Cloning And Functional Analysis Of OsACOS12 For Pollen Wall Development And Thermosensitive Genic Male Sterility In Rice

Since the male sterile varieties provide a great convenience for cross pollination,male reproductive regulation is crucial for crop breeding and agricultural production.Asian cultivated rice(Oryza sativa L.)is one of the world's the most important crop species,especially in China.The well-developed three-line and two-line hybrid systems indicated that the rice reproductive development is closely related to crop yield.These male sterile lines act as the important materials which are widely used for heterosis in agriculture production.In order to reveal the internal and external mechanisms of rice reproductive development,we isolated the male sterility osacos12 mutant and thermosensitive genic male sterility tms18 mutant.Meanwhile,the corresponding mutated genes OsACOS12 and TMS18 were characterized,and their biological functions were further analyzed.The major research results are summarized as follows:1.In Arabidopsis,acyl-CoA synthetase 5(ACOS5)has been reported to be critical for sporopollenin biosynthesis and pollen development.ACOS5 could catalyze the conversion of medium and long-chain fatty acids to the corresponding acyl-CoA ester.In this study,we characterized an orthologue of Arabidopsis ACOS5 in rice,OsACOS12(LOC?Os04g24530).Sequence analysis showed that OsACOS12 shares 63.9% amino acid sequence identity with ACOS5.The osacos12 mutant anthers had a white colour without pollen grains inside,which lead to complete male sterility.Sequence analysis and complementary experiment revealed a point mutation from A to T at position 1,000 th base of the LOC?Os04g24530 genomic sequence in the mutant,creating a premature stop codon.Cytological observation showed that no mature pollen grains were detected in the locule of osacos12 at later stages of anther development.The exine layer was mostly absence in osacos12 mutant.The anther surface of mutant was also lack of reticulate structure,and orbicules were barely detected in the inner surface.GC-MS result showed that the total cuticular wax amount was reduced by approximately 42.6% in the mutant,which contributed to the significant reduction of most wax constituents.These results demonstrated that OsACOS12 plays an important role not only in pollen exine formation but anther development in rice.RT-PCR and in situ hybridization showed that the transcripts of OsACOS12 were expressed in tapetal cells and microspores.On the protein level,OsACOS12-GFP signal was detected within the tapetal cells,which formed a circle in the anther.In the late stages of anther development,the fluorescence signal transferred into the locule.However,the GFP signal was not detected inside the microspores.OsACOS12 driven by the ACOS5 promoter could partially restore the male fertility of the acos5 mutant in Arabidopsis.However,the Pp ACOS6 driven by the ACOS5 promoter could not rescue its fertility.These results suggested that function of ACOS genes are conserved among angiosperm,but have evolutionary divergence in other species.Additionally,the ACOS5 gene triggered by OsACOS12 or Pp ACOS6 promoter could also partially rescued the fertility of the acos5 mutant.However,SEM analysis showed these pollen grains still had slight morphology defects.In the pro OsACOS12:OsACOS12 line,OsACOS12 is driven by its own promoter,and the transgenic plants exhibited a male sterile phenotype.In the transgenic lines,the expression level of OsACOS12 was lower compared with ACOS5 in wild type.RT-PCR showed that the expression of OsACOS12 was downregulated in rice gamyb and tdr mutants.Electrophoretic mobility shift assays showed that GAMYB and TDR could directly bind the promoter of OsACOS12.In Arabidopsis,the transcription factors MS188 and AMS may form a complex to activate the expression of ACOS5 for sporopollenin biosynthesis.This result suggested that the regulatory mechanism for sporopollenin synthesis related genes were slightly different between rice and Arabidopsis.2.The two-line hybrid rice is based on the discovery and application of thermosensitive genic male sterility(TGMS)lines or photoperiod-sensitive genic male sterility(PGMS)lines as the male sterile lines and maintainer lines.TMS18,a thermosensitive genic male sterility gene in Zhonghua 11 was screened by EMS treatment.The tms18 mutant exhibited normal vegetative and spikelet development.It was completely male sterile when grown under sterility-inducing high temperatures(>28?),but became male fertile when put it into lower temperatures(20-24?)during male gametogenesis.Scanning electron microscopy(SEM)analysis showed that the pollen grains of TMS18 were shrivelled and collapsed when grown under high temperatures,but became mature pollen grains when it was subjected to lower temperatures.The pattern of anther cuticle was not affected by temperature conditions.To further clarify the details of the abnormal exine development,anther samples were investigated using transmission electrion microscopy.Under high temperature,tms18 microspore exine II was lack of sporopollenin deposition and presents several gaps.However,under low temperature,breakage of exine layer of microspores was recovered.The results showed that the fertility of plant under different temperatures was closely related to exine layer structure of microspore.Gentic analysis suggested that tms18 is controlled by a single recessive gene.The gene was non-allelic with the TGMS genes in the two-line system of hybrid rice breeding in China.By map-based cloning and sequencing showed a single bases mutation(G-to-A,Gly-to-Ser transition)at position 181 of the gene LOC?Os10g38050 compared with that in WT.TMS18 encodes an evolutionarily conserved glucose-methanol-choline(GMC)oxidoreductase,which are apparently present in land plants.The SNP in the tms18 was located at the central of a conserved ADP-binding motif.TMS18 gene was highly expressed in the spikelet,but hardly detected in the leaf and stem during the pre-heading stage.Our data suggested that tms18 is a new TGMS line,which steriltiy-fertility conversion is sensitively during post-meiosis stages.This characteristic differentiate from that in current TPGMS lines,which has certain application value.