Proceedings of the XLVII Italian Society of Agricultural Genetics - SIGA Annual Congress

Verona, Italy - 24/27 September, 2003

ISBN 88-900622-4-X

Poster Abstract - 1.32

Mob-like proteins are involved in both cell cycle progression and programmed cell death in alfalfa

G. Barcaccia*, S. Varotto*, S. Citterio**, S. Sgorbati**, M. Lucchin*, P. Parrini*

*) Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Università degli Studi di Padova, Agripolis, Via dell’Università 16, 35020 Legnaro, Padova

**) Dipartimento di Scienze dell’Ambiente e del Territorio, Università degli Studi di Milano-Bicocca, Piazza della Scienza 1, 20126 Milano

Medicago sativa L., reproductive mutants, gene expression, cytokinesis, apoptosis

Reproductive mutants of Medicago spp. that form 2n eggs by restitutional apomeiosis (FDR) and failure of cytokinesis (SDR) have been discovered and cytologically characterized. The movement of chromosomes and cytokinesis are accomplished by cytoskeletal structures, the spindle and the phragmoplast, respectively. The formation of 2n eggs may depend upon alterations of the cytoskeleton, especially defects in proteins which affect the function and integrity of spindle and phragmoplast tubules. A differential display of mRNAs allowed us to select a flower-specific EST with structural homology to MOB (Mps-one-binder) gene family members. The full-length cDNA clone of the Mob-like gene of alfalfa (GenBank acc. AJ319713) showed a significant amino acid similarity with several Mob1s from a variety of eukaryote organisms. Several Mob1 homologues have been recently cloned in Arabidopsis, chickpea, rice and maize, but the function of this gene in plants remains unexplored. Alfalfa genomic clones amplified by UTR-specific and ORF-specific primers enabled to find out two introns of 394 and 105 pb in the Mob1 gene. RT-PCRs with Mob1-specific primers followed by sequencing analysis revealed a messenger from the apomeiotic mutants bearing two adjacent stop codons and an insertion of 66 nt never found in wild-types. Transcripts of this member 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 Mob1 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. The potential link between Mob1 expression and apoptosis in alfalfa reproductive tissues was investigated in flower bud sections by TUNEL: apoptotic MMCs of the apomeiotic type were never observed and DNA fragmentation was observed solely in degenerating meiotic megaspores. Polyclonal antibodies against the Mob1 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.