Structural and biochemical characterization of SmoPG1, an exo-polygalacturonase from Selaginella moellendorffii.

Polygalacturonases (PGs) can modulate chemistry and mechanical properties of the plant cell wall through the degradation of pectins, one of its major constituents. PGs are largely used in food, beverage, textile, and paper industries to increase processes' performances. To improve the use of PGs, knowledge of their biochemical, structural and functional features is of prime importance. Our study aims at characterizing SmoPG1, a polygalacturonase from Selaginella moellendorffii, that belongs to the lycophytes. Transcription data showed that SmoPG1 was mainly expressed in S. moellendorffii shoots while phylogenetic analyses suggested that SmoPG1 is an exo-PG, which was confirmed by the biochemical characterization following its expression in heterologous system. Indeed, LC-MS/MS oligoprofiling using various pectic substrates identified galacturonic acid (GalA) as the main hydrolysis product. We found that SmoPG1 was most active on polygalacturonic acid (PGA) at pH 5, and that its activity could be modulated by different cations (Ca2+, Cu2+, Fe2+, Mg2+, Mn2+, Na2+, Zn2+). In addition, SmoPG1 was inhibited by green tea catechins, including (-)-epigallocatechin-3-gallate (EGCG). Docking analyses and MD simulations showed in detail amino acids responsible for the SmoPG1-EGCG interaction. Considering its expression yield and activity, SmoPG1 appears as a prime candidate for the industrial production of GalA.

Mutation of AtPME2, a pH-Dependent Pectin Methylesterase, Affects Cell Wall Structure and Hypocotyl Elongation.

Pectin methylesterases (PMEs) modify homogalacturonan's chemistry and play a key role in regulating primary cell wall mechanical properties. Here, we report on Arabidopsis AtPME2, which we found to be highly expressed during lateral root emergence and dark-grown hypocotyl elongation. We showed that dark-grown hypocotyl elongation was reduced in knock-out mutant lines as compared to the control. The latter was related to the decreased total PME activity as well as increased stiffness of the cell wall in the apical part of the hypocotyl. To relate phenotypic analyses to the biochemical specificity of the enzyme, we produced the mature active enzyme using heterologous expression in Pichia pastoris and characterized it through the use of a generic plant PME antiserum. AtPME2 is more active at neutral compared to acidic pH, on pectins with a degree of 55-70% methylesterification. We further showed that the mode of action of AtPME2 can vary according to pH, from high processivity (at pH8) to low processivity (at pH5), and relate these observations to the differences in electrostatic potential of the protein. Our study brings insights into how the pH-dependent regulation by PME activity could affect the pectin structure and associated cell wall mechanical properties.

Plant polygalacturonase structures specify enzyme dynamics and processivities to fine-tune cell wall pectins.

Polygalacturonases (PGs) fine-tune pectins to modulate cell wall chemistry and mechanics, impacting plant development. The large number of PGs encoded in plant genomes leads to questions on the diversity and specificity of distinct isozymes. Herein, we report the crystal structures of 2 Arabidopsis thaliana PGs, POLYGALACTURONASE LATERAL ROOT (PGLR), and ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2 (ADPG2), which are coexpressed during root development. We first determined the amino acid variations and steric clashes that explain the absence of inhibition of the plant PGs by endogenous PG-inhibiting proteins (PGIPs). Although their beta helix folds are highly similar, PGLR and ADPG2 subsites in the substrate binding groove are occupied by divergent amino acids. By combining molecular dynamic simulations, analysis of enzyme kinetics, and hydrolysis products, we showed that these structural differences translated into distinct enzyme-substrate dynamics and enzyme processivities: ADPG2 showed greater substrate fluctuations with hydrolysis products, oligogalacturonides (OGs), with a degree of polymerization (DP) of ≤4, while the DP of OGs generated by PGLR was between 5 and 9. Using the Arabidopsis root as a developmental model, exogenous application of purified enzymes showed that the highly processive ADPG2 had major effects on both root cell elongation and cell adhesion. This work highlights the importance of PG processivity on pectin degradation regulating plant development.

Putative pectate lyase PLL12 and callose deposition through polar CALS7 are necessary for long-distance phloem transport in Arabidopsis.

In plants, the phloem distributes photosynthetic products for metabolism and storage over long distances. It relies on specialized cells, the sieve elements, which are enucleated and interconnected through large so-called sieve pores in their adjoining cell walls. Reverse genetics identified PECTATE LYASE-LIKE 12 (PLL12) as critical for plant growth and development. Using genetic complementations, we established that PLL12 is required exclusively late during sieve element differentiation. Structural homology modeling, enzyme inactivation, and overexpression suggest a vital role for PLL12 in sieve-element-specific pectin remodeling. While short distance symplastic diffusion is unaffected, the pll12 mutant is unable to accommodate sustained plant development due to an incapacity to accommodate increasing hydraulic demands on phloem long-distance transport as the plant grows-a defect that is aggravated when combined with another sieve-element-specific mutant callose synthase 7 (cals7). Establishing CALS7 as a specific sieve pore marker, we investigated the subcellular dynamics of callose deposition in the developing sieve plate. Using fluorescent CALS7 then allowed identifying structural defects in pll12 sieve pores that are moderate at the cellular level but become physiologically relevant due to the serial arrangement of sieve elements in the sieve tube. Overall, pectin degradation through PLL12 appears subtle in quantitative terms. We therefore speculate that PLL12 may act as a regulator to locally remove homogalacturonan, thus potentially enabling further extracellular enzymes to access and modify the cell wall during sieve pore maturation.

The specificity of pectate lyase VdPelB from Verticilium dahliae is highlighted by structural, dynamical and biochemical characterizations.

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 ß-elimination, releasing unsaturated oligogalacturonides (OGs). To understand the catalytic mechanism and structure/function of these enzymes, we characterized VdPelB from Verticillium dahliae. We first solved the crystal structure of VdPelB at 1.2 Å resolution showing that it is a right-handed parallel ß-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. Pectate lyase VdPelB was most active on non-methylesterified pectins, at pH 8.0 in presence of Ca2+ ions. The VdPelB-G125R mutant was most active at pH 9.0 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.

Auxin triggers pectin modification during rootlet emergence in white lupin.

Emergence of secondary roots through parental tissue is a highly controlled developmental process. Although the model plant Arabidopsis has been useful to uncover the predominant role of auxin in this process, its simple root structure is not representative of how emergence takes place in most plants, which display more complex root anatomy. White lupin is a legume crop producing structures called cluster roots, where closely spaced rootlets emerge synchronously. Rootlet primordia push their way through several cortical cell layers while maintaining the parent root integrity, reflecting more generally the lateral root emergence process in most multilayered species. In this study, we showed that lupin rootlet emergence is associated with an upregulation of cell wall pectin modifying and degrading genes under the active control of auxin. Among them, we identified LaPG3, a polygalacturonase gene typically expressed in cells surrounding the rootlet primordium and we showed that its downregulation delays emergence. Immunolabeling of pectin epitopes and their quantification uncovered a gradual pectin demethylesterification in the emergence zone, which was further enhanced by auxin treatment, revealing a direct hormonal control of cell wall properties. We also report rhamnogalacturonan-I modifications affecting cortical cells that undergo separation as a consequence of primordium outgrowth. In conclusion, we describe a model of how external tissues in front of rootlet primordia display cell wall modifications to allow for the passage of newly formed rootlets.

New insights into the specificity and processivity of two novel pectinases from Verticillium dahliae.

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 oligoprofiling, 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 as endo-PG releasing both methylesterified and non-methylesterified oligogalacturonides (OGs). Additionally, when flax roots were used as substrate, acetylated 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 exogenous application of AtPGLR, an endo-polygalacturonase, triggers pollen tube burst and repair.

Plant cell wall remodeling plays a key role in the control of cell elongation and differentiation. In particular, fine-tuning of the degree of methylesterification of pectins was previously reported to control developmental processes as diverse as pollen germination, pollen tube elongation, emergence of primordia or elongation of dark-grown hypocotyls. However, how pectin degradation can modulate plant development has remained elusive. Here we report the characterization of a polygalacturonase (PG), AtPGLR, the gene for which is highly expressed at the onset of lateral root emergence in Arabidopsis. Due to gene compensation mechanisms, mutant approaches failed to determine the involvement of AtPGLR in plant growth. To overcome this issue, AtPGLR has been expressed heterologously in the yeast Pichia pastoris and biochemically characterized. We showed that AtPGLR is an endo-PG that preferentially releases non-methylesterified oligogalacturonides with a short degree of polymerization (< 8) at acidic pH. The application of the purified recombinant protein on Amaryllis pollen tubes, an excellent model for studying cell wall remodeling at acidic pH, induced abnormal pollen tubes or cytoplasmic leakage in the subapical dome of the pollen tube tip, where non-methylesterified pectin epitopes are detected. Those leaks could either be repaired by new ß-glucan deposits (mostly callose) in the cell wall or promoted dramatic burst of the pollen tube. Our work presents the full biochemical characterization of an Arabidopsis PG and highlights the importance of pectin integrity in pollen tube elongation.