DETECTION OF NITRATE REDUCTASE ACTIVITY IN NITRATE REDUCTASE DEFICIENT MUTANTS OF BARLEY (HORDEUM VULGARE)
SRINIVASAN, S.K. SAWHNEY, SUSHIL KUMAR, P. MOHANTI, S.K. SINHA and M.S. NAIK
Division of Biochemistry, Indian Agricultural Research Institute, New Delhi-110012 (India)
(Received May 18th, 1982) (Revision received October 12th, 1982) (Accepted October 22nd, 1982)
SUMMARY
Nitrate reductase deficient mutants of barley Az l2 and Az l3 showed 40--50% in vivo enzyme activity as compared with the Steptoe non-mutant under strictly anaerobic conditions of assay. However, in vitro nitrate reduc- tase activity could not be detected in the mutan t extracts prepared by differ- ent methods. The extracts did not contain any inhibitory factor as judged by effect of in vitro nitrate reductase activity in the Steptoe non-mutant.
Defective functioning of the Mo cofactor in the mutants was indicated by the fact that reduced benzyl viologen was ineffective as an electron donor. Incorporation of Mo-cofactor obtained from the Steptoe non-mutant into the mutan t extract significantly reconsti tuted nitrate reductase activity. The re- const i tuted enzyme was NADH specific, indicating that coenzyme specificity is not altered in the mutants. Thus association of Mo-cofactor with the apoenzyme appears to be defective in the mutants.
Nitrate reductase is a key enzyme in the nitrate assimilation pathway and hence mutants deficient in this enzyme are incapable of normal growth with nitrate as a sole source of nitrogen. This has been demonstrated with mutants of Neurospora crassa, Aspergillus nidulans [1,2], Arabidopsis theliana [3] and Nicotiana tabacum cell lines [4,5]. However, nitrate reductase deficient
mutants of Hordeum vulgare, developed by Warner et al [6], showed normal growth in the field [7] , suggesting that they can assimilate nitrate quite efficiently, in spite of this deficiency. Subsequently, Warner and Kleinhofs [ 8] conclusively demonstrated that barley mutants A z l 2 and Az l3 produced almost as much dry matter and reduced nitrogen accumulation as the Step- toe non-mutant , although the mutants consistently showed less thm~ 4% and 8% of the in vivo nitrate reductase activity in leaves and roots respectively, as compared with Steptoe. Substantial rate of nitrate assimilation, in the absence of any demonstrable nitrate reductase activity could not be readily explained. James et al. [9] confirmed that in vitro nitrate reductase in Az l2 mutant was only 0.5% to 2% of that of the Steptoe non-mutant, but in- creased 10-fold by increasing nutr ient molybdenum ranging from 0.5 pm to 1 mM. They suggested that although aponitrate reductase and Mo containing cofactor are present in the mutant , aggregation of the subunits seems to be impaired in vivo and fails in vitro.
In order to understand how mutants of barley deficient in nitrate reduc- tase are capable of normal growth and nitrate assimilation rates, we have examined the in vivo and in vitro activity of the enzyme under different conditions. Suggestions that coenzyme specificity of the enzyme in the mutants might have changed [ 8] have also been examined. Reconst i tut ion of in vitro nitrate reductase activity in the extracts of the mutant Az l3 , by the incorporation of Mo cofactor from the extracts of the Steptoe non-mutant barley leaves, was a t tempted with a view to understand the nature of muta- tion.
MATERIALS AND METHODS
Plant material Seeds of barley Steptoe and its mutants A z l 2 and Az l3 were obtained
through the kind courtesy of Dr. A. Kleinhofs, Washington State University, Washington, U.S.A. Plants were grown in an experimental field of the Insti- tute. The soil contains abundant molybdenum and no deficiency has ever been reported. Twenty kg/ha of urea was applied at the time of sowing and 40 kg of each of single super phosphate and muriate of potash were given one week after sowing.
In vivo assay Four different methods of in vivo assay were used. In each case 0.1 g leaf
material was used. Method A. Leaves were cu t in approx. 5 mm pieces and were placed in a
50 ml beaker with 10 ml of phosphate buffer (0.1 M, pH 7.0) and KNO3, 10 mM. Beakers were kept in a vacuum dessicator and evacuated for 2 min. After infiltration the leaves were incubated in a water bath at 30°C. After 30 min suitable aliquots were withdrawn and nitrite estimated as described below.
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Methods B--D. In methods B and C, 5 mm segments of leaf discs were placed in 5 ml phosphate buffer (0.1 M, pH 7.5) containing KNO3 (10 mM). In method C, propanol was added to give a final concentrat ion of 2% but not in Method B. The tubes were infiltrated and then incubated at 30°C. After 30 min the tubes were placed in boiling water for 5 min for complete extrac- tion of nitrite. In Method D, the infiltration and incubation was done in Thunberg tubes under complete anaerobic conditions. The tubes were thoroughly evacuated with a vacuum pump to remove traces of air. After incubation at 30°C for 30 rain, the tubes were opened and placed in boiling water for 5 min for extraction of nitrite.
In vitro nitrate reductase activity Cell free extract of the leaves was prepared by homogenizing I g of tissue
in 4 ml of 0.1 M phosphate buffer (pH 7.2) containing 2 mM cysteine and 2.0% casein. The homogenate was then centrifuged at 15 000 × g for 30 min. All these operations were done in cold (1--4°C). The in vitro activity was determined as described in Kadam et al. [ 10], in a reaction mixture of 2 ml containing phosphate buffer (pH 7.5), 50 mM; KNO3 10 mM; enzyme, 0.2 ml; reduced benzyl viologen (0.5 mM) or NADH (0.25 mM).
Isolation o f Mo cofactor from the non-mutant For the isolation of the Mo cofactor from the extracts of leaves, method
of Rucklidge et al. [11] was followed. The extract was gradually treated with 1 N HC1 to adjust the pH to 2.5. After 3 min the pH was restored back to 7.0 by slowly adding 1 N NaOH. The extract was centrifuged at 3000 × g for 15 min and pellet suspended in phosphate buffer (0.1 M pH 6.2). Equal volumes of the cofactor and extract of mutan t were mixed and preincubated for 20 min before in vitro nitrate reductase assay.
Estimation o f nitrite In all these experiments the amount of nitrite formed was determined in
suitable aliquots by adding 1 ml each of 1.0% sulfanilamide in 1% HC1 and 0.02% Naphthyl ethylene diamine dihydrochloride in that order. Colour was allowed to develop for 15 min after which absorbance was recorded at 540 nm.
RESULTS
In vivo activity o f nitrate reductase by different methods In vivo activity of nitrate reductase in leaf discs was determined by differ-
ent methods (Table I). In Methods A - C strictly anaerobic conditions were not maintained,
because after vacuum infiltration in a liquid medium, the leaf discs were exposed to air during incubation, Method A, which is similar to the one used by Warner et al. [6] showed very little activity, probably also because
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TABLE I
IN VIVO NITRATE REDUCTASE ACTIVITY IN BARLEY LEAVES BY DIFFERENT METHODS
Four methods described in Materials and Methods, were used for the assay of in vivo activity.
Variety of barley Methods of assay (~mol of NO~ formed 1 h/g leaf tissue)
only nitrite released in the medium was measured and nitrite retained in the leaf cells which was not taken into account. It has been shown that for complete excretion of nitrite from the leaf discs, an alkaline pH is necessary [12,13]. In this Method A, the pH of the medium was 7.0 and hence some amount of nitrite could still have been retained in the leaf tissue.
In Methods B--D, nitrite retained in the leaf cells were extracted by placing the tubes in boiling water. Method C gave higher in vivo activity as compared with Method B because propanol was included in the former. Although stimulatory effect of propanol on in vivo nitrate reductase has been reported by several workers, the exact mechanism is not clear [14]. Subbalakshmi et al. [ 15] suggested that propanol may be diminishing the rate of diffusion of oxygen under partially aerobic conditions, or alternatively it may be inhibiting the oxidation of NADH by oxygen via the mitochon- drial electron transport chain, possibly by its effect on membranes.
In Method D strictly anaerobic conditions were maintained in a Thun- berg tube. Under these conditions maximum in vivo activity was detected in all these varieties of barley. The mutants Az l2 and Az l3 showed 40--50% of the activity as compared with the Steptoe non-mutant . James et al. [9] used an in vitro method with 0.4% Tween 20 and observed 22--37% in vivo activity in the mutan t A z l 2 as compared with the non-mutant. These results are similar to those obtained by us in Method C using propanol (Table I).
In vitro activity By the conventional extraction procedure in vitro nitrate reductase
activity could not be detected in the extracts of mutants either with NADH, NADPH or reduced benzyl viologen as electron donors, although consider- able activity was present in the non-mutant (Table II). Since the mutan t showed substantial in vivo activity, the enzyme probably gets inactivated during extraction. Modification of the extraction procedure was, therefore,
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TABLE II
IN VITRO NITRATE REDUCTASE ACTIVITY
This was determined in cell free extracts of barley leaves with different electron donors, NADH, NADPH and reduced benzyl viologen as described in Materials and Methods.
Electron donors (~mol of NO; formed 1 h/g leaf tissue)
a t tempted by including different concentrations of cysteine, casein, NADH, KNOa, NiC12, glycerol etc.
In order to check whether oxygen has any adverse effect on the stability of the enzyme in the extract, these leaves were also extracted and subse- quently assayed under anaerobic conditions in Thunberg tubes.
All these treatments could no t prevent almost complete loss of activity of the enzyme from the mutants during extraction. It was also observed that addition of extract of the mutants to the extracts of non-mutant did not result in any inhibition of nitrate reductase activity in the latter. This demon- strated that no inhibitory factors were present in the extracts of mutants.
Reconsti tution o f in vitro nitrate reductase activity Mo cofactor extracted by acid t rea tment from the Steptoe non-mutant
was added to the extract of the mutan t Az13. Significant activity could be detected in the mutan t after the addition of molybdenum cofactor extracted from the Steptoe non-mutant . Thus the mutan t which fails to show any activity in vitro in extracts obtained by different procedures described above, could be activated by the incorporation of the molybdenum cofactor to the extent of about 10% of the activity as compared to the Steptoe non-mutant. However acidified fraction from mutan t plants failed to restore activity of nitrate reductase (Table III). Rucklidge et al. [11] have shown that Mo co- factor is isolated from leaf extracts by acidification with i N HC1 followed by subsequent neutralisation. The pellet thus obtained is considerably en- riched with Mo cofactor [ 11]. In vitro reconsti tution studies have also been conducted by Mendel and Muller [4], with two types of Nicotiana tabacurn cell lines. The reconsti tuted enzyme showed less than 2% of the activity of the control. About 1% activity of~ NADPH nitrate reductase was observed by Garrett and Cove [16] in an in vitro reconsti tution experiment with extracts of n/a D and cux mutants of Aspergillus nidulans. Complementat ion in spinach leaves resulted in about 25% reconsti tution of the enzyme [ 11].
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T A B L E III
R E C O N S T I T U T I O N O F IN V I T R O N I T R A T E R E D U C T A S E A C T I V I T Y BY Mo- C O F A C T O R
Mo co fac to r was i so la ted f r o m t he ex t r ac t s of S t e p t o e n o n - m u t a n t and m u t a n t was added to t he ex t r ac t of m u t a n t A z l 3 a n d in vivo n i t r a t e reduc tase ac t iv i ty was d e t e r m i n e d wi th N A D H as an e l ec t ron d o n o r . T h e a m o u n t of n i t r i t e f o r m e d (300 n m o l ) b y 0.1 ml of the ex t r ac t of n o n - m u t a n t in 30 rain was t a k e n as 100 for compar i son .
Ni t r a t e r educ tase ac t iv i ty
N o n - m u t a n t 100 Acidi f ied n o n - m u t a n t nil Mo-cofac to r c o n t a i n i n g
f r ac t i on f r o m the n o n - m u t a n t nil M u t a n t ex t r ac t +
Mo co fac to r f r o m n o n - m u t a n t 11 M u t a n t ex t r ac t +
Mo co fac to r f r o m m u t a n t nil
DISCUSSION
The important observation of Warner and Kleinhofs [ 8] that the mutants of barley are sensitive to chlorate indicated the presence of an active nitrate reductase, because chlorate toxici ty results from the ability of nitrate reduc- tase to reduce chlorate to toxic chlorite. However, they could not detect any significant in vivo or in vitro activity of the enzyme to account for the normal growth and development of the mutants.
Activity of nitrate reductase in leaf discs in vivo is extremely sensitive to oxygen and is completely inhibited at less than 1% of the atmospheric oxygen concentrat ion [17--19] . Failure to detect significant in vivo nitrate reductase activity in these mutants by Warner and Kleinhofs [ 8] could be due to partial aerobic condit ions of assay used by them. Moreover, accumula- tion of nitrite in the leaf tissue was also probably not taken into account. Under strictly anaerobic condit ions of assay and subsequent extraction of nitrite by boiling, leaf discs of the mutants showed substantial nitrate reduc- tase activity, which was abou t 40--50% of that of the Steptoe non-mutant (Table I). This showed that a functional enzyme containing apoenzyme and Mo cofactor is present in the mutants, in vivo.
However, the enzyme in the mutants was found to be extremely unstable and lost all activity during extraction. As suggested by James et al. [9] aggregation of the subunits and the Mo cofactor in the mutants seems to be impaired. Defective functioning of the Mo cofactor of nitrate reductase is also indicated by the fact that reduced benzyl viologen, which donates elec- trons to the terminal Mo containing moiety of nitrate reductase was com- pletely ineffective in the mutan t extract (Table II). Our results in Table III demonstrate that incorporation of Mo cofactor from an active nitrate reduc-
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tase of the Steptoe non-mutant was effective in reconst i tu t ion of the nitrate activity in the mutants . However addit ion of Mo cofac tor extracted from the mutant , failed to restore activity. Therefore, it appears tha t the muta t ion has affected the Mo cofactor and its association with the enzyme. Since the reconst i tu ted enzyme accepted electrons from NADH, the coenzyme specifi- city does no t seem to have been altered in the mutants .
Even under opt imum condit ions (Table I) the mutants showed only about 50% of the in vivo activity as compared with the parent. However, this defi- ciency has in no way affected the growth, dry mat ter product ion and nitro- gen assimilation rates in the mutants as repor ted by Warner and Kleinhofs [ 8]. If nitrate reductase is a rate limiting step in nitrate assimilation pathway as proposed by Beevers and Hageman [ 20] and Nair and Abrol [ 21], then the activity in the mutants under physiological condit ions could be still higher than what could be detected by the presently available experimental techniques. Alternatively diminished activity of nitrate reductase may be sufficient to sustain normal plant growth. Alternate mechanisms of non- enzymatic photochemical nitrate reduct ion by UV componen t of sunlight could also perhaps cont r ibute to some ex ten t in the assimilation of nitrate in these mutants [22] .
REFERENCES
1 D.J. Cove, Biol. Rev., 54 (1979) 291. 2 R.H. Garret and N.N. Amy, Adv. Microbial. Physiol., 18 (1978) 1. 3 F.J. Oostindier-Braakama and W.J. Feenstra, Mutat. Res., 19 (1973) 175. 4 R.R. Mendel and A.J. Muller, Mol. Gen. Genet., 161 (1978) 77. 5 A.J. Muller and R. Grafa, Mol. Gen. Genet., 161 (1978) 67. 6 R.L. Warner, C.J. Lin and A. Kleinhofs, Nature, 269 (1977) 406. 7 J.Y. Oh, R.L. Warner and A. Kleinhofs, Crop Sci., 20 (1980) 487. 8 R.L. Warner and A. Kleinhofs, Plant Physiol., 67 (1981) 740. 9 D.M. James, E.J. Hewitt and E.F. Watson, Plant Physiol., (Suppl.) 67 (1981) 39.
