| Summary: | 碩士 === 國立陽明大學 === 生命科學暨基因體科學研究所 === 97 === Nitrate is not only an essential nutrient for plant, but also a signal molecule to regulate the downstream responses. CHL1 is a dual-affinity nitrate transporter with two action modes regulated by the phosphoryation and dephosphorylation of T101 residue. Recent studies in our lab indicated that CHL1 is also a nitrate sensor. Moreover, kinases and phophatases such as CIPK8, CIPK23, and PP2C which are rapidly induced by nitrate were identified by microarray analysis and found to be involved in nitrate signaling.
In previous studies, it was shown that PP2C is located in plasma membrane and the membrane localization was blocked by a palmitoylation inhibitor, BrPA. In this study, we further demonstrated that PP2C was located in plasma membrane by palmitoylation at Cys4 and Cys6 residues, and the interaction between PP2C and CHL1 may not be affected by palmitoylation. Furthermore, the root protoplasts isolated from the PP2C-GFP transgenic plants showed the PP2C with endogenous promoter driven was located both in plasma membrane and cytosol. The transgenic plants expressing C4A C6A mutation of PP2C-GFP were also generated to find out if palmitoylation is required for nitrate signaling.
It had shown that PP2C can interact with CHL1 and dephosphorylate CHL1 in previous works. In this study, the interaction analysis indicated that PP2C prefer to interact with phosphorylated CHL1 in yeast. Additionally, both N- and C-terminus and another putative palmitoylation site of PP2C, Cys313, are important for the interaction with CHL1. Since CIPK8, CIPK23 and PP2C are involved in primary nitrate response, the cipk8 pp2c, and cipk23 pp2c double mutants were generated to elucidate the genetic interaction between these three components. The results from Q-PCR analysis were shown that a rescue primary nitrate response in cipk8 pp2c double mutant.
Taken together, those evidences were suggested that palmitoylation of PP2C may probably help to stable the newly formed PP2C complex. Cys313 and N terminus of PP2C are important for the interaction of PP2C and CHL1. Moreover, CIPK8 and PP2C might play different roles in high-affinity and low-affinity primary nitrate response. The Km studies of cipk8pp2c and cipk23pp2c double mutant were needed to further analyze in the future.
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