Functional Analysis Of PPR Genes Emp17 And Emp18 In Seed Dvelopment Of Maize

Maize is not only the largest cereal crop in the world,but also the main source of feed and industrial processing raw materials.With the development of economy and increase of population,the demand for better quality and higher yield is stronger than before.Seed development is a key factor to influence yield and quality of maize.Therefore,it is of great significance to identify the key genes in regulating maize seed development and to elucidate the molecular mechanism underlying seed development.PPR genes are a large gene family discovered in 2000.It is widely distributed in eukaryotes,especially in terrestrial plants.PPR proteins are nuclear-encoded proteins containing multiple PPR motifs in tandem.PPR proteins are sequence-specific RNA binding proteins,often located in mitochondria or plastids where they function in post-transcriptional processes,such as RNA splicing,RNA editing,and translation.By binding to specific sequences in the RNA,PPR proteins directly regulate the expression of mitochondrial or plastid genes,thereby controlling the biogenesis and functions of plastids and mitochondria.Due to the large number of PPR genes and mutants often leading to abnormal seed development or cytoplasmic male sterility,functions of many PPR proteins are unknown and it has received increased attention from researchers.emp17 and emp18 mutants obtained from the UniformMu maize mutant library were used as materials.To determine the linkage between gene and phenotype,seed characterization,Mu transposon insertion,and allele hybridization were performed.The molecular functions of Emp17 and Emp18 involved in maize seed development were explored by subcellular localization of proteins,post-transcriptional processing analysis of mitochondrial transcripts,and were confirmed by assembly and activity detection of mitochondrial respiratory chain complex.Main conclusions are indicated as below:1)Mutation of Emp17 and Emp18 lead to seed abortion in maize.Selfed ears of emp17 and emp18 heterozygotes segregated emp kernels and WT at a ratio of 1:3,indicating that the mutation is a monogenic and recessive gene.The mutant kernels were small size and translucent white appearance.At maturity,the mutant kernels collapsed and abortion.The paraffin sections of seed from different developmental stages showed that the embryo development of emp17 and emp 18 mutants is arrested at transition stage without scutellum,shoot apical meristem and root apical meristem.Similarly,the endosperm development in emp17 and emp18 was significantly inhibited,leaving a gap between the seed coat and endosperm.2)Emp17 and Emp18 encode mitochondrion-targeted E-and DYW-PPR protein.Alignment between cDNA and gDNA showed that Emp17 is an intronless gene consisting of 1947-bp nucleotides and encods EMP 17 protein consisting of 648 amino acids.EMP 17 is predicted to contain 14 PPR motifs and an E domain,and thus can be classified as an E-PPR protein of PLS subfamily.Transient expression of GFP fusion protein in tobacco subepidermal cells indicated EMP 17 protein is targeted to mitochondria.Emp18 is an intronless gene consisting of 2421-bp nucleotides and encods EMP 18 protein consisting of 806 amino acids.EMP 18 contains 17 PPR motifs,an E domain,an E+domain and a DYW domain,and thus can be classified as a DYW-PPR protein of PLS subfamily.Transient expression of GFP fusion protein in tobacco subepidermal cells indicated EMP18 protein is targeted to mitochondria.3)Emp17 involves in the splicing of nad1 intron 4 and nad4 intron 1 in mitochondria.The mature transcripts of nadl and nad4 in emp 17-1 mutant were significantly reduced or disappeared comparing with WT by analyzing the RNA transcription levels of mitochondrial coding genes in WT and emp17-1 mutants.RT-PCR and qRT-PCR analysis showed that nad4 intron 1 is 100%spliced in WT,but almost abolished the emp17-1 mutant.The splicing efficiency of nad1 intron 4 in the emp17-1 mutant was also significantly lower than that in the WT.However,the splicing efficiency of other 20 group ? introns in mitochondria was identical between WT and emp17-1 mutant.These results suggest that Emp17 specifically participates in the splicing of mitochondrial nad1 intron 4 and nad4 intron 1,and its mutation leads to the splicing defect of them.4)Loss-of-function of EMP17 protein results in inhibited assembly and lost activity of mitochondrial complex ?.Nad1 and Nad4 are important subunits of complex ? in mitochondrial respiratory chain.The results of BN-PAGE,coomassie brilliant blue staining and NADH dehydrogenase activity staining of WT and emp17-1 mitochondrial membrane protein showed that the specific bands of mitochondrial complex ? in emp17-1 mutant were significantly weakened,and almost no NADH dehydrogenase activity was detected.It was concluded that the mutation of EMP17 caused the intron splicing defect of mitochondrial nad1 and nad4,and then the abnormality of Nad1 and Nad4 proteins,which affected the normal assembly of complex I in mitochondrial respiratory chain,and consequently led to the loss of NADH dehydrogenase activity.5)EMP18 is involved in the C to U editing at atp6-635 and cox2-449 sites.RNA editing analysis of transcripts of mitochondrial coding genes in WT and emp18 mutants revealed that the C to U editing of atp6-635 was abolished in both emp18-1 and emp18-2 alleles of Emp18.In addition,the cox2-449 site,which was 100%edited in WT,was abolished in the emp18-1 allele but remained 50%edited in the empl8-2 allele.The lack of editing in both cases caused a change of Leu212 to Pro212 in Atp6 and Met150 to Thr150 in Cox2 in the mutants.6)Loss-of-function of EMP18 leads to a dramatic reduction in assembly of F1Fo-ATPase holoenzyme.F1Fo-ATPase is composed of F1-subcomplex catalyzing ATP synthesis and Fo-subcomplex involved in proton transfer.Mitochondrial Fo-subcomplex contains a subunit a,a subunit b and a c-ring composed of several c subunits.The subunit a is encoded by mitochondrial gene atp6.Structural prediction of subunit a in WT and emp18 mutant showed that the substitution of Pro at position 212 caused the breaking of the second a helix,and conformational change of subunit a,indicating that the editing of C to U of atp6-635 is very important for maintaining the correct conformation of subunit a.The analyses of Western blot and ATP hydrolysis activity indicated that the assembly efficiency of F1Fo-ATPase was significantly reduced and the free F1-subcomplex was increased by 6-7 folds.Similarly,the ATP hydrolysis activity of F1Fo-ATPase in emp18 mutant decreased,while the activity of free F1-subcomplex increased.It is speculated that the mutation of EMP18 may result in the abortion of editing at atp6-635 site,amino acid substitution,and protein conformationnal change,resulting in the reduction of the assembly and activity of F1Fo-ATPase holoenzyme.7)Alternative respiratory pathway is induced in emp17 and emp18 mutant.Mitochondrial respiratory pathway is mainly cytochrome respiratory pathway,and the blockage of cytochrome respiratory pathway usually leads to an increase in the alternative pathway.We determined the expression levels of alternative oxidase(AOX)genes,AOX1,AOX2 and AOX3 by RT-PCR,qRT-PCR and Western blot.The transcript level of AOX2 in emp17 and emp18 mutant increased compared with the WT.Consistently,AOX protein levels were also dramatically increased in the emp17 and emp18 mutant.These results indicate that the loss-of-function in EMP17 and EMP18 blocks the cytochrome respiratory pathway and induces the alternative respiratory pathway in mitochondria.Based on the above results,it was concluded that Emp17 and Emp18 affected the assembly and activity of complex I and complex V in mitochondrial respiratory chain respectively by participating in the splicing of mitochondrial nad1 intron 4 and nad4 intronl,the editing of mitochondrial atp6-635 and cox2-449.These defects affect the biological function of mitochondria,causing severely arrested embryo and endosperm development and resulting in seed abortion.These results reveal that Emp17 and Emp18 are critical to mitochondrial RNA post-transcriptional processing,and furthermore to mitochondrial function and seed development in maize.