Physiological And Molecular Mechanisms Of Enhanced Nitrate Nutrition In Rice Cultivars With Different Nitrogen-Use-Efficiency

Rice (Oryza sativa L.) is one of the major crops in the world and it contributes 43.7% of the total national grain production in China, which represents 22.8% of the total world rice cropping area and 36.9% of its production. Rice is traditionally produced under flooded conditions, and more than 80% of the freshwater used in agriculture in China is for rice production. However, increasing water shortages are threatening the sustainability of lowland rice fields, and in the future global climate change may lead to higher potential evapotranspiration, decreasing precipitation, and increasing frequency of high intensity rains. Hence, there is an urgent need of developing water-saving technologies, such as intermittent irrigation or even aerobic cultivation of rice. In paddy field, ammonium (NH₄⁺) rather than nitrate (N(V) tends to be a better source of N for rice. However, in well-drained soils, NH₄⁺-N converts rapidly to nitrite (NO₂^-) and then to NO₃^-. Thus, the main form of N in upland soils is NO₃^--N no matter what form of N fertilizer is applied. Furthermore, rice roots can aerate the surrounding soil (rhizosphere) by excreting oxygen (O2), and this activity promotes the process of nitrification, i.e. the conversion of NH₄⁺ to NO₃^- (Rubinigg et al., 2002; Wang and Peng, 2003). Therefore, although the predominant species of mineral N in bulk soil for paddy rice field is likely to be NH₄⁺ (Revsbech et al., 1999), rice roots are actually exposed to a mixed N supply (Duan et al., 2004).Nitrogen use efficiency (NUE) is a key parameter for evaluating a rice cultivar and very important both for improving grain yield and reducing environment pollution. In order to increase the understanding of relationship between NUE and enhanced nitrate nutrition, several experiments were carried out in the dissertation, which included three parts: (1) Solution culture experiment were carried out to study the effects of different ratios of NH₄⁺-N /NO₃^--N (NH₄⁺/ NO₃^- as 100/ 0, 75/ 25, 50/ 50 and 25/ 75) on the biological characteristics of 8 genotypes of rice with different nitrogen (N) efficiency at the seedling stage, and chose three cultivars as materials for experiments below. (2) With the three rice cultivars with different NUE, rice yield, yield components, rice growth, N uptake and assimilation were examined at different growth stages to look at the mechanisms of responses of rice plants to NO₃^-. (3) Root growth, soluble sugar concentration and root morphologic characteristics of the three different NUE rice cultivars by seedling stage experiment were studied. (4) ¹⁵N-labled technique was used to study the ¹⁵NH₄⁺ accumulation and uptake efficiency of Nanguang (high-NUE) and ELIO (low-NUE). (5) Solution culture experiments were carried out to study the effect of NO₃^- on the ¹⁵N-labled NH₄⁺ uptake kinetics of Nanguang rice cultivar (high-NUE) and ELIO rice cultivar (low-NUE) at the seedling stage (30-day-old). (6) With specific primers, real-time SYBR Green quantitative PCR (RQ-PCR) was used to study the expression of OsAMT1s in Nanguang (high-NUE) and ELIO (low-NUE). The results obtained were as follows.1. Different rice genotypes have different responses to NO₃^-. Among the eight rice genotypes, the improvement of growth by NO₃^- to the rice genotypes with higher N use efficiency was greater than that with lower N use efficiency. Furthermore, we compared the growth of Nanguang (higher N efficiency) and ELIO (lower N efficiency) at the ratio of NH₄⁺ /NO₃^- as 75/25 and 100/0. The tillers and photosynthetic rate in the leaves of Nanguang were averagely increased by 35%, respectively, while those of ELIO (lower N efficiency) had no significant difference, between the treatments of 75/25 and 100/0. NO₃^-could improve the accumulation of dry matter in the roots of Nanguang, and increase the volume of roots of Nanguang and ELIO.2. Compared with the treatment of 100/0 NH₄⁺-N/NO₃^--N, a ratio of 75/25 NH₄⁺-N /NO₃^--N increased the spikelets per panicle of Nanguang cultivar (high NUE) and thus increased its grain yield by 21%, while the yield of ELIO cultivar (low NUE) had no significant differences in the two nutrient solutions. Enhanced nitrate nutrition increased total N accumulation and dry matter production in Nanguang by 36% and 30%, respectively, averaging in the four growth stages, while the increased effect of NO₃^- was not found in ELIO cultivar. In the mixture of NO₃^- and NH₄⁺ nutrient solution, the NO₃^-reductase activity in the leaves and glutamine synthetase activity in the roots of Nanguang were increased by 100% and 95%, respectively, compared to the 100% NH₄^<sub> treatment. All these results showed that the increased spikelets per panicle and improved N uptake and assimilation by some NO₃^- might contribute to the increased grain yields of Nanguang. Rice cultivar with high NUE had stronger response to enhanced nitrate nutrition than the rice cultivar with low NUE, suggesting that there might be a relationship between enhanced nitrate nutrition and NUE in rice crop.3. Results indicated a ratio of 75/25 NH₄⁺-N/ NO₃^--N, when compared with those of 100/0 NH₄⁺-N/ NO₃^--N, increased the root biomass and N accumulation in Nanguang cultivar by 32% and 38%, respectively, while the increased effect of NO₃^- on the root of ELIO cultivar was not found in the four growth stages. Results from seedling stage experiment showed that enhanced nitrate nutrition increased the dry weight and N accumulation in roots of Nanguang. Besides, the root surface area and root volume of Nanguang cultivar were also increased significantly in enhanced nitrate nutrition solution, compared with that plants in sole NH₄⁺ solution. In both treatments of the root for the two rice cultivars, there were no significant difference in soluble sugar concentration treated with different N forms, suggesting that it is not soluble sugar which stimulated the lateral root growth after NO₃^- addition.4. The dry weight and N accumulation in root of Nanguang were improved by enhanced nitrate nutrition by 18% and 31%, and those in shoot of Nanguang were improved by 18% and 17%, respectively, averaging at seedling stage, early tillering stage and maximum tillering stage. Enhanced nitrate nutrition could increase the ¹⁵NH₄⁺ uptake efficiency of Nanguang, while has no change on that of ELIO. Compared with the treatment of 100/0 NH₄⁺-N/NO₃^--N, a ratio of 75/25 NH₄⁺-N/NO₃^--N increased the ¹⁵NH₄⁺ uptake efficiency of Nanguang cultivar by 57% in root and by 46% in shoot. Both of the rice cultivars with different NUE could absorb large amount of NO₃^- which could be as much as 44% of the total N absorbed. The uptake amount of NO₃^- by two rice cultivars in different growth stages was in following order: Maximum tillering stage > Seedling stage> early tillering stage.5. NO₃^- addition could increase the uptake rate of ¹⁵NH₄⁺ by Nanguang, and improve its N uptake efficiency, while it has no change on the uptake rate of ¹⁵NH₄⁺ by ELIO. Enhanced nitrate nutrition could increase the NH₄⁺ uptake by Nanguang. This increase was mainly attributed to a significantly increased V_max (31.5%) in the treatment with NO₃^-while the K_m values for NH₄⁺ were not significantly affected by the addition of NO₃^-. This demonstrated that enhanced nitrate nutrition could significantly increase the numbers of the ammonium transporters but did not affect the affinity of the transporters to NH₄⁺.6. All three genes were expressed in greater extent in roots than in leaves. OsAMT1; 1 transcript levels were higher in both roots and leaves of rice (Fig. 3) than those of OsAMT1; 2 and OsAMT1; 3 whether in enhanced nitrate or no nitrate treatment, and expression of OsAMT1; 2 and OsAMT1; 3 were faintly expressed, especially in leaves of rice. Enhanced nitrate nutrition improved the expression of OsAMT1;1, OsAMT1;2 and 0sAMT1;3 in roots of Nanguang and ELIO, and also improved the expression of OsAMT1;2 in leaves of Nanguang and ELIO, but depressed the expression of 0sAMT1;3 in leaves of Nanguang and ELIO, compared to those in sole NH₄⁺ solution. In leaves of Nanguang, expression of OsAMT1;1 was more constitutive in both nutrient solutions while that in ELIO was depressed by 10.3%. In sum, enhanced nitrate nutrition improved expression of OsAMT1s by 14.5% in Nanguang and 0.29% in ELIO. The different effect of nitrate nutrition on OsAMT1s expression between two cultivars should be attribute to the different expression pattern of OsAMT1;1 in leaves.