Identification Of Related Genes, Markers And Studying On Physiological Mechanisms Of Cytoplasmic Male Sterility In Capsicum Annuum L.

Plant cytoplasmic male sterility (CMS) has been proved to result from the rearrangementof the mitochondrial genome. Cytoplasmic male sterility (CMS) in pepper (Capsicum annuumL.) has been associated with novel genes in the mitochondria, such as orf507and Ψatp6-2.Pollen formation is a complex process that is strictly controlled by genetic factors. Althoughmany novel mitochondrial genes have been implicated in the dysfunction of mitochondrialenzymes and the cytoplasmic male sterility (CMS), there is little empirical evidence to showthat CMS-related genes actually result in the dysfunction of enzyme and little is known aboutthe regulatory mechanisms of the aberrant mitochondrial enzymes in male sterility in CMSlines. Weak amplifications of orf507andΨatp6-2in some maintainer lines and the variouscharacters of pollen abortion in pepper indicated that in pepper CMS lines there are someother CMS related genes. More importantly, there is a potential risk related with widespreaduse of a single cytoplasm resource. All these determined the importance of the developmentof new cytoplasm and analysis of the function of the CMS related genes in cytoplasmic malesterility, which will help us understand the mechanism of plant cytoplamic male sterility andkeep the diversification of various types of cytoplasm with CMS property to provideconvenience for the agricultural production.In this study, pepper CMS line HW203A and its near-isogenic maintainer line HW203Bwere used as the materials. First the characters of the pollen abortion in HW203A wereidentified via cytological observation, then we analyzed the expression patterns of orf507andΨatp6-2. The function of the two genes was analyzed by VIGS and genetic transformation.Based on these, the mitochondrial genome from three different CMS systems was comparedand analyzed by SRAP technique to develop new CMS-specific markers and discuss themolecular mechanism of cytoplasmic male sterility. The main results were as following:1. Anther abortion occurred after the tetrad stage in the CMS line (HW203A) withshrinking anther, which was accompanied by premature programmed cell death (PCD) in thetapetum, compared with the maintainer line (HW203B) which produces plenty of viablepollen grains. The spatiotemporal expression patterns of orf507and Ψatp6-2were analyzedtogether with the corresponding enzyme activity to investigate the interactions of the genes and mitochondrial enzymes. The two genes were both highly expressed in the anther. Theorf507was down-regulated in HW203A (CMS line), with nearly no expression in HW203B(the maintainer line). In contrast, the cytochrome c oxidase activity in HW203A showed theopposite trend, reaching its highest peak at the tetrad stage when compared with HW203B atthe same stage. The Ψatp6-2in the CMS line was also down-regulated, but it wasup-regulated in the maintainer line. The corresponding F1Fo-ATPase activity in the CMS linewas gradually decreased along with the development of the anther, which showed the sametrend for Ψatp6-2gene expression. On the contrary, with up-regulated gene expression ofatp6-2in the maintainer line, the F1Fo-ATPase activity sharply decreased after the initialdevelopment stage, but gradually increased following the tetrad stage, which was contrary towhat happened in the CMS line. Taken together, all these results may provide evidence forthe involvement of aberrant mitochondrial cytochrome c oxidase and F1Fo-ATPase in CMSpepper anther abortion. Moreover, the novel orf507and Ψatp6-2genes in the mitochondriamay be involved in the dysfunction of the cytochrome c oxidase and F1Fo-ATPase,respectively, which are responsible for the abortion of anthers in the CMS line.2. Using virus-induced gene silencing (VIGS), we observed that silencing the atp6-2gene in the maintainer line resulted in an increase in ATP hydrolysis activity of themitochondrial F1Fo-ATP synthase along with pollen abortion, while silencing the truncatedΨatp6-2gene in the CMS line resulted in an inhibition of ATP hydrolysis activity andrestoration of fertility. Altered ATP hydrolysis also affected the tolerance of the gene-silencedplants to abiotic stresses. Localization experiments showed that premature ATP hydrolysisoccurred at the tetrad stage of pollen development in the CMS line, but no ATPase activitywas observed in the microspores at the later stage. These results suggest that the Ψatp6-2genecauses the alteration in ATP hydrolysis activity of the mitochondrial F1Fo-ATP synthaseduring pollen development, which eventually leads to male sterility in pepper.3. Histochemical localization experiments showed significant differences of cytochromec oxidase activity in the tapetum cell and tapetum membrane between the CMS line and themaintainer line. To analyze the effect of orf507gene on the cytoplasmic male sterility inpepper, a plant expression vector (TCON vector) was developed. The tapetum-specificexpression of the orf507gene was drived by tobacco TA29promoter, and then the protein wastargeted into mitochondria via the transit peptide of the first25amino acids of COXIV fromyeast. The transformed plants were morphologically similar to the maintainer lines, except thelower fruit setting. Moreover, we nearly could not observe the anther dehiscence in thetransformants which have lower pollen number and germination rate, higher exploded pollenrate than the untransformed plants. Meanwhile the anthers of the transformants were shrinked, not plumped and became etiolated, while the abortion characters of the transformants antherwere different from the CMS-HW203A which could not produce any pollen grains. Thebright-field microscopy demonstrated ablation of the tapetal cell induced by the prematureprogrammed cell death (PCD) at the tetrad stage. The microspores of the transformants weredistorted and degraded at the mononuclear stage. In addition, expression of orf507in themaintainer lines could inhibit the expression of atp6-2gene in certain extent, and increase theactivity of cytochrome c oxidase and the ATP hydrolysis of the mitochondrial F1Fo-ATPsynthae. This constitutes the report at demonstrating the induction of semi-male sterility byintroducing orf507gene. Expression of orf507altered the activity of cytochrome c oxidaseand the ATP hydrolysis of the mitochondrial F1Fo-ATP synthae, which resulted in the disorderof the energy metabolism and the premature PCD of tapetum cell.4. The study aimed to develope a new CMS-specific molecular marker, SCAR130, forreliable identification of S-cytoplasm in pepper. Meanwhile, the new and previous threemolecular markers were used to determine the cytoplasm types of pepper lines. Based on themitochondrial genome SRAP (sequence related amplification of polymorphism) analysis ofthe CMS lines and the maintainer lines, SCAR130was developed from a10bp deletion at theSRAP primer binding site in the CMS line (130bp) compared with that in maintainer line(140bp). S-cytoplasm could be unambiguously selected from the pepper lines by the differentlength of the marker band. Application of the four molecular markers to various pepper linesrevealed that SCAR130is more reliable than other three previous markers, orf507, ψatp6-2andaccD-U. Homology alignment with BLAST showed that the marker was located between“trnE” and “trnS” in Nicotiana tabacum mitochondrial genome. Furthermore, expression ofthe marker linked gene was significantly higher at the pollen abortive stage in CMS line(HW203A) than that in maintainer line, which indicated that the marker was closely relatedwith the male sterility. Hence, factors other than orf507and ψatp6-2may be existed for theregulation of male sterility in pepper.