Plant Growth Regul (2006) 49:85–93 DOI 10.1007/s10725-006-0023-5
ORIGINAL PAPER
The response of antioxidant enzymes in cellular organelles in cucumber (Cucumis sativus L.) leaves to methyl viologen-induced photo-oxidative stress Xing Shun Song Æ Chun Lan Tiao Æ Kai Shi Æ Wei Hua Mao Æ Joshua Otieno Ogweno Æ Yan Hong Zhou Æ Jing Quan Yu
Received: 18 October 2005 / Accepted: 8 March 2006 Springer Science+Business Media B.V. 2006
Abstract In order to clarify the response of antioxidant systems in various cellular organelles to photo-oxidative stress, the activities of superoxide dismutase (SOD) and enzymes of the ascorbate– glutathione (AsA-GSH) cycle were investigated in chloroplasts, mitochondria and cytosol of cucumber leaves subjected to methyl viologen (MV) treatment. Photo-oxidation by MV resulted in significant reductions in net photosynthetic rate (Pn) and increases in the ratio of the quantum efficiency of photosystem II (PSII), UPSII to that of the quantum efficiency of CO2 fixation (UCO2 ), followed by increased activities of SOD, and a general increase of AsA-GSH cycle enzymes in chloroplasts, mitochondria and cytosol. These increases were however, most significant in chloroplasts. There were also significant increases in dehydroascorbate (DHA), reduced glutathione (GSH), and oxidized glutathione (GSSG) X. S. Song Æ C. L. Tiao Æ K. Shi Æ W. H. Mao Æ J. O. Ogweno Æ Y. H. Zhou Æ J. Q. Yu Department of Horticulture, Huajiachi Campus, Zhejiang University, Kaixuan Road 268, Hangzhou 310029, P.R. China J. Q. Yu (&) Key Laboratory of Horticultural Plants Growth, Development and Biotechnology, Agricultural Ministry of China, Kaixuan Road 268, Hangzhou 310029, P.R. China e-mail:
[email protected] Tel.: +0086-57186971120 Fax: +0086-57186049815
except that the content of ascorbate (AsA) in chloroplasts and cytosol was slightly decreased and little effected, respectively. However, GSSG in mitochondria and GSH in cytosol were little influenced by the MV treatment. The activity of ascorbate oxidase (AO) in these organelles was independent of the MV treatment while the activity of L-galactono-1,4lactone dehydrogenase (GLDH) in mitochondria was slightly inhibited by MV treatment. These results indicate that disturbance of electron transport in chloroplasts by MV influenced the metabolism of whole cell by a crosstalk signaling system and that the AsA-GSH cycle played a primary role in sustaining the levels of AsA. Keywords Antioxidant enzymes Æ Cucumis sativus L. Æ Methyl viologen Æ Organelles
Introduction In contrast to atmospheric oxygen, reactive oxygen species (ROS) are capable of unrestricted oxidation of various cellular components and can lead to the oxidative destruction of the cell (Hammond-Kosack and Jones 1996; Asada 1999). Since ROS are generated in organelles such as chloroplasts, mitochondria, and cytosol (Noctor and Foyer 1998), subcellular compartmentation of the antioxidant system is necessary for the efficient scavenging of them (Alscher et al. 1997). Synchronous action of
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superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDAR), dehydroascorbate reductase (DHAR) and glutathione reductase (GR), the last four of which participate in the ascorbate–glutathione (AsA-GSH) cycle, are parts of the antioxidative system which protects the cells against ROS (Asada 1994; Foyer et al. 1994). Activities of these enzymes have been detected in subcellular compartments of different plant species (Jime´nez et al. 1997; Berczi and Møller 1998; Jime´nez et al. 1998). Ascorbic acid (AsA) is an abundant antioxidant in various plant tissues (Noctor and Foyer 1998). L-Galactono-1,4-lactone dehydrogenase (GLDH) and ascorbate oxidase (AO), two key enzymes in AsA synthesis and degradation, as well as enzymes in AsA-GSH cycle influence the metabolism of AsA (Smirnoff 2000). However, the integration of ascorbate synthesis, regeneration, and degradation is not fully understood (Bartoli et al. 2000). Chloroplasts and mitochondria are traditionally considered to be autonomous organelles but they are not as independent as they were once thought to be. The marked and mutually beneficial interaction between mitochondria and chloroplasts is intriguing. The key compartments within plant cells, including chloroplasts, mitochondria and cytosol, appear to be in a delicate metabolic equilibrium (Raghavendra and Padmasree 2003). Methyl viologen (MV), which is commercially known as paraquat, is a water-soluble herbicide that disrupts electron transport and catalyzes the production of O)2 within the chloroplasts (Halliwell and Gutteridge 1989). Recently, MV has been used as a model chemical to study oxidative stress (Asada and Takahashi 1987; Bowler et al. 1994; Kwon et al. 2002; Sankhalkar and Sharma 2002; Kobayashi et al. 2004; Tsukamoto et al. 2005; Rey et al. 2005). For example, transgenic plants that over-expressed both SODs and APX showed increased levels of protection against MV-mediated oxidative damage in leaf discs and less visual damage symptoms in whole plants (Kwon et al. 2002). Under photo-oxidative stress induced by MV, plants overexpressing CDSP32 (a thioredoxin induced under environmental stress conditions) exhibit decreased maximal PSII photochemical efficiency and retain much less chlorophyll compared to wild plants (Rey et al. 2005). Until now, however, no studies have characterized antioxidant enzymes in subcellular
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compartments during MV-induced photo-oxidative stress. Cucumber (Cucumis sativus L.) is a widely cultivated vegetable crop and is very vulnerable to environmental stresses. In our previous study we found that the chill-induced decrease in the proportion of electron flux for photosynthetic carbon reduction was partly compensated by an O2-dependent alternative electron flux driven by the water-water cycle, followed by significant increases in the activity of antioxidant enzymes (i.e. SOD, APX and GR) and antioxidants (i.e. ASA and GSH) (Zhou et al. 2004). Furthermore, we also showed the changes of APX isoenzymes in cucumber plants after heat, MV and H2O2 treatments (Song et al. in press). In this study, we further examined how the antioxidant system in various organelles (chloroplasts, mitochondria and cytosol) in cucumber leaves responds to MV-induced photo-oxidation stress in chloroplasts. In addition, the metabolism of ascorbate (AsA) was discussed in relation to photo-oxidation.
Material and methods Plant material Cucumber (Cucumis sativus L. cv. Jinyan No. 4) seeds were sown in a growth medium containing a mixture of soil and perlite (1:1, v:v) in 12 cm diameter plastic pots in a greenhouse. Seven days after sowing, groups of eight seedlings were transplanted into containers (40 · 25 · 15 cm) filled with half-strength Enshi nutrient solution (Yu and Matsui 1997). The environmental conditions were as follows: a 12-h photoperiod, temperature of 25/17C (day/night), and photosynthetic photon flux density (PPFD) of 600 lmol m2 s1 . Plants at the four-leaf stage were exposed to the photo-oxidation stress by spraying with 50 lM MV as described previously (Mittler and Zilinskas 1992). Water was sprayed onto cucumber leaves as a control treatment. Leaf gas exchange and chlorophyll fluorescence were measured as described below. Four days after transplanting, tissue samples were taken randomly from fully expanded leaves. The samples were frozen quickly in liquid nitrogen and stored at )86C prior to the biochemical assays.
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Leaf gas exchange and chlorophyll fluorescence measurements Leaf net photosynthetic rates (Pn) were determined by using an infrared gas analyzer based portable photosynthesis system (LI-6400, LI-COR Biosciences, USA). The air temperature, relative humidity, CO2 concentration and PPFD were maintained at 25C, 85%, 360 lmol mol1 and 1000 lmol (Photon) m2 s1 respectively. Measurements were taken at 0, 1, 2 and 4 days after treatment. The quantum efficiency of CO2 fixation (UCO2 ) was also determined simultaneously at 600 lmol m2 s1 PPFD. UCO2 was calculated by dividing the rate of Pn at 600 lmol m2 s1 PPFD by the rate at which quanta were absorbed after correction for dark respiration (Zhou et al. 2004). Leaf chlorophyll fluorescence was measured using a portable pulse modulated fluorometer (FMS-2, Hansatech, UK). Before each measurement, the leaves were dark-adapted for at least 30 min. The minimal fluorescence (Fo) was determined under a weak pulse of modulating light over a 0.8 s period and the maximal fluorescence (Fm) was induced with a high intensity saturating pulse of light applied over 0.8 s. The maximal quantum efficiency of PSII was determined as Fv/Fm, where Fv is the difference between Fm and Fo. An actinic light source was then applied to achieve a steady state of photosynthesis and to obtain Fs (steady state fluorescence yield), after which a second saturation pulse was applied for 0.7 s to obtain F0m, light adapted maximum fluorescence. Quantum efficiency of PSII (UPSII ) was calculated as (F0m)Fs)/F0m (Van Kooten and Snel 1990; Yu et al. 2002). Purification of cell organelles Organelles were isolated from fully expanded leaves of control and MV-stressed plants by differential and density gradient centrifugation as described by Mittova et al. (2000) with a modification. Briefly, leaves (10 g each) were chopped using a blender (HR2826, PHILIPS, China) with 5 volumes of medium containing 50 mM HEPES [N-(2-hydroxyethyl) piperazine-N 0-(2-ethanesulfonic acid); pH 7.5], 5 mM c-caproic acid, 0.3% bovine serum albumin (w/v), 0.4 M sucrose, 10 mM NaCl, 10 mM mercaptoethanol, 2 mM ethylenediaminetetraacetic acid (EDTA) and 1% (w/v) polyvinylpyrrolidine. The
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homogenates were filtered through four layers of gauze. The crude chloroplast fraction from leaves was sedimented by centrifugation at 1000 g for 5 min. Chloroplasts in the residues were then purified by a 10, 40, 70 and 90% Percoll discontinuous gradient and centrifugation at 4700 g for 15 min. Intact chloroplast layer was obtained in between the 40 and 70% Percoll fractions. The ferricyanide method was used to measure the intactness of chloroplasts (Takeda et al. 1995). The method is based on the principle that ferricyanide (an artificial electron acceptor) is not able to cross the chloroplast envelope and react with the electron transport system within the intact thylakoid membranes. Electron transport from water to ferricyanide results in oxygen release, which can be measured by an oxygen electrode. D,L-glyceraldehyde which inhibits CO2 fixation and NH4Cl which uncouples the electron flow from the proton gradient, are added to the reaction in order to increase the rate of oxygen evolution. The level of oxygen released by the chloroplast preparation in isotonic medium is proportional to the fraction of ruptured chloroplasts within the preparation. The level of oxygen released by the same chloroplast preparation after osmotic shock represents the total chloroplast content. The percent of intact chloroplasts is determined by comparing the rates of oxygen evolution upon illumination before and after osmotic shock of the chloroplasts. We found that about 85–95% of chloroplasts were intact. The 1000 g supernatant was re-centrifuged at 12,000 g for 15 min and the pellet was collected and re-suspended in: 20 mM HEPES-KOH (pH 7.5), 330 mM sorbitol, 10 mM NaCl, 2 mM EDTA. In this isolation procedure the 12,000 g supernatant was considered to be the cytosol fraction. The 12,000 g pellets were fractionated on a 25, 37, 45 and 57% (w/w) sucrose gradient at 68,000 g for 3.5 h and the intact mitochondria layer between the 37 and 45% sucrose fractions was removed. The integrity of the mitochondria was estimated from the Cyt c oxidoreductase activity, as described by Millenaar et al. (2002), Cyt c oxidase was measured at 550 nm in the presence of312 lM reduced Cyt c (5 ll) and extract in the cuvette with 1 ml KH2PO4 buffer at 25C. Cyt c (in KH2PO4 buffer) was reduced with sodium dithionite. Excess dithionite was removed by a gentle flow of normal air in the solution for a few minutes. The purified mitochondria had intactness rates between 75 and 90%.
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Enzymes activities and contents of AsA and GSH APX (EC 1.11.1.11) activity was determined by monitoring the decrease of AsA in A290 according to Jime´nez et al. (1997). SOD (EC 1.15.1.1) activity was measured by monitoring the inhibition of photochemical reduction of nitro tetrazolium according to Beyer and Fridovich (1987); MDAR (EC 1.6.5.4) activity was measured by monitoring the decrease in A340 due to the NADH oxidation (Arrigoni et al. 1981). DHAR (EC 1.8.5.1) was measured according to Dalton et al. (1986). DHAR and MDHAR were extracted in 50 mM Tris–HCl (pH 7.2) containing 1 mM Na2EDTA, 0.05% cysteine (w/v) and 2% PVP (w/v). DHAR activity was measured by following the formation of AsA from DHA at 265 nm. MDAR activity was measured by using 1 U ascorbate oxidase and the oxidation rate of NADH was followed at 340 nm. GR (EC 1.6.4.2) was determined following Madamanchi and Alscher (1991). GR was extracted in 1 mM potassium phosphate (pH 7.5) containing 0.4 mM Na2EDTA and 9.94 mM isoascorbate and its activity was measured by following the GSSG dependent oxidation of NADPH at 340 nm; GLDH (EC 1.3.2.3) according to De Gara et al. (2000), by following the cytochrome c reduction at 550 nm in a mixture reaction composed of 0.1 M Tris–HCl, pH 8.0, 60 lM cytochrome c, 2 mM L-galactono-1,4lactone (GL) and enzyme; AO (EC 1.10.3.3) as described by Hagashi and Morohashi (1993). AO activity was determined from the decrease in A265 at 25C in a reaction mixture containing 0.1 M sodium phosphate, pH 5.6, 0.5 mM EDTA, and 100 lmol AsA. One unit of AO activity was defined as the oxidation of 1 lmol AsA min1 at 25C. The contents of AsA and DHA were determined according to Sgherri et al. (2000); GSH and GSSG according to Sgherri and Navari-Izzo (1995). Chlorophyll was assayed according to Arnon (1949).
Results and discussion The purities of the isolated organelles from leaves of both control and MV-treated cucumber were shown in Table 1. The contamination of chloroplasts by mitochondria was within the range of 4–8%. In comparison, a higher contamination (7–10%) by chloroplasts was found in leaf mitochondria.
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Plant Growth Regul (2006) 49:85–93 Table 1 Distribution of cytochrome c oxidase (CCO), a marker enzyme for mitochondria, and chlorophyll content, the marker for chloroplasts in different organelles isolated from leaves of control (CK) and MV-treated (MV) leaves Treatment
Fraction
Markers (pigment and enzyme) Chlorophyll (lg g1 FW)
CK MV
Chloroplasts Mitochondria Chloroplasts Mitochondria
0.56 0.03 0.45 0.02
– – – –
0.02 0.01 0.03 0.01
CCO (lmol g1 FW) 0.06 0.68 0.05 0.72
– – – –
0.01 0.10 0.02 0.07
The results are the means from at least 3 independent experiments – SD
However, these values were within an acceptable range for purified chloroplasts and mitochondria fractions (Mittova et al. 2000) and indicated that these organelles were well separated. MV-treated plants displayed a significantly lower net photosynthetic rate (Pn) than control plants (Fig. 1), especially 2 days after the treatment. By day 4, Pn for MV treated plants was only 21% of the control plants. Meanwhile, a sharp decline in the maximum quantum efficiency of photosystem II (Fv/ Fm) was also observed on day 2, and by day 4 it was decreased by more than 60% as compared to control plants (Fig. 1). This suggests that photoinhibition occurred in the MV-stressed leaves. The ratio UPSII =UCO2 is an indicator for electron sink other than the photosynthetic carbon reduction. We found that it significantly increased after exposure to MV stress, especially after day 4 of the treatment (Fig. 1). Accordingly, there must be electron sinks other than CO2 assimilation operating to sustain the increased ratio of UPSII =UCO2 . Among them, Mehler reaction (AsA-GSH cycle) in chloroplasts is a potential candidate for dealing with the increase in electron flux relative to CO2 assimilation during stress (Ort and Baker 2002; Zhou et al. 2004). Plants possess a complex battery of enzymatic and non-enzymatic antioxidants that protect cells from oxidative damage by scavenging ROS. In our study, we detected the presence of SOD and enzymes of the AsA-GSH cycle in chloroplasts, mitochondria and cytosol in cucumber leaves. MV treatment resulted in a global increase in the activities of SOD (67%), APX (380%), DHAR (150%), MDAR (320%) and GR (50%) in chloroplasts (Table 2). Significant increase
Pn( µ mol CO2 m-2 s-1)
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89
15 10 5 Control
0
MV
1.0
Fv/Fm
0.8
0.6
0.4
30
φ
PSII/
φ
CO2
45
15
0
0
1
2
3
4
Days after treatment Fig. 1 Changes in net photosynthetic rate (Pn), maximal quantum efficiency of PSII (Fv/Fm) and quantum efficiency of PSII to that of CO2 fixation quantum (UPSII =UCO2 ) in cucumber leaves as influenced by MV treatment at different times. Data are the mean of at least three replicates with standard errors shown by vertical bars
in chloroplastic APX activity together with increased resistance to MV-induced photo-oxidation has also been observed in Arabidopsis lines overexpressing thylakoidal APX (tAPX) (Murgia et al. 2004). There were also significant increases in the activities of SOD, APX, DHAR and MDAR in mitochondria although the increases were less significant as compared to those in chloroplasts (Table 2). In
contrast, GR activity was slightly inhibited. However, in the cytosol, SOD, MDAR and GR activities were little affected by MV treatment while APX activity decreased by 29% and DHAR activity increased by 47%. Yabuta et al. (2004) found that cytosolic APX transcript increased by 2.4 folds after 10 lM MV treatment in leaves of spinach. This discrepancy is likely due to the difference of MV concentrations since MV was applied at a much higher concentration (50 lM) in our experiment which might inactivate cytosolic APX by superfluous production of O)2 (Halliwell and Gutteridge 1989). We also found that enzyme activities in peroxisomes, especially DHAR, MDAR and GR, were too low to be detected (data not shown). Although mitochondria showed higher SOD and AsA-GSH cycle activities than chloroplasts (Table 2), they also contained less protein than chloroplasts (about 1/20, data not shown). Therefore, it is possible that the antioxidant enzymes in chloroplasts played a more important role in weeding out redundant ROS than those in other cellular organelles during MV-induced photo-oxidative stress under our experimental conditions. MV usually exacerbates O)2 radical production only within chloroplasts (Halliwell and Gutteridge 1989). We found, however, that chloroplasts and mitochondria, even cytosol, all responded to this change (Table 2). There must be some signal(s) acting as a bridge for triggering defense responses in various subcellular compartments. ROS have been suggested to take on this responsibility (OrozcoCa´rdenas et al. 2001; Vranova´ et al. 2002; Mittler et al. 2004; Laloi et al. 2004). Although localized increases in ROS can occur in certain circumstances such as during the photo-oxidative damage in chloroplasts, redox balance would be altered in the whole plants (Foyer and Noctor 2003; Baier and Dietz 2005). Concomitantly, an intricate network for mutual regulation by anthero- and retro-grade signals has emerged to co-ordinate the activities of the different genetic and metabolic compartments. Furthermore, changes in ROS were found to trigger profound modifications in gene expression that extend far beyond the antioxidative system (Kovtun et al. 2000; Desikan et al. 2001; Vranova´ et al. 2002). Accordingly, the defence system in various subcellular compartments forms an integrated network with extensive crosstalk that can be triggered by ROS (Vranova´ et al. 2002).
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Accompanying the changes in anti-oxidant enzyme activities, significant changes in non-enzymic antioxidants were also observed. There were global increases in the concentrations of AsA, DHA, GSH and GSSG in chloroplasts, mitochondria and cytosol after MV treatment except that concentrations of AsA in chloroplasts, GSSG in mitochondria and GSH in cytosol were little influenced or even slightly decreased (Table 3). MV has also been found to decrease AsA content in spinach leaves by inhibiting the reduction of monodehydroascorbate (MDA) to AsA (Noctor and Foyer 1998; Mano et al. 2001). It should be noted that the leakage of non-enzymatic antioxidant might occur during the organelle
fractation. The absolute concentrations of AsA and GSH could be, to some extent, underestimated in present study. Accordingly, the observed changes in AsA and GSH in our study are just likely to reflect qualitatively the in vivo response. We found that MV induced an overall increase in antioxidants in various organelles (Table 3), suggesting that they cooperatively participate in eliminating excessive ROS when disturbance of electron transport in chloroplasts is produced by MV induced photo-oxidation. In agreement with our observation, Raghavendra and Padmasree (2003) also suggested that disturbance of any of these compartments might affect the metabolism of whole cell. MV treatment
Table 2 Effects of methyl viologen (MV) on antioxidant enzymes in chloroplasts, mitochondria and cytosol in cucumber leaves 4 days after treatment Organelle/ Treatment
Chloroplasts
CK MV
Mitochondria
CK MV
Cytosol
CK MV
SOD (Units mg1 protein)
APX (lmol min1 mg1 protein)
DHAR (lmol min1 mg1 protein)
MDAR (lmol min1 mg1 protein)
GR (lmol min1 mg1 protein)
4.01 – 0.26 b 6.71 – 0.48 a (167) 10.35 – 0.384 b 15.89 – 0.19 a (154) 2.63 – 0.15 a 2.45 – 0.23 a (93)
0.09 – 0.02 0.43 – 0.07 (476) 1.34 – 0.23 3.53 – 0.55 (263) 2.75 – 0.22 1.94 – 0.42 (70)
0.015 – 0.038 – (256) 0.098 – 0.120 – (122) 0.073 – 0.107 – (147)
0.005 – 0.021 – (417) 0.073 – 0.085 – (116) 0.175 – 0.176 – (101)
0.010 – 0.003 0.015 – 0.002 (149) 0.127 – 0.035 0.105 – 0.005 (83) 0.076 – 0.008 0.071 – 0.009 (93)
b a b a a b
0.003 b 0.002 a 0.010 b 0.030 a 0.010 b 0.020 a
0.001 b 0.002 a 0.001 b 0.002 a 0.006 a 0.004 a
b a a a a a
Each value represents the mean of at least three replicates. The values with the same letter are not significantly different in the same organelle between control and MV treatment (P < 0.05) One unit of SOD activity was defined as the amount of enzyme resulting in one half of maximal inhibition. CK denotes Control plants, MV denotes MV-treated plants. Values in the parentheses represent the ratio of MV/CK · 100 (%)
Table 3 Effects of methyl viologen (MV) on contents of ascorbate and glutathione in chloroplasts, mitochondria and cytosol in cucumber leaves 4 days after treatment GSH (nmol mg1 GSSG (nmol mg1 GSH/ AsA (nmol mg1 DHA (nmol mg1 AsA/ protein) protein) (AsA+DHA) protein) protein) (GSH + GSSG)
Organelle/ Treatment Chloroplasts
CK MV
Mitochondria CK MV Cytosol
CK MV
87 – 9 a 73 – 8 a (84) 1203 – 50 b 1633 – 60 a (135) 219 – 22 a 227 – 18 a (104)
13 – 2 b 31 – 3 a (238) 850 – 23 b 1005 – 12 a (118) 14 – 1 b 38 – 2 a (143)
0.87 0.70 0.59 0.62 0.94 0.86
122 – 10 b 208 – 11 a (169) 706 – 25 b 781 – 32 a (111) 28 – 4 a 26 – 4 a (93)
13 – 1 b 37 – 7 a (200) 126 – 21 a 133 – 10 a (105) 4.6 – 0.5 b 8.7 – 0.8 a (189)
0.90 0.85 0.85 0.85 0.86 0.75
Each value represents the mean of at least three replicates. The values with the same letter are not significantly different in the same organelle between control and MV treatment (P < 0.05) CK denotes Control plants, MV denotes MV-treated plants. Values in the parentheses represent the ratio of MV/CK · 100 (%)
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Table 4 Effects of methyl viologen (MV) on ascorbate oxidase (AO) in chloroplasts, mitochondria, cytosol, and L-galactono1,4-lactone dehydrogenase (GLDH) in mitochondria in cucumber leaves 4 days after treatment Organelle/Treatment Chloroplasts
CK MV
Mitochondria
CK MV
Cytosol
CK MV
AO activity (lmol min1 mg1 protein)
GLDH activity (lmol min1 mg1 protein)
0.017 – 0.010 – (59) 0.103 – 0.109 – (106) 0.140 – 0.140 – (100)
0.006 a 0.001 a
– –
0.001 a 0.011 a
0.028 – 0.002 a 0.022 – 0.004 b (85) – –
0.011 a 0.021 a
Each value represents the mean of at least three replicates. The values with the same letter are not significantly different in the same organelle between control and MV treatment (P < 0.05) CK denotes Control plants, MV denotes MV-treated plants. Values in the parentheses represent the ratio of MV/CK · 100 (%). ‘–’ no activity detected
also resulted in declines in ratios of AsA:(DHA+ AsA) and GSH:(GSH+GSSG) in various organelles, suggesting that cucumber leaves, to some extent, were damaged when extreme stress was imposed on them. Similar phenomena were also observed in maize after exposure to MV (Iturbe-Ormaetxe et al. 1998). We also investigated MV-induced changes of the activities of GLDH and AO. Negligible changes in the activity of AO were observed in all cellular organelles after MV treatment. GLDH activity, however, decreased by 21% after exposure to the photo-oxidation stress (Table 4). Since AsA is believed to be the first line of defense against oxidative stress (Barnes et al. 2002), it is necessary to sustain AsA at an optimum level. The AsA pool is generally determined by its rates of synthesis (catalyzed by GLDH) and degradation (catalyzed by AO), as well as its regeneration by the AsA-GSH cycle (Smirnoff 2000). Increased AsA levels were found in tobacco and maize leaves overexpressing DHAR, an important component of AsA recycling (Chen et al. 2003). In our study, GLDH and AO activity were decreased or little affected by MV whilst activity of AsA-GSH cycle was significantly increased. Accordingly, AsA regeneration likely played an important role in sustaining AsA level. It is worth to note that the plant AsA metabolism is complicated. AO is only one of several routes by which AsA can be degraded (Robert and Roberto 2005), and there are several routes such as oxalic acid and tartaric acid pathways (Davey et al. 2000). Moreover, the decrease of GLDH activity is also likely due to the substrate limited. The detailed mechanism remains to be further elucidated.
In conclusion, our results indicate that antioxidants and antioxidative enzymes in various organelles cooperatively function to protect whole plant cells from further destruction when MV-induced photooxidation was imposed on chloroplasts. ROS, possibly acting as a bridge, induced defense reactions in different organelles. Moreover, the pathway of AsA regeneration, namely the AsA-GSH cycle, played a primary role in sustaining AsA level. Acknowledgements We thank Dr. Demetriades-Shah of Li-Cor for his critical reading of the manuscript. This work was supported by the National Natural Science Foundation of China (30230250; 3050344) and National Outstanding Youth Scientist Foundation (30235029), 863 plan, Excellent Teacher Foundation of China, and Natural Science Foundation of Zhejiang Province as a special project.
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