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Environmental and Experimental Botany 103 (2014) 12–23

Contents lists available at ScienceDirect

Environmental and Experimental Botany

jo ur nal home p ag e: www.elsev ier .com/ locate /envexpbot

eview

hotosynthetic limitations in Mediterranean plants: A review

. Flexasa,∗,1, A. Diaz-Espejob,1, J. Gagoa, A. Galléa,2, J. Galmésa, J. Gulíasa, H. Medranoa

Research Group on Plant Biology under Mediterranean Conditions, Departament de Biologia, Universitat de les Illes Balears, Carretera de Valldemossa Km.5, 07122 Palma de Mallorca, Illes Balears, SpainIrrigation and Crop Ecophysiology Group, Instituto de Recursos Naturales y Agrobiología de Sevilla (IRNAS, CSIC), Avenida Reina Mercedes 10, 41012evilla, Spain

r t i c l e i n f o

rticle history:eceived 31 May 2013eceived in revised form 3 September 2013ccepted 13 September 2013

eywords:editerranean

tomatal limitationesophyll conductance limitation

iochemical limitationroughthilling

a b s t r a c t

The aim of the present work is to review the literature concerning photosynthesis of Mediterraneanplants. 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 photo-synthetic 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 lowerthan that of plants from other biomes. It is shown that Mediterranean plants of different life forms andleaf types present, on average, similar photosynthetic capacity to plants from any other biome. How-ever, the mechanisms potentially limiting maximum photosynthesis differ between Mediterranean andnon-Mediterranean species. For instance, Mediterranean plants compensate their lower mesophyll con-ductance to CO2 (gm) with a larger velocity of carboxylation (Vc,max) to achieve similar photosynthesisrates (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 Mediter-ranean 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 highergm/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 specificadaptations of Mediterranean plants to their environment, leading these plants to achieve high AN despitetheir large LMA, and Mediterranean ecosystems to be among those presenting the largest net primaryproductivities worldwide.

© 2013 Elsevier B.V. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.1. Environmental constraints to photosynthesis under Mediterranean conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.2. Photosynthesis limitations in winter . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131.3. Photosynthesis limitations in summer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2. Photosynthesis limitations under non-stress conditions: is the photosynthetic capacity of Mediterranean plantssmaller than in plants from other biomes? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

3. Intrinsic photosynthetic water use efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

4. Photosynthesis limitation in Mediterranean crops . . . . . . . . . . . . . . . . . . . . . . . .5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +34 971 172365; fax: +34 971 173184.E-mail address: jaume.flexas@uib.es (J. Flexas).

1 These authors contributed equally to this review.2 Present address: Bayer CropScience NV, Technologiepark 38, 9052 Zwijnaarde, Belgiu

098-8472/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.envexpbot.2013.09.002

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

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Experimental Botany 103 (2014) 12–23 13

1

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14Montpellier (France)Binifaldó (Mallorca, Spain)Puigpunyent (Mallorca, Spain)

Winter advantadge over colder sites

Summ er ad vantadge over drier sites

Fig. 1. Annual variations of photosynthesis of the evergreen sclerophyll tree Quer-cus 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 indi-cate the photosynthetic advantage of plants growing at Binifaldó over those growing

J. Flexas et al. / Environmental and

. Introduction

The landscape of Mediterranean-type ecosystems is dominatedy evergreen sclerophyll forests, woodlands of either ever-reen sclerophylls or semideciduous malacophylls, and grasslands.lthough diverse and variable, these ecosystems present lowertanding biomass per hectare than non-Mediterranean ecosystems,uch as tropical broadleaf evergreen forests and temperate decid-ous forests (Potter, 1999). Woody evergreen sclerophyll plants,hat exhibit low specific leaf area, are indeed slow-growing speciesReich et al., 1992; Galmés et al., 2005). This, coupled with cold win-ers and hot/dry summers typical of Mediterranean climate, whichestrict the favorable periods for photosynthesis to a few weeksn spring and autumn, originated the assumption that the photo-ynthetic capacity of Mediterranean plants (Folch and Camarasa,999) and the productivity of Mediterranean ecosystem were quite

ow (Ehleringer and Mooney, 1983). It is noted that most reviewsn photosynthetic performance of Mediterranean plants, as wells the techniques adopted for estimating gas exchange, are out-ated (Margaris, 1981; Ehleringer and Mooney, 1983; Ne′emannd Goubitz, 2000). The increasing popularity of portable infra-redas analysers in the 90s, and the introduction of eddy-flux tech-ique for direct assessment of gas exchange and productivity athe canopy level have allowed the accumulation of a larger amountf data. Therefore, a more precise picture of the photosyntheticimitations in Mediterranean plants can now be drawn. More-ver, the combination of gas exchange and chlorophyll fluorescenceeasurements in addition to carbon isotope discrimination allows

stimations not only of the net CO2 assimilation rate and stoma-al conductance, but also of the mesophyll conductance to CO2nd the rate of gross photosynthesis. Altogether it is now possibleo address the mechanisms that limit photosynthesis in Mediter-anean plants (Grassi and Magnani, 2005).

A review of the literature concerning photosynthesis in Mediter-anean plants is presented. First, the most important environmentalonstraints to photosynthesis, i.e. chilling winter temperaturesT) and summer drought are reviewed. Then, three main ques-ions of plants under non-stressed conditions are addressed: (1)s the photosynthetic capacity of Mediterranean plants lower thanlants of other biomes? (2) What about the intrinsic photosyntheticater use efficiency of Mediterranean plants? (3) Do Mediter-

anean plants differ from plants in other biomes for traits that favorhotosynthesis, such as stomatal (gs) and mesophyll (gm) conduc-ances to CO2 and the maximum carboxylation velocity (Vc,max)?inally, there is a brief discussion about the photosynthetic capac-ties and limitations of typical Mediterranean crops.

.1. Environmental constraints to photosynthesis underediterranean conditions

Based on annual trends of photosynthesis estimated at mid-orning, it appears evident that maximum rates occur in spring

nd autumn, whereas depressions in photosynthesis of variableagnitude are detected in winter and summer (Eckardt et al.,

977; Tenhunen et al., 1987; Tretiach, 1993; Castell et al., 1994;amesin and Rambal, 1995; Gratani, 1995; García-Plazaola et al.,997; Penuelas and Llusía, 1999; Haase et al., 2000; Méthy et al.,000; Flexas et al., 2001; Ogaya and Penuelas, 2003; Gratani andarone, 2004; Gulías et al., 2009). Therefore, chilling temperaturesuring winter and hot/dry summer (i.e. drought) are the most lim-

ting factors for photosynthesis under Mediterranean conditions.ndeed, the distribution of Mediterranean species along a latitu-

inal gradient depends on the species-specific adaptation to thesenvironmental constrains (Mitrakos, 1980). The extent to which thehotosynthesis rate of an individual species is depressed in win-er/summer may depend on both species-specific adaptations and

at the colder site Montpellier during winter, while thick arrows indicate the photo-synthetic advantage of plants growing at Binifaldó over those growing at the driersite Puigpunyent during summer. Modified after Flexas et al. (2003).

climatic conditions of individual Mediterranean sites. This is pre-sented in Fig. 1 by comparing published data of annual trends ofdaily photosynthesis rates in Quercus ilex growing at three differentlocations: 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), andPuigpunyent (Mallorca, Spain, AYP 450 mm, MMT 12 ◦C). Minimumphotosynthesis was observed in winter at the coldest site (Mont-pellier) and in summer at the hottest and driest site (Puigpunyent).Photosynthesis was much higher in Binifaldó than at Montpellierduring winter periods (indicated by thin arrows), and substantiallyhigher than in Puigpunyent during summer (indicated by thickarrows), which is characterized by mild temperatures and relativelyhigh yearly precipitation. Year-round maximum net photosyn-thesis averaged 5.5, 9.5 and 8.0 �mol CO2 m−2 s−1 at Montpellier,Binifaldó, and Puigpunyent, respectively. Using these rates as roughproxies of year carbon balance (at the leaf level) and followingMitrakos (1980), Q. ilex should be preferentially distributed at Bini-faldó, where it indeed dominates the arboreal ecosystem coverage,and less at cooler sites like Montpellier (where it is indeed less dom-inant in mixed stands with deciduous Quercus species) and at thedrier site like Puigpunyement (where there are indeed only fewtrees within a shrub macchia stand). Similar findings have beenreported by Corcuera et al. (2005) who concluded that Q. ilex ismore sensitive to winter than to summer stress.

Besides different magnitudes in the depression of photosyn-thesis during winter or summer the mechanistic causes for suchdepressions may differ between winter and summer and will bediscussed in the next sections. It is out of the aim of this review toelucidate the responses of photosynthesis in Mediterranean plantsto other stresses such as nutrient availability (Daas-Ghrib et al.,2011), ozone (Mereu et al., 2011; Velikova et al., 2005; Lombardozziet 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 con-

trolled conditions, most Mediterranean plants show an optimum Tfor photosynthesis in the range 15–30 ◦C, the most common being25–30 ◦C (Oechel et al., 1980; Larcher, 2000; Ogaya and Penuelas,2003). However, under Mediterranean field conditions low T are

14 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 fort e that although theoretically these plants have a temperature optimum around 25–30 ◦C,a ey display maximum photosynthesis at much lower temperatures, since when highert ance and water stress.

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Table 1Chilling sensitivity of different Mediterranean plants. The values of net photosyn-thesis were averaged for all data available in months having temperature minimabelow 5 ◦C, and expressed as percentage of the maximum measured photosynthe-sis 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 38–81Ceratonia siliqua 67Cistus albidus 91Cistus incanus 27Cistus monspeliensis 54–58Cistus salvifolius 62Cneorum tricoccon 45Erica arborea 51Erica multiflora 27Hypericum balearicum 58Olea europaea 47–61Phyllirea latifolia 42–62Pistacia lentiscus 46–77Quercus coccifera 81Quercus ilex 39–99Quercus suber 52Rhamnus alaternus 78

hree years and four locations in Mallorca, Spain (data from Gulías et al., 2009). Notics shown in pot experiments with irrigated plants; in practice (i.e. in the field) themperatures occur these are accompanied by additional stresses like excess irradi

sually accompanied by moderate sunlight irradiance coupled withdequate water availability whereas high T are associated withxcess sunlight irradiance and water deficit. As a consequence, theffects of T on photosynthesis may be confounded by the concomi-ant action of other environmental constraints (Gratani and Varone,004; Gulías et al., 2009). Therefore, maximum photosynthesisates in Mediterranean plants occur in ambient T range 10–20 ◦Cnder field conditions (Fig. 2), simply because when more optimumemperatures occur these are accompanied by additional stresseseducing photosynthesis.

Regarding to chilling sensitivity Mediterranean species showarge differences. For instance, Q. ilex is more chilling-resistant thanlea europaea, which in turn is more resistant than Ceratonia sili-ua (Mitrakos, 1980; Larcher, 1981). Photosynthesis may reflectpecies-specific differences in chilling sensitivity. Table 1 shows

list of Mediterranean species in which photosynthesis has beennalyzed in winter at ambient T < 5 ◦C as compared to maximumspring or autumn) rates. Decreases of net photosynthesis in win-er (as % of yearly maximum rates) range from 40% in some specieso ca. 100% in others, reflecting inter-specific differences. Cistuslbidus, Quercus coccifera or Rhamnus alaternus appear to be morehilling-resistant (less than 30% inhibition) than Cistus monspelien-is, Cneorum tricoccon, O. europaea, Phyllirea latifolia or Quercus suberphotosynthesis depressed by more than 50%).

These differences may result from the original provenance ofhe species when the Mediterranean climate originated (Mitrakos,980). On the other hand, these differences could also be as aesult of specific environmental conditions (the selected criterionf T < 5 ◦C is rough), or even to intra-specific differences. For exam-

le, photosynthesis in Arbutus unedo and Q. ilex varies between 39%nd 99% of its maximum rates. In Quercus the largest reduction wasbserved in North Italy (Larcher, 2000), while the minimum reduc-ion was recorded in Mallorca (Gulías et al., 2009). Intermediate

Rhamnus ludovici-salvatoris 60Rosmarinus officinalis 27

depressions were found at South France (44%) and Nord-East Spainand Rome (59–64%). In this case, the extent of winter inhibitionfollows a latitudinal gradient, mostly due to the prevailing effect ofclimate at each site.

Irrespective of the magnitude of the inhibition, potential mech-

anisms responsible for photosynthesis depression during winterhave been studied in Mediterranean plants. Non-stomatal factorsare mostly responsible for photosynthesis inhibition under low T.Net CO2 assimilation (AN) is indeed much more depressed under

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hese conditions than stomatal conductance (gs). In the evergreenclerophyll Pistacia lentiscus winter depression of photosynthe-is was accompanied by decreased AN/gs, whereas during earlyummer drought AN/gs increased despite similar depression of pho-osynthesis (Flexas et al., 2001). Presently, there is not sufficientata to assess the relative significance of the different mecha-isms (photochemistry, photosynthetic and Calvin-cycle enzymesr mesophyll conductance to CO2) responsible for photosynthesisepression.

Leaf photochemistry has been shown to be significantly affectedy winter chilling temperatures in many Mediterranean speciesGarcía-Plazaola et al., 1997; Karavatas and Manetas, 1999; Méthyt al., 2000; Oliveira and Penuelas, 2000; Flexas et al., 2001).ark-adapted maximum photochemical efficiency of PSII (Fv/Fm)

s generally depressed during winter in the evergreen sclerophylls. unedo, Arbutus andrachne, Juniperus phoenicea, Nerium olean-er, P. latifolia, Pinus halepensis, P. lentiscus, Q. coccifera and Q. ilexGarcía-Plazaola et al., 1999; Karavatas and Manetas, 1999; Méthyt al., 2000; Oliveira and Penuelas, 2000, 2005; Flexas et al., 2001;aquedano and Castillo, 2007). By contrast, Fv/Fm seems moreesistant to winter temperatures in the semi-deciduous species C.lbidus, Cistus creticus, Cistus salvifolius, Genista acanthoclada, Hal-mium halimifolium, Phlomis fruticosa and Sarcopterium spinosumKaravatas and Manetas, 1999; Zunzunegui et al., 1999; Oliveira andenuelas, 2000, 2005). Oliveira and Penuelas (2000) have suggestedhat the high resistance of PSII photochemistry in semi-deciduouspecies is due to their steep leaf angle, thus reducing the absorp-ion of excessive photons, a matter still to be conclusively proven.onetheless, permanent photodamage has been recorded duringinter in the semi-deciduous Cistus incanus, a condition which can-ot be alleviated by increasing the biosynthesis of anthocyaninsZeliou et al., 2009).

Biochemical mechanism(s) responsible for winter depression inv/Fm and photochemistry have been suggested. García-Plazaolat al. (1997) showed that xanthophyll de-epoxidation in Q. suberas not maintained at pre-dawn during winter, although it wasaintained during summer drought. In contrast, sustained de-

poxidation was maintained at pre-dawn during winter in Q. ilexGarcía-Plazaola et al., 1999). These results support the hypoth-sis of photodamage being responsible for reduced Fv/Fm duringinter in Q. suber, whereas sustained thermal dissipation of excess

nergy could be the cause of reductions in Fv/Fm in Q. ilex. Further-ore, winter depression in PSII photochemistry observed in several

vergreen species was associated with increases in the concentra-ion of alpha-tocopherol (chloroplast antioxidant), as well as in theanthophyll de-epoxidation and de-epoxidation retention at nightGarcía-Plazaola et al., 2003; Muller et al., 2006).

Although leaf photochemistry (i.e. the thylakoid electron trans-ort rate) is depressed in many Mediterranean plants underhilling, Flexas et al. (2001) observed that during both winter andummer leaf photochemistry was less affected than CO2 assimila-ion in P. lentiscus. These results suggest increased photorespirationnd/or electron transport to other non-carboxylating processes,ogether with photochemistry as responsible for the reductions inN during winter.

.3. Photosynthesis limitations in summer

In summer, high sunlight irradiance is coupled with high both and atmosphere vapor pressure deficits (VPD). These factors,ogether with the absence of precipitation, result in a droughteriod, the severity of which differs among sites, lasting from one to

ix months (Ehleringer and Mooney, 1983). Therefore, a plant’s per-ormance is severely constrained by the combined effect of watereficit and high atmospheric water demand. Plant tissues may suf-er from dehydration, in turn causing embolism and plant death

imental Botany 103 (2014) 12–23 15

(Vilagrosa et al., 2003, 2010; McDowell et al., 2008; Miranda et al.,2010).

Different sensitivity of photosynthesis to summer drought areexpected among Mediterranean species. In some field studies,differences among species were described for their maximum pho-tosynthesis rates during summer (Tretiach, 1993; Gratani, 1995;Gulías et al., 2009). These differences, however, do not necessar-ily reflect differences in the photosynthetic sensitivity to drought,as the different species may display different root systems and/orosmotic adjustment, heavily affecting tissue dehydration.

In order to specifically assess the photosynthetic sensitivity todrought, a correlation between photosynthesis rates and tissuewater relations (e.g. leaf water potential (LWP) or relative watercontent (RWC)) is needed. Table 2 shows a rough classificationof the ‘photosynthetic drought tolerance’ for some Mediterraneanspecies based on the criterion of the minimum measured leaf waterpotential 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 resistantgroup which, together with their chilling tolerance (see previoussection), convert them into the most tolerant group to the majorenvironmental constraints of the Mediterranean climate, as pre-viously suggested by Margaris (1981) and Suc (1984). Evergreensclerophylls follow malacophylls as the most drought resistant – ingeneral angiosperms being more resistant than conifers – whereaswinter deciduous species are the least resistant. Secondly, droughtresistance is more likely to be related with species phylogeneticcloseness than to life form or leaf types, as species belonging tothe same genus behave similarly to each other regardless of beingdeciduous or evergreen (as illustrated in Table 2 by Quercus andPistacia species). Additionally, intra-specific differences in the pho-tosynthetic sensitivity to drought have been detected in plants fromsingle species but with contrasting populations, when grown incommon 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 plantsavoid or delay tissue dehydration. Stomatal closure leads to reducedphotosynthesis, thus further compromising the plant’s carbon bal-ance (McDowell, 2011). Stomatal closure is an early response ofMediterranean plants to water stress (Martínez-Ferri et al., 2000;Gulías et al., 2002; Mediavilla and Escudero, 2003b, 2004; Gallé andFeller, 2007; Gallé et al., 2007), and it has been usually considered asthe prominent factor limiting photosynthesis under drought stress.Several Mediterranean species have their stomata inside epider-mal crypts, which allow a fine regulation of stomatal aperture ina micro-environment dampening frequent changes in VPD (Roth-Nebelsick et al., 2013) and, at the same time, shorten the mesophyllpathway for CO2 below the actual leaf thickness, thus allowing rel-atively high photosynthesis rates in species with large leaf massarea (LMA, Hassiotou et al., 2009a,b, 2010). The significance ofstomatal limitations to photosynthesis is well known, althoughnon-stomatal limitations have long been reported to be equallyimportant in reducing photosynthesis (Tenhunen et al., 1984, 1985,1987).

In semi-deciduous malacophylls, drought-induced impairmentof photochemistry is avoided by decreases in chlorophyll content inPhlomis sp. (Kyparissis et al., 1995), variations in leaf angles in Cis-tus sp. (Werner et al., 1999, 2001) or increasing leaf pubescencein some Digitalis sp. (Galmés et al., 2007a). These adjustmentsreduce the amount of light absorbed by the leaves during the unfa-vorable summer periods. Even in evergreen sclerophyll species,

which generally do not possess effective light-avoidance mech-anisms (with few exceptions, e.g. the pubescence exhibited byQ. ilex subsp. ballota, Morales et al., 2002), drought-induced con-strains on leaf photochemistry prevail in seedlings and only for few

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

Table 2Water stress tolerance of photosynthesis in different Mediterranean plants. The species were ordered based on the minimumwater 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 underlinedtext evergreen species (black underlining for angiosperms and gray underlining for gymnosperms, i.e. conifers).

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pecies and some conditions of light irradiance (Valladares et al.,005, 2008; Galmés et al., 2007b; Peguero-Pina et al., 2009; Petsasnd Grammatikopoulos, 2009). Overall, Mediterranean plants areell equipped with photoprotection mechanisms, which are fur-

her enhanced during summer: these include xanthophyll cycleigments (Munné-Bosch et al., 2003; Penuelas et al., 2004; Galmést al., 2007c; Peguero-Pina et al., 2008) and an integrated networkf antioxidant defenses (Munné-Bosch et al., 2003; Penuelas et al.,004; Munné-Bosch and Lalueza, 2007).

Similarly to leaf photochemistry, the biochemical capacity ofhotosynthesis estimated either in vivo – Vc,max – or in vitro –ubisco activity – is significantly constrained in Mediterraneanlants under very severe drought. In contrast, the mesophyll con-uctance to CO2 (gm) decreases at mild to moderate droughtGalmés et al., 2007d, 2011; Limousin et al., 2009, 2010; Fleckt al., 2010; Misson et al., 2010), although a few experiments haveeen reported showing that gm could be more resistant to drought,ot declining even under moderate to severe stress (Warren et al.,011).

A quantitative estimation of the relative significance of stoma-al conductance (gs), mesophyll (gm) conductances to CO2 and the

aximum capacity for carboxylation (Vc,max) in limiting net pho-osynthesis (AN) has been proposed by Grassi and Magnani (2005).n detail, AN and its limiting factors are measured under ‘control’i.e. non-stress) and stress conditions. The total photosynthesis lim-tation (TL) is then calculated as the reduction (%) from controlaused by the stressful conditions. Mechanistic causes of the TL areuantitatively assigned to stomatal (SL), mesophyll conductanceMCL) and biochemical (BL) limitations, the sum of SL, MCL andL equaling TL. For very different Mediterranean species (includingerbs, evergreen sclerophyll shrubs and trees, and semi-deciduousalacophyll shrubs) SL is the most important factor under mildater stress (i.e. early drought), SL and MCL co-limit photosyn-

hesis under moderate drought, while MCL is the most importantimitation (followed by BL) under severe drought (Galmés et al.,007d; Limousin et al., 2009, 2010; Misson et al., 2010; StPault al., 2012). Decreased mesophyll conductance is additionallynvolved in midday-depression of photosynthesis that occurs in

editerranean plants (Grassi et al., 2009; Bickford et al., 2010). Theignificance of drought-induced reduction of gm as a limiting factoro photosynthesis in Mediterranean plants is very relevant. Accu-ate carbon and water fluxes cannot be modeled in Mediterranean

cosystems unless accurate measurements of gm are included inhe models (Reichstein et al., 2002; Keenan et al., 2010a,b).

Mechanistic responses of photosynthesis to drought are gen-rally analyzed at the level of representative leaves (i.e. fully

developed, sun-exposed). Consequently, it is difficult to scale upto canopy level, mostly due to factors that affect the photosyn-thetic performance of leaves located at different positions in theplant. Among these factors, light availability is the most impor-tant, as drought-induced photosynthetic limitations are maximalat high light (Niinemets et al., 2006; Aranda et al., 2007). Interac-tions between drought and light availability are also evident alongthe canopy (Niinemets et al., 2004, 2006; Cano et al., 2013). Innon-stressed Quercus petraea and Fagus sylvatica, the highest ANat the top of the canopy is associated with higher gs and Vc,max,despite ‘top leaves’ display greater LMA and lower gm than leaveslocated deeper in the canopy (Cano et al., 2013). As water stressincreases, photosynthesis is mostly limited by SL in leaves at theupper canopy, while SL, MCL and BL co-limit photosynthesis in themost basal leaves (Cano et al., 2013).

Leaves, as well as plants of different age, may also respond dif-ferently to drought stress (Gratani and Ghia, 2002; Escudero andMediavilla, 2003; Mediavilla and Escudero, 2003a, 2004; Juarez-Lopez et al., 2008; Rodríguez-Calcerrada et al., 2012; Varone et al.,2012). The effects of plant age are also species-specific. In theevergreen Q. ilex and the deciduous Q. faginea, SL increased withplant age. In contrast, the significance of stomatal limitation wasgreat in young plants, whereas non-stomatal limitations prevailedin old plants in three evergreen species (Varone et al., 2012).These responses conform to (1) mesophyll conductance to CO2 pro-gressively decreasing with plant age in several evergreen species(Niinemets et al., 2005). (2) In old plants, leaves suffer from greateroxidative stress than those in young plants, when exposed toadverse climatic conditions (Munné-Bosch and Lalueza, 2007).

Other factors influencing the specific responses to droughtinclude plant’s acclimation to different altitudes (Cabrera, 2002)as well as to consecutive drought and re-watering cycles (Galléet al., 2011). In the evergreen sclerophyll Q. ilex, the photosyntheticlimitations were similar during three consecutive drought and re-watering cycles. In contrast, some acclimation was observed in thesemi-deciduous malacophyll C. albidus after the first drought cycle,consisting of a smaller decrease of gm than gs in the second andthird cycles (i.e. higher SL and lower MCL), which in turn resultedin much higher photosynthetic water use efficiency in Cistus thanin Quercus (Gallé et al., 2011).

Finally, long-term experiments combining rain exclusion withincreased temperature to simulate the likely effects of cli-

mate change, suggest that high temperature may exacerbate theobserved photosynthetic limitations imposed by drought, whichmay also be species-specific. For instance, the evergreen speciesC. siliqua (Osorio et al., 2011) and the malacophyll Tuberaria

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ajor (Osorio and Romano, 2013) showed a larger photosynthe-is decrease under the combination of both stresses. This wasue to the combination of increased transpiration rates leading toecreased water potential, and to increased oxidative stress. Sim-

larly, larger decreases of Fv/Fm under the effect of both stressesere observed in the evergreens Q. ilex and Phillyrea latifolia (Ogaya

t al., 2011) as well as in Erica multiflora (Prieto et al., 2009). Theatter also showing larger oxidative stress under the combinationf both stresses (Nogués et al., 2012). Contrarily, other evergreenpecies like Globularia alypum and P. halepensis appear to be moreesistant to the combination of high temperature and drought con-itions (Prieto et al., 2009). It is noteworthy that invasive speciesppear to be far more resistant to combination of high temperaturend drought than Mediterranean native species, for which differenthotosynthesis acclimation to climate change which could result

n substantial changes in species distribution in the future (Godoyt al., 2011).

. Photosynthesis limitations under non-stress conditions:s the photosynthetic capacity of Mediterranean plantsmaller than in plants from other biomes?

Analysis of a literature dataset including both Mediterraneannd non-Mediterranean was performed in plants where AN, gs andm had been measured under no-stress conditions (see Supplemen-al Table) using an updated published database (Flexas et al., 2013).

henever maximum capacity for carboxylation (Vc,max) was notrovided in the original reference, it was estimated as follows: (1)ay respiration rate (RD) was either computed from the original ref-rence or estimated using the empirical equation relating AN andD provided by Galmés et al. (2007e). This relationship yielded veryimilar results to that provided by Gratani et al. (2008) for a dif-erent set of species (not shown). (2) Vc,max was estimated using

single-point method based on chloroplast CO2 concentrationsescribed by Grassi and Magnani (2005). Once all required param-ters were estimated, each entry was classified in terms of life formnd leaf type, according to the following categories: (1) annualerbaceous monocotyledons, (2) annual herbaceous dicotyledons,3) perennial herbaceous monocotyledons, (4) perennial herba-eous dicotyledons, (5) conifers, (6) evergreen angiosperms, (7)emi-deciduous woody perennial angiosperms and (8) deciduousoody perennial angiosperms. These groups were further reduced

o four different leaf types: (a) herbaceous, (b) woody mesophytes,c) woody sclerophytes and (d) woody malacophytes. The lastwo categories are much related to the Mediterranean vegeta-ion. Sclerophytes leaves are characterized by thick leaves with

dense cuticle, thick cell walls, high stomata density and abun-ant sclerenchyma. The main criteria followed to label a speciess sclerophyte or mesophyte was that the former are evergreennd present a leaf mass per area (LMA) above 120 g m−2. When inoubt, species distribution and its recognition in previous literatures sclerophyte were considered (Wickens, 1998).

The obtained dataset allowed the comparison between Mediter-anean and non-Mediterranean species with respect to AN, orifferences in their inherent mechanistic determinants of AN,

.e. stomatal (gs) and mesophyll (gm) conductances to CO2 andhe maximum capacity for carboxylation (Vc,max). Average val-es of AN (Fig. 3) for the above described eight different lifeorms using the described dataset are similar to those previ-usly reported for the same life forms using larger datasetsEhleringer and Mooney, 1983; Gulías et al., 2003; Galmés et al.,

012). Herbaceous species show the largest average AN, fol-

owed by the woody semi-deciduous species, while evergreenpecies have the lowest – conifers showing somewhat smaller ANhan evergreen angiosperms (Fig. 3A). In general, Mediterranean

imental Botany 103 (2014) 12–23 17

and non-Mediterranean species show similar average AN withineach life form, except for perennial herb dicots and semi-deciduous woody perennial angiosperms, for which Mediterraneanplants show larger average AN. This is not consistent with morerecently published data compilations for plants from Mediter-ranean (Galmés et al., 2012) and temperate (Warren et al., 2012)regions. From that comparison, the average AN was indeed some-what higher in Mediterranean than in non-Mediterranean speciesfor 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 thatit includes information of gm and Vc,max. In general, Mediter-ranean plants of several life forms tend to have larger Vc,max,while differences in the two diffusive parameters were more vari-able. On the other hand, the present dataset has the handicap ofits limited data to the extent that for some of the groups (e.g.Mediterranean conifers or non-Mediterranean semi-deciduouswoody angiosperms) there is only a single measurement or speciesavailable (notice the absence of standard error for these groupsin Fig. 3). In order to strengthen the significance of differencesbetween groups, species have been re-grouped considering theirleaf type rather than their life form. Consequently, the numberof categories decreased from eight to four, with more data avail-able for each category (Fig. 4). Again, it can be observed thatherbaceous and malacophyll species show the largest averageAN, while sclerophyll species present the lowest. In all groupsAN there was a significant difference between Mediterranean andnon-Mediterranean species, although Mediterranean malacophyllsshowed much larger AN than non-Mediterraneans but with largeintra-group variability. However, there were significant differencesin some of the parameters influencing AN in both herbaceous andsclerophylls. In the two groups, gm was lower and Vc,max higher inMediterranean plants while only in evergreen sclerophylls gs wasalso lower in Mediterranean species (Fig. 4).

Following Grassi and Magnani (2005), the ‘theoretical’ maxi-mum photosynthetic performance for any given life form or leaftype, the total (TL) and its components SL, MCL and BL were esti-mated from the maximum values of AN, gs, gm and Vc,max, i.e. the‘theoretical’ maximum photosynthetic performance for any givenlife form or leaf type. Thus, the reference value was taken the sin-gle species displaying the maximum values for each parameterwithin each leaf type and TL, SL, MCL and BL from the compari-son of the rest of the species. The selected reference species wereTriticum aestivum (Tazoe et al., 2009), Banksia integrifolia (Hassiotouet al., 2009a,b), Prunus persica (Syvertsen et al., 1995) and Lavat-era maritima (Galmés et al., 2007c) for herbaceous, sclerophytes,mesophytes and malocophytes, respectively. In malacophytes, TLwas higher in non-Mediterranean species caused exclusively byits higher MCL (Fig. 5). For the other three groups, the differ-ences between Mediterranean and non-Mediterranean species inTL were small, although significant in some cases. However, themost important differences were found in their partial limitationswith SL and MCL tending to be larger and BL smaller in Mediter-ranean species (Fig. 5).

Therefore, it can be concluded that mesophyll conductanceto CO2 plays a major role in setting the photosynthetic capacityof Mediterranean plants and setting the differences with non-Mediterranean species (Figs. 4 and 5). It is worth noting that thesemi-deciduous malacophyll species, the most specifically adaptedplants to Mediterranean conditions, present higher photosyntheticcapacity than non-Mediterranean because of its larger gm. Onthe other hand, other Mediterranean species presenting lower gm

achieve similar AN than non-Mediterranean species by increasingits Vc,max (Fig. 4). Higher values of gm observed were only observedin Mediterranean malacophylls and its reasoning is unknown anddeserves further studies. Lower gm in the other groups appears to be

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

F diterra al coni ean an

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ig. 3. Comparison of the main driving variables of leaf CO2 assimilation rate in Messimilation rate (AN); (B) maximum RuBP carboxylation velocity (Vcmax); (C) stomatndicates a significant difference with P < 0.1 (*) or P < 0.05 (**) between Mediterran

elated to the significantly larger leaf mass area (LMA) observed inediterranean species (Fig. 6). Large LMA is associated to high leaf

hickness and density, which appears to be an adaption to stressful

nvironments like those in the Mediterranean climate (Niinemets,999). The fact that gm is constrained by large LMA has previouslyeen reviewed (Flexas et al., 2008), being recently established that

ts underlying reason is mostly related to the thicker cell walls

ig. 4. Comparison of the main driving variables of leaf CO2 assimilation rate in Mediterret CO2 assimilation rate (AN); (B) maximum RuBP carboxylation velocity (Vcmax); (C) stomsterisks indicates a significant difference with P < 0.01 (*) or P < 0.05 (**) between Medite

anean and non-Mediterranean species with eight different leaf forms. (A) Net CO2

ductance to CO2 (gsc); (D) mesophyll conductance to CO2 (gm). Means ± SE. Asterisksd non-Mediterranean leaves within each functional type.

observed in species with high LMA, significantly limiting CO2 diffu-sion 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 perarea among Mediterranean species, which could be induced by anincreased Rubisco content per area caused by the overall increasingin leaf volume.

anean and non-Mediterranean species with four different leaf functional types. (A)atal conductance to CO2 (gsc); (D) mesophyll conductance to CO2 (gm). Means ± SE.rranean and non-Mediterranean leaves within each functional type.

J. Flexas et al. / Environmental and Experimental Botany 103 (2014) 12–23 19

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 specieswithin 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) forh ersica

f 01 (*)f

3

Vdefpenp

FrA

erbaceous, Banksia integrifolia (Hassiotou et al., 2009a,b) for sclerophytes, Prunus por malocophytes. Means ± SE. Asterisks indicates a significant difference with P < 0.unctional type.

. Intrinsic photosynthetic water use efficiency

It has recently been highlighted that differences in gs, gm andc,max – and, specifically, differences in the gm/gs ratio – induceifferences in AN/gs, i.e. the photosynthetic intrinsic water usefficiency or WUEi (Flexas et al., 2013). Hence, the observed dif-erences in gm between Mediterranean and non-Mediterranean

lants would be expected to result in differences in WUEi. How-ver, this increase in higher WUEi between Mediterranean andon-Mediterranean species is only observed in evergreen sclero-hylls (Fig. 6). This is due to the unique feature displayed by this

ig. 6. (A) Average leaf mass per area (LMA), (B) intrinsic-water-use efficiency (AN/gs), (atio mesophyll conductance to CO2 to stomatal conductance to CO2 (gm/gs) of the four lesterisks indicates a significant difference with P < 0.1 (*) or P < 0.05 (**) between Mediter

(Syvertsen et al., 1995) for mesophytes, and Lavatera maritima (Galmés et al., 2007) or P < 0.05 (**) between Mediterranean and non-Mediterranean leaves within each

group in having both larger carboxylation efficiency (AN/Cc) andlarger diffusion efficiency (gm/gs). A similar situation is observedfor malacophylls, although larger intra-group variability results indifferences being statistically significant only for gm/gs (Fig. 6). Ina previous survey in Mediterranean species, evergreens alreadyshowed larger WUEi than semi-deciduous under non-stress con-ditions, although the latter took advantage under severe water

stress (Medrano et al., 2009). In mesophytes none of theseparameters differed significantly between Mediterranean and non-Mediterranean plants, while in Mediterranean herbs their higherAN/Cc is compensated by a lower (gm/gs). We propose that leaf

C) ratio CO2 assimilation rate to chloroplastic CO2 at ambient CO2 (AN/Cc), and (D)af functional types for Mediterranean and non-Mediterranean species. Means ± SE.ranean and non-Mediterranean leaves within each functional type.

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

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 herbaceousspecies: Triticum aestivum, Triticum durum, Hordeum vulgare and Solanum lycopersicum var. Ramallet. Non-Mediterranean herbaceous species: Phaseolus vulgaris, Nicotianat m anne an wod anean

tidi

4

sMbrlwPHcntTfcmncse

5

prdMe

abacum, Vicia faba, Oryza sativa, Lycopersicum esculentum, Spinacia oleracea, Capsicuuropea, Vitis vinífera, Citrus limon, Prunus persica and Prunus dulcis. Non-Mediterraneifference with P < 0.1 (*) or P < 0.05 (**) between Mediterranean and non-Mediterr

rait adjustments for increased WUEi may be an adaptive strategyn Mediterranean species, in particular in evergreen and semi-eciduous, which sustain green leaves during summer, when there

s less water and its efficient use more important.

. Photosynthesis limitation in Mediterranean crops

All previous sections were focused on native Mediterraneanpecies. Regarding crops, only those that can be consideredediterranean were being considered. This consideration was

ased on the time length that they have been cultivated in Mediter-anean areas for and their typical presence in the Mediterraneanandscape. Among them, the species considered were: (a) the

oody crops Olea europea, Vitis vinífera, Citrus limon, P. persica andrunus dulcis, and (b) the herbaceous T. aestivum, Triticum durum,ordeum vulgare and Solanum lycopersicum var. Ramallet. Whenompared to non-Mediterranean crops, Mediterranean crops showot only similar average values of AN, but also – and contrarilyo native vegetation – similar values for gs, gm and Vc,max (Fig. 7).he absence of differences is likely to reflect the different selectionorces that have operated during the evolution between native androp species. While native plants may have been forced to opti-ize survival and reproduction under conditions of soils poor in

utrients and climatic stresses (cold winter and summer droughts),rops are bred for maximizing production, and its cultivation oftenupposes their settlement in richer soils (or supplied with fertiliz-rs) while alleviating stress conditions (for instance by irrigation).

. Concluding remarks

The general use of portable infrared gas analysers and chloro-hyll fluorimeters has allowed for a large accumulation of data

egarding photosynthesis in Mediterranean plants in the last twoecades. A compilation and review of these bulk information onediterranean species – including herbaceous species, woody

vergreen sclerophytes, evergreen and deciduous mesophytes and

uum, Helianthus annuum and Brassica carinata. Mediterranean woody species: Oleaody species: Citrus paradise, Macadamia integrifolia. Asterisks indicates a significant

leaves within each functional type.

semi-deciduous melacophytes – provides a general picture ofthe responses of photosynthesis to environmental stresses andgives new clues on how it copes with cold winters and summerdroughts, the two most constraining environmental conditionsunder Mediterranean climate. In addition, comparative analy-sis of the photosynthetic capacity and its components combinedwith a quantitative limitation analysis between Mediterraneanand non-Mediterranean species permits the observation of specificmechanisms limiting photosynthesis.

Contrary to common assumptions, it can be concluded thatMediterranean plants present photosynthetic capacities similarto those of other biomes. With the exception of semi-deciduousmalacophytes – which are optimally adapted to the Mediter-ranean climate and which present even higher photosynthesisthan their non-Mediterranean counterparts – plants with all otherleaf types show average photosynthesis similar to those of otherbiomes. However, a reduced mesophyll conductance to CO2 (gm)is observed in herbaceous and evergreen sclerophyll Mediter-ranean 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 photosynthesisrates. In Mediterranean evergreen sclerophylls, stomatal conduc-tance (gs) is also lower resulting in higher intrinsic photosyntheticwater-use-efficiency. Moreover, Mediterranean plants are able tosustain higher photosynthesis rates in spring and autumn and,at least in one of the two most limiting periods – winter orsummer – depending on the site and species. Consequently, andnot surprisingly, eddy-flux based measurements in Mediterraneanecosystems reveal that these plants are among those presenting thehighest net primary productivities worldwide (Allard et al., 2008).

Acknowledgments

This work was partly supported by the Plan Nacional, Spain, con-tracts 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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ibas-Carbo and an anonymous reviewer for grammar corrections,nd to two anonymous reviewers for their suggestions to improvehe manuscript.

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