[Show abstract][Hide abstract] ABSTRACT: 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.
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[Show abstract][Hide abstract] ABSTRACT: Nitrate is an essential nitrogen source and serves as a signal to control growth and gene expression in plants. In rice, OsNAR2.1 is an essential partner of multiple OsNRT2 nitrate transporters for nitrate uptake over low and high concentration range. Previously, we have reported that -311 bp upstream fragment from the translational start site in the promoter of OsNAR2.1 gene is the nitrate responsive region. To identify the cis-acting DNA elements necessary for nitrate induced gene expression, we detected the expression of beta-glucuronidase (GUS) reporter in the transgenic rice driven by the OsNAR2.1 promoter with different lengths and site mutations of the 311 bp region. We found that -129 to -1 bp region is necessary for the nitrate-induced full activation of OsNAR2.1. Besides, the site mutations showed that the 20 bp fragment between -191 and -172 bp contains an enhancer binding site necessary to fully drive the OsNAR2.1 expression. Part of the 20 bp fragment is commonly presented in the sequences of different promoters of both the nitrate induced NAR2 genes and nitrite reductase NIR1 genes from various higher plants. These findings thus reveal the presence of conserved cis-acting element for mediating nitrate responses in plants.
[Show abstract][Hide abstract] ABSTRACT: Potassium (K) deficiency in plants confines root growth and decreases root-to-shoot ratio, thus limiting root K acquisition in culture medium. A WUSCHEL-related homeobox (WOX) gene, WOX11, has been reported as an integrator of auxin and cytokinin signalling that regulates root cell proliferation. Here, we report that ectopic expression of WOX11 gene driven by the promoter of OsHAK16 encoding a low-K-enhanced K transporter led to an extensive root system and adventitious roots and more effective tiller numbers in rice. The WOX11-regulated root and shoot phenotypes in the OsHAK16p:WOX11 transgenic lines were supported by K-deficiency-enhanced expression of several RR genes encoding type-A cytokinin-responsive regulators, PIN genes encoding auxin transporters and Aux/IAA genes. In comparison with WT, the transgenic lines showed increases in root biomass, root activity and K concentrations in the whole plants, and higher soluble sugar concentrations in roots particularly under low K supply condition. The improvement of sugar partitioning to the roots by the expression of OsHAK16p:WOX11 was further indicated by increasing the expression of OsSUT1 and OsSUT4 genes in leaf blades and several OsMSTs genes in roots. Expression of OsHAK16p:WOX11 in the rice grown in moderate K-deficient soil increased total K uptake by 72% and grain yield by 24%–32%. The results suggest that enlarging root growth and development by the expression of WOX11 in roots could provide a useful option for increasing K acquisition efficiency and cereal crop productivity in low K soil.
[Show abstract][Hide abstract] ABSTRACT: Plant proteins belonging to the NPF (formerly NRT1/PTR) family are well represented in every genome and function in transporting
a wide variety of substrates. In this study, we showed that rice OsNPF2.4 is located in the plasma membrane and is expressed
mainly in the epidermis, xylem parenchyma, and phloem companion cells. Functional analysis in oocytes showed that OsNPF2.4
is a pH-dependent, low-affinity N O 3 – NO3– transporter. Short-term 15 N O 3 – NO3– influx rate, long-term N O 3 – NO3– acquisition by root, and upward transfer from root to shoot were decreased by disruption of OsNPF2.4 and increased by OsNPF2.4 overexpression under high N O 3 – NO3– supply. Moreover, the redistribution of N O 3 – NO3– in the mutants in comparison with the wild type from the oldest leaf to other organs, particularly to N-starved roots, was
dramatically changed. Knockout of OsNPF2.4 decreased rice growth and potassium (K) concentration in xylem sap, root, culm, and sheath, but increased the shoot:root
ratio of tissue K under higher N O 3 – NO3–. We conclude that OsNPF2.4 functions in acquisition and long-distance transport of N O 3 – NO3–, and that altering its expression has an indirect effect on K recycling between the root and shoot.
[Show abstract][Hide abstract] ABSTRACT: Rapid improvements in DNA synthesis technology are revolutionizing gene cloning and the characterization of their encoded proteins. Xenopus laevis oocytes are a commonly used heterologous system for the expression and functional characterization of membrane proteins. For many plant proteins, particularly transporters, low levels of expression can limit functional activity in these cells making it difficult to characterize the protein. Improvements in synthetic DNA technology now make it quick, easy and relatively cheap to optimize the codon usage of plant cDNAs for Xenopus. We have tested if this optimization process can improve the functional activity of a two-component plant nitrate transporter assayed in oocytes.
We used the generally available software (http://www.kazusa.or.jp/codon/; http://genomes.urv.es/OPTIMIZER/) to predict a DNA sequence for the plant gene that is better suited for Xenopus laevis. Rice OsNAR2.1 and OsNRT2.3a DNA optimized sequences were commercially synthesized for Xenopus expression. The template DNA was used to synthesize cRNA using a commercially available kit. Oocytes were injected with cRNA mixture of optimized and original OsNAR2.1 and OsNRT2.3a. Oocytes injected with cRNA obtained from using the optimized DNA template could accumulate significantly more NO3- than the original genes after 16 h incubation in 0.5 mM Na15NO3. Two-electrode voltage clamp analysis of the oocytes confirmed that the codon optimized template resulted in significantly larger currents when compared with the original rice cDNA.
The functional activity of a rice high affinity nitrate transporter in oocytes was improved by DNA codon optimization of the genes. This methodology offers the prospect for improved expression and better subsequent functional characterization of plant proteins in the Xenopus oocyte system.
[Show abstract][Hide abstract] ABSTRACT: Root nitrate uptake is well known to adjust to the plant's nitrogen demand for growth. Long distance transport and/or root storage pools are thought to provide negative feedback signals regulating root uptake. We have characterized a vascular specific nitrate transporter belonging to high affinity NRT2 family, OsNRT2.3a in rice (Oryza sativa, ssp. Japonica, cv. Nipponbare). Localization analyses using protoplast expression, in planta promoter-β-glucuronidase assay and in situ hybridization, showed that OsNRT2.3a was located in the plasma membrane and mainly expressed in xylem parenchyma cells of the stele of nitrate supplied roots. Knock-down expression of OsNRT2.3a by RNA interference had impaired xylem loading of nitrate and decreased plant growth at low (0.5 mM) nitrate supply. In comparison to WT, the RNAi lines contained both nitrate and total nitrogen significantly higher in the roots and lower in the shoots. The short-term 15N-NO3- influx (5 min) in entire roots and NO3- ﬂuxes in root surfaces showed that the knockdown of OsNRT2.3a in comparison to WT did not affect nitrate uptake by roots. The RNAi plants showed no significant changes in the expression of some root nitrate transporters (OsNRT2.3b, OsNRT2.4 and OsNAR2.1), but transcripts for nia1 (nitrate reductase) had increased and OsNRT2.1, OsNRT2.2 decreased when the plants were supplied with nitrate. Taken together, the data demonstrate that OsNRT2.3a plays a key role in long-distance nitrate transport from root to shoot at low nitrate supply level in rice.
[Show abstract][Hide abstract] ABSTRACT: Two component high affinity nitrate transport system, NAR2/NRT2, has been defined in several plant species. In Arabidopsis, AtNAR2.1 has a role in the targeting of AtNRT2.1 to the plasma membrane. The gene knock out mutant atnar2.1 lacks inducible high-affinity transport system (IHATS) activity, it also shows the same inhibition of lateral root (LR) initiation on the newly developed primary roots as the atnrt2.1 mutant in response to low nitrate supply. In rice, OsNAR2.1 interacts with OsNRT2.1, OsNRT2.2 and OsNRT2.3a to provide nitrate uptake over high and low concentration ranges. In rice roots OsNAR2.1 and its partner NRT2s show some expression differences in both tissue specificity and abundance. It can be predicted that NAR2 plays multiple roles in addition to being an IHATS component in plants.
[Show abstract][Hide abstract] ABSTRACT: The high affinity nitrate transport system (HATS) plays an important role in rice nitrogen acquisition because, even under flooded anaerobic cultivation when NH(4)(+) dominates, significant nitrification occurs on the root surface. In the rice genome, four NRT2 and two NAR2 genes encoding HATS components have been identified. One gene OsNRT2.3 was mRNA spliced into OsNRT2.3a and OsNRT2.3b and OsNAR2.1 interacts with OsNRT2.1/2.2 and OsNRT2.3a to provide nitrate uptake. Using promoter-GUS reporter plants and semi-quantitative RT-PCR analyses, it was observed that OsNAR2.1 was expressed mainly in the root epidermal cells, differently from the five OsNRT2 genes. OsNAR2.1, OsNRT2.1, OsNRT2.2, and OsNRT2.3a were up-regulated by nitrate and suppressed by NH(4)(+) and high root temperature (37 °C). Expression of all these genes was increased by light or external sugar supply. Root transcripts of OsNRT2.3b and OsNRT2.4 were much less abundant and not affected by temperature. Expression of OsNRT2.3b was insensitive to the form of N supply. Expression of OsNRT2.4 responded to changes in auxin supply unlike all the other NRT2 genes. A region from position -311 to -1, relative to the translation start site in the promoter region of OsNAR2.1, was found to contain the cis-element(s) necessary for the nitrate-, but not light- and sugar-dependent activation. However, it was difficult to define a conserved cis-element in the promoters of the nitrate-regulated OsNRT2/OsNAR2 genes. The results imply distinct physiological functions for each OsNRT2 transporter, and differential regulation pathways by N and C status.