Why are Chloris gayana leaves shorter in salt-affected plants? Analyses in the elongation zone

Reduced hydraulic conductance calculated from growth data was suggested to be the main reason for reduced leaf expansion in salt-stressed Chloris gayana (Rhodes grass). In this work, xylem vessel cross-sections and wall enzyme activities were analysed to re-examine the effects of salinity on leaf growth in this species. Maximal segmental growth rates were 20% lower and the growth zone was 23% shorter in leaves from salinized plants than in controls; however, growth rates between 0 mm and 15 mm from the ligule were similar in both types of leaves. Xylem cross-sectional areas in this region were about 65% smaller in leaves of salinized plants, suggesting that hydraulic restrictions in the leaves of salinized plants were much higher than overall growth reductions. Extractable xyloglucan endotransglucosylase activity in this zone was twice as high in leaves of salinized plants as in leaves of controls. Nevertheless, the activity of the extracted enzyme was not affected by up to 1 M NaCl added to the reaction medium. Therefore, increased xyloglucan endotransglucosylase activity under salinity may be due to a promotion of transcription of XTH (xyloglucan endotransglucosylase/hydrolases) genes and/or translation of preformed transcripts. These results suggest that, as in drought stress, increased activity of cell wall enzymes associated with wall loosening may contribute to the maintenance of growth under saline conditions despite hydraulic restrictions.Instituto de Fisiología y Recursos Genéticos VegetalesFil: Ortega, Leandro Ismael. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales (ex IFFIVE); ArgentinaFil: Fry, Stephen C. University of Edinburgh. Institute of Molecular Plant Sciences. The Edinburgh Cell Wall Group; Gran BretañaFil: Taleisnik, Edith. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Fisiología y Recursos Genéticos Vegetales (ex IFFIVE); Argentina

Chara — a living sister to the land plants with pivotal enzymic toolkit for mannan and xylan remodelling

Land-plant transglycosylases ‘cut-and-paste’ cell-wall polysaccharides by endo-transglycosylation (transglycanases) and exo-transglycosylation (transglycosidases). Such enzymes may remodel the wall, adjusting extensibility and adhesion. Charophytes have cell-wall polysaccharides that broadly resemble, but appreciably differ from land-plants'. We investigated whether Chara vulgaris has wall-restructuring enzymes mirroring those of land-plants. Wall enzymes extracted from Chara were assayed in vitro for transglycosylase activities on various donor substrates — β-(1→4)-glucan-based [xyloglucan and mixed-linkage glucans (MLGs)], β-(1→4)-xylans and β-(1→4)-mannans — plus related acceptor substrates (tritium labelled oligosaccharides, XXXGol, Xyl6-ol and Man6-ol), thus 12 donor:acceptor permutations. Also, fluorescent oligosaccharides were incubated in situ with Chara, revealing endogenous enzyme action on endogenous (potentially novel) polysaccharides. Chara enzymes acted on the glucan-based polysaccharides with [3 H]XXXGol as acceptor substrate, demonstrating ‘glucan:glucan-type’ transglucanases. Such activities were unexpected because Chara lacks biochemically detectable xyloglucan and MLG. With xylans as donor and [3 H]Xyl6-ol (but not [3 H]Man6-ol) as acceptor, high trans-β-xylanase activity was detected. With mannans as donor and either [3 H]Man6-ol or [3 H] Xyl6-ol as acceptor, we detected high levels of both mannan:mannan homo-trans-β-mannanase and mannan:xylan hetero-trans-β-mannanase activity, showing that Chara can not only ‘cut/paste’ these hemicelluloses by homo-transglycosylation but also hetero-transglycosylate them, forming mannan→xylan (but not xylan→mannan) hybrid hemicelluloses. In in-situ assays, Chara walls attached endogenous polysaccharides to exogenous sulphorhodamine-labelled Man6-ol, indicating transglycanase (possibly transmannanase) action on endogenous polysaccharides. In conclusion, cell-wall transglycosylases, comparable to but different from those of land-plants, pre-dated the divergence of the Charophyceae from its sister clade (Coleochaetophyceae/Zygnematophyceae/land-plants). Thus, the ability to ‘cut/paste’ wall polysaccharides is an evolutionarily ancient streptophytic trai

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