Functional Study Of Circadian Clock Gene TaCOL1in Wheat

Common wheat (Triticum aestivum L.) is one of the most important staple food crops in the world and is grown in very different environments. The high yield of wheat mast due to the "green revolution", and its wide adaptability to a broad range of agricultural environments is conferred by insensitivity to photoperiod (day length neutrality). Abiotic stresses such as salt and drought seriously restrict the yielding of wheat, so it appears to be urgent to unravel the mechanisms of abiotic stress tolerance, identify stress tolerance associated genes, and finally breed stress tolerant cultivars via the molecular assistant breeding strategy. Along with improving abiotic stress tolerance, it is important to reverse the excellent traits of high yield and wide adaptability. Therefore, it is necessary to uncover shield insight into the scenario of the interacting mechanisms between photoperiod and abiotic stress response in wheat and other plants. In this study, we cloned a clock-associated gene TaCOL1that was induced by abiotic stresses, confirmed its role in regulating development and abiotic stress response, and primarily analyzed how its role is performed.1. TaCOL1is a circadian clock gene that participates in regulating photoperiodThrough screening photoperiod associated gene from the transcriptomic data of wheat responding to ionic and osmotic stresses, we cloned an abiotic stress responsive gene TaCOLl, encoding a B-box zinc finger protein. TaCOL1localizes in the nucleus, and exhibits transcriptional activation activity. This protein possesses two B-box domains in the N-terminus, and one CCT (CO, COL, TOC1) domain in the C-terminus, and the domains are characterized in CO and other COLs of Arabidopsis. TaCOL1has orthologues in Gramineaes such as barley, rice and maize, but not in Arabidopsis; the function of these orthologues in Gramineaes has not been investigated. These results indicate that TaCOL1and its homologues were produced after the divergence between monocots and dicots.TaCOL1transcribed higher in leaves than in other tissues; in leaves, the transcript abundance of TaCOL1at elongation stage was higher than that at other stages during the whole life course. TaCOLl was photoperiod responsive, and exhibited a24h circadian rhythmic expression pattern under various photoperiod conditions. Under the long-day (LD) condition, TaCOL1was expressed during from early daytime until dusk, and its transcript peaked at14h after light onset and was significantly higher than those under other photoperiod conditions. In contrast, under the short-day (SD) condition, TaCOLl transcribed earlier, with a peak at2h before light onset. Under the continuous light (CL) conditions, TaCOL1expression was similar to that under the short-day condition, but the rhythmic pattern was weakened. Under the continuous dark (CD) conditions, the circadian rhythm of TaCOL1was disturbed, resulting in no apparent rhythmicity. According to this, the circadian rhythm of TaCOL1may depend on light. Note that the high expression level of TaCOL1under the LD condition suggests that it play important role in the vigorous growth of wheat in spring.To know the role of TaCOL1in circadian clock regulation, it was ectopically expressed in Arabidopsis Col-0. In TaCOLl overexpression lines, the expression patterns of AtTOC1, AtCCA1and AtLHY, three key genes of circadian clock central regulatory machinery, were all influenced. Of them, the peak of AtTOCl transcript was shifted, and appeared occurred in the dark, nearly4h later than that of the Col-0, which reduced the expression levels of AtCCA1and AtLHY, and led to the loss of circadian rhythm of AtLHY. TaCOL1overexpression also altered the expression profiles of other circadian clock associated genes, AtELF3and AtGI. These findings demonstrate that TaCOL1is a circadian clock gene.2. TaCOLl impacted the developmentUnder either LD or SD condition, TaCOLl overexpression resulted in development-blocked phenotypes, including short primary roots, dwarf plants, small and upward-curled leaves, more branching, short siliques, which phenocopied the alteration by auxin over-production. Consistently, TaCOLl overexpression lines had lower sensitivity to exogenous IAA and higher relative growth rate, and their leaf upward-curling was erased by high concentration of exogenous IAA. A set of genes involved in auxin synthesis and metabolism as well as auxin signaling pathway were upregulated, including AtIAMTl, encoding an IAA methyltransferase that has proved to contribute leaf up-curling. These results indicate that TaCOLl modulates plant development via promoting auxin synthesis and signaling pathway.Under both SD and LD conditions, the flowering-time was delayed by TaCOLl overexpression. Further studies revealed that in TaCOLl overexpression lines, the expression of AtFT, a flowering-accelerated gene, was restricted, while that of AtFLC, a flowering-blocked gene by inhibiting AtFT transcription, was also down-regulated. Based on the previous conclusion that AtFT was positively regulated by CO, whose encoding gene had lower expression level in TaCOL1overexpression lines, we propose that TaCOL1delays flowering time via modulating the GI-CO pathway of the circadian clock system to lower the expression of CO, and consequently the expression of AtFT.In comparison with Col-0, the hypocotyl length of TaCOLl overexpression lines was longer under white light (both SD or LD photoperiod), was shorter under either blue or red light, but was comparable under the CD condition, indicating TaCOL1participate in photomorphogenesis. Unlike Col-0who had shorter hypocotyl under white light than under red or blue light, TaCOL1overexpression lines had similar hypocotyls under white, red and blue light, suggesting that TaCOLl participate in photomorphogenesis via modulating the downstream components such as COP1and HY5shared by blue and red light signaling pathways. However, yeast two hybridization and yeast one hybridization assays showed that TaCOL1did not interact with either COP1or HY5, and it also did not bind with the promoters of the two genes. This implies that TaCOL1may contribute to photomorphogenesis through modulating other component(s) of light signaling pathways or indirectly influencing COP1and HY5.TaCOL1overexpression lines had higher stomatal density, and some of stoma were agglutinated. Real-time quantitative PCR analysis showed that, in TaCOL1overexpression lines, the expression of genes in YDA pathway that governing stomatal development was increased by various extent. It has proved that YDA pathway is regulated by by BR, and the increased stomatal density by TaCOL1overexpression was similar to the phenotype of some BR mutants. This suggests that the role of TaCOL1in stomatal development is associated with BR. Exactly, we found that TaCOL1overexpression lines had lower sensitivity to BR, and produced less transcript of DWF, encoding a key enzyme for BR synthesis pathway, indicating that TaCOLl modulates stomatal development via inhibiting BR synthesis.3. TaCOLl enhanced abiotic stress tolerance via modulating redox homeostasisWhen exposed to either NaCl or PEG, the expression level of TaCOL1was drastically increased after0.5h, while descended to the normal after24h, suggesting that TaCOL1sever as a transcription factor to play roles in the response to early stage of abiotic stress. Under either NaCl or PEG treatment, TaCOLl overexpression lines had higher relative growth rates of both primary roots and shoot fresh weight. Water-withholding assay indicates that TaCOL1overexpression lines had superior drought tolerance. It has been concluded that, abiotic stress induced a mass of ABA, which triggers the downstream signaling pathway to produce abiotic stress response. However, TaCOL1was not ABA-responsive, and TaCOLl overexpression did not change the sensitivity to ABA, and TaCOL1overexpression lines had higher water loss rate that Col-0, indicating the lack of association between ABA and the role of TaCOL1in abiotic stress response. By contrast, H2O2induced the expression of TaCOL1, and TaCOLl overexpression lines had pronounced tolerance to H2O2in comparison with Col-0. Further analysis showed that in TaCOL1overexpression lines, both the expression level of RBOHC for ROS production and the activity of SOD for ROS scavenging were elevated, and ROS level was increased, indicating the enhancement of abiotic stress tolerance by TaCOL1is partially achieved via regulating redox homeostasis. Interestingly, the elevation of SOD activity by TaCOLl overexpression is accociated with photoperiod. In comparison with Col-0, the SOD activity in TaCOL1overexpression lines had no obvious difference under the SD condition, but was significantly higher under the LD condition. This demonstrates that TaCOL1modulates SOD activity in a photoperiod-dependent manner, which speculates that TaCOLl links circadian clock and downstream phytohormone and abiotic stress responsive pathways via regulating redox homeostasis to orchestrate development and abiotic stress response.