Functional Analysis Of PPR250,an Essential Gene For Seed Development In Maize

Maize is one of the most important crops,providing resources for food,feed and bioenergy.The development of maize seed is a key factor to affect yield.Therefore,functional characterization of gene regulating seed development is both a fundamental scientific question and a key step in molecular crop breeding.Nuclear-encoded PPR proteins are predominantly targeted to mitochondria or chloroplasts where they regulate gene expression at the RNA level to play a role in organelles.The function of PPR proteins includes RNA editing,intron splicing,mRNA stability and translation regulation.Mutations of PPR proteins in plants often cause defective embryo and endosperm development,small kernels,cytoplasmic male sterility or slow-growth.The full length of PPR250 gene is 1560bp and encodes a mitochondrion-targeted P-type PPR protein containing 11 PPR motifs.We obtained two alleles of ppr250 from UniformMu Seeds Stock Center.At maturity,the mutant kernels were smaller than the wild-type with white and wrinkled pericarp.The homozygote of ppr250 is embryo-lethal and could not germinate.We observed the development progress of seeds at different stages by light microscope.At 15 DAP kernels,the embryogenesis was arrested at the transition stage and endosperm was arrested at the differentiation stage.Additionally,the development of basal endosperm transfer cells was affected.Several P-type PPR proteins have been reported to have the function of RNA splicing.Because PPR250 is a bona fide mitochondrion-targeted P-type PPR protein,it is likely that PPR250 is required for the splicing of group Ⅱ introns in mitochondrion.Our results show that the expression levels of most genes are comparable between the ppr250 mutant and wild-type.But the full length of nad4 transcript was absent in ppr250 mutant.To confirm the absence of nad4 transcript is due to intron-splicing deficiency,we examined the splicing efficiency of 22 group II introns in ppr250 mutant by RT-PCR and quantitative RT-PCR.The splicing efficiency of the nad4 intronl,2 and 3 were affected in ppr250 mutant.NAD4 is an important subunit of complex I,the absence of matured nad4 transcript resulted in the deficiency of complex I activity and assembly.During the complex I assembly progress,NAD4 and NAD5 were assembled as a sub-complex together.The results of mass spectrometry show that the absence of NAD4 also leads to undetectable NAD5 in complex I.The severe deficiency of complex I inhibited the cytochrome pathway and induced the up-regulating of AOXs in the alternative respiratory pathway.In conclusion,the severely affected splicing of nad4 and abolition of complex I activity blocked the OXPHOS progress and energy supply in mitochondria in ppr250 mutant.The shortage of nutrients and energy leads to the embryo-lethal mutation in which the development of embryo and endosperm is repressed.