Journal of Experimental Botany, Vol. 51, No. 344, pp. 645-655,
March 2000
© 2000 Oxford University Press
The effects of ethylene, depressed oxygen and elevated carbon dioxide on antioxidant profiles of senescing spinach leaves
Atlantic Food and Horticulture Research Centre, Agriculture and Agri-Food Canada, 32 Main Street, Kentville, Nova Scotia, B4N 1J5 Canada
Received 29 June 1999; Accepted 2 November 1999
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Abstract |
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It has been suggested that antioxidants play a role in regulating or modulating senescence dynamics of plant tissues. Ethylene has been shown to promote early plant senescence while controlled atmospheres (CA; reduced O2 levels and elevated CO2 levels) can delay its onset and/or severity. In order to examine the possible importance of various antioxidants in the regulation of senescence, detached spinach ( Spinacia oleracea L.) leaves were stored for 35 d at 10 °C in one of three different atmospheres: (1) ambient air (0.3% CO2, 21.5% O2, 78.5% N2), (2) ambient air+10 ppm ethylene to promote senescence, or (3) CA (10% CO2, 0.8% O2 and 89.2% N2) to delay senescence. At weekly intervals, material was assessed for activities of the antioxidant enzymes ascorbate peroxidase (ASPX; EC 1.11.1.11), catalase (CAT; EC 1.11.1.6), dehydroascorbate reductase (DHAR; EC 1.8.5.4), glutathione reductase (GR; EC 1.6.4.2), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), and superoxide dismutase (SOD; EC 1.15.1.1), and concentrations of the water-soluble antioxidant compounds ascorbate and glutathione. Indicators of the rate and severity of senescence (lipid peroxidation, chlorophyll, and soluble protein levels) were also determined. Results indicated that the rate and severity of senescence was similar between the leaves stored in ambient air or CA until day 35, at which point the ambient air-stored leaves exhibited a sharp increase in lipid peroxidation. Tissues under both storage regimes demonstrated significant declines only in levels of ASPX, CAT, and ascorbate. Glutathione content in the CA-stored tissue also significantly dropped, but only on day 35. In contrast, spinach leaves stored in ambient air+ethylene experienced a rapid decrease in levels of all the antioxidants assessed except SOD. Declines in levels of ASPX, CAT, and ascorbate over the 35 d storage period regardless of the composition of the storage atmosphere suggests that regulation of H2O2 levels plays an important role in both the dynamics and severity of post-harvest senescence of spinach.
Key words: Antioxidants, controlled atmosphere, ethylene, post-harvest, senescence.
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Introduction |
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Senescence has been defined as a genetically regulated process which leads to the death of cells, organs, or whole organisms (Kawakami and Watanabe, 1988
; Noodén and Guiamét, 1989). In plants, senescence is accompanied by morphological changes and/or alterations in biochemical and biophysical properties of metabolism. Leaf senescence in particular may involve degradation of proteins (Lutts et al., 1996), chlorophyll (Fang et al., 1998), nucleic acids (Buchanan-Wollaston, 1997), and membranes (Trippi and Thiamann, 1983), followed by transport of some of the degradation products to other parts of the plant.
Many changes resembling those associated with senescence can be induced when a plant is exposed to one or more biotic or abiotic stresses such as drought (Irigoyen et al., 1992; Olsson, 1995), flooding (Hurng and Kao, 1994), ozone (Pell et al., 1997), or excessive levels of exogenous compounds such as NaCl (Lutts et al., 1996), nitrate (Escuredo et al., 1996), manganese (González et al., 1998) or ethylene (Sylvestre et al., 1989; Kim and Wills, 1995). Physical detachment of leaves can also promote or accelerate symptoms of early leaf senescence (Philosoph-Hadas et al., 1991; Bartoli et al., 1996).
Increased proliferation of potentially toxic active oxygen species (AOS) within plant systems is apparently a common denominator of many types of stress. AOS have also been implicated in induced or natural senescent processes and its dynamics (Droillard et al., 1987; Thompson et al., 1991; Philosoph-Hadas et al., 1994). Indeed, one of the major characteristics of senescence in plant tissues is increased lipid peroxidation (Kunert and Ederer, 1985; Lacan and Baccou, 1998) with the concomitant production of malondialdehyde (MDA), a secondary end-product of polyunsaturated fatty acid oxidation which acts as an estimator of the degree of oxidative stress experienced by the tissue (Hodges et al., 1999).
Plants contain complements of enzymic and non-enzymic antioxidants which play an important role in regulating levels of AOS such as superoxide (
), hydrogen peroxide (H2O2), the hydroxyl radical (·OH) and singlet oxygen (1O2) (Foyer et al,. 1994a; Hodges et al., 1996, 1997a, b). These antioxidants include the enzymes ascorbate peroxidase (ASPX; EC 1.11.1.11), catalase (CAT; EC 1.11.1.6), dehydroascorbate reductase (DHAR; EC 1.8.5.4), glutathione reductase (GR; EC 1.6.4.2), monodehydroascorbate reductase (MDHAR; EC 1.6.5.4), and superoxide dismutase (SOD; EC 1.15.1.1), the water-soluble compounds such as ascorbate, glutathione and flavonoids, and the lipid-soluble compounds such as the carotenoids and tocopherols. SOD dismutates to H2O2, which is further reduced to H2O by CAT in peroxisomes and by ASPX in the cytosol and chloroplasts through the concomitant oxidation of ascorbate. DHAR, MDHAR, and glutathione serve to reduce oxidized ascorbate, and GR reduces oxidized glutathione (Trümper et al., 1994). Both ascorbate and glutathione can also react directly with and scavenge certain AOS (Foyer et al., 1994a).
As involvement of AOS in leaf senescence has been well established, it is not surprising that the relation between leaf oxidative and antioxidative potentials has been implicated in the dynamics of senescence (Kunert and Ederer, 1985). It has been demonstrated that general leaf reductant levels were negatively correlated with the rate of senescence for a number of herbaceous species (Philosoph-Hadas et al., 1994; Meir et al., 1995). During leaf senescence some antioxidants increase while others decrease (Irigoyen et al., 1992; Olsson, 1995; Kingston-Smith et al., 1997), with the specific profiles probably dependent upon the species examined and whether senescence was natural or stress-induced. The importance of antioxidants in senescence dynamics has not only been shown for leaves. For example, high levels of antioxidant enzymes were correlated with delayed senescence in two varieties of non-netted muskmelon fruit differing in their storage life (Lacan and Baccou, 1998) and the deterioration of sunflower seeds was closely related to decreased activity of antioxidant enzymes concomitant with increased lipid peroxidation (Bailly et al., 1996).
Exposure to exogenous ethylene has been demonstrated to escalate the biosynthesis of endogenous ethylene and to enhance the rate of membrane lipid breakdown and, in general, to accelerate foliar senescence (Sylvestre et al., 1989; Kim and Wills, 1995). In contrast, detached plant material placed in controlled atmospheres (CA) containing lowered O2 levels and/or elevated CO2 concentrations often experiences reductions in respiration rates, chlorophyll loss, oxidase activities, and ethylene biosynthesis (Kader, 1986). Reduced levels of available O2 may also limit the potential of AOS production. Storage under CA conditions thus generally promotes a delay in the onset and/or a decrease in the rate of senescence, and this process has been widely adopted commercially for post-harvest storage of produce such as apples, pears, and various berries. An atmosphere of 4.0% O2 and 9.2% CO2 was reported to reduce ascorbate loss in spinach by 50% compared to air (Burgheimer et al., 1967) while spinach stored in 0.8% O2 exhibited a lower respiration rate concomitant with a superior appearance and taste than that stored in air (Platenius, 1943). Profiles of antioxidant changes in detached spinach leaves stored in ambient air at 10 °C over 35 d were analysed in order to identify particular antioxidants whose decline was concomitant with increasing senescence and lipid peroxidation. Spinach leaves stored for 35 d in either CA or air+10 ppm ethylene were also assessed to determine how senescence-inhibiting or -promoting regimes impacted upon the antioxidants profiled above.
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Materials and methods |
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Plant material
Spinach (Spinacia oleracea L. cv. Vienna ) seeds (Vesey's Seeds Ltd, York, PEI, Canada) were sown 0.5 inch deep in potting soil, 3 seeds per 5-inch standard pot. Pots were placed in a growth chamber (Econaire GR36; Econaire, Winnipeg, MB, Canada) at 18 °C and 95% relative humidity with a photocycle of 10/14 h light/dark and a photosynthetic photon flux density of 400–450 µmol m-2 s-1. Plants were watered and fed twice weekly with 15 : 15 : 18 (N : P : K) (Plant Products, Brampton, ON, Canada) as per directions. Spinach was grown for 45 d and then all leaves except for the four oldest (excluding cotyledonous leaves) were harvested and either analysed immediately (day 0) or placed under storage conditions.
Storage protocols
Harvested spinach was placed into perforated plastic bags and then into stainless steel controlled atmosphere (CA) chambers (LTB Ltd, Port Williams, NS, Canada) complete with a plastic lid which fitted into a water trough around the circumference of the chamber body to ensure a gas-tight seal. The CA containers were placed in a growth chamber (Econaire GR100; Econaire, Winnipeg, MB, Canada) at 10 °C in the dark. Bagged spinach was exposed to one of three storage atmospheres: (1) ambient air (0.3% CO2, 21.5% O2, 78.5% N2), (2) ambient air+10 ppm ethylene, or (3) CA (10% CO2, 0.8% O2 and 89.2% N2). The gas mixtures (Praxair Canada Inc, Paris, ON, Canada) were humidified to saturation by bubbling through water and were passed through the chambers at a flow rate of 10 l min-1 until flushing was complete after which the flow rate was set at 200 ml min-1. CA containers were flushed after each opening. The O2 and CO2 concentrations in the CA chambers were assessed routinely with O2 (Ametek, Pittsburgh, PA) and CO2 (Li-Cor, Lincoln, NE, USA) analysers.
Spinach was removed and analysed on day 0 (point of harvest), 7, 14, 21, 28, and 35 for all three storage protocols, each protocol being repeated twice. Fresh tissue was used immediately for all antioxidant enzyme and MDA-TBARS assays whereas frozen samples were stored for later analyses of antioxidant compound, chlorophyll and protein levels. A total of six independent harvests, two per storage treatment, were performed.
Lipid peroxidation and chlorophyll analyses
Estimates of lipid peroxidation were assessed spectrophotometrically in 7.5–10 g (fresh mass) samples of spinach tissue using a modified TBA-MDA assay (Hodges et al., 1999) which corrects for compounds other than the TBA-MDA adduct which absorb at 532 nm.
Chlorophyll was determined in 7.5–10 g (fresh mass) samples of spinach leaves as described previously (Wintermans and De Mots, 1965).
Antioxidant enzyme analyses
Enzyme extracts were prepared by homogenizing spinach leaves (7.5–10 g fresh mass) in a prechilled mortar and pestle nestled in ice along with 0.5 g inert sand, 0.5 g polyvinylpolypyrrolidone, and 30 ml chilled extraction buffer consisting of 100 mM potassium phosphate buffer (pH 7.5), 1.0 mM EDTA, and 1.0 mM ascorbate added fresh just prior to use. The extraction buffer used for the SOD assays was without ascorbate. Extracts were then centrifuged (Sorvall RC-5C plus, Dupont Instruments) at 10 000 g for 15 min at 2 °C and the supernatants analysed. For DHAR and SOD assays, the supernatants were immediately depleted of low-molecular-mass compounds by passage through a Sephadex G25 PD-10 mini-column (Pharmacia Biotech, Uppsala, Sweden) pre-equilibrated with the extraction buffer. Enzyme assays were conducted immediately following extraction.
All enzymes were assessed spectrophotometrically at 25 °C on an Ultraspec 3000 (Pharmacia Biotech, Uppsala, Sweden) equipped with an Endocal RTE-5B (Neslab, Portsmouth, NH, USA) water bath for calibrated temperature control.
ASPX was determined using a method described previously (Nakano and Asada, 1987). The assay mixture contained 90 mM potassium phosphate buffer (pH 7.0), 0.1 mM EDTA, 0.65 mM ascorbate, and 1.0 mM H2O2. The reaction was initiated with the addition of approximately 40 µg extract protein. Activity was determined by following the H2O2-dependent decomposition of ascorbate at 290 nm.
CAT activity was assayed in a reaction mixture containing 100 mM potassium phosphate buffer (pH 6.5), 1.0 mM EDTA, 60.0 mM H2O2, and approximately 40 µg extract protein in a method following Aebi (Aebi, 1983). Activity was determined by following the decomposition of H2O2 at 240 nm.
DHAR activity was determined following Doulis et al. (Doulis et al., 1997). The assay mixture contained 90 mM phosphate buffer (pH 7.0), 0.1 mM EDTA, 5.0 mM reduced glutathione (GSH), and approximately 30 µg extract protein. The reaction was initiated with the addition of freshly made 0.2 mM dehydroascorbate (DAsA). Activity was determined by following the reduction of DAsA at 265 nm after accounting for the non-enzymic reduction of DAsA by GSH.
GR activity was determined following Foyer and Halliwell (Foyer and Halliwell, 1976). The reaction mixture contained 80 mM TRIS-HCl (pH 8.5), 1.5 mM EDTA, 2.5 mM oxidized glutathione (GSSG), and up to 100 µg extract protein. The reaction was initated with the addition of 0.5 mM NADPH in 1% NaHCO3 and was followed by monitoring the oxidation of NADPH at 340 nm.
MDHAR activity was assayed in a reaction mixture consisting of 90 mM potassium phosphate (pH 7.5), 0.01 mM EDTA, 0.0125% Triton X-100, 2.5 mM ascorbate, 0.25 units ascorbate oxidase (units as defined by Sigma Chemical Co.), 0.2 mM NADH, and up to 50 µg protein extract (Hossain et al., 1984). The reaction was followed by measuring the decrease in absorbance at 340 nm due to NADH oxidation.
SOD activity was determined according to methods described earlier (McCord and Fridovich, 1969; as modified by Hodges et al., 1997a). The reaction mixture consisted of 65.0 mM potassium phosphate (pH 7.5), 0.01 mM EDTA, 0.5 mM xanthine, 0.13 mM cyctochrome c, and 0.025 units xanthine oxidase (units as defined by Sigma Chemical Co.). Activity was determined by monitoring the inhibition of the reduction rate of cyctochrome c between reaction mixtures with and without protein extract (up to 200 µg protein) at 500 nm.
Protein concentration was determined spectrophotometrically at 595 nm using the Bio-Rad Protein Assay Dye Reagent Concentrate (Bio-Rad Laboratories, Hercules, CA, USA; catalogue number 500–0006 ) in a method based on Bradford (Bradford, 1976). Bovine gamma-globulin (0.25–1.4 mg ml-1) was used as a standard reference.
Antioxidant compound analyses
Reduced ascorbate (AsA), DAsA, and total ascorbate (AsA+DAsA) were determined spectrophotometrically (according to Law et al., 1983; Hodges et al., 1996). Over ice, 7.5–10 g (fresh mass) of spinach tissue was homogenized with 0.5 g inert sand and 15 ml of ice-cold freshly-made 5% (w/v) m-phosphoric acid with a mortar and pestle. The homogenate was centrifuged at 10 000 g for 15 min at 2 °C. Total ascorbate was determined by initially incubating for 50 min in a 700 µl total volume 100 µl supernatant, 110 mM KH2PO4, 3.6 mM EDTA, and 1.5 mM dithiothreitol (DTT) to reduce all DAsA to AsA. After incubation, 100 µl of 0.5% (w/v) N-ethylmaleimide (NEM) was added to remove excess DTT. AsA was analysed in a similar manner except that 200 µl deionized H2O was substituted for DTT and NEM. Colour was developed in both series of reaction mixtures (total and reduced ascorbate) with the addition of 400 µl 10% (w/v) TCA, 400 µl 44% o-phosphoric acid, 400 µl of 65 mM 
,1-dipyridyl in 70% ethanol, and 200 µl 110 mM FeCl3. The reaction mixtures were then incubated at 40 °C for 1 h in a shaking water bath (Julabo Labortechnik, Seelbach, Germany) and quantified at 525 nm. AsA and DAsA standards were between 0 and 5 mM in 5% (w/v) m-phosphoric acid. For each sample, DAsA was estimated from the difference between total ascorbate and AsA.
Total glutathione, GSSG and GSH were determined spectrophotometrically following methods described earlier (Griffith, 1980; Hodges et al., 1996). Over ice, 7.5–10 g (fresh mass) spinach tissue was homogenized in a mortar and pestle along with 0.5 g inert sand and 15 ml of ice-cold freshly-made 5% (w/v) 5-sulphosalicylic acid. The homogenate was centrifuged at 10 000 g at 2 °C for 15 min. Two solutions were then prepared. Solution A (pH 7.2) consisted of 100 mM Na2HPO4.7H2O, 40 mM NaH2PO4.H2O, 15 mM EDTA, 1.8 mM 5,5'-dithiobis-(2-nitrobenzoic acid), and 0.04% BSA. Solution B (pH 7.2) consisted of 1.0 mM EDTA, 50 mM imidazole, 0.2% BSA, and 2 units ml-1 glutathione reductase (units as defined by Sigma Chemical Co.). Total glutathione was measured in a reaction mixture consisting of 400 µl solution A, 320 µl of solution B, 400 µl of a 1 : 25 dilution of supernatant in 0.5 M KH2PO4 (pH 7.0), and 80 µl of 3.0 mM NAPDH. The reaction rate was measured by following the change in absorbance at 412 nm for 5 min. GSSG was analysed in a similar manner except that 1.0 ml of 1 : 10 diluted supernatant in 0.5 M KH2PO4 (pH 6.5) was first incubated with 20 µl 2-vinylpyridine at 25 °C for 1 h to derivatize GSH. GSH and GSSG standards were between 0 and 18 µM in 5% (w/v) 5-sulphosalicylic acid diluted appropriately with 0.5 M KH2PO4 (pH 7.0). For each sample, GSH was estimated from the difference between total glutathione and GSSG.
Statistical analyses
All antioxidant enzyme, antioxidant compound, TBA-MDA, and chlorophyll results were based on at least three readings each of three independent replicate samples for each harvest. Harvests were repeated twice for each storage protocol. The effects of harvest, storage time, and storage protocol were analysed by a three-factor completely randomized ANOVA and lsd values then calculated at the P
0.05 level using Genstat 5 (release 4.1).
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Senescence indicators
A significant increase in MDA content of the detached spinach leaves during storage was observed regardless of the composition of the storage atmosphere; however, the timing of this increase was atmosphere-dependent (Fig. 1A
). The most rapid increase in MDA content occurred in leaves held under ambient air (0.3% CO2, 21.5% O2, 78.5% N2)+10 ppm ethylene. This increase was noted after day 7, with MDA content being maximal on and after day 21. In contrast, the material held in ambient air or CA (10% CO2, 0.8% O2 and 89.2% N2) did not experience significant increases in MDA content until after day 21. Overall, MDA content of the spinach leaves stored from day 0 to day 35 increased 361.8%, 453.5% and 211.7% under ambient air, ambient air+ethylene, and CA conditions, respectively. While the MDA content of leaves held under ambient air or CA conditions experienced a similar increase from day 0 to day 28, after this point the MDA content of the leaves stored under ambient air was significantly higher than those held under CA. Although the MDA content of the detached spinach leaves stored under ambient air+10 ppm ethylene was not significantly different from that of ambient air or CA material at day 7, thereafter it was greater than those from the other two storage regimes and remained significantly higher until day 35, at which point it was not significantly different from that of the ambient-stored leaves.
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), or CA (
) consisting of 10% CO2, 0.8% O2 and 89.2% N2. Numbers in parentheses represent the overall change from day 0 to day 35. Results represent the means and lsd values (PSpinach leaves stored under both ambient air and the CA atmospheres experienced a similar, non-significant change in Chl content over the storage term (Fig. 1B). However, the detached leaves stored under ambient air+ethylene experienced a rapid, significant, decline in total Chl after day 7 which began to level off after day 21 in a trend inversely proportional to the observed changes in MDA content of this tissue. Overall, the Chl content at day 35 was 68.7%, 59.2% and 38.9% of day 0 in the ambient air, CA, and ambient air+ethylene storage conditions, respectively.
The soluble protein content declined significantly over the 35 d storage period for spinach leaves held in all three storage atmospheres (Fig. 1C). The overall decrease in soluble protein content over storage was greatest for the ambient+ethylene-stored material (39.1% of day 0) and, similar to the trends for MDA and Chl contents, occurred after day 7, levelling off after day 21. There were no significant differences in decreasing soluble protein content between the CA- (72.9% of day 0) or ambient-stored (54.6% of day 0) material for the 35 day, but leaves from both these storage conditions maintained a significantly higher soluble protein contents than those from the ambient air+ethylene regime after day 7.
Effect of storage atmospheres on antioxidant enzyme activities
Only activities of ASPX and CAT significantly declined in detached spinach leaves stored in ambient air over 35 d (Fig. 2). Activities of DHAR, GR, MDHAR, and SOD remained unchanged over the entire storage period, although transient increases in MDHAR and SOD activities were noted early in storage (Figs 3, 4). In contrast, ASPX, CAT, DHAR, GR, and MDHAR of spinach stored in air+10 ppm ethylene all demonstrated significant decreases in activity over 35-d-long storage (Figs 2, 3, 4). ASPX activities exhibited a much more rapid decline in tissue stored in ethylene-containing air than in tissue stored in air alone, whereas a similar rate of decrease in CAT activity was observed (Fig. 2). Although ASPX and CAT declined in spinach leaves stored in both ambient air and ambient air+ethylene, the overall decrease over the 35 d storage period was greater for tissue stored in ethylene-containing air.
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As with ambient air storage, detached spinach leaves held in CA conditions demonstrated significantly decreased activities of ASPX and CAT over the storage period (Fig. 2). The rate of decrease in activities of these two enzymes was similar between tissue stored in ambient air and CA. However, the overall decrease in ASPX and CAT activities was greater in the leaves stored in CA as opposed to that held in ambient air alone. Again, as with spinach stored in ambient air, SOD exhibited a transient increase in activity on day 7, though the overall levels remained similar over the entire 35 d storage regime (Fig. 4).
Effect of storage atmospheres on ascorbate and glutathione levels
Concentrations of total ascorbate dropped significantly in spinach leaves following detachment, regardless of the storage atmosphere (Table 1). Although a significant loss of ascorbate in spinach held in the ambient air or CA atmospheres did not occur until mid-way through the storage period, the spinach held in ambient+ethylene incurred a significant reduction on and after day 7. Overall ascorbate loss was greatest in the spinach held in ambient+ethylene as opposed to the other storage atmospheres.
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Ratios of oxidized ascorbate (DAsA) to reduced ascorbate (AsA) decreased in spinach held in ambient or ambient air+ethylene conditions as the storage period progressed (Table 1). This decrease was most pronounced in the spinach stored under ambient conditions, particularly during the later stages of storage. However, the DAsA : AsA ratio of spinach held in CA increased with storage until by day 35 it was 292.5% of its initial value on day 0.
Total glutathione levels increased significantly in leaves held in ambient air for longer than 14 d (Table 1). This increase was maintained until day 35, at which point levels had declined back to initial values. Glutathione levels of tissue placed in CA did not change from day 0 to day 28, but dropped significantly on day 35. The glutathione concentrations of spinach placed into ambient air+ethylene decreased significantly after day 14, ultimately reaching values on day 35 which were 2.2% of their initial day 0 level.
Ratios of reduced glutathione (GSH) to oxidized glutathione (GSSG) did not change over the 35 d in spinach leaves stored in ambient air (Table 1). In contrast, the GSH : GSSG ratios of the tissue held in CA first declined and then climbed back to initial values. The GSH : GSSG ratios of material placed in ambient air+ethylene steadily increased from day 0 onwards until day 35, at which point the GSH : GSSG ratio represented a 304.7% increase over that of day 0.
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Discussion |
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In this study the effects of senescence on antioxidant levels of detached spinach leaves stored in ambient air at 10 °C for 35 d were examined. Comparisons were made with detached spinach leaves stored in CA and in ambient air+10 ppm exogenous ethylene in an effort to examine the effects of treatments designed to alter the rates of senescence on antioxidant profiles and capacities.
Levels of MDA, a secondary by-product of lipid peroxidation, indicate the degree of plant oxidative stress (Irigoyen et al., 1992; DeLong and Steffen, 1997; Hodges et al., 1999), while decreases in Chl and soluble protein concentrations often serve to estimate the extent of senescence (Kar and Feierabend, 1984; Philosoph-Hadas et al., 1991; Meir et al., 1995). Although changes in MDA, Chl, and soluble protein contents showed that detached spinach leaves stored 35 d experienced some degree of senescence regardless of the storage atmosphere, the application of 10 ppm exogenous ethylene led to the dramatically accelerated onset of lipid peroxidation and senescence. The involvement of ethylene as a regulator of foliar senescence has been previously documented (Philosoph-Hadas et al., 1991; Kim and Wills, 1995) though not as well as has the senescence-promoting effects of this hormone on flowers or climacteric fruit (Sylvestre et al., 1989; De Pooter and Schamp, 1989; Celikel and van Doorn, 1995). Increased ethylene biosynthesis and lipid peroxidation have been closely linked (Lesham, 1988; Bartoli et al., 1996; Pell et al., 1997) though the exact sequence of events remains to be elucidated. Lipid peroxidation and AOS have been implicated in the direct or indirect degradation and/or bleaching of Chl (Hodges et al., 1996; Toivonen and Sweeney, 1998) thus it should not be surprising that the significant increases in MDA and decreases in Chl contents in the spinach after 14 d stored in ambient air+ethylene appear concomitant. Although degradation of Chl was not accelerated in spinach subjected to a 160 ml l-1 stream of ethylene (Baardseth and Von Elbe, 1989), in contrast to 10 µl l-1 or 10 ppm used in this study, it has been demonstrated that the enhanced rates of Chl loss in spinach placed in ambient air+10 ppm ethylene at 25 °C produced a trend similar to that in this study (Yamauchi and Watada, 1991).
Exposure to CA has been demonstrated to retard senescence symptoms such as Chl loss (Kader, 1986; Fang et al., 1998) compared to storage in ambient air. Reduced O2 levels have been shown to decrease both oxidative respiration rates and oxidase kinetics and may also inhibit enzymes involved in ethylene biosynthesis (Kader, 1986; Weichmann, 1987). Elevated CO2 concentrations can also affect ethylene biosynthesis and binding dynamics, and can reduce aerobic respiration rates (Herner, 1987, Herregods, 1995). No significant differences in Chl content or soluble protein levels were observed between the ambient air- or CA-stored material for the 35 d storage period of this study. However, the significant increase in MDA on day 35 for spinach stored in ambient air as compared to CA indicated that ambient air was more conducive to generating oxidative stress towards the later stages of storage. CA may inhibit the production of AOS at the later stages of storage by retarding rates of oxidative respiration at the level of the mitochondria, which contains electron transport chains acting as sources of active oxygen (Monk et al., 1989). Furthermore, decreased availability of O2 may be beneficial in that less is available to be reduced in a variety of reactions to produce AOS including production of through the reduction of O2 by ferredoxin in the chloroplasts (Scandalios, 1993).
AOS have been implicated in induced or natural senescent processes and its dynamics (Droillard et al., 1987; Thompson et al., 1991; Philosoph-Hadas et al., 1994). Loss of membrane integrity, a common phenomenon in plant senescence (Thompson, 1988), and subsequent detrimental effects on organellar integrity can lead to enhanced production of AOS through unregulated electron transfer reactions and/or production of further substrate in the form of polyunsaturated fatty acids for lipoxygenase enzymes. The primary products of lipoxygenase activity are hydroperoxy conjugated dienes (Hildebrand, 1989), along with 1O2 often released as a by-product (Kanofsky and Axelrod, 1986). Chain reactions of hydroperoxy conjugated dienes can also promote AOS accumulation; for example, lipid hydroperoxides can react with Fe2+ to form ·OH. Conjugated dienes can exacerbate further losses in membrane integrity (Thompson et al., 1991). A cycle of membrane degradation, AOS generation and enhanced senescence results.
Declining activities of ASPX and CAT over the 35 d storage period in ambient air-held tissue suggest that the capacity for enzyme-catalysed H2O2 scavenging became increasingly degraded. The requirement for ASPX capacity may have declined over storage due to decreasing concentrations of total available ascorbate. Reduction in H2O2 -scavenging potential may have significantly contributed to enhanced oxidative stress as measured by lipid peroxidation. Reduced activities of ASPX over storage would presumably have lessened DAsA production, which, in concert with the stability of DHAR, GR and MDHAR (primarily concerned with the recycling of ascorbate), could explain the observed decrease in DAsA/AsA. Although relatively constant activities of SOD over storage suggests that the detached leaves' abilities to dismutate were unchanged, transient increases in levels of MDHAR, SOD and glutathione observed early in the storage period may have been an attempt by the leaves to accommodate burgeoning oxidative stress generated by declining activities of ASPX and CAT and overall concentrations of ascorbate. Certainly, relative stability of DHAR, GR, MDHAR, SOD, and glutathione was not enough to prevent the observed increase in lipid peroxidation.
Little difference was observed in MDA content between the ambient air- and CA-stored spinach leaves until day 35, at which point the leaves under CA exhibited significantly less lipid peroxidation. The major differences in antioxidants between the tissue stored in ambient air and that held in CA were that CA-stored leaves did not demonstrate the overall magnitude of ascorbate loss over the 35 d storage period and that DAsA/AsA levels rose instead of declining. Furthermore, glutathione concentrations in the CA material also exhibited a significant decrease on day 35 compared with those from ambient air-stored tissue. As glutathione plays an important role in the reduction of DAsA to AsA, the observed decrease in this antioxidant compound may have contributed to the manifested increase in DAsA/AsA over storage. Moreover, both glutathione and ascorbate can directly react with and neutralize 1O2 and ·OH (Foyer et al., 1994b), thus a decline in glutathione could lead to alternate oxidation of ascorbate, generating DAsA at the expense of AsA.
Why glutathione content declined in CA-stored leaves as opposed to those held in ambient air is unknown, but decreased levels of this compound may not be as detrimental if the CA environment of low O2 and elevated CO2 levels was capable of limiting O2 reduction rates (Kader, 1986). Furthermore, the relatively smaller loss of ascorbate in the CA-stored tissue compared to the ambient air-held leaves may have accommodated for the lower levels of glutathione; stable levels of DHAR and MDHAR would have ensured a viable reduction potential for DAsA. Lower oxidative stress in the CA material is supported by the observations that, although MDA content did increase significantly after 21 d of storage, indicating that CA material was experiencing some degree of escalating oxidative stress, it did so to a significantly lesser extent than in any of the other storage atmospheres.
The most rapid and ultimately greatest increase in MDA content was observed for spinach leaves held for 35 d in ambient air+10 ppm ethylene when compared to the other two storage regimes. MDA levels increased significantly after day 7, much sooner than the 28 d required by both the ambient-air and CA-stored spinach leaves, supporting previous demonstrations that applications of exogenous ethylene accelerate or induce early senescence and peroxidation of lipids (Sylvestre and Paulin, 1987; Woltering and van Doorn, 1988; Sylvestre et al., 1989). Activities of ASPX, CAT, DHAR, GR, and MDHAR and concentrations of ascorbate and glutathione in leaves stored in ambient air+ethylene became significantly lower over the storage period following detachment than in tissue held in ambient air alone. The enzymes are intrinsic components of the Mehler-peroxidase cycle which primarily functions to detoxify H2O2 and reduce oxidized ascorbate and glutathione (Foyer et al., 1994a, b). It is possible that the relatively lower antioxidant capacity of the leaves subjected to the ethylene treatment compared to that of the tissue held in ambient air led to an increased presence of AOS, inducing a higher level of lipid peroxidation and accelerated senescence. Alternatively, ethylene may have had a more direct effect on lipid peroxidation levels and the antioxidants were of secondary importance; ethylene production is known to be autocatalytic, possibly through the actions of lipoxygenases, which may contribute to enhanced lipid peroxidation and AOS generation (Lynch et al., 1985; Kacperska and Kubacka-Zebalska, 1989; Sylvestre et al., 1989; Pell et al., 1997).
Why most of the antioxidants assessed in this study declined in tissue subjected to ethylene is a matter of conjecture. Biosynthesis of the antioxidants may have directly or indirectly been affected by ethylene, which is known to be able to alter gene expression (Woodson, 1987). Alternatively, ethylene may have induced such large increases in AOS levels that the antioxidant system became overwhelmed and suffered degradation. Previous work has shown that applications of exogenous ethylene led to decreases in activities of SOD and CAT in cut carnations (Sylvestre et al., 1989), and increases in ASPX activity in mung beans and peas (Melhorn, 1990). Ethylene has been suggested to play a role in the modulation of ASPX activity (Ievinsh et al., 1995).
Declines in levels of ASPX, CAT and ascorbate over the 35 d storage period regardless of the composition of the storage atmosphere suggests that regulation of H2O2 plays an important role in both the dynamics and severity of post-harvest senescence of spinach. Hydrogen peroxide, although not in itself particularly toxic, can be detrimental to plant metabolism through such mechanisms as the metal catalysed Haber–Weiss reaction with to form the very potent oxidizer ·OH (Salin, 1988). Thus detoxification of H2O2 is extremely important in controlling levels of other AOS within plant systems (Hodges et al., 1997a, b). Decreased antioxidant potential concomitant with an increased AOS production may be an important component in the induction and sequence of senescence, and future work resolving whether the particular antioxidants decline as a result of altered biosynthesis and/or degradation promises further elucidation of the senescence phenomenon.
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Acknowledgments |
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We would like to thank Mr M Jordan and Mr D Canton for technical assistance and advice and Dr K McRae and Mr B Walker for their statistical expertise. We would also like to acknowledge Drs John Delong and Peter Hicklenton, Atlantic Food and Horticulture Research Centre, Kentville, for their critical review of this manuscript.
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Notes |
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1 To whom correspondence should be addressed. Fax: +1 902 679 2311, E-mail:HodgesM{at}em.agr.ca
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Abbreviations |
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AsA, reduced ascorbate; AOS, active oxygen species; ASPX, ascorbate peroxidase; CA, controlled atmosphere; CAT, catalase; Chl, chlorophyll; DAsA, oxidized ascorbate; DHAR, dehydroascorbate reductase; GR, glutathione reductase; GSH, reduced glutathione; GSSG, oxidized glutathione; MDA, malondialdehyde; MDHAR, monodehydroascorbate reductase; SOD, superoxide dismutase..
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