The Gene Cloning And Functional Analysis Of Maize Dek35 Mutant

Maize is world’s important food,economic and energy crop.Maize seeds are major consumable organ.Studying maize seed mutants is important for understanding maize seed development and storage compound accumulation.We isolated a new seed mutant"dek35-ref" from maize active Mutator line.dek35-ref is a lethal-seed mutant and exhibits small and flat seeds as well as defective seed development.At maturity,the 100-seed weight of mutant seeds is only 20%that of the wild type.The zein protein contents were significantly lower in mutant seed endosperm.By cytological observation,dek35-ref shows developmental delay during embryo and endosperm development.By transmission electron microscopy observation,the endosperm cells in dek3 5-ref developing seeds were significantly cytoplasmically less dense and exhibited enlarged mitochondria with severe deformations and voided internal assembly.Dek35 was cloned through Mutator tag isolation and was confirmed by four additional independent mutant alleles.Dek35 encodes a new P-type(pentatricopeptide repeat)PPR protein.The dek35 mutant showed increased dek35 transcript level and significantly decreased DEK35 protein.It is implicated that P-type PPR protein mainly involved in the splicing of organelle-encoded mRNA.By observation with the confocal laser scanning microscope,we determined that the DEK35 protein targeted to mitochondria.Among maize mitochondrial 22 group Ⅱ introns,we found only splicing efficiency of nad4 intron 1 was affected and declined.Western blot analysis further verified the significant reduction of NAD4 protein.By Blue Native Polyacrylamide Gel Electrophoresis(BN-PAGE)and in-gel NADH dehydrogenase activity test,we found the severe deficiency of mitochondrial complex I assembly and activity.The qRT-PCR result of genes related to alternative respiratory pathway also showed significantly up-regulated transcriptional activity,indicating a severe defect in mitochondrial function.Transcriptome analysis of dek35 endosperm revealed enhanced expression in the mitochondrial function and activity,as well as decreased expression related to zein protein genes.In conclusion,our results indicated that Dek35 encodes a new P-type PPR protein that affects the cis-splicing of mitochondrial nad4 intron 1,and is required for mitochondrial function and seed development.The functional characterization of DEK35 further extended our understanding of the specific biological function of the existing PPR protein family.