Manuscript accepted on : August 04, 2009
Published online on: 28-12-2009
Vinodh Ratnala2*, Dsvgk. Kaladhar1 and B. Seshagiri2
2Department of Biochemistry, M.V.R College of Post-Graduate Studies, Visakhapatnam India.
1Department of Biochemistry and Bioinformatics, GITAM University, Visakhapatnam India.
ABSTRACT: The etiolated shoots were exposed to a constant intensity of light (5000 lux supplied by a bank of white, cool, fluorescent lights) in the presence of optimal concentration of the substrate – inducer (20mM KNO3). When the etiolated shoots were exposed to light and 20mM KNO3 simultaneously, there was a progressive synthesis of photosynthetic pigments and induction of NR, assayed by the in vivo method under optimal assay conditions, with time of exposure (0,4,8,12, and 24 hours). The amount of Chl b synthesis, compared to that of Chl a, was less throughout the time of exposure to light. Chl a/b remained constant up to 8 hours of exposure to light and decreased slightly thereafter. A positive correlation was observed between the amount of total chlorophyll and the magnitude of in vivo NR activity throughout the 24 hours period. Significantly, by the 24th hour of greening, the amount of NR activity in the 3-day-old greening shoots was 1.75 micromole NO2 produced per hour per gram fresh weight, a value almost equal to that of 7-day-old green leaves induced under similar conditions. Etiolated shoots kept in darkness throughout the NR induction period did not show any enzyme activity, indicating that even in the presence of optimal external nitrate, the NR-inducer; light is still and perquisite for optimal induction of NR activity.
KEYWORDS: Etiolated Shoots; Photosynthetic Pigments; Substrate Inducer; Triticum Vulgare
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Introduction
Light as an environmental factor affecting enzyme activity in plants is widely recognized. Light has been reported to play a complex and varied role in nitrate assimilation in that it.
Stimulates nitrate uptake.1-3
Enhances transfer of nitrate from the vacuolar (storage) pool to the easily accessible cytoplasmic (metabolic) pool in the cells4.
Promotes synthesis of nitrate reductase5.
Activates the pre-existing enzyme6.
Increase the accessibility of the enzyme to nitrate via phytochrome – mediated membrane changes and /or other phytochorme effects7.
Provides the reductant via photosynthesis8-9.
However, nitrate reductase was detected in dark-grown corn seedlings10 and Chlorella11, etiolated barely leaves12, radish seedlings13, and etiolated wheat seedlings14.
The role of light in the reduction of nitrate has been considered indirect. In addition, the nitrate reductase complex from higher plants specifically requires NADH as an electron donor. For this reason, a direct involvement of chloroplast reaction in nitrate reduction is thought to be improbable. The extent of dependence of nitrate reduction on chloroplast development and metabolism therefore need to be explored.
Materials and Methods
Seed Material
Wheat (Triticum vulgare) seeds of Punjab variety were obtained from local seed stores.
Cultivation of Wheat Seedlings
Healthy seeds of wheat were surface – sterilized in 4% (v/v) sodium hypochlorite for 5 min, thoroughly washed several times in tap water and subsequently soaked for 3 hours in distilled water. The seeds were germinated in Petri dishes lined with a course filter paper (Kalpi) in distilled water in dark before being exposed to 20 mM KNO3 under a constant illumination of 5000 lux supplied by a bank of white, cool, fluorescent lamps.
Harvest of Seedlings
Unless otherwise stated, uniformly growing seven-day-old green seedlings were harvested at least 4 hours after exposure to light. For experiments involving etiolated seedlings, 3-day-old etiolated shoots raised in distilled water in dark were exposed to light and 20mM KNO3 simultaneously.
Induction of Nitrate reductase (NR) and assay of in vivo NR activity
Nitrate reductase was induced in green. Seedlings and etiolated shoots with 20mMKNO3 under constant illumination.
Unless otherwise mentioned, the standard, infiltration medium (2ml to 5ml depending on the experiment) for the in vivo NR assay was composed of
–100mM KH2 PO4 – KOH, pH 7.5
— 100mM KNO3
— 1% butanol
— 0.1% Triton X – 100
Leaf segments (1mm) equivalent to 0.1-0.2g fresh weight were incubated in dark at room temperature (300C) in 20ml glass vials with air-tight caps containing either 2ml (for greening shoots) or 5ml (for green leaf segments) infiltration medium. The contents were periodically shaken. After 1 hour incubation, 0.2ml to 0.5ml of the infiltration medium was removed for nitrite analysis. The nitrate reductase activity was expressed as µ moles nitrite produced per hour per gram fresh weight.
Extraction and Estimation of Chlorophyll and Carotenoids:
Shoot segments (100mg) were ground with a chilled pestle and mortar in diffuse light in 80% cold acetone and the homogenate was centrifuged at 3,000 X g for 2min. Aliquots of 5ml of 80% cold acetone were added to the pellet and pigments were extracted in dark and cold till the pellet was non-green. The supernatants were pooled and protected from light prior to estimation of chlorophyll and carotenoids in a UV-visible spectrophotometer (Systronics, Model 118).
The concentration of chlorophyll was measured according to15 Arnon (1949)
Chl a (mg/l) = (12.7 X A663) – (2.69 X A645)
Chl b (mg/l) = (22.9 X A645) – (4.68 X A645)
Total chlorophyll (mg/l) = (20.2 X A645) + (8.02 X A663)
The concentration of carotenoids was measured at 473 nm according to Goodwin (1954)
E1Cm1% = 2,500
Nitrate (NO2–) estimation
To nitrite solution or a known volume of the infiltration medium containing nitrite, 1ml of 1% (w/v) sulphanilamide reagent prepared in 3N HCL and 1ml of 0.02% (w/v) N – (1 – naphthyl) ethylendiamine dihydrochloride reagent were added in quick succession and the contents were thoroughly mixed. The color was allowed to develop for 15 min prior to reading at 540nm in a UV-visible spectrophotometer (Systronics, Model 118)
The amount of nitrite formed was calculated based on
E1Cm1mM Nitrite complex (540nm) = 55
Results
The role of light in the induction and activity of nitrate reductase activity has been reported to be indirect17. In the present investigation the relationship between light, the level of photosynthetic pigment synthesis indicative of extent of chloroplast development and nitrate reductase induction was investigated in 3-day-old etiolated shoots raised in distilled water. When these etiolated shoots were exposed to light and 20 mM KNO3 simultaneously, there was a photosynthetic pigments and induction of nitrate reductase activity with time of exposure to light and nitrate. The amount of enzyme activity was related to the level of total chlorophyll (Table 1& 2) and no enzyme activity was observed in the etiolated shoots induced in darkness with 20 mM KNO3.
Discussion
NR activity is the rate – limiting step in the process of nitrate reduction to ammonia18. Several workers have recommended the use of intact tissue (in vivo) assay of NR19-20. The in vivo assay described in this study is a simple and rapid way of assaying NR activity in the leaves of Punjab wheat.
The observed correlation between the increase in the levels of photosynthetic pigments and induction of nitrate reductase activity with the time of exposure of etiolated shoots to light under NR – inducing conditions indicates that induction and activity of nitrate reductase depends on the extent of chlorophyll synthesis and therefore chloroplast development. Such a dependence of induction and activity of NR on chloroplast development was shown in pigment – deficient leaves21 and mustard seedlings23 and wheat seedlings under bleaching and non-bleaching condtions24-25. The effects of light on NR induction and activity have been shown to include stimulation of de novo synthesis, activation of the enzyme, increased transcription of the NR genes by light absorbed by chlorophyll26-27.
Table 1: Effect Of Greening On Pigment Synthesis In 3-Day-Old Etiolated Shoots Exposed To Light +20 Mm Kno3 Simultaneously.
S.NO. | HRS. OF GREENING | CHL.A | CHL.B | CHL. A/B | TOTAL CHL. | CAROTENOIDS |
1 | 0 | 0.00 | 0.00 | 0.00 | 0.00 | Not estimated |
2 | 4 | 50.6 | 16.1 | 3.15 | 66.7 | 29 |
3 | 8 | 103.8 | 32.8 | 3.17 | 136.6 | 53 |
4 | 12 | 114.0 | 40.0 | 2.8 | 154.0 | 67 |
5 | 24 | 311.0 | 131.0 | 2.38 | 442.0 | 121 |
Table 2: Effect Of Greeting On Induction Of Nr Activity In 3-Day-Old Etiolated Shoots Exposed To Light + 20mm Kno3 Simultaneously.
S.NO. | HRS. OF GREENING | µ MOLES OF
NO2 ¯ FORMED /Hr/ GRAM FRESH Wt. |
1 | 0 | 0.00 |
2 | 4 | 0.19 |
3 | 8 | 0.32 |
4 | 12 | 0.65 |
5 | 24 | 1.75 |
Acknowledgement
We would like to thank the management and staff of MVR PG College and GITAM University, Visakhapatnam, India for providing material and lab space in bringing out the above literature and experimentation.
Referances
- Beevers, L and R.H. Hageman, Nitrate and nitrite reduction.(Stumpf PK, Conn EE, eds.), The Biochemistry of Plants, Academic Press, New York, 5, 115-68 (1980).
- Prasad Rao and D. William Rains, Nitrate Absorption by Barley: I. Kinetics and Energetics, Plant Physiol., 57, 55-58 (1976).
- Hallmark, W.B., and R.C. Huffaker, The influence of ambient nitrate, temperature, and light on nitrate assimilation of sudangrass seedlings. Physiologia Plantarum 44 (3), 147-152 (1978)
- Aslam Muhammad, Ann Oaks, and Ray C. Huffaker, Effect of Light and Glucose on the Induction of Nitrate Reductase and on the Distribution of Nitrate in Etiolated Barley Leaves, Plant Physiol., 58, 588-591(1976).
- Travis, R.L., R.C. Huffaker and J.L.Key, Light-induced Development of Polyribosomes and the Induction of Nitrate Reductase in Corn Leaves, Plant Physiol. , 46, 800-805 (1970).
- Tischner, R. and A. Hutterman, Light-mediated Activation of Nitrate Reductase in Synchronous Chlorella Plant Physiol., 62, 284-286 (1978).
- Jones, R.W. and Sheard, R. W, Nitrogen Assimilation of Plants (eds Hewitt, E. J. & Cutting, C. V.) Academic, London, 521−539 (1979).
- Klepper, L., D. Flesher and R.H. Hageman, Generation of Reduced Nicotinamide Adenine Dinucleotide for Nitrate Reduction in Green Leaves, Plant physiol., 48, 580-590 (1971).
- Nicholas, J.C., J.E. Harper, and R.H. Hageman, Nitrate reductase activity in soybeans (Glycine max [L.] Merr.). I. Effects of Light and Temperature, Plant Physiology, 58, 731-735 (1976).
- Travis, R.L., and J.L.Key, Correlation between Polyribosome Level and the Ability to Induce Nitrate Reductase in Dark-grown Corn Seedlings, Plant Physiology, 48, 617-620 (1971)
- Guerreno, M.G., J. Rivas, A. Paneque and M. Losad. Mechanism of nitrate and nitrate reduction in Chlorella cells grown in the dark, Biophys. Res. Commun, 45(1). 82-89 (1971).
- Roth-Bejerano, N. and S.H. Lips, Induction of Nitrate Reductase in Leaves of Barley in the Dark, New Phytologist, 72(2), 253-257 (1973).
- Stulen, I, Nitrate reduction in radish seedlings. Thesis University of Groningen, pp 85 (1974).
- Datta, N., L.V.M. Rao, S.G. Mukherjee and S.K. Sopory, Regulation of nitrate reductase activity by ammonium in wheat, Plant Sci. Let, 20, 1299-1308 (1981)
- Amon, D.I. 1949 Copper enzymes in isolated chloroplasts. polyphenoloxidase in Beta vulgaris, Plant Physiol. 24: 1-15.
- Goodwin, T.W, Carotenoids, In: Handbook of Plant Analysis (K. Paech and M.V. Tracey, eds.), Springer- Verlag, 3, 272-311 (1954).
- Campbell, W.M, nitrate reductase structure, function and regulation: bridging the gap between biochemistry and physiology, Annual Review of Plant Physiology and Plant Molecular Biology, 50, 277-303 (1999).
- Beevers, L. and R.H. Hageman, Nitrate Reduction in Higher Plants, Annual Review of Plant physiology, 20, 495 – 522 (1969).
- Jaworski, E.G, Nitrate reductase assay in intact plant tissues, Biochem. Biophys. Res. Commun., 43, 1274-1279 (1971).
- Harper, J.E. and Hageman, R.H, Canopy and seasonal. profiles of nitrate reductase in soybeans (Glycine max L. Merr.), Plant Physiol., 49, 146-154 (1972).
- Borner, T., R.R. Mendel, and J. Schiemann, Nitrate reductase is not accumulated in chloroplast-ribosome deficient mutants of higher plants, Planta, 169, 202-207 (1986).
- Deane – Drummond, C.E. and C.B.Johnson, Absence of nitrate reductase activity in San 9789 bleached leaves of barley seedlings (Hordeum vulgare Midas), Plant cell Environ., 3(5), 303-308 (1980).
- Rajasekhar, V.K. and H. Mohr, Appearance of nitrite reductase in cotyledons of the mustard (Sinapis alba) seedling as affected by nitrate, phytochrome and photooxidative damage of plastids, Planta, 168(3), 369-376 (1986).
- Rao, K.P. and D.W. Rains, Nitrate absorption by barley. II. Influence of nitrate reductase activity, Plant Physiol., 57, 59-62 (1976b).
- Campbell, W.H., Higher plant nitrate reductase and its role in regulation of nitrate assimilation, Physiologia Plantarum, 74, 214-219 (1988).
- Lillo C, Light regulation of nitrate reductase in green leaves of. higer plants, Plant, 90: 616-620 (1994)
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