In vitro precultivation of tobacco affects the response of antioxidative enzymes to ex vitro acclimation

J. Plant Physiol. 159. 781 – 789 (2002)  Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp

In vitro precultivation of tobacco affects the response of antioxidative enzymes to ex vitro acclimation Helena Synková, Jana Pospíˇsilová* Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Na Karlovce 1a, CZ-160 00 Praha 6, Czech Republic

Received November 29, 2000 · Accepted February 12, 2002

Summary Both growth under elevated CO2 (CE) conditions and/or treatment with abscisic acid (ABA) during acclimation to ex vitro conditions and precultivation in vitro in tightly closed glass vessels (G-plants) or ventilated Magenta boxes (M-plants) affected activities of antioxidative enzymes of tobacco 28 days after the transfer. Single treatment with 5 µmol/L ABA immediately after transplantation caused an increase in net photosynthetic rate and in the content of chlorophyll a, and decreases in the activities of glutathione reductase, Mn-SOD, peroxidases, malic enzyme (ME), and glucose-6-phosphate dehydrogenase (G-6P-DH) in both types of plantlets. Contrary to this, CE effects were dependent on the plant origin and promoted activities of peroxidase, ME, and G6PDH were observed more in M-plants than in G-plants. Effects of a single ABA treatment lasted throughout the whole acclimation and alleviated transplantation shock more efficiently than CE, irrespective of plant precultivation. However, under combined treatment (CE + ABA) CE effects prevailed over ABA. Key words: abscisic acid – antioxidant enzymes – carbon dioxide – ex vitro – in vitro – Nicotiana tabacum Abbreviations: ABA = abscisic acid. – CA = ambient CO2 concentration. – CAT = catalase. – CE = elevated CO2 concentration. – Chl = chlorophyll. – FM = fresh leaf mass. – G-6P-DH = glucose-6phosphate dehydrogenase. – GPOD = guaiacol peroxidase. – GR = glutathione reductase. – ME = malic enzyme. – p = protein. – PN = net photosynthetic rate. – SPOD = syringaldazine peroxidase. – SOD = superoxide dismutase

Introduction Special conditions that prevail during in vitro cultivation result in the formation of plantlets of abnormal morphology, anatomy, and physiology (e.g., Pospíˇsilová et al. 1997). After ex

* E-mail corresponding author: [email protected]

vitro transplantation, plantlets usually need some time for acclimatization under shade with gradually lowering air humidity (e.g., Bolar et al. 1998) to avoid desiccation and photoinhibition, and to repair the abnormalities. It was shown in previous experiments (e.g., Pospíˇsilová et al. 1998) that the addition of ABA to the substrate immediately after transplantation can alleviate «transplantation shock» of Nicotiana tabacum plants by decreasing stomatal conductance (gs) and transpiration 0176-1617/02/159/07-781 $ 15.00/0

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rate. ABA-treatment had slight positive or insignificant effect on photosynthetic parameters and enhanced plant growth. Another possibility for speeding the ex vitro acclimation process is to grow plants under CO2 enrichment which can promote photosynthesis and ex vitro growth (for review see Buddendorf-Joosten and Woltering 1994). Elevated CO2 concentration (CE) during acclimation of tobacco plants markedly increased net photosynthetic rate (PN) in situ, water use efficiency and growth, and slightly improved Chl a fluorescence kinetic parameters, photochemical activities, and stomatal regulation of gas exchange (Pospíˇsilová et al. 1999). However, CE introduced during ex vitro acclimation more effectively promoted the growth of plants grown in vitro under ambient CO2 concentration (CA) than that of plants grown during both growth phases under CE (Solárová and Pospíˇsilová 1997). The imposition of any condition in which cellular redox homeostasis is disrupted can cause oxidative stress, i.e. a rise of concentration of reactive and toxic oxygen species such as H2O2, superoxide radicals, and singlet oxygen (Alscher et al. 1997). Water stress and photoinhibition, often accompanying the acclimatization of in vitro grown plantlets to ex vitro conditions, are probably the main factors promoting oxidative stress (Van Huylenbroeck et al. 1995). Plants possess defense mechanisms, which can overcome oxygen toxicity and delay the deleterious effects of free radicals (Foyer et al. 1994). Superoxide dismutases (SOD) are believed to play a crucial role in antioxidant defense because they catalyse the dismutation of O2 – in H2O2. The removal of the hydrogen peroxides is catalysed by catalases (CAT) and peroxidases (Scandalios 1993). Based on the assumption that the activities of antioxidant enzymes reflect the need for detoxification of the respective active oxygen species, the increase in activities of glutathione reductase (GR), CAT, ascorbate peroxidase (APOD), and SOD in plants would be expected to indicate elevated formation of those species under particular conditions. Very little information addresses CE and ABA effects on antioxidant systems and results are contradictory. Schwanz et al. (1996) reported on a reduction of activity in SOD, APOD and CAT under CE, while Polle et al. (1997) observed an increase in SOD, which was dependent on plant mineral nutrition. Kurepa et al. (1997) found a 60 % decrease in mRNA for Cu-Zn-SOD and no effect on Fe-SOD after ABA treatment. Nevertheless, increases in SOD and APOD transcripts and activity and a decrease in GR were observed after ABA application by Bueno et al. (1998). Exogenously applied ABA significantly enhanced expression of a particular isozyme of CAT (Guan et al. 2000). The goal of the present study was to investigate whether the alleviation of water stress by a single ABA treatment and enhanced growth under elevated CO2 concentration affected antioxidant enzyme activities in tobacco plantlets during their acclimation to ex vitro conditions. Tobacco plantlets differing in in vitro cultivation were used in the experiment to find out

whether precultivation can also influence the capacity of antioxidant enzymes in those leaves that unfolded after transplantation and developed under ex vitro conditions.

Material and Methods Plant material Tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) plantlets were grown in vitro either in glass vessels tightly closed with aluminium foil (G-plants), or in polycarbonate vessels Magenta GA-7 (Sigma, Deisenhofen, Germany) covered with closures with microporous vents (M-plants; better supplied with CO2). Plants were grown at a 16-h photoperiod, photosynthetic photon flux density (PPFD) of 100 – 120 µmol m – 2 s –1, and day/night temperature of 25/20 ˚C (for detail see Haisel et al. 1999).

ABA and CO2 treatment After six weeks, the plantlets were transplanted into pots with coarse sand saturated with water (control, CA) or 5 µmol/L ABA (to avoid initial water stress). For irrigation of all plants after transplantation, only Hewitt nutrient solution and water were alternated. The plants were grown for 28 d in a naturally lit glasshouse in two polyethylene chambers: in the first chamber, the CO2 concentration was natural (CA – 350 mg m – 3), in the other one, CO2 concentration was increased to 1200 mg m – 3 (CE) during the light period (for detail see Pospíˇsilová et al. 1999). Daily maximum PPFD inside the chambers was less than 800 µmol m – 2 s –1, minimum and maximum air temperatures were 16 and 28 ˚C, respectively, and relative humidity was gradually decreased from 90 to 70 %.

Photosynthetic parameters Chl a fluorescence kinetics was measured on the adaxial surface of attached leaves after a 15-min dark period with the PAM Chlorophyll Fluorometer (Walz, Effeltrich, Germany) at room temperature and CO2 concentration. Measured PPFD was 0.35 µmol m – 2 s –1, actinic PPFD 200 µmol m – 2 s –1, and 700-ms saturating flashes of 2500 µmol m – 2 s –1 were applied at 300 s intervals (for details see Pospíˇsilová et al. 1998). PN was determined as CO2 influx in a PC-assisted closed gas exchange system with an infra-red gas analyser Infralyt IV (Junkalor, Dessau, Germany) in a CO2 concentration range from 100 to 3000 mg m – 3, leaf temperature 20 ˚C, and saturating PPFD of 860 µmol m – 2 s –1. Diffusive conductances of abaxial and adaxial epidermes for water vapour were measured with diffusion porometer Delta-T type Mk3 (Delta-T Devices, Kingston upon Thames, UK) either at the growing conditions in the greenhouse or in laboratory at constant temperature of 25 ˚C, PPFD of 860 µmol m – 2 s –1, and air humidity of 60–70 %. Content of photosynthetic pigments was determined in acetone extracts of leaf discs by HPLC (Spectra-Physics, San Jose, USA) as described in Pospíˇsilová et al. (1999).

Enzyme activities For enzyme activity determinations, leaf tissue (1g) was homogenised in 5 mL of ice cold buffer (0.1mol/L Tris-HCl, pH 7.8) containing 1% of

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polyvinylpyrrolidone, and 1 mmol/L dithiothreitol and centrifuged at 20,000 ×g for 10 min. The supernatant obtained was immediately used for enzyme activity measurements. Frozen samples were later used for soluble protein determination according to Bradford (1976). Glutathione reductase (GR, EC 1.6.4.2) activity was assayed according to Goldberg and Spooner (1984). Catalase activity (CAT, EC 1.11.1.6) was assayed by monitoring oxygen production from H2O2 by Clark-type oxygen electrode as described by del Río et al. (1977). Superoxide dismutase (SOD, EC 1.15.1.1) activities and isozymes patterns were obtained after separation by gradient 7–14 % nondenaturing acrylamide gel electrophoresis. Aliquots of supernatants corresponding to 55 µg of protein per lane were used. SOD isozymes were detected in situ in the gel by nitroblue tetrazolium staining method according to Beauchamp and Fridovich (1971). The different types of SOD were distinguished by sensitivity to inhibition by 2 mmol/L KCN and 5 mmol/L H2O2. Mn-SOD is resistant to KCN and H2O2, Fe-SOD is resistant to KCN but inhibited by H2O2, Cu-Zn-SOD is inhibited by both inhibitors (Fridovich 1986). Stained gels were scanned and activity of SOD was estimated from total intensity of individual bands. The relative SOD activities were expressed as percentages, where 100 % represented the respective values found in control M-plants. Guaiacol peroxidase (GPOD, EC 1.11.1.7) activity was assayed by the method of Amako et al. (1994). Syringaldazine peroxidase (SPOD) was determined according to Imberty et al. (1984). Malic enzyme (ME, EC 1.11.1.40) activity was assayed according to Outlaw and Springer (1984) and glucose-6-phosphate dehydrogenase (G-6P-DH, EC 1.1.1.49) was determined by the method of Deutsch (1984).

Statistical analysis Experiments were repeated 3 times. In each experimental set, young fully developed leaves from six plants were used for enzyme determi-

Table 1. Values of statistical significance (P) from analysis of variance of Fv/Fm, PN [µg(CO2) m – 2 s –1], gs [cm s –1], total Chl content [µg cm – 2], the ratio of Chl a/b, soluble protein content [mg g –1(FM)] and activities of antioxidant enzymes (for GR, GPOD, SPOD, G-6P-DH, and ME in [mUg –1 (protein)], for CAT in [Ug –1 (protein)], for SOD in [%]). The effects of plant precultivation (N = 1) and treatment (N = 3) were examined. ns = not significant. Characteristics

Plant origin

Treatment

Origin × treatment

Fv/Fm PN gs Total Chl a + b Chl a/b Protein content GR GPOD G-6P-DH ME SPOD CAT TOT SOD SOD + KCN SOD + H2O2

ns ns ns 0.0422 0.0012 0.0005 0.0008 0.0178 0.0003 ns ns 0.000 0.000 ns ns

ns 0.000 0.0103 0.000 0.000 ns 0.000 0.000 0.000 0.0004 0.000 0.0002 0.000 0.0001 0.000

ns 0.000 ns ns ns ns 0.0187 0.0206 0.0029 0.0072 0.000 0.000 ns 0.0009 0.048

Figure 1. The stomatal conductances (A), net photosynthetic rate (B), and maximal efficiency of photochemistry of photosystem II (Fv/Fm) (C) in tobacco plants precultivated in vitro in Magenta boxes (M) or in glass vessels (G) 28 days after the transfer to ex vitro. Plants were growing under ambient CO2 (CA), elevated CO2 (CE), and were treated by ABA under CE or CA conditions. The values represent means ± SE. The same letter marked values that are not significantly different at p = 0.05. nation in each treatment. Statistical analysis was done by ANOVA and statistical significance of differences was evaluated by Student’s t-test.

Results Photosynthesis and pigment content 28 d after the transfer of in vitro grown plantlets to ex vitro conditions, minor differences between M- and G-plants were

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Helena Synková, Jana Pospíˇsilová Soluble protein content was significantly higher in G-plants, namely in those grown under CE, compared to M-plants where CO2 concentration and ABA treatment had no effect (Fig. 2 A). No significant differences among plants were found in leaf total Chl content 28 d after transfer from in vitro, except ABA treatment under CA which caused an increase in both G- and M-precultivation, (Fig. 2 B). All treated plants (CE, CA + ABA, CE + ABA) exhibited a higher ratio of Chl a/b (Fig. 2 C) than control plants (CA).

Antioxidant enzyme activities

Figure 2. Protein content calculated per fresh leaf matter [mg g –1 (FM)] (A), total chlorophyll content calculated per unit of leaf area (B), and the ratio of chlorophyll a/b (C) in tobacco plants precultivated in vitro in Magenta boxes (M) or in glass vessels (G) 28 days after the transfer to ex vitro. Plants were growing under ambient CO2 (CA), elevated CO2 (CE), and were treated by ABA under CE or CA conditions. The values represent means ± SE. The same letter marked values that are not significantly different at p = 0.05.

found in their photosynthetic parameters (Figs. 1, 2, Table 1). Total stomatal conductances of both types of plantlets slightly decreased in response to ABA (Fig. 1 A). PN of control (CA) G-plants was higher than that of M-plants after 28 d of acclimatization (Fig. 1 B). ABA treatment promoted PN moderately in M-plants. Maximal photochemical efficiency of PS II (Fv/Fm) was lower in M-plants than in G-plants under CE (Fig. 1C).

Activities of antioxidant enzymes apparently were affected by plant precultivation even four weeks after ex vitro growth. CAT activity significantly increased in M-plants after ABA treatment both under CE and CA, compared with G-plants (Fig. 3 A). GR activities were more than double in all G-plants compared to M-plants under both CA and CE, except those treated by ABA. Promotion of GR activity in M-plants was observed only in CE + ABA (Fig. 3 B). In CA grown G-plants, activities of GPOD and SPOD were two and ten times higher respectively than in M-plants (Fig. 3 C, D). ABA treatment at CA considerably lowered both GPOD and SPOD activities in G-plants, but only GPOD in M-plants. Moreover, M-plants showed significant enhancement in SPOD activity under CE + ABA conditions (Fig. 3 D). Samples taken from M-plants also exhibited more and different peroxidase isozymes than those from G-plants (not shown). Generally, higher activities of SOD were observed in M-plants than in G-plants (Fig. 4 A). The highest total SOD activity was found in ABA treated M-plants grown under CA. In G-plants, growth under CE caused a moderate decrease in total SOD activity both in untreated and ABA treated plants compared to control. SOD activities (inhibited by KCN) were not significantly affected except for G-plants grown under CE, where about 40 % increase was observed (Fig. 4 B). The growth under CE caused an increase of Mn-SOD activity (visible after inhibition by H2O2) in both M and G-plants, while CA + ABA treatment resulted in lowering Mn-SOD activity (Fig. 4 C, Table 1). Significant differences among M- and G-plants and also upon the effect of ABA and CE were found in SOD isozyme patterns (not shown). M-plants usually showed lower numbers of isozymes as compared to G-plants. The most pronounced reduction in the number of isoforms was observed in M-plants treated by ABA under CA. Two enzymes of intermediary metabolism, G-6P-DH (oxidative pentose phosphate cycle), and ME related to Krebs cycle, also were affected significantly by both plant precultivation and the following treatments (Fig. 5 A, B, Table 1). G-6P-DH activity was significantly higher under both CA and CE in G-plants than in M-plants which exhibited very low activity in CA. However, in CE, and particularly in CE + ABA,

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Figure 3. Activities of glutathione reductase (GR) (B), catalase (CAT) (A), guaiacol peroxidase (GPOD) (C), syringaldazine peroxidase (SPOD) (D) calculated per unit of leaf soluble protein in tobacco plants precultivated in vitro in Magenta boxes (M) or in glass vessels (G) 28 days after the transfer to ex vitro. Plants were growing under ambient CO2 (CA), elevated CO2 (CE), and were treated by ABA under CE or CA conditions. The values represent means ± SE. The same letter marked values that are not significantly different at p = 0.05.

considerable increase of activity was observed in M-plants. High G-6P-DH activity in G-plants decreased only after ABA treatment at CA (Fig. 5 A). ME activity was higher in G compared to M-plants under CA. ABA treatment at CA caused considerable decrease in activity in both plant types, while CE increased ME activity only in M-plants. The combination of CE + ABA stimulated ME activity in G-plants (Fig. 5 B).

Discussion Photosynthesis Our previous works showed that photosynthetic parameters of the leaves formed under ex vitro conditions were moderately affected by ABA treatment and elevated concentrations of CO2 within four weeks of acclimation (Pospíˇsilová et al. 1998, 1999, 2000). As in the present paper, in vitro cultivation of plants played a significant role in the course of acclimation, particularly at the beginning of the process (Pospíˇsilová et al. 2000). The dissimilar behaviour of M- and G-plants probably

originates from different conditions during in vitro cultivation, which results in significantly different photosynthetic capacity of both plantlets (Haisel et al. 1999). Compared to G-plants, M-plants always had higher Chl content, higher photochemical activities of photosystems, and higher PN, but lower transpiration rate and stomatal conductance, and lower xanthophyll cycle pigments were observed (Haisel et al. 1999). Contrary to G-plants, M-plants often exhibited higher fractions of closed PSII, representing a PSII population that is likely to be damaged by chronic overexcitation and lower non-photochemical quenching. As Fv/Fm was maintained or increased in M-plants, dissipation of excess energy and the rapid turnover of D1 protein is probably the mechanism responsible for photoprotection in M-plants (Haisel et al. 1999). A better supply of CO2 in M-plants might be a major reason for the findings that the plants are able to maintain higher photosynthetic and metabolic efficiency. However, higher rates of photosynthesis might also mean higher concentrations of produced oxygen and this might mobilize antioxidant defense systems of M-plants in a different way than in G-plants. Moreover, this might somehow predispose their capacity after transfer from in vitro to ex vitro as shown in this paper.

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Helena Synková, Jana Pospíˇsilová mation and they appeared only at certain periods of that process. The rise in SOD, APOD and GR activity under high PPFD correlated with their protective function against photooxidative stress linked to photoinhibition (Van Huylenbroeck et al. 2000), but it was also dependent on the plant species used in the experiment (Van Huylenbroeck et al. 1998). In our experiments, strong photoinhibition has never been observed, even when we transferred plantlets from PPFD ca. 100 µmol m – 2 s –1 (in vitro) to PPFD up to 800 µmol m – 2 s –1 (ex vitro), which are values way in excess of those used by the authors mentioned above. This could mean that tobacco belongs to a plant species that is less sensitive to such a transfer. Prominent increase of CAT activity in M-plants after ABA treatment both under CA and CE could indicate a promotion of photorespiration due to the lowering of stomatal conductance (see Fig. 1 and 3) and the higher need for detoxification. This is in agreement with the results of Guan et al. (2000), who showed that higher concentrations of exogenously applied ABA (10 – 5 or 10 – 4 mol/L) cause an increase in H2O2 con-

Figure 4. Relative activities of superoxide dismutase (SOD), before (TOTAL) and after inhibition by KCN or H2O2 in tobacco plants precultivated in vitro in Magenta boxes (M) or in glass vessels (G) 28 days after the transfer to ex vitro. Plants were growing under ambient CO2 (CA), elevated CO2 (CE), and were treated by ABA under CE or CA conditions. 100 % corresponded to intensity of all bands present in control M-plants. The values represent means ± SE. The same letter marked values that are not significantly different at p = 0.05.

Antioxidant systems A progressive activation of antioxidant enzymatic systems during ex vitro acclimation has been observed by Van Huylenbroeck et al. (1998, 2000). They found a significant increase in CAT, moderate increases in APOD, GR and GPOD, a decrease in DHAR and little or no change in SOD activities. However, the changes were dependent on PPFD during accli-

Figure 5. Activity of malic enzyme (ME) (B), and glucose-6-phosphate dehydrogenase (G-6P-DH) (A) expressed in [U mg –1 (protein)] in tobacco plants precultivated in vitro in Magenta boxes (M) or in glass vessels (G) 28 days after the transfer to ex vitro. Plants were growing under ambient CO2 (CA), elevated CO2 (CE), and were treated by ABA under CE or CA conditions. The values represent means ± SE. The same letter marked values that are not significantly different at p = 0.05.

In vitro precultivation affects antioxidative enzymes tent and accumulation of one of the CAT isozymes (Cat1) in young maize leaves. It is not clear why this was not also happening in G-plants, where a similar decrease of stomatal conductance occurred. GR, together with APOD and monodehydroascorbate reductase, are involved in the cycle for regeneration of glutathione and ascorbate that are important in detoxification of oxy-radicals (Alscher et al. 1997). Relative resistance to various stresses was often correlated with an increase in GR activity (Foyer et al. 1994). Contrasting GR performance in Gand M-plants might reflect the differences between both types, which arose during in vitro cultivation, and the importance of different roles of this enzyme in plant metabolism. Treatment by ABA caused a decrease in GR activity in tobacco cell culture (Bueno et al. 1998). The authors explained this effect by a small quantity of available NADPH, which could be related to a decrease in the activity of the pentosephosphate pathways generating most of the reduced NADP. This also correlates with our results, where GR activity (Fig. 3) showed a very similar course of activity in G-6P-DH (Fig. 5), the main enzyme of the pentose-phosphate cycle. Peroxidases, which belong to a large family of enzymes able to oxidize various substrates in the presence of H2O2, are involved in several physiological processes including auxin metabolism, lignification, disease resistance and regulation of growth and cell expansion (Van Huystee 1987). However, peroxidases also are considered the most reliable indicators of plant senescence and stress (Tadeo and Primo-Millo 1990). Therefore, the reason for changes in peroxidase activity could be seen from different points of view, although none of them can be proved at this stage of our research. As peroxidases are also specifically expressed during protoplast regeneration and cell development (de Marco and RoubelakisAngelakis 1996) and play a key role in cell wall assembly and in the control of cell wall plasticity during cell elongation (Hoson et al. 1995), the elevated activity in G-plants could be an indication of an intense process of hardening the cell walls, which is more prominent or longer lasting in this type of plantlet. Van Huylenbroeck et al. (2000) offered a similar explanation for the increase in GPOD activity in Calathea. According to Lee and Lin (1996), ABA treatment caused an increase of GPOD and SPOD activity, which is sometimes contradictory to our observations. Decreasing peroxidase activities at CA in plants of both origins were found after ABA treatment. This could mean that stressful conditions after transfer to ex vitro were alleviated by the addition of ABA or that the process of cell wall lignification passed faster and earlier in acclimation due to ABA treatment. However, this hypothesis needs further investigation. SOD activities are often, but not always, related to resistance to oxidative stress (Alscher et al. 1997). Different SOD genes are differentialy regulated not only by stress factors, but also during plant development (Casano et al. 1994, Kurepa et al. 1997). ABA has been reported to promote total SOD activity and expression of Mn-SOD genes (Bueno et al.

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1998), while CuZn-SOD activity decreased (Kurepa et al. 1997). High Mn-SOD activity is often connected with improvement of tolerance to water stress (e.g. McKersie et al. 1996). In our experiment, the changes in total SOD activity were not strong and corresponded particularly to changes in Mn-SOD (see Fig. 4, + H2O2). ABA treatment caused a decline in MnSOD activities in all plants. Although there was no difference between G- and M-plants under CA, in ABA treated plants at CA, activity of CuZn-SOD (localized in chloroplasts or cytoplasm) slightly increased (Fig. 4, see Total SOD). Some authors have reported that CE caused a decrease in SOD and CAT activity (Havir and McHalle 1989, Polle et al. 1997), which was probably because of lower rates of photorespiration. However, in contrast to CAT, SOD activity in plants under various nutrient regimes increased or remained unchanged under CE (Polle et al. 1997). Contrary to ABA treatment, we have found particular stimulation of Mn-SOD in plants grown under CE, while CAT activity was unchanged. G-plants exhibited consistently higher numbers of SOD isozymes compared to M-plants. Different levels of intrinsic oxidative stress probably were experienced by M- and G-plants during in vitro cultivation and this caused different demands for induction of various SOD isozymes. ABA treatment dramatically restricted a number of isozymes, mainly in M-plants grown at CA but also CE, which is quite surprising because total activity increased. This is in contradiction to Edwards et al. (1994), who suggest that oxidative stress induces changes in SOD isoforms as the synthesis of new ones is probably a better and more efficient way for detoxification than an increased activity of constitutive SOD. The activity of several enzymes of the intermediary metabolism involved in, or closely related to, the Krebs cycle and pentose phosphate pathway is often enhanced by stress (e.g., Vangrosveld and Clijsters 1994). Their activity is possibly stimulated to compensate for the imbalance in contents of ATP and NADPH. Moreover, enzymes such as ME and G-6P-DH can increase CO2 release from malate and glucose6-phosphate, respectively, which is followed by CO2 refixation (Becker et al. 1986). This could help to overcome a situation in which CO2 supply is somehow limited. This might be relevant to G-plants, where, during in vitro cultivation, plants often experience CO2 starvation (Solárová and Pospíˇsilová 1997), or limitation caused by ABA and CE treatment, which decreased stomatal conductance. In conclusion, we have shown that conditions during precultivation could predispose the activity of the antioxidant systems in plants transferred from in vitro to ex vitro, and that this effect can last even when the photosynthesis and water regimes of those plants are fully acclimated to the new environment. Plants adjust their metabolism and defence systems up to a certain level of intrinsic oxidative stress during their growth in the original environment and this also influences their reaction to ABA and CE treatment. However, further detailed investigation of the initial conditions in both types of in vitro precultivation and probably also processes throughout

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the acclimation are needed to fully understand the observed differences. Acknowledgements. Research was supported by grant No. 501/95/ 1303 of the Grant Agency of the Czech Republic and by grant No. 100/1998/B BIO/PˇrF of the Grant Agency of the Charles University.

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