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Free radical scavenging and senescence in Iris tepals
Plant Physiol. Biochem. 39 (2001) 649−656 © 2001 Éditions scientifiques et médicales Elsevier SAS. All rights reserved S098194280101289X/FLA
Free radical scavenging and senescence in Iris tepals Christophe Baillya, Françoise Corbineaua, Wouter G. van Doornb* a
Physiologie végétale appliquée, université Pierre-et-Marie-Curie, tour 53, 4, place Jussieu, 75252 Paris cedex 01, France
b Agrotechnological Research Institute (ATO, Wageningen University and Research Centre), P.O. Box 17, 6700 AA Wageningen, the Netherlands
Received 6 December 2000; accepted 8 March 2001 Abstract – The visible symptoms of tepal senescence in Iris flowers (Iris × hollandica, cv. Blue Magic) are preceded by the death of most mesophyll cells. We investigated the role of some enzymes involved in free radical scavenging. During the 4 d following tepal unfolding, no changes occurred in superoxide dismutase (SOD, EC 1.15.11) activity, though activity had become low by the time of visible senescence (day 5). SOD produces peroxide, which may lead to further free radical production. Peroxide is reduced by catalase (EC 1.11.1.6) or ascorbate peroxidase (APX, EC 1.11.1.11). In Iris tepals, the activity of APX activity was much higher than that of catalase. Catalase activity gradually increased, whereas APX activity remained unchanged until day 4, then dropped to low values when visual senescence symptoms were expressed. Glutathione reductase (GR, EC 1.6.4.2.), involved in maintaining the antioxidant glutathione, exhibited no change in activity until day 5, but a change in isozyme activity occurred prior to senescence. Treatments known to increase free radical levels, such as elevated oxygen concentrations or placing isolated tepals in aqueous solutions containing a range of free radical scavengers/antioxidants, did not affect the time to visible tepal senescence. It is concluded that the early processes of senescence seem neither related to diminished free radical scavenging by SOD, APX and catalase, nor to diminished GR activity. As SOD and APX activities were low by the time visible senescence symptoms were expressed, diminished free radical scavenging may occur at the very last stages of senescence. © 2001 Éditions scientifiques et médicales Elsevier SAS ascorbate peroxidase / catalase / glutathione reductase / Iris × hollandica / senescence / superoxide dismutase / tepals APX, ascorbate peroxidase / AT, 3-amino-1,2,4-triazole / CHX, cycloheximide / DDTC, diethyldithiocarbamate / DMSO, dimethylsulfoxide / GR, glutathione reductase / GSH, glutathione / NBT, nitroblue tetrazolium / SOD, superoxide dismutase
1. INTRODUCTION Free radicals have been implicated in programmed cell death, both in animal and in plant cells [11, 19, 20]. During leaf senescence, for example, proteins, phospholipids and pigments may be degraded by free radicals due to a fine-tuned decrease in free radical scavenging mechanisms [22]. Superoxide dismutase (SOD) converts superoxide to hydrogen peroxide and thus maintains low superoxide concentrations. The activity of SOD determined in vitro decreased prior to petal senescence, in petals of both carnation [13] and chrysanthemum [8], sug*Correspondence and reprints: fax +31 317 475347. E-mail address: [email protected] (W.G. van Doorn).
gesting that increased superoxide anion levels may be involved in the senescence process. If not immediately converted, hydrogen peroxide readily forms potentially harmful radicals, such as the hydroxyl anion. Hydrogen peroxide is normally rapidly reduced by catalase or ascorbate peroxidase (APX) [18, 27]. Cellular antioxidants are an important buffer against free radical induced oxidations. Glutathione reductase (GR) reduces glutathione, one of the important cellular antioxidants [27]. GR activity has been shown to correlate with trapping of oxygen radicals and protection of membranes from peroxidation [2]. Tepals on flowers of Iris × hollandica cv. Blue Magic, placed in water at 20 °C, show visible senescence symptoms 4 to 5 d after the flowers emerge above the green sheath leaves, i.e. about 3 d after flower opening. Using transmission and scanning elec
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tron microscopy, we observed that the first visible signs of senescence were expressed after the death of most of the mesophyll cells. We hypothesized that the activities of the discussed enzymes would decrease prior to cell death and prior to development of the visible senescence symptoms. We only determined in vitro enzyme activity, as no tests of in vivo activity of these enzymes are as yet available. We also investigated the effect on senescence of chemicals that inhibit the action of SOD and of catalase, and we tested treatments that either purportedly elevate the levels of endogenous free radicals, or reduce free radical levels. We hypothesized that chemicals that tend to increase or reduce free radical levels would hasten or delay visible senescence, respectively.
2. RESULTS 2.1. Visible senescence symptoms and cell death Iris flowers are harvested (day 0) when less than 1 cm of the blue tepals emerge above the green sheath leaves. When placed in water at 20 °C, the flower opens on days 0–1. In most experiments, the flowers open almost completely during the first day, but in some experiments opening is slower. Iris flowers have two whorls of three tepals each. Tepals of the inner whorl grow during flower opening, but do not bend laterally. Tepals of the outer whorl grow and bend laterally both at the tepal base, and at the junction between the terminal flag area of the tepal and the more proximal part. Visible senescence starts at the distal edges of all tepals. In this study we concentrated on the edges of the flag tepals. By day 4 or 5, these become somewhat lighter, then become infiltrated with liquid and show inrolling on the adaxial surface. This inrolling proceeds towards the yellow centre of the tepal. When it has reached this centre the tepal has become discoloured. Prior to any visible symptom at the tepal edges, electron microscopy (both TEM and SEM) showed that most mesophyll cells at the distal tepal parts had collapsed. This collapse had occurred by day 3 or 4 depending on the experiment. After the collapse of the mesophyll, the visible integrity of the tepal edge was maintained only by the epidermal cells and the veins. Tepal inrolling coincided with the loss of turgor of a few cells on the adaxial epidermis, often connected in rows of 3–4 cells. 2.2. Enzyme activities Visible senescence symptoms of Iris × hollandica flag tepals are first expressed at the distal edges. In the
Figure 1. Protein content, fresh weight (FW) and dry weight (DW) of the material harvested from the flag tepals of Iris × hollandica, cv. Blue Magic flowers, during vase life at 20 °C. The tepal edges were harvested along two straight lines from the widest part of the tepal to their distal tip.
present tests, therefore, only these edges were harvested. In the experiments on enzyme activities reported here, the first senescence symptoms (infiltration with liquid, followed by inrolling) were expressed on days 4.5 to 5.0 after the onset of flower opening, although in many other experiments with flowers these symptoms occur on day 4.0. Enzyme activities were expressed per unit protein. Protein levels somewhat increased from days 0–1, then decreased from day 2 (figure 1). The activity of SOD did not change during the days prior to tepal senescence (figure 2), but was low by day 5, when visible symptoms of senescence were found. Hydrogen peroxide scavenging activity by catalase was much lower than that of ascorbate peroxidase (figure 3). Although catalase activity steadily increased during vase life (figure 3), when plotted on the same scale as that of ascorbate peroxidase little change is apparent with respect to the high ascorbate peroxidase activity (figure 3). Ascorbate peroxidase activity remained similar from days 0–1, then increased until day 3, and subsequently decreased. By the time the tepals showed wilting, ascorbate oxidase activity had sunk below the detection limit. The activity of glutathione reductase did not change throughout the experiment, i.e. it remained high even when the tepals showed visible senescence symptoms (figure 4). The fresh weight (FW) and dry weight (DW) of the harvested distal parts of the tepals increased from days 0–1 of vase life, remained constant from days 1–4, then decreased by day 5 (figure 1). Expressing the enzyme activities per unit FW or DW rather than per unit protein gives results similar to expression per unit
C. Bailly et al. / Plant Physiol. Biochem. 39 (2001) 649–656
Figure 2. Activity of superoxide dismutase (SOD) in distal edges of the tepals of Iris × hollandica, cv. Blue Magic flowers, during vase life at 20 °C. Enzyme activity is expressed in units per mg protein, one unit being 50 % inhibition of blue formazan formation.
protein (although the activities rise somewhat from days 1–4). The decrease in SOD and APX activity on day 5 is maintained when expressed per unit tepal DW or FW.
2.3. Native gel staining for GR activity In the experiments used for isozyme analysis, the first visible symptoms of senescence were expressed on day 4 following the onset of flower opening. Staining for GR activity showed five bands, of which at least three exhibited clear differential activity (figure 5). Of these, one band was present on day 0, then dropped to progressively lower activities on days 1 and 2. Two other bands exhibited high activity on day 1. One showed low activity from day 2 onward, whilst the other showed high activity on days 2 through
Figure 3. Activity of hydrogen peroxide scavenging enzymes in the distal edges of the tepals of Iris × hollandica, cv. Blue Magic flowers, during vase life at 20 °C. A, Ascorbate peroxidase (APX) and catalase expressed on the same scale. B, Catalase activity expressed on a smaller scale.
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Figure 4. Activity of glutathione reductase (GR) in distal edges of the tepals of Iris × hollandica, cv. Blue Magic flowers, during vase life at 20 °C.
4. Two faint bands at the lower end of the gel somewhat increased in activity.
2.4. Effects of elevated oxygen concentrations and chemicals Placement of the flowers in 80 % oxygen had no effect on the time to the visual symptoms of tepal senescence (table I). The same results were obtained following placement of isolated tepals in 80 % oxygen. In this experiment, the visible symptoms of senescence were expressed about 0.5 d earlier than in tepals attached to cut flowering stems (table I). DDTC, an inhibitor of SOD, and AT, an inhibitor of catalase
Figure 5. Isoenzyme pattern of glutathione reductase in the distal edges of the tepals of Iris × hollandica, cv. Blue Magic, during vase life at 20 °C.
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Table I. Effect of elevated oxygen levels, inhibitors of SOD and catalase, and a number of other chemicals interfering with free radical metabolism, on the senescence of Iris × hollandica, cv. Blue Magic flag tepals. Flowers, 15 cm long, were placed at 20 °C and 80 % oxygen. Isolated tepals were placed in aqueous solutions of the chemicals mentioned, using five concentrations (see Methods). Results (in days until wilting) refer to the (lowest) concentration giving the shortest (SOD and catalase inhibitors, and oxidizing agents) or the longest time to senescence and are the means of ten replications ± SD. Treatment Tepals on flowers (80 % oxygen) Isolated tepals SOD-inhibitor DDTC (diethyldithiocarbamate; 1 mM) Catalase inhibitor AT (3-amino-1,2,4-triazole; 0.01–5 mM) SOD inhibitor + catalase inhibitor DDTC (1 mM) and AT (5 mM) Oxidizing agents Paraquat (1 mM) DL-buthionine sulfoximide (0.01–5 mM) Antioxidants/free radical scavengers Iso-ascorbate (0.1–5 mM) DMSO (dimethyl sulfoxide; 0.7–70 mM) 2-Mercaptoethylamide (0.01–5 mM) Butylated hydroxyanisole (0.01–0.5 mM) Phorbol-12-myristate-13-acetate (0.01–5 mM) Tiron (1,2-dihydroxybenzene-3,5-disulfonate; 0.1–1 mM)
activity did not affect the time to senescence in isolated tepals. No effect was observed even when the two chemicals were applied together (table I). All oxidizing agents and all antioxidants/free radical scavengers tested also had no effect on senescence in isolated tepals (table I).
3. DISCUSSION We hypothesized that a decrease in the activity of a number of enzymes that normally prevent the build-up of elevated free radical concentrations might partially account for the processes leading to the observed death of mesophyll cells and the visible senescence symptoms. The present experiments, however, show no changes in the activities of SOD, the two hydrogen peroxide reducing enzymes investigated, or GR, prior to the onset of visible symptoms of tepal senescence. Catalase activity even increased until the time of visible senescence, but its activity was low compared to that of APX hence its contribution to hydrogen peroxide scavenging is apparently low. Cycloheximide (CHX) application delayed the onset of the visible senescence symptoms in Iris tepals [28] if applied before day 2 following harvest, i.e. 3 d prior to the senescence symptoms (Pak and van Doorn,
Treated tepals (d)
Controls (d)
4.6 ± 0.4
4.7 ± 0.3
3.8 ± 0.3
3.9 ± 0.4
3.7 ± 0.3
3.9 ± 0.4
3.6 ± 0.4
3.8 ± 0.5
3.9 ± 0.4 3.5 ± 0.1
3.8 ± 0.2 3.5 ± 0.1
3.6 ± 0.3 3.5 ± 0.4 3.4 ± 0.2 3.8 ± 0.3 4.0 ± 0.1 4.0 ± 0.3
3.7 ± 0.4 3.5 ± 0.3 3.5 ± 0.4 3.9 ± 0.4 4.0 ± 0.1 3.9 ± 0.4
unpubl. results). This small window in the CHX effect indicates the importance of processes probably related to protein synthesis, which occur relatively early after flower opening. No decrease in SOD, APX, catalase or GR activities accompanied this window of CHX activity. The present data, therefore, suggest that the early processes leading to senescence were apparently not due to a decrease in radical scavenging by these enzymes. By the time the tepal edges showed inrolling and wilting, a decrease in the activity of SOD and APX was observed. The combined decrease of these two enzymes may result in high superoxide and high hydrogen peroxide concentrations. The latter chemical apparently is the cause of hypersensitive cell death in soybean leaves [21]. It may directly, or indirectly via the formation of free radicals, be involved in the last stages of senescence and cell death in Iris tepals. We observed five GR isoforms in Iris tepals. Similarly, some or several isoforms have been detected in other plant tissues, such as needles of Eastern white pine [3] and in maize mesocotyl [4]. The presence of various GR isoforms and their response to stress has been reviewed recently [23]. The relationship between the activity of GR isoforms and senescence, if any, is as yet unclear. Our preliminary experiments with native gels showed one band each for SOD, APX and
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catalase. Each of these bands did not decline in activity until day 5. Our molecular work showed three SOD genes, one being expressed at much higher levels than the other two, and expression until visible senescence (unpubl. results). The literature reports various isoforms of SOD, APX and catalase. Our preliminary tests with native gels apparently showed the isoforms with the highest activity. The absence of a decline in their activity until day 4 is in agreement with the biochemical data shown in figures 1–4. Elevated oxygen concentrations generally induce a higher concentration of free radicals, in particular superoxide and other oxygenated species [17]. In our tests, an oxygen concentration of 80 % had no effect on the time to tepal senescence. In these experiments, the high oxygen level was maintained until the first visible symptoms of senescence were noted. A range of chemicals, that result in elevated levels of free radicals or are known either to scavenge for free radicals or to protect against free radical damage, included in the vase water also had no effect on the time to senescence. In addition, we previously found no effect of some other antioxidants, such as sodium benzoate [10]. These results are not conclusive but may indicate the absence of a role for free radicals in Iris tepal cell death. Petal senescence in carnation, in contrast, is delayed by sodium benzoate [6], Tiron [7], iso-ascorbate [25] and by 3,4,5-trichlorophenol, a scavenger not tested in the present experiments [26]. The difference between carnation and Iris may be due to a different mechanism leading to cell death. It may or may not relate to the fact that petal wilting in carnation occurs after an increase in ethylene production [26], whereas in Iris such an increase is not observed [10]. It is concluded that the activities of the main enzymes involved in scavenging superoxide ions, and the activities of two enzymes involved in hydrogen peroxide reduction, in the tepals of Iris flowers, apparently do not decrease prior to cellular death and the visible senescence symptoms. This may indicate that reduced capacity for free radical scavenging by these enzymes is not involved in the early processes leading to cell death. The very late processes, in contrast, may be causally related to the observed decrease in activities of both SOD and APX. However, the evidence for these hypotheses are so far only preliminary as no detailed assessment was made on the changes in various organelles of the senescent cells, and the enzyme activities have not been determined in vivo. The present results do, therefore, not allow a final conclusion on the role of free radicals in the
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mechanism leading to tepal cell death. Further experimentation is required to assess free radical levels, and the role of the defence against free radicals by glutathione, ascorbic acid and tocopherol. Nonetheless, the present experiments tend to question the widely held, but unsubstantiated, hypothesis that petal/tepal cell death is due to an explosion of free radical levels. This explosion is thought to be due to reduced efficiency of free radical scavenging and/or to membrane lipid peroxidation. We previously also found no clear evidence for membrane lipid peroxidation, prior to the visible senescence symptoms in Iris tepals [10]. It is also concluded that the present results do not substantiate the hypothesis that a decrease in free radical scavenging is involved in the early processes leading to tepal cell death.
4. METHODS 4.1. Plant material Iris flowers (Iris × hollandica, cv. Blue Magic), 40 cm long, were cut in the morning, placed immediately in water and transported (for 2 h) in water to the laboratory. Flowers were harvested when the petals just emerged above the green sheath leaves, i.e. at the time just before flower opening. Tepal edges senesce prior to the more proximal parts. Tepal edges were cut along a straight line from the widest area of the tepal to the top, on both sides, immediately upon arrival of the flowers in the laboratory and on five subsequent days, at the same time of day. The weight of the edges of the five flowers was determined in triplicate, immediately after severing, and after 72 h at 70 °C.
4.2. Extraction procedure Cut edges meant for enzyme assays were immediately placed in liquid nitrogen, and the collected material was stored at –80 °C, then lyophilized. Of the material 0.3 g, representing about twenty flowers, was ground with pestle and mortar, using 15 mL 50 mM potassium phosphate buffer (pH 7.8), also including 0.1 mM EDTA, 2 mM dithiothreitol and 1 % (w/v) polyvinylpyrrolidone. The homogenate was centrifuged at 25 400 × g for 25 min and the supernatant was filtered through two layers of Miracloth and passed through a PD 10 column (containing Sephadex G 25; Pharmacia, Uppsala, Sweden) to remove pigments and salts. All steps of the extraction procedure were carried out at 1–4 °C.
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During vase life at 20 °C, the flower opens on days 1 and 2, and starts to show inrolling of the tepal edges on days 4 to 5, depending on the experiment. The edges are then liquid-logged. During the next days, inrolling and the soaked appearance progresses towards the middle of the tepals.
4.3. Enzyme assays All enzyme assays were checked for unspecific background rates, and, where these occurred, were corrected for. The activity of superoxide dismutase (EC 1.15.11) was measured according to Giannopolitis and Ries [15]. The reaction mixture contained 1.3 M riboflavine, 13 mM methionine, 63 M nitroblue tetrazolium (NBT) in 0.1 M phosphate buffer (pH 7.8), and 25 µL extract in a final volume of 3 mL. SOD activity was assayed by measuring the ability of the extract to inhibit the photochemical reduction of NBT. Glass test tubes containing the mixture were immersed in a water bath at 25 °C and were illuminated for 15 min with a fluorescent lamp (Philips MLL 500 W, Eindhoven, the Netherlands). Identical tubes, held in darkness, served as blanks. The absorbance was read at 560 nm. One unit SOD was defined as 50 % inhibition of the NBT photoreduction to blue formazan and SOD activity of the extracts was expressed as units SOD per mg protein. Catalase (EC 1.11.1.6) activity was determined at 25 °C according to Clairbone [12]. The reaction mixture assay contained 3.125 mM H2O2 in 50 mM phosphate buffer (pH 7.0) and 200 µL enzyme extract, a total volume of 3 mL. Catalase activity was estimated by the decrease of absorbance of H2O2 at 240 nm. Glutathione reductase (EC 1.6.4.2.) activity was assayed at 25 °C according to Esterbauer and Grill [14], by following the rate of NADPH oxidation at 340 nm. The mixture contained 0.5 mM NADPH, 10 mM oxidized glutathione, 3 mM MgCl2 in 0.1 M phosphate buffer (pH 7.8), and 100 µL extract in a total volume of 400 µL. The activity of ascorbate peroxidase (EC 1.11.1.11) was assessed using the method described by Nakano and Asada [24]. In some species, APX is labile when extracts are prepared in the absence of ascorbate. Extraction of our material with and without added ascorbate resulted in the same APX activity. The reaction mixture contained 50 mM of a potassium phosphate buffer (pH 7.0), 0.5 mM ascorbate and 0.4 mM hydrogen peroxide, and 200 µL enzyme extract in a total volume of 1 mL. Peroxidase activity was determined by the decrease in absorbance of ascorbate at 290 nm.
4.4. Protein level The protein concentration of the extracts was determined according to Bradford [9], using the BioRad protein assay kit and bovine serum albumin as a standard. 4.5. Gel electrophoresis and activity staining All gels (precast minigels; 5 × 4 cm) were ran on a PhastSystem (Pharmacia, Uppsala, Sweden). GR isozymes were electrophoresed on native 12.5 % polyacrylamide gels (PhastGel homogeneous 12.5, Pharmacia). Activity staining was adapted from Anderson et al. [3] and was carried out in 0.4 mM NADPH, 3.4 mM oxidized glutathione (GSSG), 1.2 mM 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 0.3 mM 2,6-dichlorophenol-indophenol (DPIP) and 50 mM Tris-HCl buffer (pH 7.75). 4.6. Effects of high ambient oxygen concentrations and chemicals Flowers with a stem length of 15 cm or isolated tepals were placed in demineralized water and continuously held in stainless steel tanks at 21 and 80 % oxygen, in darkness and a relative humidity of about 80 %. The carbon dioxide concentration was held at 0.045 %. The remainder of the gas phase was nitrogen. Flowers were taken out of the oxygen treatments on days 3, 4 and 5 of treatment, and individually placed in fresh demineralized water in the climate-controlled room at 20 °C, 60 % RH; and a photon flux density of 15 µmol·m–1·s–1 from 7:00 to 19:00 hours. The base of isolated flag tepals was continuously immersed in aqueous solution of chemicals that purportedly interfere with free radical metabolism: diethyldithiocarbamate (DDTC; Sigma), an inhibitor of SOD [1]; paraquat (= methylviologen) results in elevated concentrations of the superoxide and hydroxyl anions [5]; 3-amino-1,2,4-triazole (AT), an inhibitor of catalase [11]; buthionine sulfoximide, a compound that increases endogenous free radical levels by inhibition of glutathione synthesis [1], and the following compounds that are antioxidants/free radical scavengers: isoascorbate [25], butylated hydroxyanisole [29], DMSO (dimethyl sulfoxide), which scavenges hydroxyl radicals [5], 2-mercaptoethylamide [16], phorbol-12myristate-13-acetate [1], and Tiron (1,2-dihydroxybenzene-3,5-disulfonate; [7]). These chemicals were all dissolved directly in water, and were tested at five concentrations ranging from 10–5 to 5·10–3 M. The solutions were not renewed. Tepals were placed at 20 °C, 60 % RH, and a photon flux density of 15 µmol·m–1·s–1 from 7:00 to 19:00 hours.
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4.7. Statistics All data on enzyme activity showed less than 5 % variance between repeat measurements of the same extract. The standard deviation shown in the graphs represents variance between repeated extracts (two to three) of the same lyophilised material. These experiments were repeated once. The experiment with elevated oxygen concentration was performed three times. Each oxygen concentration (21 and 80 %), at each of the sampling times, was maintained in two duplicate tanks, ten replicate flowers per tank. Experiments with chemicals included ten replicate tepals, and were repeated once. When necessary, the results were compared by analysis of variance using the GENSTAT V program (Rothamsted UK) and F-test at P < 0.05.
Acknowledgments. The authors are grateful to Harmannus Harkema (ATO-DLO, Wageningen) who repeated some of the experiments on the effect of enzyme inhibitors and free radical scavengers.
[10] [11] [12] [13]
[14] [15] [16] [17]
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Free radical scavenging and senescence in Iris tepals Christophe Baillya, Françoise Corbineaua, Wouter G. van Doornb* a
Physiologie végétale appliquée, université Pierre-et-Marie-Curie, tour 53, 4, place Jussieu, 75252 Paris cedex 01, France
b Agrotechnological Research Institute (ATO, Wageningen University and Research Centre), P.O. Box 17, 6700 AA Wageningen, the Netherlands
Received 6 December 2000; accepted 8 March 2001 Abstract – The visible symptoms of tepal senescence in Iris flowers (Iris × hollandica, cv. Blue Magic) are preceded by the death of most mesophyll cells. We investigated the role of some enzymes involved in free radical scavenging. During the 4 d following tepal unfolding, no changes occurred in superoxide dismutase (SOD, EC 1.15.11) activity, though activity had become low by the time of visible senescence (day 5). SOD produces peroxide, which may lead to further free radical production. Peroxide is reduced by catalase (EC 1.11.1.6) or ascorbate peroxidase (APX, EC 1.11.1.11). In Iris tepals, the activity of APX activity was much higher than that of catalase. Catalase activity gradually increased, whereas APX activity remained unchanged until day 4, then dropped to low values when visual senescence symptoms were expressed. Glutathione reductase (GR, EC 1.6.4.2.), involved in maintaining the antioxidant glutathione, exhibited no change in activity until day 5, but a change in isozyme activity occurred prior to senescence. Treatments known to increase free radical levels, such as elevated oxygen concentrations or placing isolated tepals in aqueous solutions containing a range of free radical scavengers/antioxidants, did not affect the time to visible tepal senescence. It is concluded that the early processes of senescence seem neither related to diminished free radical scavenging by SOD, APX and catalase, nor to diminished GR activity. As SOD and APX activities were low by the time visible senescence symptoms were expressed, diminished free radical scavenging may occur at the very last stages of senescence. © 2001 Éditions scientifiques et médicales Elsevier SAS ascorbate peroxidase / catalase / glutathione reductase / Iris × hollandica / senescence / superoxide dismutase / tepals APX, ascorbate peroxidase / AT, 3-amino-1,2,4-triazole / CHX, cycloheximide / DDTC, diethyldithiocarbamate / DMSO, dimethylsulfoxide / GR, glutathione reductase / GSH, glutathione / NBT, nitroblue tetrazolium / SOD, superoxide dismutase
1. INTRODUCTION Free radicals have been implicated in programmed cell death, both in animal and in plant cells [11, 19, 20]. During leaf senescence, for example, proteins, phospholipids and pigments may be degraded by free radicals due to a fine-tuned decrease in free radical scavenging mechanisms [22]. Superoxide dismutase (SOD) converts superoxide to hydrogen peroxide and thus maintains low superoxide concentrations. The activity of SOD determined in vitro decreased prior to petal senescence, in petals of both carnation [13] and chrysanthemum [8], sug*Correspondence and reprints: fax +31 317 475347. E-mail address: [email protected] (W.G. van Doorn).
gesting that increased superoxide anion levels may be involved in the senescence process. If not immediately converted, hydrogen peroxide readily forms potentially harmful radicals, such as the hydroxyl anion. Hydrogen peroxide is normally rapidly reduced by catalase or ascorbate peroxidase (APX) [18, 27]. Cellular antioxidants are an important buffer against free radical induced oxidations. Glutathione reductase (GR) reduces glutathione, one of the important cellular antioxidants [27]. GR activity has been shown to correlate with trapping of oxygen radicals and protection of membranes from peroxidation [2]. Tepals on flowers of Iris × hollandica cv. Blue Magic, placed in water at 20 °C, show visible senescence symptoms 4 to 5 d after the flowers emerge above the green sheath leaves, i.e. about 3 d after flower opening. Using transmission and scanning elec
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tron microscopy, we observed that the first visible signs of senescence were expressed after the death of most of the mesophyll cells. We hypothesized that the activities of the discussed enzymes would decrease prior to cell death and prior to development of the visible senescence symptoms. We only determined in vitro enzyme activity, as no tests of in vivo activity of these enzymes are as yet available. We also investigated the effect on senescence of chemicals that inhibit the action of SOD and of catalase, and we tested treatments that either purportedly elevate the levels of endogenous free radicals, or reduce free radical levels. We hypothesized that chemicals that tend to increase or reduce free radical levels would hasten or delay visible senescence, respectively.
2. RESULTS 2.1. Visible senescence symptoms and cell death Iris flowers are harvested (day 0) when less than 1 cm of the blue tepals emerge above the green sheath leaves. When placed in water at 20 °C, the flower opens on days 0–1. In most experiments, the flowers open almost completely during the first day, but in some experiments opening is slower. Iris flowers have two whorls of three tepals each. Tepals of the inner whorl grow during flower opening, but do not bend laterally. Tepals of the outer whorl grow and bend laterally both at the tepal base, and at the junction between the terminal flag area of the tepal and the more proximal part. Visible senescence starts at the distal edges of all tepals. In this study we concentrated on the edges of the flag tepals. By day 4 or 5, these become somewhat lighter, then become infiltrated with liquid and show inrolling on the adaxial surface. This inrolling proceeds towards the yellow centre of the tepal. When it has reached this centre the tepal has become discoloured. Prior to any visible symptom at the tepal edges, electron microscopy (both TEM and SEM) showed that most mesophyll cells at the distal tepal parts had collapsed. This collapse had occurred by day 3 or 4 depending on the experiment. After the collapse of the mesophyll, the visible integrity of the tepal edge was maintained only by the epidermal cells and the veins. Tepal inrolling coincided with the loss of turgor of a few cells on the adaxial epidermis, often connected in rows of 3–4 cells. 2.2. Enzyme activities Visible senescence symptoms of Iris × hollandica flag tepals are first expressed at the distal edges. In the
Figure 1. Protein content, fresh weight (FW) and dry weight (DW) of the material harvested from the flag tepals of Iris × hollandica, cv. Blue Magic flowers, during vase life at 20 °C. The tepal edges were harvested along two straight lines from the widest part of the tepal to their distal tip.
present tests, therefore, only these edges were harvested. In the experiments on enzyme activities reported here, the first senescence symptoms (infiltration with liquid, followed by inrolling) were expressed on days 4.5 to 5.0 after the onset of flower opening, although in many other experiments with flowers these symptoms occur on day 4.0. Enzyme activities were expressed per unit protein. Protein levels somewhat increased from days 0–1, then decreased from day 2 (figure 1). The activity of SOD did not change during the days prior to tepal senescence (figure 2), but was low by day 5, when visible symptoms of senescence were found. Hydrogen peroxide scavenging activity by catalase was much lower than that of ascorbate peroxidase (figure 3). Although catalase activity steadily increased during vase life (figure 3), when plotted on the same scale as that of ascorbate peroxidase little change is apparent with respect to the high ascorbate peroxidase activity (figure 3). Ascorbate peroxidase activity remained similar from days 0–1, then increased until day 3, and subsequently decreased. By the time the tepals showed wilting, ascorbate oxidase activity had sunk below the detection limit. The activity of glutathione reductase did not change throughout the experiment, i.e. it remained high even when the tepals showed visible senescence symptoms (figure 4). The fresh weight (FW) and dry weight (DW) of the harvested distal parts of the tepals increased from days 0–1 of vase life, remained constant from days 1–4, then decreased by day 5 (figure 1). Expressing the enzyme activities per unit FW or DW rather than per unit protein gives results similar to expression per unit
C. Bailly et al. / Plant Physiol. Biochem. 39 (2001) 649–656
Figure 2. Activity of superoxide dismutase (SOD) in distal edges of the tepals of Iris × hollandica, cv. Blue Magic flowers, during vase life at 20 °C. Enzyme activity is expressed in units per mg protein, one unit being 50 % inhibition of blue formazan formation.
protein (although the activities rise somewhat from days 1–4). The decrease in SOD and APX activity on day 5 is maintained when expressed per unit tepal DW or FW.
2.3. Native gel staining for GR activity In the experiments used for isozyme analysis, the first visible symptoms of senescence were expressed on day 4 following the onset of flower opening. Staining for GR activity showed five bands, of which at least three exhibited clear differential activity (figure 5). Of these, one band was present on day 0, then dropped to progressively lower activities on days 1 and 2. Two other bands exhibited high activity on day 1. One showed low activity from day 2 onward, whilst the other showed high activity on days 2 through
Figure 3. Activity of hydrogen peroxide scavenging enzymes in the distal edges of the tepals of Iris × hollandica, cv. Blue Magic flowers, during vase life at 20 °C. A, Ascorbate peroxidase (APX) and catalase expressed on the same scale. B, Catalase activity expressed on a smaller scale.
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Figure 4. Activity of glutathione reductase (GR) in distal edges of the tepals of Iris × hollandica, cv. Blue Magic flowers, during vase life at 20 °C.
4. Two faint bands at the lower end of the gel somewhat increased in activity.
2.4. Effects of elevated oxygen concentrations and chemicals Placement of the flowers in 80 % oxygen had no effect on the time to the visual symptoms of tepal senescence (table I). The same results were obtained following placement of isolated tepals in 80 % oxygen. In this experiment, the visible symptoms of senescence were expressed about 0.5 d earlier than in tepals attached to cut flowering stems (table I). DDTC, an inhibitor of SOD, and AT, an inhibitor of catalase
Figure 5. Isoenzyme pattern of glutathione reductase in the distal edges of the tepals of Iris × hollandica, cv. Blue Magic, during vase life at 20 °C.
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Table I. Effect of elevated oxygen levels, inhibitors of SOD and catalase, and a number of other chemicals interfering with free radical metabolism, on the senescence of Iris × hollandica, cv. Blue Magic flag tepals. Flowers, 15 cm long, were placed at 20 °C and 80 % oxygen. Isolated tepals were placed in aqueous solutions of the chemicals mentioned, using five concentrations (see Methods). Results (in days until wilting) refer to the (lowest) concentration giving the shortest (SOD and catalase inhibitors, and oxidizing agents) or the longest time to senescence and are the means of ten replications ± SD. Treatment Tepals on flowers (80 % oxygen) Isolated tepals SOD-inhibitor DDTC (diethyldithiocarbamate; 1 mM) Catalase inhibitor AT (3-amino-1,2,4-triazole; 0.01–5 mM) SOD inhibitor + catalase inhibitor DDTC (1 mM) and AT (5 mM) Oxidizing agents Paraquat (1 mM) DL-buthionine sulfoximide (0.01–5 mM) Antioxidants/free radical scavengers Iso-ascorbate (0.1–5 mM) DMSO (dimethyl sulfoxide; 0.7–70 mM) 2-Mercaptoethylamide (0.01–5 mM) Butylated hydroxyanisole (0.01–0.5 mM) Phorbol-12-myristate-13-acetate (0.01–5 mM) Tiron (1,2-dihydroxybenzene-3,5-disulfonate; 0.1–1 mM)
activity did not affect the time to senescence in isolated tepals. No effect was observed even when the two chemicals were applied together (table I). All oxidizing agents and all antioxidants/free radical scavengers tested also had no effect on senescence in isolated tepals (table I).
3. DISCUSSION We hypothesized that a decrease in the activity of a number of enzymes that normally prevent the build-up of elevated free radical concentrations might partially account for the processes leading to the observed death of mesophyll cells and the visible senescence symptoms. The present experiments, however, show no changes in the activities of SOD, the two hydrogen peroxide reducing enzymes investigated, or GR, prior to the onset of visible symptoms of tepal senescence. Catalase activity even increased until the time of visible senescence, but its activity was low compared to that of APX hence its contribution to hydrogen peroxide scavenging is apparently low. Cycloheximide (CHX) application delayed the onset of the visible senescence symptoms in Iris tepals [28] if applied before day 2 following harvest, i.e. 3 d prior to the senescence symptoms (Pak and van Doorn,
Treated tepals (d)
Controls (d)
4.6 ± 0.4
4.7 ± 0.3
3.8 ± 0.3
3.9 ± 0.4
3.7 ± 0.3
3.9 ± 0.4
3.6 ± 0.4
3.8 ± 0.5
3.9 ± 0.4 3.5 ± 0.1
3.8 ± 0.2 3.5 ± 0.1
3.6 ± 0.3 3.5 ± 0.4 3.4 ± 0.2 3.8 ± 0.3 4.0 ± 0.1 4.0 ± 0.3
3.7 ± 0.4 3.5 ± 0.3 3.5 ± 0.4 3.9 ± 0.4 4.0 ± 0.1 3.9 ± 0.4
unpubl. results). This small window in the CHX effect indicates the importance of processes probably related to protein synthesis, which occur relatively early after flower opening. No decrease in SOD, APX, catalase or GR activities accompanied this window of CHX activity. The present data, therefore, suggest that the early processes leading to senescence were apparently not due to a decrease in radical scavenging by these enzymes. By the time the tepal edges showed inrolling and wilting, a decrease in the activity of SOD and APX was observed. The combined decrease of these two enzymes may result in high superoxide and high hydrogen peroxide concentrations. The latter chemical apparently is the cause of hypersensitive cell death in soybean leaves [21]. It may directly, or indirectly via the formation of free radicals, be involved in the last stages of senescence and cell death in Iris tepals. We observed five GR isoforms in Iris tepals. Similarly, some or several isoforms have been detected in other plant tissues, such as needles of Eastern white pine [3] and in maize mesocotyl [4]. The presence of various GR isoforms and their response to stress has been reviewed recently [23]. The relationship between the activity of GR isoforms and senescence, if any, is as yet unclear. Our preliminary experiments with native gels showed one band each for SOD, APX and
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catalase. Each of these bands did not decline in activity until day 5. Our molecular work showed three SOD genes, one being expressed at much higher levels than the other two, and expression until visible senescence (unpubl. results). The literature reports various isoforms of SOD, APX and catalase. Our preliminary tests with native gels apparently showed the isoforms with the highest activity. The absence of a decline in their activity until day 4 is in agreement with the biochemical data shown in figures 1–4. Elevated oxygen concentrations generally induce a higher concentration of free radicals, in particular superoxide and other oxygenated species [17]. In our tests, an oxygen concentration of 80 % had no effect on the time to tepal senescence. In these experiments, the high oxygen level was maintained until the first visible symptoms of senescence were noted. A range of chemicals, that result in elevated levels of free radicals or are known either to scavenge for free radicals or to protect against free radical damage, included in the vase water also had no effect on the time to senescence. In addition, we previously found no effect of some other antioxidants, such as sodium benzoate [10]. These results are not conclusive but may indicate the absence of a role for free radicals in Iris tepal cell death. Petal senescence in carnation, in contrast, is delayed by sodium benzoate [6], Tiron [7], iso-ascorbate [25] and by 3,4,5-trichlorophenol, a scavenger not tested in the present experiments [26]. The difference between carnation and Iris may be due to a different mechanism leading to cell death. It may or may not relate to the fact that petal wilting in carnation occurs after an increase in ethylene production [26], whereas in Iris such an increase is not observed [10]. It is concluded that the activities of the main enzymes involved in scavenging superoxide ions, and the activities of two enzymes involved in hydrogen peroxide reduction, in the tepals of Iris flowers, apparently do not decrease prior to cellular death and the visible senescence symptoms. This may indicate that reduced capacity for free radical scavenging by these enzymes is not involved in the early processes leading to cell death. The very late processes, in contrast, may be causally related to the observed decrease in activities of both SOD and APX. However, the evidence for these hypotheses are so far only preliminary as no detailed assessment was made on the changes in various organelles of the senescent cells, and the enzyme activities have not been determined in vivo. The present results do, therefore, not allow a final conclusion on the role of free radicals in the
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mechanism leading to tepal cell death. Further experimentation is required to assess free radical levels, and the role of the defence against free radicals by glutathione, ascorbic acid and tocopherol. Nonetheless, the present experiments tend to question the widely held, but unsubstantiated, hypothesis that petal/tepal cell death is due to an explosion of free radical levels. This explosion is thought to be due to reduced efficiency of free radical scavenging and/or to membrane lipid peroxidation. We previously also found no clear evidence for membrane lipid peroxidation, prior to the visible senescence symptoms in Iris tepals [10]. It is also concluded that the present results do not substantiate the hypothesis that a decrease in free radical scavenging is involved in the early processes leading to tepal cell death.
4. METHODS 4.1. Plant material Iris flowers (Iris × hollandica, cv. Blue Magic), 40 cm long, were cut in the morning, placed immediately in water and transported (for 2 h) in water to the laboratory. Flowers were harvested when the petals just emerged above the green sheath leaves, i.e. at the time just before flower opening. Tepal edges senesce prior to the more proximal parts. Tepal edges were cut along a straight line from the widest area of the tepal to the top, on both sides, immediately upon arrival of the flowers in the laboratory and on five subsequent days, at the same time of day. The weight of the edges of the five flowers was determined in triplicate, immediately after severing, and after 72 h at 70 °C.
4.2. Extraction procedure Cut edges meant for enzyme assays were immediately placed in liquid nitrogen, and the collected material was stored at –80 °C, then lyophilized. Of the material 0.3 g, representing about twenty flowers, was ground with pestle and mortar, using 15 mL 50 mM potassium phosphate buffer (pH 7.8), also including 0.1 mM EDTA, 2 mM dithiothreitol and 1 % (w/v) polyvinylpyrrolidone. The homogenate was centrifuged at 25 400 × g for 25 min and the supernatant was filtered through two layers of Miracloth and passed through a PD 10 column (containing Sephadex G 25; Pharmacia, Uppsala, Sweden) to remove pigments and salts. All steps of the extraction procedure were carried out at 1–4 °C.
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During vase life at 20 °C, the flower opens on days 1 and 2, and starts to show inrolling of the tepal edges on days 4 to 5, depending on the experiment. The edges are then liquid-logged. During the next days, inrolling and the soaked appearance progresses towards the middle of the tepals.
4.3. Enzyme assays All enzyme assays were checked for unspecific background rates, and, where these occurred, were corrected for. The activity of superoxide dismutase (EC 1.15.11) was measured according to Giannopolitis and Ries [15]. The reaction mixture contained 1.3 M riboflavine, 13 mM methionine, 63 M nitroblue tetrazolium (NBT) in 0.1 M phosphate buffer (pH 7.8), and 25 µL extract in a final volume of 3 mL. SOD activity was assayed by measuring the ability of the extract to inhibit the photochemical reduction of NBT. Glass test tubes containing the mixture were immersed in a water bath at 25 °C and were illuminated for 15 min with a fluorescent lamp (Philips MLL 500 W, Eindhoven, the Netherlands). Identical tubes, held in darkness, served as blanks. The absorbance was read at 560 nm. One unit SOD was defined as 50 % inhibition of the NBT photoreduction to blue formazan and SOD activity of the extracts was expressed as units SOD per mg protein. Catalase (EC 1.11.1.6) activity was determined at 25 °C according to Clairbone [12]. The reaction mixture assay contained 3.125 mM H2O2 in 50 mM phosphate buffer (pH 7.0) and 200 µL enzyme extract, a total volume of 3 mL. Catalase activity was estimated by the decrease of absorbance of H2O2 at 240 nm. Glutathione reductase (EC 1.6.4.2.) activity was assayed at 25 °C according to Esterbauer and Grill [14], by following the rate of NADPH oxidation at 340 nm. The mixture contained 0.5 mM NADPH, 10 mM oxidized glutathione, 3 mM MgCl2 in 0.1 M phosphate buffer (pH 7.8), and 100 µL extract in a total volume of 400 µL. The activity of ascorbate peroxidase (EC 1.11.1.11) was assessed using the method described by Nakano and Asada [24]. In some species, APX is labile when extracts are prepared in the absence of ascorbate. Extraction of our material with and without added ascorbate resulted in the same APX activity. The reaction mixture contained 50 mM of a potassium phosphate buffer (pH 7.0), 0.5 mM ascorbate and 0.4 mM hydrogen peroxide, and 200 µL enzyme extract in a total volume of 1 mL. Peroxidase activity was determined by the decrease in absorbance of ascorbate at 290 nm.
4.4. Protein level The protein concentration of the extracts was determined according to Bradford [9], using the BioRad protein assay kit and bovine serum albumin as a standard. 4.5. Gel electrophoresis and activity staining All gels (precast minigels; 5 × 4 cm) were ran on a PhastSystem (Pharmacia, Uppsala, Sweden). GR isozymes were electrophoresed on native 12.5 % polyacrylamide gels (PhastGel homogeneous 12.5, Pharmacia). Activity staining was adapted from Anderson et al. [3] and was carried out in 0.4 mM NADPH, 3.4 mM oxidized glutathione (GSSG), 1.2 mM 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), 0.3 mM 2,6-dichlorophenol-indophenol (DPIP) and 50 mM Tris-HCl buffer (pH 7.75). 4.6. Effects of high ambient oxygen concentrations and chemicals Flowers with a stem length of 15 cm or isolated tepals were placed in demineralized water and continuously held in stainless steel tanks at 21 and 80 % oxygen, in darkness and a relative humidity of about 80 %. The carbon dioxide concentration was held at 0.045 %. The remainder of the gas phase was nitrogen. Flowers were taken out of the oxygen treatments on days 3, 4 and 5 of treatment, and individually placed in fresh demineralized water in the climate-controlled room at 20 °C, 60 % RH; and a photon flux density of 15 µmol·m–1·s–1 from 7:00 to 19:00 hours. The base of isolated flag tepals was continuously immersed in aqueous solution of chemicals that purportedly interfere with free radical metabolism: diethyldithiocarbamate (DDTC; Sigma), an inhibitor of SOD [1]; paraquat (= methylviologen) results in elevated concentrations of the superoxide and hydroxyl anions [5]; 3-amino-1,2,4-triazole (AT), an inhibitor of catalase [11]; buthionine sulfoximide, a compound that increases endogenous free radical levels by inhibition of glutathione synthesis [1], and the following compounds that are antioxidants/free radical scavengers: isoascorbate [25], butylated hydroxyanisole [29], DMSO (dimethyl sulfoxide), which scavenges hydroxyl radicals [5], 2-mercaptoethylamide [16], phorbol-12myristate-13-acetate [1], and Tiron (1,2-dihydroxybenzene-3,5-disulfonate; [7]). These chemicals were all dissolved directly in water, and were tested at five concentrations ranging from 10–5 to 5·10–3 M. The solutions were not renewed. Tepals were placed at 20 °C, 60 % RH, and a photon flux density of 15 µmol·m–1·s–1 from 7:00 to 19:00 hours.
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4.7. Statistics All data on enzyme activity showed less than 5 % variance between repeat measurements of the same extract. The standard deviation shown in the graphs represents variance between repeated extracts (two to three) of the same lyophilised material. These experiments were repeated once. The experiment with elevated oxygen concentration was performed three times. Each oxygen concentration (21 and 80 %), at each of the sampling times, was maintained in two duplicate tanks, ten replicate flowers per tank. Experiments with chemicals included ten replicate tepals, and were repeated once. When necessary, the results were compared by analysis of variance using the GENSTAT V program (Rothamsted UK) and F-test at P < 0.05.
Acknowledgments. The authors are grateful to Harmannus Harkema (ATO-DLO, Wageningen) who repeated some of the experiments on the effect of enzyme inhibitors and free radical scavengers.
[10] [11] [12] [13]
[14] [15] [16] [17]
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