| Summary: | 碩士 === 國立臺灣大學 === 分子與細胞生物學研究所 === 96 === Nitrate is one of the most important nutrients for plants. It not only serves as a nitrogen source but also a potent signal molecule to regulate gene expression, plant metabolism and development. Recent studies in our lab indicated that CHLorate resistant mutant 1 (CHL1), a dual affinity nitrate transporter, also functions as a nitrate sensor. In addition, two potential candidates involved in nitrate signaling, CBL-interacting protein kinase 23 (CIPK23, At1g30270) and a PP2C-type phosphatase (At4g32950), were identified by Affymetrix microarray analysis in our lab.
In cipk23 mutant, expression level of CHL1, as well as other primary nitrate response genes, were higher than that in wild-type, suggesting that CIPK23 is a negative regulator of primary nitrate response in high affinity phase. Neither 15NO3 uptake activity nor nitrate-regulated primary root growth was altered in cipk23 mutant. Transient expressing CIPK23-GFP in Arabidopsis protoplasts indicated that CIPK23 was localized in the cytoplasm. Bimolecular fluorescence complementation (BiFC) analysis suggested that under certain nitrate conditions tested, CHL1 may interact with CIPK23 at the plasma membrane of Arabidopsis protoplasts.
In addition, the role of PP2C in nitrate signaling was analyzed by GFP localization study and BiFC. Protein sequence analysis indicated that PP2C is a soluble protein, but PP2C-GFP was localized at the plasma membrane. When treated with palmitoylation inhibitor 2-bromopalmitate, PP2C-GFP became cytosolic-localized, suggesting that PP2C was targeted to the plasma membrane by palmitoylation. BiFC analysis showed that CHL1 interacted with PP2C at the plasma membrane of Arabidopsis protoplasts. Together with other studies in our lab, the data suggested that CIPK23 and PP2C participate in nitrate signaling by directly interacting with CHL1.
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