Studys On Biochemical And Biological Functions Of OsVHA-A Gene

In plant cell, there are three different types of proton pump: plasma membraneH+-ATPase, vacuolar H+-ATPase and vacuolar H+-PPase. Besides the transport ofproton, they also transport some ions and solutes. The former is termed primary activetransport, and the latter is termed secondary active transport. The normal function ofsecondary active transport is dependent on primary active transport to provide drivenforce. Before the cross-membrane transport of proton, a proton concentration gradientshould be built. Thus, it is no surprised to find that the pH value is7.5in cytoplasm but5.5in vacuole, and also5.5in intracellular region in normal plant cell. The equilibriumof proton in plant cell is dependent on these three types of proton pump. Plasmamembrane H+-ATPase, localized on plasma membrane, is a single polypeptide thatplays a key role in the process of transport proton across the plasma membrane andfunctions in nutrient uptake, stomatal movement, cell elongation, and intracellular pHhomeostasis. The source of energy is the hydrolysis of ATP and it is activated byphosphorylation. Usually, it is named “Master enzyme”. The vacuolar H+-ATPase(V-ATPase) is multi-subunit complex, which is known to be required for embryonicdevelopment and cell expansion, specifically acidifies the vacuole and otherintracellular trafficking compartments. The source of energy is also the hydrolysis ofATP, but its activation is not dependent on phosphorylation. In plant cell, it is termed“ecological enzyme”. And the vacuolar H+-pyrophosphatases (V-PPases) aresingle-subunit homodimers that also transport proton from cytosol to vacuole, similarlywith the vacuolar H+-ATPase. For one thing, the V-PPase generates protonelectrochemical gradients in vacuole energized by the hydrolysis of pyrophosphate (PPi)other than ATP, and also provide the driven force to transport ions or solutes crosstonoplast which not only maintain the equilibrium of ions in vacuole, but also alleviatethe toxicity of ions. For another thing, it makes the vacuole acidification in favor of therunning of physiological and biochemical response.Stoma serve as major gateways for both CO2influx into plants from theatmosphere and transpirational water loss of plants in physiological conditions orbiotic/abiotic conditions. Most of genes are reported in these process by the regulationof stomatal density and movement, such as subunit c1, B and C of V-ATPase. InArabidopsis det3(de-etiolated3) mutant derived from downregulation of the subunit C of V-ATPase, the ability of stomatal closure was abolished probably due to thedisruption of calcium oscillation. Transgenic introduction of subunit c1of V-ATPasefrom halophyte grass spartina alterniflora into rice plants resulted in a significantincrease of salt stress tolerance accompanied by reduced stomata density and early stageclosure of the leaf stomata. In Arabidopsis, subunit B of V-ATPase was recently foundto bind to F-actin in vivo and regulate actin reorganization, suggesting a potential roleof the subunit B in the regulation of stomatal movement. Subunit A, the criticalcomponent of V-ATPase protein complex, contains an ATP-binding region and mayrepresent a catalytic reaction center. The transcript level of subunit A of V-ATPase hasbeen shown previously to be induced by salt and osmotic stresses in Arabidopsis andbarley. However, its function related to stomatal conductance regulation andphysiological homeostasis remains largely unknown.By using inverse genetics approach, we firstly cloned the gene of OsVHA subunitA and constructedd the pHB-OsVHA-A-RNAi vector by RNAi which was transformedinto wild-type rice by Agrobacterium tumefaciens-mediated transformation, resulting inthe construction of OsVHA-A-RNAi transgenic rice. Our study found that OsVHA-Aplayed a vital role in the regulation of stomatal density and movement, resulting in thesensitive of salt and osmotic stress.By using real-time PCR, it was found that OsVHA-A was almost expressed in allrice tissue, and most expressed in leaf and flower. Moreover, we found that the proteinencoded by OsVHA-A was only localized on tonoplast by transforming theGFP-OsVHA-A fusion protein into onion epidermal cell. By using yeastcomplementation (transformed the pEYS2-OsVHA-A vector into the yeast mutantvma1Δ), it was found that Heterologous expression of OsVHA-A was able to rescue theyeast mutant vma1Δ (lacking subunit A activity) phenotype, suggesting that it partiallyrestored the activity of V-ATPase. Additionally, the analysis of V-ATPase and V-PPasewere shown that the activity of V-ATPae was downregulated in OsVHA-A RNAi linesbut that of the V-PPase was not changed obviously. Further study by the determinationof pH value was shown a upregulation of pH value in transgenic plants. Additionally, itseemed that the root length and seedling length of transgenic lines were more than thatin wild-type lines. Meanwhile, in contrast to that of the wild type, the chlorophyllcontent of the transgenic plants was increased. Furthermore, the transgenic plants wereobviously shorter and more tiller number than that of WT. T2mature seeds werestatistical analysis, as a result, grain length and thousand seed weight increased significantly compared with the wild type.Then an increase of stomatal density andaperture in transgenic rices were found by using scanning electron microscopy similarlywith the increase of water loss in transgenic lines. Besides, the OsVHA-A RNAi seemedto increase the sensitive of salt and drought stress. By using atomic absorptionspectrometry, it was found that the concentration of K+and Na+in intracellular regionswere up-regulated in transgenic rice. Moreover, it was found that H+efflux wasincreased in normal condition while the H+influx was decreased under the treatment of20%PEG in transgenic lines by using the non-invasive micro-test system. And theosmotic pressure was obviously lower in transgenic lines than that in wild-type lines,which were cultured in MS liquid medium supplemented with20%PEG or in pot.Real-time PCR showed that the expression of some genes related with the stomataldensity and movement are altered. The expression of CAM1, CAM3and YDA weredown-regulated while that of PMA3was up-regulated in OsVHA-A RNAi lines.Taken together, we tried to analyze the mechanism of increase of stomatal densityand aperture by OsVHA-A RNAi. The knockdown of OsVHA-A down-regulated theactivity of V-ATPase, which resulted in the increase of pH value in vacuole. Thus theaccumulation of proton in cytosol promoted the upregulation of PMA3expression,leading to the up-regulation of plasma membrane H+-ATPase, subsequently resulted inthe increase in the influx of Ca2+and K+, eventually promoted the open of stoma.Moreover, the down-regulated YDA gene, which was one of the negative factor in theregulation of stomatal density, maked the increase of stomatal density. And theincreased stomatal density and aperture, for one thing, increased the transpiration andthe water loss, resulting in the sensitive of salt and drought stress. For another thing,increased the absorption of CO2and the efficiency of photosynthesis, resulting yieldboost.