Novel roles of hydrogen peroxide (H2O2) in regulating pectin synthesis and demethylesterification in the cell wall of rice (Oryza sativa) root tips.

Hydrogen peroxide (H2O2) has been reported to increase lignin formation, enhance cell wall rigidification, restrict cell expansion and inhibit root elongation. However, our results showed that it not only inhibited rice (Oryza sativa) root elongation, but also increased root diameter. No study has reported how and why H2O2 increases cell expansion and root diameter. Exogenous H2O2 and its scavenger 4-hydroxy-Tempo were applied to confirm the roles of H2O2. Immunofluorescence, fluorescence probe, ruthenium red staining, histological section and spectrophotometry were used to monitor changes in the degree of pectin methylesterification, pectin content, pectin methylesterase (PME) activity and H2O2 content. Exogenous H2O2 inhibited root elongation, but increased cell expansion and root diameter significantly. H2O2 not only increased the region of pectin synthesis and pectin content in root tips, but also increased PME activity and pectin demethylesterification. The scavenger 4-hydroxy-Tempo reduced root H2O2 content and recovered H2O2-induced increases in cell expansion and root diameter by inhibiting pectin synthesis, PME activity and pectin demethylesterification. H2O2 plays a novel role in the regulation of pectin synthesis, PME activity and pectin demethylesterification. H2O2 increases cell expansion and root diameter by increasing pectin content and demethylesterification.

Differential expression of vacuolar H+-ATPase subunit c genes in tissues active in membrane trafficking and their roles in plant growth as revealed by RNAi.

Acidification of intracellular compartments by the vacuolar-type H(+)-ATPases (VHA) is known to energize ion and metabolite transport, though cellular processes influenced by this activity are poorly understood. At least 26 VHA genes encode 12 subunits of the V(1)V(o)-ATPase complex in Arabidopsis, and how the expression, assembly, and activity of the pump are integrated into signaling networks that govern growth and adaptation are largely unknown. The role of multiple VHA-c genes encoding the 16-kD subunit of the membrane V(o) sector was investigated. Expression of VHA-c1, monitored by promoter-driven beta-glucuronidase (GUS) activity was responsive to light or dark in an organ-specific manner. VHA-c1 expression in expanding cotyledons, hypocotyls of etiolated seedlings, and elongation zone of roots supported a role for V-ATPase in cell enlargement. Mutants reduced in VHA-c1 transcript using dsRNA-mediated interference showed reduction in root growth relative to wild-type seedlings. In contrast, VHA-c3 promoter::GUS expression was undetectable in most organs of seedlings, but strong in the root cap. Interestingly, dsRNA-mediated mutants of vha-c3 also showed reduced root length and decreased tolerance to moderate salt stress. The results suggest that V-ATPase functions in the root cap influenced root growth. Expression of VHA-c1 and VHA-c3 in tissues with active membrane flow, including root cap, vascular strands, and floral style would support a model for participation of the V(o) sector and V(1)V(o)-ATPase in membrane trafficking and fusion. Two VHA-c genes are thus differentially expressed to support growth in expanding cells and to supply increased demand for V-ATPase in cells with active exocytosis.

Microsomal membrane proteins and vanadate-sensitive ATPase from Vicia faba root tips after clinostat treatment.

Microsomal and soluble protein fractions from Vicia faba root tips were used for SDS-PAGE and Western-immunoblot analysis with anti-ubiquitin antibodies after 9 h clinostat treatment of the plants. In contrast to soluble proteins omnilateral gravistimulation (9 h) resulted in an enhanced proteolytic capacity for microsomal proteins. The increase of vanadate-sensitive ATPase activity was 83% after 9 h clinostat treatment, when the enzyme activity was measured directly after membrane preparation. Enhanced ATPase activity was correlated with the appearance of a polypeptide of about 100 kDa and its fragments (93 and 80 kDa). ATPases are not the only membrane bound proteins, which are changed during clinostat treatment, as several ubiquitinated polypeptides were also affected. A 1 h storage of microsomal fractions led to a shift of band intensities on ubiquitin-specific Western-blots. The demonstrated effect could not be observed, when fractions were isolated in the presence of protease inhibitors. In accordance with the polypeptide analysis omnilateral gravistimulation resulted in an enhanced capacity to degrade specific microsomal ubiquitin-conjugates, whereas the soluble ubiquitin-pool was not visibly affected.