The Expression And Regulation Of VHA-c Genes In Arabidopsis Thaliana

The plant V-ATPase is a membrane-bound transport protein located to the tonoplast and various subcellular membranes, including the Golgi, endoplasmic reticulum, intracellular vesicles, as well as the plasma membrane. The V-ATPase uses the energy released during cleavage of the y-phosphate group of cytosolic ATP to pump protons into the vacuolar lumen and acidify the vacuole, thereby creating an electrochemical H⁺-gradient which is the driving force for a variety of transport events of ions and metabolites and so influencing turgor, cell expansion and guard cell movement. On the other hand, under stress conditions such as salinity, drought, cold, survival of the cells depends strongly on maintaining or adjusting the activity of the V-ATPase. Thus V-ATPase is indispensable for plant growth, development and adaptation to changing environmental conditions.A plant V-ATPase consists of at least 13 distinct subunits organized in two large subcomplexes: the cytosolic V₁ and memebarne V₀. The V₁ sector consists of subunits A through H. The V₀ sector includes five proteins, named subunits a, c, c", d,e. Subunit c is a major component of the membranous V₀ sector, forms the proton conductance pathway and invovles in assembly of the V₀ complex.Small gene families have been detected in the plant genome for most plant V-ATPase subunits. Subunits B, E and G in the V₁ sector and all the Vo subunits are encoded by at least two genes in Arabidopsis. Subunit c is encoded by the largest gene family with five members (VHA-c1, VHA-c2, VHA-c3, VHA-c4 and VHA-c5). Yet the specific function of multiple genes of subunits in plant and their regulation are largely unknown.To understand the roles and regulation of VHA-c genes, we determined their patterns of expression under normal and stress conditions by usingRT-PCR. Then the expression pattern of VHA-c3, monitored by promoter-driven β-glucuronidase(GUS)acivity, was also investigated under normal and stress conditions. To analysis expression and regulation mechanism of VHA-c3 , a series of promoter mutants were constructed in which various extents of VHA-c3 promoter were placed upstream of the GUS gene in the vector pCambial305.1. Our results indicated that:(1) VHA-c1 VHA-c2 and VHA-c3 were expressed in Arabidopsis root, bolt, leaf, flower and silique, while basal trancription level of VHA-c4 and VHA-c5 was very low under normal conditions.(2) The expression of VHA-c genes was regulated by salt stress, acidic or alkali conditions, water stress and exogenous ABA. Expression of VHA-c4 and VHA-c5 was clearly regulated by environmental cues, suggesting their function responsive to stress conditions. To our knowledge, this is the first report on expression and function of these two genes.(3) The expression of VHA-c3 in root, cotyledon of seedlings and hypocotyls of etiolated seedlings. The expression of VHA-c3 in Arabidopsis leaf was developmental stage dependent. Its expression was also in a tissue-specific manner, expressed only in floral organ including sepal, stigma, style and anther.(4) Furthermore, NaCl stress could also alter the expression of VHA-c3 in Arabidopsis leaf in a tissue-specific manner.(5) Under pH4 condition, the expression of VHA-c3 was inhibited in Arabidopsis leaf. To our knowledge, this is the first report that pH regulates expression of plant V-ATPase genes.(6) The essential cis-element for expression of VHA-c3 was located withinthe 772bp fragments upstream of the translation start codon of VHA-c3.