The role of CIPK8 in nitrate sensing

硝酸鹽對植物而言不僅是重要的氮源，也是不可或缺的訊息分子。植物感知土壤中的硝酸鹽後，會快速誘發和硝酸鹽相關的基因表現（nitrate primary response），對硝酸鹽做出最有效率的利用。但對於植物細胞如何傳遞這一連串的訊號，仍所知甚少。本實驗室先前以微陣列（microarray）分析顯示磷酸激酶CIPK23和CIPK8皆參與在CHL1主導的硝酸鹽訊息傳遞路徑中。已知CHL1會感知外界硝酸鹽的濃度，透過CIPK23對自身第101胺基酸─蘇胺酸（Threonine）的磷酸化與否來改變訊號，造成下游基因不同程度的表現；此磷酸化也是CHL1硝酸鹽轉運能力能在高低親和性之間轉換的關鍵。本研究則針對CIPK8分析。酵母菌雙雜合試驗顯示CHL1無法和full length CIPK8結合，但可和CIPK8 kinase domain結合，推測兩者要能結合，CIPK8可能需改變構型；為了找出能幫助full length CIPK8和CHL1相互結合的蛋白，測試CBL1、CBL9、ANI、CIPK23、NLP7等等，但以上蛋白仍無法幫助兩者相互結合。爪蟾卵硝酸鹽吸收活性分析顯示，CIPK8使CHL1高親和性吸收能力下降13%，且此下降在CBL9同時表現時更明顯，顯示CIPK8會影響CHL1 T101的磷酸化狀態。檢視cipk8-1突變株中的nitrate response，secondary response以及high affinity的 primary nitrate response均與野生株一致，顯示CIPK8只參與在low affinity primary nitrate response。深入探究cipk8-1突變株中CHL1T101磷酸化的情形，發現不論高或低硝酸鹽濃度下，CHL1 T101都無法被磷酸化，說明CIPK8對CHL1的磷酸化是CHL1 T101被磷酸化的先決條件。以酵母菌雙雜合試驗分析CIPK8在CHL1上的結合位置，結果顯示CHL1 C-terminal end和第八、第九穿膜區域間的loop為可能的兩個結合位。綜合以上，我們推論CIPK8在CHL1 C-terminal區域可能有兩個磷酸化位，第一位置的磷酸化可促進T101磷酸化，而在高硝酸鹽濃度時CIPK8會將第二位置磷酸化，而誘發高劑量（low affinity level）的訊息反應。我們推測CIPK23和CIPK8會共同藉由磷酸化來影響CHL1 N-和C-terminal區域結構的動態，調控CHL1對硝酸鹽的親和性以及轉運能力。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