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.     

Effects of Local Nitrogen Supply on Water Uptake of Bean Plants in a Split Root System

To study the effects of local nitrogen supply on water and nutrient absorption, French bean （Phaseolus vulgaris L.） plants were grown in a split root system. Five treatments supplied with different nitrogen forms were compared： homogeneous nitrate （NN） and homogenous ammonium （AA） supply, spatially separated supply of nitrate and ammonium （NA）, half of the root system supplied with N-free nutrient solution, the other half with either nitrate （NO） or ammonium （AO）. The results showed that 10 d after onset of treatments, root dry matter （DM） in the nitratesupplied vessels treated with NA was more than two times higher than that in the ammonium-supplied vessels. Water uptake from the nitrate-supplied vessels treated with NA was 281% higher than under ammonium supply. In treatments NO and AO, the local supply of N resulted in clearly higher root DM, and water uptake from the nitratesupplied vessels was 82% higher than in the -N vessels. However, in AO plants, water uptake from the -N nutrient solution was 129% higher than from the ammonium-supplied vessels. This indicates a compensatory effect, which resulted in almost identical rates of total water uptake of treatments AA and AO, which had comparable shoot DM and leaf area. Ammonium supply reduced potassium and magnesium absorption. Water uptake was positively correlated with N, Mg and K uptake.     

A comparison of nitrate transport in four different rice(Oryza sativa L.) cultivars

As rice can use both nitrate (NO3-) and ammonium (NH4+), we have tested the hypothesis that the shift in the pattern of cultivars grown in Jiangsu Province reflects the ability of the plants to exploit NO3- as a nitrogen (N) source. Four rice cultivars were grown in solution culture for comparison of their growth on NO3- and NH4+ nitrogen sources. All four types of rice, Xian You 63 (XY63), Yang Dao 6 (YD), Nong Keng 57 (NK) and Si You 917 (SY917), grew well and produced similar amounts of shoot biomass with 1 mmol/L NH4+ as the only N source. However, the roots of NK were significantly smaller in comparison with the other cultivars. When supplied with 1 mmol/L NO3- YD produced the greatest biomass; while NK achieved the lowest growth among the four cultivars. Electrophysiological measurements on root rhizodermal cells showed that the NO3- elicited changes in membrane potential (△Em) of these four rice cultivars were significantly different when exposed to low external NO3- (<1 mmol/L); while they were very similar at high external NO3- (10 mmol/L). The root cell membrane potentials of YD and XY63 were more responsive to low external NO3- than those of NK and SY917. The△Em values for YD and XY63 rhizodermal cells were almost the same at both 0.1 mmol/L and 1 mmol/L NO3-; while for the NK and SY917 the values became larger as the external NO3- increased. For YD cultivar,△Em was measured over a range of NO3- concentrations and a Michaelis-Menten fit to the data gave a Km value of 0.17 mmol/L. Net NO3- uptake depletion kinetics were also compared and for some cultivars (YD and XY63) a single-phase uptake system with first order kinetics best fitted the data; while other cultivars (ND and SY917) showed a better fit to two uptake systems. These uptake systems had two affinity ranges: one had a similar Km in all the cultivars (0.2 mmol/L); the other much higher affinity system (0.03 mmol/L) was only present in NK and SY917. The expression pattern of twelve different NO3- transporter genes was tested using specific primers, but only OsNRT1.1 and OsNRT2.1 expression could be detected showing significant differences between the four rice cultivars. The results from both the physiological and molecular experiments do provide some support for the hypothesis that the more popular rice cultivars grown in Jiangsu Province may be better at using NO3- as an N source.     

Growth,Nitrogen Uptake and Flow in Maize Plants Affected by Root Growth Restriction

The objective of the present study was to investigate the influence of a reduced maize root-system size on root growth and nitrogen （N） uptake and flow within plants. Restriction of shoot-borne root growth caused a strong decrease in the absorption of root ： shoot dry weight ratio and a reduction in shoot growth. On the other hand, compensatory growth and an increased N uptake rate in the remaining roots were observed. Despite the limited long-distance transport pathway in the mesocotyl with restriction of shoot-borne root growth, N cycling within these plants was higher than those in control plants, implying that xylem and phloem flow velocities via the mesocotyl were considerably higher than in plants with an intact root system. The removal of the seminal roots in addition to restricting shoot-borne root development did not affect whole plant growth and N uptake, except for the stronger compensatory growth of the primary roots. Our results suggest that an adequate N supply to maize plant is maintained by compensatory growth of the remaining roots, increased N uptake rate and flow velocities within the xylem and phloem via the mesocotyl, and reduction in the shoot growth rate.     

Root Cluster Formation and Citrate Exudation of White Lupin （Lupinus albus L.） as Related to Phosphorus Availability

A split-root system was used to investigate whether the external or internal P concentration controls root cluster formation and citrate exudation in white lupin (Lupinus albus L.) grown under controlled conditions. In spite of low P concentrations in the shoots and roots of the -P plant, its dry weight was not reduced compared with the P plant. Supplying external P (0.25 mmol/L) to one root half resulted in an increase in P concentration not only in the shoot, but also in the P-deprived root half, indicating P cycling within the plants. Omitting P from both split-root pots stimulated root cluster formation in both root halves,whereas P supply to one root half stimulated root cluster formation at the beginning of the treatment. Neither P supply to just one root half continuously nor resupply of P to one root half after 19 d of P starvation inhibited root cluster formation on the P-deprived side, although the concentration of P in this root half and shoot increased markedly. The results indicate that root cluster formation in L. albus is controlled by both shoot and root P concentrations. The rates of citrate exudation by both root halves with P deficiency were higher than those of the one root half supplied with P only. In the treatment with one root half supplied with P, the rates of citrate exudation by either the P-supplied or -deprived root halves were almost the same,regardless of P concentration in the roots. The results suggest that internal P concentration controls root cluster formation and citrate exudation in white lupin, but these processes may be regulated by different mechanisms.     

Growth and Major Nutrient Concentrations in Brassica campestris Supplied with Different NH4^＋/NO3 Ratios

In the present study, we investigated whether growth and main nutrient ion concentrations of cabbage （Brassica campestris L.） could be increased when plants were subjected to different NH4^＋/NO3- ratios. Cabbage seedlings were grown in a greenhouse in nutrient solutions with five NH4^＋/NO3- ratios （1：0; 0.75：0.25; 0.5：0.5; 0.25：0.75; and 0：1）. The results showed that cabbage growth was reduced by 87% when the proportion of NH4^＋-N in the nutrient solution was more than 75% compared with a ratio NH4^＋/NO3- of 0.5：0.5 35 d after transplanting, suggesting a possible toxicity due to the accumulation of a large amount of free ammonia in the leaves. When the NH4＋/NO3- ratio was 0.5：0.5, fresh seedling weight, root length, and H2PO4- （P）, K^＋, Ca^2＋, and Mg^2＋ concentrations were all higher than those in plants grown under other NH4^＋/NO3- ratios. The nitrate concentration in the leaves was the lowest in plants grown at 0.5： 0.5 NH4^＋/NO3-. The present results indicate that an appropriate NH4^＋/NO3- ratio improves the absorption of other nutrients and maintains a suitable proportion of N assimilation and storage that should benefit plant growth and the quality of cabbage as a vegetable.     

Changes in Growth and Nitrogen Assimilation in Maize Plants Induced by NaCl and Growth Regulators

Experiments were conducted to determine the interactive effects of salinity and certain growth regulators on growth and nitrogen assimilation in maize (Zea mays L. cv. GS-2). 100 mM NaCl inhibited the biomass accumulation, chlorophyll and carotenoid contents in leaves, nitrate content and uptake and nitrate reductase activity. The application of kinetin, ascorbic acid and 10 and 50 μM abscisic acid in the first experiment and 50 and 100 μM abscisic acid in the second experiment induced a substantial increase in the above parameters, the effect was highest with abscisic acid in salinized as well as non-salinized plants. This revised version was published online in July 2006 with corrections to the Cover Date.     

Cultivar variation in the effect of chlorsulfuron in depressing the uptake of copper in wheat

The application of herbicides has induced symptoms of nutrient deficiencies under some circumstances. This glasshouse study examined the effect of chlorsulfuron on the uptake and utilization of copper (Cu) in four cultivars of wheat plants (Triticum aestivum L. cvs. Kulin, Cranbrook, Gamenya and Bodallin) on a Cu-responsive soil. Application of chlorsulfuron depressed the concentration of Cu in wheat plants receiving either inadequate or adequate Cu. In plants with inadequate Cu supply, chlorsulfuron increased the severity of Cu deficiency. Shoot weight was markedly decreased by chlorsulfuron at all levels of Cu, through decreasing the number of tillers and the elongation of leaves. This decreased growth of shoots occurred prior to the effect on Cu concentration in tissues. The retranslocation of Cu in old tissues over time was unaffected by chlorsulfuron. In all wheat cultivars, the decreased growth of shoots were correlated with the concentration of Cu in the youngest fully emerged leaf blade with critical levels of 1.6−1.7 at day 25 and 0.9−1.0 μg g⁻¹ d. wt. at day 60. The application of chlorsulfuron tended to increase the critical level at day 25 but not at day 60. In addition, Kulin seems to be most, and Cranbrook least, sensitive to chlorsulfuron. This sensitivity was associated with the sensitivity of the cultivars to Cu deficiency. It is suggested that chlorsulfuron application induces Cu deficiency in wheat plants mainly due to effects on the uptake of Cu. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effects of Phosphorus Nutrient on the Hydraulic Conductivity of Sorghum （Sorghum vulgare Pers.） Seedling Roots Under Water Deficiency

Hydroponic experiments were conducted in a growth chamber and changes in the hydraulic conductivity of sorghum (Sorghum vulgare Pers.) roots (Lpr) at the three-leaf stage were measured using the pressure chamber method. Water deficiency was imposed with polyethylene glycol (PEG) 6000 and the phosphorus (P) levels were controlled by complete Hoagland solution with and without P nutrient. The objective of this study was to investigate the effect of P nutrition on root Lpr under water deficiency. The results showed that the Lpr in P deficiency treatments decreased markedly, but the Lpr recovered to the same value as that of control when sufficient P was supplied for 4-24 h. Water deficiency decreased Lpr, but the hydraulic conductivity of the roots with sufficient P supply was still higher than that of plants without P supply. When resuming water supply, the Lpr of the water-deficient plants under P supply recovered faster than that of plants without P supply, which indicates that plants with sufficient P nutrient are more drought tolerant and have a greater ability to recover after drought. The treatment of HgCl2 indicated that P nutrient could regulate the Lpr by affecting the activity and the expression levels of aquaporins.     

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.     

A comparison of nitrate transport in four different rice(Oryza sativa L.) cultivars

As rice can use both nitrate (NO3-) and ammonium (NH4+), we have tested the hypothesis that the shift in the pattern of cultivars grown in Jiangsu Province reflects the ability of the plants to exploit NO3- as a nitrogen (N) source. Four rice cultivars were grown in solution culture for comparison of their growth on NO3- and NH4+ nitrogen sources. All four types of rice,Xian You 63 (XY63), Yang Dao 6 (YD), Nong Keng 57 (NK) and Si You 917 (SY917), grew well and produced similar amounts of shoot biomass with 1 mmol/L NH4+ as the only N source.However, the roots of NK were significantly smaller in comparison with the other cultivars. When supplied with 1 mmol/L NO3-, YD produced the greatest biomass; while NK achieved the lowest growth among the four cultivars. Electrophysiological measurements on root rhizodermal cells showed that the NO3--elicited changes in membrane potential (ΔEm) of these four rice cultivars were significantly different when exposed to low external NO3- (＜1 mmol/L); while they were very similar at high external NO3- (10 mmol/L). The root cell membrane potentials of YD and XY63 were more responsive to low external NO3- than those of NK and SY917. The ΔEm values for YD and XY63 rhizodermal cells were almost the same at both 0.1 mmol/L and 1 mmol/L NO3-;while for the NK and SY917 the values became larger as the external NO3- increased. For YD cultivar, ΔEm was measured over a range of NO3- concentrations and a Michaelis-Menten fit to the data gave a Km value of 0.17 mmol/L. Net NO3- uptake depletion kinetics were also compared and for some cultivars (YD and XY63) a single-phase uptake system with first order kinetics best fitted the data; while other cultivars (ND and SY917) showed a better fit to two uptake systems. These uptake systems had two affinity ranges: one had a similar Km in all the cultivars (0.2 mmol/L); the other much higher affinity system (0.03 mmol/L) was only present in NK and SY917. The expression pattern of twelve different NO3- transporter genes was tested using specific primers, but only OsNRT1.1 and OsNRT2. 1 expression could be detected showing significant differences between the four rice cultivars. The results from both the physiological and molecular experiments do provide some support for the hypothesis that the more popular rice cultivars grown in Jiangsu Province may be better at using NO3- as an N source.     

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     

Ammonium and nitrate uptake by the floating plant Landoltia punctata

BACKGROUND AND AIMS: Plants from the family Lemnaceae are widely used in ecological engineering projects to purify wastewater and eutrophic water bodies. However, the biology of nutrient uptake mechanisms in plants of this family is still poorly understood. There is controversy over whether Lemnaceae roots are involved in nutrient uptake. No information is available on nitrogen (N) preferences and capacity of Landoltia punctata (dotted duckweed), one of the best prospective species in Lemnaceae for phytomelioration and biomass production. The aim of this study was to assess L. punctata plants for their ability to take up NH4+ and NO3- by both roots and fronds. METHODS: NO3- and NH4+ fluxes were estimated by a non-invasive ion-selective microelectrode technique. This technique allows direct measurements of ion fluxes across the root or frond surface of an intact plant. KEY RESULTS: Landoltia punctata plants took up NH4+ and NO3- by both fronds and roots. Spatial distribution of NH4+ and NO3- fluxes demonstrated that, although ion fluxes at the most distal parts of the root were uneven, the mature part of the root was involved in N uptake. Despite the absolute flux values for NH4+ and NO3- being lower in roots than at the frond surface, the overall capacity of roots to take up ions was similar to that of fronds because the surface area of roots was larger. L. punctata plants preferred to take up NH4+ over NO3- when both N sources were available. CONCLUSIONS: Landoltia punctata plants take up nitrogen by both roots and fronds. When both sources of N are available, plants prefer to take up NH4+, but will take up NO3- when it is the only N 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.     

The effect of nitrogen nutrition on cluster root formation and proton extrusion by Lupinus albus

Nitrogen nutrition can influence cluster root formation in many wild species, but the effect of N form on cluster root formation and root exudation by white lupin is not known. In a solution culture study, we examined the effect of N nutrition (ammonium, nitrate, both or N2 fixation) on cluster root formation and H+ extrusion by white lupin plants under deficient and adequate P supply. The number of cluster roots increased greatly when plants were supplied with I microM P compared with 50 microM P, the increase being 7.8-fold for plants treated with (NH4)2SO4, 3-fold for plants treated with KNO3 and NH4NO3, and 2-4-fold for N2-fixing plants. Under P deficiency. NH4+-N supply resulted in production of a greater number and biomass of cluster roots than other N sources. Dry weight of cluster roots was 30 % higher than that of non-cluster roots in P-deficient plants treated with (NH4)2SO4 and NH4NO3. In plants treated with sufficient P (50 microM), the weight of non-cluster roots was approx. 90 % greater than that of cluster roots. Both total (micromol per plant h(-1)) and specific (micromol g(-1) root d. wt h(-1)) H+ extrusions were greatest from roots of plants supplied with (NH4)2SO4, followed by those supplied with NH4NO3 and N2 fixation, whereas plants receiving KNO3 had negative net H+ extrusion between the third and fifth week of growth (indicating uptake of protons or release of OH- ions). The rate of proton extrusion by NH4+-N-fed plants was similar under P-deficient and P-sufficient conditions. In contrast, proton exudation by N2-fixing plants and KNO3-treated plants was ten-fold greater under P deficiency than under P sufficiency. In comparison with P deficiency, plants treated with 50 microM P had a significantly higher concentration of P in roots, shoots and youngest expanded leaves (YEL). Compared with the N2 fixation and KNO3 treatments, total N concentration was highest in roots, shoots and YEL of plants supplied with (NH4)2SO4 and NH4NO3, regardless of P supply. Under P deficiency, K concentrations in roots decreased at all N supplies, especially in plants treated with (NH4)2SO4 and NH4NO3, which coincided with the greatest H+ extrusion at these P and N supplies. In conclusion, NH4-N nutrition stimulated cluster root formation and H+ extrusion by roots of P-deficient white lupin.     

冬小麦等4种作物对铵,硝态氮的吸收能力

采用水培试验探讨了冬小麦、大豆、油菜和莴笋４种作物对硝、铵态氮的相对吸收能力以及这两种氮源对它们生长发育的影响。试验表明：（１）不同氮源对供试作物的生长发育影响极大。供给硝态氮,这些作物生长发育良好,供给等量的ＮＯ＾－３和ＮＨ＾－４（１：１）时,蔬菜作物莴笋生长量下降幅度最大;供给铵态氨,莴笋和大豆极为敏感,供给ＮＯ＾－３时莴笋吸氮量显著高于供给等氮量ＮＯ＾－３和ＮＨ＾＋４,莴上麦供给等量ＮＯ＾－