Responses of antioxidative enzymes to elevated CO2 in leaves of ...

Plant, Cett and Environment (1997) 20, 13^7-^32•i

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Responses of antioxidative enzymes to elevated CO2 in leaves of beech {Fagus sylvatica L.) seedlings grown under a range of nutrient regimes A. POLLE,'* M. EIBLMEIER,^ L. S H E P P A R D ' & M. MURRAY^ 'Georg-Augu.st Universitat, Gottingen, Eorsthotanisches Institut, BUsgenweg 2, D-37077 Gottingen, ~A1hert-Ludwigs-Universitat Freiburg, lnstittitfiir Eorsthotatiiktmd Bawnphysioiogie, Am Elughafen 17, D-79085 Ereiburg, Gerniatiy, atid ^Institute of Terrestrial Ecology, Edinburgh Research Station, Bush Estate, Penicuik, Midlothian EH26 OQB, UK

ABSTRACT To study whether responses of antioxidative enzymes to enhanced atmospheric CO2 concentrations are affected by plant nutrition, the activities of superoxide dismutase, catalase and peroxidase were investigated in leaves of 3year-old beech trees grown with low (O-l x optimum), intermediate (0-5 x optimum) and high (2 x optimum) nutrient supply rates in open-top chambers at either ambient (=355;t;niol mol"') or elevated (700;UnioI moP') CO2 concentrations. These treatments resulted in foliar C/N ratios of about 20 in the presenee of bigli and > 30 in the presence of low nutrient supply rates. Pigment and malondialdehyde contents were determined to assess plant stress levels. Low nutrient supply rates caused pigment loss, whereas elevated CO2 bad no effect on pigmentation. Guaiacol peroxidase activities did not respond to eitber CO2 or nutrient treatment. Catalase activity decreased witb decreasing nutrient supply rate and also in response to elevated CO2. Superoxidase dismutase activity was affected by botb nutrient supply and CO2 concentration. In leaves lrom trees grown with the high-nutrient treatment, superoxide dismutase activity was low irrespective of CO2 concentration. In chlorotic leaves, superoxide dismutase activity was increased, suggesting an enhanced need for detoxification of reactive oxygen species. Leaves from plants grown under elevated CO2 witb medium nutrient supply rates showed decreased malondialdehyde contents and superoxide dismutase activities. Tbis suggests tbat tbe intrinsic oxidative stress of leaves was decreased under tbese conditions. Tbese results imply tbat intrinsic oxidative stress is modulated by tbe balance between N and C assimilation. Key-words: beech; catala.se; nutrition; oxidative stress; peroxidase; superoxide dismutase.

seedlings (Bazzaz 1990). Under elevated atmospheric COj concentrations, growth of C3 species will depend on the availability of nitrogen, because the metabolism of carbon and nitrogen are tightly linked and regulated in parallel (Foyer et al. 1995). If the supply of nitrogen or other essential elements does not meet tlie potentially enhanced requirements of the plant under elevated CO2, then the nutt-ient status may decrease, resulting in photosynthetic limitations and hence growth retardation (Petterson & McDonald 1994). Several lines of evidence suggest that severe nutrient imbalances may result in an increased production of potentially toxic oxy-products such as H2O2, O2*~ and -OH (Marsehner & Cakmak 1989; Caktnak & Marsehner 1992; Polle etaL 1992; Cakmak 1994). Reactive oxygen species at-e also produced during normal metabolism, e.g. O2-~ during photosynthesis, and H2O2 during respiration and photorespiration. To prevent accumulation of toxic oxyproducts, plants contain constituively protective enzymes such as superoxide dismutases, catalases and peroxidases. Superoxide dismutases scavenge 02-~ in chlotoplasts and other subcellular compartments (Bowler et al. 1992). Catalases degrade H2O2 to H2O and O2 in peroxisomes and mitochondria (Scandalios 1994). Peroxidases use ascorbate or phenolic compounds to remove H2O2 in various subcellular compartments (Castillo 1992). Plants suffering ixom incr-eased oxidative stress generally respond with increases in antioxidative systems, although this response appears not always to be sufficient to prevent injury. For example, in Mg-deficient leaves high light resulted in significant incteases in activities of antioxidative enzymes but also in severe chlorosis (Cakmak & Marsehner 1992). In Mg-deficient spruce needles, the tnagnitude of such an antioxidative stress response was modified by the nitrogen nutrition (Polle et al. 1994).

Leaves fr"oin various plant species grown under elevated CO2 concetitrations contained lower activities of superAtmospheric CO2 and soil nitrogen content are important oxide distnutase or catalase than foliage frotn plants grown factors controlling the growth and performance of tree under ambient CO2 concentrations (tobacco; Havir & McHale 1989; spruce; Polle et al. 1993; oak and pine; * Correspondence: A. Polte. Eax: +49 551 392705; e-mait: apotte Schwanz et al. 1996a). These observations suggest that plants grown under elevated CO2 are exposed to decreased @gwdg.de © 1997 Blackwell Science Ltd

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1318 A Po/teetal. leaf powder) as described previously (Polle et al. 1990). The extracts were passed through Sephadex G-25 (PD-10 colurrrns, Pharmacia, Freiburg) before further analyses. The soluble protein content of the purified extracts was determined with bicinchoninic acid reagent (Pierce, Miinchen, Germany) and bovine serum albumin as standard. The enzymatic activities were determined in standard assay systems according to the following methods: superoxide dismutase (EC 1.15.1.1, McCord & Fridovich 1969), guaiacol peroxidase (EC 1.11.1.7, Polle et al. 1990), and catalase (EC 1.11.1.6, Aebi 1983). Malondialdehyde was determined in crude extracts with thiobarbituric acid (Peever & Higgins 1989). Chlorophyll and carotenoids were extracted in 80% acetone and r-neasured spectr-ophotometrically at 470, 646 and 663 nrrr. The pigment concentrations were calculated using the extir-rction coefficients given by Lichtenthaler & Wellburn (1983). Dry weight was determined after drying foliage for 72 h at 60 °C. Dry foliage was used for nutrient analysis as decribed previously (Mur-ray, Leith & Friend 1995). Statistical analysis was performed with the software STATGRAPHICs (STN, St. Louis, USA) using multiple analysis of variance followed by a multiple range test (Duncan test) to evalute significant treatr-nent effects. The analytical er-ror of individual sarTrple.s representing mean values of eight trees per char-nber was generally below 5%, Data in the table and flgure indicate means of independent chamber replicates (n = 4, ± SD). Growth of beech seedlings at an enhanced CO2 concentration over two growing seasons resulted in significant decreases in foliar N as corrrpared with those grown under ambient CO2 concentrations (Table 1). High nutrient supply rates resulted in increased foliar nitrogen and also affected the C content (Table 1). It is important to note that the C/N ratios were always higher under enhanced as cor-npar-ed with ambient CO2 concentrations and increased from about 20 in leaves of trees grown with a high nutrient supply rate to ratios > 30 in trees grown with a low nutrient supply rate (cf. Table 1).

intrinsic oxidative stress as compared with plants grown under ambient CO2. However, in one study conducted with young spruce trees grown with a superabundant N supply these C02-mediated changes in the activities of protective enzymes were not observed (Schwanz et al. 1996b). Therefore, a link between plant nutrient status and the response of antioxidative enzymes to elevated CO2 concentrations may exist (Schwanz et al. 1996b). The aim of the present study was to address the effect of elevated CO2 on the activities of superoxide dismutase, catalase and guaiacol peroxidase in leaves of beech trees grown under a range of nutrient regimes. Maloridialdehyde, an indicator for lipid peroxidation, and pigment and soluble protein contents were also determined to assess oxidative stress. Three-year-old beech seedlings [Eagus sylvatica, no. 9I(439)F, provenance Hungary] were placed in open-top chambers at the Institute of Terrestrial Ecology, Edinburgh, UK during March 1994 (for a detailed description of these facilities, see Fowler et al. 1989). Four chambers received arrrbient air (=355 /imol moP' CO2) and four received air with elevated CO2 concentration (700 ± 80 /imol mol"' CO2) by direct addition of pure CO2 to the air stream as decribed by Murray et al. (1994). Each char-nber contained three groups of eight plants. Each group had one of three Ingestad nutrient solutions (high = 2 X optimum, medium = 0-5 x optimum, and low = 0-1 x optimum) applied on a weekly basis (Ingestad & Lund 1986). Optimum was taken to be the amount of nitrogen solution required to produce 2-7% nitrogen in the foliage for a given growth rate under optimum uptake rates (cf. Table 1). In August 1994, one leaf per tree was harvested from each of the eight chambers. Leaves collected within each nutrient level were pooled within each char-nber, immediately frozen in liquid nitrogen, shipped on dry ice and stored at -80 °C for analyses. Enzyme extracts were prepared in 20 cm~-^ of an extraction buffer (100 mol m^^ KH2PO4/K2HPO4, pH 7-8, 0-5% Triton X-100, 800 mg polyvinylpyrrolidone and 400 mg of

CO,

Nutrient

C

N

Protein

Chi a+b

Car

Chi a+h 1 Car

ambient ambient ambient elevated elevated elevated LSD

high medium low high medium

All 480 472 492 477 472 14

26.5 20.5 15.2 24.5 17.5 12.7

3.74 2.97 1.67 4.53 2.38

2.9

86.7 88.2 75.7 72.0 70.1 73.9 15.2

0.95

1.01 0.89 0.63 0.87 0.73 0.60 0.28

3.67 3.29 2.70 5.23 3.21 3.02 0.45

0.094 0.033 0.113

0.032 0.001 0.575

0.035 0.657 0.344

0.676 0.001 0.130

0.172 0.009 0.742

0.001 0.001 0.001

low

L83

Table 1. Carbon, nitrogen, pigrrrent and soluble protein contents of beech leaves frorrr seedlings exposed to ambient or elevated CO, at high, medium or low nutrient supply rates

Significance ^(CO2)

Interaction

CO2XN

The treatments began in March 1994. Leaves for nutrient element analyses were eolleeted in the second and for biocherTrical analyses in the third week of August 1994. Figures indicate means (mg g~'of dry weight) of four independent chamber replicates. Chi a+fc= , , Chlorophyll a and b. Car = carotenoids, LSD = least significant difference. © 1997 Blackwell Science Ltd, P/am, Cell and Environment, 20, t3t7-1321

Antioxidative enzymes and nutrition in beech

The soluble ptotein content was not significantly affected in beech seedlings grown in atnbient COT atid supplied with high, tnediutTi or low tiutrient solutions (Table 1), despite mean foliar nitrogeti contents of between 2-65 and 1-52% of dry tnatter across the three treatments (Table 1). In plants grown under elevated CO2, the soluble protein content was slightly lower than in those grown under atnbient CO2 concenttations and obviously also independent of the nutrient supply rate (Table 1). In plants gtown under elevated CO2 concentrations decreased foliar protein contetits have frequently been reported atid were generally associated with a reduction in the atnount Rubi.sco protein (Webber et al. 1994). Analysis of gtowth paratneters of the beech seedlings investigated in the present study shows that increased nitrogen supplied in the high nutrient treattnent was used for growth, resulting in apptoximately 4 titnes higher plant biotnass under high as compared with low nutrient supply rates (Murray et al. 1995), and was not stored in the fortn of ptotein within the foliage (Table 1). Enhanced CO, had no significant effects on the total chlorophyll or carotenoid contents of beech leaves, whereas a restricted nutrient supply caused significant decreases in pigment contents (Table I). The teduction in chlorophyll was larger than the reduction in carotenoids, resulting in lower chlorophyll-to-carotenoid ratios in nutrient-limited than in well-supplied platits, i.e. in chlototic leaves the atnount of catotenoids per chlotophyll was increased (Table 1). Carotenoids play an itnpottant role in protection of the photosynthetic appatatus ftotn excess light energy (Demtnig-Adatns & Adatns 1994). The relative inctease in catotenoids suggests that nutrientlimited plants might have an enhanced need for protectioti frotn light-induced stress. On the other hand, the highest chlotophyll-to-cattenoid ratios wete found in leaves frotn plants grown with high nutrient supply t ates under elevated CO2 concentrations (Table 1). This obsetvation tnight suggest that these plant convert intercepted light tnore efficiently into chetnical ctiergy and, thus, have less need for enetgy dissipation than plants grown under ambient CO2 concentrations. To corroborate the hypothesis that intrinsic sttess levels were tnodulated by the C and N nutrition of plants, we investigated the activities of catalase, superoxide distnutase and peroxidase as potential tnarkets for oxidative stress, across a range of nuttient tegitnes under atnbient and elevated atmospheric CO2 concentrations. Catalase activity was significantly lower in leaves frotn beech seedlings grown at elevated CO2 than in plants grown at ambient CO2, itrespective of the nutrient supply rate (Fig. la), A reduction in catalase in response to elevated CO2 has also been observed in other plant species (tobacco: Havir & McHale 1989; spruce: Polle etal. 1993). Plants grown under elevated CO2 show lower rates of photorespitation and respiration (Shatkey 1988; Bowes 1993; Wullsehleger et al. 1994). Since H2O2 is a product of these pathways, we suggested that the necessity for detoxification of H2O2 tnight be ditninished in plants grown ® 1997 Blackwell Scienee Ltd, P/o/U, Cell and Environment, 20, 1317-1321

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under elevated atmospheric CO2 concentrations (Polle et al. 1993; Polle 1996). In fact, a correlation between redueed rates of photorespiration and reduced catalase activities in plants grown at enhanced CO2 concentrations has been observed (Thibaud et al. 1995). The present results furthermore show that catalase activity was decreased in chlorotic leaves obtained frotn plants grown with a low nutrient supply rate (Fig. 1 a). Regression analysis revealed a litiear conelation between foliar N content and catalase 1200

(a)

P(co,)= 00002, P(N, 5 0.0001

900 -

600 .

300 •

) = 0 0199. P(N) =00055 %

2000

•g 1500

¥ 'p 1000 'a H 500 CO

0 400

I

,-

(C)

P(co,)=0 0795, P,^) =0.5644

300

100

Low

Medium

High

Nutrient supply Figure 1, (a) Catalase and (b) superoxide di.smutase activities and (c) maloiidialdehyde content in leaves from beech seedling.s exposed to ambient (black bars) or elevated (grey bars) COo at a high, medium or low nutrient supply rate. The treatments started in March 1994. Leaves for biochemical analyses were colleeted in the third week of August 1994. Figures indicate means of four independent chamber replieates (± SD). Each sample colleeted in one chamber consisted of leaves from eight individual trees, as explained in 'Materials and methods'. Data are expressed on the basis of dry matter. Different letters in bars indicate significant effects at P < 0-05.

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/\. Po//e et al.

activity at R = 0-942 and P = 0-0049. The reasons for this tight conelation are currently unknown, and such a correlation was not found for the other enzymes analysed, namely superoxide dismutase and guaiacol peroxidase. We can only speculate that high nutrient supply rates, which result in enhanced rates of photosynthesis (Petterson & McDonald 1994), might also stimulate processes such as photorespiration and, thereby, require increased detoxification of H2O2 by catalases. In C4 plants, a link between lower investment of N in Rubisco, higher photosynthetic rates and reduced photorespiration has been established (Brown & Bouton 1993). However-, because catalase is a protein with a high turn-over in the light (Feierabend & Engel 1986), the possibility cannot be excluded that low nutrient availability might sustain only low catalase production rates, resulting in decreased catalase activities in nutrient-limited plants. In contrast to catalase activity, the activity of superoxide dismuta.se was significantly increased in beech leaves from the low nutrient treatment (Fig. lb). This suggests that chlorotic leaves were exposed to elevated oxidative str-ess arising from superoxide radicals. Since the dismutation of 02-~ yields H2O2, it is conceivable that the chlorotic leaves must also cope with an increased production of H2O2. However, peroxidase activities, determined with guaiacol as substrate, did not respond to either treatment and had a mean value of 2-69 ±0-48 fikal g~' dry matter (« = 24). Furthermore, the present data do not provide any evidence that potentially enhanced production rates of H2O2 were compensated by corresponding increases in catalase activity (Fig. la). However, the protection from oxidants seemed to be sufficient since the content of malondialdehyde, which indicates lipid peroxidation, was not increased in leaves suffering from nutrient limitations (Fig. lc). It is feasible that an enhanced production of H2O2 in chloroplasts was counterbalanced by ascorbate peroxidase activity. Since ascorbate peroxidase activity is freezing-sensitive in beech leaves it could not be analysed using the techniques employed here. In plants grown under the high nutrient supply rate, superoxide dimutase activity was low and not affected by CO2 concentration (Fig. lb). In plants grown with low or medium nutrient supply, elevated CO2 caused a reduction in the activity of superoxide dismutase in comparison with that found in leaves of plants grown at the ambient CO2 level (Fig. lb). We have previously suggested that the activity of superoxide dismutase might respond to the internal utilization of photosynthetically produced reductant (Polle et al. 1993; Polle 1996). The intrinsic oxidative stress would decrease if the demand for NADPH was increased and fewer electrons were available for reduction of O2 under elevated CO2 (Polle el al. 1993; Polle 1996). The present data support this hypothesis; reductions in superoxide dismutase activity were observed (Fig. Ib), and a trend was found towards lower malondialdehyde contents in leaves from plants grown at elevated as compared to tho.se grown at at-nbient CO2 concentrations (Fig. 1 c). In summary, the present study shows that low nutrient

availability results in an enhancement of the ratio of carotenoids to chlorophyll and in an increase in supetoxide dismutase activity. This implies an increase in oxidative stress in leaves from nutrient-limited plants. However, the cor-npensation of this stress appeared to be sufficient to prevent damage to membranes because no evidence for increased lipid peroxidation was found. The decrease in superoxide dismutase under elevated CO2 and the variation of this re.sponse as a result of high or low nutrient supply rates suggest that the intrinsic oxidative stress increases when N and/or C assimilation is limited. The consequences of these results for stress tolerance and survival of plants in a future high-CO2 world ate currently unknown and need to be addressed in further experitnents. ACKNOWLEDGMENTS We are grateful to Dr H. Rennenberg (University Freiburg, Germany) for his help during the collection of the samples and critical reading of the manuscript. Financial suppor-t by the UK DoE (contract EPG1/1/3), the European Union (ECOCRAFT EV5V-CT92-0127) and a travel grant to A.P. under COST 614 is gratefully acknowledged. REFERENCES Aehi H. (1983) Catalase. In Methods of Enzymatic Analysis, Vol. 3 (ed. H. Ber-grrreyer-), pp. 273-77. Ver-|ag Chemie, Weiriheirn. Bazzaz F. A. (1990) The response of natural ecosystems to the rising global CO2 levels. Annual Reviews of Ecotogy and Systeniatics 21, 167-196. Bowes G. (1993) Facing the inevitahle: plants and incr-easing atmospheric CO2. Annuat Reviews of Plant Physiotogy and Plain Motecutar liiotogy 44, 309-332. Bowler- C , Van Montagu M. & hrze D. (1992) Superoxide disriiu.tase and stress tolerance. Annuat Reviews of Plant Physiotogy and Ptant Motecutar Biotogy 43, 83-116. Br-own R.H. & Bouton J.H. (1993) Physiology and genetics of interspecific hybrids between photosynthetic types. Annual Reviews of Plant Physiotogy and Ptant Molecular Biology 44, 43.5^56. Cakmak I. (1994) Activity of ascor-bate-dependent H2O2-scavenging enzymes and leaf chlorosis are enhanced in magnesium and in potassium-deficient leaves, but not in phosphor-us-deticient leaves. Journat of Experimenlat Botany 45, 1259-1266. Caktnak 1. & Mar-schner H. (1992) Magnesium defrciency and high light intensity enhance activities of superoxide dismutase, a.scorbate peroxidase, and glutathione reductase in bean leaves. Plant Phy.siology 98, 1222-1227. Castillo F.J. (t992) Per-oxidases and stress. In Planl Peroxidases 1980-90 (eds C. Penel, T. Gaspar & H. Greppin), pp. 187-203. University of Geneva, Geneva. Demmig-Adams B. & Adams W. (1994) Light stress and photoprotection related to the xanthophyll cycle. In Phoiooxidative Stresses in Plants: Causes and Ainetioralion (eds C. Foyer & P. Mullitieaux), pp. 105-126. CRC Press, itic, Boca Raton. Feierabend J. & Engel S. (t986) Photoinactivation of catalase in vitro and in leaves. Archives of Biochemistiy and tiiophysic 251, 567-576. Fowler D., Cape J.N., Deans J.D., Leith I.D., Murray M.B., Smith R.I., Sheppard L.J. & Unsworth M.H. (1989) Effects of acid tnist on the fr-ost hardiness of red spruce seedlings. New Phytologist

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© 1997 Blaekwell Science Ltd, P/a/K, Cell and Emit omnent, 20, 1317-1321