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Plant Physiol. Biochem. 40 (2002) 761–769 www.elsevier.com/locate/plaphy Review Effects of wounding and salicylic acid on hydroxycinnamoylmalic acids in Thunbergia alata Fatima Housti a,b, Claude Andary b,*, Annick Gargadennec b, Mohammed Amssa a aDépartement de Biologie, Laboratoire de Physiologie et de Biotechnologie végétales, Faculté des Sciences, Université My Ismail, BP 4010, Meknès, Morocco bLaboratoire de Botanique, Phytochimie et Mycologie, Faculté de Pharmacie, (UM1-CNRS, UPR 9056), 15, avenue C.-Flahault, BP 14491, 34093 Montpellier cedex 5, France Received 22 January 2002; accepted 2 April 2002 Abstract We describe the identification of three phenolic compounds (caffeoylmalic, feruloylmalic and p-coumaroylmalic acids) in the leaves of Thunbergia alata. Caffeoylmalic and feruloylmalic acids represent the majority of all the derivatives of hydroxycinnamic acid in this plant. Elicitation with 5 mM of salicylic acid (SA) after wounding produces an intense necrotic reaction which reaches a peak after 24 h. This reaction is much less intense when the leaves are subjected to wounding alone. HPLC analysis of the above three acids in leaf samples taken 24 h after treatment shows that the concentration of caffeoylmalic acid increases in the case of both wounding and SA treatment. The level of feruloylmalic acid increases principally in response to wounding while the concentration of p-coumaroylmalic acid increases essentially following elicitation by SA. The accumulation of these three compounds occurs not only in directly treated leaves, but also in untreated leaves situated above treated leaves (systemic accumulation). Wounding and SA produce a greater local accumulation of feruloyl and p-coumaroylmalic malic acid, respectively. For caffeoylmalic acid, wounding produces the greatest local accumulation whereas in response to SA, systemic accumulation of this compound is greater. Plants aged 2 months are more sensitive than younger plants to these two stress treatments. © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. Keywords: Hydroxycinnamoylmalic acids; Salicylic acid; Thunbergia alata; Wounding 1. Introduction defence reactions, are very sensitive to environmental variations and can accumulate in the plant under the Plants have the ability to develop multiple and complex influence of various factors [7,8,21,23]. More recent studies defence strategies in order to limit invasion by pathogenic have demonstrated the involvement, particularly of some agents in particular, and generally to enable them to manage hydroxycinnamic acid derivatives, in defence responses to various environmental stress situations. These defence re- different biotic and abiotic stresses [18,24,37,38]. There are, sponses involve lignification of the tissues subjected to however, no published reports on the effect of salicylic acid aggression [5,20,27]; induction of proteins with anti-fungal (SA) on the production of these compounds. actions [35] and other proteins such as chitinases and glucanases [13]; the production of phytoalexins [4] and of Thunbergia alata is a member of the Acanthaceae family secondary metabolites, in particular phenolic compounds. in which we identified various hydroxycinnamoylmalic The latter substances, whose anti-microbial properties are acids (HCM acids). To our knowledge these phenolic acids, well-documented, [3,6,18], and which are often involved in which occur in several plant families [10,12,30,31], have never been found in plants belonging to the Acanthaceae family. In addition, even though some studies demonstrated Abbreviations: CM, caffeoylmalic acid; FM, feruloylmalic acid; HCM, the anti-inflammatory [26], antioxidant [36] and antispas- hydroxycinnamoylmalic acids; HPLC, high performance liquid chromato- modic [9] effects of caffeoylmalic acid, no data are available graphy; p-CM, p-coumaroylmalic acid ; SA, salicylic acid * Corresponding author. on the behaviour of HCM acids in response to stress in E-mail address: [email protected] (C. Andary). plants. In this report, we describe the identification of these © 2002 Éditions scientifiques et médicales Elsevier SAS. All rights reserved. PII:S0981-9428(02)01427-4 762 F. Housti et al. / Plant Physiol. Biochem. 40 (2002) 761–769 Fig. 1. Variations in the relative amounts of CM, FM and p-CM in the leaves of younger (1 month) and older (2 months) plants. Fig. 2. Necrotic surfaces measured in wounded and wounded + SA leaves. Each point shows the mean surfaces ± SD for 12 plants. compounds which represent the majority of the phenolic compounds in the leaves of T. alata. We also studied the 2.2.1. Local effect of wounding effect of wounding, with or without the application of SA and of SA on the accumulation of HCM acids which causes the formation of a necrotic zone around the point of elicitation. 2.2.1.1. In 1-month-old plants Studies on the accumulation of HCM acids in the leaves of 1-month-old plants showed that wounding slightly in- 2. Results creases the concentration of both CM (16%) and FM (22%), but has no effect on p-CM. SA increases the concentration 2.1. The phenolic compounds of Thunbergia alata of p-CM (50%), but the effect on CM and FM accumulation was insignificant (Fig. 4A). Hydroxycinnamic acid derivatives are the principal phe- 2.2.1.2. In 2-month-old plants nolic compounds found in the leaves of T. alata. Three of Determination of the concentrations of the three acids in these compounds have been identified: (E)-caffeoyl-L-malic leaves of 2-month-old plants shows that wounding alone acid (CM), feruloylmalic acid (FM) and p-coumaroylmalic significantly increases the concentrations of CM (75%) and acid (p-CM). These three acids represent 76% and 53% of FM (59%) (Fig. 4B). However, wounding is not observed to all the phenolic compounds in plants aged 1 and 2 months, have any effect on the concentration of p-CM. Application respectively (Fig. 1). The levels of CM, the predominant of SA in conjunction with wounding has an additive effect, phenolic acid in young plants, decrease by half in older increasing the concentration of CM to an even greater extent plants, whereas the concentrations of FM and p-CM do not alter with age (Fig. 1). 2.2. Effect of wounding and SA on the leaves The leaves react within 5 min following treatment by developing a necrotic zone around the point of wounding. The diameter of the resulting lesions reaches a maximum 24 h after the treatments (Fig. 2). The lesions are approxi- mately 1 mm diameter in leaves subjected to wounding, and approximately 5 mm diameter in leaves subjected to both wounding and SA treatment. The cells in the treated zone lose their chlorophyll, becoming white and more or less transparent (Fig. 3). The application of SA alone without Fig. 3. Necrotic surfaces observed in wounded + SA (A) and wounded (B) prior wounding does not produce any lesion. leaves 7 days after treatment. F. Housti et al. / Plant Physiol. Biochem. 40 (2002) 761–769 763 Fig. 5. Systemic accumulation of CM, FM and p-CM 24 h after treatment in upper control, upper wounding and upper wounding + SA leaves of 2-month-old plants. The different letters indicate statistically significant differences. alone results in the systemic accumulation of FM (26%) (Fig. 5) whereas with SA, the systemic effect on the concentration of this compound is insignificant. For p-CM, SA leads to systemic accumulation (47%) whereas woun- ding has no observable effect (Fig. 5). Therefore, the systemic accumulation of HCM acids (Fig. 5) is analogous to the local accumulation observed in (Fig. 4B). 2.3. Histochemistry Fig. 4. Local accumulation of CM, FM and p-CM 24 h after treatment in control and treated leaves of 1-month-old (A) and 2-month-old (B) plants. Only those reactions observed in leaves subjected to The different letters indicate statistically significant differences. wounding followed by the application of SA are presented in this paper. The leaves subjected to wounding alone display the same reactions, but to a lesser extent since the (92%) (Fig. 4B). In contrast to wounding, SA increases the lesions produced by this procedure are less severe. concentration of p-CM (64%) and has no effect on FM (Fig. 4B). It appears that in 2-month-old plants the effect on HCM 2.3.1. Demonstration of the presence of phenols acids levels of either wounding or SA applied to the wound Neu’s reagent is a borate salt that forms complexes with is similar to the effect on 1-month-old plants (Fig. 4A, B). certain groups of phenolic compounds giving them specific However, the differences between “control”, “wounding” fluorescence. This fluorescence is blue–white under UV and “wounding + SA” plants are greater than those obser- light, which is characteristic of caffeic acid derivatives [2]. ved in 1-month-old plants. A positive reaction with Neu’s reagent is observed within a few minutes of treatment; the reaction produces a blue 2.2.2. Effect of wounding and SA on the systemic fluorescence due to the accumulation of caffeic acid deri- accumulation of HCM acids vatives in the zone surrounding the lesion (Fig. 6A) and Wounding, alone or with SA, produces a systemic effect particularly in the walls of cells situated in this area (Fig. which results in the accumulation of HCM acids at sites 6B). distant from the point of treatment. Thus, non-treated leaves situated above the treated leaves also exhibit increased 2.3.2. Demonstration of peroxidase activity concentrations of CM (47%) (Fig. 5) by comparison with Examination of the leaves four days after treatment leaves in the same position in control plants. Wounding revealed a positive reaction with TMB reagent in the cells 764 F. Housti et al. / Plant Physiol. Biochem. 40 (2002) 761–769 Fig. 6. Lower leaf surface treated with Neu’s reagent (A, UV, 100×; bar = 74 µm; B, UV, 400×; bar = 18 µm), treated with TMB (C, visible light, 400×; bar = 18 µm) and treated with phloroglucinol-HCl (D, visible light, 400×; bar = 18 µm).

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