Making and breaking of boron bridges in the pectic domain rhamnogalacturonan-II at apoplastic pH in vivo and in vitro.

Cross-linking of the cell-wall pectin domain rhamnogalacturonan-II (RG-II) via boron bridges between apiose residues is essential for normal plant growth and development, but little is known about its mechanism or reversibility. We characterized the making and breaking of boron bridges in vivo and in vitro at 'apoplastic' pH. RG-II (13-26 µm) was incubated in living Rosa cell cultures and cell-free media with and without 1.2 mm H3 BO3 and cationic chaperones (Ca2+ , Pb2+ , polyhistidine, or arabinogalactan-protein oligopeptides). The cross-linking status of RG-II was monitored electrophoretically. Dimeric RG-II was stable at pH 2.0-7.0 in vivo and in vitro. In-vitro dimerization required a 'catalytic' cation at all pHs tested (1.75-7.0); thus, merely neutralizing the negative charge of RG-II (at pH 1.75) does not enable boron bridging. Pb2+ (20-2500 µm) was highly effective at pH 1.75-4.0, but not 4.75-7.0. Cationic peptides were effective at approximately 1-30 µm; higher concentrations caused less dimerization, probably because two RG-IIs then rarely bonded to the same peptide molecule. Peptides were ineffective at pH 1.75, their pH optimum being 2.5-4.75. d-Apiose (>40 mm) blocked RG-II dimerization in vitro, but did not cleave existing boron bridges. Rosa cells did not take up d-[U-14 C]apiose; therefore, exogenous apiose would block only apoplastic RG-II dimerization in vivo. In conclusion, apoplastic pH neither broke boron bridges nor prevented their formation. Thus boron-starved cells cannot salvage boron from RG-II, and 'acid growth' is not achieved by pH-dependent monomerization of RG-II. Divalent metals and cationic peptides catalyse RG-II dimerization via co-ordinate and ionic bonding respectively (possible and impossible, respectively, at pH 1.75). Exogenous apiose may be useful to distinguish intra- and extra-protoplasmic dimerization.

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

BACKGROUND AND AIMS: Cress seeds release allelochemicals that over-stimulate the elongation of hypocotyls of neighbouring (potentially competing) seedlings 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: The 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 separated from each other, in part, 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 the former 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.

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 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.

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 [14C]glucose, then the boron bridging status of newly synthesized [14C]RG-II domains was tracked by polyacrylamide gel electrophoresis of endo-polygalacturonase digests. KEY RESULTS: Optimal culture ages for 14C-labelling were ~5 and ~1 d in Rosa and arabidopsis respectively. De-novo [14C]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 [14C]RG-II domains appeared simultaneously, both being detectable within 4 min of [14C]glucose feeding, i.e. well before the secretion of newly synthesized [14C]polysaccharides into the apoplast at ~15-20 min. The [14C]dimer : [14C]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, [14C]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 (<4 min after biosynthesis, thus while still intraprotoplasmic). This tallies with the finding that boron withdrawal causes cell wall weakening within 10-20 min, and supports a previously proposed biological role for boron/RG-II complexes specifically at the wall/membrane interface. We also discuss RG-II monomer ↔ dimer interconversion as monitored in vitro using gel electrophoresis and a novel thin-layer chromatography method to resolve monomers and dimers. Physiologically relevant acidity did not monomerise dimers, thus boron bridge breaking cannot be a wall-loosening mechanism in 'acid growth'; nevertheless, recently discovered RG-II trimers and tetramers are unstable and may thus underpin reversible wall loosening. Dimerising monomers in vitro by B(OH)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.

Effects of multiple halotolerant rhizobacteria on the tolerance, growth, and yield of rice plants under salt stress.

Halotolerant bacteria get adapted to a saline environment through modified physiological/structural characteristics and may provide stress tolerance along with enhanced growth to the host plants by different direct and indirect mechanisms. This study reports on multiple halotolerant plant growth-promoting rhizobacteria isolated from the coastal soils in Bangladesh, in fields where the halophytic wild rice Oryza coarctata is endemic. The aim was to find halotolerant bacteria for potential use as biofertilizer under normal/salt-stressed conditions. In this study, eight different strains were selected from a total of 20 rhizobacterial isolates from the saline-prone regions of Debhata and Satkhira based on their higher salt tolerance. 16S rRNA gene sequencing results of the rhizobacterial strains revealed that they belonged to Halobacillus, Bacillus, Acinetobactor, and Enterobactor genera. A total of ten halotolerant rhizobacteria (the other 2 bacteria were previously isolated and already reported as beneficial for rice growth) were used as both single inoculants and in combinations and applied to rice growing in pots. To investigate their capability to improve rice growth, physiological parameters such as shoot and root length and weight, chlorophyll content at the seedling stage as well as survival and yield at the reproductive stage were measured in the absence or presence (in concentration 40 or 80 mmol/L) of NaCl and in the absence or presence of the rhizobacteria. At the reproductive stage, only 50% of the uninoculated plants survived without setting any grains in 80 mmol/L NaCl in contrast to 100% survival of the rice plants inoculated with a combination of the rhizobacteria. The combined halotolerant rhizobacterial inoculations showed significantly higher chlorophyll retention as well as yield under the maximum NaCl concentration applied compared to application of single species. Thus, the use of a combination of halotolerant rhizobacteria as bioinoculants for rice plants under moderate salinity can synergistically alleviate the effects of stress and promote rice growth and yield.

MPDB 1.0: a medicinal plant database of Bangladesh.

UNLABELLED: The term of medicinal plants include a various types of plants used in herbalism with medicinal activities. These plants are considered as rich resources of ingredients which can be used as complementary and alternative medicines and, also in drug developments and synthesis. In addition, some plants regarded as valuable origin of nutrition. Thus, all these plants are recommended as therapeutic agents. Information related to medicinal plants and herbal drugs accumulated over the ages are scattered and unstructured which make it prudent to develop a curated database for medicinal plants. MPDB 1.0 database is dedicated to provide the first window to find the plants around Bangladesh claimed to have medicinal and/or nutritive values by accumulating data from the published literatures. This database contains 406 medicinal plants with their corresponding scientific, family and local names as well as utilized parts for treatment from different districts of Bangladesh. Information regarding ailments is available for 353 plants. In addition, we have found active compounds for 78 plants with their corresponding PubMed ID. AVAILABILITY: www.medicinalplantbd.net.