拟南芥体内硝酸盐积累差异与细胞膜H^+-ATPase的关系

The relationship between the accumulation of nitrate and plasma membrane H^+-ATPase in Arabidopsis

以野生型拟南芥(WT)和叶柄硝酸盐积累减少的拟南芥突变体atnrt1.4为材料,研究了叶柄与叶片硝酸盐的积累及细胞膜H+-ATPase。采用两相法分离了2种材料细胞膜并测定了细胞膜H+-ATPase活性,通过RT-PCR分析了WT与at-nrt1.4叶柄及叶片中各个细胞膜H+-ATPase基因的表达,并采用Western blot分析了WT与atnrt1.4叶柄及叶片细胞膜上H+-ATPase的蛋白浓度。结果显示:atnrt1.4叶柄硝酸盐积累显著低于WT叶柄,仅为WT的58.6%;atnrt1.4叶柄中细胞膜H+-ATPase基因AHA3、AHA5、AHA10和AHA11的表达比WT显著降低;atnrt1.4叶柄细胞膜H+-ATPase蛋白浓度也显著低于WT叶柄。WT叶柄硝酸盐积累显著高于叶片,相应的细胞膜H+-ATPase的水解活性也显著高于叶片。上述研究表明:atnrt1.4叶柄硝酸盐积累减少导致其细胞膜H+-ATPase活性降低;拟南芥体内硝酸盐积累差异与细胞膜H+-ATPase活性有关。

Arabidopsis nitrate transporter mutant atnrt1.4,which has a very low nitrate concentration in petiole,and wild type Arabidopsis（WT）were used as materials to investigate the relationship between the distribution of nitrate in plant tissues and plasma membrane H＋-ATPase.The plasma membrane vesicles of petiole and lamina of atnrt1.4 and WT were isolated,respectively,by two-phase system for the determination of H＋-ATPase activity.The expression of plasma membrane H＋-ATPase genes was analyzed by RT-PCR and the enzyme concentration of plasma membrane H＋-ATPase was detected by Western blot.Results showed：the nitrate concentration of atnrt1.4 petiole was only 58.6% of WT petiole,and H＋-ATPase activity of atnrt1.4 petiole was much lower than WT petiole;the decreased expression of plasma membrane H＋-ATPase genes,AHA3,AHA5,AHA10 and AHA11,were responsible for the lower plasma membrane H＋-ATPase activity in atnrt1.4 petiole;in addition,enzyme concentration of plasma membrane H＋-ATPase of atnrt1.4 petiole was evidently lower than WT petiole;the nitrate concentration of WT petiole was higher than WT lamina,and the corresponding plasma membrane H＋-ATPase activity of WT petiole was also higher than WT lamina.The results indicated that the lower activity of plasma membrane H＋-ATPase in atnrt1.4 petiole was attributed to the less accumulation of nitrate,and the accumulation of nitrate was related to plasma membrane H＋-ATPase in Arabidopsis.