Functional Analysis Of PPR-SMR1,Zm-mCSF1,PPR14,and SMK4 In Seed Development Of Maize

Group Ⅱ introns are ribozymes that can excise themselves from precursor-RNA transcripts,but plant organellar group Ⅱ introns have structural deviations that inhibit ribozyme activity.Therefore,splicing of these introns requires the assistance of nuclear-and/or organellar-encoded splicing factors;however,how these splicing factors function remains unclear.In this study,we report the functions and interactions of two splicing factors,PPR-SMR1 and Zm-mCSF1,in intron splicing in maize mitochondria.PPR-SMR1 is a SMR domain-containing pentatricopeptide repeat(PPR)protein and Zm-mCSF1 is a CRM domain-containing protein,and both are targeted to mitochondria.Loss-of-function mutations in each of them severely arrests embryogenesis and endosperm development in maize.Functional analyses indicate that PPR-SMR1 and Zm-mCSF1 are required for the splicing of most mitochondrial groupⅡ introns.Among them,the introns 2 and 3 of the NADH dehydrogenase 2(nad2)and the intron 1 of nad5 are PPR-SMR1/Zm-mCSFl-dependent introns.Our findings suggest that PPR-SMR1,a novel splicing factor,acts in the splicing of multiple groupⅡ introns in maize mitochondria,and its interaction with Zm-mCSF1 might allow the formation of large macromolecular splicing complexes.In plants,splicing of organellar group Ⅱ introns requires numerous nuclearencoded trans-factors.But,how these trans-factors function and interact is not well understood.Here we report the function of a PPR protein PPR14 and its physical relationship with other splicing factors in mitochondria.Null mutations of PPR14 severely arrest the embryo and endosperm development,causing an empty pericarp phenotype.PPR 14 is required for the splicing of nad2 intron 3 and nad7 introns 1 and 2 in mitochondria.The absence of nad2 and nad7 transcripts leads to disruption of the mitochondrial complex I assembly and abolishes its NADH dehydrogenase activity.This is accompanied with increased levels of other mitochondrial complexes and elevated expression of the alternative oxidase proteins.As the function of PPR14 overlaps with PPR-SMR1 and the CRM-domain containing protein Zm-mCSF1,we tested their interactions.Protein-protein interaction analysis indicated that PPR14 interacts with PPR-SMR1 and Zm-mCSF1,suggesting that these three proteins may form a complex.As PPR proteins and CRM-domain containing proteins have many members in mitochondria and chloroplasts,we propose that organellar group Ⅱ intron splicing is probably mediated by a dynamic complex that includes different PPR and CRM proteins in plants.In land plants,cytidine-to-uridine(C-to-U)editing of organellar transcripts is an important post-transcriptional process,which is considered to remediate DNA genetic mutations to restore the coding of functional proteins.PPR proteins have key roles in C-to-U editing.Owing to its large number,however,the biological functions of many PPR proteins remain to be identified.Through characterizing a small kernel4(smk4)mutant,here we report the function of Smk4 and its role in maize growth and development.Null mutation of Smk4 slows plant growth and development,causing small plants,delayed flowering time,and small kernels.Cloning revealed that Smk4 encodes a new E-subclass PPR protein,and localization indicated that SMK4 is exclusively localized in mitochondria.Loss of Smk4 function abolishes C-to-U editing at position 1489 of the cytochrome c oxidasel(cox1)transcript,causing an amino acid change from serine to proline at 497 in Cox1.Cox1 is a core component of mitochondrial complex Ⅳ.Indeed,complex Ⅳ activity is reduced in the smk4,along with drastically elevated expression of alternative oxidases(AOX).These results indicate that SMK4 functions in the C-to-U editing of cox1-1489,and this editing is crucial for mitochondrial complex Ⅳ activity,plant growth,and kernel development in maize.