Nitrate Re-utilization Mechanism Of Two Brassica Napus Genotypes With Different Nitrogen Use Efficiency Basis On Proton Pump In Tonoplast

Improved N fertilizer use efficiency based on plant mechanisms, and exploitation of high N use efficiency potential of plants has become very important in plant nutrition research area in recent years. High concentrations of NO3--N can accumulate in plant cell vacuoles, if it is not reduced, reutilized and transported into the cytoplasm. These are the main reasons limiting nitrogen use efficiency (NUE) in plants. Pot experiments were conducted in a greenhouse under normal (15.0mmol L-1nitrate), N-limited (7.5mmol L"1nitrate) conditions using Brassica napus genotypes that demonstrated either high (742and xiangyoul5) or low (814and H8) NUE. Using specific inhibitors for V-ATPase and V-PPase to elucidates transport mechanisms of NO3--N from the cytoplasm to vacuoles by the V-proton pump (V-ATPase and V-PPase) and their relationship with different NUE in two Brassica napus genotypes. Results showed as below:1. Distribution results of nitrate-N and ammonium-N in plant tissues at seedling stage showed that:nitrate-N and ammonium-N in L1plant tissues are higher than H2when applied the same N level, nitrate-N content in root is significantly higher than shoot under all treatments, but the differences of ammonium-N content between root and shoot are lower than nitrate-N. Consequently, there are significant differences of nitrate-N and ammonium-N content in plant tissues between genotypes.2. Comparison study of NUE between genotypes at harvesting stage:the NUE based on biomass and seed yield of H1and H2are significantly higher than L1and L2. Consequently, HI and H2defined as high NUE genotypes, but L1and L2defined as low NUE genotypes, respectively.3. Results of V-ATPase, V-PPase, NR activities and NO3--N content at seedling and flowering stages showed that:the changes of V-ATPase, V-PPase, NR activities and N03--N content are almost the same, regardless stages and genotypes, and the V-PPase activity at seedling stage is higher than flowering stage, but the V-ATPase, NR activities and NO3--N content of flowering stage are similar with seedling stage. NO3--N transport from cytoplasm into vacuolar can be accelerated by V-ATPase and V-PPase, but NO3--N transport from vacuolar into cytoplasm can be accelerated by NR and declined the content of NO3--N. The differences of NO3--N accumulation between genotypes are depend on V-ATPase, V-PPase, NR activities, and the contribution of V-PPase for NO3--N accumulation is higher than V-ATPase.4. RT-PCR results of V-ATPase, V-PPase, NR at flowering stages showed that:the essential reason of high NUE genotype with lower activities of V-ATPase, V-PPase and higher activity of NR is possess lower relative expression of V-ATPase, V-PPase genes and higher relative expression of NR major gene (nia2), but not because of lower response sensitive of V-ATPase, V-PPase and higher response sensitive of NR.5. Results of specific inhibitor at flowering stage showed that:Specific inhibitors for V-ATPase and V-PPase were effective, leading to increased nitrate reductase (NR) activity, resulting in greatly decreased N03--N in plant tissues. Correlations of NR activity and NO3--N content to V-PPase activity were higher than to V-ATPase activity, and correlation between V-PPase activity and NO3--N content was significantly higher than to V-ATPase.6. NMT measurement for H+and NO3-flux across the tonoplast:V-ATPase and V-PPase supply power for NO3--N transport from cytoplasm into vacuolar, NO3--N accumulated is more when activities of V-ATPase and V-PPase are higher. Low NUE genotype with higher activities of V-ATPase and V-PPase, leading more H+and NO3-influx into vacuolar, more nitrate is accumulated in vacuolar also. But the reverse results showed in high NUE genotype, high NUE genotype with lower activities of V-ATPase and V-PPase, leading less H+and NO3-influx into vacuolar, less nitrate is accumulated in vacuolar also. These are further to explain the reason of NO3--N accumulation.Conclusions:Genotypes with high NUE had significantly lower V-ATPase and V-PPase activities and higher NR activity than those with low NUE. We believe this is mainly related to the expression levels of V-ATPase, V-PPase and NR (nia2) genes. In the high-NUE plants, higher expression of the NR gene (nia2) and lower expression of the V-ATPase andV-PPase genes underlie mechanisms that result in significantly lower NO3--N content in plant tissues of the high NUE genotypes than those found in plant tissues ofthe low NUE genoytpes. Our results show that the proton pumps V-PPase and V-ATPase in the tonoplast strongly negatively affect NR activity and positively affect NO3--N content. V-PPase contributed more to this regulatory mechanism than did V-ATPase. These results are the essential reason of NUE differences between genotypes, it will supply theory support to improve NUE for the future study.