Regulation of nitrate assimilation in plants in light and dark

Plant Science Letters, 34 (1984) 25--34

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Elsevier Scientific Publishers Ireland Ltd.

REGULATION OF NITRATE ASSIMILATION IN PLANTS IN LIGHT AND D A R K

S. PRAKASH, PRIKHSHAYAT SINGH*, S.K. SAWHNEYand M.S. NAIK Division o f Biochemi#try, Indian Agricultural Research Institute, New Delhi -- 110012 (India)

(Received June 9th, 1983) (Revision received October 12th, 1983) (Accepted October 12th, 1983)

SUMMARY

Assimilation o f nitrate and nitrite accumulated in leaves was much more rapid in the light than in the dark-. Under light aerobic and dark anaerobic conditions the extent o f nitrate reduction was almost equal. Dark assimilation o f nitrite was drastically inhibited by 2,4
K e y w o r d s : Higher plants -- Light dark mechanisms -- Nitrate assimilation --

Pentose phosphate p a t h w a y -- Uncoupler

INTRODUCTION Whether light is absolutely essential for nitrate assimilation in green cells is still not known with certainty. Since dark heterotrophic assimilation o f nitrate in fungi and plant non-photosynthetic tissues such as roots is

*To whom correspondence should be sent. Abbreviations: BSA, bovine serum albumin; 2,4-DNP, 2,4-dinitrophenol; GOGAT, glutamate synthase; G 6-P, glucose 6-phosphate; GS, glutamine synthetase. 0304-4211/84/$03.00 © 1984 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

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known to depend on the reducing power generated by the oxidation of glucose, it might appear that the role of light if any, would be indirect, only to supply carbohydrates for nitrate assimilation. Aslam et al. [1,2] showed that barley leaves and seedlings can assimilate nitrate into amino acids in complete darkness. Similarly Yoneyama [3] reported significant dark assimilation of both nitrate and nitrite in the leaves of a number of plant species and suggested that for this the pathway would be the same as in light namely, nitrate reductase (EC 1.6.6.1), nitrite reductase (EC 1.7.7.1), glutamine synthetase (GS) (EC 6.3.1.2) and glutamate synthase (GOGAT) (EC 2.6.1.53). Lee [4] has reviewed the work on possible sources of reductants for dark nitrate assimilation in roots. Canvin and Atkins [ 5], however, showed that assimilation of nitrate and nitrite in green leaves is strictly light~lependent and ceases abruptly when the light is extinguished. The chloroplastic location of nitrite reductase, GS and GOGAT in leaf cells was demonstrated by Wallsgrove et al. [6], who suggested that activities of these enzymes are largely light~lependent, as they make use of reduced ferredoxin and ATP generated in light reactions of photosynthesis. As regards regulation by light of nitrate reductase, probably located in the cytoplasm, Sawhney et al. [7,8] proposed that a switch mechanism (light-on, dark-off) functions via inhibition of mitochondrial oxidation of NADH in light by enhanced ATP levels (reviewed by Naik et al. Ref. 9). Recently, Reed.and Canvin [10] demonstrated that in wheat leaf protoplasts,assimilation of both nitriteand nitrate is strictlylight
MATERIALS A N D M E T H O D S

Plant materials Rice (Oryza satipa L. cv. P-2-21), wheat (Triticum aestipum L. cv. R-306), cotton (Gossypium hirsutum L. cv. 9-40-50), maize (Zea mays L. cv. Ratna) and sorghum (Sorghum b/co/or L. cv. 5), seedlingswere grown for 10--15 days in pots under normal aunlight and periodicallyirri~ted with 15 m M KNO~

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Nitrate assimilation in.lightand dark Rice, wheat, cotton, maize and sorghum plants were supplied with excess of nitrate so that sufficient nitrate accumulated in the leaves. These leaves were then excised from the plants and total nitrate was determined in a sample. In each treatment 8--10 whole leaves were placed with bases dipped in a beaker containing 5 ml water. One set was exposed to direct sunlight (70--100 Klux) and the other was kept in the dark. Nitrate remaining in the leaves after these treatments at different time intervals was determined.

Treatmentwith 2, 4.DNP Solution of 2,4-DNP was prepared in minimum amount of ethanol and for further use diluted in water to required concentration. Effect of uncoupler on nitriteassimilation In order to avoid complications arising out of problems associated with the uptake of externally supplied nitrite, leaves were initially allowed to accumulate nitrite endogenously by incubating them under dark anaerobic conditions for i h. A portion of these leaves was analysed for initialnitrite concentration. Remaining leaves were floated in uncoupler solutions for 30 rain in dark at 0--4°C and then washed with water and blotted dry. These leaves were exposed to light and dark treatments as mentioned above. At different time intervals nitriteremaining in the leaves was determined.

14C02 evolution from substrates in the presence of 2,4-DNP Uptake of exogenously added substrates by leaf discs is likely to be inhibited by 2,4-DNP. Hence in order to study the effect of 2,4-DNP on 14CO2 evolution from endogenously labelled substrates, the leaves were allowed to assimilate ~4CO2 in 2 1 capacity air-tight Perspex chamber in dark or light for 30 rain at 25 + I°C. ~4CO2 was released from Na2~4CO3 by injecting 1 N HCI, through the septum into 2 ml of 0.5 M Na2CO3 (containing 1.85 X 106 Bq of Na2~4CO3 for light treatment and 1.67 × 107 Bq of Na2~4CO3 for dark treatment) placed in a small tube kept at the bottom of the chamber. These leaves were then used for the study of total ~4CO2 incorporation [12] and for the estimation of ~4CO2 evolution in the presence of 2,4-DNP, leaf discs (200 mg) were incubated in 3 ml of 100 m M phosphate buffer (pH 5.0) in Warburg flasks. Parallel control flasks contained 200 m g boiled leaf tissue. 2,4-DNP was added as indicated in Table IV. Flasks were stoppered and ~4CO2 evolved during the incubation at 30°C was absorbed on a filter paper strip (20 × 22 ram) contained in central well of the flask with 0.2 ml of 20% K O H and radioactivity was counted and determined by using sample channel ratio method as described [12 ]. Other details Mitochondria from wheat and rice leaves were isolated employing the procedure [13] as described earlier [14]. Oxygen uptake with N A D H and

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succinate was monitored using a Clark O2 electrode and calculated [15] by taking O2 concentration in air-satttrated medium as 240 pM. Nitrite and nitrate in leaf extracts were determined as described in Ref. 7 and Ref. 16, respectively. All experiments described above were done in triplicates and averages are recorded in Tables. In general variability was 5--7%. Protein was estimated by the method described in Ref. 17 as explained in Ref 14 with crystalline BSA as standard. In th~se experiments all glass double distilled water was used. All biochemicals were obtained from Sigma Chemical Co., (CA, U.S.A.). Radioactive chemicals were obtained from Bhabha Atomic Research Centre (Bombay, India). RESULTS

Reduction o f nitrate in light and dark in leaves In Table I reduction of accumulated nitrate in excised leaves of wheat, rice, sorghum and cotton directly exposed to light and dark treatments without any aqueous medium, is presented. Small but significant reduction of nitrate is observed in dark, the rate being less than 30% as compared to that in light. When a comparison was made between reduction of leaf nitrate in light aerobic and dark anaerobic conditions it was found that the rates were almost equal (Table II). In the latter treatment, nitrite accumulated in the leaves as its further assimilation in the chloroplasts is largely light dependent [6]. These rates were much faster than dark aerobic reduction of nitrate which was similar to the amount of nitrite formed when 2,4-DNP was added. In the presence of 2,4-DNP under dark aerobic conditions, nitrite accumulated in the leaves, because its further reduction to ammonia was probably inhibited as shown below. TABLE I N I T R A T E ASSIMILATION IN L I G H T AND D A R K IN LEAVES OF D I F F E R E N T PLANTS Excised whole leaves of plants which were allowed to accumulate sufficient nitrate were exposed to light or dark treatments for 1 h ~s described in Materials and Methods. Nitrate contents at the start and end of the experiment were determined and nitrate assimilated was calculated. Plants

n m o l N O ~ -I g fresh wt. "I Initial

Cotton Rice Sorghum Wheat

3200 3600 2800 6700

Assimilated after 1 h exposure to

Light

Dark

2100 2300 2000 3600

700 700 400 500

29 TABLE II COMPARISON OF NITRATE REDUCTION IN LIGHT AEROBIC, DARK ANAEROBIC AND DARK AEROBIC CONDITIONS IN RICE AND WHEAT LEAVES Leaves were treated with 5 mM DNP for dark aerobic treatment as described in Materials and Methods. In this treatment and also in dark anaerobic treatment nitrite accumulated in the tissue was taken as an index of nitrate reduction. In the remaining two treatments, light aerobic and dark aerobic, nitrate assimilation was calculated from the nitrate remaining in the leaves. Treatment

Time

nmol NO~-1 reduced g fresh wt. -1

(min) Light aerobic Dark anaerobic Dark aerobic Dark aerobic with 5 mM DNP

30 60 30 60 60 60

Rice

Wheat

-1800 -1700 ---

2700 3900 2500 3200 754 975

Effect of 2,4-DNP on the reduction of nitrite Large q u a n t i t i e s o f nitrite are k n o w n t o a c c u m u l a t e w h e n leaves o f plants are i n c u b a t e d u n d e r d a r k a n a e r o b i c c o n d i t i o n s [ 7 , 8 , 1 8 , 1 9 ] . This h a p p e n s because m i t o c h o n d r i a l o x i d a t i o n o f N A D H is inhibited and r e d u c i n g equivalents are t h e n available f o r t h e r e d u c t i o n o f nitrate. T h e d i s a p p e a r a n c e o f a c c u m u l a t e d nitrite d u r i n g s u b s e q u e n t aerobic i n c u b a t i o n is m u c h m o r e rapid in light t h a n in d a r k [ 1 8 - - 2 0 ] . Mann et al. [19] a n d R a m a r a o et al. [ 2 1 ] s h o w e d t h a t t h e d i s a p p e a r a n c e o f a c c u m u l a t e d nitrite c o m m e n c e s a f t e r a lag p e r i o d o f 2 0 - - 3 0 rain u n d e r d a r k aerobic c o n d i t i o n s . T h e slower rate o f nitrite r e d u c t i o n in d a r k as c o m p a r e d with t h a t in light is c o n f i r m e d in Table III. When t h e e f f e c t o f 5 m M 2,4-DNP was studied o n light and d a r k r e d u c t i o n o f nitrite in w h e a t and rice leaves, it was o b s e r v e d t h a t t h e unc o u p l e r i n h i b i t e d t h e r e d u c t i o n o f nitrite slightly in t h e light and substantially in t h e dark. It is interesting t o n o t e t h a t in t h e d a r k m o r e nitrite a c c u m u l a t e d t h a n was p r e s e n t at t h e start o f t h e r e a c t i o n (Table III). This increase in nitrite c o n t e n t was o b v i o u s l y d u e t o r e d u c t i o n o f n i t r a t e p r e s e n t in t h e leaves t o nitrite d u r i n g i n c u b a t i o n with 2,4-DNP in dark.

Effect of 2,4-DNP on oxidation of different substrates I n h i b i t i o n o f d a r k aerobic nitrite r e d u c t i o n b y 2,4-DNP c o u l d be d u e t o i n h i b i t i o n o f s u p p l y o f r e d u c t a n t g e n e r a t e d b y t h e o x i d a t i o n o f substrates. It was f o u n d t h a t even at l o w c o n c e n t r a t i o n s o f 2,4-DNP o f 0.01 mM, c o n s i d e r a b l e i n h i b i t i o n o f 14CO2 e v o l u t i o n f r o m e x o g e n o u s l y supplied labelled succinate, malate, p y r u v a t e and glucose was o b s e r v e d in leaves (results n o t shown). It is possible t h a t t h e u n c o u p l e r m i g h t b e inhibiting t h e u p t a k e o f t h e s e substrates b y leaf discs, since active u p t a k e o f sub-

30 TABLE III E F F E C T OF DNP ON LIGHT AND DARK N I T R I TE REDUCTION IN RICE AND WHEAT LEAVES Rice and wheat leaves were initially incubated for 1 h under vacuum and nitrite accumulated in the tissue was determined in a sample (200 rag). These leaves were then exposed to light and dark treatments and nitrite present in the tissues at different time intervals was determined. Treatment with 5 mM DNP was given as indicated in Materials and Methods. Time (min)

nmol N O = -I g fresh wt. -I -- DNP

Rice 0(initial) 30 60 120 Wheat 0 (initial) 30 60 120

TABLE

+ DNP

Light

Dark

Light

Dark

920 480 290 110

920 660 560 480

920 660 490 350

920 1080 1240 1375

1215 340 240 210

1215 1010 970 990

1215 510 440 360

1215 1350 1750 2025

IV

E F F E C T OF DNP ON '4CO2 R E L E A S E FROM WHEAT LEAVES, PREVIOUSLY ALLOWED TO ASSIMILATE 14CO2 IN LIGHT OR D A R K Whole leaves were exposed to ~4CO~ in light or dark for 30 min at 25 ± I°C, in Perspex chamber as described in Methods. Total incorporation of ~4CO= was determined in leaf samples (200 rag) after extraction with 80% h o t ethanol (v/v). 14CO2 evolved from these leaves was subsequently studied in 3 ml K-phoQphate buffer (pH 5.0) 100 raM, as in Materials and Methods. 14CO= evolved (× l0 s c p m g fresh wt. -I h -~)

Leaves allowed t° assimilate ~4CO2 in light or dark

Light

Dark

Total incorporation of ~4CO2

23054

948

1171 1195 682 319 192

327 364 360 306 210

D N P added ( m M ) Control 0.05 0.5 1.0 2.0

31 strates requires ATP. In order to label endogenous substrates such as sugars or organic acids wheat leaves were initially allowed to assimilate 14CO2 in light or dark respectively [21,22]. When these two sets of leaves were subsequently treated with 2,4-DNP, it was observed (Table IV) that the uncoupler had negligible effect on 14CO2 evolution from leaves in which organic acids were labelled in dark. This shows that 2,4-DNP did not have any effect on oxidation of endogenous organic acids. The uncoupler significantly inhibited ~4CO2 evolution from leaves in which sugars were labelled in light. Hence, the inhibitory effect of 2,4-DNP on nitrite reduction can be attributed to inhibition of glucose metabolism. State 3 oxygen uptake was not significantly affected by 2,4-DNT at 30 ~M concentration from isolated rice (14 as against 15 with succinate, and 50 as against 57 with NADH) and wheat (18 as against 25 with succinate, and 30 as against 31 with NADH) leaf mitochondria, expressed as natom O2 uptake mg protein -~ min -x. Similar results were earlier reported [23]. DISCUSSION Regulation of nitrate reduction in leaves operates through competition for reducing equivalents from NADH between cytoplasmic nitrate reductase and mitochondrial electron transfer chain to oxygen [7,8,19,24,25]. It was therefore proposed that in light, inhibitory effect of oxygen on nitrate reduction is probably abolished via photosynthesis-induced increase in cytoplasmic ATP level [ 7--9 ]. On the basis of uncoupling of photophosphorylation by nigericin, when CO2 fixation and formation of triose phosphates is inhibited, Reed and Canvin [10] have given an alternative explanation and proposed that light-induced increase of cytoplasmic reducing equivalents suppresses the competition between nitrate reductase and dark respiration. Whether under physiological conditions of photosynthesis mitochondrial oxidation of NADH is in fact inhibited in light is still controversial [26--31]. In a recent review Tager et al. [32] have shown that extramitochondrial phosphate potential and ATP/ADP ratio are not the only parameters controlling respiration, and other factors including cytochrome c oxidase, inorganic phosphate and hydrogen supply also play a significant role. Perhaps the strongest direct evidence of light inhibition of mitochondrial respiration in leaves was obtained from in vivo experiments using 1sO2 [33]. If this conclusion is correct the question of saturating the oxidative demands of mitochondria and nitrate reductase as proposed by Reed and Canvin [10] does not arise in any case. Dark anaerobic and light aerobic conditions resemble each other as the amount of nitrate reduced in leaves is almost equal (Table II). In the former treatment nitrite accumulated in the leaf tissue. Probable complications arising out of the effect of light and photosynthetic reactions on the uptake of nitrate by plants from the external medium

32 has been avoided in our experiments because decrease in the nitrate content already absorbed by the leaves was examined in light and dark. Moreover since intact leaves were not suspended in any liquid medium, the likely leakage of nitrate or nitrite in the aqueous m e d i u m was avoided. This approach is different from that used by Aslam et al. [1] who measured de, pletion of nitrate from the external m e d i u m as an indication of nitrate assimilation over a prolonged period of incubation of about 24 h. Our short duration experiments (Table I) minimise likely loss of enzyme activity [34]. Mann et al. [19] found that during 1 h incubation, lSN-enrichment from labelled nitrate in spinach leaf discs was 6-fold higher in light than in dark. Yoneyama [3] demonstrated that under anaerobic conditions the a m o u n t of nitrate reduced was several fold more than under dark aerobic conditions. In roots and leaves in darkness, while nitrate reduction takes place in the cytoplasm, the subsequent assimilation of nitrite into amino acids is largely confined to the plastids and chloroplasts, respectively [ 35--37 ]. Emes et al. [38] and Dry et al. [39] have shown that a major function o f oxidative pentose phosphate pathway in the roots is to supply NADPH necessary for nitrite assimilation, which ensures closely coupled supply of electrons as long as sufficient ATP and consequently G 6-P is available. As regards dark assimilation of nitrite in leaves, it is reasonable to expect that a similar mechanism can function in chloroplasts. In the event of adequate supply of G 6-P, the NADPH generated in the oxidative pentose phosphate pathway can be used for the reduction of ferredoxin via ferredoxin-NADP reductase [21]. Reduced ferredoxin can t h e n be used by nitrite reductase and GOGAT. Inhibition of nitrite reduction in the dark by 2,4-DNP observed by us (Table III) could be due to the deficiency of ATP which is required for the synthesis of G 6-P. In light, however, nitrite reduction continued even in the presence of 2,4-DNP because reduced ferredoxin was generated directly in the light reaction of photosynthesis [6,40] (Table III). Results in Table IV showed that 2,4-DNP inhibited 14CO2 evolution from 14C-sugars which were labelled in light. Since in the oxidative pentose phosphate pathway, 6-phosphogluconate dehydrogenase is the likely source of 14CO2 from labelled glucose, it can be concluded that the uncoupler inhibits the formation of G 6-P by depletion o f ATP supply as proposed by Dry et al. [ 39]. As a consequence dark reduction of nitrite which depends on supply of G 6-I) is inhibited. In leaves the enzymes o f nitrite assimilation are located in the chloroplast and hence nitrite assimilation is largely light dependent [6,35]. However, recent reports showed that reduction of nitrite can take place in the leaves in darkness, although at a m u c h slower rate than in the light [11,18,19,34]. The rate o f dark aerobic nitrate reduction is extremely slow as compared with the rate under light or even under dark anaerobic conditions, when mitochondrial oxidation of NADH is inhibited. In light oxidative pentose phosphate pathway is blocked in the chloroplasts as both oxidative and

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reductive (Calvin cycle) mechanisms can not function simultaneously [27]. The key enzyme glucose 6-phosphate dehydrogenase is inhibited in light by reduced thioredoxin [41--44]. Hence the dark heterotrophic nitrate assimilatory pathway is suppressed in light and superseded by regulatory reactions operating under photoautotrophic conditions. Very rapid rate of nitrate assimilation in light is obviously due to direct photosynthetic assimilation of nitrite in the chloroplast and availability of excess NADH for the reduction of nitrate in the cytosol. REFERENCES

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32

M. Aslam, R.C. Huffaker, D.W. Rains and K.P. Rao, Plant Physiol., 63 (1979) 1205. M. Aslam and R.C. Huffaker, Plant Physiol., 70 (1982) 1009. T. Yoneyama, Plant Cell Physiol., 22 (1981) 1507. R.B. Lee, Plant Cell Environ., 3 (1980) 65. D.T. Canvin and C.A. Atkins, Planta, 116 (1974) 207. R.M. Wallsgrove, P.J. Lea and B.J. Miflin, Plant Physiol., 63 (1979) 232. S.K. Sawhney, M.S. Naik and D.J.D. Nicholas, Nature, 272 (1978) 647. S.K. Sawhney, M.S. Naik and D.J.D. Nicholas, Biochem. Biophys. Res. Commun., 81 (1978) 1209. M.S. Naik, Y.P. Abrol, T.V.R. Nair and C.S. Ramarao, Phytochemistry, 21 (1982) 495. A.J. Reed and D.T. Canvin, Plant Physiol., 69 (1982) 508. A.J. Reed, D.T. Canvin, J.H. Sherrard and R.H. Hageman, Plant Physiol., 71 (1983) 291. M.S. Naik and Prikhshayat Singh, FEBS Lett., 111 (1980) 277. R. Donce, A.L. Moore and M. Neuburger, Plant Physiol., 60 (1977) 625. Prikhshayat Singh and P.S. Kirshnan, Physiol. Plant., 39 (1977) 179. R.W. Estabrook, Methods Enzymol., 10 (1967) 41. H.L. Grover, T.V.R. Nair and Y.P. Abroi, Physiol. Plant., 42 (1978) 287. O.H. Lowry, N.J. Rosebrough, A.L. Farr and R.J. Randall, J. Biol. Chem., 193 (1951) 265. R.W. Jones and R.W. Sheard, Plant Sci. Lett., 11 (1978) 285. A.F. Mann, D.P. Hucklesby and E.J. Hewitt, Planta, 146 (1979) 83. N.P. Voskresenakaya and G.G. Grishina, Soviet Plant Physiol., (Translation), 9 (1962) 4. C.S. Ramarao, Srinivasan and M.S. Naik, New Phytol., 87 (1981) 517. M.S. Naik and D.J.D. Nicholas, Aust. J. Plant Physiol., 8 (1981) 515. D.P. Hackett and D.W. Haas, Plant Physiol., 33 (1958) 27. L. Beevers and R.H. Hageman, in: B.J. Miflin (Ed.), The Biochemistry of Plants, Academic Press, New York, Vol. 5, 1980, 115. D.T. Canvin and K.C. Woo, Can. J. Bot., 57 (1979) 1155. U. Heber, Annu. Rev. Plant Physiol., 25 (1974) 393. D. Graham, in: D.D. Davies (Ed.), The Biochemistry of Plants, Academic Press, New York, Vol. 2, 1980, 526. U. Heber, U. Takahama, S. Neimanis and M. Shimizu-Takahama~ Biochim~ Biophys. Acta, 679 (1982) 287. R. Hampp, M. Goller and H. Ziegler, Plant Physiol., 69 (1982) 448. N. Stitt, R. McClilley and H.W. Heldt, Plant Physiol., 70 (1982) 971. I. Dry and J.T. Wiskich, Arch. Biochem. Biophys., 217 (1982) 72. J.M. Tager, R.J.A. Wanders, A.K. Groen, W. Kunz, R. Bohnensack, U. Kiister, G. Letko, G. B6hme, J. Duszynski and L. Wojtczak, FEBS Lett., 151 (1983) 1.

34 33 D.T~ Canvin, J.A. Berry, M.R. Badger, H. Fock and C.B. Osmond, Plant Physiol., 66 (1980) 302. 34 C.S. Ramarao, Srinivasan and M.S. Naik, Phytochemistry, 26 (1981) 1487. 35 B.J. Miflin, Plant Physiol., 54 (1974) 550. 36 M.J. Emes and M.W. Fowler, Planta, 145 (1979) 287. 37 M.J. Emes and M.W. Fowler, Planta, 158 (1983) 97. 38 M.J. Emes, H. Ashihara and M.W. Fowler, FEBS Lett., 105 (1979) 370. 39 L Dry, W. Wallace and D.J.D. Nicholas, Planta, 152 (1981) 234. 40 B.J. Miflin, Planta, 116 (1974) 187. 41 K. Lendzian and H. Ziegler, in: G. Forti, M. Avron and A. Melandri (Eds.), Photosynthesis, Two Centuries after its Discovery by Joseph Priestly, Dr. W. Junk, N.V. Publishers, The Hague, Proc. Int. Congr. Photosynth. Res., 3 (1971) 1831. 42 A.R. Ashton, T. Brennan and L,E. Anderson, Plant Physiol., 66 (1980) 605. 43 B.B. Buchanan, Annu. Rev. Plant Physiol., 31 (1980) 341. 44 R. Scheibe and L.E. Anderson, Biochim. Biophys. Acta, 636 (1981) 58.