Functional Analysis Of A Key Seed Development Gene PPR114 In Maize

PPR(pentatricopeptide repeat)proteins as one kind of RNA binding protein are widely distributed interrestrial plants.The function-loss of PPR proteins in some cases bring plant development deficiency:restricted growth,male sterility,seeds abortion,etc.It has been verified that PPR proteins mainly involve in organellarpost-transcriptional processes,including RNA precursor cleavage,ending maturation,stability,splicing and RNA editing(C?U).We found a PPR gene and name it PPR114,then we order two ppr114independent mutant alleles from UniformMu,then studied them from the following items:1)insertion idetificaiton and phenotype observation;2)subcellular localization of PPR114;3)screening the aberrance of mitochondrialgenome encoding transcripts,so as to identify the substrate of PPR114;4)analyzing mitochondrialcomplex assembly and activity;5)combining PPR code analysis to find the putative cis-elements of editing sites.Several conclusions were made as follow:1)ppr114 gives rise to an empty pericarp(emp),and embryo and endosperm abort at early development stage.The analysis of genotype and alleles test indicate that the abortion of seeds is caused by PPR 114mutation.The kernels from PPR114/ppr114 heterozygous plant segregate defective kernels with 25%proportion,indicating a recessive monogenic mutation.The observation of ppr114 mutant Paraffinsectioning shows retarded development of embryo and endosperm,and the embryo aborts at transition stage.2)PPR114 encodes an E+subgroup PPR protein targeting to mitochondria.Alignment of PPR114 with conserved PPR motifs reveals that PPR114 is an E+subgroup PPR protein.And Target P prediction result demonstrates that PPR114 localizes to mitochondria.Then we construct an transient expression vector fused with GFP(35S::PPR1 14?N136aa:GFP),which also shows a localization to mitochondria.3)PPR114 is required for the editing of nad7-169 and atp4-118.It is reported that PPR proteins from PLS subfamily involve in organelle RNA editing(C?U).By comparing RT-PCR analysis,we find that the mutation of PPR114 abolish nad7-769 and atp4-118 C?U RNA editing.In ppr114 mutant,the 257th amino acid of Nad7 converts from Cysteine to Argine,and there is also an transition from Cysteine to Argine at the 40th amino acid of Atp4.4)The loss of PPR114 impairs mitochondrial complexI assembly and activity.Nad7 is a mitochondrial complex I key subunit,and the dysfunction of it can block the electon transfer of mitochondrialcytochrome respiratorypathway,thus compromising mitochondrial function.Crude mitochondria are resolved in Blue-nativepolyacrylamide gel electrophoresis(BN-PAGE)to segregate mitochondrial protein complexes.The result reveals that inppr114-1,the abundance ofcomplex I decreases dramatically compared with that in wild type(WT),and NADH dehydrogenase activity also decrease sharply.Thus the editing abolishment of nad7-769 comprises Nad7 function,affecting complex I assembly and NADH dehydrogenase activity.5)The loss of PPR114 affects the assembly of complex V.Atp4 is a component of ATP Synthase(F0F1)peripheral stalk,participating in stabilizing F0F1 holoenzyme.we test ATP hydrolytic enzyme activity by Pb(NO3)2 dye method.It reveals that the abundance of free F1 subunit is higher in ppr114 mutant compared with that in WT siblings;meanwhile the result of western blotting with ATPaseaspecific antibody also indicates an increase of free F1 subunit in ppr114 mutant,hence,the assembly of complex V is probably impaired in ppr114 mutant.6)The putative cis-elements of PPR114.The alignment of 16 nt upstream and 3 nt downstream of nad7-769 and atp4-118 shows 60%identity.PPR code analysis of P and S motif shows high consistency with the vicinity sequence(16 nt upstream)of nad7-769 and atp4-118 editing sites,which suggests that these two 16 nt nucleotidesequences may act as cis-elements of the two editing sites recognized by PPR114.Above all,these results reveals that PPR114 is required for the editing of nad7-769 and atp4-118to regulate mitochondrial genome encoding genes expression,and thus to maize seeds development.