Physiological and Biochemical Responses of Common Bush Bean to Drought

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1 demipres ville online: Print ISSN X; Eletroni Not Bot Horti groo, 218, 46(2): DOI: /nh Originl rtile Notule Botnie Horti grootnii Cluj-Npo Physiologil nd Biohemil Responses of Common Bush Ben to Drought lefsi Dvid SÁNCHEZ-REINOSO, Gustvo dolfo LIGRRETO-MORENO, Hermnn RESTREPO-DÍZ* Universidd Nionl de Colomi, Fultd de Cienis grris, Deprtmento de gronomí, Crrer 3 No 45-3 Edifiio 5, Bogotá, Colomi; dsnhezre@unl.edu.o; gligrretom@unl.edu.o; hrestrepod@unl.edu.o (*orresponding uthor) strt griulture hs een dversely ffeted y the low wter vilility resulting from limte hnge, reting environmentl stress for the ommon en (Phseolus vulgris L.). growth room experiment ws performed to evlute the physiologil nd iohemil responses of the severl ush en genotypes to wter defiit onditions. Plnts in soil with 2 g L -1 polyethylene glyol 6 (PEG) were sujeted to drought for 15 d. The levels of photosynthesis, stomtl ondutne nd trnspirtion in ll genotypes deresed y pproximtely 65% under wter defiit onditions ompred with the orresponding vlues in the ontrols. Wter use effiieny ws enhned y wter defiit onditions, with ʻBinʼ plnts exhiiting the highest vlues (28.8 µmol mol -1 ), followed y ʻNU35ʼ, ʻBhueʼ nd ʻCerinzʼ (2.46, 2.11 nd µmol mol -1, respetively). The ʻBinʼ plnts exhiited lower reltive tolerne index (5%), nd wter defiit inresed the levels of lef photosyntheti pigments, hlorophyll nd rotenoids in this genotype y pproximtely 1%. The photosyntheti effiieny, whih ws evluted using the F v /F m rtio nd rpid light-derived prmeters (the mximum eletron trnsport rte nd light sturtion prmeter), deresed due to wter defiit onditions, prtiulrly in the ʻBinʼ plnts, in whih these prmeters were redued y pproximtely 6%. The proline nd mlondildehyde (MD) ontents were inresed y the ddition of PEG, primrily in the ʻBtáʼ nd ʻBinʼ plnts. In onlusion, our results suggest tht rpid light-response urves n e useful for hrterizing genotypes euse they represent n esy nd non-destrutive tool for understnding limtiztion mehnisms under wter defiit onditions. In ddition, ll genotypes exhiited suseptiility to wter defiit onditions, nd the most suseptile genotype ws ʻBinʼ, s refleted y signifint redution in the eletron trnsport rte nd the presene of oxidtive dmge (high MD ontent nd eletrolyte lekge), suggesting tht this ultivr ould not dpt well to lndsping situtions in whih periods of extreme wter defiit n e expeted. Keywords: lipid peroxidtion; photosynthesis; proline; rpid light-response urves; reltive tolerne index Introdution The ommon en (Phseolus vulgris L.) is trditionl rop of the neotropis nd primry soure of protein in the diets of popultions in developing ountries (Beee et l., 212). signifint prodution onstrint of ommon en rops is wter defiit, whih ffets up to 6% of enproduing regions (Beee et l., 212). In the nden region, P. vulgris is minly grown y smll holder frmers, often under unfvorle onditions due to drought nd het (Beee et l., 28; Ome et l., 212). Climte hnge dversely ffets food prodution due to inresed drought in prodution res (Di, 211). Glol wrming n generte hnges in rinfll ptterns in the nden ountries, ffeting the vilility of wter for griulture (Brdley et l., 26). By 25, limte hnge will ffet 8% of rops in Colomi, using hnges in rop phenology nd ultimtely the produt hin of ommon ens (Rmirez-Villegs et l., 212). Plnts disply dverse effets in growth, development nd yield in response to wter defiit (Loell nd Gourdji, 212). Knowledge of plnt physiology hs helped identify strtegies used y drought-resistnt genotypes to ope with wter defiits (Polni et l., 216). P. vulgris plnts under wter defiit onditions dopt higher wter use effiieny (WUE) (De Lt, 214; Polni et l., 216). Polni et l. (216) lso stted tht hrteristi of en genotypes Reeived: 11 Jul 217. Reeived in revised form: 19 De 217. epted: 6 Jn 218. Pulished online: 24 Jn 218.

2 Sánhez-Reinoso D et l / Not Bot Horti groo, 218, 46(2): with drought tolerne is the ility to moilize photosynthtes to pods nd seeds. Others hve reported tht greter root length under wter defiit onditions ontriutes to improved drought resistne of the ommon en (White nd Cstillo, 1992; Polni et l., 29). The hlorophyll fluoresene tehnique hs eome importnt in plnt eophysiologil studies euse photosystem II (PSII) is sensitive to wter defiit (Lu nd Zhng, 1999; Mxwell nd Johnson, 2). Consequently, hlorophyll fluoresene prmeters re used to sreen for tolerne to ioti stress (Syed, 23). Rpid lightresponse urves llow mesurement of the effetive quntum yield s funtion of irrdine. This tehnique provides omplete informtion on the sturtion hrteristis of eletrons nd is powerful tool for ssessing photosyntheti tivity (Rlph et l., 22). The rpid light urve-derived prmeters [the mximum reltive eletron trnsport rte (mx), minimum sturting irrdine (Ek) nd initil slope of the urve (α)] indite photosyntheti effiieny in plnts under stress (Xu et l., 213). Plnts exposed to wter defiit exhiit other limtion mehnisms, suh s (i) deresed lef hlorophyll ontent (Chvez et l., 22), (ii) inresed proline prodution (Rosles et l., 212), nd (iii) ell memrne dmge due to lipid peroxidtion, using the genertion of peroxide ions nd mlondildehyde (MD) (Sánhez-Rodríguez et l., 21). Biohemil prmeters re importnt mrkers for tegorizing suseptile nd/or tolernt genotypes with respet to responses to wter defiit (Ghnri et l., 213; Siddiqui et l., 215). physiologil pproh n help inrese the ville knowledge regrding the ehvior of ommon en genotypes to limte hnge (Restrepo-Diz et l., 21; Polni et l., 216). The im of this study ws to evlute phenotypi differenes in the wter defiit tolernes of ommon ush en genotypes growing in regions where periods of drought n e expeted sed on physiologil nd iohemil trits. Mterils nd Methods Biologil mterils nd growth onditions The experiment ws performed from Septemer to Novemer 214 in growth room of the Fulty of griulturl Sienes t Universidd Nionl de Colomi, Bogotá Cmpus (4 35'56" N nd 74 4'51" W). Seeds of the following ush en genotypes were used: ʻCerinzʼ nd ʻBhueʼ, whih hve een used in trditionl Colomin griulture for 2 yers, ʻNU35ʼ, whih is genotype developed y CIT tht hs een ommerilized for nine yers sine its relese, nd ʻBinʼ nd ʻBtáʼ, whih re ultivrs tht hve een relesed for less thn two yers. Seeds were pre-germinted in growth hmer for 7 dys in the drk t reltive humidity (RH) of 75% nd n mient temperture of 25 C nd then trnsplnted into floting hydroponi system in 8-L glss ontiners. The following environmentl onditions were mintined in the growth room throughout the experiment: dy/night temperture of 26/22 C, RH of 6 to 8 %, nd 12-h rtifiil photoperiod using inndesent lmps supplying 8-µmol m -2 s -1 photosynthetilly tive rdition (PR). The plnts were grown in nutrient solution using liquid 4N-4P-2K fertilizer ontining mironutrients (Nutri- Poni, Wlo S.., Bogotá D.C., Colomi) t rte of 2.5 ml L -1 ; the ph rnged from 5.5 to 6.. The nutrient solution ws refilled every other dy to mintin onstnt volume (42 L) in the ontiners nd ws lwys erted y n eletri om. Defiit tretments Wter defiit ws imposed when plnts hd three or four fully expnded trifolite leves, pproximtely 4 dys fter trnsplnting (DT). The wter defiit tretment ws indued y the ddition of polyethylene glyol 6 (PEG) (PnRe, Brelon, Spin) to the nutrient solution t rte of 2 g L -1 (De Lt et l., 214). The plnts were exposed to the solution ontining PEG from 4 to 55 DT. Physiologil mesures The reltive wter ontent (RWC) in fully expnded leves from the upper prt of the nopy ws determined. The leves were olleted t 55 DT for ll genotypes. The RWC ws lulted ording to the eqution desried y Clvijo-Sánhez et l. (215) using fresh nd turgid weights fter sumergene in distilled wter for 24 h t 4 C in the drk. The levels of photosynthesis (Pn), stomtl ondutne (g s) nd trnspirtion (E) in fully expnded trifolite lef from the upper hlf of the plnts were mesured t 54 nd 55 DT etween 1: nd 15: using portle photosynthesis meter (LSPro-SD, DC BioSientifi Ltd., UK). The hmer onditions during lef gs exhnge mesurements were the following: PR, 8 μmol m -2 s -1 ; lef temperture, 25 ± 2 C; nd CO2 onentrtion, 4 ± 1 μmol mol -1. The extrinsi wter use effiieny (WUE) ws lulted s the Pn/E rtio. The plnts were hrvested t 55 DT nd dried in ompressed ir oven t 7 C for 48 h to otin the lef, stem, root nd totl plnt dry weights. The shoot:root rtio (S/R) ws lulted sed on the reltionship etween the dry weight of the eril prts (leves nd stem) to tht of the roots. dditionlly, the reltive tolerne index (RTI) ws otined using the eqution desried y Dutt Gupt et l. (1995). The Fv/Fm rtio nd rpid light-response urves (RLCs) t 54 nd 55 DT were determined using modulted hlorophyll fluoresene meter (MINI-PM, Wlz, Effeltrih, Germny). fter mesurement of the lef-gs exhnge, the sme leves were dpted to the drk for 15 min. The Fv/Fm mesurements were performed y pplying pulse with mximum light intensity of up to 2.6 µmol m -2 s -1 to the surfe of lef smples. The RLCs were onstruted y plotting the eletron trnsport rte () versus the inresing tini irrdine (from 1 to µmol m -2 s -1 ) with 1-s intervls etween the irrdine levels. The prmeters α (initil slope), mx (mximum ) nd Ik ( light-sturtion prmeter) were estimted using the model desried y Xu et l. (214).

3 The lef photosyntheti pigments were mesured t 55 DT, nd pproximtely 3 mg of the seond fully expnded trifolite lef ws homogenized in 4 ml of 8% etone. Susequently, the smples were entrifuged (Model 4211, Beton Dikinson Primry Cre Dignostis, MD, US) t 3 g to remove prtiles. The superntnt ws diluted to finl volume of 6 ml y dding etone. The lef hlorophyll ontent ws mesured t 663 nd 646 nm, nd the rotenoid levels were determined t 47 nm using spetrophotometer (Spetroni BioMte 3 UV-VIS, Thermo, WI, US). The equtions desried y Wellurn (1994) were used to lulte the lef photosyntheti pigment ontents. Eletrolyte lekge, lipid peroxidtion nd proline ontent Dmge to the plsm memrne ws estimted s the perentge of eletrolyte lekge t 55 DT following the method desried y Snhez-Reinoso et l. (214). Five diss (.5 m dimeter) from the seond fully expnded trifolite lef were extrted in 5-mL Flon tues using 25 ml of deionized wter s the medium. The initil eletril ondutivity of the smples (CE 1) ws reorded with ondutivity meter (Model P7, Okton Instruments, Vernon Hills, IL, US). The smples were pled in wter th (Model B-48, Bühi Lortehnik G, Switzerlnd) t 3 C for 2 h, nd the finl eletril ondutivity (CE 2) ws then mesured fter 2 min in wter th t 9 C. The thiorituri id (TB) method desried y Hodges et l. (1999) ws used to ssess lipid oxidtion sed on the MD onentrtion. pproximtely.3 g of homogenized plnt mteril ws olleted t 55 DT nd stored in liquid nitrogen. The smples were entrifuged t 3 g, nd the sorne vlues t 44, 532 nd 6 nm were estimted using spetrophotometer (Spetroni BioMte 3 UV-VIS, Thermo, WI, US). n extintion Sánhez-Reinoso D et l / Not Bot Horti groo, 218, 46(2): oeffiient ws used (157 M ml -1 ) to lulte the MD onentrtion. pproximtely.3 g of homogenized plnt mteril ws olleted from the seond fully expnded trifolite lef t 55 DT nd stored in liquid nitrogen. The sorne ws mesured t 52 nm using spetrophotometer (Spetroni), nd the proline onentrtion ws determined using stndrd urve nd the eqution desried y Btes et l. (1973). Experimentl design nd sttistil nlysis ftoril design, in whih the first ftor ws the wter stress tretment nd the seond ftor ws the ultivrs evluted, resulting in totl of 1 tretments with four replites (4 seedlings in totl), ws dopted. Perentge vlues were trnsformed using the rsine funtion. vrine nlysis followed y the omprtive Tukey s test ws performed. The dt were nlyzed using Sttistix (ver. 9., nlytil Softwre, Tllhssee, FL, US), nd SigmPlot (version 1.; Systt Softwre, Sn Jose, C, US) ws used to drw three-dimensionl plots nd to perform luster nlysis. Results nd Disussion Physiologil mesures Most of the vriles were ffeted y the wter tretment or genotype nd their intertion (Tle 1); however, the reltive wter ontent ws ffeted y the wter tretment ut not the genotype or their intertion, nd stomtl ondutne ws not ffeted y genotype. The plnts under wter defiit onditions hd lower RWC ompred with the ontrol plnts (Fig. 1). RWC is n importnt physiologil mesurement tht helps quntify plnt s wter sttus under norml or drought 395 Tle 1. Effets of wter stress on the physiologil ehvior of Phseolus vulgris L. genotypes sed on NOV Vrile revition Vrine soure Wter tretment (W) Genotype (G) Intertion (W G) Reltive wter ontent RWC *** NS NS Photosynthesis Pn *** ** * Stomtl ondutne gs *** NS *** Trnspirtion E *** *** * Wter use effiieny WUE *** *** *** eril dry weight DW ** *** * Root dry weight RDW *** *** * Totl dry weight TDW *** *** ** Shoot:root rtio P/R NS *** *** Chlorophyll Chl ** *** *** Chlorophyll Chl * *** *** Totl hlorophyll ontent Chl totl * *** *** Crotenoids Cx+ * *** *** Mximum effiieny of PSII Fv/Fm *** * * Eletrolyte lekge *** * ** Mlondildehyde MD *** ** * Proline *** *** *** Initil slope α *** *** ** Mximum reltive eletron trnsport rte mx *** * *** Light sturtion prmeter EK *** *** *** NS, *, **, nd *** indite not signifint t P.5 or signifint t proility levels of.5,.1 nd.1, respetively.

4 396 Reltive wter ontent (%) Sánhez-Reinoso D et l / Not Bot Horti groo, 218, 46(2): Fig. 1. Effet of wter defiit on the reltive wter ontent (RWC) in leves of ommon en (Phseolus vulgris L.). The rs represent the mens of 2 dt points ± stndrd errors Pn (µmol. m -2. s -1 ) g s (µmol. m -2. s -1 ) Cerinz Bhue NU35 Btá Bin B onditions (Keyvn, 21). Redued RWC vlues indite tht plnt-wter reltionships might e ffeted y dverse ioti onditions, whih ould redue the photosyntheti rte (Cost-Frnç et l., 2). The intertion etween wter tretment nd genotype ffeted Pn (Tle 1). In response to wter defiit, the Pn level in ʻBhueʼ deresed y pproximtely 6%, wheres tht in ʻBtáʼ, ʻBinʼ, ʻCerinzʼ nd ʻNU35ʼ deresed y pproximtely 8% (Fig. 2). The wter defiit ondition used the gretest g s redution in ʻBinʼ (Fig. 2B). dditionlly, E deresed y pproximtely 7% ross ll genotypes in response to the imposed wter defiit (Fig. 2C). Lnn et l. (216) nd Mthoo et l. (217) found tht moderte wter defiit led to severe redution ( 45%) in photosynthesis. Wter defiit n inhiit lef photosynthesis due to ltertions in the photosyntheti pprtus, redued stomtl perture, deresed trnspirtion, hnges in light sorption, nd ltertions of the iohemil pthwys ssoited with CO2 fixtion (Frooq, 29). WUE ws signifintly ffeted y the intertion etween wter tretment nd genotype (Tle 1). In generl, differenes were not oserved etween genotypes under ontrol onditions. However, when the plnts were sujeted to wter defiit, the WUE of ll the genotypes inresed. ʻBinʼ hd the highest vlues, followed y the ʻNU35ʼ, ʻBhueʼ nd ʻCerinzʼ ultivrs (Fig. 3). Inresed WUE in P. vulgris genotypes hs een reported to our s physiologil response to wter-stress WUE (µmol m -2 s -1 /mmol m -2 s -1 ) Estrés. Cerinz Bhue NU35 Btá Bin d d d d d Cerinz Bhue NU35 Btá Bin E (mol. m -2. s -1 ) C RTI (%) B 2 2 Cerinz Bhue NU35 Btá Bin Fig. 2. Effets of the intertion etween genotype nd wter tretment on net photosynthesis (Pn) (), stomtl ondutne (g s ) (B) nd lef trnspirtion (E) (C) in the ush en genotypes. The dt represent the verge of four plnts per tretment (n = 4). The mens followed y the sme letter re not signifintly different ording to Tukey s test t P.5 Cerinz Bhue NU35 Btá Bin Fig. 3. Effets of the intertion etween genotype nd wter defiit on wter use effiieny (WUE) () nd the reltive tolerne index (RTI) (B) in the ommon en genotypes (Phseolus vulgris L). The dt represent the verge of four plnts per tretment (n = 4). The mens followed y the sme letter re not signifintly different ording to Tukey s test t P.5

5 onditions (Munoz-Pere et l., 27; Lnn et l., 216), suggesting tht greter WUE under wter defiit onditions n likely led to redutions in seed prodution nd resistne to wter stress (Polni et l., 216). The intertion lso ffeted the plnts eril prts nd roots, the totl plnt dry weights nd the shoot:root rtio (Tle 2). In generl, the ʻCerinzʼ plnts under the wter defiit onditions hd the lowest eril prt dry mtter ompred with the other genotypes. The root dry weights of ʻBinʼ, ʻBtáʼ, ʻCerinzʼ nd ʻNU35ʼ were redued y the wter defiit, wheres the root dry weight of ʻBhueʼ plnts inresed. The totl plnt dry weights of ʻBinʼ, ʻBtáʼ nd ʻCerinzʼ umulted less iomss under the wter defiit onditions, ut the iomss of ʻBhueʼ nd ʻNU35ʼ did not vry etween tretments. The ʻCerinzʼ, ʻBinʼ, nd ʻBhueʼ plnts hd lower shoot:root rtios ompred with the ʻBtáʼ nd ʻNU35ʼ plnts. It hs een reported tht wter defiit results in less iomss umultion in P. vulgris genotypes (Munoz-Pere et l., 27; Lnn et l., 216, Mthoo et l., 217). However, Polni et l. (216) reported tht higher root dry weight under drought onditions is n importnt physiologil trit tht ould e used to hrterize tolernt genotype. ʻBhueʼ produes vigorous root system, suggesting tht this genotype might e tolernt to drought. indies, suh s the RTI, hve een used to sreen genotypes in response to wter defiits. Tolernt genotypes exhiit the highest RTI sores (Fernndez, 1993). The RTI results reveled different drought-tolernt responses mong the genotypes nd in response to the wter tretment. The ʻCerinzʼ nd ʻBinʼ plnts hd the lowest vlues, inditing tht these genotypes re suseptile to drought (Fig. 4). Suseptile genotypes hve een reported to exhiit lower iomss umultion, s refleted y lower RTI (Drkw et l., 216). In generl, the hlorophyll nd nd the totl hlorophyll ontents of the plnts sujeted to wter defiit Sánhez-Reinoso D et l / Not Bot Horti groo, 218, 46(2): did not vry ompred with the orresponding vlues in the ontrol plnts, exept for ʻBinʼ. In Bin, the lef hlorophyll ontents (, nd totl) were 2-fold higher thn those in the ontrol plnts (Fig. 5, B, C). ʻBinʼ plnts exposed to wter defiit exhiited higher lef rotenoid ontent thn the ontrol plnts (Fig. 5D). The ility to mintin the lef hlorophyll ontent under ioti stress hs een used s prmeter in the seletion of tolernt ultivrs (Kini-Pouy nd Rsouli, 214). The lef rotenoid ontent (nother lef photosyntheti pigment) lso plys role in tolerne to drought (Frooq et l., 29). The levels of lef photosyntheti pigments were fvorle under moderte wter defiit, prtiulrly in 'Bin'. n inrese in the ontents of these pigments n e onsidered response mehnism to redue the dverse effets of wter defiitindued photoinhiition (Silv et l., 27). ʻBinʼ plnts hd lower Fv/Fm rtio thn the ʻCerinzʼ, ʻBhueʼ, ʻNU35ʼ nd ʻBtáʼ under wter defiit onditions (Fig. 6). The RLCs showed differenes etween wter tretments (Fig. 7). The vlues of ll genotypes under wter defiit onditions generlly deresed due to n inrese in tini irrdine. Higher α vlues were otined for ll genotypes under wter defiit onditions, wheres mx nd I K deresed. Similr results hve een found for ush lover (Lespedez dvuri (Lx.) Shindl) (Xu et l., 214). RLCs represent tool for estimting photosyntheti tivity euse these mesurements provide omplete informtion on the sturtion hrteristis of eletrons (Rlph et l., 22), nd dereses in mx nd I K indite redution in the photosyntheti effiieny of plnts under ioti stress (Song et l., 213). ʻBinʼ plnts onsistently exhiited lower rpid light-derived prmeters (mx nd I K) nd Fv/Fm rtios ompred with the other genotypes, inditing tht the photosyntheti tivity ws severely ffeted y the wter defiit. 397 Tle 2. Effets of the intertion etween wter defiit nd genotype on the initil slope of the urve (α), the mximum eletron trnsport rte (mx) nd minimum sturtion irrdine (IK) Tretment α (µmol m -2 s -1 ) mx IK (µmol m -2 s -1 ) Cerinz Bhue NU d Btá Bin Cerinz e e Bhue e e NU de e Btá d d Bin e 16.9 e Signifine ** *** *** ** nd *** refer to intertion dt nlyzed using the lest squres mens nd mens seprted t P <.5 nd P <.1, respetively. The dt represent the verge of four plnts per tretment (n = 4). The mens followed y the sme letter re not signifintly different ording to Tukey s test t P.5.

6 Sánhez-Reinoso D et l / Not Bot Horti groo, 218, 46(2): Chl (mg.g -1 FW) d d Chl (mg.g -1 FW) B Cerinz Bhue NU35 Bt Bin 2 Cerinz Bhue NU35 Bt Bin Totl Chl (mg.g -1 FW) C Crotenoids (mg.g -1 FW) D Cerinz Bhue NU35 Bt Bin Cerinz Bhue NU35 Bt Bin Fig. 4. Effets of the intertion etween genotype nd wter defiit on the totl hlorophyll (), hlorophyll (B), hlorophyll (C) nd totl rotenoid (D) ontents in the leves of ommon ush en (Phseolus vulgris L.). The dt represent the verge of four plnts per tretment (n = 4). The mens followed y the sme letter re not signifintly different ording to Tukey s test t P.5 F v /F m Cerinz Bhue NU35 Bt Bin Fig. 5. Effet of the intertion etween genotype nd wter defiit on the PSII effiieny (F v /F m ) in leves of ommon ush en (Phseolus vulgris L). The dt represent the verge of four plnts per tretment (n = 4). The mens followed y the sme letter re not signifintly different ording to Tukey s test t P.5 Eletrolyte lekge, lipid peroxidtion nd proline ontent Eletrolyte lekge nd the MD nd proline ontents were ffeted y the intertion etween wter tretment nd genotype. Eletrolyte lekge in the ʻCerinzʼ, ʻNU35ʼ, ʻBtáʼ nd ʻBinʼ genotypes inresed under wter defiit onditions, nd ʻBhueʼ plnts mintined levels similr to those mesured in the ontrols (Fig. 7). higher MD ontent ws oserved in ll the genotypes sujeted to wter defiit onditions, nd the highest vlues were otined in the ʻBinʼ nd ʻBtáʼ plnts (Fig. 7B). dditionlly, the lef proline ontent inresed under wter defiit onditions: ʻCerinzʼ nd ʻBinʼ hd the highest vlues of this osmolyte, followed y ʻBtáʼ, ʻNU35ʼ nd ʻBhueʼ (Fig. 7C). Wter defiits hve een reported to enhne the proline nd MD ontents of ommon en nd f en (Vii f L.) (Ghnri et l., 213; Siddiqui et l., 215). Inreses in the proline ontent under wter defiit onditions help mintin hydrtion in ells nd prevent dmge. This mino id ts s n osmolyte nd s storge soure for ron nd nitrogen, whih re susequently used to stilize mromoleules, proteins nd ell memrnes in plnt tissues (George et l., 215). Proline hs een proposed s iohemil mrker to determine the tolerne of genotype to wter defiit onditions (Nser et l., 21). Higher MD umultion indites greter dmge t the ellulr level due to inresed memrne lipid peroxidtion (Snhez-Reinoso et l., 214). It hs een reported tht higher proline ontent, lower MD prodution nd eletrolyte lekge under wter defiit onditions re useful inditors of plnt tolerne, nd these fetures n e used for the seletion of tolernt genotypes (Liu et l., 213; Snhez-Reinoso et l., 214). The results otined from the nlyses of the lef proline ontent, eletrolyte lekge nd MD ontent plots indite tht Btáʼ nd ʻCerinzʼ re drought-suseptile genotypes sed on (Fig. 8). The luster nlysis identified ʻBinʼ s drought-suseptile genotype, ʻCerinzʼ, ʻBtáʼ nd ʻNU35ʼ s modertely suseptile genotypes, nd ʻBhueʼ s modertely tolernt to wter defiit onditions (Fig. 9).

7 Sánhez-Reinoso D et l / Not Bot Horti groo, 218, 46(2): B C Irrdine (µmol. m 12 1 Irrdine (µmol. m D E Irrdine (µmol. m Irrdine (µmol. m Irrdine (µmol. m Fig. 6. Rpid light-response urves of ʻCerinzʼ (), ʻBtáʼ (B), ʻBinʼ (C), ʻBhueʼ nd ʻNU35ʼ (D) genotypes due to wter defiit. The dt represent the men of four dt points ± stndrd errors Eletrolytes lekge (%) Proline (µmol.g -1 FW) Bin stress Cerinz stress Btá stress NU35 stress MD (µmol.g -1 FW) Proline (µmol.g -1 FW) d d Cerinz Bhue NU35 Bt Bin Fig. 7. Effets of the intertion etween genotype nd wter defiit on eletrolyte lekge (), MD ontent (B) nd proline ontent (C) mesured in the leves of ommon ush en (Phseolus vulgris L.) genotypes. The dt represent the verge of four plnts per tretment (n = 4). The mens followed y the sme letter re not signifintly different ording to Tukey s test t P.5 B C Bhue stress MD (µmol.g -1 FW) Bin ontrol Cerinz ontrol Bhue ontrol Btá ontrol NU35 ontrol Eletrolytes lekge (%) Fig. 8. Three-dimensionl plot (proline ontent, MD ontent nd eletrolyte lekge) for ommon ush en (Phseolus vulgris L.) genotypes under wter defiit onditions. The dt represent the men of four dt points intil Versión Estudintil Ver Bin wter defiit sión Estudintil Versión Estudi intil Versión Estudintil Ver Cerinz wter defiit sión Estudintil Versión Estudi intil Versión Estudintil Ver NU35 wter defiit sión Estudintil Versión Estudi intil Versión Estudintil Ver Btá wter defiit sión Estudintil Versión Estudi intil Versión Estudintil Ver Bhue wter defiit sión Estudintil Versión Estudi Versión Estudintil Versión Estudintil Versión Estudintil Bin ontrol Versión Estudintil Versión Estudintil Versión Estudintil NU35 Versión ontrol Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Cerinz Versión ontrol Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Bhue Versión ontrol Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Btá Versión ontrol Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Versión Estudintil. Versión Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Versión Estudintil Fig. 9. Dendrogrm using the Euliden distne to hrterize ommon ush en (Phseolus vulgris L.) genotypes under ontrol nd wter defiit onditions

8 Sánhez-Reinoso D et l / Not Bot Horti groo, 218, 46(2): Conlusions ʻBinʼ ws identified s the most suseptile genotype to wter defiit, nd ʻBhueʼ n e onsidered modertely tolernt to short periods of wter defiit nd n dpt to rid regions or to situtions in whih periods of moderte drought n e expeted. Our results indite tht physiologil vriles suh s Pn, the Fv/Fm rtio, RLCs nd the MD nd proline ontents n e used to hrterize genotypes tht re tolernt to wter defiit onditions. Referenes Btes L, Wldren R, Tere I (1973). Rpid determintion of free proline for wter-stress studies. Plnt nd Soil 39: Beee S, Ro IM, Cjio C, Grjles M (28). Seletion for drought resistne in ommon en lso improves yield in phosphorus limited nd fvorle environments. Crop Siene 48: Beee S, Ro IM, Muknkusi C, Buruhr R (212). Improving resoure use effiieny nd reduing risk of ommon en prodution in fri, Ltin meri nd the Crien. In: Hershey C (Ed). Issues in tropil griulture. I. Eo-effiieny: From vision to relity. 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9 Sánhez-Reinoso D et l / Not Bot Horti groo, 218, 46(2): Polni J, Ro IM, Cjio C, River M, Rtz B, Beee S (216). Physiologil trits ssoited with drought resistne in nden nd Mesomerin genotypes of ommon en (Phseolus vulgris L.). Euphyti 21: Rlph PJ, Polk SM, Moore K, Orth RJ, Smith, Jr. WO (22). Opertion of the xnthophyll yle in the segrss (Zoster mrin) in response to vrile irrdine. Journl of Experimentl Mrine Biology nd Eology 271: Rmírez-Villegs J, Slzr M, Jrvis, Nvrro-Rines CE (212). wy forwrd on dpttion to limte hnge in Colomin griulture: perspetives towrds 25. Climti Chnge 115: Restrepo-Díz H, Melgr JC, Lomrdini L (21). Eophysiology of hortiulturl rops: n overview. gronomí Colomin 28: Rosles M, Ompo E, Rodríguez-Vlentín R, Olver-Crrillo Y, ost- Gllegos J, Covrruis (212). Physiologil nlysis of ommon en (Phseolus vulgris L.) ultivrs unovers hrteristis relted to terminl drought resistne. Plnt Physiology nd Biohemistry 56: Sánhez-Reinoso D, Grés-Vrón G, Restrepo-Díz H (214). Biohemil nd physiologil hrteriztion of three rie ultivrs under different dytime temperture onditions. Chilen Journl of griulturl Reserh 74: Sánhez-Rodríguez E, Ruio-Wilhelmi M, Cervill LM, Blso JJ, Rios JJ, Rosles M, Romero L, Ruiz JM (21). Genotypi differenes in some physiologil prmeters symptomti for oxidtive stress under moderte drought in tomto plnts. Plnt Siene 178: Syed OH (23). Chlorophyll fluoresene s tool in erel rop reserh. Photosyntheti 41: Siddiqui MH, l-khishny MY, l-qutmi M, l-whii MH, Grover, li HM, l-whii MS, Bukhri N (215). response of different genotypes of F en plnt to drought stress. Interntionl Journl of Moleulr Sienes 16: Silv M, Jifon JL, D Silv JG, Shrm V (27). Use of physiologil prmeters s fst tools to sreen for drought tolerne in sugrne. Brzilin Journl of Plnt Physiology 19: Song R, Zho CY, Liu J, Zhng J, Du YX, Li JZ, Sun HZ, Zho HB, Zho QZ (213). Effet of sulphte nutrition on rseni trnslotion nd photosynthesis of rie seedlings. t Physiologi Plntrum 35: Wellurn R (1994). The spetrl determintion of hlorophylls nd, s well s totl rotenoids, using vrious solvents with spetrophotometers of different resolution. Journl of Plnt Physiology 144: White JW, Cstillo J (1992). Evlution of diverse shoot genotypes on seleted root genotypes of ommon en under soil wter defiits. Crop Siene 32: Xu WZ, Deng XP, Xu BC, Go ZJ, Ding WL (214). Photosyntheti tivity nd effiieny of Bothriohlo ishemum nd Lespedez dvuri in mixtures ross growth periods under wter stress. t Physiologi Plntrum 36: