Nucleo-cytoplasmic interactions affect RNA editing of cox2, atp6 and atp9 in alloplasmic male-sterile rice (Oryza sativa L.) lines

RNA editing plays an important role in the regulation of mitochondrial gene expression in flowering plants. In this study, we examined RNA editing of the mitochondrial genes cox2, atp6 and atp9 in five isonuclear alloplasmic male-sterile lines (IAMSLs) of rice to investigate whether different cytoplasmic types affect RNA editing. Although many editing sites were conserved among the three genes, we found that the editing efficiency of certain sites was significantly different between different IAMSLs or between IAMSLs and their corresponding cytoplasmic donor CMS lines. Furthermore, several editing sites were found to be either present or absent in certain IAMSLs and their corresponding CMS lines. These results indicate that nuclear loci, as well as unknown editing factors within the mitochondria of different cytoplasmic types, may be involved in RNA editing, and they suggest that RNA editing in plant mitochondria is affected by nucleo-cytoplasmic interactions.     

A CMS-Related Gene, Ψatp6-2, Causes Increased ATP Hydrolysis Activity of the Mitochondrial F1Fo-ATP Synthase and Induces Male Sterility in Pepper (Capsicum annuum L.)

Pollen formation is a complex process that is strictly controlled by genetic factors. Although many novel mitochondrial genes have been implicated in the dysfunction of mitochondrial enzymes and the cytoplasmic male sterility (CMS), there is little empirical evidence to show that CMS-related genes actually result in the dysfunction of enzyme and little is known about the regulatory mechanisms of the aberrant mitochondrial enzymes in male sterility in CMS lines. Here, we report the characterization of a novel mitochondrial gene, Ψatp6-2, which is hypothesized to play a role in male sterility in pepper. Using virus-induced gene silencing (VIGS), we observed that silencing the atp6-2 gene in the maintainer line resulted in an increase in ATP hydrolysis activity of the mitochondrial F₁F_o-ATP synthase along with pollen abortion, while silencing the truncated Ψatp6-2 gene in the CMS line resulted in an inhibition of ATP hydrolysis activity and restoration of fertility. Altered ATP hydrolysis also affected the tolerance of the gene-silenced plants to abiotic stresses. Localization experiments showed that premature ATP hydrolysis occurred at the tetrad stage of pollen development in the CMS line, but no ATPase activity was observed in the microspores at the later stage. These results suggest that the Ψatp6-2 gene causes the alteration in ATP hydrolysis activity of the mitochondrial F₁F_o-ATP synthase during pollen development, which eventually leads to male sterility in pepper.     

红莲型细胞质雄性不育水稻线粒体atp6基因转录本的编辑位点研究

以红莲（HL）型水稻细胞质雄性不育系A、保持系B及杂种一代F₁为材料,首次比较研究了红莲型水稻线粒体atp6基因转录本的编辑位点及各位点的编辑频率.结果表明atp6基因的转录本有18个编辑位点,其中有15个发生在密码子的第一和第二位点上,这些位点的编辑最终会导致氨基酸种类的变化.18个编辑位点在A、B和F₁中没有差异,但各位点的编辑频率在引入了核恢复基因的条件下发生了较大的变化,完全编辑的比例增加.这些结果首次证明HL型细胞质雄性不育与线粒体atp6转录本的编辑有一定相关性,编辑不充分的转录产物最终会干扰线粒体功能的正常发挥.     

Identification of mitochondrial DNA sequence variation and development of single nucleotide polymorphic markers for CMS-D8 in cotton

Cytoplasmic male sterility (CMS), which is a maternally inherited trait and controlled by novel chimeric genes in the mitochondrial genome, plays a pivotal role in the production of hybrid seed. In cotton, no PCR-based marker has been developed to discriminate CMS-D8 (from Gossypium trilobum) from its normal Upland cotton (AD1, Gossypium hirsutum) cytoplasm. The objective of the current study was to develop PCR-based single nucleotide polymorphic (SNP) markers from mitochondrial genes for the CMS-D8 cytoplasm. DNA sequence variation in mitochondrial genes involved in the oxidative phosphorylation chain including ATP synthase subunit 1, 4, 6, 8 and 9, and cytochrome c oxidase 1, 2 and 3 subunits were identified by comparing CMS-D8, its isogenic maintainer and restorer lines on the same nuclear genetic background. An allelic specific PCR (AS-PCR) was utilized for SNP typing by incorporating artificial mismatched nucleotides into the third or fourth base from the 3′ terminus in both the specific and nonspecific primers. The result indicated that the method modifying allele-specific primers was successful in obtaining eight SNP markers out of eight SNPs using eight primer pairs to discriminate two alleles between AD1 and CMS-D8 cytoplasms. Two of the SNPs for atp1 and cox1 could also be used in combination to discriminate between CMS-D8 and CMS-D2 cytoplasms. Additionally, a PCR-based marker from a nine nucleotide insertion–deletion (InDel) sequence (AATTGTTTT) at the 59–67 bp positions from the start codon of atp6, which is present in the CMS and restorer lines with the D8 cytoplasm but absent in the maintainer line with the AD1 cytoplasm, was also developed. A SNP marker for two nucleotide substitutions (AA in AD1 cytoplasm to CT in CMS-D8 cytoplasm) in the intron (1,506 bp) of cox2 gene was also developed. These PCR-based SNP markers should be useful in discriminating CMS-D8 and AD1 cytoplasms, or those with CMS-D2 cytoplasm as a rapid, simple, inexpensive, and reliable genotyping tool to assist hybrid cotton breeding.     

Cytoplasmic-nuclear male sterility in pearl millet: comparative RFLP and transcript analyses of isonuclear male-sterile lines

The identification of diagnostic cytoplasmic molecular markers is of prime interest to pearl millet breeders wishing to identify sources of cytoplasmic-nuclear male sterility (CMS) which can be used as an alternative to the single source currently used in the production of F₁ hybrid seed. Here, we report the classification of five pearl millet CMS sources based on RFLP analysis of isonuclear lines carried out using mitochondrial gene-specific DNA probes in combination with eight restriction endonucleases. On the basis of RFLP data, the five CMS cytoplasms can be distinguished from each other and from the isonuclear fertile cytoplasm. In addition, based on cox1, cox3, atp6 and atp9 polymorphisms, these lines can be classified into two major groups: one corresponds to A₅, A_egp, A_v and A₁ cytoplasms, and the other consists of the A₄ cytoplasm. Our results suggest that a rearrangement involving the cox1 gene might be related to CMS in the first group (A₅, A_egp, A_v and A₁), whereas a rearrangement within the atp6/cox3 cluster region might be related to CMS in the second group (A₄).