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     

2′‐Deoxymugineic acid promotes growth of rice (Oryza sativa L.) by orchestrating iron and nitrate uptake processes under high pH conditions

Poaceae plants release 2′‐deoxymugineic acid (DMA) and related phytosiderophores to chelate iron (Fe), which often exists as insoluble Fe(III) in the rhizosphere, especially under high pH conditions. Although the molecular mechanisms behind the biosynthesis and secretion of DMA have been studied extensively, little information is known about whether DMA has biological roles other than chelating Fe in vivo. Here, we demonstrate that hydroponic cultures of rice (Oryza sativa) seedlings show almost complete restoration in shoot height and soil‐plant analysis development (SPAD) values after treatment with 3–30 μm DMA at high pH (pH 8.0), compared with untreated control seedlings at normal pH (pH 5.8). These changes were accompanied by selective accumulation of Fe over other metals. While this enhanced growth was evident under high pH conditions, DMA application also enhanced seedling growth under normal pH conditions in which Fe was fairly accessible. Microarray and qRT‐PCR analyses revealed that exogenous DMA application attenuated the increased expression levels of various genes related to Fe transport and accumulation. Surprisingly, despite the preferential utilization of ammonium over nitrate as a nitrogen source by rice, DMA application also increased nitrate reductase activity and the expression of genes encoding high‐affinity nitrate transporters and nitrate reductases, all of which were otherwise considerably lower under high pH conditions. These data suggest that exogenous DMA not only plays an important role in facilitating the uptake of environmental Fe, but also orchestrates Fe and nitrate assimilation for optimal growth under high pH conditions.     

Cloning of a cDNA for a constitutive NRT1 transporter from soybean and comparison of gene expression of soybean NRT1 transporters

We have isolated a cDNA for a putative transporter, named GmNRT1-3, in the NRT1 family from soybean. It was predicted to have a similar topological structure not only to both GmNRT1-1 and GmNRT1-2 reported previously, but also to other members of the family. Two other cDNAs isolated have parts of the sequence for putative NRT1 transporters, GmNRT1-4 and GmNRT1-5, suggesting that at least five NRT1 transporters occur in soybean. These GmNRT1 genes and the GmNRT2 gene, encoding a soybean NRT2 nitrate transporter, showed different expression patterns to each other under various nitrogen conditions. Specifically, GmNRT1-3 was constitutively expressed in both roots and leaves, while GmNRT1-2 was gradually expressed as the roots developed in the presence of ammonium as a nitrogen source, but not in the presence of both ammonium and nitrate. Based on these results, we discussed the possible regulation in the expression and role of these transporters in nitrate uptake.     

A two-component high-affinity nitrate uptake system in barley

The analysis of genome databases for many different plants has identified a group of genes that are related to one part of a two-component nitrate transport system found in algae. Earlier work using mutants and heterologous expression has shown that a high-affinity nitrate transport system from the unicellular green algae, Chlamydomonas reinhardtii required two gene products for function. One gene encoded a typical carrier-type structure with 12 putative trans-membrane (TM) domains and the other gene, nar2 encoded a much smaller protein that had only one TM domain. As both gene families occur in plants we investigated whether this transport model has more general relevance among plants. The screening for nitrate transporter activity was greatly helped by a novel assay using (15)N-enriched nitrate uptake into Xenopus oocytes expressing the proteins. This assay enables many oocytes to be rapidly screened for nitrate transport activity. The functional activity of a barley nitrate transporter, HvNRT2.1, in oocytes required co-injection of a second mRNA. Although three very closely related nar2-like genes were cloned from barley, only one of these was able to give functional nitrate transport when co-injected into oocytes. The nitrate transport performed by this two-gene system was inhibited at more acidic external pH and by acidification of the cytoplasm. This specific requirement for two-gene products to give nitrate transport function has important implications for attempts to genetically manipulate this fundamental process in plants.     

A MODEL APPROACH FOR SIZE-SELECTIVE COMPETITION OF MARINE PHYTOPLANKTON FOR FLUCTUATING NITRATE AND AMMONIUM1

Phytoplankton size-selective competition for fluctuating nutrients was studied with the use of a numerical model, which describes nitrate and ammonium uptake, nitrate reduction to ammonium, and growth as a function of cell she under fluctuating nitrogen limitation. The only size-dependent parameter in the model was the cell nutrient quota. Related to this, the cell surface area per biomass was negatively correlated to cell volume, and the vacuole volume per biomass ratio was positively correlated to cell volume. Simulations showed an inverse correlation between the maximum specific growth rate and cell size under steady-state conditions. With nitrate as the limiting nitrogen source, nitrogen quotas were always higher than with ammonium at the same specific growth rate. Net passive transport of ammonium due to unspecific diffusion of ammonia across the plasma membrane decreased the affinity for ammonium, whereas the affinity for nitrate was not influenced. Transient state-specific ammonium uptake was not dependent on cell size. However, small algae always have the highest specific growth rate in ammonium-controlled systems according to our model. Transient state nitrate uptake rate was positively correlated to cell size because larger algae have a higher vacuole volume per biomass, in which nitrate can be stored. Despite their lower maximum growth rate, larger algae became dominant during simulations under fluctuating nitrate supply when amplitude of and the period between nitrate pulses were high enough. Results from model simulations were qualitatively validated by earlier observations that large diatoms become dominant under fluctuating conditions when nitrate is the main nitrogen source.     

Gene structure and expression of the high-affinity nitrate transport system in rice roots

Rice has a preference for uptake of ammonium over nitrate and can use ammonium-N efficiently. Consequently, transporters mediating ammonium uptake have been extensively studied, but nitrate transporters have been largely ignored. Recently,some reports have shown that rice also has high capacity to acquire nitrate from growth medium, so understanding the nitrate transport system in rice roots is very important for improving N use efficiency in rice. The present study identified four putative NRT2 and two putative NAR2 genes that encode components of the high-affinity nitrate transport system (HATS) in the rice (Oryza sativa L. subsp, japonica cv. Nipponbare) genome. OsNRT2.1 and OsNRT2.2 share an identical coding region sequence, and their deduced proteins are closely related to those from monocotyledonous plants. The two NAR2 proteins are closely related to those from mono-cotyledonous plants as well. However, OsNRT2.3 and OsNRT2.4 are more closely related to Arabidopsis NRT2 proteins. Relative quantitative reverse tranecdption-polymerase chain reaction analysis showed that all of the six genes were rapidly upregulated and then downregulated in the roots of N-starved rice plants after they were re-supplied with 0.2 mM nitrate, but the response to nitrate differed among gene members.The results from phylogenetic tree, gene structure and expression analysis implied the divergent roles for the individual members of the rice NRT2 and NAR2 families. High-affinity nitrate influx rates associated with nitrate induction in rice roots were investigated and were found to be regulated by external pH. Compared with the nitrate influx rates at pH 6.5, alkaline pH (pH 8.0) inhibited nitrate Influx, and acidic pH (pH 5.0) enhanced the nitrate influx In I h nitrate induced roots, but did not significantly affect that in 4 to 8 h nitrate induced roots.     

沼泽红假单胞菌(Rhodopseudomonas palustris)CQV97对无机三态氮共存水体中氮素的去除效率及其影响因素

研究水体环境因素(温度、光照和pH)、小分子有机碳和有机氮化合物对一株具有高效脱氮潜力的沼泽红假单胞菌(Rhodopseudomonas palustris)CQV97在无机三态氮共存体系中脱除无机三态氮的影响规律。结果显示,该菌株在20~40℃,500~5 000lux,pH 6.0~9.0环境条件下,能够脱除高浓度无机三态氮(其中亚硝氮不低于40mg·L-1),表明该菌株具有较强的适应复杂环境的能力;以乙酸钠/乙醇为唯一碳源时,该菌株能有效地去除无机三态氮,而以葡萄糖为唯一碳源时,能有效去除硝氮,但不能去除氨氮,亚硝氮明显积累,表明环境中小分子有机碳源影响菌体对无机三态氮的去除能力;体系中添加高浓度(120mg·L-1)蛋白胨或尿素时,由于有机氮降解的释氨作用,菌体对氨氮的去除能力受到明显抑制,氨氮积累明显,13d时氨氮去除率仅分别为16%(蛋白胨)和6%(尿素),但硝氮和亚硝氮的去除能力并没有受到明显影响。异位处理实际水体结果表明,菌株可使水体中氨氮含量明显降低、硝氮和亚硝氮被完全去除。综上,沼泽红假单胞菌CQV97菌株环境适应能力强,具有高效脱除水体无机三态氮的应用潜力,这为进一步开发高效脱氮微生物制剂及其合理使用奠定了基础。     

重庆市蔬菜硝酸盐、亚硝酸盐含量及其与环境的关系

在重庆市的主要蔬菜中,硝酸盐含量依次为根菜类＞叶菜类＞葱蒜类＞瓜类＞豆类＞茄果类,前３类的硝酸盐含量超标,但各种蔬菜的亚硝酸盐含量一般较低,未超过卫生标准。就同一种蔬菜的不同样品而言,硝酸盐和亚硝酸盐含量的差异很大,说明品种或环境条件可显著影响其含量。重庆市多雾少光,冬春低温,土壤含氮量和钠高子含量较高,有效钾含量较低可能是导致蔬菜大量积累硝酸盐的环境因素。蔬菜不同器官硝酸盐和亚硝酸盐含量各异,根＞茎＞叶柄＞叶片。     

The Chlamydomonas reinhardtii Nar1 gene encodes a chloroplast membrane protein involved in nitrite transport

A key step for nitrate assimilation in photosynthetic eukaryotes occurs within chloroplasts, where nitrite is reduced to ammonium, which is incorporated into carbon skeletons. The Nar1 gene from Chlamydomonas reinhardtii is clustered with five other genes for nitrate assimilation, all of them regulated by nitrate. Sequence analysis of genomic DNA and cDNA of Nar1 and comparative studies of strains having or lacking Nar1 have been performed. The deduced amino acid sequence indicates that Nar1 encodes a chloroplast membrane protein with substantial identity to putative formate and nitrite transporters in bacteria. Use of antibodies against NAR1 has corroborated its location in the plastidic membrane. Characterization of strains having or lacking this gene suggests that NAR1 is involved in nitrite transport in plastids, which is critical for cell survival under limiting nitrate conditions, and controls the amount of nitrate incorporated by the cells under limiting CO(2) conditions.     

Acclimation of Chlamydomonas reinhardtii to its nutrient environment

To cope with low nutrient availability in nature, organisms have evolved inducible systems that enable them to scavenge and efficiently utilize the limiting nutrient. Furthermore, organisms must have the capacity to adjust their rate of metabolism and make specific alterations in metabolic pathways that favor survival when the potential for cell growth and division is reduced. In this article I will focus on the acclimation of Chlamydomonas reinhardtii, a unicellular, eukaryotic green alga to conditions of nitrogen, sulfur and phosphorus deprivation. This organism has a distinguished history as a model for classical genetic analyses, but it has recently been developed for exploitation using an array of molecular and genomic tools. The application of these tools to the analyses of nutrient limitation responses (and other biological processes) is revealing mechanisms that enable Chlamydomonas to survive harsh environmental conditions and establishing relationships between the responses of this morphologically simple, photosynthetic eukaryote and those of both nonphotosynthetic organisms and vascular plants.     

Nitrate reduction and compartmentation in tomato leaves. II. Nitrate loss by leaf sections in a gaseous atmosphere and the size of the metabolic nitrate pool

Nitrate disappearance in tomato ( (ycopersicon esculentum Mill. cv. Azes) leaf sections kept under a stream of gas (nitrogen or air) has been studied, using leaf sections from plants supplied with low (7.5 mM) or high (17.5 mM) nitrate levels in their nutrient solution. Cessation of nitrate loss occurred in leaf sections taken from plants irrigated with low (7.5 mM) nitrate-containing nutrient solution. Resumption of nitrate disappearance occurred upon addition of exogenous nitrate by vacuum infiltration to leaf sections, suggesting that cessation of nitrate loss was due to exhaustion of the metabolic pool. We estimated that 53% of the total nitrate in leaf sections from low nitrate plants was located in a storage pool, probably the vacuole. The remainder was located in a pool, readily available for reduction (the metabolic pool). This pool is composed of nitrate in the free space as well as in the cytoplasm which was estimated to contain about 20% of the total nitrate.
Either under air or nitrogen, less nitrite was accumulated than nitrate assimilated suggesting that nitrite accumulation was not an adequate parameter for the estimation of nitrate utilization. Anaerobic conditions inhibited nitrite reduction whereas nitrate assimilation was not blocked. Nitrate loss from endogenous pool in leaf sections placed under aerobic conditions is suggested as an adequate method for the estimation of the metabolic pool of nitrate.     

Denitrifying ability of thirteen Rhizobium meliloti strains

The denitrifying ability of thirteen strains of Rhizobium meliloti was tested. Most of the strains were able to reduce nitrate to nitrous oxide or dinitrogen. However, they failed to use nitrate as electron acceptor for ATP generation or growth at low oxygen tensions. Under micro-aerobic conditions, free-living cells of R. meliloti 102-F-51 strain exhibited a constitutive nitrate reductase activity independent of the presence of nitrate. On the other hand, nitrite reductase activity was dependent not only on low levels of oxygen but also on the presence of a high nitrate concentration in the medium. Denitrification activity proceeded immediately once a threshold level of nitrite was accumulated in the medium or in cells incubated with 1mM nitrite. However, a lag period was required when cells were incubated with nitrate.     

Microbial oxidation of 1,2-dichloroethane under anoxic conditions with nitrate as electron acceptor in mixed and pure cultures

Many organisms have been found to readily oxidize the prevalent contaminant 1,2-dichloroethane (1,2-DCA) to CO2 under aerobic conditions. Some organisms have also been isolated that can reduce 1,2-DCA to ethene via dihaloelimination under anaerobic, fermentative conditions. However, none have been described that can metabolize 1,2-DCA under anoxic, nitrate-reducing conditions. In microcosms prepared from aquifer material and groundwater samples from a contaminated site in eastern Louisiana, USA, 1,2-DCA was observed to degrade with nitrate as the terminal electron acceptor. Nitrate-dependent enrichment cultures were developed from these microcosms that sustained rapid 1,2-DCA degradation rates of up to 500 microM day(-1). This degradation was tightly coupled to complete reduction of nitrate via nitrite to nitrogen gas. A novel 1,2-DCA-degrading organism belonging to the Betaproteobacteria (affiliated with the genus Thauera) was isolated from this enrichment culture. However, degradation rates were much slower in cultures of the isolate than observed in the parent mixed culture. Complete mineralization of 1,2-DCA to CO2 was linked to cell growth and to nitrate reduction in both enrichment and isolated cultures. Monochloroacetate, a putative metabolite of 1,2-DCA degradation, could also be mineralized by these cultures.