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Multiple functions and forms of higher plant nitrate reductase
Phytochemistry, Vol. 31, No. 9, pp. 2941-2947, 1992
0031-9422192 $5.00+0.00 0 1992 PergamonPress Ltd
Printedin Great Britain.
REVIEW ARTICLE NUMBER 69 MULTIPLE FUNCTIONS AND FORMS OF HIGHER PLANT NITRATE REDUCTASE H. S.
SRIVASTAVA
Department of Plant Science, Rohilkhand University, Bareilly 243 005, India
(Receiued19 Aiqrust 1991) Key Word Index-Nitrate
reductase; enzyme activity; nitrogen assimilation; isozymes.
Abstract-The enzyme nitrate reductase is a multimeric protein facilitating the flow of electrons from NADH to nitrate through its prosthetic groups FAD, heme and molybdenum cofactor. It also shows partial activities involving one or two of the prosthetic groups. Some mutants known to be deficient in NADH specific nitrate reductase activity are known to assimilate and grow on nitrate. Some alternate functions of the enzyme such as reductive cleavage of the @on siderophores, dissimilatory release of molecular oxygen and chlorate reduction are also known. Unknown functions of the enzyme, especially in the storage tissues, point to the possibility that nitrate reductase may have roles other than the assimilation of nitrate, at least in some tissues, which may perhaps be linked to different isoforms of the enzyme.
INTRODUCTION
Reduction of nitrate to nitrite by the enzyme nitrate reductase is the first step in the assimilation of nitrate and is often considered to be the rate-limiting step in the process [ 11. Consistent with this consideration, a positive correlation between nitrate reductase (NR) activity and organic nitrogen contents and/or growth has been reported in several systems [2,3]. In the leaves of Zea mays seedlings, NR activity can be taken as an index of protein and Kjeldahl nitrogen, irrespective of the form of inorganic nitrogen (nitrate, ammonium or ammonium nitrate) supplied to the seedlings [4]. However, in many other cases, a positive correlation between NR activity and protein/total nitrogen has not been observed. For example, in storage tissue (endosperm, scutellum), while nitrate supply increases NR activity substantially, it has no effect on total Kjeldahl nitrogen [S]. Customarily nitrate reductase activity is determined by the measurement of either in oiuo or in vitro production of nitrite from nitrate. Even using this step as a criterion for NR function, a strict stoichiometry between nitrate loss and nitrite production has not been observed. There are several instances where observed nitrate loss is in excess of nitrite production [6-lo]. Although, this discrepancy to some extent may arise due to the interference from different components of in uivo or in vitro assay mixtures [ll], other possibilities may exist as well. One such possibility is the gaseous evolution of nitrogen oxides (NOx) during in uiuo NR activity assay, as has been found in Glycine rncx [12, 133. Although Nelson et al. [14] and Dean and Harper [15] have suggested that NO, gas evolution is the function of constitutive NR, the physiological signiticance of such a phenomenon is not understood. Some other unexplained roles of NR are also known, which are described in this article, which aims to
assess the significance of this enzyme in -overall plant metabolism. PARTIAL ACTIVITIESOF NITRATE REDUCTASF,
The possibility of multiple functions of NR is apparent from its multimeric nature and several partial activities of electron transfer from NADH to NO;. It is at least a dimer; each monomer containing an active site and three redox prosthetic groups, FAD, heme and a molybdenum cofactor (MoCo) in 1: 1: 1 ratio. The electron flow from NADH to nitrate through these prosthetic groups can be shunted by artificial electron donors and acceptors. Thus, NR shows partial activities also involving one or two of its prosthetic groups [16,17]. One of the partial activities of the enzyme is its dehydrogenase (diaphorase) action, which is perhaps catalysed by the proximal part of the electron transport chain, encompassing the flavin domain [18]. The electrons from NAD(P)H can be passed on to some acceptors, other than the redox system of the enzyme and thus the enzyme can act as NADH-ferricyanide reductase, NADH-dichlorophenol indophenol reductase and NADH-cytochrome c reductase [19-211. The enzyme can reduce nitrate to nitrite by using electron donors other than those comprising the component electron transport chain of the enzyme. Such activities usually termed as ‘terminal activities’ or nitrate reducing activities are believed to be catalysed by the distal (MoCocontaining) part of the enzyme. Reduced flavin nitrate reductase and reduced methyl viologen nitrate reductase are examples of terminal activities. Hoarau et al. [22] demonstrated that bromophenol blue (3,3’,5,5’-tetrabromophenol sulphonaphthalein) can also act as an electron donor for nitrate reduction by NR from cultured Nieoriana cells. In fact, the velocity of
2941
2942
H. S. SRIVASTAVA
nitrate reduction with bromophenol blue was five to 10 times higher than that with NADH. In Spinacea, all partial activities are faster than the full NADH-NR activity [23]. Cytochrome c reductase has been shown to be functional in many crude and purified enzyme preparations [24 -281. By employing labelled and peptide mapping procedure, Kuo et al. [28] have demonstrated that NADH-cytochrome c reductase is the same as NADH:NR, in Hordeurn. It is possible to block the nitrate reduction with cyanide but still carry out dehydrogenase activity of the enzyme [29]. Not only this, the molybdenum cofactor-deficient mutants, which do not have a functional NR activity, do have a nitrate-inducible cytochrome c reductase activity [27]. Daniel-Vedele et al. [30] have suggested that NR, a three redox centre enzyme, is probably evolved from gene fusion between sequences coding for one redox centre proteins. In this light, it will be interesting to search for certain wild ancestors of modern plants, which may exhibit quite active partial activities of NR, but not the full NADH : NR.
DISSIMILATORY
FUNCTIONS
OF NITRATE REDUCTASE
In several micro-organisms, including green algae, the utilization of nitrate by NR activity is not only assimilatory but dissimilatory also, as these organisms can use nitrate as an electron sink to unload their excess of reductant [31-331. These organisms excrete nitrite and sometimes ammonium to the culture medium. The dissimilatory function also enables micro-organisms to grow anaerobically while still gaining energy via oxidative phosphorylation. In higher plants also, reduction of nitrate seems to perform a dissimilatory function in poorly aerated culture solutions. In some early studies by Arnon [34] and by Gilbert and Shive [35] supply of nitrate to poorly aerated culture solutions was found to promote growth in Hordeum and Avena plants, respectively. In some later studies, Garcia-Novo and Crawford [36] and Reggiani et al. [37] suggested a dissimilatory role of nitrate reduction in Oryza (flood tolerant) plants, during annoxia. Perhaps the oxygen generated from reduction of nitrate to nitrite oxidizes NADH to regenerate NAD’, which is essential for the continuation of glycolysis. In Oryza roots, supply of 7.0 mM nitrate gave a better adenylate energy charge and also increased the NAD’/NADH ratio [37,38]. However, no such increase was seen in Zea roots [39], although anaerobiosis in_creased nitrate utilization in this plant also [40]. Increased nitrate reduction during anaerobiosis may also have some link with the regulation of intracellular pH. Restricted oxygen availability or annoxia causes acidification of the cell sap, which is much more apparent in sensitive plants such as Triticum and Hordeum, than in flood tolerant species of Zea, Oryza and Echinocloa C41-431. The resistant species, however, had an efficient mechanism of cellular pH regulation, involving some complex mechanism includiqg carboxylic acid production and alcoholic fermentation [43]. Nitrate reduction may contribute towards cell alkalization, as it consumes protons and produces OH-. However, in one study with Hordeum, decreased nitrate reduction under anaerobiosis has been reported [44]. Apparently more rigorous experiments with clearly defined and sterile experimental conditions are required to assess the impact of anaerobiosis on nitrate reduction in a variety of tissues.
CHLORATE
REDUCTION
Another manifestation of nitrate reductase function is the reduction of chlorate to chlorite, as has been observed in many bacteria, algae and higher plants [4547]. In fact, the reduction of chlorate to chlorite is considered to be the basis of chlorate toxicity, and this property has been used for selecting NR-deficient mutants. The organisms lacking the capability of reducing chlorate to chlorite lack NRA as well, and are resistant to chlorate toxicity [48]. The enzyme from Spinacea leaves is able to reduce bromate and iodate in addition to chlorate [23]. Although, the K, for chlorate, bromate and iodate are several-fold greater than that for nitrate, the V,,,., of the enzyme with chlorate is almost as high as that for nitrate, under saturating concentrations of NADH [23]. This shows that the halogenates compete with nitrate for the active site in the enzyme. However, chlorate does not prevent the induction of NR activity by nitrate, although it decreases the effectiveness of nitrate reduction by NR at low levels of nitrate [49]. The physiological significance of chlorate reduction is not understood, although the carrier protein for chlorate and nitrate seems to be the same [SO]. The concentration of chlorate in most soils is very low, but in some localities, where KClO, is manufactured for its use in matches, fireworks, photographic flash powders, disinfectants and generation of oxygen, it can accumulate to toxic levels in soils. These soils may be unfit for crop production, especially with nitrate as a source of nitrogen. However, in Glycine, mechanism(s) other than the reduction of chlorate to chlorite by NR, seem to be involved in chlorate toxicity, as Glycine mutants lacking constitutive NR and grown on urea show toxic symptoms of chlorate toxicity (J. E. Harper, Personal communication). NITRATE ACQUISITION
AND ION TRANSPORT
BY NITRATE
REDUCTASE
Similarities between the induction of nitrate reductase and nitrate accumulation led Butz and Jackson [Sl] to propose that a membrane-associated NR was involved in the acquisition of nitrate from the medium. Jones and Morel [52] have demonstrated that NR present in the plasmalemma in the diatom Thalossiosira sp. acts as a transplasmalemma proton pump. For the involvement of NR in ion acquisition and transport, the enzyme must be located in the membrane. Although there are a few reports for NR location on membranes [53-551, most of the workers have indicated the cytoplasmic location of the enzyme [S&59]. By employing aqueous two-phase partioning, Ward et al. [60, 613, have demonstrated that 4-19% of the total NR in Hordeum roots is associated with the membranes. Immunochemical studies with marine flagellates and diatoms indicate that NR is located in the plasmalemma, as well as inside the cell [62]. The situation in higher plants, however, seems to be different. Vaughan and Campbell [63], by using electron microscopic analysis using antibodies for NR and immunogold labelling, demonstrated that NR in Zea mesophyll cells was exclusively in the cytoplasm. Therefore, the subcellular location of NR remains controversial, although the bulk of data support a cytoplasmic location. NITRATE REDUCTASE
AND IRON NUTRITION
Nitrate reductase is believed to play an important role in the supply of iron to the plants by the reductive
Multiple functions of nitrate reductase cleavage of iron siderophores [64]. The dehydrogenase part of the enzyme can reduce ferric citrate and thus, the enzyme can play a role in iron assimilation [65]. In Sorghum, the iron content in the roots of the plants supplied with NO; and NHf as nitrogen source, increased with the increasing NO; :NH; ratio [66]. This is expected if we assume that the iron-acquiring system was nitrate inducible and/or ammonium repressible. The most likely agent with such a property is NADH-NR. Perhaps it would also be interesting to examine the role of NR in internal mobilization of iron in higher plants. Most iron is stored in the leaf chloroplasts as hydrous ferric oxide in the protein phytoferritin [67]. The phytoferritins are not as well characterized as animal ferritins. However, reducing agents such as ascorbate [68] and dihydroflavins [69, 703 promote the release of ferritin iron. Plant phenol& which induce NR activity [71, 721 also promote reductive release of ferritin iron [73]. Whether this correlation between the increase in NR activity and release of ferritin iron by phenolics is just a coincidence or a real link between the two processes needs to be examined. Iron assimilation is one of the key aspects of building effective photosynthetic units, and thus NR may have an important role in overall plant productivity through this nutrient as well. NITRATE REDUCTASE
DEFICIENT
MUTANTS
A number of variant plant lines have been obtained which have either significantly reduced or totally absent nitrate reductase activity, such as Arabidopsis thaliana [74], Datura innoxia [48], Glycine max [47, 75, 761, Hordeum oulgare [77,78], Nicotiana tabncum [7!9-811, N. plumbagini$ofolia [82] and Pisum satiuum [83-85]. In ethylmethanesulphonate-induced albino mutants of Hordeum, no in vitro NR activity was detected but a significant
amount of NiR, the second enzyme in the assimilation of nitrate, was detected in the same leaves [86]. In the same species, NR-deficient mutants possessed substantial amounts of nitrate-inducible cytochrome c reductase activity and FMNH,-NR activity, although they did not possess NADH-NR activity [28]. The NR-deficient mutants seem to differ in their ability to absorb and assimilate nitrate. Nicotiana plumbaginifilia mutants lacking NR activity could not grow on nitrate as sole nitrogen source, but they could be cultivated as graftings on wild type N. tabacum plants [87]. The mutants underwent senescence earlier than the wild type. By employing X-ray irradiation, 13 mutants of Cklorella sorokiniana were produced which were all incapable of using nitrate as nitrogen source [SS]. In the nar la mutant of Hordeum, nitrate accumulation has also been reported during nitrate supply [78]. Some NR-deficient mutants however, are able to assimilate and grow on nitrate. Nitrate reductase-deficient Hordeum mutants AZ12 and AZ13 produced good growth in the field, suggesting that plants could utilize nitrate [89]. The plants could also be grown in vermiculite with nitrate as the sole nitrogen source [90]. In Pisum also, the mutant A334, which had less than 10% of the NR activity of the wild-type parent Juneau, grew as well as the wild-type parent with nitrate as the nitrogen source, although mutants A300 and A317 produced very little growth [SS]. By using a strictly anaerobic in uiuo assay, Srinivasan et al. [91] demonstrated that AZ12 and AZ13 Hordeum mutants contained 40-50% NR activity, as compared to that in Steptoe wild type, which could
2943
account for the required nitrate assimilation. A bispecific NAD(P)H-NR has also been shown to be present in some of these mutants, which lack NADH-NR [92]. In the leaves of nar la Hordeum mutants, the activity of NAD(P)H-NR is about 15 times higher than in wild type Steptoe, while the activity of NADH-NR in the mutants is only about 5% of the activity in wild type [93]. However, NADH-NR is active in the roots of both wild type and mutants. The NADH and NAD(P)H-NR are apparently different isoforms [94] and they have been shown to possess different kinetic, immunological and physical characteristics [95]. Thus, in Hordeum mutants lacking NADH-NR, NAD(P)H-NR may play an active role in nitrate reduction. However, Vigue and Warner [SS] were unable to demonstrate the presence of any NAD(P)H-NR in A334 mutants of Pisum. THE PRESENCE OF NITRATE REDUCTASE ASSIMILATING
IN NON-
TISSUES
Nitrate is primarily assimilated in roots or leaves, depending on the species [96]. However, the presence of nitrate reductase activity has been demonstrated in almost all the plant parts examined, including aleurone layers [97], endosperm [98], scutellum [98, 993, cotyledons [lOO-1021, stems [103-1051, petioles [106], fruits [107], ovules [lOS], developing pods of legumes [109], etc. The enzyme activity has been reported in human saliva as well, although its exact role is uncertain [ 1lo]. In Zea endosperm and scutellum, the NR activity increases with nitrate supply, as is the case with root and leaf enzyme [98]. The concentration of nitrate required to induce maximum NR activity however, is higher in endosperm and scutella, as compared to that in root and leaf tissues [3]. This, of course, may be partly related to the differential rates of absorption of ions by different tissues. In spite of the fact that these storage tissues (endosperm and scutella) have appreciable amounts of inducible NR activity, they are unable to assimilate nitrate into organic nitrogen [S]. It is possible that NR activity plays some dissimilatory role in these tissues, especially before seed germination. The seeds are often impermeable to oxygen due to the seed coat, and reduction of nitrate to nitrite deep inside the seed may generate oxygen for the oxidation of NADH. INCREASE
IN NITRATE
REDUCTASE
ACTIVITY
BY META-
BOLITES OTHER THAN GROWTH REGULATORS
Several endogenous and exogenous metabolites are known to influence nitrate reductase activity, although their physiological significance in nitrogen metabolism is not understood. One such metabolite is ammonium. Ammonium as a potential product of nitrate reduction is expected to inhibit NR activity. Although this expectation is realized in some micro-organisms [20], the effect of ammonium on higher plant NR is quite variable. It has either a slight inhibitory effect [111-1131 or no effect [114-1163. In many plants however, the supply of ammonium increases NR activity either in the absence or presence of nitrate (Table 1). Some attempts have been made to understand the mechanism of this increase [ 1191. Among other possibilities, it has been suggested that aminonium is oxidized to nitrate either by the plant tissue itself or by contaminating microbes, which in turn acts as an inducer of NR activity [ 1251. As there is no convincing evidence for the oxidation of ammonium to nitrate by the
H. S. SRIVASTAVA
2944 Table
1. Effect of ammonium
supply
on nitrate reductase activity absence of nitrate
Chenopodium rubrum cultured cells Cuscuta rejkxa cultured seedlings Phaseolus vulgaris excised leaves Rosa sp. (Paul’s scarlet suspension culture Sinapis alba cotyledons incubated in far-red light
(mM)
rn vitro in vitro
10 20
+540
Cl171 Ul71
in vi00
50
0
Cl181
in vivo
10
+ 248
Cl191
+260
rose)
in far-red light Zea mays excised roots excised shoots excised leaves intact leaves intact leaves intact shoot
m the presence
Assay method
ammonium species and tissues
plants
Percentage increase ( +) or decrease ( - ) Over no nitrogen Over NO;
Cont. of Plant
in higher
in vitro
0.91
+100
m vitro
15
+90
in vitro
15
+500
in in tn m in in
10 10 10 10 10 5
-31 -6 t175 +38 +457 +73
vivo vivo vi00 vivo vitro vitro
or
Reference
._
cm
143 +182
Cl211 Cl221 cl231 cl231 cl231 c41 L4l
f4
~1241
The percentage values are approximate. Not determined.
higher plant tissues, and as most of the systems where an increase in NR activity in the presence of ammonium have been reported are not properly sterilized, one would be inclined to believe that the microbes were the real agents of ammonium oxidation and NR induction. However, Schuster et al. [ 1261 have suggested that ammonium induces a specific isoform of NR. But synergistic action of nitrate and ammonium in inducing NR activity, in some systems, indicates that both nitrogen sources act independently in the induction of NR [122]. Nitrite, the immediate product of nitrate reduction, is also reported to increase NR activity in many tissues, the increase in some cases being equal to or even more than that observed with nitrate [127-1291. Aslam et al. Cl303 have suggested that the increase in Hordeum leaves is the result of nitrate, which is produced by the oxidation of nitrite. However, evidence for oxidation of nitrite or ammonium is certainly lacking in higher plants. Experiments with NR induction by nitrite or ammonium, performed in completely sterile conditions, may provide insight to this problem. It would be also worthwhile to compare kinetic properties and electrophoretic mobility of nitrite/ammonium-induced NR to that with nitrateinduced NR. CONCLLIBING
REMARKS: POLYMORPHIC ENZYME
NATURE OF THE
Although the significance of various functions of nitrate reductase is not understood, the origin of such functions in some cases may be traced to the different isoforms of the enzyme. In Glycine tissues three isoforms
of NR have been demonstrated [131, 1321: (1) a nitrateinducible NADH-NR (EC 1.6.6.1) with a pH optimum of 7.5; (2) a constitutive bispecific NADH/NADPH [NAD (P) HI-NR (EC 1.6.6.2) with a pH optimum of 6.5; and (3) a constitutive NADH-NR (EC number not yet assigned) with a pH optimum of 6.5. There is evidence for the occurrence of more than one isoform of NR in other species also, and it is likely that polymorphism in NR is not limited to Glycine only [16, 29, 1333. The presence and relative activities of different isoforms depend apparently on the species, tissues and nutritional factors. The first two forms are usually found in close association, and perhaps both contribute to the reduction and assimilation of nitrate. There is a vast literature on NADH-NR, emphasizing its role in nitrate assimilation, although NAD(P)H bispecific NR also seems to have an important role in the process, and it may be more widespread than currently believed [ 1341. As described earlier, in Hordeum mutants lacking NADH-NR, the NAD(P)H bispecificNR may assume the role of nitrate reduction [93]. The constitutive NR isoform in Glycine constitutes about 12-20% of the total NR activity and its absence does not impede normal nitrate utilization [47]. In Chlorella, the constitutive isoform is plasmamembrane-bound and appears to be involved primarily in nitrate uptake [135], although in other systems a component of nitrate transporter may be inducible as well [136, 1373. In another study [138] “NO; influx in non-induced (nitrate starved) Hordeum roots has also been shown to be mediated by a constitutive rather than inducible transport system. However, Warner and Huffaker [139], by analysing Hordeum mutants, have demonstrated that genetic loci
Multiple functions of nitrate reductase regulating
nitrate
transport
are distinct
from those
for
NR and NiR, although it may be under the same promoter region. NO, evolution activity and reduction of chlorate to chlorite are also believed to be the function of constitutive NR [ 14,473. The inducibility of NR by nitrate might have been selected by evolutionary pressure and agricultural practices and it is possible that the wild ancestors of cultivated plants had constitutive NR only [140]. Oumidou et al. [ 1411 have demonstrated that while the inducible form of NR is the major form in cultivated tetraploid and hexaploid Triticum aestivum, the constitutive form of the enzyme is predominant in their diploid ancestor, Aegilops squarrosa. Sensitive techniques for purification and characterization of NR are now available. By combining the cellular and molecular techniques with genetic approaches, it may be possible to detect different isoforms of NR in a variety of species [ 1261 and to relate them with a specific function. In many cases, it would also be necessary to culture plant tissues in completely sterile conditions, to ensure that one was dealing with the plant enzyme and not with the contaminating microbial enzyme. AcRnowbBgernents-The author thanks Dr P. V. Sane for his encouragement and initial interest in the article and Drs Ann Oaks and J. E. Harper for their critical reading of the article.
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H. S.
SRIVASTAVA
57. Dalling, M. J., Tolbert, N. E. and Hageman, R. H. (1972) Biochem. Biophys. Acta 283, 513. 58. Suzuki, A., Gadal, P. and Oaks, A. (1981) Planta 151,457. 59. Oaks, A. and Hirel, B. (1985) Ann. Rev. Plant Physiol. 36, 345. 60. Ward, M. R., Tischner, R. and Hutfaker, R. C. (1988) Plant Physiol. 88, 1141. 61. Ward, M. R., Grimes, H. D. and Hutfaker, R. C. (1989) Planta 177, 470. 62. Batch, W. M., Yentsch, C. M., Reguera, B. and Campbell, W. H. (1987) in Immunological Approach to Oceanography (Yentsch, C. M., Mague, F. C. and Horendo,P. K., eds), Springer, New York. 63. Vaughn, K. C. and Campbell, W. H. (1988) Plant Physiol. 88, 1354. 64. Castignetti, D. and Smarelli, J. (1984) Biochem. Biophys. Res. Commun. 125, 25. 65. Campbell, W. H. and Redinbaugh, M. G. (1984) .I. Plant Nutr. 7, 799. 66. Bernardo, L. M., Clark, R. B. and Naranwille, J. W. (1984) J. Plant Nutr. 7, 1401. 67. Sekbach, J. C. (1982) J. Plant Nutr. 5, 369. 68. Bienfait, H. and Vanden Brief, M. (1980) B&him. Biophys. Acta 631, 507. 69. Jones, T., Spencer, R. and Walsch, C. (1978) Biochemistry 17, 4011. 70. Funk, E., Lenders, J., Crichton, R. and Schneider, W. (1985) Eur. J. Biochem. 152, 167. 71. Knypl, J. S. (1974) in Mechanism of Regulation of Plant Growth (Bieleski, R. L. et al., eds), p. 71. Royal Society of New Zealand, Wellington. 72. Jain, A. and Srivastava, H. S. (1981) Physiol. Plant. 51,334. 73. Boyer, R. F., McArthur, J. S. and Cary, T. M. (1990) Phytochemistry 29, 3717. 74. Oostindier-Brasksma, F. J. and Feenstra, W. J. (1973) Mutat. Res. 19, 715. 75. Nelson, R. S., Ryan, S. A. and Harper, J. E. (1983) Plant Physiol. 72, 503. 76. Caroll, B. J. and Gresshoff, P. M. (1986) Plant Physiol. 81, 572. 77. Tokarevo, B. J. and Shumny, V. K. (1977) Genetika 13,2097. 78. Warner, R. L., Lin, C. J. and Kleinhofs, A. (1977) Nature 269,406. 79. Muller, A. J. and Grafe, R. (1978) Mol. Gen. Genet. 161, 67. 80. Mendel, R. R. and Muller, A. J. (1979) Mol. Gen. Genet. 177, 145. 81. Mendel, R. R., Ahkulov, Z. A., Lvov, N. P. and Muller, A. J. (1981) Mol. Gen. Genet. 181, 395. 82. Marton, L., Dung, T. M., Mendel, R. R. and Maliga, P. (1982) Mol. Gen. Genet. 182, 301. 83. Kleinhof, A., Warner, R. L., Muehlbauer. F. J. and Nrlan, R. A. (1978) Mutat. Res. 51, 29. 84. Feenstra, W. J. and Jacobsen, E. (1980) Theor. .4ppl. Genet. 58, 39. 85. Vigue, G. T. and Warner, R. L. (1987) Crop Sci. 27, 548. 86. Sawhney, S. K., Parkash, V. and Naik, M. S. (1977) FEBS Letters 22, 200. 87. Saux, C., Lemoine, Y., Morion-Poll, A., Voladier, M. H., Leng, M. and Norot-Gaudry, J. F. (1987) Plant Physiol. 84, 67. 88. Knobloch, 0. and Tischner, H. (1989) Plant Physiol. 89, 786. 89. Oh, J. Y., Warner, R. L. and Kleinhoffs, A. (1980) Crop Sci. 20,487. 90. Warner, R. L. and Kleinhof, A. (1981) Plant Physiol. 67,740. 91. Srinivasan, Sawhney, S. K., Kumar, S., Mohanty, P., Sinha, S. K. and Naik, M. S. (1983) Plant Sci. Letters 30, 17.
92. Dailey, F. A., Warner, R. L., Somers, D. A. and Klein, A. (1982) Plant Physiol. 69, 1200. 93. Warner, R. L., Narayanan, K. R. and Kleinhofs, A. (1987) Theor. Appl. Genet. 74, 714. 94. Heath-Pagliuso. S., Huffaker, R. C. and Allard. R. W. (1984) Plant Physiol. 76, 353. 95. Harker, A. R., Narayanan. K. R., Warner, R. L. and Kleinhofs, A. (1986) Phytochemlstry 25, 1275. 96. Radin, J. W. (1978) Plant Sci. Letters 13, 21. 97. Ferrari, T. E. and Varner, J. E. (1969) Plant Phvsiol. 44,85. 98. Srivastava, H. S., Asthana, J. S. and Jain, A. (1979) Physiol. Plant. 47, 119. 99. Wallace, W. (1973) Plant Physiol. 92, 191. 100. Orihuel-Ivanzo, B. and Campbell, W. H. (1980) Plant Physiol. 65, 595. 101. Carelli, M. L. C. and Meghalhaes, A. C. (1981) Z. Pjlanzenphysiol. 104, 17. 102. Kakefuda, G., Duke, S. H. and Duke, S. 0. (1983) Plant Physiol. 73, 56. 103. Andrews, M., Sutherland, J. M., Thomas, R. J. and Sprent, J. J. (1984) New Phytol. 98, 301. 104. Hunter, W. J. (1985) Ann. Botany 55, 759. 105. Wallace, W. (1986) Physiol. Plant. 66, 630. 106. Crafts-Brandner, S. J. and Harper, J. E. (1982) Plant Physiol. 69, 1293. 107. Menary, R. C. and Jones, R. H. (1972) Aust. J. Biol. Sci. 25, 531. 108. Smgh, 0. S. and Singh, 0. (1976) Curr. Sci. 45, 632. 109. Schlesier, G. (1977) Biochem. Physlol. Pflanzen 171. 525. 110. Srivastava, R. C.. Siddiqui, M. H.. Bose, B., Mathur. A. and Mathur, S. N. (1982) Naturwissen schafen 69, 400. 111. Smith, F. W. and Thompson, J. F. (1971 Plant Physiol. 48, 219. 112. Frith. G. J. T. (1972) Plant Cell Physiol. 20. 1085. 113. Radin, J. W. (1975) Plant Physzol. 55, 178. 114. Beevers, L., Schrader, L. E. and Hageman. R. H. (1965) Plant Physiol. 40. 691. 115. Schrader, L. E. and Hageman. R. H. (1967) Plant Physiol. 84.67. 116. Oaks, A., Aslam, M. and Boessel, I. (1977) Plant Physd. 59, 391. 117. Renner, U. and Beck, E. (1988) Plant Cell Physiol. 29, 1123. 118. Chauhan. J. S. and Srrvastava, H. S. (1980) Plant Blochem J. 7, 25. 119. Srivastava, H. S. and Mehta. P. (1983) Tropical Plant Sci. Res. 1. 75. 120. Mohanty, B. and Fletcher. J. S. (1976) Plant Physiol. 58, 152. 121. Rajasekhar, V. K. and Mohr, H. (1986) Planta 169, 594. 122. Mehrer, I. and Mohr, H. (1989) Physiol. Plant. 77, 545. 123. Mehta, P., Jain, A. and Srtvastava. H. S. (1980) Physiol. Plant. 49, 417. 124. Oaks, A. and Long, D. M. (1992) in Nrtrogen Metabohsm in Plants (Pilbeam, D. J. and Mengel, K., eds). Oxford University Press (in press). 125. Mishra, S. N. and Srivastava. H. S. (1983) Brochem. Physiol. Pflanzen 178,449. 126. Schuster, C, Schmidt, S. and Mohr, H. (1989) Planta 177. 74. 127. Kelker, H. C. and Frlner, P. (1971) Biochim. Biophys. Acta 252, 69. 128. Lips, H.. Kaplan, D. and Roth-Bejerano. N. (1973) Eur. J. Biochem. 37, 589. 129. Kaplan, D., Roth-Bejerano, N. and Lips, H. (1974) Eur. J. Biochem. 49, 393.
Multiple functions of nitrate reductase 130. Aslam, M., Rosichan, J. L. and Huffaker, R. C. (1987) Plant Physiol. 83, 519.
(1986) in Biochemical Nitrogen Metabolism
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131. Streit, L., Nelson, R. S. and Harper, J. E. (1985) Plant Physiol. 78, 80. 132. Streit, L., Martin, B. A. and Harper, J. E. (1987) Plant Physiol. 84, 654. 133. Wray, J. L. (1988) Plant Cell Emiron. 11, 369.
Boca Raton. 137. Lee, R. B. and Drew, M. C. (1986) J. Exp. Botany 37,1768. 138. Siddiqui, M. Y., Glass, A. D. M., Ruth, T. J. and Rufty, T. W., Jr (1990) Plant Physiol. 93, 1426. 139. Warner, R. L. and Huffaker, R. C. (1989) Plant Physiol. 91,
134. Kleinhofs, A., Warner, R, L. and Narayanan, K. R. (1985) Oxford Survey Plant Mol. Cell. Biol. 2, 91. 135. Tischner, R., Ward, M. R. and Hutfaker, R. C. (1989) Planta 178, 19. 136. Jackson, W. A., Pan, W. L., Moll, R. H. and Kamprath, E. J.
140. Oaks, A. (1992) Bioscience (in press). 141. Ouhmidou, B., Cauderon, Y., Cherel, I. and Champigny, M. L. (1990) 7’heor. Appl. Genet. 79, 8.
941.
0031-9422192 $5.00+0.00 0 1992 PergamonPress Ltd
Printedin Great Britain.
REVIEW ARTICLE NUMBER 69 MULTIPLE FUNCTIONS AND FORMS OF HIGHER PLANT NITRATE REDUCTASE H. S.
SRIVASTAVA
Department of Plant Science, Rohilkhand University, Bareilly 243 005, India
(Receiued19 Aiqrust 1991) Key Word Index-Nitrate
reductase; enzyme activity; nitrogen assimilation; isozymes.
Abstract-The enzyme nitrate reductase is a multimeric protein facilitating the flow of electrons from NADH to nitrate through its prosthetic groups FAD, heme and molybdenum cofactor. It also shows partial activities involving one or two of the prosthetic groups. Some mutants known to be deficient in NADH specific nitrate reductase activity are known to assimilate and grow on nitrate. Some alternate functions of the enzyme such as reductive cleavage of the @on siderophores, dissimilatory release of molecular oxygen and chlorate reduction are also known. Unknown functions of the enzyme, especially in the storage tissues, point to the possibility that nitrate reductase may have roles other than the assimilation of nitrate, at least in some tissues, which may perhaps be linked to different isoforms of the enzyme.
INTRODUCTION
Reduction of nitrate to nitrite by the enzyme nitrate reductase is the first step in the assimilation of nitrate and is often considered to be the rate-limiting step in the process [ 11. Consistent with this consideration, a positive correlation between nitrate reductase (NR) activity and organic nitrogen contents and/or growth has been reported in several systems [2,3]. In the leaves of Zea mays seedlings, NR activity can be taken as an index of protein and Kjeldahl nitrogen, irrespective of the form of inorganic nitrogen (nitrate, ammonium or ammonium nitrate) supplied to the seedlings [4]. However, in many other cases, a positive correlation between NR activity and protein/total nitrogen has not been observed. For example, in storage tissue (endosperm, scutellum), while nitrate supply increases NR activity substantially, it has no effect on total Kjeldahl nitrogen [S]. Customarily nitrate reductase activity is determined by the measurement of either in oiuo or in vitro production of nitrite from nitrate. Even using this step as a criterion for NR function, a strict stoichiometry between nitrate loss and nitrite production has not been observed. There are several instances where observed nitrate loss is in excess of nitrite production [6-lo]. Although, this discrepancy to some extent may arise due to the interference from different components of in uivo or in vitro assay mixtures [ll], other possibilities may exist as well. One such possibility is the gaseous evolution of nitrogen oxides (NOx) during in uiuo NR activity assay, as has been found in Glycine rncx [12, 133. Although Nelson et al. [14] and Dean and Harper [15] have suggested that NO, gas evolution is the function of constitutive NR, the physiological signiticance of such a phenomenon is not understood. Some other unexplained roles of NR are also known, which are described in this article, which aims to
assess the significance of this enzyme in -overall plant metabolism. PARTIAL ACTIVITIESOF NITRATE REDUCTASF,
The possibility of multiple functions of NR is apparent from its multimeric nature and several partial activities of electron transfer from NADH to NO;. It is at least a dimer; each monomer containing an active site and three redox prosthetic groups, FAD, heme and a molybdenum cofactor (MoCo) in 1: 1: 1 ratio. The electron flow from NADH to nitrate through these prosthetic groups can be shunted by artificial electron donors and acceptors. Thus, NR shows partial activities also involving one or two of its prosthetic groups [16,17]. One of the partial activities of the enzyme is its dehydrogenase (diaphorase) action, which is perhaps catalysed by the proximal part of the electron transport chain, encompassing the flavin domain [18]. The electrons from NAD(P)H can be passed on to some acceptors, other than the redox system of the enzyme and thus the enzyme can act as NADH-ferricyanide reductase, NADH-dichlorophenol indophenol reductase and NADH-cytochrome c reductase [19-211. The enzyme can reduce nitrate to nitrite by using electron donors other than those comprising the component electron transport chain of the enzyme. Such activities usually termed as ‘terminal activities’ or nitrate reducing activities are believed to be catalysed by the distal (MoCocontaining) part of the enzyme. Reduced flavin nitrate reductase and reduced methyl viologen nitrate reductase are examples of terminal activities. Hoarau et al. [22] demonstrated that bromophenol blue (3,3’,5,5’-tetrabromophenol sulphonaphthalein) can also act as an electron donor for nitrate reduction by NR from cultured Nieoriana cells. In fact, the velocity of
2941
2942
H. S. SRIVASTAVA
nitrate reduction with bromophenol blue was five to 10 times higher than that with NADH. In Spinacea, all partial activities are faster than the full NADH-NR activity [23]. Cytochrome c reductase has been shown to be functional in many crude and purified enzyme preparations [24 -281. By employing labelled and peptide mapping procedure, Kuo et al. [28] have demonstrated that NADH-cytochrome c reductase is the same as NADH:NR, in Hordeurn. It is possible to block the nitrate reduction with cyanide but still carry out dehydrogenase activity of the enzyme [29]. Not only this, the molybdenum cofactor-deficient mutants, which do not have a functional NR activity, do have a nitrate-inducible cytochrome c reductase activity [27]. Daniel-Vedele et al. [30] have suggested that NR, a three redox centre enzyme, is probably evolved from gene fusion between sequences coding for one redox centre proteins. In this light, it will be interesting to search for certain wild ancestors of modern plants, which may exhibit quite active partial activities of NR, but not the full NADH : NR.
DISSIMILATORY
FUNCTIONS
OF NITRATE REDUCTASE
In several micro-organisms, including green algae, the utilization of nitrate by NR activity is not only assimilatory but dissimilatory also, as these organisms can use nitrate as an electron sink to unload their excess of reductant [31-331. These organisms excrete nitrite and sometimes ammonium to the culture medium. The dissimilatory function also enables micro-organisms to grow anaerobically while still gaining energy via oxidative phosphorylation. In higher plants also, reduction of nitrate seems to perform a dissimilatory function in poorly aerated culture solutions. In some early studies by Arnon [34] and by Gilbert and Shive [35] supply of nitrate to poorly aerated culture solutions was found to promote growth in Hordeum and Avena plants, respectively. In some later studies, Garcia-Novo and Crawford [36] and Reggiani et al. [37] suggested a dissimilatory role of nitrate reduction in Oryza (flood tolerant) plants, during annoxia. Perhaps the oxygen generated from reduction of nitrate to nitrite oxidizes NADH to regenerate NAD’, which is essential for the continuation of glycolysis. In Oryza roots, supply of 7.0 mM nitrate gave a better adenylate energy charge and also increased the NAD’/NADH ratio [37,38]. However, no such increase was seen in Zea roots [39], although anaerobiosis in_creased nitrate utilization in this plant also [40]. Increased nitrate reduction during anaerobiosis may also have some link with the regulation of intracellular pH. Restricted oxygen availability or annoxia causes acidification of the cell sap, which is much more apparent in sensitive plants such as Triticum and Hordeum, than in flood tolerant species of Zea, Oryza and Echinocloa C41-431. The resistant species, however, had an efficient mechanism of cellular pH regulation, involving some complex mechanism includiqg carboxylic acid production and alcoholic fermentation [43]. Nitrate reduction may contribute towards cell alkalization, as it consumes protons and produces OH-. However, in one study with Hordeum, decreased nitrate reduction under anaerobiosis has been reported [44]. Apparently more rigorous experiments with clearly defined and sterile experimental conditions are required to assess the impact of anaerobiosis on nitrate reduction in a variety of tissues.
CHLORATE
REDUCTION
Another manifestation of nitrate reductase function is the reduction of chlorate to chlorite, as has been observed in many bacteria, algae and higher plants [4547]. In fact, the reduction of chlorate to chlorite is considered to be the basis of chlorate toxicity, and this property has been used for selecting NR-deficient mutants. The organisms lacking the capability of reducing chlorate to chlorite lack NRA as well, and are resistant to chlorate toxicity [48]. The enzyme from Spinacea leaves is able to reduce bromate and iodate in addition to chlorate [23]. Although, the K, for chlorate, bromate and iodate are several-fold greater than that for nitrate, the V,,,., of the enzyme with chlorate is almost as high as that for nitrate, under saturating concentrations of NADH [23]. This shows that the halogenates compete with nitrate for the active site in the enzyme. However, chlorate does not prevent the induction of NR activity by nitrate, although it decreases the effectiveness of nitrate reduction by NR at low levels of nitrate [49]. The physiological significance of chlorate reduction is not understood, although the carrier protein for chlorate and nitrate seems to be the same [SO]. The concentration of chlorate in most soils is very low, but in some localities, where KClO, is manufactured for its use in matches, fireworks, photographic flash powders, disinfectants and generation of oxygen, it can accumulate to toxic levels in soils. These soils may be unfit for crop production, especially with nitrate as a source of nitrogen. However, in Glycine, mechanism(s) other than the reduction of chlorate to chlorite by NR, seem to be involved in chlorate toxicity, as Glycine mutants lacking constitutive NR and grown on urea show toxic symptoms of chlorate toxicity (J. E. Harper, Personal communication). NITRATE ACQUISITION
AND ION TRANSPORT
BY NITRATE
REDUCTASE
Similarities between the induction of nitrate reductase and nitrate accumulation led Butz and Jackson [Sl] to propose that a membrane-associated NR was involved in the acquisition of nitrate from the medium. Jones and Morel [52] have demonstrated that NR present in the plasmalemma in the diatom Thalossiosira sp. acts as a transplasmalemma proton pump. For the involvement of NR in ion acquisition and transport, the enzyme must be located in the membrane. Although there are a few reports for NR location on membranes [53-551, most of the workers have indicated the cytoplasmic location of the enzyme [S&59]. By employing aqueous two-phase partioning, Ward et al. [60, 613, have demonstrated that 4-19% of the total NR in Hordeum roots is associated with the membranes. Immunochemical studies with marine flagellates and diatoms indicate that NR is located in the plasmalemma, as well as inside the cell [62]. The situation in higher plants, however, seems to be different. Vaughan and Campbell [63], by using electron microscopic analysis using antibodies for NR and immunogold labelling, demonstrated that NR in Zea mesophyll cells was exclusively in the cytoplasm. Therefore, the subcellular location of NR remains controversial, although the bulk of data support a cytoplasmic location. NITRATE REDUCTASE
AND IRON NUTRITION
Nitrate reductase is believed to play an important role in the supply of iron to the plants by the reductive
Multiple functions of nitrate reductase cleavage of iron siderophores [64]. The dehydrogenase part of the enzyme can reduce ferric citrate and thus, the enzyme can play a role in iron assimilation [65]. In Sorghum, the iron content in the roots of the plants supplied with NO; and NHf as nitrogen source, increased with the increasing NO; :NH; ratio [66]. This is expected if we assume that the iron-acquiring system was nitrate inducible and/or ammonium repressible. The most likely agent with such a property is NADH-NR. Perhaps it would also be interesting to examine the role of NR in internal mobilization of iron in higher plants. Most iron is stored in the leaf chloroplasts as hydrous ferric oxide in the protein phytoferritin [67]. The phytoferritins are not as well characterized as animal ferritins. However, reducing agents such as ascorbate [68] and dihydroflavins [69, 703 promote the release of ferritin iron. Plant phenol& which induce NR activity [71, 721 also promote reductive release of ferritin iron [73]. Whether this correlation between the increase in NR activity and release of ferritin iron by phenolics is just a coincidence or a real link between the two processes needs to be examined. Iron assimilation is one of the key aspects of building effective photosynthetic units, and thus NR may have an important role in overall plant productivity through this nutrient as well. NITRATE REDUCTASE
DEFICIENT
MUTANTS
A number of variant plant lines have been obtained which have either significantly reduced or totally absent nitrate reductase activity, such as Arabidopsis thaliana [74], Datura innoxia [48], Glycine max [47, 75, 761, Hordeum oulgare [77,78], Nicotiana tabncum [7!9-811, N. plumbagini$ofolia [82] and Pisum satiuum [83-85]. In ethylmethanesulphonate-induced albino mutants of Hordeum, no in vitro NR activity was detected but a significant
amount of NiR, the second enzyme in the assimilation of nitrate, was detected in the same leaves [86]. In the same species, NR-deficient mutants possessed substantial amounts of nitrate-inducible cytochrome c reductase activity and FMNH,-NR activity, although they did not possess NADH-NR activity [28]. The NR-deficient mutants seem to differ in their ability to absorb and assimilate nitrate. Nicotiana plumbaginifilia mutants lacking NR activity could not grow on nitrate as sole nitrogen source, but they could be cultivated as graftings on wild type N. tabacum plants [87]. The mutants underwent senescence earlier than the wild type. By employing X-ray irradiation, 13 mutants of Cklorella sorokiniana were produced which were all incapable of using nitrate as nitrogen source [SS]. In the nar la mutant of Hordeum, nitrate accumulation has also been reported during nitrate supply [78]. Some NR-deficient mutants however, are able to assimilate and grow on nitrate. Nitrate reductase-deficient Hordeum mutants AZ12 and AZ13 produced good growth in the field, suggesting that plants could utilize nitrate [89]. The plants could also be grown in vermiculite with nitrate as the sole nitrogen source [90]. In Pisum also, the mutant A334, which had less than 10% of the NR activity of the wild-type parent Juneau, grew as well as the wild-type parent with nitrate as the nitrogen source, although mutants A300 and A317 produced very little growth [SS]. By using a strictly anaerobic in uiuo assay, Srinivasan et al. [91] demonstrated that AZ12 and AZ13 Hordeum mutants contained 40-50% NR activity, as compared to that in Steptoe wild type, which could
2943
account for the required nitrate assimilation. A bispecific NAD(P)H-NR has also been shown to be present in some of these mutants, which lack NADH-NR [92]. In the leaves of nar la Hordeum mutants, the activity of NAD(P)H-NR is about 15 times higher than in wild type Steptoe, while the activity of NADH-NR in the mutants is only about 5% of the activity in wild type [93]. However, NADH-NR is active in the roots of both wild type and mutants. The NADH and NAD(P)H-NR are apparently different isoforms [94] and they have been shown to possess different kinetic, immunological and physical characteristics [95]. Thus, in Hordeum mutants lacking NADH-NR, NAD(P)H-NR may play an active role in nitrate reduction. However, Vigue and Warner [SS] were unable to demonstrate the presence of any NAD(P)H-NR in A334 mutants of Pisum. THE PRESENCE OF NITRATE REDUCTASE ASSIMILATING
IN NON-
TISSUES
Nitrate is primarily assimilated in roots or leaves, depending on the species [96]. However, the presence of nitrate reductase activity has been demonstrated in almost all the plant parts examined, including aleurone layers [97], endosperm [98], scutellum [98, 993, cotyledons [lOO-1021, stems [103-1051, petioles [106], fruits [107], ovules [lOS], developing pods of legumes [109], etc. The enzyme activity has been reported in human saliva as well, although its exact role is uncertain [ 1lo]. In Zea endosperm and scutellum, the NR activity increases with nitrate supply, as is the case with root and leaf enzyme [98]. The concentration of nitrate required to induce maximum NR activity however, is higher in endosperm and scutella, as compared to that in root and leaf tissues [3]. This, of course, may be partly related to the differential rates of absorption of ions by different tissues. In spite of the fact that these storage tissues (endosperm and scutella) have appreciable amounts of inducible NR activity, they are unable to assimilate nitrate into organic nitrogen [S]. It is possible that NR activity plays some dissimilatory role in these tissues, especially before seed germination. The seeds are often impermeable to oxygen due to the seed coat, and reduction of nitrate to nitrite deep inside the seed may generate oxygen for the oxidation of NADH. INCREASE
IN NITRATE
REDUCTASE
ACTIVITY
BY META-
BOLITES OTHER THAN GROWTH REGULATORS
Several endogenous and exogenous metabolites are known to influence nitrate reductase activity, although their physiological significance in nitrogen metabolism is not understood. One such metabolite is ammonium. Ammonium as a potential product of nitrate reduction is expected to inhibit NR activity. Although this expectation is realized in some micro-organisms [20], the effect of ammonium on higher plant NR is quite variable. It has either a slight inhibitory effect [111-1131 or no effect [114-1163. In many plants however, the supply of ammonium increases NR activity either in the absence or presence of nitrate (Table 1). Some attempts have been made to understand the mechanism of this increase [ 1191. Among other possibilities, it has been suggested that aminonium is oxidized to nitrate either by the plant tissue itself or by contaminating microbes, which in turn acts as an inducer of NR activity [ 1251. As there is no convincing evidence for the oxidation of ammonium to nitrate by the
H. S. SRIVASTAVA
2944 Table
1. Effect of ammonium
supply
on nitrate reductase activity absence of nitrate
Chenopodium rubrum cultured cells Cuscuta rejkxa cultured seedlings Phaseolus vulgaris excised leaves Rosa sp. (Paul’s scarlet suspension culture Sinapis alba cotyledons incubated in far-red light
(mM)
rn vitro in vitro
10 20
+540
Cl171 Ul71
in vi00
50
0
Cl181
in vivo
10
+ 248
Cl191
+260
rose)
in far-red light Zea mays excised roots excised shoots excised leaves intact leaves intact leaves intact shoot
m the presence
Assay method
ammonium species and tissues
plants
Percentage increase ( +) or decrease ( - ) Over no nitrogen Over NO;
Cont. of Plant
in higher
in vitro
0.91
+100
m vitro
15
+90
in vitro
15
+500
in in tn m in in
10 10 10 10 10 5
-31 -6 t175 +38 +457 +73
vivo vivo vi00 vivo vitro vitro
or
Reference
._
cm
143 +182
Cl211 Cl221 cl231 cl231 cl231 c41 L4l
f4
~1241
The percentage values are approximate. Not determined.
higher plant tissues, and as most of the systems where an increase in NR activity in the presence of ammonium have been reported are not properly sterilized, one would be inclined to believe that the microbes were the real agents of ammonium oxidation and NR induction. However, Schuster et al. [ 1261 have suggested that ammonium induces a specific isoform of NR. But synergistic action of nitrate and ammonium in inducing NR activity, in some systems, indicates that both nitrogen sources act independently in the induction of NR [122]. Nitrite, the immediate product of nitrate reduction, is also reported to increase NR activity in many tissues, the increase in some cases being equal to or even more than that observed with nitrate [127-1291. Aslam et al. Cl303 have suggested that the increase in Hordeum leaves is the result of nitrate, which is produced by the oxidation of nitrite. However, evidence for oxidation of nitrite or ammonium is certainly lacking in higher plants. Experiments with NR induction by nitrite or ammonium, performed in completely sterile conditions, may provide insight to this problem. It would be also worthwhile to compare kinetic properties and electrophoretic mobility of nitrite/ammonium-induced NR to that with nitrateinduced NR. CONCLLIBING
REMARKS: POLYMORPHIC ENZYME
NATURE OF THE
Although the significance of various functions of nitrate reductase is not understood, the origin of such functions in some cases may be traced to the different isoforms of the enzyme. In Glycine tissues three isoforms
of NR have been demonstrated [131, 1321: (1) a nitrateinducible NADH-NR (EC 1.6.6.1) with a pH optimum of 7.5; (2) a constitutive bispecific NADH/NADPH [NAD (P) HI-NR (EC 1.6.6.2) with a pH optimum of 6.5; and (3) a constitutive NADH-NR (EC number not yet assigned) with a pH optimum of 6.5. There is evidence for the occurrence of more than one isoform of NR in other species also, and it is likely that polymorphism in NR is not limited to Glycine only [16, 29, 1333. The presence and relative activities of different isoforms depend apparently on the species, tissues and nutritional factors. The first two forms are usually found in close association, and perhaps both contribute to the reduction and assimilation of nitrate. There is a vast literature on NADH-NR, emphasizing its role in nitrate assimilation, although NAD(P)H bispecific NR also seems to have an important role in the process, and it may be more widespread than currently believed [ 1341. As described earlier, in Hordeum mutants lacking NADH-NR, the NAD(P)H bispecificNR may assume the role of nitrate reduction [93]. The constitutive NR isoform in Glycine constitutes about 12-20% of the total NR activity and its absence does not impede normal nitrate utilization [47]. In Chlorella, the constitutive isoform is plasmamembrane-bound and appears to be involved primarily in nitrate uptake [135], although in other systems a component of nitrate transporter may be inducible as well [136, 1373. In another study [138] “NO; influx in non-induced (nitrate starved) Hordeum roots has also been shown to be mediated by a constitutive rather than inducible transport system. However, Warner and Huffaker [139], by analysing Hordeum mutants, have demonstrated that genetic loci
Multiple functions of nitrate reductase regulating
nitrate
transport
are distinct
from those
for
NR and NiR, although it may be under the same promoter region. NO, evolution activity and reduction of chlorate to chlorite are also believed to be the function of constitutive NR [ 14,473. The inducibility of NR by nitrate might have been selected by evolutionary pressure and agricultural practices and it is possible that the wild ancestors of cultivated plants had constitutive NR only [140]. Oumidou et al. [ 1411 have demonstrated that while the inducible form of NR is the major form in cultivated tetraploid and hexaploid Triticum aestivum, the constitutive form of the enzyme is predominant in their diploid ancestor, Aegilops squarrosa. Sensitive techniques for purification and characterization of NR are now available. By combining the cellular and molecular techniques with genetic approaches, it may be possible to detect different isoforms of NR in a variety of species [ 1261 and to relate them with a specific function. In many cases, it would also be necessary to culture plant tissues in completely sterile conditions, to ensure that one was dealing with the plant enzyme and not with the contaminating microbial enzyme. AcRnowbBgernents-The author thanks Dr P. V. Sane for his encouragement and initial interest in the article and Drs Ann Oaks and J. E. Harper for their critical reading of the article.
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