Using Biochemical Approaches to Verify the Role of the Nitrate Transporter CHL1 and Its Homologue, AtNRT1:5 in Plant Cell and to Elucidate Their Regulatory Mechanisms

• Main Authors: Shin-Chen Hou, 侯信成
• Other Authors: Chi-Ying Huang
• Format: Others
• Language: zh-TW
• Published: 2001
• Online Access: http://ndltd.ncl.edu.tw/handle/17274064037057506045

碩士 === 國立臺灣大學 === 植物學研究所 === 89 === 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, CHL1 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 25 mM 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-B1 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.