Tetratricopeptide-containing SMALL KERNEL 11 is essential for the assembly of cytochrome c oxidase in maize mitochondria.

Assembly of the functional complexes of the mitochondrial respiratory chain requires sophisticated and efficient regulatory mechanisms. In plants, the subunit composition and assembly factors involved in the biogenesis of cytochrome c oxidase (complex IV) are substantially less defined than in mammals and yeast. In this study, we cloned maize (Zea mays) Small kernel 11 (Smk11) via map-based cloning. Smk11 encodes a mitochondria-localized tetratricopeptide repeat protein. Disruption of Smk11 severely affected the assembly and activity of mitochondrial complex IV, leading to delayed plant growth and seed development. Protein interactions studies revealed that SMK11 might interact with four putative complex IV assembly factors, Inner membrane peptidase 1A (ZmIMP1A), MYB domain protein 3R3 (ZmMYB3R-3), cytochrome c oxidase 23 (ZmCOX23), and mitochondrial ferredoxin 1 (ZmMFDX1), among which ZmMFDX1 might interact with subunits ZmCOX6a and ZmCOX-X1; ZmMYB3R-3 might also interact with ZmCOX6a. The mutation of SMK11 perturbed the normal assembly of these subunits, leading to the inactivation of complex IV. The results of this study revealed that SMK11 serves as an accessory assembly factor required for the normal assembly of subunits into complex IV, which will accelerate the elucidation of the assembly of complex IV in plant mitochondria.

The pentatricopeptide repeat protein EMP603 is required for the splicing of mitochondrial Nad1 intron 2 and seed development in maize.

Intron splicing is an essential event in post-transcriptional RNA processing in plant mitochondria, which requires the participation of diverse nuclear-encoded splicing factors. However, it is presently unclear how these proteins cooperatively take part in the splicing of specific introns. In this study, we characterized a nuclear-encoded mitochondrial P-type pentatricopeptide repeat (PPR) protein named EMP603. This protein is essential for splicing of intron 2 in the Nad1 gene and interacts with the mitochondria-localized DEAD-box RNA helicase PMH2-5140, the RAD52-like proteins ODB1-0814 and ODB1-5061, and the CRM domain-containing protein Zm-mCSF1. Further study revealed that the N-terminal region of EMP603 interacts with the DEAD-box of PMH2-5140, the CRM domain of Zm-mCSF1, and OBD1-5061, but not with OBD1-0814, whereas the PPR domain of EMP603 can interact with ODB1-0814, ODB1-5061, and PMH2-5140, but not with Zm-mCSF1. Defects in EMP603 severely disrupt the assembly and activity of mitochondrial complex I, leading to impaired mitochondrial function, and delayed seed development. The interactions revealed between EMP603 and PMH2-5140, ODB1-0814, ODB1-5061, and Zm-mCSF1 indicate a possible involvement of a dynamic 'spliceosome-like' complex in intron splicing, and may accelerate the elucidation of the intron splicing mechanism in plant mitochondria.

Maize Defective Kernel605 Encodes a Canonical DYW-Type PPR Protein that Edits a Conserved Site of nad1 and Is Essential for Seed Nutritional Quality.

Pentatricopeptide repeat (PPR) proteins involved in mitochondrial RNA cytidine (C)-to-uridine (U) editing mostly result in stagnant embryo and endosperm development upon loss of function. However, less is known about PPRs that are involved in farinaceous endosperm formation and maize quality. Here, we cloned a maize DYW-type PPR Defective Kernel605 (Dek605). Mutation of Dek605 delayed seed and seedling development. Mitochondrial transcript analysis of dek605 revealed that loss of DEK605 impaired C-to-U editing at the nad1-608 site and fails to alter Ser203 to Phe203 in NAD1 (dehydrogenase complex I), disrupting complex I assembly and reducing NADH dehydrogenase activity. Meanwhile, complexes III and IV in the cytochrome pathway, as well as AOX2 in the alternative respiratory pathway, are dramatically increased. Interestingly, the dek605 mutation resulted in opaque endosperm and increased levels of the free amino acids alanine, aspartic acid and phenylalanine. The down- and upregulated genes mainly involved in stress response-related and seed dormancy-related pathways, respectively, were observed after transcriptome analysis of dek605 at 12 d after pollination. Collectively, these results indicate that Dek605 specifically affects the single nad1-608 site and is required for normal seed development and resulted in nutritional quality relevant amino acid accumulation.

Maize Empty Pericarp602 Encodes a P-Type PPR Protein That Is Essential for Seed Development.

Pentatricopeptide repeat (PPR) proteins play crucial roles in intron splicing, which is important for RNA maturation. Identification of novel PPR protein with the function of intron splicing would help to understand the RNA splicing mechanism. In this study, we identified the maize empty pericarp602 (emp602) mutants, the mature kernels of which showed empty pericarp phenotype. We cloned the Emp602 gene from emp602 mutants and revealed that Emp602 encodes a mitochondrial-localized P-type PPR protein. We further revealed that Emp602 is specific for the cis-splicing of mitochondrial Nad4 intron 1 and intron 3, and mutation of Emp602 led to the loss of mature Nad4 transcripts. The loss of function of Emp602 nearly damaged the assembly and accumulation of complex I and arrested mitochondria formation, which arrested the seed development. The failed assembly of complex I triggers significant upregulation of Aox expression in emp602 mutants. Transcriptome analysis showed that the expression of mitochondrial-related genes, e.g. the genes associated with mitochondrial inner membrane presequence translocase complex and electron carrier activity, were extensively upregulated in emp602 mutant. These results demonstrate that EMP602 functions in the splicing of Nad4 intron 1 and intron 3, and the loss of function of Emp602 arrested maize seed development by disrupting the mitochondria complex I assembly.

ZmnMAT1, a nuclear-encoded type I maturase, is required for the splicing of mitochondrial Nad1 intron 1 and Nad4 intron 2.

Maturases can specifically bind to intron-containing pre-RNAs, folding them into catalytic structures that facilitate intron splicing in vivo. Plants possess four nuclear-encoded maturase-related factors (nMAT1-nMAT4) and some maturases have been shown to involve in the splicing of different mitochondrial group II introns; however, the specific biological functions of maturases in maize are largely uncharacterized. In this study, we identified a maize ZmnMAT1 gene, which encodes a mitochondrion-localized type I maturase with an RT domain at N-terminus and an X domain at C-terminus. Loss-of-function mutation in ZmnMAT1 significantly reduced the splicing efficiencies of Nad1 intron 1 and Nad4 intron 2, and showed arrested embryogenesis and endosperm development, which may be related to impaired mitochondrial ultrastructure and function due to the destruction of the assembly and activity of complex I. Direct physical interaction was undetectable between ZmnMAT1 and the proteins associated with the splicing of Nad1 intron 1 and/or Nad4 intron 2 by yeast two-hybrid assays, suggesting the complexity of group II intron splicing in plants.

Generation of Transgene-Free Semidwarf Maize Plants by Gene Editing of Gibberellin-Oxidase20-3 Using CRISPR/Cas9.

The "green revolution" gene gibberellin oxidase contributes to the semidwarf phenotype, improving product and lodging resistance. Dissecting the function of GA biosynthetic genes would be helpful for dwarf maize breeding. In this study, we edited the maize GA20ox3 gene and generated semidwarf maize plants using CRISPR/Cas9 technology. Application of exogenous gibberellin can recover the dwarf phenotype, indicating that the mutants are gibberellin deficient. The contents of GA12 and GA53 were elevated in the mutants due to the disruption of GA20 oxidase, whereas the contents of other GA precursors (GA15, GA24, GA9, GA44, and GA20) were decreased in the mutants, and the accumulation of bioactive GA1 and GA4 was also decreased, contributing to the semidwarf phenotype. Transgene-free dwarf maize was selected from T2-generation plants and might be useful for maize breeding in the future.

Mutations in the maize zeta-carotene desaturase gene lead to viviparous kernel.

Preharvest sprouting reduces the maize quality and causes a significant yield loss in maize production. vp-wl2 is a Mutator (Mu)-induced viviparous mutant in maize, causing white or pale yellow kernels, dramatically reduced carotenoid and ABA content, and a high level of zeta-carotene accumulation. Here, we reported the cloning of the vp-wl2 gene using a modified digestion-ligation-amplification method (DLA). The results showed that an insertion of Mu9 in the first intron of the zeta-carotene desaturase (ZDS) gene results in the vp-wl2 mutation. Previous studies have suggested that ZDS is likely the structural gene of the viviparous9 (vp9) locus. Therefore, we performed an allelic test using vp-wl2 and three vp9 mutants. The results showed that vp-wl2 is a novel allele of the vp9 locus. In addition, the sequences of ZDS gene were identified in these three vp9 alleles. The vp-wl2 mutant gene was subsequently introgressed into four maize inbred lines, and a viviparous phenotype was observed with yield losses from 7.69% to 13.33%.