Scientia Horticulturae 95 (2002) 277284

Catalase activity, hydrogen peroxide content and thermotolerance of pepper leaves

Jeffrey A. Anderson*
Department of Horticulture and Landscape Architecture, Oklahoma State University, 360 Ag Hall, Stillwater, OK 74078, USA Accepted 2 April 2002

Abstract Activated forms of oxygen, including hydrogen peroxide (H2O2), have been implicated in plant responses to stress. Catalases (CAT) and peroxidases are the primary enzymatic detoxiers of H2O2 in most plant tissues. Pepper (Capsicum annuum L.) leaf disks oated on 0100 mM H2O2 solutions in the dark were not affected or showed minimal effects depending on the assay. Changes in electrolyte leakage (EL) and evolution of ethylene and methanol from H2O2-treated disks were slight compared to freeze-killed tissues, indicating that pepper leaves had considerable capacity to detoxify exogenous H2O2. H2O2 concentration in leaf tissue was not signicantly affected by injurious (48 8C) or lethal (54 8C) temperature treatments. As plants aged from 6 to 10 weeks, thermotolerance increased from 45 to 50 8C based on calculated inection points (Tmid) of sigmoidal EL response curves. A further increase of about 1 8C occurred from 10 to 14 weeks after sowing. However, CAT activity decreased as plants aged from 6 to 14 weeks old. Although 11-week-old plants had lower baseline CAT activity in controls, activity was stable to a higher temperature than in 6-week-old plants (53.1 versus 48.4 8C). CAT activity was more stable than membrane integrity since an increase in EL occurred at a lower temperature than a decline in CAT activity. Thermotolerance of plants exposed to the acclimating regime of 38/30 8C (day/night temperatures) increased from 50.7 to 53.9 8C. CAT activity also showed an adaptive response with the inection point increasing from 52.6 to 56.8 8C. Changes in H2O2 levels do not appear to have a direct role in injury to pepper leaves exposed to high temperatures in the dark. Thermal inactivation of CAT may be a consequence, rather than a cause of high temperature injury. # 2002 Elsevier Science B.V. All rights reserved.
Keywords: Activated oxygen; Capsicum annuum; Free radical; Heat stress; Scavengers

* Tel.: 1-405-744-5414; fax: 1-405-744-9709. E-mail address: jander@okstate.edu (J.A. Anderson).

0304-4238/02/$ see front matter # 2002 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 2 ) 0 0 0 7 6 - 6

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1. Introduction Plant responses to environmental stress have been associated with activated forms of oxygen, including hydrogen peroxide (H2O2), singlet oxygen, superoxide, and the hydroxyl radical. Free radicals and nonradical forms of activated oxygen (AO) are formed by fatty acid b-oxidation (del Rio et al., 1998), membrane-associated oxidases (Desikan et al., 1996), photorespiration (Faria et al., 1999) and as byproducts from electron transport chains (Shewfelt and Purvis, 1995). While excessive levels of AO cause injury (Prasad et al., 1994), smaller amounts trigger acclimation (Foyer et al., 1997). Whether AO molecules are benecial or damaging depends on the amount, type and location of AO, and the status of defense systems. Uncontrolled production of AO can inactivate biomolecules or initiate chain reactions destroying membrane structure and function. Benecial roles for AO include cell wall polymerization and generation of secondary messengers for signal transduction pathways. Many cellular compounds act as preemptive scavengers, detoxifying AO before they react with lipids, proteins and nucleic acids. Enzymatic defenses include superoxide dismutases (EC 1.15.1.1) which convert the superoxide radical to H2O2, and the catalases (CAT) (EC 1.11.1.6) and peroxidases (primarily ascorbate peroxidase: EC 1.11.1.11) which trigger the conversion of H2O2 to water and oxygen. While cellular defenses are adequate to handle the oxidative load under normal conditions, temperature stresses may tip the scales in favor of oxidation by increasing the concentration of prooxidants and/or impairing defenses (Purvis and Shewfelt, 1993; Walker and McKersie, 1993). Chilling injury can increase the level of AO in plants, especially when tissues are exposed to low temperatures at high light intensity (Wise and Naylor, 1987). Lower activity of defensive enzymes at chilling temperatures can impair the plants ability to detoxify products of oxygen photoreduction (Baker, 1994). Damage to maize (Zea mays L.) seedlings exposed to chilling temperatures in the dark was also related to AO (Prasad et al., 1994). Acclimated seedlings had modestly increased H2O2 levels and CAT activity increased several fold. Non-acclimated seedlings exhibited large increases in H2O2, but not in CAT activity following a 4 8C exposure in the dark which killed 98% of the plants. Hydrogen peroxide can also increase in plant tissues exposed to heat stress in the dark. Although the source of hydrogen peroxide was not known, levels increased about 65% in mustard seedlings after exposure to 55 8C for 1 h, a treatment that killed about 80% of the plants (Dat et al., 1998). Although a role of hydrogen peroxide in acclimation has been established (Dat et al., 1998; Lopez-Delgado et al., 1998), it is not clear whether H2O2 is directly involved in high temperature injury. My objectives were to: (1) determine the toxicity of exogenous H2O2 to pepper leaf disks based on membrane selective permeability and evolution of stress volatiles; (2) determine whether H2O2 increased in heat-stressed leaves; (3) determine the relationship between thermotolerance and CAT activity of pepper leaves ranging in age, and whether thermal deactivation of CAT activity occurred over the same temperature range in tissues differing in thermotolerance; (4) determine if changes in thermal stability of pepper CAT activity occurred during temperatureinduced acclimation.

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2. Materials and methods Early Calwonder pepper plants were grown in commercial mix (Universal Mix, Strong Lite, Pine Bluff, Ark.) in a growth chamber at 24/20 8C day/night cycles. A photosynthetic photon ux of %350 mmol m2 s1 at canopy height was maintained during the 14 h photoperiod. Plants were fertilized at each watering with 0.75 g l1 20N9P17K soluble fertilizer. Pepper plants were acclimated by transfer to a paired growth chamber set at 38/ 30 8C. The soil was kept saturated at eld capacity during the 24 h acclimation period to prevent secondary water stress. Thermotolerance of leaf tissue was determined by measuring electrolyte leakage (EL) following exposure to high temperatures. Each subsample consisted of three leaf disks (8 mm diameter) placed in a 25 150 mm test tube containing 2 ml distilled water to prevent secondary water stress. Three subsamples per treatment combination were placed into a circulating bath for 15 min at the prescribed treatment temperature. Twenty milliliters of distilled water was added to each tube after temperature treatments. Initial EL measurements were recorded with a conductance meter (Model 35, Yellow Springs Instrument Company, Yellow Springs, OH) after 22 h incubation at 24 8C on an orbital shaker. Final readings were taken following autoclaving and an additional 22 h incubation at 24 8C. EL data were expressed as initial readings/nal readings 100. The midpoint of the temperature response curve, Tmid, was calculated according to Ingram (1985). Leaf disks were exposed to exogenous H2O2 by oating disks on H2O2 solutions or water for 1 h in petri dishes. Disks were maintained at 24 8C in the dark. Additional samples were exposed to 70 8C for 1 h to provide a reference for killed tissues. Disks were rinsed in distilled water, then blotted dry before measurement of stress volatiles or EL. Disks (10 per subsample) used to measure stress-related volatiles were placed into 14.7 ml serum bottles with septa for hydrocarbon measurements after temperature treatments. After 18 h incubation at 24 8C in the dark, 1 ml headspace samples were introduced into a 30 m 0:55 mm GS-Q Megabore column (J & W Scientic, Folsom, CA) using a gastight syringe with Teon valve and plunger. The Tracor 540 (Tracor Instruments, Austin, TX) gas chromatograph was equipped with a ame ionization detector. Injector and detector temperatures were 180 and 200 8C, respectively. The column was held at 60 8C for 2 min, then raised at 10 8C/min to 160 8C. Mean production rates were calculated from response factors derived from standards. Tissue levels of endogenous H2O2 were measured based on the Ngo and Lenhoff (1980) procedure, with modications (Kerdnaimongkol and Woodson, 1999). Leaf tissue (1 g) was frozen in liquid N2, then homogenized in 10 ml TCA (5% by volume) and 0.2 g activated carbon. The homogenate was ltered through two layers of Miracloth (Calbiochem-Novabiochem, San Diego, CA), then centrifuged at 16,000 gn for 15 min. Three hundred microliters of the supernatant was introduced into a cuvette containing 9.9 mmol 3-(dimethylamino)benzoic acid, 0.2 mmol 3-methyl-2-benzothiazoline hydrazone and 0.25 units type 1 horseradish peroxidase (Sigma-Aldrich, St. Louis, MO) in 0.375 M sodium phosphate buffer (pH 6.5) in a total reaction volume of 3 ml. The colorimetric assay was conducted at 24 8C in the dark for 24 h before measuring absorbance at 590 nm. CAT activity was measured spectrophotometrically according to Aebi (1983). Leaf disks (8 mm diameter) were homogenized in 1 ml 50 mM phosphate buffer (pH 7.0) containing

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5 mg polyvinylpolypryrolidone in micro-centrifuge tubes using a Teon pestle. Extracts were centrifuged at 16,000 gn for 15 min. An aliquot of the supernatant was diluted 1:14 with buffer before assay. The 3 ml reaction volume contained 1.5 ml crude extract and 1.5 ml 30 mM H2O2. The decrease in absorbance at 240 nm was monitored in kinetic mode as H2O2 was degraded. APOX activity was measured from the same crude extract as CAT, except without dilution. Oxidation of ascorbate was determined by the decrease in absorbance at 290 nm as described by Mittler and Zilinskas (1991). All experiments consisted of at least three subsamples per treatment combination and were conducted on at least three dates. Mean separations by Duncans multiple range test using treatment date as the error term were conducted as appropriate following analysis of variance.

3. Results Pepper leaves were not affected, or slightly affected, by exogenous H2O2 depending on the assay (Table 1). Although disks oated on 100 mM H2O2 had greater EL than disks oated on 0 or 0.1 mM H2O2, values did not exceed 33%. In contrast, samples exposed to 70 8C had 93% EL. Ethylene, a sensitive indicator of plant stress, increased with increasing H2O2 concentration, but the change was not signicant at P 0:05. Ethylene was not detected from 70 8C samples. Methanol evolution increased following treatment with 100 mM H2O2, but the values were more than an order of magnitude less than freezekilled samples. A previous study indicated that evolution of methanol was closely correlated with EL in pepper leaves exposed to temperature stresses (Anderson, 1994). The source of methanol appeared to be de-esterication reactions of pectin associated with changes in pH in heat-stressed tissue (Anderson, 1994) and in plantpathogen interactions (Mundodi et al., 1998). Tissue levels of H2O2 were not signicantly affected by exposure to 48 or 54 8C (Table 2). Based on EL, membrane integrity was moderately affected by heating for 15 min at 48 8C and severely damaged by the 54 8C treatment (Table 2). CAT activity decreased by about one-third following 48 8C treatment and was barely detectable following 54 8C. APOX activity was not affected by exposure to 48 8C, but was reduced to 6% of the control value following a 15 min exposure to 54 8C.

Table 1 Hydrogen peroxide toxicity to pepper leaf disks floated on solutions for 1 h in the darka,b H2O2 (mM) 0 0.1 100 EL (%) 24 a 26 a 33 b Ethylene (nmol l1) 14 a 16 a 19 a Methanol (nmol l1) 32 a 38 a 193 b

a Means within a column followed by the same letter are not significantly different at P 0:05 using Duncans multiple range test. b EL, ethylene and methanol values for samples killed by exposure to 70 8C were 93%, not detectable and 3297 nmol l1, respectively.

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Table 2 Hydrogen peroxide concentration, CAT activity, ascorbate peroxidase (APOX) activity, and EL from 8-week-old pepper leaves exposed to 24, 48 or 54 8C for 15 mina Temperature (8C) 24 48 54 H2O2 (mmol/g FW) 6.9 a 6.8 a 6.6 a CAT activity (mmol H2O2/s/g FW) 128 a 104 a 15 b APOX activity (nmol ASC/s/g FW) 122 a 140 a 7b EL (%) 23 a 61 b 92 c 0:05 using

a Means within a column followed by the same letter are not significantly different at P Duncans multiple range test.

Tmid values increased from 45 to 50 8C as plants aged from 6 to 10 weeks old and were relatively stable at 5051 8C in plants 1014 weeks old (Fig. 1). CAT activity decreased as plants aged from 6 to 14 weeks old. Since CAT activity declined markedly in heat-damaged tissues (Table 2), temperature stability of CAT was determined in tissues of different hardiness level (6 and 11 weeks old). Six-week-old leaves were susceptible to high temperature-induced loss of selective permeability with an EL Tmid of about 45 8C (Fig. 2). CAT activity was also thermo-labile with an activity midpoint of about 48 8C. Membrane and CAT stability were greater in 11-week-old leaves with EL and CAT stability midpoint values of 51.5 and 53.5 8C, respectively. CAT activity was more stable than EL in both 6 and 11-week-old leaves. Baseline CAT activity (controls) was less in the older leaves, consistent with results reported in Fig. 1. As plants acclimated to tolerate higher temperatures (EL Tmid increased from 50.7 to 53.9 8C), CAT also exhibited increased stability (52.6 to 56.8 8C). Activity levels were 19% higher (not signicant at P 0:05) in non-stressed acclimated tissues, compared with nonstressed controls after a 24 h acclimation period (data not presented).

Fig. 1. Midpoints (Tmid) of EL versus temperature curves and CAT activity of pepper leaves 614 weeks old. Mean S:E: of at least nine measurements are reported.

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Fig. 2. EL (solid lines) and CAT activity (dashed lines) of pepper leaf disks exposed to temperatures from 24 to 56 8C for 15 min. Mean S:E: of nine measurements are reported.

4. Discussion While EL and methanol evolution indicated a measurable response to H2O2, it was clear that leaves were resistant to high levels (100 mM) of exogenous H2O2. Even though H2O2 can pass through membranes and participate in reactions at sites remote from production, exogenous application may be detoxied at a different site or by a different mechanism than endogenous H2O2. Nevertheless, AO defenses in pepper leaves were sufcient to detoxify a heavy H2O2 load. Similar results were reported by Kauss and Jeblick (1996), who found that exogenous H2O2 was rapidly degraded by cucumber (Cucumis sativus L.) hypocotyls. Lethal damage to plants via H2O2 may require impaired defenses or conversion to AO with greater toxicity than H2O2. In contrast to increases in H2O2 observed in heat-stressed mustard seedlings (Dat et al., 1998), heat-injured and heat-killed pepper leaves had similar levels of H2O2 as controls (Table 2). Data suggest that there is not a simple relationship between H2O2 concentration and activity of CAT and APOX. Tissues exposed to 54 8C had very little CAT or APOX activity, but concentrations of H2O2 were similar to control levels. MacRae and Ferguson (1985) reported decreased CAT activity and H2O2 levels in several species exposed to chilling temperatures. If H2O2 contributes to tissue damage at high temperatures and CAT is the primary detoxier, it may be expected that increases in thermotolerance would be paralleled by increases in CAT activity. When this relationship was explored by examining plants of different age, and therefore different thermotolerance, the opposite trend was observed. Older plants were more thermotolerant, but had lower CAT activity (Fig. 1). Although it has been known that CAT activity decreases during senescence (Dhindsa et al., 1982), CAT activity in pepper leaves declined before visible symptoms of senescence were observed. In addition to baseline CAT activity, CAT thermal stability was also examined in leaves with different thermotolerance. Although older leaves had lower baseline CAT activity, the activity was maintained at higher temperatures in 11-week-old plants compared with

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6-week-old plants (Fig. 2). The enzyme was not puried and characterized from 6 and 11week-old plants, so it is not known if there were differences in protein properties. Alternatively, the observed differences in thermal stability may be related to CAT inactivation being a secondary event or related to changes in protective compounds such as molecular chaperones. Examination of acclimated plants indicated that adaptation was primarily associated with enzyme stability to higher temperatures, rather than higher baseline levels. A timecourse study of CAT activity during acclimation may have shown signicant treatment effects, as observed in oak (Quercus suber L.) seedlings exposed to a 45 8C heat shock (Faria et al., 1999). However, transient changes in CAT activity during acclimation may have more to do with signal transduction leading to induction of heat shock genes than directly preventing H2O2-induced injury. It is possible that acclimation-induced heat shock proteins stabilized CAT activity since protection of bovine liver CAT activity was provided by molecular chaperones (Hook and Harding, 1997). The greater CAT stability in acclimated pepper leaves could also have involved a shift to more stable isozymes. Scandalios (1994) reported different thermal stabilities of CAT isozymes from maize. High temperature injury may involve different mechanisms depending on environmental conditions before or during the heat stress. Genetic differences may also be important, with elevated levels of H2O2 contributing to heat stress injury in some plants, such as mustard seedlings (Dat et al., 1998), but not in pepper leaves. Unless there are changes in tissue sensitivity, it does not appear that H2O2 plays a direct role in high temperature injury to pepper leaves. Loss of CAT activity at high temperatures may be a consequence, rather than a cause, of membrane dysfunctions in dark-treated pepper leaves.

Acknowledgements The technical assistance of Lynda Wells and the use of Shiping Dengs equipment is gratefully acknowledged. Approved for publication by the Director, Okla. Agricultural Experiment Station. Mention of a trademark, proprietary product, or vendor does not constitute a guarantee or warranty of the product and does not imply its approval to the exclusion of other products or vendors that also may be suitable. References
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