Effects of fumigating rice plants with sulphur dioxide on photosynthetic pigments and nonstructural carbohydrates

Agriculture, Ecosystems and Environment, 18 (1986) 53-62 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

EFFECTS OF FUMIGATING RICE PLANTS WITH SULPHUR ON PHOTOSYNTHETIC PIGMENTS AND NONSTRUCTURAL CARBOHYDRATES

53

DIOXIDE

P.K. NANDI, MADHOOLIKA AGRAWAL and D.N. RAO Ecology Research Laboratory, Centre of Advanced Study in Botany, Banaras Hindu University, Varanasi 221 005 (India) (Accepted for publication 2 June 1986)

ABSTRACT Nandi, P.K., Agrawal, M. and Rao, D.N., 1986. Effects of fumigating rice plants with sulphur dioxide on photosynthetic pigments and nonstructural carbohydrates. Agric. Ecosystems Environ., 18: 53-62. Exposure of Oryza sativa plants to 670 and 1330 pg mm” SO, during tillering and flowering decreased the concentrations of photosynthetic pigments and starch, and increased peroxidase activity and amounts of reducing sugar. Chlorophyll b was more sensitive to SO, damage than chlorophyll a. It is suggested that peroxidase-mediated oxidation of chlorophyll is possible in SO,-exposed plants and that this change may ultimately affect plant development.

INTRODUCTION

Sulphur dioxide (SO,), a major atmospheric pollutant, adversely affects various physiological and biochemical processes which may lead to growth and yield reductions in plants (Last, 1982; Malhotra and Khan, 1984). A number of workers have reported the injurious effects of SO* on photosynthetic pigments in many plant species (Rao and LeBlanc, 1966; Puckett et al., 1973; Nandi et al., 1984a). It has been suggested that increased acidity accompanying SO, absorption is responsible for chlorophyll degradation in these plants. It is also noted that excess free oxygenradicals (0, ‘Oz and OH’), formed in SO,-exposed plants, may affect cellular components including chlorophyll pigments (Shimazaki et al,, 1980) and that HzOz, a dismutation product of 0; in chloroplasts may inactivate several enzymes involved in the reductive pentose phosphate cycle (Tanaka et al., 1982a,b). Furthermore, Nandi et al. (1984b) found that SO* induced changes in the activities of enzymes catalysing HzOz breakdown in plants. These SOzinduced changes must have bearing on the photosynthetic efficiency of plants. Koziol and Jordan (1978) noted that in Phaseolus uulgaris, the amount of sugar and starch increased in response to SOz concentrations

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which did not induce SO* visible injury, but decreased at SO* concentrations which caused development of necrotic lesions. Total nonstructural carbohydrate level was also decreased following SO,-exposure of Ulmus americana seedlings (Constantinidou and Kozlowski, 1979). Jones and Mansfield (1982) noted that the reduced availability of photosynthates limited root and tiller production in SOz-exposed Phleum pratense. Furthermore, Japanese and Korean cultivars of rice were noted to be most sensitive to SO* during their tillering and flowering stages of growth (Taniyama et al., 1972; Kim and Han, 1980). The concentrations of SO, used in the latter studies were exceptionally large, and caused the development of acute, visible injuries, In Western countries, ambient SOz concentrations have decreased considerably since the 1960’s, but in India daily average concentrations of pollutant in areas close to emission source may be as large as 920 ccg mm3 (Dubey et al., 1982). Therefore, the present study was designed to assess the potential effects of such episodic and exceptionally large concentrations of SO? for a long period on photosynthetic pigments and carbohydrate levels in an Indian cultivar of rice (Oryza satiua L. cv. Ratna) during its tillering and flowering stages of growth. MATERLALS AND METHODS

Plant material Rice plants (Oryza satiua L. cv. Ratna) were raised in nursery conditions and when 20 days old were transplanted in 27cm diameter plastic pots (2 plants per pot) filled with sandy loam soil (organic carbon = 0.86%, pH 7.4-7.6, cation exchange capacity = 15.4 meq. lOO- ’ g). The plants were cultured in an environmentally controlled glasshouse maintained at 30 f 2°C during the day and 23 f 2°C at night, with a relative humidity of 75 f 5% under natural light. AR plants received optimal watering conditions. Fumigation Fumigation of plants (with about 28 leaves) with 670 f 15 pg me3 and 1330 * 18 pg mV3 SO, was started when the plants were 40 days old. It was performed for 1.5 h daily (between 08 00 and 10 00 h) for 20 days (41-60 day plant ages) during their tillering stages and again for 10 days (71-80 day plant ages) during their flowering stages (Table I). During fumigation, plants in 25 pots were placed within perspex chambers, 1.5 X 1.5 X 1.5 m having the environmental conditions already enumerated. Sulphur dioxide was generated in a continuous manner by bubbling air through a l.O-1.5% aqueous solution of sodium metabisulphite (Nandi et al., 1984a), and the desired concentrations within the chambers were a-

55 TABLE I Experimental schedule for fumigating and sampling rice plants

Experimental condition

Plant age (days)

Sampling order

No treatment Treatment Treatment No treatment Treatment Recuperation

l-40 41-50 51-60 61-70 71-80 81-90

I (40 day) II (50th day) III (60th day) IV (70th day) V (80th day) VI (90th day)

thieved by dilution with carrier air (56 1 s-l). The gas was dispersed within the fumigation chambers through a network of perforated alkathene pipes arranged at the base (Nandi et al., 1984a). Control plants were placed in identical chambers, but flushed with activated charcoal filtered air. Air in the chambers was mixed with a small turbulence fan. Sulphur dioxide within the chambers was continuously monitored by means of a CEA 555 calorimetric SO* analyser (CEA Instruments, Inc., U.S.A.). Plant analyses Fully expanded leaves were collected from randomly selected plants of all treatments at 50, 60, 70, 80 and 90 days old. Additionally, controls were collected at 40 days old, prior to the commencement of SOz treatments. They were taken at mid-day for quantitative estimations of photosynthetic pigments, non-structural carbohydrates and peroxidase activity (Table I). Plants were also frequently examined for the presence of visible injuries. Photosynthetic pigments For the estimation of chlorophyll and carotenoid contents, a 0.25-g leaf sample was homogenized in cold 80% acetone and centrifuged at 5000 g for 15 min. The final volume of the supernatant was made-up to 25 ml by adding 80% acetone. The optical densities of the extract were measured at wavelengths of 480 and 510 nm for carotenoids and at 645 and 663 nm for chlorophyll a and b on a spectronic 20 spectrophotometer. The amounts of carotenoids were calculated using the fomulae of Duxbury and Yentsch (1956); and those of chlorophyll a and b by the formulae of Maclachlan and Zalik (1963). The amounts of chlorophyll a and b were added together to obtain estimates of total chlorophyll. The chlorophyll a/b ratio was calculated to determine the relative sensitivity of these pigments to SO,.

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Enzyme assay For the determination of peroxidase activity, a lOO-mg leaf sample was homogenized with 10 ml 0.1 M cold phosphate buffer containing 5 mM cysteine at pH 6.8 in a chilled mortar and pestle. The homogenate was centrifuged at 10 000 g for 15 min at 04°C and the supernatants were used for enzyme assays. A 5ml assay mixture for peroxidase activity, containing 125 E.tmol phosphate buffer (pH 6.8), 50 pmol pyrogallol, 50 pmol HzOz and 1 ml of diluted enzyme extract was incubated at 25°C for 5 min after which the reaction was terminated by adding 0.5 ml of 5% HzSO+ The coloured end-product (purpurogallin) was extracted in ether and the quantity determined spectrophotometrically at 430 nm, where its extinction coefficient is 2.47 cm-’ mM_’ (B&ton and Mehley, 1955). Carbohydrates Soluble sugars were extracted from 50 mg dried (105°C for 8 h) and powdered leaf samples with boiling 80% ethanol (v/v). Determinations of reducing and total soluble sugars were performed with reference to glucose standards using the calorimetric copper method of Somogyi (1952) and phenol/HzS04 calorimetric assay of Dubois et al. (1956), respectively. The differences between amounts of total soluble sugars and reducing sugars were calculated to obtain the values of non-reducing sugars. The ethanol-washed pellets were dried and the starch in them hydrolysed to sugars using 52% perchloric acid. Sugar contents in hydrolysed extracts were determined following the method of Dubois et al. (1956). Values of starch were obtained by multiplying the sugar content by 0.92 (McCready et al., 1950). Statistics All analyses for each treatment were made with the samples taken from five separate plants. Data were analysed using two way analysis of variance and Duncan’s multiple range test (Little and Hills, 1978). RESULTS

Visible chlorotic followed by necrotic foliar injury appeared first in plants treated with 1330 pg SO, rnw3 on the 47th day and then in plants treated with 670 c(g SO2 mm3 when 52 days old that is 7 and 12 days after starting fumigation. When 90 days old, the percentages of acute leaf area injury in these plants were 52.0 and 34.0, respectively. The chlorophyll content of the control plants gradually decreased upto an age of 80 days. In contrast, there was a fall in the content of the SO,-

57 TABLE II Effects of SO,[67O(T,) or 1330(T,) pg me31 on the absolute and relative (values in parentheses) amounts of chlorophyll (I and b in rice plants’ Plant age (days)

Plant

Chlorophyll (mg g-’ dry leaf) a

b

Total

Chlorophyll

a/b

60

Control T, T, Control T, T, Control

70

T, T, Control

80

T, T* Control

90

T, T, Control

40

50

T, T,

2.09s 2.12s 2.07a 2.35a(100.0) 1.999 84.7) 1.85c( 72.7) 2.69a(100.0) 1.76b( 65.5) 1.599 59.1) 3.05s(100.0) 2.489 81.3) 2.07c( 67.9) 3.35s(loo.o) 2.589 77.0) 2.16c( 64.5) 3.00s( 100.0) 2.30”( 76.7) 2.05c( 68.4)

3.0ga 3.10s 3.06a 3.478( 100.0) 2.77b( 81.8) 2.44c( 70.3) 4.10a(100.0) 2.47”( 60.3) 2.28”( 55.6) 5.35s(loo.o) 3.2gb( 61.5) 2.8gc( 54.0) 5.82a( 100.0) 3.56b( 61.2) 3.17q 54.5) 5.23a(100.0) 3.64b( 69.6) 3.2gc( 62.9)

5.17s 5.22a 5.13s 5.82a(100.0) 4.77b( 82.0) 4.29C(73.7) 6.7ga( 100.0) 4.23b( 62.3) 3.88b( 57.2) 8.3ga( 100.0) 5.77b( 68.7) 4.96c( 59.1) 9.17s(loo.o) 6.14”( 67.0) 5.33C( 58.0) 8.23a( 100.0) 5.94b( 72.2) 5.33c( 64.8)

0.68 0.68 0.67 0.68 0.72 0.76 0.66 0.71 0.70 0.57 0.75 0.72 0.58 0.72 0.68 0.57 0.63 0.62

‘Values followed by the same letter in the same column for each harvest are not significantly different (P < 0.05).

treated plants during the first treatment phase (41-60 days), followed by some evidence of recovery, although it remained below that of the control plants (Table II). Such changes in quantities of chlorophyll a and chlorophyll b due to SO,-treatment, plant age and their interaction were highly significant (P < 0.001). The concentrations of chlorophyll a decreased by 15.0-34.5s in the 670 pg SOP my3 treatment (in T1 plants) and 21.240.8% in 1330 Mg SOz me3 (in Tz plants). Similarly, chlorophyll b decreased by 20.1-30.9% in T, and 29.6-45.6s in Tz plants. In contrast, the ratios of chlorophyll a/b in SO*-treated plants were larger than that in the control. Although these differences due to SOz-treatment (P < 0.01) and plant age (P < 0.05) were significant, there was not a significant interaction. Changes in amounts of carotenoid pigments followed more or less the same trends as those of chlorophyll pigments. With respect to control, amounts of carotenoid pigments decreased by 11.5-23.1% in T1 and 20.236 .O% in Tz plants (Table III). In contrast to the effects on photosynthetic pigments, SO? increased peroxidase activity by 102.0 and 179.0% in treatments T1 and Tz, respective-

58 TABLE III Effects of S0,[670(T,) or 1330(T,) pg m-j] on the absolute and relative (values in parentheses) amounts of carotenoids in rice plants’

Carotenoids (mg g-’ dry leaf)

Plant age (days)

40 (Start of SO, exposure) 50 60 70 80 90

C

T,

T2

1.41a 1.52a( 100.00) 1.53*(100.00) 1.55a( 100.00) 1.3Ba( 100.00) 1.34~(100.00)

1 .43a 1.34b(88.20) 1.1Bb(77.20) 1.24b(80.00) 1.09b(79.00) 1.06b(81.20)

1.3Ba 1.21c(81.60) 1.03c(67.30) l.llb(71.60) 0.92c(66.70) 0.86c(64.20)

‘Values followed by the same letter in the same row do not differ significantly (P < 0.01).

3O

1

I

I

I

I

40

50

60

70

80

PLANT

I 90

AGE (DAYS)

Fig. 1. Effects of S0,[670(T,) or 1330(T,) ug m-“1 on the peroxidase activity of rice plants (bars represent standard errors; C = Contr&I).

ly (Fig. 1). A significant interaction (P < 0.01) due to SO,-treatment and plant age on peroxidase activity was also detected (Table IV). Although amounts of foliar starch decreased while those of reducing sugar increased significantly (P < O.OOl), there was no significant effect of SOz-treatment on non-reducing sugars (Table V). With respect to the control, the maximum decreases in starch concentrations of 80-day old plants due to T1 and Tz treatments were 16.8 and 29.6%, respectively, and increases in reducing sugars were 26.6 and 35.1%, respectively. DISCUSSION

Sulphur dioxide reduced the rate of increment in photosynthetic pigments with age and interfered with carbohydrate metabolism of rice plants. The

59

TABLE IV Results of analysis of variance of the effects of SO,-treatment and plant age on the photosynthetic pigments, nonstructural carbohydrates and peroxidase activity of Oiyza sativa plants’

Plant parameter

Factor SO,-treatment

(S)

*** *** ** *** *** ***

Chlorophyll a Chlorophyll b Chlorophyll a/b ,Carotenoid Starch Reducing sugar Non-reducing sugar Peroxidase activity

NS ***

‘*P < 0.05; **P < 0.01; ***P < 0.001;

Plant age (A)

Sx

*** *** * *** *** ** *** **

** ***

A

NS ** ** *** NS **

NS = not significant.

TABLE V Effects of S0,[670(T,) or 1330(T,) c(g m-‘I on the absolute and relative (values in parentheses) amounts of starch and reducing and non-reducing sugars in rice plants’

Carbohydrate

Starch (mg g-’ dry leaf)

Reducing sugar (mg g-r dry leaf)

Non-reducing sugar (mg 8-l dry leaf)

Plant

Plant age (days) 40=

60

60

70

80

90

T,

124.0’ (100.00) 125.6’

T,

126ZZa

183.1a (100.00) 172.Qb ( 94.40) 149.0c ( 81.40) 30.94a (100.00) 33 .soa (108.30b 39.75 (128.50) 27.16 30.06 31.26

197.ba (100.00) 166.8b ( 84.60) 152.3’ ( 77.30) 31.13a (100.00~ 37.50 (120.60) 41.5oc (133.30) 21.87 22.50 22.60

200.1a (100.00) 183.1b ( 91.50) 176.bb ( 88.40) 31.76a (100.00~ 36.15 (113.80) 39.50c (124.40) 9.06 11.65 10.00

125.8” (100.00) 104.4b ( 83.00) 88.3’ ( 70.30) 33.76a (100.00~ 42.76 (126.60& 45.60 (135.10) 9.06 9.26 8.40

101.28 (100.00) 85.6b ( 84.60) 76.8’ ( 75.90) 33.13a (100.00~ 39.00 (117.70) 42.26’ (127.60) 13.13 12.60 15.35

Control

Control

28 .50a

T,

28 .50a

T,

29.00a

Control T,

30.06 31.26 31.76

Ts

‘Values followed by the same letter in the same column do not differ significantly for starch (P < 0.01) and for reducing sugars (P < 0.05). ‘Start of SO, exposure.

main effects and interactions of SO, and plant age were statistically significant (Table IV). The results show that chlorophyll b was more sensitive in rice plants to SO2 damage than chlorophyll a. This observation contradicts earlier observations (Williams et al., 1971; Kondo et al., 1980; Shimazaki et al., 1980). The higher SO,-sensitivity of chlorophyll b in rice plants, could be

60

due to the increase of chlorophyllase activity (Malhotra, 1977) and/or inhibition of chlorophyll b synthesis (Aronoff and Kwok, 1977; Castelfranco, 1983). Furthermore, from a significant correlation between increasing peroxidase activities and decreasing amounts of chlorophyll in 1330 pg me3 SOz-exposed plants (r = 0.835, P < 0.05), it may be inferred that at higher SOz concentration, peroxidase-mediated oxidation of chlorophyll is possible. This interpretation is supported from in vitro studies by Huff (1982), where peroxidasecatalysed oxidation of chlorophyll by HzOz was reported. It is already known that with the decrease in catalase activity, there is accumulation of HzOz in chloroplasts of SO,-exposed plants (Tanaka et al., 1982a). Carotenoid pigments of SOz-exposed rice plants seem to be less sensitive than chlorophyll. This is possibly because the carotenoids, unlike chlorophylls, are more resistant to destruction under adverse conditions (Kramer and Kozlowski, 1979). Such changes in amounts of photosynthetic pigments, however, may affect carbohydrate metabolism, as has been noted in the present study, subsequently leading to reduced growth and yield (Nandi et al., 1985). Koziol and Jordan (1978) correlated decreases in photosynthesis with visible injury and with the reduction of starch in SO* -exposed leaves. However, in rice leaves decrease of starch was noted even before the development of visible injuries. Possibly the inactivation of certain enzymes of the reductive pentose phosphate cycle by HzOz (Tanaka et al., 1982b) is responsible for small amounts of starch in SO,-exposed leaves. Malhotra and Sarkar (1979) have reported increased reducing sugars and decreased nonreducing sugars in S02exposed pine needles. In the present study, increases in reducing sugars were not accompanied by significant changes in nonreducing sugars. It has been suggested that polyhydric sugars may act as scavengers of free oxygen radicals (Asada, 1980). However, with increases in SO* concentrations, decreases in chlorophyll and increases in reducing sugars accompanied by increases in visible injury indicate that accumulations of sugars were the result of either their reduced utilization for repair processes (Koziol and Jordan, 1978) or inhibition in synthesis of other metabolites, rather than an adaptive response against free radical toxicity. This explanation lends support to the possible hypothesis of peroxidase-mediated oxidation of chlorophyll in SO+xposed rice plants. Thus, it may be concluded that the mechanism involved in SOz-induced destruction or chlorophyll may depend upon pollutant concentrations and the plant species under study, and that reductions in photosynthetic pigments and disturbed carbohydrate metabolism during tillering and flowering may affect their vegetative growth and grain yields, respectively. ACKNOWLEDGEMENTS

The financial knowledged .

assistance

extended

by CSIR, New Delhi is gratefully

ac-

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