Research articleEffects of long-term UV-exposure and plant sex on the leaf phenoloxidase activities and phenolic concentrations of Salix myrsinifolia (Salisb.)
Introduction
Ultraviolet radiation (UV, 200-400 nm) reaching the surface of the earth has been increasing at a rate of about 5% a year since the 1970′s due to depletion of the ozone layer (Madronich et al., 1998). It was earlier predicted that ozone depletion will result in a 50–60% increase in the spring time UV-B radiation in 2010–2020 (Taalas et al., 2000). More recent calculations, including climate feedbacks, suggest that ozone is expected to return to pre-1980 levels (McKenzie et al., 2007). However, in the year 2011, an ozone hole over the Arctic was comparable to that over Antarctica, and significantly elevated UV irradiances may extend to northern mid- and high-latitudes, with consequences for ecosystems (EEAP, 2011).
Reports of UV-B radiation often concentrate on the negative effects of enhanced UV-B (Jansen and Bornman, 2012). However, UV radiation also has a wider role as an important regulator of many physiological and morphological processes in plants and in animals (Jenkins, 2009; Jansen and Bornman, 2012). Many early studies (in 1980–2000) show that enhanced UV-B radiation causes, among other things, growth retardation, oxidative stress, DNA damage and a decrease in photosynthetic rates; however, several of these studies, have been performed under unnatural conditions (Jansen and Bornman, 2012). Photosynthetically active radiation (PAR) that is too low in relation to the level of UV-B radiation reduces the plants' defence against UV radiation, thus overemphasizing the damaging effects of UV-B radiation. The current study uses a modulated system that provides designed supplemental irradiances proportional to ambient light conditions. This is reckoned to be the ecologically most relevant way to study the effects of elevated UV-B radiation (Sullivan, 2005).
Enhanced UV-B radiation is often related to an increased accumulation of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), superoxide anion radicals (•O2-) and hydroxyl ions (•OH) (Strid et al., 1994). However, plants have efficient systems for dissipating ROS that involve phenolic compounds, small molecular antioxidants such as ascorbate and glutathione, and a variety of enzymes, such as superoxide dismutase (SOD), catalase (CAT), ascorbate (APX) and guaiacol peroxidase (POD). The accumulation of protective phenolic compounds in plant leaves as a response to enhanced UV-B radiation has been described thoroughly in the literature (e.g. Li et al., 2010). Flavonoids and phenolic acids strongly absorb radiation in the UV-B range, acting as a protective shield in the epidermis and thus reducing the penetration of UV radiation in the lower tissues of leaves (Sullivan, 2005). In addition, flavonoids are well-known antioxidants that suppress ROS (e.g. Agati et al., 2013).
Guaiacol peroxidases belonging to class III PODs (EC 1.11.1.7) form a large multigene family and are involved in many important physiological processes in plants. The best-known role of PODs is the synthesis of lignin (e.g. Hiraga et al., 2001). POD-mediated biosynthesis of lignin, which results in increased cell wall and leaf toughness, would contribute to the UV tolerance of plants (Jansen et al., 2001). The oxidation of phenolics catalyzed by PODs either consumes or produces H2O2, through the peroxidative or hydroxylic cycle, depending on the circumstances (Passardi et al., 2004). The pro-oxidative activity of PODs, i.e. the ability to produce ROS, is linked to defence against herbivores (e.g. Barbehenn et al., 2010). The ability to consume ROS, on the other hand, is related to the PODs' antioxidative capacity and defence against environmental stresses (Ruuhola et al., 2009). There are many reports on UV-B-induced alterations in POD activity in several non-woody plant species (e.g. Jansen et al., 2001), but there are few studies on woody-plants.
It has been suggested that polyphenoloxidases (PPOs) act as quenchers of photo-oxidation in chloroplasts. PPO enzymes are encoded by a small multigene family, and this family contains two types of enzymes. Catechol oxidases or diphenolases (EC 1.10.2.2) oxidize o-diphenols to produce o-quinones, while tyrosinases or monophenoloxidases (EC 1.14.18.1) convert monophenols to o-diphenols (Sullivan, 2015 and references therein). PPOs play an important role in defence against herbivores in several classes of plants (Felton et al., 1989; Thipyapong et al., 2007; Ruuhola et al., 2008). In addition, PPOs play an important role in disease resistance (e.g. Thipyapong et al., 2007). PPOs may also be important in defence against abiotic stresses, such as water stress (Thipyapong et al., 2004, 2007). We also found a slight induction in PPOs in a deciduous tree species, Betula pubescens ssp. czerepanovii, as a response to elevated UV-B radiation (Anttila et al., 2010). Furthermore, due to the chloroplastic location of PPO protein in contrast to the vacuolar location of PPO substrates, PPOs could have potential roles in the biosynthesis of specialized metabolites and in plant responses to environmental factors, but so far these topics remain largely unexplored (Boeckx et al., 2015; Sullivan, 2015).
Female plants of deciduous species, such as willows, are often better defended against herbivores than are male plants, which are, in turn, more growth-oriented (Cornelissen and Stiling, 2005; Nybakken et al., 2012). Such gender-related differences in response to abiotic stresses might explain the often found, yet unexplained female-biased sex ratio in Salix populations (Ueno et al., 2007; Myers-Smith and Hik, 2012). Salix myrsinifolia (Salisb.) is rich in species-specific phenolic glycosides (salicylates). The effects of UV-B radiation on the phenolic compounds of S. myrsinifolia have been investigated in several earlier studies (e.g. Tegelberg et al., 2001; Randriamanana et al., 2015a), but no long-term study investigating the possible roles of PPO and POD enzymes in plants' tolerance to UV-B has yet been conducted. In particular, no earlier study has investigated the sex-related differences and the importance of phenoloxidase activities in response to long-term UV-B exposure in woody temperate plants.
The purpose of this study was to follow how a long-term (three growth seasons) exposure to enhanced UV-B radiation affects the phenolic metabolism and phenoloxidase activities of a dioecious species, S. myrsinifolia. We hypothesized that after three years exposure, concentrations of certain secondary chemicals (particularly quercetins) would increase, as has been found for different boreal deciduous tree species (Tegelberg et al., 2001; Nybakken et al., 2012; Randriamanana et al., 2015a,b). In addition, we expected an enhancement in the plants' ability to quench ROS by antioxidative defences such as PPOs and PODs.
Section snippets
Experimental setup
We used clonal Salix myrsinifolia plants grown in the field at the Botanical Garden of the University of Eastern Finland for three growth seasons, starting from June 2009 (see Nybakken et al., 2012). The S. myrsinifolia clones originated from cuttings that we collected from different areas in Eastern Finland. During the present experiment, the plants were 3 years old (sampling time in early August) and had an average height of 2.15 m in 2011 (Randriamanana et al., 2015a). We did not fertilize,
Results
Effects of long-term UV-A and UV-B radiation on foliar phenoloxidase activities in female and male plants of S. myrsinifolia.
The long-term UV-B treatment had significant interactive effects with plant sex on the PPO activities of the S. myrsinifolia leaves (Table 1). Male plants had higher specific PPO activity (U mg−1 protein) than did female plants, but this difference was significant only in the control treatment (Table 1, Fig. 1A and B). A similar tendency was detected in the PPO activity
Discussion
After three years' exposure to UV-A and enhanced UV-B, the PPO activity of male S. myrsinifolia plants was higher than that of female plants, but only in the control treatment. It has been shown that male and female plants of dioecious species differ in their investments in growth and defence (Cornelissen and Stiling, 2005; Nybakken et al., 2012). The growth of male S. myrsinifolia plants responded more vigorously to elevated temperature than did female plants during the two first years (
Contribution
T Ruuhola performed the chemistry and enzyme analyses. T Randriamanana and AL contributed to the field experiment. The experiment was designed by RJT and LN. T Randriamanana was the corresponding author and has taken the lead in revising the manuscript. All the authors contributed to the writing of the manuscript.
Acknowledgements
We wish to thank Matti Savinainen and Kari Määttä for all their help with the planning, construction and maintenance of the field. Our thanks are also due to Minna Kosonen, Merja Maukonen, Katri Nissinen, Milla Ratilainen, Tarja Parkkinen, Riitta Pietarinen and Virpi Virjamo for their help with the field work, and to Mervi Kupari and Sinikka Sorsa for their help with field and laboratory work. We also wish to thank Rosemary Mackenzie for her help in revising the English text. The work was
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