A study of the microtubule associated protein family, MAP65, in Arabidopsis
Microtubules (MTs) play a key role in cell division and morphogenesis. Plant cells form four distinct MT arrays through the cell cycle; the interphase cortical array, the preprophase band, the spindle and the phragmoplast. The cortical array, the preprophase band and the phragmoplast are unique to plant cells. Microtubule Associated Proteins (MAPs) must be involved in the organisation of the MT structures. The most abundant plant MAPs are the MAP65 proteins. A database search has revealed the presence of nine MAP65 genes in the Arabidopsis genome. Homologues of MAP65 proteins are present in vertebrates and yeast. Using prediction servers, putative phosphorylation and post-translation modification sites were identified in AtMAP65 protein sequences and transcription regulatory elements in AtMAP65 promoter sequences. The temporal and spatial expression of all nine AtMAP65 proteins was examined by transforming Arabidopsis with AtMAP65 promoter: :GUS reporter gene transcriptional fusion constructs. The data revealed that the AtMAP65 isoforms were expressed differentially in Arabidopsis seedling and flower tissues. The abundance of the each of the AtMAP65 transcripts in various Arabidopsis tissues was investigated by Reverse Transcription-PCR. The full length of AtMAP65-6 coding sequence was cloned by screening cDNA libraries and subsequently, it was expressed in E.coli. The recombinant AtMAP65-6 protein was shown to bind MTs and to increase the turbidity of MT solutions. The possible protein- protein interactions between AtMAP65-l, AtMAP65-6 and AtMAP65-5 were assessed with yeast two hybrid assays and it was shown that AtMAP65-6 interacts with itself An antibody was raised against AtMAP65-6 and used for investigating the AtMAP65-6 localisation to MT structures through the cell cycle. AtMAP65-6 does not bind to cortical MTs, however localises to preprophase band, anaphase spindle and phragmoplast. The anti-AtMAP65-6 antibody recognises three isoforms on a western blot of a 2D SDS-PAGE gel of total protein extract of Arabidopsis tissue culture cells.