Importance of Physiological Traits Vulnerability in Determine Halophytes Tolerance to Salinity Excess: A Comparative Assessment in Atriplex halimus

plan s A icle Impo ance o Physiological T ai s Vulne abili y in De e mine Halophy es Tole ance o Salini y Excess: A Compa a i e Assessmen in A iplex halimus Jesús Albe o Pé ez-Rome o 1,* , En ique Ma eos-Na anjo 1, Ja ie López-Ju ado 1, Susana Redondo-Gómez 1and JoséM. To es-Ruiz 2 1Dp o. de Biología Vege al y Ecología, Facul ad de Biología, Uni e sidad de Se illa, A Reina Me cedes s/n, 41012 Se illa, Spain; [email p o ec ed] (E.M.-N.); [email p o ec ed] (J.L.-J.); [email p o ec ed] (S.R.-G.) 2INRA, PIAF, Uni e si éCle mon -Au e gne, 63000 Cle mon -Fe and, F ance; o es [email p o ec ed] *Co espondence: jpe [email p o ec ed]; Tel.: +34-95-4557064; Fax: +34-95-4615780 Recei ed: 7 May 2020; Accep ed: 26 May 2020; Published: 28 May 2020   Abs ac : Many halophy ic physiological ai s ela ed o he ole ance o plan s o salini y excess ha e been ex ensi ely s udied, wi h a ocus on biomass and/o gas exchange pa ame e s. To gain a mo e comple e unde s anding o whe he salini y excess a ec s he physiological pe o mance o halophy es, an expe imen was pe o med using he halophy e A iplex halimus L. as a model. A. halimus plan s we e subjec ed o wo salini y ea men s (171 and 513 mM NaCl) o e 60 days in a con olled en i onmen . A e his pe iod, d y biomass, speci ic s em conduc i i y, wa e po en ial a u go loss poin , osmo ic po en ial, gas exchange pa ame e s, and he luo escence o chlo ophyll a de i ed pa ame e s we e assessed in o de o ob ain knowledge abou he di e ences in ulne abili y ha hese pa ame e s can show when subjec ed o salini y s ess. Ou esul s showed a dec ease in belowg ound and abo eg ound biomass. The dec emen in biomass seen a 513 mM NaCl was ela ed o pho osyn he ic limi a ions and speci ic s em conduc i i y. Tu go loss poin did no a y signi ican ly wi h he inc emen o salini y. The e o e, he pa ame e ha showed less ulne abili y o saline s ess was he u go loss poin , wi h only a 5% dec ease, and he mo e ulne able ai was he s em conduc i i y, wi h a educ ion o nea ly 50%. Keywo ds: halophy e; speci ic conduc i i y; u go loss poin ; pho osyn hesis; chlo ophyll luo escence 1. In oduc ion Halophy es a e plan species wi h he abili y o comple e hei li e cycles unde a leas 200 mM NaCl [ 1 ]. The e a e many physiological mechanisms in ol ed in he capaci y o halophy es o cope wi h salini y excess, and hese esponses and mechanisms a e species-speci ic in many cases [2]. Among speci ic ole ance mechanisms, i is well ecognized ha halophy es use osmop o ec i e and ion-de oxi ica ion s a egies [ 3 – 5 ] o egula e hei issues’ Na concen a ion pa e ns o K/Na a io a ia ions [ 2 ]. Many s udies ha e been ca ied ou o e alua e he e ec o sal excess ca boxyla ion capaci y and ene gy use e iciency in halophy es [ 6 – 12 ], showing how hese plan s a e able o main ain me abolic p ocesses despi e being exposed o high sal concen a ion. Howe e , s udies ocused on he e ec o sal on xylem ana omy and unc ioning, plan –wa e ela ions, and pho osyn he ic capaci y a e sca ce [ 13 ], despi e he ele ance o hese ai s o de e mine plan ole ance o en i onmen al s ess [ 14 – 16 ]. The e ha e been ew a emp s o assess he e ec o salini y on pa ame e s such as u go loss poin in halophy e species. These s udies ha e assessed he impo ance o soil-sal wa e on he wa e s a us o Juncus oeme ianus [ 17 ] and di e ences in xylem speci ic conduc i i y among di e en mang o e species, in which L ó pez-Po illo e al. [ 18 ] ound di e en esponses in glycophy e and halophy e species. Plan s 2020,9, 690; doi:10.3390/plan s9060690 www.mdpi.com/jou nal/plan s Plan s 2020,9, 690 2 o 11 Cell u go loss has an impac on cellula s uc u al in eg i y, me abolism, and whole-plan pe o mance [ 19 , 20 ], being a ai ha has been adi ionally conside ed an indica o o plan wa e s ess [ 21 ]. Due o he simila i ies be ween wa e and Salini y s ess [ 22 ], s udying how u go loss poin a ies in halophy es exposed o salini y s ess is impo an o imp o e ou knowledge abou which mechanisms a ec hese changes. E alua ing how di e en sal concen a ions a ec he ana omical p ope ies o xylem and i s hyd aulic e iciency would also allow us o be e unde s and he esponse o halophy es o salini y [13]. In addi ion, i is a e o ind a comple e analysis o hese ai s aking in o accoun plan pho osyn he ic pe o mance pa ame e s o acqui e a b oade iew o some o he main physiological esponses o halophy es o excess salini y. To gain a mo e comple e unde s anding o whe he salini y excess a ec s he physiological pe o mance o halophy es, he e ec o di e en sal concen a ions on he ana omical and unc ional p ope ies o xylem, plan wa e s a us, gas exchange, and chlo ophyll luo escence we e e alua ed in he halophy e A iplex halimus L. The A iplex genus is composed o mo e han 400 species wo ldwide [ 23 ]. Se e al species o his genus a e well adap ed o ex eme en i onmen al condi ions such as A. ho ensis, which is ole an o high salini y, A. canescens a . angus i olia, which is a d ough - ole an sh ub, o A. len i o mis, which can li e in seawa e i iga ion and xe ic condi ions [ 24 ]. A iplex species could se e as ools o he phy o emedia ion o pollu ed, a id, o semi-a id soils [ 22 , 25 – 27 ] o as o age species [ 27 – 29 ]. Among hem, A iplex halimus L. is one o he mos plan ed A iplex species wo ldwide [ 22 ], and he e o e ep esen s one o he mos impo an species wi hin his genus. I is a pe ennial sh ub wi h a C4 pho osyn he ic me abolic pa hway ha is able o g ow in a wide ange o salini y condi ions [ 22 ]. This cha ac e is ic makes i a good candida e o he exhaus i e analysis o he e ec s o salini y excess on plan physiological pe o mance. He e, we hypo hesized ha di e en salini y le els would exe a di e en ial e ec on he main physiological plan ai s ela ed o ole ance o salini y. The e o e, he possible exis ence o di e en ulne abili y le els o sal s ess in hese ai s should be conside ed when de e mining he salini y ole ance o his species. Thus, he aim o his wo k was o e alua e how di e en le els o NaCl concen a ion in g owing solu ion a ec ed he u go loss poin , xylem speci ic heo e ical conduc i i y, hyd aulic mean diame e , essel densi y, osmo ic po en ial, and he main lea gas exchange and chlo ophyll luo escence pa ame e s in o de o achie e a mo e global ision o he physiological pe o mance o A. halimus unde di e en salini y concen a ions. The wo selec ed le els we e 171 mM NaCl (o 10 g L−1) and a h ee old highe one (i.e., 513 mM o 30 g L−1). 2. Resul s and Discussion 2.1. Plan De elopmen and Xylem Ana omical and Func ional Fea u es The e we e signi ican e ec s o salini y on he g ow h, xylem speci ic heo e ical conduc i i y, and hyd aulic mean diame e o A iplex halimus L. a e 60 days o ea men . Mo e speci ically, ae ial pho osyn he ic, ae ial non-pho osyn he ic, and oo d y mass dec eased by 24%, 51%, and 41%, espec i ely, in plan s g own in 513 mM NaCl compa ed o he con ol ea men (171 mM NaCl) (one-way ANOVA, p<0.05; Table 1). Ou esul s ag ee wi h o he o me s udies showing ha NaCl le els g ea e han 200 mM induce a signi ican dec ease in he biomass o A. halimus [ 29 – 31 ]. Howe e , no e ec on g ow h has been desc ibed o da e o NaCl concen a ions below 600 mM [ 22 , 32 ]. These disc epancies be ween s udies ega ding he e ec o NaCl concen a ions on plan g ow h ha e been asc ibed o in aspeci ic a iabili y in g ow h esponses o salini y excess ac oss A iplex popula ions, which a e dis ibu ed in di e en habi a s [32–36]. Plan s 2020,9, 690 3 o 11 Table 1. Pho osyn he ic d y mass, non-pho osyn he ic d y mass, oo d y mass (R ), ne pho osyn he ic a e (A N ), s oma al conduc ance (g s ), in e cellula CO 2 concen a ion, (C i ), in insic wa e use e iciency ( i WUE), and maximum quan um e iciency o pho osys em II (PSII) pho ochemis y (F /F m ) and quan um e iciency o PSII ( ΦPSII ) o A iplex halimus L. in esponse o ea men wi h 171 and 510 mM NaCl o 60 d. Biomass and physiological pa ame e alues ep esen mean ± SE, n=16 and n=12, espec i ely. Di e en le e s indica e means ha a e signi ican ly di e en be ween bo h salini ies (ANOVA es , p<0.05). Salini y Concen a ion Pa ame e s 171 mM 510 mM Pho osyn he ic d y mass (g) 5.98 ±0.44 a4.55 ±0.53 b Non-pho osyn he ic d y mass (g) 5.41 ±0.53 a2.73 ±0.44 b Roo d y mass (g) 1.48 ±0.12 a0.89 ±0.13 b AN(µmoL m−2s−1)4.35 ±0.67 a2.74 ±0.32 b gs(mmoL m−2s−1)53.4 ±7.47 a46.6 ±6.49 a Ci (µmoL mol−1)252.1 ±30.0 a274.6 ±23.5 a iWUE (µmoL mol−1)84.3 ±12.6 a69.1 ±11.6 a F /Fm 0.70 ±0.02 a0.70 ±0.02 a ΦPSII 0.18 ±0.03 a0.14 ±0.02 a a, b show he signi ican di e ences be ween salini ies I is well known ha sal s ess can a ec he ana omy o xylem, which can exe a limi a ion on plan hyd aulic unc ioning [ 13 , 37 ]. Highe NaCl concen a ion a ec ed he ana omy, educing he essel diame e (Figu e 1) and p oducing a non-signi ican inc ease o essel densi y (VD) (Figu e 2). Mo eo e , he pi h and co ex a ea inc eased, and a xylem essel numbe dec ease was seen a 510 mM NaCl (Figu e 1). Thus, no signi ican di e ences in VD we e obse ed be ween he wo NaCl concen a ions (Figu e 2). Howe e , he e was a signi ican educ ion in hyd aulic mean diame e (dh) o plan s g own a 513 mM NaCl (one-way ANOVA, p<0.05; Figu e 2). These a ia ions in he diame e o he xylem essels be ween ea men s also led o a signi ican di e ence in xylem hyd aulic unc ioning be ween hem. The lowe alues o xylem speci ic heo e ical conduc i i y (K S ) o hose plan s exposed o highe NaCl concen a ion (one-way ANOVA, p<0.05; Figu e 2) illus a ed his di e ence in xylem unc ioning. In line wi h ou esul s, Boughalleb e al. [ 13 ] obse ed a dec ease in essel diame e o A. halimus g own a 800 mM NaCl, and L ó pez-Po illo e al. [ 18 ] epo ed highe K S alues o halophy e plan s when g own be ween 3 and 253 mM NaCl and lowe alues o plan s g own beyond ha ange o salini y. Figu e 1. T ans e sal sec ions o he main s em o A iplex halimus L. s ained wi h 1% oluidine blue g own a 171 ( A ) and 513 ( B ) mM NaCl a e 60 d ea men showing E (epide mis), Co (co ex), Xy (xylem essels), and P (pi h cells). Plan s 2020,9, 690 4 o 11 Figu e 2. Xylem speci ic heo e ical conduc i i y, K S ( A ), essel densi y, VD ( B ), and hyd aulic mean diame e , dh ( C ) in andomly selec ed p ima y lea es o A iplex halimus L. in esponse o ea men wi h wo NaCl concen a ions (171 and 513 mM) a e 60 d o ea men . Values ep esen mean ± SE, n=3. Di e en le e s indica e means ha a e signi ican ly di e en om each o he (ANOVA es , p<0.05) and ns indica es non-signi ican di e ences. 2.2. Plan –Wa e Rela ions Analysis The e we e signi ican e ec s o salini y on some plan –wa e ela ions o A iplex halimus L. a e 60 days o ea men , wi h lowe osmo ic po en ial alues ( ΨO ) o highe NaCl ea men . Plan s g own a 171 and 513 mM NaCl showed ΨO alues o − 4.5 and − 6.9 MPa, espec i ely (one-way ANOVA, p<0.05; Figu e 3B), in conco dance wi h p e iously epo ed alues o his species [ 33 ]. Meanwhile, lea u go loss poin ( ΨTLP ) did no a y wi h NaCl concen a ion, wi h mean alues o ~ − 1.70 MPa o bo h ea men s (Figu e 3A). ΨTLP has been used o assess physiological abio ic s ess ole ance o d ough [ 21 ], wi h lowe alues measu ed in plan s occu ing in high salinized habi a s [ 17 ]. Lowe u go loss poin alues indica e a wide ange o lea wa e po en ials wi hin which he lea emains u gid and main ains i s unc ion [ 38 , 39 ]. In his sense, lowe ΨTLP alues ha e been ela ed o plan abili y o main ain s oma al conduc ance, pho osyn he ic gas exchange and g ow h a low soil wa e Plan s 2020,9, 690 5 o 11 po en ial [ 38 , 40 – 43 ]. The osmo egula o y capaci y ep esen s an impo an adap ion by plan s o sal s ess since i allows hem o keep aking wa e om he soil e en when exposed o high le els o sal s ess [ 17 ]. Thus, he capaci y o A. halimus o main ain he ΨTLP while educing ΨO a high NaCl concen a ions e lec s he capaci y o his species o cope wi h high concen a ions o sal . Figu e 3. Wa e po en ial o u go loss poin , ΨTLP ( A ) and osmo ic po en ial, Ψ0 ( B ) in andomly selec ed p ima y lea es o A iplex halimus L. in esponse o ea men wi h wo NaCl concen a ions (171 and 513 mM) a e 60 d o ea men . Values ep esen mean ± SE, n=5. Di e en le e s indica e means ha a e signi ican ly di e en om each o he (ANOVA es , p<0.05) and ns indica es non-signi ican di e ences. 2.3. Plan Pho osyn he ic Pe o mance Analysis Salini y a ec ed some ai s ela ed o gas exchange. Thus, ne pho osyn he ic a e (A N ) alues we e signi ican ly lowe in plan s g own a 513 mM NaCl han in he con ol plan s (one-way ANOVA, p<0.05; Table 1), while simila alues o s oma al conduc ance (g s ), in e cellula CO 2 concen a ion (C i ), and in insic wa e use e iciency ( i WUE) we e obse ed be ween NaCl concen a ion ea men s (Table 1). The lack o di e ences in C i e en wi h a signi ican dec emen o A N could indica e a g ea e e ec o NaCl inc emen on he ubisco ac i i y a he han plan CO 2 di usion capaci y, as has been desc ibed o o he halophy es [12,44–46]. I has been epo ed ha he impac o NaCl excess on plan pho osyn he ic pe o mance is ela ed o al e a ions in pho osys em II (PSII) pho ochemis y machine y e iciency [ 47 ]. In ac , se e al s udies Plan s 2020,9, 690 6 o 11 ha e al eady shown how salini y excess dec eased elec on chain e iciency along wi h he abili y o use he inciden pho ons [ 10 , 48 , 49 ], which can lead o a signi ican dec ease in he maximum yield o p ima y pho ochemis y [ 50 ]. In con as o hese obse a ions, ou luo escence esul s indica ed ha F /F m and ΦPSII alues did no a y signi ican ly be ween salini y le els (ANOVA, p>0.05; Table 1), indica ing he high in eg i y and unc ionali y o i s pho ochemical appa a us unde sal excess, as has been p e iously desc ibed o o he halophy ic species [9]. 2.4. Analysis o Di e en ial T ai s Vulne abili y o Salini y Excess The ole ance o halophy es o salini y has been in ensely s udied wo ldwide, and he cha ac e iza ion o his ole ance has usually been based on speci ic physiological ai s, o en combined wi h de ailed plan de elopmen analyses [ 6 – 12 ]. Many s udies ha e based hei ole ance in e ences on de ailed analyses o plan wa e s a us. In ac , ΨTLP has been widely used o assess physiological abio ic s ess ole ance, as p e iously men ioned. Based on his, ou ΨTLP alues indica ed a a ia ion o 5% be ween 171 and 513 mM NaCl. Toge he wi h small a ia ions in o he eco ded ai s linked wi h plan wa e s a us and use e iciency, such as ΨO, g s and i WUE, his indica ed a g ea capaci y o A. halimus o ole a e salini y. This was also suppo ed by ou esul s on PSII pho ochemis y e iciency, since F /F m and ΦPSII did no a y wi h he inc emen o salini y. Howe e , hese esul s con as ed wi h a d as ic educ ion in plan g ow h (i.e., almos − 40%) unde ele a ed NaCl concen a ion. This g ow h esponse is likely ela ed o he a ia ions obse ed in o he physiological ai s, such as plan CO 2 assimila ion capaci y. In his sense, we ound ha A N dec eased ci ca 30% in plan s g own a 513 mM NaCl, his pe cen age being simila o ha egis e ed o plan pho osyn he ic and oo g ow h. Simila ly, he xylem ana omical p ope ies showed a good ela ionship wi h plan g ow h esponses. Thus, a educ ion in K S o almos 50% was obse ed o plan s g own a 513 mM NaCl compa ed wi h plan s g own a 171 mM NaCl. The e o e, ou esul s showed di e en esponses o salini y o hose physiological ai s in ol ed in ca bon assimila ion and hyd aulic unc ioning, which we e e lec ed in di e en g ow h a es o plan s exposed o he highe NaCl concen a ion. Thus, he capaci y o A. halimus o main ain he ΨTLP while educing ΨO a high NaCl concen a ion e lec s he capaci y o his species o cope wi h high concen a ions o sal . Mo e s udies e alua ing di e en halophy e species exposed o di e en NaCl concen a ions a e he e o e equi ed o ob ain a mo e comp ehensi e unde s anding o how his abio ic s ess a ec s halophy e pe o mance and o de e mine plan sal ole ance mo e p ecisely. This is i al o a oid possible masking e ec s ha occu when s udies a e based on assessing a small numbe o ai s ocusing exclusi ely on ce ain me abolic p ocesses. 3. Ma e ial and Me hods 3.1. Plan Ma e ial Cu ings o A iplex halimus L. we e collec ed in Ap il 2018 om di e en adul indi iduals (n=20) ha we e andomly selec ed om a well-es ablished popula ion in Odiel Ma shes (37 ◦ 15 0 N, 6 ◦ 58 0 O; SW Spain). Cu ings we e anspo ed o he labo a o y in a e ige a ed chambe (4 ◦ C) and immedia ely plan ed in indi idual plas ic po s (9 cm high × 11 cm diame e ) using pe li e as subs a e. Then, po s we e placed in a g eenhouse unde con olled condi ions: empe a u e be ween 21 and 25 ◦ C, 40%–60% ela i e humidi y, and na u al dayligh o 250 µ mol m −2 s −1 as he minimum and 1000 µ mol m −2 s −1 as he maximum ligh lux. Po s we e alloca ed o shallow ays and wa e ed wi h 20% Hoagland’s solu ion [ 51 ] and 171 mM NaCl. Plan s we e kep unde hese condi ions un il he expe imen al se up. 3.2. Expe imen al T ea men s In July 2018, a e 3 mon hs o cu ing cul u e, 17 cm high plan s showing a comple ely de eloped oo sys em we e andomly di ided in o wo blocks o 30 plan s each. Each block was exposed o Plan s 2020,9, 690 7 o 11 di e en NaCl concen a ions: con ol (171 mM) and ele a ed concen a ion (513 mM) o 60 days. Chosen salini ies we e based on A. halimus soil salini y concen a ion ole ance, [ 29 – 31 ] and can be ound in he na u al dis ibu ion o his species [ 7 ]. These NaCl concen a ions we e es ablished by combining Hoagland’s solu ion wi h app op ia e amoun s o NaCl. A he beginning o he expe imen , he po s we e placed in plas ic ays con aining app op ia e solu ions o a dep h o 1 cm. Du ing he expe imen , g eenhouse condi ions we e con olled wi h a empe a u e o 21–25 ◦ C, 40%–60% ela i e humidi y, and na u al dayligh o 250 µ mol m −2 s −1 as he minimum and 1000 µ mol m −2 s −1 as he maximum ligh lux. NaCl concen a ion in he g ow h medium was moni o ed con inuously o a oid changes caused by wa e e apo a ion om he nu ien solu ion. In addi ion, he en i e solu ion (including NaCl) in he ays was enewed weekly. A e 60 days o exposu e o salini y ea men s, an exhaus i e e alua ion o xylem ana omical and unc ional ea u es we e made. This analysis was complemen ed wi h measu emen s o lea wa e ela ions and pho osyn he ic appa a us pe o mance. Finally, he emaining plan s (n=16, six een samples pe ea men ) we e ha es ed, and belowg ound and abo eg ound (pho osyn he ic and non-pho osyn he ic) ac ions we e sepa a ed, d ied a 80 ◦ C o 48 h, and weighed o d y mass de e mina ion. 3.3. E alua ion o Xylem Speci ic Theo e ical Conduc i i y, Hyd aulic Mean Diame e , and Vessel Densi y Fo xylem ana omy cha ac e iza ion, 5 cm long b anch samples we e andomly collec ed om plan s g own in bo h salini y ea men s (n=3, h ee samples pe ea men ). All samples we e collec ed 10 cm abo e he base o he ille and we e w apped in mois pape o keep hem well hyd a ed un il sample p epa a ion o sec ioning. Samples we e i s ly ixed in FAA (3.7% o maldehyde, 50% e hanol, 5% ace ic acid, and 41.3% wa e ) and hen dehyd a ed h ough a g adual e hanol se ies (50%, 70%, 80%, and 95%). Then, samples we e p og essi ely embedded in LR Whi e esin (Sigma-Ald ich) o 30 min a 4 ◦ C in di e en esin:e hanol 100% combina ions (1/3:2/3; 1/2:1/2; 2/3:1/3) o 1 h a 4 ◦ C in pu e LR Whi e esin. Finally, samples embedded in he esin we e encapsula ed in gela in capsules (size 4) and le o 48 h a 50–55 ◦ C o achie e he polyme iza ion o he esin. In o de o ob ain hin sec ions (2 and 3 µ m hick), an OmU2 o a y mic o ome (Reiche , Vienna, Aus ia) equipped wi h a his o diamond kni e was used. Sec ions we e s ained wi h 1% oluidine blue (w/ ). C oss sec ions we e obse ed unde an op ical mic oscope ( ansmi ed ligh , Zeiss Axioplan 2, Zeiss, Jena, Ge many) a × 40 magni ica ion. Images we e eco ded using a digi al came a (AxioCam HR, Zeiss) wi h AxioVision digi al imaging so wa e. By using he “mosaic” ool, a single image pe sample was cons uc ed by joining images wi h he same magni ica ion. A e spa ial calib a ion, ana omical and unc ional measu emen s we e pe o med by image analysis in o de o de e mine essel densi y (VD), hyd aulically weigh ed essel diame e (dh), and heo e ical speci ic xylem hyd aulic conduc ance (K S ) o h ee c oss sec ions pe sample using ImageJ so wa e [ 52 ]. K S (mmol m −2 MPa −1 s −1 ) was calcula ed by adding up he conduc i i ies o he condui s ound in he c oss sec ion, using he Hagen–Poiseuille equa ion o calcula e he conduc i i y o e e y single condui : Ks = πd4 128η As whe e dis he in e nal diame e o he condui , η is he dynamic iscosi y o wa e aken as 10 −9 MPa s a 20 ◦C and As is he c oss-sec ional a ea. VD was calcula ed as he quo ien be ween he xylem a ea and he numbe o essels. Hyd aulic mean diame e (dh) was calcula ed ollowing he Spe y e al. [53] o mula: dh = 2P 5 P 4 whe e is he a io o he essels. Plan s 2020,9, 690 8 o 11 3.4. Lea Tu go Loss Poin and Osmo ic Po en ial To es he e ec o salini y inc emen on plan –wa e ela ions, we quan i ied lea u go loss poin ( ΨTLP ) and osmo ic po en ial ( ΨO ) (n=5, i e samples pe ea men ). Fo ΨTLP , p essu e– olume cu es we e pe o med on andomly selec ed ully de eloped lea es. Thus, lea es we e collec ed wi h a azo blade and ully hyd a ed du ing 24 h in he da k a 4 ◦ C. A e ha , lea wa e po en ial and esh lea weigh we e measu ed e e y 3 min using a Scholande - ype chambe (PMS) and a scale (Me le -Toledo), espec i ely, un il he lea wa e po en ial eached ca. − 4 MPa. Then, lea es we e d ied a 80 ◦ C o 24 h, and he d y mass was measu ed [ 54 ]. Lea ela i e wa e con en (RWC) was de e mined along wi h he measu emen s by he a io be ween he d y and esh weigh acco ding o Sack and Pasque -Kok [ 55 ]. The a ea o each indi idual lea was de e mined by using an LI-3100C a ea me e (LICOR). Finally, ΨTLP alues we e ob ained a e plo ing lea wa e po en ial s. 100 RWC alue o de e mine he poin a which he ansi ion be ween cu ed and linea po ions occu ed. Lea osmo ic po en ial ( ΨO ) was de e mined by eezing small po ions o lea issue in liquid ni ogen, le ing hem haw, and cen i uging (12,000 × g, 10 min) a 4 ◦ C in 2 mL ubes. To sepa a e a minimum o 10 µ L o lea sap, we inse ed he ops o il e ips in o hese ubes. ΨO was measu ed om he ex ac ed sap using he psych ome ic echnique wi h a apo p essu e osmome e (5600 Vap o, Wesco , Logan, UT, USA). 3.5. Lea Gas Exchange Lea gas exchange and chlo ophyll luo escence pa ame e s we e measu ed in ully expanded lea es (n=12, wel e samples pe ea men ) using an in a ed gas analyze (LI-6400-XT, Li-COR Inc., Lincoln, NE, USA) and a modula ed luo ime e (FMS-2; Hansa ech Ins umen s L d., King’s Lynn, UK), espec i ely. Thus, ne pho osyn he ic a e (AN), s oma al conduc ance (gs), in e cellula CO 2 concen a ion (C i ), and ins an aneous wa e use e iciency ( i WUE; a io be ween A N and g s ) we e ob ained. The ollowing se ings we e applied: lux ligh densi y o 1000 µ moL pho ons m −2 s −1 (wi h 15% blue ligh o maximize s oma al ape u e), ambien CO 2 concen a ion (C a ) o 400 µ moL moL −1 ai , lea empe a u e o 25 ± 2 ◦ C, 50% ± 5% ela i e humidi y, and apo p essu e de ici o 2.0–3.0 kPa. As Sch eibe e al. [ 56 ] desc ibed, ligh ene gy yields o pho osys em II (PSII) eac ion cen e s we e de e mined wi h a sa u a ion pulse me hod. Thus, he maximum quan um e iciency o PSII pho ochemis y (F /F m ) and quan um e iciency o PSII ( ΦPSII ) we e ob ained in ligh and 30 min da k-adap ed lea es a midday (1500 µ moL pho ons m −2 s −1 ) using a sa u a ing ligh pulse o 0.8 s wi h an in ensi y o 10,000 µ moL m −2 s −1 acco ding o he p o ocol ollowed by Ma eos-Na anjo e al. [25]. 3.6. S a is ical Analysis The e ec o NaCl ea men s on xylem ana omical and unc ional ea u es, as well as on wa e po en ial measu emen s, pho osyn he ic pe o mance, and g ow h was de e mined by using one-way analysis o a iance (F- es ). Be o e s a is ical analysis, Kolmogo o –Smi no and Le ene es s we e used o e i y he assump ions o no mali y and homogenei y o a iances, espec i ely. All he s a is ical es s we e pe o med using he s a is ical so wa e package R. Au ho Con ibu ions: Concep ualiza ion, me hodology, o mal analysis, w i ing—o iginal d a , w i ing— e iew and edi ing, J.A.P.-R.; concep ualiza ion, me hodology, unding acquisi ion, w i ing— e iew and edi ing, E.M.-N.; me hodology, o mal analysis, w i ing— e iew and edi ing, J.L.-J.; concep ualiza ion, unding acquisi ion, w i ing— e iew and edi ing, S.R.-G.; concep ualiza ion, me hodology, o mal analysis, esou ces, w i ing— e iew and edi ing, J.M.T.-R. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . 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