Map-based Cloning And Functional Analysis Of Maize Empty Pericarp601 Gene

Maize(Zea mays L.)is important for the food security of the world as feed and grain.Many genes are involved in maize grain growth,indicating the complexity of genetic regulation of maize kernel development,so cloning and functional analysis of genes that are involved in maize grain development would be helpful for breeding..In this study,an empty pericarp mutant emp601 was identified.The emp601 mutant kernels were distinguished from the WT kernels at 15 days after pollination(DAP)due to a white pericarp and half-translucent appearance.Mature emp601 mutant kernels are unviable and could not germinate.Paraffin sectioning showed that compared with WT,at the early stage,the development of embryo and endosperm of emp601 were both strongly delayed;and the endosperm had degenerated at the later stage of kernel development.Semi thin sectioning showed that the BETL region in the emp601 kernels was severely impacted compared to the WT kernels.The differentiation of the cell wall ingrowth in emp601 BETL was delayed and less.To generate a mapping population,an emp601 heterozygote(Emp601/emp601)was crossed with inbred line B73.F1 plants with Emp601/emp601 genotype were selfed to generate the F2 segregation population.The F2 heterozygous plants showed the 3:1 segregation ratio between WT and empty pericarp(emp)kernels,indicating that the mutant trait is controlled by a recessive nuclear gene.Bulked segregant RNA-seq(BSR-seq)showed that the Emp601 gene was located on the long arm of the chromosome 6.The Emp601 gene was further mapped in a 248 kb region with 6 genes by fine mapping.The candidate gene5 had a single base deletion in emp601 mutant,causing a frameshift and a premature stop codon.This result was further confirmed by Emp601 transgene plants.The transgene can complement the emp phenotype and confirmed that this gene was responsible for the emp601 mutant phenotype,named it Emp601.Emp601 gene encodes an E-type pentatricopeptide repeat(PPR)protein that is targeted to mitochondria.Sequencing 35 mitochondrial transcripts of emp601 and WT kernels results showed the editing efficiencies at ccmFN-137,ccmFN-190,ccmFN-743,ccmFN-790 and ccmFN-824 loci were decreased significantly in emp601 mutant(P<0.05)and circular-RT-PCR results showed that the 5'-end of the ccmFN was extended in emp601.Thus ccmFN protein was significantly decreased.Compared to the wild-type(WT)kernels,the emp601 showed significantly reduced cytochrome c1,Complex ? and complex ?+?2 and NADH dehydrogenase activity of complex ?+?2,while the NADH dehydrogenase activity of complex I was the same,the transcription levels and protein of alternative oxidase(AOX)were upregulated.In addition,the emp601 mitochondria showed a poorly developed membrane system without normal cristae structures by examining the endosperm cells with transmission electron microscopy.Metabolism profile showed that the loss of function of Emp601 resulted in a significant accumulation of amino acids.Taken together,the loss of function of Emp601 impaired the editing of ccmFN 13 7,ccmFN-190,ccmFN-743,ccmFN-790 and ccmFN-824 and the 5'-end processing of ccmFN,hence the function of mitochondria and the energy supply for kernel development and resulting in the empty pericarp phenotype.