Plant cell wall patterning and expansion mediated by protein-peptide-polysaccharide interaction.

Assembly of cell wall polysaccharides into specific patterns is required for plant growth. A complex of RAPID ALKALINIZATION FACTOR 4 (RALF4) and its cell wall-anchored LEUCINE-RICH REPEAT EXTENSIN 8 (LRX8)-interacting protein is crucial for cell wall integrity during pollen tube growth, but its molecular connection with the cell wall is unknown. Here, we show that LRX8-RALF4 complexes adopt a heterotetrametric configuration in vivo, displaying a dendritic distribution. The LRX8-RALF4 complex specifically interacts with demethylesterified pectins in a charge-dependent manner through RALF4's polycationic surface. The LRX8-RALF4-pectin interaction exerts a condensing effect, patterning the cell wall's polymers into a reticulated network essential for wall integrity and expansion. Our work uncovers a dual structural and signaling role for RALF4 in pollen tube growth and in the assembly of complex extracellular polymers.

Contrasting Cd accumulation of Arabidopsis halleri populations: a role for (1→4)-ß-galactan in pectin.

Cadmium (Cd) accumulation is highly variable among Arabidopsis halleri populations. To identify cell wall (CW) components that contribute to the contrasting Cd accumulation between PL22-H (Cd-hyperaccumulator) and I16-E (Cd-excluder), Cd absorption capacity of CW polysaccharides, CW mono- and poly- saccharides contents and CW glycan profiles were compared between these two populations. PL22-H pectin contained 3-fold higher Cd concentration than I16-E pectin in roots, and (1→4)-ß-galactan pectic epitope showed the biggest difference between PL22-H and I16-E. CW-related differentially expressed genes (DEGs) between PL22-H and I16-E were identified and corresponding A. thaliana mutants were phenotyped for Cd tolerance and accumulation. A higher Cd translocation was observed in GALACTAN SYNTHASE1 A. thaliana knockout and overexpressor mutants, which both showed a lengthening of the RG-I sidechains after Cd treatment, contrary to the wild-type. Overall, our results support an indirect role for (1→4)-ß-galactan in Cd translocation, possibly by a joint effect of regulating the length of RG-I sidechains, the pectin structure and interactions between polysaccharides in the CW. The characterization of other CW-related DEGs between I16-E and PL22-H selected allowed to identify a possible role in Zn translocation for BIIDXI and LEUNIG-HOMOLOG genes, which are both involved in pectin modification.

A dive into the cell wall with Arabidopsis.

One of the many legacies of the work of Michel Caboche is our understanding of plant cell wall synthesis and metabolism thanks to the use of Arabidopsis mutants. Here I describe how he was instrumental in initiating the genetic study of plant cell walls. I also show, with a few examples for cellulose and pectins, how this approach has led to important new insights in cell wall synthesis and how the metabolism of pectins contributes to plant growth and morphogenesis. I also illustrate the limitations of the use of mutants to explain processes at the scale of cells, organs or whole plants in terms of the physico-chemical properties of cell wall polymers. Finally, I sketch how new approaches can cope with these limitations.

L'un des nombreux héritages des travaux de Michel Caboche est notre compréhension de la synthèse et du métabolisme des parois cellulaires végétales grâce à l'utilisation de mutants d'Arabidopsis. Je décris ici comment il a joué un rôle déterminant dans le lancement de l'étude génétique des parois cellulaires végétales. Je montre également, avec quelques exemples pour la cellulose et les pectines, comment cette approche a conduit à de nouvelles connaissances importantes sur la synthèse de la paroi cellulaire et comment le métabolisme des pectines contribue à la croissance et à la morphogenèse des plantes. J'illustre également les limites de l'utilisation de mutants pour expliquer des processus à l'échelle de cellules, d'organes ou de plantes entières en termes de propriétés physico-chimiques de polymères de parois cellulaires. Enfin, j'esquisse comment de nouvelles approches peuvent faire face à ces limitations.

Optimal BR signalling is required for adequate cell wall orientation in the Arabidopsis root meristem.

Plant brassinosteroid hormones (BRs) regulate growth in part through altering the properties of the cell wall, the extracellular matrix of plant cells. Conversely, feedback signalling from the wall 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. Furthermore, both depletion of endogenous BRs and exogenous supply of BRs triggered these defects. Cell wall signalling-induced alterations in the orientation of newly placed walls appear to occur late during cytokinesis, after initial positioning of the cortical division zone. 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.

Oligogalacturonide production upon Arabidopsis thaliana-Botrytis cinerea interaction.

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.

The Control of Growth Symmetry Breaking in the Arabidopsis Hypocotyl.

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. 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 but also anisotropic cell growth in animal cells.

The yin and yang of cell wall integrity control: brassinosteroid and FERONIA signaling.

Understanding how developmental and environmental signals control plant cell expansion requires an intimate knowledge of the architecture of the primary cell wall and the chemo-rheological processes that underlie cell wall relaxation. In this review I discuss recent findings that reveal a more prominent role than previously suspected for covalent bonds and pectin cross-links in primary cell wall architecture. In addition, genetic studies have uncovered a role for receptor kinases in the control of cell wall homeostasis in growing cells. The emerging view is that, upon cell wall disruption, compensatory changes are induced in the cell wall through the interplay between the brassinosteroid signaling module, which positively regulates wall extensibility and receptor kinases of the CrRLKL1 family, which may act as negative regulators of cell wall stiffness. These findings lift the tip of the veil of a complex signaling network allowing normal homeostasis in walls of growing cells but also crisis management under stress conditions.

Tracking developmentally regulated post-synthetic processing of homogalacturonan and chitin using reciprocal oligosaccharide probes.

Polysaccharides are major components of extracellular matrices and are often extensively modified post-synthetically to suit local requirements and developmental programmes. However, our current understanding of the spatiotemporal dynamics and functional significance of these modifications is limited by a lack of suitable molecular tools. Here, we report the development of a novel non-immunological approach for producing highly selective reciprocal oligosaccharide-based probes for chitosan (the product of chitin deacetylation) and for demethylesterified homogalacturonan. Specific reciprocal binding is mediated by the unique stereochemical arrangement of oppositely charged amino and carboxy groups. Conjugation of oligosaccharides to fluorophores or gold nanoparticles enables direct and rapid imaging of homogalacturonan and chitosan with unprecedented precision in diverse plant, fungal and animal systems. We demonstrated their potential for providing new biological insights by using them to study homogalacturonan processing during Arabidopsis thaliana root cap development and by analyzing sites of chitosan deposition in fungal cell walls and arthropod exoskeletons.

A receptor-like protein mediates the response to pectin modification by activating brassinosteroid signaling.

The brassinosteroid (BR) signaling module is a central regulator of plant morphogenesis, as indicated by the large number of BR-responsive cell wall-related genes and the severe growth defects of BR mutants. Despite a detailed knowledge of the signaling components, the logic of this auto-/paracrine signaling module in growth control remains poorly understood. Recently, extensive cross-talk with other signaling pathways has been shown, suggesting that the outputs of BR signaling, such as gene-expression changes, are subject to complex control mechanisms. We previously provided evidence for a role of BR signaling in a feedback loop controlling the integrity of the cell wall. Here, we identify the first dedicated component of this feedback loop: a receptor-like protein (RLP44), which is essential for the compensatory triggering of BR signaling upon inhibition of pectin de-methylesterification in the cell wall. RLP44 is required for normal growth and stress responses and connects with the BR signaling pathway, presumably through a direct interaction with the regulatory receptor-like kinase BAK1. These findings corroborate a role for BR in controlling the sensitivity of a feedback signaling module involved in maintaining the physico-chemical homeostasis of the cell wall during cell expansion.

Arabidopsis PECTIN METHYLESTERASE17 is co-expressed with and processed by SBT3.5, a subtilisin-like serine protease.

BACKGROUND AND AIMS: In Arabidopsis thaliana, the degree of methylesterification (DM) of homogalacturonans (HGs), the main pectic constituent of the cell wall, can be modified by pectin methylesterases (PMEs). In all organisms, two types of protein structure have been reported for PMEs: group 1 and group 2. In group 2 PMEs, the active part (PME domain, Pfam01095) is preceded by an N-terminal extension (PRO part), which shows similarities to PME inhibitors (PMEI domain, Pfam04043). This PRO part mediates retention of unprocessed group 2 PMEs in the Golgi apparatus, thus regulating PME activity through a post-translational mechanism. This study investigated the roles of a subtilisin-type serine protease (SBT) in the processing of a PME isoform. METHODS: Using a combination of functional genomics, biochemistry and proteomic approaches, the role of a specific SBT in the processing of a group 2 PME was assessed together with its consequences for plant development. KEY RESULTS: A group 2 PME, AtPME17 (At2g45220), was identified, which was highly co-expressed, both spatially and temporally, with AtSBT3.5 (At1g32940), a subtilisin-type serine protease (subtilase, SBT), during root development. PME activity was modified in roots of knockout mutants for both proteins with consequent effects on root growth. This suggested a role for SBT3.5 in the processing of PME17 in planta. Using transient expression in Nicotiana benthamiana, it was indeed shown that SBT3.5 can process PME17 at a specific single processing motif, releasing a mature isoform in the apoplasm. CONCLUSIONS: By revealing the potential role of SBT3.5 in the processing of PME17, this study brings new evidence of the complexity of the regulation of PMEs in plants, and highlights the need for identifying specific PME-SBT pairs.

Plant cell wall homeostasis is mediated by brassinosteroid feedback signaling.

Brassinosteroid (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, 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. 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, which can be modified in the wall by the enzyme pectin methylesterase (PME). 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.

Probing the mechanical contributions of the pectin matrix: insights for cell growth.

The plant cell wall has a somewhat paradoxical mechanical role in the plant: it must be strong enough to resist the high turgor of the cell contents, but at the right moment it must yield to that pressure to allow cell growth. The control of the cell wall's mechanical properties underlies its ability to regulate growth correctly. Recently, we have reported on changes in cell wall elasticity associated with organ formation at the shoot apical meristem in Arabidopsis thaliana. These changes in cell wall elasticity were strongly correlated with changes in pectin matrix chemistry, and we have previously shown that changes in pectin chemistry can dramatically effect organ formation. These findings point to a important role of the cell wall pectin matrix in cell growth control of higher plants. In this addendum we will discuss the biological significance of these new observations, and will place the scientific advances made possible through Atomic Force Microscopy-based nano-indentations in a relatable context with past experiments on cell wall mechanics.

Growth control and cell wall signaling in plants.

Plant cell walls have the remarkable property of combining extreme tensile strength with extensibility. The maintenance of such an exoskeleton creates nontrivial challenges for the plant cell: How can it control cell wall assembly and remodeling during growth while maintaining mechanical integrity? How can it deal with cell wall damage inflicted by herbivores, pathogens, or abiotic stresses? These processes likely require mechanisms to keep the cell informed about the status of the cell wall. In yeast, a cell wall integrity (CWI) signaling pathway has been described in great detail; in plants, the existence of CWI signaling has been demonstrated, but little is known about the signaling pathways involved. In this review, we first describe cell wall-related processes that may require or can be targets of CWI signaling and then discuss our current understanding of CWI signaling pathways and future prospects in this emerging field of plant biology.

Pectin-induced changes in cell wall mechanics underlie organ initiation in Arabidopsis.

Tissue 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.

A role for pectin de-methylesterification in a developmentally regulated growth acceleration in dark-grown Arabidopsis hypocotyls.

• 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.

Abscisic acid deficiency causes changes in cuticle permeability and pectin composition that influence tomato resistance to Botrytis cinerea.

A mutant of tomato (Solanum lycopersicum) with reduced abscisic acid (ABA) production (sitiens) exhibits increased resistance to the necrotrophic fungus Botrytis cinerea. This resistance is correlated with a rapid and strong hydrogen peroxide-driven cell wall fortification response in epidermis cells that is absent in tomato with normal ABA production. Moreover, basal expression of defense genes is higher in the mutant compared with the wild-type tomato. Given the importance of this fast response in sitiens resistance, we investigated cell wall and cuticle properties of the mutant at the chemical, histological, and ultrastructural levels. We demonstrate that ABA deficiency in the mutant leads to increased cuticle permeability, which is positively correlated with disease resistance. Furthermore, perturbation of ABA levels affects pectin composition. sitiens plants have a relatively higher degree of pectin methylesterification and release different oligosaccharides upon inoculation with B. cinerea. These results show that endogenous plant ABA levels affect the composition of the tomato cuticle and cell wall and demonstrate the importance of cuticle and cell wall chemistry in shaping the outcome of this plant-fungus interaction.

Pectin may hinder the unfolding of xyloglucan chains during cell deformation: implications of the mechanical performance of Arabidopsis hypocotyls with pectin alterations.

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.

Arabidopsis phyllotaxis is controlled by the methyl-esterification status of cell-wall pectins.

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. Some studies propose a key role for the mechanical properties of the cell walls in the control of organ outgrowth. A major cell-wall component is the linear alpha-1-4-linked D-GalAp pectic polysaccharide homogalacturonan (HG), which plays a key role in cell-to-cell cohesion. 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. PME action modulates cell-wall-matrix properties and plays a role in the control of cell growth. 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.

Reduced number of homogalacturonan domains in pectins of an Arabidopsis mutant enhances the flexibility of the polymer.

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.