Nitrate reductase: a target for molecular and cellular studies in higher plants

BEVIEWS

Nitrate reductase: a target for molecular and cellular studies in higher plants

N i t r a t e can be used as the sole nitrogen source to sustain growth, in both microorganisms and higher #ants. However, the requirement for nitrate can be bypassed by supplying nitrogen in reduced form, such as ammonia. The discovery of chlorate as a potent selective agent against cells expressing a nitrate-reducing activity led Cove and co-workers to develop a ROUZ~ genetic approach to study nitrate assimilation in the MICHEL c a m m m Am) m fungus AsJX,rgiUus nidulan#. Nitrate metabolism has in the ussimff~iml been characterized in other fungi and in other organ- Niiraie mduciase (NR) is a key ~ isms by a sinfilar approach; the corresponding studies of t~trate by plam~. NR expvessioa c n be selected either have been reviewed in the proceedings of a recent for or a&aimg both at the cellular level and at the level of meeting2 and more specifically for higher plants by the who~ plng attd manwrous mataffts affected at the Wray3. We will focus this review on recent advances in Iocusfor the ala structural geK-whtch etgodes the NR the molecular and genetic analysis of plant nitrate apoemTme-bave beea tdeatifleat The ~a gene, which has reductase (NR), the enzyme that catalyses the fast step tow bees cloae~ is a useful toolfor moleculargenetic stud/es ta M&herplaats,'furthermore, a combhwdgeztet/c of the nitrate assimilatory pathway. aml biochemical approach to studFlag NR should allow an Structure, functions and isoforms of nitrate reductase iasight iato the catalytic process of a mutltceater redox Plant nitrate reductases catalyse the reduction of eazym~ nitrate to nitrite, using NADH or NADPH as reducing substrate. They have been shown to be multimeric enzymes, each monomer containing three redox prosthetic groups - FAD, heine and a molybdenum cofactor (MoCo) (stoichiometry 1:1:1) - which, in that order, catalytically transfer two electrons from NAD(P)H to nitrate't. In vitro, the electron flow can be shunted by artificial donors or acceptors (Fig. 1). Thus NR 'partial' activities can be assayed that require only one or two of the three prosthetic groups. These activities are classified as diaphorases ff they use NAD(P)H

Artificial e- acoeptors ~W~m~iill~~ e-donors[

~]~

FMNH2 ~ Viologens-I'~

~

NADH

~ /~..._.p"

~ i ~ W

HAD+

i NH~

W

~

BPBI

NO3 j

~

-

NO

as electron donor, or as 'terminal' activities when they use nitrate as acceptor. NADH:NR (EC 1.6.6.1) is the most common form of nitrate reductase in higher plants, and it appears to be a homodimer (monomer size, between 100 and 120 kDa). It is also one of the best characterized plant enzymes, mainly because of the biochemical studies carried out on the Chlorella NADH:NR. This algal enzyme is more abundant and stable than higher plant NRs, and active dimers tend to aggregate Lnto tetramers at high concentration4. A bispecific NAD(P)H:NR (EC 1.6.6.2) is found in eukaryotic algae; this form is also found in several plants (barley, corn, rice) as a second isoform predominantly present in roots. Both the NADH-specific and the bispecific enzymes have a pH optimum of 7.5 and are nitrate inducible3. In nitrogenfixing plants, peculiar situations have been described: a so-called 'constitutive' NR (expressed in the absence of nitrate) was identified in several leguminous !r~,~ specie# and in aide#. In soybean, three isoforms were isolated: the ubiquitous, inducible NADH:NR and two constitutive "" NRs, one NADH dependent and one bispecific; both had an unusual pH optimum for catalytic activity of 6.53.

protease sensitive HINGE

HaO Structure and properties of higher plant NR. In this hypothetical model of a dhneric NR molec~Ae,the electron donor and acceptor sites for NADH:NR and p,agial enzymatic a~vities are shown on one subunit (lefC, on the other subunit (right), the main structural featu~msare shown: the three catalyticdomains with their respective redox prosthetic groups, the amino-terminaldomain (here shown involved in the interaction between subunits), the amino and carboxy termini of the polypeptide chain. T[GJUNE1990 VOW.6 NO. 6 ©1990 EP_,cvierScience Publishers Lid (UK) 0168 - 9479/90/$02.00

/

Selection and phenotypes of mutants defldent for a ~ structural gene Three methods have been used to identiff mutant plants impaired in expression of nitrate reductase. The most laborious approach involves assaying the enzyme directly in the leaves of plants derived from mutagenized seeds. Mutants can also be selected on the basis of chlorate resistance or by screening for the inability to grow on nitrate as sole nitrogen source, either at the whole plant level, or in cell culture followed by regeneration into plants.

EVIEWS Among the mutants that have been obtained, complementation groups corresponding to the NR structural gene (which encodes the NR apoenzyme) have been identified in several plant species and given the general name nia. These complementation groups are characterized by the presence of mutants defective for some, or sometimes all, of the partial catalytic activities normally carried out by NR. In Nicotiana plumbaginifolia, nfa mutants completely lacking NR activity and therefore unable to grow on nitrate as the sole nitrogen source have been identified. These mutants can develop and set seeds, though poorly, when provided with ammonium and calcium carbonate as substitutes for potassium nitrate 7. In our laboratory, routine analysis of more than 130 nia mutant plants, after their grafting onto wild-type stocks, revealed that NR deficiency was systematically correlated with a chlorotic phenotype and with a severe defect in the production

of organic acids in the leavesS. Chlorosis is not due to poisoning by non-metabolized nitrate or to nitrogen depletion. Although NR has been postulated to be involved in iron metabolism9, we observed that NRdeficient mutants do not display the chlorotic phenotype typical of iron-starved plants and can use Fe3÷ ions from the environment. It has been suggested by Huffaker and co-worker# 0 that NR is coupled with the nitrate carrier system located at the plasma membrane. However, NR-deficient mutants of barley n or N. plumbaginifolia are able to accumulate nitrate into their leaves.

Mutants deficient for the biosynthesis of the molybdenum cofactor of Nit The molybdenum cofactor is a complex of the metal molybdenum with an organic moiety, molybdopterin. The structure of MoCo has recently been elucidatedl2,

T~tE 1. Genetic a n d moleotl-r data o n nia genes and nitrate reductase lsoforms in various plant species

Species

Loam a

Genomlc done b

eDNA

Arabfdops~ tbaliana

chl-2 (1)

-

-

chr. 2

cbl-3 (4) ?

nfa2 nial

Full length Partial

chr. 1 chr. 1

nft-1 1d

ND

Linked to pfl3

Map/RFI.P

Comments n/a gene? regulatory? Not linked to nfal Near gl2 NAD(P)H:NR

Cblamydomonas refnbardttf

nft-1c

Cblorella spp.

nft-1c (8)

ND

ND

ND

NADH:NR

Glycine max (soybean)

X (2)

ND

ND

ND

?

ND

ND

ND

ND

ND

ND

ND

ND

ND

'Constitutive' NADH:NR 'Constitutive' NAD(P)H:NR Both cNRe absent, regulatory? Inducible NADH:NR

? -

Partial -

chr. 6 Not linked to narl

n/a 1

ND

ND

NADH:NR

Id

Partial

ND

nta2 I d

Partial

ND

From N. tomentoaformfs From N. sylvestr/s ancestor

ND I d

Full length

ND

NADH:NR

nial ? ? ?

ND ND

ND ND

NADH:NR NAD(P)H:NR

nrl (3)

? Hordeum vulsare (barley) Lycopemcon esculenturn (tomato) Nicotiana tabacum ftobacco)

narl (22) nat7 (1)

?

nial

nial

(39~ nfa2

N. plumbasinifolta Oryza satWa (flee)

nk~c (65) ? ?

aName of the locus and, in brackets, number of mutants tested. bName and number of nfa genes per haploid genome. cOb,sewed intragenic complementatton. '~-,omplementation obtained by transformation with an isolated n~ gene.

ecNR, 'constitutivet NR.

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NADH:NR NAD(P)H:NR

g~EVIEWS but its biosynthetic pathway remains largely Genes TATATAA unknown. Since the enzyme xanthine dehyI .ATG TAA drogenase (XDH) also requires MoCo, t o m a t o nla ~mm~ mutants impaired in the biosynthesis of t~/4398102774141 ~0277i~i~s4s e40233437 ~ 1~ ~ ~ MoCo have been isolated and identified on the basis of simultaneous loss of XDH and NR activity. The corresponding loci were ~obacco nla.1 ~ ~ ~ . - , . , - • ,• 89 1012 2/4141 912 233 652 1328 300? named cnx. In A. nidulans, in which intensive investigations have been performed, six cnx loci have been identifiedL The most ttobacc© o b a c c o nla.2 m m ~ detailed genetic analysis of the MoCo -II 1~ 1012 ,4 141 1298 . 3 ~ . biosynthesis pathway in higher plants has been performed in N. plumbaginifolia, where six cnx complementation groups a-1 m~ have been identified among 54 mutants rice ni nia simultaneously deficient for NR and XDH13. Miiller and co-worker# 4 have shown that one of them, cnxA, has similar characterisIRNA i ~ : ~ l / tics to the chlG locus of E. coil and cnxE of mRNI A. nidulans, since it is not defective in Exons molybdate uptake and can be restored to ....=m Introns the wild-type phenotype in the presence of Protei )rein domains wa2.4~z~\\\\\,,k~////~.coo. high molybdate concentrations. The cn.xA r--! ilflralIslatedmRNA locus is thought to be involved in the inseremmmi untranscribed DNA Subslrates, colaclors ~ tion of the molybdate ion into molyband electron flux dopterin. Other cnx loci are presumed to be involved in the biosynthesis of the FIGM molybdopterin. Structure of higher plant nfa genes. Note that intron positions do not Apart from being impaired in nitrate metabolism, cnx mutants are also defective correlate with the boundaries of functional domains in the proteins. For each gene, the lengths of the mRNAleader sequence, and of exons and in other functions involving the molyb- introns in the coding sequence are given as numbers of nucleotides. denum cofactor. Walker-Simmons and coworker#5 have described a barley cnx Molecular probes specific for the nla genes can mutant with a thermosensitive wilty phenotype. The basis for this phenotype has been attributed to a also be used to establish the copy number of these defect in abscisic acid (ABA) biosynthesis, possibly genes. NR apoenzyme cDNAs have been cloned in linked to the absence of a MoCo-dependent aldehyde barley, squash, tobacco, A. thalian# s and corna; nla genes have been recently isolated and sequenced in oxidase activity. Grafted MoCo-deficient N. plumbaginifolia plants do not display the characteris- tobacco19, tomato 20, rice 21, and the green alga Chlamydomonas reinhardtiz22. Southern hybridization tics of ABA-deficient mutants. However, these cnx mutants, unlike nia mutants, systematically display studies with NR eDNA probes generally conf'Lrmgenetic developmental abnormalities in vitro, including data, with the exception that restriction fragment polymorphism (RFLP) mapping of the nla genes in the A. reduced leaf size and long intemodes. thaliana genome suggests that chl-2 is probably not a nfa locusm. How many functional tda genes? The general organization of the plant-derived nia Molecular and genetic analysis confirms that the number of nia genes varies in different species. The genes was found to be very similar (Fig. 2), their codgenetic analysis of mutants deficient in the NR apo- ing sequenc.~ .being interrupted by three introns of enzyme, by complementation and biochemical tests, variable siz-~ but located at strictly conserved positions. allows one to establish the, number of loci involved in Sequence analysis of the C. reinhardtil nit-1 gene, apoenzyme synthesis (Table 1). Only one ne':~ locus however, indicates that the intron positions differ from those found in higher plants; this was also observed was found in N. plumbaginifolia7. Tobacco (N. tabacum), an amphidiploid species resulting from the for the A. mdulans niaD gene 23. In tobacco, N. plumbaginifolia 24 and C. reqnhardtiz22, nfa mutants hybridization between iV. sylvestris and N. tomentosiformis, has two functional homologous nfa loci (nial, were successfully complemented by genetic transfornia2)~4. In barley, the narl and nar7 loci were found mation with a cloned n/a gene, thus providing further to correspond to NADH:NR and NAD(P)H:NR, respec- evidence that the nia genes that have been identified tively. All nat1 and nat7 mutants can grow on nitrate, are functional. but a narl nat7 double mutant is completely devoid of NR activity16.This is a good indication that there are Genetic analysis of Hit s t m ~ c t i o n ~lstionships no other functional nia loci in barley. In A. tbaliana, Comparison of the coding sequences of n/a genes where NR-deficient mutants fall into seven complementation groups, cbl-2 and cbl-3 have been assumed also shows that the overall structure of NR proteins is quite highly conserved among plants~ suggesting that, to be nia loci17.

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TIG .JUNE 1990 VOL. 6 NO. 6

BEVIEWS despite conflicting biochemical results, one structural model (see Fig. 1) can represent every eukaryotic assimilatory NR. This justifies the extension of detailed functional information on NR from one species (e.g. Chloreila or spinach) to others, provided a critical eye is kept on the specific traits of each enzyme. Each monomer of the enzyme is organized into three catalytic domains linked one to another by proteasesensitive hinges. The catalytic domains are well defined in the primary sequenceZ0,25 by homology with redox proteins: cytochrome bs reductase, cytochrome bs and suifite oxidase. Being homologous to cytochrome bs reductase, the FAD-containing carboxyterminal domain of NR is thought to transfer electrons from NADH to the heme domain, as the cytochrome bs reductase does to cytochrome b5. Surprisingly, the MoCo-binding domain has no clear homology with other molybdoproteins, apart from sulfite oxidase 25, with which it shares a similar amino-terminal sequence z6 except for the first 80 amino acids. Since this extreme amino-terminal sequence is less well conserved among NR from higher plants, and is even partially absent in the A. nidulans NR23, it is thought not to be involved in catalysis. Limited proteolysis ~-.xperiments z7 are in agreement with this model and also indicate that dimerization does not involve the FAD or the heme domains. Apoenzyme-deficient mutants are helpful for studying structure-function relationships. This has been done in N. plumbaginifolia by measuring amounts of NR protein and partial activities of the enzyme in the nia mutants, which have been classified according to

these biochemical tests 2~. Most of the mutants (40 out of 65) were negative in enzymatic activities and in inununological tests for the presence of the enzyme (class 1, Fig. 3). The remaining nia mutants were divided into three other classes according to which NR redox center - FAD, heme or MoCo - was functionally deficient (Fig. 3). The analysis of nia activities, in addition to immunological data, suggests that reduction of nitrate by the artificial electron donor bromphenol blue requires only MoCo as cofactor, while reduction by methyl-vio!ogen or by ravin mononucleotide needs both MoCo and heme. Intragenic cc;mplementation was observed in N. plumbaginifoliaz9 as well as in tobacco ]'i and C. reinbardtii3o apoenzyme mutants. A detailed analysis of 22 N. plumbaginifolia nia mutants showed that in vivo complementation occurs only between mutants that are from different classes, and thus mutated in different domains (F. Pelsy and M. Gonneau, submitted). Complementation also occurs in vitro (by mixing protein extracts from each mutant), demonstrating that complementation does not require the transcriptional or translational machinery. Such complementation shows that electron transfer may occur between functional domains belonging to separate subunits or molecules, through intersubunit and intermolecular contacts.

Regulation of Nit expression NR activity has been found to be inducible by nitrate in a number of plant specie#. NR mRNA accumulates in the leaves of A. tbaliana z5 or barley plants grown on nitrate. Nitrate-starved tobacco plants rapidly accumulate NR mRNA into their leaves within E.L.I.S.A. 0 E.L.I,S.A. , E.L.I.S.A. + E.U.S'AI ....... o minutes of nitrate replenishment3~, NADH:CytoR 0 NADH:CytcR 0 NADH:CytcR + while NR protein accumulation proNADH:CytcR 0 MVr:NR 0 MVr:NR MVr:NR 0 MVr:NR 0 ceeds more slowly after a lag of FMNH2:NR 0 FMNH2:NR FMNH2:NR 0 FMNH2:NR 0 several hours. BPBr:NR 0 BPBr:NR BPBr:NR 0 BPBr:NR ÷ Light is also required for the expression of a high level of leaf NR and a plastid factor has been postuFAD lated to be involved in this regulation32. In tobacco leaves, the kinetics of light-induced accumulation of mRNAs coding for NR or for the small subunit of ribulose bisphosphate carboxylase are very simila#3. Preliminary experiments on squash Class 1 Class 2 Class 3 Class 4 suggest that this light regulation is under phytochrome control34. When F/GIn tobacco or tomato plants are grown Genetic and biochemical analysis of nia mutants in N. plumbagfnffolia: location under a dark-light regime, the conof mutations in catalytic domains. 65 nia mutants have been classified into four centrations of NR transcripts are high classes (40 in class 1, 14 in class 2, eight in class 3, three in class 4) according to at the beginning of the day period an immunological (ELISA)test for a nitrate reductase epitope, and to partial but almost undetectable at the end enzymatic activities28. Intragenic complementation has only been observed between nfa mutants from separate classes - 2, 3 or 4 (F. Pelsy and M. of this day period31. This fluctuation of NR mRNA expression appears to Gonneau, submitted). In classes 2, 3 and 4, the domains assumed to be be under the control of a circadian deficient in catalytic activityare shaded. In class 1, the site(s) of mutation rhythm33 that can be abolished by cannot be predicted. I, site of interaction between subunits in the dimer; NADH:CytcR, NADH:cytochrome c reductase (diaphorase); MVr:NR,reduced biochemical or genetic impairment methyl viologen: nitrate reductase (terminal activity); FMNH2:NR, ravin of NR catalytic activity. Mutations in mononucleotide: nitrate reductase (terminal activity); BPBr:NR, reduced nia or cn#5, as well as tungstate36, bromphenol blue: nitrate reductase (terminal activity). an inhibitor of NR activity, lead to H

i !l

TIGJUNE1990 VOL.6 NO. 6

high and stable expression of NR mRNA; however, the accumulation of the transcript is still nitrate inducible. In A. nidulan# and Neurospora crassa37, an autocatalytic model of NR regulation has been postulated on the basis of genetic and molecular evidence. In the absence of nitrate, NR is thought to prevent the transcription of its own structural gene by interfering with the positive regulator encoded by the gene nirA (A. nidulans) or nit-4 (N. crassa). Structural defects in NR, including the absence of MoCo in the apoenzyme, can lead to constitutive expression of nitrate-regulated genes in these organisms. However, since plant cnx mutants, as well as nia mutants that are unable to produce detectable NR protein, express a nitrate-inducible NR mRNA35, the validity of the autocatalytic model is questionable for higher plants. Overexpression of NR mRNA it'. NR-deficient plants suggests that NR activity may indirectly contribute to its own regulation: NR expression could be downregulated by a nitrogen metabolite derived from the assimilated nitrate. In two cases, nitrogen metabolite regulation of NR has been shown to involve a positive regulator, encoded by areA (A. nidulan#) and nit-2 (N. crass#7). Physiological studies of higher plants have led to conflicting results. Ammonium, for instance, does not significantly affect the accumulation of leaf NR mRNA in A. thaliana plantlets grown on nitrate 25, but in tissue culture, ammonium reduces the level of NR activity~. We found that in tobacco plants grown on nitrate, NR mRNA accumulation in roots was strongly reduced by ammonium succinate or glutamine, whereas it was not significantly affected in the leaves of the same plants (M. Deng et aL, submitted). Perhaps differences in translocation and intracellular compartmentation of nitrogen metabolites, processes about which little is known, might explain these apparently conflicting results. Identification of plant genes involved in nitrate induction or nitrogen metabolite regulation will be needed to answer the question of whether regulation of NR occurs at these levels. Several putative regulatory mutants have been identified in A. tbalian# 7, but their leaky phenotype makes it difficult to classify them properly as regulatory mutants, since leaky mutations in either nia or cnx genes would also lead to a deregulated expression of the NR apoenzyme. Comparison of the expression of reporter genes fused to the NR promoter in a wild-type or mutant background could help to identify true regulatory mutants.

verspmives Many aspects of the regulation of the nia gene by light, nitrate and nitrogen metabolites need further characterization. The enzymology of the metalloprotein NR, involving at least four successive steps of electron transfer, is a further fascinating topic for future study. The occurrence of the nitrate assimilatory pathway in a variety of microorganisms and higher eukaryotes offers a unique opportunity to identify the common features of the catalytic process and of its regulation, as well as atypical regulatory circuits that may be involved in speciation. In higher plants, cytokinins exert regulatory effects on NR expression; if we knew which aspect of NR expression they affected

we might be able to identify their mode of action, which remains essentially unknGwn. The nia gene appears in many respects to be a unique tool for plant molecular biology studies. The combination of nia mutations together with dominant resistance markers in the same genome offers the possibility of using the corresponding cells as universal hybridizers for somatic hybridization experiments: only hybrids between the marker line and nitrate-utilizing cells will simultaneously express the dominant marker and grow on nitrate as sole nitrogen source39. The nia gene can be used as a transposon trap for the identification of new transposable elements. This possibility has recently been demonstrated by the cloning of Tnt-1, a retrotransposon active in the tobacco genome 4°, and this approach could be extended to other plant species. The nia locus can also be envisaged as a target for homologous recombination studies. However, these last two approaches require the presence of only one functional nia gene in the genome of interest, in a plant species amenable to cell manipulation in vitro.

Acknowledgements We wish to thank colleagues in our laboratory for many helpful comments during the preparation of the manuscript.

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