Regulatory Roles Of Jasmonate Receptor Protein COI1 In Nic Otiana Tabacum And Brassica Napus

Male sterility is of great value for crop breeding. Phytohormone jasmonate (JA), a regulator of pest resistance and secondary metabolism, plays important roles in the regulation of plant fertility. Studies in Arabidopsis thaliana revealed that the JA receptor is a complex composed of COI1 (Coronatine-insensitive 1), JAZ (Jasmonate ZIM-Domain Protein) and inositol phosphate, and COI1 is the core component of JA receptor. Studies in the regulation of male sterility by JA signaling pathway have recently made great progress. However, the underling regulatory mechanism of male sterility occurrence upon blocking of JA singnaling pathway remains unclear. In this study, roles of COI1 in regulating pollen abortion were extensively investigated in tobacco(Nicotiana tabacum L.) and oilseed rape (Brassica napus L.).Tobacco is an important model plant for secondary metabolism studies, with unique advantages in the regulation of secondary metabolism by JA signaling pathway. In this study, a conserved fragment of tobacco NtCOI1 was cloned to construct the RNA interference (RNAi) vector, and to develop NtCOI1 silenced tobacco plants. Results of this study showed that the silencing of NtCOIl in tobacco resulted in male sterility, JA insensitivity, and loss of floral anthocyanin production, which mimicked the hallmarks of the Arabidopsis coil mutant. NtCOIl-silencing tobacco also lost anthocyanin synthesis in corolla and carotenoids accumulation in floral nectary, and had the expression of anthocyanin synthetic genes in corolla and carotenoid synthetic genes in floral nectary suppressed. Carotenoid synthesis and carbohydrate metabolism are closely related with starch metabolsism, therefore, the carbohydrate metabolism of floral nectaries were further investigated. Results showed that starch accumulation in the NtCOI1-silenced tobacco nectary was significantly inhibited, and the expression of starch metabolic genes as well as a starch metabolism regulatory gene NtMYB305 was significantly altered. This study provided essential evidence that JA receptor protein COI1 is involved in the regulation of starch metabolism.Study on Arabidopsis mutant coil showed that the loss of function of COI1 had no effect on tapetum and microspore development. And, normal microspore could be formed in coil mutant, but aborted at late microspore development stage. A typical attribute of the abortive pollens formed by coil mutant is the reduction in carbohydrate accumulation. The cause of pollen abortion in coil mutant remains unclear. In this study, it was found that NtCOI1-silenced tobacco could also form normal microspore as the Arabidopsis mutant with loss of function of COI1, these microspores did not accumulate starch as the control and then aborted. Interestingly, a large amount of starch granules were observed in the anther wall of NtCOI1-silenced tobacco at late microspore stage. Analysis of gene expression demonstrated that silencing of NtCOI1 changed the expression of starch metabolic genes in tobacco anther wall, and inhibited the expression of starch metabolism related transcription factor gene NtMYB305 in the anther wall. These results proved the roles of COI1 in regulating carbohydrate metabolism, and provided important clues to reveal the regulation mechanism of pollen-formation-related carbohydrate metabolism by JA signaling pathway.Genome sequencing of B. napus and its parental species B. oleracea and B. rapa has been completed, but the gene function research progresses relatively slow. Study on JA receptor protein COI1 of B. napus has not been reported, neither has this gene been cloned. In this study, COI1 genes in B. napus as well as its parental species B. oleracea and B. rapa were analyzed based on the genomic data. Spatial and temporal transcription patter of COI1 in B. napus was analyzed using quantitative and semi-quantitative RT-PCR. Then, roles of COI1 in regulating male fertility and pest resistance in B. napus were investigated via VIGS (virus-induced gene silencing) technology. Genomic data analyses showed that B. napus possess 8 COI1 genes, which were highly homologous to their homologues B. oleracea and B. rapa and could be classified into 4 categories COI1a, COI1b.1, COI1b.2 and COI1c according to their sequence similarity. In the transcriptional assay, the lowest expression level of COI1 was observed in the stem of in B. napus. Silencing of COI1 with VIGS method,25 B. napus lines with the expression of COI1 suppressed more than 70% were obtained. And, the ten lines with the greatest knockdown of COI1 were used for fertility regulation and aphid resistance test. Fertility analysis results showed that the fertility of COI1-silenced B. napus plants was seriously affected. Siliques of COI1-silenced B. napus at 20 days after pollination were significantly shorter than that of control at 5 days after pollination, furthermore, no seeds were observed in the siliques of COI1-silenced B. napus. Shorter stamens and delayed anther dehiscence were also observed in the COI1-silenced B. napus plants. More than 80% pollens produced by the COI1-silenced B. napus plants were irregular in shape, while most of the pollens produced by control plants were normal in shape. Aphid resistance test indicated that the aphid populations on the COI1-silenced B. napus plants grew much faster than those on the control plants. These results provided important information for the regulatory roles of COI1 in Brassica species.