Data were compiled from peer-reviewed journals only, using the “ISI Web of Sciences” source. Only original articles written in English and published in the last 20 years, covering the time laps between January 1996 and May 2016, were considered. Key words used in the search included (i) antioxidant-related terms: “tocopherol OR glutathione OR ascorb^* OR zeaxanthin OR lutein OR caroten^* OR antioxidant OR ^*oxid^*,” (ii) Mediterranean ecosystems-related terms “Mediterranean OR scrub OR chaparral OR shrubland OR woodland OR Quercus OR Arbutus OR Cistus OR Rosmarinus OR Pistacia OR Lavandula),” and (iii) terms related to photosynthetic tissues: “leaf OR leaves OR foliar OR chloroplast^*.” The search was also filtered by areas of knowledge: “Plant Sciences OR Agriculture OR Environmental Sciences Ecology OR Forestry.” A total number of 840 articles fitted to these criteria.

From the initial search, manuscripts were partitioned according to the following principles: only wild species original from the Mediterranean bioclimatic region were included. Field studies with Mediterranean species conducted out of the Mediterranean bioclimatic region or with aquatic plants (i.e., Posidonia), or with crops, were excluded. Works performed under growth chamber or greenhouse conditions were excluded. Finally, only papers using high-performance liquid chromatography for carotenoid and/or tocopherol quantification were included. After all these criteria our final database comprised data from 89 papers, 73 species, and 32 families (Table S1).

Numeric data were extracted from text, tables and figures of the 89 selected manuscripts (Table S2). Data from figures were obtained by using the “scale tool” from the software Adobe PhotoShop, CS5 Extended, v 12.1 ×64. Different type of numeric data were obtained from the manuscripts: (1) foliar content of photosynthetic pigments and small molecular weight antioxidants: anteraxanthin (Ant), neoxanthin (Neo), violaxanthin (Vio), lutein (Lut), lutein epoxide (Lx), zeaxanthin (Zea), chlorophyll a (Chl a), chorophyll b (Chl b), β-carotene (β-Car), α-tocopherol (α-Toc), total ascorbate (tAsc), total glutathione (tGSH) and/or sum or ratios of these parameters i.e., (A+Z)/(V+A+Z) (so far referred to as AZ/VAZ), Chl a+b, pigments or antioxidants per Chl, etc.; (2) parameters indicative of stress: maximal photochemical efficiency of PSII (Fv/Fm), predawn water potential (Ψ), relative water content (RWC), midday stomatal conductance (g_s); (3) parameters relating leaf mass and area: leaf mass per area (LMA), specific leaf area (SLA). In the case of photosynthetic pigments and antioxidants, values expressed per fresh weight (FW), dry weight (DW), leaf area or Chl content were obtained. When any of these data was missing, and whenever possible, they were calculated from the original data from the paper. In some cases, units were converted.

Further details collected from each paper regarded plant developmental stage (seedling, adult), leaf age (young, mature), time of the day (predawn, artificial predawn (> 12 h darkness), morning, midday, evening). Additionally, information about growth type (woody: tree, shrub; non-woody: herb), taxonomic group (angiosperm, gymnosperm), leaf texture (membranous, sclerophyllous, malacophyllous, succulent), leaf attributes (none, waxy, pubescent, hairy-abaxial) and leaf phenology (evergreen, winter-deciduous, summer-deciduous) were compiled from the available literature.

Every manuscript was first labeled with regard to its main stress or topic (i.e., drought, salinity, climate-comparison, high temperature, low temperature, species comparison, etc.). Secondly, each data-row within the manuscript was labeled as control or treatment. When a treatment was designated as “control” in the study, it was included as such in the database. Nevertheless, there were some manuscripts in which there were not real controls. In those cases, the less stressful condition was considered as control for each study. Apart from this division, and taking into consideration all the manuscripts together, data from non-treated plants, together with real controls, were considered as “non-stress” values (data were excluded from this category if their Fv/Fm values were < 0.6, predawn AZ/VAZ > 0.4 and/or predawn Ψ < −2 MPa). Data from “non-stress” category were used for the characterization of photosynthetic pigment and antioxidant composition in Mediterranean species (Table 1), and for the assessment of their composition according to plant biological traits (Figure 1), and leaf traits (Figures 2, 3, 6).

To evaluate the influence of the main Mediterranean climatic factors over photosynthetic pigments and antioxidants, manuscripts dealing with drought (D), low temperature (LT), high temperature (HT), and/or seasonality (S) were analyzed in detail (total manuscripts = 43). Within each manuscript, an index of variation with respect to the control was calculated for each of the climatic stressors independently. Hence, all data values were normalized to their respective controls. After data transformation, controls resulted as 1, while the deviation from the value 1 indicated the variation of the parameters analyzed. Reference values (controls) were considered the ones cataloged as “control” in the original papers.

To study the effect of leaf age on photosynthetic pigment and antioxidant content, only those studies where both mature and young leaves were simultaneously reported were included (total articles = 8). The young leaves were taken as reference values and the fold-changes were calculated as follows: Fold-change = (value of mature leave/value of young leave) −1. Thus, reference value was equaled to zero.

Plasticity Index (PI) for each parameter (Fv/Fm, pigments, antioxidants and LMA) was estimated according to Valladares et al. (2006) with the following formula: (Max-Min)/Max (within each paper of origin and parameter). This index generates values between 0 (no plasticity) and 1 (highest plasticity) comparable among parameters. A minimum of three different papers for each parameter was used as source of data for the calculation of average PI values. Only papers with two or more data for at least one species and one parameter where selected.

One-way ANOVA (for normally distributed data) and a Kruskal-Wallis ANOVA (for non-normally distributed data) were applied to test for significant differences among categories within functional groups, growth forms and plant developmental stages (Figure 1). Non-parametric Mann Whitney-U test was used to check for significant differences in the content of antioxidants and pigments among different leaf traits (Figure 2). Pearson's correlation was used to check for relationship between LMA and photosynthetic pigments or antioxidants (Figure 3). Student's t-test was performed in order to assess significant differences between control and climatic stresses (Figure 4). Student's t-test (or single sample Kolmogorov-Smirnov-test, for non-normally distributed data) was performed to assess differences between young and mature leaves (Figure 6). The resulting P-values were considered to be statistically significant at α = 0.05. All statistical analyses were performed with SPSS 24.0 (IBM Corp., Armonk, NY, USA).

Table 1 depicts reference values of photosynthetic pigments and antioxidants from a representative selection of native Mediterranean species living under non-stressful conditions (see Table S3 for an extended data set including parameters with n < 3). Average Chl a+b content was remarkably stable, fluctuating between 278 (μmol m⁻²) in Rosmarinus officinalis, and 547 in Quercus ilex, with the highest values being found in sclerophyllous species from Fagaceae and Rhamnaceae families. Among Fagaceae, sclerophyllous Quercus showed, additionally, the highest Chl a/b ratio. The content of α-Toc was the most variable among lipophilic antioxidants, varying up to two orders of magnitude among species (e.g., Pistacia lentiscus vs. Rosmarinus officinalis) and tGSH among hydrophilic antioxidants, varying also up to two orders of magnitude (e.g., Cistus salviifolius vs. Buxus sempervirens, Table 1). The intraspecific variability within a species (i.e., maximal amplitude between minimum and maximum values) was the highest for most of the metabolites in the case of Quercus species (except for the α-Toc, that appeared as extremely variable in Pistacia lentiscus).

The internal plant factors considered in the present work were the taxonomic class, the functional group, the growth form and the plant age (Figure 1). Our analysis revealed that pigment and antioxidant content in Mediterranean wild plant species under non-stressed conditions was differentially distributed depending on taxonomic class (gymnosperm or angiosperm) and functional group (herbaceous or woody) (Figure 1). In this regard, we identified two critical gaps of knowledge that introduce uncertainty to data interpretation: the lack of information about photoprotective features in gymnosperms and in herbs. Even so, when gymnosperms and angiosperms were compared, significantly higher values of AZ/VAZ and α-Toc were observed in the latter (Figure 1, left). After subdividing this taxonomic class into growth types, significant differences were found for α-Toc, being much more abundant in trees and lianas than herbs. Total Chl and Chl a/b were also significantly higher in trees and lianas than in the rest of angiosperm groups.

In agreement with this, when the functional group (herbaceous vs. woody) of Mediterranean plants was considered (Figure 1, right), levels of Chl a/b and α-Toc were significantly higher in woody species. Within them, we also analyzed the effect of plant age on the antioxidant load. Interestingly, VAZ and AZ/VAZ decreased while α-Toc increased with plant age (Figure 1, right).

The influence of different leaf traits (i.e., attributes, morphology, or texture) on the biochemical demand for photoprotection is shown in Figure 2. Foliar hairs efficiently decreased the biochemical demand for photoprotection (i.e., lower VAZ, α-Toc, and tAsc) even if they were present at the abaxial side of the leaf only. Higher requirement of biochemical photoprotection was found in evergreen than in deciduous leaves (this was remarkably evident in the higher contents of VAZ, α-Toc, and tAsc) and in summer-deciduous than in winter-deciduous leaves (evidenced in significantly higher Lut and tAsc, and higher, although not significantly, β-Car and AZ/VAZ). Besides having strongly developed structural photoprotective mechanisms, sclerophyllous leaves showed higher values of antioxidants L, VAZ, α-Toc, and tAsc than membranous leaves (Figure 2).

Associated to this, the predawn content of carotenoids and antioxidants (expressed in mmol mol⁻¹ Chl) increased with leaf mass per area (LMA) (Figure 3). This relationship was significant for all the metabolites (including tGSH, which was represented by a low number of cases: n = 6), except for α-Toc. At lesser extent, Chl content also followed a similar trend, although the correlation was no significant (P > 0.05). No correlation at all was found between Chl a/b ratio and LMA, or between AZ/VAZ and LMA (Figure 3).

The most characteristic climatic stresses in Mediterranean areas, and as a consequence, the stresses more deeply studied in the literature were: LT, HT, D, and seasonality. Variation of the main photosynthetic pigments and antioxidants (by comparison with controls) in response to Mediterranean climatic stressors are shown in Figure 4. The results show that these stresses provoked an enhancement in the content of all the parameters, with the exception of Chl a+b under HT (Figure 4). Drought significantly enhanced Neo, Lut, β-Car, VAZ, AZ/VAZ and it was the stress that more intensively triggered the accumulation of α-Toc (Figure 4C). Low temperature increased the Chl a/b ratio, and Lut, β-Car and α-Toc contents, while HT enhanced the total VAZ pool, AZ/VAZ, Chl a/b, and α-Toc, and induced a significant decrease in Chl a+b. Seasonality provoked an augmentation in the contents of Neo, Chl a/b, β-Car, AZ/VAZ, and α-Toc (Figure 4). Interestingly, the total VAZ and the antioxidant α-Toc (Figures 4B,C) showed more responsiveness to all the stresses than the other parameters, being the amplitude of the change higher than in the other compounds. The hydrophilic antioxidants tGSH and tASC did not show any significant change under any of the climatic stressors (Figure 4C).

To tackle the question of whether A+Z can be retained overnight under particular stress conditions in Mediterranean plants, frequencies of predawn values of AZ/VAZ in non-stressed and stressed plants were compared (Figure 5A). Predawn AZ/VAZ values higher than 0.4 were reported for 24% of stressed plants while only 1% of the non-stressed plants gave values higher than this threshold. Furthermore, photochemical efficiency (measured as predawn Fv/Fm) decreased with increasing overnight retention of Ant+Zea (measured as AZ/VAZ) (Figure 5B). This effect was particularly noticeable in winter, and to a lesser extent in other seasons, and correlated with the size of the VAZ pool (Figure 5C). However, apart from winter low temperatures, water stress also triggered the sustained retention of Ant+Zea (Figure 5D). Accordingly, data shown in Figure 5D evidenced that to attain high values of predawn AZ/VAZ, a low g_s is required and conversely, at high g_s, overnight Ant+Zea is always low. Relationship between water potential (ψ) and predawn AZ/VAZ (Figure 5E) showed the same trend but less markedly.

In order to assess the response capacity of main photosynthetic pigments and antioxidants in Mediterranean plants, a Plasticity Index (PI) was calculated for each of those parameters (Table 2). Higher plasticity was found in antioxidants than in pigment contents. Low variability in pigment content was generally found in Chl a+b, Chl a/b, Neo and β-Car. Among pigments, only VAZ and AZ/VAZ showed PI values above 0.5. Among antioxidants α-Toc showed higher PI than tAsc and tGSH suggesting a functional and important role for this metabolite in response to fluctuations in the environment.

Within the 73 total species included in this study, higher number of cases concerned sclerophyllous Quercus, which is actually one of the most preponderant tree and an important model genus within the Mediterranean Basin ecosystems. The sclerophyllous leaves from oak contain relatively high levels of Chl a+b per area, when compared to other Mediterranean species (Table 1). The rest of photosynthetic pigments, (i.e., carotenoids per Chl ratios) however, followed a quite stable stoichiometry, being similar for most of the studied species (Table 1). Besides the well-known phenotypic/morphological plasticity of oak leaves, biochemical plasticity appeared also higher than in other species, e.g., higher plasticity indexes were obtained for Chl a/b, AZ/VAZ, α-Toc, and tGSH in Quercus than in the other well represented Mediterranean genus Cistus (Table 2). Regarding leaf age and development, mature leaves of sclerophyllous Quercus species had significantly higher content of all photosynthetic pigments and antioxidants (specially, Neo, tGSH, and tAsc) than young leaves (Figure 6). This fact, contrasts with the different influence of plant age over the antioxidant pool: i.e., VAZ and AZ/VAZ were lower in older than in younger plants, but higher in mature than in young leaves (Figure 1 vs. Figure 6).

Mediterranean-type ecosystems are characterized by a period of a summer drought, extreme temperatures, both in winter and summer, and a marked seasonality. This type of climate can be found in all continents, but the largest area is the Mediterranean basin. This biome, one of the richest in terms of plant diversity with 25,000 native species, is recognized as a Global Biodiversity Hotspot (Myers et al., 2000). The succession of these stresses requires the acclimation of plant species living in this ecosystem. In this sense, the plant photoprotective mechanisms are key strategies in the resilience of Mediterranean species to the harsh environment. Such is the case of evergreens with long lived leaves, which compared to deciduous are more likely to suffer and cope with more stressful conditions during their life duration. As expected, the morphological and biochemical characteristics should them adjust along seasons and years. For this reason, evergreens require more rigid mesophyll structure (Onoda et al., 2017) or greater investment in photoprotection (with higher contents of VAZ and α-Toc) than deciduous, as we shown in this study (Figure 2). Sclerophyllous leaves, a trait that is closely related to evergreen strategy, also showed the same pattern.

Leaf pubescence (presence of trichomes) and glaucescence (waxes) are two of the most conspicuous foliar traits in Mediterranean plants, giving these ecosystems one of their signs of identity. These structures represent an example of evolutionary convergence. They reduce water loss (Peguero-Pina et al., 2017) and they may be highly reflective, limiting the penetration of light to the photosynthetic apparatus and reaching the mesophyll (Agrawal et al., 2009; Camarero et al., 2012). Thereby they reduce the need of photoprotective pigments and antioxidants, supporting the photoprotective role of leaf pubescence in Mediterranean species (Morales et al., 2002). Alternatively, trichomes and waxes can act as protective response since their accumulation is triggered after extenuation of biochemical photoprotection, as has been evidence for the waxes in Juniperus thurifera, where their accumulation represented a symptom of species decline (Esteban et al., 2014). Here, we show that biochemical traits (antioxidants and pigments) compensate the absence of morphological protective traits in Mediterranean species (Figure 2).

Another characteristic of Mediterranean flora is, compared to other temperate biomes, the higher leaf thickness (Peguero-Pina et al., 2012) and LMA (Traiser et al., 2005). Despite structural filtering of light and efficient protection against water lost of most sclerophyllous leaves, it has been proposed the existence of a trade-off between photosynthesis and LMA in relation with Leaf Economics Spectrum (Wright et al., 2004). It seems that mesophyll conductance (g_s) may be the most limiting factor for carbon assimilation, showing those leaves with highest LMA values, a reduction in CO₂ diffusion to the carboxylation sites (Flexas et al., 2014; Onoda et al., 2017; Peguero-Pina et al., 2017). This implies that plants with high LMA will generally show lower photosynthetic capacity and thus, may require higher levels of photoprotective metabolites. Interestingly, we found a positive relationship between LMA and photoprotective compounds, confirming that thicker leaves had more photoprotective demand (Figure 3). The cost associated with greater investment in photoprotection compounds in high LMA leaves underpin longer lifespan in these species (Wright et al., 2004; Díaz et al., 2015), meaning that long-lasting leaves may cope with higher cumulative number of stressful events along their life-span.

Pigment composition in Mediterranean plants does not differ substantially from the standard stoichiometry of higher plants of other biomes. Thus, with a few exceptions, photosynthetic pigment composition of non-stressed Mediterranean plants (Table 1) fitted within the 95% percentile of the control values reported by Esteban et al. (2015a) in a wider survey, including more than 800 plant species from diverse biomes. Most of these exceptions were found in the genera Quercus and Cistus, with the high Chl a/b and β-Car values being singularly frequent. Stress responses diverged from some of the general patterns described by Esteban et al. (2015a) in intensity and direction. In general, most stress factors induced increases on protective carotenoid pools (Figures 4A,B) such as β-Car (Munné-Bosch et al., 2003), VAZ and Lut (Li et al., 2009), but also of structural components such as Neo (Figure 4A) that are supposed to be strongly stable (Schmid, 2008). Among the environmental stressors, drought was the factor that affected more strongly pigment composition followed by seasonality and low temperatures. This corresponds with the Mediterranean dual stress model, in which summer drought and, in some places winter low temperatures, determines plant responses and distribution (Mitrakos, 1982). Besides, a high degree of plasticity was observed in all these responses (Table 2). This is in agreement with the large phenotypic plasticity as one of the typical strategies of Mediterranean plants to cope with their changing environmental conditions (Hormaetxe et al., 2007).

Under severe stress conditions, specially low temperature, the VAZ cycle does not reach a complete relaxation (Demmig-Adams and Adams, 1996; Demmig-Adams et al., 2006), and AZ/VAZ is typically higher than 0.5 before dawn (Míguez et al., 2015). This process has been characterized in detail in boreal conifers and alpine evergreens (Verhoeven, 2014; Míguez et al., 2017) involving profound change in the organization of photosynthetic apparatus. The overnight retention of AZ/VAZ is usually concurrent with a proportional decrease of photochemical efficiency (measured as Fv/Fm) (Demmig-Adams et al., 2006), also known as “winter photoinhibition.” In Mediterranean evergreens, the existence of an inverse relationship between AZ/VAZ and Fv/Fm, notably in winter (Figure 5B), suggests that Mediterranean evergreens employ the same mechanism of down-regulation of photochemical efficiency as boreal and alpine evergreens. Furthermore, present analysis suggests that this relationship extends to other stress factors rather than low temperature (Figure 5B, white dots), particularly water stress, which is the main environmental driver in Mediterranean ecosystems. Thus, high predawn AZ/VAZ values were observed only in plants with a low stomatal conductance (Figure 5D). Drought-stress induction of overnight retention of A+Z has been described in desert and desiccation-tolerant plants (Barker et al., 2002; Fernández-Marín et al., 2011, 2013). Here, we confirm that this mechanism is also activated at milder stress levels, such as that exerted by Mediterranean summer, being more universal than previously expected. Nevertheless, the high dispersion of values shown in Figure 5 indicates that the retention of A+Z does not always ensure a decrease in photochemical efficiency, likely playing other roles in the responses to stress (Havaux and Niyogi, 1999; Dall'Osto et al., 2010).

In contrast to the constricted stoichiometry of pigment composition in chloroplasts caused by the binding of pigments to thylakoid proteins (Antal et al., 2013), antioxidants display a much higher degree of variation, as we show in this study (Table 1). Although all species have available the same set of low molecular weight lipophilic (α-Toc, β-Car, and Zea) and hydrophilic antioxidants (Asc and GSH) to cope with oxidative stress, strong differences exist depending on taxonomy, plant growth and leaf characteristics (phenology, texture, age, attributes) (Figures 1, 2) and in response to stressful conditions (Figure 4). Thus, such differences can reach several orders of magnitude for α-Toc and tAsc (Table 1, Figures 1, 4). Curiously, despite Asc is the most abundant antioxidant in leaves (Table 1), neither tAsc nor tGSH was induced by any climatic stressors (Figure 4). This result suggests that most photo-oxidative damage undergone by Mediterranean species comes from the chloroplast, where α-Toc and β-Car are embedded in thylakoid membranes (Antal et al., 2013).

Interestingly, α-Toc and VAZ contents in angiosperms significantly differed from those found in gymnosperms (Figure 2). In angiosperms, there was a higher α-Toc and a lower VAZ contents than in gymnosperm. Within angiosperm, the higher content of α-Toc in woody species suggests an increased demand for α-Toc associated to the woody trait. This result is verified by an increment of α-Toc from seedling to adults, showing at the same time a decrease in VAZ (Figure 1). As stated before by Esteban et al. (2009), α-Toc increases progressively in the phylogeny from green algae to gymnosperms and even more to angiosperms, whereas the size of total VAZ pool shows the opposite pattern. This supports the idea that VAZ pigments and α-Toc share some photoprotective functions, which may lead to some degree of inter-compensation (Havaux and García-Plazaola, 2014). The cooperative function of VAZ and α-Toc is accounted because both are lipophilic compounds synthesized in the plastids being able to interact with membranes where they share several functions. Both are antioxidants (Falk and Munne-Bosch, 2010) and quenchers of ¹O₂ (Triantaphylidès and Havaux, 2009) and carotenoids may act also as membrane stabilizers (Havaux, 1998) and as quenchers of triplet Chl (Esteban et al., 2015b). Consequently, in agreement with Wujeska et al. (2013) VAZ and α-Toc exhibit the highest plasticity among pigments and antioxidants, respectively (Table 2). The paramount importance of α-Toc in the photoprotective response of Mediterranean plants is confirmed by the fact that it was the parameter that showed the highest responsiveness under all stresses analyzed in this manuscript (Figure 4 and Table 2). Among stressors, drought induced the highest α-Toc increase, followed by low and high temperatures, highlighting the importance of climatic stressors on the performance of plants in the Mediterranean ecosystems.

Throughout this work several gaps of knowledge related to photoprotection in Mediterranean plants have been identified: (i) herbs have been much less studied than woody species, (ii) and gymnosperms than angiosperms; and (iii) much less data are available about hydrophilic antioxidants than about lipophilic ones. Despite of these limitations in the literature, several conclusive statements have arisen from this analysis. The traits woody and high LMA, were generally related with a high demand for photoprotection. This can be explained by the evergreen strategy of most Mediterranean plants that implies high need of protection because of the long lifespan of their leaves. tGSH and tAsc showed the highest amplitude in their content. Nevertheless, α-Toc and VAZ were more responsive against Mediterranean climatic stressors. Thus, α-Toc responded mainly to drought and also to high temperature, while VAZ responded mainly to low temperature and also to drought. Therefore, the powerful photoprotective system of Mediterranean species pivots on lipophilic antioxidants, in particular Z and α-Toc, which play multiple roles in the plastid. In the context of a global climatic change, in which drought and extreme temperatures events are foreseen to increase their frequency in the Mediterranean area, the plasticity of photoprotective mechanisms to cope with such enhancing stressors will be fundamental. Resilience of Mediterranean species, compared with the template flora will likely benefit from such higher plasticity. To fully address these questions a complete understanding of this issue covering ecoregions other than the Mediterranean Basin (e.g., Western and South Australia, Cape Region, coastal California, and Chile) is needed.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer LLVJ and handling Editor declared their shared affiliation, and the handling Editor states that the process nevertheless met the standards of a fair and objective review