Physiological and biochemical responses to drought stress in Barbados cherry

Transcription

1 2012 BRAZILIAN SOCIETY OF PLANT PHYSIOLOGY RESEARCH ARTICLE Physiologicl nd iochemicl responses to drought stress in Brdos cherry Dvid Bros Medeiros 1 *, Elizmr Cirico d Silv 2, Hugo Rfel Bentzen Sntos 3, Cinthy Mirell Pcheco 4, Rosimr dos Sntos Musser 1, Rejne Jurem Mnsur Custódio Nogueir 5 1 Deprtmento de Agronomi, Universidde Federl Rurl de Pernmuco, Recife, PE, Brzil. 2 Deprtmento de Biologi, Centro de Ciêncis Biológics e d Súde, Universidde Federl de Sergipe, São Cristóvão, SE, Brzil. 3 Deprtmento de Tecnologi Rurl, Universidde Federl Rurl de Pernmuco, Recife, PE, Brzil. 4 Deprtmento de Genétic, Centro de Ciêncis Biológics, Universidde Federl de Pernmuco, Recife, PE, Brzil. 5 Deprtmento de Biologi, Universidde Federl Rurl de Pernmuco, Recife, PE, Brzil. *Corresponding uthor: dvidmedeiros3@hotmil.com Received: 25 My 2012; Accepted: 3 Septemer 2012 ABSTRACT To evlute physiologicl genotypic differences etween two Brdos cherry genotypes (13- nd 14-CPA) under wter deficit, initil growth, wter reltions, nd orgnic solute ccumultion were evluted in n experiment performed using four-month-old seedlings, which were sujected to four wter tretments (100, 75, 50, nd 25% of field cpcity), with five replictions. Severe wter deficit (25% of field cpcity) negtively ffected plnt height, stem dimeter, lef re, dry mtter of the leves nd stem to oth genotypes, nd root dry mtter to genotype 13-CPA. Predwn (Y wpd ) nd middy lef wter potentils (Y wmd ) were reduced in plnts grown under 25% of field cpcity, only in the genotype 14-CPA. There ws not chnge in reltive wter content, even with the reduction in the lef wter potentil. Severe wter deficit did not induce orgnic solutes ccumultion, insted it reduced crohydrte content in leves of genotypes nd minocids, proline nd proteins, in genotype 13-CPA. In the roots ccumultion of ll orgnic solutes studied, it ws verified genotype 13-CPA under 25% of field cpcity, ut only crohydrtes incresed in plnts under 25% of field cpcity to 14-CPA. These results suggest two different mechnisms used y Brdos cherry genotypes to mintin the wter sttus. To the 13-CPA one, the ccumultion of solule orgnic solutes in the roots is the min mechnism used to mintin the tissue hydrtion. However, the 14-CPA genotype chnged the root to shoot rtio in order to void desicction. Despite the mechnism used y oth genotypes, moderte drought stress does not induce significnt morphophysiologicl chnges in Brdos cherry. Keywords: Mlpighi emrgint DC, solule orgnic solutes, iomss lloction, wter reltions, wter deficit. Brz. J. Plnt Physiol., 24(3): , 2012

2 182 MEDEIROS D. B. et l. INTRODUCTION Brdos cherry (Mlpighi emrgint DC) is crop with gret economic potentil in Brzil due to its high nutritionl vlue. Its fruit hs high concentrtion of scoric cid (AA), which mkes it n importnt species in regions with low-income popultions. The wide cceptnce of its fruit in foreign mrkets hs led to its exporttion s fresh nd processed product. Severl environmentl fctors limit the production of this crop, including the rinfll shortge, since wter ffects plnt growth nd development. During drought seson, the inflow of wter diminishes due the soil drying, which consequently ffects ll the growth processes dependent on turgor pressure, such s cell elongtion nd division (Lrcher, 2006). To evlute plnt wter sttus, the lef wter potentil (Ψ w ) nd reltive wter content (RWC) re essentil prmeters generlly used to study plnt physiologicl responses to drought stress (Silv et l., 2009; Mrchese et l., 2010; Silv et l., 2010). Silv et l. (2009) stted tht these prmeters decrese in most plnts under wter deficit. Among the vrious mechnisms used y plnts to reduce the negtive effects of wter stress, mny plnt species ccumulte solule orgnic compounds, such s osmoregultors. This process is known s osmotic djustment nd it is considered s n importnt tolerncemechnism, which llows the mintennce of cellulr turgor nd fvors the sorption of wter (White et l., 2000; Chves et l., 2003). In this process, the iosynthesis nd ccumultion of nontoxic molecules of low-moleculr weight in the vcuole nd cytosol, such s inorgnic ions, solule sugrs, minocids, proline, glycine-etine, mong others, contriute to keep the integrity of cellulr memrnes nd proteins, which re necessry for metolic ctivities. Therefore, studies on the ccumultion of these molecules hve een used s physiologicl indictors in the evlution of drought tolernce in severl species (Sho et l., 2006; Frooq et l., 2009). The different physiologicl mechnisms used y different plnt species to tolerte drought re lso found within one species or mong genotypes, vrieties, or cultivrs. In this context, studies hve een done iming t comprehending how some species or cultivrs support drought periods nd how the physiologicl nd metolic processes modulte this tolernce in severl species, such s Brdos cherry (Nogueir et l., 2001), citrus (Peixoto et l., 2006; Al-Asi, 2009; Nscimento et l., 2010), umu tree (Silv et l., 2009, ), grpe (Toumi et l., 2008; Sntesten et l., 2009), whet (Sho et l., 2006), nd numer of others. These uthors hve found intrspecific differences in drought tolernce evidencing different responses under wter stresses s result of cscde in physiologicl nd iochemicl events, which culminte in chnges on growth ptterns. As oserved y Peixoto et l. (2006), chnges on growth prmeters (lef re rtio LAR, reltive growth rte, nd net ssimiltion rte) in citrus genotypes support tht short periods of drought help to mintin positive lnce of photosynthesis nd dry mss production. Specificlly in Brdos cherry, some physiologicl chnges in gs exchnge nd wter reltions were oserved fter 20 dys of wtering disruption, with the restriction on trnspirtion rte y strictly increse in stomtl resistnce nd decrese in Ψ w nd RWC (Nogueir et l., 2001). Intrspecific differences were lso oserved in umu tree genotypes during intermittent drought, which present n isohydric ehvior, ut it hs gret vriility in the production of orgnic solutes etween genotypes (Silv et l., 2009). The Universidde Federl Rurl de Pernmuco hs cultivted n Active Germplsm Bnk of Brdos cherry (BAG Acerol UFRPE) since 1998, locted in Crpin City, North Forest Zone of Pernmuco Stte, Brzil, which is currently composed of 42 genotypes. The nnul rinfll nd temperture men in this region re 1,702.1 mm nd 25ºC, respectively (Mscrenhs et l., 2005). However, there re lrge res suject to drought during the yer, nd to increse the re under cultivtion y this crop is so importnt tht requires knowledge of the physiologicl responses to drought, especilly during the initil phse of development, this eing the most criticl in estlishing ny culture. Therefore, the ims of this work were: to evlute two Brdos cherry genotypes under wter deficit in order to verify if there re physiologicl differences etween them reltive to initil growth, wter reltions, nd orgnic solute ccumultion, since there is no informtion out them; nd to test the hypothesis tht Brdos cherry tolerte drought through the mintennce of wter sttus y reducing its lef wter potentil, due to the ccumultion of osmoregultors tht help to mintin the growth processes. MATERIAL AND METHODS Plnt mteril nd growth conditions: The experiment ws crried out in greenhouse from Jnury Brz. J. Plnt Physiol., 24(3): , 2012

3 PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES TO DROUGHT STRESS IN BARBADOS CHERRY 183 to My, in The verge dily temperture nd reltive ir humidity vried from 24.3 to 31ºC nd from 50.2 to 75.0%, respectively. The plnt mteril ws selected considering the high productivity, high AA content, nd some different spects such s tree cnopy nd height, 13-CPA eing tll nd 14-CPA eing short (Musser et l., 2005). According to Musser et l. (2005), the nnul production in 2000 ws 32.2 nd 44.7 kg kg plnt-1 nd AA of 2,068.5 nd 1,686.2 mg AA for ech 100 ml of pulp, respectively. The seedlings were propgted y cutting nd trnsferred 60 dys lter to polyethylene pots contining 8 kg of soil. The soil properties re presented in Tle 1. Thirty dys fter the trnsfer (cclimtiztion period), wter tretments (100, 75, 50 nd 25% of field cpcity FC) were pplied in the sis of FC. The FC ws determined y the grvimetric method following the methodology descried y Souz et l. (2000), which consists on the difference etween the wet soil fter sturtion nd free dringe, nd the weight of the dry soil. Mintennce of the wter tretments ws mde y dily weighing of the pots replcing the wter lost y trnspirtion using precision scle, up to 15 kg of cpcity. Growth mesurements: Plnt height, numer of leves, nd stem dimeter were weekly mesured fter strting the wter tretments. At the end of the experimentl period, the plnts were hrvested nd the orgns were seprted (leves, stem, nd roots). LA ws determined following the methodology descried y Mielke et l. (1995) y weighing lef discs. Six of them were detched from sl, medin, nd picl leves. Using the known re of the detched lef discs, their dry mtter nd totl dry mtter of the leves, totl LA ws estimted. Leves (LDM), stem (SDM), roots (RDM), nd totl (TDM) dry mtter were lso determined fter drying the mteril in n oven (65ºC) until constnt weight. From the vlues of dry mtter nd LA, LA rtio (LAR), iomss lloction to leves (BAL), stem (BAS) nd roots (BAR), nd root to shoot rtio were clculted ccording to Benincs (2003). Wter potentil nd reltive wter content mesurements: Ψ w nd RWC were determined t predwn (Ψ wpd ) nd t middy (Ψ wmd ), 90 dys fter the eginning of the wter tretment. The Ψ w ws mesured using pressure chmer (model 3035, Soil Moisture Equipment Corp, CA, USA) in rnches contining two pirs of leves, locted in the middle portion of the plnts, following the model descried y Scholnder et l. (1964). Tle 1. Soil chemicl nd physicl chrcteristics. The soil ws collected in deep of 0 to 20 cm from the Estção Experimentl de Cn-de-Açúcr do Crpin in Crpin City, North Forest Zone of Pernmuco Stte, Brzil. Anlysis Chemicl Soil depth 0-20 cm Electricl conductivity 0.83 ds m -1 ph 4.0 C cmolc kg -1 Mg cmolc kg -1 N cmolc kg -1 N 1.80 g kg -1 P 41 mg kg -1 K cmolc kg -1 Al cmolc kg -1 Orgnic mtter g kg -1 Physicl Totl porosity 45.0% Bulk density 1.42 g cm- 3 Rel density 2.58 g cm- 3 Snd 69.1% Silt 10.4% Cly 20.5% Texturl clssifiction Frnco sndy cly Moisture retention curve Moisture t 1.5 MP 14.0% Moisture t 0.03 MP 18.9% Useful wter 4.9% RWC ws determined in the sme leves used for Ψ w mesurements using six discs of ech lef, which were weighed to otin fresh mss weight (FMW). Then, the discs were plced in Petri dishes contining filter pper with 10 ml of deionized wter. The dishes were kept in refrigertor for period of 24 hours nd fterwrds they were weighed gin to otin the turgid mss weight (TMW). Finlly, the discs were plced in n oven t 65 C until they reched constnt weight, thus otining the dry mss weight (DMW). According to Ciro (1995), RWC ws clculted using the following formul: RWC={[(FMW-DMW)/(TMW-DMW)] x 100%}. Solule crohydrte, free minocids, free proline, nd solule proteins contents: At the end of the experimentl period, smples of leves nd roots were collected to perform iochemicl nlysis. Aout 1 g of fresh tissue ws tken nd frozen to -10ºC. The extrcts were prepred y grinding the mteril with 5 (leves) or 4 ml (roots) of 0.1 M monosic phosphte uffer. The homogenized extrcts were filtered in nylon tissue nd centrifuged in cold centrifuge 3,000 g n for 15 min nd the superntnt ws used for the nlysis. Solule crohydrtes were determined y the phenol-sulfuric cid method, using D (+)-glucose s stndrd (Duois Brz. J. Plnt Physiol., 24(3): , 2012

4 184 MEDEIROS D. B. et l. et l., 1956). Free minocids were nlyzed y the ninhydrin method using L-leucine s the stndrd (Yemm nd Cocking, 1955). Free proline content ws determined y oth the ninhydrin nd the phosphoric cid methods, using proline s stndrd (Btes, 1973). The solule protein determintion ws performed y the protein-dye inding method using ovine serum lumin s stndrd (Brdford, 1976). All procedures were crried out using the colorimetric technique nd spectrophotometer, BIOSpectro, model SP220. The wve lengths used for totl solule crohydrtes, free proline, free minocid nd solule protein were 490, 520, 570 nd 595 nm, respectively. Experimentl design nd sttisticl nlysis: A completely rndom design ws used in fctoril rrngement 2 x 4 corresponding to two genotypes (13-CPA nd 14-CPA) nd four wter tretments (100, 75, 50, nd 25% of FC) with five replictions. Ech replicte ws composed of one plnt per pot of ech genotype, totling 40 experimentl prcels. Dt were sumitted for nlysis of vrince (ANOVA), nd the verges using. Tukey s multiple rnge test (p<0.05) with the help of the ASSISTAT 7.5 softwre. RESULTS Growth: The extension nd severity of the wter deficit (p<0.05) significntly ffected lmost ll growth prmeters in Brdos cherry genotypes (Figure 1, Tles 2 nd 3). Reduction in stem dimeter hd een oserved firstly to genotype 13-CPA nd then to 14-CPA. This reduction ws out 22.7% in plnts under 25% of FC in comprison with control plnts to genotype 13-CPA fter 42 dys of wter tretment. After 91 dys, this reduction ws 21%, with n verge of 6.1 mm. For genotype 14-CPA, similr vlues of stem dimeter were verified mong the tretments until 77 dys, when reductions strted to e oserved in plnts under 25% of FC, nd continuing until 91 dys, with totl decrese of 10.9% when compred with control plnts (Figure 1). The Brdos cherry displyed similr growth in oth genotypes in reltion to the plnt height of the control ones, nd those cultivted t 75% nd 50% of FC. Although finl plnt height ws similr (c. 440 mm) for oth genotypes under control, 75 nd 50% of FC, the development differed etween the two long the experimentl period ccording to tretment (Figure 1). Both rected positively to lower wter vilility. After 42 dys, 13-CPA showed incresed height under 50% of FC compred to control nd 75% of FC. However, 14-CPA (shorter genotype) presented etter height vlues under 50 nd 75% of FC thn control throughout ll the experiment. After 28-dy period in 13-CPA nd 21 dys in 14-CPA, significnt reductions were oserved in response to severe wter deficit. At the end of the experimentl period (91 dys), these reductions were out 33.8 nd 23.0%, respectively. Severe wter stress significntly reduced the LA (Tle 2). For 13-CPA, the reductions oserved were 38.2 nd 57.4% in 14-CPA for plnts cultivted under 25% FC when compred with the Control ones. LAR ws not ffected y wter tretments pplied (Tle 2), nd there ws no difference mong genotypes either. Thus, genotypes 13-CPA nd 14-CPA used out 3,231.2 mm 2 of their lef surfce to produce 1 g of dry mtter (mm 2 g -1 DM). The production of dry mtter ws significntly reduced y wter stress in oth genotypes (Tle 3), with the exception to the RDM in 14-CPA, which ws not ffected y wter deficit. Genotype 13-CPA showed RDM reduction of out 54% in plnts grown under 25% of FC. In the leves nd stem, the stress pplied induced reductions of 61.4 nd 46.9% in 14-CPA, nd 42.3 nd 40.0% in 13-CPA, respectively. These results represent lrge reduction of the TDM in plnts under severe stress (25% of FC), corresponding to 46.3 nd 45.2% in genotypes 13-CPA nd 14-CPA, respectively. When evluting the iomss lloction (Figure 2), similr ehvior ws oserved in oth genotypes, since the shoot hd higher iomss lloction thn the roots in ll wter tretments. Nevertheless, wter deficit induced decrese in iomss lloction to roots in 13- CPA in plnts under 50% of FC, nd n increse in 14-CPA in plnts under 25% of FC. Confirming the dt on iomss lloction, the genotypes presented different physiologicl ehvior to root to shoot rtio, s oserved in Figure CPA reduced this rtio in stressed plnts, while 14-CPA incresed in plnts under severe stress (25% of FC). Wter potentil nd reltive wter content: The genotypes presented differences reltive to wter reltions. The most severe tretment (25% of FC) induced significnt reductions in wter potentil t predwn (Ψ wpd ) nd middy (Ψ wmd ) in 14-CPA when compred Brz. J. Plnt Physiol., 24(3): , 2012

5 PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES TO DROUGHT STRESS IN BARBADOS CHERRY CPA 14-CPA 10 Stem dimeter (mm) % FC 75% FC 50% FC 25% FC * Stem dimeter (mm) Plnt height (mm) Plnt height (mm) DAYS OF EXPOSURE TO STRESS Figure 1. Stem dimeter nd plnt height in two genotypes of Brdos cherry cultivted under four wter tretments in greenhouse conditions. Lower cse equl letters mong tretments do not differ y Tukey s test (p<0.05). FC: field cpcity. Tle 2. Lef re nd lef re rtio in two Brdos cherry genotypes cultivted under four wter tretments in greenhouse conditions. Wter tretments Lef re (mm 2 ) Lef re rtio (mm 2 gdm -1 ) 100% of FC 43,662.5 A 43,859.8 A 2,885.6 A 3,608.7 A 75% of FC 55,659.6 A 40,441.0 B 3,775.2 A 3,343.7 A 50% of FC 44,359.4 A 34,855.4 A 3,327.2 A 2,825.3 A 25% of FC 27,096.5 A 18,683.2 A 3,376.5 A 2,707.1 A FC: field cpcity. Equl letters, lower cse mong tretments nd upper cse etween genotypes do not significntly differ y Tukey s test (p<0.05). with control plnts (Figure 3). Ψ wpd ws reduced 33.3% in stressed plnts with vlues of MP. At noon, the reduction ws out 36.9%, with vlues of -2.5 MP. Nevertheless, these reductions were not oserved mong tretments to genotype 13-CPA. RWC did not significntly differ (p<0.05) mong wter tretments nor etween genotypes. The verge vlues found in predwn RWC rnged from 76.1 to 93.3%, nd t noon they rnged from 75.0 to 92.9% (Figure 4). Solule crohydrte, free minocids, free proline, nd solule proteins contents: In generl, the content of orgnic compounds ws ffected y severe wter deficit in the leves (Tle 4) nd roots (Tle 5). Solule crohydrtes were reduced in the leves of plnts Brz. J. Plnt Physiol., 24(3): , 2012

6 186 MEDEIROS D. B. et l. Tle 3. Leves, stem, roots, nd totl dry mtter production in two Brdos cherry genotypes cultivted under four wter tretments in greenhouse conditions. Wter tretments LDM (g) SDM (g) 100% of FC 2.6 A 2.7 A 6.5 A 6.4 A 75% of FC 2.9 A 2.6 A 6.1 A 6.2 A 50% of FC 2.5 A 2.3 A 6.8 A 6.8 A 25% of FC 1.5 A 1.0 A 3.9 A 3.4 A Wter tretments RDM (g) TDM (g) 100% of FC 5.9 A 3.4 B 15.1 A 12.5 B 75% of FC 5.7 A 3.3 B 14.7 A 12.0 B 50% of FC 4.0 A 3.3 A 13.3 A 12.5 A 25% of FC 2.7 ca 2.4 A 8.1 A 6.9 A LDM: leves dry mtter; SDM: stem dry mtter; RDM: roots dry mtter; TDM: totl dry mtter; FC: field cpcity.equl letters, lower cse mong tretments nd upper cse etween genotypes do not significntly differ y Tukey s test (p<0.05). Biomss lloction (%) BAR BAS BAL 13-CPA 14-CPA Biomss lloction (%) Root:Shoot Rtio Root:Shoot Rtio %FC 75%FC 50%FC 25%FC WATER TREATMENTS 100%FC 75%FC 50%FC 25%FC 0.0 Figure 2. Biomss lloction to roots (BAR), stem (BAS), nd leves (BAL) nd root to shoot rtio in two genotypes of Brdos cherry cultivted under four wter tretments in greenhouse conditions. Lower cse equl letters mong tretments do not differ y Tukey s test (p<0.05). FC: field cpcity. under 25% of FC in comprison with the other tretments y out 51.8 nd 38.4% to genotypes 13-CPA nd 14-CPA, respectively. Free minocids were reduced 46.3% in 13-CPA (25% of FC), ut they did not differ significntly in 14-CPA (p<0.05) etween tretments (100 nd 25% of FC). However, there ws difference etween Brz. J. Plnt Physiol., 24(3): , 2012

7 PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES TO DROUGHT STRESS IN BARBADOS CHERRY ψ w (MP) A A A A B Predwn A A A 13-CPA 14-CPA B A A A Middy B A A A ψw (MP) %FC 75%FC 50%FC 25%FC 100%FC 75%FC 50%FC 25%FC -3.0 WATER TREATMENTS Figure 3. Wter potentil (Ψ w ) in two genotypes of Brdos cherry cultivted with four wter tretments in greenhouse conditions. Equl letters, lower cse mong tretments nd upper cse etween genotypes do not significntly differ y Tukey s test (p<0.05). FC: field cpcity A A A Predwn A A A A A Middy A A A A A A A A RWC (%) CPA 14-CPA RWC (%) %FC 75%FC 50%FC 25%FC 100%FC 75%FC 50%FC 25%FC 0 WATER TREATMENTS Figure 4. Reltive wter content (RWC) in two Brdos cherry genotypes cultivted with four wter tretments in greenhouse conditions. Equl letters, lower cse mong tretments nd upper cse etween genotypes do not significntly differ y Tukey s test (p<0.05). FC: field cpcity. 25% of FC tretment nd the 75 nd 50 of FC, which showed higher vlues, verging 18.7 µmol g -1 FW. Free proline in the leves ws lso reduced in plnts under 25% FC in genotype 13-CPA (55.6%). However, there were no differences mong tretments to genotype 14-CPA. The sme ehvior occurred with solule protein. Genotype 13-CPA reduced protein content y out 55.23% in plnts grown under 25% of FC in comprison with the other tretments. However, 14-CPA showed tendency to increse the protein content in stressed plnts. The test to compre the verges showed tht the tretment of moderte stress (50% of FC) incresed the protein in 50.03% when compred to the control plnts. In the roots (Tle 5), the pttern of solule solutes ccumultion ws not the sme s in the leves. Increse of 40.3% in crohydrte content ws verified in plnts under 25% of FC to 13-CPA nd 30.7% to 14-CPA. Free minocids differed only in 13-CPA, incresing 25% of FC pronouncedly to tretment y out 67.3% (verge of µmol g -1 FW) when compred with 100% of FC. In reltion to free proline in the roots, increses were verified in ll stress tretments of the genotype 13-CPA. Plnts under 75% presented n increse of 36.4% in free proline, while plnts of genotype 14-CPA did not show significnt differences etween wter tretments. The verge proline vlues of 14-CPA were 0.14 µmol g -1 FW. Brz. J. Plnt Physiol., 24(3): , 2012

8 188 MEDEIROS D. B. et l. Tle 4. Solule crohydrte, free minocids, free proline nd solule proteins contents in leves of two Brdos cherry genotypes cultivted under four wter tretments in greenhouse conditions. Wter tretments Crohydrte (μmol g -1 FM) Aminocids (μmol g -1 FM) 100% of FC A A A A 75% of FC B A B A 50% of FC A A B A 25% of FC A A 6.09 A 5.65 A Wter tretments Proline (μmol g -1 FM) Proteins (μmol g -1 FM) 100% of FC 0.18 A 0.10 A 1, A 1,076.2 A 75% of FC 0.16 A 0.12 A 2, A 1,175.7 B 50% of FC 0.20 A 0.19 A 1, B 2,253.3 A 25% of FC 0.08 A 0.14 A cb A FC: field cpcity. Equl letters, lower cse mong tretments nd upper cse etween genotypes do not significntly differ y Tukey s test (p<0.05). Tle 5. Solule crohydrte, free minocids, free proline nd solule protein contents in roots of two Brdos cherry genotypes cultivted under four wter tretments in greenhouse conditions. Wter tretments Crohydrte (μmol g -1 FM) Aminocids (μmol g -1 FM) 100% of FC A A 5.44 cb 7.52 A 75% of FC A A A 6.86 B 50% of FC B A 9.53 A 8.98 A 25% of FC A B A 7.96 B Wter tretments Proline (μmol g -1 FM) Proteins (μmol g -1 FM) 100% of FC 0.07 B 0.15 A 1,972.4 B 3, A 75% of FC 0.12 A 0.13 A 2, B 3, A 50% of FC 0.10 B 0.13 A 1, B 3, A 25% of FC 0.10 B 0.14 A 5, A 3, B FC: field cpcity. Equl letters, lower cse mong tretments nd upper cse etween genotypes do not significntly differ y Tukey s test (p<0.05). Protein content lso incresed in plnts under 25% of FC (58.13%) in genotype 13-CPA. However, no significnt differences were oserved mong tretments to 14-CPA, which presented verge vlues of 3, µmol g -1 FW. DISCUSSION It is well-known tht wter deficits generlly ffect plnt growth nd development negtively. When wter deficit develops grdully, sequence of physiologicl nd iochemicl events will occur. The first nd most sensitive response to wter deficit in plnts is the reduction in growth processes due to loss of cell turgor (Lrcher, 2006). In this reserch, oth genotypes presented similr growth in height, stem dimeter, nd LA during the experimentl period, regrdless of whether they were cultivted t 100, 75, nd 50% of FC. This demonstrtes tht neither mild nor moderte stress cuses significnt growth ltertion. The reductions in growth prmeters oserved in these studies to plnts grown with 25% of FC seem to e ssocited with decreses in cell turgor, nd the tller genotype 13-CPA ws ffected erlier thn 14-CPA, considered the shorter genotype. The reduction oserved in plnts grown under 25% of FC must e explined due to the lower turgor pressure cused y the low-soil wter vilility, which involves processes such s cell division nd elongtion (Sho et l., 2008; Al-Asi, 2009). Thus, plnts tend to e smller in height, LA, stem dimeter, nd ll growth processes dependent on turgor. This fct ws oserved in the present study with Brdos cherry genotypes only etween plnts grown under severe stress (25% of FC). However, these plnts hve no prlyzed growth during the experimentl period nd for lmost ll prmeters, plnts under mild (75% of FC) nd moderte Brz. J. Plnt Physiol., 24(3): , 2012

9 PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES TO DROUGHT STRESS IN BARBADOS CHERRY 189 (50% of FC) stresses did not significntly differ from the Control ones. These results demonstrte certin degree of drought tolernce in the Brdos cherry. Severl uthors hve found similr results, verifying reductions in plnt growth with the severity or extent of the wter stress. Exmples include citrus genotypes cultivted with 25% of FC (Nscimento et l., 2010) nd rootstock of citrus sumitted to 16 dys without wter (Donto et l., 2007). In ddition, Al-Asi (2009) verified reductions in plnt height of other citrus cultivted t less thn 50% of FC. The sme results were oserved with LA, plnts under 25% of FC reduced it in oth genotypes. However, LAR ws not ffected y wter deficit. This result suggests tht dry mtter reduced t the sme proportion s LA s reduction, once LAR is the rtio etween LA nd TDM. In mny situtions, the reduction in LA occurs due to the reductions in the numer of leves y senescence nd leves flling, s oserved in citrus (Al-Asi, 2009). Al-Asi (2009) reports tht the leves flling in the three genotypes studied should e proposed s droughtvoidnce mechnism in itself. In the present study, however, leves were not oserved flling during the experimentl period, suggesting tht the totl LA ws reduced in prt y the lower emission of new leves, ut lso to smller leves due to reduced cell expnsion. LA is fctor of gret importnce in physiologicl studies, minly to cultivted plnts, since it is one of the determinnt fctors in the crop production. Leves re the source of ssimiltes to the shoots s well s to roots, nd they provide the structure responsile for nutrient ssimiltion, plying n importnt role in plnt s growth (Bonfim-Silv et l., 2011). Peixoto et l. (2006) oserved reductions of 40% in the LA fter 12 dys of withholding wter nd decreses in LAR fter period of re-wtering in citrus genotypes. The uthors stted tht these reductions occurred due to the tendency of LAR to diminish with the plnt growth, since in the initil phse of development most ssimiltes re converted into leves iming t hrvesting the solr rdition through photosynthetic processes. Although photosynthesis ws not evluted in this work, weight gin hppens due to cron ssimiltion. TDM ws not ffected y wter deficit in the tretments of oth genotypes t 75 or 50% of FC, demonstrting tht these levels of wter (mild nd moderte stresses) did not ffect cron gin in the initil phse of development. However, in generl, 14-CPA hd lower TDM thn 13-CPA when compred to the control plnts nd those t 75% of FC. In situtions of moderte or severe stress, oth genotypes showed similr vlues of TDM (Tle 3). Only plnts under 25% of FC hd reductions in dry mtter of leves nd stem in oth genotypes (Tle 3). However, the roots of 13-CPA grown under 50 nd 25% of FC showed significnt reduction in dry mtter compred with control plnts, while 14-CPA ws not ffected. These results demonstrte the physiologicl differences etween the genotypes, in reltion to root growth s response to drought. The roots re in contct with the soil nd re the first orgn to feel the effects of soil desicction. The ehvior oserved in 14-CPA is xeromorphic chrcteristic, which could e clssified s droughttolernce mechnism tht mintins root growth during drought conditions. At the sme time, the fct tht oth genotypes of Brdos cherry did not reduce y more thn 50% of the TDM demonstrtes certin degree of drought-tolernce. Similr results were found y Cerqueir et l. (2004) upon nlyzing the response of some citrus rootstock to wter deficit. The uthors verified tht the hyrids did not show reduction in the shoot dry mtter with the increse of wter stress. Mglhães Filho et l. (2009), while evluting the wter deficit in two ornge tree rootstocks, verified reductions in CO 2 ssimiltion rte fter 56 dys of withholding wter. The uthors stted tht this decrese reflected smller dry mtter ccumultion in those plnts. In ddition, the uthors oserved different responses to wter deficit in lemon genotypes. The dry mss of roots in lemon Crvo ws similr for oth tretments with nd without stress, which is similr to the response found to genotype 14-CPA in the present study, while in lemon Trifolit RDM ws smller thn the control, similr response to genotype 13-CPA in the current work. The iomss distriution pttern reveled different physiologicl ehvior in 14-CPA genotype, which incresed iomss lloction to the roots nd reduced iomss lloction to the leves. This ehvior hs een previously reported nd is ccepted s either drought voidnce or survivl strtegy. The root depth helps the plnt to survive during times of low-soil wter vilility. This preferentil lloction to the roots indictes n increse in the sorptive re nd represents etter wy to explore soil moisture nd, t the sme time, reduces wter loss y using smller trnspirtion re. This ehvior ws confirmed y exmining the root: shoot rtio dt, which did not occur with 13-CPA. Medeiros et l. (2009) upon sumitting six-month-old seedlings of the Brdos cherry to four wter irrigtion regimes Brz. J. Plnt Physiol., 24(3): , 2012

10 190 MEDEIROS D. B. et l. oserved similr results with genotype 14-CPA. Seedlings under severe wter stress (25% of FC) llocted more photossimiltes to roots thn shoots in comprison with control plnts (100% of FC) fter 35 dys of stress, modifying the root to shoot rtio. Wter reltions re generlly directly ffected y wter deficit, ecuse they reduce the wter flow in the soil-plnttmosphere system. Ψ w decreses minly in times of high evportive tmospheric demnd. Other fctors, such s high temperture nd low reltive humidity, increse the vpor pressure deficit etween the lef nd the ir, therey incresing the loss of wter y trnspirtion, which fvors the decline of lef wter potentil. Ψ wpd mesurement is known to e more sensitive when evluting wter deficit in plnts ecuse significnt wter loss y trnspirtion is not oserved t this time, nd the vlues of Ψ w in soil-plnt system tends towrds equilirium. Thus, the predwn evlution should provide etter demonstrtion of the wter potentil in the root zone (Ciro, 1995; Améglio et l., 1999; Sircelj et l., 2005). However, this equilirium in the wter sttus is currently eing questioned, since some plnts present nocturnl trnspirtion (Bucci et l., 2005), nd this loss of wter overnight induces imlnce in predwn lef wter potentil. In this reserch, Ψ wpd ws reduced only in plnts under severe stress (25% of FC) to 14-CPA, ut it ws not significnt to 13-CPA, lthough oth genotypes showed tendency to reduce the solute vlue overll. Such reduction in Ψ w should e induced y reduction in RWC, which ws not ffected y wter deficit of 25% of FC (Figure 4). On the other hnd, the ccumultion of osmoregultors contriutes to reducing Ψ w, helping to mintin the wter sttus, ut the orgnic solutes studied here did not ccumulte in sufficient mounts to induce ltertions in lef Ψ w to genotype 14-CPA. Nogueir et l. (2000), while studying two genotypes of the Brdos cherry in the field, oserved vritions in the Ψ w throughout the dy. Similr to these studies, the uthors verified the most negtive vlues of wter potentil t noon in oth genotypes, independent of sesonlity, lthough the minimum vlues occurred in dry seson nd the genotype UFRPE 8 showed the lowest vlue (-4.32 MP). Donto et l. (2006) found vlues of Ψ wpd of nd MP in citrus sumitted to 12 nd 16 dys without wter respectively, while dily-irrigted plnts hd vlues of nd MP t the sme time of evlution. RWC ws not significntly ffected in the present reserch s function of wter deficit. However, contrsting results were verified y Nogueir et l. (2001) in three-month-old plnts of the Brdos cherry which were irrigted nd under stress, nd propgted y seeds nd cutting. The uthors oserved vritions in RWC etween the hours of 1000 nd 1100 m with the extent of wter deficit, reching 38.4% in plnts propgted y seeds nd 48.2% in those originted y stkes fter 20 dys without irrigtion. This plnt response vrition, when compred with these studies, could e ssocited with the lower soil volume used y the uthors (6 kg). Also in the present studies, the plnts under severe stress kept receiving 25% of FC, while the soil used y the uthors must hve een extremely dry, denoting desicction in those plnts, since the stress pplied to them ws for wter suppression. Comptile orgnic solute ccumultion in response to drought is n importnt mechnism to mintin cell turgor, contriuting to reduction in the plnt wter potentil (Frooq et l., 2009; Silv et l., 2009). However, ccording to Hsegw et l. (2000), only solute ccumultion does not confer complete drought tolernce, ut the pth tht conducts the mechnism of drought tolernce is modulted y this fctor. Silv et l. (2009), while studying the wter reltions nd orgnic solute ccumultion in six-month-old seedlings of four umu tree genotypes under intermittent drought, verified reductions in solule crohydrtes in leves fter 31 dys of wter stress. In the roots, the uthors oserved n increse of 7.14% only in genotype GBU48. According to Pimentel (1999), the increses in solule crohydrte content my occur in the eginning of the stress period for two resons: s result of the stoppge of growth nd y the wter deficit intensity ecuse of strch degrdtion. In the present study, solule crohydrtes decresed in the leves nd incresed in the roots with the extent of the wter deficit, suggesting trnsloction of photossimiltes from leves to roots s stted y Pimentel (1999) due to the reduction in growth processes. Free minocids were lso ffected y drought, ut the rel mens of its ccumultion during stress periods re still uncertin (Sircelj et l., 2005). Current studies indicte tht the min function of minocids could e osmotic djustment. Altertions in free minocid content were verified in two pple cultivrs under wter deficit, incresing more thn 40% in comprison with control plnts (Sircelj et l., 2005). Rodrigues et l. (2010) lso verified n increse in minocids in leves of six-monthold jck-plnts nd sugr pple plnts when sujected to eight dys without wter. These increses were out 61 nd 66% respectively. Brz. J. Plnt Physiol., 24(3): , 2012

11 PHYSIOLOGICAL AND BIOCHEMICAL RESPONSES TO DROUGHT STRESS IN BARBADOS CHERRY 191 The increse in minocid content in genotype 13-CPA long with the increse in protein content demonstrtes tht there ws no proteolysis, ut insted of tht, synthesis in roots s response to wter deficit. On the other hnd, in genotype 14-CPA, this solute could not e considered s fundmentl to osmotic djustment ecuse the stress did not induce increses in the roots s oserved with other compounds of the sme genotype. The results found in these studies re similr to those found y Silv et l. (2009) in four umu tree genotypes under intermittent drought, where the uthors verified no differences mong genotypes nd wter tretments reltive to protein content in leves, ut increses were found in the roots of one genotype (GBU 44). In ddition to the contriution of osmoregultors, proline is widely found in higher plnts nd generlly is ccumulted in lrge mounts during situtions of wter stress. However, this ccumultion ws seen only in genotype 13-CPA in the roots, ut not in the leves, while 14-CPA hd no chnges in leves or roots from wter stress. Genotypic differences reltive to proline ccumultion during drought periods s found in these studies hve een relted to severl crops. It is ccepted tht proline hs n importnt nd complex role in drought tolernce of plnts nd the ssistnce of osmotic djustment (Kvi Kishor et l., 2005). Therefore, ccording to Ashrf nd Foold (2007), side from its role s n osmoregultor, proline lso contriutes to memrne stiliztion nd rdicl oxygen scvenger, preventing plnts from eing dmged y environmentl stresses. Nogueir et l. (2001) working with the Brdos cherry seedlings sujected to 20 dys without wter oserved ltertions in the proline content, recording 38.1% more proline in stressed plnts thn in the control ones when propgted y seeds, nd 26.4% more proline in stressed plnts thn control ones, when originted y stkes, totling n mount of 18.0 nd 10.2 mg g -1 DM, respectively. In these studies, proline ccumultion ws oserved only in the roots nd ws not close to the mount oserved y Nogueir et l. (2001). However, it certinly contriuted to mintining wter inflow nd thus growth processes dependent on turgor. Accumultion of orgnic solutes in the roots, s oserved with 13-CPA, incresing crohydrtes, protein, minocids nd proline, hs n importnt role in mintining wter inflow from the soil. Consequently, it helps the growth nd metolic processes since there is no increse in root growth in this genotype. The incresed root growth in 14-CPA llows it to find wter t greter depths in the soil. Therefore, the increse in ccumultion of orgnic solutes, with the im of soring wter, ws not necessry for 14-CPA in this sitution. The results otined in this study confirm the hypothesis tht the Brdos cherry genotypes present different physiologicl mechnisms to overcome periods of wter deficit in the initil phse of development. The wter sttus is kept in genotype 13-CPA through the ccumultion of solule orgnic solutes, such s minocids, protein, crohydrtes nd proline in the roots, which help to mintin the inflow of wter from the soil to the plnt. The mechnisms used y genotype 14-CPA to void desicction re reltive to chnges in growth ptterns, modifying root to shoot rtio, nd the reduction in LA seems to ssist the tolernce for periods of wter deficit. Despite the mechnisms used y the genotypes, moderte stress does not induce morphophysiologicl chnges in the Brdos cherry. This demonstrtes drought-tolernce in such species in its initil phse of development. Acknowledgments The uthors would like to thnk the Conselho Ncionl de Desenvolvimento Científico e Tecnológico (CNPq) for their finncil support, nd collegues Dnúi Rmos (iologist), Mrcelo Rieiro (gronomist), nd grdution student Ntáli Vz for their technicl support during the experiment. REFERENCES Al-Asi KM (2009) Gs Exchnge, Chlorophyll nd Growth Response of Three Ornge Genotypes (Citrus sinensis [L.] Oseck) to Ascisic Acid under Progressive Wter Deficit. Jordn J. Agric. Sci. 5: Améglio T, Archer P, Cohen M, Vlncogne C, Dudet FA, Dyu S, Cruizit P (1999) Significnce nd limits in the use of predwn lef wter potentil for tree irrigtion. Plnt Soil 207: Ashrf M, Foold MR (2007) Roles of glycine etine nd proline in improving plnt iotic stress resistnce. Environ. Exp. Bot. 59: Btes LS (1973) Rpid determintion of free proline for wter-stress studies. Plnt Soil 39: Benincs MMP (2003). Análise de crescimento de plnts (noções ásics). FUNEP, Joticl. Bonfim-Silv EM, Silv TJA, Crl CEA, Kroth BE, Rezende D (2011) Desenvolvimento inicil de grmínes sumetids o estresse hídrico. Rev. Cting 24: Brdford MM (1976) A rpid nd sensitive method for the quntittion of microgrm quntities of protein utilizing the principle of protein-dye inding. Anl. Biochem. 72: Brz. J. Plnt Physiol., 24(3): , 2012

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