The Roles Of AT14A Protein In The Mediation Of Cell Wall-Plasma Membrane-Cytoskeleton Continnum In Arabidopsis

In animal cells, there exists extracellular matrix (ECM)-plasma membrane-cytoskeleton continuum, which mediates cell signal transduction and plays vital roles in cell differentiation, the responses to extracellular stimuli, etc. Unlike animal cells, the extracellular matrix of plant cells is the cell wall which is composed of fibrin, hemi-celluloses and pectin. Recent research suggested that there exists similar cell wall-plasma membrane-cytoskeleton continuum, which also plays important roles in the responses to fluctuating environment factors. AT14A protein is a partial similarity to integrin at the amino acid level, however, there is no direct evidence at present that AT14A is the integrin-like molecule in plant cells. In this study, we investigated the functions of AT14A in the mediation of the cell-cell attachment and the cell wall-plasma membrane-cytoskeleton continuum. The main results are as follows:1. The constructed AT14A-GFP fused gene was transferred into wild type Arabidopsis, col, and AT14A null-mutant, at14a, respectively by Agrobacterium-mediated method. We first successfully constructed the suspension cultured cell lines of four genotypes for further research, i.e, Col, at14a mutant, AT14A-overexpressing and AT14A-rescued transgenic cell lines. The results of western blotting and semi-quantitative analysis proved transgenic success.2. The phenotype of the mutant cells was similar to that of the complementary genotype cells, however, cells of both genotypes become smaller and more round than the wild-type and AT14A-overexpressing genotypes. Fluorescent analysis showed that the actinic filaments of the mutant cells were thicker and arranged more sparsely than those of wild type and over-expressing mutant cells. The arrangement of microtubules of the mutant cells was irregular, but the regular arrangement of microtubules was observed in wildtype and AT14A-overexpressing cell lines, suggesting that AT14A might involve in the arrangement and assembly of cytoskeleton.3. Pharmacological studies showed that treatment with RGD, a synthetic tripeptide containing an RGD domain, which interacted specifically with the integrin protein, can induce a mechnical separation between cell wall and plasma membrane. Upon exposure to osmotic stress, the cell wall and the plasma membrane also become mechanically separated, a process known as plasmolysis, however, the degree of plasmolysis varied greatly between genotypes and the plasmolysis of at14a mutant cells was much more serious than other genotypes. Furthermore, as compared with the cases treated with osmotic stress only, when cells were pre-treated with RGD and then treated with osmotic stress, the plasmolysis of at14a mutant cells was affected little, but those of other genotypes became much more serious.4. The cell-to-cell interaction (indicated as adhension degree) of at14a mutant was ginificantly low than those of wildtype and AT14A-overexpressing genotype. The cell-cell adhesion degree of AT14A-rescued transgenic cell lines was recovered fully as compared with those of at14a mutant and wildtype cells. AT14A protein plays a direct role of the adhesion between cells.5. Significant differences in cytoskeleton dynanmics were observed among varied genotypes after treated with RGD or depolymerized agent of microfilaments or microtubule, colchicine or cytochalasin D, or stabilized agent of microtubule, taxol. The effects of thses treatments on the arrangement of microfilaments or microtubule were more significant in at14A mutant cells than in other genotype cell lines.6. Protein-protein interaction analysis showed that there was no interaction between AT14A protein and actin 8 protein. We think AT14A protein could indirectly affect the cytoskeleton form and stability.In summary, AT14A protein had important roles in the mediation of cell-to-cell interaction and in mediating cell wall-plasma membrane-cytoskeleton continuum.