Function Analysis Of Two Microtubule-Associated Proteins Of The Arabidopsis MAP65 Family

The microtubule (MT) organization and dynamics are regulated by microtubule-associated proteins (MAPs). Tireless quest of plant MAPs has recovered a class of MAPs of approximately 65-kDa in both tobacco and carrot suspension cells, thus called MAP65. Recent research showed that the function of one member with MT is really different from others in NtMAP65-l family.In Arabidopsis thaliana, nine genes encode proteins of the evolutionarily conserved MAP65 family. We proposed that different MAP65s might also have distinct roles in the regulation of MT functions. In this study, two AtMAP65 proteins, AtMAP65-l and AtMAP65-6 with the amino affinity about 41%, were chosen to test this hypothesis in vitro.Genes of AtMAP65-l and AtMAP65-6 were successfully cloned by RT-PCR and PCR. The GST fusion proteins were purified in vitro, respectively. Although both fusion proteins were able to co-sediment with MTs in vitro, different properties on tubulin polymerization and MT bundling were observed. AtMAP65-l was able to promote tubulin polymerization, enhance MT nucleation, and decrease the critical concentration for tubulin polymerization. It also induced the formation of large MT bundles by forming cross-bridges between MTs, evenly along the whole length of the MTs. In the presence of AtMAP65-l, MT bundles were more resistant to cold and dilution treatments. AtMAP65-6, however, was demonstrated no activity in promoting tubulin polymerization and stabilizing preformed MTs. AtMAP65-6 induced MTs to form a mesh-like network with individual MTs. Cross-bridges were only found at regional sites between MTs. The MT network induced by AtMAP65-6 was more resistant to high concentration of NaCl, than the bundles induced by AtMAP65-l. Imuno-fluoresence observation with purified monospecific anti-AtMAP65-6 antibody revealed that AtMAP65-6 was associated with mitochondria in Arabidopsis suspension cultured cells. It was concluded that these two MAP65 proteins are targeted to distinct sites, thus perform distinct functions in Arabidopsis cells.AtMAP65-6 overexpression Arabidopsis showed the phenotype of enhanced lateral roots development, suggesting that AtMAP65-6 probably participates in the development of lateral roots.The study demonstrated that the members of AtMAP65 family have distinct properties with MT, and therefore may play different functions in cells. It provides important evidences for further functional analysis of the AtMAP65 family.