The Cellular Localization Of AT14A Protein And Its Functions In Mediating Cell Wall-Plasmam Membrane-Cytoskeleton Continuum In Arabidopsis

In animal cells, integrins are a family of transmembran proteins, which is the receptor of extracellular matrix (ECM), mediates signal transduction and plays vital roles in cell differentiation, the responses to extracellular stimuli, etc. At present, we know little whetherthere exist the same molecules in plant cells. AT14A protein of arabidopsis has some homolous sequence to integrin. However, there is no direct evidence that AT14A is the integrin or integrin-like molecule in plant cells.In the present study, we first cloned AT14A from Arabidopsis genome, and constructed suspension culture cell lines of at14a mutant and AT14A over-expressing transgenic lines. And then, we studied the functions of AT14A in mediating cell wall-plasma membrane-cytoskeleton continuum. The main results were summarized as follows:1. AT14A was cloned from Arabidopsis thaliana (Col) by RT-PCR and TOPO-cloning approaches. The AT14A then was fused with GFP coding sequences, and introduced into wild type Arabidopsis by Agrobacterium-mediated method. Expression of AT14A driven by 35S promoter was analyzed by detection of GFP fluorescence. Western blotting analysis, using a monoclonal antibody against GFP, showed that AT14A-GFP fused protein localized in the plasma membrane and its molecular weight is about 70 kDa. The molecular weight of GFP is 27kDa, so the molecular weight of AT14A is about 43 kDa, which was consistent with the predicated result according to the cDNA sequence.2. We successfully constructed the suspension cultured cell lines of at14a mutant and AT14A over-expressing transgenic lines.3. The results showed that AT14A played vital important roles in the regulation of cell morphology and cell adhesion. More than 60% of at14a null mutant suspension cells were in round-shape, whereas wildtype or overespressing transgenic cells were in acerose or oval-shape. Most of at14a null mutant suspension cells existed as single cell, nevertheless, most of overexpressing AT14A-GFP transformant suspension cells have more than 10 cells of every cell aggregate. This result showed that AT14A was involved in cell-cell adhesion.4. AT14A also involeved in controlling cytoskeleton arrangement. We used fluorescent localization method as well as confocal laser microscope to study the arrangement of actin microfilaments and microtubules. Significant differences in arrangement of cytoskeleton were observed and actin microfilaments of at14a null mutant suspension cells became thicker and the arrangements were sparser, while those of overexpression AT14A-GFP transformant suspensioned cells became denser in arrangments. The microtubules of at14a null mutant suspension cells changed into unorderly arrangments.5. Under osmotic stess, more than 90% wild type suspension cells were partially plasmolyzed, however, 62% of at14a null mutant suspension cells have been completely plasmolyzed.Based on the results above, it was suggested that AT14A was localized in plasma membrane and played vital roles in regulating cell adhesion and mediating cell wall-plasma membrane -cytoskeleton continuum, which was very similar to the functions of integrin in animal cells.