Functional Analysis Of A Key Gene PPR329 In Maize Seed Development

Maize is one of the important food crops in my country and plays an important role in food security,animal husbandry,industry and other fields.The development of maize embryo and endosperm has an important influence on the increase of maize yield and the nutrients of internal protein.Studying the development process of maize seeds is very important for increasing maize yield and improving people’s food structure.Pentatricopeptide Repeat(PPR)Protein is a kind of RNA binding protein encoded by nuclear genes and located in mitochondria,chloroplasts and other organelles.It is involved in the process of RNA editing,intron splicing,RNA stability and maturation after transcription.The amount of PPR protein in maize reaches more than 400,which plays a key role in the post-transcriptional processing of organelles,which in turn affects the development process of maize embryo and endosperm.In this paper,ppr329 mutant with PPR329 loss of function was identified.The phenotype of the mutants was empty pericarp(emp).There was only one small embryo proper in the early development stage of ppr329.The development of embryo and endosperm of the mutant seed was stagnant.From the late development stage to the mature stage,the mutant seeds were squeezedinto thin flakes or shrunk by the normal seeds,and the seeds were not fertile.PPR329 encodes a P-type PPR protein harboring 18 P-motifs.PPR329 is highly conserved in monocotyledons.Subcellular localization showed that PPR329 was located in mitochondria.RT-PCR showed that the mature transcripts of nad5,the key gene of mitochondrial complex I,were missing in ppr329 mutant.Nad5 is composed of five exons and four introns.Introns 1 and 4 are cis-spliced,introns 2 and 3 are trans-spliced.The results showed that the splicing efficiency of the first intron and the fourth intron of nad5 was significantly lower than that of wild-type,and the trans-splicing efficiency of the third intron was significantly lower than that of the wild-type.The qRT-PCR of 22 group Ⅱ introns in mitochondria showed that the splicing efficiency of 1,3,4 introns of nad5 was significantly lower than that of wild-type.Nad5 is an important subunit of mitochondrial oxidative phosphorylation complex I.The Blue Native-PAGE active gel experiment showed that the assembly of mitochondrial respiratory chain complex I was significantly reduced in the mutants,and the activity of complex I was seriously decreased.The accumulation of the complexes in the mutants was detected by the antibody and Western,blot tests.It was found that the complex I in the mutants decreased significantly,while the other complexes increased.Moreover,the expression of AOX2 gene in the mutants were significantly up-regulated,and the AOX protein accumulated in the mutant,indicating that the AOX pathway was activated in the mutants.In conclusion,PPR329 is involved in the splicing process of introns 1,3 and 4 of mitochondrial nad5.The deletion of PPR329 gene leads to the failure of the formation of mature nad5 transcripts and the synthesis of functional Nad5 protein subunits,which causes the assembly and activity of mitochondrial respiratory chain complex I to be affected,the development of seed embryo and endosperm to be blocked,and the formation of normal maize kernels is not possible.Therefore,PPR329 is necessary for the development of maize seeds.The splicing of nad5 in maize is rarely reported at present.The intron splicing of nad5 in the maize ppr329 mutant found in this paper is affected,so it provides materials for studying the mechanism of splicing of intron of nad5 and can further deepen the mitochondrial group Ⅱ exploration of intron splicing and assembly of mitochondrial complex I.