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.
Highlights
Stomatal pores, surrounded by a pair of guard cells, play a crucial role in controlling gaseous exchange and water release by transpiration [1]
By using inverse genetics approach, we demonstrated that a rice (Oryza sativa L.) vacuolar H+-ATPase subunit A (OsVHA-A) gene plays an important role in the regulation of stomatal movement and determination of stomatal density, which is associated with increase or inhibition of growth of rice plants under nonstress or salt/osmotic stress conditions, respectively
To investigate the expression pattern of OsVHA-A in different tissues, root, stem, leaf, intenode and flower were collected from wild type plants and immediately frozen in liquid nitrogen stored at 280uC until further analyses
Summary
Stomatal pores, surrounded by a pair of guard cells, play a crucial role in controlling gaseous exchange and water release by transpiration [1]. Stomatal aperture is regulated by the reversible swelling and shrinking of guard cells, which sense environmental signals and endogenous hormonal stimuli, such as light, atmospheric CO2 levels, humidity, temperature, pathogens and hormones [1], [2]. In response to these stimuli, transport of ions and water through channel proteins across the plasma and vacuolar membranes changes the turgor and volume of guard cell, thereby regulating stomatal aperture [3]. Stomata are produced by a series of cell divisions which starts with an asymmetric division and ends with a symmetric division. In the initial stage of stomata biogenesis, several genes encoding putative receptors, proteases or kinases, such as TMM (TOO MANY MUTHS), SDD1
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