| Vacuolar-type H+-ATPase (Y-ATPase, VHA) is a multisubunit endomembrane proton pump that is conserved in eukaryotes. These pumps play an important role in various biological processes, including organelle acidification, ion homeostasis, and vesicular trafficking. In plants, V-ATPase is localized to the tonoplast and membranes of the secretory system, including the endoplasmic reticulum (ER), trans-Golgi network (TGN) or endosomes, intracellular vesicles and provacuole (PVC). V-ATPases consist of the cytosolic ATP-hydrolyzing V1 complex (subunits A to H) and the membrane-bound proton-translocating Vo complex (subunits a, c, c’, c", d, and e). The symmetrical arrangement of four or five copies of the V-ATPase c subunit (VHA-c) forms the core of the Vo complex. VHA-c contains four putative transmembrane helices (TM1 to TM4), which consist of half of the total length of the protein. A highly conserved glutamate (Glu) residue in TM4 acts as the proton-binding site.Plant species from which genes for the V-ATPase subunit c have been cloned include Mesembryanthemum crystallinum, Pennisetum glaucum, and Tamarix hispida. The expression of these genes was shown to be affected by various environmental stresses. However, the functions of the VHA-c in plant growth, development, and stress responses are not well defined. In this study, we characterized V-ATPase subunit c (PutVHA-c) from Puccinellia tenuiflora, which can grow in the saline alkali soil of the Songnen Plains in northeast China.Based on the research on PutVHA-c, we also investigated function of five AtVHA-c genes in Arabidopsis. Using real-time quantitative PCR and promoter-driving GUS expression method, we investigated the expression patterns of five AtVHA-c genes and response relationship in salt stress. Using mutant and transgenic Arabidopsis, we also investigated the function of five AtVHA-c genes in plant growth, development and response to abiotic stress; Using colocalization of GFP markers with FM4-64 and immune electron microscopy method, study on the subcellular localization of AtVHA-c proteins.1) The 872-bp putative full-length cDNA of PutVHA-c contains a 495-bp putative open reading frame (ORF). PutVHA-c has four putative transmembrane regions, with the conserved Glu residue in the fourth transmembrane region. The tissue-specific expression of PutVHA-c in P. tenuiflora was examined using semi-quantitative RT-PCR. PutVHA-c was expressed in almost all of the tissues studied, and its expression was slightly higher in leaf, sheath, and anther tissues than in the other tissues assayed. The effects of salt and alkali stresses on PutVHA-c expression were investigated using Northern blot analysis. An increase in levels of Put VHA-c transcripts in roots after exposure to NaCl stress.2) In Arabidopsis, three AtVHA-c proteins encoded by five genes, they are highly conserved in the amino acid sequence. Study of the quantitative real-time PCR and promoter showed that the five AtVHA-c genes are highly expressed in all tissues of Arabidopsis and has tissue specificity, also found that five AtVHA-c genes are different degrees induced by salt stress.3) Phenotype analysis showed that overexpression of PutVHA-c or AtVHA-c genes can promote plant growth and development, and improve the salt tolerance of transgenic Arabidopsis. The Arabidopsis atvha-c5 mutant reduced root growth, and enhanced salt sensitivity.4) Activity analysis showed that overexpression of PutVHA-c enhanced the V-ATPase activity in Arabidopsis. The inhibitor of V-ATPase activity (ConcA) treatment experiments showed that ConcA can inhibit the root growth of Arabidopsis, and with the increase of concentration of inhibitory effect of strengthening. Decreased V-ATPase activity in Arabidopsis atvha-c5 mutant. These results indicated that the expression level of VHA-c gene affects the activity of V-ATPase, and the level of V-ATPase activity affects the growth of plants.5) Colocalization experiments of GFP with endosome markers of FM4-64 as well as immunogold labeling showed that PutVHA-c and AtVHA-c localized to endosomal vesicles of different-sizes in Arabidopsis cells.6) In WT Arabidopsis, ConcA induced the accumulation of large Golgi-derived secretory vesicles in the root cells. In PutVHA-c-GFP transgenic Arabidopsis, ConcA induced the accumulation of endosomal vesicles and plasma membrane protein PIN1. These results probably suggested that reduction of V-ATPase activity by ConcA blocked endosomal vesicle trafficking.Together, these results indicated that VHA-c protein may effects vesicle trafficking by enhancing V-ATPase activity, thus play a function in plant growth and response to salt stress. |
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