AT14A Mediates Cell Wall Synthesis And The Response Of Arabidopsis Thaliana To Drought Stress

AT14A is a membrane protein with unknown function in Arabidopsis thaliana and has a small domain that has sequence similarities to integrins from animals. As a substantial middle member of the cell wall-plasma membrane-cytoskeleton continuum on plasma membrane, integrins mediates signal transduction. Some work provided implication that basic mechanisms of integrin function were well similar between animals and plants. There also is a cell wall-plasma membrane-cytoskeleton continuum in plant cells , and this continuum plays important roles in the regulation of plant responses to environmental cues. But its molecular basis mediated by the membrane protein is largely unknown. However, there is no direct evidence that AT14A is the integrin or integrin-like molecule in plant cells until now. In addition, what are the roles of cell wall-plasma membrane-cytoskeleton continuum in the responses of plant to drought stress are still unknown.In this study, we studied the possible functions of AT14A, especially in the regulation of cell wall biosynhesis and the responses to drought stress, using biochemical, cell biological and molecular biological techniques. The main results were summarized as follows:1. AT14A was cloned from Arabidopsis thaliana (Col) by RT-PCR and TOPO-cloning methods. The AT14A then was fused with GFP coding sequences, and introduced into at14a null-mutant and wild type, respectively. Expression of AT14A driven by 35S promoter was analyzed by detection of GFP fluorescence. The results showed that AT14A-GFP fused protein was localized in the plasma membrane and root tips.2. The results showed that AT14A played vital roles in the regulation of cell morphology. More than 60% of at14a mutant suspension cells were in round-shape. Most of at14a mutant suspension cells existed as single cell while most of overexpressing AT14A-GFP transformant suspension cells had more than 10 cells of every cell aggregate. Cell wall thicknesses of three types suspension cells were altered, and results showed: Col (wild type) > AT14A (overexpression transformant) > at14a (null mutant). The ultrastructure cross sections of Arabidopsis hypocotyl were observed by electronic microscope. The results showed that the mutant cells of hypocotyl were bigger than wild type and overexpression transformant. The analysis of cell wall thicknesses showed the same results in hypocotyl and stomata as the suspension cells.3. AT14A may also involve in the regulation of cell wall biosynthesis. Compared with wild type, cellulose content of at14a null mutant was lower and that of overexpression transformants was higher, but there was little difference in other cell wall component content among four genetypes. Using two-dimensional electrophoresis (2-DE), we also analysed the difference of cell wall proteins among four genetypes.4. Under drought stess, both of at14a null mutant and AT14A overexpression transformant seedlings improved drought stress tolerance. Measurations of leaves water potential and stomatal conductance of wild type, at14a null mutant, overexpression AT14A-GFP transformant seedlings showed that the stomatal conductance of overexpression AT14A-GFP transformant seedlings was the lowest, while the changed extent of at14a null mutant stomatal conductance by drought stress was the biggest. The result of leaves water potential showed the similar change. AT14A lackness or overexpression mediated drought stess-induced ABA biosynthesis. These results indicated that AT14A plays important roles in response to drought stress.According to the results above, AT14A might be the middle member of cell wall-plasma membrane-cytoskeleton continuum, and it has close relations to cell wall. Also, AT14A is involved in the response of Arabidopsis to drought stress.