| Nitrogen is one of the most important nutritional elements in crop and it is also an important limiting factor in crop production.In order to increase the crop yield,excessive nitrogen fertilizer application leads to the decline of crop nitrogen use efficiency,which not only triggers a series of ecological problems,but also negatively affects the grain quality of crops.Improving nitrogen use efficiency of crops can not only promote the absorption of trace elements,but also increase the translocation of trace elements in grains.Therefore,there is a coordinated interaction mechanism between nitrogen and trace elements.Besides,protein content in grains was correlated with trace elements such as manganese and iron.To understand the molecular regulation mechanism about is great great significance to improve crop yield and quality.Previous studies have shown that high-affinity nitrate transporter OsNRT2s gene family plays a key role in improving nitrogen use efficiency in rice.We first found NO3-can promote Mn absorption and the concentration of Mn only increased in seeds and husks of OsNRT2.1 overexpression line.Overexpression of OsNRT2.1 could not increase the nitrogen accumulation,but improved the manganese accumulation in rice grains.Therefore,we focused on the overexpression of OsNRT2.1 rice line to explore the molecular regulation of NO3-absorption and Mn accumulation.Besides,OsNRT2.3b,which an important gene related to nitrogen use efficiency can perform nitrate transport function independently.Previous studies have shown that overexpression of OsNRT2.3b not only improved nitrate transportation capability,but also increased ammonium uptake to enhance nitrogen use efficiency in rice.Simultaneously,the content of total P and Fe in overexpression of OsNRT2.3b rice lines also increased significantly.Overexpression of OsNRT2.3b in barley,which grows on soil with NO3--N has an effect on barley phenotypes,nitrogen use efficiency and nutrient absorption balance applying the transgenic technology.The main results were listed as follows:1.Wild type rice seedlings were planted under different conditions of N supply.Mn uptake and OsNRAMP3/OsNRAMP5/OsNRAMP6 expression are increased by NO3Therefore,NO3-positively regulates the absorption of Mn in rice.When Mn concentrations were assessed in OsNRT2.1/OsNRT2.3a/b transgenic lines planted in normal field which was WL(waterlogged)condition,respectively,we observed increased Mn in the seeds and husk of OsNRT2.1 overexpression line,but no enhanced uptake in OsNRT2.3a/b transgenic lines.In addition,the expression of OsNRT2.1 and OsNAR2.1 is enhanced in overexpression of OsNRT2.1 lines,allowing an efficient transfer of NO3-to enhance total N accumulation and also promote Mn absorption under 0.5 mM NO3-conditions.2.To simulate hydroponic conditions in the presence of different N treatments,we designed a field experiment under different irrigation conditions.Under AWD(alternating wet and dry)condition,we observed a 31%reduction in grain yields of wild type(WT)plants compared to WL(waterlogged)condition.The overexpression of OsNRT2.1 could recover this loss,as OsNRT2.1 transgenic line displayed higher grain yields than WT plants.Comparison of the Mn concentrations in rice grain under different irrigation conditions,we observed 60%higher grain Mn in the transgenic lines under AWD(alternating wet and dry)condition and approximately 30%higher Mn in the grain of transgenic lines in WL(waterlogged)condition.We further found that the overexpression of OsNRT2.1 did not alter Mg and Fe in the seeds in either growth condition.The reasons for the increased Mn content in OsNRT2.1 transgenic seed under AWD condition could be explained by the elevated expression of OsNRAMP family genes including OsNRAMP3,OsNRAMP5,and OsNRAMP6 in node Ⅰ,the panicle-neck,and the flag leaves.3.The functional analysis of the transporter in Xenopus laevis Oocytes heterologous expression system showed that Mn transporter protein OsNRAMP3 need partner protein OsNAR2.1 to uptake Mn2+in Xenopus laevis Oocytes.Besides,Mn2+uptake and transport by OsNRAMP3 and OsNAR2.1 are pH dependent.The Mn2+ binding activity of OsNAR2.1 can only be observed under NO3-and Mn2+ treatment at pH 7.4.However,it didn’t show Mn2+binding activity when treated with Mn2+alone.4.Overexpression of OsNRT2.3b,which is an important gene related to nitrogen use efficiency,can improve grain yield and nitrogen use efficiency of barley in plot experiment.We demonstrated that the NRT2 family protein and the partner protein HvNAR2.3 were also up-regulated by 0.2 mM NO3-treatment in transgenic barley lines.This suggests that the expression of OsNRT2.3b and other HvNRT2 family genes were all up-regulated in the transgenic barley to increase the efficiency of N uptake and usage.Simultaneously,ovexpression of OsNRT2.3b can increase the distribution ratio of N,P and Fe to the aboveground organ of barley.5.Analysis of OsNRT2.3b efflux function by xenogeneic expression system of Xenopus oocyte.At pH 7.4,OsNRT2.3a and OsNRT2.3b both have the NO3-efflux function,and co-injection of OsNAR2.1 and OsNRT2.3a is more significant.However,NO3-efflux function disappeared in Xenopus laevis oocytes injected with genes at pH 5.5 condition.In summary,OsNRT2.1 has been shown to interact with OsNAR2.1 in the regulation of nitrate uptake.We also demonstrated that OsNAR2.1 with Mn2+ binding activity participated in manganese absorption and transport through the interaction with manganese transport OsNRAMP3 under nitrate supply condition.The efflux function of OsNRT2.3b can facilitate the transport of NO3-,which in turn coordinates the distribution of iron in the shoot of barley. |
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