How do Mediterranean shrub species cope with shade? Ecophysiological response to different light intensities

1 This is the peer-reviewed version of the article accepted for publication in PLANT BIOLOGY Volume 20, 1 296-306. 2018, which has been published in final form at https://doi.org/10.1111/plb.12661 2 3 4 How do Mediterranean shrub species cope with shade? Ecophysiological response 5 to different light intensities 6 Díaz-Barradas MCa, Zunzunegui Ma, Alvarez-Cansino Lb, Esquivias MPa, Valera Ja, 7 Rodríguez Hc 8 aDepartamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, 9 Apartado 1095, 41080 - Sevilla, Spain 10 bDepartment of Plant Ecology, University of Bayreuth, Universitätsstrasse 30, 95440 Bayreuth, 11 Germany 12 cInstituto de Bioquímica Vegetal y Fotosíntesis, Universidad Sevilla y CSIC, Centro de 13 Investigaciones Científicas Isla de la Cartuja, Avenida de Américo Vespucio 49, 41092-Sevilla, 14 Spain 15 16 ABSTRACT 17  Under natural conditions, light exposition for Mediterranean shrubs can be highly 18 variable, especially during cloudy days or under a canopy, and can interfere with 19 other environmental factors such as temperature and water availability. 20  With the aim of decoupling the effect of radiation and temperature from water 21 availability, we conducted an experiment where 2 perennial and 3 summer semi- 22 deciduous shrub species were subjected to different levels of irradiation. In order 23 to follow plant responses to light exposure, we measured gas exchange, 24 photosystem II photochemical efficiency, photosynthetic pigments, and leaf mass 25 area in spring and summer. 26  Results showed that all study species presented a plastic response to different light 27 conditions and that light-related traits varied in a coordinated manner. Summer 28 semi-deciduous species exhibited a more opportunistic response, with higher 29 photosynthetic rates under full sun, but under shade conditions, the two strategies 30 2 presented similar assimilation rates. Stomatal conductance did not show such a 31 drastic response as photosynthetic rate did, giving place to changes in water use 32 efficiency. Daily cycles of Fv/Fm revealed a slight photoinhibitory response during 33 summer, mainly in perennial species. In all cases photosynthetic pigments 34 adjusted to the radiation level; leaves presented lower chlorophyll content, higher 35 pool of xanthophylls and higher proportion of the de-epoxydaded state of 36 xanthophylls under sun conditions. Lutein content increased in relation to the 37 xanthophyll pool under shade conditions. 38  Our results evidenced that radiation is an important driving factor controlling 39 morphological and physiological status of Mediterranean shrub species, 40 independently of water availability. Summer semi-deciduous species exhibit a set 41 of traits, with higher response variability, maximizing their photosynthetic 42 assimilation under different sun conditions. 43 44 Keywords: Chlorophyll; light availability; lutein; photochemical efficiency; photosynthetic rate; 45 xanthophyll cycle 46 47 48 Corresponding Author: Mari Cruz Díaz-Barradas, 49 Departamento de Biología Vegetal y Ecología, Facultad de Biología, Universidad de Sevilla, 50 Apartado 1095, 41080 - Sevilla, Spain 51 Email: [email protected] 52 Telephone: +34954557062; Fax: +34954626308 53 54 55 56 3 ABSTRACT 57  Under natural conditions, light exposition for Mediterranean scrubs can be highly 58 variable, especially during cloudy days or under a canopy, and can interfere with 59 other environmental factors such as temperature and water availability. 60  With the aim of decoupling the effect of radiation and temperature from water 61 availability, we conducted an experiment where 2 perennial and 3 summer semi- 62 deciduous species were subjected to different levels of irradiation. In order to 63 follow plant responses to light environment, we measured gas exchange, 64 photosystem II photochemical efficiency, photosynthetic pigments, and leaf mass 65 area in spring and summer. 66  Results showed that all study species presented a plastic response to different light 67 conditions and that light-related traits varied in a coordinated manner. Summer 68 semi-deciduous species exhibited a more opportunistic response, with higher 69 photosynthetic rates under full sun, but under shade conditions, both strategies 70 presented similar assimilation rates. Stomatal conductance did not show such a 71 drastic response as photosynthetic rate, giving place to changes in water use 72 efficiency. Daily cycles of Fv/Fm revealed a slight photoinhibitory response during 73 summer, mainly in perennial species. In all cases photosynthetic pigments 74 adjusted to the radiation level; leaves presented lower chlorophyll content, higher 75 pool of xanthophylls and higher proportion of the de-epoxydaded state of 76 xanthophylls under sun conditions. Lutein content increased in relation to the 77 xanthophyll pool under shade conditions. 78  Our results evidenced that radiation is an important driving factor controlling 79 morphological and physiological status of Mediterranean species, independently 80 of water availability. Summer semi-deciduous species exhibit a set of traits, with 81 4 higher response variability, maximizing their photosynthetic assimilation under 82 different sun conditions. 83 84 INTRODUCTION 85 Mediterranean plants regularly cope with severe drought stress, together with high 86 sunlight irradiance, high temperatures, and elevated vapour pressure deficits (VPD) over 87 the long summer season (Ehleringer & Mooney 1983). In this sense, they have evolved a 88 set of morpho-anatomical, biochemical, and physiological traits that enable their 89 adaptation and survival in such harsh conditions (Salleo et al. 1997; Tattini et al. 2005; 90 Galmés et al. 2007; Flexas et al. 2014; Tattini 2014). In general, Mediterranean scrub 91 species present two contrasting strategies to face such environmental conditions: Summer 92 semi-deciduous species (which experiment an important decrease of leaf area index in 93 summer), generally with a drought tolerance strategy, and perennial species (which have 94 sclerophyllous leaves), that mainly show a drought avoidance strategy (Levitt 1980; 95 Werner et al. 1999, Zunzunegui et al. 2011). 96 Although plants are adapted to cope with Mediterranean summers, radiation input 97 during this season is greatly over the level that can be derived for photosynthesis. 98 Radiation excess, together with stomatal closure to avoid water loss at the leaf level, can 99 cause an excess of excitation energy, ultimately leading to photoinhibition or even photo- 100 oxidation processes (Kyparisis et al. 2000). 101 Plants have to maintain equilibrium between the availability of radiation and its use 102 (Flexas et al. 2002). In this context, they exhibit an array of different photoprotective 103 mechanisms to avoid damage in the photosynthetic systems (Das 2004). Such 104 morphological or structural photoprotective mechanisms consist of changes in leaf angle, 105 leaf pubescence, decrease in leaf area index decrease (Long et al. 1994; Werner et al. 106 5 1999; Zunzunegui et al. 1999), leaf level structural changes (Terashima et al. 2001; 107 Weston et al. 2000), structure and movement of chloroplasts (Kasahara et al. 2002);, and 108 physiological or biochemical changes, such as regulation of light absorption by changes 109 in pigment content and composition, or in energy dissipation, antioxidant compounds or 110 elimination of different Reactive Oxygen Species (ROS) produced during photosynthesis 111 (Ort 2001). Within these mechanisms, the most generalized one is the dissipation of 112 energy excess as heat through the xanthophyll cycle. The xanthophyll cycle is 113 differentiated into two types: the ubiquitous Violaxanthin cycle (VAZ) cycle (Demmig- 114 Adams et al. 1996; Demmig-Adams & Adams 2002; Peñuelas et al. 2004; Munné-Bosch 115 & Lalueza 2007), and the less known Lx-L cycle which is not present in all plants, but 116 plays an important role in the acclimation of shade plants to light (Bungard et al. 1999; 117 Matsubara et al. 2005; García-Plazaola et al. 2007; Esteban & García-Plazaola 2014). 118 Plants from extreme environments also exhibit an integrated network of antioxidant 119 defenses to prevent against ROS species (Munné-Bosch et al. 2003; Peñuelas et al. 2004; 120 Munné-Bosh & Lalueza 2007). 121 Under natural light conditions, especially when vegetation is structured in a canopy 122 cover and a shrub cover, the light environment is highly variable for plants growing in 123 the understorey (Zunzunegui et al. 2016). Light availability is the most important 124 photosynthetic limitation for leaves located at different positions in the plant (Aranda et 125 al. 2007) and competition for light is a major determinant of forest structure and growth 126 (Coomes et al. 2011). At the stand level, the coexistence of species with different crown 127 architecture, leaf and phenological strategies results in vertically structured canopies that 128 intercept a greater portion of incoming solar radiation (Morin et al. 2011; Seidel et al. 129 2013; Jucker et al. 2014). However, in Mediterranean or semiarid regions, some authors 130 have recently questioned if shade conditions can be of benefit for understorey shrub 131 6 vegetation, concluding that the potential benefit of shade is overlapped by water 132 competition with trees (Valladares et al. 2008). 133 Although the response to light excess in Mediterranean vegetation has been 134 subjected to deep research, less attention has been drawn to the effect of shade on such 135 species. Moreover, studies have failed to distinguish the effects of water deficit and 136 radiation separately, a crucial issue in the study of Mediterranean traits combinations. In 137 a revision article, Flexas et al. (2013) have shown that Mediterranean plants exhibit a 138 series of biochemical traits that allow them to achieve similar photosynthetic rates (An) 139 to than non-Mediterranean plants, even though they exhibit a larger leaf mass area (LMA) 140 than non-Mediterranean species. 141 Under field conditions, plant responses to stress can be modified by the 142 synergistically or antagonistically effects of different factors (Chaves et al. 2002; 143 Zunzunegui et al. 2016). To solve this problem, we designed an experiment to untangle 144 the effects of light from drought stress. With this aim, we subjected different 145 Mediterranean shrub species to three greenhouse treatments: plants growing in full sun 146 and plants exposed to two levels of radiation reduction. The experiment was performed 147 under full irrigation conditions during spring and summer. 148 To assess if whether the response to light intensity is dependent on the functional 149 type strategy, we based species selection on the two main functional strategies of 150 Mediterranean scrub, perennials, and summer semi-deciduous. 151 We hypothesized that under lack of water deficit conditions, sun-exposed plants 152 should have higher photosynthetic assimilation rates, increasing their mesophyll 153 resistance through an increase of their LMA (Flexas 2008; Niinemets et al. 2009), and 154 being at the same time more protected through the different components of the VAZ 155 xanthophyll cycle. 156 7 Additionally, we hypothesized that facing variation in light environment 157 (Zunzunegui et al. 2011), perennial species -with an avoider strategyshould have a more 158 homogeneous response, while summer semi-deciduous species -exhibiting a tolerance 159 strategyshould take the maximum profit when conditions were more favourable and 160 consequently they should exhibit a more plastic response. 161 162 MATERIAL AND METHODS 163 Study species and experimental design 164 We performed the experiment in an open-air greenhouse of the University of Sevilla, with 165 5 Mediterranean shrub species belonging to both perennial and summer semi-deciduous 166 strategies. Perennial species were: Myrtus communis and Pistacia lentiscus. Summer 167 semi-deciduous were: Halimium halimifolium, Rosmarinus officinalis, and Cistus 168 salvifolius. Plants came from an official forestry nursery run by Junta de Andalucía (San 169 Jerónimo, Sevilla) with an average age of one year, and 30 cm of height. They were 170 transplanted to 2 L capacity pots, with a mixed substrate of peat (60%), perlite (40%), 171 and stimulating root factor AGROSIL (once per month). Plants were exposed to three 172 light treatments by shading with commercial sun shelters. 173  Sun treatment, with a naturally occurring light intensity of 100% (5 species x 9 plants 174 = 45). 175  Semi-shade treatment, with 30% of natural light intensity (4 species x 9 plants = =36). 176 Only 4 species were used in this treatment due to a lack of stock of Cistus salvifolius 177 plants. 178  Shade treatment, with 10% of natural light intensity (5 species x 9 plants =45). 179 8 Plants were maintained for one month under experimental conditions prior to the 180 beginning of measurements. The experiment lasted 28 weeks (from March to September 181 2008). 182 183 Gas exchange 184 Gas exchange measurements were performed by means of an open IRGA LCi (ADC, 185 UK), for the determination of net photosynthetic rate (An), transpiration rate (E), and 186 stomatal conductance (gs). Intrinsic water use efficiency (WUE) was calculated as An/gs. 187 Measurements were conducted on fully expanded leaves from terminal shoots and 188 all results were expressed on an area basis, that was calculated using Midebmp (R. 189 Ordiales, CSIC, Spain, 2000) from scanned images of the leaves. Two measurements 190 were conducted per plant, in 5 individuals per species and treatment, and the mean value 191 per plant was calculated (N=70; 4 species x 3 lights intensities x 5 individuals + 1 species 192 x 2 light intensities x 5 individuals). Measurements were taken during the morning (7-9 193 h, solar time), and at midday (11-13h, solar time). 194 Gas exchange measurements were performed in spring (April) and summer (July). 195 196 Fluorescence measurements 197 Chlorophyll fluorescence kinetics of leaves was assessed in situ by means of the pulse- 198 amplitude modulation technique using a portable fluorometer (mini-PAM, Walz, 199 Effeltrich, Germany). Fluorescence was excited by a pulse of modulated red light from a 200 light-emitting diode (LED, type H-3000 Stanley) connected to a fibre optic following 201 Bilger et al. (1995). Maximum (or potential) photochemical efficiency of photosystem II 202 was measured in leaves that were pre-adapted to darkness for 20 min (a time-span 203 9 considered sufficient for allowing complete reoxidation of all reaction centres), and 204 calculated as Fv/Fm = (Fm- F0)/Fm (Shreiber et al. 1995), where F0 is the basal minimum 205 fluorescence, corresponding to the relaxed reaction centres and Fm is the maximum 206 fluorescence in dark-adapted leaves obtained with a pulse of saturating light 207 (approximately 3000 µmol m-2s-1 for 800 ms). 208 Effective photochemical efficiency of photosystem II (фPSII) was measured in light- 209 adapted leaves as ΔF/F’m = (F’m-F)/F’m (Genty et al. 1989), where F’m is the maximum 210 fluorescence of light-adapted leaves and F is the steady-state fluorescence of irradiated 211 leaves (Maxwell & Johnson 2000). From the different fluorescence parameters and the 212 incident radiation of on the leaf, we calculated the effective electron transport rate (ETR) 213 as ETR = (ΔF/F’m) x PAR x (PSII/PSI) x ah, where (PSII/PSI) = 0.5, considering an even 214 distribution of excitation of PSI and PSII (Krall & Edwards 1992) and leaf absorption, ah 215 = 0.84, assuming that 16% of PAR is not absorbed by photosystems (Lüttge et al. 2003; 216 Bjorkman & Demming 1987). Non-photochemical energy dissipation (NPQ) was 217 calculated as NPQ = Fm-F’m/F’m (Shreiber et al. 1995). 218 Two fluorescence measurements were conducted per plant, in 5 individuals per 219 species and treatment, and the mean value per plant was calculated (4 species x 3 220 treatments x 5 individuals; but 2 treatments x 5 individuals in C. salvifolius, where there 221 were only plants for sun and shade treatments). 222 Measurements were performed during a full day cycle, with a periodicity of 3 hours 223 from 6h to 18h solar time (5 measurements per day per plant). Day cycles were conducted 224 in spring (April), early summer (July) and late summer (September), to evaluate 225 differences between species and the effect of light treatment during the summer season. 226 For statistical analysis, we used Fv/Fm values from 8h and 14h to analyse the widest 227 contrasted responses during the day. 228 16 Fluorescence measurements 367 All analysed variables showed significant differences between treatments and sampling 368 seasons. There were significant differences between species in all variables related to 369 photochemical efficiency (Fv/Fm at 8h and 14h and ΦPSII at 14h), but such differences 370 were not significant for NPQ, nor for ETR, measured at midday (14h). The interaction 371 between species and treatments was significant for all variables except for ETR, the 372 interactions between species and season and between treatment and season showed varied 373 responses (Table 2). 374 Table 2. Three-way ANOVA results (species, treatment and season) for LMA values. F 375 and P-values are shown, bold indicates significant differences. 376 377 378 Perennial species: During spring, all sun treatment plants of M. communis showed 379 lower Fv/Fm values than P. lentiscus during the whole day cycle (P<0.01 for both 8 and 380 14h measurements). However, in summer, the daily Fv/Fm curves were similar in both 381 species and close to optimum values. At the end of summer, under optimum suboptimum 382 Fv/Fm values were recorded at midday, without significant differences between species 383 (Fig. 4a). 384 For sun treatment plants, фPSII showed a characteristic daily curve, with the lowest 385 values at the maximum radiation hours, ranging between 0.1 and 0.2. M. communis 386 showed lower values than P. lentiscus, with significant differences (P<0.05) in sun- 387 exposed plants and full-shade treatment (Figure 4a). 388 Summer semi-deciduous species: The daily Fv/Fm curve showed a similar pattern in 389 the three study species and treatments, only showing a slight decrease during the 390 afternoon in the sun treatment at the end of summer (Figure 4b). 391 17 Solar time 810 12 14 16 18 20 Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 SUN SUN Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 SUN Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 SEMISHADE SEMISHADE Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 SEMISHADE Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 SHADE SHADE Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 FvFm M. communis Yield M. communis FvFm P. lentiscus Yield P. lentiscus SHADE Solar time 810 12 14 16 18 20 Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 SHADE Solar time 810 12 14 16 18 20 FvFm H. halimifolium Yield H. halimifolium FvFm R. officinalis Yield R. officinales Fv/Fm C. salvifolius Yield C. salvifolius SHADE SHADE SUN SUN SUN Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 SEMISHADE SEMISHADE Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 SEMISHADE SPRING SUMMER END SUMMER a a a a a a a a a b b b b b b b b b In sun treatment plants, ΦPSII of summer semi-deciduous species showed, similarly 392 to perennial plants, a daily curve with a midday depression. In the semi-shade treatment, 393 there was a midday depression, with a different daily pattern in the study species. In the 394 shade treatment, the daily curve was very similar to Fv/Fm, without any clear midday 395 depression (Figure 4b). 396 397 398 399 400 401 402 403 404 405 406 407 408 409 410 411 18 Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 SUN Fv/Fm and PSII 0.2 0.3 0.4 0.5 0.6 0.7 0.8 SEMISHADE Solar time 810 12 14 16 18 20 Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 FvFm M. communis Yield M. communis FvFm P. lentiscus Yield P. lentiscus SHADE PERENNIALS Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 SUN Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 SEMISHADE SUMMER-SEMIDECIDUOUS Solar time 810 12 14 16 18 20 Fv/Fm and PSII 0.0 0.2 0.4 0.6 0.8 FvFm H. halimifolium Yield H. halimifolium FvFm R. officinalis Yield R. officinalis Fv/Fm C. salvifolius Yield C. salvifolius SHADE Figure 4. Daily cycle of maximum photochemical efficiency (Fv/Fm) and effective 412 photochemical efficiency (ФPSII) in the study species under the different light treatments. 413 a) perennial species in grey colour; b) summer semi-deciduous species in white. 414 Strategy comparison: Integrating the Fv/Fm curves of the three study periods, it is 415 evident that there were no differences between the species of the same functional group, 416 but there were clear differences between plant strategies. In the sun treatment, perennial 417 species showed lower daily values to those of summer semi-deciduous species (P<0.001, 418 both for 8h values and for midday values) (Figure 5). 419 At the same time, ΦPSII curves showed stronger midday depression in perennials 420 in comparison to summer semi-deciduous plants in sun treatment (P<0.05), while there 421 were no differences in any shade treatments (Fig. 5). 422 423 424 425 426 427 428 429 430 431 432 433 434 19 Figure 5. Average daily cycle of maximum photochemical efficiency (Fv/Fm) and 435 effective photochemical efficiency (ФPSII) in perennial and summer semi-deciduous 436 species. 437 Leaf mass per area (LMA) 438 LMA values were significantly different between species and treatments, without 439 significant differences between seasons (spring and summer). The interactions between 440 species and treatment and between treatment and seasons were significant (Table 3). 441 LMA was significantly higher in the sun treatment (P<0.05), without differences between 442 semi-shade and shade treatments for all species. 443 Table 3. Results of two-way ANOVA (Species and Treatment) for photosynthetic 444 pigment data. Total a + b chlorophyll. Chlorophyll a/b ratio. Carotenoid content. The 445 indices (V+A+Z)/Total chlorophyll, De-epoxidised index ((Z+A)/V+A+Z) and 446 Lutein/V+A+Z. (A) antheraxanthin, (Z) zeaxanthin, (V) violaxanthin, (L) lutein. F and 447 P-values are shown, bold indicates significant differences. 448 449 In the perennial species, LMA was positively correlated positively with stomatal 450 conductance (r=0.301, P<0.001), and with intrinsic WUE values (r=0.239, P<0.01); for 451 20 summer semi-deciduous species, such correlation was only significant for intrinsic WUE 452 (r=0.576, P<0.001). 453 454 Photosynthetic pigments 455 Total chlorophyll content was significantly different between species and treatments. 456 Although the species x treatment interaction was not significant, the chlorophyll a/b ratio 457 was significantly different in all cases. Total carotenoid content did not show significant 458 differences between species, treatments, and species x treatment. Nevertheless, the ratios 459 VAZ/Chl, DEI, and L/VAZ showed significant differences in all cases (table 4). 460 In all study species, total chlorophyll content was significantly higher in the shade 461 treatments in relation to sun-exposed plants (P<0.05). In perennial species, chlorophyll 462 content increased proportionally to shade intensity, while in summer semi-deciduous 463 species, chlorophyll content was similar in both shade treatments. Comparing all 464 treatments, chlorophyll a/b ratio was significantly higher in sun plants in comparison to 465 shade treatments (P<0.05) (Fig. 6). 466 467 468 469 470 471 472 473 474 21 sun semi shade sun semi shade sun semi shade sun semi shade sun semi shade VAZ/Chl a+b 0.00 0.02 0.04 0.06 0.08 0.10 Carotenoids (mg·g-1 DM) 0.0 0.1 0.2 0.3 0.4 Violaxanthin Antheraxanthin Zeaxanthin Lutein carotene Chlorophyll a/b 1.0 1.2 1.4 1.6 1.8 2.0 2.2 Chlorophylls a+b (mg·g-1 DM) 0.0 2.0 4.0 6.0 8.0 Chlorophyll a Chlorophyll b L/VAZ 0 2 4 6 8 10 12 14 16 18 AZ / VAZ 0.2 0.4 0.6 0.8 1.0 1.2 1.4 M. communisP. lentiscus H. halimifolium R. officinalisC. salvifolius sun semishade shade sun semishade shade 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 Figure 6. Mean values and standard deviation in the study species under the different 494 light treatments for Total chlorophyll (a+b), Chlorophyll a/b ratio, Total carotenoid 495 22 (antheraxanthin (A), zeaxanthin (Z), violaxanthin (V), lutein (L) and β-carotene), de- 496 epoxidation index (AZ)/VAZ) and ratios (VAZ)/Total chlorophyll and Lutein/VAZ. 497 Total carotenoid content increased in the shade treatments in all species, except for 498 P. lentiscus, without general differences between treatments (Table 4). When the different 499 carotenoids were analysed separately, L was always the major component, and only in 500 the sun treatment plants, it had a significant amount of Z. Accordingly, DEI ranged 501 between 0.7 and 0.87 in all sun treatment plants, while in shade plants, this index was 502 around 0.2 in H. halimifolium plants of semi-shade treatment and close to 0 in all the rest, 503 therefore, indicating that all xanthophyll cycle components were in the epoxidized state, 504 mainly as V (Fig. 6). 505 The ratios L/VAZ and VAZ/Chl showed significantly lower values in sun plants 506 than in shade plants (P<0.05) (Fig. 6). 507 508 DISCUSSION 509 Any kind of mild stress induces acclimation in plants through morphological, 510 physiological and biochemical changes, which are transient and persist only during the 511 stress. Plant acclimation reduces the effects of stress and produces mechanisms of 512 homeostasis (Tapia et al. 2013, 2016). 513 Mediterranean species present an elevated acclimation capacity, adjusting their leaf 514 physical and chemical traits to obtain a positive photosynthetic balance in response to 515 changes in the light environment. Our study demonstrated how different functional 516 groups responded differently without water limitation, showing that summer semi- 517 deciduous and perennial species exhibit different acclimation capacities to light 518 availability. 519 23 Our results show that the highest differences between species and functional groups 520 occurred under the sun-treatment and that such differences were reduced progressively 521 when the available radiation decreased. 522 Many field studies have pointed out that under Mediterranean summer conditions, 523 most summer semi-deciduous species usually suffer from marked water stress, presenting 524 a drought-tolerant strategy, while responses of perennial species are in many cases 525 species-specific, generally showing drought-avoidance strategies (Kyparissis et al. 2000; 526 Zunzunegui et al. 2011). However, under field conditions plant responses to drought or 527 radiation excess can be synergistically or antagonistically modified by other stress factors 528 (Chaves et al. 2002; Zunzunegui et al. 2016). The co-occurrence of multiple stresses can 529 dramatically alter the capacity of a given species to tolerate low light (Valladares & 530 Niinemets 2008). 531 In spite of the vast information on Mediterranean species responses to summer 532 drought, the independent effect of radiation excess has rarely been investigated. In the 533 sun treatment, summer semi-deciduous species showed the highest photosynthetic 534 assimilation during summer months, although they presented slight symptoms of 535 photoinhibition with drops in Fv/Fm in the first afternoon hours. Under shade treatments, 536 such species responded markedly, with a strong decrease in An that was not clearly 537 followed by drops in gs. In the perennial species, the response was different in the two 538 study species, M. communis exhibiting a lower An than P. lentiscus, especially under the 539 sun treatment. Such response was concomitant to a higher reduction in Fv/Fm under the 540 sun treatment. In our study, An decreased faster than stomatal conductance. Such results 541 evidence that all species were limited by light income and not by stomatal conductance 542 under the shade treatments (Valladares et al. 2008). 543 24 As expected, summer semi-deciduous species were in overall more opportunistic: 544 they maximised the use of available resources when these were favourable. Thus, in the 545 sun treatment plants, An was significantly higher than in perennial plants, with equal 546 figures in both functional groups under the full shade treatment. Such opportunistic 547 strategy has also been highlighted by other authors, such as Werner et al. (2002). Summer 548 semi-deciduous species present malacophyllous leaves, with low LMA (Correia et al. 549 1992) and are associated with early successional stages, with high transpiration and 550 photosynthetic rates during spring (Correia & Catarino 1994; Grant & Incoll 2005). For 551 example, in a study at a global scale, Wright et al. (2004) described that, on average, a 552 tenfold decrease in LMA coincided with a 21-fold increase in photosynthetic capacity. 553 Such results agree with the traditional idea of higher plasticity to light conditions in early 554 successional species (Bazzaz 1979; Abrams & Mostoller 1995). Even though perennial 555 woody species’ plasticity has been reported to decrease with increasing leaf age in some 556 leaf traits (Keenan & Niinemets 2016), these species are expected to have longer leaf 557 lifespan and higher survival in the face of abiotic and biotic hazards (Díaz et al. 2015). 558 Alternatively, in other studies, the lack of such pattern has equally been described 559 (Chazdon 1992; Nicotra et al. 1997). 560 The positive correlation found between LMA and intrinsic WUE is showing that 561 under sun-exposed conditions, where leaves were more sclerophyllous, photosynthesis 562 increased in a bigger proportion than did stomatal conductance did, and such effect was 563 more marked in summer semi-deciduous species. Werner & Máguas (2010) found a 564 negative relationship between leaf LMA and Δ13C ( a proxy for integrated WUE) in 565 Mediterranean shrub species, indicating a positive relationship with WUE that can be 566 associated with leaf structural characteristics, such as an increase in mesophyll resistance 567 (Díaz-Espejo et al. 2007). Leaf morphology has an important role in explaining the 568 increased LMA with high light intensity (Poorter et al. 2009). Higher LMA at higher 569 25 irradiance is generally associated with a greater fraction of palisade parenchyma, whereas 570 the thickness of the epidermis remains remarkably constant. An analysis comparing 571 deciduous and perennial leaves showed that evergreen species had considerably thicker 572 mesophyll tissue (Poorter et al. 2009). Mediterranean species exhibit an elevated LMA 573 which might be associated to a low gm (mesophyll conductance) (Flexas et al. 2014), 574 although other studies support the idea that LMA determines the limits to maximum gm 575 and it is not indicative of its real value (Flexas et al. 2008; Pérez-Martín et al. 2009). 576 Fv/Fm is an index of the maximum efficiency of energy absorption by the opened 577 reaction centres in PSII and decreases in this parameter reflect a photoinhibition situation 578 (Öquist et al. 1992; Carvalho et al. 2001). In our study, perennial species, mainly M. 579 communis, showed the highest signs of photoinhibition, especially at the end of summer. 580 In spite of such reduction, and the low values of ΦPSII, under 0.1 at midday, Fv/Fm showed 581 a good recovery, with optimum values in the morning (8h). Such results suggest that the 582 photoinhibition in perennial species was a dynamic response of the photosynthetic 583 apparatus, without permanent damage of PSII (Werner et al. 2002; Álvarez-Cansino et al. 584 2010). We only found chronic photoinhibition symptoms during spring in M. communis. 585 As the available radiation decreased from sun exposure to semi-shade and shade 586 treatments, the daily ΦPSII curve became closer to that of Fv/Fm, showing optimal values 587 throughout the day. Such results are indicating that the available radiation is being used 588 efficiently through the photochemical pathway, with a significant reduction of its 589 dissipation as heat. 590 In our study, the lack of Photosystem II damage in all sun treatment species could 591 be attributed to the activation of effective photoprotective mechanisms (Oliván & Munné- 592 Bosch 2010). 593 32 Flexas J., Scoffoni C., Gago J., Sack L. 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