| Plant cytoplasmic male sterility (CMS) is a maternally inherited trait that is unable to produce viable pollens, which plays an important role in utilization of hybrid vigor. There are not many corresponding reports on the mechanism of cotton CMS, which is far behind the study of the CMS mechanism of maize and rice, etc. Therefore, it has theoretical and practical significance for investigating the molecular mechanism of cotton CMS in order to utilize the cotton hybrid vigor better. Cotton CMS with the cytoplasm of G. harknessii Brandegee is a genetic trait that controlled by cytoplasmic genes and repressed by nuclear restorer genes. Sterile-line anthers begin to degenerate in the sporogenous cell division stages and reach abortion peak in the pollen mother cell meiosis stage, so that it can not form tetrad, which is named as non-pollen male-sterility, and is appropriate for the hybrid seed production and study of the CMS mechanism.So in this paper, the anthers of CMS line and its maintainer were employed for trying to find the cotton CMS-associated genes and the possible molecular mechanism of CMS in cotton, by comparing the ultrastructure of anther mitochondria, sequences and gene expression of CMS-associated mitochondrial genes, biochemical assays for the enzymes of mitochondrial metabolism, changes of mitochondrial ROS metabolism and gene expression of ROS-scavenging enzyme. The results were shown as follows:1. Through observing the anther ultrastructure of cotton CMS line and its maintainer in the different developmental stages, CMS anther mitochondria began to show the abnormality in the sporogenous cell division stage, which was gently swelled and had electron translucent area. During the microspore mother cell stage, sterile anthers presented the abnormal phenomena with the more swelled mitochondria, blurred cristae, vacuoled mitochondria and diffused matrix. When it was at the stage of meiosis, the organelles such as mitochondria and vacuole gradually disassembled and disappeared. At the different microspore developmental stages, mitochondria with non-cristae, or undeveloped cristae, or vacuolation, could not fulfill the normal metabolism function, and the production of ATP was restrained so that the energy supply for the anther development was insufficient to cause the anther abortion. Therefore, the structural abnormality of mitochondria was an important cytology characteristic for the microspore abortion of cotton CMS line.2. CMS-associated mitochondrial genes were cloned from the mitochondrial genome of cotton CMS line and its maintainer line using the method of homological cloning. Nucleotide sequences of eight mitochondrial gene fragments, atp6, atp9, nad3, nad6, nad9, cob, cox1, and coxII, had no changes between the CMS line and its maintainer line. However, the mitochondrial gene coxIII presented mutation of five nucleotides in CMS line although there was no difference of the size of full coxIII coding sequences between cotton CMS line and maintainer line, which was 798bp. In these five mutation nucleotides, three mutation nucleotides caused variation of coding amino acids. Compared with maintainer line, five nucleotides of A, C, A, C and G at positions 184, 255, 366, 442 and 516, in coxIII coding region of CMS line, were replaced with C, T, C, A and C, respectively. In terms of the nucleotide sequences, cotton coxIII gene was deduced that it could code 265 amino acids. Consequently, the deduced amino acids of Ser62, Lys122 and Thr148 were substituted for Arg, Asn and Asn in cotton CMS line, while Ser85 and Leu172 were not changed. Therefore, there were both of missense and nonsense mutant in the coding region of coxIII gene in cotton CMS line. By the assay of similarity alignment, the nucleotide sequences of coxIII gene in cotton CMS line had homology with those in Arabidopsis thaliana, Brassica napus, Nicotiana tabacum and Zea mays, all showing 95% similarity. Analyzing the CMS-associated gene expression with the method of RT-PCR and fluorescent quantitative RT-PCR, the CMS-associated gene expression level in cotton CMS line was lower than that in maintainer line as a whole during the anther developmental stages. At the peak of anther abortion (Pollen mother cell meiosis stage), the expression level of CMS-associated mitochondrial gene in sterile anther was significantly lower than that in maintainer anther.3. By measuring the cytochrome oxidase activity and its isozymic activity and assaying its cytochemistry location in the leaves and anthers of cotton CMS line and its maintainer line, activities of cytochrome oxidase and its isozyme presented no obvious difference in the leaves of CMS line and its maintainer line, but significant differences were detected in cotton anthers. During the abortion preliminary stage (sporogenous cell division stage), there were no significant differences in the activities of cytochrome oxidase and its isozyme and the nurnber of osmiophilic globules in the mitochondrial membrane between the CMS and maintainer anther. At the stage of abortion peak, differences of cytochrome oxidase activities and its isozyme in CMS anther were significant. The accumulation of osmiophilic globules in the mitochondrial cristae and membrane in CMS anther was obviously less than that in maintainer anther. In the following anther developmental stages, the cytochrome oxidase activities in CMS anther had no more significant differences compared with those in maintainer anther. The reduction of cytochrome oxidase activities in cotton CMS line perhaps had an important relation with the abortion of microspore and occurrence of male-sterility.4. There were no significant differences in ROS content and ROS-scavenging enzymic activity among the cotton leaves of CMS line, maintainer line and hybrid F1, through analyzing the content of ROS, activities of ROS-scavenging enzymes, and gene expression of antioxidant enzymes in the anthers, leaves and theirs mitochondria of CMS line, maintainer line and hybrid F1. During the anther preliminary abortion stage, CMS anthers had a little higher ROS content than those of maintainer, meanwhile activities of ROS-scavenging enzymes and gene expression level of antioxidant enzymes in CMS anther presented no obvious difference compared with maintainer anther. But at the stage of abortion peak (PMC meiosis stage), on one hand, content of ROS was extraordinarily high in CMS anthers, on the other hand, the activities of ROS-scavenging enzymes and expression level of antioxidant enzymes were excessively low, which disturbed the balance between the generation and elimination of ROS. Once ROS metabolism loses balance, it will cause excessive ROS accumulation and led to oxygen stimulation, which is toxic to the cell development. During the anther abortion, not like the sterile line, hybrid F1 always kept normal ROS levels and stable transcripts and protein activities of antioxidant enzymes with the maintainer. When the restorer gene was introduced to sterile line, excessive production of ROS could be eliminated in the anthers of hybrid F1, which likely resulted from the regulation role of nuclear restorer gene in the dynamic balance between the generation and elimination of ROS.According to the above results, one hypothesis would be made about the mechanism of CMS in cotton with the cytoplasm of G harknessii Brandegee. The mitochondrial gene coxIII mutation and abnormality of mitochondrial structure, lead to the pathway of mitochondrial electron transfer not smooth and result in the abundant generation of ROS in cotton CMS anthers. Because fertile anther and other tissue cells have higher ROS-scavenging enzymic activities and are not more sensitive to ROS than male cells (PMCs) in the anther, cotton fertile anthers and other tissues can develop normally. But in the CMS anthers, the abundant production of mitochondrial ROS, the significant reduction of activities and gene expression level of mitochondrial antioxidant enzymes, and very low activities of ROS-scavenging enzymes in the cytoplasm during the anther abortion, cause the formation of ROS with a great deal in the cell of CMS anther not to be eliminated rapidly and effectively, which results in the excessive accumulation of ROS. Because of the misbalance of ROS metabolism and toxicity resulting from the ROS excessive accumulation, large numbers of male cells (PMCs) are dying inside the CMS anthers, and finally the CMS anthers are aborted. |
