Plant Growth Regulation 30: 157–161, 2000. © 2000 Kluwer Academic Publishers. Printed in the Netherlands.
157
Acetyl salicylic acid (Aspirin) and salicylic acid induce multiple stress tolerance in bean and tomato plants Tissa Senaratna∗ , Darren Touchell, Eric Bunn & Kingsley Dixon Kings Park and Botanic Garden, West Perth, WA 6005, Australia (∗ author for correspondence; e-mail:
[email protected]) Received 1 June 1999; accepted 24 August 1999
Key words: stress tolerance, acetyl salicylic acid, salicylic acid, heat, drought, cold
Abstract The hypothesis that physiologically active concentrations of salicylic acid (SA) and its derivatives can confer stress tolerance in plants was evaluated using bean (Phaseolus vulgaris L.) and tomato (Lycopersicon esculentum L.). Plants grown from seeds imbibed in aqueous solutions (0.1–0.5 mM) of salicylic acid or acetyl salicylic acid (ASA) displayed enhanced tolerance to heat, chilling and drought stresses. Seedlings acquired similar stress tolerance when SA or ASA treatments were applied as soil drenches. The fact that seed imbibition with SA or ASA confers stress tolerance in plants is more consistent with a signaling role of these molecules, leading to the expression of tolerance rather than a direct effect. Induction of multiple stress tolerance in plants by exogenous application of SA and its derivatives may have a significant practical application in agriculture, horticulture and forestry. Abbreviations: SA – salicylic acid; ASA – acetyl salicylic acid; SAR – systemic acquired resistance
1. Introduction One of the most important factors that dictates the distribution of many plant species is their ability to withstand environmental stress including seasonal variations in temperature and available moisture. Plants generally respond to environmental stress by activating defence mechanisms and adjusting their cellular metabolism [8, 16]. Plants perceive the stress condition and signal to alter the metabolic flux for the activation/synthesis of defence mechanisms [16]. Many molecules, for example, calcium, jasmonic acid, ethylene and salicylic acid have been suggested as signal transducers or messengers [11]. Salicylic acid (SA) has received much attention after the discovery of its ability to induce resistance (systemic acquired resistance or SAR) to pathogens [13, 19, 20, 25]. Exogenous application of SA induced pathogenesis-related gene expression and systemic acquired resistance [2]. Extensive studies have been undertaken to elucidate the molecular biology of SA induced SAR [2, 5,
17]. However, the physiological and biochemical basis for this phenomenon is not clear at present. Pathological disorders caused by microbial agents usually promote the development of hypersensitive reactions within the infected plant tissues. If the pathogen is allowed to develop unchecked, necrotic lesions develop, resulting in cell and tissue death [1]. It has been demonstrated [10] that this sequence involves destructive attack by free radicals mediated through oxidative degradation of membrane lipids. Similarly there is considerable evidence to suggest that irreversible injury due to environmental stress is caused by increased free radical titre and consequent oxidation events which lead to degradation of biomolecules such as membrane lipids and proteins [15, 22, 23]. The similarity of the injury mechanism between pathogenesis and stress leads us to hypothesise that salicylic acid which induces resistance to disease also confers tolerance to environmental stress. In this communication, evidence is provided that salicylic acid
158 Table 1. Survival (%) of SA or ASA – treated tomato and bean plants after heat, cold and drought stress Heat SI
Chilling SD
SI
Drought SD
SI
SD
Conc(mM)
Tomato
Bean
Tomato
Bean
Tomato
Bean
Tomato
Bean
Tomato
Bean
Tomato
Bean
SA
0 0.05 0.1 0.5 1.0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
ASA
0 0.05 0.1 0.5 1.0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
0 0 100 100 0
SI Seed Imbibed. SD Soil Drenched.
(SA) and acetyl salicylic acid (ASA) provide multiple stress tolerance in plants and that salicylic acid and its derivatives regulate the expression of stress tolerance. This is the first report to suggest that SA or ASA imbibed into a seed impart tolerance to a variety of stresses in plants.
2. Materials and methods To evaluate the hypothesis that SA and derivatives can confer stress tolerance, experimentation was conducted using bean (Phaseolus vulgaris L. cv. brown beauty) and tomato (Lycopersicon esculentum L. cv. romano) as test species. Plants were grown in 135 ml pots in a glasshouse maintained at ambient temperature and humidity (in Perth, Western Australia). Fourteen day-old plants were soil-drenched with 20 ml of distilled water or 0.05, 0.1, 0.5, 1.0 and 5.0 mM ASA or SA (dissolved in distilled water). Alternatively, seeds were imbibed in the solutions or in distilled water for 24 h and sown in pots. One week after soil-drenching or three weeks after the seed treatment, seedlings were subjected to heat, cold and drought stresses. For heat treatment, seedlings were exposed to 54 ± 0.5 ◦ C for 3 h with an average light intensity of 40 µMol m−2 sec−1 and then returned to room temperature. For chilling stress, plants were exposed to 0 ± 0.5 ◦ C in an incubator with an average light intensity of 35 µMol m−2 sec−1 and 16/8 h light/dark photoperiod for two days. All pots were saturated with water 2 h prior to and after
the imposition of heat and chilling stress treatments. Drought stress was imposed by withholding water for 7 days, then on the 8th day all pots were watered until saturation. To evaluate the benefit of foliar spray applications plants were sprayed with either 0.5 mM ASA or 0.5 mM SA, and subjected to stress treatments as above. The nozzle of the hand held sprayer was adjusted to deliver 0.5 ml of solution per spray and each plant received 1 ml of solution or distilled water for control. One week after spraying, plants were exposed to stress and scored for survival. All treatments were assessed after 48 h and survival was determined by the ability of plants to regain turgidity and resume normal growth after the stress treatment. Damage was recorded as irreversible wilting, desiccation of leaves and presence of necrotic leaf lesions. Randomized complete block design with 5 replicates was used in all experiments.
3. Results and discussion Concentrations over 1 mM ASA or SA appeared to have adverse effects and therefore lower concentrations were used in further experiments. There were no observable differences in plant height, vigour or leaf number between non treated and SA or ASA treated plants (data not shown). Heat treatment at 54 ◦ C for 3 h induced severe wilting in controls of tomato and bean plants and in plants treated with 0.05 mM SA or ASA. The plants, seed imbibed or drenched with
159 0.1 mM or 0.5 mM SA or ASA did not exhibit signs of wilting. After two days, plants which were not treated with SA or ASA or which were treated with 0.05 mM or 1.0 mM SA or ASA had not regained turgor in any leaves and exhibited no signs of resprouting (Table 1). The plants which were soil drenched with 0.5 mM SA or ASA did not display any observable injury symptoms (Figure 1). Minor damage was visible in the leaves of 0.1 mM treated plants, evident as desiccation of leaf tips and some margins; however the injury was transitory and the plants exhibiting these symptoms recovered and were able to revive and resume growth. Similar suppression of stress injury following chilling treatment was observed in plants treated with SA and ASA. Both tomato and bean plants subjected to soil-drenching or seed treatment with 0.5 mM SA or ASA did not display any injury symptoms after cold stress treatment. Plants which received the 0.1 mM SA or ASA treatments displayed necrotic lesions on some leaves, but resumed growth after stress treatment (Table 1). The plants not treated with SA or ASA, or treated with 0.05 mM or 1.0 mM SA or ASA as soil-drench or seed treatment showed typical symptoms of chilling injury with wilting of leaves, necrosis and subsequent desiccation of most leaves (Figure 1). One week after withholding water, the plants not treated with SA or ASA as well as those treated with 0.05 mM or 1.0 mM SA or ASA lost turgor and wilted, whereas plants subjected to soildrench or seed treatment with 0.1 mM or 0.5 mM SA or ASA retained a relatively high degree of turgidity. Most importantly, upon watering, 0.1 mM and 0.5 mM treated plants recovered completely within a few hours, whereas untreated plants or those treated with 0.05 mM or 1.0 mM SA or ASA retained wilted leaves which subsequently desiccated (Table 1). Spray treatment with 0.5 mM SA or ASA was also effective. The concentration was selected based on the results of previous experiments. All the SA or ASA treated plants survived (100% survival) after heat, chilling or drought stress while all the control plants died. Enhancing stress tolerance in plants has major implications in agriculture, horticulture, forestry as well as in the re-establishment of natural vegetation. However a simple method for inducing multiple stress tolerance in plants without undesirable side effects has not been available till now. Seed imbibition or drenching with triazole compounds such as paclobutrazol, triademophone and uniconazole have been demonstrated to induce multiple stress tolerance in plants [9, 12, 21]. Unfortunately, these compounds also
Figure 1. Bean plants A) exposed to heat stress B) pre-treated as a soil drench with 0.5 mM ASA and exposed to heat stress C) exposed to chilling D) pre-treated as a soil drench with 0.5 mM ASA and subjected to chilling E) subjected to drought F) pre-treated as a soil drench with 0.5 mM ASA and subjected to drought. Immediately after the completion of heat, chilling or drought treatments all pots were saturated with water (photographs were taken 48 h after the completion of stress treatments).
160 inhibit gibberellin biosynthesis in plants and hence have growth retarding effects. Salicylic acid and acetyl salicylic acid are not known to retard plant growth and no evidence of growth impairment of treated plants over untreated controls was observed in this study. The fact that seed imbibition of SA and ASA confers tolerance to plants suggests that these molecules trigger the expression of the potential to tolerate stress rather than having any direct effect as a protectant. Some reports suggest that SA induces an oxidative burst involving H2 O2 (hydrogen peroxide) accumulation which acts as the signal transducer for SAR [3]. Neuenschwander et al. [18] did not observe major changes in H2 O2 levels during the onset of SAR. The exact mechanism of action of SA in inducing SAR and its role in the transient increase of H2 O2 is still cause for debate [4]. Prevention of oxidative damage to cells during stress has been suggested as one of the mechanisms of stress tolerance [12, 24] and this level of protection is attributed to enhanced antioxidant activity [6, 21, 24]. Enhanced thermotolerance has been observed in potato microplants grown on media containing acetyl salicylic acid [14]. Increased thermotolerance in mustard seedlings sprayed with SA has also been reported [7] and the tolerance was associated with changes in antioxidants such as glutathione reductase, dehydroascorbate reductase and monodehydroascorbate reductase [6]. In pea seedlings SA treatment decreased catalase and peroxidase levels with concomitant increase in glutathione reductase [26]. SA treatment also increased the level of reduced glutathione (GSH) with an increase in the ratio of reduced to oxidised glutathione (GSSG) indicating higher antioxidant potential [26]. There are suggestions that salicylic acid acts as an antioxidant [4]. However the fact that seed imbibition with SA or ASA provides stress tolerance in plants is more consistent with a signalling role for the expression of tolerance rather than a direct effect. The other mechanisms of tolerance may involve avoidance of lethal stress by altering cellular metabolism, synthesis of stabilising molecules to maintain integrity and function of cellular membranes during stress [27]. Stomatal resistance in SA treated mustard seedlings with enhanced thermotolerance was not different from controls [7]. However detailed studies on plant water relations after SA and ASA treatments are necessary to understand the events leading to stress tolerance. Although the physiological and biochemical basis for SA induced tolerance is not clearly understood, we believe that a cascade of events
is triggered to provide multiple stress tolerance in plants. Further research is warranted to elucidate the physiological and biochemical mechanisms by which SA induces tolerance to a variety of environmental stresses. In addition research is required to elucidate the reasons why plants treated with higher concentrations of SA or ASA (eg. 1.0 mM) were susceptible to stress injury. However, the phenomenon that common aspirin can be utilised to prevent crop losses during stress may have significant practical application.
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