Functional Analysis Of Spr2 In Maize Seed Development

Seed development is essential to cereal yield.However,the fundamental mechanisms governing these processes are obscure.Plant seed is originated from double fertilization.Many genes participate in the regulation of seed development.Unfortunately,mutants in many of these genes are embryo lethality or pollen sterile,obstructing further study.We uncovered the maize seed mutant system that allows functional study of these genes.Recent studies revealed that PPR genes play critical roles in embryogenesis processe.Given the size and complexity of the PPR protein family and embryo lethality in mutants,the function of many PPR genes and the fundamental mechanisms governing these processes are obscure.Hence identifying these genes and elucidating their functions is the key to a better understanding of the underlying mechanisms.PPR proteins are encoded by nucleus,consisting of 35 peptides repeats.Based on the motifs,PPR proteins can be divided into P subclass and PLS subclass.PPR proteins recognize RNA in a modular and sequence specific manner(PPR code):each PPR motif recognizes one nucleotide in RNA via interaction with the residues at 5th and 35th positions in the motif.PPRs are mainly targeted to chloroplasts or mitochondria,functioning in organellar RNA processing,including RNA editing,RNA maturation and stabilization,and intron splicing.Intron splicing is essential to expression of mitochondrial genes.Almost all of the mitochondrial introns in plants belong to group Ⅱ.In bacteria,ancient group Ⅱ introns can self-splice,which is facilitated by the intron-encoded cognate maturase(Mat)-However,mitochondrial group Ⅱ introns in land plants have lost the self-splicing activity,caused by a loss of maturase genes in the introns and compounded by accumulated mutations in the intron sequences.Instead,splicing of these group Ⅱ introns ia facilitated by many nucleus-encoded proteins from multiple families.Despite recent studies have uncovered proteins from several families that are required for splicing of mitochondrial introns,the splicing of a specific intron may require the participation of multiple splicing factors.How these splicing factors function in the splicing event and the underlying mechanism remains largely unclear.This study addresses the function of PPRs with a few PPR repeats.To answer this question,we attempted to identify the function of PPR proteins with less than four PPR repeats.This led us to reveal the function of GRMZM2G122344 designated as Small PPR protein 2,SPR2,which contains merely four PPR repeats.Spr2 is an intronless gene encoding a 35 kDa protein with 320 amino acid residues.SPR2 is a canonical P-type PPR protein as predicted by the TPRpred algorithm.Spr2 mutants are obtained from the Maize Genetics Stock Center.Two insertional mutants were isolated with Mu insertions at+21 bp and+529 bp from the translation start codon ATG of Spr2,named spr2-1 and spr2-2,respectively.The selfed progeny of spr2-1/+and spr2-2/+heterozygous plants segregated wild-type(WT)and empty pericarp(emp)kernels at a 3:1 ratio,suggesting that the emp phenotype is caused by a monogenic and recessive mutation.Linkage analysis showed that the Mu insertion is tightly linked to the emp phenotype.Reciprocal crosses between spr2-1/+and spr2-2/+heterozygotes.produced approximately a quarter emp mutant kernels,and over-expression of Spr2 rescues the embryo-lethality of spr2,confirming that the emp phenotype is indeed caused by the loss of SPR2.Paraffin sectioning showed that the Spr2 mutation arrests the embryo and endosperm development in spr2.Protein localization by fusing the N-terminal 291 amino acids of SPR2 with GFP at the C-terminus and transfecting Nicotiana benthamiana leaves showed that SPR2 is targeted to mitochondria.Western blotting analysis on fractions of different cellular compartments of the transgenic Spr2-OE1 plants demonstrates that SPR2 is localized in mitochondria.The cytochrome respiratory rate is substantially decreased,alternative respiratory rate is sharply increased,and the alternative oxidase transcripts of AOX2 and AOX3 and the AOX abundance were dramatically increased in the spr2 mutants compared with WT,indicating that Spr2 loss of function impairs the cytochrome pathway and enhances the alternative oxidase pathway.Coomassie Brilliant Blue(CBB)staining and In-gel NBT-NADH activity staining showed that the complex Ⅰ and super-complex CⅠ+CⅢ2 were completely deficient in spr2-1,indicating that the loss of SPR2 inhibits the assembly and activity of complex Ⅰ in mitochondria.RT-PCR results indicate that the transcripts of nad1,nad2,nad4,nad5,and nad7 are remarkably reduced and the splicing of 15 group Ⅱ introns(nad1-intron 1 to 4;nad2-intron 1 to 4;nad4-intron 1,3;nad5-intron 1,2,4 and nad7-intron 1,2)is impaired in both spr2 alleles.Meanwhile,the splicing efficiency of these introns is dramatically decreased in spr2,demonstrating that SPR2 is required for the splicing of these 15 introns in maize mitochondria.The splicing efficiency of these introns in Spr2 transgenic lines and complemented lines is comparable to that in the WT,indicating that 1)over-expression of SPR2-HA restores the splicing defects in the mutants;2)over-expression of SPR2-HA does not increase the splicing efficiency.Y2H,LCI,BiFC,and semi-in vivo pulled-down analyses indicate that SPR2 interacts with Small MutS-related domain protein PPR-SMR1,both of which are required for the splicing of 13 introns(nad1-intron 1 to 4;nad2-intron 1 to 4;nad4-intron 1,3;nad5-intron 1,4;nad7-intron 2).In addition,SPR2 interacts with other splicing factors,including PPR proteins EMP16,PPR14,and Chloroplast RNA splicing and ribosome Maturation(CRM)domain-containing protein Zm-mCSF1,which is known to participate in the splicing of specific intron(s)of the 13 introns.PPR-SMR1 was reported to interact with PPR 14 and Zm-mCSF1,but not EMP16.However,PPR-SMR1 can interact with EMP16 through the bridge of SPR2.Based on the results,we propose a model for the SPR2 and PPR-SMR1 function in the Group Ⅱ intron splicing in maize mitochondria.SPR2 interacts with PPR-SMR1 to form a core complex,which binds to other intron-specific splicing factors.SPR2 and PPR-SMR1 can interact with various splicing factors(CRM proteins,P-type PPR protein,et al.)to facilitate the splicing of an individual intron and possibly exerts the splicing function through a dynamic interaction with specific substrate recognizing proteins in mitochondria.Although the splicing mechanism of mitochondrial introns is still unclear,uncovering the core role of SPR2/PPR-SMR1 will facilitate the complete elucidation of the mitochondrial intron-splicing mechanism in plants.The above study substantially broadens our understanding of PPR gene functions,molecular mechanisms and genetic networks of kernel development and provide theoretical support and genetic resources for molecular breeding of maize.