Investigation on the Potential Functions of ZmEPF/EPFL Family Members in Response to Abiotic Stress in Maize.

Maize is an important crop used for food, feed, and fuel. Abiotic stress is an important factor affecting maize yield. The EPF/EPFL gene family encodes class-specific secretory proteins that play an important role in the response to abiotic stress in plants. In order to explore and utilize the EPF/EPFL family in maize, the family members were systematically identified, and their chromosomal localization, physicochemical properties, cis-acting element prediction in promoters, phylogenetic tree construction, and expression pattern analysis were carried out using bioinformatics techniques. A total of 18 ZmEPF/EPFL proteins were identified in maize, which are mostly alkaline and a small portion acidic. Subcellular localization results showed that ZmEPF6, ZmEPF12, and ZmEPFL2 are localized in the nucleus and cytoplasm. Analysis of cis-acting elements revealed that members of the ZmEPF/EPFL family contain regulatory elements such as light response, anoxic, low temperature, and hormone response regulatory elements. RT-qPCR results showed that these family members are indeed responding to cold stress and hormone treatments. These results of this study provide a theoretical basis for improving the abiotic stress resistance of maize in future research.

PPR596 Is Required for nad2 Intron Splicing and Complex I Biogenesis in Arabidopsis.

Mitochondria are essential organelles that generate energy via oxidative phosphorylation. Plant mitochondrial genome encodes some of the respiratory complex subunits, and these transcripts require accurate processing, including C-to-U RNA editing and intron splicing. Pentatricopeptide repeats (PPR) proteins are involved in various organellar RNA processing events. PPR596, a P-type PPR protein, was previously identified to function in the C-to-U editing of mitochondrial rps3 transcripts in Arabidopsis. Here, we demonstrate that PPR596 functions in the cis-splicing of nad2 intron 3 in mitochondria. Loss of the PPR596 function affects the editing at rps3eU1344SS, impairs nad2 intron 3 splicing and reduces the mitochondrial complex I's assembly and activity, while inducing alternative oxidase (AOX) gene expression. This defect in nad2 intron splicing provides a plausible explanation for the slow growth of the ppr595 mutants. Although a few P-type PPR proteins are involved in RNA C-to-U editing, our results suggest that the primary function of PPR596 is intron splicing.

The small PPR protein SPR2 interacts with PPR-SMR1 to facilitate the splicing of introns in maize mitochondria.

Splicing of plant mitochondrial introns is facilitated by numerous nucleus-encoded protein factors. Although some splicing factors have been identified in plants, the mechanism underlying mitochondrial intron splicing remains largely unclear. In this study, we identified a small P-type pentatricopeptide repeat (PPR) protein containing merely four PPR repeats, small PPR protein 2 (SPR2), which is required for the splicing of more than half of the introns in maize (Zea mays) mitochondria. Null mutations of Spr2 severely impair the splicing of 15 out of the 22 mitochondrial Group II introns, resulting in substantially decreased mature transcripts, which abolished the assembly and activity of mitochondrial complex I. Consequently, embryogenesis and endosperm development were arrested in the spr2 mutants. Yeast two-hybrid, luciferase complementation imaging, bimolecular fluorescence complementation, and semi-in vivo pull-down analyses indicated that SPR2 interacts with small MutS-related domain protein PPR-SMR1, both of which are required for the splicing of 13 introns. In addition, SPR2 and/or PPR-SMR1 interact with other splicing factors, including PPR proteins EMPTY PERICARP16, PPR14, and chloroplast RNA splicing and ribosome maturation (CRM) protein Zm-mCSF1, which participate in the splicing of specific intron(s) of the 13 introns. These results prompt us to propose that SPR2/PPR-SMR1 serves as the core component of a splicing complex and possibly exerts the splicing function through a dynamic interaction with specific substrate recognizing PPR proteins in mitochondria.

EMP80 mediates the C-to-U editing of nad7 and atp4 and interacts with ZmDYW2 in maize mitochondria.

RNA C-to-U editing is important to the expression and function of organellar genes in plants. Although several families of proteins have been identified to participate in this process, the underlying mechanism is not fully understood. Here we report the function of EMP80 in the C-to-U editing at the nad7-769 and atp4-118 sites, and the potential recruitment of ZmDYW2 as a trans deaminase in maize (Zea mays) mitochondria. Loss of EMP80 function arrests embryogenesis and endosperm development in maize. EMP80 is a PPR-E+ protein localised to mitochondria. An absence of EMP80 abolishes the C-to-U RNA editing at nad7-769 and atp4-118 sites, resulting in a cysteine-to-arginine (Cys→Arg) change in Nad7 and Atp4 in the emp80 mutant. The amino acid change consequently reduces the assembly of complexes I and V, leading to an accumulation of the F1 subcomplex of complex V. EMP80 was found to interact with atypical DYW-type PPR protein ZmDYW2, which interacts with ZmNUWA. Co-expression of ZmNUWA enhances the interaction between EMP80 and ZmDYW2, suggesting that EMP80 potentially recruits ZmDYW2 as a trans deaminase through protein-protein interaction, and ZmNUWA may function as an enhancer of this interaction.

DEK48 Is Required for RNA Editing at Multiple Mitochondrial Sites and Seed Development in Maize.

In flowering plants, C-to-U RNA editing can be critical to normal functions of mitochondrion-encoded proteins. Mitochondrial C-to-U RNA editing is facilitated by many factors from diverse protein families, of which the pentatricopeptide repeat (PPR) proteins play an important role. Owing to their large number and frequent embryo lethality in mutants, functions of many PPRs remain unknown. In this study, we characterized a mitochondrion-localized DYW-type PPR protein, DEK48, functioning in the C-to-U RNA editing at multiple mitochondrial transcripts in maize. Null mutation of Dek48 severely arrests embryo and endosperm development, causing a defective kernel (dek) phenotype, named dek48. DEK48 loss of function abolishes the C-to-U editing at nad3-185, -215, and nad4-376, -977 sites and decreases the editing at 11 other sites, resulting in the alteration of the corresponding amino acids. Consequently, the absence of editing caused reduced assembly and activity of complex I in dek48. Interestingly, we identified a point mutation in dek48-3 causing a deletion of the Tryptophan (W) residue in the DYW motif that abolishes the editing function. In sum, this study reveals the function of DEK48 in the C-to-U editing in mitochondrial transcripts and seed development in maize, and it demonstrates a critical role of the W residue in the DYW triplet motif of DEK48 for the C-to-U editing function in vivo.

Regulator of Chromosome Condensation 1-Domain Protein DEK47 Functions on the Intron Splicing of Mitochondrial Nad2 and Seed Development in Maize.

In flowering plants, mitochondrial genes contain approximately 20-26 introns. Splicing of these introns is essential for mitochondrial gene expression and function. Recent studies have revealed that both nucleus- and mitochondrion-encoded factors are required for intron splicing, but the mechanism of splicing remains largely unknown. Elucidation of the mechanism necessitates a complete understanding of the splicing factors. Here, we report the identification of a regulator of chromosome condensation 1 (RCC1)-domain protein DEK47 that is required for mitochondrial intron splicing and seed development in maize. Loss of function in Dek47 severely arrests embryo and endosperm development, resulting in a defective kernel (dek) phenotype. DEK47 harbors seven RCC1 domains and is targeted to mitochondria. Null mutation of DEK47 causes a deficiency in the splicing of all four nad2 introns, abolishing the production of mature nad2 transcript and resulting in the disassembly and severely reduced activity of mitochondrial complex I. In response, the expression of the alternative oxidase AOX2 is sharply increased in dek47. These results indicate that Dek47 is required for the splicing of all the nad2 introns in mitochondria, and essential for complex I assembly, and kernel development in maize.

The Mitochondrial Pentatricopeptide Repeat Protein PPR18 Is Required for the cis-Splicing of nad4 Intron 1 and Essential to Seed Development in Maize.

Pentatricopeptide repeat (PPR) protein comprises a large family, participating in various aspects of organellar RNA metabolism in land plants. There are approximately 600 PPR proteins in maize, but the functions of many PPR proteins remain unknown. In this study, we defined the function of PPR18 in the cis-splicing of nad4 intron 1 in mitochondria and seed development in maize. Loss function of PPR18 seriously impairs embryo and endosperm development, resulting in the empty pericarp (emp) phenotype in maize. PPR18 encodes a mitochondrion-targeted P-type PPR protein with 18 PPR motifs. Transcripts analysis indicated that the splicing of nad4 intron 1 is impaired in the ppr18 mutant, resulting in the absence of nad4 transcript, leading to severely reduced assembly and activity of mitochondrial complex I and dramatically reduced respiration rate. These results demonstrate that PPR18 is required for the cis-splicing of nad4 intron 1 in mitochondria, and critical to complex I assembly and seed development in maize.

The DYW-subgroup pentatricopeptide repeat protein PPR27 interacts with ZmMORF1 to facilitate mitochondrial RNA editing and seed development in maize.

C-to-U RNA editing in plant mitochondria requires the participation of many nucleus-encoded factors, most of which are pentatricopeptide repeat (PPR) proteins. There is a large number of PPR proteins and the functions many of them are unknown. Here, we report a mitochondrion-localized DYW-subgroup PPR protein, PPR27, which functions in the editing of multiple mitochondrial transcripts in maize. The ppr27 mutant is completely deficient in C-to-U editing at the ccmFN-1357 and rps3-707 sites, and editing at six other sites is substantially reduced. The lack of editing at ccmFN-1357 causes a deficiency of CcmFN protein. As CcmFN functions in the maturation pathway of cytochrome proteins that are subunits of mitochondrial complex III, its deficiency results in an absence of cytochrome c1 and cytochrome c proteins. Consequently, the assembly of mitochondrial complex III and super-complex I+III2 is decreased, which impairs the electron transport chain and respiration, leading to arrests in embryogenesis and endosperm development in ppr27. In addition, PPR27 was found to physically interact with ZmMORF1, which interacts with ZmMORF8, suggesting that these three proteins may facilitate C-to-U RNA editing via the formation of a complex in maize mitochondria. This RNA editing is essential for complex III assembly and seed development in maize.

Two Pentatricopeptide Repeat Proteins Are Required for the Splicing of nad5 Introns in Maize.

Mitochondrial genes in flowering plants contain predominantly group II introns that require precise splicing before translation into functional proteins. Splicing of these introns is facilitated by various nucleus-encoded splicing factors. Due to lethality of mutants, functions of many splicing factors have not been revealed. Here, we report the function of two P-type PPR proteins PPR101 and PPR231, and their role in maize seed development. PPR101 and PPR231 are targeted to mitochondria. Null mutation of PPR101 and PPR231 arrests embryo and endosperm development, generating empty pericarp and small kernel phenotype, respectively, in maize. Loss-of-function in PPR101 abolishes the splicing of nad5 intron 2, and reduces the splicing of nad5 intron 1. Loss-of-function in PPR231 reduces the splicing of nad5 introns 1, 2, 3 and nad2 intron 3. The absence of Nad5 protein eliminates assembly of complex I, and activates the expression of alternative oxidase AOX2. These results indicate that both PPR101 and PPR231 are required for mitochondrial nad5 introns 1 and 2 splicing, while PPR231 is also required for nad5 intron 3 and nad2 intron 3. Both genes are essential to complex I assembly, mitochondrial function, and maize seed development. This work reveals that the splicing of a single intron involves multiple PPRs.

Em pty Pericarp24 and Empty Pericarp25 Are Required for the Splicing of Mitochondrial Introns, Complex I Assembly, and Seed Development in Maize.

RNA splicing is an essential post-transcriptional regulation in plant mitochondria and chloroplasts. As the mechanism of RNA splicing remains obscure, identification and functional elucidation of new splicing factors are necessary. Through a characterization of two maize mutants, we cloned Empty pericarp 24 (Emp24) and Empty pericarp 25 (Emp25). Both Emp24 and Emp25 encode mitochondrion-targeted P-type PPR proteins. EMP24 is required for the splicing of nad4 introns 1 and 3, which was reported (Ren Z. et al., 2019), and EMP25 functions in the splicing of nad5 introns 1, 2, and 3. Absence of either Nad4 or Nad5 proteins blocks the assembly of mitochondrial complex I, resulting in the formation of a sub-sized complex I of similar size in both mutants. Mass spectrometry identification revealed that the subcomplexes in both mutants lack an identical set of proteins of complex I. These results indicate that EMP24 and EMP25 function in the splicing of nad4 and nad5 introns, respectively, and are essential to maize kernel development. The identification of the subcomplexes provides genetic and molecular insights into the modular complex I assembly pathway in maize.