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.

Metabolomics-Based Profiling via a Chemometric Approach to Investigate the Antidiabetic Property of Different Parts and Origins of Pistacia lentiscus L.

Pistacia lentiscus L. is a medicinal plant that grows spontaneously throughout the Mediterranean basin and is traditionally used to treat diseases, including diabetes. The aim of this work consists of the evaluation of the α-glucosidase inhibitory effect (i.e., antidiabetic activity in vitro) of different extracts from the leaves, stem barks and fruits of P. lentiscus harvested on mountains and the littoral of Tizi-Ouzou in Algeria. Metabolomic profiling combined with a chemometric approach highlighted the variation of the antidiabetic properties of P. lentiscus according to the plant's part and origin. A multiblock OPLS analysis showed that the metabolites most involved in α-glucosidase inhibition activity were mainly found in the stem bark extracts. The highest inhibitory activity was found for the stem bark extracts, with averaged inhibition percentage values of 84.7% and 69.9% for the harvested samples from the littoral and mountain, respectively. On the other hand, the fruit extracts showed a lower effect (13.6%) at both locations. The UHPLC-ESI-HRMS characterization of the metabolites most likely responsible for the α-glucosidase-inhibitory activity allowed the identification of six compounds: epigallocatechin(4a>8)epigallocatechin (two isomers), (epi)gallocatechin-3'-O-galloyl-(epi)gallocatechin (two isomers), 3,5-O-digalloylquinic acid and dihydroxy benzoic acid pentoside.

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.

Isolation and Identification of Flavones Responsible for the Antibacterial Activities of Tillandsia bergeri Extracts.

Plants are an everlasting inspiration source of biologically active compounds. Among these medicinal plants, the biological activity of extracts from some species of the Tillandsia genus has been studied, but the phytochemistry of the hardy species Tillandsia bergeri remains unknown. The aim of the present study was to perform the first phytochemical study of T. bergeri and to identify the compounds responsible for the antibacterial activity of T. bergeri extracts. Soxhlet extraction of predried and grinded leaves was first performed using four increasing polarity solvents. A bio-guided fractionation was performed using agar overlay bioautography as a screening method against 12 Gram-positive, Gram-negative, sensitive, and resistant bacterial strains. The results showed the inhibition of Gram-positive methicillin-sensitive Staphylococcus aureus ATCC 29213 (MSSA), methicillin-resistant S. aureus N-SARM-1 (MRSA), and Staphylococcus caprae ATCC 35538 by the dichloromethane fraction. A phytochemical investigation led to the isolation and identification by high-resolution mass spectrometry and nuclear magnetic resonance of the two flavones penduletin and viscosine, responsible for this antibacterial activity. For viscosine, the minimum inhibitory concentration (MIC) value is equal to 128 µg/mL against MSSA and is equal to 256 µg/mL against MRSA and S. caprae. The combination of these compounds with vancomycin and cloxacillin showed a decrease in MICs of the antibiotics. Penduletin showed synergistic activity when combined with vancomycin against MSSA (FICI < 0.258) and S. caprae (FICI < 0.5). Thus, unexplored Tillandsia species may represent a valuable source for potential antibiotics and adjuvants.

Integument-Specific Transcriptional Regulation in the Mid-Stage of Flax Seed Development Influences the Release of Mucilage and the Seed Oil Content.

Flax (Linum usitatissimum L.) seed oil, which accumulates in the embryo, and mucilage, which is synthesized in the seed coat, are of great economic importance for food, pharmaceutical as well as chemical industries. Theories on the link between oil and mucilage production in seeds consist in the spatio-temporal competition of both compounds for photosynthates during the very early stages of seed development. In this study, we demonstrate a positive relationship between seed oil production and seed coat mucilage extrusion in the agronomic model, flax. Three recombinant inbred lines were selected for low, medium and high mucilage and seed oil contents. Metabolite and transcript profiling (1H NMR and DNA oligo-microarrays) was performed on the seeds during seed development. These analyses showed main changes in the seed coat transcriptome during the mid-phase of seed development (25 Days Post-Anthesis), once the mucilage biosynthesis and modification processes are thought to be finished. These transcriptome changes comprised genes that are putatively involved in mucilage chemical modification and oil synthesis, as well as gibberellic acid (GA) metabolism. The results of this integrative biology approach suggest that transcriptional regulations of seed oil and fatty acid (FA) metabolism could occur in the seed coat during the mid-stage of seed development, once the seed coat carbon supplies have been used for mucilage biosynthesis and mechanochemical properties of the mucilage secretory cells.

Optimizing 1D 1H-NMR profiling of plant samples for high throughput analysis: extract preparation, standardization, automation and spectra processing.

INTRODUCTION: Proton nuclear magnetic resonance spectroscopy (1H-NMR)-based metabolomic profiling has a range of applications in plant sciences. OBJECTIVES: The aim of the present work is to provide advice for minimizing uncontrolled variability in plant sample preparation before and during NMR metabolomic profiling, taking into account sample composition, including its specificity in terms of pH and paramagnetic ion concentrations, and NMR spectrometer performances. METHODS: An automation of spectrometer preparation routine standardization before NMR acquisition campaign was implemented and tested on three plant sample sets (extracts of durum wheat spikelet, Arabidopsis leaf and root, and flax leaf, root and stem). We performed 1H-NMR spectroscopy in three different sites on the wheat sample set utilizing instruments from two manufacturers with different probes and magnetic field strengths. The three collections of spectra were processed separately with the NMRProcFlow web tool using intelligent bucketing, and the resulting buckets were subjected to multivariate analysis. RESULTS: Comparability of large- (Arabidopsis) and medium-size (flax) datasets measured at 600 MHz and from the wheat sample set recorded at the three sites (400, 500 and 600 MHz) was exceptionally good in terms of spectral quality. The coefficient of variation of the full width at half maximum (FWHM) and the signal-to-noise ratio (S/N) of two selected peaks was comprised between 5 and 10% depending on the size of sample set and the spectrometer field. EDTA addition improved citrate and malate resonance patterns for wheat sample sets. A collection of 22 samples of wheat spikelet extracts was used as a proof of concept and showed that the data collected at the three sites on instruments of different field strengths and manufacturers yielded the same discrimination pattern of the biological groups. CONCLUSION: Standardization or automation of several steps from extract preparation to data reduction improves data quality for small to large collections of plant samples of different origins.