Characterizing Physiological Responses And Regulated Expression Of Relevant Genes In Rice To Supply Of Different Nitrogen Sources

Rice is one of the most important crops both in China and in the world, and its growth and development are largely depending on the supply of nitrogen(N). In flooded paddy soils, rice takes up ammonium as the preferred N source. However, rice roots can transport oxygen into the rhizosphere, thus, oxidizing part of ammonium to nitrate by nitrobacteria in the root surface of wetland plants. Therefore, rice roots may take up substantial quantities of N in the form of nitrate even when grown in waterlogged paddy soil. Previously reporters had shown that a mixed supply of ammonium and nitrate stimulates N uptake and growth of rice, and improves N use efficiency. However, how rice regulates the uptake and utilization of different N sources is still not clear in molecular physiological level. Dissecting the genes responsible for uptake, assimilation and translocation of ammonium and nitrate are, therefore, essential for improving the rice production and efficient use of N by bio-technological approaches in near future. In the present work, physiologic differences and regulated expressions of the genes which are involved in N uptake and assimilation were determined by re-supply of different sources of N (NH₄⁺, NO₃^-, NH₄NO₃) to N-starved rice plants(Oryza sativa L. ssp. Japonics cv. wu yun jing 7#) for 4 days(d). The gene expression profiling of the rice cultivar to changes in the N-supply form for 4 d was obtained by analyses of cDNA microarrays. In addition, the expression patterns of four putative high affinity nitrate transporter genes(OsNRT2; 3,OsNRT2; 4,OsNAR2; 1 and OsNAR2; 2) were characterized by transforming the fused genes of their respective promoter and GUS reporter genes into the model rice cultivar(Oryza sativa L. ssp. Japonic cv. Nipponbare). The main results are shown as in follows.The N deprivation stimulated the growth of roots and increased the ratios of roots to shoots in the rice. In contrast, the sole NH₄⁺ nutrition supressed the growth of root and decreased the ratios of roots to shoots. In comparison to nitrate as sole N source, ammonium tended to significantly improved shoot growth and total N uptake. In comparison to either ammonium or nitrate alone, mixture supply of the two forms of N increased total N uptake and translocation of N to above ground parts, as well as activation of glutamine synthetase in the roots and nitrate reductase in the leaves. It suggests that balanced supply of both ammonium and nitrate could improve both uptake and use efficiency of N by rice crop. Additional, the root activity which may relate the ability of nutrient uptake was significant improved by NO₃^- and NH₄NO₃ in comparison to only NH₄⁺ nutrition.The expression patterns in transcriptional levels of the genes which may involve in N uptake, reduction and assimilation were analyzed in the rice roots by semi-quantitative reverse transcription polymerase chain reaction(RT-PCR). A putative high affinity ammonium transporter gene, OsAMT1; 1(accession number: AF289477), is constitutive and highly expressed in the rice roots independent of the supply of N forms and status. However, both OsAMT1; 2(accession number: AF289478), and OsAMT1; 3(accession number: AK107204), other two putative high affinity ammonium transporter genes, were expressed, sustained at low abundance in the N-starved rice roots. Expression of OsAMT1; 2 was enhanced by re-supply of NH₄⁺ but suppressed rapidly by both NO₃^- -only and NH₄NO₃, indicating that the nitrate in the culture solution inhibiting the expression of OsAMT1; 2 in rice. In contrast, expression of OsAMT1; 3 was transiently enhanced in the first few hours(2 h) by re-supply NH₄⁺-only or NO₃^- -only, and sustained enhanced by NH₄NO₃. It suggests that expression of the ammonium transporter genes in the rice might have been not only regulated by ammonium concentration, but also by nitrate concentration, as well as by the balance between ammonium and nitrate both in the external medium and inside the plant.A putative high affinity nitrate transporter, OsNRT2; 3(accession number: AF289479/AK072215), was highly expressed in transcriptional level in N-starved rice roots, however, its expression was strongly suppressed by re-supply of 2.5 mM N whatever the N forms were in the external medium. Moreover, a putative nitrate reductase(NR) gene, OsNiR1 (accession number: AK102178), has been induced expression immediately by exposure to nitrate or nitrate ammonium. It is very interesting that co-provision of NH₄⁺ and NO₃^- in the culture solution resulted in more abundant steady-state expression of OsNiR1 in the roots than the provision of nitrate alone. However, a putative glutamine synthetase gene, OsGS1;1(accession number: AK109397/AB037595), was not largely affected by N form or starvation in the rice roots.Using the mixed supply of ammonium nitrate as a control, we had analyzed the expression profiling of genes responsible for re-supply NH₄⁺ -N, NO₃^- -N and N starvation treatments for 4 d by cDNA microarray(including about 21, 495 oligonucleotide). The results suggested that the genes in the roots were more sensitive than that in the shoot to the re-supply of N in the different forms to the N-starved dee plants. The total number of root genes that were more than two fold up-or down-regulated after 96 h was 445 for ammonium and 324 for nitrate out of a total of 22, 000 genes. In the shoot, the equivalent numbers were much smaller only 32 and 58, respectively. These genes were not only involved in N uptake and metabolism, but also involved in chlorophyll metabolism, carbon fixation and assimilation. We analyzed the cis-elements in putative promoter regions of the rice genes that were specifically regulated by the three N-treatments relative to the mixed supply of ammonium and nitrate. There are six and eight such cis-elements which are very abundantly contained in the promoters of ammonium only and nitrate only regulated genes, respectively. Notably, a cis-element ARECOREZMGAPC4, defined as a transcription factor Myb-binding site, is present in very high frequency in the promoters of all "nitrate only" regulated genes. In addition, 18 transcription factors(TFs) genes were regulated by more than two-fold by the change of N supply forms and/or N starvation, among which 16 TFs in the root and only two TFs in the shoot. Moreover, six genes encoding NO₃^-transporter and eight genes encoding NH₄⁺ transporter were significantly regulated by the change of N supply forms and/or N starvation.By being detected the expression of GUS reporter gene in transgenic dee, the specific spatio and temporal expression of four putative nitrate transporter genes, OsNRT2; 3,OsNRT2; 4,OsNAR2; 1 and OsNAR2; 2 were characterized. It was found that OsNRT2; 3 is expressed mainly in the root stele and leaf nervure, possibly playing the role in transport of NO₃^- in the long-distance translocation and reutilization inside the plant, but not in the direct uptake of NO₃^- from external growth medium. OsNAR2; 1 is very strongly expressed in the whole root, particularly in the root cap, which indicates that OsNAR2; 1 is not only helpful for NO₃^- uptake and translocation, but may also play a role as a signal recepter in the NO₃^- signal pathways to regulate plant nutrient uptake. OsNRT2; 4 and OsNAR2; 2 are weakly expressed in roots stele and leaves nervure。Their functions both in sensing N supply status and uptake and/or translocation of N needs further investigated in details.