DNA alleles in THESEUS1 correlate with the expression of a predicted functional THE1 protein lacking the kinase domain and antisense transcripts, respectively., Perturbation of cellulose synthesis in plants triggers stress responses, including growth retardation, mediated by the cell wall integrity-sensing receptor-like kinase (RLK) THESEUS1 (THE1). The analysis of two alleles carrying T-DNA insertions at comparable positions has led to conflicting conclusions concerning the impact of THE1 signaling on growth. Here we confirm that, unlike the1-3 and other the1 alleles in which cellular responses to genetic or pharmacological inhibition of cellulose synthesis are attenuated, the1-4 showed enhanced responses, including growth inhibition, ectopic lignification, and stress gene expression. Both the1-3 and the1-4 express a transcript encoding a predicted membrane-associated truncated protein lacking the kinase domain. However, the1-3, in contrast to the1-4, strongly expresses antisense transcripts, which are expected to prevent the expression of the truncated protein as suggested by the genetic interactions between the two alleles. Seedlings overexpressing such a truncated protein react to isoxaben treatment similarly to the1-4 and the full-length THE overexpressor. We conclude that the1-4 is a hypermorphic allele; that THE1 signaling upon cell wall damage has a negative impact on cell expansion; and that caution is required when interpreting the phenotypic effects of T-DNA insertions in RLK genes.

Plants actively perceive and respond to perturbations in their cell walls which arise during growth, biotic and abiotic stresses. However, few components involved in plant cell wall integrity sensing have been described to date. Using a reverse-genetic approach, we identified the Arabidopsis thaliana leucine-rich repeat receptor kinase MIK2 as an important regulator of cell wall damage responses triggered upon cellulose biosynthesis inhibition. Indeed, loss-of-function mik2 alleles are strongly affected in immune marker gene expression, jasmonic acid production and lignin deposition. MIK2 has both overlapping and distinct functions with THE1, a malectin-like receptor kinase previously proposed as cell wall integrity sensor. In addition, mik2 mutant plants exhibit enhanced leftward root skewing when grown on vertical plates. Notably, natural variation in MIK2 (also named LRR-KISS) has been correlated recently to mild salt stress tolerance, which we could confirm using our insertional alleles. Strikingly, both the increased root skewing and salt stress sensitivity phenotypes observed in the mik2 mutant are dependent on THE1. Finally, we found that MIK2 is required for resistance to the fungal root pathogen Fusarium oxysporum. Together, our data identify MIK2 as a novel component in cell wall integrity sensing and suggest that MIK2 is a nexus linking cell wall integrity sensing to growth and environmental cues., Author summary Plants are constantly exposed to external stresses of biotic and abiotic nature, as well as internal stresses, resulting from growth and mechanical tension. Feedback information about the integrity of the cell wall can enable the plant to perceive such stresses, and respond adequately. Plants are known to perceive signals from their environment through receptor kinases at the plant cell surface. Here, we reveal that the Arabidopsis thaliana receptor kinase MIK2 regulates responses to cell wall perturbation. Moreover, we find that MIK2 controls root growth angle, modulates cell wall structure in the root tip, contributes to salt stress tolerance, and is required for resistance against a root-infecting pathogen. Our data suggest that MIK2 is involved in sensing cell wall perturbations in plants, whereby it allows the plant to cope with a diverse range of environmental stresses. These data provide an important step forward in our understanding of the mechanisms plants deploy to sense internal and external danger.

SummaryBrassinosteroid (BR) signaling is required for normal plant growth as shown by the dwarf phenotype of loss-of-function BR biosynthetic or perception mutants. Despite a detailed understanding of the BR signaling network [1–3], it is not clear how exactly BRs control growth. For instance, genetic sector analysis shows that BRs, in contrast to most other growth regulators, act locally, presumably in an autocrine and/or paracrine mode, suggesting that they have some role in feedback regulation [4, 5]. Here, we show that at least one role for BRs in growth control is to ensure pectin-dependent cell wall homeostasis. Pectins are complex block cell wall polymers [6], which can be modified in the wall by the enzyme pectin methylesterase (PME) [7]. Genetic or pharmacological interference with PME activity causes dramatic changes in growth behavior, which are primarily the result of the activation of the BR signaling pathway. We propose that this activation of BR signaling is part of a compensatory response, which protects the plant against the loss of cell wall integrity caused by the imbalance in pectin modification. Thus, feedback signaling from the cell wall is integrated by the BR signaling module to ensure homeostasis of cell wall biosynthesis and remodeling.

SummaryTissue mechanics have been shown to play a key role in the regulation of morphogenesis in animals [1–4] and may have an equally important role in plants [5–9]. The aerial organs of plants are formed at the shoot apical meristem following a specific phyllotactic pattern [10]. The initiation of an organ from the meristem requires a highly localized irreversible surface deformation, which depends on the demethylesterification of cell wall pectins [11]. Here, we used atomic force microscopy (AFM) to investigate whether these chemical changes lead to changes in tissue mechanics. By mapping the viscoelasticity and elasticity in living meristems, we observed increases in tissue elasticity, correlated with pectin demethylesterification, in primordia and at the site of incipient organs. Measurements of tissue elasticity at various depths showed that, at the site of incipient primordia, the first increases occurred in subepidermal tissues. The results support the following causal sequence of events: (1) demethylesterification of pectin is triggered in subepidermal tissue layers, (2) this contributes to an increase in elasticity of these layers—the first observable mechanical event in organ initiation, and (3) the process propagates to the epidermis during the outgrowth of the organ.

International audience; The growth of plants, like that of other walled organisms, depends on the ability of the cell wall to yield without losing its integrity. In this context, plant cells can sense the perturbation of their walls and trigger adaptive modifications in cell wall polymer interactions. Catharanthus roseus receptor-like kinase 1-like (CrRLK1L) THESEUS1 (THE1) was previously shown in Arabidopsis to trigger growth inhibition and defense responses upon perturbation of the cell wall, but so far, neither the ligand nor the role of the receptor in normal development was known. Here, we report that THE1 is a receptor for the peptide rapid alkalinization factor (RALF) 34 and that this signaling module has a role in the fine-tuning of lateral root initiation. We also show that RALF34-THE1 signaling depends, at least for some responses, on FERONIA (FER), another RALF receptor involved in a variety of processes, including immune signaling, mechanosensing, and reproduction [1]. Together, the results show that RALF34 and THE1 are part of a signaling network that integrates information on the integrity of the cell wall with the coordination of normal morphogenesis.

Summary Background A major challenge is to understand how the walls of expanding plant cells are correctly assembled and remodeled, often in the presence of wall-degrading micro-organisms. Plant cells, like yeast, react to cell-wall perturbations as shown by changes in gene expression, accumulation of ectopic lignin, and growth arrest caused by the inhibition of cellulose synthesis. Results We have identified a plasma-membrane-bound receptor-like kinase (THESEUS1), which is present in elongating cells. Mutations in THE1 and overexpression of a functional THE1-GFP fusion protein did not affect wild-type (WT) plants but respectively attenuated and enhanced growth inhibition and ectopic lignification in seedlings mutated in cellulose synthase CESA6 without influencing the cellulose deficiency. A T-DNA insertion mutant for THE1 also attenuated the growth defect and ectopic-lignin production in other but not all cellulose-deficient mutants. The deregulation of a small number of genes in cesA6 mutants depended on the presence of THE1. Some of these genes are involved in pathogen defense, in wall crosslinking, or in protecting the cell against reactive oxygen species. Conclusions The results show that THE1 mediates the response of growing plant cells to the perturbation of cellulose synthesis and may act as a cell-wall-integrity sensor.

Pectins are a family of complex cell-wall polysaccharides, the biosynthesis of which remains poorly understood. We identified dwarf mutants with reduced cell adhesion at a novel locus, QUASIMODO2 (QUA2). qua2-1 showed a 50% reduction in homogalacturonan (HG) content compared with the wild type, without affecting other cell-wall polysaccharides. The remaining HG in qua2-1 showed an unaltered degree of methylesterification. Positional cloning and GFP fusions showed that QUA2, consistent with a role in HG synthesis, encodes a Golgi-localized protein. In contrast to QUA1, another Golgi-localized protein required for HG-synthesis, QUA2 does not show sequence similarity to glycosyltransferases, but instead contains a putative methyltransferase (MT) domain. The Arabidopsis genome encodes 29 QUA2-related proteins. Interestingly, the transcript profiles of QUA1 and QUA2 are correlated and other pairs of QUA1 and QUA2 homologues with correlated transcript profiles can be identified. Together, the results lead to the hypothesis that QUA2 is a pectin MT, and that polymerization and methylation of homogalacturonan are interdependent reactions.

Early ViewTechnical advance; The localization of proteins in specific domains or compartments in the 3D cellular space is essential for many fundamental processes in eukaryotic cells. Deciphering spatial organization principles within cells is a challenging task, in particular because of the large morphological variations between individual cells. We present here an approach for normalizing variations in cell morphology and for statistically analyzing spatial distributions of intracellular compartments from collections of 3D images. The method relies on the processing and analysis of 3D geometrical models that are generated from image stacks and that are used to build representations at progressively increasing levels of integration, ultimately revealing statistical significant traits of spatial distributions. To make this methodology widely available to end-users, we implemented our algorithmic pipeline into a user-friendly, multi-platform, and freely available software. To validate our approach, we generated 3D statistical maps of endomembrane compartments at subcellular resolution within an average epidermal root cell from collections of image stacks. This revealed unsuspected polar distribution patterns of organelles that were not detectable in individual images. By reversing the classical ‘measure-then-average’ paradigm, one major benefit of the proposed strategy is the production and display of statistical 3D representations of spatial organizations, thus fully preserving the spatial dimension of image data and at the same time allowing their integration over individual observations. The approach and software are generic and should be of general interest for experimental and modeling studies of spatial organizations at multiple scales (subcellular, cellular, tissular) in biological systems.

Cellulose-deficient Arabidopsis mutants were identified using FT-IR microspectroscopy. The study of these mutants not only led to the identification of actors in cellulose synthesis, but also provided insights in the organization of the hexameric terminal complex from CESA mutants and identified unsuspected accessory proteins with so far unknown roles in the synthesis and/or assembly of cellulose microfibrils. Finally, mutant analysis established a role for protein glycosylation in cellulose synthesis and provided new perspectives on the developmental regulation of cell wall synthesis and the role that cellulose synthesis plays in the control of cell elongation.

in press; Complex shapes in biology depend on the ability of cells to shift from isotropic to anisotropic growth during development. In plants, this growth symmetry breaking reflects changes in the extensibility of the cell walls. The textbook view is that the direction of turgor-driven cell expansion depends on the cortical microtubule (CMT)-mediated orientation of cellulose microfibrils [1, 2]. Here, we show that this view is incomplete at best. We used atomic force microscopy (AFM) to study changes in cell-wall mechanics associated with growth symmetry breaking within the hypocotyl epidermis. We show that, first, growth symmetry breaking is preceded by an asymmetric loosening of longitudinal, as compared to transverse, anticlinal walls, in the absence of a change in CMT orientation. Second, this wall loosening is triggered by the selective de-methylesterification of cell-wall pectin in longitudinal walls, and, third, the resultant mechanical asymmetry is required for the growth symmetry breaking. Indeed, preventing or promoting pectin de-methylesterification, respectively, increased or decreased the stiffness of all the cell walls, but in both cases reduced the growth anisotropy. Finally, we show that the subsequent CMT reorientation contributes to the consolidation of the growth axis but is not required for the growth symmetry breaking. We conclude that growth symmetry breaking is controlled at a cellular scale by bipolar pectin de-methylesterification, rather than by the cellulose-dependent mechanical anisotropy of the cell walls themselves. Such a cell asymmetry-driven mechanism is comparable to that underlying tip growth in plants [3] but also anisotropic cell growth in animal cells [4].

International audience; Pectins are a highly complex family of cell wall polysaccharides. As a result of a lack of specific mutants, it has been difficult to study the biosynthesis of pectins and their role in vivo. We have isolated two allelic mutants, named quasimodo1 (qua1-1 and qua1-2), that are dwarfed and show reduced cell adhesion. Mutant cell walls showed a 25% reduction in galacturonic acid levels compared with the wild type, indicating reduced pectin content, whereas neutral sugars remained unchanged. Immersion immunofluorescence with the JIM5 and JIM7 monoclonal antibodies that recognize homogalacturonan epitopes revealed less labeling of mutant roots compared with the wild type. Both mutants carry a T-DNA insertion in a gene (QUA1) that encodes a putative membrane-bound glycosyltransferase of family 8. We present evidence for the possible involvement of a glycosyltransferase of this family in the synthesis of pectic polysaccharides, suggesting that other members of this large multigene family in Arabidopsis also may be important for pectin biosynthesis. The mutant phenotype is consistent with a central role for pectins in cell adhesion.

The cell wall is the major limiting factor for plant growth. Wall extension is thought to result from the loosening of its structure. However, it is not known how this is coordinated with wall synthesis. We have identified two novel allelic cellulose-deficient dwarf mutants, kobito1-1 and kobito1-2 (kob1-1 and kob1-2). The cellulose deficiency was confirmed by the direct observation of microfibrils in most recent wall layers of elongating root cells. In contrast to the wild type, which showed transversely oriented parallel microfibrils, kob1 microfibrils were randomized and occluded by a layer of pectic material. No such changes were observed in another dwarf mutant, pom1, suggesting that the cellulose defect in kob1 is not an indirect result of the reduced cell elongation. Interestingly, in the meristematic zone of kob1 roots, microfibrils appeared unaltered compared with the wild type, suggesting a role for KOB1 preferentially in rapidly elongating cells. KOB1 was cloned and encodes a novel, highly conserved, plant-specific protein that is plasma membrane bound, as shown with a green fluorescent protein–KOB1 fusion protein. KOB1 mRNA was present in all organs investigated, and its overexpression did not cause visible phenotypic changes. KOB1 may be part of the cellulose synthesis machinery in elongating cells, or it may play a role in the coordination between cell elongation and cellulose synthesis.

An 8.5-kb cosmid containing the KORRIGAN gene complements the cellulose-deficient rsw2-1 mutant of Arabidopsis. Three temperature-sensitive alleles of rsw2show single amino acid mutations in the putative endo-1,4-β-glucanase encoded by KOR. The F1 from crosses betweenkor-1 and rsw2 alleles shows a weak, temperature-sensitive root phenotype. The shoots ofrsw2-1 seedlings produce less cellulose and accumulate a short chain, readily extractable glucan resembling that reported forrsw1 (which is defective in a putative glycosyltransferase required for cellulose synthesis). The double mutant (rsw2-1 rsw1) shows further reductions in cellulose production relative to both single mutants, constitutively slow root growth, and enhanced temperature-sensitive responses that are typically more severe than in either single mutant. Abnormal cytokinesis and severely reduced birefringent retardation in elongating root cell walls of rsw2 link the enzyme to cellulose production for primary cell walls and probably cell plates. The Rsw2− phenotype generally resembles the Kor−and cellulose-deficient Rsw1− phenotypes, but anther dehiscence is impaired in Rsw2-1−. The findings link a second putative enzyme activity to cellulose synthesis in primary cell walls of Arabidopsis and further increases the parallels to cellulose synthesis in Agrobacterium tumefaciens where thecelA and celC genes are required and encode a putative glycosyltransferase and an endo-1,4-β-glucanase related to RSW1 and KOR, respectively.

Korrigan (kor) is a dwarf mutant of Arabidopsis thaliana (L.) Heynh. that is deficient in a membrane-bound endo-1,4-beta-glucanase. The effect of the mutation on the pectin network has been studied in kor by microscopical techniques associated with various probes specific for different classes of pectic polysaccharides. The localisation of native crystalline cellulose was also examined using the cellobiohydrolase I-gold probe. The investigations were focused on the external cell walls of the epidermis, a cell layer that, in a number of plant species, has been shown to be growth limiting. Anionic sites associated with pectic polymers were quantified using the cationic gold probe. Homogalacturonans were quantified using polyclonal anti-polygalacturonic acid/rhamnogalacturonan I antibodies recognising polygalacturonic acid, and monoclonal JIM7 and JIM5 antibodies recognising homogalacturonans with a high or low degree of methyl-esterification, respectively. Rhamnogalacturonans were quantified with two monoclonal antibodies, LM5, recognising beta-1,4 galactan side chains of rhamnogalacturonan I, and CCRCM2. Our results show a marked increase in homogalacturonan epitopes and a decrease in rhamnogalacturonan epitopes in kor compared to the wild type. A substantial decrease in cellobiohydrolase I-gold labelling was also observed in the mutant cell walls. These findings demonstrate that a deficiency in an endo-1,4-beta-glucanase, which is in principle not directly implicated in pectin metabolism, can induce important changes in pectin composition in the primary cell wall. The changes indicate the existence of feedback mechanisms controlling the synthesis and/or deposition of pectic polysaccharides in primary cell walls.

Endo‐1,4‐β‐d‐glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non‐crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN ( KOR ) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane‐anchored member of the EGase family, which is highly conserved between mono‐ and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane–cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark‐grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR ‐mRNA levels. However, reduced KOR ‐mRNA levels were observed in det2 , a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose–hemicellulose network in the expanding cell wall.

Cellulose synthesis is driven by large plasma membrane-inserted protein complexes, which in plants have 6-fold symmetry. In Arabidopsis (Arabidopsis thaliana), functional cellulose synthesis complexes (CSCs) are composed of at least three different cellulose synthase catalytic subunits (CESAs), but the actual ratio of the CESA isoforms within the CSCs remains unresolved. In this work, the stoichiometry of the CESAs in the primary cell wall CSC was determined, after elimination of CESA redundancy in a mutant background, by coimmunoprecipitation and mass spectrometry using label-free quantitative methods. Based on spectral counting, we show that CESA1, CESA3, and CESA6 are present in a 1:1:1 molecular ratio.

The synthesis and composition of cell walls is dynamically adapted in response to many developmental and environmental signals. In this respect, cell wall proteins involved in controlling cell elongation are critical for cell development. Transcriptome analysis identified a gene in Arabidopsis thaliana, which was named proline-rich protein-like, AtPRPL1, based on sequence similarities from a phylogenetic analysis. The most resemblance was found to AtPRP1 and AtPRP3 from Arabidopsis, which are known to be involved in root hair growth and development. In A. thaliana four proline-rich cell wall protein genes, playing a role in building up the cross-connections between cell wall components, can be distinguished. AtPRPL1 is a small gene that in promoter:GUS (β-glucuronidase) analysis has high expression in trichoblast cells and in the collet. Chemical or mutational interference with root hair formation inhibited this expression. Altered expression levels in knock-out or overexpression lines interfered with normal root hair growth and etiolated hypocotyl development, but Fourier transform-infrared (FT-IR) analysis did not identify consistent changes in cell wall composition of root hairs and hypocotyl. Co-localization analysis of the AtPRPL1green fluorescent protein (GFP) fusion protein and different red fluorescent protein (RFP)-labelled markers confirmed the presence of AtPRPL1GFP in small vesicles moving over the endoplasmic reticulum. Together, these data indicate that the AtPRPL1 protein is involved in the cells elongation process. How exactly this is achieved remains unclear at present.

Cellulose is synthesized by the so called rosette protein complex and the catalytic subunits of this complex are the cellulose synthases (CESAs). It is thought that the rosette complexes in the primary and secondary cell walls each contains at least three different non-redundant cellulose synthases. In addition to the CESA proteins, cellulose biosynthesis almost certainly requires the action of other proteins, although few have been identified and little is known about the biochemical role of those that have been identified. One of these proteins is KORRIGAN (KOR1). Mutant analysis of this protein in Arabidopsis thaliana showed altered cellulose content in both the primary and secondary cell wall. KOR1 is thought to be required for cellulose synthesis acting as a cellulase at the plasma membrane-cell wall interface. KOR1 has recently been shown to interact with the primary cellulose synthase rosette complex however direct interaction with that of the secondary cell wall has never been demonstrated. Using various methods, both in vitro and in planta, it was shown that KOR1 interacts specifically with only two of the secondary CESA proteins. The KOR1 protein domain(s) involved in the interaction with the CESA proteins were also identified by analyzing the interaction of truncated forms of KOR1 with CESA proteins. The KOR1 transmembrane domain has shown to be required for the interaction between KOR1 and the different CESAs, as well as for higher oligomer formation of KOR1.

Summary Arabinogalactan proteins (AGPs) are a complex family of cell-wall proteoglycans that are thought to play major roles in plant growth and development. Genetic approaches to studying AGP function have met limited success so far, presumably due to redundancy within the large gene families encoding AGP backbones. Here we used an alternative approach for genetic dissection of the role of AGPs in development by modifying their glycan side chains. We have identified an Arabidopsis glycosyltransferase of CAZY family GT31 (AtGALT31A) that galactosylates AGP side chains. A mutation in the AtGALT31A gene caused the arrest of embryo development at the globular stage. The presence of the transcript in the suspensor of globular-stage embryos is consistent with a role for AtGALT31A in progression of embryo development beyond the globular stage. The first observable defect in the mutant is perturbation of the formative asymmetric division of the hypophysis, indicating an essential role for AGP proteoglycans in either specification of the hypophysis or orientation of the asymmetric division plane.

Organ growth depends on two distinct, yet integrated, processes: cell proliferation and post-mitotic cell expansion. Although the regulatory networks of plant cell proliferation during organ growth have begun to be unveiled, the mechanisms regulating post-mitotic cell growth remain mostly unknown. Here, we report the characterization of three EXIGUA (EXI) genes that encode different subunits of the cellulose synthase complex specifically required for secondary cell wall formation. Despite this highly specific role of EXI genes, all the cells within the leaf, even those that do not have secondary walls, display small sizes in the exi mutants. In addition, we found a positive correlation between cell size and the DNA ploidy levels in exi mutant leaves, suggesting that both processes share some regulatory components. Our results are consistent with the hypothesis that the collapsed xylem vessels of the exi mutants hamper water transport throughout the plant, which, in turn, limits the turgor pressure levels required for normal post-mitotic cell expansion during leaf growth.

It is generally believed that cell elongation is regulated by cortical microtubules, which guide the movement of cellulose synthase complexes as they secrete cellulose microfibrils into the periplasmic space. Transversely oriented microtubules are predicted to direct the deposition of a parallel array of microfibrils, thus generating a mechanically anisotropic cell wall that will favor elongation and prevent radial swelling. Thus far, support for this model has been most convincingly demonstrated in filamentous algae. We found that in etiolated Arabidopsis thaliana hypocotyls, microtubules and cellulose synthase trajectories are transversely oriented on the outer surface of the epidermis for only a short period during growth and that anisotropic growth continues after this transverse organization is lost. Our data support previous findings that the outer epidermal wall is polylamellate in structure, with little or no anisotropy. By contrast, we observed perfectly transverse microtubules and microfibrils at the inner face of the epidermis during all stages of cell expansion. Experimental perturbation of cortical microtubule organization preferentially at the inner face led to increased radial swelling. Our study highlights the previously underestimated complexity of cortical microtubule organization in the shoot epidermis and underscores a role for the inner tissues in the regulation of growth anisotropy.

Plant leaves and flowers are positioned along the stem in a regular pattern. This pattern, which is referred to as phyllotaxis, is generated through the precise emergence of lateral organs and is controlled by gradients of the plant hormone auxin. This pattern is actively maintained during stem growth through controlled cell proliferation and elongation. The formation of new organs is known to depend on changes in cell wall chemistry, in particular the demethylesterification of homogalacturonans, one of the main pectic components. Here we report a dual function for the homeodomain transcription factor BELLRINGER (BLR) in the establishment and maintenance of the phyllotactic pattern in Arabidopsis. BLR is required for the establishment of normal phyllotaxis through the exclusion of pectin methylesterase PME5 expression from the meristem dome and for the maintenance of phyllotaxis through the activation of PME5 in the elongating stem. These results provide new insights into the role of pectin demethylesterification in organ initiation and cell elongation and identify an important component of the regulation mechanism involved.

As part of the goal to generate a detailed transcript map for Arabidopsis thaliana, 1152 single run sequences (expressed sequence tags or ESTs) have been determined from cDNA clones taken at random in libraries prepared from different sources of plant material: developing siliques, etiolated seedlings, flower buds, and cultured cells. Eight hundred and ninety-five different genes could be identified, 32% of which showed significant similarity to existing sequences in Arabidopsis and an array of other organisms. These sequences in combination with their positioning on the Arabidopsis genetic map will not only constitute a new set of molecular markers for genome analysis in Arabidopsis but also provide a direct route for the in vivo analysis of their gene products. The sequences have been made available to the public databases.

International audience; A recent report shows that cells in the Arabidopsis apical meristem orientate their cortical microtubules along mechanical stress patterns generated during tissue morphogenesis. This in turn is expected to influence the mechanical properties of the cell via the modification of the cortical microtubule network and the cell wall. This feedback loop controlling the shape of the meristem may act in parallel with auxin signalling, which determines the site of organ primordium formation.

* We focused on a developmentally regulated growth acceleration in the dark-grown Arabidopsis hypocotyl to study the role of changes in cell wall metabolism in the control of cell elongation. * To this end, precise transcriptome analysis on dissected dark-grown hypocotyls, Fourier transform infrared (FT-IR) microspectroscopy and kinematic analysis were used. * Using a cellulose synthesis inhibitor, we showed that the growth acceleration marks a developmental transition during which growth becomes uncoupled from cellulose synthesis. We next investigated the cellular changes that take place during this transition. FT-IR microspectroscopy revealed significant changes in cell wall composition during, but not after, the growth acceleration. Transcriptome analysis suggested a role for cell wall remodeling, in particular pectin modification, in this growth acceleration. This was confirmed by the overexpression of a pectin methylesterase inhibitor, which caused a delay in the growth acceleration. * This study shows that the acceleration of cell elongation marks a developmental transition in dark-grown hypocotyl cells and supports a role for pectin de-methylesterification in the timing of this event.

SummaryHow are the different secondary cell wall deposition patterns generated in the vascular cells of plants? The use of a novel Arabidopsis mesophyll cell culture that transdifferentiates into vascular cells shows a crucial role for a complex of two microtubule-binding proteins.

Plant shoots have thick, polylamellate outer epidermal walls based on crossed layers of cellulose microfibrils, but the involvement of microtubules in such wall lamellation is unclear. Recently, using a long-term movie system in which Arabidopsis seedlings were grown in a biochamber, the tracks along which cortical microtubules move were shown to undergo slow rotary movements over the outer surface of hypocotyl epidermal cells. Because microtubules are known to guide cellulose synthases over the short term, we hypothesised that this previously unsuspected microtubule rotation could, over the longer term, help explain the cross-ply structure of the outer epidermal wall. Here, we test that hypothesis using Arabidopsis plants expressing the cellulose synthase GFP-CESA3 and show that cellulose synthase trajectories do rotate over several hours. Neither microtubule-stabilising taxol nor microtubule-depolymerising oryzalin affected the linear rate of GFP-CESA3 movement, but both stopped the rotation of cellulose synthase tracks. Transmission electron microscopy revealed that drug-induced suppression of rotation alters the lamellation pattern, resulting in a thick monotonous wall layer. We conclude that microtubule rotation, rather than any hypothetical mechanism for wall self-assembly, has an essential role in developing cross-ply wall texture.

Plant cell walls, like a multitude of other biological materials, are natural fiber-reinforced composite materials. Their mechanical properties are highly dependent on the interplay of the stiff fibrous phase and the soft matrix phase and on the matrix deformation itself. Using specific Arabidopsis thaliana mutants, we studied the mechanical role of the matrix assembly in primary cell walls of hypocotyls with altered xyloglucan and pectin composition. Standard microtensile tests and cyclic loading protocols were performed on mur1 hypocotyls with affected RGII borate diester cross-links and a hindered xyloglucan fucosylation as well as qua2 exhibiting 50% less homogalacturonan in comparison to wild-type. As a control, wild-type plants (Col-0) and mur2 exhibiting a specific xyloglucan fucosylation and no differences in the pectin network were utilized. In the standard tensile tests, the ultimate stress levels (approximately tensile strength) of the hypocotyls of the mutants with pectin alterations (mur1, qua2) were rather unaffected, whereas their tensile stiffness was noticeably reduced in comparison to Col-0. The cyclic loading tests indicated a stiffening of all hypocotyls after the first cycle and a plastic deformation during the first straining, the degree of which, however, was much higher for mur1 and qua2 hypocotyls. Based on the mechanical data and current cell wall models, it is assumed that folded xyloglucan chains between cellulose fibrils may tend to unfold during straining of the hypocotyls. This response is probably hindered by geometrical constraints due to pectin rigidity.

International audience; Pectins are a family of highly complex multifunctional cell wall polysaccharides. Little is known on the relation between pectin structure, hydrodynamic properties, and cellular function. In this study, we took advantage of the Arabidopsis pectin mutant quasimodo2 (qua2), which specifically lacks half of its homogalacturonan blocks, to study the relationship between the amount of homogalacturonan blocks and the hydrodynamic properties of pectins. It was first shown that, in qua2 pectins, homogalacturonans had maintained the same size as those in the wild type. The persistence lengths of isolated homogalacturonan and rhamnogalacturonan-I blocks were then measured and it was shown that homogalacturonan was over 4-fold more rigid than rhamnogalacturonan-I. WT and qua2 pectins were next compared and it appeared that the specific reduction of the number of homogalacturonan blocks leads to an increased flexibility of qua2 pectins. These results show for the first time how mutant pectins can be used to demonstrate the opposite influence of rhamnogalacturonan-I and homogalacturonan blocks on the hydrodynamic properties of pectins.

Summary Plant organs are produced from meristems in a characteristic pattern. This pattern, referred to as phyllotaxis, is thought to be generated by local gradients of an information molecule, auxin [1–6]. Some studies propose a key role for the mechanical properties of the cell walls in the control of organ outgrowth [7–12]. A major cell-wall component is the linear α-1-4-linked D-GalAp pectic polysaccharide homogalacturonan (HG), which plays a key role in cell-to-cell cohesion [13, 14]. HG is deposited in the cell wall in a highly (70%–80%) methyl-esterified form and is subsequently de-methyl-esterified by pectin methyl-esterases (PME, EC 3.1.1.11). PME activity is itself regulated by endogenous PME inhibitor (PMEI) proteins [15]. PME action modulates cell-wall-matrix properties and plays a role in the control of cell growth [16–18]. Here, we show that the formation of flower primordia in the Arabidopsis shoot apical meristem is accompanied by the de-methyl-esterification of pectic polysaccharides in the cell walls. In addition, experimental perturbation of the methyl-esterification status of pectins within the meristem dramatically alters the phyllotactic pattern. These results demonstrate that regulated de-methyl-esterification of pectins is a key event in the outgrowth of primordia and possibly also in phyllotactic patterning.

In all land plants, cellulose is synthesized from hexameric plasma membrane complexes. Indirect evidence suggests that in vascular plants the complexes involved in primary wall synthesis contain three distinct cellulose synthase catalytic subunits (CESAs). In this study, we show that CESA3 and CESA6 fused to GFP are expressed in the same cells and at the same time in the hypocotyl of etiolated seedlings and migrate with comparable velocities along linear trajectories at the cell surface. We also show that CESA3 and CESA6 can be coimmunoprecipitated from detergent-solubilized extracts, their protein levels decrease in mutants for either CESA3 , CESA6 , or CESA1 and CESA3, CESA6 and also CESA1 can physically interact in vivo as shown by bimolecular fluorescence complementation. We also demonstrate that CESA6-related CESA5 and CESA2 are partially, but not completely, redundant with CESA6 and most likely compete with CESA6 for the same position in the cellulose synthesis complex. Using promoter-β-glucuronidase fusions we show that CESA5 , CESA6 , and CESA2 have distinct overlapping expression patterns in hypocotyl and root corresponding to different stages of cellular development. Together, these data provide evidence for the existence of binding sites for three distinct CESA subunits in primary wall cellulose synthase complexes, with two positions being invariably occupied by CESA1 and CESA3, whereas at least three isoforms compete for the third position. Participation of the latter three isoforms might fine-tune the CESA complexes for the deposition of microfibrils at distinct cellular growth stages.

Pectins are a family of complex cell-wall polysaccharides, the biosynthesis of which remains poorly understood. We identified dwarf mutants with reduced cell adhesion at a novel locus, QUASIMODO2 (QUA2). qua2-1 showed a 50% reduction in homogalacturonan (HG) content compared with the wild type, without affecting other cell-wall polysaccharides. The remaining HG in qua2-1 showed an unaltered degree of methylesterification. Positional cloning and GFP fusions showed that QUA2, consistent with a role in HG synthesis, encodes a Golgi-localized protein. In contrast to QUA1, another Golgi-localized protein required for HG-synthesis, QUA2 does not show sequence similarity to glycosyltransferases, but instead contains a putative methyltransferase (MT) domain. The Arabidopsis genome encodes 29 QUA2-related proteins. Interestingly, the transcript profiles of QUA1 and QUA2 are correlated and other pairs of QUA1 and QUA2 homologues with correlated transcript profiles can be identified. Together, the results lead to the hypothesis that QUA2 is a pectin MT, and that polymerization and methylation of homogalacturonan are interdependent reactions.

The composition and permeability of the cuticle has a large influence on its ability to protect the plant against various forms of biotic and abiotic stress. WAX INDUCER1 (WIN1) and related transcription factors have recently been shown to trigger wax production, enhance drought tolerance, and modulate cuticular permeability when overexpressed in Arabidopsis thaliana. We found that WIN1 influences the composition of cutin, a polyester that forms the backbone of the cuticle. WIN1 overexpression induces compositional changes and an overall increase in cutin production in vegetative and reproductive organs, while its downregulation has the opposite effect. Changes in cutin composition are preceded by the rapid and coordinated induction of several genes known or likely to be involved in cutin biosynthesis. This transcriptional response is followed after a delay by the induction of genes associated with wax biosynthesis, suggesting that the regulation of cutin and wax production by WIN1 is a two-step process. We demonstrate that at least one of the cutin pathway genes, which encodes long-chain acyl-CoA synthetase LACS2, is likely to be directly targeted by WIN1. Overall, our results suggest that WIN1 modulates cuticle permeability in Arabidopsis by regulating genes encoding cutin pathway enzymes.

Quantitative trait loci (QTL) analysis was used to identify genes underlying natural variation in primary cell wall composition in Arabidopsis (Arabidopsis thaliana). The cell walls of dark-grown seedlings of a Bay-0 × Shahdara recombinant inbred line population were analyzed using three miniaturized global cell wall fingerprinting techniques: monosaccharide composition analysis by gas chromatography, xyloglucan oligosaccharide mass profiling, and whole-wall Fourier-transform infrared microspectroscopy. Heritable variation and transgression were observed for the arabinose-rhamnose ratio, xyloglucan side-chain composition (including O-acetylation levels), and absorbance for a subset of Fourier-transform infrared wavenumbers. In total, 33 QTL, corresponding to at least 11 different loci controlling dark-grown hypocotyl length, pectin composition, and levels of xyloglucan fucosylation and O-acetylation, were identified. One major QTL, accounting for 51% of the variation in the arabinose-rhamnose ratio, affected the number of arabinan side chains presumably attached to the pectic polysaccharide rhamnogalacturonan I, paving the way to positional cloning of the first gene underlying natural variation in pectin structure. Several QTL were found to be colocalized, which may have implications for the regulation of xyloglucan metabolism. These results demonstrate the feasibility of combining fingerprinting techniques, natural variation, and quantitative genetics to gain original insight into the molecular mechanisms underlying the structure and metabolism of cell wall polysaccharides.

The synthesis of cellulose microfibrils requires the presence of a membrane-bound endo-1,4-β-d-glucanase, KORRIGAN1 (KOR1). Although the exact biochemical role of KOR1 in cellulose synthesis is unknown, we used the protein as a marker to explore the potential involvement of subcellular transport processes in cellulose synthesis. Using immunofluorescence and a green fluorescent protein (GFP)–KOR1 fusion that complemented the phenotype conferred by the kor1-1 mutant, we investigated the distribution of KOR1 in epidermal cells in the root meristem. KOR1 was localized in intracellular compartments corresponding to a heterogeneous population of organelles, which comprised the Golgi apparatus, FM4-64–labeled compartments referred to as early endosomes, and, in the case of GFP-KOR1, the tonoplast. Inhibition of cellulose synthesis by isoxaben promoted a net redistribution of GFP-KOR1 toward a homogeneous population of compartments, distinct from early endosomes, which were concentrated close to the plasma membrane facing the root surface. A redistribution of GFP-KOR1 away from early endosomes was also observed in the same cells at later stages of cell elongation. A subpopulation of GFP-KOR1–containing compartments followed trajectories along the plasma membrane, and this motility required intact microtubules. These observations demonstrate that the deposition of cellulose, like chitin synthesis in yeast, involves the regulated intracellular cycling of at least one enzyme required for its synthesis.

Sulphorhodamine-labelled oligosaccharides of xyloglucan are incorporated into the cell wall of Arabidopsis and tobacco roots, and of cultured Nicotiana tabacum cells by the transglucosylase (XET) action of XTHs. In the cell wall of diffusely growing cells, the subcellular pattern of XET action revealed a 'fibrillar' pattern, different from the xyloglucan localization. The fibrillar fluorescence pattern had no net orientation in spherical cultured cells. It changed to transverse to the long axis when the cells started to elongate, a feature mirroring the rearrangements of cortical microtubules and the accompanying cellulose deposition. Interference with the polymerization of microtubules and with cellulose deposition inhibited this strong and 'fibrillar'-organized XET-action, whereas interference with actin-polymerization only decreased the intensity of enzyme action. Epidermal cells of a mutant with reduced cellulose synthesis also had low XET action. Root hairs (tip-growing cells) exhibited high XET-action over all their length, but lacked the specific parallel pattern. In both diffuse- and tip-growing cell types extraction of the incorporated fluorescent xyloglucans by a xyloglucan-specific endoglucanase reduced the fluorescence, but the 'fibrillar' appearance in diffuse growing cells was not eliminated. These results show that XTHs act on the xyloglucans attached to cellulose microfibrils. After incorporation of the fluorescent oligosaccharides, the xyloglucans decorate the cellulose microfibrils and become inaccessible to hydrolytic enzymes.

Cell wall polysaccharides are some of the most complex biopolymers known, and yet their functions remain largely mysterious. Advances in imaging methods permit direct visualisation of the molecular architecture of cell walls and the modifications that occur to polymers during growth and development. To address the structural and functional relationships of individual cell wall components, we need to better characterise a broad range of structural and architectural alterations in cell walls, appearing as a consequence of developmental regulation, environmental adaptation or genetic modification. We have developed a rapid method to screen large numbers of plants for a broad range of cell wall phenotypes using Fourier transform infrared microspectroscopy and Principal Component Analysis. We are using model systems to uncover the genes that encode some of the cell-wall-related biosynthetic and hydrolytic enzymes, and structural proteins.

54 ref.; International audience; Mutants at the PROCUSTE1 (PRC1) locus show decreased cell elongation, specifically in roots and dark-grown hypocotyls. Cell elongation defects are correlated with a cellulose deficiency and the presence of gapped walls. Map-based cloning of PRC1 reveals that it encodes a member (CesA6) of the cellulose synthase catalytic subunit family, of which at least nine other members exist in Arabidopsis. Mutations in another family member, RSW1 (CesA1), cause similar cell wall defects in all cell types, including those in hypocotyls and roots, suggesting that cellulose synthesis in these organs requires the coordinated expression of at least two distinct cellulose synthase isoforms.

An ever growing collection of cell wall mutants is yielding new insights into the mechanisms underlying the synthesis and assembly of cell walls in plants. In this review, we will provide an update on the use of genetic tools in plant cell wall research and we will discuss the lessons that can be drawn from the study of the first generation of mutants.

Mutants at the BOTERO1 locus are affected in anisotropic growth in all non-tip-growing cell types examined. Mutant cells are shorter and broader than those of the wild type. Mutant inflorescence stems show a dramatically reduced bending modulus and maximum stress at yield. Our observations of root epidermis cells show that the cell expansion defect in bot1 is correlated with a defect in the orientation of the cortical microtubules. We found that in cells within the apical portion of the root, which roughly corresponds to the meristem, microtubules were loosely organized and became much more highly aligned in transverse arrays with increasing distance from the tip. Such a transition was not observed in bot1. No defect in microtubule organization was observed in kor-1, another mutant with a radial cell expansion defect. We also found that in wild-type root epidermal cells, cessation of radial expansion precedes the increased alignment of cortical microtubules into transverse arrays. Bot1 roots still show a gravitropic response, which indicates that ordered cortical microtubules are not required for differential growth during gravitropism. Interestingly, the fact that in the mutant, these major changes in microtubule organization cause relatively subtle changes in cell morphology, suggest that other levels of control of growth anisotropy remain to be discovered. Together, these observations suggest that BOT1 is required for organizing cortical microtubules into transverse arrays in interphase cells, and that this organization is required for consolidating, rather than initiating, changes in the direction of cell expansion.

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.