Photochemistry and Photobiology Vol. 39, No. 4, pp. 491 - 493, 1984 Printed in Great Britain. All rights reserved

003 1-8655184 SO3 .00+0.00 Copyright 0 1984 Pergamon Press Ltd

INDEPENDENT EFFECTS OF PHYTOCHROME AND NITRATE ON NITRATE REDUCTASE AND NITRITE

REDUCTASE ACTIVITIES IN MAIZE

ARUN KUMAR SHARMA and SUDHIR K. SOPORY* Plant Research Laboratory, School of Life Sciences, Jawaharlal Nehru University, New

Delhi-110067, India

(Received 11 July 1983; accepted 2 November 1983)

Abstract-Involvement of phytochrome in the regulation of nitrate reductase (NR) and nitrite reductase (NIR) activities in excised, etiolated leaves of Zea mays (L.) variety ‘Ganga-5’ is demonstrated using low energy and high irradiance responses of phytochrome action. Photoreversibility by far-red light of red light stimulated increases in NR and NIR activities was lost by 2 h. Red light given to the leaves, when induction by NO, was saturated, further increased both enzyme activities. Even if red light was given 4-8 h before NO,, it still increased both NR and NIR activities.

INTRODUCTION

Although phytochrome regulation of nitrate reduc- tase (NR) activity has been demonstrated for several plants (Jones and Sheard, 1972,1975; Johnson, 1976; Vijayaraghavan et al., 1979; Rao et al., 1980; Johnson and Whitelam, 1982; Ramaswamy et al., 1983), information on phytochrome regulation of nitrite reductase (N1R)t is scant (Ramirez and Vicente, 1979; Rao et al., 1981). Since both enzymes are inducible by NO:, any factor that influences the availability of NOT is likely to affect their activities. However, Jones and Sheard (1975) repor- ted that phytochrome mediated increases in NO, accumulation and NR activity in pea terminal buds, but sugar induced an increase in NO, accumula- tion without an increase in NR activity. Further, they could not detect any increase in a ‘metabolic pool’ of NO, (Jones and Sheard, 1979), the existence of which is itself a debatable question (Aslam, 1981; Naik et al., 1982). Alternatively, phytochrome has been reported to increase the activity of NR either by direct activation (Johnson, 1976) or by de now synthesis of the enzyme (Jones and Sheard, 1975; Rao et al., 1980). We report here on an interaction between red light and NO; in regulating NR and NIR activities by separating the two treatments in time. We also describe the escape from photo- reversibility of the red light effects.

MATERIALS AND METHODS

Seeds of maize (Zea mays L., variety ‘Ganga-5’) were soaked in deionized water for 24 h and grown at 27°C on moist absorbant paper for 8 days in darkness in a seed germinator. Seedlings were supplied daily with nitrate-free Hoagland’s nutrient medium (Hoagland and Amon, 1950). All manipulations were carried out under green safe light (0.01 W m-2) obtained by filtering the light from a cool

‘To whom correspondence should be addressed. tAbbreviations: NR, nitrate reductase; NIR, nitrite

reductase; Pfr, far-red light absorbing form of phyto- chrome.

white fluorescent lamp through several layers of green cellophane (emission maximum, 500 nm). Red light (1.47 W m-2) was obtained by filtering the light from four 100-W tungsten lamps through a CBS-650 filter (Carolina Biological Supply Co., USA; emission maximum, 650 nm). Far-red light (1.50 W m-*) was obtained from a 300-W tungsten reflector lamp (Westinghouse. USA), the output of which was filtered through a CBS-750 filter (emission maximum, 750 nm) and 8 cm of constantly flowing tap water. The emission maxima of CBS filters mentioned were given by the supplier and were confirmed using a spectro- radiometer. The emission maximum of green cellophane was measured by transmission spectrum. The light intensi- ties were measured with a radiometer.

The procedure for the extraction of NR and NIR was the same as followed earlier (Rao et al., 1980). The activity of NR was measured as described earlier (Sihag et al., 1979). Nitrite reductase was assayed according to the method of Ramirez ei al. (1966). Protein was measured according to Bradford (1976). Percent photoreversibility was calculated by taking the enzyme activities after far-red light treatment (5 min) as the basal level. All experiments were repeated at least three times. Data presented are the averages of three or more experimental values.

RESULTS AND DISCUSSION

The involvement of phytochrome in the regulation of NR and NIR activities has been demonstrated in 2. mays (L.) variety ‘A 5154‘ (Rao e taf . , 1980,1981). In ‘Ganga-5’ also, the variety of maize used in the present study, the involvement of phytochrome in the regulation of NR and NIR activities was demon- strated by using low energy and high irradiance responses of phytochrome action (Table 1). Red light increased the activities of NR and NIR by 150 and 49‘70, respectively, over the dark controls. The increase in these enzyme activities was reversed by subsequent exposure to far-red light. Continuous far-red light increased NR and NIR activities by 198 and 100‘70, respectively, over the dark controls (Table 1).

In order to delineate the effect of red light and NOT on NR and NIR activities, red light and NO, treatments were separated in time. In one

49 1

492 ARUN KUMAR SHARMA AND SUDHIR K. % ~ R Y

Table 1. Effect of various light treatments on the activities of NR and NIR

Treatment NR activity NIR activity

D 51 f l(100) 3.2 f O.Z(l00) FR 73 f 3(144) 3.2 f 0.2(104) R 128 rt 9(250) 4.5 f 0.4(149) R + FR 93 f 7(182) 3.3 f 0.2(108) R + FR + R 142 f 4(278) 4.6 f 0.3(150) Cont FR 152 f 2(298) 6.1 f 0.1(200)

Red (R), far-red (FR) and continuous far-red (Cont FR) light treatments of 5 min, 5 min and 18 h duration, respectively, were given to excised etiolated maize leaves floating on 60 mM KN03. After light treatments, the leaves were kept in darkness (D). Enzyme activities were assayed 18 h after the light treatments. The specific activities of NR and NIR are expressed as nmol NO, roduced h-' mg protein-' and pmol NO; utilized h- mg protein -', respectively. Relative activity (Yo) is given in parentheses.

P

- NR

Time (h)

Figure 1. Effect of separation in time of red light (5 min) and NO, (60 mM) treatment on NR and NIR activities. Nitrate was given at increasing time periods after red light irradiation to leaves floating on water alone. Enzyme activities were measured 12 h after onset of nitrate treatment. For each point, the standard error was never

more than 5%.

experiment red light irradiation was given after NOT pre-treatment (Table 2), while in the other NO, was given at different times after red light treatment (Fig. 1). We have observed, in the system studied earlier (maize, variety 'A5154'), that the effect of NO, on NR (Rao et al., 1980) and NIR (Rao et af., 1981) activities was saturated by 4 and 12 h, respectively. In the present system, the effect of NOT on both enzyme activities was saturated by 12 h. There was no significant change in the enzyme activities during the next 6 h (data not shown). If red light is given after the 12 h preincubation in NO,, it increased NR and NIR activities by 78 and 51%, respectively, over the dark controls after 4 h. The increase was reversible by far-red light (Table 2). Thus, in a situation where any further exposure to NOT did not result in an increase in NR and NIR activities, red light stimulated the response further, suggesting thereby that it may be acting at a site other than the site of action of NO,. When NO, was provided at different times after red light, the stimulatory effect of red light was retained up to 8 h, although the intensity of stimulation decreased with time (Fig. 1). These experiments also suggest that the effect of red light and nitrate on NR and NIR activities could be independent of one another. Since the effect of red light was realized 4 h after, when NO: was given, it seems that Pfr makes the system 'more potent' for the increase in NR and NIR activities by NO, (Fig. 1). The biochemical basis of this 'potential' needs to be worked out.

Photoreversibility by far-red light of red light induced increases in NR and NIR activities de- creased with an increase in intervening dark period between red light and far-red light treatments. Both enzymes escaped photoreversibility completely by 2 h (Fig. 2). The Pfr, therefore, induced a 'biochemical signal' within 2 h. This 'biochemical signal' was found to persist for about 12 h (Fig. 1). Similarity in the pattern of decay of the 'biochemical signal' for the F'fr-stimulated increase in NR and NIR activities suggests that Pfr regulation of these enzymes could be linked at some level (Fig. 1).

Table 2. Effect of red (R) and far-red (FR) light on NR and NIR activities after pre-incubation in NO;

Time NR activity NIR activity (h)

D R R+FR D R R+FR

0 47 f 4(100) 47 f 4(100) 47 f 4(100) 2.08 f O.OS(l00) 2.08 +_ 0.08(100) 2.08 f 0.08(100) 1 46 k 2( 98) 61 f 6(130) 47 f 4(100) 1.99 f O M ( 96) 2.48 f 0.05(119) 2.11 f 0.09(101) 4 47 f 6(100) 84 f l(178) 55 f 3(117) 2.17 f 0.03(104) 3.15 f 0.03(151) 2.11 f 0.09(101)

Etiolated leaves, which were pre-incubated for 12 h in NO; (60 mM) and maintained in NO;, were treated with R or R followed by FR of 5 min duration each. Enzyme activities were measured at the time indicated after R treatment. The specific activities of NR and NIR are expressed as nmol NO; produced h-' mg protein-' and pmol NO, utilized h-' mg protein-'. Relative activity (%) has been given in parentheses.

Phytochrome regulation 493

I I Y E t r n l " l

Figure 2. Escape of NR and NIR from photoreversibility. Far-red light (5 min) was given at the time indicated after red light treatment. Enzyme activities were measured 18 h after red light treatment. For each point, the standard error

was never more than 5%.

The data suggest that Pfr and NOT act at different levels to increase NR and NIR activities. The potentiating effect of red light is quite stable and independent of the presence of NO; in the induction medium. This observation suggests that the phytochrome effect may be of a more general nature, which becomes specific under certain condi- tions, depending upon the system and environment.

Acknowledgements-One of us (AKS) is thankful for a research fellowship awarded by U.G.C. The research was partially,supported by a grant from U.G.C. We thank Dr S. Guha-Mukherjee for her help.

Aslam, M. (1981) Plant Physiol. 68, 305-308. Bradford, M. M. (1976) Anal. Biochem. 72,248-254. Hoagland, D. R. and D. I. Arnon (1950) Calif. Agric.

Johnson, C. B. (1976) Planru 123, 127-131. Johnson, C. B. and G. C. Whitelarn (1982) Photochem.

Phorobiol. 35, 251-254. Jones, R. W. and R. W. Sheard (1972) Nature (New

Biol.) 283, 221-222. Jones, R. W. and R. W. Sheard (1975) Plant Physiol.

55, 954-959. Jones, R. W. and R. W. Sheard (1979) In Nitrogen

Assimilation of Plants (Edited by E. J. Hewitt and C. V. Cuttings), pp. 521-535. Academic Press, London.

Naik, M. S., Y. P. Abrol, T. V. R., Nair and C. S. Ramarao (1982) Phytochembtry 21,495-504.

Ramaswamy, O . , I. M. Saxena, P. Guha-Mukherjee and S. K. Sopory (1983) J . Biosci. 5, 63-70.

Ramirez, R. and C. Vicente (1979) Phyron 37,25-28. Rao, L. V. M.. N. Datta, S. K. Sopory and S. Guha-

Mukherjee (1980) Physiol. Plant. 50, 208-212. Rao, L. V. M., V. K. Rajasekhar, S. K. Sopory and S.

Guha-Mukherjee (1981) Plant Cell Physiol. 22,

Sihag, R. K., S. Guha-Mukherjee and S. K. Sopory (1979) Physiol. Plant, 45, 281-287.

Vijayaraghavan, S. J., S. K. Sopory and S. Guha- Mukherjee (1979) Plant Cell Physiol. 20, 1251-1261.

REFERENCES

Exp. St. Circ. 347, 1-39.

577-582.

rw 3914-D