Research article Altitudinal and seasonal changes of phenolic compounds in Buxus sempervirens leaves and cuticles M. Bernal a, * , L. Llorens a , R. Julkunen-Tiitto b , J. Badosa c , D. Verdaguer a a Environmental Sciences Department, Faculty of Sciences, University of Girona, C/M a Aurèlia Capmany 69, 17071 Girona, Spain b Natural Product Research Laboratory, Department of Biology, University of Eastern Finland, P.O. Box 111, 80101 Joensuu, Finland c Laboratoire de Météorologie Dynamique, Ecole Polytechnique, 91128 Palaiseau, France article info Article history: Received 22 March 2013 Accepted 17 June 2013 Available online 25 June 2013 Keywords: Phenolic compounds Buxus sempervirens UV-B Cuticle Flavonoids Altitudinal gradient abstract The variation in the leaf content of phenolic compounds has been related to the UV-B changes of the environment in which plants grow. In this context, we aimed to investigate: a) whether the seasonal and altitudinal changes in the content of phenolic compounds of Buxus sempervirens L. leaves and cuticles could be related to the natural fluctuations in UV-B levels and b) the possible use of specific phenolic compounds as biomarkers of ambient UV-B levels. To achieve these goals we sampled, every three months during one year, leaves of B. sempervirens along an altitudinal gradient. At the lowest and the highest altitudes, we also conducted a UV-exclusion experiment to discern whether the observed changes could be attributed to the natural variation in UV-B. Results show that total phenolic content of leaves was lower in June than in the other sampling dates, which suggests a leaf ontogenic rather than a UV-B effect on the leaf content of these compounds. Regarding the elevational gradient, the overall amount of phenolic acids and neolignan of entire leaves increased with altitude while the total amount of flavonoids in leaf cuticles decreased. However, the lack of a significant effect of our UV-exclusion treatment on the content of these compounds suggests that the observed variations along the altitu- dinal gradient would respond to other factors rather than to UV-B. Concomitantly, we did not find any phenolic compound in leaves or cuticles of B. sempervirens that could be considered as a biomarker of ambient UV-B levels. Ó 2013 Elsevier Masson SAS. All rights reserved. 1. Introduction The amount of UV-B radiation reaching the Earth’s surface de- pends on many factors, among them, ozone, clouds, aerosols and surface albedo [1]. Levels of UV-B radiation also change seasonally and altitudinally [2,3]. Indeed, while the highest levels of UV-B in European ecosystems are recorded in summer, especially in June, the lowest are in winter [4]. In addition, while total irradiance and UV-A radiation increase on average 9% and 11% per 1000 m of elevation, respectively, UV-B radiation increases 19% [5]. Conse- quently, the altitudinal migration of plant species in response to global warming [6,7] will expose them to higher doses of UV ra- diation, especially UV-B. Taking into account that climate models predict increases in the UV-B levels for the Mediterranean Basin in summer [8], it is essential to improve our knowledge on how Mediterranean and sub-Mediterranean plant species respond to the altitudinal and seasonal changes in UV-B radiation in order to predict their capacity to cope with future increases in UV-B radiation. Ultraviolet-B radiation is the most energetic component of sunlight reaching the Earth’s surface and can be a photo-oxidative stress factor for plants that affects their physiological activity and morphology [9,10]. Among plant responses to increases in UV-B radiation, there are changes at the morphological (e.g. increases in leaf thickness and specific leaf weight or decreases in plant height) and biochemical levels (e.g. the synthesis of phenolic compounds) [10,11]. It is well known that a general response to enhanced levels of UV-B radiation is the biosynthesis of phenolic compounds [12], since they can help filter UV radiation avoiding or minimizing penetration of UV-B into internal tissues [13]. Phenolic compounds can also act as antioxidants by scavenging the free radicals produced under oxidative stress [14]. Therefore, variations in the leaf phenolic content of plants have been related to the natural changes in UV-B radiation of the environment in which plants grow. Hence, higher leaf phenolic content is expected with increasing altitudes [15e24] or in the seasons with the highest UV- * Corresponding author. Tel.: þ34 658333082. E-mail address: meritxell.bernal@gmail.com (M. Bernal). Contents lists available at SciVerse ScienceDirect Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy 0981-9428/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved. http://dx.doi.org/10.1016/j.plaphy.2013.06.012 Plant Physiology and Biochemistry 70 (2013) 471e482