Biology subjects

Senechal, F.

Publications and source records attributed to Senechal, F..

7 recordsLinked to original sources

bioRxiv2023

Pectin methylesterification modulates cell wall properties to promote neighbour proximity-induced hypocotyl growth

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 the cgr2cgr3 double 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

plant biology↗

bioRxiv2022

The structural, dynamical and biochemical characterizations of Verticillium dahliae pectate lyase, VdPelB, highlight its specificities

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 {beta}-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB from Verticillium dahliae, a plant pathogen. We first solved the crystal structure of VdPelB at 1.2[A] resolution showing that it is a right-handed parallel {beta}-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 from Verticillium-partially tolerant and sensitive flax cultivars differed which could facilitate the identification VdPelB-mediated elicitors of defence responses.

biochemistry↗

bioRxiv2022

Differences in the structure of plant polygalacturonases specify enzymes dynamics and processivities to fine-tune pectins and root development

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.

biochemistry↗

bioRxiv2022

Robustness of organ morphology is associated with modules of co-expressed genes related to plant cell wall

Control of organ morphology is a fundamental feature of living organisms. There is, however, observable variation in organ size and shape within a given genotype. Taking the sepal of Arabidopsis as a model, we investigated whether we can use variability of gene expression alongside variability of organ morphology to identify gene regulatory networks potentially involved in organ size and shape determination. We produced a dataset composed of morphological parameters and genome-wide transcriptome obtained from 27 individual sepals from wild-type plants with nearly identical genetic backgrounds, environment, and developmental stage. Sepals exhibited appreciable variability in both morphology and transcriptome, with response to stimulus genes and cell-wall related genes displaying high variability in expression. We additionally identified five modules of co-expressed genes which correlated significantly with morphology, revealing biologically relevant gene regulatory networks. Interestingly, cell-wall related genes were overrepresented in two of the top three modules. Overall, our work highlights the benefit of using coupled variation in gene expression and phenotype in wild-type plants to shed light on the mechanisms underlying organ size and shape determination. Although causality between gene expression and sepal morphology has not been established, our approach opens the way to informed analysis for mutant characterization and functional studies.

plant biology↗

bioRxiv2022

Biochemical characterization of Pectin Methylesterase Inhibitor 3 from Arabidopsis thaliana

The Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR 3 (PMEI3) gene is frequently used as a tool to manipulate PME activity in vivo, in studies assessing the role of pectin de-methylesterification in the control of cell expansion. One limitation of these studies is that the exact biochemical activity of this protein has not yet been determined. In this manuscript we produced the protein in Pichia pastoris and characterized its activity in vitro. Like other PMEIs, PMEI3 inhibits PME activity in acidic pH conditions for a variety of cell wall extracts and for purified PME preparations, but doesnt affect PME activity at neutral pH. This suggests that the previously observed in vivo effects reflect the inhibition of PME activity at low pH. The protein is remarkable heat stable and shows higher activity against PME3 than against PME2, illustrating how different members of the large PMEI family can differ in their specificities towards PME targets. Finally, application of purified PMEI3 on Arabidopsis thaliana seedlings showed a dose-dependent inhibition of homogalacturonan de-methylesterification and root growth. Purified recombinant PMEI3 is therefore a powerful tool to study the connection between pectin methylesterification and cell expansion.

plant biology↗

bioRxiv2021

Pectin remodeling belongs to a homeostatic system and triggers transcriptomic and hormonal modulations

O_LIHere, we focused on the biological modifications arisen from a strong and transient variation of the pectin methylesterification status during the seed-to-seedling transition. C_LIO_LIA reverse genetic approach was used to trigger specific reduction of pectin de-methylesterification during the seed maturation stage and the related physiological effects were assessed using a combination of biochemical, transcriptomic and microscopic analyses. C_LIO_LIArabidopsis PME36 is required to implement the characteristic pattern of de-methylesterified pectin in the mature seed. While this pattern is strongly impaired in pme36-1 and pme36-2 mature seed, no phenotypical effect is observed in the knockout mutant during seed germination. By analyzing hormone homeostasis and gene expression regulation, we show a strong and dynamic physiological disorder in the mutant, which reveals the existence of a complex compensatory mechanism overcoming the defect in pectin de-methylesterification. C_LIO_LIOur results reveal that pectin methylesterification status acts as upstream modulator involved in an undescribed homeostatic system in which pectin remodeling, hormone signaling and transcriptomic regulations interact to ensure the maintenance of a normal seed-to-seedling developmental program. C_LI

plant biology↗

bioRxiv2021

The pH-dependent processivity of Arabidopsis AtPME2 can control cell wall mechanical properties

Pectin methylesterases (PMEs) modify homogalacturonans (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 summaryThe 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.

plant biology↗