THE BLUE LIGHT EFFECT AND ITS INTERACTION WITH PHYTOCHROME IN THE CONTROL OF NITRITE REDUCTASE ACTIVITY IN SORGHUM BICOLOR WILLD.

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1 New Phytol (1985) 101, I THE BLUE LIGHT EFFECT AND ITS INTERACTION WITH PHYTOCHROME IN THE CONTROL OF NITRITE REDUCTASE ACTIVITY IN SORGHUM BICOLOR WILLD. BY V. K. RAJASEKHAR* AND S U D H I R K. SOPORYf Plant Research Laboratory, School of Life Sciences, Jawaharlal Nehru University, New Delhi , India {Accepted 31 May 1985) SUMMARY Irradiation with red light for 5 min stimulated nitrite reductase activity in 5-d-old etiolated seedlings of Sorghum bicolor. The stimulation was less when measured after a subsequent dark period of 12 h than after one of 24 h. A short period of irradiation with blue light did not alter the enzyme activity nor did it elicit any effect even if given for 4 6 h prior to the red light irradiation. Exposure to blue light for 12 h prior to the red light treatment increased the enzyme activity, when measured after 12 h of darkness, to the same level as that obtained by red light irradiation alone followed by 24 h of darkness. Blue light given continuously for 24 h increased nitrite reductase activity and tbis increase was inbibited by 6-methylpurine (5 fig ml~^) or cyclobeximide (25 /*g ml~^). Transfer of seedlings to continuous white ligbt for 6 b after treatment witb a pulse of red ligbt followed by 24 b of darkness, or witb continuous blue ligbt for 24 b, revealed an independent action of blue ligbt and ligbt operating via pbytocbrome, in tbe control of nitrite reductase activity. Tbe accumulation of nitrate was unaffected eitber by a red ligbt pulse followed by a subsequent dark period, or by continuous blue ligbt. In contrast, continuous wbite ligbt led to an increased accumulation of nitrate. Key words: Blue ligbt, nitrite reductase, pbytocbrome. Sorghum bicolor, wbite ligbt. INTRODUCTION Phytochrome (Pfr) is involved in the regulation of several enzymes of nitrate metabolism namely nitrate reductase (Jones & Sheard, 1972, 1977; Johnson, 1976; Duke & Duke, 1978; Vijayaraghavan, Sopory & Guha-Mukherjee, 1979; Rao et al, 1980; Johnson & Whitelam, 1982; Ramaswamy et al., 1983; Sharma & Sopory, 1984), nitrite reductase (Ramirez & Vincente, 1979; Rao et al., 1981; Rajasekhar, 1983; Sharma & Sopory, 1984) and glutamine synthase (Takebe, 1983). Evidence also indicates the involvement of blue light in the light mediated control of nitrate reduction (Jones & Sheard, 1974, 1977; Rao et al., 1983a). However, the effect of blue light and its possible interaction with white light as well as with the light wavelengths operating via Pfr has not been studied with regard to the regulation of nitrite reductase activity. The present investigation attempts such a study. M A T E R I A L S AND M E T H O D S Seeds of Sorghum bicolor Willd. var. Pusa Chari-6 were obtained from the Indian * Present address: Biology Institute II, University of Freiburg, FRG. t To whom correspondence should be addressed X/85/10O $03.00/0 ^^^^ '^^^ ^ ^ ^ Phytologist

2 252 V. K. RAJASEKMAR AND S. K. SOPORY Agricultural Research Institute, New Delhi and were grown on moist germination paper at C as described previously (Rajasekhar et al., 1981). Unless otherwise mentioned 5-d-old etiolated seedlings were normally used. The sources of red light (A^ax'"" """; 5 W m-^), far red light (A^ax'"** """; 1-4 W m-^) and green safe light (0-01 W m~^) were as described by Sharma, Sopory & Guha Mukherjee (1976). The blue light source (A^nax*^" ""^) was as described earlier (Rao et al., 1983a). The intensity of blue light for short term (i.e. minutes) irradiation was 5 W m~^ and the prolonged illumination (i.e. hours) was 1-4 W m~^. White light (8 W m^) was obtained by using eight cool white fluorescent tubes (Sylvania and Laxman Ltd., fluorescent, daylight 6500 K, India). Nitrite reductase was extracted from etiolated shoot apices (3 to 4 cm length from apex, 250 mg f. wt) by homogenizing for 60 s in a mortar and pestle with 2 ml of 50 mm Tris-HCl buffer (ph 8) containing 3 mm Nag-EDTA, 3 mm cysteine, 20 mm NiClg and 1 % polyvinylpolypyrrolidone. The homogenate was centrifuged at 20000^ for 30 min at 0 to 4 C. The resulting clear supernatant was assayed for nitrite reductase activity as the amount of NOg" utilized in comparison with the amount of NOg" present at zero time of reaction (Rao et al., 1981). T h e concentration of NOg" was calculated from a standard curve. Protein estimation was according to Lowry et al. (1951). Estimation of NO3" was described by Wooley, Hicks & Hageman (1960). For the determination of ph]uridine incorporation into RNA and L-[^H]leucine into protein, the etiolated seedlings were incubated 12 h before the start of continuous blue light illumination in ^H-uridine (0-18 MBq ml~^, speciflc activity 0-5 TBq mmol~^) or L-pH]leucine (0-18 MBq ml~\ specific activity 0 2 TBq mmol"^) with or without the respective inhibitor. At the end of 24 h of continuous blue light the incorporation of [^HJuridine and L-[^H]leucine was determined from the shoot apices according to Rao et al. (1980). A Packard Liquid Scintillation counter and a Shimadzu UV-visible recording spectrophotometer. Model UV-240, were used for the radioactivity determinations and absorption measurements respectively. Unless mentioned otherwise the results given are the average of at least three independent determinations. R E S U L T S AND D I S C U S S I O N Irradiation by red light for 5 min increased nitrite reductase activity (Fig. 1). It was shown that red light operated through the involvement of Pfr (Rajasekhar, 1983; Rajasekhar & Sopory, unpublished). However, a similar short duration irradiation with blue light did not change the enzyme activity; nor did irradiation with far-red light (Fig. 1). However, in contrast to far-red light, a pulse of blue light if given immediately after red light did not reverse its effect. While ineffective in itself, pretreatment with blue light for 6 h strongly increased the effectiveness of Pfr in controlling the mesocotyl longitudinal growth in Sorghum vulgare (Mohr, 1980). To test for such an effect, we irradiated seedlings with blue light for 4 or 6 h before giving the red light pulse; such pretreatments were ineffective (Fig. 2). The effect of red light on nitrite reductase activity, therefore, may not be a consequence of either an obligatory or a facultative sequential interaction between blue light and light operating via Pfr (see Mohr, 1980). Irradiation with blue light for 24 h enhanced nitrite reductase activity by 35 % but exposure for only 12 h had no effect (Table 1). However, if after 12 h of blue

3 Blue light and nitrite reductase in Sorghum D.E 3 o Q. ICVJ O "o e o a cr Control R+B B+R R+FR Fig. 1. The effect of short term irradiation with red, far red, or blue light on nitrite reductase (NIR) activity. Five-day-old etiolated seedlings were given red (R), far-red (FR) or blue (B) light for 5 min each followed by 24 h dark period. When two light treatments were supplied, one was given after the other in the order shown on the figure. Controls were in darkness throughout. Seedlings were incubated with KNO3 (60 mm) 12 h before start of the preirradiation and maintained in this until the end of experiment. Relative values are given on top of the vertical bars. light irradiation, seedlings were left in darkness for another 12 h, enzyme activity increased. Following a pulse of red light, enzyme activity was higher after a subsequent dark period of 24 h (50%) than after one of 12 h (30%). When interaction between red and blue light was studied, pretreatment with blue light for 12 h enhanced the effect of a red light pulse as measured after a subsequent 12 h dark period, almost to the effectiveness of red light followed by a 24 h dark period (Table 1). There thus appears to be an additive effect of blue light and red light. However, if another pulse of red light was given in the middle of the 24 h period of darkness following an initial red light treatment, there was no further enhancement of the enzyme activity. These results suggest that the increase in nitrite reductase activity by short irradiation of red light or by continuous blue light is mediated via different receptors. Blue light has been shown to regulate high irradiation responses independent of Pfr action (Gaba & Black, 1979); (Koorneef, Rolff & Spruit, 1980), and also in the control of nitrate reductase activity in etiolated excised leaves of maize (Rao et al. 1983a). The increase in nitrite reductase activity induced by irradiation with blue light was inhibited completely by 6-methylpurine (5 fig mv^) whereas inhibitors of plastid RNA and protein synthesis such as rifampicin and D-threochloramphenicol showed no effect (Table 2). General incorporation of [^Hjuridine and L-[»H]leucine was also greater under continuous blue light irradiation than in dark grown seedlings. 6-Methyl purine inhibited RNA synthesis by 50% and cycloheximide

4 254 V. K. RAJASEKMAR AND S. K. SOPORY D Control Fig. 2. The effect of prolonged blue light pretreatment on the red light mediated increase of nitrite reductase. Five day old etiolated seedlings were given 5 min red light (R) followed by a 24 h dark period. Blue (B) light pretreatments were of 4 h and 6 h before exposure to red light. Controls were in darkness throughout. Seedlings were incubated in KNO3 (60 mm), 12 h before start of red irradiation and maintained in this until the end of the experiment. Results are the means of two independent determinations. Relative values are given on top of the vertical bars. Table 1. Effect of red light (R) and continuous blue light (B) on nitrite reductase (NIR) activity NIR activity (/tmol utilized) Treatments (mg protein * h ') (g f. Dark control 24hB 12hdarkness + 12h B 12hB + 12h darkness R + 24 h darkness 12 h darkness -I- R h darkness 12hB + R + 12h darkness R- -12h darkness+ R -I-12 h darkness R + 12 h darkness 4-12hB 1-89 (100) 2-55(135) 1-94(103) 2-49(132) 2-81 (149) 2-47(131) 2-88(151) 2-94(156) 2-77 (147) 7-65 (100) 12-27(160) 7-82(102) 12-36(162) 13-30(174) 11-86(155) 13-18(172) 13-85(181) 13-22(173) Five-day-old etiolated seedlings were given 5 min red light (R) followed by 12 or 24 h in darkness. Continuous blue light (B) was given for 12 or 24 h. The 12 h of blue light was either preceded by R irradiation with a subsequent 12 h in darkness or followed by the R irradiation. Controls were maintained in darkness throughout. Seedlings were incubated with KNO3 (60 mm) 12 h before start of light treatment and maintained in this until the end of the experiment. Results are the mean of two independent determinations. Relative values are given in parentheses.

5 Blue light and nitrite reductase in Sorghum 255 Table 2. Effects of inhibitors of RNA and protein synthesis of the increase in nitrite reductase (NIR) activity induced by continuous blue light NIR activity (/imol of NOg" utilized) Treatments Dark control 24 h blue light + 6-Methylpurine {5 fig ml-') + Cycloheximide (25 fig ml-i) + D-thereo-chloramphenicol (400 fig mr») -1- Rifampicin (400 fig ml-i) (mg protein ' h ') (g f. wt 1 h 1) (100) 1-91 (41) (100) 5-92 (41) 1-08 (38) 5-32(37) 3-03 (105) (104) 2-97 (103) 15-14(105) Five-day-old etiolated seedlings were incubated with KNO3 (60 mm) 12 h before start of the blue light treatment and maintained in this until the end of the experiment. Relative values are given in parentheses. Table 3. Effect of 6-methyl purine and cycloheximide on the incorporation of [^Hjuridine into RNA and [^H]L-leucine into protein Incorporation (cpmg f. wt ') Dark Continuous blue light PH]uridine + 6-methylpurine 940 (0) 416(56) 1490 (0) 796(47) PH]leucine -h Cycloheximide (25 fig ml-i) 6436 (0) 564(91) 9080 (0) 772(91) Treatments Five-day-old etiolated seedlings were given continuous blue light (B) for 24 h. The inhibitors and radioactive substrates were supplied 12 h before the start of the blue light treatment and the seedlings remained in these solutions until the end of the experiment. Appropriate control sets were maintained without the inhibitors. The incorporation of radioactivity was then determined in shoot apices m each of the sets and expressed as cpm per g f. wt. Results are the means of two mdependent determmations. Percentage inhibitions are given in parentheses. inhibited protein synthesis by 90% (Table 3). Chloramphenicol and rifampicin, although they did not inhibit the increase in nitrite reductase activity, did inhibit the incorporation of leucine and uridine respectively. Blue light has been suggested toamplify the fresh synthesis of RNA (Gressel, 1980; Hundrieser & Richter, 1982; Rao et al., 1983a), and soluble proteins (Voskresenskaya, 1972; Conradt & Ruyters, 1980; Kulandaivelu & Sarojini, 1980). These observations are comparable to that made earlier during Pfr mediated increase in nitrite reductase activity (Rajasekhar, 1983). Despite the observation that blue light and Pfr appear to act independently, both appear to control the increase in nitrite reductase activity at the level of transcription and/or translation especially in the nucleo-cytoplasmic system, a situation similar to that controlling nitrate reductase activity in maize (Rao et al, 1983b).

6 256 V. K. RAJASEKMAR AND S. K. SOPORY Table 4. The effect of white light {WL) subsequent to continuous blue light (B) and red light (R) pre-irradiation followed by darkness on nitrite reductase (NIR) activity NIR activity (/tmol N'Og utilized) Treatments (mg protein"* h~*) (g f. wt-> h->) 1-75 (100) 2-67(153) 2-82(161) 3-16(181) 3-60 (206) 2-69(154) 2-49 (142) 8-91 (100) 14-34(161) 13-22(148) 16-16(199) 21-59(242) (179) 13-04(151) Dark control 24 h darkness H-6 h W L R + (24 h + 6 h) darkness (24h- -6h) WL R + 24 h darkness + 6 WL 24hB-l-6h WL 24 h B + 6 h darkness Five-day-old etiolated seedlings were given 5 min red light followed by 24 h darkness or 24 h blue light. Seedlings were incubated with KNO3 (60 mm) 12 h before the start of light treatment. All the sets were then transferred to continuous white light (WL) for 6 h. Corresponding control sets were maintained in darkness. The results are the means of the two independent determinations. Relative values are given in parentheses. Table 5. Effect of light on nitrate accumulation Treatments Dark control 24 h blue light Red light 5 min -f- 24 h darkness 24 h white light accumulated, /tmol (g f. wt~') 1-86 (100) 1-81 (97) 1-92(103) 7-45 (401) Five-day-old etiolated seedlings were given 5 min red light followed by 24 h darkness or continuous illumination with blue light or white light. Controls were in darkness throughout. Seedlings were incubated in KNO3 (60 mm), 12 h before the start of light treatment and maintained in this until the end of the experiment. Relative values are given in parentheses. The effect, on nitrate reductase activity, of further exposure of plants to white light after pre-irradiation with a pulse of red light or continuous blue light was studied. White light given for 6 h increased the enzyme activity as compared with similarly irradiated seedlings left in darkness for 30 h. Plants exposed to white light for 30 h showed higher activity and a yet higher activity could be induced with only 6 h of white light exposure if the plants had earlier received a red light pulse of 5 min. In contrast to these effects of red light, pre-exposure to continuous blue light had no effect of nitrite reductase activity. From the data in Table 1 and Table 4, it is clear that the effect of a pulse of red light was influenced by a subsequent period of 6 h white light (Table 4) but not by interposition of blue light or by red light given again (Table 1). White light must affect nitrite reductase activity in some additional way. This may be connected with an increase in the accumulation of nitrate (Table 5) which was the substrate used in the present study for enzyme induction. It seems, therefore, that light regulation of nitrite reductase is mediated by more than one photoreceptor. In white light both uptake or nitrate as well as the enzyme synthesis might be affected but in blue light only the transcriptional machinery might be altered.

7 Blue light and nitrite reductase in Sorghum 257 ACKNOWLEDGEMENTS V.K.R. is a recipient of post-doctoral Research Fellowship from CSIR, New Delhi. The research was partly supported by a grant from UGC, New Delhi to S.K.S. REFERENCES CoNRADT, W. & RuYTERS, G. (1980). Blue light effects on enzymes of the carbohydrate metabolism in Chlorella. In: The Blue Light Syndrome (Ed. by H. Senger), pp Springer-Verlag, Berlin. DUKE, S. H. & DUKE, S. O. (1978). In vitro nitrate reductase activity and in vivo phytochrome measurements of maize seedlings as affected by various light treatments. Plant and Cell Physiology, 19, 481^89. GABA, V. & BLACK, M. (1979). Two separate photoreptors control hypocotyl growth in green seedlings. Nature 278, GRESSEL, J. (1980). Blue light and transcription. In: The Blue Light Syndrome (Ed. by H. Senger), pp Springer-Verlag, Berlin. HuNDRiESER, J. & RiCHTER, G. (1982). Blue light induced synthesis of ribulose bisphosphate carboxylase in cultured plant cells. Plant Cell Reports, 1, JOHNSON, C. B. & WHITELAM, G. C. (1982). Phytochrome action in light grown plants the control of nitrate reduction as a model response. Photochemistry and Photobiology, 35, JONES, R. W. & SHEARD, R. W. (1972). Nitrate reductase: phytochrome mediation of induction in etiolated peas. Nature New Biology, 238, JONES, R. W. & SHEARD, R. W. (1974). Blue light enhanced nitrate reductase in etiolated pea terminal buds. Candian Journal of Botany, 52, JONES, R. W. & SHEARD, R. W. (1977). Effects of blue and red light on nitrate reductase level in leaves of maize and pea seedlings. Plant Science Letters, 8, KooRNEEF, M., RoLEF, E. & SPRUIT, C. J. P. (1980) Genetic control of light inhibited hypocotyl elongation in Arabidopsis thaliana L. Heynth. Zeitschrift fur Pfianzenphysiologie, 100, KuLANDAiVELU, G. & SAROJINI, G. (1980). Blue light induced enhancement in activity of certain enzymes in heterotropically grown cultures of Scenedesmus obliques. In: The Blue Light Syndrome (Ed. by H. Senger), pp Springer-Verlag, Berlin. LowRY, O. H., RosEBROUGH, N. J., FARR, A. L. & RANDALL, R.J. (1951). Protein measurement with folin phenol reagent, ^ourno/ of Biological Chemistry, 193, MANCINELLI, A. L. (1980). The photoreceptors of the high irradiance responses of plant photomorphogenesis. Photochemistry and Photobiology, 32, MoHR, H. (1980). Interaction between blue light and phytochrome in photomorphogenesis. In: The Blue Light Syndrome (Ed. by H. Senger), pp , Springer-Verlag, Berlin. RAJASEKHAR, V. K. (1983). Phytochrome regulation of chloroplast development in Sorghum bicolor. Ph.D. thesis, Jawaharlal Nehru University, New Delhi, India. RAJASEKHAR, V. K., GUHA-MUKHERJEE, S. & SOPORY, S. K. (1983). The effect of y-aminolevulinic acid and inhibitors of RNA and protein synthesis on phytochrome mediated chlorophyll accumulation m Sorghum bicolor. Journal of Experimental Botany, 34, 1444-^1454. RAJASEKHAR, V. K., RAO, L. V. M., GUHA-MUKHERJEE, S. & SOPORY, S. K. (1981). Phytochrome control of chlorophyll and carotenoid accumulation in Sorghum bicolor. Plant and Cell Physiology, 22, RAMASWAMY, O., SAXENA, I. M., GUHA-MUKHERJEE, S. & SOPORY, S. K. (1983). Phytochrome regulation of nitrate reductase in wheat. Journal of Bioscience, 5, ^,, RAMIREZ, R. & VICENTE, C. (1979). Photocontrol of nitrite reductase in cotyledons of Litrulus vulgaris L. Phyton, 37, ,.,.._ RAO, L. V. M., RAJASEKHAR, V. K., SOPORY, S. K. & GUHA-MUKHERJEE, S. (1981) Phytochrome regulation of nitrite reductase - a chloroplast enzyme - in etiolated maize leaves. Plant and Cell Physiology, 22, RAO, L. V. M., DATTA, N., GUHA-MUKHERJEE, S. & SOPORY, S. K. (1983a). The effect of blue light on the induction of nitrate reductase in etiolated excised maize leaves. Plant Science Letters, 28, RAO, L. V. M., DATTA, N., SOPORY, S. K. & GUHA-MUKHERJEE, S. (1980) Phytochrome mediated induction of nitrate reductase activity in etiolated maize leaves. Physiologia Plantarum, 50, RAO, L. V. M., RAJASEKHAR, V. K., SOPORY, S. K. & GUHA-MUKHERJEE, S. (1983b)^ Studies on the involvement of RNA synthesis and polyribosome formation in phytochrome mediated nitrate reductase induction in excised maize leaves. Plant Science Letters, 29, SHARMA, A. K. & SoPORY_S.JMi984). Independent effects of phytochronie and nitrate on nitrate reductase and nitrite reductase activities in maize. Photochemistry and Photobiology, 39, 491-^93.

8 258 V. K. RAJASEKMAR AND S. K. SOPORY SHARMA, R., SOPORY, S. K. & GUHA-MUKHERJEE, S. (1976). Phytochrome regulation of peroxidase activity in maize. Plant Science Letters, 6, TAKEBE, G. (1983). Phytochrome mediated increase in glutamine synthetase activity in photosensitive New York lettuce seeds. Plant and Cell Physiology, 24, 1477-^1483. VijAYARAGHAVAN, S. J., SopORY, S. K. & GuHA-MuKHERjEE, S. (1979). Role of light in the regulation of nitrate reductase level in wheat. Plant and Cell Physiology, 20, VosKRESENSKAYA, N. R. (1972). Blue light and carbon metabolism. Annual Reviews of Plant Physiology, 23, WooLEY, J. T., HICKS, G. P. & HAGEMAN, R. H. (1960) Rapid determination of nitrate and nitrite in plant material. Journal of Agriculture and Food Chemistry, 8,

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