AMT1;1 transgenic rice plants with enhanced NH4(+) permeability show superior growth and higher yield under optimal and suboptimal NH4(+) conditions.
ABSTRACT: The major source of nitrogen for rice (Oryza sativa L.) is ammonium (NH4(+)). The NH4(+) uptake of roots is mainly governed by membrane transporters, with OsAMT1;1 being a prominent member of the OsAMT1 gene family that is known to be involved in NH4(+) transport in rice plants. However, little is known about its involvement in NH4(+) uptake in rice roots and subsequent effects on NH4(+) assimilation. This study shows that OsAMT1;1 is a constitutively expressed, nitrogen-responsive gene, and its protein product is localized in the plasma membrane. Its expression level is under the control of circadian rhythm. Transgenic rice lines (L-2 and L-3) overexpressing the OsAMT1;1 gene had the same root structure as the wild type (WT). However, they had 2-fold greater NH4(+) permeability than the WT, whereas OsAMT1;1 gene expression was 20-fold higher than in the WT. Analogous to the expression, transgenic lines had a higher NH4(+) content in the shoots and roots than the WT. Direct NH4(+) fluxes in the xylem showed that the transgenic lines had significantly greater uptake rates than the WT. Higher NH4(+) contents also promoted higher expression levels of genes in the nitrogen assimilation pathway, resulting in greater nitrogen assimilates, chlorophyll, starch, sugars, and grain yield in transgenic lines than in the WT under suboptimal and optimal nitrogen conditions. OsAMT1;1 also enhanced overall plant growth, especially under suboptimal NH4(+) levels. These results suggest that OsAMT1;1 has the potential for improving nitrogen use efficiency, plant growth, and grain yield under both suboptimal and optimal nitrogen fertilizer conditions.
Project description:OsNRT1.1a is a low-affinity nitrate (NO3 (-) ) transporter gene. In this study, another mRNA splicing product, OsNRT1.1b, putatively encoding a protein with six transmembrane domains, was identified based on the rice genomic database and bioinformatics analysis. OsNRT1.1a/OsNRT1.1b expression in Xenopus oocytes showed OsNRT1.1a-expressing oocytes accumulated (15) N levels to about half as compared to OsNRT1.1b-expressing oocytes. The electrophysiological recording of OsNRT1.1b-expressing oocytes treated with 0.25?mM NO3 (-) confirmed (15) N accumulation data. More functional assays were performed to examine the function of OsNRT1.1b in rice. The expression of both OsNRT1.1a and OsNRT1.1b was abundant in roots and downregulated by nitrogen (N) deficiency. The shoot biomass of transgenic rice plants with OsNRT1.1a or OsNRT1.1b overexpression increased under various N supplies under hydroponic conditions compared to wild-type (WT). The OsNRT1.1a overexpression lines showed increased plant N accumulation compared to the WT in 1.25?mM NH4 NO3 and 2.5?mM NO3 (-) or NH4 (+) treatments, but not in 0.125?mM NH4 NO3 . However, OsNRT1.1b overexpression lines increased total N accumulation in all N treatments, including 0.125?mM NH4 NO3 , suggesting that under low N condition, OsNRT1.1b would accumulate more N in plants and improve rice growth, but also that OsNRT1.1a had no such function in rice plants.
Project description:The NO3 - transporter plays an important role in rice nitrogen acquisition and nitrogen-use efficiency. Our previous studies have shown that the high affinity systems for nitrate uptake in rice is mediated by a two-component NRT2/NAR2 transport system. In this study, transgenic plants were successful developed by overexpression of OsNAR2.1 alone, OsNRT2.3a alone and co-overexpression of OsNAR2.1 and OsNRT2.3a. Our field experiments indicated that transgenic lines expressing p35S:OsNAR2.1 or p35S:OsNAR2.1-p35S:OsNRT2.3a constructs exhibited increased grain yields of approximately 14.1% and 24.6% compared with wild-type (cv. Wuyunjing 7, WT) plants, and the agricultural nitrogen use efficiency increased by 15.8% and 28.6%, respectively. Compared with WT, the 15N influx in roots of p35S:OsNAR2.1 and p35S: OsNAR2.1-p35S:OsNRT2.3a lines increased 18.9%?27.8% in response to 0.2 mM, 2.5 mM 15NO3 -, and 1.25 mM 15NH4 15NO3, while there was no significant difference between p35S:OsNAR2.1 and p35S:OsNAR2.1-p35S:OsNRT2.3a lines; only the 15N distribution ratio of shoot to root for p35S:OsNAR2.1-p35S:OsNRT2.3a lines increased significantly. However, there were no significant differences in nitrogen use efficiency, 15N influx in roots and the yield between the p35S:NRT2.3a transgenic lines and WT. This study indicated that co-overexpression of OsNAR2.1 and OsNRT2.3a could increase rice yield and nitrogen use efficiency.
Project description:Nitrogen (N) is a major essential nutrient for plant growth, and rice is an important food crop globally. Although ammonium (NH4+) is the main N source for rice, nitrate (NO3-) is also absorbed and utilized. Rice responds to NO3- supply by changing root morphology. However, the mechanisms of rice root growth and formation under NO3- supply are unclear. Nitric oxide (NO) and auxin are important regulators of root growth and development under NO3- supply. How the interactions between NO and auxin in regulating root growth in response to NO3- are unknown. In this study, the levels of indole-3-acetic acid (IAA) and NO in roots, and the responses of lateral roots (LRs) and seminal roots (SRs) to NH4+ and NO3-, were investigated using wild-type (WT) rice, as well as osnia2 and ospin1b mutants. NO3- supply promoted LR formation and SR elongation. The effects of NO donor and NO inhibitor/scavenger supply on NO levels and the root morphology of WT and nia2 mutants under NH4+ or NO3- suggest that NO3--induced NO is generated by the nitrate reductase (NR) pathway rather than the NO synthase (NOS)-like pathway. IAA levels, [3H] IAA transport, and PIN gene expression in roots were enhanced under NO3- relative to NH4+ supply. These results suggest that NO3- regulates auxin transport in roots. Application of SNP under NH4+ supply, or of cPTIO under NO3- supply, resulted in auxin levels in roots similar to those under NO3- and NH4+ supply, respectively. Compared to WT, the roots of the ospin1b mutant had lower auxin levels, fewer LRs, and shorter SRs. Thus, NO affects root growth by regulating auxin transport in response to NO3-. Overall, our findings suggest that NO3- influences LR formation and SR elongation by regulating auxin transport via a mechanism involving NO.
Project description:As glutamate dehydrogenases (GDHs) of microorganisms usually have higher affinity for NH4 + than do those of higher plants, it is expected that ectopic expression of these GDHs can improve nitrogen assimilation in higher plants. Here, a novel NADP(H)-GDH gene (TrGDH) was isolated from the fungus Trichurus and introduced into rice (Oryza sativa L.). Investigation of kinetic properties in vitro showed that, compared with the rice GDH (OsGDH4), TrGDH exhibited higher affinity for NH4 + (K m = 1.48 ± 0.11 mM). Measurements of the NH4 + assimilation rate demonstrated that the NADP(H)-GDH activities of TrGDH transgenic lines were significantly higher than those of the controls. Hydroponic experiments revealed that the fresh weight, dry weight and nitrogen content significantly increased in the TrGDH transgenic lines. Field trials further demonstrated that the number of effective panicles, 1,000-grain weight and grain weight per plant of the transgenic lines were significantly higher than those of the controls, especially under low-nitrogen levels. Moreover, glutelin and prolamine were found to be markedly increased in seeds from the transgenic rice plants. These results sufficiently confirm that overexpression of TrGDH in rice can improve the growth status and grain weight per plant by enhancing nitrogen assimilation. Thus, TrGDH is a promising candidate gene for maintaining yields in crop plants via genetic engineering.
Project description:BACKGROUND: Ammonium is one of the major forms in which nitrogen is available for plant growth. OsAMT1;1 is a high-affinity ammonium transporter in rice (Oryza sativa L.), responsible for ammonium uptake at low nitrogen concentration. The expression pattern of the gene has been reported. However, variations in its nucleotides and the evolutionary pathway of its descent from wild progenitors are yet to be elucidated. In this study, nucleotide diversity of the gene OsAMT1;1 and the diversity pattern of seven gene fragments spanning a genomic region approximately 150 kb long surrounding the gene were surveyed by sequencing a panel of 216 rice accessions including both cultivated rice and wild relatives. RESULTS: Nucleotide polymorphism (Pi) of OsAMT1;1 was as low as 0.00004 in cultivated rice (Oryza sativa), only 2.3% of that in the common wild rice (O. rufipogon). A single dominant haplotype was fixed at the locus in O. sativa. The test values for neutrality were significantly negative in the entire region stretching 5' upstream and 3' downstream of the gene in all accessions. The value of linkage disequilibrium remained high across a 100 kb genomic region around OsAMT1;1 in O. sativa, but fell rapidly in O. rufipogon on either side of the promoter of OsAMT1;1, demonstrating a strong natural selection within or nearby the ammonium transporter. CONCLUSIONS: The severe reduction in nucleotide variation at OsAMT1;1 in rice was caused by a selective sweep around OsAMT1;1, which may reflect the nitrogen uptake system under strong selection by the paddy soil during the domestication of rice. Purifying selection also occurred before the wild rice diverged into its two subspecies, namely indica and japonica. These findings would provide useful insights into the processes of evolution and domestication of nitrogen uptake genes in rice.
Project description:Nitrogen (N) is an important element required for plant growth and development, which also affects plant yield and quality. Autophagy, a conserved pathway in eukaryotes, degrades and recycles cellular components, thus playing an important role in N remobilization. However, only a few autophagy genes related to N remobilization in rice (Oryza sativa) have been reported. Here, we identified a core autophagy gene in rice, OsATG8b, with increased expression levels under N starvation conditions. It was investigated the function of OsATG8b by generating three independent homozygous 35S-OsATG8b transgenic Arabidopsis thaliana lines. The overexpression of OsATG8b significantly enhanced autophagic flux in the transgenic Arabidopsis plants. It was also showed that over-expressing OsATG8b promoted growth and development of Arabidopsis, in which the rosette leaves were larger than those of the wild type (WT), and the yield increased significantly by 25.25%. In addition, the transgenic lines accumulated more N in seeds than in the rosette leaves. Further examination revealed that overexpression of OsATG8b could effectively alleviate the growth inhibition of transgenic Arabidopsis under nitrogen (N) stress. N partitioning studies revealed that nitrogen-harvest index (NHI) and nitrogen use efficiency (NUE) were significantly increased in the transgenic Arabidopsis, as well as the 15N-tracer experiments revealing that the remobilization of N to seeds in the OsATG8b-overexpressing transgenic Arabidopsis was high and more than WT. Based on our findings, we consider OsATG8b to be a great candidate gene to increase NUE and yield, especially under suboptimal field conditions.
Project description:The nitrate (NO3-) transporter has been selected as an important gene maker in the process of environmental adoption in rice cultivars. In this work, we transferred another native OsNAR2.1 promoter with driving OsNAR2.1 gene into rice plants. The transgenic lines with exogenous pOsNAR2.1:OsNAR2.1 constructs showed enhanced OsNAR2.1 expression level, compared with wild type (WT), and 15 N influx in roots increased 21%-32% in response to 0.2 mm and 2.5 mm 15NO3- and 1.25 mm 15 NH415 NO3 . Under these three N conditions, the biomass of the pOsNAR2.1:OsNAR2.1 transgenic lines increased 143%, 129% and 51%, and total N content increased 161%, 242% and 69%, respectively, compared to WT. Furthermore in field experiments we found the grain yield, agricultural nitrogen use efficiency (ANUE), and dry matter transfer of pOsNAR2.1:OsNAR2.1 plants increased by about 21%, 22% and 21%, compared to WT. We also compared the phenotypes of pOsNAR2.1:OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines in the field, found that postanthesis N uptake differed significantly between them, and in comparison with the WT. Postanthesis N uptake (PANU) increased approximately 39% and 85%, in the pOsNAR2.1:OsNAR2.1 and pOsNAR2.1:OsNRT2.1 transgenic lines, respectively, possibly because OsNRT2.1 expression was less in the pOsNAR2.1:OsNAR2.1 lines than in the pOsNAR2.1:OsNRT2.1 lines during the late growth stage. These results show that rice NO3- uptake, yield and NUE were improved by increased OsNAR2.1 expression via its native promoter.
Project description:affy_nitrogen_medicago - affy_nitrogen_medicago - Experiment has been designed to characterize the molecular expression patterns associated to a contrasted modification of the nitrogen status of the whole plant. The systemic effects of nitrogen status modifications are investigated and compared on non nodulated plant supplied with NO3, NH4 or nodulated plants (Sinorhizobium meliloti 2011) supplied with air. The root systems were separated in two compartments of unequal sizes (split root system). Two treatments were applied on the larger compartment in order to modulate the nitrogen status of the plant: for the S treatment, roots are supplied with nutrient solution containing 10 mM NH4NO3,, whereas for the C treatment, roots are supplied with nitrogen free medium. In the case of N2 fixing plants, N limitation was obtained by replacing air by a mixture of Ar and O2 80 per cent and 20 per cent. The effects of these treatments were investigated on roots of the minor compartment supplied continuously with either NO3 1 mM, NH4 1 mM or air (N2) and on the shoots. We were also interested in the molecular expression patterns associated to the roots deprived of N.-The root system of non-nodulated (NO3- and NH4+) or nodulated (N2) plants is split into two unequal parts and each one is installed in a separate compartment. For the S treatement, the major root part is supplied with NH4NO3 10 mM whereas the minor part is supplied with either NO3- 1mM, NH4+ 1mM or N2. For the C treatement, the major root part is supplied with nitrogen-free nutrient solution whereas the minor part is supplied with either NO3- 1mM, NH4plus 1mM or N2. Each treatement is four days long. Samples of roots of six biological types (NO3S, NO3C, NH4S, NH4C, N2S and N2C) were collected. Two biological repeats per biological types have been analyzed. The effect of the S and C treatments were investigated for each N sources by comparing Affymetrix transcriptomes (NO3C vs NO3S, NH4C vs NH4S, N2C vs N2S). Keywords: treatement (nitrogen-sufficient) vs treatement (nitrogen-limited) Overall design: 26 arrays - medicago
Project description:As a vital beverage crop, tea has been extensively planted in tropical and subtropical regions. Nitrogen (N) levels and forms are closely related to tea quality. Based on different N levels and forms, we studied changes in NO3- and NH4+ fluxes in tea roots utilizing scanning ion-selective electrode technique. Our results showed that under both single and mixed N forms, influx rates of NO3- were much lower than those of NH4+, suggesting a preference for NH4+ in tea. With the increase in N concentration, the influx rate of NO3- increased more than that of NH4+. The NH4+ influx rates in a solution without NO3- were much higher than those in a solution with NO3-, while the NO3- influx rates in a solution without NH4+ were much lower than those in a solution with NH4+. We concluded that (1) tea roots showed a preference for NH4+, (2) presence of NO3- had a negative effect on NH4+ influx, and (3) NH4+ had a positive effect on NO3- influx. Our findings not only may help advance hydroponic tea experiments but also may be used to develop efficient fertilization protocols for soil-grown tea in the future.
Project description:Oryza sativa cv. Nipponbare was engineered to over-express a barley alanine aminotransferase (alaAT) gene using the promoter (OsANT1) from a rice aldehyde dehydrogenase gene that expresses in roots. We use biotechnology to improve the nitrogen use efficiency of rice by over-expressing alaAT in a tissue specific (root) manner. The AlaAT enzyme is a reversible aminotransferase that is linked to both C and N metabolism since it uses pyruvate plus glutamate to produce alanine and 2-oxoglutarate, and visa versa. Transcriptome data from the roots and shoots of rice plants at maximum tillering, grown hydroponically on either 0.5, 2 or 5 mM NH4+ as the nitrogen source. Wildtype rice (Nipponbare) and two independent OsANT1:HvAlaAT rice transgenic lines (AGR1/7, and AGR3/8) were grown hydroponically with either 0.5, 2 or 5mM NH4+ as the nitrogen source, to the reproductive stage. Tissue samples were taken at active and maximum tillering from root and shoot, at mid-day of the plants' day/night cycle. The RNA from root and shoot at maxiumum tillering was used for mcroarray analysis. Please read Beatty et al., 2009, PLant Biotechnology Journal 7, pp562-576 for detailed about these transgenic lines. The results from this variable N study were reported in a manuscript submitted to Botany, July 2013