Photoprotection processes under water stress and recovery in Mediterranean plants with different growth forms and leaf habits

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1 Physiologi Plntrum 13: Copyright ª Physiologi Plntrum 7, ISSN Photoprotetion proesses under wter stress nd reovery in Mediterrnen plnts with different growth forms nd lef hits Jeroni Glmés, *, Anuniión Adí, Josep Cifre, Hipólito Medrno nd Jume Flexs Grup de Reer en Biologi de les Plntes en Condiions Mediterrànies, Universitt de les Illes Blers, Crreter de Vlldemoss Km.7.5, 7122 Plm de Mllor, Spin Deprtment of Plnt Nutrition, Estión Experimentl de Aul Dei, CSIC, Zrgoz, Spin Correspondene *Corresponding uthor, e-mil: jeroni.glmes@ui.es Reeived 22 Deemer 6; revised 8 Mrh 7 doi: /j x The response of photoprotetion mehnisms to short-term wter stress period followed y rewtering, to simulte ommon episodi wter stress periods ourring in Mediterrnen res, ws studied in 1 potted plnts representtive of different growth forms nd lef hits. During wter stress nd reovery, reltive wter ontent, stomtl ondutne, lef pigment omposition, eletron trnsport rtes, mximum quntum effiieny of PSII photohemistry ( ), therml energy dissiption nd photorespirtion rtes (P r ) were determined. All the speies nlyzed proved to e strongly resistnt to photointivtion of PSII under the imposed wter stress onditions. The responses of the nlyzed prmeters did not differ lrgely mong speies, suggesting tht Mediterrnen plnts hve similr needs nd pity for photoprotetion under episodi wter stress periods regrdless of their growth form nd lef hit. A generl pttern of photoprotetion emerged, onsisting in mintenne or inrese of P r t mild stress nd the inrese of the therml energy dissiption t more severe stress. Adjustments in pigment pool sizes were not n importnt short-term response to wter stress. The inrese of therml energy dissiption euse of wter stress depended mostly on the deepoxidtion stte of xnthophylls, lthough the slope nd kinetis of suh reltionship strongly differed mong speies, suggesting speies-dependent dditionl roles of de-epoxidted xnthophylls. Also, smll dereses in t predwn during wter stress were strongly orrelted with mintined deepoxidtion of the xnthophylls yle, suggesting tht form of xnthophylldependent sustined photoprotetion ws developed during short-term wter stress not only in evergreen ut lso in semideiduous nd nnul speies. Arevitions A N, net CO 2 ssimiltion rte; DPS, de-epoxidtion stte; DPS MD, middy de-epoxidtion stte; DPS PD, predwn de-epoxidtion stte; ETR, eletron trnsport rte; F m, mximum fluoresene; F m #, stedy-stte mximum fluoresene yield; F o, kground fluoresene signl; Fs, stedy-stte fluoresene signl;, mximum quntum effiieny of PSII photohemistry; g s, stomtl ondutne; MiWS, mild wter stress; MoWS, moderte wter stress;, non-photohemil quenhing of hlorophyll fluoresene; P r, photorespirtion rte; R L, rte of non-photorespirtory CO 2 evolution in the light; RW, rewtering; RWC PD, reltive wter ontent t predwn;, severe wter stress; VAZ, sum of violxnthin, ntherxnthin nd zexnthin. Physiol. Plnt. 13, 7 495

2 Introdution Summer wter defiit is onsidered the min environmentl onstrint for plnt growth nd survivl in Mediterrnen type eosystems. In these environments, nturl vegettion hs developed n rry of dpttions to drought, resulting in high diversity of life hits nd growth forms. The resulting vegettion onsists mostly of deep-rooted evergreen slerophyll trees nd shrus whih mintin green leves during the summer drought period, semideiduous shrus whih lose prt of their leves during summer, nd geophytes nd winter nnul hers whih espe drought y finishing their nnul yle efore summer (Ehleringer nd Mooney 1982). Low soil wter vilility during summer is ompnied y high temperture nd exessive rdition, whih imposes multiple stress to plnts (Di Cstri 1973). The omintion of these stresses n led to photoinhiition nd photodmge of the photosyntheti pprtus, whih my result in deresed photosyntheti pity nd, eventully, in plnt deth (Chves et l. 2, Peñuels et l. 4). Beuse of this, nd tking into ount the lrge vriility of hitt mirolimtes in the Mediterrnen region, s well s the stohsti distriution of rinfll, it is likely tht Mediterrnen plnts my hve evolved lrge diversity of photoprotetive mehnisms to ope with exess light, prtiulrly during the summer drought period. Mny photoprotetive mehnisms hve een desried in higher plnts (Björkmn nd Demmig- Adms 1994, Niyogi 1999), inluding reduing light sorption through lef or hloroplst movements, deresed Chl ontents or refletive strutures suh s hirs; regultion of energy dissiption through photohemil (e.g. photorespirtion) nd non-photohemil (e.g. sfe therml dissiption of exess sored light energy, vi the xnthophyll yle) mehnisms; svenging of retive oxygen speies formed euse of exess light nd repir nd resynthesis of photodmged omponents of the photosyntheti pprtus (e.g. D1 protein). Mny of these mehnisms hve een desried in Mediterrnen plnts. For instne, steep lef ngles hve een shown s effiient struturl photoprotetive fetures in perennil grsses like Stip tenissim (Vlldres nd Pugnire 1999), semideiduous shrus like Cistus lidus nd evergreen slerophyll shrus like Arutus unedo (Werner et l. 1999, 1). Some semideiduous shrus present nother mehnism to redue light sorption during summer, onsisting of prtil lef loss in prllel to sustntil loss of Chl in the remining leves (Kyprissis et l. 1995, ; Munné-Bosh nd Alegre, ). In Phlomis frutios, Chl loss during summer is not ompnied y deresed photohemil pity, whih suggests it s photoprotetive feture (Kyprissis et l. 1995). In the tussok grss S. tenissim, whih inhits more rid environments thn Phlomis, sustntil loss of Chl is ompnied y lrge redution in photohemil pity nd mrked derese in lef wter ontent, ut leves totlly reover fter utumn rinflls. This hs een interpreted s poikilohydri-type response llowing for greter tolerne to wter shortge in the most extreme Mediterrnen environments (Blguer et l. 2). Redued light sorption through umultion of red rotenoids in lef surfes hs lso een reently desried s prtiulr photoprotetive mehnism of the evergreen shru Buxus sempervirens (Hormetxe et l. 5). Mehnisms leding to retive oxygen svenging nd ntioxidnt protetion hve lso een desried in Mediterrnen plnts, prtiulrly in evergreen nd semideiduous shrus. These mehnisms inlude rotenoids (Munné-Bosh nd Peñuels 3), isoprene (Affek nd Ykir 2), toopherols (Munné- Bosh nd Peñuels 4), diterpenes (Munné-Bosh et l. 1) nd enzymti ntioxidnts (Kyprissis et l. 1995). Besides these mehnisms, therml energy dissiption in the pigment ed, ssoited with the so-lled xnthophyll yle, is usully regrded s the most importnt photoprotetion mehnism in higher plnts (Demmig et l. 1987, Björkmn nd Demmig-Adms 1994). The first evidenes tht wter stress inresed deepoxidtion of the xnthophyll yle were in ft desried in the Mediterrnen evergreen slerophylls Nerium olender (Demmig et l. 1988) nd A. unedo (Demmig-Adms et l. 1989). Sine then, sustntil evidene hs een umulted for inresed deepoxidtion of the xnthophyll yle during summer in Mediterrnen evergreens (Gulís et l. 2, Peñuels et l. 4), semideiduous (Munné-Bosh et l. 3) nd perennil hers (Blguer et l. 2). An inrese in the totl xnthophyll pool during summer is lso usully oserved (Grí-Plzol et l. 1997, Fri et l. 1998), lthough not in ll the speies (Munné-Bosh et l. 3). However, most of these studies hve een foused on evergreen shrus nd trees nd semideiduous shrus, while muh less informtion is ville for semishrus or perennil hers. On the other hnd, most of these studies hve nlyzed the vrition of photoprotetive mehnisms during the seson. The short-term response (i.e. dys to weeks), whih my e most relevnt euse of the undne of episodi drought periods in Mediterrnen res, hs een less evluted, prtiulrly in reltion to reovery fter rewtering (RW). In the present study, we ssessed the reltionship etween the xnthophyll yle nd therml dissiption nd deresed 496 Physiol. Plnt. 13, 7

3 quntum effiieny of PSII during short-term wter stress nd reovery in Mediterrnen plnts with different lef hits nd growth forms. The ojetives were (1) to study how photoprotetion responds to wter stress in Mediterrnen plnts differing in growth forms, nd (2) to investigte the vriility in the reovery of photoprotetion nd photoinhiition fter RW. Mterils nd methods Plnt mteril Ten Mediterrnen speies nturlly ourring in the Bleri Islnds, five of them endemi to these islnds, were seleted for this study (Tle 1). Speil re ws tken in the seletion of the speies, in order to inlude txons representing different growth forms nd lef hits: two evergreen slerophyll shrus (Pisti lentisus nd Hyperium lerium), two evergreen slerophyll semishrus (Limonium giertii nd Limonium mgllufinum), three summer semideiduous shrus (Lvter mritim, Phlomis itli nd C. lidus), two perennil hers (Bet mritim ssp. mritim nd Bet mritim ssp. mrosii) nd n nnul her (Diplotxis iiensis). Seeds of eh speies were olleted in the field from nturl popultions nd tken from severl prent plnts to otin representtive smple of popultions in the nture. Seeds were germinted on filter pper moistened with deionized wter in ontrolled environment (germintion hmer, t 18 C in drkness). After germintion nd emergene of one true lef, 1 seedlings were trnsplnted into pots (25 l volume, m height) ontining :: mixture of ly-lreous soil, hortiulturl sustrte (pet) nd perlite (grnulometry A13). Plnts were grown outdoors t the University of the Bleri Islnds (Mllor, Spin). The experiment ws performed in five rounds, eh one with one ouple of speies t the sme time during the lte spring erly summer 3 nd 4. Four weeks efore strting the experiment, plnts were pled in ontrolled growth hmer with 12-h photoperiod (26 C dy/ C night) nd photon flux density t the top of the leves of out mmol m 22 s 21. Plnts were dily irrigted with 5% Hoglnd s solution. Mesurements orresponding to ontrol tretments were mde during the first dy of the experiment, when ll the plnts were well wtered. Therefter, irrigtion ws stopped in five plnts for eh speies. Pots were dily weighed to determine the mount of wter ville for plnts with respet to tht in ontrol plnts. Mesurements were mde on dys 4, 8 nd fter wter withholding, when plnts were sujeted to mild wter stress (MiWS), moderte wter stress (MoWS) nd severe wter stress () intensities, respetively. Eh experiment ws stopped when the stomtl ondutne (g s ) ws lose to zero. At this time, pots were irrigted t field pity, nd mesured the following dy, onsidering it the RW tretment. Control plnts (C) were wtered dily to field pity throughout the experiment nd mesured every 5 6 dys to ensure tht they mintined onstnt vlues of eh prmeter during the experiment. Plnt wter sttus Reltive wter ontent t predwn (RWC PD ) ws determined s follows: RWC ¼ (FW 2 DW)/(turgid weight 2 DW) 1. Turgid weight ws determined fter pling the smples in distilled wter in drkness t 4 C to minimize respirtion losses, until they rehed onstnt weight (full turgor, typilly fter 24 h). DW ws otined fter 48 h t C in n oven. Four replites per speies nd tretment were otined from different individuls. Chl fluoresene mesurements Chl fluoresene prmeters were mesured on tthed leves using portle pulse mplitude modultion fluorometer (PAM-, Wlz, Effeltrih, Germny). For eh smpling time, tretment nd speies, four mesurements were mde on different plnts. A mesuring light of out.5 mmol photon m 22 s 21 ws set t frequeny of Hz to determine, t predwn, the kground fluoresene signl (F o ), the mximum fluoresene (F m ) nd the mximum quntum effiieny of PSII photohemistry ( ¼ (F m 2 F o )/F m ). At middy, stedy-stte fluoresene signl (F s ) ws mesured on the sme leves with photon flux density round 15 mmol m 22 s 21. To otin the stedy-stte mximum fluoresene yield (F m #), sturtion pulses of out 1 mmol photon m 22 s 21 nd.8 s durtion were pplied. The Stern Volmer non-photohemil quenhing of Chl fluoresene () t middy ws lulted using the expression ¼ (F m 2 F m #)/F m #. The PSII photohemil effiieny (Genty et l. 1989) ws then lulted s DF=F m # ¼ F m # 2F s =Fm # nd used for the lultion of the liner eletron trnsport rte (ETR) ording to Krll nd Edwrds (1992): ETR ¼ DF=F m # PPFD ; where PPFD is the photosynthetilly tive photon flux density, is the lef sorptne nd is ftor tht Physiol. Plnt. 13, 7 497

4 Tle 1. List of speies onsidered with their growth form, fmily nd rief desription. The numer of plnts used ws 1 per speies, nd the ge differed euse of the different phenology of the speies seleted. Plnts of Pisti lentisus, Hyperium lerium, Cistus lidus, Phlomis itli nd Lvter mritim were 3 yers old, plnts of Limonium mgllufinum nd Limonium giertii were yers old nd plnts of Diplotxis iiensis, Bet mritim ssp. mrosii nd B. mritim ssp. mritim were 6 months old t the onset of the experiments. Growth form Speies Code Fmily Desription Hers B. mritim L. ssp. mrosii A Jun nd MB Crespo MC Chenopodiee Perennil her. Endemi of the Bleri Islnds, inhiting few smll islets sujeted to strong sline spry. B. mritim L. ssp. mritim MT Chenopodiee Perennil her inhiting ostl eosystems. Widespred in Mediterrnen nd temperte limtes. D. iiensis Pu DI Brssiee Annul her, endemi of the Bleri Islnds nd inhiting few ostl lotions. Semideiduous shrus L. mritim Goun LA Mlvee Semideiduous shru up to 2 m, densely overed y hirs. Inhits in ostl lotions. P. itli L. PI Lite Semideiduous shru up to 1 m, densely overed y hirs. Endemi of the Bleri Islnds. The iggest popultions re found 5 m ove the se level, where they oexist with C. lidus. C. lidus L. CA Cistee Semideiduous shru up to 1 m. Commonly found in the Mediterrnen grigue. Its leves re densely overed y hirs. Woody evergreen shrus Woody evergreen semishrus H. lerium L. HB Guttifere Woody evergreen shru up to 2 m, endemi of the Bleri Islnds. The iggest popultions re found in the grigue 5 m ove the se level, where ompetes with P. lentisus. P. lentisus L. PL Anrdiee Woody evergreen shru up to 5 m, ommonly found in the Mediterrnen grigue. L. mgllufinum L. Llorens LM Plumginee Woody evergreen semishru, in ushion-like rosettes. Endemi of the Bleri Islnds, inhiting just in one ostl mrsh loted in Mglluf, Mllor. L. giertii (Sennen) Sennen LG Plumginee Woody evergreen semishru, in ushion-like rosettes. Ourring in West Mediterrnen roky nd sndy ostl res. ssumes equl distriution of energy etween the two photosystems (the tul ftor hs een desried to e etween.4 nd.6; Lisk nd Loreto 1996). Lef sorptnes were determined for ll 1 speies in 1 replites on leves of well-irrigted plnts with spetrordiometer oupled to n integrtion sphere (Uni- Spe, PP-Systems, Amesury, MA). A vlue of.84 ws otined for ll speies, exept for C. lidus nd P. itli, whih presented lef sorptne vlues of.74 nd.77, respetively. Potentil hnges in lef sorptne with wter stress were not ssessed ut, euse hnges in Chl ontent were smll or nonsignifint depending on the speies, they were ssumed to e negligile, induing no importnt ises in the lultions of ETR. Gs exhnge mesurements Light-sturted net CO 2 ssimiltion rtes (A N ) nd g s were mesured t midmorning in one tthed, fully developed young lef of four plnts per speies nd tretment with gs exhnge system (Li-, Li-Cor In., Linoln, NE). Environmentl onditions in the hmer used for lef mesurements onsisted in photosyntheti photon flux density of 15 mmol m 22 s 21, vpor pressure defiit of kp, n ir temperture of 25 C nd n mient CO 2 onentrtion (C )ofmmol mol ir 21. After induing stedy-stte photosynthesis, four photosynthesis response urves to vrying sustomtl CO 2 onentrtion (C i ) were performed per speies nd tretment, nd used to determine the rte of 498 Physiol. Plnt. 13, 7

5 non-photorespirtory CO 2 evolution in the light (R L )on the sme tretment, s in Grssi nd Mgnni (5). Photorespirtion estimtions From omined gs exhnge nd Chl fluoresene mesurements, the photorespirtion rte (P r ) ws lulted ording to Vlentini et l. (1995). In their model, they ssumed tht ll the reduing power generted y the eletron trnsport hin is used for photosynthesis nd photorespirtion, nd tht Chl fluoresene gives relile estimte of the quntum yield of eletron trnsport. Thus, P r n e solved from dt of A N,R L nd ETR, nd from the known stoihiometries of eletron use in photorespirtion, s follows (Vlentini et l. 1995): P r ¼ 1/12 [ETR 2 4(A N 1 R L )]. Pigment nlyses Immeditely fter Chl fluoresene mesurements (t predwn nd middy), diss were punhed from leves of the sme plnts showing the sme orienttion s those used for fluoresene mesurements nd sumersed into liquid nitrogen. Four smples per tretment nd speies were tken from different plnts (four leves per smple). Pigments were extrted y grinding lef tissue in mortr with etone in the presene of sodium sorte. Pigments were identified nd quntified y high-performne liquid hromtogrphy ording to Adí nd Adí (1993) with modifitions s desried in Lri et l. (4). The de-epoxidtion stte (DPS) of the xnthophylls yle ws lulted s (Z 1.5A)/(V 1 Z 1 A), where Z is zexnthin, A is nterxnthin nd V is violxnthin. Sttistil nlysis Simple liner regression oeffiients were lulted using SPSS 1 softwre pkge (Anon 199). A set of simple ANOVA were mde to ompre the different speies nd tretments. Differenes etween mens were reveled y Dunn nlyses (P <.5) performed with the SPSS 1 softwre pkge. For eh tretment, luster nlysis nd prinipl omponent nlysis were performed using STATGRAPHICS PLUS 5.1 softwre pkge (Mnugistis 1998) in order to group oth the speies nd prmeters nlyzed in few more omprehensive vriles. Results Plnt wter sttus Lef RWC PD deresed s wter stress intensified (Tle 2). Under optiml onditions, RWC PD rnged from.2% for D. iiensis to 94.8% for P. lentisus. Under, RWC PD rnged from 37.9% for P. itli to 69.5% for L. mgllufinum. g s strongly differed mong speies nd growth forms, pproximtely in 1-fold rnge (Tle 2). Under well-wtered onditions, L. mritim showed the highest g s (22 mol H 2 O m 22 s 21 ), while P. lentisus hd the lowest (.122 mol H 2 Om 22 s 21 ). g s deresed in ll the speies to vlues etween nd.6 mol H 2 Om 22 s 21 s wter limittion inresed. Pigment omposition under wter stress nd reovery Under well-wtered onditions, lef Chl ontent expressed on n re sis t middy ws found to e higher in the two Limonium speies, with nd mmol m 22 for L. mgllufinum nd L. giertii, respetively (Fig. 1), while C. lidus presented the lowest vlues (281.1 mmol m 22 ). Inresing wter stress tretment influenes on Chl lrgely depended on the speies. In some speies (the two Bet, L. mritim nd C. lidus), Chl ws inresed under with respet to ontrol plnts (P <.5). In ddition to this diversity in the speies response to wter limittion, high vriility in the intensity nd the timing of the Chl evolution euse of wter stress ws lso found. For instne, the two Bet speies, whih inresed their Chl s wter stress intensified, presented different pttern: B. mritim ssp. mrosii inresed Chl t MoWS, while B. mritim ssp. mritim t. Twenty-four hours fter refilling pots t field pity, Chl evolution lso depended strongly on the speies (Fig. 1). Hene, four speies mintined similr Chl t RW tretment when ompred with, nd the remining six speies deresed Chl fter RW (P <.5). It is remrkle tht in B. mritim ssp. mritim nd P. itli, dereses of Chl fter RW resulted in vlues signifintly lower thn those mesured under well-wtered onditions (P <.5). The sum of violxnthin, ntherxnthin nd zexnthin (VAZ) per unit lef re t middy under wellwtered onditions rnged from 11.2 mmol m 22 for C. lidus to 5.3 mmol m 22 for L. mritim (Fig. 1). VAZ per unit lef re ws ffeted y wter stress only in C. lidus, eing signifintly inresed (P <.5). As ourred with Chl, refilling wter t field pity fter resulted in speifi pttern strongly dependent on speies. Under ontrol onditions, lutein ontent t middy expressed on Chl sis ws found to e more similr mong speies thn other pigments, rnging etween 94 nd 127 mmol mol 21 Chl. Lutein ontent on Chl sis Physiol. Plnt. 13, 7 499

6 Tle 2. RWC PD nd g s for the 1 seleted speies under different tretments: ontrol (C), MiWS, MoWS, nd RW. Vlues re mens SE of four replites per speies nd tretment. C MiWS MoWS RW Bet mritim ssp. mrosii RWC PD g s B. mritim ssp. mritim RWC PD g s Diplotxis iiensis RWC PD g s Lvter mritim RWC PD g s Phlomis itli RWC PD g s Cistus lidus RWC PD g s Hyperium lerium RWC PD g s Pisti lentisus RWC PD g s Limonium mgllufinum RWC PD g s Limonium giertii RWC PD g s ws unffeted or enhned y inresing wter stress intensity (Fig. 2). D. iiensis, P. itli, C. lidus nd L. giertii presented n inrese in lutein ontent under when ompred with well-wtered plnts. Other rotenoids suh s neoxnthin did not show ny speifi trend of response to wter stress (dt not shown). VAZ ontent expressed on Chl sis inresed under t middy only in C. lidus nd L. giertii (P <.5), while for the remining speies it ws found to e unffeted (Fig. 2). Agin, the intensity nd timing of the VAZ/Chl evolution euse of inresing wter stress were highly speies dependent. Fig. 3 shows the evolution of violxnthin, ntherxnthin nd zexnthin t middy throughout the wter stress experiment. Among well-wtered plnts, the woody evergreen shrus, with out 65% of VAZ pigments eing violxnthin, presented the lowest perentge of violxnthin with respet to totl VAZ pool. On the ontrry, in ll the remining speies violxnthin ounted for pproximtely 9% of totl VAZ pool in ontrol plnts, exept for B. mritim ssp. mrosii with n intermedite ehvior. In ll the speies, exept the two Limonium, violxnthin ontent deresed with deresing wter vilility (P <.5), with proportionl inrese of zexnthin (Fig. 3). Antherxnthin ws kept t lmost onstnt onentrtion through the entire experiment, nd signifint inreses euse of wter stress were only oserved in D. iiensis, L. mritim nd L. giertii (P <.5). After RW, ll the speies exept L. mgllufinum inresed violxnthin nd deresed zexnthin k to ontrol vlues. All these hnges in xnthophylls omposition indued similr trends in the DPS of xnthophylls. Both t predwn nd middy, DPS ws lrgely inresed in prllel with inresing wter stress intensity (dt not shown). However, lrge differenes were otined in DPS mong speies. For instne, under middy de-epoxydtion stte (DPS MD ) rnged from.1 in L. mgllufinum to more thn.6 in H. lerium nd B. mritim ssp. mrosii, 5 Physiol. Plnt. 13, 7

7 Chl + (µmol m 2 ) B. mritim ssp. mrosii B. mritim ssp. mritim VAZ (µmol m 2 ) Chl + (µmol m 2 ) D. iiensis L. mritim VAZ (µmol m 2 ) Chl + (µmol m 2 ) P. itli C. lidus VAZ (µmol m 2 ) Chl + (µmol m 2 ) Chl + (µmol m 2 ) H. lerium WES L. mgllufinum WESS P. lentisus WES L. giertii WESS VAZ (µmol m 2 ) VAZ (µmol m 2 ) C MiWS MoWS RW Tretment C MiWS MoWS RW Tretment Fig. 1. Totl Chl ontent (Chl 1, d) nd the sum of violxnthin, ntherxnthin nd zexnthin (VAZ, s), expressed in mmol m 22,t middy under different tretments: ontrol (C), MiWS, MoWS, nd RW. Vlues re mens SE of four replites per speies nd tretment. Different letters denote sttistil differenes y Dunn test (P <.5) mong tretments for eh prmeter. Growth form revitions:, hers;, semideiduous shrus; WES, woody evergreen shrus; WESS, woody evergreen semishrus. P. itli nd H. lerium. After RW, the extent of reovery of DPS MD ws highly speies dependent, from no reovery (D. iiensis) to totl reovery (L. giertii). Pigment omposition t predwn (not shown) followed pttern similr to tht oserved t middy. In Fig. 2. Lutein (d) nd the VAZ (s) t middy, expressed in mmol mol 21 Chl, t middy under different tretments: ontrol (C), MiWS, MoWS, nd RW. Vlues re mens SE of four replites per speies nd tretment. Different letters denote sttistil differenes y Dunn test (P <.5) mong tretments for eh prmeter. Growth form revitions:, hers;, semideiduous shrus; WES, woody evergreen shrus; WESS, woody evergreen semishrus. ft, orreltions mong vlues mesured t middy nd predwn were highly signifint (P <.1) for ll the pigments onsidered in the study. As expeted, for ll the speies, VAZ pool ws lwys in highly epoxidted stte t predwn for most tretments. This ws euse of lower onentrtion of zexnthin t expenses of n inrese in the violxnthin ontent, while the nterxnthin ontent did not hnge Physiol. Plnt. 13, 7 51

8 B. mritim ssp. mrosii C D. iiensis H. lerium WES L. mgllufinum WESS MiWS MoWS P. itli Tretment d RW B. mritim ssp. mritim L. mritim Tretment signifintly. Finlly, vritions of Chl nd VAZ onentrtions from predwn to middy, when ourred, were speies speifi nd no-generl pttern ws oserved. d C d MiWS C. lidus P. lentisus WES L. giertii WESS MoWS d Fig. 3. Violxnthin (V, d), ntherxnthin (A, s) nd zexnthin (Z, ;) t middy, expressed in mmol mol 21 Chl, under different tretments: ontrol (C), MiWS, MoWS, nd RW. Vlues re mens SE of four replites per speies nd tretment. Different letters denote sttistil differenes y Dunn test (P <.5) mong tretments for eh pigment. Growth form revitions:, hers;, semideiduous shrus; WES, woody evergreen shrus; WESS, woody evergreen semishrus. RW Photoprotetion nd photoinhiition under wter stress nd reovery As wter stress intensified, P r ws kept t ontrol vlues or inresed, depending on the speies, nd only t MoWS to did photorespirtion deline (Fig. 4). P r (% ontrol) P r (% ontrol) P r (% ontrol) P r (% ontrol) P r (% ontrol) B. mritim ssp. mrosii D. iiensis P. itli H. lerium WES C MiWS MoWS RW d d d L. mgllufinum WESS Tretment B. mritim ssp. mritim L. mritim C. lidus P. lentisus WES L. giertii WESS C MiWS MoWS RW Tretment Fig. 4. P r (expressed s % in respet to ontrol tretment vlues, d) nd middy (s) under different tretments: ontrol (C), MiWS, MoWS, nd RW. Vlues re mens SE of four replites per speies nd tretment. Different letters denote sttistil differenes y Dunn test (P <.5) mong tretments for eh prmeter. Growth form revitions:, hers;, semideiduous shrus; WES, woody evergreen shrus; WESS, woody evergreen semishrus. d 52 Physiol. Plnt. 13, 7

9 inresed progressively s wter stress intensified, prtiulrly t MoWS to (Fig. 4), while it ws negtively orrelted with P r (r ¼.476, P <.1). Both P r nd orrelted with soil wter vilility (r ¼.386, P <.1, nd r ¼.591, P <.1, respetively). The mximum vlues rehed under stress were similr in ll speies (etween nd ). After RW, ll the speies exept C. lidus showed some relxtion of, ut only in B. mritim ssp. mritim, H. lerium nd P. lentisus the relxtion ws omplete (Fig. 4). By ontrst, the, mesured t predwn, followed pttern tht differed mong speies (Fig. 5). The lowest vlues of mximum were generlly higher thn.75, nd only in P. lentisus deresed to.. All the speies exept L. mgllufinum progressively deresed s wter stress intensified nd presented signifintly lower vlues t with respet to ontrol (P <.5). The semideiduous shrus L. mritim nd P. itli showed signifint dereses t MoWS (P <.5). In the other speies, ws mintined t ontrol vlues or delined only slightly (nd often nonsignifintly) from MiWS to MoWS nd delined to some extent t. The only exeption ws L. mgllufinum, whih mintined high vlues through the entire experiment. A lrge vriility ws lso oserved in the reovery of fter RW, from totl (L. mritim) or ner totl (H. lerium, C. lidus) to lmost no reovery (D. iiensis, P. lentisus, B. mritim ssp. mrosii). Finlly, in four speies (B. mritim ssp. mritim, the two Limonium nd P. itli), there ws some deline of fter RW. In well-wtered plnts, the rte of liner ETR rnged from 294 mmol e 2 m 22 s 21 for L. mritim to 122 mmol e 2 m 22 s 21 C. lidus (Fig. 5). ETR deresed signifintly in ll speies euse of wter stress imposition, with the lowest vlues oserved under, where P. lentisus presented the lowest ETR, with 35 mmol e 2 m 22 s 21,ndL. mritim the highest, with 167 mmol e 2 m 22 s 21. After RW ll speies inresed ETR, ut the extent of suh reovery ws speies-speifi response nd did not depend on the extent of previous derese in ETR. Disussion Pigment omposition under wter stress nd reovery Although Chl loss hs een onsidered negtive onsequene of stress, deresed Chl ontent hs lso een desried in some Mediterrnen speies s regultory mehnism to redue the mount of photons sored y leves, onferring some degree of photoprotetion B. mritim ssp. mrosii P. itli D. iiensis H. lerium WES L. mgllufinum WESS C MiWS MoWS RW Tretment B. mritim ssp. mritim L. mritim C. lidus P. lentisus WES L. giertii WESS C MiWS MoWS RW Tretment under drought (Blguer et l. 2, Kyprissis et l., Munné-Bosh nd Alegre ). However, in the present study, lef Chl ontent ws generlly unffeted y wter stress, with few exeptions (Fig. 1). This suggests tht djusting Chl my not e mjor photoprotetive response to short-term wter stress or tht the presene of suh response is strong speies-dependent Fig. 5. of PSII mesured t predwn (d) nd the liner ETR (s) under different tretments: ontrol (C), MiWS, MoWS, nd RW. Vlues re mens SE of four replites per speies nd tretment. Different letters denote sttistil differenes y Dunn test (P <.5) mong tretments for eh prmeter. Growth form revitions:, hers;, semideiduous shrus; WES, woody evergreen shrus; WESS, woody evergreen semishrus. 5 ETR (µmol e-m 2 s 1 ) ETR (µmol e-m 2 s 1 ) ETR (µmol e-m 2 s 1 ) ETR (µmol e-m 2 s 1 ) ETR (µmol e-m 2 s 1 ) Physiol. Plnt. 13, 7 53

10 feture. This ft seems to e in ordne with previous studies tht questioned the role of hnges in Chl ontent in regulting light intereption (Björkmn nd Demmig- Adms 1987). The VAZ per unit lef re hs een shown to inrese (Grí-Plzol et l. 1997), remin onstnt (Kyprissis et l. 1995, Munné-Bosh et l. 3) or even derese (Blguer et l. 2, Munné-Bosh nd Peñuels 3) in different Mediterrnen speies from spring to lte summer. Our results seem to support those works suggesting tht VAZ per unit lef re is not signifintly ffeted y wter stress (Fig. 1), exept in C. lidus. Furthermore, in ordne with previous reports in Mediterrnen plnts (Mrtínez-Ferri et l. ) nd tropil evergreens (Demmig-Adms nd Adms 6), no inverse reltionship etween VAZ size nd photosyntheti pity mong speies ws found. Inresing VAZ pool per Chl my e nother mehnism to inrese photoprotetion pity in leves, nd it hs een shown to generlly our during summer in oth evergreen slerophylls nd semideiduous shrus (Fri et l. 1998, Kyprissis et l. 1995, ). In short-term wter stress experiments, VAZ/Chl rtio hs een shown to inrese in the evergreen slerophylls A. unedo (Munné-Bosh nd Peñuels 4) nd Phillyre ngustifoli (Munné-Bosh nd Peñuels 3, Peñuels et l. 4). In the present study, none of the two woody evergreen shrus nlyzed (P. lentisus nd H. lerium) presented suh n inrese (Fig. 2), suggesting tht this my not e speifi feture of evergreen slerophyll speies. An inresed VAZ/Chl rtio in response to wter stress, ompnied y inreses in the lutein ontent, ws oserved only in three of the speies nlyzed (Fig. 2), n evergreen semishru (L. giertii) nd two semideiduous shrus (C. lidus nd P. itli). Therefore, in the speies nlyzed neither Chl nor totl VAZ pool djustments seemed to e importnt responses to short-term wter stress. However, hnges in the de-epoxydtion stte of the xnthophylls yle were generl fte in ll the speies (Fig. 3). In generl, wter stress tivted the de-epoxidtion of violxnthin to zexnthin, while ntherxnthin ws kept t more onstnt onentrtion through the entire experiment (Fig. 3). Zexnthin formtion is relted to the dissiption of exess sored light s het, s indited y the strong orreltions found etween the DPS of the xnthophylls yle nd (s disussed in the next setion). Photoprotetion nd photoinhiition under wter stress nd reovery When photosynthesis progressively delines with drought, photorespirtion nd therml energy dissiption re regrded s the most importnt photoprotetive mehnisms leding to dissiption of exess sored light (Powles nd Osmond 1978, Demmig et l. 1988, Flexs nd Medrno 2). The importne of lterntive eletron sinks, suh s the Mehler-sorte peroxidse retion, hs een shown to e minor oth under wellwtered onditions nd drought in other Mediterrnen speies (Flexs nd Medrno 2). In the present study, photorespirtion delined when wter stress eme MoWS to, onomitntly with inreses in the, n inditor of therml energy dissiption in the pigment ed (Björkmn nd Demmig-Adms 1994) (Fig. 4). These results demonstrte tht, s lredy shown for some Mediterrnen evergreen slerophylls (Grí- Plzol et l. 1997, Gulís et l. 2), mintining photorespirtion nd inresing re ommon responses to wter stress in other Mediterrnen speies, inluding hers nd semideiduous shrus. Remrkly, most of the speies inresed the photorespirtory oxygen metolism from ontrol to MiWS, in ordne with Flexs nd Medrno (2). Björkmn nd Demmig- Adms (1994) lredy pointed tht the rte of photorespirtion nd hene its ontriution to energy dissiption would e expeted to e greter in temporrily wterstressed leves in whih the intrinsi photosyntheti pity remined unhnged nd the deline in A N is minly euse of derese in interellulr CO 2 pressure, s ourred in the present study (dt not shown). Role of the photorespirtory oxygen metolism in photoprotetion hs een lrgely desried (Flexs nd Mendrno 2, Niyogi 1999). Remrkly, the only speies tht did not present the initil inrese t MiWS ws L. giertii, whih hs een shown to present the highest vlue of Ruiso speifiity for CO 2 up to now desried in higher plnts (Glmés et l. 5). As usully desried in Mediterrnen (Dmesin nd Rml 1995, Vlldres et l. 5) s well s in non- Mediterrnen speies (Flexs et l. 4), dereses in were generlly smll (exept for P. lentisus) lthough signifint, inditing only minor photoinhiitory effet of wter stress. Although the mximum extent of deline did not differ mong growth forms, there ws ertin effet of growth form in the pttern of response to wter stress euse the semideiduous speies delined progressively from erly stges of stress, while in other groups it delined only t. This wter stress-indued deline in ws not ompnied y deresed Chl ontent (Fig. 1), nd hene my not e ssoited with the photoprotetive mehnism onsisting of deresing light sorption, s desried for P. frutios or S. tenissim (Blguer et l. 2, Kyprissis et l. 1995). Although the semideiduous speies inluded in this study provide severl morphologil 54 Physiol. Plnt. 13, 7

11 dpttions, e.g. lef trihomes, high lef refletne, these were not suffiient to prevent some derese of F v / F m in plnts sujeted to stress. Different mehnisms my operte to derese, depending on the speies. For instne, in L. mritim, deresing ws prlleled y progressive inrese of F o (dt not shown), therefore suggesting progressive photointivtion of PSII enters from erly wter stress stges. By ontrst, F o progressively delined in P. itli nd C. lidus, suggesting tht deresed ws refleting sustined photoprotetion in these two speies. Alterntively, deresed ould e refleting other uses, suh s hnges in PSI fluoresene, unoupling of externl ntenne, et. Whtever the reson, dereses in were low nd did not limit ETR (Fig. 5). The extent of reovery of ws not dependent on plnt growth form. For instne, mong the evergreens, reovery ws null in P. lentisus while lmost omplete in H. lerium. Also, the extent of reovery did not depend on the mximum extent of depression hieved during wter stress. For instne, reovery ws omplete in L. mritim nd null in D. iiensis, while oth speies hd hieved similr. In four speies (B. mritim ssp. mritim, the two Limonium nd P. itli), there ws some deline of fter RW, whih in three of them ws ompnied y deresed hlorophyll ut not xnthophyll ontent (Fig. 1). This hs een lredy oserved fter wter stress in Vign unguiult (De Souz et l. 4) nd fter photoinhiitory experiments in different speies (J. Flexs, J. Glmés, nd H. Medrno, Universitt de les Illes Blers, Plm, unpulished dt). Proly, this effet my e relted to some memrne dmge used y RW, or it my e euse of the ft tht reovery of PSII fter photoinhiition requires degrdtion nd de novo synthesis of dmged omponents, prtiulrly D1 protein (Aro et l. 1994). Assuming tht this is generl proess tht my our in ll speies, differenes in the oserved ehvior of t fixed time (24 h) fter RW my reflet interspeifi differenes in the veloity with whih they n reover PSII. In this sense, L. mritim would e the speies with the fstest pity for reovery, followed y H. lerium nd C. lidus. The four speies showing deline of during reovery mesurements my hve n intermedite speed, hving lredy strted the proess 24 h fter RW, while those showing no hnge (D. iiensis, P. lentisus, B. mritim ssp. mrosii) my e regrded s hving the lowest pity nd/or veloity for reovery. It is remrkle tht lso in speies showing no wter stress-indued deline of, suh s L. mgllufinum, ws deresed fter RW. Generl pttern of photoprotetive responses to stomtl losure Despite the oserved differenes etween speies in photoprotetion nd photoinhiition response to wter stress, they ll respond to generl pttern desried for C 3 plnts when g s is used s referene for wter stress intensity (Flexs nd Medrno 2, Flexs et l. 4). This pttern is hrterized y two phses of response to wter stress, first phse orresponding to g s delining from mximum to out.15. mol H 2 Om 22 s 21, nd seond phse orresponding to further dereses in g s (Fig. 6). During the first phse, photorespirtion ts s mjor photoprotetive mehnism, eing kept t ontrol vlues or even inresed (Fig. 6A), while inreses only slightly (Fig. 6B) nd is mintined ove.8 (Fig. 6C), inditing little or no photoinhiition. During the seond phse, photorespirtion dereses (Fig. 6A) nd lrgely inreses (Fig. 6B), suggesting tht therml dissiption eomes the mjor photoprotetive mehnism during this phse. In this phse, dereses of eventully our, to n extent tht lrgely differs mong speies (Fig. 6C). The deline of under severe wter stress my e understood s onsequene of wter stress-indued photosynthesis deline rther thn its use euse ETR (Fig. 5) nd net photosynthesis (not shown) strt to deline well efore nd hieve muh lrger redutions. Another indition tht wter stress-indued vritions in did not limit photosynthesis omes from the ft tht despite ws further redued fter RW in four speies, ETR nd net photosynthesis simultneously reovered y out 5% in these speies, s well s in those showing no reovery exept P. lentisus (Fig. 5 nd dt not shown). These results re onsistent with the ft tht leves present muh lrger PSII onentrtion thn needed for photosynthesis, so tht up to 5% of PSII units n e dmged efore ny effet is detetle in photosynthesis (Lee et l. 1999). Reltionship etween therml dissiption nd pigment omposition Highly signifint orreltions were found etween DPS MD nd, whih suggest tht lrge prt of the is relted to DPS in Mediterrnen speies (Fig. 7). However, the slope nd kinetis of suh reltionship strongly differed mong speies, suggesting speiesdependent dditionl roles of de-epoxidted xnthophylls (Demming-Adms nd Adms 6). PSII effiieny did not return to optimum vlues efore dwn under in ny speies. However, the signifint orreltions oserved etween predwn de-epoxidtion Physiol. Plnt. 13, 7 55

12 B. vulgris ssp. mrosii r 2 =.941 B. vulgris ssp. mritim r 2 =.549 D. iiensis r 2 =.672 L. mritim r 2 =.99 P. itli r 2 =.93 C. lidus r 2 =.971 H. lerium r 2 =.718 P. lentisus r 2 =.51 L. mgllufinum r 2 =.925 L. giertii r 2 = Fig. 6. Reltionship etween g s nd (A) P r (expressed s % in respet to ontrol tretment vlues), (B) middy nd (C). Vlues re mens SE of four replites per speies nd tretment. RW vlues were not inluded. Symols nd speies re s follows: d, Diplotxis iiensis; s, Bet mritim ssp. mrosii; n, Bet mritim ssp. mritim; h, Limonium mgllufinum; :, Limonium giertii; n, Phlomis itli; ;, Lvter mritim;,, Cistus lidus;, Pisti lentisus; ), Hyperium lerium. stte (DPS PD ) nd efore dwn for most of the speies (Fig. 8) suggest tht the low PSII ws proly the result of sustined inreses in the DPS of the xnthophyll yle rther thn result of photoinhiitory dmge to the leves. This my reflet one of the two forms of the zexnthin-relted sustined photoprotetion forms, desried in evergreen speies (Demming-Adms nd DPS MD DPS MD Fig. 7. Reltionship etween the middy nd the DPS MD for the five tretments studied. Regression oeffiients re shown for eh one of the speies. Mesurements orresponding to RW tretment re indited y s nd were not onsidered for the regression djustment. Vlues re mens SE of four replites per speies nd tretment. Adms 6). Remrkly, the present results suggest tht this form of sustined photoprotetion my lso our in some Mediterrnen semideiduous shrus, perennils nd nnuls. As lredy shown for the reltionship etween nd DPS MD, the slope of the reltionship etween nd DPS PD ws lso dependent on the speies, with no pprent reltion to speies growth forms (Fig. 8). It is lso worth nothing tht in oth 56 Physiol. Plnt. 13, 7

13 B. mritim ssp. mrosii r 2 =.323 D. iiensis r 2 =.887 P. itli r 2 =.584 H. lerium r 2 =.611 L. mgllufinum r 2 = DPS PD B. mritim ssp. mritim r 2 =.776 L. mritim r 2 =.5 C. lidus r 2 =.565 P. lentisus r 2 =.937 L. giertii r 2 = DPS PD reltionships, the vlue fter RW does not lwys fit the sme regression line desried for the remining tretments, phenomenon tht my deserve further ttention in future studies. Differenes in photoprotetion mong growth forms nd evolutionry groups Fig. 8. Reltionship etween the mesured t predwn nd DPS PD of the xnthophyll yle, for the five tretments studied. Regression oeffiients re shown for eh one of the speies. Mesurements orresponding to RW tretment re indited y s nd were not onsidered for the regression djustment. Vlues re mens SE of four replites per speies nd tretment. Note the different y-xis sle for Pisti lentisus. Five of the 1 speies inluded in the present work were endemi of the Bleri Islnds. The geogrphilly limited distriution of the endemi speies my e euse of underlying negtive eophysiologil trits tht impede these speies eoming more widespred. In other insulr systems with high perentge of endemiity, ntive speies hve een shown to exhiit greter photoinhiition nd lower performne thn invsive speies when sujeted to high light levels (Durnd nd Goldstein 1, Ymshit et l. ). Although no previous ttempts exist in ompring photoinhiition nd photoprotetive strtegies etween endemi nd non-endemi speies in the Mediterrnen sin, Gulís et l. (3) showed tht endemi speies presented in generl % lower photosyntheti pity when ompred with widely distriuted speies. In the present work, set of ANOVA s ws mde to investigte possile differenes in photoprotetion mehnisms nd photoinhiition proesses etween endemi nd non-endemi speies. Differenes were exmined for eh prmeter nlyzed nd onsidering ll tretments nd smpling time (i.e. predwn nd middy). Nevertheless, differenes etween oth evolutionry groups were only signifint (P <.5) for lutein, - rotene nd totl xnthophyll ontent under MoWS t middy.therefore,tlestfor the speies inluded in the present survey, differenes in the photoprotetion mehnisms re not the min use tht limits distriution nd ompetitiveness of endemi speies in the Bleri Islnds. A series of luster nlysis of the speies onsidered in the present survey ws performed for eh tretment (dt not shown). Suh nlysis, whih inluded the min prmeters mesured t middy, reflets the existene of ontinuum of ehvior in response to wter stress tht is independent of growth form. Effetively, in ny of the tretments, no ler grouping of speies ording to their growth form ws oserved. One it hs een shown tht no importnt differenes mong growth forms ould e estlished, the next step ws to summrize the generl vrine in few omponents. In this wy, the prinipl omponent nlysis of the different vriles mesured t middy distinguished three min groups explining pproximtely 7% of totl vrine. The first omponent (pproximtely % of totl vrine) ws formed y ll pigments exept those representing de-epoxidted sttes, i.e. rotenes, hlorophylls, lutein epoxide, neoxnthin nd violxnthin ontent. The seond omponent (pproximtely % of totl vrine) ws formed y pigments nd indexes refleting the de-epoxidted stted of the xnthophylls yle (i.e. DPS, ntherxnthin nd zexnthin) nd tretment. Finlly, the third omponent (pproximtely 1% of totl vrine) ws formed y ETR, nd P r. The remining omponents, whih inluded speies, growth form nd evolutionry history, ounted for less thn 7% of totl vrine. Physiol. Plnt. 13, 7 57

14 To further onfirm the no existene of ler differenes mong growth forms, speies were plotted ginst the two min prinipl omponents (Fig. 9). Suh nlysis, in ddition, llowed visulizing how the different speies re differentilly distriuted vs these prinipl omponents, nd how suh distriution vried ording to the wter stress tretments. Conluding remrks The present study shows tht Mediterrnen plnts, regrdless of their growth form, re sustntilly resistnt to wter stress-indued photoinhiition. However, lthough ll these speies hieve photoprotetion y omintion of photohemil (photorespirtion) nd non-photohemil (therml dissiption) mehnisms, the mehnisms nd/or pigments involved in the ltter my differ mong speies, in mnner tht is independent of the plnt growth form. Similrly, the veloity of Component CO CA PI PL MT MC DI Component 1 HB LA LG LM PSII reovery from photoinhiition or sustined photoprotetion lso differs mong speies. These fetures my reflet dpttions to prtiulr environments nd re in greement with the different speies distriution. For instne, very effetive photoprotetion under wter stress my e of dptive vlues for Limonium, C. lidus or P. itli euse they ll inhit sun-exposed res, while eing shllow-rooted speies tht retin some green leves in summer. Therefore, they my hve the pity to respond to frequent short episodes of drought in ddition to the long summer drought period. An lterntive dpttion for speies inhiting similr res, suh s L. mritim, my e to possess high plstiity, whih inludes high pity for rpid reovery. By ontrst, the speies elonging to other growth form groups my hve to endure less frequent periods of omined drought nd high light intensity, the hers euse they do not retin leves during summer nd the lrge woody perennils euse they use to live under the shde of dult plnts when young, nd e deep-rooted when dult. Heterogeneity in the eologil performne of Mediterrnen speies my e onsequene of the funtionl omplexity of Mediterrnen eosystems nd likely reflets the ft tht ny speies in this environment hs to endure temporry drought periods, whih hs led to n rry of different dptive strtegies. Aknowledgements Dr M Ris-Cró is knowledged for his helpful omments on previous version of the mnusript nd grmmtil orretions. This work ws prtly funded y Projets REN1-356-CO2-O2 nd BFU5-312/BFI (Pln Nionl, Spin). Component PL CA PI MC MT DI HB Component 1 Fig. 9. Distriution of the 1 speies ording to the first two prinipl omponents of vrine under ontrol (C) nd tretments. Speies odes s in Tle 1. Further explntion in the text. LG LM LA Referenes Adí J, Adí A (1993) Iron nd plnt pigments. In: Brton LL, Hemmig BC (eds) Iron Cheltion in Plnts nd Soil Miroorgnisms. Ademi Press, Sn Diego, pp Affek HP, Ykir D (2) Protetion y isoprene ginst singlet oxygen in leves. Plnt Physiol 29: Anon (199). SPSS Bse System User s Guide. SPSS In., Chigo, IL. 949 p Aro EM, MCffery S, Anderson JM (1994) Reovery from photoinhiition in pes (Pisum stivum L.) limted to vrying growth irrdines role of D1 protein-turnover. Plnt Physiol 14: Blguer L, Pugnire FI, Mrtínez-Ferri E, Arms C, Vlldres F, Mnrique E (2) Eophysiologil signifine of hlorophyll loss nd redued photohemil effiieny under extreme ridity in Stip tenissim L. Plnt Soil 2: Physiol. Plnt. 13, 7

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