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Plants growing with neighbours compete for light and consequently increase growth of their vegetative organs to enhance access to sunlight. This response, called shade avoidance syndrome (SAS), involves photoreceptors such as phytochromes as well as phytochrome interacting factors (PIFs), which regulate the expression of growth-mediating genes. Numerous cell wall-related genes belong to the putative targets of PIFs, and the importance of cell wall modifications for enabling growth was extensively shown in developmental models such as dark-grown hypocotyl. However, the role of the cell wall in the growth of de-etiolated seedlings regulated by shade cues remains poorly established. Through analyses of mechanical and biochemical properties of the cell wall coupled with transcriptomic analysis of cell wall-related genes, we show the importance of cell wall modifications in neighbour proximity-induced elongation. Further analysis using loss-of-function mutants impaired in the synthesis and remodeling of the main cell wall polymers corroborated this. We focused on thecgr2cgr3double mutant that is defective in homogalacturonan (HG) methyltransferase activity required for methylesterification of HG-type pectins. By following hypocotyl growth kinetically and spatially and analyzing the mechanical and biochemical properties of cell walls, we found that methylesterification of HG-type pectins was required to enable global cell wall modifications. Moreover, HG-class pectin modification was needed for plant competition-induced hypocotyl growth. Collectively our work suggests that in the hypocotyl PIFs orchestrate changes in the expression of numerous cell wall genes to enable neighbour proximity-induced growth.One sentence summaryThe degree of methylesterification of pectins modulates global changes in the cell wall and its mechanical properties that contribute to the neighbour proximity-induced hypocotyl growth in Arabidopsis

Pectins, complex polysaccharides and major components of the plant primary cell wall, can be degraded by pectate lyases (PLs). PLs cleave glycosidic bonds of homogalacturonans (HG), the main pectic domain, by β-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB fromVerticillium dahliae, a plant pathogen. We first solved the crystal structure of VdPelB at 1.2Å resolution showing that it is a right-handed parallel β-helix structure. Molecular dynamics (MD) simulations further highlighted the dynamics of the enzyme in complex with substrates that vary in their degree of methylesterification, identifying amino acids involved in substrate binding and cleavage of non-methylesterified pectins. We then biochemically characterized wild type and mutated forms of VdPelB. VdPelB was most active on non-methylesterified pectins, at pH 8 in presence of Ca2+ions. VdPelB-G125R mutant was most active at pH 9 and showed higher relative activity compared to native enzyme. The OGs released by VdPelB differed to that of previously characterized PLs, showing its peculiar specificity in relation to its structure. OGs released fromVerticillium-partially tolerant and sensitive flax cultivars differed which could facilitate the identification VdPelB-mediated elicitors of defence responses.

International audience; Pectin, the major non-cellulosic component of primary cell wall can be degraded by polygalacturonases (PGs) and pectin methylesterases (PMEs) during pathogen attack on plants. We characterized two novel enzymes,VdPG2 and VdPME1, from the fungal plant pathogen Verticillium dahliae. VdPME1 was most active on citrus methylesterified pectin (55–70%) at pH 6 and a temperature of 40 °C, while VdPG2 was most active on polygalacturonic acid at pH 5 and a temperature of 50 °C. Using LC-MS/MS oligo profiling, and various pectins,the mode of action of VdPME1 and VdPG2 were determined. VdPME1 was shown to be processive, in accordance with the electrostatic potential of the enzyme. VdPG2 was identified asendo-PG releasing both methylesterified and non-methylesterified oligogalacturonides (OGs). Additionally, whenflax roots were used as substrate, acet-ylated OGs were detected. The comparisons of OGs released from Verticillium-susceptible and partially resistant flax cultivars identified new possible elicitor of plant defence responses.

The fine-tuning of pectins by polygalacturonases (PGs) plays a key role in modulating plant cell wall chemistry and mechanics, impacting plant development. In plants, the high number of PGs encoded in the genome questions the regulation of pectin depolymerization and the roles of distinct isozymes in the control of development. Here we report the first crystal structures of two PGs from Arabidopsis, PGLR and ADPG2 whose expression overlap in roots. Albeit having overall conserved folds and active sites, PGLR and ADPG2 differed in the structure of their binding grooves and in the amino-acids of the subsites. We determined the structural features that explain the absence of inhibition of the plant PGs by endogenous PG-Inhibiting Proteins (PGIPs). By combining molecular dynamic simulations, analysis of enzymes’ kinetics and hydrolysis products, we showed that subtle differences in PGLR and ADPG2 structures translated into distinct enzyme-substrate dynamics and enzymes’ processivities. Using the plant root as a developmental model, exogenous application of purified enzymes showed that these distinct PGLR/ADPG2 processivities ultimately translated into different impacts on development. The highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. Our study suggests that, in plants, gene redundancy is unlikely to reflect redundant biochemical specificities. Isozymes of distinct specificities and processivities are likely to be of major importance for the fine spatial and temporal regulation of pectin structure.Significance StatementPlant polygalacturonases (PG) are enzymes that play a key role in the regulation of cell wall pectin chemistry by controlling the degree of polymerization of the HG chains. The high number of genes encoding PG in Arabidopsis questions the rationale for such abundance. We solved the crystal structure of two PG (PGLR and ADPG2) whose expression overlap in roots and showed, using combined computational and experimental approaches, that they differ in their enzyme-substrate dynamics, leading to distinct processivities. The highly processive ADPG2 can generate digestion products of shorter degree of polymerization, and upon exogenous application on developing roots, induced drastic developmental defects. Our study suggests that gene redundancy is unlikely to reflect redundant biochemical specificities of isozymes.

Efficient design of functional proteins with higher thermal stability remains challenging especially for highly diverse sequence variants. Considering the evolutionary pressure on protein folds, sequence design optimizing evolutionary fitness could help designing folds with higher stability. Using a generative evolution fitness model trained to capture variation patterns in natural sequences, we designed artificial sequences of a proteinaceous inhibitor of pectin methylesterase enzymes. These inhibitors have considerable industrial interest to avoid phase separation in fruit juice manufacturing or reduce methanol in distillates, averting chromatographic passages triggering unwanted aroma loss. Six out of seven designs with up to 30 % divergence to other inhibitor sequences are functional and two have improved thermal stability. This method can improve protein stability expanding functional protein sequence space, with traits valuable for industrial applications and scientific research.

Pectins occur in primary cell walls and consist of multiblock co-polymers among which homogalacturonan (HG) is the simplest and most abundant form. Methylesterification patterns of HG are tuned by pectin methylesterases (PMEs), the activities of which are controlled by specific inhibitors (PMEIs). By impacting cell wall mechanical properties, PME-mediated regulation of HG methylesterification plays a major role in several developmental processes, including seed germination and dark-grown hypocotyl elongation. Arabidopsis PME36 is preferentially expressed during the late stage of seed development and, using the knock-out mutant pme36-1, we show here that PME36 is required to implement the characteristic pattern of de-methylesterified pectin in the mature seed. Surprisingly, while this pattern is strongly impaired in pme36-1 mature seed, no phenotypical effect is observed in the mutant during seed germination and dark-grown hypocotyl elongation, suggesting the existence of a compensatory mechanism overcoming the defect in pectin de-methylesterification. By analyzing hormone contents and gene expression, a strong, dynamic, and long-lasting physiological disorder is revealed in the mutant. These results suggest the existence of complex connections between pectin remodeling, transcriptomic regulations and hormonal homeostasis, modulating several physiological parameters to ensure the maintenance of a normal seed-to-seedling developmental program in pme36-1. Considered for a long time as an end-point passive effector mainly involved in modification of cell wall mechanics, the role of pectin methylesterification needs to be reconsidered as a modulator acting upstream of diverse regulatory pathways involved in plant development.

Pectin methylesterases (PMEs) modify homogalacturonan’s (HG) chemistry and play a key role in regulating primary cell wall mechanical properties. How PME activity can fine-tune pectin structure in the growing plant has remained elusive. Here we report on the Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum suitable for detecting recombinant and native enzyme independent of species source. At neutral pH AtPME2 is preferentially active on pectins with a degree of 55-70% methylesterification and can be inhibited by PME inhibitor protein (PMEI). We show that the mode of action for AtPME2 can switch from full processivity (at pH 8), creating large blocks of unmethylated galacturonic acid, to low processivity (at pH 5) and relate these observations to the differences in electrostatic potential of the protein at acidic and alkaline pH. To assess the role of AtPME2 in development, we characterized two knock-out lines. We show that in the context of acidified apoplast, low-processive demethylesterification by AtPME2 can loosen the cell wall, with consequent increase in cell elongation and etiolated hypocotyl length. Our study brings insights into how the pH-dependent regulation by PME activity could affect pectin structure and associated cell wall mechanical properties in expansion. One sentence summary The processivity of AtPME2, a pectin methylesterase that fine-tunes cell wall pectins is modulated by pH in vitro and impacts the mechanical properties of the wall, affecting development in planta.

Pectin methylesterases (PMEs) catalyze the demethylesterification of homogalacturonan domains of pectin in plant cell walls and are regulated by endogenous pectin methylesterase inhibitors (PMEIs). In Arabidopsis dark-grown hypocotyls, one PME (AtPME3) and one PMEI (AtPMEI7) were identified as potential interacting proteins. Using RT-quantitative PCR analysis and gene promoter::GUS fusions, we first showed that AtPME3 and AtPMEI7 genes had overlapping patterns of expression in etiolated hypocotyls. The two proteins were identified in hypocotyl cell wall extracts by proteomics. To investigate the potential interaction between AtPME3 and AtPMEI7, both proteins were expressed in a heterologous system and purified by affinity chromatography. The activity of recombinant AtPME3 was characterized on homogalacturonans (HGs) with distinct degrees/patterns of methylesterification. AtPME3 showed the highest activity at pH 7.5 on HG substrates with a degree of methylesterification between 60 and 80% and a random distribution of methyl esters. On the best HG substrate, AtPME3 generates long non-methylesterified stretches and leaves short highly methylesterified zones, indicating that it acts as a processive enzyme. The recombinant AtPMEI7 and AtPME3 interaction reduces the level of demethylesterification of the HG substrate but does not inhibit the processivity of the enzyme. These data suggest that the AtPME3·AtPMEI7 complex is not covalently linked and could, depending on the pH, be alternately formed and dissociated. Docking analysis indicated that the inhibition of AtPME3 could occur via the interaction of AtPMEI7 with a PME ligand-binding cleft structure. All of these data indicate that AtPME3 and AtPMEI7 could be partners involved in the fine tuning of HG methylesterification during plant development.

International audience; Pectin methylesterases (PMEs) catalyze the demethylesterification of pectin, one of the main polysaccharides in the plant cell wall, and are of critical importance in plant development. PME activity generates highly negatively charged pectin and mutates the physiochemical properties of the plant cell wall such that remodeling of the plant cell can occur. PMEs are therefore tightly regulated by proteinaceous inhibitors (PMEIs), some of which become active upon changes in cellular pH. Nevertheless, a detailed picture of how this pH-dependent inhibition of PME occurs at the molecular level is missing. Herein, using an interdisciplinary approach that included homology modeling, MD simulations, and biophysical and biochemical characterizations, we investigated the molecular basis of PME3 inhibition by PMEI7 in Arabidopsis thaliana. Our complementary approach uncovered how changes in the protonation of amino acids at the complex interface shift the network of interacting residues between intermolecular and intramolecular. These shifts ultimately regulate the stability of the PME3–PMEI7 complex and the inhibition of the PME as a function of the pH. These findings suggest a general model of how pH-dependent proteinaceous inhibitors function. Moreover, they enhance our understanding of how PMEs may be regulated by pH and provide new insights into how this regulation may control the physical properties and structure of the plant cell wall.

The fine-tuning of the degree of methylesterification of cell wall pectin is a key to regulating cell elongation and ultimately the shape of the plant body. Pectin methylesterification is spatiotemporally controlled by pectin methylesterases (PMEs; 66 members in Arabidopsis [Arabidopsis thaliana]). The comparably large number of proteinaceous pectin methylesterase inhibitors (PMEIs; 76 members in Arabidopsis) questions the specificity of the PME-PMEI interaction and the functional role of such abundance. To understand the difference, or redundancy, between PMEIs, we used molecular dynamics (MD) simulations to predict the behavior of two PMEIs that are coexpressed and have distinct effects on plant development: AtPMEI4 and AtPMEI9. Simulations revealed the structural determinants of the pH dependence for the interaction of these inhibitors with AtPME3, a major PME expressed in roots. Key residues that are likely to play a role in the pH dependence were identified. The predictions obtained from MD simulations were confirmed in vitro, showing that AtPMEI9 is a stronger, less pH-independent inhibitor compared with AtPMEI4. Using pollen tubes as a developmental model, we showed that these biochemical differences have a biological significance. Application of purified proteins at pH ranges in which PMEI inhibition differed between AtPMEI4 and AtPMEI9 had distinct consequences on pollen tube elongation. Therefore, MD simulations have proven to be a powerful tool to predict functional diversity between PMEIs, allowing the discovery of a strategy that may be used by PMEIs to inhibit PMEs in different microenvironmental conditions and paving the way to identify the specific role of PMEI diversity in muro.

In response to neighbor proximity, plants increase the growth of specific organs (e.g., hypocotyls) to enhance access to sunlight. Shade enhances the activity of Phytochrome Interacting Factors (PIFs) by releasing these bHLH transcription factors from phytochrome B-mediated inhibition. PIFs promote elongation by inducing auxin production in cotyledons. In order to elucidate spatiotemporal aspects of the neighbor proximity response, we separately analyzed gene expression patterns in the major light-sensing organ (cotyledons) and in rapidly elongating hypocotyls of Arabidopsis thaliana PIFs initiate transcriptional reprogramming in both organs within 15 min, comprising regulated expression of several early auxin response genes. This suggests that hypocotyl growth is elicited by both local and distal auxin signals. We show that cotyledon-derived auxin is both necessary and sufficient to initiate hypocotyl growth, but we also provide evidence for the functional importance of the local PIF-induced response. With time, the transcriptional response diverges increasingly between organs. We identify genes whose differential expression may underlie organ-specific elongation. Finally, we uncover a growth promotion gene expression signature shared between different developmentally regulated growth processes and responses to the environment in different organs.

International audience; The degree of methylesterification (DM) of homogalacturonans (HGs), the main constituent of pectins in Arabidopsis thaliana, can be modified by pectin methylesterases (PMEs). Regulation of PME activity occurs through interaction with PME inhibitors (PMEIs) and subtilases (SBTs). Considering the size of the gene families encoding PMEs, PMEIs and SBTs, it is highly likely that specific pairs mediate localized changes in pectin structure with consequences on cell wall rheology and plant development. We previously reported that PME17, a group 2 PME expressed in root, could be processed by SBT3.5, a co-expressed subtilisin-like serine protease, to mediate changes in pectin properties and root growth. Here, we further report that a PMEI, PMEI4, is co-expressed with PME17 and is likely to regulate its activity. This sheds new light on the possible interplay of specific PMEs, PMEIs and SBTs in the fine-tuning of pectin structure.

Understanding the changes affecting the plant cell wall is a key element in addressing its functional role in plant growth and in the response to stress. Pectins, which are the main constituents of the primary cell wall in dicot species, play a central role in the control of cellular adhesion and thereby of the rheological properties of the wall. This is likely to be a major determinant of plant growth. How the discrete changes in pectin structure are mediated is thus a key issue in our understanding of plant development and plant responses to changes in the environment. In particular, understanding the remodelling of homogalacturonan (HG), the most abundant pectic polymer, by specific enzymes is a current challenge in addressing its fundamental role. HG, a polymer that can be methylesterified or acetylated, can be modified by HGMEs (HG-modifying enzymes) which all belong to large multigenic families in all species sequenced to date. In particular, both the degrees of substitution (methylesterification and/or acetylation) and polymerization can be controlled by specific enzymes such as pectin methylesterases (PMEs), pectin acetylesterases (PAEs), polygalacturonases (PGs), or pectate lyases-like (PLLs). Major advances in the biochemical and functional characterization of these enzymes have been made over the last 10 years. This review aims to provide a comprehensive, up to date summary of the recent data concerning the structure, regulation, and function of these fascinating enzymes in plant development and in response to biotic stresses.

International audience; Here, we focused on the biochemical characterization of the Arabidopsis thaliana pectin methylesterase 3 gene (AtPME3; At3g14310) and its role in plant development. A combination of biochemical, gene expression, Fourier transform-infrared (FT-IR) microspectroscopy and reverse genetics approaches were used. We showed that AtPME3 is ubiquitously expressed in A. thaliana, particularly in vascular tissues. In cell wall-enriched fractions, only the mature part of the protein was identified, suggesting that it is processed before targeting the cell wall. In all the organs tested, PME activity was reduced in the atpme3-1 mutant compared with the wild type. This was related to the disappearance of an activity band corresponding to a pI of 9.6 revealed by a zymogram. Analysis of the cell wall composition showed that the degree of methylesterification (DM) of galacturonic acids was affected in the atpme3-1 mutant. A change in the number of adventitious roots was found in the mutant, which correlated with the expression of the gene in adventitious root primordia. Our results enable the characterization of AtPME3 as a major basic PME isoform in A. thaliana and highlight its role in adventitious rooting.

Plant cell wall plays a key role in the control of cell elongation and differentiation as well as in defence against pathogens. Homogalacturonan (HG)-type pectin, one of the major components of the primary cell wall in dicotyledonous plants, comprises 1,4 linked α-D galacturonic acids (GalA) that can be methylesterified. HG is modified by homogalacturonan modifying enzymes (HGME), such as pectin methylesterase (PME), polygalacturonases (PG) and pectin/pectate lyases like (PLLs), that control their degree of methylesterification (DM) and polymerization (DP), respectively. Plant enzymes fine-tune the cell wall pectins during development, while pathogens’ enzymes degrade the pectin matrix to facilitate infection. These enzymes are encoded, in plants and pathogens, by large multigenic families, which questions the rationale for such abundance. Thus, to understand the biochemistry, catalytic mechanisms and structure/function relationships of these enzymes, we expressed and characterized isoforms from Verticillium dahliae (VdPME1, VdPG2 and VdPelB) and Arabidopsis thaliana (PGLR, ADPG2). VdPME1 had processive mode of action at pH 7 for demethylesterification, that could be related to the surface electrostatic properties of the protein, and which creates a substrate for VdPG2. VdPG2, act as an endo-enzyme and was most active at pH 5 and temperature of 50°C. VdPelB had surprising PNL-PL biochemical characteristics, being most active at alkaline pH in presence of Ca2+ ions and on high DM pectins. Using X-ray diffraction, we resolved the VdPelB structure at 1.2 Å and showed that it indeed differed from previously reported fungal enzymes. We further characterized Arabidopsis PGLR and ADPG2, whose expression overlaps in roots. LC-MS/MS analyses of digested pectins showed that the oligogalacturonides (OGs) released by both enzymes differed. To understand these differences in processivity, we resolved the structures of PGLR and ADPG2 at 1.3 and 2.03 Å, respectively. The two enzymes had right-handed parallel β-helix fold, characteristic for pectinases with the tunnel like active site allowing the release of OGs of distinct DP. If the amino acids at the active site were conserved, those in subsites differed, partly explaining substrate specificities. These first structures of plant PGs shed new light on the functional diversity of enzymes in plants and the differences with that of pathogens; La paroi cellulaire des plantes joue un rôle clé dans le contrôle de l'élongation et de la différenciation, ainsi que dans la défense des plantes face aux pathogènes. Les pectines de type homogalacturonanes (HG), un des composants majeurs de la paroi primaire des plantes dicotylédones, sont constituées d'acides galacturoniques (α-D GalA) liés en 1,4, qui peuvent être méthylestérifiés. Les HG sont modifiés par des enzymes telles que les pectine-méthylestérases (PME), les polygalacturonases (PG) et pectine-pectate lyases (PLL), qui contrôlent respectivement leur degré de méthylestérification (DM) et de polymérisation (DP). Alors que les enzymes de plantes permettent une régulation fine de la structure des pectines de la paroi lors du développement, celles de pathogènes sont impliquées dans la dégradation de celle-ci lors de l'infection. Elles sont par ailleurs codées par des familles multigéniques, ce qui questionne les raisons d'une telle abondance. Ainsi, pour comprendre leurs spécificités biochimiques, des isoformes de Verticillium dahliae (VdPME1, VdPG2 et VdPelB) et d'Arabidopsis thaliana (PGLR, ADPG2) ont été exprimées et caractérisées. VdPME1 déméthylestérifie les pectines de façon processive à pH 7, ce qui peut être relié au potentiel électrostatique de surface de la protéine, et créé des substrats préférentiels pour VdPG2, une endo-polygalacturonase. VdPelB présente des caractéristiques biochimiques intermédiaires entre une PNL et une PL : elle est plus active à pH alcalin et agit préférentiellement sur des pectines de fort DM en présence de calcium. La structure 3D de la protéine, ré’solue à 1,2 Å par diffraction au rayon X, permet de confirmer les particularités structurales de VdPelB en regard d'enzymes fongiques. PGLR et ADPG2, qui présentent un patron d'expression similaire dans les racines d'Arabidopsis, produisent des oligogalacturonides (OGs) distincts. Afin de comprendre ces différences de mode d'action, les structures 3D des protéines ont été déterminées à 1.3 (PGLR) et 2.03Å (ADPG2). Si les deux enzymes présentent une structure caractéristique des pectinases et un site actif conservé, la séquence particulière de leurs sous-sites, peut expliquer leurs différences de processivité. Ces structures de PG, les premières obtenues chez les plantes, permettent de comprendre leur diversité fonctionnelle ainsi que les différences majeures avec les enzymes de phytopathogènes