Photosynthetic limitations in Mediterranean plants: A review

Environmental and Experimental Botany 103 (2014) 12–23

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Environmental and Experimental Botany journal homepage: www.elsevier.com/locate/envexpbot

Review

Photosynthetic limitations in Mediterranean plants: A review J. Flexas a,∗,1 , A. Diaz-Espejo b,1 , J. Gago a , A. Gallé a,2 , J. Galmés a , J. Gulías a , H. Medrano a a Research Group on Plant Biology under Mediterranean Conditions, Departament de Biologia, Universitat de les Illes Balears, Carretera de Valldemossa Km 7.5, 07122 Palma de Mallorca, Illes Balears, Spain b Irrigation and Crop Ecophysiology Group, Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS, CSIC), Avenida Reina Mercedes 10, 41012 Sevilla, Spain

a r t i c l e

i n f o

Article history: Received 31 May 2013 Received in revised form 3 September 2013 Accepted 13 September 2013 Keywords: Mediterranean Stomatal limitation Mesophyll conductance limitation Biochemical limitation Drought Chilling

a b s t r a c t The aim of the present work is to review the literature concerning photosynthesis of Mediterranean plants. First, we briefly review the most important environmental constraints to photosynthesis, i.e. chilling winter temperatures and summer drought. Then, the review specifically focus on the photosynthetic capacity and photosynthetic limitations of Mediterranean plants under non-stress conditions, to test the general assumption that that the photosynthetic capacity of Mediterranean plants is lower than that of plants from other biomes. It is shown that Mediterranean plants of different life forms and leaf types present, on average, similar photosynthetic capacity to plants from any other biome. However, the mechanisms potentially limiting maximum photosynthesis differ between Mediterranean and non-Mediterranean species. For instance, Mediterranean plants compensate their lower mesophyll conductance to CO2 (gm ) with a larger velocity of carboxylation (Vc,max ) to achieve similar photosynthesis rates (AN ) to non-Mediterranean plants, both factors being associated to a larger leaf mass area (LMA) in Mediterranean species. In contrast, stomatal conductance (gs ) was found to be lower only in Mediterranean sclerophytes. On the other hand, Mediterranean sclerophytes and malacophytes (but not herbs and mesophytes) show higher mean intrinsic water use efficiency (AN /gs ) due to a combination of higher gm /gs and AN per unit CO2 concentration in the chloroplasts, i.e. carboxylation efficiency. The described variations in the mechanistic components of photosynthesis may represent specific adaptations of Mediterranean plants to their environment, leading these plants to achieve high AN despite their large LMA, and Mediterranean ecosystems to be among those presenting the largest net primary productivities worldwide. © 2013 Elsevier B.V. All rights reserved.

Contents 1.

2. 3. 4. 5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Environmental constraints to photosynthesis under Mediterranean conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Photosynthesis limitations in winter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3. Photosynthesis limitations in summer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Photosynthesis limitations under non-stress conditions: is the photosynthetic capacity of Mediterranean plants smaller than in plants from other biomes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Intrinsic photosynthetic water use efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Photosynthesis limitation in Mediterranean crops . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +34 971 172365; fax: +34 971 173184. E-mail address: jaume.fl[email protected] (J. Flexas). 1 These authors contributed equally to this review. 2 Present address: Bayer CropScience NV, Technologiepark 38, 9052 Zwijnaarde, Belgium. 0098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.envexpbot.2013.09.002

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J. Flexas et al. / Environmental and Experimental Botany 103 (2014) 12–23

1. Introduction

1.1. Environmental constraints to photosynthesis under Mediterranean conditions Based on annual trends of photosynthesis estimated at midmorning, it appears evident that maximum rates occur in spring and autumn, whereas depressions in photosynthesis of variable magnitude are detected in winter and summer (Eckardt et al., 1977; Tenhunen et al., 1987; Tretiach, 1993; Castell et al., 1994; Damesin and Rambal, 1995; Gratani, 1995; García-Plazaola et al., ˜ and Llusía, 1999; Haase et al., 2000; Méthy et al., 1997; Penuelas ˜ 2000; Flexas et al., 2001; Ogaya and Penuelas, 2003; Gratani and Varone, 2004; Gulías et al., 2009). Therefore, chilling temperatures during winter and hot/dry summer (i.e. drought) are the most limiting factors for photosynthesis under Mediterranean conditions. Indeed, the distribution of Mediterranean species along a latitudinal gradient depends on the species-specific adaptation to these environmental constrains (Mitrakos, 1980). The extent to which the photosynthesis rate of an individual species is depressed in winter/summer may depend on both species-specific adaptations and

AN (μ μmol CO2 m-2 s-1)

The landscape of Mediterranean-type ecosystems is dominated by evergreen sclerophyll forests, woodlands of either evergreen sclerophylls or semideciduous malacophylls, and grasslands. Although diverse and variable, these ecosystems present lower standing biomass per hectare than non-Mediterranean ecosystems, such as tropical broadleaf evergreen forests and temperate deciduous forests (Potter, 1999). Woody evergreen sclerophyll plants, that exhibit low specific leaf area, are indeed slow-growing species (Reich et al., 1992; Galmés et al., 2005). This, coupled with cold winters and hot/dry summers typical of Mediterranean climate, which restrict the favorable periods for photosynthesis to a few weeks in spring and autumn, originated the assumption that the photosynthetic capacity of Mediterranean plants (Folch and Camarasa, 1999) and the productivity of Mediterranean ecosystem were quite low (Ehleringer and Mooney, 1983). It is noted that most reviews on photosynthetic performance of Mediterranean plants, as well as the techniques adopted for estimating gas exchange, are outdated (Margaris, 1981; Ehleringer and Mooney, 1983; Ne eman and Goubitz, 2000). The increasing popularity of portable infra-red gas analysers in the 90s, and the introduction of eddy-flux technique for direct assessment of gas exchange and productivity at the canopy level have allowed the accumulation of a larger amount of data. Therefore, a more precise picture of the photosynthetic limitations in Mediterranean plants can now be drawn. Moreover, the combination of gas exchange and chlorophyll fluorescence measurements in addition to carbon isotope discrimination allows estimations not only of the net CO2 assimilation rate and stomatal conductance, but also of the mesophyll conductance to CO2 and the rate of gross photosynthesis. Altogether it is now possible to address the mechanisms that limit photosynthesis in Mediterranean plants (Grassi and Magnani, 2005). A review of the literature concerning photosynthesis in Mediterranean plants is presented. First, the most important environmental constraints to photosynthesis, i.e. chilling winter temperatures (T) and summer drought are reviewed. Then, three main questions of plants under non-stressed conditions are addressed: (1) is the photosynthetic capacity of Mediterranean plants lower than plants of other biomes? (2) What about the intrinsic photosynthetic water use efficiency of Mediterranean plants? (3) Do Mediterranean plants differ from plants in other biomes for traits that favor photosynthesis, such as stomatal (gs ) and mesophyll (gm ) conductances to CO2 and the maximum carboxylation velocity (Vc,max )? Finally, there is a brief discussion about the photosynthetic capacities and limitations of typical Mediterranean crops.

Quercus ilex L. 14 12

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Montpellier (France) Binifaldó (Mallorca, Spain) Puigpunyent (Mallorca, Spain)

10 8 6 4 2 0

Winter advantadge over colder sites

Summer advantadge over drier sites

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC JAN Fig. 1. Annual variations of photosynthesis of the evergreen sclerophyll tree Quercus ilex (holm oak) at three different locations: Montpellier (data from Méthy et al., 2000), Binifaldó and Puigpunyent (data from Gulías et al., 2009). Thin arrows indicate the photosynthetic advantage of plants growing at Binifaldó over those growing at the colder site Montpellier during winter, while thick arrows indicate the photosynthetic advantage of plants growing at Binifaldó over those growing at the drier site Puigpunyent during summer. Modified after Flexas et al. (2003).

climatic conditions of individual Mediterranean sites. This is presented in Fig. 1 by comparing published data of annual trends of daily photosynthesis rates in Quercus ilex growing at three different locations: Montpellier (South of France, average year precipitation –AYP– of 750 mm, and minimum monthly temperature –MMT– of 1 ◦ C), Binifaldó (Mallorca, Spain, AYP 1050 mm, MMT 8 ◦ C), and Puigpunyent (Mallorca, Spain, AYP 450 mm, MMT 12 ◦ C). Minimum photosynthesis was observed in winter at the coldest site (Montpellier) and in summer at the hottest and driest site (Puigpunyent). Photosynthesis was much higher in Binifaldó than at Montpellier during winter periods (indicated by thin arrows), and substantially higher than in Puigpunyent during summer (indicated by thick arrows), which is characterized by mild temperatures and relatively high yearly precipitation. Year-round maximum net photosynthesis averaged 5.5, 9.5 and 8.0 ␮mol CO2 m−2 s−1 at Montpellier, Binifaldó, and Puigpunyent, respectively. Using these rates as rough proxies of year carbon balance (at the leaf level) and following Mitrakos (1980), Q. ilex should be preferentially distributed at Binifaldó, where it indeed dominates the arboreal ecosystem coverage, and less at cooler sites like Montpellier (where it is indeed less dominant in mixed stands with deciduous Quercus species) and at the drier site like Puigpunyement (where there are indeed only few trees within a shrub macchia stand). Similar findings have been reported by Corcuera et al. (2005) who concluded that Q. ilex is more sensitive to winter than to summer stress. Besides different magnitudes in the depression of photosynthesis during winter or summer the mechanistic causes for such depressions may differ between winter and summer and will be discussed in the next sections. It is out of the aim of this review to elucidate the responses of photosynthesis in Mediterranean plants to other stresses such as nutrient availability (Daas-Ghrib et al., 2011), ozone (Mereu et al., 2011; Velikova et al., 2005; Lombardozzi et al., 2012), UV-radiation (Llusía et al., 2012) or excess soil salinity (Redondo-Gomez et al., 2008). 1.2. Photosynthesis limitations in winter Based on response curves of photosynthesis to air T under controlled conditions, most Mediterranean plants show an optimum T for photosynthesis in the range 15–30 ◦ C, the most common being ˜ 25–30 ◦ C (Oechel et al., 1980; Larcher, 2000; Ogaya and Penuelas, 2003). However, under Mediterranean field conditions low T are

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J. Flexas et al. / Environmental and Experimental Botany 103 (2014) 12–23

Fig. 2. Relationship between net photosynthesis (AN ) and air temperature (T, ◦ C) in Pistacia lentiscus, O. europaea, Cistus monspelliensis and Quercus ilex, combining data for three years and four locations in Mallorca, Spain (data from Gulías et al., 2009). Notice that although theoretically these plants have a temperature optimum around 25–30 ◦ C, as shown in pot experiments with irrigated plants; in practice (i.e. in the field) they display maximum photosynthesis at much lower temperatures, since when higher temperatures occur these are accompanied by additional stresses like excess irradiance and water stress.

usually accompanied by moderate sunlight irradiance coupled with adequate water availability whereas high T are associated with excess sunlight irradiance and water deficit. As a consequence, the effects of T on photosynthesis may be confounded by the concomitant action of other environmental constraints (Gratani and Varone, 2004; Gulías et al., 2009). Therefore, maximum photosynthesis rates in Mediterranean plants occur in ambient T range 10–20 ◦ C under field conditions (Fig. 2), simply because when more optimum temperatures occur these are accompanied by additional stresses reducing photosynthesis. Regarding to chilling sensitivity Mediterranean species show large differences. For instance, Q. ilex is more chilling-resistant than Olea europaea, which in turn is more resistant than Ceratonia siliqua (Mitrakos, 1980; Larcher, 1981). Photosynthesis may reflect species-specific differences in chilling sensitivity. Table 1 shows a list of Mediterranean species in which photosynthesis has been analyzed in winter at ambient T < 5 ◦ C as compared to maximum (spring or autumn) rates. Decreases of net photosynthesis in winter (as % of yearly maximum rates) range from 40% in some species to ca. 100% in others, reflecting inter-specific differences. Cistus albidus, Quercus coccifera or Rhamnus alaternus appear to be more chilling-resistant (less than 30% inhibition) than Cistus monspeliensis, Cneorum tricoccon, O. europaea, Phyllirea latifolia or Quercus suber (photosynthesis depressed by more than 50%). These differences may result from the original provenance of the species when the Mediterranean climate originated (Mitrakos, 1980). On the other hand, these differences could also be as a result of specific environmental conditions (the selected criterion of T < 5 ◦ C is rough), or even to intra-specific differences. For example, photosynthesis in Arbutus unedo and Q. ilex varies between 39% and 99% of its maximum rates. In Quercus the largest reduction was observed in North Italy (Larcher, 2000), while the minimum reduction was recorded in Mallorca (Gulías et al., 2009). Intermediate

Table 1 Chilling sensitivity of different Mediterranean plants. The values of net photosynthesis were averaged for all data available in months having temperature minima below 5 ◦ C, and expressed as percentage of the maximum measured photosynthesis rate for each species. Combined data from Larcher (2000), Varone and Gratani (2007) and Gulías et al. (2009). Species

Winter photosynthesis (% of maximum)

Arbutus unedo Ceratonia siliqua Cistus albidus Cistus incanus Cistus monspeliensis Cistus salvifolius Cneorum tricoccon Erica arborea Erica multiflora Hypericum balearicum Olea europaea Phyllirea latifolia Pistacia lentiscus Quercus coccifera Quercus ilex Quercus suber Rhamnus alaternus Rhamnus ludovici-salvatoris Rosmarinus officinalis

38–81 67 91 27 54–58 62 45 51 27 58 47–61 42–62 46–77 81 39–99 52 78 60 27

depressions were found at South France (44%) and Nord-East Spain and Rome (59–64%). In this case, the extent of winter inhibition follows a latitudinal gradient, mostly due to the prevailing effect of climate at each site. Irrespective of the magnitude of the inhibition, potential mechanisms responsible for photosynthesis depression during winter have been studied in Mediterranean plants. Non-stomatal factors are mostly responsible for photosynthesis inhibition under low T. Net CO2 assimilation (AN ) is indeed much more depressed under

J. Flexas et al. / Environmental and Experimental Botany 103 (2014) 12–23

these conditions than stomatal conductance (gs ). In the evergreen sclerophyll Pistacia lentiscus winter depression of photosynthesis was accompanied by decreased AN /gs , whereas during early summer drought AN /gs increased despite similar depression of photosynthesis (Flexas et al., 2001). Presently, there is not sufficient data to assess the relative significance of the different mechanisms (photochemistry, photosynthetic and Calvin-cycle enzymes or mesophyll conductance to CO2 ) responsible for photosynthesis depression. Leaf photochemistry has been shown to be significantly affected by winter chilling temperatures in many Mediterranean species (García-Plazaola et al., 1997; Karavatas and Manetas, 1999; Méthy ˜ et al., 2000; Oliveira and Penuelas, 2000; Flexas et al., 2001). Dark-adapted maximum photochemical efficiency of PSII (Fv /Fm ) is generally depressed during winter in the evergreen sclerophylls A. unedo, Arbutus andrachne, Juniperus phoenicea, Nerium oleander, P. latifolia, Pinus halepensis, P. lentiscus, Q. coccifera and Q. ilex (García-Plazaola et al., 1999; Karavatas and Manetas, 1999; Méthy ˜ 2000, 2005; Flexas et al., 2001; et al., 2000; Oliveira and Penuelas, Baquedano and Castillo, 2007). By contrast, Fv /Fm seems more resistant to winter temperatures in the semi-deciduous species C. albidus, Cistus creticus, Cistus salvifolius, Genista acanthoclada, Halimium halimifolium, Phlomis fruticosa and Sarcopterium spinosum (Karavatas and Manetas, 1999; Zunzunegui et al., 1999; Oliveira and ˜ ˜ 2000, 2005). Oliveira and Penuelas (2000) have suggested Penuelas, that the high resistance of PSII photochemistry in semi-deciduous species is due to their steep leaf angle, thus reducing the absorption of excessive photons, a matter still to be conclusively proven. Nonetheless, permanent photodamage has been recorded during winter in the semi-deciduous Cistus incanus, a condition which cannot be alleviated by increasing the biosynthesis of anthocyanins (Zeliou et al., 2009). Biochemical mechanism(s) responsible for winter depression in Fv /Fm and photochemistry have been suggested. García-Plazaola et al. (1997) showed that xanthophyll de-epoxidation in Q. suber was not maintained at pre-dawn during winter, although it was maintained during summer drought. In contrast, sustained deepoxidation was maintained at pre-dawn during winter in Q. ilex (García-Plazaola et al., 1999). These results support the hypothesis of photodamage being responsible for reduced Fv /Fm during winter in Q. suber, whereas sustained thermal dissipation of excess energy could be the cause of reductions in Fv /Fm in Q. ilex. Furthermore, winter depression in PSII photochemistry observed in several evergreen species was associated with increases in the concentration of alpha-tocopherol (chloroplast antioxidant), as well as in the xanthophyll de-epoxidation and de-epoxidation retention at night (García-Plazaola et al., 2003; Muller et al., 2006). Although leaf photochemistry (i.e. the thylakoid electron transport rate) is depressed in many Mediterranean plants under chilling, Flexas et al. (2001) observed that during both winter and summer leaf photochemistry was less affected than CO2 assimilation in P. lentiscus. These results suggest increased photorespiration and/or electron transport to other non-carboxylating processes, together with photochemistry as responsible for the reductions in AN during winter. 1.3. Photosynthesis limitations in summer In summer, high sunlight irradiance is coupled with high both T and atmosphere vapor pressure deficits (VPD). These factors, together with the absence of precipitation, result in a drought period, the severity of which differs among sites, lasting from one to six months (Ehleringer and Mooney, 1983). Therefore, a plant’s performance is severely constrained by the combined effect of water deficit and high atmospheric water demand. Plant tissues may suffer from dehydration, in turn causing embolism and plant death

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(Vilagrosa et al., 2003, 2010; McDowell et al., 2008; Miranda et al., 2010). Different sensitivity of photosynthesis to summer drought are expected among Mediterranean species. In some field studies, differences among species were described for their maximum photosynthesis rates during summer (Tretiach, 1993; Gratani, 1995; Gulías et al., 2009). These differences, however, do not necessarily reflect differences in the photosynthetic sensitivity to drought, as the different species may display different root systems and/or osmotic adjustment, heavily affecting tissue dehydration. In order to specifically assess the photosynthetic sensitivity to drought, a correlation between photosynthesis rates and tissue water relations (e.g. leaf water potential (LWP) or relative water content (RWC)) is needed. Table 2 shows a rough classification of the ‘photosynthetic drought tolerance’ for some Mediterranean species based on the criterion of the minimum measured leaf water potential at which positive AN is measurable. This classification, far from being completed, reveals several general patterns. First, malacophyll semi-deciduous plants appear to be the most resistant group which, together with their chilling tolerance (see previous section), convert them into the most tolerant group to the major environmental constraints of the Mediterranean climate, as previously suggested by Margaris (1981) and Suc (1984). Evergreen sclerophylls follow malacophylls as the most drought resistant – in general angiosperms being more resistant than conifers – whereas winter deciduous species are the least resistant. Secondly, drought resistance is more likely to be related with species phylogenetic closeness than to life form or leaf types, as species belonging to the same genus behave similarly to each other regardless of being deciduous or evergreen (as illustrated in Table 2 by Quercus and Pistacia species). Additionally, intra-specific differences in the photosynthetic sensitivity to drought have been detected in plants from single species but with contrasting populations, when grown in common gardens (Lauteri et al., 2004; Ramírez-Valiente et al., 2010; Sánchez-Gómez et al., 2011; De Miguel et al., 2012; StPaul et al., 2012). Although interesting, this is still an open research area. Stomatal closure is the main mechanism through which plants avoid or delay tissue dehydration. Stomatal closure leads to reduced photosynthesis, thus further compromising the plant’s carbon balance (McDowell, 2011). Stomatal closure is an early response of Mediterranean plants to water stress (Martínez-Ferri et al., 2000; Gulías et al., 2002; Mediavilla and Escudero, 2003b, 2004; Gallé and Feller, 2007; Gallé et al., 2007), and it has been usually considered as the prominent factor limiting photosynthesis under drought stress. Several Mediterranean species have their stomata inside epidermal crypts, which allow a fine regulation of stomatal aperture in a micro-environment dampening frequent changes in VPD (RothNebelsick et al., 2013) and, at the same time, shorten the mesophyll pathway for CO2 below the actual leaf thickness, thus allowing relatively high photosynthesis rates in species with large leaf mass area (LMA, Hassiotou et al., 2009a,b, 2010). The significance of stomatal limitations to photosynthesis is well known, although non-stomatal limitations have long been reported to be equally important in reducing photosynthesis (Tenhunen et al., 1984, 1985, 1987). In semi-deciduous malacophylls, drought-induced impairment of photochemistry is avoided by decreases in chlorophyll content in Phlomis sp. (Kyparissis et al., 1995), variations in leaf angles in Cistus sp. (Werner et al., 1999, 2001) or increasing leaf pubescence in some Digitalis sp. (Galmés et al., 2007a). These adjustments reduce the amount of light absorbed by the leaves during the unfavorable summer periods. Even in evergreen sclerophyll species, which generally do not possess effective light-avoidance mechanisms (with few exceptions, e.g. the pubescence exhibited by Q. ilex subsp. ballota, Morales et al., 2002), drought-induced constrains on leaf photochemistry prevail in seedlings and only for few

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J. Flexas et al. / Environmental and Experimental Botany 103 (2014) 12–23 Table 2 Water stress tolerance of photosynthesis in different Mediterranean plants. The species were ordered based on the minimum water potential at which they still display positive values of net photosynthesis, so that a species at the left side of a ‘>’ symbol shows some positive AN at a lower leaf water potential than the species at the right side. Based on literature data, modified after Flexas et al. (2003), with more recent literature: Bombelli and Gratani (2003), Gratani and Varone (2004), Levizou et al. (2004), Baquedano and Castillo (2007), Ladjal et al. (2007), Rubio-Casal et al. (2010), and Quero et al. (2011). Plain italics represent semi-deciduous malacophyll species, bold italics represent winter deciduous species, and underlined text evergreen species (black underlining for angiosperms and gray underlining for gymnosperms, i.e. conifers).

species and some conditions of light irradiance (Valladares et al., 2005, 2008; Galmés et al., 2007b; Peguero-Pina et al., 2009; Petsas and Grammatikopoulos, 2009). Overall, Mediterranean plants are well equipped with photoprotection mechanisms, which are further enhanced during summer: these include xanthophyll cycle ˜ et al., 2004; Galmés pigments (Munné-Bosch et al., 2003; Penuelas et al., 2007c; Peguero-Pina et al., 2008) and an integrated network ˜ of antioxidant defenses (Munné-Bosch et al., 2003; Penuelas et al., 2004; Munné-Bosch and Lalueza, 2007). Similarly to leaf photochemistry, the biochemical capacity of photosynthesis estimated either in vivo – Vc,max – or in vitro – Rubisco activity – is significantly constrained in Mediterranean plants under very severe drought. In contrast, the mesophyll conductance to CO2 (gm ) decreases at mild to moderate drought (Galmés et al., 2007d, 2011; Limousin et al., 2009, 2010; Fleck et al., 2010; Misson et al., 2010), although a few experiments have been reported showing that gm could be more resistant to drought, not declining even under moderate to severe stress (Warren et al., 2011). A quantitative estimation of the relative significance of stomatal conductance (gs ), mesophyll (gm ) conductances to CO2 and the maximum capacity for carboxylation (Vc,max ) in limiting net photosynthesis (AN ) has been proposed by Grassi and Magnani (2005). In detail, AN and its limiting factors are measured under ‘control’ (i.e. non-stress) and stress conditions. The total photosynthesis limitation (TL) is then calculated as the reduction (%) from control caused by the stressful conditions. Mechanistic causes of the TL are quantitatively assigned to stomatal (SL), mesophyll conductance (MCL) and biochemical (BL) limitations, the sum of SL, MCL and BL equaling TL. For very different Mediterranean species (including herbs, evergreen sclerophyll shrubs and trees, and semi-deciduous malacophyll shrubs) SL is the most important factor under mild water stress (i.e. early drought), SL and MCL co-limit photosynthesis under moderate drought, while MCL is the most important limitation (followed by BL) under severe drought (Galmés et al., 2007d; Limousin et al., 2009, 2010; Misson et al., 2010; StPaul et al., 2012). Decreased mesophyll conductance is additionally involved in midday-depression of photosynthesis that occurs in Mediterranean plants (Grassi et al., 2009; Bickford et al., 2010). The significance of drought-induced reduction of gm as a limiting factor to photosynthesis in Mediterranean plants is very relevant. Accurate carbon and water fluxes cannot be modeled in Mediterranean ecosystems unless accurate measurements of gm are included in the models (Reichstein et al., 2002; Keenan et al., 2010a,b). Mechanistic responses of photosynthesis to drought are generally analyzed at the level of representative leaves (i.e. fully

developed, sun-exposed). Consequently, it is difficult to scale up to canopy level, mostly due to factors that affect the photosynthetic performance of leaves located at different positions in the plant. Among these factors, light availability is the most important, as drought-induced photosynthetic limitations are maximal at high light (Niinemets et al., 2006; Aranda et al., 2007). Interactions between drought and light availability are also evident along the canopy (Niinemets et al., 2004, 2006; Cano et al., 2013). In non-stressed Quercus petraea and Fagus sylvatica, the highest AN at the top of the canopy is associated with higher gs and Vc,max , despite ‘top leaves’ display greater LMA and lower gm than leaves located deeper in the canopy (Cano et al., 2013). As water stress increases, photosynthesis is mostly limited by SL in leaves at the upper canopy, while SL, MCL and BL co-limit photosynthesis in the most basal leaves (Cano et al., 2013). Leaves, as well as plants of different age, may also respond differently to drought stress (Gratani and Ghia, 2002; Escudero and Mediavilla, 2003; Mediavilla and Escudero, 2003a, 2004; JuarezLopez et al., 2008; Rodríguez-Calcerrada et al., 2012; Varone et al., 2012). The effects of plant age are also species-specific. In the evergreen Q. ilex and the deciduous Q. faginea, SL increased with plant age. In contrast, the significance of stomatal limitation was great in young plants, whereas non-stomatal limitations prevailed in old plants in three evergreen species (Varone et al., 2012). These responses conform to (1) mesophyll conductance to CO2 progressively decreasing with plant age in several evergreen species (Niinemets et al., 2005). (2) In old plants, leaves suffer from greater oxidative stress than those in young plants, when exposed to adverse climatic conditions (Munné-Bosch and Lalueza, 2007). Other factors influencing the specific responses to drought include plant’s acclimation to different altitudes (Cabrera, 2002) as well as to consecutive drought and re-watering cycles (Gallé et al., 2011). In the evergreen sclerophyll Q. ilex, the photosynthetic limitations were similar during three consecutive drought and rewatering cycles. In contrast, some acclimation was observed in the semi-deciduous malacophyll C. albidus after the first drought cycle, consisting of a smaller decrease of gm than gs in the second and third cycles (i.e. higher SL and lower MCL), which in turn resulted in much higher photosynthetic water use efficiency in Cistus than in Quercus (Gallé et al., 2011). Finally, long-term experiments combining rain exclusion with increased temperature to simulate the likely effects of climate change, suggest that high temperature may exacerbate the observed photosynthetic limitations imposed by drought, which may also be species-specific. For instance, the evergreen species C. siliqua (Osorio et al., 2011) and the malacophyll Tuberaria

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major (Osorio and Romano, 2013) showed a larger photosynthesis decrease under the combination of both stresses. This was due to the combination of increased transpiration rates leading to decreased water potential, and to increased oxidative stress. Similarly, larger decreases of Fv /Fm under the effect of both stresses were observed in the evergreens Q. ilex and Phillyrea latifolia (Ogaya et al., 2011) as well as in Erica multiflora (Prieto et al., 2009). The latter also showing larger oxidative stress under the combination of both stresses (Nogués et al., 2012). Contrarily, other evergreen species like Globularia alypum and P. halepensis appear to be more resistant to the combination of high temperature and drought conditions (Prieto et al., 2009). It is noteworthy that invasive species appear to be far more resistant to combination of high temperature and drought than Mediterranean native species, for which different photosynthesis acclimation to climate change which could result in substantial changes in species distribution in the future (Godoy et al., 2011).

2. Photosynthesis limitations under non-stress conditions: is the photosynthetic capacity of Mediterranean plants smaller than in plants from other biomes? Analysis of a literature dataset including both Mediterranean and non-Mediterranean was performed in plants where AN , gs and gm had been measured under no-stress conditions (see Supplemental Table) using an updated published database (Flexas et al., 2013). Whenever maximum capacity for carboxylation (Vc,max ) was not provided in the original reference, it was estimated as follows: (1) day respiration rate (RD ) was either computed from the original reference or estimated using the empirical equation relating AN and RD provided by Galmés et al. (2007e). This relationship yielded very similar results to that provided by Gratani et al. (2008) for a different set of species (not shown). (2) Vc,max was estimated using a single-point method based on chloroplast CO2 concentrations described by Grassi and Magnani (2005). Once all required parameters were estimated, each entry was classified in terms of life form and leaf type, according to the following categories: (1) annual herbaceous monocotyledons, (2) annual herbaceous dicotyledons, (3) perennial herbaceous monocotyledons, (4) perennial herbaceous dicotyledons, (5) conifers, (6) evergreen angiosperms, (7) semi-deciduous woody perennial angiosperms and (8) deciduous woody perennial angiosperms. These groups were further reduced to four different leaf types: (a) herbaceous, (b) woody mesophytes, (c) woody sclerophytes and (d) woody malacophytes. The last two categories are much related to the Mediterranean vegetation. Sclerophytes leaves are characterized by thick leaves with a dense cuticle, thick cell walls, high stomata density and abundant sclerenchyma. The main criteria followed to label a species as sclerophyte or mesophyte was that the former are evergreen and present a leaf mass per area (LMA) above 120 g m−2 . When in doubt, species distribution and its recognition in previous literature as sclerophyte were considered (Wickens, 1998). The obtained dataset allowed the comparison between Mediterranean and non-Mediterranean species with respect to AN , or differences in their inherent mechanistic determinants of AN , i.e. stomatal (gs ) and mesophyll (gm ) conductances to CO2 and the maximum capacity for carboxylation (Vc,max ). Average values of AN (Fig. 3) for the above described eight different life forms using the described dataset are similar to those previously reported for the same life forms using larger datasets (Ehleringer and Mooney, 1983; Gulías et al., 2003; Galmés et al., 2012). Herbaceous species show the largest average AN , followed by the woody semi-deciduous species, while evergreen species have the lowest – conifers showing somewhat smaller AN than evergreen angiosperms (Fig. 3A). In general, Mediterranean

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and non-Mediterranean species show similar average AN within each life form, except for perennial herb dicots and semideciduous woody perennial angiosperms, for which Mediterranean plants show larger average AN . This is not consistent with more recently published data compilations for plants from Mediterranean (Galmés et al., 2012) and temperate (Warren et al., 2012) regions. From that comparison, the average AN was indeed somewhat higher in Mediterranean than in non-Mediterranean species for woody deciduous and evergreen species, as well as for herbs, shrubs and trees (and within herbs, in particular for therophytes). The advantage of the present dataset over previous ones is that it includes information of gm and Vc,max . In general, Mediterranean plants of several life forms tend to have larger Vc,max , while differences in the two diffusive parameters were more variable. On the other hand, the present dataset has the handicap of its limited data to the extent that for some of the groups (e.g. Mediterranean conifers or non-Mediterranean semi-deciduous woody angiosperms) there is only a single measurement or species available (notice the absence of standard error for these groups in Fig. 3). In order to strengthen the significance of differences between groups, species have been re-grouped considering their leaf type rather than their life form. Consequently, the number of categories decreased from eight to four, with more data available for each category (Fig. 4). Again, it can be observed that herbaceous and malacophyll species show the largest average AN , while sclerophyll species present the lowest. In all groups AN there was a significant difference between Mediterranean and non-Mediterranean species, although Mediterranean malacophylls showed much larger AN than non-Mediterraneans but with large intra-group variability. However, there were significant differences in some of the parameters influencing AN in both herbaceous and sclerophylls. In the two groups, gm was lower and Vc,max higher in Mediterranean plants while only in evergreen sclerophylls gs was also lower in Mediterranean species (Fig. 4). Following Grassi and Magnani (2005), the ‘theoretical’ maximum photosynthetic performance for any given life form or leaf type, the total (TL) and its components SL, MCL and BL were estimated from the maximum values of AN , gs , gm and Vc,max , i.e. the ‘theoretical’ maximum photosynthetic performance for any given life form or leaf type. Thus, the reference value was taken the single species displaying the maximum values for each parameter within each leaf type and TL, SL, MCL and BL from the comparison of the rest of the species. The selected reference species were Triticum aestivum (Tazoe et al., 2009), Banksia integrifolia (Hassiotou et al., 2009a,b), Prunus persica (Syvertsen et al., 1995) and Lavatera maritima (Galmés et al., 2007c) for herbaceous, sclerophytes, mesophytes and malocophytes, respectively. In malacophytes, TL was higher in non-Mediterranean species caused exclusively by its higher MCL (Fig. 5). For the other three groups, the differences between Mediterranean and non-Mediterranean species in TL were small, although significant in some cases. However, the most important differences were found in their partial limitations with SL and MCL tending to be larger and BL smaller in Mediterranean species (Fig. 5). Therefore, it can be concluded that mesophyll conductance to CO2 plays a major role in setting the photosynthetic capacity of Mediterranean plants and setting the differences with nonMediterranean species (Figs. 4 and 5). It is worth noting that the semi-deciduous malacophyll species, the most specifically adapted plants to Mediterranean conditions, present higher photosynthetic capacity than non-Mediterranean because of its larger gm. On the other hand, other Mediterranean species presenting lower gm achieve similar AN than non-Mediterranean species by increasing its Vc,max (Fig. 4). Higher values of gm observed were only observed in Mediterranean malacophylls and its reasoning is unknown and deserves further studies. Lower gm in the other groups appears to be

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Fig. 3. Comparison of the main driving variables of leaf CO2 assimilation rate in Mediterranean and non-Mediterranean species with eight different leaf forms. (A) Net CO2 assimilation rate (AN ); (B) maximum RuBP carboxylation velocity (Vcmax ); (C) stomatal conductance to CO2 (gsc ); (D) mesophyll conductance to CO2 (gm ). Means ± SE. Asterisks indicates a significant difference with P < 0.1 (*) or P < 0.05 (**) between Mediterranean and non-Mediterranean leaves within each functional type.

related to the significantly larger leaf mass area (LMA) observed in Mediterranean species (Fig. 6). Large LMA is associated to high leaf thickness and density, which appears to be an adaption to stressful environments like those in the Mediterranean climate (Niinemets, 1999). The fact that gm is constrained by large LMA has previously been reviewed (Flexas et al., 2008), being recently established that its underlying reason is mostly related to the thicker cell walls

observed in species with high LMA, significantly limiting CO2 diffusion inside the leaves (Peguero-Pina et al., 2012; Tosens et al., 2012; Tomás et al., 2013). Consequently, it can be hypothesize that high LMA is also responsible for the compensatory greater Vc,max per area among Mediterranean species, which could be induced by an increased Rubisco content per area caused by the overall increasing in leaf volume.

Fig. 4. Comparison of the main driving variables of leaf CO2 assimilation rate in Mediterranean and non-Mediterranean species with four different leaf functional types. (A) Net CO2 assimilation rate (AN ); (B) maximum RuBP carboxylation velocity (Vcmax ); (C) stomatal conductance to CO2 (gsc ); (D) mesophyll conductance to CO2 (gm ). Means ± SE. Asterisks indicates a significant difference with P < 0.01 (*) or P < 0.05 (**) between Mediterranean and non-Mediterranean leaves within each functional type.

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Fig. 5. Quantitative limitation analysis of photosynthetic CO2 assimilation in Mediterranean and non-Mediterranean (A) herbs; (B) sclerophytes, (C) mesophytes and (D) malacophytes. The percentage of total (TL), stomatal (SL), mesophyll (MCL) and biochemical (BL) are shown, estimated for each group in comparison to a selected species within each specific group displaying control (i.e. maximum) values of AN , gs , gm , and Vcmax . The selected reference control species were: T. aestivum (Tazoe et al., 2009) for herbaceous, Banksia integrifolia (Hassiotou et al., 2009a,b) for sclerophytes, Prunus persica (Syvertsen et al., 1995) for mesophytes, and Lavatera maritima (Galmés et al., 2007) for malocophytes. Means ± SE. Asterisks indicates a significant difference with P < 0.01 (*) or P < 0.05 (**) between Mediterranean and non-Mediterranean leaves within each functional type.

3. Intrinsic photosynthetic water use efficiency It has recently been highlighted that differences in gs , gm and Vc,max – and, specifically, differences in the gm /gs ratio – induce differences in AN /gs , i.e. the photosynthetic intrinsic water use efficiency or WUEi (Flexas et al., 2013). Hence, the observed differences in gm between Mediterranean and non-Mediterranean plants would be expected to result in differences in WUEi . However, this increase in higher WUEi between Mediterranean and non-Mediterranean species is only observed in evergreen sclerophylls (Fig. 6). This is due to the unique feature displayed by this

group in having both larger carboxylation efficiency (AN /Cc ) and larger diffusion efficiency (gm /gs ). A similar situation is observed for malacophylls, although larger intra-group variability results in differences being statistically significant only for gm /gs (Fig. 6). In a previous survey in Mediterranean species, evergreens already showed larger WUEi than semi-deciduous under non-stress conditions, although the latter took advantage under severe water stress (Medrano et al., 2009). In mesophytes none of these parameters differed significantly between Mediterranean and nonMediterranean plants, while in Mediterranean herbs their higher AN /Cc is compensated by a lower (gm /gs ). We propose that leaf

Fig. 6. (A) Average leaf mass per area (LMA), (B) intrinsic-water-use efficiency (AN /gs ), (C) ratio CO2 assimilation rate to chloroplastic CO2 at ambient CO2 (AN /Cc ), and (D) ratio mesophyll conductance to CO2 to stomatal conductance to CO2 (gm /gs ) of the four leaf functional types for Mediterranean and non-Mediterranean species. Means ± SE. Asterisks indicates a significant difference with P < 0.1 (*) or P < 0.05 (**) between Mediterranean and non-Mediterranean leaves within each functional type.

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Fig. 7. Comparison of the main driving variables of leaf CO2 assimilation rate in both Mediterranean and non-Mediterranean crop species. (A) CO2 assimilation rate (AN ); (B) maximum RuBP carboxylation velocity at 25 ◦ C (Vcmax ); (C) stomatal conductance to CO2 (gsc ); (D) mesophyll conductance to CO2 (gm ). Means ± SE. Mediterranean herbaceous species: Triticum aestivum, Triticum durum, Hordeum vulgare and Solanum lycopersicum var. Ramallet. Non-Mediterranean herbaceous species: Phaseolus vulgaris, Nicotiana tabacum, Vicia faba, Oryza sativa, Lycopersicum esculentum, Spinacia oleracea, Capsicum annuum, Helianthus annuum and Brassica carinata. Mediterranean woody species: Olea europea, Vitis vinífera, Citrus limon, Prunus persica and Prunus dulcis. Non-Mediterranean woody species: Citrus paradise, Macadamia integrifolia. Asterisks indicates a significant difference with P < 0.1 (*) or P < 0.05 (**) between Mediterranean and non-Mediterranean leaves within each functional type.

trait adjustments for increased WUEi may be an adaptive strategy in Mediterranean species, in particular in evergreen and semideciduous, which sustain green leaves during summer, when there is less water and its efficient use more important. 4. Photosynthesis limitation in Mediterranean crops All previous sections were focused on native Mediterranean species. Regarding crops, only those that can be considered Mediterranean were being considered. This consideration was based on the time length that they have been cultivated in Mediterranean areas for and their typical presence in the Mediterranean landscape. Among them, the species considered were: (a) the woody crops Olea europea, Vitis vinífera, Citrus limon, P. persica and Prunus dulcis, and (b) the herbaceous T. aestivum, Triticum durum, Hordeum vulgare and Solanum lycopersicum var. Ramallet. When compared to non-Mediterranean crops, Mediterranean crops show not only similar average values of AN , but also – and contrarily to native vegetation – similar values for gs , gm and Vc,max (Fig. 7). The absence of differences is likely to reflect the different selection forces that have operated during the evolution between native and crop species. While native plants may have been forced to optimize survival and reproduction under conditions of soils poor in nutrients and climatic stresses (cold winter and summer droughts), crops are bred for maximizing production, and its cultivation often supposes their settlement in richer soils (or supplied with fertilizers) while alleviating stress conditions (for instance by irrigation). 5. Concluding remarks The general use of portable infrared gas analysers and chlorophyll fluorimeters has allowed for a large accumulation of data regarding photosynthesis in Mediterranean plants in the last two decades. A compilation and review of these bulk information on Mediterranean species – including herbaceous species, woody evergreen sclerophytes, evergreen and deciduous mesophytes and

semi-deciduous melacophytes – provides a general picture of the responses of photosynthesis to environmental stresses and gives new clues on how it copes with cold winters and summer droughts, the two most constraining environmental conditions under Mediterranean climate. In addition, comparative analysis of the photosynthetic capacity and its components combined with a quantitative limitation analysis between Mediterranean and non-Mediterranean species permits the observation of specific mechanisms limiting photosynthesis. Contrary to common assumptions, it can be concluded that Mediterranean plants present photosynthetic capacities similar to those of other biomes. With the exception of semi-deciduous malacophytes – which are optimally adapted to the Mediterranean climate and which present even higher photosynthesis than their non-Mediterranean counterparts – plants with all other leaf types show average photosynthesis similar to those of other biomes. However, a reduced mesophyll conductance to CO2 (gm ) is observed in herbaceous and evergreen sclerophyll Mediterranean plants caused by a significantly higher leaf mass area. Lower gm is compensated by larger velocity of carboxylation – i.e. biochemical capacity – to achieve similar photosynthesis rates. In Mediterranean evergreen sclerophylls, stomatal conductance (gs ) is also lower resulting in higher intrinsic photosynthetic water-use-efficiency. Moreover, Mediterranean plants are able to sustain higher photosynthesis rates in spring and autumn and, at least in one of the two most limiting periods – winter or summer – depending on the site and species. Consequently, and not surprisingly, eddy-flux based measurements in Mediterranean ecosystems reveal that these plants are among those presenting the highest net primary productivities worldwide (Allard et al., 2008). Acknowledgments This work was partly supported by the Plan Nacional, Spain, contracts BFU2011-23294 (J.F and J.G), AGL2009-11310/AGR (A. D-E) and AGL2011-30408-C04-01 (H.M). We are indebted to Dr. Miquel

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Ribas-Carbo and an anonymous reviewer for grammar corrections, and to two anonymous reviewers for their suggestions to improve the manuscript.

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