- Email: [email protected]
Nitrate transport: a key step in nitrate assimilation
235
Nitrate transport: a key step in nitrate assimilation Franpise
The nitrate assimilation intensive involved identified
research
pathway
during
has been the matter of
the past decade.
in low and high affinity
nitrate
Many genes
uptake
in fungi, algae and, more recently,
plant genes SO far isolated inducibility
Sophie Filleur and Michel Caboche
Daniel-Vedele”,
by nitrate seems
by their homologs
are transcriptionally to be a common
regulated; feature,
in fungi and algae. A number
remain to be elucidated these transporters
have been in plants. The
regarding
of questions
the physiological
and the regulation
their
shared roles of
Physiology of nitrate uptake
of their expression.
Nitrate is the common form of inorganic nitrogen in most soils and the available concentration can vary enormously depending on pH and oxygen availability [B]. Active
Addresses Laboratoire de Biologic Cellulaire, INRA, Route de St Cyr, 78026 Versailles Cedex, France *e-mail: [email protected] Current Opinion in Plant Biology
nitrate transport across the plasmalemma and cortical cells of the root is the first acquisition and use.
1998, 1:235-239
http://biomednet.com/elecref/1369526600100235 0 Current Biology Ltd ISSN 1369-5266 Abbreviations high affinity transport system HATS low affinity transport system LATS nitrite reductase NiR nitrate reductase NR
Introduction
.
Nitrate is the major source of nitrogen for the vast majority of plants. It is first reduced to nitrite by nitrate reductase (NR), nitrite being further reduced into ammonium by nitrite reductase (NiR). Nitrate assimilation has been the matter of many studies and has been reviewed by Hoff et al. [l], Crawford [Z] and more recently by Daniel-Vedele and Caboche [3] and Campbell [4]. Of particular interest, is the study of the post-transcriptional control of nitrate reductase activity. nitrate metabolism
sensing and nitrate uptake. In this review, we will begin by describing the physiology of nitrate uptake and then go on to discuss the genes involved in low and high affinity transport, and how nitrate transport and reduction is regulated. We will then highlight the many questions that have been brought to light by recent advances in these areas.
This enzyme represents a key step which involves phosphorylation.
in
Nitrate itself also has to be considered as a signal; recent data suggest that nitrate may be a signal for the regulation of carbon metabolism, for example by modulating the expression of genes involved in the biosynthesis of organic acids [SO]. Nitrate also functions as a morphogenetic signal, governing shoot:root balance [6**]. Very recently, the Arabidopsis thaliana ANRl gene was isolated on the basis of its nitrate inducibility. This gene belongs to the MADS box family, whose members code for transcription factors mostly expressed in flowers, affecting the floral organ identity. ANR, specifically expressed in roots, was shown to be a key determinant of developmental plasticity in A. dzaliatanaroots in response to nitrate [7**]. The duality of nitrate, as both a nutrient and a signal, reinforces the need to increase our knowledge of the two primary steps in nitrate acquisition, namely nitrate
of epidermal step in nitrate
Net uptake of nitrate is the difference between its influx and efflux across the plasma membrane. On the basis of physiological studies of nitrate influx, three different types of electrogenic H+/N03symporters have been postulated to exist [9]. At high external nitrate concentration, a low affinity transport system (LATS, Km>05 mM) operates and appears to be constitutively expressed and essentially unregulated. At low external concentrations (O-O.5 mM), two high affinity transport systems (HATS, S
Genes involved in low affinity nitrate transport (LATS) Chlorate, a nitrate analog which to chlorite by nitrate reductase, to select for mutants impaired
is toxic when reduced has been widely used in nitrate uptake or
metabolism. The genes encoding nitrate transporters have been renamed according to the recommended nomenclature: Nrt (nitrate transport) 1 or 2 (for low and high affinity respectively): member number, plant abbreviation. In higher plants, the first mutants defective in nitrate transport were identified in Arabidopsis thahaiana, and were all found to belong to the same complementation group termed c/r/l. A screening of chlorate resistant plants among
236
Physiology and metabolism
T-DNA [14]
tagged
populations
allowed
Tsay and collaborators
to
isolate a disrupted allele of C/VI, renamed NrtZ:lAt according to the agreed nomenclature [1.5], and to demonstrate its role in chlorate toxicity. Nrtl:lAt protein contains 12 putative transmembrane-spanning segments. The expression of Nrtl:IAt is preferentially located in root cells, and is pH dependent and nitrate inducible. In addition, expression of the Nrtl:lAt protein in Xenopus ooqtes exposed to nitrate at acidic pH leads to a depolarization of the membrane potential, which is also the initial response observed when plant cells are exposed to nitrate, to an inward current or to an accumulation of nitrate. The role of a matter of depletion in mutant than
Nrtl:lAt in low affinity nitrate uptake is still debate. Nitrate uptake, measured by nitrate the medium, is found to be lower in the c/z11 in the wild-type or in transgenic C/Z/Zmutant
CMamydomonas reinfiardtii, restoration of nitrate transport in a mutant strain was achieved by co-transformation with the genes nar-2 and NrtZ:lCr or nar-2 and Nrt2:ZCr but not with single plasmids containing the individual genes. In addition, in contrast with ?iar-2 whose function is not yet identified, the deduced amino acid sequences of Nrt2:lCr and Nrt2:ZCr showed significant similarity with the A. niduians CRNA protein (Figure 1 [Zl]). Physiological studies of the complemented strains suggest that three HATS operate in C. reinhardtii: one, encoded by a gene sharing strong similarities with the other members of the family (A Quesada, J Hidalgo, E Fernandez, unpublished data), is specific for nitrite; a second one is encoded by
nar2/Nfl2:2Cr specific for nitrate; and a third one encoded for both anions [22]. by nar2/Nrt2:lCr, which is bispecific CRNA, Nrt2:lCr and Nrt2:2Cr share a common structural motif of 12 transmembrane a-helical segments and a number of conserved sequence motifs [23]. They belong to the major facilitator superfamily which contains five
plants constitutively expressing 35s:: Nrtl:ZAt only when these different genotypes are grown on an ammonium nitrate containing medium. When plants are grown on potassium nitrate alone, however, the depletion rates for
clusters proteins,
the wild-type and the mutant are similar [16**]. This surprising result is confirmed by short-term measurements
intermediates, phosphate ester-phosphate a distinct group of oligosaccharide-proton
of 13N03- influx [ 171. Clearly, the presence of ammonium during the growth period is required to reveal differences between the two genotypes. One explanation for these findings is the possible existence of a second, low affinity nitrate transporter, encoded by an ammonium-repressible gene. The two-gene model for the LATS nitrate uptake system would explain the apparent conflict between the observed constitutive low affinity nitrate transport and the nitrate inducibility of NrtZ:ZAt expression. Recently, to isolate
Nrtl:lAt homologues
was
used as a heterologous probe from tomato [18]. Interestingly,
two different but homologous genes a root hair-specific tomato cDNA
were isolated from library: Nrtl:ILe is
expressed in root hairs as well as other root tissues under all nitrogen treatments applied and may correspond to a constitutively expressed LATS. In contrast, Nrtl:ZLe mRNA accumulation is restricted to root hairs that had been exposed to nitrate. Functional studies remain to be performed, however, to determine characteristics of these two proteins.
the
transport
Genes involved in high affinity nitrate transport (HATS) Recent HATS analysis
progress made in isolating genes involved in the in higher plants originates from the molecular of nitrate uptake in fungi and algae.
The cr~A mutant of Aspergihs nidulam was isolated on the basis of chlorate resistance: this mutant is defective in nitrate uptake at the conidiospore and young mycelium stages. The corresponding gene was isolated [19] and it encodes a 507 amino acid protein, containing 12 putative membrane-spanning domains [ZO]. In the green alga,
of transport proteins, including drug-resistant sugar facilitators, facilitators for Kreb’s cycle antiporters symporters
and [23].
A PCR approach using degenerate oligonucleotides derived from these conserved motifs allowed Trueman and collaborators to isolate barley cDNAs (Nrtz:IHv and Nrtz:ZHv) encoding polypeptides similar to the algal genes Nrt2:lCr and Nrt2:lCr. They have lesser, but significant, similarity to the CRNA protein (Figure 1). As in the case of Nrtl:lAt, these genes are expressed preferentially in barley roots and are induced by nitrate [24**]. Recently, a full length cDNA, N~2:2Np, was isolated from the dicot species Nicotiafza plumbaginifolia, also using a PCR approach [25]. The expression of this gene is root-specific, nitrate-inducible and negatively metabolites. This last observation
regulated by nitrogen is especially striking as
it suggests that the high affinity transporter is under the same regulatory control operating on nitrate and nitrite reduction. Furthermore, nitrate inducibility of Nrt2:ZNp in plants starved of a nitrogen source is only transient; after a rapid initial induction, the Nrt2:INp level decreases even though the internal nitrate concentration remains high (A Krapp et al., unpublished data). This suggests that the process of nitrate uptake by root cells is tightly controlled by the plant nitrogen status. In contrast to algae, there is not yet direct evidence for the physiological role of the plant Nrtz genes. A first interesting correlation, however, comes from the study of a methylammonium resistant mutant of N. plumbaginifoha in the negative [26]. This mutant, MeaR, is impaired feedback regulation of nitrate uptake by methylammonium. It is also impaired in the repression mechanism of Nrf2:ZNp expression by methylammonium (A Krapp et al., unpublished data). A Hansenula pol’ymorpha nitrate transport mutant was recently isolated [27] and, consistent
Nitrate transport: a key step in nitrate assimilation
Figure
Daniel-Vedele, Filleur and Caboche
237
1
231
Sequence alignment and comparison of higher plants, algae and fungi CRNA related proteins: Nrt2:l Hv and Nrt2:2Hv: H. vulgare sequences [24**]; Nrt2:l Np: N. plumbaginifolia sequence [251; Nrt2:lAt: A. fhaliana sequence (this work); NrtP:lCr: C. reinhardtii sequence [211; Nrt2:l Hp: H. polymorpha sequence [271; CRNA : A. nidulans sequence [201. The alignment was performed using the PILEUP program, identities of at least five residues among seven are boxed and shaded, gaps generated in the alignment are indicated by dots. Stretches of sequence homology, represented by shaded areas do not correspond only to membrane spanning regions defined by Trueman et a/. [24*1, but rather are dispersed throughout the protein.
with a nitrate transport function, the Nt&?:Zffv cDNA from barley was able to complement this yeast mutant (M Dunn and B Forde, abstract 682, 5th International Congress of Plant Molecular Biology, Singapore, 21-27 September 1997). In the future, two parallel approaches should give further insights into the function of the plant Nrt genes. The physiological characterization of transgenic plants specifically overexpressing or underexpressing these genes will be of great interest. Loss-of-function mutations affecting these genes would be extremely useful, but cannot be easily screened for, due to the redundancy of such genes; two copies exist in N. pkmbaginifo~ia [ZS], and between 7 and 10 related genes are found in barley [24”]. This goal can be achieved in the model species Arabidopsis thaliana, however, using a reverse genetic approach with PCR experiments performed on a T-DNA insertional library. For this purpose, we have recently isolated by differential display a full length cDNA from A. thaliana, Nd’:ZAt,
which we presume to code for one of the HATS elements (Figure 1; S Filleur, F Daniel-Vedele, unpublished data).
Regulation
of nitrate transport and reduction
It has been known for a long time that the nitrate assimilatory pathway is under tight regulation by the available nitrate and reduced nitrogen. Several of the LATS- and HATS-related genes, apart from being root specific, are also inducible by nitrate, and there is evidence that at least one HATS-related gene, Nd’:ZNp, is also repressible by reduced nitrogen ([ZS], A Krapp et a/., unpublished data). This suggests that regulatory mechanisms are shared by components of the transport system and of the assimilatory pathway. In algae, the N@ and Nrg2 genes are proposed to be responsible for a specific negative control by ammonium (or its derivatives) of the expression of proteins involved in nitrate assimilation, namely NR, NiR and nitrate
238
Physiology
and metabolism
transporters [28]. It will be interesting MeaR loci are related to these regulatory
to test whether elements.
The search for such regulatory loci has led to the identification of AreAINS and NirAJNif4 in fungi [29]. In these organisms, the regulation of NR and NiR genes is governed, on the one hand, by pathway-specific control genes, N&l/Nit4 for A. nidulatzs and Neurospora respectively, involved in nitrate inducibility. On the other hand, AreAIN&, the major positive control genes are involved in N-metabolite repression. It has turned out to be more of a challenge to identify their plant homologs. Putative homologs of Nit2 were isolated [30] from tobacco, but their role in nitrate metabolism is not yet proven. A functional complementation approach aimed at identifying a homolog of the yeast Cltz3 gene, which is related to AreA and Nit2, led to the finding of a new class of genes named RCA1 (restore growth otz ammot&n 1) and RCA2 [31*] in Arabidopsis. These genes are members of a multigene family, a member of which, SCARECROW, is involved in regulating pattern formation in roots [32]. More recently, RGAZ and RCA2 were found to be, respectively, the GAI [33*] and RCA [34] genes involved in giberellin signal transduction. Work is, therefore, under progress in our laboratory to evaluate the putative role of RCA2 and RCA2 in N metabolite regulation.
do not affect inducible, high affinity or constitutive, low affinity nitrate uptake. To which family does the corresponding gene belong? Is there, as found for algae, a two-component high affinity transport system in plants? If so, does a homolog of the nar-2 gene exist in plants? Is there a specific role for each copy of the ctxA related genes found in N. plumbaginifolia or barley? A common feature to all these plant genes is their preferential expression in roots. One additional challenge for the future will be identifying the molecular components of nitrate translocation in shoots and its storage in the vacuole.
Note added in proof The papers referred to in the text as (A Quesada, J Hidalgo, E Fernandez, unpublished data) and (A Krapp et al., unpublished data) have now been accepted for publication [36,37].
References
. *
??
Conclusion: genes involved in nitrate transport, the beginning of a long story? Nrtl and Nrt2 genes define two classes of membrane proteins, probably involved in low and high affinity nitrate transport respectively. At the molecular level, it is still unclear whether they function alone as nitrate transporters or as part of a more sophisticated transport system involving other polypeptides. A number of questions remain unanswered regarding their physiological roles and their regulation. What are the molecular components of the constitutive low affinity transport system? Is there an Nrtl related gene such as the Nrtl:ZLe in tomato? In A. thaliana, an Nrtl:lAt homolog, NTLZ, fits the criteria of a second, constitutive low affinity nitrate transporter [16**]. It has also been shown that Nrtl:lAt is able to transport peptides (T Miller, persona1 communication). Is this latter observation of physiological significance? Finally, are these low affinity transporters also involved in nitrate efflux? The observation of differential turnovfl rates for the uptake and efflux does not support this hypothesis, but suggests rather that’ influx and efflux are independent processes [ll]. It would be of great interest to identify the proteins involved in nitrate efflux and to compare their structural characteristics to that of NRTl and NRTZ proteins. One class of mutants impaired in constitutive high affinity nitrate transport has been identified [35]. These mutants
and recommended
Papers of particular interest, published have been highlighted as:
reading
within the annual period of review,
of special interest of outstanding interest
1.
Hoff T, Truong HN, Caboche M: The use of mutants and transgenic plants to study nitrate assimilation. Plant Cell Environment 1994, 17:489-506.
2.
Crawford NM: Nitrate: Cell 1995, 7:859-868.
3.
Daniel-Vedele F, Caboche M: Molecular analysis of nitrate assimilation in higher plants. CR Acad Sci Paris 1996, 319:961968.
4.
Campbell WH: Nitrate reductase Plant Physiol 1996, 111:355-361.
nutrient
and signal for plant growth.
biochemistry
comes
Plant
of age.
5. .
Scheible WR, Gonzales-Fontes A, Lauerer M, Muller-Rober B, Caboche M, Stitt M: Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. Plant Cell 1997, 9:1-l 7. These authors have investigated the effects of nitrate on carbon and nitrogen metabolism in wild-tvoe tobacco slants and low nitrate reductase exoressina transformants. The& experimenk allowed the distinction betweek events triggered by nitrate itself and changes produced more indirectly as a result of nitrate assimilation. Nitrate initiates an extensive program of gene expression, resulting in co-ordinate alterations in the activities of enzymes in several metabolic pathways. 6. ..
Scheible WR, Lauerer M, Schulze ED, Caboche M, Stitt M: Accumulation of nitrate in the shoot acts as a signal to regulate shoot-root allocation in tobacco. Plant J 1997, Ii:671 691. Tobacco genotypes with low expression of nitrate reductase resemble a nitrate-deficient wild-type with respect to their growth rates and content of organic nitrogenous compounds, but accumulate high levels of nitrate. Nitrate accumulation in the shoots is accompanied by inhibition of root growth and an increase in the shoot:root ratio. 7. ..
Zhang H, Forde BG: An Arabidopsis MADS Box gene that controls nutrient-induced changes in root architecture. Science 1998, 279:407-409. In the course of screening Arabidopsis roots for nitrate-inducible genes, a cDNA clone was identified that has homology to the MADS Box family of transcription factors. The expression of the gene in antisense orientation leads to an increased sensitivity of lateral roots to nitrate inhibition, when nitrate is ubiquitously supplied, and to an inhibition of lateral root elongation
Nitrate
transport:
by localized applications of nitrate. These results demonstrate acts as a signal in the development of the plant root system, nutrition
in
Marschner Academic
9.
Glass ADW, Siddiqui MY: Nitrogen absorption by plant roots. ln Nitrogen Nutrition in Higher F’lants. Edited by Srivastava HS, SinghRP. New Dehli: Associate Publishers; 1995:21-56.
10.
Aslam M, Travis RL, Huffaker RC: Comparative kinetics and reciprocal inhibition of nitrate and nitrite uptake in roots of uninduced and induced barley seedlings. P/ant fhysiol 1992, 99:l 124-l 133.
1 I.
M: Miners/
that nitrate
8.
!%?SS;
a key step in nitrate
higher plants, edn 2. London:
i 9%.
Aslam M, Travis RL, Rains WD: Evidence for substrate induction of a nitrate efflux system in barley roots. Plant Physiol 1996, I 12:i 167-l 175.
12.
King BJ, Siddiqui MY, Ruth T, Warner RL, Glass AD: Feedback regulation of nitrate influx in barley roots by nitrate, nitrite and ammonium. P/ant Physiol 1993, 102:1279-l 286.
13.
lsmande J, Touraine B: N demand and the regulation uptake. Plant Physiol 1994, 105:3-7.
14.
Tsay YF, Schroeder JI, Feldmann KA, Crawford NM: The herbicide sensitivity gene CHLl of Arabidopsis encodes a nitrateinducible nitrate transporter. Cell 1995, 72:705-713.
15.
Caboche M, Campbell W, Crawford NM, Fernhndez E, Kleinhofs Ida S, Mendel R, Omata T, Rothstein S, Wray J: Genes involved in nitrate assimilation. Plant MO/ Biol Rep 1994, 12:545-S49.
Huang NC, Chiang CS, Crawford NM, Tsay YI: C/I/~ encodes a component of the low affinity nitrate uptake system in arabidopsis and shows cell type-specific expression in roots. Plant Cell 1996, 8:2183-2191. In viva uptake measurements show that the amount of nitrate taken up by chll mutants was less than that in the wild-type plants and this uptake deficiency was effectively corrected in 35S:CHL7 transgenic plants of the ch/7 mutant. CHL 1 mRNA is found primarily in the epidermal cells near the root tip and in cells beyond the epidermis when moving basipetally. The low affinity transport system system of Arabidopsis is proposed to be comprised of both inducible and constitutive elements encoded by two different genes. Touraine B, Glass ADM: NO,- and Clo,- fluxes in the chll-5 mutant of Arabidopsis thaliana. Does the Chll-5 gene encode a low affinity NO,- transporter? Plant Physiol 1997, 114:i 37s 144.
18.
Lauter FR, Ninnemann 0, Bucher M, Riesmeier JW, Frommer WB: Preferential expression of an ammonium transport and of two putative nitrate transporters in root hairs of tomato. Proc Nat/ Acad Sci USA 1996, 93:8139-8144.
19.
Unkles SE, Hawker KL, Grieve C, Campbell El, Montague P, Kinghorn JR: crnA encodes a nitrate transporter in Aspergillus nidulans. Proc Nat/ Acad Sci USA 1991, 88:204-208.
20.
Unkles SE, Hawker KL, Grieve C, Campbell El, Montague P, Kinghorn JR: crnA encodes a nitrate transporter in Aspergillus nidulans (correction). Proc Nat/ Acad Sci USA 1995, 82:3076.
21.
Quesada A, Galvan A, Ferngndez E: Identification of nitrate transporter genes in Chlamydomonas reinhardtii. Plant J 1994, 5:407-419.
22.
Galvan A, Quesada A, Ferngndez E: Nitrate and nitrite are transported by different specific transport system and by a bispecific transporter in Chlamydomonas reinhardtii. J Biol Chem 1996, 271:2088-2092.
23.
Trueman U, Onyeocha I, Forde B: Recent advances in molecular biology of a family of eukaryotic high affinity P/ant Physiol Biochem 1996, 34:621-627. transporters.
24. ..
nitrate
Trueman U, Richardson A, Forde B: Molecular cloning of higher plants homologues of the high affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergiilus nidulans Gene 1996, 175:223-231. Degenerate oligonucleotides corresponding to crnA were used in PCR to amplify related sequences from barley root RNA. Two full-length cDNAs were isolated and found to code for hydrophobic proteins with twelve predicted transmembrane domains. NARK from E. co/i, CRNA, NRT2:l Cr and barley
Filleur and Caboche
239
25.
Quesada A, Krapp A, Trueman L, Daniel-Vedele F, Fernbndez E, Forde B, Caboche M: PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high affinity nitrate transporters of the crnA family. Plant MO/ Biol 1997, 34:265274.
26.
Godon C, Krapp A, Leydecker MT, Daniel-Vedele F, Caboche M: Methylammonium-resistant mutants of Nicotiana plumbaginifolia are affected in nitrate transport MO/ Gen Genet 1996, 250:357-366.
27.
Perez MD.‘Gonzalez C. Avila J. Brito N. Siverio J: The YNTl gene encoding the n&ate tr&sporte; in the yeast Hansenula polymorpha is clustered with genes YN/l and YNR7 encoding nitrite reductase and nitrate reductase, and its disruption causes inability to grow in nitrate. Biochem J 1997, 321:397403.
28.
Pietro R, Dubus A, Galvan A, Fernendez E: Isolation and characterization of two negative regulatory mutants for reinhardtii obtained nitrate assimilation in Chlamydomonas insertional mutagenesis. MO/ Gen Genet 1996, 251:461-471.
29.
Marzluf GA: Genetic regulation of nitrogen metabolism fungi. Microbial MO/ Biol Rev 1997, 61 :I -17.
30.
Daniel-Vedele F, Caboche M: A tobacco cDNA clone encoding a GATA-1 zinc finger protein homologous to regulators of nitrogen metabolism in fungi. MO/ Gen Genet 1993, 240:365373.
16. ..
1 7.
Daniel-Vedele,
homologs are members of a subclass within the major facilitator superfamily. The expression of the barley gene was induced by nitrate but not by ammonium. This paper describes for the first time the molecular cloning of putative high affinity nitrate transporters from higher plants.
of nitrate
A,
assimilation
by
in the
31. .
Truong HN, Caboche M, Daniel-Vedele F: Sequence and characterization of two Arabidopsis thaliana cDNAs isolated by functional complementation of a yeast g/n3 gdhl mutant. FEBS Lett 1997, 410:213-218. The expression of two Arabidopsis thaliana cDNAs was found to restore growth on ammonium containing medium of a yeast strain, mutated in both the Gdhl gene encoding the NADP(H)-glutamate dehydrogenase and the regulatory G/n3 gene. The two clones are homologous to each other and define, with the SCARECROW gene, a new family of putative transcription factors. Their expression in plants appears to be constitutive with respect to nitrogen source or organ specificity. 32.
Di Laurenzio I, Wysocka-Diller J, Malamy JE, Pysh L, Helariutta Y, Freshour G, Hahn MG, Feldmann K, Benfey P: The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 1996, 86:423-433.
33. .
Peng J, Carol P, Richards DE, King KE, Cowling R, Murphy GP, Harberd NP: The Arabidopsis GA/ gene defines a signaling pathway that negatively regulates giberellin responses. Genes Develop 1997, 1 I :3194-3205. This reports the molecular cloning of GA/ and a closely related gene GRS, both of which are involved in giberellin signaling. The authors demonstrate that, like the pathway that operates as a negative regulator of the ethylene response, a negative and derepressible regulatory system is the mechanism by which giberellin regulates plant development. It is rather surprising that the GA/ and GRS genes correspond to genes that are able to restore growth on ammonium (RGAI and RGAP) of a yeast mutant strain [31]. 34.
Silverstone AL, Ciampaglio CN, Sun TP: The Arabidopsis RGA gene encodes a transcriptional regulator repressing the giberellin signal transduction pathway. P/ant Cell 1998, 1O:in press.
35.
Wang R, Crawford NM: Genetic identification of a gene involved in constitutive, high-affinity nitrate transport in higher plants. F’roc Nat/ Acad Sci USA 1996, 93:9297-9301,
36.
Quesada A, Hidalgo J, Fernandez E: Three Nr2 genes are differentially regulated in Chlamydomonas reinhardtii. MO/ Gen Genet 1998, in press.
37.
Krapp A, Fraisier V, Scheible A, Queseda A, Gojon A, Stitt M, Caboche M, Daniel-Vedele F: Expression studies of NrtZ:fNp, a putative high affinity nitrate transporter: evidence for its role in nitrate uptake. P/ant J 1998, in press.
Nitrate transport: a key step in nitrate assimilation Franpise
The nitrate assimilation intensive involved identified
research
pathway
during
has been the matter of
the past decade.
in low and high affinity
nitrate
Many genes
uptake
in fungi, algae and, more recently,
plant genes SO far isolated inducibility
Sophie Filleur and Michel Caboche
Daniel-Vedele”,
by nitrate seems
by their homologs
are transcriptionally to be a common
regulated; feature,
in fungi and algae. A number
remain to be elucidated these transporters
have been in plants. The
regarding
of questions
the physiological
and the regulation
their
shared roles of
Physiology of nitrate uptake
of their expression.
Nitrate is the common form of inorganic nitrogen in most soils and the available concentration can vary enormously depending on pH and oxygen availability [B]. Active
Addresses Laboratoire de Biologic Cellulaire, INRA, Route de St Cyr, 78026 Versailles Cedex, France *e-mail: [email protected] Current Opinion in Plant Biology
nitrate transport across the plasmalemma and cortical cells of the root is the first acquisition and use.
1998, 1:235-239
http://biomednet.com/elecref/1369526600100235 0 Current Biology Ltd ISSN 1369-5266 Abbreviations high affinity transport system HATS low affinity transport system LATS nitrite reductase NiR nitrate reductase NR
Introduction
.
Nitrate is the major source of nitrogen for the vast majority of plants. It is first reduced to nitrite by nitrate reductase (NR), nitrite being further reduced into ammonium by nitrite reductase (NiR). Nitrate assimilation has been the matter of many studies and has been reviewed by Hoff et al. [l], Crawford [Z] and more recently by Daniel-Vedele and Caboche [3] and Campbell [4]. Of particular interest, is the study of the post-transcriptional control of nitrate reductase activity. nitrate metabolism
sensing and nitrate uptake. In this review, we will begin by describing the physiology of nitrate uptake and then go on to discuss the genes involved in low and high affinity transport, and how nitrate transport and reduction is regulated. We will then highlight the many questions that have been brought to light by recent advances in these areas.
This enzyme represents a key step which involves phosphorylation.
in
Nitrate itself also has to be considered as a signal; recent data suggest that nitrate may be a signal for the regulation of carbon metabolism, for example by modulating the expression of genes involved in the biosynthesis of organic acids [SO]. Nitrate also functions as a morphogenetic signal, governing shoot:root balance [6**]. Very recently, the Arabidopsis thaliana ANRl gene was isolated on the basis of its nitrate inducibility. This gene belongs to the MADS box family, whose members code for transcription factors mostly expressed in flowers, affecting the floral organ identity. ANR, specifically expressed in roots, was shown to be a key determinant of developmental plasticity in A. dzaliatanaroots in response to nitrate [7**]. The duality of nitrate, as both a nutrient and a signal, reinforces the need to increase our knowledge of the two primary steps in nitrate acquisition, namely nitrate
of epidermal step in nitrate
Net uptake of nitrate is the difference between its influx and efflux across the plasma membrane. On the basis of physiological studies of nitrate influx, three different types of electrogenic H+/N03symporters have been postulated to exist [9]. At high external nitrate concentration, a low affinity transport system (LATS, Km>05 mM) operates and appears to be constitutively expressed and essentially unregulated. At low external concentrations (O-O.5 mM), two high affinity transport systems (HATS, S
Genes involved in low affinity nitrate transport (LATS) Chlorate, a nitrate analog which to chlorite by nitrate reductase, to select for mutants impaired
is toxic when reduced has been widely used in nitrate uptake or
metabolism. The genes encoding nitrate transporters have been renamed according to the recommended nomenclature: Nrt (nitrate transport) 1 or 2 (for low and high affinity respectively): member number, plant abbreviation. In higher plants, the first mutants defective in nitrate transport were identified in Arabidopsis thahaiana, and were all found to belong to the same complementation group termed c/r/l. A screening of chlorate resistant plants among
236
Physiology and metabolism
T-DNA [14]
tagged
populations
allowed
Tsay and collaborators
to
isolate a disrupted allele of C/VI, renamed NrtZ:lAt according to the agreed nomenclature [1.5], and to demonstrate its role in chlorate toxicity. Nrtl:lAt protein contains 12 putative transmembrane-spanning segments. The expression of Nrtl:IAt is preferentially located in root cells, and is pH dependent and nitrate inducible. In addition, expression of the Nrtl:lAt protein in Xenopus ooqtes exposed to nitrate at acidic pH leads to a depolarization of the membrane potential, which is also the initial response observed when plant cells are exposed to nitrate, to an inward current or to an accumulation of nitrate. The role of a matter of depletion in mutant than
Nrtl:lAt in low affinity nitrate uptake is still debate. Nitrate uptake, measured by nitrate the medium, is found to be lower in the c/z11 in the wild-type or in transgenic C/Z/Zmutant
CMamydomonas reinfiardtii, restoration of nitrate transport in a mutant strain was achieved by co-transformation with the genes nar-2 and NrtZ:lCr or nar-2 and Nrt2:ZCr but not with single plasmids containing the individual genes. In addition, in contrast with ?iar-2 whose function is not yet identified, the deduced amino acid sequences of Nrt2:lCr and Nrt2:ZCr showed significant similarity with the A. niduians CRNA protein (Figure 1 [Zl]). Physiological studies of the complemented strains suggest that three HATS operate in C. reinhardtii: one, encoded by a gene sharing strong similarities with the other members of the family (A Quesada, J Hidalgo, E Fernandez, unpublished data), is specific for nitrite; a second one is encoded by
nar2/Nfl2:2Cr specific for nitrate; and a third one encoded for both anions [22]. by nar2/Nrt2:lCr, which is bispecific CRNA, Nrt2:lCr and Nrt2:2Cr share a common structural motif of 12 transmembrane a-helical segments and a number of conserved sequence motifs [23]. They belong to the major facilitator superfamily which contains five
plants constitutively expressing 35s:: Nrtl:ZAt only when these different genotypes are grown on an ammonium nitrate containing medium. When plants are grown on potassium nitrate alone, however, the depletion rates for
clusters proteins,
the wild-type and the mutant are similar [16**]. This surprising result is confirmed by short-term measurements
intermediates, phosphate ester-phosphate a distinct group of oligosaccharide-proton
of 13N03- influx [ 171. Clearly, the presence of ammonium during the growth period is required to reveal differences between the two genotypes. One explanation for these findings is the possible existence of a second, low affinity nitrate transporter, encoded by an ammonium-repressible gene. The two-gene model for the LATS nitrate uptake system would explain the apparent conflict between the observed constitutive low affinity nitrate transport and the nitrate inducibility of NrtZ:ZAt expression. Recently, to isolate
Nrtl:lAt homologues
was
used as a heterologous probe from tomato [18]. Interestingly,
two different but homologous genes a root hair-specific tomato cDNA
were isolated from library: Nrtl:ILe is
expressed in root hairs as well as other root tissues under all nitrogen treatments applied and may correspond to a constitutively expressed LATS. In contrast, Nrtl:ZLe mRNA accumulation is restricted to root hairs that had been exposed to nitrate. Functional studies remain to be performed, however, to determine characteristics of these two proteins.
the
transport
Genes involved in high affinity nitrate transport (HATS) Recent HATS analysis
progress made in isolating genes involved in the in higher plants originates from the molecular of nitrate uptake in fungi and algae.
The cr~A mutant of Aspergihs nidulam was isolated on the basis of chlorate resistance: this mutant is defective in nitrate uptake at the conidiospore and young mycelium stages. The corresponding gene was isolated [19] and it encodes a 507 amino acid protein, containing 12 putative membrane-spanning domains [ZO]. In the green alga,
of transport proteins, including drug-resistant sugar facilitators, facilitators for Kreb’s cycle antiporters symporters
and [23].
A PCR approach using degenerate oligonucleotides derived from these conserved motifs allowed Trueman and collaborators to isolate barley cDNAs (Nrtz:IHv and Nrtz:ZHv) encoding polypeptides similar to the algal genes Nrt2:lCr and Nrt2:lCr. They have lesser, but significant, similarity to the CRNA protein (Figure 1). As in the case of Nrtl:lAt, these genes are expressed preferentially in barley roots and are induced by nitrate [24**]. Recently, a full length cDNA, N~2:2Np, was isolated from the dicot species Nicotiafza plumbaginifolia, also using a PCR approach [25]. The expression of this gene is root-specific, nitrate-inducible and negatively metabolites. This last observation
regulated by nitrogen is especially striking as
it suggests that the high affinity transporter is under the same regulatory control operating on nitrate and nitrite reduction. Furthermore, nitrate inducibility of Nrt2:ZNp in plants starved of a nitrogen source is only transient; after a rapid initial induction, the Nrt2:INp level decreases even though the internal nitrate concentration remains high (A Krapp et al., unpublished data). This suggests that the process of nitrate uptake by root cells is tightly controlled by the plant nitrogen status. In contrast to algae, there is not yet direct evidence for the physiological role of the plant Nrtz genes. A first interesting correlation, however, comes from the study of a methylammonium resistant mutant of N. plumbaginifoha in the negative [26]. This mutant, MeaR, is impaired feedback regulation of nitrate uptake by methylammonium. It is also impaired in the repression mechanism of Nrf2:ZNp expression by methylammonium (A Krapp et al., unpublished data). A Hansenula pol’ymorpha nitrate transport mutant was recently isolated [27] and, consistent
Nitrate transport: a key step in nitrate assimilation
Figure
Daniel-Vedele, Filleur and Caboche
237
1
231
Sequence alignment and comparison of higher plants, algae and fungi CRNA related proteins: Nrt2:l Hv and Nrt2:2Hv: H. vulgare sequences [24**]; Nrt2:l Np: N. plumbaginifolia sequence [251; Nrt2:lAt: A. fhaliana sequence (this work); NrtP:lCr: C. reinhardtii sequence [211; Nrt2:l Hp: H. polymorpha sequence [271; CRNA : A. nidulans sequence [201. The alignment was performed using the PILEUP program, identities of at least five residues among seven are boxed and shaded, gaps generated in the alignment are indicated by dots. Stretches of sequence homology, represented by shaded areas do not correspond only to membrane spanning regions defined by Trueman et a/. [24*1, but rather are dispersed throughout the protein.
with a nitrate transport function, the Nt&?:Zffv cDNA from barley was able to complement this yeast mutant (M Dunn and B Forde, abstract 682, 5th International Congress of Plant Molecular Biology, Singapore, 21-27 September 1997). In the future, two parallel approaches should give further insights into the function of the plant Nrt genes. The physiological characterization of transgenic plants specifically overexpressing or underexpressing these genes will be of great interest. Loss-of-function mutations affecting these genes would be extremely useful, but cannot be easily screened for, due to the redundancy of such genes; two copies exist in N. pkmbaginifo~ia [ZS], and between 7 and 10 related genes are found in barley [24”]. This goal can be achieved in the model species Arabidopsis thaliana, however, using a reverse genetic approach with PCR experiments performed on a T-DNA insertional library. For this purpose, we have recently isolated by differential display a full length cDNA from A. thaliana, Nd’:ZAt,
which we presume to code for one of the HATS elements (Figure 1; S Filleur, F Daniel-Vedele, unpublished data).
Regulation
of nitrate transport and reduction
It has been known for a long time that the nitrate assimilatory pathway is under tight regulation by the available nitrate and reduced nitrogen. Several of the LATS- and HATS-related genes, apart from being root specific, are also inducible by nitrate, and there is evidence that at least one HATS-related gene, Nd’:ZNp, is also repressible by reduced nitrogen ([ZS], A Krapp et a/., unpublished data). This suggests that regulatory mechanisms are shared by components of the transport system and of the assimilatory pathway. In algae, the N@ and Nrg2 genes are proposed to be responsible for a specific negative control by ammonium (or its derivatives) of the expression of proteins involved in nitrate assimilation, namely NR, NiR and nitrate
238
Physiology
and metabolism
transporters [28]. It will be interesting MeaR loci are related to these regulatory
to test whether elements.
The search for such regulatory loci has led to the identification of AreAINS and NirAJNif4 in fungi [29]. In these organisms, the regulation of NR and NiR genes is governed, on the one hand, by pathway-specific control genes, N&l/Nit4 for A. nidulatzs and Neurospora respectively, involved in nitrate inducibility. On the other hand, AreAIN&, the major positive control genes are involved in N-metabolite repression. It has turned out to be more of a challenge to identify their plant homologs. Putative homologs of Nit2 were isolated [30] from tobacco, but their role in nitrate metabolism is not yet proven. A functional complementation approach aimed at identifying a homolog of the yeast Cltz3 gene, which is related to AreA and Nit2, led to the finding of a new class of genes named RCA1 (restore growth otz ammot&n 1) and RCA2 [31*] in Arabidopsis. These genes are members of a multigene family, a member of which, SCARECROW, is involved in regulating pattern formation in roots [32]. More recently, RGAZ and RCA2 were found to be, respectively, the GAI [33*] and RCA [34] genes involved in giberellin signal transduction. Work is, therefore, under progress in our laboratory to evaluate the putative role of RCA2 and RCA2 in N metabolite regulation.
do not affect inducible, high affinity or constitutive, low affinity nitrate uptake. To which family does the corresponding gene belong? Is there, as found for algae, a two-component high affinity transport system in plants? If so, does a homolog of the nar-2 gene exist in plants? Is there a specific role for each copy of the ctxA related genes found in N. plumbaginifolia or barley? A common feature to all these plant genes is their preferential expression in roots. One additional challenge for the future will be identifying the molecular components of nitrate translocation in shoots and its storage in the vacuole.
Note added in proof The papers referred to in the text as (A Quesada, J Hidalgo, E Fernandez, unpublished data) and (A Krapp et al., unpublished data) have now been accepted for publication [36,37].
References
. *
??
Conclusion: genes involved in nitrate transport, the beginning of a long story? Nrtl and Nrt2 genes define two classes of membrane proteins, probably involved in low and high affinity nitrate transport respectively. At the molecular level, it is still unclear whether they function alone as nitrate transporters or as part of a more sophisticated transport system involving other polypeptides. A number of questions remain unanswered regarding their physiological roles and their regulation. What are the molecular components of the constitutive low affinity transport system? Is there an Nrtl related gene such as the Nrtl:ZLe in tomato? In A. thaliana, an Nrtl:lAt homolog, NTLZ, fits the criteria of a second, constitutive low affinity nitrate transporter [16**]. It has also been shown that Nrtl:lAt is able to transport peptides (T Miller, persona1 communication). Is this latter observation of physiological significance? Finally, are these low affinity transporters also involved in nitrate efflux? The observation of differential turnovfl rates for the uptake and efflux does not support this hypothesis, but suggests rather that’ influx and efflux are independent processes [ll]. It would be of great interest to identify the proteins involved in nitrate efflux and to compare their structural characteristics to that of NRTl and NRTZ proteins. One class of mutants impaired in constitutive high affinity nitrate transport has been identified [35]. These mutants
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reading
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Scheible WR, Gonzales-Fontes A, Lauerer M, Muller-Rober B, Caboche M, Stitt M: Nitrate acts as a signal to induce organic acid metabolism and repress starch metabolism in tobacco. Plant Cell 1997, 9:1-l 7. These authors have investigated the effects of nitrate on carbon and nitrogen metabolism in wild-tvoe tobacco slants and low nitrate reductase exoressina transformants. The& experimenk allowed the distinction betweek events triggered by nitrate itself and changes produced more indirectly as a result of nitrate assimilation. Nitrate initiates an extensive program of gene expression, resulting in co-ordinate alterations in the activities of enzymes in several metabolic pathways. 6. ..
Scheible WR, Lauerer M, Schulze ED, Caboche M, Stitt M: Accumulation of nitrate in the shoot acts as a signal to regulate shoot-root allocation in tobacco. Plant J 1997, Ii:671 691. Tobacco genotypes with low expression of nitrate reductase resemble a nitrate-deficient wild-type with respect to their growth rates and content of organic nitrogenous compounds, but accumulate high levels of nitrate. Nitrate accumulation in the shoots is accompanied by inhibition of root growth and an increase in the shoot:root ratio. 7. ..
Zhang H, Forde BG: An Arabidopsis MADS Box gene that controls nutrient-induced changes in root architecture. Science 1998, 279:407-409. In the course of screening Arabidopsis roots for nitrate-inducible genes, a cDNA clone was identified that has homology to the MADS Box family of transcription factors. The expression of the gene in antisense orientation leads to an increased sensitivity of lateral roots to nitrate inhibition, when nitrate is ubiquitously supplied, and to an inhibition of lateral root elongation
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Huang NC, Chiang CS, Crawford NM, Tsay YI: C/I/~ encodes a component of the low affinity nitrate uptake system in arabidopsis and shows cell type-specific expression in roots. Plant Cell 1996, 8:2183-2191. In viva uptake measurements show that the amount of nitrate taken up by chll mutants was less than that in the wild-type plants and this uptake deficiency was effectively corrected in 35S:CHL7 transgenic plants of the ch/7 mutant. CHL 1 mRNA is found primarily in the epidermal cells near the root tip and in cells beyond the epidermis when moving basipetally. The low affinity transport system system of Arabidopsis is proposed to be comprised of both inducible and constitutive elements encoded by two different genes. Touraine B, Glass ADM: NO,- and Clo,- fluxes in the chll-5 mutant of Arabidopsis thaliana. Does the Chll-5 gene encode a low affinity NO,- transporter? Plant Physiol 1997, 114:i 37s 144.
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Lauter FR, Ninnemann 0, Bucher M, Riesmeier JW, Frommer WB: Preferential expression of an ammonium transport and of two putative nitrate transporters in root hairs of tomato. Proc Nat/ Acad Sci USA 1996, 93:8139-8144.
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Unkles SE, Hawker KL, Grieve C, Campbell El, Montague P, Kinghorn JR: crnA encodes a nitrate transporter in Aspergillus nidulans. Proc Nat/ Acad Sci USA 1991, 88:204-208.
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Unkles SE, Hawker KL, Grieve C, Campbell El, Montague P, Kinghorn JR: crnA encodes a nitrate transporter in Aspergillus nidulans (correction). Proc Nat/ Acad Sci USA 1995, 82:3076.
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Trueman U, Richardson A, Forde B: Molecular cloning of higher plants homologues of the high affinity nitrate transporters of Chlamydomonas reinhardtii and Aspergiilus nidulans Gene 1996, 175:223-231. Degenerate oligonucleotides corresponding to crnA were used in PCR to amplify related sequences from barley root RNA. Two full-length cDNAs were isolated and found to code for hydrophobic proteins with twelve predicted transmembrane domains. NARK from E. co/i, CRNA, NRT2:l Cr and barley
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Quesada A, Krapp A, Trueman L, Daniel-Vedele F, Fernbndez E, Forde B, Caboche M: PCR-identification of a Nicotiana plumbaginifolia cDNA homologous to the high affinity nitrate transporters of the crnA family. Plant MO/ Biol 1997, 34:265274.
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Truong HN, Caboche M, Daniel-Vedele F: Sequence and characterization of two Arabidopsis thaliana cDNAs isolated by functional complementation of a yeast g/n3 gdhl mutant. FEBS Lett 1997, 410:213-218. The expression of two Arabidopsis thaliana cDNAs was found to restore growth on ammonium containing medium of a yeast strain, mutated in both the Gdhl gene encoding the NADP(H)-glutamate dehydrogenase and the regulatory G/n3 gene. The two clones are homologous to each other and define, with the SCARECROW gene, a new family of putative transcription factors. Their expression in plants appears to be constitutive with respect to nitrogen source or organ specificity. 32.
Di Laurenzio I, Wysocka-Diller J, Malamy JE, Pysh L, Helariutta Y, Freshour G, Hahn MG, Feldmann K, Benfey P: The SCARECROW gene regulates an asymmetric cell division that is essential for generating the radial organization of the Arabidopsis root. Cell 1996, 86:423-433.
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Peng J, Carol P, Richards DE, King KE, Cowling R, Murphy GP, Harberd NP: The Arabidopsis GA/ gene defines a signaling pathway that negatively regulates giberellin responses. Genes Develop 1997, 1 I :3194-3205. This reports the molecular cloning of GA/ and a closely related gene GRS, both of which are involved in giberellin signaling. The authors demonstrate that, like the pathway that operates as a negative regulator of the ethylene response, a negative and derepressible regulatory system is the mechanism by which giberellin regulates plant development. It is rather surprising that the GA/ and GRS genes correspond to genes that are able to restore growth on ammonium (RGAI and RGAP) of a yeast mutant strain [31]. 34.
Silverstone AL, Ciampaglio CN, Sun TP: The Arabidopsis RGA gene encodes a transcriptional regulator repressing the giberellin signal transduction pathway. P/ant Cell 1998, 1O:in press.
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