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Antioxidant capacity is correlated with susceptibility to leaf spot caused by a rapid temperature drop in Saintpaulia (African violet)
Scientia Horticulturae 88 (2001) 59±69
Antioxidant capacity is correlated with susceptibility to leaf spot caused by a rapid temperature drop in Saintpaulia (African violet) S.J. Yanga,*, M. Hosokawaa, Y. Mizutaa, J.G. Yunc, J. Manob, S. Yazawaa a
Laboratory of Vegetable and Ornamental Horticulture, Department of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Kyoto 606-8502, Japan b The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan c Department of Horticulture, Chinju National University, Chinju City, South Korea Accepted 13 October 2000 Abstract The relationship between the susceptibility to rapid temperature drop induced leaf injury, i.e. ``leaf spot'', and the antioxidant capacities of several Saintpaulia (African violet) cultivars was examined. Cultivars `Ritali' and `Tamiko', that are more susceptible to leaf spot caused by a rapid drop in leaf temperature from 30 to 158C than are cultivars `Maui' and `New Jersey'. The susceptible cultivars were also more susceptible to oxidative stress caused by H2O2 and active chlorine than the tolerant cultivars. Reduction of available chlorine, considered to be accelerated by antioxidants of leaf tissue, was rapid in `Maui' and `New Jersey'. Activity of the antioxidant enzymes superoxide dismutase and catalase were higher in the leaves of `Maui' and `New Jersey' than in the susceptible cultivars. These ®ndings show a correlation between leaf spot injury and antioxidant capacity in Saintpaulia leaves. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Saintpaulia; Reactive oxygen species; Leaf injury; Rapid temperature drop; Antioxidant capacity
1. Introduction Leaf spot of Saintpaulia is caused by a rapid drop in leaf temperature during irrigation (Poesch, 1942; Elliot, 1946; Maekawa et al., 1987) or exposure to cold * Corresponding author. Tel.: 81-75-753-6048; fax: 81-75-753-6068. E-mail address: [email protected] (S.J. Yang).
0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 2 3 5 - 1
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air (Maekawa et al., 1987). Leaf spot reduces the ornamental value of the plants and is a problem in commercial production. Research on the mechanism of leaf spot has focused on the palisade cells (Yun et al., 1996a, 1997, 1998), the main site of injury (Elliot, 1946; Maekawa et al., 1987). A rapid drop in leaf temperature for a few seconds drastically decreases photosynthetic activity (Yun et al., 1997, 1998) and destroys the ultrastructural composition of palisade cells (Yun et al., 1996a). This injury is irreversible and can be caused even by immersion in 258C water if the leaf temperature before the immersion was higher than 358C. Thus, leaf spot is caused by a temperature difference, and does not always require chilling temperature (i.e. below 108C). On this point and on the rapidness of its development, leaf spot injury can be distinguished from typical chilling injury. Yasuda et al. (1997) showed the induced production of reactive oxygen species (ROS) in the leaf extracts following a rapid temperature drop. Also, the morphological changes including shrinkage of cytoplasm, condensation of chromatin and disarrangement of organelles, were observed in the palisade cells of Saintpaulia `Ritali' after the temperature drop (Yun et al., 1996a; Yasuda et al., 1997). These symptoms are very similar to the apoptotic morphological changes induced by ROS (Fuchs et al., 1997). However, there has been no study on the relationship between the leaf spot and antioxidant capacity in Saintpaulia. When a plant is exposed to stress, the production of active oxygen can exceed the capacity of the antioxidant system, resulting in oxidative damage. Thus, antioxidant capacity is a major factor in determining the stress tolerance of plants, and the plants having higher antioxidant capacity by acclimation or transformation show stress tolerance (Gupta et al., 1993; Allen, 1995; Anderson et al., 1995). Superoxide dismutase (SOD), the ®rst enzyme in the detoxifying process, converts O2ÿ to O2 and H2O2, which is further decomposed to O2 and H2O by catalase (Fridovich, 1978). NaOCl has a disinfecting or sterilizing effect as active chlorine, which is a powerful anti-fungal agent, and is used for sterilization of plant material in in vitro culture. H2O2 also has a toxic effect on organisms. Both are ROS. We observe that the occurrence of leaf spot varies among cultivars in Saintpaulia, but it is unknown what is the main factor to determine the sensitivity for generating leaf spot. The objective of this research is to determine the relationship between the susceptibility to leaf spot and the antioxidant capacity in Saintpaulia. 2. Materials and methods 2.1. Plant material Saintpaulia ionantha (African violet), cultivars `Ritali', `Tamiko', `Maui' and `New Jersey', were grown in a glasshouse in which the temperature was
S.J. Yang et al. / Scientia Horticulturae 88 (2001) 59±69
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maintained at 17±258C, and the irradiation below 252 mmol mÿ2 sÿ1, during the daytime. Fully expanded leaves were detached from each four cultivars before experiment and immediately used. 2.2. Rapid temperature drop treatment and quanti®cation of leaf spot Each detached leaf was immersed in 308C water for 3 min, and then dipped in 158C water for 3 s to make a rapid drop in leaf surface temperature by 158C. In our preliminary experiment, leaf surface temperature was detected suddenly after the immersion by thermography so that it is almost same as water temperature. The room temperature was 20±258C. The leaf spot severity was quanti®ed by an image analysis technique as described previously (Yun et al., 1996b). The leaf images were captured using a digital camera and the images were transferred to a personal computer to quantify the area of leaf spot by using image software (Adobe PhotoShop Version 5.0). The severity of leaf spot was shown by the percentage of the damaged leaf area to the total leaf area. 2.3. Quanti®cation of leaf injury caused by H2O2 and NaOCl The leaves were rinsed lightly with a detergent, and the leaf discs were excised using a leaf punch (Disposable Biopsy Trepan). Midrib parts were not included in the sampled leaf discs. The super¯uous leakage from the cut section of leaf discs was removed using ®lter paper. Then, ®ve leaf discs, 8 mm in diameter, were immersed in 50 ml of 0.2 M mannitol solution containing 1, 5 or 10 mM H2O2, and agitated (50 rpm) at room temperature in light (39 mmol mÿ2 sÿ1). Then, the electric conductivity of the solution was measured with a conductivity meter (CM-1K, Toa Electrics Ltd., Tokyo, Japan). The solution of 0.2 M mannitol with the leaf discs but without H2O2 was used as a control, and the relative injury due to H2O2 was calculated as the ratio of the conductivity of H2O2 solution to that of control solution, both measured after 3 h treatment. To elucidate the toxic effect of chlorine itself, we immersed ®ve leaf discs of `Ritali' in the 50 ml of NaOCl solution containing 20 ppm available chlorine, itself, in the presence or absence of sodium thiosulfate (100 ppm) that detoxi®es active chlorine (Dychdala, 1991) and agitated under the same condition as above. After a 3 h agitation, the leaf discs were washed with distilled water and incubated in 50 ml of 0.2 M mannitol solution for 3 h and electrolyte conductivity was measured. The control was agitated in distilled water for 3 h and then immersed in mannitol solution for 3 h. The relative injury was calculated as the ratio of the conductivity of NaOCl solution to that of control solution. The relative leaf injury caused by active chlorine (5 and 10 ppm of available chlorine) was calculated as same as above, measured after 4 h treatment.
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2.4. OClÿ reduction by leaf discs Ten leaf discs, 8 mm in diameter, were immersed in 30 ml of NaOCl solution (4 ppm) with agitation (50 rpm) at room temperature in light (39 mmol mÿ2 sÿ1) for 210 min. `Available chlorine' is expressed in terms of the equivalent amount of elemental chlorine (Dychdala, 1991). The residual-available chlorine in the NaOCl solution in which the samples were immersed was measured by using chlorine comparator (Water tester, Sibata, Japan) according to the N,N-diethyl-pphenylenediamine (DPD) method (Gordon et al., 1991). Seven replicates of the solution containing leaf discs were prepared for each cultivar, and the available chlorine was assayed at 30 min intervals using one of the replications chosen at random. 2.5. Determination of enzymatic activities of SOD and catalase Leaf extract was prepared at 08C as follows. Leaf samples of 0.5 g fresh weight from the fully expanded leaves were ground rapidly in a mortar with a pestle in the presence of 1 ml of 50 mM potassium phosphate (pH 7.0). Following centrifugation at 8000 g for 10 min at 48C the supernatant was removed for enzymatic analysis. The supernatant was gel-®ltered over Sephadex G-25 (NAP-5 column, Pharmacia, Sweden), equilibrated with 50 mM potassium phosphate (pH 7.0), to remove small molecules. SOD activity was determined spectrophotometrically by measuring the inhibition of O2ÿ-dependent reduction of nitroblue tetrazolium (NBT), according to the xanthine±xanthine oxidase system (Beyer and Fridovich, 1987). One unit of SOD was de®ned as the quantity of enzyme required to inhibit the reduction of NBT by 50% in a reaction volume of 1 ml. Catalase activity was determined by monitoring the production of O2 from H2O2 with a Clark-type oxygen electrode at 258C by the method of Kerdnaimongkol and Woodson (1999) with some modi®cations. The assay was carried out in a stirred reaction cuvette containing 50 mM HEPES (pH 7.6) and 100 mM H2O2. The decomposition of H2O2 was initiated with 100 ml of the enzyme extract. 3. Results 3.1. Susceptibility for generating leaf spot and oxidative stress caused by NaOCl and H2O2 in four cultivars Leaves pre-incubated at 308C were exposed to a rapid temperature drop by being immersed in water at 158C. This treatment lowers the leaf temperature from 30 to 158C in 3 s, as determined by thermography in our preliminary experiment. By this treatment, more than 80% of the leaves in `Ritali' and `Tamiko' were
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Fig. 1. Injury of leaf spot caused by exposure to water of 308C for 3 min followed by 158C for 3 s in Saintpaulia cultivars `Maui', `New Jersey', `Ritali' and `Tamiko'. z: The details in Section 2. Bars represent means S:E: of seven replicates.
injured, whereas less than 20% in `Maui' and `New Jersey'(Fig. 1). Leaf spot was not caused by the immersion in water either at 30 or 158C in any of these cultivars. This result corresponds with the observation that the leaf spot caused by overhead irrigation with low temperature water in a glasshouse was severer in `Ritali' and `Tamiko' than in `Maui' and `New Jersey'. To evaluate the susceptibility to oxidative stress, we immersed and agitated the leaf discs of four cultivars in the H2O2 solution at a low concentration for 3 h. As shown in Fig. 2, `Ritali' and `Tamiko' were injured more severely than `Maui'
Fig. 2. Leaf injury induced by H2O2 in four cultivars of Saintpaulia. z: Leaf discs were incubated in each solution for 3 h and relative injury was calculated as conductivity of treatment after 3 h/ conductivity of control after 3 h. Bars represent means S:E: of ®ve replicates.
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Fig. 3. Leaf injury induced by available chlorine (HOCl, OClÿ) in four cultivars of Saintpaulia. A, Leaf injury of four cultivars in each of NaOCl solution at 5 and 10 ppm available chlorine. z: The relative injury was calculated as (conductivity of treatment after 4 h ± conductivity of treatment after 0 h/conductivity of control after 4 h ± conductivity of control after 0 h). The conductivity of treatment contains not only ion leakage from leaf tissue but also contains ion of sodium and chlorine in all cultivars. Bars represent means S:E: of three replicates. B, Amelioration of leaf injury by decomposition of available chlorine using sodium thiosulfate. y: Described in Section 2. Bars represent means S:E: of ®ve replicates.
and `New Jersey' at 5 and 10 mM H2O2. The relative injury in the H2O2 solution were discernible, the most in 10 mM H2O2 between the tolerant and sensitive cultivars to rapid temperature drop (Fig. 2). `Ritali' and `Tamiko' were more severely injured than `Maui' and `New Jersey' also in the NaOCl solution at 5 and 10 ppm (available chlorine) (Fig. 3A). `Ritali' and `Tamiko' were injured 3±4-fold more severely than `Maui' and `New Jersey' at 5 ppm of this solution. The injury induced by NaOCl was alleviated by the presence of sodium thiosulfate, a dechlorinating chemical (Fig. 3B). This indicates that the leaf injury by NaOCl solution was not caused by sodium ion but mainly by available chlorine, a powerful oxidant. These results showed that `Maui' and `New Jersey' are more susceptible to both reactive oxidants, H2O2 and active chlorine, than `Ritali' and `Tamiko'. Thus, the sensitivity order of the four cultivars to the oxidants corresponded with that to the leaf spot. 3.2. Reduction rate of available chlorine in the presence of leaf discs of four cultivars Available chlorine, OClÿ or HOCl, is a powerful oxidant and generates other ROS, 1 O2 or OH , provoking cell damage (Khan and Kasha, 1994). This assay
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Fig. 4. Reduction rate of available chlorine by the leaf discs in four cultivars of Saintpaulia. z: Ten leaf discs of each cultivar were incubated in NaOCl solution at 4 ppm of available chlorine for 210 min and the residual available chlorine was measured at the interval of 30 min.
was conducted to compare the capacity to reduce available chlorine among the cultivars, which showed different susceptibility for available chlorine. Ten leaf discs of each cultivar were immersed in 30 ml NaOCl solution containing 4 ppm available chlorine. The available chlorine in the solution decreased rapidly during the ®rst 30 min, and then gradually thereafter (Fig. 4). In the absence of the leaf discs, the concentration of available chlorine in the NaOCl solution did not change for 210 min. This shows that the reduction of available chlorine in NaOCl solution was caused by the presence of leaf discs. In the presence of the leaf discs of `Maui' and `New Jersey', most of the available chlorine (4 ppm) disappeared within 120 min, but in the presence of the leaf discs of `Ritali' and `Tamiko', 0.7 and 0.9 ppm of available chlorine, respectively, remained after a 120 min incubation. Thus, `Maui' and `New Jersey' have higher capacities to reduce available chlorine than `Ritali' and `Tamiko'. From the correlation between reducing capacity of available chlorine and susceptibility to active chlorine (Figs. 3A and 4), the cultivars show tolerance against active chlorine may depend on the detoxify-capacity of active chlorine by reduce it rapidly. 3.3. Leaf-SOD, catalase activity of four cultivars SOD activity in `Maui' and `New Jersey' was higher than that of `Ritali' and `Tamiko' (Fig. 5). Catalase activity in `Maui' and `New Jersey' were higher also
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Fig. 5. Leaf-SOD activity in four cultivars of Saintpaulia. Bars represent means S:E: of ®ve replicates.
compared with `Ritali' and `Tamiko'(Fig. 6). `Maui' showed twofold of SOD and catalase activities compared with `Ritali' and `Tamiko'. Both SOD and catalase activities in the leaves of the leaf spot-tolerant cultivars were higher than that of the susceptible cultivars. Thus, the higher tolerance to leaf spot injury and oxidative stress are signi®cantly correlated to the cultivar's SOD and catalase activities.
Fig. 6. Leaf-catalase activity in four cultivars of Saintpaulia. Bars represent means S:E: of ®ve replicates.
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4. Discussion In Saintpaulia, the sensitivity to a rapid drop in leaf temperature varied among cultivars. `Maui' and `New Jersey' were relatively more tolerant to a rapid temperature drop by 158C (30±158C) than `Ritali' and `Tamiko' (Fig. 1). Bodner and Larcher (1989) reported the chilling susceptibility of seven wild species of Saintpaulia, but there are no reports on the susceptibility for generating leaf spot in Saintpaulia cultivars. From the reason that leaf spot is a highly acute injury and from the characteristic of its morphological changes of palisade cells after the rapid temperature drop (Yun et al., 1996a), it is considered that the leaf spot is mediated by oxidative stress. However, since the development of leaf spot in Saintpaulia is extremely rapid, which the ¯uorescence intensity drops within a few seconds after the rapid drop in leaf temperature (Yun et al., 1997), direct assay of ROS related to leaf spot injury is dif®cult. In our research, leaf injury caused by H2O2 or active chlorine was severer, the higher the susceptibility of the cultivar to leaf spot (Figs. 2 and 3). The active chlorine and H2O2 are included in ROS and their cytotoxicity is largely due to their oxidizing effects (Halliwell and Gutteridge, 1999). In our research, also, the cultivars tolerant for generating leaf spot were relatively tolerant to ROS. Although, the mechanism of resistance to active chlorine is unknown, it is presumably related to the detoxi®cation of active chlorine in plant systems. Available chlorine in an aqueous solution exhibits high disinfecting power as active chlorine, which is considered to generate other radicals (Hauptmann and Cadenas, 1997). Available chlorine in NaOCl solution was reduced more rapidly in the presence of the leaf discs of `Maui' and `New Jersey' than in the presence of the leaf discs of `Ritali' and `Tamiko' (Fig. 4). In our study, reducing capacity of available chlorine has correlation with the susceptibility to active chlorine (Figs. 3A and 4). It suggest that the tolerance against active chlorine may depend on the detoxify-capacity of active chlorine by several kinds of antioxidants such as ascorbic acid, glutathione and a-tocopherol in leaf tissue (Halliwell and Gutteridge, 1999). `Maui' and `New Jersey' showed the high activities of SOD and catalase than those in `Ritali' and `Tamiko' (Figs. 5 and 6). The combined action of SOD and catalase ef®ciently eliminates O2ÿ and H2O2, and consequently, protects cellular components against the more reactive hydroxyl radical (OH ), which results from the interaction between O2ÿ and H2O2 (Fridovich, 1978). Yasuda et al. (1997) indicated that the oxygen radicals participate in the acute irreversible morphological changes, resulting in apoptotic cell death of Saintpaulia palisade cells. In our study, the cultivars showing higher SOD and catalase activities were tolerant to not only oxidative stress, but also to a rapid temperature drop. This suggests that the susceptibility to leaf spot in Saintpaulia be closely related to the
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antioxidant capacities. In addition, the measurement of chlorine-reduction capacity of leaf discs can be used as a rapid method for screening the susceptibility of leaf spot injury among cultivars in Saintpaulia. In conclusion, ROS is considered to be involved in the process causing leaf spot and the tolerance to leaf spot is correlated with the high capacity of the antioxidant defense system in Saintpaulia. Since the susceptibility of Saintpaulia cultivars to leaf spot is related to the antioxidant capacity, the enhancement of the capacity by modern breeding methods will result in the improvement of the tolerance to leaf spot in Saintpaulia. References Allen, R.D., 1995. Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol. 107, 1049±1054. Anderson, M.D., Prasad, T.K., Stewart, C.R., 1995. Change in isozyme pro®les of catalase, peroxidase, and glutathione reductase during acclimation to chilling in mesocotyls of maize seedlings. Plant Physiol. 109, 1247±1257. Beyer Jr., W.F., Fridovich, I., 1987. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Analytical Biochemistry 161, 559±566. Bodner, M., Larcher, W., 1989. Chilling susceptibility of wild Saintpaulia species of different altitudinal orign. Angew. Botanik 63, 501±512. Dychdala, G.R., 1991. Chlorine and chlorine compounds. In: Block, S.S. (Ed.), Disinfection, Sterilization, and Preservation, 4th Edition. Lea & Febiger, Philadelphia, London, PA, pp. 131± 151. Elliot, F.H., 1946. Saintpaulia leaf spot and temperature differential. Proc. Amer. Soc. Hort. Sci. 47, 511±514. Fridovich, I., 1978. The biology of oxygen radicals. Science 201, 875±880. Fuchs, D., Baier-Bitterlich, G., Wede, I., Wachter, H., 1997. Reactive oxygen and apoptosis. In: Scandalios, J.G. (Ed.), Oxidative Stress and the Molecular Biology of Antioxidant Defense, Cold Spring Harbor Laboratory Press, NY, pp. 139±167. Gordon, G., Sweetin, L., Smith, K., Pacey, G.E., 1991. Improvements in the N,N-diethyl-pphenylenediamine method for the determination of free and combined residual chlorine through the use of FIA. Talanta 38, 145±150. Gupta, A.S., Webb, R.P., Holaday, A.S., Allen, R.D., 1993. Overexpression of superoxide dismutase protects plants from oxidative stress. Plant Physiol. 103, 1067±1073. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine, 3rd Edition. Oxford University Press, Oxford, pp. 27, 456±462. Hauptmann, N., Cadenas, E., 1997. The oxygen paradox: biochemistry of active oxygen. In: Scandalios, J.G. (Ed.), Oxidative Stress and the Molecular Biology of Antioxidant Defense. Cold Spring Harbor Laboratory Press, pp. 1±20. Kerdnaimongkol, K., Woodson, W.R., 1999. Inhibition of catalase by antisense RNA increases susceptibility to oxidative stress and chilling injury in transgenic tomato plants. J. Amer. Soc. Hort. Sci. 124, 330±336. Khan, A.U., Kasha, M., 1994. Singlet molecular oxygen evolution upon simple acidi®cation of aqueous hypochlorite: application to studies on the deleterious health effects of chlorinated drinking water. Proc. Natl. Acad. Sci. U.S.A. 91, 12362±12364.
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Maekawa, S., Torisu, Y., Inagaki, N., Terabun, M., 1987. Leaf injury caused by drop in leaf temperature of Saintpaulia ionantha (in Japanese, with English abstract). J. Jpn. Soc. Hort. Sci. 55, 484±489. Poesch, G.H., 1942. Ring spot on Saintpaulia. Proc. Am. Soc. Hort. Sci. 41, 381±382. Yasuda, Y., Yun, J.G., Miyoshi, N., Hayashi, T., Yazawa, S., Fujita, S., 1997. Production of radicals and acute apoptotic changes of palisade cells in early steps of Saintpaulia leaf spot appearance. Acta Histochem. Cytochem. 30, 303±307. Yun, J.G., Hayashi, T., Yazawa, S., Katoh, T., Yasuda, Y., 1996a. Acute morphological changes of palisade cells of Saintpaulia leaves induced by a rapid temperature drop. J. Plant Res. 109, 339±342. Yun, J.G., Hayashi, T., Yazawa, S., 1996b. Precise quanti®cation of leaf spot in Saintpaulia by image analysis with transmitted light. Environ. Control Biol. 34, 329±334. Yun, J.G., Hayashi, T., Yazawa, S., Yasuda, Y., Katoh, T., 1997. Degradation of photosynthetic activity of saintpaulia leaf by sudden temperature drop. Plant Sci. 127, 25±38. Yun, J.G., Hayashi, T., Yazawa, S., Katoh, T., Yasuda, Y., 1998. Abrupt and irreversible reduction of chlorophyll ¯uorescence associated with leaf spot in Saintpaulia (African violet). Sci. Horti. 72, 157±169.
Antioxidant capacity is correlated with susceptibility to leaf spot caused by a rapid temperature drop in Saintpaulia (African violet) S.J. Yanga,*, M. Hosokawaa, Y. Mizutaa, J.G. Yunc, J. Manob, S. Yazawaa a
Laboratory of Vegetable and Ornamental Horticulture, Department of Agronomy and Horticultural Science, Graduate School of Agriculture, Kyoto University, Oiwake-cho, Kitashirakawa, Kyoto 606-8502, Japan b The Research Institute for Food Science, Kyoto University, Uji, Kyoto 611-0011, Japan c Department of Horticulture, Chinju National University, Chinju City, South Korea Accepted 13 October 2000 Abstract The relationship between the susceptibility to rapid temperature drop induced leaf injury, i.e. ``leaf spot'', and the antioxidant capacities of several Saintpaulia (African violet) cultivars was examined. Cultivars `Ritali' and `Tamiko', that are more susceptible to leaf spot caused by a rapid drop in leaf temperature from 30 to 158C than are cultivars `Maui' and `New Jersey'. The susceptible cultivars were also more susceptible to oxidative stress caused by H2O2 and active chlorine than the tolerant cultivars. Reduction of available chlorine, considered to be accelerated by antioxidants of leaf tissue, was rapid in `Maui' and `New Jersey'. Activity of the antioxidant enzymes superoxide dismutase and catalase were higher in the leaves of `Maui' and `New Jersey' than in the susceptible cultivars. These ®ndings show a correlation between leaf spot injury and antioxidant capacity in Saintpaulia leaves. # 2001 Elsevier Science B.V. All rights reserved. Keywords: Saintpaulia; Reactive oxygen species; Leaf injury; Rapid temperature drop; Antioxidant capacity
1. Introduction Leaf spot of Saintpaulia is caused by a rapid drop in leaf temperature during irrigation (Poesch, 1942; Elliot, 1946; Maekawa et al., 1987) or exposure to cold * Corresponding author. Tel.: 81-75-753-6048; fax: 81-75-753-6068. E-mail address: [email protected] (S.J. Yang).
0304-4238/01/$ ± see front matter # 2001 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 2 3 8 ( 0 0 ) 0 0 2 3 5 - 1
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air (Maekawa et al., 1987). Leaf spot reduces the ornamental value of the plants and is a problem in commercial production. Research on the mechanism of leaf spot has focused on the palisade cells (Yun et al., 1996a, 1997, 1998), the main site of injury (Elliot, 1946; Maekawa et al., 1987). A rapid drop in leaf temperature for a few seconds drastically decreases photosynthetic activity (Yun et al., 1997, 1998) and destroys the ultrastructural composition of palisade cells (Yun et al., 1996a). This injury is irreversible and can be caused even by immersion in 258C water if the leaf temperature before the immersion was higher than 358C. Thus, leaf spot is caused by a temperature difference, and does not always require chilling temperature (i.e. below 108C). On this point and on the rapidness of its development, leaf spot injury can be distinguished from typical chilling injury. Yasuda et al. (1997) showed the induced production of reactive oxygen species (ROS) in the leaf extracts following a rapid temperature drop. Also, the morphological changes including shrinkage of cytoplasm, condensation of chromatin and disarrangement of organelles, were observed in the palisade cells of Saintpaulia `Ritali' after the temperature drop (Yun et al., 1996a; Yasuda et al., 1997). These symptoms are very similar to the apoptotic morphological changes induced by ROS (Fuchs et al., 1997). However, there has been no study on the relationship between the leaf spot and antioxidant capacity in Saintpaulia. When a plant is exposed to stress, the production of active oxygen can exceed the capacity of the antioxidant system, resulting in oxidative damage. Thus, antioxidant capacity is a major factor in determining the stress tolerance of plants, and the plants having higher antioxidant capacity by acclimation or transformation show stress tolerance (Gupta et al., 1993; Allen, 1995; Anderson et al., 1995). Superoxide dismutase (SOD), the ®rst enzyme in the detoxifying process, converts O2ÿ to O2 and H2O2, which is further decomposed to O2 and H2O by catalase (Fridovich, 1978). NaOCl has a disinfecting or sterilizing effect as active chlorine, which is a powerful anti-fungal agent, and is used for sterilization of plant material in in vitro culture. H2O2 also has a toxic effect on organisms. Both are ROS. We observe that the occurrence of leaf spot varies among cultivars in Saintpaulia, but it is unknown what is the main factor to determine the sensitivity for generating leaf spot. The objective of this research is to determine the relationship between the susceptibility to leaf spot and the antioxidant capacity in Saintpaulia. 2. Materials and methods 2.1. Plant material Saintpaulia ionantha (African violet), cultivars `Ritali', `Tamiko', `Maui' and `New Jersey', were grown in a glasshouse in which the temperature was
S.J. Yang et al. / Scientia Horticulturae 88 (2001) 59±69
61
maintained at 17±258C, and the irradiation below 252 mmol mÿ2 sÿ1, during the daytime. Fully expanded leaves were detached from each four cultivars before experiment and immediately used. 2.2. Rapid temperature drop treatment and quanti®cation of leaf spot Each detached leaf was immersed in 308C water for 3 min, and then dipped in 158C water for 3 s to make a rapid drop in leaf surface temperature by 158C. In our preliminary experiment, leaf surface temperature was detected suddenly after the immersion by thermography so that it is almost same as water temperature. The room temperature was 20±258C. The leaf spot severity was quanti®ed by an image analysis technique as described previously (Yun et al., 1996b). The leaf images were captured using a digital camera and the images were transferred to a personal computer to quantify the area of leaf spot by using image software (Adobe PhotoShop Version 5.0). The severity of leaf spot was shown by the percentage of the damaged leaf area to the total leaf area. 2.3. Quanti®cation of leaf injury caused by H2O2 and NaOCl The leaves were rinsed lightly with a detergent, and the leaf discs were excised using a leaf punch (Disposable Biopsy Trepan). Midrib parts were not included in the sampled leaf discs. The super¯uous leakage from the cut section of leaf discs was removed using ®lter paper. Then, ®ve leaf discs, 8 mm in diameter, were immersed in 50 ml of 0.2 M mannitol solution containing 1, 5 or 10 mM H2O2, and agitated (50 rpm) at room temperature in light (39 mmol mÿ2 sÿ1). Then, the electric conductivity of the solution was measured with a conductivity meter (CM-1K, Toa Electrics Ltd., Tokyo, Japan). The solution of 0.2 M mannitol with the leaf discs but without H2O2 was used as a control, and the relative injury due to H2O2 was calculated as the ratio of the conductivity of H2O2 solution to that of control solution, both measured after 3 h treatment. To elucidate the toxic effect of chlorine itself, we immersed ®ve leaf discs of `Ritali' in the 50 ml of NaOCl solution containing 20 ppm available chlorine, itself, in the presence or absence of sodium thiosulfate (100 ppm) that detoxi®es active chlorine (Dychdala, 1991) and agitated under the same condition as above. After a 3 h agitation, the leaf discs were washed with distilled water and incubated in 50 ml of 0.2 M mannitol solution for 3 h and electrolyte conductivity was measured. The control was agitated in distilled water for 3 h and then immersed in mannitol solution for 3 h. The relative injury was calculated as the ratio of the conductivity of NaOCl solution to that of control solution. The relative leaf injury caused by active chlorine (5 and 10 ppm of available chlorine) was calculated as same as above, measured after 4 h treatment.
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2.4. OClÿ reduction by leaf discs Ten leaf discs, 8 mm in diameter, were immersed in 30 ml of NaOCl solution (4 ppm) with agitation (50 rpm) at room temperature in light (39 mmol mÿ2 sÿ1) for 210 min. `Available chlorine' is expressed in terms of the equivalent amount of elemental chlorine (Dychdala, 1991). The residual-available chlorine in the NaOCl solution in which the samples were immersed was measured by using chlorine comparator (Water tester, Sibata, Japan) according to the N,N-diethyl-pphenylenediamine (DPD) method (Gordon et al., 1991). Seven replicates of the solution containing leaf discs were prepared for each cultivar, and the available chlorine was assayed at 30 min intervals using one of the replications chosen at random. 2.5. Determination of enzymatic activities of SOD and catalase Leaf extract was prepared at 08C as follows. Leaf samples of 0.5 g fresh weight from the fully expanded leaves were ground rapidly in a mortar with a pestle in the presence of 1 ml of 50 mM potassium phosphate (pH 7.0). Following centrifugation at 8000 g for 10 min at 48C the supernatant was removed for enzymatic analysis. The supernatant was gel-®ltered over Sephadex G-25 (NAP-5 column, Pharmacia, Sweden), equilibrated with 50 mM potassium phosphate (pH 7.0), to remove small molecules. SOD activity was determined spectrophotometrically by measuring the inhibition of O2ÿ-dependent reduction of nitroblue tetrazolium (NBT), according to the xanthine±xanthine oxidase system (Beyer and Fridovich, 1987). One unit of SOD was de®ned as the quantity of enzyme required to inhibit the reduction of NBT by 50% in a reaction volume of 1 ml. Catalase activity was determined by monitoring the production of O2 from H2O2 with a Clark-type oxygen electrode at 258C by the method of Kerdnaimongkol and Woodson (1999) with some modi®cations. The assay was carried out in a stirred reaction cuvette containing 50 mM HEPES (pH 7.6) and 100 mM H2O2. The decomposition of H2O2 was initiated with 100 ml of the enzyme extract. 3. Results 3.1. Susceptibility for generating leaf spot and oxidative stress caused by NaOCl and H2O2 in four cultivars Leaves pre-incubated at 308C were exposed to a rapid temperature drop by being immersed in water at 158C. This treatment lowers the leaf temperature from 30 to 158C in 3 s, as determined by thermography in our preliminary experiment. By this treatment, more than 80% of the leaves in `Ritali' and `Tamiko' were
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Fig. 1. Injury of leaf spot caused by exposure to water of 308C for 3 min followed by 158C for 3 s in Saintpaulia cultivars `Maui', `New Jersey', `Ritali' and `Tamiko'. z: The details in Section 2. Bars represent means S:E: of seven replicates.
injured, whereas less than 20% in `Maui' and `New Jersey'(Fig. 1). Leaf spot was not caused by the immersion in water either at 30 or 158C in any of these cultivars. This result corresponds with the observation that the leaf spot caused by overhead irrigation with low temperature water in a glasshouse was severer in `Ritali' and `Tamiko' than in `Maui' and `New Jersey'. To evaluate the susceptibility to oxidative stress, we immersed and agitated the leaf discs of four cultivars in the H2O2 solution at a low concentration for 3 h. As shown in Fig. 2, `Ritali' and `Tamiko' were injured more severely than `Maui'
Fig. 2. Leaf injury induced by H2O2 in four cultivars of Saintpaulia. z: Leaf discs were incubated in each solution for 3 h and relative injury was calculated as conductivity of treatment after 3 h/ conductivity of control after 3 h. Bars represent means S:E: of ®ve replicates.
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Fig. 3. Leaf injury induced by available chlorine (HOCl, OClÿ) in four cultivars of Saintpaulia. A, Leaf injury of four cultivars in each of NaOCl solution at 5 and 10 ppm available chlorine. z: The relative injury was calculated as (conductivity of treatment after 4 h ± conductivity of treatment after 0 h/conductivity of control after 4 h ± conductivity of control after 0 h). The conductivity of treatment contains not only ion leakage from leaf tissue but also contains ion of sodium and chlorine in all cultivars. Bars represent means S:E: of three replicates. B, Amelioration of leaf injury by decomposition of available chlorine using sodium thiosulfate. y: Described in Section 2. Bars represent means S:E: of ®ve replicates.
and `New Jersey' at 5 and 10 mM H2O2. The relative injury in the H2O2 solution were discernible, the most in 10 mM H2O2 between the tolerant and sensitive cultivars to rapid temperature drop (Fig. 2). `Ritali' and `Tamiko' were more severely injured than `Maui' and `New Jersey' also in the NaOCl solution at 5 and 10 ppm (available chlorine) (Fig. 3A). `Ritali' and `Tamiko' were injured 3±4-fold more severely than `Maui' and `New Jersey' at 5 ppm of this solution. The injury induced by NaOCl was alleviated by the presence of sodium thiosulfate, a dechlorinating chemical (Fig. 3B). This indicates that the leaf injury by NaOCl solution was not caused by sodium ion but mainly by available chlorine, a powerful oxidant. These results showed that `Maui' and `New Jersey' are more susceptible to both reactive oxidants, H2O2 and active chlorine, than `Ritali' and `Tamiko'. Thus, the sensitivity order of the four cultivars to the oxidants corresponded with that to the leaf spot. 3.2. Reduction rate of available chlorine in the presence of leaf discs of four cultivars Available chlorine, OClÿ or HOCl, is a powerful oxidant and generates other ROS, 1 O2 or OH , provoking cell damage (Khan and Kasha, 1994). This assay
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Fig. 4. Reduction rate of available chlorine by the leaf discs in four cultivars of Saintpaulia. z: Ten leaf discs of each cultivar were incubated in NaOCl solution at 4 ppm of available chlorine for 210 min and the residual available chlorine was measured at the interval of 30 min.
was conducted to compare the capacity to reduce available chlorine among the cultivars, which showed different susceptibility for available chlorine. Ten leaf discs of each cultivar were immersed in 30 ml NaOCl solution containing 4 ppm available chlorine. The available chlorine in the solution decreased rapidly during the ®rst 30 min, and then gradually thereafter (Fig. 4). In the absence of the leaf discs, the concentration of available chlorine in the NaOCl solution did not change for 210 min. This shows that the reduction of available chlorine in NaOCl solution was caused by the presence of leaf discs. In the presence of the leaf discs of `Maui' and `New Jersey', most of the available chlorine (4 ppm) disappeared within 120 min, but in the presence of the leaf discs of `Ritali' and `Tamiko', 0.7 and 0.9 ppm of available chlorine, respectively, remained after a 120 min incubation. Thus, `Maui' and `New Jersey' have higher capacities to reduce available chlorine than `Ritali' and `Tamiko'. From the correlation between reducing capacity of available chlorine and susceptibility to active chlorine (Figs. 3A and 4), the cultivars show tolerance against active chlorine may depend on the detoxify-capacity of active chlorine by reduce it rapidly. 3.3. Leaf-SOD, catalase activity of four cultivars SOD activity in `Maui' and `New Jersey' was higher than that of `Ritali' and `Tamiko' (Fig. 5). Catalase activity in `Maui' and `New Jersey' were higher also
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Fig. 5. Leaf-SOD activity in four cultivars of Saintpaulia. Bars represent means S:E: of ®ve replicates.
compared with `Ritali' and `Tamiko'(Fig. 6). `Maui' showed twofold of SOD and catalase activities compared with `Ritali' and `Tamiko'. Both SOD and catalase activities in the leaves of the leaf spot-tolerant cultivars were higher than that of the susceptible cultivars. Thus, the higher tolerance to leaf spot injury and oxidative stress are signi®cantly correlated to the cultivar's SOD and catalase activities.
Fig. 6. Leaf-catalase activity in four cultivars of Saintpaulia. Bars represent means S:E: of ®ve replicates.
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4. Discussion In Saintpaulia, the sensitivity to a rapid drop in leaf temperature varied among cultivars. `Maui' and `New Jersey' were relatively more tolerant to a rapid temperature drop by 158C (30±158C) than `Ritali' and `Tamiko' (Fig. 1). Bodner and Larcher (1989) reported the chilling susceptibility of seven wild species of Saintpaulia, but there are no reports on the susceptibility for generating leaf spot in Saintpaulia cultivars. From the reason that leaf spot is a highly acute injury and from the characteristic of its morphological changes of palisade cells after the rapid temperature drop (Yun et al., 1996a), it is considered that the leaf spot is mediated by oxidative stress. However, since the development of leaf spot in Saintpaulia is extremely rapid, which the ¯uorescence intensity drops within a few seconds after the rapid drop in leaf temperature (Yun et al., 1997), direct assay of ROS related to leaf spot injury is dif®cult. In our research, leaf injury caused by H2O2 or active chlorine was severer, the higher the susceptibility of the cultivar to leaf spot (Figs. 2 and 3). The active chlorine and H2O2 are included in ROS and their cytotoxicity is largely due to their oxidizing effects (Halliwell and Gutteridge, 1999). In our research, also, the cultivars tolerant for generating leaf spot were relatively tolerant to ROS. Although, the mechanism of resistance to active chlorine is unknown, it is presumably related to the detoxi®cation of active chlorine in plant systems. Available chlorine in an aqueous solution exhibits high disinfecting power as active chlorine, which is considered to generate other radicals (Hauptmann and Cadenas, 1997). Available chlorine in NaOCl solution was reduced more rapidly in the presence of the leaf discs of `Maui' and `New Jersey' than in the presence of the leaf discs of `Ritali' and `Tamiko' (Fig. 4). In our study, reducing capacity of available chlorine has correlation with the susceptibility to active chlorine (Figs. 3A and 4). It suggest that the tolerance against active chlorine may depend on the detoxify-capacity of active chlorine by several kinds of antioxidants such as ascorbic acid, glutathione and a-tocopherol in leaf tissue (Halliwell and Gutteridge, 1999). `Maui' and `New Jersey' showed the high activities of SOD and catalase than those in `Ritali' and `Tamiko' (Figs. 5 and 6). The combined action of SOD and catalase ef®ciently eliminates O2ÿ and H2O2, and consequently, protects cellular components against the more reactive hydroxyl radical (OH ), which results from the interaction between O2ÿ and H2O2 (Fridovich, 1978). Yasuda et al. (1997) indicated that the oxygen radicals participate in the acute irreversible morphological changes, resulting in apoptotic cell death of Saintpaulia palisade cells. In our study, the cultivars showing higher SOD and catalase activities were tolerant to not only oxidative stress, but also to a rapid temperature drop. This suggests that the susceptibility to leaf spot in Saintpaulia be closely related to the
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antioxidant capacities. In addition, the measurement of chlorine-reduction capacity of leaf discs can be used as a rapid method for screening the susceptibility of leaf spot injury among cultivars in Saintpaulia. In conclusion, ROS is considered to be involved in the process causing leaf spot and the tolerance to leaf spot is correlated with the high capacity of the antioxidant defense system in Saintpaulia. Since the susceptibility of Saintpaulia cultivars to leaf spot is related to the antioxidant capacity, the enhancement of the capacity by modern breeding methods will result in the improvement of the tolerance to leaf spot in Saintpaulia. References Allen, R.D., 1995. Dissection of oxidative stress tolerance using transgenic plants. Plant Physiol. 107, 1049±1054. Anderson, M.D., Prasad, T.K., Stewart, C.R., 1995. Change in isozyme pro®les of catalase, peroxidase, and glutathione reductase during acclimation to chilling in mesocotyls of maize seedlings. Plant Physiol. 109, 1247±1257. Beyer Jr., W.F., Fridovich, I., 1987. Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Analytical Biochemistry 161, 559±566. Bodner, M., Larcher, W., 1989. Chilling susceptibility of wild Saintpaulia species of different altitudinal orign. Angew. Botanik 63, 501±512. Dychdala, G.R., 1991. Chlorine and chlorine compounds. In: Block, S.S. (Ed.), Disinfection, Sterilization, and Preservation, 4th Edition. Lea & Febiger, Philadelphia, London, PA, pp. 131± 151. Elliot, F.H., 1946. Saintpaulia leaf spot and temperature differential. Proc. Amer. Soc. Hort. Sci. 47, 511±514. Fridovich, I., 1978. The biology of oxygen radicals. Science 201, 875±880. Fuchs, D., Baier-Bitterlich, G., Wede, I., Wachter, H., 1997. Reactive oxygen and apoptosis. In: Scandalios, J.G. (Ed.), Oxidative Stress and the Molecular Biology of Antioxidant Defense, Cold Spring Harbor Laboratory Press, NY, pp. 139±167. Gordon, G., Sweetin, L., Smith, K., Pacey, G.E., 1991. Improvements in the N,N-diethyl-pphenylenediamine method for the determination of free and combined residual chlorine through the use of FIA. Talanta 38, 145±150. Gupta, A.S., Webb, R.P., Holaday, A.S., Allen, R.D., 1993. Overexpression of superoxide dismutase protects plants from oxidative stress. Plant Physiol. 103, 1067±1073. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine, 3rd Edition. Oxford University Press, Oxford, pp. 27, 456±462. Hauptmann, N., Cadenas, E., 1997. The oxygen paradox: biochemistry of active oxygen. In: Scandalios, J.G. (Ed.), Oxidative Stress and the Molecular Biology of Antioxidant Defense. Cold Spring Harbor Laboratory Press, pp. 1±20. Kerdnaimongkol, K., Woodson, W.R., 1999. Inhibition of catalase by antisense RNA increases susceptibility to oxidative stress and chilling injury in transgenic tomato plants. J. Amer. Soc. Hort. Sci. 124, 330±336. Khan, A.U., Kasha, M., 1994. Singlet molecular oxygen evolution upon simple acidi®cation of aqueous hypochlorite: application to studies on the deleterious health effects of chlorinated drinking water. Proc. Natl. Acad. Sci. U.S.A. 91, 12362±12364.
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Maekawa, S., Torisu, Y., Inagaki, N., Terabun, M., 1987. Leaf injury caused by drop in leaf temperature of Saintpaulia ionantha (in Japanese, with English abstract). J. Jpn. Soc. Hort. Sci. 55, 484±489. Poesch, G.H., 1942. Ring spot on Saintpaulia. Proc. Am. Soc. Hort. Sci. 41, 381±382. Yasuda, Y., Yun, J.G., Miyoshi, N., Hayashi, T., Yazawa, S., Fujita, S., 1997. Production of radicals and acute apoptotic changes of palisade cells in early steps of Saintpaulia leaf spot appearance. Acta Histochem. Cytochem. 30, 303±307. Yun, J.G., Hayashi, T., Yazawa, S., Katoh, T., Yasuda, Y., 1996a. Acute morphological changes of palisade cells of Saintpaulia leaves induced by a rapid temperature drop. J. Plant Res. 109, 339±342. Yun, J.G., Hayashi, T., Yazawa, S., 1996b. Precise quanti®cation of leaf spot in Saintpaulia by image analysis with transmitted light. Environ. Control Biol. 34, 329±334. Yun, J.G., Hayashi, T., Yazawa, S., Yasuda, Y., Katoh, T., 1997. Degradation of photosynthetic activity of saintpaulia leaf by sudden temperature drop. Plant Sci. 127, 25±38. Yun, J.G., Hayashi, T., Yazawa, S., Katoh, T., Yasuda, Y., 1998. Abrupt and irreversible reduction of chlorophyll ¯uorescence associated with leaf spot in Saintpaulia (African violet). Sci. Horti. 72, 157±169.