Cloning And Functional Analysis Of The Key Gene Emp21 In Maize Kernel Development

Maize is one of the three major food crops in the world,and is also the main raw material for animal feed and industrial production.Maize is widely grown all over the world and the total production of maize surpassing that of wheat or rice.The yield and quality of corn are directly affected by the seed development.Therefore,the cloning and functional analysis of the key genes in maize seed development and the construction of genetic network regulating seed development have important theoretical significance to understand the molecular mechanism of maize seed development,and provide important theoretical basis for further molecular breedingPlant mitochondrion possesses its own genome which retains-5%genes from its prokaryotic ancestor.These genes encode proteins for oxidative phosphorylation,ribosomal RNAs and transfer RNAs,and ribosomal proteins.RNA C-to-U editing is widespread in land plant organelles.Many angiosperms habor more than 300 editing sites in mitochondria.RNA editing often restores evolutionarily conserved amino acids,generates start/stop codons,promotes intron splicing,and increases the efficiency of transfer RNA processing.Thus,RNA editing is an important event in organelle post-transcriptional RNA processing.Although multiple factors in RNA editing have been identified,the functions of these factors in the editing process remain to be elucidated The DYW-subclass PPR is one of these editing factors.There are 82 predicted PPR-DYW proteins in maize genome,only a few of which have been studied(EMP5,EMP18 and PPR2263).Hence,the cloning and study of PPR-DYW protein is important to exploring the RNA C-to-U editing process,and elucidation of the components of editosomeAn empty pericarp mutant(emp21-1)was charcterized from the UniformMu mutagenic population.The selfed progenies of the emp21-1 heterozygotes segregated about 1/4 emp kernels.Emp21 was cloned by using Mu-seq analysis.Emp21 encodes a canonical DYW-subclass PPR protein,possessing 11 PPR motifs,E1,E2 and DYW motifs.To confirm the causal gene,a second mutant(emp21-2)with a Mu ineriton in Emp21 was isolated from the UniformMu population.Linkage and allelism analysis confirmed that the emp phenotype is caused by mutation of the PPR-DYW gene.The function of EMP21 in maize kernel development and in the editosomes were analyzed by observing the kernel phenotype,detecting the subcellular localization of EMP21,analyzing the editing efficiency of mitochondrial editing sites and the assembly and activity of mitochondrial complexes in emp21 and WT,analyzing the genetic relationship between Emp21 and Emp5,and detecting the interaction between EMP21 and ZmMORF8,as well as between EMP5 and ZmMORF8.The completion of this work plays important theoretical significance in promoting the understanding of the relationship between PPR-DYW protein and maize embryogenesis and endosperm development,and the resolution of editosome.These results are as follows:1)Loss-of-founction of EMP21 severely arrests maize embryogenesis and endosperm development.The selfed progenies of emp21-1 and emp21-2 heterozygotes segregated about 1/4 empty pericaip(emp)kernels,indicating a nuclear and recessive mutation.The mutant kernels at 12 days after pollination(DAP)were smaller than the wild type,and harbored a tiny embryo and transparent endosperm while the wild type kernels developed all the seed structures.At maturity,the mutant kernels were collapsed.Sectioning confirmed that both embryogenesis and endosperm development were defective in the mutants.2)Emp21 encodes a mitochondrion-targeted PPR-DYW protein.Emp21 encodes a canonical DYW-subclass PPR protein,possessing 11 PPR motifs,E1,E2 motifs,and a DYW motif which contains the conserved CDAs-like signature residues(HxE(x)nCxxC).The GFP signals generated by expressing EMP21N511:GFP were detected in the mitochondria indicating the mitochondrional localization of EMP21.3)EMP21 is required for the C-to-U editing at 81 mitochondrial target Cs.The strand-and transcript-specific RNA-seq(STS-PCRseq)method was used to analyze the editing sites in predicted 35 protein-encoding mitochondrial transcripts Based on a total of 600 Mb sequence data,493 C-to-U editing sites were identified in these transcripts in maize.Two independent methods,the STS-PCRseq and direct sequencing of the RT-PCR amplified transcripts,analyzed the editing of mitochondrial editing sites revealed that the editing is completely abolished at the nad7-77,atp1-1292,atp8-437,nad3-275 and rps4-870 sites and is substantially reduced at a further 76 sites in both the emp21-1 and emp21-2 mutants4)The assembly and activity of the mitochondrial complexes I and V are compromised in the emp21-1 mutant.Blue Native-PAGE(BN-PAGE)assay indicated that the abundance and activity of complex I in emp21-1 was greatly reduced,while that of supercomplex ?+?2 was below the level of detection.Similarly,assays targeting F1Fo-ATPase hydrolysis activity and assembly showed that neither F1Fo-ATPase nor the free F and F1 moieties were formed in the mutant,indicating that the assembly and activity of complex V were both compromised by the loss of Emp21 function.Western blotting assay gave a consistent result.Thus,the mutation of Emp21 severely inhibits the assembly and activity of mitochondrial complexes I and V in maize5)A portion(-30%)of the rpl16-458 sites are edited by EMP21 and EMP5 jointly.EMP5 is found to be required for the editing of 10 sites in maize mitochondrial transcripts,and six(rpl16-458,nad9-1 90,nad9-356,cox3-245,cox3-257,and rps12-71)of these sites overlap with those of EMP21.The editing efficiency rpl16-458 site in the single mutants was approximately 73%(cmp5-4)and 80%(emp21-1)falling to 35%in the emp21-1 emp5-4 double mutant revealing that a portion(?30%)of the rpl16-458 sites are edited by EMP21 and EMP5 jointly6)The editing of certain sites by EMP21 and EMP5 involves interactions with ZmMORF8.MORFs/RIPs are responsible for the editing at most of the editing sites in the mitochondrial and plastidial transcripts in Arabidopsis.Functions of all the maize MORFs/RIPs are not identified.34 editing sites controlled by EMP21 require the editing function of MORF8 in Arabidopsis.Eight mediated by MORF8 overlap with those mediated by EMP5.Y2H and BIFC assays indicated that both EMP21 and EMP5 are able to directly interact with ZmMORF8.Thus,the editing of certain sites controlled by EMP21 and EMP5 involves interactions with ZmMORF8 in maize.