RNAi-Directed Downregulation of Vacuolar H+-ATPase Subunit A Results in Enhanced Stomatal Aperture and Density in Rice

Abstract

Stomatal movement plays a key role in plant development and response to drought and salt stress by regulating gas exchange and water loss. A number of genes have been demonstrated to be involved in the regulation of this process. Using inverse genetics approach, we characterized the function of a rice (Oryza sativa L.) vacuolar H+-ATPase subunit A (OsVHA-A) gene in stomatal conductance regulation and physiological response to salt and osmotic stress. OsVHA-A was constitutively expressed in different rice tissues, and the fusion protein of GFP-OsVHA-A was exclusively targeted to tonoplast when transiently expressed in the onion epidermal cells. Heterologous expression of OsVHA-A was able to rescue the yeast mutant vma1Δ (lacking subunit A activity) phenotype, suggesting that it partially restores the activity of V-ATPase. Meanwhile, RNAi-directed knockdown of OsVHA-A led to a reduction of vacuolar H+-ATPase activity and an enhancement of plasma membrane H+-ATPase activity, thereby increasing the concentrations of extracellular H+ and intracellular K+ and Na+ under stress conditions. Knockdown of OsVHA-A also resulted in the upregulation of PAM3 (plasma membrane H+-ATPase 3) and downregulation of CAM1 (calmodulin 1), CAM3 (calmodulin 3) and YDA1 (YODA, a MAPKK gene). Altered level of the ion concentration and the gene expression by knockdown of OsVHA-A probably resulted in expanded aperture of stomatal pores and increased stomatal density. In addition, OsVHA-A RNAi plants displayed significant growth inhibition under salt and osmotic stress conditions. Taken together, our results suggest that OsVHA-A takes part in regulating stomatal density and opening via interfering with pH value and ionic equilibrium in guard cells and thereby affects the growth of rice plants.

Expressing Arabidopsis thaliana V-ATPase subunit C in barley (Hordeum vulgare) improves plant performance under saline condition by enabling better osmotic adjustment

“…5). The association between expressing VHA subunits and stomatal opening and closing has been reported by (Allen et al 2000) using VHA-C mutant (det3), and also by RNAi knockdown of OsVHA-A (Zhang et al 2013). Two to 3-fold higher g s values in transgenic lines compared with WT ( Fig.…”

Phosphatidylinositol 3-Kinase Promotes V-ATPase Activation and Vacuolar Acidification and Delays Methyl Jasmonate-Induced Leaf Senescence

“…Plant V-ATPase down-regulation results in an enhanced stomatal aperture (Allen et al, 2000;Zhang et al, 2013), which leads to rapid water loss and premature leaf senescence (Zhang et al, 2012). Given these findings, we speculated that leaf senescence proceeds in the following order: Glc-PI3K-activation of V-ATPase-vacuolar acidification-stomatal closure-reduction of water lossalleviation of leaf senescence.…”

“…E00777.1)-specific primers (59-TAGGAGGGCGTGGATATGTC-39 and 59-TACACAGCCATC GGTCCAGA-39; Yue et al, 2012;Zhang et al, 2013). The overexpression of AtVPS34 gene in 35S:AtVPS34-YFP transgenic Arabidopsis was determined by qRT-PCR (Supplemental Fig.…”

Interaction between the signaling molecules hydrogen sulfide and hydrogen peroxide and their role in vacuolar H+‐ATPase regulation in cadmium‐stressed cucumber roots

“…Trans‐tonoplast proton gradient generated by V‐ATPase and H + ‐pyrophosphatase (V‐PPase) can be used as a driving force by active antiporters, such as CDF (cation diffusion facilitators) and CAX (cation/proton exchangers) proteins, to remove harmful heavy metals from the cytoplasm (Singh et al ). On the other hand, V‐ATPase, as the main tonoplast proton pump of mature plant cells, takes part in growth processes by regulating expansion of the central vacuole, and consequently cell turgor (Seidel et al ), as well as stomatal density and opening (Zhang et al ). Surprisingly, we have demonstrated previously that Cd inhibits activity of V‐ATPase, indicating that this enzyme is not an essential element of Cd tolerance in Cucumis sativus roots (Kabała et al , , ).…”