RMS2 Encoding A GDSL Lipase Functions In Rice Male Fertility

Rice is one of the most important food crops.In rice production,fertility is a key factor in determining rice yield.In addition,male sterility lines are also important resources for utilization of heterosis in rice with improved yield.Therefore,cloning male sterility genes in rice and elucidating the underlying regulatory mechanism have great theoretical and application value.Plant male fertility is a coordinated effort involving both reproductive tissues and sporophytic tissues,in which lipid metabolism plays essential roles.GDSL esterases/lipases are a class of enzymes with a conserved Gly-Asp-Ser-Leu motif.Although GDSL esterases/lipases have been well known as key enzymes for plant development and stress response,their functions in reproductive development remain unclear.In this study,we carried out phenotypical observation,map-based cloning and transcriptional regulation characterization on a novel mutant rice male sterile 2?rms2?,which showed complete male sterility and normal female fertility during reproduction.The obntained results are shown as follows:1.Compared with the wild type,the mutant rms2 had a normal phenotype during vegetative growth.Agronomic traits in rms2 such as plant height,tiller number,panicle length,and grain number were not significantly different from those of the wild type.However,rms2 had a shorter,pale anther without fertile pollen grains,and the male sterility was stable and independent from the environmental factors.2.Cytological observations found that in rms2,the tapetum and middle layers of anther wall did not degrade,the anticle and ubisch body formation was delayed,the nexine of pollen wall was discontinuous,and the central vacuole of microspores was defective,which eventually gave rise to aborted pollens.3.F₂ population derived from the cross between rms2 and Minghui 63 was used for map-based cloning,and RMS2 was located on the short arm of chromosome 2.By sequencing 40-kb genome,the mutant rms2 showed TTGT to A in the third exon on ORF3?LOC?Os02g18870?,which caused amino acid deletion and substitution.LOC?Os02g18870 encodes a GDSL lipase with typical GDSL motif,and belongs to the SGNH hydrolase subfamily.The function of this gene has not been reported.CRISPR/Cas9-derived mutants of LOC?Os02g18870 exhibited identical male sterility as rms2,while genetic complementation could rescue the male fertility of rms2,which demonstrated that LOC?Os02g18870 is RMS2.4.RMS2 was predominantly transcribed during early anther development,and mainly expressed in the tapetum and microspores,while transcription levels were lower in other tissues.Transient expression in protoplasts indicated that RMS2 is localized in the endoplasmic reticulum.5.RMS2 displayed enzyme activity in hydrolyzing esters on the general substrate p-nitrophenyl butyrate,and the enzyme activity was reduced in rms2 crude extracts when compared with the WT.Comparative metabolomic analysis in wild type and rms2 indicated significant change in content of 16lipid components and numerous other metabolites.Above results indicated that RMS2 can control pollen fertility by regulating lipid metabolism in anthers.6.Key male fertility Regulators Rice Male Fertility UDT1?Undeveloped Tapetum 1?and PTC1?Persistent Tapetal Cell 1?are bHLH and PHD-finger transcription factors,respectively.UDT1 and PTC1 can directly bind to the E-box motif-containing region in the RMS2 promoter and activate its transcription both in vitro and in vivo as indicated by the EMSA,ChIP and luciferase activation assays.Furthermore,the transcriptional level of RMS2 were significantly decreased in the anther of udt1 and ptc1 mutants.Our research suggested that RMS2 may serve as a key node in the rice male fertility regulatory network.In this study,RMS2,a GDSL lipase gene,that regulates male fertility was cloned.It affected the development of anther and pollen walls and vacuole morphology by regulating lipid metabolism in anthers,thereby determining pollen fertility.The findings of the current study shed novel light in the function of GDSLs in reproductive development and provided promising gene resource for hybrid rice breeding.