Improving Nitrogen Use Efficiency in Rice through Enhancing Root Nitrate Uptake Mediated by a Nitrate Transporter,NRT1.1B

<正>Nitrogen(N)is one of most important nutrients for crop production,which makes up 1%-5%of total plant dry matter(Marschner,2012).Due to the limited availability of N in soil,application of N fertilizers has been an important agronomic practice to increase crop yield.However,over-application of N fertilizers has caused pollution of N in soil,water and air.It     

The Arabidopsis Nitrate Transporter NRT1.8 Functions in Nitrate Removal from the Xylem Sap and Mediates Cadmium Tolerance

Long-distance transport of nitrate requires xylem loading and unloading, a successive process that determines nitrate distribution and subsequent assimilation efficiency. Here, we report the functional characterization of NRT1.8, a member of the nitrate transporter (NRT1) family in Arabidopsis thaliana. NRT1.8 is upregulated by nitrate. Histochemical analysis using promoter-β-glucuronidase fusions, as well as in situ hybridization, showed that NRT1.8 is expressed predominantly in xylem parenchyma cells within the vasculature. Transient expression of the NRT1.8:enhanced green fluorescent protein fusion in onion epidermal cells and Arabidopsis protoplasts indicated that NRT1.8 is plasma membrane localized. Electrophysiological and nitrate uptake analyses using Xenopus laevis oocytes showed that NRT1.8 mediates low-affinity nitrate uptake. Functional disruption of NRT1.8 significantly increased the nitrate concentration in xylem sap. These data together suggest that NRT1.8 functions to remove nitrate from xylem vessels. Interestingly, NRT1.8 was the only nitrate assimilatory pathway gene that was strongly upregulated by cadmium (Cd²⁺) stress in roots, and the nrt1.8-1 mutant showed a nitrate-dependent Cd²⁺-sensitive phenotype. Further analyses showed that Cd²⁺ stress increases the proportion of nitrate allocated to wild-type roots compared with the nrt1.8-1 mutant. These data suggest that NRT1.8-regulated nitrate distribution plays an important role in Cd²⁺ tolerance.     

Uptake of Nitrate by Mutants of Arabidopsis thaliana, Disturbed in Uptake or Reduction of Nitrate

A study of nitrate and chlorate uptake by Arabidopsis thaliana was made with a wildtype and two mutant types, both mutants having been selected by resistance to high chlorate concentrations. All plants were grown on a nutrient solution with nitrate and/or ammonium as the nitrogen source. Uptake was determined from depletion in the ambient solution. Nitrate and chlorate were able to induce their own uptake mechanisms. Plants grown on ammonium nitrate showed a higher subsequent uptake rate of nitrate and chlorate than plants grown on ammonium alone. Mutant B25, which has no nitrate reductase activity, showed higher rates of nitrate and chlorate uptake than the wildtype, when both types were grown on ammonium nitrate. Therefore, the uptake of nitrate is not dependent on the presence of nitrate reductase. Nitrate has a stimulating effect on nitrate and chlorate uptake, whereas some product of nitrate and ammonium assimilation inhibits uptake of both ions by negative feedback. Mutant B 1, which was supposed to have a low chlorate uptake rate, also has disturbed uptake characteristics for nitrate.     

Uptake of Nitrate by Mutants of Arabidopsis thaliana, Disturbed in Uptake or Reduction of Nitrate

The uptake of nitrate by wildtype plants and chlorate-resistant mutants of Arabidopsis thaliana (L.) Heynh. was studied by intermittent or continuous measurement of the nitrate concentration of the ambient solution. The uptake rate in the wildtype and the nitrate reductase-less mutant B 25 showed a dual-phase relation ship with concentration. Each phase showed Michaelis-Menten kinetics although multiphasic patterns within each phase could not be excluded. A dual-phase relationship was also found in the uptake mutant B I. Here, however, phase II did not follow Michaelis-Menten kinetics and uptake rate of nitrate in the phase II concentration range was considerably lower in the B 1 mutant than in the wildtype. It is concluded that the mutation in B I has disturbed phase II of the nitrate uptake, without affecting phase I, which leads to the suggestion that uptake of nitrate in Arabidopsis is mediated by at least two independent uptake mechanisms. The nitrate uptake rate showed an optimum at pH 8, and it was not stimulated by the presence of calcium. Ammonium had different effects on nitrate uptake: a direct effect, when it was present during the uptake of nitrate, resulting in a release of nitrate and a reduced rate of uptake, and an indirect inhibitory effect, possibly caused by assimilation products of ammonium, which is most pronounced after growth on ammonium as the sole nitrogen source or in long-lasting uptake experiments in the presence of ammonium. Chlorate also showed a multiple effect, an inhibiting one which proved to be competitive and, at very low concentrations of chlorate, a stimulating one. Evidence was obtained that chlorate and nitrate arc taken up by the same carrier.     

Trichlorfon-Induced Inhibition of Nitrate and Ammonium Uptake in Cyanobacteria

The possibility that the primary effect of the toxic insecticidetrichlorfon is an inhibition of nitrate uptake in cyanobactenahas been investigated. A drastic reduction in the rate of uptakeis detected 3 h after the addition of the insecticide to batchcultures of nabaena PCC 7119. The dose-response curves indicatea relationship between the degree of inhibition of nitrate uptakeand the reduction of chlorophyll content and growth. Nitratereductase (ferredoxin : nitrate reductase, EC 1.7.99.4 [EC] ) activityis also lowered as a result of insecticide action. When AnabaenaPCC 7119 cells are grown with ammonium as a source of combinednitrogen, trichlorfon reduces the rate of ammonium uptake. Therate of uptake of both nitrate and ammonium is restored uponwashing the cells. Ultrastructural analysis of Anabaena nitrate-growncells shows that trichlorfon does not damage thylakoid membranes,but brings about the accumulation of enlarged cyanophycin granulesand the increase of carboxysome number. Nitrate uptake rateand chlorophyll and phycobiliprotein contents are also reducedby insecticide treatment in the cyanobacteria SynechococcusUAM 211, GloeothecePCC 6501, Plectonema calothricoides, NostocUAM 205 and Chlorogloeopsis PCC 6912. These results are consistentwith the inhibition of nitrate uptake due to weak adsorptionof trichlorfon to the plasmalemma being the main effect of theinsecticide on cyanobacterial metabolism. Key words: Nitrate uptake, cyanobacteria, Anabaena, ammonium uptake, trichlorfon     

拟南芥K+转运蛋白AtKup1基因的DNA改组

采用同源重组法制备钾离子转运蛋白基因TRKI和TRK2缺失的酿酒酵母钾营养缺陷型。通过RNA反转录PCR方法从拟南芥幼根扩增获得片段长度为2 139bp的AtKup1基因,以此片段为膜板,采用DNA重排技术,经DNase I降解,Primerless PCR和Primer PCR建立AtKup1基因突变库。将突变库和未经DNA重排处理的AtKup1基因分别构建酵母穿梭载体,并导入K 转运蛋白基因TRK1和TRK2缺失的酿酒酵母中,分别在低钾(5．0mmoL／L KC1)不合色氨酸的培养基上筛选转化子,从突变基因库酵母转化子中获得2株长势明显优于AtKup1基因转化子的突变基因转化菌株,菌株质粒上的突变AtKup1基因核苷酸测序结果表明突变基因AtKup1发生了2个碱基的置换,造成了2个氨基酸的改变。转化烟草烟叶化学成分分析证实突变基因的吸钾活性显著提高。     

Characterization of the Arabidopsis Nitrate Transporter NRT1.6 Reveals a Role of Nitrate in Early Embryo Development

This study of the Arabidopsis thaliana nitrate transporter NRT1.6 indicated that nitrate is important for early embryo development. Functional analysis of cDNA-injected Xenopus laevis oocytes showed that NRT1.6 is a low-affinity nitrate transporter and does not transport dipeptides. RT-PCR, in situ hybridization, and β-glucuronidase reporter gene analysis showed that expression of NRT1.6 is only detectable in reproductive tissue (the vascular tissue of the silique and funiculus) and that expression increases immediately after pollination, suggesting that NRT1.6 is involved in delivering nitrate from maternal tissue to the developing embryo. In nrt1.6 mutants, the amount of nitrate accumulated in mature seeds was reduced and the seed abortion rate increased. In the mutants, abnormalities (i.e., excessive cell division and loss of turgidity), were found mainly in the suspensor cells at the one- or two-cell stages of embryo development. The phenotype of the nrt1.6 mutants revealed a novel role of nitrate in early embryo development. Interestingly, the seed abortion rate of the mutant was reduced when grown under N-deficient conditions, suggesting that nitrate requirements in early embryo development can be modulated in response to external nitrogen changes.     

The Arabidopsis Nitrate Transporter NRT1.7, Expressed in Phloem,Is Responsible for Source-to-Sink Remobilization of Nitrate

Several quantitative trait locus analyses have suggested that grain yield and nitrogen use efficiency are well correlated with nitrate storage capacity and efficient remobilization. This study of the Arabidopsis thaliana nitrate transporter NRT1.7 provides new insights into nitrate remobilization. Immunoblots, quantitative RT-PCR, β-glucuronidase reporter analysis, and immunolocalization indicated that NRT1.7 is expressed in the phloem of the leaf minor vein and that its expression levels increase coincidentally with the source strength of the leaf. In nrt1.7 mutants, more nitrate was present in the older leaves, less ¹⁵NO₃⁻ spotted on old leaves was remobilized into N-demanding tissues, and less nitrate was detected in the phloem exudates of old leaves. These data indicate that NRT1.7 is responsible for phloem loading of nitrate in the source leaf to allow nitrate transport out of older leaves and into younger leaves. Interestingly, nrt1.7 mutants showed growth retardation when external nitrogen was depleted. We conclude that (1) nitrate itself, in addition to organic forms of nitrogen, is remobilized, (2) nitrate remobilization is important to sustain vigorous growth during nitrogen deficiency, and (3) source-to-sink remobilization of nitrate is mediated by phloem.     

Arabidopsis NRTI.1 Is a Bidirectional Transporter Involved in Root-to-Shoot Nitrate Translocation

Dear Editor, In most plants, nitrogen （N） is acquired by roots in the form of nitrate （NO3-）. In many species, NO3- is not assimi- lated in the roots, but is secreted into the xylem sap for translocation to the shoot, where it enters the cells to be metabolized and/or stored in the vacuoles. Several plasma membrane transporters involved in NO3- influx into the cell have been identified in Arabidopsis （Wang et ai., 2012）, especially in the roots where members of the NPF （NRTI/PTR Family, L~ran et al., 2013） and NRT2 transporter families are predominantiy implicated. Concerning efflux to the xylem sap, only one transporter, NPF7.3/NRT1.5, has been shown to be involved. However, physiological characterization of npf7.31nrtl.5 knockout mutant plants demonstrated that other transporter（s） is （are） also contributing to xylem Ioad- inq of NO~- （Lin et al., 2008）.     

Nitrate Signaling and the Two Component High Affinity Uptake System in Arabidopsis

Nitrate transporters are important for nitrogen acquisition by plants and in algae some require two gene products, NRT2 and NAR2, for function. The NRT2 family was already described and the recent identification of a family of the NAR2-type genes in higher plants showed that there was a homologue in Arabidopsis, AtNAR2.1. Using heterologous expression in yeast and oocytes we showed that the two Arabidopsis AtNRT2.1 and AtNAR2.1 proteins interacted to give a functional high affinity nitrate transport system (HATS). The gene knock out mutant atnar2.1-1 is deficient specifically for HATS activity and the resulting growth phenotype on low nitrate concentration is more severe than for the atnrt2.1-1 knock out mutant. Physiological characterisation of the plant N status and gene expression revealed a pattern that was characteristic of severe nitrogen deficiency. Consistent with the down regulation of AtNRT2.1 expression, the atnar2.1-1 plants also displayed the same phenotype as atnrt2.1 mutants in lateral root (LR) response to low nitrate supply. Using atnar2.1-1 plants constitutively expressing the NpNRT2.1 gene, we now show a specific role for AtNAR2.1 in LR response to low nitrate supply. AtNAR2.1 is also involved in the repression of LR initiation in response to high ratios of sucrose to nitrogen in the medium. Therefore the two component system itself is likely to be involved in the signaling pathway integrating nutritional cues for LR architecture regulation. Using a green fluorescent protein-NRT2.1 protein fusion we show the essential role of AtNAR2.1 for the presence of AtNRT2.1 to the plasma membrane.Key Words: high affinity nitrate transport, nitrate transporter, nitrate signalling, root growth     

The HvNramp5 Transporter Mediates Uptake of Cadmium and Manganese,But Not Iron

The Natural Resistance Associated Macrophage Protein (Nramp) represents a transporter family for metal ions in all organisms. Here, we functionally characterized a member of Nramp family in barley (Hordeum vulgare), HvNramp5. This member showed different expression patterns, transport substrate specificity, and cellular localization from its close homolog in rice (Oryza sativa), OsNramp5, although HvNramp5 was also localized to the plasma membrane. HvNramp5 was mainly expressed in the roots and its expression was not affected by Cd and deficiency of Zn, Cu, and Mn, but slightly up-regulated by Fe deficiency. Spatial expression analysis showed that the expression of HvNramp5 was higher in the root tips than that in the basal root regions. Furthermore, analysis with laser microdissection revealed higher expression of HvNramp5 in the outer root cell layers. HvNramp5 showed transport activity for both Mn²⁺ and Cd²⁺, but not for Fe²⁺ when expressed in yeast. Immunostaining with a HvNramp5 antibody showed that this protein was localized in the root epidermal cells without polarity. Knockdown of HvNramp5 in barley resulted in a significant reduction in the seedling growth at low Mn supply, but this reduction was rescued at high Mn supply. The concentration of Mn and Cd, but not other metals including Cu, Zn, and Fe, was decreased in both the roots and shoots of knockdown lines compared with the wild-type barley. These results indicate that HvNramp5 is a transporter required for uptake of Mn and Cd, but not for Fe, and that barley has a distinct uptake system from rice.Transport of mineral elements from soil to different organs and tissues of plants requires different types of transporters (Hall and Williams, 2003; Nevo and Nelson, 2006; Yokosho et al., 2009; Olsen and Palmgren, 2014; Sasaki et al., 2016), which include the zinc-regulated transporters, iron-regulated transporter-like protein family; the natural resistance-associated macrophage protein (Nramp) family of transporters; the multidrug and toxic compound extrusion protein transporters; the heavy metal ATPase transporters; the oligopeptide transporters family; the ATP-binding cassette family of transporters; and the cation-diffusion facilitator family of transporters. Among them, Nramp represents a transporter family for metal ion in all organisms including bacteria, animals, and plants (Curie et al., 2000; Nevo and Nelson, 2006). Some members of this family in plants have been functionally characterized, especially in model plants such as Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa). In Arabidopsis, there are six members of Nramp transporter proteins. AtNramp1 is localized to the plasma membrane of root cells and functions as a high-affinity transporter for Mn uptake (Cailliatte et al., 2010). Both AtNramp3 and AtNramp4 are localized to the tonoplast and play redundant roles in Fe, exporting from the vacuole during seed germination and in Mn homeostasis at the adult stage (Thomine et al., 2000; Lanquar et al., 2005, 2010). AtNramp6 is targeted to a vesicular-shaped endomembrane compartment and is implicated in the distribution or availability of Cd within cells (Cailliatte et al., 2009). However, the function of AtNramp2 and AtNramp5 has not been characterized.On the other hand, there are seven members of Nramp transporter family in the rice genome, of which four have been functionally characterized. They all are localized to the plasma membrane but show different roles. OsNramp1 shows transport activity for Fe and Cd in yeast and is proposed to be involved in Cd accumulation (Takahashi et al., 2011). OsNramp3 is localized at the vascular tissues of nodes and plays an important role in distribution of Mn, but not Fe and Cd (Yamaji et al., 2013). On the other hand, OsNrat1 (OsNramp4) transports trivalent Al ion (Xia et al., 2010) and is required for high Al tolerance in rice roots. Finally, OsNramp5 functions as a major transporter responsible for root Mn and Cd uptake (Ishimaru et al., 2012; Ishikawa et al., 2012; Sasaki et al., 2012). However, the function of OsNramp2, OsNramp6, and OsNramp7 is unknown.In addition to Nramp members characterized in rice and Arabidopsis, some members in other plant species have also been characterized. For example, a soybean (Glycine max) Nramp transporter, GmDMT1 is implicated in the ferrous iron transport (Kaiser et al., 2003). Nramp1 isolated from Noccaea caerulescens, a Zn/Cd hyperaccumulator, is involved in the influx of Cd across the endodermal plasma membrane and plays a key role in Cd flux into the stele and root-to-shoot Cd transport (Milner et al., 2014). In Malus baccata, Nramp1 is capable of mediating the distribution of ions as well as transport of Fe, Mn, and Cd (Xiao et al., 2008). Besides, Nramp1 and Nramp3 in tomato (Solanum lycopersicum) have also been suggested to be involved in Mn transport (Bereczky et al., 2003). When NcNramp3 and NcNramp4 from Noccaea caerulescens were expressed in yeast, NcNramp3 transported Fe, Mn, and Cd, while NcNramp4 also transported Zn in addition to Fe, Mn, and Cd (Oomen et al., 2009). However, Nramp4 isolated from Thlaspi japonicum, a Ni hyperaccumulator, showed transport activity for Ni but not for Zn, Cd, or Mn in yeast (Mizuno et al., 2005). These findings indicate that Nramp members have a diverse role in metal transport in plants.Barley (Hordeum vulgare) is the fourth most important cereal crop in the world; however, less progress has been made in understating of molecular mechanisms on mineral element transport in barley due to its large genome size. For example, no Nramp members in barley have been functionally characterized so far. In this study, we first isolated barley Nramp member, HvNramp5, which is a close homolog of rice OsNramp5. Detailed functional analysis revealed that HvNramp5 is involved in the uptake of both Mn and Cd, but not of Fe in barley roots. Furthermore, we found that different from OsNramp5, HvNramp5 showed a distinct pattern in the gene expression, cellular localization, and transport substrate.     

Inhibition of Assimilatory Nitrate Uptake by Ammonium Ions in Azorhizobium caulinodans IRBG 46

Addition of ammonium sulphate at low concentrations to Azorhizobium caulinodans IRBG 46 cells caused an immediate cessation of nitrate uptake activity, which was restored when the added ammonium ions were exhausted from the medium. Blockage of ammonium assimilation by L-methionine sulfoximine did not prevent the negative effect of ammonium on the assimilatory nitrate uptake, thus indicating that ammonium ions per se and not its assimilatory product(s) are actual regulators of assimilatory nitrate uptake.     

Feedback Inhibition of Ammonium Uptake by a Phospho-Dependent Allosteric Mechanism in Arabidopsis

The acquisition of nutrients requires tight regulation to ensure optimal supply while preventing accumulation to toxic levels. Ammonium transporter/methylamine permease/rhesus (AMT/Mep/Rh) transporters are responsible for ammonium acquisition in bacteria, fungi, and plants. The ammonium transporter AMT1;1 from Arabidopsis thaliana uses a novel regulatory mechanism requiring the productive interaction between a trimer of subunits for function. Allosteric regulation is mediated by a cytosolic C-terminal trans-activation domain, which carries a conserved Thr (T460) in a critical position in the hinge region of the C terminus. When expressed in yeast, mutation of T460 leads to inactivation of the trimeric complex. This study shows that phosphorylation of T460 is triggered by ammonium in a time- and concentration-dependent manner. Neither Gln nor l-methionine sulfoximine–induced ammonium accumulation were effective in inducing phosphorylation, suggesting that roots use either the ammonium transporter itself or another extracellular sensor to measure ammonium concentrations in the rhizosphere. Phosphorylation of T460 in response to an increase in external ammonium correlates with inhibition of ammonium uptake into Arabidopsis roots. Thus, phosphorylation appears to function in a feedback loop restricting ammonium uptake. This novel autoregulatory mechanism is capable of tuning uptake capacity over a wide range of supply levels using an extracellular sensory system, potentially mediated by a transceptor (i.e., transporter and receptor).     

Differential Inhibition by Ferulic Acid of Nitrate and Ammonium Uptake in Zea mays L

The influence of the allelopathic compound ferulic acid (FA) on nitrogen uptake from solutions containing both NO₃⁻ and NH₄⁺ was examined in 8-day-old nitrogen-depleted corn (Zea mays L.) seedlings. Concurrent effects on uptake of Cl⁻ and K⁺ also were assessed. The presence of 250 micromolar FA inhibited the initial (0-1 hours) rate of NO₃⁻ uptake and also prevented development of the NO₃⁻-inducible accelerated rate. The pattern of recovery when FA was removed was interpreted as indicating a rapid relief of FA-restricted NO₃⁻ uptake activity, followed by a reinitiation of the induction of that activity. No inhibition of NO₃⁻ reduction was detected. Ammonium uptake was less sensitive than NO₃⁻ uptake to inhibition by FA. An inhibition of Cl⁻ uptake occurred as induction of the NO₃⁻ transport system developed in the absence of FA. Alterations of Cl⁻ uptake in the presence of FA were, therefore, a result of a beneficial effect, because NO₃⁻ uptake was restricted, and a direct inhibitory effect. The presence of FA increased the initial net K⁺ loss from the roots during exposure to the low K, ammonium nitrate uptake solution and delayed the recovery to positive net uptake, but it did not alter the general pattern of the response. The implications of the observations are discussed for growth of plants under natural conditions and cultural practices that foster periodic accumulation of allelopathic substances.     

Post-Flowering Nitrate Uptake in Wheat Is Controlled by N Status at Flowering,with a Putative Major Role of Root Nitrate Transporter NRT2.1

In bread wheat (Triticum aestivum L.), the simultaneous improvement of both yield and grain protein is difficult because of the strong negative relationship between these two traits. However, some genotypes deviate positively from this relationship and this has been linked to their ability to take up nitrogen (N) during the post-flowering period, regardless of their N status at flowering. The physiological and genetic determinants of post-flowering N uptake relating to N satiety are poorly understood. This study uses semi-hydroponic culture of cv. Récital under controlled conditions to explore these controls. The first objective was to record the effects of contrasting N status at flowering on post-flowering nitrate (NO₃ ^-) uptake under non-limiting NO₃ ^- conditions, while following the expression of key genes involved in NO₃ ^- uptake and assimilation. We found that post-flowering NO₃ ^- uptake was strongly influenced by plant N status at flowering during the first 300–400 degree-days after flowering, overlapping with a probable regulation of nitrate uptake exerted by N demand for growth. The uptake of NO₃ ^- correlated well with the expression of the gene TaNRT2.1, coding for a root NO₃ ^- transporter, which seems to play a major role in post-flowering NO₃ ^- uptake. These results provide a useful knowledge base for future investigation of genetic variability in post-flowering N uptake and may lead to concomitant gains in both grain yield and grain protein in wheat.     

Arabidopsis Transporter MGT6 Mediates Magnesium Uptake and Is Required for Growth under Magnesium Limitation

Although magnesium (Mg²⁺) is the most abundant divalent cation in plant cells, little is known about the mechanism of Mg²⁺ uptake by plant roots. Here, we report a key function of Magnesium Transport6 (MGT6)/Mitochondrial RNA Splicing2-4 in Mg²⁺ uptake and low-Mg²⁺ tolerance in Arabidopsis thaliana. MGT6 is expressed mainly in plant aerial tissues when Mg²⁺ levels are high in the soil or growth medium. Its expression is highly induced in the roots during Mg²⁺ deficiency, suggesting a role for MGT6 in response to the low-Mg²⁺ status in roots. Silencing of MGT6 in transgenic plants by RNA interference (RNAi) resulted in growth retardation under the low-Mg²⁺ condition, and the phenotype was restored to normal growth after RNAi plants were transferred to Mg²⁺-sufficient medium. RNAi plants contained lower levels of Mg²⁺ compared with wild-type plants under low Mg²⁺ but not under Mg²⁺-sufficient conditions. Further analysis indicated that MGT6 was localized in the plasma membrane and played a key role in Mg²⁺ uptake by roots under Mg²⁺ limitation. We conclude that MGT6 mediates Mg²⁺ uptake in roots and is required for plant adaptation to a low-Mg²⁺ environment.     

Structure and Mechanism of a Nitrate Transporter

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Highlights? Structure of the nitrate transporter NarU in substrate-free and -bound states ? Distinct conformations of NarU in substrate-free and -bound states ? Identification of a substrate binding site and transport path ? Transport mechanism may deviate from the rocker-switch model