Springer-VerlagTokyohttp://www.springer.de102650918-94401618-0860Journal
of Plant ResearchJ
Plant ResLife Sciences27510.1007/s10265-006-0275-1
J Plant Res (2006) 119:321–327 Digital Object Identifier (DOI) 10.1007/s10265-006-0275-1
© The Botanical Society of Japan and Springer-Verlag Tokyo 2006
REGULAR PAPER
Antonio Cano • Josefa Hernández-Ruiz • Marino B. Arnao
Changes in hydrophilic antioxidant activity in Avena sativa and Triticum aestivum leaves of different age during de-etiolation and high-light treatment
Received: December 28, 2005 / Accepted: February 15, 2006 / Published online: April 21, 2006 The Botanical Society of Japan and Springer-Verlag 2006
Abstract The steady-state of reactive oxygen species (ROS) in plant cells is controlled by ROS-producing and scavenging agents. A large cellular pool of antioxidant metabolites is involved in their control. Variations in this antioxidant pool may be monitored by measuring changes in hydrophilic antioxidant activity (free radical-quenching activity of water-soluble components) and ascorbic acid levels. The de-etiolation process and induction of light stress in Avena sativa and Triticum aestivum leaves were used as physiological models to study the antioxidant status at different ages. The data showed that five-day-old green plants and de-etiolated plants of the same age have similar hydrophilic antioxidant activity (∼8 µmol ASC equivalents g FW−1), which increases during the de-etiolation process. In oat and wheat, young leaves (five days old) had higher antioxidant status (hydrophilic antioxidant activity and ascorbic acid level) than old leaves (10 and 20 days old). High-light treatment caused a decrease in antioxidant status, especially in young leaves. Hydrophilic antioxidant activity and ascorbic acid levels recovered totally or partially after 30 or 60 min in the dark. This capacity also depends on age and species. The ascorbic acid/hydrophilic antioxidant activity ratio is presented as an indicator of antioxidant variations in response to stress, but taking into account the absolute levels of antioxidants. Key words Antioxidant activity · Ascorbic acid · Deetiolation · Light stress · Oat · Wheat
Introduction Photoautotrophic transition (the de-etiolation process) is an interesting effect of light in which dark-grown seedlings
A. Cano · J. Hernández-Ruiz · M.B. Arnao (*) Department of Plant Physiology, Faculty of Murcia, University of Murcia, 30100 Murcia, Spain Tel. +34-968-367001; Fax +34-968-363963 e-mail:
[email protected]
(etiolated) take on the morphology of a light-grown plants (de-etiolated). This transition can be regarded as light stress, because the new environmental situation (light) requires chloroplasts to mature and causes other dramatic phenotypical changes, for example inhibition of shoot elongation, active root growth, and change in colour (synthesis of chlorophylls, carotenoids, and anthocyanins), etc. (Briggs and Olney 2001; Kendrick and Kronenberg 1994; McNellis and Deng 1995; Quail 2002). Irradiance in excess of that required for photosynthesis saturation may cause thylakoid photoinhibition, however, especially in photosystem II. Excess irradiance may be converted into heat for thermal dissipation via the violaxanthin–antheraxanthin–zeaxanthin system (VAZ cycle), which can be regarded as a protective mechanism against light stress (Cleland et al. 1986; Demming-Adams et al. 1995; Eskling et al. 1997; Prasil et al. 1992). Chloroplasts, and other organelles with highly oxidizing metabolic activity, for example mitochondria and microbodies (peroxisomes, glyosyxomes) are a major source of reactive oxygen species (ROS) in plant cells. The production of ROS in plants is enhanced by limiting conditions such as drought, salinity, light and temperature stress, and, in general, by biotic and abiotic-induced stress (Asada 1999; Moller 2001). For each situation, the steady-state level of ROS in cells must be precisely regulated. In Arabidopsis thaliana (L.) Heynh, a network of at least 152 genes is involved in managing levels of ROS. This network is highly dynamic and encodes ROS-producing and ROS-scavenging agents (Mittler et al. 2004). The main ROS-scavenging enzymes in plants include superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, catalase, glutathione peroxidase, glutathione reductase, NADPH oxidase, and peroxiredoxins (a family of hydroperoxide-detoxifying enzymes recently described in plants) (Apel and Hirt 2004; Asada 1999; Dietz 2003; Mittler et al. 2004). Together with ROSscavenging enzymes, a cellular pool of antioxidants, ascorbic acid (ASC), and glutathione, are essential to their activity. The reduced state of these antioxidant metabolites is maintained by enzymes capable of using NADP(H) to
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regenerate oxidized forms of glutathione and ascorbic acid. In addition, protection of membrane integrity against oxidative stress is guaranteed by the α-tocopherol (vitamin E) pool, which is kept in its reduced state by the pool of ascorbic acid. Ascorbic acid and glutathione are also metabolites involved in the VAZ cycle. Thus, in the antioxidant pool of cellular metabolites, compounds other than ascorbic acid and glutathione, for example phenolic compounds or other secondary metabolites, could also act on antioxidant enzymes in specific organelles (Conklin and Barth 2004; Noctor and Foyer 1998; Smirnoff et al. 2001; Valpuesta and Botella 2004). One way of monitoring the antioxidant status, or the antioxidant pool of cells, tissues, organs or of the entire plant, could be to determine antioxidant activity. Methods that measure antioxidant activity have usually been based on free-radical-scavenging activity, which is related to the antioxidant metabolite level present in the sample. One of the most widely used methods uses the ABTS radical as a chromogen; this reacts with many antioxidant compounds, for example ascorbic acid, glutathione, flavonoids, tocopherols, carotenoids, etc. (Arnao 2000; Arnao et al. 1999, 2001; Cano et al. 1998, 2002). We have developed and applied this method to the study and characterization of some physiological situations, for example the on-vine ripening of tomatoes, the re-emergence of leaf sprouts of Quercus ilex after fire, and the gradient of growth and development for different varieties of lettuce. The method has also been used for characterization of different varieties of grapes and processes related to wine-making (Alcolea et al. 2002, 2003; Cano et al. 2003; Cano and Arnao 2005; El Omari et al. 2003). In this study we have used our antioxidant activity method to monitor the antioxidant status of seedlings of oat (Avena sativa L.) and wheat (Triticum aestivum L.) and changes in their antioxidant pool during two physiological processes – the de-etiolation transition of seedlings and induction of light stress in leaves. The correlation observed between antioxidant status and ascorbic acid levels demonstrates that this method can be used to obtain interesting data for study of the ROS-scavenging system in plants.
Materials and methods Plant material and growth conditions Oat (A. sativa L.) and wheat (T. aestivum L.) leaves were harvested from 5, 10, and 20-day-old plants grown in vermiculite, in a controlled chamber at 25°C, HR 70%, and a photoperiod (16 h light:8 h dark) of low-light 80 µmol photon m−2 s−1 photosynthetic photon flux density (PPFD) supplied by 36 W Sylvania Grolux fluorescent tubes. The PPFD was measured with a quantum sensor Delta OHM HD 9021 (Padova, Italy). In addition, etiolated plants were grown in vermiculite at 25°C in the dark for 5 days, after which the plants were transferred to the low-light chamber described above.
High-light treatment High-light stress was induced by a halogen lamp (1,000 W) in 2-cm sections of leaves in Petri dishes with distilled water. The PPFD reaching the surface of the leaves was approximately 2,800 µmol photon m−2 s−1. To avoid overheating, a 10-cm deep circulating ice-cold water bath was placed under the lamp, so that the leaf-surface temperature remained at 22–25°C. Four situations were studied: Control (C), leaf sections without high-light treatment; high-light (HL), leaf sections submitted to high-light treatment for 20 min; HL30, HL with a 30-min recovery period in the dark, and HL60, HL with a 60-min recovery period in the dark.
Reagents 2,2′-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) as the crystalline diammonium salt and L-ascorbic acid were purchased from Sigma (Madrid, Spain). H2O2 (30%, v/v) was obtained from Aldrich (Madrid, Spain). The concentrations of ABTS, hydrogen peroxide, and ASC were determined by measuring their absorbance using ε340 nm = 36 mmol−1 L cm−1 for ABTS, ε240 nm = 43.6 mol−1 L cm−1 for H2O2, and ε290 nm = 2.8 mmol−1 L cm−1 for ASC. Horseradish peroxidase (HRP) type VI was obtained from Sigma (ε403 nm = 100 mmol−1 L cm−1). Spectrophotometric measurements were recorded with a Perkin–Elmer Lambda-2S UV– visible spectrophotometer interfaced on-line with a PC. The temperature was controlled at 25 ± 0.1°C by use of a Haake D1G circulating bath with a heater/cooler. Solvents (HPLC grade) were obtained from Baker (Deventer, the Netherlands). The reagents and salts (analytical grade) used to prepare the solutions were obtained from Merck (Darmstadt, Germany).
Determination of antioxidant activity Hydrophilic antioxidant activity after the four treatments described (C, HL, HL30, and HL60) was determined by the method of Cano et al. (1998). This method is based on the capacity of biological samples to scavenge the ABTS radical compared with a standard antioxidant (for example ascorbic acid) in a dose–response curve. Antioxidants were extracted from 0.1 g leaf. The tissue was homogenized for 2 min with an Euroturrax T20 homogeniser (IKA, Germany) in extraction solution containing 5 mL metaphosphoric acid (3%), 1 mmol L−1 EDTA-Na2, and 2 mL ethyl acetate. The mixture was then centrifuged at 1,000g, for 10 min, in a refrigerated Sorvall RC-5B Plus centrifuge (Dupont, USA). The aqueous phase was used immediately to determine the HAA and the ascorbic acid content. To measure HAA, the reaction mixture contained 2 mmol L−1 ABTS, 30 µmol L−1 H2O2, and 0.25 µmol L−1 HRP in 50 mmol L−1 sodium phosphate buffer (pH 7.5) in a total volume of 1 mL. The assay temperature was 25°C. The reaction was monitored at 730 nm until absorbance became stable. The aqueous extraction phase (10 µL) was added to the
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reaction medium and the decrease in absorbance, which is proportional to the ABTS radical quenched, was determined after 5 min. The antioxidant activity was expressed as moles equivalent of ascorbic acid per gram of fresh weight.
times, the HAA of the leaves was measured. It is apparent from Table 1 that HAA values increased with the length of light exposure (maximum at 10 h). Both oat and wheat leaves slowly turned green; the de-etiolation process also progressively increased HAA levels.
Ascorbic acid determination by HPLC–EC
HAA in high-light treatment of leaves of different age
Ascorbic acid was determined by high-performance liquid chromatography with electrochemical detection (HPLC– EC). A Beckman (CA, USA) System Gold instrument with programmable injector and electrochemical detector (Hewlett–Packard model 1049A) with glassy-carbon working electrode and a liquid Ag/AgCl reference electrode was used. Samples of aqueous extraction phase (10 µL) were filtered and injected into a RP-ODS2-Spherisorb (5-µm particle size) column. The mobile phase, in isocratic mode, was 100 mmol L−1 ammonium acetate and 1 mmol L−1 EDTA-Na2 at a flow rate of 0.5 mL min−1. The presence of ASC (retention time of 7.7 min) was detected selectively at +600 mV with excellent sensitivity.
Figures 1 and 2 show HAA values of oat and wheat leaves harvested at different ages. HAA values of five-day-old oat and wheat plants grown in the low-light chamber (80 µmol photon m−2 s−1) were very similar to those of de-etiolated plants after 10 h of low light (data in Table 1 and Figs. 1 and 2). Maximum HAA values were observed in leaves of 5day-old oat and wheat plants. In oat leaves of 10 and 20day-old plants HAA values were 75 and 50%, respectively, of the values recorded for 5-day-old leaves (Figs. 1a–1c); for wheat leaves the value was 50% (Figs. 2a–2c). Thus, for both species studied, HAA values were highest for young leaves (five days old); in older plants HAA decreased, with the lowest values being reached in 20-day-old plants. We then studied the effect of high-light treatment on the leaves of oat and wheat plants of different ages. To test the recovery capacity of leaves after high-light treatment, we determined the HAA of leaves after 30 min (HL30) and after 60 min (HL60) (see Material and methods section). Figure 1 shows that the HAA decreased in both HL and HL30 oat leaves but recovered in HL60 leaves (i.e. after treatment for 60 min). The decrease in HAA was more pronounced in young leaves. The HAA decrease was lowest for 20-day-old leaves, however, which also had the highest recovery capacity – HAA values (∼5 µmol g FW−1) were higher than for the control leaves (∼3.8 µmol g FW−1). Similar behaviour was observed for wheat (Fig. 2), HAA recovering in HL60, especially in 20-day-old leaves, although HAA recovery never exceeded the initial HAA status of control leaves.
Statistical analysis Differences between results were determined by use of SPSS software (SPSS, Chicago, IL, USA), applying the Fisher’s LSD multiple range test to establish significant differences at P < 0.05.
Results Hydrophilic antioxidant activity (HAA) in the de-etiolation of oat and wheat The hydrophilic antioxidant activity (free radical-quenching activity of water-soluble components) of leaves was determined by means of an established assay that uses the ABTS radical cation (ABTS+) as chromogen (Arnao 2000; Cano et al. 1998). The presence of an antioxidant leads to the disappearance of this species. Table 1 shows HAA values (expressed as micromoles ASC equivalents) for five-day-old etiolated leaves of oat and wheat plants. It is apparent that etiolated oat and wheat seedlings had similar antioxidant capacity. The seedlings were then transferred to the lowlight chamber (80 µmol photon m−2 s−1) and, at different
ASC in high-light treatment of leaves of different age The level of ASC in plant tissues is an important property for studying stress situations. ASC measurements which do not involve selective methods (for example titration) are not always accurate, however. Although HPLC methods enable more selective determination of ASC, we have observed that UV detection tends to overestimate ASC
Table 1. Hydrophilic antioxidant activity (HAA) of five-day-old oat and wheat seedlings during the de-etiolation process in low-light conditions HAA (micromoles ASC equivalents g FW−1) De-etiolation (hours of low light) Etiolated (0 h) Oat Wheat a
2.82 (±0.08) 2.62 (±0.06)
a
Mean value ± SE (in parentheses), n = 3
1h
4h
8h
10 h
14 h
3.84 (±0.17) 5.55 (±0.22)
6.30 (±0.21) 7.81 (±0.40)
7.51 (±0.32) 8.36 (±0.39)
8.22 (±0.38) 8.53 (±0.44)
8.18 (±0.41) 8.22 (±0.42)
324 a
a
b
b
c
Fig. 1. Hydrophilic antioxidant activity (HAA) and ascorbic acid (ASC) content (filled squares) of oat leaves 5 days old (a), 10 days old (b), and 20 days old (c) in different situations: control; HL (high-light treatment); HL30 (HL with a 30-min dark-recovery period), and HL60 (HL with a 60-min dark-recovery period). Error bars represents standard errors of the mean (n = 5). The different superscripts represent statistically significant differences at P < 0.05 for each situation. The percentage data are the ASC/HAA ratio for each situation
levels in fruits and leaves. We therefore measured the ASC levels by HPLC using electrochemical detection (HPLC– EC), which is a selective and extremely sensitive technique for determination of ASC. Figure 3a shows representative chromatograms obtained from an ASC standard and from the ASC detected in a leaf extract, whereas Fig. 3b shows
c
Fig. 2. Hydrophilic antioxidant activity (HAA) and ascorbic acid (ASC) content (filled squares) of wheat leaves 5 days old (a), 10 days old (b), and 20 days old (c) in different situations: control; HL (highlight treatment); HL30 (HL with a 30-min dark-recovery period), and HL60 (HL with a 60-min dark-recovery period). Error bars represents standard errors of the mean (n = 5). The different superscripts represent statistically significant differences at P < 0.05 for each situation. The percentage data are the ASC/HAA ratio for each situation
the excellent linearity of the response. It is apparent that it is possible to determine ASC in amounts as low as 5 pmol. Figures 1 and 2 show ASC levels in oat and wheat leaves. In the control leaves, the youngest leaves contained the highest ASC levels, wheat leaves containing approximately double the ASC of oat leaves. ASC levels decreased after
325 Fig. 3. Representative chromatograms showing HPLC– EC analysis of ascorbic acid (ASC). a ASC standard (dashed line) and wheat leaf extract diluted 100-fold (solid line) recorded at +600 mV (scale 0– 500 nA) using an isocratic elution program. b Linear response of electrochemical detection of ASC in the range 2–20 ng
a
HL treatment, but recovered after 60 min (HL60), in a similar way to HAA. The 20-day-old leaves suffered the greatest decrease in ASC level, but also had the highest recovery capacity, the initial ASC levels being restored or even exceeded, as occurs in 20-day-old oat leaves. We can contrast HAA and ASC levels because both are represented as mole equivalents, which is a significant advantage of the HAA method used in this study (Cano et al. 2002, 2003). Figures 1 and 2 reflect the similar behaviour of HAA and ASC levels. Figure 4 shows the linear regressions for these properties (HAA vs. ASC levels) for each species. Good linear regression coefficients were obtained (r2 = 0.919 for oat and 0.923 for wheat), indicating that variations in ASC and HAA are appropriate indicators of HL-induced stress in leaves. The similar behaviour of the HAA and ASC levels and the excellent correlation between them indicate that the changes in HAA were because of variations in ASC levels. Thus, by calculating the contribution of ASC to HAA in each case we can determine whether other antioxidant molecules changed significantly during the treatment. Figures 1 and 2 show, in percentage terms, the contribution of ASC to HAA. As can be seen, in oat leaves (Fig. 1) ASC contributes 20–30% of the HAA in control plants; in HL60 the ASC contribution increased, illustrating that ASC is the principal molecule involved in the recovery of the antioxidant status in leaves. It is worthy of note that in 5, 10, and 20-day-old leaves 33% of HAA was attributable to ASC, irrespective of the previous ASC/HAA ratio for control leaves. In wheat leaves, the ASC/HAA ratio (%) was higher than in oat, ranging from 40% in older leaves to 51% in younger leaves. As in oat, ASC increased its contribution to HAA after HL-treatment, demonstrating that ASC was the most important antioxidant to change in this stress situation.
b
Discussion Hydrophilic antioxidant activity has been used to study changes in the antioxidant status of oat and wheat leaves during two commonly occurring environmental processes: de-etiolation and high-light stress. Our previous data on other physiological situations, for example ripening of tomatoes (Cano et al. 2003), re-emergence of Q. ilex leaf sprouts after fire (El Omari et al. 2003), and the gradient of growth and development in different varieties of lettuce (Cano and Arnao 2005), revealed that determination of HAA, with other related properties, for example ASC levels, phenolic compounds, etc., can be a useful approach to studying situations in which variation of the levels of antioxidant metabolites may be expected. For five day-old de-etiolated plants (oat and wheat) HAA values (approx. 8 µmol ASC equivalents g FW−1) were similar to those of five-day-old green plants (Table 1 and Figs. 1, 2, control). HAA values were highest during the first 20 days of plant growth. In 10 and 20-day-old plants HAA values decreased in both oat and wheat leaves to reach ∼4 µmol ASC equivalents g FW−1. Behaviour of ASC levels was similar. ASC levels were highest in the youngest leaves, and higher in wheat leaves than in oat leaves. Compared with control leaves, young leaves were more efficiently prepared, from the standpoint of antioxidant capacity (high HAA and ASC levels), to confront stress. HAA decreased in response to high-light treatment, especially in younger leaves, i.e. the decrease in HAA was lower in older leaves, for both oat and wheat. HAA recovery capacity differed according to age and species, however. In oat, whereas HAA values for 20-day-old leaves were higher than control values, five-day-old leaves did not recover their initial HAA status, For both five and 20-day-
326
a
ASC consumption in leaves. This process is clearly mirrored by the decreases in HAA observed. In addition, HAA recovery is probably a consequence of activation of the antioxidant enzymatic system, enzymes of the ascorbate– glutathione cycle that operate both in the chloroplasts and in the cytosol (ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase, and glutathione reductase). Thus, during the dark recovery period significant regeneration of ASC occurs, which is in accordance with changes in the ASC/HAA ratio and which demonstrates the relative contribution of ASC (or other antioxidants) to the antioxidant pool in leaves under stress conditions. Acknowledgements This work was supported by project MCyTAGL2003-00638 (co-financed by FEDER) and by project F. SENECA 502/PI/04. J.H.R. has a contract with the University of Murcia.
b
References
Fig. 4. Linear regression analysis of hydrophilic antioxidant activity (HAA) and ascorbic acid (ASC) values in the high-light study of oat (a) and wheat (b) leaves. The linear regression coefficients were: r2 = 0.919 for oat and 0.923 for wheat. Data were taken from Figs. 1 and 2
old leaves, however, HAA after 60 min of high-light treatment recovered to reach 5–6 µmol ASC equivalents g FW−1 for oat and 3–6 µmol ASC equivalents g FW−1 for wheat. These data indicate that a minimum HAA threshold was present in older leaves; young leaves had high HAA status (surplus) that enabled them to buffer stress-induced situations more gently. These interpretations are supported by the data for wheat leaves, which contained twice as much ASC as oat leaves, whereas HAA and ASC variations were less pronounced. A possible indicator of these antioxidant variations was the ASC/HAA ratio (%), which indicates how much of the HAA was attributable to ASC levels, but taking into account the absolute levels of ASC or other relevant antioxidant molecules. Thus, variations in this ratio were lower for oat leaves than for wheat leaves, and so the antioxidant pool against stress in oat was more effective than in wheat. To summarize, our results show that overproduction of ROS during high-light treatment provokes a high level of
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