Nitrate reutilization mechanisms in the tonoplast of two Brassica napus genotypes with different nitrogen use efficiency

Abstract

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.