Annual Report 2003

Fig. 1Antisense expression of OsDMC1A gene in rice results in the productionof aborted and sterile pollens.Pollens were differentially stained according to Alexander (1969).Photographs were taken under same magnification. Red/purple pollens:nonaborted; transparent or blue/green pollens: aborted. Alsonote that sizes of pollens from transgenic rice expressing antisenseDMC1A gene are quite variable.0Gy400GyWT rad51b-1 rad51b-2Fig. 2Plants with disruption of AtRAD51B gene are hypersensitive togamma-ray irradiation.A. Schematic representation of AtRAD51B gene with the positions ofT-DNA insertions.B. Sensitivity to gamma rays of rad51b mutants. Imbibed seeds wereirradiated with gamma rays and grown for 2 weeks under white light.C. Sensitivity to gamma rays of rad51b mutants under various dosagesof irradiation. Sensitivity was estimated by the percentage of plantshaving true leaves.ray irradiation with higher induction level inOsRAD51A2. Expression of OsRAD51A2promoter::GUS chimeric gene in rice calliwas strongly up-regulated by double-strandbreaks (DSB) inducers, gamma rays andbleomycin.DMC1 is one of the key players in meioticrecombination. Interestingly, both OsDMC1-A and -B genes were expressed not only inanthers, but in cultured cells, suggesting arole in mitotic recombination in plants.Transgenic rice plants bearing antisenseexpression of 35S::OsDMC1A/B genes producedpollens with abnormal shape and verylow fertility (Fig. 1), though they did not giveany obvious phenotypes at vegetative stages.Among the five members of Rad51 paralogs,Rad51B is suggested to promote theassembly of Rad51 nucleoprotein filamentsduring homologous recombination repair.RT-PCR analysis of Arabidopsis RAD51Bgene (AtRAD51B) indicated that this genewas expressed in young stage of flower budsand the transcript level was increased aftergamma-ray irradiation. Yeast two-hybridanalysis showed AtRad51B interacted withanother member of Arabidopsis Rad51-paralogRad51C (AtRad51C). To elucidate thebiological function of the AtRad51B, we alsoisolated T-DNA knockout (KO) mutants ofthis gene, and named them rad51b-1 andrad51b-2. As shown in RAD51B KO inchicken DT40 cells, Arabidopsis rad51bmutant plants were hypersensitive togamma rays that induce DSBs (Fig. 2).These results demonstrated that AtRAD51Bplays a role in DNA repair in Arabidopsis.Rad54 is a SWI2/SNF2-related proteinthat possesses double-stranded DNA-dependentATPase activity and changes DNAtopology in an ATP hydrolysis-dependentmanner. Most recently, it was shown thatRad54 facilitated chromatin remodeling andwas suggested to enhance protein clearing atthe homology search step of HR. RT-PCRanalysis indicated that Arabidopsis RAD54(AtRAD54) transcripts are expressed in alltissues with the highest level of expressionin flower buds, induced by gamma rays.110 Annual Report 2003