in the dark with nitrate or sucrose solution or nitrate + sucrose solution helped to ... thirty days old potted plants, raised under natural ... zero time control samples.
lQdian Journal of Experimental Biology "'Vol. 37, May 1999, pp. 515-518
Regulation of leaf nitrate reductase activity in spinach (Spinacea oleracea L.) during light and dark transition Bhupinder Singh, M S Kaim, P K Hari Kumar, S R Chatterjee & T V R Nair Nuclear Research Laboratory, Indian Agricultural Research Institute, New Delhi 110 012, India Received JOJune 1998; revised 12 F~brll(Jry 1999
experiments were conducted on spinach variety jyoti to understand the role of light in regulation of nitrate reductase (NR) activity. The plants were grown under three different light intensities (a) >1200 ~ (under natural conditions), (b) about 300 ~ (in a growth chamber) and (c) about 100 ~ (in an open growth chamber). Highest in vivo NR activity was rt!corded in leaf samples harvested from (a) followed by (b) and (c). Photosynthetic' activity, chlorophyll content and tissue sugar content also showed a similar trend . Leaf tissues of plants raised under natural conditions showed a decline in NR activity when incubated in distilled water in the dark. The activity disappeared almost totally after 36hr of incubation in dark. However, incubation of the leaves in the dark with nitrate or sucrose solution or nitrate + sucrose solution helped to maintain the activity even after 3hr. Potted plants also showed a decrease in the enzyme activity when kept in the dark. Intact plants which had lost all activity by 36hr in the dark showed recovery of NR activity only after 48hr in light. Apparently light does not regulate NR activity by any switch-on, switch-off mechanism.
Intensity, duration and quality of light influences plant growth and development, differentiation and reproduction by altering metabolic profiles like bioreduction of carbon and ' nitrate, the two important processes in a growing plant. One of the most important enzymes involved in reduction of nitrate is nitrate reductase (NR) . A stimulatory role of light on process of nitrate assimilation has long been known l ,2. In green plants, nitrate assimilation involves two reductive steps located in different subcellular compartments. In cytosol, nitrate is reduced to nitrite via NADH-dependant nitrate reductase and nitrite formed after transport into chloroplast is further reduced to ammonia by nitrite reductase (NiR) located in the stroma3 . NADH required as substrate for NR in cytosol could be provided by multiple ways; either by photosynthetic electron transport via malate-oxaloacetate or triose phosphate-34 phosphoglycerate shuttle , or from mitochondria again via malate-oxaloacetate shuttleS. While NiR linked nitrite reduction needs reduced form of ferredoxin which is provided by PS-I, and its regeneration is essentially light dependent6 . Despite these reports our understanding of the precise role of light on nitrate assimilation is still far from complete. Even the basic question of direct or indirect involvement of light in the regulation of this enzyme
remains un-resolved . There have been reports about instantaneous ceaser of NR when leaves are transferred to dark? Coupled to it are the reports which indicate that leaves of higher plants have the ability to reduce nitrate even in the dark though at substantially lower rates than in lighe and that this attribute appears species dependant 8 .9 . Kaim et using IS KN0 3', observed dark reduction of nitrate in seedlings of wheat, barley, sorghum, bajra, pea, chenopodium and lathyrus. Ability of.leaves to reduce nitrate under dark is also greatly influenced by the availability of reducing sugars lO . Investigations were, therefore, conducted to ascertain the relationship ,between light, photosynthetic rate, other ,related biochemical attributes and NR activity. Effect of light-dark transition on NR activity and its recovery was also studied. Spinach seeds (Spinacea oleracea L.) variety Jyoti, procured frQm the Division of Vegetables, Indian Agricultural Research Institute, New Delhi were grown in sandy loam soil in plastic pots (6.6 inches diam) . Available N,P,K and nitrate-N contents in the soil were 97,11,148 and 8 ppm respectively. Leaves harvested at 3-4 leaf stage of plant growth, as otherwise mentioned, were used for different investigations under the following experimental conditions.
at,
516
INDIAN J. EXP. BIOL., MAY 1999
Experiment I-Inunediately after seed gennination, potted plants were exposed to three different light flux viz., natural light condition with light flux exceeding 1200 IlE (I,); in growth chamber with light flux of about 300 IlE (12) and in open growth chamber with light flux of about 100 JlE (13), for 8hr day"' . Each treatment was replicated thrice and the data presented is mean of six observations. NR activity was assayed in vivo as detailed earlier" by modification of the methods suggested by Hageman and Hucklesby'2 and Nair and Abrol 13 • Photosynthetic rate of these leaves was measured with an Infra-red gas analyzer (LI-6000, LICOR Instruments Inc.). Chlorophyll pigments (a and b) and total sugars were estimated by the methods of Hiscox and Israelstam'4 and Dubois et a/'5 respectively. Experiment II-Nitrate reductase (NR) activity was assayed in the excised leaves of spinach plants, raised under natural light conditions and incubated under different experimental conditions (Table 3). Excised leaves were incubated in dark for 3hlf with the petiole dipped in solutions of 15mM KN0 3, I per cent sucrose and a combination of these two. Leaves were also incubated in distilled water as one of the treatments. NR activity assayed in freshly harvested leaf tissues (Zero incubation time) was taken as control. Experiment Ill-In a separate set of experiments, thirty days old potted plants, raised under natural light, were transferred to dark chamber and nitrate reductase acti vity was recorded periodically for 36hr, after which the pots were shifted to natural light conditi on and re-activation of the enzyme was monitored . To authenticate the findings, in vitro NR act ivity was also recorded in the above plants, after 3hr of dark resi dence, as per method of Chalifour and Nelspn '6. It is well conceded that a much faster rate of nitrate assimilation is operative under light than under dark condition . Various factors li ke accumulation of photolabile inhibitors in the dark, influence of light on uptake, translocation of nitrate (an inducer of NR) to the leaves and its transfer from the metabolically inert storage pools to the active pools, increased availability of the enzyme protein during light, photosynthetic reactions involving generation of reductants, and availability of haemoprotein, have been reported to influence, solely or collectively, the activity of this enzyme under light-dark transition2. The present investigations reveal that a reduction in
light intensity severely impaired NR activity, photosynthesis, chlorophyll pigments and soluble sugars in leaves. The decline in NR activity was 40.2 and 78.5 per cent respectively under h and 13 as compared to natural light (I,) condition (Table 1). The photosynthetic activity also suffered reduction by 47.0 and 67.3 per cent under 12 and 13 respectively . Though there was a decline in the chlorophyll content, for both a and b, it was not proportionate with the reduction of photosynthetic rate and NR activity. Chlorophyll a and b contents were reduced by 17-35 and 14-33 per cent for h-13 conditions respectively. Decline in the content of soluble sugars under low light intensities was observed with inhibition of NR and photosynthesis (Table 1). NR activity of excised leaves reduced after 3hr of dark incubation in distilled water (Table 2). Nitrate and sucrose supplementation significantly protected the enzyme activity. NR activity rec'o rded under the above conditions was comparable with the activity of zero time control samples. The results suggest substrate induction of NR by nitrate while sucrose may be regulating the enzyme activi ty by mimicking the light induction of nitrate reductase gene transcription 17. ' 8. Inhibition in NR actIvIty was observed when potted plants were kept in the dark (Table 3A) . Though the extent of loss in activity was relati vely lower than that observed in excised leaves, this co.uld be due to initiation of degradation of NR proteins in excised tissues during incubation . Decrease in barley root and shoot NR activity on light-dark transition has Table I-Effect of different light regimes on in vivo nitrate reductase (N R) activity. photosynthetic activi ty, total sugar and chlorophyll contents in spinach . Parameter studied NR activity
CI-l mol N02 g· 1
fwt h· l) Photosynthetic activity CI-l mo l CO2 m·2 S· I) Chloroph yll a content b (mg g·lfwt) Total sugar (mg g.1 fwl)
Light conditions used Natural light Growth chamber (>12001' E) (-3001-< E) (- 1001' E) 3.9 1 2.34 0.84 (40.2) (78.5) 13 .29
7.00 (46.7)
4.34 (67.3)
1.11 0.49
0.92(1 7.1 ) 0.420 4.3)
0.72(35 . 1) 0.33(32.7)
14.95
11.87 (20.6)
8.95 (40. 1)
(Values in parenthesis indicate per cent inhibition over natural light observati ons)
517
NOTES
Table 2-ln vivo nitrate reductase (NR) activity in excised leaves of spinach under different experimental conditions. Experimental conditions used
NR activity (}..tmoleN~·
g.1 f wt hr· l ) Leaves incubated for 3h in dark a) in distilled water b) in 15 mM KN03 solution c) in I % sucrose solution d) in nitrate (l5mM) + sucrose (I %) Zero time control
1.85 (34.9) 2.75 (3.2) 2.67 (5.9) 2.60 ( 8.4)
2.84
(Values in parenthesis indicate per cent inhibition over zero time control ob~ation) Table 3--Reactivation of nitrate reductase in light after dark residence of spinach plants • Experimental conditions used
In Vivo NR activity (}..t moleN0"2 g.1 f wt.hr· l)
A Potted plants kept in the dark a) 5hr
b)7hr c) 36hr
Zero TIme Control B. Potted plants from A (c) above when transferred to light a) Ihr b) 2hr c) 3hr d)5hr e)48hr Control (36hr in Dark)
0.95 (-33.6) 0.58 (-54.7) 0.00 1.28 (100.0)
0.09 (+ 7.0) 0.11 (+ 8.6) 0.16 (+12.5) 0.26 (+20.3) 1.06 (+82.8) 0.00 In Vitro NR activity
(pmoleNO' 2 Potted plants kept in dark for 3hr Control (plants from natural light)
.g"1 fwt.hr"l) 0.562 (-34.8) 0.862 (100.0)
(Values in parenthesis indicate per cent change over zero time control observation)
been reported to be related to degradation of NR protein molecules l9 • In potted plants the leaf NR activity declined by 26 and 55 per cent after 5 and 7 hr of dark residence respectively. While near zero enzyme activity was recorded at 36hr. In vitro NR activity revealed an inhibition of only 34.8 per cent even after 3 hr dark treatment. These results are in total disagreement with the observations made by Remmler and Campbell20 who have shown a 30 per cent decline in NR activity in corn leaves within lhr of dark treatment, which is not accompanied by degradation of NR proteins. Riens and Heldt7 have also shown a rapid decline in NR activity (in vitro)in spinach leaves under dark. They reported a 50 per .cent decline in NR activity within 2 min of light to
dark transition, with activity reducing to 15 per cent within a 60 min duration. Our results have, however, indicated 35 per cent inhibition of in vitro NR activity even after 3 hr dark "treatment. This discrepancy in the abo~ observations could be linked to tlie species differences and/or quality of light in which plants are grown 8•9 • When potted plants with negligible NR activity (36hr dark treated plants) were transferred back to natural light conditions, there was a recovery of enzyme activity at slow rate during the initial phase (Table 3B). NR activity compared to zero time control recovered completely after 48hr of residence in light. It indicated that t.he inactivation of NR activity under dark was reversible and was related to de-novo synthesis of the enzyme rather than reactivation of enzyme molecules, which is exPected to take place in a short span. An increase in \ NR activity on transfer of, dark grown, etiolated barley seedlings to light has been shown to be regulated by de-novo synthesis of NR protein and has been confirmed though immunological cross reactivity studies l9 . In conclusion, the present results indicateq that light did not regulate NR activity by switch-on, switch-off mechanism. Inadequate light intensity affects the photosynthetic apparatus and hence the concentration of tissue sugars decreased with decrease in light intensity. Further, since there is a considerable lag period. before NR becomes maximally active in light after dark residence of the plants, light appears to be promoting fresh synthesis of enzyme rather than causing activation or modulation of the preformed enzyme. The authors wish to thank the Project Director, Nuclear Research Laboratory for the facilities and showing keen interest in the work.
References I 2
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