Proceedings of the XLVI Italian Society of Agricultural Genetics - SIGA Annual Congress
Giardini Naxos, Italy - 18/21 September, 2002
CHARACTERIZATION OF MOB1, A CANDIDATE GENE FOR APOMEIOSIS IN MEDICAGO MUTANTS
BARCACCIA G., VAROTTO S., PALLOTTINI L., LUCCHIN M.
Dipartimento di Agronomia Ambientale e Produzioni Vegetali, Università di Padova, Agripolis, Via Romea 16, 35020 Legnaro, Padova, Italy
alfalfa, 2n eggs, Mps-one-binder, RT-PCR, in situ hybridization
Reproductive mutants of Medicago spp. that form 2n-eggs by restitutional apomeiosis have been discovered and cytologically characterized. Studies on the inheritance of this trait revealed that it is controlled by a single recessive major gene and a few minor genes. An approach of reverse genetics was carried out to clone candidate genes for apomeiosis by isolating and characterizing transcripts selected because of their expression pattern or sequence information. To this purpose, a differential display of mRNAs that combines cDNA-AFLP markers and bulked segregant analysis allowed us to select a number of flower-specific ESTs from a segregating progeny (Barcaccia et al. 2001 Sex. Plant Reprod. 14:233-38). One of the cloned ESTs proved to have 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 organisms. So far it is known that Mob1 is a gene required for duplication of spindle pole body, separation of chromosomes and completion of cytokinesis during both mitotic and meiotic cell divisions (Straight et al. 2000 Mol. Biol. Cell. 11:3525-37; Luca et al. 2001 Mol. Cell. Biol. 21:6972-83). The Mob1 gene of alfalfa was chosen for further molecular characterization because of its cDNA-AFLP expression pattern and BLAST protein homology results. Semi-quantitative PCR analysis with Mob-specific primers followed by sequencing analysis revealed 6 distinct members characterized by one to two mis-sense point mutations each. Interestingly all members from the apomeiotic mutants displayed two adjacent stop codons and one of them differed for an insertion of 66 nt never found in wild-type members. RT-PCR experiments were performed to study temporal gene expression patterns of Mob1 using a wild-type member specific primer or a primer designed in the putative mutated member region in combination with a 3’-end conserved primer. The former primer combination showed a constitutive transcript, while the latter primer combination yielded a 270 bp-transcript highly expressed in the mutant and poorly expressed in the wild-type in flower buds at the megasporogenesis stage. Transcripts of this member were detected neither in roots, stems and leaves nor in pods confirming the organ-specificity revealed by Northern blot hybridization. In order to evaluate Mob1 involvement in apomeiotic processes, in situ hybridization experiments were carried out. Flower buds of the mutant at different developmental stages, from the beginning of meiosis to the mature embryo sac, were hybridized with DIG-labeled Mob1 riboprobes. Two different antisense hybridization patterns were observed in ovaries: in ovules undergoing a normal megagametogenesis the signal was detected in apoptotic megaspores after meiosis was regularly occurred. In apomeiotic ovules the signal was visualized in either megaspore mother cells and embryo sacs. The hybridization signal was also detected in anther tapetal cells during microgametogenesis, from young microspores to pollen grains, which are the developmental stages characterized by tapetum programmed cell death, PCD (Wu and Cheung 2000 Plant Mol. Biol. 44:267-81). Although the genetic basis underlying PCD in plants remains to be discovered, many of the cytological and biochemical markers of mammalian apoptosis have been observed also in plants (Balk and Leaver 2001 13:1803-13). As a matter of fact, gene bank searches suggest that MOB can be combined (e.g. in Arabidopsis) with the NB-ARC domain, a signaling motif shared by animal cell death gene regulators (Van der Biezen and Jones 1998 Curr. Biol. 8:226-7). To further investigate the potential link between Mob1 expression and PCD in alfalfa reproductive tissues a detailed DNA fragmentation analysis in flower buds sections is in progress.