The role of CIPK8 in nitrate sensing

• Main Authors: Yu-Ting Chou, 周于婷
• Other Authors: Yi-Fang Tsay
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/27374396467505745456

碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 100 === Nitrate is not only an important nitrogen source for plants, but also a signaling molecule. Nitrate can rapidly induce the transcriptional expression of nitrate-related genes, such as CHL1 and AtNRT2.1. This response is called the primary nitrate response. Through the previous microarray analyses, several signaling molecules such as CIPK8 and CIPK23(CBL-interacting protein kinase) were identified and found to participate in the primary nitrate response,. Previously study showed that CIPK23 can interacts with CHL1 and phosphorylates the threonine 101 of CHL1 when exposed to low nitrate concentration. By this phosphorylation, the uptake activity of CHL1 can be switched between high or low affinity, and the gene expression level of the primary nitrate response can be regulated. In this study, we investigated the role of CIPK8 in regulating the nitrate uptake and the nitrate signaling. CHL1 only interacts with the kinase domain of CIPK8; it suggested a conformation change of CIPK8 is necessary for the interaction between CHL1 and CIPK8, but all of the candidate proteins we analyzed can’t help the interaction between CHL1 and CIPK8. Oocyte uptake activity assay showed that CIPK8 can reduce the high affinity nitrate uptake activity of CHL1. Q-PCR analysis showed that the cipk8-1 mutant was only defective in low affinity phase of primary nitrate response. However, the western analysis using a CHL1 T101-P specific antibody indicated that CHL1T101 can’t be phosphorylated at low nitrate concentration in cipk8-1 mutant. The yeast-two hybrid analysis showed that both the loop between TM8 -TM9 and the C-terminal end of CHL1 are the binding sites of CIPK8 kinase domain. These result suggested that CHL1 may have two CIPK8 binding sites, one is required for T101 phosphorylation; another one is response to different nitrate concentration. We speculate that CIPK8 and CIPK23 can work together to regulate the nitrate sensing ability and nitrate uptake activity of CHL1 by phosphorylation at different sites.