Map-based Cloning And Functional Analysis Of Rice PPR Gene FGR1

Pentatricopeptide repeat(PPR)proteins constitute one of the largest protein families in land plants,with more than 400 members in most species.Recent studies revealed the functions of PPR proteins in organellar RNA metabolism and play physiological roles during plant growth and development.A typical PPR protein is targeted to mitochondria or chloroplasts,binds one or several organellar transcripts and involves in the progress of splicing,cleavge,translation,expresstion and editing,consequently,has profound effects on photosynthesis and respiration.But the functions of most PPR proteins,especially PPRs localized in the nuclear,remain largely unknown.Therefore,it has important significance and application value to identify new family members of PPR proteins.Our results provide novel insights into coordinated interaction between nuclear-localized PPR proteins and mitochondrial function.The main results are as follows:1.The fgr1 mutant has opaque endosperm and slightly retarded seedling growth.The fgr1 mutant exhibited significantly slower grain-filling after fertilization and the mature grain weight was dramatically reduced.The endosperm in the mutant produced an opaque grain appearance indicating starch synthesis was damaged.The germination seeds from the fgr1 mutant was 2 days later than wild type and juvenile leaves and roots were significantly shorter at 9 days post-germination.Mutant plants gradually recovered and grew into normal adult plants.2.Starch grain development is delayed in fgr1 mutant.Semi-thin sections found that numerous smaller,single starch grains(SGs)were present in endosperm cells with extra air spaces in the fgr1 mutant compared with the almost homogeneous compound SGs in wild type.The reduction in SG number in endosperm cells led to decreased starch content in the fgr1 mutant endosperm.Scanning electron microscopy(SEM)analysis of mature grains further showed that fgr1 mutant endosperm cells were packed with loosely-arranged starch granules that were much rounder than the irregular polyhedral ones in wild-type.Amylose content was significantly lower in the mutant,whereas total lipid contents were dramatically increased.3.Map-based cloning of FGR1.Map-based cloning was undertaken to identify the FGR1 allele.FGR1 was narrowed down to 273 kb region based on analysis of 2,716 individuals with the recessive mutant phenotype.Compared with wild-type genomic sequences,Os08g0290000 in fgr1 mutant contained a single nucleotide substitution,that led to transition of a highly conserved phenylalanine(I)to threonine(T).Os08g0290000 contained a single exon that encoded a PPR protein with 16 PPR repeats;it belonged to the P-type PPR protein group.Mature seeds from the transformed complementation plants were similar to the wild type and SGs from 6 DAF endosperm were also restored to wild-type appearance.4.Subcellular localization and expression analysis.FGR1-GFP and GFP-FGR1,which contain the full length FGR1 coding region in front of,or behind,the GFP,respectively,and N181aa-GFP containing only the N-terminal region(1-181 aa,in front of the PPR repeat)in front of the GFP were transformed into rice protoplasts,all co-localized with nuclear maker signals.qRT-PCR and GUS analysis showed that FGR1 was constitutively expressed in various organs.5.Mitochondrial function is defective in fgr1 mutant.We checked the expression levels of 20 different expressed genes(DEGs)in RNA-seq results related to mitochondrial function.All showed remarkable differences between fgr1 mutant and wild type.The phenotypes of opaque endosperm and growth retardation in fgr1 are very similar to those of mutants defective in mitochondrially localized PPR proteins in rice.Thus,mitochondrial function appeared to be affected in the fgr1mutant.Expression of mitochondrially encoded genes was significantly higher in fgr1 mutant.Accumulation levels of AOXs in both mRNA and protein were highly increased in fgr1 mutant.Further measurement showed that ATP contents,mitochondria membrane potential and respiratory rate were both significantly decreased.Transmission electron micrographs of mitochondrial ultrastructure of 6 DAF endosperm cells indicated an incomplete cristae structure in fgr1 mutant.Blue native polyacrylamide gel electrophoresis(BN-PAGE)found a slight but clear decrease in Complex Ⅰ activity and protein profile.Thus,despite its nuclear localization,FGR1 still indirectly regulates mitochondrial function.6.FGR1 binds to the CUCAC motif.RNA-SELEX was used to found the RNA substrate of FGR1.A CUCAC consensus motif was obtained.We further verified the binding between the fusion protein and selected motif through an RNA-electrophoretic mobility shift assay(RNA-EMSA).7.FGR1 is required for a subset of RNA splicing events.Combined with the binding motif identified by the SELEX assay,we analyzed the retained intron(RI)genes containing the CUCAC motif in their pre-mRNAs to ascertain whether FGRl is required for splicing of nuclear transcripts.The presumed function of the 22 most affected genes showing differences between N22 and fgrl mutant in RNA-seq analysis are listed.They contain transcription factors Flo2,BIM2,and bHLH056,transcription regulator FNBP4,transcriptional silencing protein MORC6,splicing factor SCL33,atranscription complex subunit,nuclear proteins UBNl,PCO3 and RCD1 that can interact with transcription factors affecting gene expression in diffierent metabolic pathways,mitochondrial proteins or proteins involved in activities related to this organelle(pyruvate dehydrogenase ATG18h that decreases ROS in mitochondrial,and MICU1 that protects mitochondria from oxidative burden).Thus,proteins involved in transcription,RNA processes and mitochondrial function were enriched.FGR1 localizes in nuclear and regulates the mitochondria function.Our findings suggested the closely connection between nuclear localized PPR protein and mitochondria.FGR1 also affects a subset of nuclear encoded mRNA splicing events and revolves in the transcription and post-transcription regulation.Our findings provide a new direction of PPR proteins in different organelles.