Enzymic and metabolic changes in bean leaves during continuous pollution by subnecrotic levels of SO2

Environmental Pollution (Series A) 25 (1981) 41 51

ENZYMIC AND METABOLIC CHANGES IN BEAN LEAVES DURING CONTINUOUS POLLUTION BY SUBNECROTIC LEVELS OF SO2t

MICH/:LE PIERREd~, ORLANDOQUE1ROZ

Laboratoire du Phytotron, CNRS, 91190-Gif-sur-Yvette, France

ABSTRACT A rapid increase in enzyme capacity appears to be theprimary response to subnecrotic (0"lppm, 300#gm-3), continuous pollution by SO 2 in the leaves of Phaseolus vulgaris. The process is not restricted to a specific metabolic function but affects the key-enzymes of the main central metabolic pathways, and also peroxidases. Changes in the pools of organic acids, amino acids and polyamines are concomitant, and consistent, with the enzymic changes. In contrast, concentration in sulphurcontaining amino acids is not modified. The results suggest a co-ordinated increase in the metabolic potentiality of the cell affording a temporarily increased capacity of resist ing pollution by afaster metabolisation of SO z and a stabilisation of internal p H (postulated role of the variation in polyamines). The temporary (about 2-3 weeks) character of this readjustment and its physiological cost are discussed in connection with recent work.

ABBREVIATIONS

AAT: GDH: IDH: ME:

aspartate aminotransferase E.C. 2.6.1.1. glutamate dehydrogenase E.C. 1.4.1.2. isocitrate dehydrogenase E.C. 1.1.1.42. malic enzyme (malate dehydrogenase (NADP) (decarboxylating)) E.C.

POD:

peroxidases E.C. 1.11.1.7.

1.1.1.40.

t Work supported by the Minist&e de r Environnementet du Cadre de Vie, France, and the Commission of the European Communities. 41 Environ. Pollut.Ser. A. 0143-1471/81/0025-0041/$02.50© Applied SciencePublishers Ltd, England, 1981 Printed in Great Britain

42

MICHI~LE PIERRE, ORLANDO QUEIROZ INTRODUCTION

Biochemical changes occur in leaves during long term pollution by SO z at nonnecrotic concentrations in the air. These metabolic effects can be observed for concentrations of pollutant lower than those required to produce ultrastructural changes (Perez-Rodriguez, 1976). Therefore, it should be considered that the primary site for SO 2 effects is at the metabolic level. Pollution can be expected to modify the metabolism in two main ways: (1)

(2)

by diverting the metabolic operation towards a kind of pathological pattern through accumulation or consumption of a specific metabolite or group of substances, by a quantitative, rather than qualitative, modification of the general metabolic level in order to achieve a faster metabolisation of the pollutant. Less dramatic, but more insidious, this latter possibility requires the action of mechanisms capable of coordinating the operation of different levels of metabolic organisation.

Changes in the operation of the intermediary metabolism have been reported as a result of long course treatments with non-necrotic ( < 1 ppm) concentrations of SO 2. Pahlich (1972, 1973), J/iger & Pahlich (1972) and Pahlich et al. (1972) showed an increase in G D H capacity and a change in its isozymic pattern in pea seedlings during a three-week exposure to 0.3 ppm SO 2, Other authors reported variations in the pools of metabolites during non-necrotic pollution by SO 2 : changes in amino acid pools (J/iger et al., 1972; Godzik & Linskens, 1974), increase in sugar content in bean leaves, and in 14CO2 incorporation into sucrose and fructose in ryegrass under 400/ag S O2/m 3 (Koziol & Jordan, 1978; Koziol & Cowling, 1978), increase in the pool of polyamines in pea under 0.3 ppm (Priebe et al., 1978). In contrast, decreases in the concentration of lipids (Malhotra & Khan, 1978) and of ATP (Harvey & Legge, 1979) have been reported as the result of subnecrotic SO 2 treatments. Work in this laboratory with bean leaves (Pierre, 1975, 1977), provided evidence that the capacity of the key enzymes controlling several pathways increases substantially after only a few days of pollution by 0.1 ppm SO 2. These results and those reported in the present paper are consistent with the type (2) response to pollution as defined above.

MATERIALS AND METHODS

Experiments on pollution were carried out with plants of Phaseolus vulgaris var. Menil in a specially designed growth chamber, as reported in earlier papers (Pavlid6s, 1977; Pierre, 1975, 1977). Plants were 21 days old at the beginning of a three-week experiment with 0" 1 ppm SO2. The first three-lobed leaf was sampled

EFFECTS OF SUBNECROTIC LEVELS OF S O 2 ON BEAN LEAVES

43

from 6-8 plants at different times during the experiment, frozen in liquid nitrogen and lyophylised.

Enzyme measurements Extraction and measurement of enzyme capacity (i.e. total enzyme activity extractable from the tissues) were as previously described (Pierre, 1975, 1977) except for utilising phosphate buffer pH 7.7 instead of tris-HC1 buffer. Peroxidase capacity was measured spectrophotometrically (Beckman D 24) at 460 nm (Henry et al., 1974, modified) in a reaction medium containing for 1 ml phosphate buffer 0.05 M pH 7-7, 1.5 ml gaiacol (0-7 ~o), 0.4 ml HzO z (0.7 ~o), 0.1 ml plant extract. Peroxidase activity is expressed in OD units/min/g lyophylised leaf powder. Organic acids Total content of organic acids in the lyophylised powder was extracted and measured titrimetrically after silica gel column chromatography (Queiroz, 1965). Identification of the separated organic acids was achieved by using several methods: unidirectional thin-layer chromatography on silica gel (ether: formic acid, 7: 0.9 at water saturation; staining with bromophenol blue in ethanol); spectrophotometrical measurement by specific enzyme reactions; co-chromatography with labelled exogenous organic acids added to the lyophylised powder (label was detected in elution fractions by liquid scintillation). Amino acids Lyophylised powder (50 mg) was suspended in 1.5 ml 5 ~o trichloroacetic acid in 0.05 M HC1, homogenised in a Vortex and sonicated (MSE 150 watts, Scientific Instruments). After 15 min centrifugation at 5000 g, an aliquot of the supernatant (400#I) was utilised for automatic analysis by ion exchange chromatography (Beckman Unichrome). Separation and hydrolysis of peptides and proteins Lyophylised powder was boiled in 30ml water for 10min, ground in a potter grinder and subjected to 25 min of centrifugation at 45000g. The supernatant containing the small peptides was dried and the pellet utilised for the insoluble protein analysis. To avoid degradation of cysteine during hydrolysis, this amino acid is transformed into cysteic acid by oxidation of the extract for 2.5 h at - 5 °C with performic acid (formic acid: 30~o HzO/9:1): methanol: formic acid, 5:1:10, 80 #l/mg of protein. After washing three times with water, the extracts were dried and hydrolysed in a sealed vial by 1 to 2ml 6y HC1 at II0°C for 24h. Polyamines Extraction for the study of polyamines is similar to that utilised for amino acids. Analyses of 20 to 40#1 extracts were performed automatically (Adlakha & Villanueva, 1980).

44

MICHELE PIERRE, ORLANDO QUEIROZ RESULTS AND DISCUSSION

The first three-lobed leaf utilised for the experiment was in the exponential growth phase during the first week of pollution, when it attained its full expansion. After 21 days of exposure to 0.1 ppm SO 2 there was no visible necrosis of the plant tissues. For technical reasons (the surface of the experimental chamber is 0.9 m z) each experiment utilised no more than 10-12 plants, selected for homogenous development from a larger batch grown under controlled conditions; slight but significant differences were observed between the mean level of the metabolism of plants from batches sown at different times; but the variations of enzyme capacity and of metabolic pools as a function of time followed very similar patterns in all the different experiments. For these reasons results of a typical experiment are reported in the present paper. Only differences of at least 15 ~o between results obtained with polluted and non-polluted plants are taken into account for the discussion.

Enzyme capacity In order to detect the extent of the effect of subnecrotic pollution on the central metabolism of the cell several metabolic pathways have been analysed for changes in the capacity of key-enzymes: respiration (isocitrate dehydrogenase, IDH), organic acid synthesis (malic enzyme, ME), amino acid synthesis (aspartate aminotransferase, AAT and glutamate dehydrogenase, GDH). Variation in peroxidases was also measured. Results confirmed early work (Pierre, 1977), establishing an increase in the capacity of all the enzymes studied (Fig. 1). This is also in agreement with results of Jfiger & Pahlich (1972) and Pahlich et al. (1972) on GDH of pea seedlings, and with more recent work by Rabe & Kreeb (1979) in different experimental conditions. At 0.1 ppm SO 2 it appears that the increase in enzyme capacity is transitory and starts diminishing after two weeks of continuous pollution. Organic acids The pools of the seven main organic acids in bean leaves show different behaviour under pollution (a net separation between succinate and ~-ketoglutarate, and citrate and isocitrate was not obtained). Data in Fig. 2 show that the concentration of malate increases significantly, and that of malonate decreases; in all cases, these two acids are present in high concentration in the leaves. The size of the citrate-isocitrate pool shows a transitory change. The pools of those organic acids which are at low levels in the leaves were not modified by the pollution treatment. The increase in IDH capacity and the decrease in the citrate-isocitrate pool are consistent with an acceleration of the degradation of these acids, while malate accumulates. These results strongly suggest that the tricarboxylic acid cycle operates at a higher rate under pollution, in agreement with results by Cowling & Koziol (1978) showing increased respiration in ryegrass under low pollution.

EFFECTS OF SUBNECROTIC LEVELS OF S O 2 ON BEAN LEAVES

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Fig. 2. Variationsin organicacid content in polluted(S) and control(C) plants.

45

46

MICHI~LE PIERRE, ORLANDO QUEIROZ

Malonate has been shown to be a precursor of lipids in beans (Hatch & Stumpf, 1962). The decrease in malonate (Fig. 2) could be related to the inhibition of lipid synthesis observed in the case of pine needles after 0.2ppm SO2 during 24h (Malhotra & Khan, 1978). Amino acids Sulphur-containing amino acids. Measurements at picomole level showed that free

cystein and methionine are present at non-detectable levels both in polluted and non-polluted bean leaves. This could result from severe feedback control of synthesis as suggested by Smith (1972), Schiff & Hodson (1973) and Ziegler & Hampp (1977). After hydrolysis, cysteine has been shown in similar amounts in nonpolluted and in polluted leaves (respectively 10.1 and l l.7/tmoles/g dry wt in peptides, 340 and 291 #moles/g dry wt in proteins); methionine was not detected. Acidic and neutral amino acids. Results shown in Fig. 3 establish that among the five main amino acids of this group present in the leaves only the pool of alanine is not modified by pollution. The pools of aspartate and of glutamate decrease, those of serine and glycine increase. These results agree with those of J~iger et al. (1972) on glutamate, and those of Godzik & Linskens (1974), these latter after a 24 h pollution by 0.7 to 0.8 ppm SO 2.

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47

EFFECTS OF SUBNECROTIC LEVELS OF S O 2 ON BEAN LEAVES

The observed decrease in aspartate and glutamate pools are consistent with an increase in their metabolism as suggested by the increase in the capacity of AAT and GDH (Fig. 1). In contrast, the increase in serine and glycine levels appears to differ from the decrease in photorespiration observed by different authors; but it should be noted that inhibition of photorespiration was assumed by these authors after results obtained by utilising either SO2-derivates (Osmond & Avadhani, 1970; Liittge et al., 1972; Libera et al., 1975) or very high levels of SO 2, e.g. 5 ppm (Spedding & Thomas, 1973) or even 100 ppm (Tanaka et al., 1972). Koziol & Cowling (1978) utilising less than 400 pg/m 3 of SO 2 obtained a small but statistically significant decrease of ~4C incorporation into the group serine + glycine and also proposed that it could result from inhibition of photorespiration. In experiments in this laboratory incorporation of ~4C was also observed into serine and glycine from ~4C-bicarbonate injected into the leaf; pollution resulted in a slight but statistically non-significant decrease in ~4C-serine. In our experimental subnecrotic conditions (0.1 ppm) it is necessary to ascertain whether the capacity of glycollate oxidase also increases as does that of the other enzymes, which would be consistent with the increase in glycine and serine pools. Basic' amino acids. Variations in histidine, lysine and arginine are shown in Fig. 4: after about one week of pollution, their pools increase first, then decrease back to the level in the control plant. Maximum increase occurs simultaneously with the maximum increase in enzyme capacity. Po lyaDlines

Polyamines derived from arginine are a widespread group of substances which appear to play diverse functions in cellular metabolism. In particular, their polycation structure enables polyamines to play a role in pH stabilisation during the acidifying processes. For this reason, changes in polyamine content were

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48

MICHELE PIERRE, ORLANDO QUEIROZ

investigated in connection to SO2 pollution. The main polyamines present in bean leaves are putrescine and its derivatives spermidine and spermine. A substantial increase in putrescine was observed during the second week of pollution (Fig. 5), consistent with the variation in arginine (Fig. 4). The pool of spermine changes in the opposite way. In both cases, a level similar to that of the control plants is restored during the third week.

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It should be emphasised that, as in the case of organic and amino acids, maximum variation in polyamines appears during the period of rapid increase in enzyme capacity. CONCLUSIONS

Subnecrotic continuous pollution appears to trigger a generalised quantitative readjustment of the intermediary metabolism rather than to affect specifically limited metabolic areas. Other authors have reported on specific metabolic modifications, e.g. increase in the synthesis of sugars (Koziol & Cowling, 1978; Koziol & Jordan, 1978), in photosynthesis (Cowling et al., 1973), in respiration (Cowling & Koziol, 1978) and also in growth (Heitschmidt et al., 1978). But the results reported in the present and in a preceding paper (Pierre, 1977) clearly show that the general increase in the capacity of the key-enzymes of several pathways (Fig. 1), including those of the primary fixation of CO 2 for photosynthesis (Pierre, 1977), are consistent with the variations of different groups of metabolites (Figs 2 to 5). Therefore, the general picture appears to be an increase in the metabolic potentialities of the cell as a response to the onset of low levels of pollution. We assume that this generalised, but temporary, readjustment in metabolic capability will enable the cell to achieve:

EFFECTS OF SUBNECROTIC LEVELS OF S O 2 ON BEAN LEAVES (1)

(2)

49

an efficient metabolisation of the pollutant (the production of H2S by the leaves, observed by De Cormis, 1968 and De Cormis & Bonte, 1970, could be part of this process); a mechanism of stabilisation of the cellular pH, through an increase in organic bases (basic amino acids, polyamines). In contrast to the hypothesis of Priebe et al., 1978, which combines a decrease in organic acids with the increase in polyamines they observed for 0.3 ppm SO 2, our results (Fig. 6), obtained with 0.1 ppm, clearly show a parallel increase in those metabolites which would tend to decrease local pH (organic acids) and in metabolites capable of countering this tendency, achieving a kind of pH-stabilisation mechanism.

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Fig. 6. Difference in the content of organic acids, basic and acidic amino acids and polyamines between polluted and control plants as a function of the n u m b e r of days of pollution. Inserts: correlation between the variation of content in organic acids and content in polyamines and basic amino acids.

50

MICHELE PIERRE, ORLANDO QUEIROZ

The temporary character of this generalised metabolic readjustment is not as yet clearly understood. It could intervene as a relatively rapid metabolic and energetic response to the onset of low pollution, allowing time for a long-term adaptation. Results in this laboratory (to be published later in detail) show that the kinetics of the enzymic and metabolic readjustment depend on the physiological level of the leaf at the moment of the onset of pollution. An obviously fundamental question concerns the mechanism through which such a generalised increase in the capacity of different enzymes is achieved after a few hours of pollution. This problem is under current investigation. Another interesting problem deals with the physiological cost of the adaptive metabolic readjustment. The increase in metabolic potentialities could be expected to involve energetic imbalances and, in the long run, to result in earlier senescence. This hypothesis appears to be supported by the observed accumulation of serine (Fig. 3), which probably accelerates senescence (Martin & Thimann, 1972a,b), by the prolonged increase in the level of peroxidases (Fig. 1) and by morphological observations by Heitschmidt et al. (1978), on wheat grass in field, and by ourselves on bean (not yet published). REFERENCES ADLAKHA, R. C. & VILLANUEVA,V. R. (1980). Automated ion exchange chromatographic analysis of usual and unusual natural polyamines, J. Chromat., 187, 442-6. COWLING, D., JONES, L. & LOCKYER,D. (1973). Increased yield through correction of sulphur deficiency in rye grass exposed to sulphur dioxide, Nature, Lond., 243, 479-80. COWLING, D. & KOZIOL, M. (1978). Growth of ryegrass (Lolium perenne L.) exposed to SO 2. I. Effect on photosynthesis and respiration, J. exp. Bot., 29, 1029-36. DE CORMlS L. (1968). D6gagement d'hydrog~ne sulfur~ par des plantes soumises ~ une atmosphere contenant de ranhydride sulfureux, C.r. hebd. Seanc. Acad. Sci., Paris, 266, 683-5. DE CORMIS, L. & BONn, J. (1970). Etude du d6gagement d'hydrog6ne sulfur6 par des feuilles de plantes ayant re~u du dioxyde de soufre, C.r. hebd. Seanc. Acad. Sci., Paris, 270, 2078-80. GODZIK, S. & LINSKENS, H. (1974). Concentration changes of free amino-acids in primary bean leaves after continuous and interrupted SO 2 fumigation and recovery, Environ. Pollut., 7, 25-32. HARVEY, G. & LEGGE, A. (1979). The effect of sulphur dioxide upon the metabolic level of adenosine triphosphate, Can. J. Bot., 57, 759-64. HATCH, M. & STUMPF, P. (1962). Fat metabolism in higher plants. XVII. Metabolism of malonic acid and its ~t-substituted derivatives in plants. Pl. Physiol., Lancaster, 37, 121 6. HEITSCHMIDT, R., LAt~NROTH, W. & DODD, J. 0978). Effects of controlled levels of sulphur dioxide on western wheatgrass in a south-eastern Montana Grassland, J. appl. Ecol., 14, 859-68. HENRY, E., VALDOVINOS,J. & JENSEN,T. (1974). Peroxidases in tobacco abscission zone tissue. If. Time course studies of peroxidase activity during ethylene induced abscission, Pl. Physiol., Lancaster, 54, 192-6. J;~GER,H. & PAHLICn,E. (1972). EinfluB yon SO 2 aufden Aminos~iurestoffwechsel yon Erbsenkleimlinger, Oecologia, 9, 135-40. J~GER, H. J., PAHLICn, E. & STEUaING,L. (1972). Effect of sulphur dioxide on the amino acid and protein content of pea seedlings, Angew. Bot., 46, 199-211. KOZlOL, M. & COWLING, D. 0978). Growth ofryegrass (Loliumperenne L.) exposed to SO 2. II. Changes in the distribution of photoassimilated 14C, J. exp. Bot., 29, 1431-9. KozloL, M. & JORDAN,C. (1978). Changes in carbohydrate levels in red kidney bean (Phaseolus vulgaris L.) exposed to sulphur dioxide, J. exp. Bot., 29, 1037-43. LIBERA,W., ZIEGLER,|. & ZIEGLER,H. (1975). The action of sulfite on the HCO~- fixation and the fixation pattern of isolated chloroplasts and leaf tissue slices, Z. Pflanzenphysiol., 74, 420-33.

EFFECTS OF SUBNECROTIC LEVELS OF S O 2 ON BEAN LEAVES

51

LOTTGE, U., OSMOND, C., BALL, E., BRINCKMANN, E. & KINZE, G. (1972). Bisulfite compounds as metabolic inhibitors: non specific effects on membranes, PI. Cell Physiol., Tokyo, 13, 505-15. MALHOTRA, S. 8/. KHAN, A. (1978). Effects of sulphur dioxide fumigation on lipid biosynthesis in pine needles, :Phytochemistr),, 17, 241-4. MARTIN, C. & THIMANN, K. (1972a). The role of protein synthesis in the senescence of leaves. I. The formation of protease, PI. Physiol., Lancaster, 49, 64-71. MARTIN, C. & THIMANN, K. (1972b). Role of protein synthesis in the senescence of leaves. II. The influence of amino acids on senescence, PI. Physiol., Lancaster, 50, 432-7. OSMOND,C. B. & AVADHANI,P. N. (1970). Inhibition of the fl carboxylation pathway of CO 2 fixation by bisulfite compounds, PI. Physiol., Lancaster, 42, 228-30. PAHLICH, E. (1972). Sind die Multiplen Formen der glutamat dehydrogenase aus Erbsenkeimlingen ionformer? Planta, 104, 78 88. PAHLICH, E. (1973). On the mechanism of inhibition of mitochondrial glutamate oxaloacetate transaminase in SO 2 fumigated peas, Planta, ll0~ 267 78. PAHLICH, E., J.~GER, H . . J. & STEURING, L. (1972). Beinflussung des Aktivit/iten yon Glutamatdehydrogenase und Glutaminsynthetase aus Erbsenkeimligen durch SO 2, Angew. Bot., 46, 183 97. PAVLID~S, D. (1977). Action du SO 2 sur le m&abolisme interm~diaire. I. Utilisation d'une cellule de culture classique en caisson climatis6 sous atmosphere contr616e en SO2, Physiolog. Vkg., 15, 187-94. PEREZ-RODRIGUEZ,D. (1976). Effet de doses subnkcrotiques et/ou nkcrotiques de SO z sur I'ultrastructure cellulaire des feuilles de Phaseolus vulgaris. Paris, Dipl6me d'Etudes Approfondies, Universit6 Pierre et Marie Curie. PIERP~, M. (1975). Modifications mOtaboliques et enzymatiques dans lesJeuilles de Haricot (Phaseolus vulgaris) sournises (t pollution par des doses subn~crotiques et/ou nbcrotiques de SO z dans l'air. Th6se de Doctorat de 3e cycle. Paris, Universit6 Pierre et Marie Curie. PIERRE, M. (1977). Action du SO z sur le m&abolisme interm6diaire. I1. Effet de doses subn&:rotiques de SO 2 sur les enzymes de feuilles de Haricot, Physiolog. Vbg., 15, 195-205. PRIE~E,A., KLEIN,H. & J~,GER,H. (1978). Role of polyamines in SO2-polluted pea plants, J. exp. Bot., 29, 1045 50. QUEIROZ, O. (1965). Sur le metabolisme acide des Crassulac+es. Action ~ilong terme de la temperature de nuit sur la synth6se d'acide malique par Kalanchoe blossfeldiana "Tom Thumb' plac~e en jours courts, Physiolog. VOg., 3, 203-13. RABE, R. & KREEB, K. (1979). Enzyme activities and chlorophyll and protein content in plants as indicators of air pollution, Environ. Pollut., 19, 119-37. SCHIFV, J. A. & HODSON, R. C. (1973). The metabolism of sulfate. A. Rev. PI. PhysioL, 24, 381-414. SMITH, 1. K. (1972). Studies of L cysteine biosynthetic enzymes in Phaseolus vulgaris L., PI. Physiol., Lancaster, 50, 477 9. SPEDDING, D. J. & THOMAS,W. J. (1973). Effect of sulphur dioxide on metabolism of glycolic acid by barley (Hordeum vulgare) leaves, Aust. J. biol. Sci., 26, 281-6. TANAr,A, H., TAKANASHI,T. & YATAZAWA,M. (1972). Experimental studies on SO 2 injuries in higher plants. II. Disturbance of amino acid metabolism in plants exposed to sulphur dioxide, Water, Air & Soil Pollut., 1,343 6. ZIEGLER, I. & HAMPP,R. (1977). Control of asSO]- and 35SO~- incorporation in spinach chloroplasts during photosynthetic CO 2 fixation, Planta, 137, 303 7.