Pentatricopeptide repeat (PPR) proteins comprise a large family of sequence-specific RNA binding proteins in land plants. Because of its large family size and frequent embryo lethality in the mutants, molecular functions and physiological roles of many PPR proteins are unknown. Through characterization of an empty pericarp9 (emp9) mutant in maize (Zea mays), we defined the functions of EMP9 in mitochondrial RNA editing, respiratory complex formation and seed development. Mu insertions in different regions of Emp9 facilitated dissection of the domain functions of the EMP9. Through genetic and functional analyses of multiple alleles, we showed that deletions of two N-terminal PPR motifs and partial E+ domain do not eliminate the editing function of EMP9. Emp9 encodes an E+ subclass PPR protein that is localized in mitochondria. Loss of EMP9 function abolishes the C-to-U editing of ccmB-43 and rps4-335 sites in mitochondria. The loss of editing at ccmB-43 and rps4-335 affects the maturation of cytochrome c and impairs the biogenesis of mitochondrial respiratory complexes, particularly complex III. This work extends our understanding of PPR-E+ protein in editing function and seed development, and provides insights into the molecular function of mitochondrial CcmB protein in higher plants.

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).

SUMMARYTelomere truncation has been shown to be an efficient technology for the creation of mini-chromosomes that can be used as artificial chromosome platforms for genetic engineering. Artificial chromosome-based genetic engineering is considered to be superior to the existing techniques of randomized gene integration by Agrobacterium or biolistic-mediated genetic transformation. It organizes multiple transgenes as a unique genetic linkage block for subsequent manipulations in breeding. Telomere truncation technology relies on three components: the telomere sequence that mediates chromosomal truncation, a selection marker that allows the selection of transgenic events, and a site-specific recombination system that can be used to accept future genes into the mini-chromosome by gene targeting. These elements are usually pre-assembled before transformation, a process that is both time and labor consuming. We found in this research that the three elements could be mixed to transform plant cells in a biolistic transformation, and produced efficient chromosomal truncations and mini-chromosomes in rice. This system will allow rapid construction of minichromosomes with a flexible selection of resistant markers, site-specific recombination systems and other desirable elements. In addition, a rice telotrisomic line was used as the starting material for chromosomal truncations. Mini-chromosomes from the truncations of both the telocentric chromosome and other chromosomes were recovered. The mini-chromosomes remained stable during 2 years of subculture. The construction of mini-chromosomes in rice, an economically important crop, will provide a platform for future artificial chromosome-based genetic engineering of rice for stacking multiple genes.

Two maize nucleus-encoded splicing factors, PPR-SMR1 and Zm-mCSF1, are required for the splicing of most mitochondrial group II introns and subsequent complex I biogenesis, and therefore play important roles in seed development.

RNA editing plays an important role in organellar gene expression in plants, and pentatricopeptide repeat (PPR) proteins are involved in this function. Because of its large family size, many PPR proteins are not known for their function and roles in plant growth and development. Through genetic and molecular analyses of the empty pericarp18 (emp18) mutant in maize (Zea mays), we cloned the Emp18 gene, revealed its molecular function, and defined its role in the mitochondrial complex assembly and seed development. Emp18 encodes a mitochondrial-localized DYW-PPR protein. Null mutation of Emp18 arrests embryo and endosperm development at an early stage in maize, resulting in embryo lethality. Mutants are deficient in the cytidine (C)-to-uridine (U) editing at atp6-635 and cox2-449, which converts a Leu to Pro in ATP6 and a Met to Thr in Cox2. The atp6 gene encodes the subunit a of F F -ATPase. The Leu to Pro alteration disrupts an α-helix of subunit a, resulting in a dramatic reduction in assembly and activity of F F -ATPase holoenzyme and an accumulation of free F -subcomplex. These results demonstrate that EMP18 functions in the C-to-U editing of atp6 and cox2, and is essential to mitochondrial biogenesis and seed development in maize.

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A salinity stress upregulated expressed sequence tag (EST) was selected from a suppression subtractive hybridization cDNA library, constructed from the salinity-tolerant wheat cultivar Shanrong No. 3. Sequence analysis showed that the corresponding gene (named TaDi19A) belonged to the Di19 family. TaDi19A was constitutively expressed in both the root and leaf of wheat seedlings grown under nonstressed conditions, but was substantially up-regulated by the imposition of stress (salinity, osmotic stress and cold), or the supply of stress-related hormones [abscisic acid (ABA) and ethylene]. The heterologous over-expression of TaDi19A in Arabidopsis thaliana increased the plants' sensitivity to salinity stress, ABA and mannitol during the germination stage. Root elongation in these transgenic lines showed a reduced tolerance to salinity stress and a reduced sensitivity to ethophon. The expression of the ABA signal pathway genes ABI1, RAB18, ERD15 and ABF3, and SOS2 (SOS pathway) was altered in the transgenic lines. TaDi19A plays a role in the plant's response to abiotic stress, and some possible mechanisms of its action are proposed.

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