Regulation of Nitrate Reductase during Early Seedling Growth (A Role for Asparagine and Glutamine)

Growth systems that either permit (wet system) or prevent (dry system) the hydrolysis of endosperm reserves in maize (Zea mays) seedlings were developed to study the effect of endosperm reserves on the acquisition of external nitrogen. Three-day-old seedlings treated with 5 mM KNO3 for 24 h had higher levels of nitrate reductase (NR) activity and protein in shoot and root tissues in the dry relative to the wet system. This suggests that the induction of NR is sensitive to products of hydrolysis of endosperm reserves. Asparagine (1 mM) or glutamine (1 mM), potential products of that hydrolysis, inhibited the induction of NADH-dependent root NR in the dry system by about 70%. The inhibition of the induction of NR activity in the wet system was only about 35%, suggesting that the enzyme in the wet system was already partially repressed at 3 d. At 5 d, when asparagine and glutamine levels in the plant tissue had decreased, the induction of root NR activity was inhibited to a similar extent in the two growth systems by amide additions. The shoot enzyme was less sensitive to amide additions, and 10 mM concentrations of either amide was required for a 65% inhibition.     

Amino Acid interactions in the regulation of nitrate reductase induction in cotton root tips

Glycine, asparagine, and glutamine inhibited the induction by nitrate of nitrate reductase activity in root tips of cotton (Gossypium hirsutum L.). This inhibition was partially or entirely prevented when the inhibitor was applied in combination with any of several other amino acids. Studies of ¹⁴C-labeled amino acid uptake showed that, in most cases, the apparent antagonism resulted simply from competition for uptake. However, certain antagonists did not curtail uptake. The most effective of these were leucine (against all three inhibitors), and isoleucine and valine (against asparagine or glutamine, but not glycine). These results show that interactions among amino acids in the regulation of nitrate reductase induction result from at least two mechanisms, one acting on uptake of inhibitory amino acids, and the other involving true antagonism.     

Diverse transporters for neutral amino acids in Ricinus communis L. seedlings

Uptake mechanisms for neutral amino acids were investigated by expression of mRNA isolated from seedlings of Ricinus communis L. in Xenopus laevis oocytes. After injection of mRNA from root, hypocotyl and cotyledon currents elicited by saccharose and neutral amino acids ranged from 0.3 nA up to 2 nA depending on the respective substrate and the source of mRNA. These currents were due to expression of low affinity uptake mechanisms and the KM values found for amino acid induced charge flow range from 1 to 2 mM. The abundance and/or the specificity of the expressed mechanisms differ in the various tissues. Currents of similar magnitude were recorded for alanine and glutamine with mRNA isolated from root, hypocotyl and cotyledons. Serine and proline induced currents after injection of mRNA from hypocotyl and roots, in case of α-aminoisobutyric acid (AIB) induced currents were generally small with mRNA from all tissues tested. In addition, differential sensitivity of glutamine and AIB uptake in the high affinity range was evident towards the amino acid analogue 2-chloro-aminophenoxybutyric acid which indicated an additional set of carriers operating in the micromolar concentration range. The results suggest that multiple transporters for neutral amino acids exist in various tissues of the plant differing in specificity of charge flow and in sensitivity towards the inhibitor 2-chloro-aminophenoxybutyric acid. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Rhizospheric NO affects N uptake and metabolism in Scots pine (Pinus sylvestris L.) seedlings depending on soil N availability and N source

We investigated the interaction of rhizospheric nitric oxide (NO) concentration (i.e. low, ambient or high) and soil nitrogen (N) availability (i.e. low or high) with organic and inorganic N uptake by fine roots of Pinus sylvestris L. seedlings by ¹⁵N feeding experiments under controlled conditions. N metabolites in fine roots were analysed to link N uptake to N nutrition. NO affected N uptake depending on N source and soil N availability. The suppression of nitrate uptake in the presence of ammonium and glutamine was overruled by high NO. The effects of NO on N uptake with increasing N availability showed different patterns: (1) increasing N uptake regardless of NO concentration (i.e. ammonium); (2) increasing N uptake only with high NO concentration (i.e. nitrate and arginine); and (3) decreasing N uptake (i.e. glutamine). At low N availability and high NO nitrate accumulated in the roots indicating insufficient substrates for nitrate reduction or its storage in root vacuoles. Individual amino acid concentrations were negatively affected with increasing NO (i.e. asparagine and glutamine with low N availability, serine and proline with high N availability). In conclusion, this study provides first evidence that NO affects N uptake and metabolism in a conifer.     

Regulation of sulfate uptake and expression of sulfate transporter genes in Brassica oleracea as affected by atmospheric H(2)S and pedospheric sulfate nutrition

Demand-driven signaling will contribute to regulation of sulfur acquisition and distribution within the plant. To investigate the regulatory mechanisms pedospheric sulfate and atmospheric H(2)S supply were manipulated in Brassica oleracea. Sulfate deprivation of B. oleracea seedlings induced a rapid increase of the sulfate uptake capacity by the roots, accompanied by an increased expression of genes encoding specific sulfate transporters in roots and other plant parts. More prolonged sulfate deprivation resulted in an altered shoot-root partitioning of biomass in favor of the root. B. oleracea was able to utilize atmospheric H(2)S as S-source; however, root proliferation and increased sulfate transporter expression occurred as in S-deficient plants. It was evident that in B. oleracea there was a poor shoot to root signaling for the regulation of sulfate uptake and expression of the sulfate transporters. cDNAs corresponding to 12 different sulfate transporter genes representing the complete gene family were isolated from Brassica napus and B. oleracea species. The sequence analysis classified the Brassica sulfate transporter genes into four different groups. The expression of the different sulfate transporters showed a complex pattern of tissue specificity and regulation by sulfur nutritional status. The sulfate transporter genes of Groups 1, 2, and 4 were induced or up-regulated under sulfate deprivation, although the expression of Group 3 sulfate transporters was not affected by the sulfate status. The significance of sulfate, thiols, and O-acetylserine as possible signal compounds in the regulation of the sulfate uptake and expression of the transporter genes is evaluated.     

Tissue and Cellular Distribution of Glutamine Synthetase in Roots of Pea (Pisum sativum) Seedlings

The effect of nitrate application on glutamine synthetase activity in roots of pea (Pisum sativum L.) seedlings (2 weeks old) was studied. Separation of organelles from root fragments by sucrose density-gradient centrifugation revealed that both nitrite reductase and glutamine synthetase activities increased in root plastids as a response to nitrate application and that no such response was induced by ammonium application. Glutamine synthetase activity was also found to increase in plastids with distance from apex in nitrate-treated plants, the highest specific activity being located in the fourth 1-centimeter segment. Separation by SDS-PAGE and characterization by Western blotting showed that cytosolic glutamine synthetase contains one subunit polypeptide (28 kilodaltons) and that plastid glutamine synthetase contains both the 38-kilodalton subunit and a heavier subunit. When nitrate was present in the nutrient solution, the heavier subunit increased in abundance in protein fractions obtained from purified root plastids.     

Ammonium and amino acids as regulators of nitrate reductase in corn roots

When amino acids or ammonia are added to plant systems, the effects on the development of nitrate-dependent nitrate reductase activity are variable. In addition, amino acids added singly or as casein hydrolysate may not support a normal growth. A physiologically correct mixture of amino acids, one similar in composition to amino acids released by the endosperm, has been shown to support normal growth and protein synthesis in corn (Zea mays) embryos. In this investigation, we have used the mixture of corn amino acids to determine whether amino acids have an effect on the appearance or disappearance of nitrate reductase activity. The results show that these amino acids partially inhibit the induction of nitrate reductase in corn roots. The effect is more pronounced in mature root than in root tip sections. When glutamine and asparagine are included along with the "corn amino acid mixture," the inhibition is more severe. Amino acids or amino acid analogues added singly to the induction medium have a similar effect: i.e. when the induction of nitrate reductase is inhibited in the root tips (lysine, canavanine, azaserine, azetidine-2-carboxylic acid, dl-4-azaleucine, asparagine, and glutamine), that inhibition is more severe in mature root sections. Arginine enhanced the recovery of nitrate reductase in root tips but inhibited it in mature root sections. The effect of the amino acids is apparently on some phase of the induction processes (i.e. the uptake or distribution of nitrate or a direct effect on the synthesis of the enzyme) and not on the turnover of the enzyme.     

Leaf developmental stage affects sulfate depletion and specific sulfate transporter expression during sulfur deprivation in Brassica napus L

BRASSICA NAPUS was grown under hydroponic conditions and responses to the removal of the external supply of sulfur (S) were analysed in roots and in leaves of different developmental age. The concentrations of sulfate and nitrate were greatest in the older leaves and least in younger leaves, whilst phosphate was greatest in roots and youngest leaves and least in old leaves. S-deprivation resulted in decreases in tissue sulfate concentrations at variable rates in the order: roots and young leaves > middle-aged leaves > oldest leaves. Phosphate concentrations were unaffected and nitrate concentrations were only depleted in the oldest leaves. Expression of representative members of the sulfate transporter gene family was assessed by Northern blotting in the respective tissues. Group 1 transporters (high affinity type) were induced in response to S-deprivation in all tissues except old leaves, where no expression was detected, and to the greatest extent in roots. Groups 2 and 5 (a BRASSICA Group 5 sulfate transporter is reported here, accession number: AJ311389) transporters showed either no or only a small induction by S-deprivation. Group 4 transporters (localised in the tonoplast membrane and thought to be involved in vacuolar sulfate efflux) were induced by S-deprivation with a complex pattern: 4;1 was expressed in root and mature leaves, was strongly induced by sulfur-deprivation in roots, and was also induced in the middle-aged leaves alone; 4;2 was only expressed under S-deprivation in parallel with the observed pattern of tissue sulfate concentrations. Expression patterns indicated that both differences in intracellular sulfate pools and localised aspects of the signal transduction pathway link tissue sulfate-status and sulfur-nutrition regulated gene expression.     

Differential regulation of nitrate reductase induction in roots and shoots of cotton plants

The induction of nitrate reductase activity in root tips of cotton (Gossypium hirsutum L.) was regulated by several amino acids and by ammonium. Glycine, glutamine, and asparagine strongly inhibited induction of activity by nitrate and also decreased growth of sterile-cultured roots on a nitrate medium. Methionine, serine, and alanine weakly inhibited induction, and 11 other amino acids had little or no effect. Ammonium also decreased induction in root tips, but was most effective only at pH 7 or higher. The optimum conditions for ammonium regulation of induction were identical to those for growth of sterile-cultured roots on ammonium as the sole nitrogen source. Aspartate and glutamate strongly stimulated induction, but several lines of evidence indicated that the mechanism of this response was different from that elicited by the other amino acids. The effects of amino acids on induction appeared to be independent of nitrate uptake.     

小麦NO3-转运蛋白基因TaNRT2.3的克隆和表达分析

用RT-PCR和RACE技术在NO3-诱导处理的小麦(Triticum aestivum L.)根中克隆到一个硝酸根转运蛋白基因的cDNA,命名为TaNRT2.3(GenBank登录号AY053452).序列分析表明,TaNRT2.3全长1 744 bp,其中含有1 521bp的ORF,编码507个氨基酸,具有12个跨膜区,属于MFS超基因家族中的NNP家族.TaNRT2.3与其他植物中已知的NRT2具有很高的同源性.Northern杂交表明:TaNRT2具有在根中表达的组织特异性,而在叶中未检测到.TaNRT2的表达受NO3-诱导,在含NH4 介质中不表达.NO3-在低浓度(5～200μmol/L)和高浓度(2.0 mmol/L)时均起作用.通过研究小麦在0.2 mmol/LNO3-条件下TaNRT2的表达水平及对NO3-的吸收效率,表明TaNRT2在小麦高效吸收NO3-方面起着重要的作用.分根实验表明植物中N循环本身可以作为吸收N的调节信号.     

Functional analysis of <Emphasis Type="Italic">OsPUT1</Emphasis>, a rice polyamine uptake transporter

Polyamines are nitrogenous compounds found in all eukaryotic and prokaryotic cells and absolutely essential for cell viability. In plants, they regulate several growth and developmental processes and the levels of polyamines are also correlated with the plant responses to various biotic and abiotic stresses. In plant cells, polyamines are synthesized in plastids and cytosol. This biosynthetic compartmentation indicates that the specific transporters are essential to transport polyamines between the cellular compartments. In the present study, a phylogenetic analysis was used to identify candidate polyamine transporters in rice. A full-length cDNA rice clone AK068055 was heterologously expressed in the Saccharomyces cerevisiae spermidine uptake mutant, agp2∆. Radiological uptake and competitive inhibition studies with putrescine indicated that rice gene encodes a protein that functioned as a spermidine-preferential transporter. In competition experiments with several amino acids at 25-fold higher levels than spermidine, only methionine, asparagine, and glutamine were effective in reducing uptake of spermidine to 60% of control rates. Based on those observations, this rice gene was named polyamine uptake transporter 1 (OsPUT1). Tissue-specific expression of OsPUT1 by semiquantitative RT-PCR showed that the gene was expressed in all tissues except seeds and roots. Transient expression assays in onion epidermal cells and rice protoplasts failed to localize to a cellular compartment. The characterization of the first plant polyamine transporter sets the stage for a systems approach that can be used to build a model to fully define how the biosynthesis, degradation, and transport of polyamines in plants mediate developmental and biotic responses.     

Inhibition of nitrate uptake by aluminium in maize

Experiments with two maize (Zea mays L.) hybrids were conducted to determine (a) if the inhibition of nitrate uptake by aluminium involved a restriction in the induction (synthesis/assemblage) of nitrate transporters, and (b) if the magnitude of the inhibition was affected by the concurrent presence of ambient ammonium. At pH 4.5, the rate of nitrate uptake from 240 μM NH₄NO₃ was maximally inhibited by 100 μM aluminium, but there was little measurable effect on the rate of ammonium uptake. Presence of ambient aluminium did not eliminate the characteristic induction pattern of nitrate uptake upon first exposure of nitrogen-depleted seedlings to that ion. Removal of ambient aluminium after six hours of induction resulted in recovery within 30 minutes to rates of nitrate uptake that were similar to those of plants induced in absence of aluminium. Addition of aluminium to plants that had been induced in absence of aluminium rapidly restricted the rate of nitrate uptake to the level of plants that had been induced in the presence of aluminium. The data are interpreted as indicating that aluminium inhibited the activity of nitrate transporters to a greater extent than the induction of those transporters. When aluminium was added at initiation of induction, the effect of ambient ammonium on development of the inhibition by aluminium differed between the two hybrids. The responses indicate a complex interaction between the aluminium and ammonium components of high acidity soils in their influence on nitrate uptake. ei]{gnA C}{fnBorstlap}