Functional Analysis Of PPR Genes PPR101 And PPR231 In Seed Development Of Maize

In recent years,maize has surpassed wheat and rice to be the largest cereal crop in the world.With the increase of population and the development of economy,people pay more attention to the quality and yield of maize.Seed development is a key factor to maize quality and yield.Moreover,as a typical monocot,maize has big-size kernels and abundant seed mutants,which provide a lot of experimental materials for research and become an important model plant for the study of seed development.Hence,cloning genes involved in maize seed development and studying their molecular function,and thus revealing the molecular mechanism of seed development are crucial to constructing the network of seed development regulation and increasing the quality and yield of maize.Mitochondria,as the core of cellular metabolism and energy homeostasis,are highly metabolically active organelles and play a vital role in seed production.In flowering plants,mitochondrial genes contain mostly group II introns which require to be spliced before translation into functional proteins.The precise splicing of introns is essential to the growth and development of plants.However,the molecular mechanism of intron splicing has not been revealed.As nuclear encoded factors,PPR proteins can regulate mitochondrial genes expression in post-transcriptional processing level,including RNA intron splicing,RNA editing,RNA maturation and translation.In maize genome,there are more than 450 PPR genes and the molecular functions of a large number of PPR genes have not been studied.Therefore,it is of great significance to identify more PPR genes that control the development of maize seed and the function of mitochondria,which are essential to the directional improvement of maize seeds.Via reverse genetics,we obtained two seed development mutants-ppr101 and ppr231 from the UniformMu stocks.Seed phenotype observation,linkage identification and crossing between alleles confirm that the Mu insertion in PPR101 and PPR231 genes is the cause for genes mutation,thus resulting in defects of seed development.Through subcellular localization of proteins,expression level of protein coding genes in mitochondria,analysis of intron splicing efficiency,the assembly and activity detection of mitochondrial complex I,we explored the molecular functions of PPR101 and PPR231 involved in maize seed development.Results indicate that both genes are essential to group II introns splicing,the assembly and activity of mitochondrial complex 1,the function of mitochondrial respiratory chain,and the development of maize seed.Our research implies that the splicing of one groupⅡ intron needs the participation of multiple PPR proteins,which is of theoretical significance to the elucidation of intron splicing mechanism.The main results are listed as follows:1)Loss-of-function mutation in PPR101 and PPR231 impairs maize seed development.The selfed progeny of ppr101 and ppr231 heterozygous plants segregates wildtype and mutant kernels at a ratio of 3:1.The phenotypes of ppr101 and ppr231 mutant kernels are empty pericarp(emp)and small kernel(smk),respectively.Results indicate that the mutations are monogenic and recessive nuclear genes.The ppr101 mutant kernels are small during the entire seed development process and the pericarps are white,shrunken and collapsed.In the mutants,the embryogenesis is arrested and the endosperm development is hindered.With the maternal pericarp continues growing,an obvious cavity is caused between the endosperm and pericarp.The ppr231 mutant kernels are small and show partially developed embryo and endosperm.No clear differentiation of shoot apex is detected in the embryo and the endosperm development is also delayed compared with the WT.Both the ppr101 and ppr231 mutants are embryo-lethal and cannot germinate,implying that the two PPRs are essential to maize embryogenesis and endosperm development.2)PPR101 and PPR231 are P-type PPR proteins targeted to mitochondria.PPR101 and PPR231 are all P-type PPR proteins.PPR101 contains 647 amino acids with 16 PPR motifs,and PPR231 contains 526 amino acids with 10 PPR motifs.Each protein contains a signal peptide in the N-terminus and no other domains in the C-teraminus.Transient expression of PPR101-GFP and PPR231-GFP fusion proteins in tobacco epidermal cells indicates that PPR101 and PPR231 are targeted to mitochondria.3)PPR101 and PPR231 are required for the splicing of mitochondrial introns.We compared the expression level of 35 protein coding genes in mitochondria between the WT and mutants.RT-PCR analysis indicates that the level of nad5 transcript is dramatically reduced and nearly undetectable in ppr 101-1.And the nad2 and nad5 transcript levels are decreased substantially in ppr231-1.The intron splicing defects in nad2 and nad5 were examined by RT-PCR and qRT-PCR.Results indicate that the splicing of nad5 intron 1 is reduced and the splicing of nad5 intron 2 is nearly abolished in the ppr 101 mutants,and the splicing of nad5 introns 1,2 and 3,and nad2 intron 3 is decreased in the ppr231 mutants.4)Loss of function in PPR101 and PPR231 impairs mitochondrial complex Ⅰ assembly and activity.Complex Ⅰ is composed of two arms,the ’membrane arm’ that embeds in the membrane and ’peripheral arm’ that protrudes into the matrix.Nad2 and Nad5 are key subunits of the membrane arm of complex I.We performed BN-PAGE,CBB staining and in-gel NADH dehydrogenase activity assays to detect mitochondrial complexes assembly and complex Ⅰactivity.Results show that complex Ⅰ is dramatically decreased while complex Ⅲ and V are increased in two mutants,indicating that the assembly of complex Ⅰ is impaired.In-gel NADH dehydrogenase activity assays indicate that the complex Ⅰ activity is nearly abolished in ppr101-1 and is significantly reduced in ppr231-1.It is concluded that the mutation of PPR101 and PPR231 causes the intron splicing defects of mitochondrial nad5 and nad2,and then the deficiency of Nad5 and Nad2 proteins,thus affecting the assembly of complex I and consequently resulting in the decrease of NADH dehydrogenase activity.5)The mutation of PPR101 and PPR231 activates the alternative respiratory pathway.The defect of complex I can block the oxidative phosphorylation pathway and activate the alternative oxidase pathway.The maize genome contains three alternative oxidase(AOX)genes,AOXl,AOX2 and AOX3.We then analyzed the expression of A OX at the RNA and protein level.RT-PCR and qRT-PCR results indicate that the expression level of AOX2 transcript is upregulated in two mutants compared with the WT.Consistently,Western blot by specific antibody indicate that the abundance of AOX protein is increased dramatically in two mutants,while is undetectable in the WT.These results provide further evidence that the mutation of PPR101 and PPR231 impairs complex I biogenesis and leads to dysfunction of mitochondrial respiratory chain,thus blocking the oxidative phosphorylation pathway and activating the alternative respiratory pathway.6)PPR101 and PPR231 have no direct interaction.PPR101 and PPR231 are required for multiple introns splicing,and meanwhile,they have functional overlap in the splicing of specific introns.Specifically,they are all required for the splicing of nad5 introns 1 and 2.PPR proteins have the capability to form complexes,and they may cooperate to form a specific spliceosome in mitochondrial intron splicing Therefore,we performed yeast two-hybrid assay to detect the interaction between PPR101 and PPR231.However,results indicate that they have no direct interaction,implying that they may not function by directly physical contact in the splicing of nad5 introns 1 and 2.Based on the above results,we conclude that PPR101 is required for the splicing of nad5 introns 1 and 2,and PPR231 for the splicing of nad5 introns 1,2,3,and nad2 intron 3 in mitochondria.The mutation of PPR101 and PPR231 causes the intron splicing defect of mitochondrial nad5 and nad2 transcripts,and then the deficiency of Nad5 and Nad2 proteins,thus affecting the assembly and activity of complex 1,and blocking the oxidative phosphorylation pathway in mitochondrial respiratory chain.The consequences give rise to a series of feedback regulation,including the accumulation of other complexes in mitochondrial respiratory chain,the upregulation of ZmAOX2 and the activation of alternative oxidase pathway,which arrest the embryogenesis and endosperm development,resulting in the abortion of maize seed.These results reveal that PPR101 and PPR231 are essential to mitochondrial RNA post-transcriptional processing,mitochondrial function,and maize seed development.