MOB-like proteins are involved in septum formation during cell division and can play a role in programmed cell death in plants

The movement of chromosomes and cytokinesis are accomplished by cytoskeletal structures, the spindle and the phragmoplast, respectively. Pathways of meiotic and mitotic polyploitidization may depend upon alterations of the cytoskeleton, especially defects in proteins which affect the function and integrity of spindle and phragmoplast tubules. A Mob-like partial gene of alfalfa (GenBank acc. AJ319713) was isolated by mRNA profiling in a reproductive mutant of alfalfa and its full-length was cloned by RACE. Several Mob homologues have been recently cloned in Arabidopsis, chickpea, rice and maize, but the function of this gene in plants remains unexplored. Transcripts of one of the member of the Mob multi-gene family were detected only in flower buds at the mega-sporogenesis stage confirming the organ-specificity revealed by Northern blot hybridization, while roots, stems, leaves and pods revealed constitutive transcripts of a distinct Mob member. In situ localization of mRNAs with DIG-labeled Mob1 riboprobes in ovaries at different developmental stages revealed two different antisense hybridization patterns: in ovules undergoing a regular meiosis the signal was detected in apoptotic megaspores, whereas in apomeiotic ovules the signal was visualized in either MMCs and embryo sacs. The hybridization signal was also detected in anther tapetal cells when PCD takes place to allow pollen grain dispersal and in degenerating ovules with non-viable embryo sacs. Interestingly protein database searches revealed the existence of ovary-specific Mob1 gene products in human and mouse tumor tissues. It has been shown that the Phocein-MOB domain (pfam 03637) can be combined (e.g. in Arabidopsis and rice) with the NB-ARC domain (pfam 00931), a signaling motif shared by animal cell death gene regulators as well as with Leucine Rich Repeats (pfam 00560), short sequence motifs involved in protein-protein interactions. Polyclonal antibodies against the Mob proteins were produced and tested for their specificity of recognition by immunoblotting and immunocytochemical assays. Western blotting of alfalfa proteins isolated from 3-day old plantlet roots and cotyledons revealed two distinct doublets of about 28 KDa and 55 KDa providing that Mob1 may be a component of multi-domain proteins. Along with a cortical localization in synchronized root tip cells, the Mob1 proteins were visualized at the cell plate site during cytokinesis by marking the progressive formation of the division septum. Further experiments are in progress to better understand the Mob1 protein function during mitotic and meiotic spindle assembling and activity, and to check whether in alfalfa mutants Mob1 expression is correlated to either an apomeiotic or apoptotic fate of the ovule cells.