Different regions of the world are characterized by the so-called Mediterranean-type ecosystems, with generally warm and dry summers, and wet and mild winters, which determines a great diversity in vegetation that is specifically known as “chaparral” in California, “fynbos” in the Cape Province of South Africa, “matorral” in Chile, “malle” in Australia, and “macchia” (or “maquis”) in the Mediterranean basin (Cody and Mooney, 1978). Plant species distribution in Mediterranean macchias appear to be mainly limited by drought stress, that is a combination of water deficit, high temperatures and high solar radiation during the summer, but it has been suggested that low temperatures in winter may also play a role in plant adaptation and fitness (Mitrakos, 1982). However, most of the ecophysiological research on this macchia vegetation has been focused on the effects of drought stress during summer, and very few studies have investigated thus far the response and adaptation of Mediterranean plants to low-temperature winter stress (Martínez-Ferri et al., 2004; Verhoeven, 2014; Esteban et al., 2015; Míguez et al., 2015).

Although Mediterranean ecosystems are generally considered to be characterized by hot and dry summers, and wet, mild winters, previous studies have already shown sustained decreases in the maximum efficiency of photosystem II (PSII) photochemistry (F_v/F_m ratio) not only during summer drought, but also during winter, the so-called “winter photoinhibition”, in some evergreen species (Kyparissis et al., 1995; García-Plazaola et al., 1999b; Oliveira and Peñuelas, 2000; Martínez-Ferri et al., 2004; Valladares et al., 2005). Low temperatures during winter may lead to an impairment of the photosynthetic apparatus leading to reductions in PSII efficiency (Berry and Bjorkman, 1980; Adams et al., 1994; Demmig-Adams and Adams, 2000; Yamori et al., 2014), since chilling temperatures strongly reduce photosynthetic activity (Huner et al., 1998; Du et al., 1999; Allen and Ort, 2001; Foyer et al., 2002; D’Ambrosio et al., 2006; Grennan and Ort, 2007; Mohanty et al., 2007), an effect that may be exacerbated on bright days (Öquist and Huner, 1993; Huner et al., 1998). Despite solar radiation decreases considerably during winter (approximately by one-half compared to yearly maxima during late June), Mediterranean plants can still absorb more solar energy that they can use for photosynthesis, particularly when low temperatures are interacting with other stressors, such as drought or abrasive damage due to wind that can strongly enhance foliage cuticular water loss, increasing potential desiccation stress (Grace, 1977; Larcher, 2003). Indeed, drought events are becoming more and more unpredictable and are increasingly occurring during winters in Mediterranean-type ecosystems in the frame of global change (Intergovernmental Panel on Climate Change [IPCC], 2014).

Aside from seasonal variations in environmental conditions that determine plant performance in the Mediterranean macchia, it is also important to consider that some of these plant species show sexual dimorphism. It is generally thought, that females, due to a higher investments in reproductive structures may suffer more photoinhibition and photo-oxidative stress than males under environmental stresses, particularly drought stress (Li et al., 2004, 2007; Rozas et al., 2009; Simancas et al., 2016), but also low temperatures (Xu et al., 2008b; Zhang et al., 2011). However, some exceptions exist, in which no differences between sexes can be observed or even that females outperform males in their tolerance to abiotic stress, so that more studies are required to better understand secondary sexual dimorphism in plants (Barrett and Hough, 2013; Juvany and Munné-Bosch, 2015; Munné-Bosch, 2015).

Mediterranean plants have evolved complex mechanisms of photoprotection to prevent photoinhibition and irreversible damage to the photosynthetic apparatus. When excited states of chlorophyll (¹Chl^∗) are not readily processed by photochemistry (photosynthesis), they are converted to triplet excited states (³Chl^∗) and the energy may be transferred to oxygen producing singlet oxygen (¹O₂). This reactive oxygen species may eventually cause photo-oxidative stress, thus leading, among other processes, to the inactivation of PSII and/or an increased peroxidation of membrane lipids (Pintó-Marijuan and Munné-Bosch, 2014). To prevent ³Chl^∗ and the subsequent singlet oxygen (¹O₂) formation, a carotenoid (xanthophyll cycle)-dependent dissipating pathway is activated to safely return ³Chl^∗ to its ground state. The excess excitation energy is thereby dissipated as heat, directly within the carotenoid protein complexes, bound to the light-collecting chlorophylls (Demmig-Adams and Adams, 2000). Since low temperatures decrease the rate of photosynthesis and increase the excitation energy in chloroplasts; xanthophyll cycle-dependent energy dissipation, which operates in the antenna complexes of PSII, is considered one of the most efficient mechanisms to protect the photosynthetic apparatus (Adams et al., 1994; Ottander et al., 1995; Demmig-Adams and Adams, 2000). Therefore, the role of carotenoids, particularly those of the xanthophyll cycle and zeaxanthin in photoprotection, are considered to be essential in excess energy dissipation and are generally associated with reversible photoinhibition (García-Plazaola et al., 1997, 1999b; Kyparissis et al., 2000).

Singlet oxygen is formed under adverse climatic conditions when the xanthophyll cycle-dependent energy dissipation system is not activated or when it cannot dissipate more excess energy in chloroplasts. ¹O₂ can then be eliminated both physically and chemically (the so-called “quenching” and “scavenging” of ¹O₂) by the action of antioxidants, which react more quickly than lipids with ¹O₂. Carotenoids, and most particularly β-carotene and zeaxanthin, and α-tocopherol (which belongs to the vitamin E group of compounds) eliminate ¹O₂. α-Tocopherol, in turn, inhibits the propagation of lipid peroxidation in thylakoid membranes by reacting with lipid peroxyl radicals and therefore preventing the oxidation of poly-unsaturated fatty acids in cascade (Munné-Bosch and Alegre, 2002). Indeed, previous studies have shown that β-carotene and α-tocopherol act synergistically to protect PSII efficiency from ¹O₂-induced damage (Trebst, 2003).

The Mediterranean dwarf palm (Chamaerops humilis L.) is one of the only two native palms in Europe and the only one in the Iberian Peninsula, where it is autochthonous. Distinctively, this palm is also one of the very few palms originally from a temperate zone and not from the tropics, where most palms are abundant and grow naturally, being able to reach latitudes of up to 44°N (Merlo et al., 1993). This palm is native from continental Europe being mainly found in the western Mediterranean basin, all over the Mediterranean coast of Spain and Portugal, central and southern Italy, some parts of the southern coast of France, islands of the western Mediterranean and northwest Africa (from Morocco to Libya, Global Biodiversity Information Facility [GBIF], 2016). C. humilis appears in all phases of the succession of the degradation of Mediterranean ecosystems due to its tolerance to disturbances, such as deforestation, fires, or grazing (Santiesteban et al., 1992; Götzenberger et al., 2003; Alados et al., 2004). Furthermore, it is generally used in gardening, in preference to other palms of alien origin.

Here, we hypothesized that C. humilis is very well adapted to the Mediterranean climate and will therefore not suffer photoinhibition and photo-oxidative stress due to the activation of efficient photoprotection mechanisms neither during summer drought nor during winter-associated low-temperature stress. An emphasis was put on the study of photoinhibition and photoprotection mechanisms throughout seasonal variations and the possible influence of plant size, maturity and sexual dimorphism during reproductive events. Our final goal was to improve our knowledge about the ecophysiological response of this unique, native palm from Europe, which is autochthonous in the Iberian Peninsula and is considered a protected species in certain parts of its distribution area (European Food Safety Health [EFSA], 2014).

In their post-embryonary stage, plants are sessile and cannot escape from adverse environmental conditions, so they have evolved a number of complex, interconnected mechanisms of adaptation to allow their survival. Plants living in Mediterranean-type ecosystems are subjected to marked seasonal variations throughout the year and, consequently, exposed to multiple stresses. Here, it is shown that C. humilis can withstand both summer droughts and low temperatures during winter, despite a transient photoinhibition was observed during the winter months. Previous studies have shown that there are two mechanisms to counteract the potential damaging effects of excess solar radiation: (i) prevent or avoid photoinhibition, which is found in species that are able to maintain a sustained PSII photochemical efficiency, and (ii) tolerate photoinhibition, which is observed in species displaying dynamic photoinhibition, which is in turn associated with fast reversible mechanisms (Demmig-Adams and Adams, 1992; Long et al., 1994). The latter include activation of the synthesis of non-epoxy xanthophylls (typically zeaxanthin, although some species additionally accumulate lutein from lutein epoxide), leading to the development of non-photochemical quenching (Martínez-Ferri et al., 2000, 2004; Jahns and Holzwarth, 2012).

Reversible, transient photoinhibition during the winter was manifested by F_v/F_m ratios below 0.75 during the cold temperatures of January–March in the present study, which was followed by a full recovery to optimal values (F_v/F_m ratios of 0.8) during spring. C. humilis is described here, to our knowledge for the first time, as a photoinhibition-tolerant palm. Furthermore, we show that this tolerance is achieved through the activation of xanthophyll cycle de-epoxidation, leading to an enhanced accumulation of zeaxanthin, as it occurs in other evergreen species from Mediterranean-type ecosystems (García-Plazaola et al., 1999a,b, 2003) and other habitats (Baker, 1995; Verhoeven et al., 1996). In these species, low predawn F_v/F_m values during winter have been associated with the overnight retention of high amounts of zeaxanthin, which has been attributed to the inhibitory effect of chilling temperatures on the enzymatic conversion of zeaxanthin from violaxanthin in the xanthophyll cycle (Adams and Demmig-Adams, 1994; Adams et al., 1994; Leipner et al., 2000). Thereby, the winter photoinhibition observed in C. humilis in the present study can be interpreted as an adaptive photoprotection mechanism of the photosynthetic apparatus, as it occurs in some evergreen conifers, which are adapted to extreme environments, showing the greatest degree of photoinhibition during the winter months (Adams et al., 2004; Demmig-Adams and Adams, 2006).

In an attempt to get some insights into the possible causes and consequences of winter photoinhibition, we correlated the F_v/F_m ratio with all other measured parameters (Table 3). It turned out that the F_v/F_m ratio not only negatively correlated with the DPS, VZA and zeaxanthin levels, but also with the levels of lutein and β-carotene per chlorophyll unit, and it positively correlated with the leaf water content (see “Seasonal” column, Table 3). This indicates that water deficit had some influence on the development of the transient photoinhibition during winter months, which has important biological implications in the framework of global change, in which drought events are becoming more unpredictable. Furthermore, it is interesting to note that this transient photoinhibition was achieved with parallel decreases in the content of chlorophylls, particularly that of chlorophyll b (as indicated by the negative correlation with the chlorophyll a/b ratio, Table 3), but increases in the levels of lutein and β-carotene per unit of chlorophyll. Both lutein and β-carotene protect the photosynthetic apparatus from ¹O₂-induced damage (Telfer, 2005; Dall’Osto et al., 2006; Triantaphylidès and Havaux, 2009; Ramel et al., 2012, 2013), thus suggesting photoprotection to PSII is achieved by decreasing the amount of photons that enter the photosynthetic electron transport (by reducing chlorophyll levels, particularly those found in the antenna), and by increasing the amount of photoprotective molecules that protect from ¹O₂-induced damage. It appears, therefore, that xanthophyll cycle-dependent energy dissipation operates in parallel with mechanisms that increase the elimination of ¹O₂. Prevention and elimination operate therefore simultaneously to avoid chronic photoinhibition of the photosynthetic apparatus in C. humilis.

The dwarf palm also showed strategies for overcoming severe drought conditions, such as keeping leaf water content always above 80% and the activation of xanthophyll cycle-dependent excess energy dissipation during the summer. It has already been shown that this palm is well adapted to the Mediterranean summer conditions. In a comparative study between dominant species of a coastal Mediterranean macchia ecosystem, the dwarf palm showed greater water potential values, thus indicating a better tolerance to water deficit (Rotondi et al., 2003). As July might be a critical period for this species due to high temperatures, high solar radiation and drought, it is the month with maximum activation of xanthophyll cycle-dependent excess energy dissipation, which acts as a photoprotection mechanism helping to tolerate summer drought as in other typically Mediterranean species (Míguez et al., 2015). These results emphasize the importance of understanding adaptive mechanisms to drought tolerance in these habitats (Bermúdez and Retuerto, 2014; Catoni and Gratani, 2014; Gratani et al., 2014; Lloret et al., 2016). In other palms, it has also been shown enhanced drought tolerance related to greater efficiency in preventing oxidative damage by activating antioxidant mechanisms (e.g., see Gomes and Prado, 2007 for the coconut palm Cocus nucifera; Al-Khayri and Ibraheem, 2014 and Arab et al., 2016 for the date palm Phoenix datilifera; Silva et al., 2016 for the oil palms Elaeis guineensis and Elaeis oleifera).

Sexual dimorphism can lead to profound differences in secondary sexual characters, such as vegetative growth and plant response to both biotic and abiotic stresses; but the extent to which these stresses can affect photoinhibition, photoprotection, and photo-oxidative stress in dimorphic plants is still very poorly understood. Previous studies have shown that females are generally more sensitive to photoinhibition that males when subjected to either drought stress (Li et al., 2004, 2007; Xu et al., 2008a,b; Rozas et al., 2009; Chen et al., 2010, 2014; Zhang et al., 2010, 2014; Kumar et al., 2016) or low temperatures (Li et al., 2007; Zhang et al., 2011; Juvany et al., 2014), although some exceptions exist (e.g., see Oñate et al., 2012; Morales et al., 2013; He et al., 2016). In the present study, it was found that females are more sensitive than males to photoinhibition only during the summer. It is interesting to note that sex-related differences disappeared during winter, when plants were not reproducing, thus indicating photoinhibition in C. humilis is a clear secondary sexual character associated to an increased reproductive effort in females. This may be associated with sex-related differences in trade-offs in life-history traits, so that plants may allocate resources on growth, reproduction or maintenance (metabolism or defense) (Yang et al., 2014). Since females allocate a greater investment in reproduction, generally showing distinct nutrient requirements (Zhao et al., 2012; Zhang et al., 2014; Chen et al., 2015; Simancas et al., 2016), they decrease resources for other functions like defenses, and this will be the reason why females are more sensitive to photoinhibition that males when they are subjected to drought stress. Despite this, females are able to recover from photoinhibition and sex differences disappear after the reproductive period. This is particularly interesting since it suggests that females simply respond differently than males, showing compensatory mechanisms to overcome enhanced nutrient requirements during reproductive events (Obeso, 2002; Barrett and Hough, 2013; Juvany and Munné-Bosch, 2015). Previous studies have shown sex-related differences in flowering response to photoperiod (Zhao et al., 2009) and source-to-sink transitions (Zhao et al., 2012), which may indeed reflect complex sex-related differences in the physiology of dioecious plants (Ashman, 2006). If environmental stress is mild and/or transient, such compensatory mehanisms may be effective to overcome higher nutrient requirements during reproductive events in females than in males, but when stress is too severe or persists in time, more damage occurs in females than males. Our results suggest that transient photoinhibition during reproductive events in summer in females of C. humilis is indeed an adaptive mechanism to prevent irreversible damage. In this respect, it is also noteworthy that C. humilis did not show spatial segregation between sexes, as shown in Figure 2 Previous studies indicate lower ability for competition in females than males, which is associated with increased nutrient requirements in the former (Chen et al., 2014, 2015). In C. humilis, at least in our field site in the Garraf Natural Park, sex competition may occur and play a role determining sexual differences in physiological traits, but competition with other plant species, particularly some invasive ones, such as Cortaderia selloana, may be indeed even more important. This aspect requires, however, further investigations.

The present study demonstrates the photoprotective capacity of the unique native Mediterranean palm during winter photoinhibition, in which plants are able to overcome the harmful effects of chilling stress combined with high light. Furthermore, it is shown here that females are more sensitive than males to photoinhibition during reproductive events, although these effects are transient and reversible. Due to the ecological importance of the Mediterranean dwarf palm, the high capacity of photoprotection described here supports the use of this species in reforestation programs, both after fires and to recover degraded soils.

MM, MP-M, and SM-B designed the experiments; MM and MP-M performed experiments; SM-B contributed materials and reagents; MM and SM-B wrote the manuscript.

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.