关键词：
N use efficiency;Proton pumps in the tonoplast;N reutilization;Oilseed rape (Brassica napus)
摘要：
Nitrate (NO3-) can accumulate in high concentrations in plant cell vacuoles if it is not reduced, reutilized or transported into the cytoplasm. Such accumulation of NO3- in the vacuole occurs when mechanisms for NO3- assimilation in the cytoplasm are saturated. Moreover, other processes such as efflux across the plasma membrane might affect NO3- accumulation in the vacuole. These are the main reasons limiting nitrogen use efficiency (NUE) in plants. This study elucidates mechanisms for NO3- transport from the cytoplasm to vacuoles by the V-proton pump (V-ATPase and V-PPase) and their relationship with different NUE in four Brassica napus genotypes. Pot experiments were conducted in a greenhouse under normal (15.0 mmol L-1) and limited N (7.5 mmol L-1) concentrations of nitrate using B. napus genotypes that demonstrated either high (742 and Xiangyou 15) or low (814 and H8) NUE (g g(-1)). Specific inhibitors of V-ATPase and V-PPase increased nitrate reductase (NR) activity, resulting in greatly decreased NO3- in plant tissues. Nitrate reductase activity and NO3- content correlated more highly to V-PPase activity than they did to V-ATPase activity, and correlation between V-PPase activity and NO3- content was significantly higher than it was to V-ATPase. Genotypes with high NUE had significantly lower activities of V-ATPase and V-PPase than those with low NUE. In the high-NUE plants, lower activities of V-proton pump underlie mechanisms that result in significantly lower NO3- content in plant tissues of the high-NUE genotypes than those found in plant tissues of the low-NUE genotypes. Our results show that the tonoplast proton pumps V-PPase and V-ATPase strongly negatively affect NR activity and positively affect NO3- content. V-PPase contributed more to this regulatory mechanism than did V-ATPase.
作者机构：
[荣湘民; 袁丹; 宋海星; 张振华; 刘强; Zeng J.; 王文明] College of Resources and Environmental Sciences, Hunan Agricultural University/Hunan Provincial Key Laboratory of Farmland Pollution Control and Agricultural Resources Use/Hunan Provincial Key Laboratory of Plant Nutrition in Common University, Changsha, 410128, China;[官春云] Hunan Branch of National Center of Oilseed Crops Improvement, Changsha, 410128, China
通讯机构：
[Song, H.-X.] C;College of Resources and Environmental Sciences, China
关键词：
N-use efficiency;Nitrate;Proton pumps in the tonoplast;Nitrate reductase;Vacuole;Brassica napus
摘要：
Nitrate, once taken up by plants, can either be stored in vacuoles or reduced by nitrate reductase in the cytoplasm. High accumulation of NO3
− in the vacuole occurs when assimilation into the cytoplasm is saturated. This study elucidates how proton pumps at the tonoplast (V-ATPase and V-PPase) affect the NO3
− content of Brassica napus by controlling the distribution of NO3
− between the cytoplasm and vacuole. Pot experiments were conducted in a greenhouse under normal N (15.0 mM nitrate) conditions using B. napus genotypes that demonstrated either high (Xiangyou15) or low (814) nitrogen use efficiency (NUE). The NO3
− content of the high NUE genotype was significantly lower than that of the low NUE genotype, whereas the total N per plant of the two genotypes was almost the same, suggesting that the different NUE between the two genotypes is not due to the difference of NO3
− uptake. The relative expression levels of V-ATPase (vha-a3) and V-PPase (avp1) genes in the high NUE genotype were significantly lower than in the low NUE genotype, resulting in lower V-ATPase and V-PPase activities in the high NUE genotype. The transport of NO3
− and protons from the cytoplasm to the vacuole is powered by V-ATPase and V-PPase, so their lower activities increase H+ efflux from and reduce NO3
− influx into the vacuoles of the high NUE genotype. We conclude that the lower activity of proton pumps at the tonoplast is the main reason the high NUE genotype possesses lower NO3
− content and higher N-use efficiency.
摘要：
[Objective] This assay was to explore the contribution of enzymes ac- counting for nitrogen reutilization in two Brassica napus varieties No.6 （low nitrogen use efficiency） and No.2 （high nitrogen use efficiency）. [Method] We measured the yield, transportation and accumulation of grain nitrogen, loss of leaf nitrogen and ni- trogen use efficiency （NUE） in the two rape varieties, by inhibiting proteolytic en- zyme （PE）, glutamine synthetase （GS） and glutamate synthetase （GOGAT） and la- beling with lSN. [Result] Under GOGAT inhibitor treatment, both of the two varieties presented minimum NUE, yield and nitrogen transportation in grain and maximum ni- trogen loss in leaf. The effect of PE inhibitor was the second greatest, and that of GS inhibitor was the lowest. Moreover, 80% of the nitrogen that had been accumu- lated in leaf was transported out during late growth stage, and 50% to 70% of the grain nitrogen derived from the nitrogen in vegetative organs. The two varieties ex- hibited the same tendency. Rape variety No.2 had higher yield, grain nitrogen accu- mulation and lower nitrogen loss compared with No.6. [Conclusion] GOGAT has the greatest effect on the accumulation of grain nitrogen, yield and nitrogen reutilization in rape. Different enzyme activity may be the major factor resulting in different nitro- gen rautilization in the two rape varieties. Nitrogen stored in leaf during early growth stage is mainly used for nitrogen remobilization. A large proportion of grain nitrogen derives from vegetative tissues.
