JcLEA, Novel LEA-Like Protein from Jatropha curcas, Confers High Level Tolerance to Dehydration and Salinity in Arabidopsis thaliana Jing Liang., Mingqi Zhou., Xin Zhou, Yuanjie Jin, Ming Xu, Juan Lin* State Key Laboratory Genetic Engineering, Institute Plant Biology, School Life Sciences, Fudan University, Shanghai, People's Republic China Abstract Jatropha curcas L. is highly drought and salt tolerant plant species that is typically used as traditional folk medicine and biofuel crop in many countries. Understanding molecular mechanisms that underlie response to various abiotic environmental stimuli, especially to drought and salt stresses, in J. curcas could be important to crop improvement efforts. In this study, we cloned and characterized gene for late embryogenesis abundant (LEA) protein from J. curcas that we designated JcLEA. Sequence analyses showed that JcLEA protein belongs to group 5, subgroup LEA protein family. In young seedlings, expression JcLEA is significantly induced by abscisic acid (ABA), dehydration, and salt stress. Subcellular localization analysis shows that that JcLEA protein is distributed in both nucleus and cytoplasm. Moreover, based on growth status and physiological indices, overexpression JcLEA in transgenic Arabidopsis plants conferred increased resistance to both drought and salt stresses compared to WT. Our data suggests that group 5 JcLEA protein contributes to drought and salt stress tolerance in plants. Thus, JcLEA is potential candidate gene for plant genetic modification. Citation: Liang J, Zhou M, Zhou X, Jin Y, Xu M, et al. (2013) JcLEA, Novel LEA-Like Protein from Jatropha curcas, Confers High Level Tolerance to Dehydration and Salinity in Arabidopsis thaliana. PLoS ONE 8(12): e83056. doi:10.1371/journal.pone.0083056 Editor: Haibing Yang, Purdue University, United States America Received June 3, 2013; Accepted October 30, 2013; Published December 31, 2013 Copyright: ß 2013 Liang et al. This is an open-access article distributed under terms Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided original author and source are credited. Funding: The authors are grateful for financial support from Natural Science Foundation China (31170287)(¥560000), Major Program for Fundamental Research Shanghai, China (09JC1401700)(¥300000) and National High Technology Research and Development Program China (2008AA10Z105) (¥500000). The funders had no role in study design, data collection and analysis, decision to publish, or preparation manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: linjuan@fudan.edu.cn . These authors contributed equally to this work. [18,19]. They function as protecting factor associated with dessication tolerance seeds, pollen and anhydrobiotic tissues [20,21]. Over past two decades, remarkable research progress has been achieved in characterization LEA proteins, which are considered to be functional effectors in generation abiotic tolerance in plants. The LEA protein super families have diversity sequences and functions. Based on nomenclature introduced by Battaglia, LEA proteins are categorized into seven distinctive groups, which include groups 1 (LEA-5), 2 (dehydrin), 3 (LEA-4), 4 (LEA-1), 5 (A: SMP; B: LEA-3; C: LEA-2), 6 (LEA-6), and 7 (ABA-WDS) [22]. Groups 1, 2, 3, and 4, which share specific sequence motifs within each group, are considered to be typical or genuine hydrophilic LEA proteins, while group 5 LEAs lack significant signature motifs or consensus sequences and are regarded as atypical LEA proteins, containing significantly higher proportion hydrophobic residues than typical LEA proteins [22]. It is noteworthy that group 5 proteins are natively folded and are not soluble after boiling, suggesting that they are probably not heat stable [23]. The average molecular weight group 5 proteins is 18.1 kDa, and most group 5 LEA proteins are acidic. Based on these features, it is predicted that group 5 proteins play roles in seed maturation, dehydration, and combining concentrated ions [8]. At present, number group 5 LEA genes have been cloned and characterized from many plant species, including cotton LEA D- Introduction Water and ion concentrations in soil are important abiotic elements for living organisms. Unlike animals, plants are sessile organisms that are exposed to seasonal and local environmental variations, and their survival and growth are strongly influenced by local stress factors. It has been estimated that 70% crop yield losses are caused by abiotic stresses, with drought and high salinity being most serious threats to crop production in many areas world [1]. Plants have developed multi-pathway survival strategies, at multiple-levels and on multiple-scales to allow them to grow and reproduce successfully. The late embryogenesis abundant (LEA) proteins are involved in one type selfprotection mechanism. LEA proteins belong to large protein family that is closely associated with resistances to abiotic stresses, especially to drought, in range organisms [2]. The LEA proteins were originally identified in terrestrial plants, including wheat (Triticum aestivum) and cotton (Gossypium hirsutum), and were subsequently detected in many other species higher plants [3– 8]. LEA proteins mainly accumulate in late stages embryogenesis in plant seeds under dehydration stress [5], and have also been found in bacteria [9], slime molds [10], nematodes [11–15], fungi [16], and humans [17]. LEA proteins are localized to various subcellular compartments, including nucleus, mitochondria, chloroplast, endoplasmic reticulum, vacuole peroxisome, plasma membrane and cytoplasm PLOS ONE | www.plosone.org 1 December 2013 | Volume 8 | Issue 12 | e83056 Overexpression JcLEA Gene in Arabidopsis stresses, especially functions LEA proteins in protecting seedlings from effects dehydration and high salinity, are still poorly understood. In present report, we document molecular cloning an LEA-like gene from J. curcas by RACEPCR. Bioinformatics and expression analyses reveal that J. curcas LEA protein strongly resembles group 5 LEAs from other plant species. The results physiological assays under drought and salt treatments revealed that JcLEA gene confers significantly enhanced tolerance to drought and salt in transgenic Arabidopsis plants. Based on these results, potential application JcLEA gene in genetic engineering crops is discussed. 34 [24], maize RAB28 [25], carrot ECP31 [26], Arabidopsis AtECP31 and AtRAB28 [27,28], Craterostigma plantagineum pcC2745 [29], tomato ER5 [30], cayenne pepper CaLEA6 [31], Medicago truncatula MtPM25 [32], soybean GmPM22, GmPM24, GmPM25, and GmPM26 [33], and OsLEA5 from rice [34]. Nevertheless, group 5 still attracts fewer investigations compared with other LEA subcategories mentioned above. Jatropha curcas L. is multi-purpose shrub belonging to plant family Euphorbiaceae. The species is widespread throughout arid and semi-arid tropical regions world, and has been utilized as source traditional folk medicines. J. curcas is so highly tolerant to dehydration that it can survive 30% field capacity (FC) without drought injury [35]. It has been reported that J. curcas has an efficient adaptive mechanism that enables it to tolerate severe drought situations by maintaining leaf water status and making effective osmotic adjustments [36]. Some evidence also suggests that J. curcas possesses moderate tolerance to salinity, because seedlings can tolerate up to 30 mM NaCl (in irrigation water) without negative effects on their growth parameters. Salttreated plants (irrigated with NaCl levels over 30 mM NaCl) showed significant reduction in growth (by 5.82% for every 10 mM increment in NaCl concentration). This finding shows that J. curcas was more saline-tolerant than most typical Mediterranean crops [37,38]. However, molecular mechanisms involved in response J. curcas to environmental Materials and Methods Plant Materials and Sampling Locations Seeds Jatropha curcas were obtained from Jatropha curcas Germplasm Resources Preservation Center. The plants were grown in natural environment on farmland in Renhe (Panzhihua, Sichuan Province, China). No specific permission was required for any locations or activities, and field studies did not involve endangered or protected species. Seeds were germinated in soil in greenhouse. Roots, leaves and stems were collected and stored at 270uC. Seeds Arabidopsis thaliana accession Columbia (Col-0) were obtained from Arabidopsis Biological Resource Center (ABRI: Columbus, OH, USA). Table 1. Oligonucleotide primers and their uses in this study. Primers Sequence Usage LEAF 59-ATHACNATHGGWGARGCWTTRGAR-39 (H is A, C and T; N is A, C, G and T; W is and T; R is and G) Cloning JcLEA cDNA LEAR 59-RSWAGCAGCAACDCCDCCWGG-39 (W is and T; D is A, G and T) Cloning JcLEA cDNA JcLEAF1 59-GACTACCCTCTCTGATGTTTTAGCGGAC-39 Cloning JcLEA cDNA JcLEAF2 59-GATGCTGAAGGTGTGATTGGAGCGGAAA-39 Cloning JcLEA cDNA JcLEAR1 59-CAGCATCATCCCGAGTTACCGTCTTGTC-39 Cloning JcLEA cDNA JcLEAR2 59-CAGCCACAGAAGCAGCAACTCCACCAGG-39 Cloning JcLEA cDNA JcLEAfull-F 59-ATGAGCCAGGGGCAACCACGAAGAA-39 Cloning JcLEA cDNA, Semiquantitative PCR, Transformants identification JcLEAfull-R 59-TTATGGGTTCTGATTAAGCCTAGCAGCTGC-39 Cloning JcLEA cDNA, Semiquantitative PCR, Transformants identification JcLEAQ1 59-ACGCGGGAACAGAGGTGGCCACTGC-39 Cloning JcLEA cDNA JcLEA-B 59-GCggatccATGAGCCAGGGGCAACCAC-39 Cloning JcLEA cDNA JcLEA-X 59-GCtctagaTTATGGGTTCTGATTAAGC-39 Cloning JcLEA cDNA LEA-GFPF 59-AAccatggTAATGAGCCAGGGGCAACCA-39 Construction JcLEA-GFP LEA-GFPR 59-GGactagtTGGGTTCTGATTAAGCCTAGCA-39 Construction JcLEA-GFP 39-RACE CDS Primer 59-AAGCAGTGGTATCAACGCAGAGTAC(T)30N-1N-39 Cloning 59-RACE CDS Primer 59-(T)25N-1N-39 Cloning JcLEA cDNA UPM Long: 59-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-39 Short: 59CTAATACGACTCACTATAGGGC-39 Cloning JcLEA cDNA Cloning JcLEA cDNA NUP 59-AAGCAGTGGTATCAACGCAGAGT-39 SMARTIIA Oligo 59-AAGCAGTGGTATCAACGCAGAGTA-39 Cloning JcLEA cDNA AP 59-GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT-39 Cloning JcLEA cDNA JcActin-rF 59-TAATGGTCCCTCTGGATGTG-39 Quantitative Real-time PCR JcActin-rR 59-AGAAAAGAAAAGAAAAAAGCAGC-39 Quantitative Real-time PCR LEA-R1 59-AGTGATGTTGTTAGAGAA-39 Quantitative Real-time PCR LEA-R2 59-CCATTGTAATATCCATACC-39 Quantitative Real-time PCR doi:10.1371/journal.pone.0083056.t001 PLOS ONE | www.plosone.org 2 December 2013 | Volume 8 | Issue 12 | e83056 Overexpression JcLEA Gene in Arabidopsis 150 mM, or 200 mM NaCl for 25 days and photographed and analyzed for survival rates at 25th day salt treatment. For drought treatment, water was withheld from 4-week-old plants for 14 days, after which they were rewatered for 10 days. The survival rate determinations and physiological measurements were carried out at 14th day under drought treatment and 10th day after rewatering. Physiological measurements including relative water content, electrolyte leakage, glucose content, sodium content, and potassium content were measured at 14th day dehydration treatment, 10th day after rewatering, and 25th day salt treatment. Whole plants were used for investigation sodium content and potassium content, and leaves were used for other physiological measurements. Plant Growth Conditions For gene expression analysis, 30-day-old J. curcas plants were treated with 300 mM NaCl for 1 day, 100 mM ABA (Abscisic acid) for 3 hours and 30% PEG-4000 for 3 days, respectively. For root length determination, surface-sterilized Arabidopsis seeds were germinated on MS (Murashige and Skoog) medium (pH 5.8) containing 2% phytagel with 50 mM, 100 mM, and 150 mM NaCl or 10% and 15% PEG-4000. The seedlings were grown under 16 h light/8 h dark photoperiod at temperature 22uC with light intensity 150 mmol m22 s21 in plant growth cabinet. Plants were exposed to drought and salt stress for 14 days and then photographed, and root length was measured using ImageJ software. Two transgenic Arabidopsis lines overexpressing JcLEA were used for physiological tests tolerance to high salinity and dehydration. Seedlings growing in greenhouse under 16 h light/8 h dark cycle at 22uC for 4 weeks were watered with 50 mM, 100 mM, RNA Extraction Total RNA J. curcas was extracted as previously reported [39]. Total RNA was extracted from A. thaliana leaves using Figure 1. Nucleotide and deduced protein sequences cloned JcLEA gene. The start and stop codons are indicated in bold. doi:10.1371/journal.pone.0083056.g001 PLOS ONE | www.plosone.org 3 December 2013 | Volume 8 | Issue 12 | e83056 Overexpression JcLEA Gene in Arabidopsis Figure 2. Alignment JcLEA with other plant group 5 LEA proteins sequences. The alignment was performed using published sequences LEAs including RcLEA-D34 from Ricinus communis (XP_002520716), AhLEA-5 from Arachis hypogaea (ADQ91843), MtLEA-D34 from Medicago truncatula (XP_003590628), CsLEA-D34 from Cucumis sativus (XP_004146999), GmLEA-D34 from Glycine max (XP_003536910), VvLEA-D34 PLOS ONE | www.plosone.org 4 December 2013 | Volume 8 | Issue 12 | e83056 Overexpression JcLEA Gene in Arabidopsis from Vitis vinifera (XP_003632304), LEA34_GOSHI from Gossypium hirsutum (p09444), AtLEA-5 from Arabidopsis thaliana (BAB01464), and DcECP31 from Daucus carota (BAD86645). Amino acid residues conserved in all sequences are boxed in black, while similar amino acids are boxed in gray. The three SMP-like motifs are indicated by red, blue, and green boxes. doi:10.1371/journal.pone.0083056.g002 (2.16 g l21 MS powder, 0.5 g l21 2-(N-Morpholino) ethanesulfonic acid, 50 g l21 sucrose, 10 mg l21 6- benzylaminopurine and 0.02% v/v silwet L-77, pH 5.7). The T1 plants were selected on hygromycin (30 mg l21) and further confirmed by PCR using primers JcLEA-full-F and JcLEA-full-R. T2 lines showing 3:1 segregation were carried forward to T3 generation. PCR positive homozygous T3 and T4 lines were used for functional analyses JcLEA. For transient gene expression in Nicotiana tabacum cells, leaves 4-week-old sterilized tobacco plants were infiltrated with A. tumefaciens GV3101 carrying JcLEA-GFP fusion vector. The leaves were placed on MS solid medium in dark at 25uC for two days before imaging analysis. Plant RNA Mini Kit (Aidlab Biotechnologies Co., Inc., Ltd, China). RNA quality and concentration were analyzed by agarose gel electrophoresis (EC250-90, E-C Apparatus Corporation) and spectrophotometry (WFZUV-2100, UnicoTM Instruments Inc.). The RNA samples were stored at 270uC prior to use. Cloning JcLEA Gene from J. curcas Primers LEAF and LEAR were used in reverse transcription polymerase chain reaction (RT-PCR) to obtain partial fragment JcLEA cDNA. This pair primers corresponded was designed from highly conserved amino acid sequences LEA proteins from Ricinus communis (XP_002520716), Glycine max (XP_003536910), Medicago truncatula (XP_003590628) and Gossypium hirsutum (P09444). Amplifications were performed at 94uC for 4 min, followed by 30 cycles amplification (94uC for 40 s, 55uC for 30 s, and 72uC for 40 s). Following this, 59- and 39-rapid amplifications cDNA ends were conducted using SMART technology (SMARTTM RACE cDNA Amplification Kit) to produce full-length JcLEA cDNA. The JcLEA cDNA was sequenced using DYEnamic Direct dGTP Sequencing Kit (Amersham Pharmacia, UK) and 373A DNA sequencing instrument. The sequences were then compared with known sequences in NCBI database using blastp (Standard ProteinProtein BLAST) on NCBI (www.ncbi.nlm.nih.gov). The conserved domains were searched with RPS-BLAST (Search Conserved Domain Database) in NCBI database. The phylogenetic tree LEA proteins sequences was constructed based on their conserved properties and homology. The representative protein sequences seven previously reported groups LEA proteins were obtained from NCBI database (http://www.ncbi.nlm. nih.gov/). MEGA 5.0 was used as computational phylogenetics tool to construct maximum likelihood tree [40]. The classification LEA groups was performed as previously described [22]. The sequences all primers used in this paper are listed in Table 1. Fluorescence and Luminescence Imaging Fluorescence signals were imaged using confocal laser scanning microscope (Zeiss 710, Germany) at magnification 206 and analyzed with Zen software. Before detection fluorescence signals, tobacco leaf pieces were stained with DAPI for 40 min to facilitate observation leaf cell nuclei. Western Blot Analysis Plant nuclear and cytosolic proteins were extracted from N. tabacum leaf disks using Nuclear and Cytoplasmic Protein Extraction Kit (Shanghai Sangon Biotech, China). 50 ng sample extracted protein was boiled in SDS gel loading buffer and electrophoresed on 15% polyacrylamide gel for three hours. Proteins were then electroblotted onto PVDF membrane using Mini Trans-BlotH Electrophoretic Transfer Cell System (Bio-Rad). The membranes were then blocked in Tris-HCl-buffered saline (Tris-buffered saline (TBS): 10 mM Tris-HCl, 150 mM NaCl, pH 7.5) containing 1% bovine serum albumin (BSA) for $2 h. The anti-GFP antibody (#G1112 Santa Cruz Biotechnology Inc. CA, USA) was incubated with membranes at 1:200 dilution in TBS containing 3% BSA overnight at 4uC. After washing, 1:5000 dilution anti-rabbit IgG HRP secondary antibody (#E1012 Santa Cruz Biotechnology) was incubated for 1 hour at room temperature in TBST containing 3% BSA. The filter was then washed and incubated with WestDura Femto ECL kit (Fisher Scientific, Leicestershire, UK), and bands were visualized using ChemiScope 3400 Mini Imaging System (Clinx Science Instruments Co., Ltd., China). Plant Transformation Constructs The open reading frame (ORF) fragment JcLEA was amplified using gene-specific primers JcLEA-B and JcLEA-X and Pfu DNA polymerase (Promega). The resulting fragment was digested with BamHI/XbaI and ligated into corresponding restriction sites pCAMBIA1304 vector under control CaMV35S promoter. For production JcLEA-GFP fusion protein, JcLEA coding region was amplified using primers LEA-GFPF and LEA-GFPR. The PCR product was digested with NcoI/SpeI and inserted into pCAMBIA1302 vector. Expression JcLEA-GFP protein fusion was driven by CaMV35S promoter. These constructs were sequenced to confirm amplification fidelity and exact insertion point JcLEA in pCAMBIA vectors. Quantitative Real-time PCR and Semi-quantitative RTPCR Quantitative real-time PCR (qPCR) was used to analyze gene expression levels as described previously [41]. cDNA was synthesized using PrimeScriptH RT Master Mix (TaKaRa, China) in 20 ml-volume according to manufacturer's instructions. The qPCR was carried out using primers JcLEA-F and JcLEA-R in SYBRH Premix Ex TaqTM II (Perfect Real-Time; TaKaRa, China) on StepOnePlusTM Real-Time PCR System (Applied Biosystems) with three replicates. The PCR procedure was 95uC for 30 s, 40 cycles 95uC for 5 s, and 60uC for 34 s, followed by 95uC for 15 s, 60uC for 1 min, and 95uC for 15 s. The actin gene from J. curcas was used as internal control. The relative expression JcLEA gene was analyzed in four transformed lines Arabidopsis by semi-quantitative RT-PCR Plant Transformation and Confirmation For stable transformation Arabidopsis, Arabidopsis Col-0 was transformed using Agrobacterium-mediated floral dip method. Agrobacterium tumefaciens strain LBA4404 carrying plasmid 35S::JcLEA was cultured overnight to an OD600 1.2 to 1.6. The Agrobacterium cells were collected by centrifugation for 10 min at room temperature at 5000 rpm and then resuspended to an OD600 approximately 0.8 in floral dip inoculation medium PLOS ONE | www.plosone.org 5 December 2013 | Volume 8 | Issue 12 | e83056 Overexpression JcLEA Gene in Arabidopsis Figure 3. Phylogenetic tree LEA protein sequences from different plant species. MEGA 5.0 was used to create an alignment protein sequences downloaded from NCBI database (http://www.ncbi.nlm.nih.gov/). The JcLEA protein cloned from J. curcas shown boxed in group 5. doi:10.1371/journal.pone.0083056.g003 PLOS ONE | www.plosone.org 6 December 2013 | Volume 8 | Issue 12 | e83056 Overexpression JcLEA Gene in Arabidopsis using primers JcLEA-full-F and JcLEA-full-R with following PCR protocol: 94uC for 5 min, followed by 28 cycles 94uC for 30 s, and 55uC for 30 s. The tubulin gene from Arabidopsis was used as internal control. conserved amino acid sequences known plant LEA proteins in NCBI database using SMARTTM RACE cDNA Amplification Kit (Clontech, Palo Alto, CA, USA). The full-length JcLEA cDNA comprised 765 bp open reading frame (ORF), 58 bp 59-untranslated region (UTR), and 247 bp 39-UTR with poly (A) tail (Fig. 1). The ORF encoded polypeptide 254 amino acids, for which calculated isoelectric point and molecular mass were predicted to be 4.64 and 26.5 kDa, respectively (pI/Mw Tool at www.expasy.org). The predicted JcLEA protein contained 33.46% hydrophobic residues, including alanine, isoleucine, leucine, phenylalanine, and valine residues, and no cysteine, histidine or tryptophan residues. Electrolyte Leakage Measurement Ion leakage was determined by method Zhou et al. [42]. Leaves from four plants each line were sampled for each treatment and rinsed with distilled deionized water to remove surface ions before being incubated in 8 ml deionized water at room temperature for 12 h. The sample conductivities (C1) were measured with DDS-11A conductivity meter (Shanghai SUOSHEN Electrical Equipment Co. Ltd., China). The samples were then boiled for 1 h at 100uC and conductivities (C2) were measured when they had cooled to room temperature. The relative electrolyte leakage was calculated using following formula: C1/C2 6100%. The whole assay was repeated three times and data analyzed using Student's t-test. Homology Analysis Amino Acid Sequences database search using Blastx (http://www.ncbi.nlm.nih.gov/) showed that there was relatively high similarity between predicted JcLEA protein and other plant LEA proteins. Notably, LEAs identified in blast analysis all belonged to group 5 subfamily, and included following nine plant proteins: RcLEA-D34 from R. communis (XP_002520716), AhLEA-5 from Arachis hypogaea (ADQ91843), MtLEA-D34 from M. truncatula (XP_003590628), CsLEA-D34 from Cucumis sativus (XP_004146999), GmLEA-D34 from Glycine max (XP_003536910), VvLEA-D34 from Vitis vinifera (XP_003632304), LEA34_GOSHI from G. hirsutum (p09444), AtLEA-5 from A. thaliana (BAB01464) and DcECP31 from Daucus carota (BAD86645). number gaps and insertions were needed to optimize alignment. The amino acid identities between JcLEA and RcLEA-D34, AhLEA-5, MtLEA-D34, CsLEA-D34, GmLEA-D34, VvLEA-D34, LEA34_GOSHI, AtLEA-5, and DcECP31 were found to be 76%, 64%, 64%, 64%, 63%, 62%, 61%, 57%, and 57%, respectively. Moreover, three SMP-like motifs that are generally present in LEA proteins were also detected (Fig. 2). Relative Water Content Measurement Leaves from four plants each Arabidopsis line were collected for each treatment and fresh weights (FW) were determined. The samples were then incubated in 8 ml deionized water at room temperature for 12 h at room temperature. The turgid weights (TW) leaf samples were measured. All samples were ovendried at 65uC for 30 h, and dry weights were measured. The relative water content (%) was calculated as = 100%6(FW-DW)/ (TW-DW) [43]. Three replicates were performed, and data was analyzed by Student's t-test. Glucose Content Assay Glucose content was assayed with Glucose (HK) Assay kit (Sigma-Aldrich, Inc.). Leaves from four different plants each Arabidopsis line for each treatment were collected and incubated in 1.5 ml distilled deionized water and absorbance NADH at 340 nm versus distilled deionized water was measured using BioPhotometer Plus (Eppendorf, Germany) [43]. Three replicates performed, and data were analyzed by Student's t-test. Phylogenic Analysis As there was relatively high degree similarity between JcLEA and other group 5 LEA proteins, we constructed phylogenetic tree to determine specific group to which JcLEA belongs. The representative protein sequences seven groups LEA protein were used to perform Blast analyses so that LEA proteins from various plant species in each group could be identified. The maximum likelihood tree constructed with MEGA 5.0 showed that JcLEA protein is related to group 5 LEA proteins and clustered together with representative proteins this group. In addition, we observed from phylogenetic tree Potassium and Sodium Ion Contents minimum 30 plants from each transgenic line were treated with 50 mM, 100 mM, and 150 mM NaCl. Whole plants were washed three times with distilled deionized water to remove any possible surface ions before drying, and 0.5 g to 1 g tissues samples were then used for measurements. Samples were digested in 30 ml mixed acid digestion solution (HNO3:HClO4) and mixed solutions were heated on electro-thermal board to clarification. The potassium or sodium standard concentration liquid was diluted and added into flame photometer with samples and blank control. Potassium production was determined at 766 nm and sodium at 589 nm using flame photometer (FP6410, China). The potassium and sodium contents samples were calculated against standard curves for potassium and sodium. Results Molecular Cloning Full-length cDNA JcLEA J. curcas belongs to botanical family Euphorbiaceae and grows widely in tropical and sub-tropical areas Central and South America, Africa, India, and Southeast Asia. J. curcas has been considered to be type crop that is highly tolerant to both dehydration [44] and high levels salinity [37]. To understand role LEA protein from J. curcas, we first cloned and sequenced full-length JcLEA cDNA based on highly PLOS ONE | www.plosone.org Figure 4. Quantitative real-time PCR analysis JcLEA gene expression. Leaves 30-day-old J. curcas plants were treated with 300 mM NaCl for one day, 100 mM ABA for three hours, and 30% PEG4000 for three days, respectively. doi:10.1371/journal.pone.0083056.g004 7 December 2013 | Volume 8 | Issue 12 | e83056 Overexpression JcLEA Gene in Arabidopsis Figure 5. Subcellular localization JcLEA protein in tobacco leaf cells. GFP fluorescence JcLEA: GFP and GFP control are shown. DAPI staining was used to visualize nucleus. doi:10.1371/journal.pone.0083056.g005 that proteins within one group shared high levels similarity, but that similarities between groups were relatively low (Fig. 3). Preparation JcLEA-overexpressing Arabidopsis Lines To study contributions JcLEA to drought and salt tolerance, we produced JcLEA-overexpressing lines Arabidopsis Col-0 that were verified by genomic DNA PCR and semiquantitative RT-PCR (Fig. 7a–c). The transgenic lines had different JcLEA expression levels. Among these, lines 25–29 and 25–33 showed higher expression levels JcLEA, and JcLEA transcripts were not detected in WT (Wild type) plants. Homozygous seeds lines 25–29 and lines 25–33 were collected for further investigation. One set was germinated on soil for physiological measurements, and another set was germinated on MS medium for root length observation under conditions drought and salt stress. Expression Pattern JcLEA under Different Abiotic Stress Treatments Previous reports have shown that expression LEA-like genes could be induced by ABA, cold, drought, and high salt treatments [45,46]. As J. curcas can tolerate dehydration and high levels salinity, we have tested possibility that JcLEA is involved in plant response to drought and high salt. The gene expression patterns JcLEA in response to 30% PEG for 3 days [47], 100 mM ABA for 3 hours [42], and 300 mM NaCl for 1 day [48] were investigated in young leaves J. curcas seedlings. The relative expression JcLEA was significantly increased in response to drought, ABA, and NaCl treatments, suggesting that JcLEA might play role in dehydration and high salt tolerance (Fig. 4). Overexpression JcLEA Enhanced Drought Tolerance in Arabidopsis When transgenic Arabidopsis seedlings were grown on MS medium containing 10% PEG and 15% PEG to be exposed to drought conditions, they exhibited much better growth status than did WT Arabidopsis plants, especially for root length, which is sensitive to dehydration (Fig. 8a & b). After water was withheld from Arabidopsis plants for 14 days, transgenic lines suffered less from dehydration compared with WT plants, in which Subcellular Localization JcLEA Protein Information on subcellular localization proteins can be important to elucidate functional roles proteins play in plant cells [43]. In order to determine localization JcLEA protein, JcLEA-GFP translational fusion was constructed and transiently expressed under control 35S promoter in tobacco leaves. Confocal laser fluorescence microscopy showed that JcLEA-GFP fusion protein was localized to cytosol and nucleus (Fig. 5). In order to localize JcLEA-GFP fusion protein more precisely, we tested 35S-JcLEA-GFP and 35S-GFP protein expression in transiently transformed tobacco, by extracting proteins and performing western blot analysis using GFP polyclonal antibody (Fig. 6). These data showed that JcLEA-GFP was localized to both cytosol and nucleus, similar to GFP control. Figure 7. Incorporation and expression JcLEA in transgenic Arabidopsis. (a) Schematic diagram 35S::JcLEA DNA vector construct. (b) Verification transformants from T3-generation regenerated Arabidopsis plants by PCR amplification from genomic DNA. Lane 1 contains fragment size marker. (c) Expression levels JcLEA in four transgenic lines as determined by semi-quantitative RT-PCR. doi:10.1371/journal.pone.0083056.g007 Figure 6. Detection LEA-GFP fusions proteins extracted from tobacco leaves. Western blot analysis proteins extracted from LEAGFP or GFP transiently expressed in tobacco leaf cells, respectively. doi:10.1371/journal.pone.0083056.g006 PLOS ONE | www.plosone.org 8 December 2013 | Volume 8 | Issue 12 | e83056 Overexpression JcLEA Gene in Arabidopsis Figure 8. Phenotypes and physiological indices transgenic Arabidopsis plants bioassayed for drought tolerance. (a and b) Phenotypes and root lengths WT and transgenic plants treated with 10% and 15% PEG on MS medium (pH 5.8) at 22uC for two weeks. Error bars represent standard deviation (SD, n = 6). (c and d) Phenotypes and survival rates plants after withholding water for 14 days, and plants that were then rewatered for 10 days. Representatives typical plants are shown (SD, n = 3). (e and f) Measurement relative water content, electrolyte leakage, and glucose content in plants on 14th day dehydration treatment and 10th day after commencing rewatering (SD, n = 3). * P,0.05; ** P,0.01. doi:10.1371/journal.pone.0083056.g008 content transgenic Arabidopsis was significantly higher than that WT drought treatment and drought plus rewatering, indicating that JcLEA could help to prevent water loss in plants (Fig. 8e). Electrolyte leakage, which is an indicator capacity to protect plasma membrane integrity under stress, was also significantly lower in transgenic plants, demonstrating that JcLEA reduced damage to cell membranes in transgenic plants (Fig. 8f). Moreover, many leaves were bleached and withered (Fig. 8c). After watering again for 10 days, several withered leaves on transgenic plants recovered and turned green, and survival rates 35S::JcLEA seedlings were higher than those WT (Fig. 8c & 8d). For further physiological investigation, relative water content, electrolyte leakage, and glucose content were analyzed as markers drought adaptation. The relative water PLOS ONE | www.plosone.org 9 December 2013 | Volume 8 | Issue 12 | e83056 Overexpression JcLEA Gene in Arabidopsis Figure 9. Phenotypes and physiological indices transgenic Arabidopsis plants bioassayed for high salt tolerance. (a and b) Phenotypes and root lengths WT and transgenic plants treated with 0, 50, 100, 150, and 200 mM NaCl on MS medium (pH 5.8) at 22uC for two weeks. Error bars represent standard deviation (SD, n = 6). (c and d) Phenotypes and survival rates plants treated with different concentrations NaCl (SD, n = 3). (e and f) Measurements relative water content (e), electrolyte leakage (f) and glucose content (g) in plants treated with different PLOS ONE | www.plosone.org 10 December 2013 | Volume 8 | Issue 12 | e83056 Overexpression JcLEA Gene in Arabidopsis concentrations NaCl (SD, n = 3). (h) The Na/K ratio plants treated with 150 mM and 200 mM NaCl. * P,0.05; ** P,0.01. doi:10.1371/journal.pone.0083056.g009 in response to dehydration stress, transgenic plants accumulated more glucose, which contributed to stability internal milieu plant cells (Fig. 8g). These physiological measurements indicated that overexpression JcLEA significantly improved drought tolerance in Arabidopsis. group 5 LEA proteins from plants. The ABA-, drought-, and saltinduced expression patterns JcLEA are also typical other LEAlike genes. Treatments with ABA, drought, salt, and extreme temperatures caused accumulation many type LEA proteins [19,34,57,58]. ABA is key signaling phytohormone involved in drought and salt responses in plants [8]. Previous reports have shown that promoter regions group 5 LEA genes generally contain ABA responsive element (ABRE), which causes induction these genes in response to various abiotic stress conditions [59]. The group 5 JcLEA genes are strongly induced by ABA, drought, and salinity, indicating potential role for JcLEA in response to drought and salt stress in J. curcas, which is consistent with functions characterized LEAs from other plant species [25]. In Arabidopsis, protective function JcLEA was confirmed, based on plant growth status and physiological indices. JcLEA effectively prevents loss water and cytoactive components to maintain integrity plasma membranes and homeostasis plant cells under dehydration as well as high salt stress. The contribution JcLEA to drought tolerance is consistent with strong dehydration resistance observed in J. curcas. During salt stress, accumulated excess Na+, which is toxic to enzymes, is extruded or compartmentalized in vacuole to prevent growth cessation or cell death [60]. The measurements potassium and sodium contents show that JcLEA may regulate Na+ transport and maintain intracellular K+ concentration to stabilize ionic balance plant cells. The function JcLEA is associated with its subcellular localization. It has been reported that LEA proteins, which are non-trans membrane proteins, are widely localized in multiple subcellular compartments in various plant species [19]. The PsLEAm protein from pea is localized in mitochondrial matrix seeds, and is first mitochondrial presequence in plant LEA protein [61]. Group 1 (AfrLEA1-1) and group 3 (AfrLEA3-4) LEA proteins from Artemia franciscana are also localized in mitochondria [62], while many kinds group 2 LEA proteins are localized in nucleus [22]. The respective subcellular locations LEA proteins may be correlated with different aspects cellular protection during various abiotic stresses. JcLEA appears to be localized mainly to nucleus and cytoplasm, which suggests that it has comprehensive protective functions in plant cells. This finding is also consistent with water and ionic homeostasis in transgenic Arabidopsis plants that express JcLEA. In summary, JcLEA is novel LEA protein belonging to group 5. The constitutive expression JcLEA does not cause growth repression in host plants. From results this study, it can be concluded that JcLEA has huge potential for transgenic breeding based on (1) fact that gene is induced by exposure to drought and salt, (2) subcellular localization JcLEA and (3) improved tolerance to dehydration and high salt levels observed in transgenic Arabidopsis plants. Overexpression JcLEA Enhanced Salt Tolerance in Arabidopsis Similar to investigation drought resistance, phenotypes and physiological indices in 35S::JcLEA plants treated with NaCl were evaluated to test whether JcLEA protein functions in salt tolerance. When treated with NaCl solutions, transgenic lines showed significantly higher levels tolerance and higher survival rates than did WT plants (Fig. 9a–d). WT plants displayed severe damage at $100 mM NaCl, while nearly 50% 35S::JcLEA plants survived even when treated with 200 mM NaCl. The physiological indices including relative water content, electrolyte leakage, and glucose content also indicated that JcLEA contributed to cellular protection during salt stress (Fig. 9e–g). When exposed to high levels NaCl, plant cells attempt to maintain high concentrations K+ and low concentrations Na+ in cytosol in order to establish intracellular K+ and Na+ homeostasis, which is crucial for protection cytosolic enzymes and osmotic pressure stability [49]. We evaluated Na/K ratio in transgenic and control seedlings based on Na+ and K+ contents. Our results showed that presence JcLEA reduced Na/K ratio below 150 mM NaCl, suggesting protective function for this protein through exclusion extra Na+. However, JcLEA failed to exclude Na+ in 200 mM NaCl treatment, which might be an upper limit for capacity ionic adjustment (Fig. 9h). Discussion The LEA proteins are family hydrophilic proteins that are presumed to play protective role during exposure to different abiotic stresses. This large protein family can be classified into seven groups, designated groups one to seven [22]. Among these, roles groups 1, 2, and 3 have been extensively studied in relation to abiotic stress response. HVA1 from Hordeum vulgare, belonging to group 3, confers increased drought tolerance in wheat and rice [50,51], and enhanced drought, salinity, and cold tolerance when expressed in mulberry [52]. In group 2 LEAs, PMA80 from wheat increases dehydration tolerance transgenic rice [53], and DHN24 from Solanum sogarandinum improves cold tolerance transgenic cucumber [54]. Similarly, group 1 protein PMA1959 from wheat enhances tolerance to dehydration and salinity in transgenic rice [53]. On other hand, some LEA proteins appear to contribute little to plant stress tolerance. The overexpression two cold-induced LEA proteins CAP160 and CAP85 from Spinacia oleracea and three desiccation-induced pcC proteins from C. plantagineum failed to change freezing or drought tolerance in transgenic tobacco plants [55,56]. Thus, LEA family members may have different functions in plant stress responses. In comparison, role group 5 LEA proteins in abiotic stress tolerance in plants is not well known. Here, we show that novel JcLEA protein isolated from J. curcas possesses high level amino acid sequence identity and has similarity to other PLOS ONE | www.plosone.org Author Contributions Conceived and designed experiments: J. Lin J. Liang MZ. Performed experiments: J. Liang MZ XZ YJ MX. Analyzed data: J. Liang XZ. Contributed reagents/materials/analysis tools: MZ XZ. Wrote paper: J. Lin J. Liang MZ. 11 December 2013 | Volume 8 | Issue 12 | e83056 Overexpression JcLEA Gene in Arabidopsis References 1. 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