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.

PHB3 Is Required for the Assembly and Activity of Mitochondrial ATP Synthase in Arabidopsis.

Mitochondrial ATP synthase is a multiprotein complex, which consists of a matrix-localized F1 domain (F1-ATPase) and an inner membrane-embedded Fo domain (Fo-ATPase). The assembly process of mitochondrial ATP synthase is complex and requires the function of many assembly factors. Although extensive studies on mitochondrial ATP synthase assembly have been conducted on yeast, much less study has been performed on plants. Here, we revealed the function of Arabidopsis prohibitin 3 (PHB3) in mitochondrial ATP synthase assembly by characterizing the phb3 mutant. The blue native PAGE (BN-PAGE) and in-gel activity staining assays showed that the activities of ATP synthase and F1-ATPase were significantly decreased in the phb3 mutant. The absence of PHB3 resulted in the accumulation of the Fo-ATPase and F1-ATPase intermediates, whereas the abundance of the Fo-ATPase subunit a was decreased in the ATP synthase monomer. Furthermore, we showed that PHB3 could interact with the F1-ATPase subunits ß and δ in the yeast two-hybrid system (Y2H) and luciferase complementation imaging (LCI) assay and with Fo-ATPase subunit c in the LCI assay. These results indicate that PHB3 acts as an assembly factor required for the assembly and activity of mitochondrial ATP synthase.

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.

Comparative assessment of chromate bioremediation potential of Pantoea conspicua and Aspergillus niger.

The recent work aims at the use of Pantoea conspicua (MT5) and Aspergillus niger (CRS3) to assess their bioremediation potential and growth restoration of Helianthus annuus L. under chromate (Cr+6) stress. The growth of the P. conspicua and A. niger was tested in Cr+6 supplemented media. The strains can withstand up to 1200 and 900 ppm respectively in the media and effectively bio-transform it to nontoxic form. Supplemented metal's levels significantly decreased the growth attribute of H. annuus (p< 0.05). On the other hand, P. conspicua and A. niger rescued the host plant by establishing higher colonization frequency with the host roots. Moreover, MT5 bio-transformed the toxic Cr+6 to non-toxic Cr+3 form in the rhizosphere. It also enhanced the host plant growth by producing phytohormones and ceasing Cr uptake and accumulation. Contrarily, CRS3 tends to accumulate and bio-transform metal in their hyphae. Nonetheless, both of the microbes tend to modulate phytohormones production and strengthening antioxidant system of the host. Improvement in the antioxidant system enabled the host plant to produce higher phenolics and flavonoids, and lower peroxidase. The associated plant species also exhibited higher ROS scavenging and lower ROS accumulation. Besides, the strains were able to produce higher amounts of phytohormones, including IAA, GA, and SA. Such activities rendered them as excellent phytostimulants, that can be used as biofertilizers in chromium polluted soils.

PPR-DYW Protein EMP17 Is Required for Mitochondrial RNA Editing, Complex III Biogenesis, and Seed Development in Maize.

The conversion of cytidines to uridines (C-to-U) at specific sites in mitochondrial and plastid transcripts is a post-transcriptional processing event that is important to the expression of organellar genes. Pentatricopeptide repeat (PPR) proteins are involved in this process. In this study, we report the function of a previously uncharacterized PPR-DYW protein, Empty pericarp17 (EMP17), in the C-to-U editing and kernel development in maize. EMP17 is targeted to mitochondria. The loss-function of EMP17 arrests maize kernel development, abolishes the editing at ccmF C -799 and nad2-677 sites, and reduces the editing at ccmF C -906 and -966 sites. The absence of editing causes amino acid residue changes in CcmFC-267 (Ser to Pro) and Nad2-226 (Phe to Ser), respectively. As CcmFC functions in cytochrome c (Cytc) maturation, the amount of Cytc and Cytc 1 protein is drastically reduced in emp17, suggesting that the CcmFC-267 (Ser to Pro) change impairs the CcmFC function. As a result, the assembly of complex III is strikingly decreased in emp17. In contrast, the assembly of complex I appears less affected, suggesting that the Nad2-226 (Phe to Ser) change may have less impact on Nad2 function. Together, these results indicate that EMP17 is required for the C-to-U editing at several sites in mitochondrial transcripts, complex III biogenesis, and seed development in maize.

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.

SMALL KERNEL4 is required for mitochondrial cox1 transcript editing and seed development in maize.

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. Pentatricopeptide repeat (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 oxidase1 (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.

Empty Pericarp21 encodes a novel PPR-DYW protein that is required for mitochondrial RNA editing at multiple sites, complexes I and V biogenesis, and seed development in maize.

C-to-U editing is an important event in post-transcriptional RNA processing, which converts a specific cytidine (C)-to-uridine (U) in transcripts of mitochondria and plastids. Typically, the pentatricopeptide repeat (PPR) protein, which specifies the target C residue by binding to its upstream sequence, is involved in the editing of one or a few sites. Here we report a novel PPR-DYW protein EMP21 that is associated with editing of 81 sites in maize. EMP21 is localized in mitochondria and loss of the EMP21 function severely inhibits the embryogenesis and endosperm development in maize. From a scan of 35 mitochondrial transcripts produced by the Emp21 loss-of-function mutant, the C-to-U editing was found to be abolished at five sites (nad7-77, atp1-1292, atp8-437, nad3-275 and rps4-870), while reduced at 76 sites in 21 transcripts. In most cases, the failure to editing resulted in the translation of an incorrect residue. In consequence, the mutant became deficient with respect to the assembly and activity of mitochondrial complexes I and V. As six of the decreased editing sites in emp21 overlap with the affected editing sites in emp5-1, and the editing efficiency at rpl16-458 showed a substantial reduction in the emp21-1 emp5-4 double mutant compared with the emp21-1 and emp5-4 single mutants, we explored their interaction. A yeast two hybrid assay suggested that EMP21 does not interact with EMP5, but both EMP21 and EMP5 interact with ZmMORF8. Together, these results indicate that EMP21 is a novel PPR-DYW protein required for the editing of ~17% of mitochondrial target Cs, and the editing process may involve an interaction between EMP21 and ZmMORF8 (and probably other proteins).