HORT640 - Metabolic Plant Physiology
Nitrate uptake and reduction
Nitrate and ammonium transport
The nitrate uptake system of higher plants consists of a constitutive, low affinity transport system (LATS) (possibly a carrier system or an anion channel), and an inducible, high affinity transport system (HATS) regulated by cellular energy supply, and by intracellular nitrate consumption, and whose activity depends on the proton electrochemical gradient. The latter system is regarded as an H+/anion co-transport carrier mechanism that produces transient plasmamembrane depolarization upon addition of nitrate. The depolarization is counteracted by the plasmamembrane H+-ATPase (Ullrich, 1992). The plasma membrane proton ATP-ase is induced by nitrate (Santi et al, 1995).
In addition to the nitrate uptake system, plants have an inducible nitrate efflux system, requiring both RNA and protein synthesis. The efflux system, however, has a much slower turnover rate than the uptake system (Aslam et al, 1996).
Nitrite is also transported by two systems, of which the low-affinity system may play a greater role than in nitrate uptake. In some, but not in all cases, the high-affinity system has been shown to be identical with that of nitrate transport, by uptake competition studies as well as voltage changes (Ullrich, 1992).
Movement of nitrite from the cytosol to the chloroplast appears to involve a proton-linked transport of nitrite. The rapid movement of HNO2 across the chloroplast inner envelope would require some active proton transport from the stroma into the external space. The presence of an H+-ATPase on the chloroplast inner envelope which pumps H+ out of the stroma into the cytosol may fulfill this role (Shingles et al, 1996).
Ammonium ions are taken up by a saturable, but apparently constitutive, carrier system with high substrate affinity, which may carry out ammonium uniport as long as H+-ATPases restore Em. The low-affinity component of transport is stimulated by high external pH and probably reflects diffusion of uncharged NH3 across the lipid phase of the plasmalemma. Both the high affinity and low affinity ammonium transport systems appear to be constitutive (Kronzucker et al, 1996). In rice roots the Km for ammonium uptake is about 32 uM (Kronzucker et al, 1996). A transient induction of the ammonium transport system (increased Vmax) occurs upon exposure of rice seedlings to oxygen deprivation; this may occur in response to hypoxia-induced cytoplasmic acidosis (Kronzucker et al, 1998). Three ammonium transporters have been identified in Arabidopsis roots; constitutive, diurnally regulated, and starvation-induced (Gazzarrini et al, 1999).
If ammonium enters the cell at high rates, it causes strong membrane depolarization and will block anion/H+ co-transport. This may be the primary mechanism by which ammonium ions inhibit nitrate uptake (Ullrich, 1992). Alternatively competition between the nitrate uptake system and glutamine synthetase (GS) for ATP may in part account for ammonium inhibition of nitrate uptake (occurs only when GS is active) (Andriesse et al, 1989).
Butz and Jackson (1977) proposed that a nitrate reductase (NR) dimer that spans a unit membrane, plus an ATP-ase, is responsible for both nitrate transport and reduction. Antibodies against Chlorella nitrate reductase detect a protein in the plasma membrane of barley roots, and IgG fragments of these antibodies inhibited nitrate transport by barley roots (Ward et al, 1988).
The plasma membrane nitrate reductase represents a small fraction of the total nitrate reductase, it has a much low molecular weight than the cytosolic form of the enzyme, and does not appear to represent the inducible nitrate transporter or the inducible nitrate reductase; its role is presently unclear (Ullrich, 1992).
Warner and Huffaker (1989) deny a connection between NR and nitrate transport. Genotypes of barley lacking both the NADH-specific and NAD(P)H-NRs show the same kinetics of nitrate uptake as the wildtype (Warner and Huffaker, 1989). However, in these NR-deficient mutants there was still a trace of NR activity which could have been due to the low molecular weight plasma membrane form of the enzyme postulated to be involved in nitrate transport (Ullrich, 1992).
A mutant of Arabidopsis (chl1) that is resistant to chlorate was found that is partially defective in nitrate uptake. The CHL1 gene was cloned from a T-DNA-tagged line identified by Ken Feldmann. This gene encodes a hydrophobic protein with 12 membrane spanning segments that confers nitrate transport activity to Xenopus oocytes. The CHL1 gene is expressed primarily in roots and is induced by nitrate and low pH. The CHL1 protein appears to be a low affinity nitrate/chlorate transporter.
A new allele of chl1 was discovered in a line of Arabidopsis carrying an active Ac element. This mutant had an insertion in the 4th intron of the CHL1 gene. The insertion was shown to be an active, endogenous transposable element, which was named Tag1 (Tsay et al, 1993).
Touraine and Glass (1997) show that in chl1-5 deletion mutants nitrate uptake is impaired only when plants are grown on ammonium nitrate; nitrate uptake is not impaired when KNO3 is the sole N source. However chlorate uptake was significantly lower than in wildtype when grown on KNO3. Touraine and Glass (1997) suggest that a second low affinity transport system (LATS) is able to compensate for the chl1-5 deficiency in KNO3-grown plants; growth on NH4NO3 may down-regulate the second LATS enough that the anticipated difference in nitrate uptake becomes apparent. As reviewed recently by Chrispeels et al (1999), CHL1 may also contribute to both the inducible and constitutive high-affinity nitrate transport systems when ammonium is present and/or the pH is low (see Wang et al, 1998). Thus, in addition to being a low-affinity nitrate transporter, CHL1 is also involved in both the inducible and constitutive phases of high-affinity nitrate uptake in Arabidopsis (Liu et al, 1999). CHL1 is a member of the NRT1 family of nitrate transporters. Members of the NRT2 family are high-affinity nitrate transporters, root specific, nitrate inducible and ammonium/glutamine repressible (Chrispeels et al, 1999). Two mutations have been found in a gene (NRT2) of Arabidopsis thaliana that specifically impair constitutive, high-affinity nitrate uptake (Wang and Crawford, 1996). These mutants were selected for resistance to 0.1 mM chlorate in the absence of nitrate (Wang and Crawford, 1996).
The evidence that Arabidopsis CHL1 (AtNRT1) gene encodes an inducible component of low-affinity nitrate uptake, has necessitated a "two-component" model to account for constitutive low-affinity uptake. The CHL1 homolog, AtNRT1:2 (originally named NTL1) encodes a constitutive component of low-affinity nitrate uptake (Huang et al, 1999).
A rice homolog of the Arabidopsis CHL1 (AtNRT1) protein, OsNRT1, has been identified (Lin et al, 2000). In contrast to the dual-affinity nitrate transport activity of CHL1, OsNRT1 displayed only low-affinity nitrate transport [Km = 9 mM] activity in Xenopus oocytes. OsNRT1 is constitutively expressed in the most external layer of the root, epidermis and root hair, and encodes a constitutive component of a low-affinity nitrate uptake system in rice (Lin et al, 2000).
Lejay et al (1999) have examined root nitrate uptake and expression of two root nitrate transporter genes (Nrt2;1 and Nrt1) in response to changes in the N- or C-status of hydroponically grown Arabidopsis thaliana plants. Expression of Nrt2;1 is up-regulated by nitrate starvation in wild-type plants and by N-limitation in a nitrate reductase (NR) deficient mutant transferred to nitrate as sole N source (Lejay et al, 1999). Thus, expression of Nrt2;1 is under feedback repression by N-metabolites resulting from nitrate reduction (Lejay et al, 1999). Expression of Nrt1 is not subject to such a repression. However, Nrt1 is over-expressed in the NR mutant even under N-sufficient conditions (growth on NH4NO3 medium), suggesting that expression of this gene is affected by the presence of active NR, but not by N-status of the plant (Lejay et al, 1999).
A full-length cDNA, GmNRT2, encoding a putative high-affinity nitrate transporter has been isolated from a Glycine max root cDNA library (Amarasinghe et al, 1998). The deduced GmNRT2 protein is 530 amino acids in length and contains 12 putative membrane-spanning domains and a long, hydrophilic C-terminal domain (Amarasinghe et al, 1998). 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 (Amarasinghe et al, 1998). GmNRT2 mRNA levels were barely detectable in ammonium-grown plants, higher in nitrogen-deprived plants, and highest in nitrate-grown plants (Amarasinghe et al, 1998). In Nicotiana plumbaginifolia the NpNRT2.1 gene encodes a putative inducible component of the high-affinity nitrate uptake system (Fraisier et al, 2000). Ammonium, or a further downstream metabolite, may exert a repressive effect on nitrate influx at both transcriptional and post-transcriptional levels (Fraisier et al, 2000).
In the marine angiosperm Zostera marina nitrate (present at micromolar concentrations in seawater) is absorbed against a steep electrochemical potential difference across the plasma membrane via a high-affinity Na+-symport system (Garcia-Sanchez et al, 2000).
The genes nrtP and narB encode nitrate/nitrite permease and nitrate reductase, respectively, in the marine cyanobacterium Synechococcus sp. strain PCC 7002. NrtP is a member of the major facilitator superfamily and is unrelated to the ATP-binding cassette-type nitrate transporters described for freshwater strains of cyanobacteria. However, NrtP is similar to the NRT2-type nitrate transporters found in diverse organisms (Sakamoto et al, 1999).
Amarasinghe BH, de Bruxelles GL, Braddon M, Onyeocha I, Forde BG, Udvardi MK 1998 Regulation of GmNRT2 expression and nitrate transport activity in roots of soybean (Glycine max). Planta 206: 44-52.
Andriesse AJ, Weisbeek PJ, van Arkel GA 1989 Biochemistry and regulatory aspects and genetics of nitrate assimilation in cyanobacteria. In (JL Wray, JR Kinghorn eds) "Molecular and Genetic Aspects of Nitrate Assimilation", Oxford Science Publications, Oxford, pp. 40-50.
Aslam M, Travis RL, Rains DW 1996 Evidence for substrate induction of a nitrate efflux system in barley roots. Plant Physiol. 112: 1167-1175.
Butz RG, Jackson WA 1977 A mechanism for nitrate transport and reduction. Phytochem. 16: 409-417.
Chrispeels MJ, Crawford NM, Schroeder JI 1999 Proteins for transport of water and mineral nutrients across the membranes of plant cells. Plant Cell 11: 661-675.
Fraisier V, Gojon A, Tillard P, Daniel-Vedele F 2000 Constitutive expression of a putative high-affinity nitrate transporter in Nicotiana plumbaginifolia: evidence for post-transcriptional regulation by a reduced nitrogen source. Plant J. 23: 489-496.
Garcia-Sanchez MJ, Jaime MP, Ramos A, Sanders D, Fernandez JA 2000 Sodium-dependent nitrate transport at the plasma membrane of leaf cells of the marine higher plant Zostera marina L. Plant Physiol. 122: 879-886.
Gazzarrini S, Lejay L, Gojon A, Ninnemann O, Frommer WB, von Wiren N 1999 Three functional transporters for constitutive, diurnally regulated, and starvation-induced uptake of ammonium into Arabidopsis roots. Plant Cell 11: 937-948.
Huang NC, Liu KH, Lo HJ, Tsay YF 1999 Cloning and functional characterization of an Arabidopsis nitrate transporter gene that encodes a constitutive component of low-affinity uptake. Plant Cell 11: 1381-1392.
Lejay L, Tillard P, Lepetit M, Olive Fd, Filleur S, Daniel-Vedele F, Gojon A 1999 Molecular and functional regulation of two NO3- uptake systems by N- and C-status of Arabidopsis plants. Plant J. 18: 509-519.
Lin CM, Koh S, Stacey G, Yu SM, Lin TY, Tsay YF 2000 Cloning and functional characterization of a constitutively expressed nitrate transporter gene, OsNRT1, from rice. Plant Physiol. 122: 379-388.
Liu KH, Huang CY, Tsay YF 1999 CHL1 is a dual-affinity nitrate transporter of Arabidopsis involved in multiple phases of nitrate uptake. Plant Cell 11: 865-874.
Sakamoto T, Inoue-Sakamoto K, Bryant DA 1999 A novel nitrate/nitrite permease in the marine cyanobacterium Synechococcus sp. strain PCC 7002. J. Bacteriol. 181: 7363-7372.
Santi S, Locci G, Pinton R, Cesco S, Varanini Z 1995 Plasma membrane H+-ATPase in maize roots induced for NO3- uptake. Plant Physiol. 109: 1277-1283.
Shingles R, Roh MH, McCarty RE 1996 Nitrite transport in chloroplast inner envelope vesicles. I. Direct measurement of proton-linked transport. Plant Physiol. 112: 1375-1381.
Touraine B, Glass ADM 1997 NO3- and ClO3- fluxes in the chl1-5 mutant of Arabidopsis thaliana: does the CHL1-5 gene encode a low-affinity NO3- transporter? Plant Physiol. 114: 137-144.
Tsay Y-F, Schroeder JI, Feldmann KA, Crawford NM 1993 The herbicide sensitivity gene CHL1 of Arabidopsis encodes a nitrate-inducible transporter. Cell 72: 705-713.
Ullrich WR 1992 Transport of nitrate and ammonium through plant membranes. In (K Mengel, DJ Pilbeam eds) "Nitrogen Metabolism of Plants", Clarendon Press, Oxford, pp 121-137.
Wang R, Crawford NM 1996 Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants. Proc. Natl. Acad. Sci. U.S.A. 93: 9297-9301.
Wang R, Liu D, Crawford NM 1998 The Arabidopsis CHL1 protein plays a major role in high-affinity nitrate uptake. Proc. Natl. Acad. Sci. U.S.A. 95: 15134-15139.
Ward MR, Tischner R, Huffaker RC 1988 Inhibition of nitrate transport by anti-nitrate reductase IgG fragments and the identification of plasma membrane associated nitrate reductase in roots of barley seedlings. Plant Physiol. 88: 1141-1145.
Warner RL, Huffaker RC 1989 Nitrate transport is independent of NADH and NAD(P)H nitrate reductases in barley seedlings. Plant Physiol. 91: 947-953.
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