Plant Physiology and Biochemistry 83 (2014) 243e249Contents lists available at ScienceDirectPlant Physiology and Biochemistryjournal homepage: www.elsevier.com/locate/plaphyResearch articlePhysiological and molecular responses to drought stress in rubber tree(Hevea brasiliensis Muell. Arg.)Li-feng Wang*Danzhou Investigation & Experiment Station of Tropical Crops, Ministry of Agriculture, Rubber Research Institute, Chinese Academy of Tropical AgriculturalSciences, Danzhou, Hainan 571737, Chinaa r t i c l e i n f oArticle history:Received 4 April 2014Accepted 14 August 2014Available online 23 August 2014Keywords:Antioxidative enzymeDroughtHevea brasiliensisOsmoregulationPhotosynthesisReactive oxygen species1. Introduction a b s t r a c tPlant drought stress response and tolerance are complex biological processes. In order to reveal thedrought tolerance mechanism in rubber tree, physiological responses and expressions of genes involvedin energy biosynthesis and reactive oxygen species (ROS) scavenging were systematically analyzedfollowing drought stress treatment. Results showed that relative water content (RWC) in leaves wascontinuously decreased with the severity of drought stress. Wilting leaves were observed at 7 daywithout water (dww). Total chlorophyll content was increased at 1 dww, but decreased from 3 dww.However, the contents of malondialdehyde (MDA) and proline were signicantly increased underdrought stress. Peroxidase (POD) and superoxide dismutase (SOD) activities were markedly enhanced at1 and 3 dww, respectively. Meanwhile, the soluble sugar content was constant under drought stress.These indicated that photosynthetic activity and membrane lipid integrity were quickly attenuated bydrought stress in rubber tree, and osmoregulation participated in drought tolerance mechanism inrubber tree. Expressions of energy biosynthesis and ROS scavenging systems related genes, includingHbCuZnSOD, HbMnSOD, HbAPX, HbCAT, HbCOA, HbATP, and HbACAT demonstrated that these genes weresignicantly up-regulated by drought stress, and reached a maximum at 3 dww, then followed by adecrease from 5 dww. These results suggested that drought stress adaption in rubber tree was governedby energy biosynthesis, antioxidative enzymes, and osmoregulation. 2014 Elsevier Masson SAS. All rights reserved.associated with low transpiration co efciency and osmoticadjustment, etc. Osmotic adjustment involves the accumulationWater decit is a major constraint to plant growth and pro-ductivity (Monclus et al., 2006). Prolonged drought stress leads tosevere problems, such as decrease in water ux, closing of stomataand reduction in carbon dioxide xation. Tree can die of both hy-draulic failure and carbon starvation during drought stress (Zeppelet al., 2013). Inhibition of photosynthesis and energy dissipation arecommon features under drought stress in many plant species,which reect as Photosystem II thermostability and electrontransport changes (Zhou et al., 2007; Brestic et al., 2012; Yan et al.,2013; Zivcak et al., 2014). Plant anti-drought characters are mainlyAbbreviations: ACAT, acetyl-CoA C-acetyltransferase; APX, ascorbate peroxidase;CAT, catalase; CDPK, calcium-dependent protein kinase; COA, a long-chain-fatty-acyl-CoA reductase; dww, day without water; MDA, malondialdehyde; POD,peroxidase; ROS, reactive oxygen species; RWC, relative water content; SOD,superoxide dismutase.* Tel.: 86 898 23300459; fax: 86 898 23300315.E-mail address: lfngwang@yahoo.com.http://dx.doi.org/10.1016/j.plaphy.2014.08.0120981-9428/ 2014 Elsevier Masson SAS. All rights reserved. of compatible solutes (low-molecular-weight organic osmolytes),such as proline, mannitol, sorbitol, fructans, sucrose and oligosac-charides (Rhodes and Hanson, 1993). These large amounts ofcompounds play a key role in maintaining the osmotic equilibriumand protecting membranes as well as macromolecules (Hoekstraet al., 2001; Couee et al., 2006). These regulations were related toabscisic acid (ABA), calcium-dependent protein kinase (CDPK),NADP-malic enzyme (Shao et al., 2013) and phospholipid signalingpathways (Zhu, 2002). Overexpression of key genes in these path-ways, such as DREB transcription factor, enhanced drought toler-ance in Arabidopsis and Lotus corniculatus (Zhou et al., 2012). Inaddition, overexpression of soybean ubiquitin-conjugating enzymegene GmUBC2 can enhance drought tolerance by modulatingabiotic stress-responsive genes expression in Arabidopsis (Zhouet al., 2010). Since drought stress doubtless generates reactive ox-ygen species (ROS) in chloroplasts and mitochondria (Apel and Hirt,2004; Asada, 2006), so ROS-scavenging enzymes play importantroles in drought tolerance responses. ROS-scavenging systems244 L.-f. Wang / Plant Physiology and Biochemistry 83 (2014) 243e249included superoxide dismutase (SOD), peroxidase (POD), ascorbateperoxidase (APX), monodehydroascorbate reductase (NADH),catalase (CAT), etc. Natural rubber is obtained from para rubber tree(Hevea brasiliensis Muell. Arg.). Rubber tree originated from theAmazon basin in South America. This area falling between equatorand 15 S is characterized by a wet equatorial climate (Gonalveset al., 2009). The optimal growth conditions of rubber tree arehigh temperature around 28 2 C and high humidity about2000e4000 mm rainfall per annum (Webster and Baulkwill, 1989;Priyadarshan et al., 2005). However, unlike traditional plantationsin south America and southeast Asia, rubber tree planting in thesemarginal areas or non-traditional rubber-growing regions, such asnortheastern states of India, south China, north and northeastThailand, usually faces abiotic stress like drought, strong winds, andlow temperature, etc. Drought stress results in growth retardationof both rubber tree seedlings and mature tapping trees, shorteningtapping period, blocking latex ow for low water supply, decreasingdry latex contents, increasing TPD (tapping panel dryness) occur-rence, and even causes tree death at severe conditions (Huang andPan, 1992).Many strategies and indices were used for selecting andbreeding drought-tolerant rubber tree clones, such as drought-tolerant rootstock (Ahamad, 1999), leaves with more epicuticularwaxes (Gururaja Rao et al., 1988), etc. Hydraulic mechanism wasused for explaining drought tolerance mechanism in rubber tree(Ayutthaya et al., 2011). The development of molecular biologicaltechniques in rubber tree provides new functional genes to extendour insights of drought tolerance mechanism. Recently, HbCuZnSODand HbMnSOD have been cloned in rubber tree, and over-expression of HbCuZnSOD in rubber tree clone PB260 conferredenhanced drought tolerance (Leclercq et al., 2012). These resultsindicated that ROS-scavenging enzymes played crucial roles indrought tolerance mechanisms. Representative genes in mito-chondria, such as HbAPX, an ascorbate peroxidases gene (Mai et al.,2009), and HbATP (Chye and Tan, 1992) were cloned. However, inrubber tree, the functions of most ROS related genes in droughtresistance mechanism were not well identied. In this study, ex-pressions of 8 genes involved in energy biosynthesis and ROSscavenging systems were characterized under drought treatment inseedlings of rubber tree clone GT1. The underlying drought toler-ance mechanism in rubber tree was discussed.2. Methods2.1. Plant material and drought treatmentRubber tree clone GT1 (original clone breed in Indonesia)seedlings were grown in the plastic pots in the chamber withvermiculite and turfy soil (1:3) at the experimental farm of theChinese Academy of Tropical Agricultural Sciences in Danzhou city,Hainan province, China (19 51051N; 109 55063E). In growing sea-son, the average temperature was about 30 C, precipitation wasabout 180 mm, and humidity was around 97.5%. Seedlings with twogrowth units of leaves were subjected to progressive drought bywithholding water, and the leaves in dark green stage werecollected at different time points after treatment and used forfollowing assay.2.2. Relative water contentThe fresh weight, dry weight and saturated weight of treatedleaves were measured. RWC (relative water content) of leaves wascalculated according to formula: 100 [(fresh weightedry weight)/(saturated weight e dry weight)]. 2.3. Determination of chlorophyll (Chl) contentChlorophyll was extracted with 80% ice cold acetone from 0.1 gleaves samples. The extract was measured spectrophotometricallyat 475, 645 and 663 nm with spectrophotometer (GE Ultrospec2100 pro UV/visible, USA), respectively. Specic chlorophyll andb-carotene contents were determined according to the reportedmethod (Lichtenthaler, 1987).2.4. Measurements of activities of SOD and PODSOD (EC 1.15.1.1) was prepared by rst freezing 0.5 g of leavessample in liquid nitrogen to prevent proteolytic activity, followedby grinding with 5 ml extraction buffer (0.1 M phosphate buffer, pH7.5, containing 0.5 mM EDTA, and 1 mM ascorbic acid). Brie wascentrifuged for 20 min at 15 000 g and the supernatant was used asan enzyme. The soluble proteins concentration in the supernatantwere determined using the method of Bradford with bovine serumalbumin (BSA) as standard (Bradford, 1976). The per unit activity ofSOD was estimated by recording the decrease in optical density ofnitro blue tetrazolium (NBT) induced by the enzyme (Dhindsa et al.,1981). 3 ml of the reaction mixture contained 13 mM methionine,75 mM nitroblue tetrazolium chloride, 0.1 mM EDTA, 50 mMphosphate buffer (pH 7.8), 50 mM sodium carbonate, and 0.1 mlenzyme solution. The reaction was started by adding 2 mM ribo-avin. The reaction mixtures were illuminated for 15 min at90 mmol m2 s1 (placing the test tubes under two 15 W uorescentlamps). A complete reaction mixture without enzyme, which gavethe maximal colour, was served as the control. The reaction wasstopped by switching off the light and putting the tubes into dark.A non-irradiated complete reaction mixture was served as a blank.POD (EC 1.11.1.7) activity was determined with spectropho-tometer. 0.5 g leaves sample was extracted with 5 ml 100 mMphosphate buffer (pH 6.0). Homogenate was centrifuged at 4000 gfor 10 min. Reaction mixture was 50 ml 100 mM phosphate buffer(pH 6.0) with 23 mM guaiacol and 1.8 mM hydrogen peroxide. 1 mlsupernatant was added into 3 ml reaction mixture. The change ofOD was recorded at 470 nm. The per unit activity of enzyme wasdened as the increase of 0.1 DOD per minute.2.5. Measurement of malondialdehyde (MDA) contentMDA content was determined by the thiobarbituric acid reac-tion (Peever and Higgins, 1989). 1.0 g freshleaves sample was ho-mogenized in 5 ml 0.1% (w/v) trichloroacetic acid (TCA). Thehomogenate was centrifuged at 10 000 g for 5 min and 4 ml of 20%TCA containing 0.5% (w/v) thiobarbituric acid (TBA) were added to1 ml of the supernatant. The mixture was heated at 95 C for 30 minand then quickly cooled on ice. The contents were centrifuged at10 000 g for 15 min and absorbance of the supernatant at 532 and600 nm was read. After subtracting the non-specic absorbance at600 nm, the MDA concentration was determined by its extinctioncoefcient of 155 mM1 cm1.2.6. Analysis of soluble sugar contentSoluble sugar content was measured by referring to (Creelmanet al., 1990). Take 0.1 g of leaf samples and put it into centrifugingtubes with a volume of 10 ml. Add 5 ml of 80% alcohol to the tubeand heat it in water for 30 min at 80 C. Then cool down the tubeand centrifuge it at 1000 g for 10 min. Soluble sugar content wasdetermined by the phenol-sulfuric acid method.L.-f. Wang / Plant Physiology and Biochemistry 83 (2014) 243e2492.7. Determination of free proline contentProline was determined following (Bates et al., 1973). Briey,0.5e1.0 g leaves was homogenized in 10 ml of 3% sulfosalicylic acidand the homogenate ltered. The ltrate (2 ml) was treated with2 ml acid ninhydrin and 2 ml of glacial acetic acid, then with 4 ml oftoluene. Absorbance of the colored solutions was read at 520 nmwith spectrophotometer.2.8. Gene expression analysis by real-time PCRTotal RNA was extracted from leaves according to the methodsof (Qin, 2013). The quality and concentrations of the extracted RNAwere detected by agarose gel electrophoresis and measured bya spectrophotometer. First strand cDNA was synthesized from 2 mgof total RNA with MMLV reverse transcriptase and random hexamerprimer (Takara) according to the manufacturer's instruction. The 245cDNA was diluted 1:20 with nuclease-free water. Aliquots of thesame cDNA sample were used for real-time PCR with primersdesigned for the selected genes, and 18S rRNA (Hb18SRNA) was usedas a house-keeping gene (Table 1). The PCR reaction was performedin a 20 mL reaction mixture containing 200 nM of each primer,1 SYBER Green PCR Master Mix (Takara), and about 30 ng cDNA.Real-time RT-PCR was performed using the Bio-RAD CFX96 system(BioRAD, Hercules, CA, USA). The reactions were carried out asfollows: 3 min at 95 C for denaturation, 10 s at 94 C, 20 s at 60 C,and 30 s at 72 C for amplication for 45 cycles. The relativeabundance of transcripts was calculated according to the Softwareinstructions in Bio-RAD CFX96 Manager. The specicity of eachprimer pairs was veried by determining the melting curve at theend of each run and sequencing the amplied bands from gelelectrophoresis.2.9. Statistical analysisAll data were analyzed with IBM-SPSS analytical softwarepackage version 20.0 (IBM Corporation, USA). One-way ANOVA andTukey text were used to assess the different level. P < 0.01 (prob-ability level) was considered signicant difference. Figures weredrawn by Origin data analysis and graphing software, OriginPro 9.1(OriginLab Corporation, USA). For real-time PCR analysis, eachvalue was the average of two biological replicates tested in tripli-cate, and for the other analyses, 6 replicate samples tested inreplicate were used.Table 1Information of primers used in this study. Fig. 1. Relative water content in rubber tree GT1 seedling leaves after withholdingwater Values represent the mean SD of 6 replicate samples tested in replicate.3. Results3.1. The effect of drought stress on relative water, chlorophyll, andb-carotene contents in the leaves of rubber treeThe relative water content (RWC) is a key indice for droughtstress study. As showed in Fig. 1, the RWC in the seedling leaves ofrubber tree clone GT1 was continuously decreased with the severityof drought stress. It decreased by almost 20% at 9 day without water(dww) compared with that at 0 dww. A wilting phenotype wasobserved in leaves at 7 dww. Since photosynthesis in plants isdependent on capturing light energy in the pigment chlorophyll,and b-carotene (b-Car) is a pigment which assists in light absorptionand energy dissipation in chloroplasts. So drought tolerance ofrubber tree was tested by evaluating photosynthesis, especiallycontents of chlorophylls and b-carotene under drought stress. Totalchlorophyll content was signicantly increased at 1 dww, butshowed a sharp decrease at 3 dww, and kept a low level until 9 dww.This variation was associated with both Chl a and Chl b, since sig-nicant change was observed in Chl a and b during drought stress.The ratio of Chl a/b was increased until 5 dww, and then decreasedfrom 7 to 9 dww. Since most Chl a located in the reaction centerchlorophylleprotein complex, and most Chl b located in light har-vesting chlorophylleprotein complex, the attenuations of Chl a, ChlGenes Accession Primer sequences (50 e30 ) Amplication Amplication Referencenumber length (bp) efciencyHbCOA AY461413 Forward: GGTGACATGGTGGTGAAT 145 1.872 0.0183 (Deng et al., 2012)Reverse: TGAAGTGACGAATGAGGTAAHbACAT AF429387 Forward: GAGTATCCAGTTAGGCATCA 119 1.918 0.0099 Direct submissionReverse: CTAGTGAATCATGTCCAAGTCHbAPX AF457210 Forward: CCAACTGACACCGTTCTT 164 1.815 0.0067 (Mai et al., 2009)Reverse: CAGCACCATCCTCTACATCHbATP X58498 Forward: GCTTCACGCAGACTATTATC 112 1.809 0.0079 (Chye and Tan, 1992)Reverse: TAGAGGATGGAGATGAGGAAHbCAT AF151368 Forward: GGTATTGTGGTTCCTGGTAT 153 1.877 0.0093 Direct submissionReverse: ATGGTGATTGTTGTGATGAGHbCuZnSOD AF457209 Forward: GTCCAACCACCGTAACTG 200 1.901 0.0109 Direct submissionReverse: GCCATCATCACCAACATTGHbMnSOD L11707 Forward: TGTGCTGTAATGTTGACCTA 128 1.873 0.00139 (Miao and Gaynor, 1993)Reverse: GTTCACCTGTAAGTAGTATGCHbRbsS M60274 Forward: GCCAAGGAAGTTGAATACC 123 1.794 0.0256 (Chye et al., 1991)Reverse: CCAGTAACGACCATCATAGTHb18SRNA AY435212 Forward: GCTCGAAGACGATCAGATACC 146 2.062 0.011 Direct submissionReverse: TTCAGCCTTGCGACCATAC246 L.-f. Wang / Plant Physiology and Biochemistry 83 (2014) 243e249content in the leaves of rubber tree was determined to reveal thelevel of ROS under drought stress. As showed in Fig. 3, MDA contentin leaves was continuously increased as prolonged drought stress.However, proline content increased slightly at 1 dww, but droppedat 3 dww, and then underwent a sudden increase at 9 dww. As forthe soluble sugar content, it reduced by nearly 50% at 1 dww, butrecovered to the untreated level (0 dww) at 3 dww, then decreasedfrom 5 to 7 dww, and suddenly increased at 9 dww. Under droughtstress condition, the accumulation of MDA usually leads to thedamage of cell membrane in plant and animal. Changes of MDA andRWC suggested that drought induced osmotic stress responsein rubber tree seedlings. However, the soluble sugar took part indrought response as we previously found in chilling stress response(Luo et al., 2012). The plant POD enzyme can decompose hydrogenperoxide, decrease oxygen radical production, and prevent plantdamaged by peroxide. Under drought stress, POD activity increasedslightly at 1 dww, but experienced a continuous decrease from 3 to9 dww. The SOD activity increased at 3 dww, but decreased from 5to 9 dww. These suggested that rubber tree seedling was suscep-Fig. 2. Contents of chlorophylls and becarotene after withholding water Valuesrepresent the mean SD of 6 replicate samples tested in replicate.b, and Chl a/b were resulted by the degradation of chlor-ophylleprotein complex under severe drought condition. The b-Carcontent was increased slightly at 1 dww, but quickly decreased from3 dww (Fig. 2). These results suggested that b-Car did not take part inquenching excess excited energy after chlorophylleprotein complexbroken down under drought stress in rubber tree.3.2. The effect of drought stress on membrane oxidation andosmosis indicesSince MDA, a cytotoxic product of lipid peroxidation is generallytaken as an index of ROS level. Therefore, the change of MDA tible to drought stress, and the protection role of physiologicalresponses only lasted for 3e5 days after withholding water.3.3. Expressions of key genes involved in energy biosynthesis andROS scavenging under drought stressAs showed in Fig. 4, drought stress induced the transcripts ofantioxidative enzyme genes HbAPX, HbCAT, HbCuZnSOD, andHbMnSOD with nearly the same pattern. Their expressions reacheda maximum (100-fold over the untreated control) at 3 dww afterwithholding water. The expression patterns of ROS scavengingsystems related genes were coincided with variations in theirenzyme activities. For instance, the gene expression of HbCuZnSODand HbMnSOD were reached their peaks at 3 dww, while SODenzyme activities were highest at 3 dww.Fig. 3. Changes of physiological indices in rubber tree after withholding water Values represent the mean SD of 6 replicate samples tested in replicate. Bars with differentuppercase letters show signicant differences at the P < 0.01 level.L.-f. Wang / Plant Physiology and Biochemistry 83 (2014) 243e249 247Fig. 4. Expressions of ROS scavenging systems related genes HbAPX, HbCAT, HbCuZnSOD, and HbMnSOD after withholding water Values represent the mean SD of twobiological replicates tested in triplicate. Bars with different uppercase letters show signicant differences at the P < 0.01 level.Expressions analyses of energy biosynthesis related genesrevealed that HbCOA, HbRbsS, and HbACAT transcripts wereincreased at the rst 3 dww, but decreased from 5 dww. HbATPshowed instant response at 1 dww then attenuate its geneexpression from 3 dww. These suggested that these genes involvedin energy biosynthesis and ROS scavenging were response todrought stress, and drought stress responses were occurred at 3e5days after withholding water in rubber tree seedling.4. Discussion4.1. Short-term drought stress caused leaves dysfunction in rubbertree seedlingsIn the sub-optical rubber tree plantation in China, droughtinhibits rubber tree growth. Research on rubber tree droughtresistance mechanism mainly concentrated in the area of anatomyand physiological response (Nair et al., 1996). The effect of droughtvaried on different physiological metabolism in various growth anddevelopment stages of rubber tree (Devakumar et al., 1988). Tran-spiration coefcient (Nair et al., 1996), membrane integrity (Reddy,2000), osmoregulation, laticifer turgor pressure (Ranasinghe andMilburn, 1995), low solute potential (Ayutthaya et al., 2011) werefound related to drought tolerance in rubber tree. Drought signi-cantly reduced the relative growth rate and RWC, and inhibitedphotosynthesis in plant seedlings (Li et al., 2011). Our studies foundsimilar physiological responses in rubber tree seedlings underdrought stress (Figs. 1 and 3). Under stresses conditions, the accu-mulation of MDA usually leads to the damage of cell membrane inplant and animal. The increase of MDA content was coincided tobroken down in rubber tree seedlings. Besides of RWC, changes ofchlorophyll contents also indicated that drought reshaped thestructure of chloroplasts, and inuenced photosynthesis andHbRbsS gene expression (Figs. 2 and 5). Similar results wereconrmed by wheat leaves under moderate drought stress, which found that thylakoid lumen acidication in drought-stressed leavescould be associated with the activity of an enhanced fraction of PSI.4.2. Osmoregulation was a physiological response to drought stressin rubber treeThe accumulation of proline was involved in regulating the os-motic. The accumulation of proline in leaves of rubber tree seedlingsat later stage after withholding water suggested that rubber treeseedlings had the ability to regulate the osmotic under droughtstress. Stress situations where soluble sugars are involved, such aschilling, herbicide injury, or pathogen attack, are related to impor-tant changes in reactive oxygen species balance (Couee et al., 2006).Fluctuations of soluble sugar content in rubber tree seedlings underdrought stress suggested that soluble sugar involved in the droughttolerance. These results were similar with our previous study inrubber tree seedling under chilling stress. These results suggestedits soluble sugar play an important role in osmoregulation underdrought stress in rubber tree seedlings rather than proline.4.3. ROS scavenging systems related genes function earlier thanphysiological responses under drought stress but limited by ATPformationThe effect of drought on chloroplasts and mitochondria werewell documented (Bigras, 2005). Changes of chlorophyll contentsand gene expression of HbRbsS indicated that the integrity ofchloroplast had been broken down under drought stress. Theimportant role of chloroplasts and mitochondria is ATP generation.HbATP gene encodes the beta subunit of mitochondrial ATP syn-thase (EC 3.6.3.14), which is the most commonly used energycurrency of cells in most organisms. HbCOA encodes a long-chain-fatty-acyl-CoA reductase (EC 1.2.1.50), which takes part in biosyn-thesis of secondary metabolites and cuticular wax biosynthesis.HbACAT encodes an acetyl-CoA C-acetyltransferase (EC 2.3.1.9),248 L.-f. Wang / Plant Physiology and Biochemistry 83 (2014) 243e249Fig. 5. Expressions of energy biosynthesis related genes HbCOA, HbATP, HbRbsS, and HbACAT after withholding water Values represent the mean SD of two biologicalreplicates tested in triplicate. Bars with different uppercase letters show signicant differences at the P < 0.01 level.which mainly takes part in fatty acid and amino acid metabolism.Drought inuenced mitochondria function since decreases ofHbATP, HbCOA and HbACAT transcripts were occurred from 3 to5 dww (Fig. 5.). These suggested that drought induced severalmetabolism pathways synchronously but rst inhibited energyformation.ROS may play two different roles: exacerbating damage oractivating defense responses. The numerous ROS generationsources and complex scavenging systems provide the exibilitynecessary for these functions (Dat et al., 2000). Mitochondria isan important place for ROS production in cell (Mller, 2001). Theintimate relationship between antioxidant enzyme activities anddrought stress were found in woody plant in karst habitats inSouthern China. Transcripts of CAT, MnSOD, and CuZnSOD arelikely to reecting the ability of mitochondria to scavenging ROSand delaying the aging process. In this study, we found that thechanges of ROS scavenging related genes expressions was tightlyrelated to the changes of corresponding enzymes activities.MnSOD is an integral mitochondrial protein known as a rst-lineantioxidant defense against superoxide radical anions producedas by-products of the electron transport chain. In our study,HbMnSOD gene expression was later than that of HbCuZnSODgene expression. These suggested that HbCuZnSOD was moreimportant for drought resistance in rubber tree clone GT1, whichwas similar with previous study in rubber tree clone PB260(Leclercq et al., 2012).5. ConclusionTaken together, these results suggested that rubber tree seedlingwas susceptible to drought stress, and the protection role ofphysiological and molecular responses only lasted for 3e5 daysafter withholding water. Moreover, adaptation to drought stresswas a complex process involved in osmoregulation, antioxidativeenzymes and energy biosynthesis related genes in mitochondriaand chloroplasts in rubber tree seedling. AcknowledgmentsThis work was supported by the National Natural ScienceFoundation of China (31270643); the Earmarked Fund for ModernAgro-industry Technology Research System (CARS-34-GW5) andNatural Science Foundation of Hainan province (310096).ContributionsConceived and designed the experiments: LF Wang. Performedthe experiments: LF Wang. Analyzed the data: LF Wang. Wrote thepaper: LF Wang.ReferencesAhamad, B., 1999. Effect of rootstock on growth and water use efciency of Heveaduring water stress. J. Rubber Res. 2, 99e119.Apel, K., Hirt, H., 2004. 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