Boron bridging of rhamnogalacturonan-II in Rosa and arabidopsis cell cultures occurs mainly in the endo-membrane system and continues at a reduced rate after secretion

BACKGROUND AND AIMS: Rhamnogalacturonan-II (RG-II) is a domain of primary cell-wall pectin. Pairs of RG-II domains are covalently cross-linked via borate diester bridges, necessary for normal cell growth. Interpreting the precise mechanism and roles of boron bridging is difficult because there are conflicting hypotheses as to whether bridging occurs mainly within the Golgi system, concurrently with secretion or within the cell wall. We therefore explored the kinetics of RG-II bridging. METHODS: Cell-suspension cultures of Rosa and arabidopsis were pulse-radiolabelled with [(14)C]glucose, then the boron bridging status of newly synthesized [(14)C]RG-II domains was tracked by polyacrylamide gel electrophoresis of endo-polygalacturonase digests. KEY RESULTS: Optimal culture ages for (14)C-labelling were ~5 and ~1 d in Rosa and arabidopsis respectively. De-novo [(14)C]polysaccharide production occurred for the first ~90 min; thereafter the radiolabelled molecules were tracked as they ‘aged’ in the wall. Monomeric and (boron-bridged) dimeric [(14)C]RG-II domains appeared simultaneously, both being detectable within 4 min of [(14)C]glucose feeding, i.e. well before the secretion of newly synthesized [(14)C]polysaccharides into the apoplast at ~15–20 min. The [(14)C]dimer : [(14)C]monomer ratio of RG-II remained approximately constant from 4 to 120 min, indicating that boron bridging was occurring within the Golgi system during polysaccharide biosynthesis. However, [(14)C]dimers increased slightly over the following 15 h, indicating that limited boron bridging was continuing after secretion. CONCLUSIONS: The results show where in the cell (and thus when in the ‘career’ of an RG-II domain) boron bridging occurs, helping to define the possible biological roles of RG-II dimerization and the probable localization of boron-donating glycoproteins or glycolipids

Arabinogalactan-Proteins as Boron-Acting Enzymes, Cross-Linking the Rhamnogalacturonan-II Domains of Pectin

Most pectic rhamnogalacturonan-II (RG-II) domains in plant cell walls are borate-bridged dimers. However, the sub-cellular locations, pH dependence, reversibility and biocatalyst involvement in borate bridging remain uncertain. Experiments discussed here explored these questions, utilising suspension-cultured plant cells. In-vivo pulse radiolabelling showed that most RG-II domains dimerise extremely quickly (3 required the simultaneous presence of RG-II-binding ‘chaperones’: co-ordinately binding metals and/or ionically binding cationic peptides. Natural chaperones of the latter type include highly basic arabinogalactan protein fragments, e.g., KHKRKHKHKRHHH, which catalyse a reaction [2 RG-II + B(OH)3 → RG-II–B–RG-II], suggesting that plants can ‘enzymically’ metabolise boron

Allelochemical root-growth inhibitors in low-molecular-weight cress-seed exudate

Background and aims. Cress seeds release allelochemicals that overstimulate the elongation of neighbouring (potentially competing) seedlings’ hypocotyls and inhibit their root growth. The hypocotyl promoter is potassium, but the root inhibitor was unidentified; its nature is investigated here.Methods. Low-molecular-weight cress-seed exudate (LCSE) from imbibed Lepidium sativum seeds was fractionated by phase partitioning, paper chromatography, high-voltage electrophoresis and gel-permeation chromatography (on Bio-Gel P-2). Fractions, compared with pure potassium salts, were bioassayed for effects on Amaranthus caudatus seedling growth in the dark for 4 days.Key results. LCSE robustly promoted amaranth hypocotyl elongation and inhibited root growth. The hypocotyl inhibitor was non-volatile, hot-acid-stable, hydrophilic, and resistant to incineration — as expected for K+. The root inhibitor(s) had similar properties but were organic (activity lost on incineration). The root inhibitor(s) remained in the aqueous phase (at pH 2.0, 6.5 and 9.0) when partitioned against butan-1-ol or toluene, and were thus hydrophilic. Activity was diminished after electrophoresis, but the remaining root-inhibitors were neutral. They became undetectable after paper chromatography; therefore, they probably comprised multiple compounds, which partially separated from each other during fractionation. On gel-permeation chromatography, the root inhibitor co-eluted with hexoses. Conclusions. Cress-seed allelochemicals inhibiting root growth are different from the agent (K+) that over-stimulates hypocotyl elongation, and probably comprise a mixture of small, non-volatile, hydrophilic, organic substances. Abundant components identified chromatographically and by electrophoresis in cress-seed exudate fitting this description include glucose, fructose, sucrose and galacturonic acid. However, none of these sugars co-chromatographed and co-electrophoresed with the root-inhibitory principle of LCSE, and none of them (in pure form at naturally occurring concentrations) inhibited root growth. We conclude that the root-inhibiting allelochemicals of cress seed exudate remain unidentified. <br/

An Arabidopsis thaliana arabinogalactan-protein (AGP31) and several cationic AGP fragments catalyse the boron bridging of rhamnogalacturonan-II

Rhamnogalacturonan-II (RG-II) is a complex pectic domain in plant primary cell walls. In vivo, most RG-II domains are covalently dimerised via borate diester bridges, essential for correct cell-wall assembly, but the dimerisation of pure RG-II monomers by boric acid in vitro is extremely slow. Cationic ‘chaperones’ can promote dimerisation, probably by overcoming the mutual repulsion between neighbouring anionic RG-II molecules. Highly effective artificial chaperones include Pb(2+) and polyhistidine, but the proposed natural chaperones remained elusive. We have now tested cationic peptide fragments of several Arabidopsis thaliana arabinogalactan-proteins (AGPs) as candidates. Fragments of AGP17, 18, 19 and 31 were effective, typically at ∼25 µg/ml (9–19 µM), promoting the boron bridging of 16–20 µM monomeric RG-II at pH 4.8 in vitro. Native AGP31 glycoprotein was also effective, and hexahistidine was moderately so. All chaperones tested interacted reversibly with RG-II and were not consumed during the reaction; thus they acted catalytically, and may constitute the first reported boron-acting enzyme activity, an RG-II borate diesterase. Many of the peptide chaperones became less effective catalysts at higher concentration, which we interpret as due to the formation of RG-II–peptide complexes with a net positive charge, as mutually repulsive as negatively charged pure RG-II molecules. The four unique AGPs studied here may serve an enzymic role in the living plant cell, acting on RG-II within Golgi cisternae and/or in the apoplast after secretion. In this way, RG-II and specific AGPs may contribute to cell-wall assembly and hence plant cell expansion and development

Boron cross-linking of rhamnogalacturonan-II in vivo and in vitro: effects of pH and cationic chaperones.

Rhamnogalacturonan-II (RG-II) is a ~5 kDa pectic polysaccharide domain, which typically represents 1–4% of the total polysaccharide of the primary cell wall in dicots. It has a complex structure with a backbone of 8-10 galacturonic acid (GalA) residues, to which are attached 6 different sidechains (A–F). In vivo, RG-II is prevalent in its dimeric form, which is produced by borate-diester bridge formation between two sidechain A apiose residues from two RG-II molecules. RG-II dimerisation is important for maintaining cell wall porosity, thickness, and biophysical properties for cell growth. But dimerisation is not inevitable even in the presence of boric acid; it also requires the presence of a cationic ‘chaperone’. In this project, the effect of acidic pH on RG-II was studied in vitro, to understand the role of RG-II and its dimerisation during auxin induced acid growth of cells. The effect of alkaline pH on RG-II was investigated to understand the importance of ester groups affecting the charge:mass ratio of RG-II. In-vivo 14C-radiolabelling using Paul’s scarlet Rosa and Arabidopsis cultures was conducted to understand the kinetics of RG-II dimerisation. RG-II monomers and dimers were separated and observed using polyacrylamide gel electrophoresis and silver nitrate staining, and newly synthesised RG-II domains were traced in vivo by pulse-labelling with [14C]glucose. In-vitro study on the effect of acidic pH showed that RG-II dimerisation is favoured at physiologically acidic pH (~4.0–5.0, mimicking the apoplastic pH during auxin-induced growth) in presence of boron and cationic chaperones. Alkaline pH affected the esterification status of RG-II, hence controlling the sites accessible to pectolytic enzymes and affecting the charge:mass ratio of RG-II. The in-vivo 14C-radiolabelling kinetic study on living Rosa and Arabidopsis cells showed that [14C]RG-II is synthesised and dimerised intra-cellularly, rather than after secretion. Another study supported this intracellular localisation of dimerisation by showing the inability of added apoplastic apiose (which cannot enter the symplast) to prevent RG-II dimerisation. A study conducted on glycosylinositol phosphorylceramide (GIPCs) gave inconclusive results regarding its hypothesized role as a boron-donor. Alongside these studies, it was found that the three classical lysine-rich arabinogalactan proteins of A. thaliana (AtAGP17, AtAGP18 and AtAGP19) are potential biological chaperones for promoting RG-II dimerisation: specifically, certain highly basic peptide fragments (DP 10– 13) of these three AGPs promoted RG-II dimerisation in vitro. Overall, these studies open the possibility of conducting future studies for revealing the detailed biochemical character and biological roles of RG-II and the proposed AGPs as cationic chaperones for RG-II dimerisation in vivo

An Arabidopsis thaliana arabinogalactan-protein (AGP31) and several cationic AGP fragments catalyse the boron bridging of rhamnogalacturonan-II

International audienceRhamnogalacturonan-II (RG-II) is a complex pectic domain in plant primary cell walls. In vivo, most RG-II domains are covalently dimerised via borate diester bridges, essential for correct cell-wall assembly, but the dimerisation of pure RG-II monomers by boric acid in vitro is extremely slow. Cationic ‘chaperones’ can promote dimerisation, probably by overcoming the mutual repulsion between neighbouring anionic RG-II molecules. Highly effective artificial chaperones include Pb2+ and polyhistidine, but the proposed natural chaperones remained elusive. We have now tested cationic peptide fragments of several Arabidopsis thaliana arabinogalactan-proteins (AGPs) as candidates. Fragments of AGP17, 18, 19 and 31 were effective, typically at ~25 µg/ml (9–19 µM), promoting the boron bridging of 16–20 µM monomeric RG-II at pH 4.8 in vitro. Native AGP31 glycoprotein was also effective, and hexahistidine was moderately so. All chaperones tested interacted reversibly with RG-II and were not consumed during the reaction; thus they acted catalytically, and may constitute the first reported boron-acting enzyme activity, an RG-II borate diesterase. Many of the peptide chaperones became less effective catalysts at higher concentration, which we interpret as due to the formation of RG-II–peptide complexes with a net positive charge, as mutually repulsive as negatively charged pure RG-II molecules. The four unique AGPs studied here may serve an enzymic role in the living plant cell, acting on RG-II within Golgi cisternae and/or in the apoplast after secretion. In this way, RG-II and specific AGPs may contribute to cell-wall assembly and hence plant cell expansion and development

Plant growth-promoting endophytic fungi of the wild halophytic rice Oryza coarctata

Abstract Background Plant growth-promoting endophytic fungi (PGPEF) that are associated with halophytes have the potential to boost crop salinity tolerance and productivity. This in turn has the potential of enabling and improving cultivation practices in coastal lands affected by salt stress. Methods Endophytic fungi from the wild halophytic rice Oryza coarctata were isolated, characterized, identified, and studied for their effects on all developing stages of rice plant growth and their yields both with and without salt stress. Key results In this study, three different fungal endophytes were isolated from the halophytic wild rice Oryza coarctata. Two isolates were identified as Talaromyces adpressus (OPCRE2) and Talaromyces argentinensis (OPCRh1) by ITS region sequencing. The remaining isolate NPCRE2 was confirmed as a novel strain named Aspergillus welwitschiae Ocstreb1 (AwOcstreb1) by whole genome sequencing. These endophytes showed various plant growth-promoting (PGP) abilities in vitro (e.g., IAA, ACC-deaminase and siderophore production, phosphate, and zinc solubilization as well as nitrogen fixation), where AwOcstreb1 was significantly more efficient compared to the other two isolates at high salinity (900 mm). Independent application of these fungi in commercial rice (Oryza sativa) showed significant elevation in plant growth, especially in the case of the AwOcstreb1 inoculants, which had enhanced metabolite and chlorophyll content at the seedling stage in both no-salt control and 100-mm salt-stressed plants. At the same time, AwOcstreb1-treated plants had a significantly lower level of H2O2, electrolyte leakage, and Na+/K+ ratio under saline conditions. Higher expression (1.6 folds) of the SOS1 (salt overly sensitive 1) gene was also observed in these plants under salinity stress. This strain also improved percent fertility, tillering, panicle number, and filled grain number in both no-salt control and 45-mm salt-stressed inoculated plants at the reproductive stage. Consequently, the differences in their yield was 125.16% and 203.96% (p < 0.05) in colonized plants in normal and saline conditions, respectively, compared to uninoculated controls. Conclusions We propose that AwOcstreb1 is a potential candidate for an eco-friendly biofertilizer formula to improve the cultivation and yield of rice or any other crop in the highly saline coastal regions of Bangladesh