Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max)

A full-length cDNA, GmNRT2, encoding a putative high-affinity nitrate transporter was isolated from a Glycine max (L.) root cDNA library and sequenced. The deduced GmNRT2 protein is 530 amino acids in length and contains 12 putative membrane-spanning domains and a long, hydrophilic C-terminal domain. GmNRT2 is related to high-affinity nitrate transporters in the eukaryotes Chlamydomonas reinhardtii and Aspergillus nidulans, and to putative high-affinity nitrate transporters in barley and tobacco. Southern blot analysis indicated that GmNRT2 is part of a small, multigene family in soybean. Expression of GmNRT2 in roots was regulated by the type of nitrogen source provided to plants: GmNRT2 mRNA levels were barely detectable in ammonium-grown plants, higher in nitrogen-deprived plants, and highest in nitrate-grown plants. Induction of GmNRT2 mRNA levels in roots occurred within 1 h after exposure of plants to nitrate. Nitrate induction of GmNRT2 mRNA levels was accompanied by a fourfold increase in net nitrate uptake by soybean roots at 100 μM external nitrate. The molecular and physiological evidence indicates that GmNRT2 is probably a high-affinity nitrate transporter involved in nitrate uptake by soybean roots. Received: 22 November 1997 / Accepted: 26 January 1998     

Expression analysis of a high-affinity nitrate transporter isolated from Arabidopsis thaliana by differential display

We used the differential display technique on total RNAs from roots of Arabidopsis thaliana (L.) Heynh. plants which had or had not been induced for 2 h by nitrate. One isolated cDNA clone, designated Nrt2:1At, was found to code for a putative high-affinity nitrate transporter. Two genomic sequences homologous to Nrt2:1At were found to be localized on the same fragment of chromosome 1 in the Arabidopsis genome. Expression analyses of both low- and high-affinity nitrate transporter genes, respectively Nrt1:1At (previously named Chl1) and Nrt2:1At, were carried out on plants grown under different nitrogen regimes. In this paper, we show that both genes are induced by very low levels of nitrate (50 μM KNO₃). However, stronger induction was observed with Nrt2:1At than with Nrt1:1At. Moreover, these two genes, although both over-expressed in a nitrate-reductase-deficient mutant, were differently regulated when N-sufficient wild-type or mutant plants were transferred to an N-free medium. Indeed, the steady-state amounts of Nrt1:1At mRNA declined whereas the amount of Nrt2:1At mRNA increased, probably reflecting the de-repression of the high-affinity transport system during N-starvation. Received: 4 May 1998 / Accepted: 26 August 1998     

Characterization of a silicon transporter gene family in Cylindrotheca fusiformis : sequences, expression analysis, and identification of homologs in other diatoms

The transport of silicon is an integral part of the synthesis of the silicified cell wall of diatoms, yet knowledge of the number, features, and regulation of silicon transporters is lacking. We report the isolation and sequence determination of five silicon transporter (SIT) genes from Cylindrotheca fusiformis, and examine their expression patterns during cell wall synthesis. The encoded SIT amino acid sequences are highly conserved in their putative transmembrane domains. Nine conserved cysteines in this domain may account for the sensitivity of silicon uptake to sulfhydryl blocking agents. A less conserved C-terminal domain is predicted to form coiled-coil structures, suggesting that the SITs interact with other proteins. We show that SIT gene expression is induced just prior to, and during, cell wall synthesis. The genes are expressed at very different levels, and SIT1 is expressed in a different pattern from SIT 2–5. Hybridization experiments show that multiple SIT gene copies are present in all diatom species tested. From the data we infer that individual transporters play specific roles in silicon uptake, and propose that the cell regulates uptake by controlling the amount or location of each. The identification of all SIT genes in C. fusiformis will enhance our understanding of the mechanism and control of silicon transport in diatoms. Received: 17 June 1998 / Accepted: 22 September 1998     

ACCLIMATION OF NITRATE UPTAKE BY PHYTOPLANKTON TO HIGH SUBSTRATE LEVELS1

A literature review of data on nitrate uptake by phytoplankton suggests that nitrate levels above 20 μmol N·L^?1 generally stimulated uptake rates in cultured unicellular algae and natural phytoplankton communities. This phenomenon indicates that phytoplankton cells acclimate to elevated nitrate levels by increasing their uptake capacity in a range of concentrations previously considered to be saturating. Cyanobacteria and flagellates were found to present a considerable capacity for acclimation, with low (0.1–2 μmol N·L^?1) half‐saturation values (K_s) at low (5–20 μmol N·L^?1) substrate levels and high (1–80 μmol N·L^?1) K_s values at high (30–100 μmol N·L^?1) substrate levels. However, some diatom genera (Rhizosolenia, Skeletonema, Thalassiosira) also appeared to possess a low affinity nitrate uptake system (K_s between 18 and 120 μmol N·L^?1), which can help resolve the paradox of their presence in enriched seas. It follows that present models of nitrate uptake can severely underestimate the effects of high nitrate concentrations on phytoplankton dynamics and development. A more adequate approach would be to consider the possibility of multiphasic uptake involving several phase transitions as nitrate concentrations increased. Because it is a nonlinear phenomenon featuring strong thresholds, this effect appears to override that of other variables, such as irradiance, temperature, and cell size. Within the present context of eutrophication and for a range of concentrations that is becoming more and more ecologically relevant, equations are tentatively presented as a first approach to estimate K_s from ambient nitrate concentrations.     

Expression studies of Nrt2:1Np, a putative high‐affinity nitrate transporter: evidence for its role in nitrate uptake

Expression of the gene Nrt2Np, which encodes a putative high-affinity nitrate transporter of Nicotiana plumbaginifolia was studied under variable physiological conditions. Nrt2Np is rapidly induced by very low nitrate concentrations and repressed by reduced nitrogen metabolites. Furthermore, Nrt2Np is expressed in coordination with other genes involved in nitrate assimilation (Nia, Nii). A deficiency in nitrate reductase activity, which is accompanied by high internal nitrate concentration and low levels of nitrogen metabolites, e.g. glutamine, leads to an overexpression of Nrt2Np, showing that high nitrate concentration per se does not repress Nrt2Np expression. By investigating plants with altered nitrate uptake properties, we showed a correlation between Nrt2 mRNA accumulation and 15N nitrate influx rates, providing the first evidence that the expression of Nrt2 correlates with the rate of nitrate uptake. In situ hybridization revealed a tissue-specific expression pattern. Nrt2Np mRNA accumulation is localized throughout all layers of the root tip, being highest in epidermal and endodermal cells. However, in mature root tissue, Nrt2 expression was detected mainly in the lateral root primordia and in the epidermis.     

CHANGES IN CELL COMPOSITION AND LIPID METABOLISM MEDIATED BY SODIUM AND NITROGEN AVAILABILITY IN THE MARINE DIATOM PHAEODACTYLUM TRICORNUTUM (BACILLARIOPHYCEAE)1

The effects of nitrogen starvation in the presence or absence of sodium in the culture medium were monitored in batch cultures of the marine diatom Phaeodactylum tricornutum Bohlin. During nitrogen starvation in the presence of sodium, cell nitrogen and chlorophyll a decreased, mainly as a consequence of continued cell division. These decreases were accompanied by decreases in the rates of photosynthesis and respiration. There was no change in either cell volume or carbohydrate, but both carbon and lipid increased. During nitrogen starvation in the absence of sodium, cell division ceased. Cell nitrogen and chlorophyll a remained constant, and respiration did not decrease, but the changes in the photosynthetic rate and the lipid content per cell were similar to cultures that were nitrogen-starved in the presence of sodium. The carbon-to-nitrogen ratio increased in both cultures. Nitrogen, in the form of nitrate, and sodium were resupplied to cultures that had been preconditioned in nitrogen- and sodium-deficient medium for 5 d. Control cultures to which neither nitrate or sodium were added remained in a static state with respect to cell number, volume, and carbohydrate but showed slight increases in lipid. Cells in cultures to which 10 mM nitrate alone was added showed a similar response to cultures where no additions were made. Cells in cultures to which 50 mM sodium alone was added divided for 2 d, with concomitant small decreases in all measured constituents. Cell division resumed in cultures to which both sodium and nitrate were added. The lipid content fell dramatically in these cells and was correlated to metabolic oxidation via measured increases in the activity of the glyoxylate cycle enzyme, isocitrate lyase. We conclude that lipids are stored as a function of decreased growth rate and are metabolized to a small extent when cell division resumes. However, much higher rates of metabolism occur if cell division resumes in the presence of a nitrogen source.     

利用抑制差减杂交技术分离三角褐指藻在缺氮条件下上调表达的基因

为了阐明硅藻利用氮源的分子机制, 以三角褐指藻为材料, 利用抑制差减杂交技术, 分离鉴定了16个在缺氮诱导条件下上调表达的基因片段。其中, 与已知功能基因具有较高相似性的有7种, 都是跟氮源的吸收利用相关的。Northern blotting验证其中5个基因, 包括硝酸盐转运蛋白基因、铁氧化还原蛋白亚硝酸还原酶基因、铵盐转运蛋白基因、结合ATP盒的转运蛋白基因和嘌呤透过酶基因, 在缺氮诱导条件下转录水平有明显上调。     

Intron features of key functional genes mediating nitrogen metabolism in marine phytoplankton

Introns are widespread and variable in eukaryotic genomes. Although their histories and functions, or even whether all of them have any function, remain largely unknown, analysis of intron sequences and genomic contexts may shed light on the evolutionary history of genes and organisms. The number and frequency of introns vary widely in the small number of published genomes of protists and algae suggesting that the same is true of the vast diversity of protists and algae that remain uncultivated. The objective of this study were to investigate introns in sequences of functional genes of phytoplankton, both in published genomes and in sequences obtained from environmental clone libraries. We examined the introns of the genes involved in nitrogen uptake and assimilation pathways in the genome sequences of cultivated phytoplankton as well as in environmental clone libraries of nitrate reductases (NR), nitrite reductase (NiR), nitrate transporter (Nrt2) and ammonium transporter (AMT) genes constructed from pelagic phytoplankton communities in Monterey Bay (CA, USA) and Onslow Bay (NC, USA). Here we describe the most extensive set to date of intron sequences from uncultivated marine algae and report important differences for diatom vs. non-diatom sequences. The majority of the introns in NR, NiR, Nrt2 and AMT from cultured phytoplankton and environmental libraries showed canonical splice patterns. Introns found in diatom-like NR environmental libraries had lower GC content than the respective exons. The green algal-like NR and Nrt2 environmental sequences had introns and exons of much more similar GC content, and both higher than in diatoms. These patterns suggest a different evolutionary history and recent acquisition of diatom introns compared to other algae.     

Photorespiration in diatoms

Cylindrotheca fusiformis is shown to be able to convert glycolate to glycerate via tartronic semialdehyde as well as by the better known route involving transamination to glycine. Enzymes related to photorespiration were compared in light-dark synchronized cultures ofC. fusiformis kept in continuous light in a complete synthetic seawater medium or starved for nitrogen or silicon. Glycolate oxidation remained constant throughout the cell cycle and was unaflected by starvation. Transamination of glyoxylate was stimulated by light, inhibited during nitrogen starvation, and dramatically stimulated by reintroduction of nitrate to the medium. Glyoxylate carboligase was also stimulated by light and inhibited during nitrogen-starvation but only partially recovered activity after reintroduction of nitrate.Non Standard Abbreviations DCIP dichlorophenol indophenol - ETS electron transport system     

PHYSIOLOGICAL AND OPTICAL PROPERTIES OF RHIZOSOLENIA FORMOSA (BACILLARIOPHYCEAE) IN THE CONTEXT OF OPEN-OCEAN VERTICAL MIGRATION1

Cultures of Rhizosolenia formosa H. Peragallo were studied to assess whether or not physiological and optical characteristics of this large diatom were consistent with the ability to migrate vertically in the open ocean. Time-course experiments examined changes in chemical composition and buoyancy of R. formosa during nitrate (N)–replete growth, N starvation, and recovery. Cells could maintain unbalanced growth for at least 53 h after depletion of ambient nitrate. Increases in C:N and carbohydrate: protein ratios observed during N starvation reversed within 24 h of reintroduction of nitrate to culture medium. Buoyancy was related to nutrition: Upon N depletion, the percentage of positively buoyant cells decreased to 4% from 11% but reverted to 9% within 12 h of nitrate readdition. Cells took up nitrate in the dark. Nitrogen-specific uptake rates averaged 0.48 d^?1; these rates were higher than N-specific growth rates (0. 15 d^?1), indicating the potential for luxury consumption of nitrate, which can be stored for later use. Measurements of photosynthesis vs. irradiance, chlorophyll-specific absorption (a_ph*(λ)), and pigment composition showed that cells may be adapted for growth under a wide range of irradiances. Values of a_ph*(λ) were lower for N-depleted cells than for N-replete cells, and N-depleted cells had higher ratios of total carotenoids to chlorophyll a. Aggregation of chloroplasts was more pronounced in N-depleted cells. These are possibly photoprotective mechanisms that would be an advantage to N-depleted cells in surface waters. Compounds that absorb in the ultraviolet region were detected in N-replete cells but were absent in N-depleted cultures. Overall, these results have important implications for migrations of Rhizosolenia in nature. Cells may survive fairly long periods in N-depleted surface waters and will continue to take up carbon; then they can resume nitrate uptake and revert to positive buoyancy upon returning to deep, N-rich water. Uncoupled uptake of carbon and nitrogen during migrations of Rhizosolenia is a form of new production that may result in the net removal of carbon from oceanic surface waters.     

STIMULATION OF NITRATE NET UPTAKE AND REDUCTION BY RED AND BLUE LIGHT AND REVERSION BY FAR-RED LIGHT IN THE GREEN ALGA ULVA RIGIDA1

The steady-state levels of nitrate, nitrite, and ammonium were estimated in the green alga Ulva rigida C. Agardh in darkness after addition of 0.5 mM KNO₃ and irradiation with red (R) and blue (B) light pulses of different duration (5 and 30 min). The net uptake of nitrate was very rapid. Seventy-five percent of the nitrate added was consumed after 60 min in darkness. Although uptake was stable after R or B, efflux of nitrate occurred within 3 h in the dark control and when R or B were followed by far-red (FR) irradiation. The internal nitrate concentration after 3 h in darkness was similar after R and B light pulses; however, the intracellular ammonium was higher after R than after B. The intracellular nitrate and ammonium decreased when FR tight pulses were applied immediately after R or B. Thus, the involvement of phytochrome in the transport of nitrate and ammonium is proposed. Nitrate reductase activity, measured by the in situ method, was increased by both R and B light pulses. The effect was partially reversed by FR light. Nitrate reductase activity was higher after 5 min of R light than after 5 min of B. However, after 30-min light pulses, the relative increase in activity was reversed for R and B. We propose that phytochrome and a blue-light photoreceptor are involved in regulation of nitrogen metabolism. Nitrate uptake and reduction correlates with previously detected light-regulated accumulation of protein in Ulva rigida under the same experimental conditions.     

Characterization of a silicon transporter gene family in Cylindrotheca fusiformis: sequences, expression analysis, and identification of homologs in other diatoms

The transport of silicon is an integral part of the synthesis of the silicified cell wall of diatoms, yet knowledge of the number, features, and regulation of silicon transporters is lacking. We report the isolation and sequence determination of five silicon transporter (SIT) genes from Cylindrotheca fusiformis, and examine their expression patterns during cell wall synthesis. The encoded SIT amino acid sequences are highly conserved in their putative transmembrane domains. Nine conserved cysteines in this domain may account for the sensitivity of silicon uptake to sulfhydryl blocking agents. A less conserved C-terminal domain is predicted to form coiled-coil structures, suggesting that the SITs interact with other proteins. We show that SIT gene expression is induced just prior to, and during, cell wall synthesis. The genes are expressed at very different levels, and SIT1 is expressed in a different pattern from SIT 2–5. Hybridization experiments show that multiple SIT gene copies are present in all diatom species tested. From the data we infer that individual transporters play specific roles in silicon uptake, and propose that the cell regulates uptake by controlling the amount or location of each. The identification of all SIT genes in C. fusiformis will enhance our understanding of the mechanism and control of silicon transport in diatoms.     

INFLUENCE OF LOW LIGHT AND A LIGHT: DARK CYCLE ON NO3– UPTAKE,INTRACELLULAR NO3–, AND NITROGEN ISOTOPE FRACTIONATION BY MARINE PHYTOPLANKTON1

The nitrogen isotope enrichment factor (ɛ) of four species of marine phytoplankton grown in batch cultures was determined during growth in continuous saturating light, continuous low light, and a 12:12‐h light:dark cycle, with nitrate as a nitrogen source. The low growth rate that resulted from low irradiance caused an increased accumulation of the intracellular nitrate pool and/or a reduction in cell volume and was correlated to a species‐specific increase in the measured ɛ value, compared with the saturating light conditions. The largest response was in the diatom Thalassiosira weissflogii (Grun.) Fryxell et Hasle, which showed a nearly 3‐fold increase between high and low light conditions (6.2–15.2‰). The smallest response was in T. pseudonana (Hustedt) Hasle et Heimdal, which showed no change in the ɛ value of approximately 5‰ in both high and low light conditions. There was significant but smaller increases in the ɛ value for the diatom T. rotula Meunier (2.7–5.6‰) and the prymnesiophyte Emiliania huxleyi (Lohm.) Hay et Mohler (4.5–9.4‰) between high and low light levels. In the light:dark experiments, all three diatoms but not the prymnesiophyte exhibited an increase in ɛ. This increase was linked to the ability of diatoms to assimilate nitrate at night. The results of the these experiments suggest that the light regime influences the relative uptake, assimilation, and efflux rates of nitrate and results in differences in the expression of the isotope effect by the enzyme nitrate reductase. Therefore, variations in nitrate isotope fractionation in nature can be more accurately interpreted when the light regime and species composition are taken into consideration.     

Regulation of the High-Affinity Nitrate Transport System in Wheat Roots by Exogenous Abscisic Acid and Glutamine

Nitrate is a major nitrogen （N） source for most crops. Nitrate uptake by root cells is a key step of nitrogen metabolism and has been widely studied at the physiological and molecular levels. Understanding how nitrate uptake is regulated will help us engineer crops with improved nitrate uptake efficiency. The present study investigated the regulation of the high-affinity nitrate transport system （HATS） by exogenous abscisic acid （ABA） and glutamine （Gin） in wheat （Triticum aestivum L.） roots. Wheat seedlings grown in nutrient solution containing 2 mmol/L nitrate as the only nitrogen source for 2weeks were deprived of N for 4d and were then transferred to nutrient solution containing 50 μmol/L ABA, and 1 mmol/L Gin in the presence or absence of 2 mmol/L nitrate for 0, 0.5, 1, 2, 4, and 8 h. Treated wheat plants were then divided into two groups. One group of plants was used to investigate the mRNA levels of the HATS components NRT2 and NAR2 genes in roots through semi-quantitative RT-PCR approach, and the other set of plants were used to measure high-affinity nitrate influx rates in a nutrient solution containing 0.2 mmol/L ^15N-labeled nitrate. The results showed that exogenous ABA induced the expression of the TaNRT2.1, TaNRT2.2, TaNRT2.3, TaNAR2.1, and TaNAR2.2 genes in roots when nitrate was not present in the nutrient solution, but did not further enhance the induction of these genes by nitrate. Glutamine, which has been shown to inhibit the expression of NRT2 genes when nitrate is present in the growth media, did not inhibit this induction. When Gin was supplied to a nitrate-free nutrient solution, the expression of these five genes in roots was induced. These results imply that the inhibition by Gin of NRT2 expression occurs only when nitrate is present in the growth media. Although exogenous ABA and Gin induced HATS genes in the roots of wheat, they did not induce nitrate influx.