Research On Molecular Mechanism Of Sa-mediated Thermo-sensitive Male Sterility Of TMS13

Two-line hybrid rice breeding is a key technology for utilization of rice heterosis,and a major strategy to ensure worldwide food security.Application of two-line hybrid rice benefits from the development of photo/thermo-sensitive genic male sterility（P/TGMS）lines.Therefore,discovery and investigation of new P/TGMS lines will be of meaning and great significance for hybrid breeding and revealing molecular mechanism underlying environmental cues and plant reproductive development.Sa is a complex locus conferring hybrid male sterility in rice,which consists of two closely linked,interacting genes SaF and Sa M.In this study,a thermo-sensitive sterility line TMS13 was identified from the recombinants of a cross between a japonica variety Taichung 65（T65,with the japonica allele SaF^-）and a near isogenic line of Sa（E4,with the indica allele SaF⁺）.Using genetic,cytological,biochemical and molecular biological methods,this study revealed that the thermo-sensitive male sterility of TMS13 is regulated by a cis element Cis17 of SaF⁺through the action of the Sa complex locus.The main results are as follows:1)TMS13 was identified from the recombinants of the cross T65×E4.Under high temperature conditions,anthers of TMS13 were thin and whitish without pollen grain,and spikelets of TMS13 were completely sterile.While under low temperature conditions,TMS13 anthers became yellowish and generated some viable pollen grains,and the spikelets of TMS13 were partially fertile.2)Temperature treatments detemined that the sterility-fertility-conversion temperature of TMS13 is between 23℃and 24℃,namely TMS13 shows fertility below 23℃and sterility above 24℃.3)Cytological analysis of anther development showed that delayed degradation of tapetum of TMS13 may cause abnormal microspores leading to no-pollen sterility phenotype.4)By genetic mapping,a cis element Cis17 located in the 5’UTR of SaF⁺was determined as key factor to control no-pollen sterility phenotype in TMS13.In TMS13,the promoter and 5’UTR（containing Cis17）of SaF⁺was replaced by that（lacking Cis17）from T65,by a genetic recombination.5)Knocking out Cis17 in E4 background produced partial pollen fertility,and knocking out the recombinant gene SaF^+ΔCis17R in TMS13 rescued the pollen fertility.6)Quantitative RT-PCR analysis showed that the SaF⁺expression in Cis17-lacking genetic materials was significantly higher than that containing Cis17,especially in high temperatures.In addition,the SaF expression was promoted by high temperature during meiosis stage.7)Both over-expression lines SaF⁺-OX-E4 and SaF⁺-Sa M⁺-OX-T65 exhibited no-pollen sterility phenotype.However,both single gene and two genes-knockout lines Sa M-KO and SaF-Sa M-KO became fully male fertile.8)The sequence analysis showed that Cis17 contains DRE and GCC-box elements.A candidate transcription-repressor factor Os DREB6 was identified as a binding factor of Cis17 in yeast one-hybrid screening.Based on above results,this study assumed that Cis17 in the SaF⁺UTR region may recruit Os DREB6 to inhibit SaF⁺mediating thermo-sensitive male sterility in rice.In the indica rice,Cis17 strongly inhibits SaF⁺expression,producing less sterile signal,resulting in full fertility phenotype.In the japonica rice,SaF^-and Sa M^-are nonfunctional thus can not generate sterile signal.In TMS13,the lack of Cis17 allows high level expression of SaF⁺,while high temperature futher promotes SaF⁺expression.Therefore,elevated SaF⁺coordinates with Sa M⁺to trigger a strong sterile signal in the anther,resulting in a no-pollen phenotype.Under low temperature conditions,the expression of SaF⁺is decreased in TMS13,and the lower SaF⁺/Sa M⁺complex levels can only trigger a weak sterile signal,leading to partial fertility phenotype.This study focuses on the characterization and molecular mechanism of thermo-sensitivity of TMS13.It is the first time to report the connection between molecular mechanisms of hybrid sterility and thermo-sensitive male sterility,providing new insight in the regulation of environmental cue and plant reproductive development.The novel understanding of the thermo-sensitive sterility mechanism may accelerate heterosis utilization in two-line hybrid rice breeding.