Functional Analysis Of A Novel Microtubule Associated Protein

Microtubules (MTs) play a critical role in cell expansion and division, cell morphology and intracellular transport. MT arrays in plant cells are different from that in animal cells. For example, the cortical MT array, MT preprophase band and phragmoplast are unique to plant cells, MT arrays transit in turn from one array to the another during the cell cycle. Microtubule-associated proteins (MAPs) are involved in the transformation of MT arrays. Study of MAPs is now the cutting-edge research in the field of the cytoskeleton researches.MAP1B is a known MAPs from mammal nerve cells. Blast of the gene data base of Arabidopsis gene bank, with the imperfect repetitive motif in the MT-binding domain of MAP1B, resulted in finding a function-unknown gene (At5g44610). Its open reading frame encodes 168 amino acid and has a predicted protein molecular weight of 18.5 kDa. The protein sequence contains 8 VVEKKN/EE, which assembles the repetitive motif in MAP1B.By gene cloning and prokaryotic expression, we purified the recombinant protein. Co-sedimentation assay showed that the recombinant protein bound to MTs, Furthermore, by labeling the recombinant protein with Aleax 488 and incubated with rhodamine-conjugated MTs, the observation of confocal microscopy also demonstrated that the green fluorescent recombinant protein was co-localized with red fluorescent MTs, Therefore, we concluded that it is a novel MAPs form Arabidopsis, and thus termed it as AtMAP18.Confocal microscopy observation showed that AtMAP18 had no effect on MT bundling. The data form experiments with in vitro MT polymerization system showed that AtMAPl 8 affected tubulin polymerization differently according to its concentrations. AtMAPl 8 enhanced tubulin polymerization at low concentrations, while inhibited tubulin polymerization at high concentrations. Nevertheless, EDC cross-link assay showed that AtMAP18 did not bind to tubulin dimmer.By transformation of Arabidopsis with AtMAPl 8 promoter tagged with GUS, the analysis of GUS activity showed AtMAP18 was expressed in root, flower, fruit, cotyledon, hypocotyls and trichmoe. By raised a rabbit anti-AtMAP18 polyclonal antibody, the western blot assay also showed that AtMAPl 8 mainly existed in root and flower tissues.In order to analyze the function of AtMAP18, AtMAP18 overexpression and RNAi plasmids were reconstructed, and transformed into Arabidopsis. Although the RNAi transgenic plants showed no obvious phenotypes, the the transgenic plants of AtMAPl 8 overexpression exhibited general abnormities with cells related to directional expansion, such as pavement cells of leaf and cotyledon, epidermal cells of roots, root hairs, and epidermal cells and cortex cells of hypocotyls. The pollen of AtMAPl 8 overexpression was aberrant and aborted, too.Furthermore, double-staining of anti-ATMAP18 antibody and anti-tubublin showed that AtMAPl 8 was co-localized with cortical MTs in root cells. Observation of transgenic RFP-AtMAP18 Arabidopsis with GFP-tubulin background also showed that AtMAP18 was co-localized with corticalMTs in cells of cotyledon and hypocotyls. In addition, The MT array in cotyledon pavement cells and hypocotyls epidermal cells of AtMAP18 overexpression was altered, and the transition of MT array was hindered by the overexpression of AtMAP18, as a result, leading to abnormalities of cell growth.In present study, we demonstrated that AtMAP18 is a novel microtubule associated protein form Arabidopsis, and analyzed it functions. Our investigation indicated that AtMAP18 has functions in MT organization and regulation of MT dynamics, thus, may have important roles in regulation of cell growth and morphology.