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Chloroplast superoxide and hydrogen peroxide scavenging systems from pea leaves: Seasonal variations
Plant Science, 50 (1987) 105-109
105
Elsevmr Sclentffic P u b h s h e r s Ireland Ltd
CHLOROPLAST S U P E R O X I D E A N D HYDROGEN P E R O X I D E S C A V E N G I N G S Y S T E M S FROM P E A L E A V E S : S E A S O N A L V A R I A T I O N S
DAVID J GILLHAM and ALAN D DODGE*
School of Bmlog~cal Sciences, Unwers~ty of Bath, Bath, Avon, BA2 7A Y (U K ) (Received October 8th, 1986) (Rewsmn received J a n u a r y 23rd, 1987) (Accepted J a n u a r y 30th, 1987)
Levels of chloroplast antmxldants and enzymes t h a t scavenge oxygen radicals were followed in the leaves of pea plants
(P~sum satwum L cv Meteor) grown under glasshouse condltmns between Apml 1984 and May 1985 While h t t l e vamatlon m pigment levels or superoxlde dlsmutase activity was detected during this pemod, plants grown in early summer ( M a y June) contained appreciably higher levels of ascorbate, ascorbate peroxldase and glutathlone reductase t h a n plants grown in winter ( D e c - J a n ) The role of h g h t intensity in regulating levels of chloroplast antmxldants was examined further using pea plants grown m a constant environment chamber under 100 or 400 #mol m - 2 s ~ photon flux density Chloroplasts isolated from plants grown at the higher h g h t lntenmty contained mgmficantly higher levels of ascorbate, ascorbate peroxldase, glutathlone reductase and dehydroascorbate reductase These data suggest t h a t h g h t intensity may have an important influence on the level and activity of chloroplast antmxldants and oxygen radical scavenger enzymes
Key words ascorbate, ascorbate peroxldase, dehydroascorbate reductase, glutathmne reductase, P~sum satwum, superoxlde dlsmutase
Introduction Chloroplasts of higher plants are protected against damaging oxygen radicals by the enzymes superoxlde dlsmutase (SOD), ascorbate perox~dase, dehydroascorbate reductase and glutathmne reductase. These enzymes efficmntly scavenge both superoxlde (O~-), whmh may be formed as a consequence of the reductmn of O2 by PSI (the Mehler reactmn), and hydrogen peroxide (H202) formed by the dmmutatmn of O~- In addltmn the chloroplast stroma contmns mHhmolar concentratmns of ascorbate and glutathmne [1,2] Chloroplast membranes also contain carotenoid p~gments t h a t quench smglet oxygen (102), generated ff
*To whom correspondence should be sent Abbreviations H202, hydrogen peroxide, ~Oz, smglet oxygen, O~-, superoxlde, OH', hydroxyl radical, SOD, superoxide dlsmutase
h g h t energy absorbed by chlorophyll is not dmmpated through photosynthesis [3] Failure to scavenge these active oxygen specms can result m damage to chloroplasts H202 is strongly inhibitory towards CO2 fixation [4], while both 102 and hydroxyl radmals (OH'), formed from interaction between O~- and H202, can reduce hpld peroxldatlon and oxldatmn of protein amino acids and nucleic acids
[11 Previous studies have demonstrated that much of the leaf SOD, [5] ascorbate peroxldase and glutathlone reductase [2] is chloroplasUc Modulatmn of the act~wty of these enzymes may be ~mportant m plant resistance to envlronmental stress or air pollutants [6-9] In this laboratory we have been interested m the role of these antloxldant mechamsms m protecting pea leaves against photo-oxidative damage [2,10,11]. As a consequence of routine analyses of SOD, ascorbate peroxldase, glutathmne reductase and ascorbate m glasshouse grown pea
0618-9452/87/$03 50 ~, 1987 Elsevmr Scmntffic Pubhshers Ireland Ltd P u b h s h e d and Printed m Ireland
106
leaves, we have observed seasonal changes m their levels, which we now report. Materials and m e t h o d s
Plant mater~al Pea (P~sum s a t w u m L. cv. Meteor) seeds were plante~ in moist L e w n g t o n Umversal Compost (Flsons Ltd, Levington, U K.) and grown for 14-21 days m a glasshouse with a mean air temperature of 20°C and natural dayhght cond~tmns Plants were watered dmly. Pea plants were also grown m a constant envyronment chamber at 20°C under continuous l l l u m m a t m n (400/~mol m - 2 s - 1 photosynthetlcally active r a d m t m n provided by warm white fluorescent tubes, 65/85 W; Thorn, London, U K ) Light intensity m half the chamber was reduced to 100 #tool m - 2 s - 1 using layers of Kodak neutral density filter. Chloroplast preparatwn and enzyme extracts For enzyme assays and ascorbate determlnatron, cell-free homogenates were prepared. Approximately 0 5 g of the first fully expanded leaves below the apex were ground m 10ml of cold 50 mM potassmm phosphate buffer (pH 7.6), with a glass homogemzer. The homogenate was strained through four layers of m u s h n and centrifuged at 4000 × g for 5 mm. The resulting supernatant fractmn was used to estimate enzyme and ascorbate levels Isolated intact chloroplasts were prepared from 25-g samples of pea leaves according to the method of Walker [12] as described previously [2] Enzyme assays Activities of ascorbate peroxldase, glut a t h m n e reductase (EC 1.6.4.2) and dehydroascorbate reductase (EC 1.85.1) were determined m ahquots of the enzyme extract or chloroplast preparatmn according to the methods of Nakano and Asada [13] and Jablonskl and Anderson [14], as described prewously [2], SOD (EC 1.15 1.1) was determined by its inhlbitmn of formazan productmn from mtro-blue tetrazohum based on the method descmbed by
Beauchamp and Frldovich [15]. The 3-ml reaction mixture contained an aliquot of leaf homogenate or chloroplast, 22 mM potassmm phosphate (pH 7 8), 0 1 mM EDTA, 25 #M nitro blue tetrazohum and 0.1 mM xanthme. The reactmn was imtiated by additmn of x a n t h m e oxldase (Sigma) representing 60 #g of protein.
Determ~natmns Ascorbate was determined according to the method of Mukherjee and C h o u d h u n [16] as descmbed previously [2]. Glutathlone was assayed by the method of Law et al. [17] as descmbed previously [2]. Pigments were extracted from leaf samples w~th ethanol. Chlorophyll and carotenoid m the ethanol extract were quantified using the extraction coefficients devised by Lmhtenthaler and Wellburn [18]. Rephcatmn and statistics All results presented m this paper are the means of at least three replicate samples prepared from three independent batches of leaf material sampled at the same time. Leaf samples were routinely taken from plants at the same time each day throughout the samphng pemod. The S E M was less than 10% of the mean m each experiment. R e s u l t s and d i s c u s s i o n Ascorbate peroxldase, glutathlone reductase and ascorbate levels all showed a marked peak m early summer (May~lune), but then dechned m winter (Dec.~Jan.) as shown in Fig. 1. Our samphng procedure entailed using the youngest fully expanded leaves to prepare homogenates for analysis, and therefore activity changes were not due to the leaf samples being of different age SOD activity (Fig. 1) and chlorophyll and carotenoid levels (Fig. 2) showed no marked change between summer and winter. One environmental factor hkely to influence levels of antloxldant and antioxidant enzymes was light. Growth m early summer corresponded to the highest total lrradlance
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L e v e l s of a s c o r b a t e p e r o x l d a s e (a). a s c o r b a t e (b), g l u t a t h m n e r e d u c t a s e (c) a n d SOD (d) m p e a l e a v e s g r o w n u n d e r g l a s s h o u s e c o n d i t i o n s a n d s a m p l e d e a c h m o n t h b e t w e e n A p r i l 1984 a n d M a y 1985 E a c h d a t a p o i n t r e p r e s e n t s t h e m e a n of t h r e e s e p a r a t e d e t e r m m a t m n s m a d e from s e p a r a t e b a t c h e s of p l a n t m a t e r i a l o b t a i n e d on t h e s a m e s a m p l e d a t e The s t a n d a r d e r r o r of t h e m e a n w a s less t h a n 10% of t h e m e a n m e a c h c a s e
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F i g 2. L e v e l s of c h l o r o p h y l l (a) a n d c a r o t e n o l d s (b) m pea l e a v e s g r o w n u n d e r g l a s s h o u s e c o n d i t i o n s b e t w e e n Apml 1984 a n d M a y 1985 E a c h d a t a p o i n t r e p r e s e n t s t h e m e a n of t h r e e s e p a r a t e d e t e r m i n a t i o n s
for each month of the samphng pemod falhng at Long Ashton Research Station, 25 KM WNW of Bath U m versl t y (Fig. 3) By contrast growth in winter matched the period of lowest lrradlance To examine the effects of h g h t intensity on the a c n w t y of chloroplast antloxldants and antmxldant enzymes, pea plants were grown m a constant environment chamber for 14-21 days under d l u m m a t l o n of either 400 or 100 t~mol m 2 s 1 photon flux density Intact chloroplasts were molated, and then analysed for achvltles of chloroplast annoxl dant s and enzymes (Table I) Chloroplasts from plants grown at the higher hght intensity contained enhanced act~vlt~es of ascorbate perox~dase, glutathlone reductase and dehyroascorbate reductase, and higher levels of ascorbate than chloroplasts from plants grown at the lower
108 T a b l e I. Effect of g r o w t h a t 100 or 400 t~mol m - 2 s - 1 p h o t o n flux d e n s i t y on t h e a c t i v i t y of e n z y m e s s c a v e n g i n g 02 a n d HsO 2 a n d t h e levels of a n t m x l d a n t s m i s o l a t e d p e a c h l o r o p l a s t s (80-85% i n t a c t ) A c t l w t l e s g i v e n m u m t s ( m g chl) - 1 for SOD, l~mol (rag chl) - 1 h - i for a s c o r b a t e perox~dase, g l u t a t h m n e r e d u c t a s e a n d d e h y d r o a s c o r b a t e reductase, p g ( m g chl) - ~ for a s c o r b a t e a n d g l u t a t h m n e or m g (g f r e s h wt ) - 1 for c h l o r o p h y l l R e s u l t s a r e t h e m e a n s of at l e a s t five i n d e p e n d e n t e x t r a c t m n s f r o m different b a t c h e s of p l a n t m a t e r m l S E M were less t h a n 10% of t h e m e a n Irradlance ( u m o l m - 2 s - 1 PAR)
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h g h t intensity. No difference m SOD act ]w t y or g l u t a t h m n e levels from the two sets of plants was detected The effect of increased levels of a n t m x l d a n t and oxygen scavenging enzymes on the capac]ty of plants to scavenge oxygen radicals is
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F i g . 4. P a r a q u a t m d u c e d c h l o r o p h y l l b l e a c h z n g m p e a leaf dmcs c u t from p l a n t s g r o w n m a c o n s t a n t e n v i r o n m e n t c h a m b e r at 100 ( • [~) or 400 (@ O) #tool m - ~ s - 1 p h o t o n flux d e n s i t y Discs were i n c u b a t e d m g l a s s p e t n d i s h e s cont a m i n g HsO ( • • ) or 10 p M p a r a q u a t ( [] O)
shown m Fig. 4. Leaf discs were excised from plants grown at 100 or 400 pmol m - 2 s - 1 photon flux density and chlorophyll ret ent i on was tested in the presence of paraquat (10 pM), a chemical known to promote O~- generation [19]. Plants grown at the lower m t e n m t y and, therefore, with lower antloxldant and oxygen radmal scavenging enzyme activltms, showed a greater rate of paraquat-mduced chlorophyll bleaching than leaf d~scs cut from plants grown at the higher light intensity. This difference was probably not due to the rate of herbicide uptake, because CO 2 fixation was mh~blted by paraquat m both sets of plant material after a similar time period (data not shown) This result indicates t hat the increased protect]on against damaging oxygen radicals m plants grown at the higher hght mtens]ty may have been caused by enhanced chloroplast levels of ascorbate, ascorbate perox]dase, glutath]one reductase and dehydroascorbate reductase.
109 The results presented in this paper indicate t h a t h g h t ls a n i m p o r t a n t f a c t o r m t h e r e g u l a t i o n of c h l o r o p l a s t h y d r o g e n peroxide scavenging systems from pea leaves While fluctuatmns m h g h t i n t e n s i t y m a y h a v e c o n t r i b u t e d to t h e c h a n g e s m a c t i v i t y o f a s c o r b a t e , a s c o r b a t e peroxldase and glutathmne reductase m pea l e a v e s g r o w n m s u m m e r a n d w i n t e r , ~t is possible that annual fluctuatmns in temperature, w a t e r s t a t u s o r p h o t o p e m o d o r f l u x e s of a i r pollutants may also exert some control Reports m the h t e r a t u r e i n d i c a t e t h a t low t e m p e r a t u r e [8,20], d r o u g h t [9], o z o n e [21,22], SO2 f u m l g a t m n [6,23], o r a l t e r a t i o n s m o x y g e n t e n s m n [24], c a n a f f e c t t h e a s c o r b a t e , g l u t a t h m n e , glutathlone reductase, ascorbate peroxldase and SOD levels m leaves These reports, a n d the r e s u l t s i n t h i s s t u d y m d m a t e t h a t a w~de vame t y of e n v i r o n m e n t a l f a c t o r s c a n m o d u l a t e t h e a c t i v i t y of c h l o r o p l a s t O~- a n d H 2 0 2 s c a v e n g i n g s y s t e m s I n w e w of t h e i n c r e a s i n g i n t e r e s t m these protective mechamsms m the defence of p l a n t s a g a i n s t s t r e s s , i n v e s t i g a t o r s s h o u l d b e a w a r e of t h e w i d e v a m e t y of f a c t o r s t h a t c o u l d affect enzyme a n d a n t m x l d a n t levels
Acknowledgements The a u t h o r s are grateful to M a r t i n H u x l e y of L o n g A s h t o n R e s e a r c h Station, Bristol, U K for s u p p l y i n g t h e l r r a d l a n c e d a t a T h i n w o r k w a s s u p p o r t e d b y a U m v e r s l t y of B a t h r e s e a r c h s t u d e n t s h l p to D . J G
References 1 B Halhwell, Chloroplast Metabohsm The Structure and Function of Chloroplasts m Green Leaf Cells, Clarendon Press, Oxford, 1981, p 179
2 D J Glllham and A D Dodge, Planta, 167 (1986) 246 3 C S Foote, Scmnce, 162 (1986) 963 4 J M Robinson, M G Smith and M Gibbs, Plant Physlol, 65 (1980) 755 5 C Jackson, J Dench, AL Moore, B Halhwell, C Foyer and D O Hall, Eur J Blochem, 91 (1978) 339 6 K Tanaka and K Sugahara, Plant Cell Physlol, 21 (1980) 601 7 E H Lee and J H Bennett, Plant Physlol, 69 (1982) 1449 8 L J de Kok and F A Oosterhms, Physlol Plant, 58 (1983) 47 9 P E Gamble and J J Burke, Plant Physlol, 76 (1984) 615 10 D J Glllham and A D Dodge, Plant Physlol, 75 (1984) P 51 11 D J Glllham and A D Dodge, Physlol Plant, 65 (1985) 393 12 D A Walker, Meth Enzymol, 69 (1980) 94 13 Y Nakano and D Asada, Plant Cell Physlol, 22 (1981) 867 14 P P Jablonskl and J W Anderson, Plant Physlol, 61 (1978) 221 15 C Beauchamp and I Fmdovlch, Anal Blochem, 44 (1971) 276 16 S P MukheDee and M A Choudhun, Physlol Plant, 58 (1983) 166 17 M Y Law, S A Charles and B Halhwell, Blochem J , 210 (1983) 899 18 H K Llchtenthaler and A R Wellburn, Blochem Soc Trans, 11 (1983) 591 19 A D Dodge, Toxin oxygen spemes and herbmlde action, m J Mlyamoto and P C Kearney (Eds), Human Welfare and the Environment, IUPAC Pesticide Chemistry, Pergamon Press. Oxford, 1983, p 59 20 C L Guay and J V Carter, Cryobiology, 21 (1984) 454 21 B D McKersle, W D Beversdorf and P Huch, Can J Bot, 60 (1982) 2686 22 K Tanaka, Y Suda, N Kondo and D Sugahara, Plant Cell Physlol, 26 (1985) 1425 23 D Gmll, H Esterbaur and U Klosch, Environ Pollut, 19 (1979) 187 24 J G Foster and J L Hess, Plant Physlol, 66 (1980) 482
105
Elsevmr Sclentffic P u b h s h e r s Ireland Ltd
CHLOROPLAST S U P E R O X I D E A N D HYDROGEN P E R O X I D E S C A V E N G I N G S Y S T E M S FROM P E A L E A V E S : S E A S O N A L V A R I A T I O N S
DAVID J GILLHAM and ALAN D DODGE*
School of Bmlog~cal Sciences, Unwers~ty of Bath, Bath, Avon, BA2 7A Y (U K ) (Received October 8th, 1986) (Rewsmn received J a n u a r y 23rd, 1987) (Accepted J a n u a r y 30th, 1987)
Levels of chloroplast antmxldants and enzymes t h a t scavenge oxygen radicals were followed in the leaves of pea plants
(P~sum satwum L cv Meteor) grown under glasshouse condltmns between Apml 1984 and May 1985 While h t t l e vamatlon m pigment levels or superoxlde dlsmutase activity was detected during this pemod, plants grown in early summer ( M a y June) contained appreciably higher levels of ascorbate, ascorbate peroxldase and glutathlone reductase t h a n plants grown in winter ( D e c - J a n ) The role of h g h t intensity in regulating levels of chloroplast antmxldants was examined further using pea plants grown m a constant environment chamber under 100 or 400 #mol m - 2 s ~ photon flux density Chloroplasts isolated from plants grown at the higher h g h t lntenmty contained mgmficantly higher levels of ascorbate, ascorbate peroxldase, glutathlone reductase and dehydroascorbate reductase These data suggest t h a t h g h t intensity may have an important influence on the level and activity of chloroplast antmxldants and oxygen radical scavenger enzymes
Key words ascorbate, ascorbate peroxldase, dehydroascorbate reductase, glutathmne reductase, P~sum satwum, superoxlde dlsmutase
Introduction Chloroplasts of higher plants are protected against damaging oxygen radicals by the enzymes superoxlde dlsmutase (SOD), ascorbate perox~dase, dehydroascorbate reductase and glutathmne reductase. These enzymes efficmntly scavenge both superoxlde (O~-), whmh may be formed as a consequence of the reductmn of O2 by PSI (the Mehler reactmn), and hydrogen peroxide (H202) formed by the dmmutatmn of O~- In addltmn the chloroplast stroma contmns mHhmolar concentratmns of ascorbate and glutathmne [1,2] Chloroplast membranes also contain carotenoid p~gments t h a t quench smglet oxygen (102), generated ff
*To whom correspondence should be sent Abbreviations H202, hydrogen peroxide, ~Oz, smglet oxygen, O~-, superoxlde, OH', hydroxyl radical, SOD, superoxide dlsmutase
h g h t energy absorbed by chlorophyll is not dmmpated through photosynthesis [3] Failure to scavenge these active oxygen specms can result m damage to chloroplasts H202 is strongly inhibitory towards CO2 fixation [4], while both 102 and hydroxyl radmals (OH'), formed from interaction between O~- and H202, can reduce hpld peroxldatlon and oxldatmn of protein amino acids and nucleic acids
[11 Previous studies have demonstrated that much of the leaf SOD, [5] ascorbate peroxldase and glutathlone reductase [2] is chloroplasUc Modulatmn of the act~wty of these enzymes may be ~mportant m plant resistance to envlronmental stress or air pollutants [6-9] In this laboratory we have been interested m the role of these antloxldant mechamsms m protecting pea leaves against photo-oxidative damage [2,10,11]. As a consequence of routine analyses of SOD, ascorbate peroxldase, glutathmne reductase and ascorbate m glasshouse grown pea
0618-9452/87/$03 50 ~, 1987 Elsevmr Scmntffic Pubhshers Ireland Ltd P u b h s h e d and Printed m Ireland
106
leaves, we have observed seasonal changes m their levels, which we now report. Materials and m e t h o d s
Plant mater~al Pea (P~sum s a t w u m L. cv. Meteor) seeds were plante~ in moist L e w n g t o n Umversal Compost (Flsons Ltd, Levington, U K.) and grown for 14-21 days m a glasshouse with a mean air temperature of 20°C and natural dayhght cond~tmns Plants were watered dmly. Pea plants were also grown m a constant envyronment chamber at 20°C under continuous l l l u m m a t m n (400/~mol m - 2 s - 1 photosynthetlcally active r a d m t m n provided by warm white fluorescent tubes, 65/85 W; Thorn, London, U K ) Light intensity m half the chamber was reduced to 100 #tool m - 2 s - 1 using layers of Kodak neutral density filter. Chloroplast preparatwn and enzyme extracts For enzyme assays and ascorbate determlnatron, cell-free homogenates were prepared. Approximately 0 5 g of the first fully expanded leaves below the apex were ground m 10ml of cold 50 mM potassmm phosphate buffer (pH 7.6), with a glass homogemzer. The homogenate was strained through four layers of m u s h n and centrifuged at 4000 × g for 5 mm. The resulting supernatant fractmn was used to estimate enzyme and ascorbate levels Isolated intact chloroplasts were prepared from 25-g samples of pea leaves according to the method of Walker [12] as described previously [2] Enzyme assays Activities of ascorbate peroxldase, glut a t h m n e reductase (EC 1.6.4.2) and dehydroascorbate reductase (EC 1.85.1) were determined m ahquots of the enzyme extract or chloroplast preparatmn according to the methods of Nakano and Asada [13] and Jablonskl and Anderson [14], as described prewously [2], SOD (EC 1.15 1.1) was determined by its inhlbitmn of formazan productmn from mtro-blue tetrazohum based on the method descmbed by
Beauchamp and Frldovich [15]. The 3-ml reaction mixture contained an aliquot of leaf homogenate or chloroplast, 22 mM potassmm phosphate (pH 7 8), 0 1 mM EDTA, 25 #M nitro blue tetrazohum and 0.1 mM xanthme. The reactmn was imtiated by additmn of x a n t h m e oxldase (Sigma) representing 60 #g of protein.
Determ~natmns Ascorbate was determined according to the method of Mukherjee and C h o u d h u n [16] as descmbed previously [2]. Glutathlone was assayed by the method of Law et al. [17] as descmbed previously [2]. Pigments were extracted from leaf samples w~th ethanol. Chlorophyll and carotenoid m the ethanol extract were quantified using the extraction coefficients devised by Lmhtenthaler and Wellburn [18]. Rephcatmn and statistics All results presented m this paper are the means of at least three replicate samples prepared from three independent batches of leaf material sampled at the same time. Leaf samples were routinely taken from plants at the same time each day throughout the samphng pemod. The S E M was less than 10% of the mean m each experiment. R e s u l t s and d i s c u s s i o n Ascorbate peroxldase, glutathlone reductase and ascorbate levels all showed a marked peak m early summer (May~lune), but then dechned m winter (Dec.~Jan.) as shown in Fig. 1. Our samphng procedure entailed using the youngest fully expanded leaves to prepare homogenates for analysis, and therefore activity changes were not due to the leaf samples being of different age SOD activity (Fig. 1) and chlorophyll and carotenoid levels (Fig. 2) showed no marked change between summer and winter. One environmental factor hkely to influence levels of antloxldant and antioxidant enzymes was light. Growth m early summer corresponded to the highest total lrradlance
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L e v e l s of a s c o r b a t e p e r o x l d a s e (a). a s c o r b a t e (b), g l u t a t h m n e r e d u c t a s e (c) a n d SOD (d) m p e a l e a v e s g r o w n u n d e r g l a s s h o u s e c o n d i t i o n s a n d s a m p l e d e a c h m o n t h b e t w e e n A p r i l 1984 a n d M a y 1985 E a c h d a t a p o i n t r e p r e s e n t s t h e m e a n of t h r e e s e p a r a t e d e t e r m m a t m n s m a d e from s e p a r a t e b a t c h e s of p l a n t m a t e r i a l o b t a i n e d on t h e s a m e s a m p l e d a t e The s t a n d a r d e r r o r of t h e m e a n w a s less t h a n 10% of t h e m e a n m e a c h c a s e
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F i g 2. L e v e l s of c h l o r o p h y l l (a) a n d c a r o t e n o l d s (b) m pea l e a v e s g r o w n u n d e r g l a s s h o u s e c o n d i t i o n s b e t w e e n Apml 1984 a n d M a y 1985 E a c h d a t a p o i n t r e p r e s e n t s t h e m e a n of t h r e e s e p a r a t e d e t e r m i n a t i o n s
for each month of the samphng pemod falhng at Long Ashton Research Station, 25 KM WNW of Bath U m versl t y (Fig. 3) By contrast growth in winter matched the period of lowest lrradlance To examine the effects of h g h t intensity on the a c n w t y of chloroplast antloxldants and antmxldant enzymes, pea plants were grown m a constant environment chamber for 14-21 days under d l u m m a t l o n of either 400 or 100 t~mol m 2 s 1 photon flux density Intact chloroplasts were molated, and then analysed for achvltles of chloroplast annoxl dant s and enzymes (Table I) Chloroplasts from plants grown at the higher hght intensity contained enhanced act~vlt~es of ascorbate perox~dase, glutathlone reductase and dehyroascorbate reductase, and higher levels of ascorbate than chloroplasts from plants grown at the lower
108 T a b l e I. Effect of g r o w t h a t 100 or 400 t~mol m - 2 s - 1 p h o t o n flux d e n s i t y on t h e a c t i v i t y of e n z y m e s s c a v e n g i n g 02 a n d HsO 2 a n d t h e levels of a n t m x l d a n t s m i s o l a t e d p e a c h l o r o p l a s t s (80-85% i n t a c t ) A c t l w t l e s g i v e n m u m t s ( m g chl) - 1 for SOD, l~mol (rag chl) - 1 h - i for a s c o r b a t e perox~dase, g l u t a t h m n e r e d u c t a s e a n d d e h y d r o a s c o r b a t e reductase, p g ( m g chl) - ~ for a s c o r b a t e a n d g l u t a t h m n e or m g (g f r e s h wt ) - 1 for c h l o r o p h y l l R e s u l t s a r e t h e m e a n s of at l e a s t five i n d e p e n d e n t e x t r a c t m n s f r o m different b a t c h e s of p l a n t m a t e r m l S E M were less t h a n 10% of t h e m e a n Irradlance ( u m o l m - 2 s - 1 PAR)
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h g h t intensity. No difference m SOD act ]w t y or g l u t a t h m n e levels from the two sets of plants was detected The effect of increased levels of a n t m x l d a n t and oxygen scavenging enzymes on the capac]ty of plants to scavenge oxygen radicals is
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F i g . 3. M e a n daffy i r r a d m n c e f a l h n g a t L o n g A s h t o n Res e a r c h S t a t m n , Bristol for e a c h m o n t h b e t w e e n April 1984 a n d M a y 1985
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F i g . 4. P a r a q u a t m d u c e d c h l o r o p h y l l b l e a c h z n g m p e a leaf dmcs c u t from p l a n t s g r o w n m a c o n s t a n t e n v i r o n m e n t c h a m b e r at 100 ( • [~) or 400 (@ O) #tool m - ~ s - 1 p h o t o n flux d e n s i t y Discs were i n c u b a t e d m g l a s s p e t n d i s h e s cont a m i n g HsO ( • • ) or 10 p M p a r a q u a t ( [] O)
shown m Fig. 4. Leaf discs were excised from plants grown at 100 or 400 pmol m - 2 s - 1 photon flux density and chlorophyll ret ent i on was tested in the presence of paraquat (10 pM), a chemical known to promote O~- generation [19]. Plants grown at the lower m t e n m t y and, therefore, with lower antloxldant and oxygen radmal scavenging enzyme activltms, showed a greater rate of paraquat-mduced chlorophyll bleaching than leaf d~scs cut from plants grown at the higher light intensity. This difference was probably not due to the rate of herbicide uptake, because CO 2 fixation was mh~blted by paraquat m both sets of plant material after a similar time period (data not shown) This result indicates t hat the increased protect]on against damaging oxygen radicals m plants grown at the higher hght mtens]ty may have been caused by enhanced chloroplast levels of ascorbate, ascorbate perox]dase, glutath]one reductase and dehydroascorbate reductase.
109 The results presented in this paper indicate t h a t h g h t ls a n i m p o r t a n t f a c t o r m t h e r e g u l a t i o n of c h l o r o p l a s t h y d r o g e n peroxide scavenging systems from pea leaves While fluctuatmns m h g h t i n t e n s i t y m a y h a v e c o n t r i b u t e d to t h e c h a n g e s m a c t i v i t y o f a s c o r b a t e , a s c o r b a t e peroxldase and glutathmne reductase m pea l e a v e s g r o w n m s u m m e r a n d w i n t e r , ~t is possible that annual fluctuatmns in temperature, w a t e r s t a t u s o r p h o t o p e m o d o r f l u x e s of a i r pollutants may also exert some control Reports m the h t e r a t u r e i n d i c a t e t h a t low t e m p e r a t u r e [8,20], d r o u g h t [9], o z o n e [21,22], SO2 f u m l g a t m n [6,23], o r a l t e r a t i o n s m o x y g e n t e n s m n [24], c a n a f f e c t t h e a s c o r b a t e , g l u t a t h m n e , glutathlone reductase, ascorbate peroxldase and SOD levels m leaves These reports, a n d the r e s u l t s i n t h i s s t u d y m d m a t e t h a t a w~de vame t y of e n v i r o n m e n t a l f a c t o r s c a n m o d u l a t e t h e a c t i v i t y of c h l o r o p l a s t O~- a n d H 2 0 2 s c a v e n g i n g s y s t e m s I n w e w of t h e i n c r e a s i n g i n t e r e s t m these protective mechamsms m the defence of p l a n t s a g a i n s t s t r e s s , i n v e s t i g a t o r s s h o u l d b e a w a r e of t h e w i d e v a m e t y of f a c t o r s t h a t c o u l d affect enzyme a n d a n t m x l d a n t levels
Acknowledgements The a u t h o r s are grateful to M a r t i n H u x l e y of L o n g A s h t o n R e s e a r c h Station, Bristol, U K for s u p p l y i n g t h e l r r a d l a n c e d a t a T h i n w o r k w a s s u p p o r t e d b y a U m v e r s l t y of B a t h r e s e a r c h s t u d e n t s h l p to D . J G
References 1 B Halhwell, Chloroplast Metabohsm The Structure and Function of Chloroplasts m Green Leaf Cells, Clarendon Press, Oxford, 1981, p 179
2 D J Glllham and A D Dodge, Planta, 167 (1986) 246 3 C S Foote, Scmnce, 162 (1986) 963 4 J M Robinson, M G Smith and M Gibbs, Plant Physlol, 65 (1980) 755 5 C Jackson, J Dench, AL Moore, B Halhwell, C Foyer and D O Hall, Eur J Blochem, 91 (1978) 339 6 K Tanaka and K Sugahara, Plant Cell Physlol, 21 (1980) 601 7 E H Lee and J H Bennett, Plant Physlol, 69 (1982) 1449 8 L J de Kok and F A Oosterhms, Physlol Plant, 58 (1983) 47 9 P E Gamble and J J Burke, Plant Physlol, 76 (1984) 615 10 D J Glllham and A D Dodge, Plant Physlol, 75 (1984) P 51 11 D J Glllham and A D Dodge, Physlol Plant, 65 (1985) 393 12 D A Walker, Meth Enzymol, 69 (1980) 94 13 Y Nakano and D Asada, Plant Cell Physlol, 22 (1981) 867 14 P P Jablonskl and J W Anderson, Plant Physlol, 61 (1978) 221 15 C Beauchamp and I Fmdovlch, Anal Blochem, 44 (1971) 276 16 S P MukheDee and M A Choudhun, Physlol Plant, 58 (1983) 166 17 M Y Law, S A Charles and B Halhwell, Blochem J , 210 (1983) 899 18 H K Llchtenthaler and A R Wellburn, Blochem Soc Trans, 11 (1983) 591 19 A D Dodge, Toxin oxygen spemes and herbmlde action, m J Mlyamoto and P C Kearney (Eds), Human Welfare and the Environment, IUPAC Pesticide Chemistry, Pergamon Press. Oxford, 1983, p 59 20 C L Guay and J V Carter, Cryobiology, 21 (1984) 454 21 B D McKersle, W D Beversdorf and P Huch, Can J Bot, 60 (1982) 2686 22 K Tanaka, Y Suda, N Kondo and D Sugahara, Plant Cell Physlol, 26 (1985) 1425 23 D Gmll, H Esterbaur and U Klosch, Environ Pollut, 19 (1979) 187 24 J G Foster and J L Hess, Plant Physlol, 66 (1980) 482