Toxic action of zinc on growth and enzyme activities of rice oryza sativa L. Seedlings

Environmental Pollution (Series A) 36 (1984) 45-59

Toxic Action of Zinc on Growth and Enzyme Activities of Rice Oryza sativa L. Seedlings

Pratima Nag, Parimal Nag, A. K. Paul

& S. Mukherji Plant Physiology Laboratory, Department of Botany, University of Calcutta, Calcutta 700 019, India

ABSTRACT This paper provides inJormation on the effects of toxic concentrations of zinc sulphate (ZnSO 4. 7H20 ) on the growth and metabolism of rice Oryza sativa L. seedlings. Root growth inhibition was always more pronounced than was shoot growth inhibition. Root growth was completely inhibited at 40 m M concentration, whereas the magnitude of reduction of shoot length was only 56 % at this concentration. Gibberellic acid ( GA 3) was partially capable of relieving zinc inhibition. The activities of peroxidase, IAA oxidase and ascorbic acid oxidase of seedlings increased in response to zinc addition, whereas catalase and IAA synthetase decreased. All the hydrolysing enzymes, viz., or-amylase and phytase of endosperm together with RNase and A TPase of the embryo, showed distinct inhibition from the control, the exception being endosperm RNase which was stimulated under zinc treatment.

INTRODUCTION Zinc is one of the essential elements required by plants in trace amounts for their growth and development. Zinc is necessary for the biosynthesis of the plant auxin indole acetic acid (IAA) through its involvement in the synthesis of tryptophan, a precursor of auxin, by influencing the activity of the enzyme tryptophan synthetase (Tsui, 1948; Nason, 1950). Zn participates in the metabolism of plants as a n a c t i v a t o r of several 45 Environ. Pollut. Ser. A. 0143-1471/84/$03"00 ~_'~Elsevier Applied Science Publishers Ltd, England, 1984. Printed in Great Britain

46

Pratima Nag, Parimal Nag, A. K. Paul, S. Mukherji

enzymes. For instance, Zn is a constituent of the enzyme carbonic anhydrase (Keilin & Mann, 1940), whereas enzymes such as alcohol dehydrogenase and pyridine nucleotide dehydrogenases are dependent on the presence of Zn (Hooh & Vallee, 1958). Zn is highly toxic to plants except in very dilute concentrations. Application of Zn fertilisers in excessive amounts to alkaline soil caused decreased yields .to a varying degree in grass, Alaska pea, tomato, lettuce, spinach, sugar beet and field beans (Boawn, 1971 ; Boawn & Rasmussen, 1971). Zn toxicity symptoms appeared in the form of leaf necrosis, and reduction in growth and flowering with an application as low as 100-200 ppm Zn in sand culture experiment (Debruyn & Mcllrath, 1966). Ernst (1972) studied the extent of pollution, caused by airborne Zn, of soils and plants growing near the vicinity of Zn smelters where the metabolic activities of higher plants and soil micro-organisms were adversely affected. Recently, Zn has been shown to impair water-uptake capacity, to promote leakage of metabolites, to reduce GA-induced ~amylase production in de-embryonated rice half-seeds, and to cause disturbed mitosis in onion root-tip cells with a series of abnormalities (Nag et al., 1980). It has been further reported that chlorophyll development and Hill reaction activity of chloroplasts in rice seedlings are depressed by Zn treatment (Nag et al., 1981). The present work was undertaken to measure the extent of injury on rice-seedling growth caused by the presence of excessive amounts of Zn in the growing medium. The effects of deleterious concentrations of this metal on the activities of some enzymes, viz. catalase, peroxidase, IAA oxidase, IAA synthetase, ascorbic acid oxidase, ~-amylase, RNase, ATPase and phytase, have been studied.

MATERIALS AND METHODS Test material and growth measurements

Rice Oryza sativa L. cv. Rupsail seeds were surface sterilised with 0.1% (w/v) HgCI 2 solution and then washed several times with distilled water. The seeds were then spread over Petri dishes lined with filter papers containing distilled water. After germination for 48 h in the dark at 30 °C, these were transferred to Petri dishes containing ZnSO 4. 7H20 (10, 20 and 40mM) and GA 3 (0.1 mM) solutions and kept under the same

Effects of zinc on rice seedlings

47

conditions for another three days. At the end of the total five days germination, lengths of shoot and root, and fresh weights of entire seedlings were measured. Water controls were run parallel to each experiment.

Assay of enzymes Seedlings (embryonic axis only) were homogenised with cold 0"05M phosphate buffer (pH 7.0 for catalase and pH 6.1 for peroxidase, IAA oxidase, IAA synthetase and ascorbic acid oxidase) and centrifuged cold at 10 000g for 15 min. The supernatants were used for assay, the total volume being adjusted to 12 ml in each case. The H 2 0 2 from the catalasic mixtures was transferred to 10~o (v/v) H2SO 4 and then evaluated by titration using a 0.01 N K M n O 4 solution (Gasper & Lacoppe, 1968). Peroxidase activity was estimated according to the method of Chance & Maehly (1955), with colorimetric determination of the change in the colour intensity of oxidised catechol at 420 nm. IAA oxidase activity was measured according to the method of Hillman & Galston (1957), where residual IAA was determined in reaction mixtures incubated at 26°C. Optical density measurements were calculated as micrograms of residual IAA according to a standard curve. Salkowski reagent was employed for colorimetric determination of IAA (Gordon & Weber, 1951). IAA synthetase was assayed according to the method described by Phelps & Sequeira (1967). IAA produced by tryptophan conversion was measured colorimetrically with the Salkowski reagent. Ascorbic acid oxidase was assayed according to the method described by Olliver (1967). Residual ascorbic acid in the reaction mixture was measured by titration with dichlorophenolindophenol (DPI P). Endosperm tissues were extracted with distilled water and then the homogenates were centrifuged in the cold. The supernatant was heated to 70 °C to inactivate fl-amylase, and ~t-amylase was assayed according to the method described by Bernfeld (1955) and modified by Dure (1960). Maltose released from starch in the reaction mixture was allowed to react with dinitrosalicylic acid and the absorbance was measured at 510 nm. For adenosine triphosphatase (ATPase) and phytase, the endosperm tissues were extracted with cold 0"01M Tris-HCl buffer (pH 7.0), centrifuged cold, and the assay was done according to the method of Young & Varner (1959). ATPase was assayed at pH 9.0 using 0.2 M glycine-NaOH buffer with I mM A T P as the substrate; phytase was

48

Pratima Nag, Parirnal Nag, A. K. Paul, S. Mukherji

assayed at pH 5.0 using 0.2 u citrate buffer with 10 mM phytin (sodium phytate) as the substrate. Phosphate released was measured according to Fiske & Subbarow (1925). Ribonuclease (RNase) activity was measured according to Cherry (1962) with some modifications. Both embryo and endosperm tissues were homogenised with cold sucrose-citrate buffer (0"05M, pH 6.0), centrifuged cold, and the supernatant was used as the enzyme source. The reaction mixture included yeast RNA. At the end of the incubation period absorbance was measured at 260 nm.

RESULTS A N D DISCUSSION With the application of ZnSO 4, both shoot length and root length of rice seedlings were inhibited and the degree of inhibition increased with the increase in concentration of the metal (Fig. 1). The toxicity caused by this heavy metal was found to be more severe for root than for shoot. At the lowest dose of ZnSO 4, i.e. 10 mM, shoot inhibition was about 30 ~o from control, whereas at the same concentration root inhibition was 80Vo. Root growth was almost completely inhibited at 40mM concentration which could elicit only 56 ,°/o shoot inhibition. The difference in responsiveness between shoot and root towards Zn finds a close parallel in seedlings treated with other heavy metals, viz. copper (Mukherji & Dasgupta, 1972; Dasgupta & Mukherji, 1977), lead (Mukherji & Maitra, 1976, 1977), chromium (Mukherji & Roy, 1977) and mercury (Mukherji & Ganguly, 1974; Mukherji & Nag, 1977). Although there was a gradual decrease in fresh weight with increasing concentrations of ZnSO4, the acceleration in the rate of fresh weight decrease was less than the rate of seedling-growth inhibition. In the case of joint application of ZnSO4 with GA 3, a remarkable reduction of Zn-induced inhibition of shoot elongation was effected by GA 3, whereas its influence on root-growth inhibition was relatively less marked. With lower concentrations of ZnSO4, reversal of shoot inhibition by GA 3 was not only complete but the shoot lengths exceeded those of the control values by an appreciable amount. A variable degree of reduction of fresh-weight inhibition was also observed when GA 3 was applied with different concentrations of ZnSO4. With higher concentrations of ZnSO 4, the growth-stimulating effect of GA 3 became progressively less pronounced. The protective effect provided by GA 3 against the toxic influence of Zn may be associated with

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SHOOT LENGTH ( - G A ) = r, SHOOT LENGTH (4- GA) E)-'-'O ROOT LENGTH (-GA') H ROOT LENGTH (+GA) FRESH WEIGHT ( - GA') "- -" FRESH WEIGHT('rGA)

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the well-known GA effect on the enhancement of synthesis of enzyme proteins and thereby stimulation of germination vigour by bringing about hydrolysis of storage materials in germinating seeds (Paleg, 1961: Chrispeels & Varner, 1967; Paul et al., 1970a). In ZnSO4-treated seedlings, catalase activity was inhibited by about 34 and 26 % at 10 and 40 mM, respectively (Fig. 2(A)). The role ofcatalase in plant metabolism is not clear (Burris, 1960). In studies on the effects of growth-retarding chemicals on catalase activity, results reported by different workers are variable, ranging from both stimulation and inhibition to a situation showing almost no response. For instance, catalase activity in cucumber seedlings has been shown to be stimulated by Amo- 1618, whereas other growth-retardants, viz. carvadan, CCC, did not

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Pratima Nag, Parimal Nag, A. K. Paul, S. Mukherji IO

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Fig. 2. Effect of different concentrations of ZnSO4 on catalase (A) and peroxidase (B) activities of rice seedlings after five days germination. Catalase expressed as/tmol H 2 0 z destroyed per 15min per seedling. Reaction mixture for catalase: I ml extract, 5ml phosphate buffer (pH 7.0) and I ml H202 (0.2 vol). Peroxidase expressed as increase in optical density at 420 nm per min per 10 seedlings. Reaction mixture for peroxidase: 0.5 ml extract, 5 ml phosphate buffer (pH 6-1), 1 ml H202 (0.2 vol) and 1 ml catechol (0.1 'I~,).C means water control.

affect enzyme activity at all, and one, B-995, behaved like the growth inhibitor MH and even reduced activity (Halevy, 1964). In barley seedlings, however, Amo-1618 and CCC have been reported to produce no effect on catalase activity (Gasper & Lacoppe, 1968). It is postulated that H 2 0 2 may be produced in plant tissues at various stages of metabolic reactions and the removal of this toxic chemical seems to be essential to prevent the oxidation of important metabolites. It is possible that, in a normal seedling growing in water, catalase helps decomposing H202 and thus removes its toxic effect; likewise it is equally possible that growth inhibition caused by Zn toxicity is associated with reduced catalase

Effects of zinc on rice seedlings

51

activity. In contrast to catalase, the activity of peroxidase increased at all the concentrations of ZnSO 4 (Fig. 2(B)). At the highest concentration, viz. 40 mM, peroxidase activity increased by about 30 ~o above the control seedlings. Concurrent with peroxidase, the activity of IAA oxidase also showed an overall stimulation at all the applied ZnSO4 levels (Fig. 3). A 2.5-fold enhanced activity was recorded at the highest dosage (40 mM). Our results seem to be comparable with those of Halevy (1962), who reported that the inhibition of growth by means of growth retardants was associated with increased peroxidase and IAA oxidase activities. This led him to suggest that growth retardants may exert their effect by lowering the auxin content through enhanced auxin destruction (Halevy, i 963). In the present work, it seems that Zn inhibits growth by acting in a similar

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Fig. 3. Effect of different concentrations of ZnSO 4 on IAA oxidase (A) and IAA synthetase (B) activities of rice seedlings after five days germination. IAA oxidase expressed as/~g IAA destroyed per hour per seedling. Reaction mixture for IAA oxidase: I ml extract, 0. I ml MnC! 2 (0-2 mM), 5 ml phosphate buffer (pH 6. I) and I ml IAA (l mM). IAA synthetase expressed as/~g IAA synthesised per hour per seedling. Reaction mixture for 1AA synthetase: I ml extract, 0.1 ml MnCI 2 (0.2mM), 0.1 ml MgSO4 (1 mM), 5ml phosphate buffer (pH 6- I) and I ml tryptophan (250 mg m l - ~). C means water control.

52

Pratima Nag, Parimal Nag, A. K. Paul, S. Mukherji

manner, although the present findings do not exclude other physiological parameters. As opposed to the promotion of the activities of peroxidase and IAA oxidase, the IAA synthetase system was less active in the presence of toxic levels of Zn (Fig. 3). In spite of the fact that the requirement of Zn as a micronutrient for IAA formation has been established (Tsui, 1948; Nason, 1950), it is noteworthy that the IAAsynthesising system suffers a decline at the growth-inhibitory concentrations of Zn. Thus promotion of IAA oxidase coupled with inhibition of IAA synthetase is expected to reduce IAA in a seedling to a sub-optimal level and thereby inhibit growth. Ascorbic acid oxidase in Zn-treated seedlings was also maintained at a much higher level than in the control (Fig. 4). About 33 ~o increased activity was noted at 20 mM. There was, however, no further increase at the maximum concentration, i.e. 40 mM: instead, the activity fell slightly to a level that was about 23 ~o above the control. These results are consistent with the existence of a Zn-induced growth inhibition (Fig. 1). This seems likely in the light of the facts that abundance and reactivity of this enzyme indicate the rapid conversion of ascorbic acid to its oxidised form, dehydroascorbic acid--the latter probably being less important in plant metabolism. It is already known that ascorbic acid oxidase has been held to be the terminal oxidase in respiration and, as the plants grow older, cytochrome oxidase may be replaced by ascorbic acid oxidase as the terminal oxidase of respiration of senescing tissues or organs (James, 1953; Stiles, 1961 ; Gauch, 1972). Studies concerning the metabolism of ascorbic acid indicate that H 2 0 2 is produced during enzymatic oxidation by ascorbic acid oxidase (Szent-Gyorgyi, 1939). In this case, there is an additional pool of H 2 0 2 that can have some effect on the control of the healthy growth of seedlings. Simultaneous increment of ascorbic acid oxidase on the one hand and depressed status of catalase on the other serve to indicate that both are linked in some manner, and it is suggested that H202 formed as an end product of ascorbate oxidation tends to accumulate in the tissue and remains in an undecomposed state. Application of ZnSO4 produced a remarkable inhibition of a-amylase in rice endosperm, the degree of inhibition increasing with increasing concentrations of the metal (Fig. 4). The data show that about 50~o enzyme activity was lost at 10 mM, the lowest concentration tried here. The reduction was further intensified at the next highest dosage, and maximum inhibition (to an extent of 80 %) was effected by the highest concentration of ZnSO4, i.e. 40 mu. In rice seedlings, a-amylase has been

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Fig. 4. Effect of different concentrations of ZnSO 4 on ascorbic acid oxidase (A) and ~amylase (B) activities of rice seedlings after five days germination. Ascorbic acid oxidase expressed as mg ascorbic acid destroyed per 30min per seedling. Reaction mixture for ascorbic acid oxidase: I ml extract, 5 ml phosphate buffer (pH 6' 1) and 1 ml ascorbic acid (2 mM). s-Amylase expressed as mg maltose liberated per endosperm per 5 min. Reaction mixture for ~-amylase: 0.8 ml extract, 0.5 ml citrate buffer (pH 5.0) and 0.5ml starch (I %). C means water control.

shown to be similarly depressed in response to growth-inhibitory concentrations of copper and lead (Mukherji & Maitra, 1976; Dasgupta & Mukherji, 1977). In the present context, inhibition of ~t-amylase formation by heavy metals may be either directly due to the inactivation of enzyme protein or indirectly due to the interference with hormonal control ofhydrolase formation. In cereal grains, gibberellin is synthesised early in the germinating process in the embryo or the scutellum and is released to the aleurone layers where it induces the de novo synthesis of several hydrolysing enzymes, such as ~-amylase (Radley, 1967; Filner et al., 1969). Some workers also believe that GA-stimulation of hydrolase

54

Pratima Nag, Parimal Nag, A. K. Paul, S. Mukherji

formation helps in shifting the metabolic pathways of tissues from a catabolic type to an anabolic one essential for growth (Jones, 1972, 1973). Here the toxic action of zinc may therefore be attributed to binding to a receptor site required by GA for the induction of s-amylase synthesis, resulting in a limited supply of food materials for embryo growth. RNase activity of rice embryo was inhibited to a considerable extent in the presence of ZnSO 4 (Fig. 5). The inhibition was more or less progressive with increasing concentrations of Zn and measured about 33 % at the highest concentration, viz. 40 mM. It is interesting to note that endosperm RNase behaved in a manner opposite to that of embryo RNase, thereby showing progressive increments as the concentrations of ZnSO 4 were increased. At 40 m~, about 50 % stimulation of endosperm RNase was noted. In view of the fact that RNase plays a dual role, being active both in degradation and in synthesis (Barker & Douglas, 1960), it is likely that a relatively high level of RNase in the control embryo 0"7

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Fig. 5. Efect of different concentrations of ZnSO 4 on RNase activity of rice embryo (A) and endosperm (B) after five days germination. Activity expressed as absorbency at 260nm per embryo or endosperm per hour. Reaction mixture: l ml extract, 1 ml phosphate buffer (pH 6.0) and 1 ml yeast RNA (1 mg m l - t). C means water control.

Effects of zinc on rice seedlings

55

contributed much towards the building up of new RNA in this tissue. On the contrary, a low level of RNase in metal-treated tissue correlates with deficient building-up processes. In the present work, Zn had an increasing effect on endosperm RNase whereas the effect on ~-amylase of the same tissue was decreased. A similar situation was previously described in maize endosperm where increased RNase activity was due to the activation of a pre-existing enzyme and did not depend on the presence of gibberellin (Ingle & Hageman, 1964, 1965). Furthermore, RNase activity in vivo is known to be directly controlled by the ratio of potassium, calcium and magnesium ions (Hanson, 1960). Zn possibly influences RNase activity by altering the ratio of these free ions. Thus endospermic RNase activity may be due to enzyme activation by Zn.

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Fig. 6. Effect of different concentrations of ZnSO 4 on ATPase activity of embryo (A) and phytase activity of endosperm (B) of rice seedlings after 5 days germination. ATPase expressed as /~g phosphorus liberated per 30min per seedling. Reaction mixture for ATPase: 0-7ml extract, 0.2ml glycine-NaOH buffer (pH 9.0) and 0.1 ml ATP (I mM). Phytase expressed as #g phosphorus liberated per hour per l0 endosperms. Reaction mixture for phytase: 0.7 ml extract, I mt citrate buffer (pH 5.0) and 0" l ml sodium phytate (l mM). C means water control.

56

Pratima Nag, Parimal Nag, A. K. Paul, S. Mukherji

Activities of both ATPase of embryo and phytase of endosperm were drastically inhibited by Zn toxicity (Fig. 6). Loss of ATPase activity was about 90 ~ at the highest dose of ZnSO4 and the corresponding loss of phytase amounted to more than 80~o from the control levels. ATP provides the necessary energy in biological systems for the driving of the synthetic processes. In Zn-treated tissues, ATPase is expected to be less active since cell division and cell elongation with accompanying synthesis of food matters proceed at a slow rate in these tissues, which should demand less energy release. Phytin, i.e. the calcium or magnesium salt of inositol hexaphosphoric acid, is the primary reserve phosphate in ungerminated seeds and the enzyme phytase catalyses the hydrolysis of inorganic phosphate from phytin during germination (Koller et al., 1962; Mukherji et al., 1971). On the other hand, in old seeds germinating with reduced vigour, phytase like other hydrolases is very feeble (Paul et al., 1970b). A diminished level of phytase, as observed in the present work, is related to the state of low biological activity coincident with growth inhibition by zinc application when less inorganic phosphorus seems to be needed. Thus it may be concluded that growth inhibition caused by ZnSO4 may, in part, be due to the decreasing efficiency of these enzymes involved in food and energy utilisation.

ACKN O W L E D G E M ENTS We thank Professor A. K. Sharma for his interest and encouragement. Financial assistance from the CSIR, New Delhi is gratefully acknowledged.

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Eff'ects of zim' on rice seedlings

57

Burris, R. H. (1960). Hydroperoxidases (peroxidases and catalases). Encycl. PI. Physiol., XII/I, 365-400. Berlin, Springer Verlag. Chance, B. & Maehly, A. C. (1955). Assay of catalases and peroxidases. In Methods in enzymology, ed. by S. P. Colowick and N.O. Kaplan, 2, 764-75. Academic Press, New York. Cherry, J. H. (1962). Nucleic acid determination in storage tissues of higher plant. PI. Physiol., Lancaster, 37, 670-8. Chrispeels, M. J. & Varner, J. E. (I 967). Gibberellic acid enhanced synthesis and release of 0t-amylase and ribonuclease by isolated barley aleurone layers. Pl. Physiol., Lancaster, 42, 396-406. Dasgupta, B. & Mukherji, S. (1977). Effects of toxic concentrations of copper on growth and metabolism of rice seedlings. Z. Pflanzenphysiol., 82, 95-106. Debruyn, J. A. & Mclllrath, W. J. (1966). Effects of boron, manganese, copper and zinc upon the growth of Setaria sphacelata. J. S. African Bot., 32, 313-24. Dure, L. S. (1960). Site of origin and extent of activity of amylases in maize germination. PI. Physiol., Lancaster, 35, 925-34. Ernst, W. (1972). Zinc and cadmium pollution of soils and plants in the vicinity of a zinc smelting plant. Bet. dt. Bot. Gez., 85, 295-300. Filner, P., Wray, J. L. & Varner, J. E. (1969). Enzyme induction in higher plants. Science, N.Y., 165, 358-67. Fiske, C. H. & Subbarow, Y. (1925). The colorimetric determination of phosphorus. J. biol. Chem., 66, 375-400. Gasper, T. & Lacoppe, J. (1968). The effect of CCC and Amo-1618 on growth, catalase, peroxidase and IAA oxidase of young barley seedling. Physiologia PI., 21, 1104-9. Gauch, H. G. (1972). Inorganic plant nutrition. Stroudsburg, Dowden, Hutchinson & Ross. Gordon, S. A. & Weber, R. P. (1951). Colorimetric estimation of indoleacetic acid. PI. Physiol., Lancaster, 26, 192-5. Halevy, A. H. (1962). Inverse effect of gibberellin and Amo-1618 on growth, catalase and peroxidase activity in cucumber seedlings. Experientia, 18, 74 -6. Halevy, A. H. (1963). Interaction of growth-retarding compounds and gibberellin on indoleacetic acid oxidase and peroxidase of cucumber seedlings. PI. Physiol., Lancaster, 38, 731-7. Halevy, A. H. (1964). Effects of gibberellin and growth-retarding chemicals on respiration and catalase activity in various organs of cucumber seedlings. J. exp. Bot., 15, 546-55. Hanson, J. B. (1960). Impairment of respiration, ion accumulation and ion retention in root tissues treated with ribonuclease and ethylenediaminetetraacetic acid. PI. Physiol., Lancaster, 35, 372-9. Hillman, W. S. & Galston, A. W. (1957). Inductive control of IAA oxidase by red and near infrared light. PI. Physiol., Lancaster, 32, 129-35. Hooh, F. L. & Vallee, B. L. (1958). The metabolic role of zinc. In Trace elements, ed. by C. A. Lamb, O. C. Bentley and J. M. Beattie, 337-63. New York, Academic Press.

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Ingle, J. & Hageman, R. H. (1964). Studies on the relationship between ribonucleic acid content and rate of growth of corn roots. PI. Physiol., Lancaster, 39, 730-4. Ingle, J. & Hageman, R. H. (1965). Metabolic changes associated with the germination of corn. PI. Physiol., Lancaster, 40, 48-53. James, W. O. (1953). The terminal oxidase in the respiration of the embryos and young roots of barley. Proc. Roy. Soc., 141, 280-99. Jones, R. L. (1972). Fractionations of the enzymes of the barley aleurone layer: evidence for a soluble mode of enzyme release. Planta, 103, 95-109. Jones, R. L. (1973). Gibberellins: their physiological role. A. Rev. PI. Physiol., 24, 571-98. Keilin, D. & Mann, T. (1940). Carbonic anhydrase. Biochem. J., 34, 1163-76. Koller, D., Mayer, A. M., Poljakoff-Mayber, A. & Klein, S. (1962). Seed germination. A. Rev. PI. Physiol., 13, 437-61. Mukherji, S. & Dasgupta, B. (1972). Characterization of copper toxicity in lettuce seedlings. Physiologia PI., 27, 126-9. Mukherji, S. & Ganguly, G. (1974). Toxic effects of mercury in germinating rice seeds and their reversal. Indian J. exp. Biol., 12, 432-4. Mukherji, S. & Maitra, P. (1976). Toxic effects of lead on growth and metabolism of germinating rice seeds and on mitosis of onion root tip cells. Indian J. exp. Biol., 14, 519-21. Mukherji, S. & Maitra, P. (1977). Growth and metabolism of germinating rice seeds as influenced by toxic concentrations of lead. Z. Pflanzenphysiol. 81, 26-33. Mukherji, S. & Nag, P. (1977). Characterization of mercury toxicity in rice seedlings. Biochem. Physiol. Pflanzen, 17, 235. Mukherji, S. & Roy, B. K. (1977). Toxic effect of chromium on germinating seedlings and potato tuber slices. Biochem. Physiol. Pflanzen, 17, 238. Mukherji, S., Dey, B., Paul, A. K. & Sircar, S. M. (1971). Changes in the phosphorus fractions and phytase activity of rice seeds during germination. Physiologia PI., 25, 94~7. Nag, P., Paul, A. K. & Mukherji, S. (1980). Effects of mercury, copper and zinc on growth, cell division, GA-induced a-amylase synthesis and membrane permeability of plant tissues. Indian J. exp. Biol., 18, 822-7. Nag, P., Paul, A. K. & Mukherji, S. (1981). Heavy metal effects in plant tissues involving chlorophyll, chlorophyllase, Hill reaction activity and gel electrophoretic patterns of soluble proteins. Indian J. exp. Biol., 19, 702-6. Nason, A. (1950). Effect of zinc deficiency on the synthesis of tryptophan by Neurospora extracts. Science, N.Y., 112, 111-12. Olliver, M. (1967). Ascorbic acid estimation. In The vitamins, ed by W. H. Sebrell and R. S. Harris, 338-59. New York, Academic Press. Paleg, L. G. (1961). Physiological effects of gibberellic acid, III. Observations on the mode of action of barley endosperm. PI. Physiol., Lancaster, 36, 829-37. Paul, A. K., Mukherji, S. & Sircar, S. M. (1970a). Enzyme activities in germinating mungbean seeds and their relation with promoters and inhibitors of protein synthesis. Osterr. Bot. Z., 118, 311-20.

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Paul, A. K., Mukherji, S. & Sircar, S. M. (1970b). Metabolic changes in rice seeds during storage. Indian J. agric. Sci., 40, 1031-6. Phelps, R. H. & Sequeira, L. (1967). Synthesis ofindoleacetic acid via tryptamine by cell free systems from tobacco terminal bud. Pl. Physiol., Lancaster, 42, ! 161 -3.

Radley, M. (1967). Site of production of gibberellin like substances in germinating barley embryos. Planta, 75, 164-71. Stiles, W. (1961). Trace elements in plants. Cambridge, Cambridge University Press. Szent-Gyorgyi, A. (1939). On oxidation, fermentation, vitamins, health and disease. Baltimore, Williams and Wilkins. Tsui, C. (1948). The role of zinc in auxin synthesis in the tomato plant. Am. J. Bot., 35, 173-9. Young, J. L. & Varner, J. E. (1959). Enzyme synthesis in the cotyledons of germinating seeds. Arch. Biochem. Biophys., 84, 71-8.