Studies On The RF5's Restoration Of Fertility Complex And Restoration Of Complex Factor2in Honglian-CMS Rice

The restoration of fertility and cytoplasmic male sterility (CMS) is a classical crosstalk of nucleus-mitochondrion in plants. CMS is caused by the genes carried by mitochondrial genomes, performs as the abortion of pollen development. Generally, CMS is caued by disorder of pollens development in spatial and temporal way, finally performs as abortion of pollens. CMS can be restored by nucleus-encoded Rf (restorer of fertility) genes, several of which were map-based cloned already. Rf5/Rf6in HL type rice and other7Rf genes belong to a large, multi-functional family of pentatricopeptide repeat (PPR). The restoration of fertility accomplished by PPR Rf genes mainly included processing of CMS co-transcripts, or depression of CMS genes translation or accumulation of CMS proteins. Besides the Rf genes restore CMS in higher plants, PPR proteins participate in almost all stages of gene expression, including transcription, RNA cleavage, RNA splicing, RNA editing and RNA stabilization, all rely on binding of RNA. PPR's divergent roles might due to they are sequence-specific RNA binding adaptors recruiting effector enzymes to the target RNA. In these studies, we are trying to indentify and characterize RF5's restoration of fertility complex, and new factors in this complex.1. RF5plays its role in a protein complex, we extracted Rf5NIL (near-isogenic line)'s mitochondria for size-exclusion chromatography, and incubated these fractions with HL-CMS line YtA's mitochondrial RNA to check whether HL-CMS transcript atp6-orfH79was processed or not. The result showed that RF5did exist in a protein complex size-about500-600kD, and processed atp6-orfH79in restoration of fertility complex (RFC).2. Then4RFC's candidate genes RNAi transgenic lines were constructed:the expression of Restoration of Fertility Factor2(Os01g61780) in RNAi lines was decreased to10%-15%, and some Rcf2TO RNAi lines were partly sterile. Half of the RCF2RNAi in each independent selfed T1population produced about50%sterile pollen, and rest of them produced completely fertile pollen. But in the NIL/RCF2i lines all the F1populations were fully-fertile, while RCF2i/NIL showed the same pollen viability segregation as RCF2i. The1:1segregation of these fully-fertile and half-fertile populations in RCF2i presented expected gametophytic way. Further, CMS transcript and protein were detected in some of the RCF2i T1, implied that RCF2was a newly found factor in RFC besides GRP162.3. Sequence-based analysis of RCF2showed that it encoded "a" subunit of vacuolar ATPase, had some substitutions of nucleotides compared with Os01g61780. Unlike other reported subunits of V-ATPase, RCF2located to mitochondria via its N-terminal's signal peptides; Proteinase K treatment showed that RCF2was a mitochondrial inner membrane part-integral protein. So RCF2possibly was not a typical V-ATPase a subunit. RCF2has interactions with RF5and GRP162, confirmed by co-immunoprecipitation assay, GST pull-down and far-western assay. CMS transcript and protein accumulated abnormally when NIL was treated with V-ATPase inhibitor concanamycin A. suggesting V-ATPase's activity of RCF2was important for restoration of fertility in HL-CMS. Moreover, GRP162could, not imported into mitochondria in some of the RCF2RNAi T2populations, the same as in YTA. Considering that GRP162fused with mitochondrial targeting peptides could restore HL-CMS, RCF2's role in RFC might involve in assisting transporting GRP162into mitochondria when RF5presents, all together with other factors assemble into RFC, process the atp6-orfH79to restore fertility in HL-CMS.