利用生化的方法研究硝酸鑒轉運蛋白CHL1及其同源蛋白AtNRT1：5在植物體內所扮演的角色及其調控機制

Abstract

硝酸鹽的攝取對植物的生長是不可或缺的，首先，植物將硝酸鹽從土壤中吸收到根部的細胞中，除了儲存在根部外，硝酸鹽於植物中也可被運送至地上部。CHL1是第一個在高等植物中被找到的硝酸鹽轉運蛋白，其mRNA表現於根部並且可被硝酸鹽誘導，且CHL1的突變株對硝酸鹽的吸收明顯的下降。因此CHL1被認為是在根部細胞中負責運送硝酸鹽通過細胞膜，但是一直缺乏直接的證據證明CHL1於細胞內所位於的膜系。於本論文研究中利用生化的方法證明瞭CHL1和它的一個同源蛋白AtNRT1:5皆位於細胞膜上。這是第一個直接的證據證明CHL1負責將硝酸鹽於細胞膜的運送，並且也分析了兩者於蛋白質層次上的表現情形。結果顯示，CHL1蛋白質於硝酸鹽誘導前就有高量的表現，並且於硝酸鹽誘導處理之後並沒有顯著的增加。CHL1mRNA及CHL1所貢獻的硝酸鹽吸收可個別受硝酸鹽誘導增加8~9倍及1.7~1.8倍。這暗示著CHL1的功能可於轉錄、轉譯、及轉譯後三個層次被調控。於AtNRT1:5，我們發現它可被乾旱及鹽害處理所誘導，如同AtNRT1:5mRNA一樣。但有趣的是，AtNRT1:5蛋白質被誘導表現的時問於乾旱處理時較鹽害處理早了2個小時。除此之外，細胞中多餘的硝酸鹽可以儲存在液胞內以供未來的使用，近來對於硝酸鹽的吸收上的研究已經有顯著的進展；然而在硝酸鹽液胞的運輸卻少有分子層次上的研究。因此，為了要對硝酸鹽液胞運輸的分子層次有更深入的瞭解，我們利用阿拉伯芥由T-DNA插入所造成的突變株系來篩選硝酸鹽之液胞儲存出現缺陷的突變株。植株25mM KN○3培養基上生長五天後，移至缺氮的培養基上生長另外五天，若植物於液胞硝酸鹽的儲存出現缺陷，理論上於缺氮的培養基中應會有生長上的缺陷。我們發現了一個株系40-B1移至缺氮的培養基後主根停止生長，然而其側根卻有稍微較野生型長的趨勢，並且此性狀是專一於缺氮的生長情況。於硝酸鹽含量的分析中40-B1與野生型並沒有顯著的差異，這暗示40-B1可能於硝酸鹽進入液胞儲存的機制沒有發生缺陷，可能是硝酸鹽由液胞回到細胞質的機制或者是與硝酸鹽有關的訊息傳導途徑出現缺陷。

Nitrate assimilation is a critical process for plant growth. To assimilate nitrate, plants must first take up nitrate from soil into plant cells. Nitrate can be either assimilated in the root or transported to the shoot. CHL1 is the first nitrate transporter isolated in higher plants. CHL1 mRNA is expressed predominantly in root and displays nitrate-dependent regulation. The uptake capacity was decreased when CHL1 was mutated. Therefore, CHL1 is hypothesized to transport nitrate across the plasma membrane in root cell. However, there is no direct evidence about the subcellular localization of CHL1 protein. In this study, we demonstrate that CHL1 protein and its homologue AtNRT1:5 are localized on the plasma membrane of Arabidopsis root cell and shoot cell by biochemical approach. It is the first subcellular localization data to confirm the hypothesis directly that CHL1 is involved in transporting nitrate across plasma membrane. The regulation of CHL1 and AtNRT1:5 on protein level were also studied. We observed that CHL1 protein has high level expression at the beginning of nitrate induction but without any significant increase after that. Unlikely, CHLI mRNA and the nitrate uptake that can be induced 8~9 and 1.7~1.8 folds respectively, after nitrate induction. This indicates that the function of CHL1 might be regulated on transcriptional, translational and post-translational levels. For AtNRT1:5 protein, we demonstrate that it can be up-regulated by drought and salt stresses like their mRNA does. Interestingly, the induction time of AtNRT1:5 protein under salt stress is slower than it induction time for drought stress. In addition, excess nitrate taken into the plant cell can be stored in vacuolar for future use. Extensive studies have been focused on the nitrate uptake step; In contrast, little is known about vacuolar storage of nitrate at the molecular level. Therefore, In order to probe the molecular mechanisms of vacuolar nitrate transportation, we have used Arabidopsis T-DNA tagged lines to screen for mutants defective in vacuolar storage. Plants were grown in the medium with 25mM nitrate for five days and then transferred to nitrogen-free medium for another five days. Mutants defective in vacuolar storage might show retarded root development in response to nitrogen starvation. For one of the candidates 40-B1, the primary root stops growing when plants were transferred to N-free medium. However, under this condition, lateral roots of the mutant were longer than those of wild type. This phenotype is specific for nitrogen starvation. However, nitrate accumulation of 40-Bi is as typical as that of wild type plant. This indicated that 40-B1 is not defective in nitrate vacuolar storage. More likely, 40-B1 is defective in either vacuolar retrieving or nitrate signaling pathway.