CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake.

Higher plants have both high- and low-affinity nitrate uptake systems. These systems are generally thought to be genetically distinct. Here, we demonstrate that a well-known low-affinity nitrate uptake mutant of Arabidopsis, chl1, is also defective in high-affinity nitrate uptake. Two to 3 hr after nitrate induction, uptake activities of various chl1 mutants at 250 microM nitrate (a high-affinity concentration) were only 18 to 30% of those of wild-type plants. In these mutants, both the inducible phase and the constitutive phase of high-affinity nitrate uptake activities were reduced, with the inducible phase being severely reduced. Expressing a CHL1 cDNA driven by the cauliflower mosaic virus 35S promoter in a transgenic chl1 plant effectively recovered the defect in high-affinity uptake for the constitutive phase but not for the induced phase, which is consistent with the constitutive level of CHL1 expression in the transgenic plant. Kinetic analysis of nitrate uptake by CHL1-injected Xenopus oocytes displayed a biphasic pattern with a Michaelis-Menten Km value of approximately 50 microM for the high-affinity phase and approximately 4 mM for the low-affinity phase. These results indicate that in addition to being a low-affinity nitrate transporter, as previously recognized, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis.     

Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRT1, from rice

Elucidating how rice (Oryza sativa) takes up nitrate at the molecular level could help improve the low recovery rate (<50%) of nitrogen fertilizer in rice paddies. As a first step toward that goal, we have cloned a nitrate transporter gene from rice called OsNRT1. OsNRT1 is a new member of a growing transporter family called PTR, which consists not only of nitrate transporters from higher plants that are homologs of the Arabidopsis CHL1 (AtNRT1) protein, but also peptide transporters from a wide variety of genera including animals, plants, fungi, and bacteria. However, despite the fact that OsNRT1 shares a higher degree of sequence identity with the two peptide transporters from plants (approximately 50%) than with the nitrate transporters (approximately 40%) of the PTR family, no peptide transport activity was observed when OsNRT1 was expressed in either Xenopus oocytes or yeast. Furthermore, contrasting the dual-affinity nitrate transport activity of CHL1, OsNRT1 displayed only low-affinity nitrate transport activity in Xenopus oocytes, with a K(m) value of approximately 9 mM. Northern-blot and in situ hybridization analysis indicated that OsNRT1 is constitutively expressed in the most external layer of the root, epidermis and root hair. These data strongly indicate that OsNRT1 encodes a constitutive component of a low-affinity nitrate uptake system for rice.     

The nitrate transporter AtNRT1.1 (CHL1) functions in stomatal opening and contributes to drought susceptibility in Arabidopsis

The movement of guard cells in stomatal complexes controls water loss and CO(2) uptake in plants. Examination of the dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) revealed that it is expressed and functions in Arabidopsis guard cells. CHL1 promoter-beta-glucuronidase and CHL1 promoter-green fluorescent protein constructs showed strong expression in guard cells, and immunolocalization experiments with anti-CHL1 antibody confirmed these results. To assess CHL1 function, chl1 mutant plants grown in the presence of nitrate were examined. Compared with wild-type plants, chl1 mutants had reduced stomatal opening and reduced transpiration rates in the light or when deprived of CO(2) in the dark. These effects result in enhanced drought tolerance in chl1 mutants. At the cellular level, chl1 mutants showed reduced nitrate accumulation in guard cells during stomatal opening and failed to show nitrate-induced depolarization of guard cells. In wild-type guard cells, nitrate induced depolarization, and nitrate concentrations increased threefold during stomatal opening. These results identify an anion transporter that functions in stomatal opening and demonstrate that CHL1 supports stomatal function in the presence of nitrate.     

CHL1 encodes a component of the low-affinity nitrate uptake system in Arabidopsis and shows cell type-specific expression in roots.

The Arabidopsis CHL1 (AtNRT1) gene confers sensitivity to the herbicide chlorate and encodes a nitrate-regulated nitrate transporter. However, how CHL1 participates in nitrate uptake in plants is not yet clear. In this study, we examined the in vivo function of CHL1 with in vivo uptake measurements and in situ hybridization experiments. Under most conditions tested, the amount of nitrate uptake by a chl1 deletion mutant was found to be significantly less than that of the wild type. This uptake deficiency was reversed when a CHL1 cDNA clone driven by the cauliflower mosaic virus 35S promoter was expressed in transgenic chl1 plants. Furthermore, tissue-specific expression patterns showed that near the root tip, CHL1 mRNA is found primarily in the epidermis, but further from the root tip, the mRNA is found in the cortex or endodermis. These results are consistent with the involvement of CHL1 in nitrate uptake at different stages of root cell development. A functional analysis in Xenopus oocytes indicated that CHL1 is a low-affinity nitrate transporter with a K(m) value of approximately 8.5 mM for nitrate. This finding is consistent with the chlorate resistance phenotype of chl1 mutants. However, these results do not fit the current model of a single, constitutive component for the low-affinity uptake system. To reconcile this discrepancy and the complex uptake behavior observed, we propose a "two-gene" model for the low-affinity nitrate uptake system of Arabidopsis.     

Functional characterization of the Arabidopsis thaliana nitrate transporter CHL1 in the yeast Hansenula polymorpha

CHL1 (AtNRT1.1) is a dual-affinity nitrate transporter of Arabidopsis thaliana, in which phosphorylation at Thr 101 switches CHL1 from low to high nitrate affinity. CHL1 expressed in a Hansenula polymorpha high-affinity nitrate-transporter deficient mutant (Deltaynt1) restores nitrate uptake and growth. These events take place at nitrate concentrations as low as 500 muM, suggesting that CHL1 has a high-affinity for nitrate in yeast. Accordingly, CHL1 expressed in H. polymorpha presents a K (m) for nitrate of about 125 muM. The absence of nitrate, the CHL1 gene inducer, showed the high turnover rate of CHL1 expressed in yeast, which is counteracted by nitrate CHL1 induction. Furthermore, H. polymorpha strains expressing CHL1 become sensitive to 250 muM chlorate, as expected for CHL1 high-affinity behaviour. Given that CHL1 presented high affinity by nitrate, we study the role of CHL1 Thr101 in yeast. Strains producing CHL1Thr101Ala, unable to undergo phosphorylation, and CHL1Thr101Asp, where CHL1 phosphorylation is constitutively mimicked, were used. Yeast strains expressing CHL1Thr101Ala, CHL1Thr101Asp and CHL1 at the same rate showed that Deltaynt1CHL1Thr101Ala is strikingly unable to transport nitrate and contains a very low amount of CHL1 protein; however, Deltaynt1CHL1Thr101Asp restores nitrate uptake and growth, although no significant changes in nitrate affinity were observed. Our results show that CHL1-Thr101 is involved in regulating the levels of CHL1 expressed in yeast and suggest that the phosphorylation of this residue could be involved in targeting this nitrate transporter to the plasma membrane. The functional expression of CHL1 in H. polymorpha reveals that this yeast is a suitable tool for evaluating the real nitrate transport capacity of plant putative nitrate transporters belonging to different families and study their regulation and structure function relationship.     

Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake.

The Arabidopsis CHL1 (AtNRT1) gene encodes an inducible component of low-affinity nitrate uptake, which necessitates a "two-component" model to account for the constitutive low-affinity uptake observed in physiological studies. Here, we report the cloning and characterization of a CHL1 homolog, AtNRT1:2 (originally named NTL1), with data to indicate that this gene encodes a constitutive component of low-affinity nitrate uptake. Transgenic plants expressing antisense AtNRT1:2 exhibited reduced nitrate-induced membrane depolarization and nitrate uptake activities in assays with 10 mM nitrate. Furthermore, transgenic plants expressing antisense AtNRT1:2 in the chl1-5 background exhibited an enhanced resistance to chlorate (7 mM as opposed to 2 mM for the chl1-5 mutant). Kinetic analysis of AtNRT1:2-injected Xenopus oocytes yielded a K(m) for nitrate of approximately 5.9 mM. In contrast to CHL1, AtNRT1:2 was constitutively expressed before and after nitrate exposure (it was repressed transiently only when the level of CHL1 mRNA started to increase significantly), and its mRNA was found primarily in root hairs and the epidermis in both young (root tips) and mature regions of roots. We conclude that low-affinity systems of nitrate uptake, like high-affinity systems, are composed of inducible and constitutive components and that with their distinct functions, they are part of an elaborate nitrate uptake network in Arabidopsis.     

Nitrate transporters and peptide transporters

In higher plants, two types of nitrate transporters, NRT1 and NRT2, have been identified. In Arabidopsis, there are 53 NRT1 genes and 7 NRT2 genes. NRT2 are high-affinity nitrate transporters, while most members of the NRT1 family are low-affinity nitrate transporters. The exception is CHL1 (AtNRT1.1), which is a dual-affinity nitrate transporter, its mode of action being switched by phosphorylation and dephosphorylation of threonine 101. Two of the NRT1 genes, CHL1 and AtNRT1.2, and two of the NRT2 genes, AtNRT2.1 and AtNRT2.2, are known to be involved in nitrate uptake. In addition, AtNRT1.4 is required for petiole nitrate storage. On the other hand, some members of the NRT1 family are dipeptide transporters, called PTRs, which transport a broad spectrum of di/tripeptides. In barley, HvPTR1, expressed in the plasma membrane of scutellar epithelial cells, is involved in mobilizing peptides, produced by hydrolysis of endosperm storage protein, to the developing embryo. In higher plants, there is another family of peptide transporters, called oligopeptide transporters (OPTs), which transport tetra/pentapeptides. In addition, some OPTs transport GSH, GSSH, GSH conjugates, phytochelatins, and metals.     

植物根系对氮胁迫的形态学响应

NO3-不仅是植物营养的主要N源,而且也是调节植物新陈代谢和生长发育的信号。植物根系对N供给的形态学适应表现在4个方面:(1)植物侧根(LR)响应体外硝酸盐的局部刺激而伸长,AtNRT1.1在侧根响应体外硝酸盐的局部刺激中,作用于ANR1 MADS box基因上游;(2)植物LR分裂组织活动受到组织中高浓度的硝酸盐抑制,RNA结合蛋白FCA是高硝酸盐/ABA诱导侧根发育抑制的信号传输途径成分;(3)植物侧根的发生受到体外高C∶N比抑制,AtNRT2.1参与高C∶N比抑制侧根发生;(4)植物根响应体外L-谷氨酸盐的刺激而分枝,初生根的生长受到体外L-谷氨酸盐抑制,植物根系对L-谷氨酸盐的响应可能与一种同源于哺乳动物离子型谷氨酸盐受体的植物蛋白的感知作用有关。植物根形态对N供给的响应具有重要生理和生态意义。     

Signalling mechanisms underlying the morphological responses of the root system to nitrogen in Arabidopsis thaliana

Plants display considerable developmental plasticity in response to changing environmental conditions. The adaptations of the root system to variations in N supply are an excellent example of such developmental plasticity. In Arabidopsis, four morphological adaptations to the N supply have been characterized: (i) a localized stimulatory effect of external nitrate on lateral root elongation; (ii) a systemic inhibitory effect of high tissue nitrate concentrations on the activation of lateral root meristems; (iii) a suppression of lateral root initiation by high C:N ratios, and (iv) an inhibition of primary root growth and stimulation of root branching by external L-glutamate. These responses have provided valuable experimental systems for the study of N signalling in plants. This article will highlight some recent progress made in this direction from studies using the Arabidopsis root system. One recent development of note has been the emerging evidence of a regulatory role of nitrate transporters in some of the responses. It has been reported that the AtNRT1.1 (CHL1) dual-affinity nitrate transporter acts upstream of the ANR1 MADS box gene in mediating the stimulatory effect of a localized nitrate supply on lateral root proliferation. The AtNRT2.1 high-affinity nitrate transporter seems to be involved in the repression of lateral root initiation by high C:N ratios. The systemic inhibitory effect of high nitrate supply on lateral root development, which is mediated by abscisic acid (ABA), may be linked to the recently identified ABA receptor, FCA. The newly discovered root architectural response to external L-glutamate potentially offers a valuable experimental tool for studying the biological function of plant glutamate receptors and amino acid signalling.     

The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) is regulated by auxin in both shoots and roots

The AtNRT1.1 (CHL1) gene of Arabidopsis encodes a dual-affinity nitrate transporter and contributes to both low and high affinity nitrate uptake. Localization studies have shown that CHL1 expression is preferentially targeted to nascent organs and growing regions of roots and shoots in Arabidopsis. In roots, CHL1 expression is concentrated in the tips of primary and lateral roots and is activated during lateral root initiation. In shoots, strong CHL1 expression is found in young leaves and developing flower buds. These findings suggest that CHL1 expression might be regulated by a growth signal such as the phytohormone auxin. To test this, auxin regulation of CHL1 was examined. Using transgenic Arabidopsis plants containing CHL1::GUS/GFP DNA constructs, it was found that treatment with exogenous auxin or introduction of the auxin overproducing mutations (yucca and rooty) resulted in a strong increase in CHL1::GUS/GFP signals in roots and leaves. When mature roots were treated with auxin to induce lateral root formation, CHL1::GFP signals were dramatically enhanced in dividing pericycle cells and throughout primordia development. RNA blot analysis showed that CHL1 mRNA levels in whole seedlings increase within 30 min of auxin treatment. The distribution of CHL1 expression in Arabidopsis roots and shoots was found to be similar to that of DR5::GUS, a synthetic, auxin-responsive gene. These results indicate that auxin acts as an important signal regulating CHL1 expression and contributes to the targeting of CHL1 expression to nascent organs and root tips in Arabidopsis.     

Mutation of the Arabidopsis NRT1.5 nitrate transporter causes defective root-to-shoot nitrate transport

Little is known about the molecular and regulatory mechanisms of long-distance nitrate transport in higher plants. NRT1.5 is one of the 53 Arabidopsis thaliana nitrate transporter NRT1 (Peptide Transporter PTR) genes, of which two members, NRT1.1 (CHL1 for Chlorate resistant 1) and NRT1.2, have been shown to be involved in nitrate uptake. Functional analysis of cRNA-injected Xenopus laevis oocytes showed that NRT1.5 is a low-affinity, pH-dependent bidirectional nitrate transporter. Subcellular localization in plant protoplasts and in planta promoter-β-glucuronidase analysis, as well as in situ hybridization, showed that NRT1.5 is located in the plasma membrane and is expressed in root pericycle cells close to the xylem. Knockdown or knockout mutations of NRT1.5 reduced the amount of nitrate transported from the root to the shoot, suggesting that NRT1.5 participates in root xylem loading of nitrate. However, root-to-shoot nitrate transport was not completely eliminated in the NRT1.5 knockout mutant, and reduction of NRT1.5 in the nrt1.1 background did not affect root-to-shoot nitrate transport. These data suggest that, in addition to that involving NRT1.5, another mechanism is responsible for xylem loading of nitrate. Further analyses of the nrt1.5 mutants revealed a regulatory loop between nitrate and potassium at the xylem transport step.     

Integrating fluctuating nitrate uptake and assimilation to robust homeostasis

Nitrate is an important nitrogen source used by plants. Despite of the considerable variation in the amount of soil nitrate, plants keep cytosolic nitrate at a homeostatic controlled level. Here we describe a set of homeostatic controller motifs and their interaction that can maintain robust cytosolic nitrate homeostasis at fluctuating external nitrate concentrations and nitrate assimilation levels. The controller motifs are divided into two functional classes termed as inflow and outflow controllers. In the presence of high amounts of environmental nitrate, the function of outflow controllers is associated to efflux mechanisms removing excess of nitrate from the cytosol that is taken up by low-affinity transporter systems (LATS). Inflow controllers on the other hand maintain homeostasis in the presence of a high demand of nitrate by the cell relative to the amount of available environmental nitrate. This is achieved by either remobilizing nitrate from a vacuolar store, or by taking up nitrate by means of high-affinity transporter systems (HATS). By combining inflow and outflow controllers we demonstrate how nitrate uptake, assimilation, storage and efflux are integrated to a regulatory network that maintains cytosolic nitrate homeostasis at changing environmental conditions.     

External Nitrogen and Carbon Source-Mediated Response on Modulation of Root System Architecture and Nitrate Uptake in Wheat Seedlings

Nitrogen uptake efficiency is an important component trait that could be targeted for improving nitrogen use efficiency of crop plants. To understand the responses of different nitrate transport systems and the influence of root system architecture on nitrate uptake under limited nitrate conditions in wheat (Triticum aestivum L.) at the seedling stage, we studied nitrate uptake, root system architecture, and expression of different nitrate transporter genes in induced and non-induced wheat seedlings. Further, effects of inclusion of sucrose and two amino acids (glutamine and asparagine) in induction medium on these parameters were also studied. We observed that the induced wheat root system took up more nitrate as compared to non-induced root system in a dose-dependent manner. Gene expression of both high- and low-affinity nitrate transporter gene showed differential expression in the induced root tissues, as compared to non-induced tissues, depending on the concentration of nitrate present in induction medium. External nutrient media containing sucrose, glutamine, and asparagine reduce nitrate concentration in both root and shoot tissues and also influence the gene expression of these transporters. Our observations indicate that upon induction with milder external nitrate concentrations, the root architecture is modulated by changing overall lateral root size and 1st order lateral root numbers along with activation of nitrate transporters which acquire and transport nitrate in roots and shoots, respectively, depending on the carbon and nitrogen source available to seedlings.

     

AtKuP1: a dual-affinity K+ transporter from Arabidopsis.

Plant roots contain both high- and low-affinity transport systems for uptake of K+ from the soil. In this study, we characterize a K+ transporter that functions in both high- and low-affinity uptake. Using yeast complementation analysis, we isolated a cDNA for a functional K+ transporter from Arabidopsis (referred to as AtKUP1 for Arabidopsis thaliana K+ uptake). When expressed in a yeast mutant, AtKUP1 dramatically increased K+ uptake capacity at both a low and high [K+] range. Kinetic analyses showed that AtKUP1-mediated K+ uptake displays a "biphasic" pattern similar to that observed in plant roots. The transition from the high-affinity phase (K(m) of 44 microM) to the low-affinity phase (K(m) of 11 mM) occurred at 100 to 200 microM external K+. Both low- and high-affinity K+ uptake via AtKUP1 were inhibited by 5 mM or higher concentrations of NaCl. In addition, AtKUP1-mediated K+ uptake was inhibited by K+ channel blockers, including tetraethylammonium, Cs+, and Ba2+. Consistent with a possible function in K+ uptake from the soil, the AtKUP1 gene is primarily expressed in roots. We conclude that the AtKUP1 gene product may function as a K+ transporter in Arabidopsis roots over a broad range of [K+] in the soil.     

The Arabidopsis dual-affinity nitrate transporter gene AtNRT1.1 (CHL1) is activated and functions in nascent organ development during vegetative and reproductive growth

The AtNRT1.1 (CHL1) transporter provides a primary mechanism for nitrate uptake in Arabidopsis and is expected to localize to the epidermis and cortex of the mature root, where the bulk of nitrate uptake occurs. Using fusions to GFP/GUS marker genes, we found CHL1 expression concentrated in the tips of primary and lateral roots, with very low signals in the epidermis and cortex. A time-course study showed that CHL1 is activated in the primary root tip early in seedling development and at the earliest stages of lateral root formation. Strong CHL1 expression also was found in shoots, concentrated in young leaves and developing flower buds but not in the shoot meristem. These expression patterns were confirmed by immunolocalization and led us to examine CHL1 function specifically in the growth of developing organs. chl1 mutants showed a reduction in the growth of nascent roots, stems, leaves, and flower buds. The growth of nascent primary roots was inhibited in the mutants even in the absence of added nitrate, whereas elongation of lateral root primordia was inhibited specifically at low nitrate and acidic pH. Interestingly, chl1 mutants also displayed a late-flowering phenotype. These results indicate that CHL1 is activated and functions in the growth of nascent organs in both shoots and roots during vegetative and reproductive growth.     

Effects of the crude venom of the social wasp Agelaia vicina on gamma-aminobutyric acid and glutamate uptake in synaptosomes from rat cerebral cortex

Glutamate (L-glu) is the most important excitatory neurotransmitter in the mammalian central nervous system. Its action is terminated by transporters located in the plasma membrane of neurons and glial cells, which have a critical role in preventing glutamate excitotoxicity under normal conditions. The neurotransmitter gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system. Venoms of solitary wasps and orb-spiders are composed of large proteins, medium-size peptides, polyamine amides (PAs), and other neuroactive components that are highly selective to nervous tissues. The abnormal operation of uptake systems is involved in several failures. Several studies indicate alterations in extracellular GABA and glutamate concentrations in epilepsy conditions that may relate to transporter functions. The effects of the crude and boiled venom of the social wasp Agelaia vicina, "cassununga," on GABA and L-glu uptake in rat cerebral cortex synaptosomes are related. The venom uncompetitively inhibited high- and low-affinity GABA uptake by 91.2% and by 76%, respectively. This kind of inhibition was also found to affect high- (99.6%) and low-affinity (90%) uptake of L-glu. These results suggest that the effects observed in these experiments indicate the venom of A. vicina to be a useful tool to further characterize GABA- and L-glu-uptake systems.     

Synthesis and High Affinity Uptake of Serotonin and Dopamine by Human Y79 Retinoblastoma Cells

Human Y79 retinoblastoma cells are capable of synthesizing the putative retinal neurotransmitters dopamine and serotonin. Separation of the catecholamines and indolamines by high performance liquid chromatography combined with electrochemical detection showed that the cells readily convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA) and, to a lesser extent, dopamine. When DOPA was added, a large quantity of dopamine was produced, as well as norepinephrine, epinephrine, and 3,4-dihydroxyphenylacetic acid. Exogenous tryptophan added to the cells was partially converted to 5-hydroxytryptophan and serotonin. A larger quantity of serotonin was produced when 5-hydroxytryptophan was added. Y79 cells have a high- and low-affinity uptake system for dopamine and serotonin. The K'm and V'max for the high-affinity uptake of dopamine and serotonin are 2.34 +/- 0.64 and 3.63 +/- 1.15 microM and 4.77 +/- 1.12 and 3.20 +/- 1.20 pmol min-1 mg protein-1, respectively. These kinetic parameters are similar to those reported for other retinal preparations where dopamine and serotonin have been suggested to function as neurotransmitters. Tyrosine and tryptophan, the physiologic precursors of dopamine and serotonin, respectively, and phenylalanine are also taken up by high- and low-affinity transport systems. The kinetic parameters for their high-affinity uptake systems are all very similar, suggesting that they may be taken up by the same transporter. These studies show that a tumor cell line derived from the human retina synthesizes dopamine and serotonin and has high-affinity uptake systems for these compounds and their precursors.(ABSTRACT TRUNCATED AT 250 WORDS)     

植物寡肽运输与硝酸根运输基因家族的研究进展

氮素是植物生长发育的重要营养元素,也是限制植物生物量尤其是经济产量的关键营养元素之一.植物不仅能从外界获取无机氮素(硝酸根、铵根和尿素等),还能以氨基酸、寡肽等形式获取有机氮素.植物已进化出复杂的运输系统来吸收与运输这些含氮化合物.硝酸根运输基因家族分为低亲和力硝酸根运输基因(low-affmity nitrate t...