Characterization of the roles of CIPK23 and PP2C in nitrate signaling in Arabidopsis

硝酸鹽不但是植物的主要氮源，也是一訊息分子，調控相關基因的表現及植物的發育。前人研究發現，阿拉伯芥的硝酸鹽雙親合性轉運蛋白CHLorate resistant mutant 1 (CHL1)可能同時扮演感應硝酸鹽的角色，並參與在硝酸鹽傳訊機制中。從本實驗室先前的Affymetrix microarray 實驗，篩選可能參與在硝酸鹽傳訊機制的基因。本篇研究針對 CBL-Interacting Protein Kinase 23 (CIPK23, At1g30270)及PP2C-type phosphatase(At4g32950)，分析兩者在硝酸鹽傳訊機制所扮演的角色。 本篇研究先以逆遺傳策略，分離出不具CIPK23基因表現的T-DNA 插入突變株 (cipk23 mutant)。由qRT-PCR 分析顯示，早期受硝酸鹽誘導反應的基因CHL1及NRT2.1在cipk23 mutant中較wild-type有較高表現，推測CIPK23在早期硝酸鹽誘導反應中可能扮演抑制者的角色。而cipk23 mutant在短期或長期對硝酸鹽的吸收能力與野生型無明顯差異。以高濃度或低濃度硝酸鹽誘導cipk23 mutant根部生長，其主根長與野生型無顯著差異。此外，在阿拉伯芥葉肉原生質細胞中表現CIPK23-GFP，證實CIPK23表現於細胞質。由Bimolecular fluorescence complementation (BiFC)實驗，推測在特定濃度硝酸鹽處理下， CIPK23與CHL1在阿拉伯芥原生質細胞的細胞膜上有蛋白質交互作用。 此外，本篇以細胞學的方法分析PP2C在硝酸鹽傳訊機制所扮演的角色。在阿拉伯芥葉肉原生質細胞中表現PP2C-GFP，顯示PP2C位於細胞膜上，但比對PP2C的蛋白質序列，發現其不具有跨膜區域 (transmembrane domain)。以Palmitoylation 的抑制劑 2-bromopalmitate (2-BrPA) 處理，發現部分原生質細胞的PP2C-GFP轉而堆積在細胞質中，推測PP2C藉由palmitoylation附著到細胞膜上。BiFC 實驗顯示PP2C與CHL1在原生質細胞的細胞膜上有蛋白質交互作用。由本研究與實驗室先前的研究成果，推測CIPK23 與PP2C可能直接與CHL1進行蛋白質交互作用而參與在硝酸鹽傳訊機制中。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.致謝………………………………………………………………………....i文摘要...………………………………………………………………... ii文摘要……………………………………………………………….iiiHAPTER 1. INTRODUCTION…………………………………………1HAPTER 2. MATERIALS AND METHODS………………………….8. Plant materials 8. Primer sequence 8. Media 9. General techniques 9. Isolation of cipk23 knock out mutant 12. Gene expression analysis 12.15N content in roots and 15N uptake activity 14. Primary root length analysis 15. Subcellular localization and BiFC assay constructions 15. Protoplast isolation, PEG transfection and Confocal Microscopy 19HAPTER 3. RESULTS………………………….……………………22HAPTER 4. DISCUSSION……………………………………………..31EFERENCE………………………………………………….…………6