Effects of enhanced UV radiation and water availability on performance, biomass production and photoprotective mechanisms of Laurus nobilis seedlings

Accepted Manuscript Title: Effects of enhanced UV radiation and water availability on performance, biomass production and photoprotective mechanisms of Laurus nobilis seedlings Author: Meritxell Bernal Dolors Verdaguer Jordi Badosa Anunciaci´on Abad´ıa Joan Llusi`a Josep Pe˜nuelas Encarnaci´on N´un˜ ez-Olivera Laura Llorens PII: DOI: Reference:

S0098-8472(14)00166-X http://dx.doi.org/doi:10.1016/j.envexpbot.2014.06.016 EEB 2825

To appear in:

Environmental and Experimental Botany

Received date: Revised date: Accepted date:

3-2-2014 29-5-2014 16-6-2014

Please cite this article as: Bernal, Meritxell, Verdaguer, Dolors, Badosa, Jordi, Abad´ia, Anunciaci´on, Llusi`a, Joan, Pe˜nuelas, Josep, N´un˜ ez-Olivera, Encarnaci´on, Llorens, Laura, Effects of enhanced UV radiation and water availability on performance, biomass production and photoprotective mechanisms of Laurus nobilis seedlings.Environmental and Experimental Botany http://dx.doi.org/10.1016/j.envexpbot.2014.06.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Effects of enhanced UV radiation and water availability on performance, biomass production and photoprotective mechanisms of Laurus nobilis seedlings

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Meritxell Bernal1, ([email protected]) 1 Environmental Sciences Department, Faculty of Sciences, University of Girona, C/ MªAurèlia Campmany 69, 17071, Girona (Spain),

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Dolors Verdaguer1* ([email protected]) 1 Environmental Sciences Department, Faculty of Sciences, University of Girona, C/ MªAurèlia Campmany 69, 17071, Girona (Spain),

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Jordi Badosa2 ([email protected]) 2 Laboratoire de Météorologie Dynamique, Ecole Polytechnique, 91128 Palaiseau (France),

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Anunciación Abadía3 ([email protected]) 3 CSIC, Department of Plant Nutrition, Aula Dei Experimental Station, Apdo. 13034, 50080 Zaragoza (Spain),

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Joan Llusià 4,5 ([email protected]), 4 CSIC, Global Ecology Unit CREAF-CEAB-UAB, Cerdanyola del Vallès 08193, Catalonia (Spain), 5 CREAF, Cerdanyola del Vallès 08193, Catalonia (Spain),

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Josep Peñuelas4,5 ([email protected]) 4 CSIC, Global Ecology Unit CREAF-CEAB-UAB, Cerdanyola del Vallès 08193, Catalonia (Spain), 5 CREAF, Cerdanyola del Vallès 08193, Catalonia (Spain), Encarnación Núñez-Olivera6 ([email protected]) 6 Complejo Científico-Tecnológico, Universidad de La Rioja, Av.Madre de Dios 51, 26006 Logroño (Spain). Laura Llorens1 ([email protected]) 1 Environmental Sciences Department, Faculty of Sciences, University of Girona, C/ MªAurèlia Campmany 69, 17071, Girona (Spain), Corresponding author: Dolors Verdaguer [email protected] Telf: 0034972418174; FAX: 0034972418150 Environmental Sciences Department, Faculty of Sciences, University of Girona, C/ Highlights ► MªAurèlia Campmany 69, 17071, Girona (Spain), Highlights

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Enhanced UVA increased plant biomass under low water availability.

Abstract Climate models predict an increase in ultraviolet (UV) radiation and a reduction in precipitation in the Mediterranean region in the coming decades. High levels of UV

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radiation and water shortage can both cause photo-oxidative stress in plants. The aim of this study was to investigate the effects of enhanced UV radiation and its interaction

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with low water availability on seedling performance, biomass production, and

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photoprotective mechanisms of the sclerophyllous evergreen species Laurus nobilis L. (laurel). To achieve this goal, one-year-old seedlings of L. nobilis were grown outdoors

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under three UV conditions (ambient UV, enhanced UV-A, and enhanced UV-A+UV-B) and under two watering regimes (watered to field capacity and reduced water supply).

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The results show that plants produced more biomass when exposed to above ambient levels of UV-A or UV-A+UV-B radiation, especially under low water availability. This

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was probably related to a UV-induced increase in leaf relative water content and in leaf

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water use efficiency under water shortage. Even though our results suggest that UV-A supplementation may play an important role in the stimulation of biomass production, plants grown under enhanced UV-A plots showed higher levels of energy dissipation as heat (measured as NPQ) and a higher de-epoxidation state of the violaxanthin cycle. This suggests a greater excess of light energy under UV-A supplementation, in accordance with the observed reduction in the foliar content of light-absorbing pigments in these plants. Strikingly, the addition of UV-B radiation mitigated these effects. In conclusion, UV enhancement might benefit water status and growth of L. nobilis seedlings, especially under low water availability. The results also indicate the activation of different plant response mechanisms to UV-A and UV-B radiation, which would interact to produce the overall plant response.

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Keywords: UV radiation, drought, phenolic compounds, xanthophyll cycle, photosynthetic pigments, Mediterranean species.

Acknowledgements: This research was supported by the following projects: CGL2007-

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64583, CGC2010-17172, Consolider Ingenio Montes (CSD2008-00040) and CGL201126977 funded by the Spanish government and by the SGR 2009-458 project funded by

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the Catalan government. We are also grateful to the Gavarres Consortium for allowing

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1. Introduction

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us to perform the experiment in Can Vilallonga.

The levels of UV-B radiation reaching the Earth’s surface have increased in recent decades due to the depletion of the ozone layer, which is not expected to recover until 2025-2040 at mid-latitudes (World Meteorological Organization, WMO, 2010). Furthermore, in the Mediterranean Basin, decreases in the mean cloudiness (Giorgi et al., 2004) will likely expose plants in terrestrial ecosystems to higher fluxes of UV-B and UV-A radiation in the near future. In addition, Mediterranean plants are expected to experience longer dry periods in the forthcoming decades (Intergovernmental Panel of Climate Change, IPCC, 2012). Considered individually, increases in UV-B radiation and drought have often been related to reductions in plant growth (Chaves et al., 2003; Llorens et al., 2004;

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Caldwell et al., 2007; Ballaré et al., 2011). However, the interactive effects of a combination of these two factors on plant growth are still largely unknown, and most of the available information is on crops or high latitude species (see Caldwell et al., 2003; Caldwell et al., 2007 and Ballaré et al., 2011 for reviews). Several studies have reported

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that plants growing under enhanced UV-B radiation are more tolerant to water stress, which has been related to an increase in leaf water potential (Hofmann et al., 2003) or in

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leaf relative water content (Poulson et al., 2006) in response to increased levels of UV-

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B. However, little is known about the mechanisms that would explain an improved plant water status under a combination of enhanced UV-B radiation and drought. In

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Arabidopsis, a higher leaf water content in response to such a combination was attributed to the production of osmolytes and stress-related proteins such as dehydrins

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(Schmidt et al., 2000), an increase in proline content, and a decrease in stomatal conductance (Poulson et al., 2006). A reduced leaf area production upon exposure to

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enhanced UV radiation and drought has also been described in wheat (Feng et al., 2007)

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and in peas, in this latter case together with a decrease in stomatal conductance (Nogués et al., 1998), which would reduce plant water loss. Unfortunately, very few data are available on Mediterranean species, and even less on woody evergreen sclerophyllous species (Paoletti, 2005). Nevertheless, it is surprising that in most of the Mediterranean species studied, such as Nerium oleander (Drilias et al., 1997), Olea europaea, Rosmarinus officinalis and Lavanda stoechas (Nogués and Baker, 2000) or Ceratonia siliqua (Kyparissis et al., 2001), no interactive effects between UV-B radiation and drought on plant growth have been reported. Only in some pine species, a beneficial effect of supplemental UV-B radiation under water stress was found. In this case, it was attributed to an increase in cuticle thickness, which would restrict cuticular transpiration thereby improving leaf water content (Manetas et al., 1997). Taking into account the

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climate model predictions for the Mediterranean Basin, it is clear that more studies are needed to understand how enhanced UV radiation can modulate plant responses to drier conditions. Among the most common plant mechanisms described to avoid or counteract the

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damaging effects of UV-B radiation are those aimed at preventing UV-B penetration into the leaf, either by developing thicker, hairier, or waxier leaves (Jansen 2002,

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Karabourniotis et al., 1993), or by increasing their content in UV-B-absorbing

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compounds, mainly phenols such as hydroxycinnamic acids and flavonoids (Jansen 2002, Julkunen-Tiitto et al., 2005). Recent studies suggest that hydroxycinnamic acids

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are predominantly involved in UV-B screening, whereas flavonoids, especially dihydroxy B-ring substituted flavonoids, are mainly involved in counteracting the

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generation of reactive oxygen species (ROS) (Agati et al., 2012; Brunetti et al., 2013). Increases in phenols have also been related to other environmental stresses, such as

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drought (see Selmar and Kleinwächter 2013 for a recent review). Indeed, in most of the

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species analysed till now - mainly crops - a synergistic effect between UV-B radiation and drought on the accumulation of foliar flavonoids and phenols has been found (e.g., Balakumar et al., 1993; Feng et al., 2007; Hofmann et al., 2003; Nogués et al., 1998). However, other studies have reported contrasting results. For instance, Alexieva et al. (2001) and Sangtarash et al. (2009) found no interactive effect between these two factors on total foliar phenol content, whereas Turtola et al. (2005) reported a lower UV-B-induced accumulation of phenols when willow seedlings were simultaneously stressed by drought. For Mediterranean sclerophyllous species, the few studies available have reported no interactive effect between UV-B radiation and drought on total leaf phenol content (Kyparissis et al., 2001; Nogués and Baker, 2000). However, these studies did not give information on possible changes in specific phenols.

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Under conditions of high irradiance or water shortage (which can limit CO2 fixation), an excess of absorbed light by plants can cause photo-oxidative damage (Foyer et al., 1994). To counteract these stressful conditions, Mediterranean plants have developed different photoprotective mechanisms, some of them involving reductions in

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the foliar pool of chlorophylls (Munné-Bosch and Alegre, 2000), or changes in carotenoids. In terrestrial plants, two carotenoid cycles have been associated with the

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thermal dissipation of excess light: the ubiquitous violaxanthin cycle (V-cycle), also

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known as the xanthophyll cycle (Demmig-Adams et al., 1996), and the lutein-epoxide cycle (Lx-cycle), which seems to be specific to certain species (García-Plazaola et al.,

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2002, Llorens et al., 2002). In the V-cycle, zeaxanthin (Z), the most effective xanthophyll in the dissipation of excess energy as heat (Demmig-Adams et al., 1996,

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García-Plazaola et al., 2007), is formed by de-epoxidation of violaxanthin (V) via the intermediate antheraxanthin (A). The de-epoxidation state of the V-cycle has been

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reported to increase under enhanced UV-B radiation in leaves of Fagus sylvatica

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(Šprtová et al., 2003, Láposi et al., 2009) or to decrease under UV exclusion in leaves of Vitis vinifera (Núñez-Olivera et al., 2006) and in pine needles (Martz et al., 2007). Other studies have failed to find any effect of ambient UV-B radiation on the V-cycle de-epoxidation of some species, such as pea or spruce, compared to plant growth without UV-B (Bolink et al., 2001, Kirchgebner et al., 2003). The de-epoxidation of lutein epoxide to lutein in the Lx-cycle has also been associated with the thermal dissipation of energy in some species (Llorens et al., 2002, García-Plazaola et al., 2007), but, to our knowledge, it has never been investigated in relation to changes in UV-B radiation. Neither is it known whether UV-B modulates water availability effects on the de-epoxidation state of the V- and Lx-cycles in sclerophyllous species.

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Therefore, the aim of this study was to examine whether above ambient UV radiation levels (specifically, a 20-25% increase in erythemally weighted UV doses) would affect growth, foliar gas exchange, and water relations of Laurus nobilis L. (laurel) seedlings, and whether these effects would improve the tolerance of this species

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to water limitation. We also investigated the effects of these two abiotic factors on the foliar content of specific phenolic compounds and chloroplastic pigments of this

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species. L. nobilis was selected because it is a native Mediterranean evergreen tree of

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great ecological and economical interest, which has been widely studied due to its

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pharmaceutical and alimentary properties.

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2. Materials and methods

2.1. Plant material and experimental design

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One-year-old seedlings of Laurus nobilis L. with a root ball were planted in 2 L pots (5

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cm wide x 20 cm deep) with 530 g of a mixture containing composted pine bark and Sphagnum peat (1:1 by volume). The growth medium was fertilised with Osmocote (4 kg m-3), basal dressing (1 kg m-3), and dolomite (4 kg m-3) to avoid nutritional deficiencies during the experiment. Seedlings were grown under controlled irrigation and supplemented with UV radiation in an outdoor setting to obtain more realistic and balanced ratios of UV radiation/PAR (photosynthetically active radiation). Seedlings were distributed in nine plots built with 1.3 x 1.2 m metallic frames

equipped with four fluorescent lamps mounted overhead. These nine plots were organised in three blocks, with each block having one plot with the following UV radiation conditions: ambient UV radiation, enhanced UV-A radiation, and enhanced UV-A+UV-B radiation. Within each plot, two irrigation regimes were applied (see

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below). Each combination of UV radiation and irrigation was, thus, replicated three times in a randomised complete block design. The experiment was conducted in an experimental field (Can Vilallonga) in the vicinity of Cassà de la Selva (Girona, northeastern Iberian Peninsula, 41º53’ N, 2º52’ E)

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from May 29, 2009 to January 21, 2010. Before the UV treatment began, plants were allowed to acclimate to the environmental UV and PAR conditions of the site for 2

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weeks. On May 25, immediately prior to starting the experiment, we measured the

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length and basal diameter (at 2 cm above the cotyledonary node) of the main stem of four plants from each UV radiation and irrigation treatment. Statistical analyses

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indicated that initial differences in these parameters among plants in the different UV radiation and irrigation treatments were not significant (data not shown). All samples

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were obtained around noon (± two hours) under clear-sky days from randomly chosen

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2.1.1. UV-radiation treatment

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seedlings, and, unless otherwise noted, they were collected in September 2009.

The three UV-radiation conditions (Table 1) were:

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Enhanced UV-A+UV-B: Solar UV radiation (UV-A+UV-B) was supplemented with four 40 W fluorescent lamps of 1.2 m in length (TL 40W/12 RS, with a peak at 313 nm; Phillips, Spain) mounted in metal frames (1.3 x 1.2 m). Ultraviolet radiation was applied daily for 2.5-3.5 h (depending on the month) centered at solar noon. The fluorescent lamps were wrapped with cellulose diacetate foil (Ultraphan URT, 0.1 mm, Digefra GmbH, Munich, Germany) to remove wavelengths < 280 nm (UV-C radiation). Cellulose diacetate films were

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pre-burned for 3 h before being applied to the lamps and were replaced after 36 h of use to avoid the effects of plastic photodegradation.

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Enhanced UV-A: These plots served as a control for the effect of UVA in the

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UV-A+UV-B plots. Ultraviolet radiation was supplied as described in the UVA+UV-B plots, but the lamps were wrapped with polyester instead of cellulose

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diacetate films (Mylar D, 0.13 mm; PSG Group, England) to block both UV-B

Ambient UV: Seedlings in these plots received only solar UV radiation. Instead

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•

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and UV-C radiation (transmittance > 315 nm).

of fluorescent holders, plots were equipped with wood strips to ensure that

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two radiation conditions.

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plants were exposed to the same conditions of shading than plants in the other

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Within each plot, plants were rotated every week to minimise microenvironmental and border effects. To prevent UV radiation contamination among plots, two 120 x 30 cm clear sheets of polycarbonate (no transmission below 400 nm) were fastened along the two sides of the metal frames parallel to the fluorescent lamps. Erythemally weighted UV doses (UVE, McKinlay and Diffey, 1987; Mckenzie et al., 2004) were measured using an UVS-E-T radiometer (Kipp and Zonen, The Netherlands) at the experimental field within enhanced UV-A+UV-B plots. Measurements were always taken at the top of plant canopies. The UVS-E-T radiometer used has a peak response around 300 nm and the response decreases by three orders of magnitude at 330 nm and an extra order of magnitude at 400 nm. UVE doses within ambient plots were estimated subtracting fluorescent’s contribution to UVE values obtained in UV-A+UV-B plots. UVE doses

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within enhanced UV-A plots were calculated by adding the UV-A contribution, which was estimated from field spectroradiometric measurements, to ambient UVE doses. On average, UV supplementation in enhanced UV-A+UV-B plots increased UVE doses a 23% above ambient levels, although increases ranged from 19% to 38% depending on

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the month (Table 1). Despite UVE is the irradiance weighted according to the action spectrum for UV-induced sunburn in human skin (CIE, International Committee of

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Lightening), UVE measures on plant growth studies have been widely used giving

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reliable estimates of UV doses (Kotilainen et al., 2011). In enhanced UV-A plots, UVE calculated values were similar to that of ambient UV plots (Table 1) due to the very

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weak spectral response of the UVS-E-T sensor in the UV-A range, as commented above. To illustrate this, while irradiance in the UV-B range contributes 83% to UVE,

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UV-A’s contribution is only 17% (although UV-A represents 94% and UV-B 6% of the global radiation that reaches the ground). In addition, UV-A doses emitted by the

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fluorescents lamps were small (less than 2% of the ambient UV-A under clear-sky

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conditions, according to our field measurements) in comparison to ambient UV-A radiation, as it has been previously reported (e.g. Aphalo 2013). Photosynthetically active radiation (PAR) was measured with a LI-190SA Quantum Sensor (Li-Cor, USA) in the same plots and at the same height than UVE doses (Table 1).

2.1.2. Irrigation treatment

Plants received water from rainfall and from a controlled drip-irrigation system, which was programmed according to the established irrigation conditions and monthly rainfall (Table 1). In June 11, two irrigation regimes were applied: half of the seedlings of each plot (chosen randomly) were irrigated to field capacity (well-watered plants, WW), which involved adding from 0.2 to 1.34 L of water per day depending on the

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precipitation, while the other half (low-watered plants, LW) received approximately 40% and 25% less water from June to September and from October to December, respectively, than WW plants.

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2.2. Biomass production and foliar morphological traits

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Above- and below-ground biomass were determined for four seedlings from each plot

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and irrigation treatment (three were harvested in September and one in January). The leaves, stems, and roots were separated and oven-dried at 70 ºC for 72 h to assess their

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dry mass (DM).

One fully expanded and light-exposed leaf from four individual plants per plot

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and irrigation condition was sampled to determine leaf thickness and leaf mass per area (LMA, mg cm-2). Foliar thickness was measured with a portable micrometer (mod.

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4000DIG, Baxlo, Spain). The leaves were then scanned (Epson perfection 1250, USA),

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and leaf areas determined using an image-analysis programme (ImageTool, University of Texas Health Science Center, USA). Subsequently, the DM of leaves was assessed and the LMA calculated as the quotient between foliar DM and foliar area for each leaf.

2.3. Relative water content

The relative water content (RWC, %) was measured in the same leaves used to analyse morphological traits. After collection, leaves were weighed to obtain leaf fresh mass (FM) and then they were stored in darkness with distilled water for 48 h to determine leaf turgid mass (TM). The DM was subsequently measured (as described above), and the RWC was calculated as: (FM-DM/TM-DM) x 100.

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2.4. Gas-exchange measurements

Foliar gas exchange was measured in one plant per plot and irrigation condition, using one young, fully expanded light-exposed leaf located at the top of the canopy.

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Measurements were taken using a gas-exchange system (CI-340 Hand-Held Photosynthesis System, CID, Inc., Camas, WA 98607 USA) as described in Llusià et al.

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(2012). With this system, L. nobilis net photosynthesis (A, µmol CO2 m–2 s–1) and

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stomatal conductance (gs, mmol H2O m–2 s–1) were determined under environmental conditions of CO2 concentration, humidity and temperature, but fixing PAR to 1000

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µmol m-2 s-1 to saturate the photosystems and to avoid possible light differences among

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2.5. Chlorophyll fluorescence

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measurements.

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At predawn and midday, the components of foliar chlorophyll fluorescence were quantified by means of a PAM-2100 portable modulated fluorometer (Heinz Walz GmbH, Effeltrich, Germany) for four seedlings per plot and irrigation condition, using one exposed and fully developed leaf per seedling. Minimum (Fo) and maximum (Fm) dark-adapted fluorescence was determined and used to calculate the potential photochemical efficiency of photosystem II as Fv/Fm, where Fv is variable fluorescence and Fv = Fm-Fo. The actual photochemical efficiency of photosystem II in the lightadapted state was calculated as ∆F/Fm’ = (Fm’-F)/Fm’, where F is the steady-state fluorescence yield under the given environmental conditions and Fm’ is the maximum level of fluorescence obtained during a saturating flash of light. The apparent electron transport rate (ETR) was then calculated as ETR = ∆F/Fm’ x PAR x 0.84 x 0.5, where

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PAR was the incident photosynthetically active radiation (expressed in µmol m-2 s-1), 0.84 was the assumed coefficient of absorption of the leaves, and 0.5 was the assumed distribution of absorbed energy between the two photosystems (Galmés et al., 2007).

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The non-photochemical quenching coefficient (NPQ) was determined as (Fm-Fm’)/Fm’.

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2.6. Photosynthetic pigments

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At predawn and midday at least three foliar discs of 0.64 cm2 per plot and irrigation condition were sampled, always from fully expanded light-exposed leaves from

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different plants. Leaf discs were immediately frozen in liquid nitrogen, and stored at -80 ºC until analysis. Photosynthetic pigments were extracted with 1 mL of acetone in the

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presence of liquid nitrogen and ascorbate by grinding the foliar discs in a mortar. Four mL of acetone were then added, and the mixture was stored at -20 ºC, as previously

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described (Abadía and Abadía, 1993). Pigment extracts were thawed on ice, filtered

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through a 0.45-µm filter, and analyzed by high-performance liquid chromatography (HPLC) following the method described by Larbi et al. (2004). Two phases were pumped instead of three: phase A [acetonitrile:methanol 7/1 (v/v)] was pumped for 3.4 min, and phase B [acetronitrile:methanol:water:ethyl acetate 7/0.96/0.04/8 (v/v/v/v)] was pumped for 4.6 min. Triethylamine was added to each phase to obtain a final concentration of 0.7% in the mixture. The injection volume was 20 µL, and the analysis time for each sample was 15 min, including the equilibration time, which consisted of flushing phase A for 5 min at the beginning. Results were expressed in µg of pigment per g of foliar DM using LMA conversion.

2.7. Total phenols

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One fully expanded light-exposed leaf from four plants per plot and irrigation condition was sampled. From each leaf, 10 mg of dried material was ground to a powder and mixed with 2.5 mL of 50% methanol. The extract was then shaken for 1 h and subsequently centrifuged for 5 min at 800 g. Total phenolic content was determined

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following the method described in Rozema et al. (2006). A fraction of the extract (50 µL) was mixed with 3.5 mL of distilled water and 250 µL of Folin-Ciocalteu reagent

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(Merck, Germany). After 8 min, 750 µL of Na2CO3 (20%) was added. Absorbance was

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measured 2 h later at 760 nm with a spectrophotometer (U-2000, Hitachi, USA). The total phenolic content of leaves was calculated from the standard curve for gallic acid,

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prepared from 50 µL of a standard solution of gallic acid (40, 80, 150, 200, 400, 600,

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2.8. UV-absorbing compounds (UACs)

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800, and 1000 mg L-1) and expressed as mg of gallic acid equivalents per g of DM.

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Methanol-soluble and methanol-insoluble (alkali-extractable cell wall-bound) UVabsorbing compounds were analyzed for four plants per plot and irrigation condition. Three foliar discs of 0.64 cm2 were collected from fully expanded and light-exposed leaves of each plant, frozen in liquid nitrogen, and stored at -80 ºC until analysis. Total foliar content of UACs and foliar concentrations of several phenolic compounds were measured by spectrophotometry (Perkin-Elmer, Wilton, CT) and HPLC (Agilent HP1100 HPLC system, Agilent Technologies, Palo Alto, CA), respectively. The extraction and analytical methods were as reported in Fabón et al. (2010). Briefly, after grinding the plant material in a tissue lyser (Qiagen, Hilden, Germany), 5 ml of methanol:water:7 M HCl (70:29:1) was added and the mixture stored for at least 20 h at 4 ºC in the dark. The extract was centrifuged at 6000 g for 15 min at 10 ºC, and the

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supernatant was removed and used for the determination of methanol-soluble UACs (mainly located in the vacuoles). The pellet was stored at -80 ºC and later used for the determination of methanol-insoluble UACs (mainly located in the cell wall). For the determination of methanol-soluble phenols by HPLC, 250 µL of the

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supernatant were filtered (0.22 µm) and pumped into the HPLC. The remainder of the supernatant was used to determine total methanol-soluble UACs spectrophotometrically

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in arbitrary units, as the area under the absorbance curve in the intervals 280-315 nm

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and 280-400 nm (UAC280-315 and UAC280-400) per unit of DM. For the methanolinsoluble UACs, the pellet remaining from the methanol extraction was hydrolysed with

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2 mL of 1 M NaOH, and the mixture was heated at 80 ºC for 3 h. One mL of 5.6 N HCl was added, and the UACs were extracted three times with 2 mL of ethyl acetate. The

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supernatants from each extraction were collected and evaporated. The residue was dissolved in methanol, and the amount of phenols specific to the cell wall were

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determined by HPLC, while the total content of UACs was determined

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spectrophotometrically in the same units as above. Specific phenolic compounds were identified taking into account their absorption spectra and retention times, and quantification was made by calibration curves using appropriate commercial external standards.

2.9. Statistical analyses

The main effects of UV radiation treatment, irrigation treatment, block, and their interactions, on all variables, except those obtained from foliar gas-exchange measurements, were assessed by three-way analysis of variance (ANOVA). When the overall effect of UV treatment and/or the interaction between UV and irrigation

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treatments were/was significant, we tested the effects of our UV treatment within each watering regime using two-way ANOVAs, with block and UV treatment as fixed factors. Pairwise comparisons between UV conditions were analyzed using Duncan’s test. For the foliar gas-exchange data, the effects of the UV and irrigation treatments

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were analyzed using two-way ANOVAs, since only one plant per plot and irrigation treatment was sampled. Kolmogorov-Smirnov and Levene’s tests were used to test

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normality and homoscedasticity, respectively, and data was log-transformed when

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necessary. When normally distributed data did not meet the assumption of homoscedasticity, the Games-Howell post-hoc test was applied. A significance level of

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P ≤ 0.05 was used for all statistical tests.

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3. Results

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3.1. Seedling biomass and leaf morphological traits

Exposure to enhanced levels of UV-A and UV-A+UV-B radiation increased the production of biomass in L. nobilis seedlings. Seedlings grown under these conditions had, respectively, 36% and 41% greater stem biomass and 26% and 30% greater root biomass than seedlings grown under ambient levels of UV radiation (Table 2). However, when the analyses were conducted within each irrigation condition, differences were only significant for low-watered plants. Indeed, low-watered plants exposed to enhanced UV-A and UV-A+UV-B radiation had greater foliar, stem and root biomass than control plants (F2,36 = 3.90, P = 0.03; F 2,36 = 5.56, P = 0.01 and F 2,36 = 3.55, P = 0.04, respectively) (Figure 1), although they did not show changes in the rootto-shoot ratio (data not shown).

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Leaves from UV-A+UV-B-supplemented plants were about 11% thicker than those of control plants, but neither foliar area nor LMA changed significantly in response to our UV treatment (Table 2). Overall, plant biomass and leaf morphological

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traits did not differ significantly between the two irrigation conditions (Table 2).

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3.2. Leaf physiological parameters

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A significant interaction between the effects of UV and irrigation treatments was found for leaf RWC (Table 2). For control plants, well-watered seedlings had a 3.4% higher

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foliar RWC than did low-watered seedlings (F 1,23 = 9.01, P < 0.01) (Figure 2). Besides, while the UV treatment did not affect the foliar RWC of well-watered plants, low-

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watered seedlings had 2.2% and 2.6% higher foliar RWCs under enhanced UV-A and UV-A+UV-B radiation, respectively, than control seedlings, although the difference

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was only significant in the UV-A+UV-B-supplemented plants (Figure 2).

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The effect of the irrigation treatment on leaf gas exchange parameters differed

among UV-conditions, which would explain the significant interaction found between treatments (Table 2). Basically, low-irrigated plants supplemented with UV-A radiation had higher photosynthetic rates (A) and stomatal conductance (gs) than those grown under optimal irrigation (Figure 3). In contrast, no significant differences were observed in leaf gas exchange parameters between low- and well-watered plants supplemented with UV-A+UV-B radiation (Figure 3). Overall, A and water-use efficiency (WUE or A/gs) were significantly higher in low- than in well-watered plants (Table 2), which was basically due to the higher values found in plants supplemented with UV-A and UVA+UV-B radiation (100% and 56%, respectively, in the case of WUE, despite differences were not significant; Figure 3).

Page 17 of 53

Parameters derived from foliar chlorophyll fluorescence measurements (ETR, Fv/Fm, and NPQ) were globally unaffected by the treatments, either at predawn (data not shown) or at midday (Table 2). Nevertheless, low-watered plants exposed to enhanced UV-A radiation fluxes had significantly higher NPQ values at midday than plants

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grown under ambient UV levels or under UV-A+UV-B supplementation (Figure 4).

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3.3. Leaf photosynthetic pigments

Results consistently indicated that, at predawn and midday, L. nobilis seedlings growing

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in enhanced UV-A plots had the lowest foliar content of most of the photosynthetic pigments related to the absorption of light: chlorophyll a+b, neoxanthin, β-carotene and

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lutein (although differences in this last pigment were not significant) (Table 3). At predawn, however, UV radiation effects on the foliar content of chlorophylls and β- and

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α-carotene were dependent on the amount of water supplied to the plants (Table 3),

Ac ce pt e

since differences were only significant for low-watered seedlings (Figure 5). Plants grown in enhanced UV-A-plots had significantly higher zeaxanthin

content than control plants at predawn or than UV-A+UV-B irradiated plants at midday (Table 3). Accordingly, under reduced irrigation, UV-A-supplemented plants showed the highest de-epoxidation state of the V-cycle (AZ/VAZ) at predawn (Figure 6). These plants also had the lowest foliar content of lutein and lutein-5,6-epoxide (Figure 6). At midday, UV-A-supplemented leaves showed higher AZ/VAZ values, but only in comparison to leaves under enhanced UV-A+UV-B (Table 3).

3.4. UV-absorbing compounds

Page 18 of 53

Eight methanol-soluble compounds were detected. Two belonged to the quercetin family (quercetin 3-O-glucuronide, quercetin-O-glycoside) and two to the kaempferol family (cis-trans-kaempferol 3-O-glucoside, kaempferol 3,7-O-diglucoside) (Table 4). There were also three unidentified compounds, named as C1, C2, and C3, with highly

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similar spectra to that of quercetins, which were, thus, considered from the quercetin family. Finally, another unidentified compound, C4, had a spectrum with a high

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similarity to that of kaempferols and it was considered from the kaempferol family

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(Table 4). Regarding cell-wall compounds, only two were detected: p-coumaric (a hydroxycinnamic acid) and one kaempferol derivative (Table 4).

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Neither the total leaf content of phenols nor the total leaf content of methanol-soluble (mainly located in vacuoles) and methanol-insoluble (from the cell

400)

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wall) UV-B-absorbing compounds (UAC280-315) or UV-absorbing compounds (UAC280varied in response to our experimental treatments or their interaction (Table 4). The

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total amount of quercetins and kaempferols was lower in plants exposed to enhanced

Ac ce pt e

UV-A or enhanced UV-A+UV-B radiation than in control plants, although the kaempferol content only differed significantly between UV-A-supplemented and control plants (Table 4). Among quercetins, the leaf content of quercetin 3-O-glucuronide was reduced in UV-A- and UV-A+UV-B-supplemented plants compared to controls, while the foliar amount of C1 was significantly lower only in UV-A-supplemented plants (Table 4). Leaves exposed to enhanced-UV-A radiation had also smaller amounts of cistrans-kaempferol 3-O-glucoside than control plants (Table 4). Any of the two cell-wall compounds detected responded significantly to the UV radiation treatment (Table 4). The irrigation treatment did not affect significantly the leaf content of phenols or UACs.

4. Discussion

Page 19 of 53

4.1. Effects of enhanced UV radiation and low irrigation on plant biomass and leaf gas exchange

Plant biomass accumulation is an integrated parameter used commonly as an indicator

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of sensitivity to stressful conditions (Smith et al., 2000). In our experiment, enhanced UV radiation (UV-A and UV-A+UV-B) positively affected the biomass accumulation

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of L. nobilis seedlings, indicating that this sclerophyllous species has effective

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mechanisms for dealing with increased levels of UV radiation. Our results also suggest that this UV-stimulation of growth is mainly a response to enhanced UV-A radiation,

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since no significant differences were found between plants grown under enhanced UVA radiation and those grown under enhanced UV-A+UV-B radiation. Similar studies

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have also reported no effect of UV-B radiation on plant biomass accumulation (de la Rosa et al., 2001, Kostina et al., 2001, Hakala et al., 2002, Zaller et al., 2004, Bassman

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and Robberecht, 2006, Wang et al., 2008), although negative effects have been shown in

Ac ce pt e

many other studies, mainly on crop species (Yuan et al., 2002, Zavala and Ravetta, 2002, Feng et al., 2003, Gao et al., 2003, Zheng et al., 2003, Yao et al., 2006, Kadur et al., 2007, Surabhi et al., 2009, Tsormpatsidis et al., 2010, Yao and Liu, 2009). The effect of UV-A radiation on plant biomass has received less attention than that of UV-B, but, like UV-B radiation, this effect appears to be species-specific. Indeed, while UV-A radiation negatively affected root biomass in Quercus robur (Newsham et al., 1999) or total biomass in cucumber (Krizek et al., 1997), it promoted overall growth in radish, probably due to an increase in the foliar content of chlorophylls, the photosynthetic activity, and the nitrogen metabolism (Tezuka et al., 1994). In line with a previous study we performed using seedlings of six Mediterranean species (Bernal et al., 2013), the possible beneficial effect of UV-A supplementation on

Page 20 of 53

biomass accumulation in L. nobilis appears to occur principally when the plants are growing under low water availability (Figure 1). In fact, our results suggest that plant exposure to enhanced UV-A radiation, may have mitigated the negative impact of water deficit on leaf water status, since while reduced irrigation decreased the leaf RWC of

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control plants, it did not modify leaf RWC of plants grown under enhanced UV-A or UV-A+UV-B (Figure 2). Several previous studies have reported an amelioration of

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water deficit in UV-B-irradiated plants grown under low water availability (Feng et al.,

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2007; Schmidt et al., 2000; Poulson et al., 2006; Nogués et al., 1998; Manetas et al., 1997). However, in our study, the observed increase in leaf RWC in response to

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enhanced UV radiation under water shortage (Figure 2) seems to be mediated mainly by UV-A radiation. In this case, the improvement in water relations is not associated with a

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decrease in leaf area or stomatal conductance (gs) (Figure 3) (Feng et al., 2007; Nogués et al., 1998; Poulson et al., 2006). Instead, it might be explained, at least partially, by a

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greater leaf thickness (Table 2), since thicker leaves have been often associated with an

Ac ce pt e

improvement in water relations (Gitz and Liu-Gitz, 2003; Ennajeh et al., 2010). In addition, UV-A- and UV-A+UV-B-irradiated plants tended to have, under low irrigation, higher leaf WUE values than plants exposed to ambient UV levels, which resulted from proportionally greater increases in leaf photosynthetic rates than in gs (86% and 30%, respectively; Figure 3). Hence, under low water availability, improved WUE and leaf RWC in UV-irradiated plants, compared to controls, might explain their higher biomass production. As a consequence, our results suggest that future increases in UV doses similar to the ones applied in this experiment might benefit the growth of L. nobilis seedlings under moderately drier conditions.

Page 21 of 53

4.2. The role of photosynthetic pigments in the response of plants to enhanced UV radiation and low watering

The xanthophyll V- and Lx-cycles have been associated with thermal dissipation

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of excess light energy in the antenna of photosystem II (García-Plazaola et al., 2007) and in fact, this has been reported for L. nobilis in particular (Esteban et al., 2007 and

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2008). The results from our study suggest that, under low water availability, small

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increases in above ambient levels of UV-A radiation might enhance the thermal dissipation of excess energy (measured as NPQ) in this species (Figure 4). This would

an

be in agreement with the higher levels of zeaxanthin (at predawn and midday) and the higher AZ/VAZ values (at midday) found in UV-A irradiated plants of L. nobilis (Table

M

3). Under reduced irrigation, UV-A irradiated seedlings also showed the highest deepoxidation state of the V-cycle (AZ/VAZ) and the lowest leaf content in lutein-5,6-

d

epoxide at predawn (Figure 6), which would support the idea that these plants would

Ac ce pt e

require a higher thermal dissipation of light energy in the antenna (García-Plazaola et al., 2002, Llorens et al., 2002). At the same time, the observed reduction in the foliar content of most of the main light-absorbing pigments in UV-A-irradiated leaves (Table 3, Figure 5) might have contributed to avoiding the imbalance between light absorption and the energy required for photosynthesis (Munné-Bosch and Alegre, 2000). Nevertheless, taking into account, the low amount of UV-A supplied by our lamps in relation to ambient levels, this point clearly deserves further investigation. Interestingly, we did not find significant differences between controls and UVA+UV-B-irradiated leaves in the NPQ, de-epoxidation of the xanthophyll cycles or the amount of light-absorbing pigments (Table 3 and Figures 4-6). In the case of the deepoxidation of the V-cycle, this might be due to a UV-B-induced inhibition of

Page 22 of 53

violaxanthin de-epoxidase, as has been observed in isolated thylakoids and in intact leaves of pea (Pfündel et al. 1992). Yang et al. (2007) reported higher levels of violaxanthin and lower levels of zeaxanthin in leaves of wheat plants subjected to UV-A plus UV-B radiation relative to those irradiated with UV-A radiation only. At the same

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time, they found an increase in the components of the cellular antioxidant system, such as superoxide dismutase, catalase, ascorbate peroxidase, and glutathione reductase, in

cr

UV-A+UV-B-supplemented plants. These authors suggested that in UV-A+UV-B-

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irradiated plants the endogenous antioxidant defense system is enhanced while in UV-

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A-irradiated plants it is the thermal dissipation of light energy that is enhanced.

4.3. Response of specific phenolic compounds to enhanced UV radiation and low

M

irrigation

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An increase in foliar UACs, especially phenols, is a common plant response mechanism

Ac ce pt e

for dealing with increased UV radiation (Julkunen-Tiitto et al., 2005, Caldwell et al., 2007). However, in the present study, the experimental increases in UV radiation did not change the total foliar content of UACs or phenolic compounds in L. nobilis seedlings (Table 4), as was the case with previous studies on other Mediterranean species (Bernal et al., 2013¸ Manetas, 1999; Stephanou and Manetas, 1997). Despite the lack of observed UV effects on the total amount of these compounds, changes in the content of specific phenols upon UV exposure were found. Indeed, plants of L. nobilis grown in enhanced UV-A plots had a lower content of quercetin and kaempferol derivatives in the vacuoles compared to control plants (Table 4). The presence of similar amounts of these compounds in UV-A+UV-B- and UV-A-supplemented plants suggests

Page 23 of 53

that UV-B supplementation did not exert a significant additional effect on the foliar accumulation of these compounds (Table 4). The greater need for energy dissipation in low-watered plants grown in enhanced UV-A plots would suggest a possible photo-oxidative stress. However, these plants had

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the lowest levels of quercetins and kaempferols which, especially in the case of quercetins, are expected to increase under oxidative stress (Agati et al., 2012; Pollastri

cr

and Tattini, 2011). Very few data are available about the effect of UV-A radiation on

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the foliar amount of phenols, but Wilson et al. (2001), in a controlled lab experiment with Brassica napus, also reported a general down-regulation of extractable flavonoids

an

mediated by UV-A radiation affecting quercetins more than kaempferols. They suggested that this decrease could be a photobiological response involving

M

activation/inactivation of leaf photoreceptors, while rejecting the idea of direct photochemical degradation. In our study, the photoprotective mechanisms detected in

d

UV-A-irradiated leaves (i.e., a reduction in the amount of light-harvesting pigments and

Ac ce pt e

an increase in the dissipation of excess energy as heat) may have lowered the production of ROS and, in consequence, the requirement for antioxidant compounds. This would be consistent with the significantly lower levels of leaf β-carotene found in these plants both at predawn and midday (Table 3). Overall, it seems evident that L. nobilis has the ability to use different mechanisms to cope with and even to benefit from UV radiation increases.

5. Conclusions

Laurus nobilis plants produced more biomass when grown under fluorescent lamps that provided above ambient levels of UV-A or UV-A+UV-B radiation, especially under

Page 24 of 53

low water availability. This was probably related to a UV-induced improvement of leaf water status under water shortage, which seems to be a response to enhanced UVA exposition. Our results also suggest that UV-A-supplemented plants grown under low water availability had an excess of light, since they activated leaf photoprotective

ip t

mechanisms, such as the de-epoxidation of the xanthophyll cycles for the thermal dissipation of energy. Accordingly, these plants also had lower foliar contents of light-

cr

harvesting pigments, which would reduce the absorption of light by the antennae.

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Additional exposure to UV-B radiation seemed to counteract UV-A-induced effects on the studied photoprotective mechanisms without altering the beneficial effects of this

an

radiation on plant biomass accumulation. Our results, therefore, point to different plant responses to UV-A and UV-B radiation and highlight the importance of taking UV-A

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Page 39 of 53

Page 40 of 53

us

cr

UV conditions did not show significant differences in the studied parameters.

watered (WW) and low-watered (LW) plants grown under the same UV conditions (P ≤ 0.05). Within each irrigation level, plants under different

different UV and irrigation conditions. Values are means ± S.E. (N = 3). The asterisks indicate statistically significant differences between well-

ip t

Figure 3 a) Net photosynthetic rate (A), b) stomatal conductance (gs), and c) water-use efficiency (WUE) of Laurus nobilis seedlings exposed to

exposed to ambient UV conditions.

(upper and lower case letters for WW and LW plants, respectively). The asterisk indicates a significant difference between WW and LW plants

an

means ± S.E. (N = 12). Different letters indicate statistically significant differences (P ≤ 0.05) among UV conditions within each irrigation level

M

Figure 2 Foliar RWC for well- (WW) and low-watered (LW) seedlings of Laurus nobilis grown under three UV radiation conditions. Values are

d

conditions within each irrigation level (upper and lower case letters for WW and LW plants, respectively).

conditions. Values are means ± S.E. (N = 12). Different letters indicate statistically significant differences (P ≤ 0.05) among UV

Figure 1 Leaf, stem, and root biomass for well- (WW) and low-watered (LW) seedlings of Laurus nobilis grown under three UV

Ac ce pt e

Page 41 of 53

an

respectively). A, antheraxanthin; V, violaxanthin; Z, zeaxanthin.

cr

ip t

significant differences (P ≤ 0.05) among UV conditions within each irrigation level (upper and lower case letters for WW and LW plants,

us

low-watered (LW) seedlings of Laurus nobilis under three UV conditions. Values are means ± S.E. (N = 9). Different letters indicate statistically

Figure 6 Predawn foliar content of lutein and lutein-5,6-epoxide and foliar de-epoxidation state of the V-cycle (AZ/VAZ) for well- (WW) and

M

among UV conditions within each irrigation level (upper and lower case letters for WW and LW plants, respectively).

d

grown under three UV conditions. Values are means ± S.E. (N = 9). Different letters indicate statistically significant differences (P ≤ 0.05)

Figure 5 Predawn foliar content of chlorophyll a+b and α- and β-carotene for well- (WW) and low-watered (LW) seedlings of Laurus nobilis

each irrigation level (upper and lower case letters for WW and LW plants, respectively).

midday. Values are means ± S.E. (N = 12). Different letters indicate statistically significant differences (P ≤ 0.05) among UV conditions within

Ac ce pt e

Figure 4 Non-photochemical quenching (NPQ) for well- (WW) and low-watered (LW) seedlings of Laurus nobilis under three UV conditions at

Page 42 of 53

ambient UV plots

4.191 ± 0.156

3.875 ± 0.230 2.759 ± 0.081 2.086 ± 0.081 1.724 ± 0.082 0.934 ± 0.057 0.398 ± 0.057 0.494 ± 0.046

(L m-2)

11

4.8

14.6

43.2

73.7

21.5

18.4

71.1

June

July

August

September

October

November

December

January

Month

UVE (kJ m-2 day-1)

Rainfall

M

0.601 ± 0.048

0.525 ± 0.072

1.263 ± 0.074

2.261 ± 0.104

2.603 ± 0.103

3.510 ± 0.094

2.092 ± 0.081

2.767 ± 0.081

3.884 ± 0.230

4.199 ± 0.156

enhanced UV-A plots

UVE (kJ m-2 day-1)

ip t

0.495 ± 0.046

0.399 ± 0.057

cr

0.937 ± 0.057

1.730 ± 0.082

us

an

4.658 ± 0.249

4.976 ± 0.175

d

enhanced UV-A+UV-B plots

UVE (kJ m-2 day-1)

enhanced UV-A plots, and monthly average photosynthetically active radiation (PAR) doses (kJ m-2 day-1) ± S.E.

Ac ce pt e

1427 ± 235

458 ± 173

2364 ± 198

4524 ± 268

5041 ± 253

6828 ± 369

7910 ± 644

9874 ± 427

(kJ m-2 day-1)

PAR

Table 1 Total monthly rainfall in Cassà de la Selva, monthly average UVE doses (kJ m-2 day-1) in ambient UV, enhanced UV-A+UV-B and

Page 43 of 53

3.42 ± 0.28 89.73 ± 0.84

6.08 ± 0.31 a 2.94 ± 0.21 89.93 ± 0.62

Root biomass (g)

Leaf biomass (g)

RWC (%)

59.69 ± 6.34

-2 -1

69.13 ± 7.88 0.66 ± 0.01 1.82 ± 0.21

68.30 ± 9.22 0.66 ± 0.01 1.72 ± 0.19

ETR midday

Fv/Fm midday

NPQ midday

1.52 ± 0.04

0.66 ± 0.01

71.06 ± 9.33

0.11 ± 0.01

53.06 ± 4.13

5.99 ± 0.92

90.79 ± 0.52

3.56 ± 0.28

ns

ns

ns

ns

ns

ns

ns

ns

0.02

0.01

0.03

ns

2.84 ± 0.20 6.96 ± 0.38

3.30 ± 0.25 7.49 ± 0.49

90.14 ± 0.62

1.78 ± 0.18

1.59 ± 0.06

0.65 ± 0.01

ns

ns

ns

0.03

ns

0.05

ns

ns

ns

ns

ns

ns

ns

ns

p-value

ip t

63.16 ± 7.00

0.13 ± 0.01

59.14 ± 5.06

7.63 ± 1.15

cr 0.66 ± 0.01

75.84 ± 7.17

0.09 ± 0.01

56.73 ± 4.64

4.49 ± 0.74

90.14 ± 0.48

3.12 ± 0.18

12.93 ± 0.73

14.28 ± 0.94

3.49 ± 0.24

12.93 ± 0.20

12.91 ± 0.30

10.84 ± 0.40

0.43 ± 0.011

0.42 ± 0.010 10.73 ± 0.62

LW

WW

us

an

M

7.92 ± 0.61 b

3.45 ± 0.31 b

14.94 ± 1.14 b

13.15 ± 0.41

ns

0.04

p- value

Irrigation treatment

ns

ns

ns

ns

0.05

0.05

0.02

ns

ns

ns

ns

ns

ns

ns

p-value

UV*irrigation

Table 2 Overall mean ± S.E. for plant biomass and leaf morphological and physiological parameters of Laurus nobilis seedlings grown under three different UV radiation conditions (ambient UV, enhanced UV-A, and enhanced UV-A+UV-B) and two irrigation levels (WW, well-

0.10 ± 0.02

0.10 ± 0.02

61.06 ± 7.03

6.76 ± 1.87

3.32 ± 0.03 b

WUE (µmol CO2 mmol H2O)

gs (mmol H2O m s )

6.11 ± 1.07

A (µmol CO2 m s )

-1

7.67 ± 0.59 b

2.44 ± 0.17 a

Stem biomass (g)

-2 -1

11.15 ± 0.69

0.45 ± 0.015 b

UV-A+UV-B

Enhanced

d

14.41 ± 1.15 b

11.46 ± 0.64 a

Total biomass (g)

13.12 ± 0.27

10.14 ± 0.56

0.42 ± 0.012 ab

12.50 ± 0.21

11.08 ± 0.65

0.40 ± 0.009 a

UV-A

UV

LMA (mg cm )

-2

Leaf area (cm )

-2

Leaf thickness (mm)

Enhanced

Ambient

UV radiation treatment

Ac ce pt e

Page 44 of 53

Violaxanthin plus Anteraxanthin plus Zeaxanthin; ns, not significant.

us

an

cr

ip t

and values in boldface indicate statistical differences (p ≤ 0.05) in pairwise comparisons. AZ, Anteraxanthin plus Zeaxanthin; VAZ,

M

tested by three-way ANOVA, with a total of 18 degrees of freedom for the UV treatment and 27 for the irrigation treatment. Different letters

watered and LW, low-watered). The effects (p-values) of the UV and irrigation treatments and the interactions between both factors were

d

grown under three different UV conditions (ambient UV, enhanced UV-A, and enhanced UV-A+UV-B) and two irrigation levels (WW, well-

Table 3 Overall means ± S.E. at predawn (A) and midday (B) for the foliar content of photosynthetic pigments of Laurus nobilis seedlings

Ac ce pt e

watered and LW, low-watered). Different letters and values in boldface indicate statistical differences (p ≤ 0.05) in pairwise comparisons. N= 72 for all variables except for A, gs and WUE (N=18). A, photosynthetic rate; ETR, apparent electron transport rate; gs, stomatal conductance; LMA, leaf mass per area; ns, not significant; NPQ, non-photochemical quenching; WUE, water-use efficiency.

Page 45 of 53

-1

-1

Chlorophyll a+b (mg g DM ) Chlorophyll a/b -1 Neoxanthin (mg g DM ) -1 β-carotene (mg g DM ) -1 α-carotene (mg g DM ) -1 Violaxanthin (mg g DM ) -1 Anteraxanthin (mg g DM ) -1 Zeaxanthin (mg g DM ) VAZ pool AZ/VAZ -1 Lutein (mg g DM ) -1 Lutein-5,6-epoxide (mg g DM ) -1 Lutein+Lutein-5,6-epoxide (mg g DM )

B) MIDDAY

Chlorophyll a+b (mg g DM ) Chlorophyll a/b -1 Neoxanthin (mg g DM ) -1 β-carotene (mg g DM ) -1 α-carotene (mg g DM ) -1 Violaxanthin (mg g DM ) -1 Anteraxanthin (mg g DM ) -1 Zeaxanthin (mg g DM ) VAZ pool AZ/VAZ -1 Lutein (mg g DM ) -1 Lutein-5,6-epoxide (mg g DM ) -1 Lutein+Lutein-5,6-epoxide (mg g DM )

(A) PREDAWN

UV-A

UV

3.50 ± 0.19 b 3.68 ± 0.07 0.09 ± 0.01 b 0.22 ± 0.01 b 0.01 ± 0.002 0.15 ± 0.01 b 0.04 ± 0.004 0.05 ± 0.01 a 0.24 ± 0.01 0.35 ± 0.06 a 0.34 ± 0.02 0.03 ± 0.004 a 0.37 ± 0.02 b

4.11 ± 0.10 0.10 ± 0.01 b 0.23 ± 0.01 b 0.01 ± 0.003 0.23 ± 0.01 0.02 ± 0.002 0.008 ± 0.001 b 0.26 ± 0.01 0.12 ± 0.01 0.34 ± 0.01 0.03 ± 0.004 b 0.37 ± 0.02 b

3.89 ± 0.06 0.12 ± 0.005 a 0.28 ± 0.01 a 0.01 ± 0.003 0.26 ± 0.01 0.02 ± 0.002 0.005 ± 0.001 a 0.28 ± 0.01 0.09 ± 0.01 0.39 ± 0.01 0.04 ± 0.004 a 0.43 ± 0.02 a

4.16 ± 0.16 a 3.63 ± 0.06 0.11 ± 0.01 ab 0.27 ± 0.01 a 0.01 ± 0.003 0.19 ± 0.01 a 0.04 ± 0.003 0.03 ± 0.01 ab 0.26 ± 0.01 0.26 ± 0.03ab 0.39 ± 0.01 0.04 ± 0.005ab 0.43 ± 0.01 a

3.33 ± 0.22 b

4.32 ± 0.24 a

4.42 ± 0.24 a 3.77 ± 0.09 0.12 ± 0.01 a 0.29 ± 0.01 a 0.02 ± 0.003 0.22 ± 0.01 a 0.03 ± 0.003 0.02 ± 0.003 b 0.27 ± 0.01 0.19 ± 0.02 b 0.39 ± 0.02 0.05 ± 0.004 b 0.43 ± 0.02 ab

0.01 ns 0.03 <0.01 ns <0.01 ns 0.03 ns 0.02 ns 0.01 0.02

4.07 ± 0.30

LW

ip t

4.03 ± 0.15 3.71 ± 0.06 0.11 ± 0.01 0.26 ± 0.01 0.02 ± 0.003 0.19 ± 0.01 0.03 ± 0.003 0.03 ± 0.01 0.26 ± 0.01 0.25 ± 0.03 0.37 ± 0.01 0.04 ± 0.004 0.41 ± 0.01

4.02 ± 0.07 0.12 ± 0.01 0.28 ± 0.01 0.01 ± 0.002 0.25 ± 0.01 0.02 ± 0.001 0.007 ± 0.001 0.28 ± 0.01 0.10 ± 0.01 0.38 ± 0.02 0.04 ± 0.004 0.42 ± 0.02

cr 4.05 ± 0.20 3.68 ± 0.06 0.11 ± 0.01 0.26 ± 0.01 0.01 ± 0.002 0.18 ± 0.01 0.03 ± 0.003 0.03 ± 0.01 0.25 ± 0.01 0.28 ± 0.04 0.37 ± 0.02 0.04 ± 0.04 0.41 ± 0.02

3.89 ± 0.06 0.12 ± 0.01 0.26 ± 0.01 0.01 ± 0.002 0.24 ± 0.01 0.02 ± 0.002 0.007 ± 0.001 0.27 ± 0.01 0.11 ± 0.01 0.37 ± 0.01 0.03 ± 0.003 0.40 ± 0.01

4.04 ± 0.20

WW

Irrigation treatment

us

ns <0.01 <0.01 ns ns ns 0.05 ns ns ns 0.02 0.03

0.01

p-value

an

M

4.52 ± 0.33 a

UV-A+UV-B

Enhanced

3.88 ± 0.07 0.13 ± 0.01 a 0.29 ± 0.01 a 0.01 ± 0.002 0.25 ± 0.01 0.02 ± 0.002 0.006 ± 0.001 ab 0.28 ± 0.01 0.10 ± 0.01 0.39 ± 0.02 0.04 ± 0.003 a 0.43 ± 0.02 a

d

Enhanced

Ambient

UV treatment

Ac ce pt e

ns ns ns ns ns ns ns ns ns ns ns ns ns

ns ns ns ns ns ns ns ns ns ns ns ns

ns

p-value

ns ns ns ns ns ns ns ns ns ns ns ns ns

ns ns 0.01 0.02 ns ns ns ns ns ns <0.01 0.03

0.02

p-value

UV * irrigation

Page 46 of 53

d

M

(B) Cell-wall compounds

ns ns 0.01 0.01 ns <0.01 ns ns 0.05 0.03 ns ns

ns

p-value

WW

LW

Irrigation treatment

cr

ip t

0.75 ± 0.02 0.77 ± 0.02 1.19 ± 0.03 1.13 ± 0.03 24.95 ± 2.58 23.65 ± 2.18 10.01 ± 1.05 9.78 ± 1.06 6.88 ± 0.86 6.11 ± 0.61 3.65 ± 0.44 3.37 ± 0.33 2.99 ± 0.48 3.16 ± 0.42 1.41 ± 0.19 1.23 ± 0.12 12.91 ± 1.38 12.73 ± 1.04 9.50 ± 1.13 8.75 ± 0.68 0.49 ± 0.04 0.49 ± 0.05 2.91 ± 0.24 3.49 ± 0.44

72.22 ± 3.91 71.04 ± 3.02

us

an

0.74 ± 0.03 0.79 ± 0.02 0.75 ± 0.02 1.73 ± 0.07 1.16 ± 0.03 1.15 ± 0.04 30.17 ± 3.81 a 20.50 ± 2.35 b 22.10 ± 1.89 b 12.77 ± 1.75 a 7.98 ± 0.90b 8.86 ± 0.77b 7.54 ± 0.86 5.67 ± 0.80 6.25 ± 1.06 4.69 ± 0.57 a 2.61 ± 0.38 b 3.21 ± 0.34 ab 3.58 ± 0.73 3.06 ± 0.55 2.58 ± 0.30 1.59 ± 0.28 1.17 ± 0.15 1.20 ± 0.09 14.65 ± 1.72 a 11.16 ± 1.30 b 12.58 ± 1.38 ab 10.55 ± 1.35 a 7.55 ± 0.85 b 9.22 ± 1.12 ab 0.59 ± 0.07 0.41 ± 0.05 0.46 ± 0.04 3.51 ± 0.37 3.20 ± 0.61 2.89 ± 0.29

75.07 ± 4.39

(A) Methanol-soluble compounds UAC280-315 UAC280-400 Quercetins Quercetin 3-O-glucuronide Quercetin-O-glycoside C1 (Quercetin family) C2 (Quercetin family) C3 (Quercetin family) Kaempferols Cis-trans-kaempferol 3-O-glucoside Kaempferol 3,7-O -diglucoside C4 (Kaempferol family)

70.35 ± 4.77

Enhanced UV-A+UV-B

69.58 ± 3.55

Enhanced UV-A

Total phenols

Ambient UV

UV treatment

absorbing compounds; UAC280-400, UV absorbing compounds; ns, not significant.

ns ns ns ns ns ns ns ns ns ns ns ns

ns

p-value

interaction between UV radiation and irrigation treatments was not significant for any of the variables analysed. UAC280-315, UV-B

low-watered; N = 36). Different letters and values in boldface indicate statistical differences (p ≤ 0.05) in pairwise comparisons. The

conditions (ambient UV, enhanced UV-A, and enhanced UV-A+UV-B, N = 24) and two irrigation levels (WW, well-watered and LW,

Ac ce pt e

DM-1), and (B) methanol-insoluble cell-wall compounds (mg g DM-1) of Laurus nobilis seedlings grown under three different UV

Table 4 Overall means ± S.E. of total foliar phenolic content (mg AG equivalents g DM-1), (A) methanol-soluble compounds (mg g

Page 47 of 53

UAC280-315 UAC280-400 p-coumaric Kaempferol derivative

d

0.58 ± 0.01 1.42 ± 0.02 0.43 ± 0.02 0.67 ± 0.042

Ac ce pt e

0.56 ± 0.02 1.36 ± 0.06 0.49 ± 0.02 0.74 ± 0.02

ns ns ns ns

cr

0.59 ± 0.02 1.43 ± 0.04 0.46 ± 0.01 0.71 ± 0.03

ip t

0.57 ± 0.01 1.39 ± 0.02 0.47 ± 0.02 0.72 ± 0.02

us

an

M

0.60 ± 0.01 1.45 ± 0.03 0.48 ± 0.01 0.74 ± 0.03

ns ns ns ns

Bernal et al., Figure 1

a) b

4

A A

ip t

ab A a

3 2

cr

Leaf biomass (g)

5

us

1

b)

A

A

A

3

a

M

2 1

c)

WW

10

ab A A

8 A

b

Ac ce pt e

Root biomass (g)

LW

X Data

d

Stem biomass (g)

4

b b

an

Ambient UV Enhanced UV-A Enhanced UV-A+UV-B

5

a

6 4 2

WW

LW

Irrigation treatment

Page 48 of 53

Bernal et al., Figure 2

A 90

A

A

a*

ab b

ip t

RWC (%)

100

Ambient UV Enhanced UV-A Enhanced UV-A+UV-B

cr

80

70 WW

LW

Ac ce pt e

d

M

an

us

Irrigation treatment

Page 49 of 53

Bernal et al., Figure 3

*

10

ip t

Ambient UV Enhanced UV-A Enhanced UV-A+UV-B

8

2

Net photosynthetic rate (A) (µ µmol CO m-2s-1)

a) 12

6

cr

4

us

2

*

an

80

60

2

Stomatal conductance (gs) (mmol H O m-2s-1)

b)

M

40

20

a* A c)

d

Ac ce pt e

WUE (A/gs ) (µ µmol CO2 mmol H2O)

0,16

0,12

0,08

0,04

WW

LW

Irrigation treatment

Page 50 of 53

Bernal et al.,

2,0

Ambient UV Enhanced UV-A Enhanced UV-A+UV-B b

A 1,6

ip t

1,8

A

A

a

a

cr

1,4

WW

us

1,2

LW

d

M

an

Irrigation treatment

Ac ce pt e

Non-photochemical quenching (NPQ)

Figure 4

Page 51 of 53

Bernal et al., Figure 5

A A

a

a

ip t

3

A

2

cr

b

1

us

Chlorophyll a+b (mg g DW-1)

a)

b) a A

ab

A

A

an

0,28

b

0,21

M

β -carotene (mg g DW-1)

0,35

0,14

Ambient UV Enhanced UV-A Enhanced UV-A+UV-B

d

0,03

c)

Ac ce pt e

α -carotene (mg g DW-1)

0,04

a

A

0,02

ab

A

0,01

A

b

WW

LW

Irrigation treatment

Page 52 of 53

Bernal et al, Figure 6

a)

A A

ab

A

b

ip t

0,4

a

cr

0,2

b)

a 0,12

a

an

A

A A

0,08

b 0,04

c)

0,20

Ambient UV Enhanced UV-A Enhanced UV-A+UV-B

d

0,25

b

A

0,15

Ac ce pt e

AZ/VAZ

us

0,16

M

Lutein-5,6-epoxide (mg g DW-1)

Lutein (mg g DW-1)

0,6

A

A

ab

a

0,10

0,05

WW

LW

Irrigation treatment

Page 53 of 53