RESEARCH ARTICLE
Overexpression of EcbHLH57 Transcription Factor from Eleusine coracana L. in Tobacco Confers Tolerance to Salt, Oxidative and Drought Stress K. C. Babitha☯, Ramu S. Vemanna☯, Karaba N. Nataraja, M. Udayakumar* Department of Crop Physiology, University of Agricultural Sciences, Bangalore, Karnataka, India ☯ These authors contributed equally to this work. * [email protected]
Abstract OPEN ACCESS Citation: Babitha KC, Vemanna RS, Nataraja KN, Udayakumar M (2015) Overexpression of EcbHLH57 Transcription Factor from Eleusine coracana L. in Tobacco Confers Tolerance to Salt, Oxidative and Drought Stress. PLoS ONE 10(9): e0137098. doi:10.1371/journal.pone.0137098 Editor: Keith R. Davis, Indiana University, UNITED STATES Received: December 25, 2014 Accepted: August 12, 2015 Published: September 14, 2015 Copyright: © 2015 Babitha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Department of Biotechnology, Centre of Excellence programme support (BT/01/COE/05/03), the Indian Council of Agricultural Research Niche area of Excellence programme (F.No. 10-(6)/2005 EP&D), and the Department of Biotechnology- University of Agricultural Sciences Biotech HUB (Ref No. BT/INF/ UAS Bangalore/2011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Basic helix-loop-helix (bHLH) transcription factors constitute one of the largest families in plants and are known to be involved in various developmental processes and stress tolerance. We report the characterization of a stress responsive bHLH transcription factor from stress adapted species finger millet which is homologous to OsbHLH57 and designated as EcbHLH57. The full length sequence of EcbHLH57 consisted of 256 amino acids with a conserved bHLH domain followed by leucine repeats. In finger millet, EcbHLH57 transcripts were induced by ABA, NaCl, PEG, methyl viologen (MV) treatments and drought stress. Overexpression of EcbHLH57 in tobacco significantly increased the tolerance to salinity and drought stress with improved root growth. Transgenic plants showed higher photosynthetic rate and stomatal conductance under drought stress that resulted in higher biomass. Under long-term salinity stress, the transgenic plants accumulated higher seed weight/pod and pod number. The transgenic plants were also tolerant to oxidative stress and showed less accumulation of H202 and MDA levels. The overexpression of EcbHLH57 enhanced the expression of stress responsive genes such as LEA14, rd29A, rd29B, SOD, APX, ADH1, HSP70 and also PP2C and hence improved tolerance to diverse stresses.
Introduction The environmental stress signals such as drought, salinity, high temperature perceived by the plants results in diverse stress responses. These responses are regulated by many complex mechanisms which are controlled by transcriptional regulation. Transcription factors (TFs) play important regulatory roles in stress responses by regulating their target gene expression by binding to the conserved cis-acting elements [1, 2, 3]. Several stress responsive TFs belonging to APETELA2 (AP2), bHLH, bZIP, NAC, ZF, MYB and WRKY families have been well elucidated for their regulatory roles under various stress conditions. Transgenic plants overexpressing these groups of genes have shown improved tolerance to different stresses [4, 5, 6, 7].
PLOS ONE | DOI:10.1371/journal.pone.0137098 September 14, 2015
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EcbHLH57 Improves Abiotic Stress Tolerance in Tobacco
Competing Interests: The authors have declared that no competing interests exist
The bHLH proteins are one of the largest families of transcription factors both in animals and plants and are well characterized in mammalian system [8]. The bHLH domain contains approximately 60 amino acids, with two functionally distinctive regions, the basic region and the HLH region. The basic region consists of 15 amino acids and functions as a DNA binding domain and HLH region contains two amphipathic α- helices linked by a loop. This allows the formation of homodimers or heterodimers [9, 10]. bHLH proteins bind to sequences containing a consensus core elements of the E-box (5’-CANNTG-3’), and the G box (5’-CACGTG-3’) cis elements. In Arabidopsis and rice, 162 and 167 bHLH encoding genes have been identified respectively [11, 12]. Many of these genes have been characterized for their role in developmental processes such as flavonoid and anthocyanin biosynthesis [10, 13], trichome and tapetum development [14, 15], shoot branching [16], controlling grain length and width [17, 18]. Some of the genes are also involved in signaling processes [19, 20, 21]. A number of bHLH genes have been shown to be involved in stress tolerance. The microarray analysis of NaCl treated Arabidopsis roots showed a differential regulation of 29 bHLH genes [22]. In soybean, out of 45 bHLH genes, 14 are responsive to abiotic/biotic stresses. Improved tolerance to diverse abiotic stresses has been reported in transgenic plants overexpressing AtbHLH92 [22], AtICE1 [23], OsbHLH1 [24], MdCIbHLH1 [25]. Both, AtICE1 in Arabidopsis and MdCIbHLH1 in apple enhanced the expression of C-repeat binding factor (CBF) regulons. Across the species there is a significant variation in the threshold levels of tolerance. One of the reasons attributed was the stress responsive functional or regulatory gene transcripts/proteins from tolerant species are either stable or functionally more efficient under stress [26, 3]. Comparative transcriptome studies in susceptible and tolerant rice varieties showed upregulation of 1900 genes with 77 TFs in drought tolerant N22 genotype as compared to drought susceptible IR64 variety which showed significant downregulation of regulatory genes. Hence tolerant variety may have better mechanisms to reprogram the expression of genes under stress conditions [27]. Similarly superoxide dismutase from drought tolerant Arabidopsis thaliana ecotype Cvi showed better stability under high photo-oxidative stress than from drought susceptible ecotype ler [28]. Hence stress tolerant genotype/species may have better alleles which may contribute to survival under adverse climatic conditions [27]. Hence recent research focus has been on validating genes from stress adapted species such as such as Thellungiella halophila, a salt-tolerant relative of Arabidopsis, Atriplex hortensis and Tamarix hispida due to their extreme stress adaptive nature [29, 30, 31, 32]. Overexpression of OrbHLH001, a close homolog of ICE1 from wild rice Oryza rufipogan in Arabidopsis resulted in higher salinity tolerance [33]. Even OrbHLH2 also showed increased tolerance to salt and osmotic stress by upregulating several dehydrin group of genes [34]. We also showed that overexpression of EcNAC1 transcription factor from finger millet in tobacco improved the tolerance to abiotic stresses [35]. In the present study our focus is to characterize a bHLH family gene from finger millet. Finger millet (Eleusine coracana (L.) Gaertn) is a drought hardy crop and widely grown in the arid areas of Africa and Asia and thrives well under disturbed soils, can grow in hot climates and survives severe water deficit and osmotic stress [36]. Comparative analysis for drought stress tolerance among different crop species showed that finger millet was highly drought tolerant with nearly 90% survivability similar to drought tolerant species peanut and horse gram. However, sunflower, beans and tomato were found to be highly susceptible to drought with only 20% survival rate [37]. Further, extensive screening of finger millet germplasms under drought conditions showed that GPU-28 belonged to tolerant group with a low drought susceptibility index (DSI) of 0.9–1 compared to susceptible genotype of DSI of 1.9. Also, in a study using induction response technique (34), GPU-28 had better acquired tolerance, hence this genotype was chosen for the present study. Due to this extreme hardiness, genes from finger millet would be a valuable resource to improve stress tolerance. From this
PLOS ONE | DOI:10.1371/journal.pone.0137098 September 14, 2015
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EcbHLH57 Improves Abiotic Stress Tolerance in Tobacco
context, a drought stress cDNA library of finger millet cv.GPU28 was constructed and several stress responsive transcription factors were identified [35, 38]. In the finger millet stress cDNA library a few bHLH ESTs were identified and the emphasis in this study was on cloning and characterization of stress responsive EcbHLH57 from finger millet, a homolog of OsbHLH057 and AtILR3 in model plant tobacco. Overexpression of EcbHLH57 in tobacco resulted in improved tolerance to diverse abiotic stresses.
Materials and Methods Plant material and stress treatments Finger millet genotype GPU-28 used in this study belongs to tolerant group of genotypes with a low drought susceptibility index (DSI) of 0.9 compared to susceptible genotypes with DSI of 1.9–2.0. Finger millet seedlings (var. GPU-28) were grown in small pots for one month and subjected to drought stress imposed gravimetrically [37]. The leaf material was collected at 100, 80, 60 and 40% field capacities (FC). For seedling level stress treatments, GPU-28 seeds were germinated in petridishes and 7-day-old seedlings were treated with 10 μM ABA, -12 bars polyethylene glycol (PEG)-6000, 300 mM NaCl and 10 μM MV. The treated seedlings were harvested at 12 and 24 h for expression analysis.
Isolation of full-length sequence of EcbHLH57 from finger millet The partial length cDNA of EcbHLH57 was isolated from finger millet stress cDNA library. The 5’ end was amplified using RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) kit (Invitrogen Corporation, Carlsbad, CA, USA) according to manufacturer’s instructions. The full length sequence of 1053 bp was amplified using gene specific primers and the reaction was carried out under standard PCR conditions. Amplified product was cloned into T/A vector (pTZ57/R, MBI Fermentas) and sequenced. The protein sequence was analysed for potential motifs/domains using PLANTsP software tool (http://plantsp.genomics.purdue. edu/index.html). The protein sequences were multiple aligned with close homolog from different species using CLUSTALW multiple alignment tool (http://www.genome.jp/tools/clustalw/) and phylogenetic tree was deduced. These aligned sequences were submitted to SMS2 (http:// www.bioinformatics.org/sms2/) to arrive at the conserved regions within the close homologs. The interaction of EcbHLH57 ortholog AtILR3 with other genes was studied using GeneMANIA tool (www.genemania.org) Generation of transgenic tobacco overexpressing EcbHLH57. The pTZ57/R vector was digested with XbaI and BamHI to release EcbHLH57 cDNA fragment and subcloned into cloning vector IM1.1RBCS. Subsequently PRBCS::EcbHLH57:Trbcs fragment was released by digesting IM1.1RBCS: EcbHLH57 with AscI and PacI and cloned into pBINplus vector. The plasmid was then mobilized into Agrobacterium tumefaciens strain EHA105 by electroporation. Tobacco var. KST-19 was transformed by using Agrobacterium mediated tranformation method [39]. The transformants were selected on Murashige and Skoog (MS) agar medium [40] containing 100 mg/L kanamycin, 350 mg/L cefotaxime. The seeds obtained from T0 transformants were germinated on MS agar medium supplemented with 100 μg/mL kanamycin. Based on the phenotype of the seedlings under the selection pressure for 15-days, segregation ratio was calculated (S2 Table).
Gene expression analysis Total RNA was extracted according to the protocol described by Datta et al. [41] and first strand cDNA was synthesized by oligo (dT) primers using Molony Murine Leukaemia Virus
PLOS ONE | DOI:10.1371/journal.pone.0137098 September 14, 2015
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EcbHLH57 Improves Abiotic Stress Tolerance in Tobacco
reverse transcriptase (MMLV-RT; MBI Fermentas, Hanover, MD, USA) according to manufacturer’s instructions. The cDNA pool was used as a template to perform RT- PCR analysis. PCR conditions were 94°C for 2 min, 25 cycles of 94°C for 45 s, 52–58°C for 30 s, 72°C for 30 s and a final extension of 72°C for 10 min. The quantitative real-time RT-PCR was performed in a qPCR machine (RT-PCR; Opticon2, MJ research, USA) with the fluorescent dye SYBR-green (TAKARA SYBR-green qPCR Kit) following the manufacturer’s protocol. The conditions for the PCR were as follows: 95°C for 2 min, 25 cycles of denaturation at 94°C for 45 s, annealing for 30 sec (56°C and 58°C for genes and elongation factor, respectively), polymerization for 45 s (72°C) followed by plate reading at 72°C for 5 min, estimation of melting curve from 50°C to 95°C and incubation at 72°C for 4 min. The relative expression levels of the selected genes under a given stress condition was calculated using comparative threshold method. Elongation factor was used as internal control for tobacco samples and actin was used as internal control for finger millet samples to normalize qRT- PCR (For primer list See S1 Table). The reaction efficiency for each gene was calculated using software LinRegPCR [42] (S3 Table).
Drought and salinity stress tolerance assays For drought stress imposition, tobacco transgenic and wild type plants were grown in pots and when the plants reached 90 days, soil water status was gradually reduced by gravimetric approach. Gradual moisture stress was imposed by weighing the pots and the loss of water in the pots was replenished with required amount of water to arrive at the desired field capacity (FC) of soil. At the end of stress period when pots reached the required FC (60 and 40%), they were maintained in that particular FC for about 3 days [37]. One set of plants was maintained at 100% FC. Gas exchange parameters such as photosynthetic rate (μmolm−2s−1) and stomatal conductance (mmol−2s−1) were measured using a portable photosynthetic system (LICOR 6400, USA). The plants were assessed for dry matter accumulation by measuring the stem, leaves and root biomass. 30-day-old transgenic and wild type plants were subjected to drought stress by withholding water for 1 week and analysed for relative water content, electrolyte leakage, chlorophyll content and MDA levels. Salinity stress was imposed on 30-day-old transgenic and wild type plants grown hydroponically by supplementing 300 mM NaCl for 7 days. At the end of stress period, the shoot and root fresh weight of plants, electrolyte leakage and chlorophyll content was recorded. For longterm salinity stress, 90-day-old pot grown plants were treated with 100 mM NaCl for 25 days and later grown upto maturity and yield parameters were recorded.
Seedling level stress imposition The germinated seeds of transgenic and wild type were placed on half MS agar medium supplemented with 100 mM NaCl. The seedling growth was observed 7 days after stress treatment. The fresh weight, root length and cotyledon greening of seedlings were recorded. Photographs were taken at the end of the stress period.
Estimation of total chlorophyll content Chlorophyll was extracted from 200 mg of leaf tissue in acetone:DMSO (1:1) and the supernatant was made up to a known volume. The absorbance was recorded at 663 nm and 645 nm using UV-visible spectrophotometer (UV 2450, Shimadzu Corporation, Kyoto, Japan). Total
PLOS ONE | DOI:10.1371/journal.pone.0137098 September 14, 2015
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EcbHLH57 Improves Abiotic Stress Tolerance in Tobacco
chlorophyll was estimated by using the following formula and expressed as mg/g FW [43]. CHLa ¼ ð12:7ðA663 Þ 2:69ðA645 Þ VÞ=ðW 1000Þ CHLb ¼ ð22:9ðA645 Þ 4:68ðA663 Þ VÞ=ðW 1000Þ TCHLðmg=gFWÞ ¼ CHLa þ CHLb Where, CHL = chlorophyll, V = volume, W = weight and TCHL = Total chlorophyll
Electrolyte leakage assay Leaf discs from 30-day-old transgenic and wild type plants subjected to NaCl treatment and drought stress were analyzed for membrane damage by electrolyte leakage assay using conductivity meter (Elico-India, CM183, EC-TDS analyser) [37]. The leaf discs were transferred to deionised water and incubated for 2 h and initial electrical conductivity (EC) was recorded. The samples were then boiled for 15 min and final EC of the resultant solution was recorded. The electrolyte leakage was calculated using the following formula: EC (%) = (IEC/FEC) x 100 where, IEC = initial EC, FEC = final EC
Estimation of relative water content Leaf discs from 30-day-old transgenic and wild type plants subjected to drought stress were analysed for relative water content. Leaf discs were initially weighed to record the fresh weight. Later they were floated in deionized water and incubated for 5 h. The discs were blotted on filter paper to remove excess water and recorded the turgid weight. The leaf discs were ovendried and recorded the dry weight. The relative water content was estimated using the formula: RWC (%) = ((FW−DW) / (TW−DW)) x 100 where FW = fresh weight, DW = dry weight, TW = Turgid weight.
Excised leaf disc assays Leaf discs were made from identical leaves of 3-month-old transgenic and wild type plants were placed on half MS medium containing -6 bars PEG 6000, 200 mM NaCl, and 2 μM Methyl viologen (MV) and incubated for 72 h. After stress period, leaf discs were analysed for total chlorophyll content as described previously [43]
Estimation of H2O2 levels The leaf discs treated with MV was extracted in phosphate buffer of pH 7.5 and 25 μl supernatant was taken and mixed with 275 μl of xylenol orange reagent. The reaction mix was incubated for 30 min at room temperature and absorbance was measured at 560 nm against blank (xylenol orange reagent) [44]. The xylenol orange reagent was prepared in 50 ml of distilled water containing 1ml of 50 mM ferrous ammonium sulphate in 2.5 M H2SO4 and 62.5 μl of 125 μM xylenol orange along with 0.9019 g sorbitol.
Estimation of malondialdehyde (MDA) content MDA content was estimated by using TBARS assay. About 0.5–1.0 g of tissue was homogenized in 5 ml of 5% (w/v) trichloroacetic acid and the homogenate was centrifuged at 12,000 g for 15 min at room temperature. The supernatant was mixed with an equal volume of thiobarbituric acid (0.5% in 20% (w/v) trichloroacetic acid), and the mixture was boiled for 25 min at 100°C, followed by centrifugation for 5 min at 7,500 g to clarify the solution. Absorbance of the
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EcbHLH57 Improves Abiotic Stress Tolerance in Tobacco
supernatant was measured at 532 nm and corrected for nonspecific turbidity by subtracting the A600. MDA content in leaf tissue was calculated using standard graph [45].
Analysis of stomatal characteristics In order to observe the stomatal characters, leaves from same aged plants and from same relative position was selected. The epidermal impressions were taken by applying a thin layer of thermocole paste in xylene on abaxial and adaxial surface of leaves. The imprint was removed and placed on glass slide and observed under light microscope with 40X magnification. The stomatal number and epidermal cell number was counted and calculated the stomatal index. The stomatal index was calculated by dividing number of stomata with total number of cells (i, e epidermal cells and stomatal cells) and multiplied by 100.
Statistical analysis The data obtained in different experimental results was analysed using two-way analysis of variance (ANOVA) as per the procedure given by Fischer [46]. Data points with different lowercase letters indicate significant differences (P
Overexpression of EcbHLH57 Transcription Factor from Eleusine coracana L. in Tobacco Confers Tolerance to Salt, Oxidative and Drought Stress K. C. Babitha☯, Ramu S. Vemanna☯, Karaba N. Nataraja, M. Udayakumar* Department of Crop Physiology, University of Agricultural Sciences, Bangalore, Karnataka, India ☯ These authors contributed equally to this work. * [email protected]
Abstract OPEN ACCESS Citation: Babitha KC, Vemanna RS, Nataraja KN, Udayakumar M (2015) Overexpression of EcbHLH57 Transcription Factor from Eleusine coracana L. in Tobacco Confers Tolerance to Salt, Oxidative and Drought Stress. PLoS ONE 10(9): e0137098. doi:10.1371/journal.pone.0137098 Editor: Keith R. Davis, Indiana University, UNITED STATES Received: December 25, 2014 Accepted: August 12, 2015 Published: September 14, 2015 Copyright: © 2015 Babitha et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Department of Biotechnology, Centre of Excellence programme support (BT/01/COE/05/03), the Indian Council of Agricultural Research Niche area of Excellence programme (F.No. 10-(6)/2005 EP&D), and the Department of Biotechnology- University of Agricultural Sciences Biotech HUB (Ref No. BT/INF/ UAS Bangalore/2011). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Basic helix-loop-helix (bHLH) transcription factors constitute one of the largest families in plants and are known to be involved in various developmental processes and stress tolerance. We report the characterization of a stress responsive bHLH transcription factor from stress adapted species finger millet which is homologous to OsbHLH57 and designated as EcbHLH57. The full length sequence of EcbHLH57 consisted of 256 amino acids with a conserved bHLH domain followed by leucine repeats. In finger millet, EcbHLH57 transcripts were induced by ABA, NaCl, PEG, methyl viologen (MV) treatments and drought stress. Overexpression of EcbHLH57 in tobacco significantly increased the tolerance to salinity and drought stress with improved root growth. Transgenic plants showed higher photosynthetic rate and stomatal conductance under drought stress that resulted in higher biomass. Under long-term salinity stress, the transgenic plants accumulated higher seed weight/pod and pod number. The transgenic plants were also tolerant to oxidative stress and showed less accumulation of H202 and MDA levels. The overexpression of EcbHLH57 enhanced the expression of stress responsive genes such as LEA14, rd29A, rd29B, SOD, APX, ADH1, HSP70 and also PP2C and hence improved tolerance to diverse stresses.
Introduction The environmental stress signals such as drought, salinity, high temperature perceived by the plants results in diverse stress responses. These responses are regulated by many complex mechanisms which are controlled by transcriptional regulation. Transcription factors (TFs) play important regulatory roles in stress responses by regulating their target gene expression by binding to the conserved cis-acting elements [1, 2, 3]. Several stress responsive TFs belonging to APETELA2 (AP2), bHLH, bZIP, NAC, ZF, MYB and WRKY families have been well elucidated for their regulatory roles under various stress conditions. Transgenic plants overexpressing these groups of genes have shown improved tolerance to different stresses [4, 5, 6, 7].
PLOS ONE | DOI:10.1371/journal.pone.0137098 September 14, 2015
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EcbHLH57 Improves Abiotic Stress Tolerance in Tobacco
Competing Interests: The authors have declared that no competing interests exist
The bHLH proteins are one of the largest families of transcription factors both in animals and plants and are well characterized in mammalian system [8]. The bHLH domain contains approximately 60 amino acids, with two functionally distinctive regions, the basic region and the HLH region. The basic region consists of 15 amino acids and functions as a DNA binding domain and HLH region contains two amphipathic α- helices linked by a loop. This allows the formation of homodimers or heterodimers [9, 10]. bHLH proteins bind to sequences containing a consensus core elements of the E-box (5’-CANNTG-3’), and the G box (5’-CACGTG-3’) cis elements. In Arabidopsis and rice, 162 and 167 bHLH encoding genes have been identified respectively [11, 12]. Many of these genes have been characterized for their role in developmental processes such as flavonoid and anthocyanin biosynthesis [10, 13], trichome and tapetum development [14, 15], shoot branching [16], controlling grain length and width [17, 18]. Some of the genes are also involved in signaling processes [19, 20, 21]. A number of bHLH genes have been shown to be involved in stress tolerance. The microarray analysis of NaCl treated Arabidopsis roots showed a differential regulation of 29 bHLH genes [22]. In soybean, out of 45 bHLH genes, 14 are responsive to abiotic/biotic stresses. Improved tolerance to diverse abiotic stresses has been reported in transgenic plants overexpressing AtbHLH92 [22], AtICE1 [23], OsbHLH1 [24], MdCIbHLH1 [25]. Both, AtICE1 in Arabidopsis and MdCIbHLH1 in apple enhanced the expression of C-repeat binding factor (CBF) regulons. Across the species there is a significant variation in the threshold levels of tolerance. One of the reasons attributed was the stress responsive functional or regulatory gene transcripts/proteins from tolerant species are either stable or functionally more efficient under stress [26, 3]. Comparative transcriptome studies in susceptible and tolerant rice varieties showed upregulation of 1900 genes with 77 TFs in drought tolerant N22 genotype as compared to drought susceptible IR64 variety which showed significant downregulation of regulatory genes. Hence tolerant variety may have better mechanisms to reprogram the expression of genes under stress conditions [27]. Similarly superoxide dismutase from drought tolerant Arabidopsis thaliana ecotype Cvi showed better stability under high photo-oxidative stress than from drought susceptible ecotype ler [28]. Hence stress tolerant genotype/species may have better alleles which may contribute to survival under adverse climatic conditions [27]. Hence recent research focus has been on validating genes from stress adapted species such as such as Thellungiella halophila, a salt-tolerant relative of Arabidopsis, Atriplex hortensis and Tamarix hispida due to their extreme stress adaptive nature [29, 30, 31, 32]. Overexpression of OrbHLH001, a close homolog of ICE1 from wild rice Oryza rufipogan in Arabidopsis resulted in higher salinity tolerance [33]. Even OrbHLH2 also showed increased tolerance to salt and osmotic stress by upregulating several dehydrin group of genes [34]. We also showed that overexpression of EcNAC1 transcription factor from finger millet in tobacco improved the tolerance to abiotic stresses [35]. In the present study our focus is to characterize a bHLH family gene from finger millet. Finger millet (Eleusine coracana (L.) Gaertn) is a drought hardy crop and widely grown in the arid areas of Africa and Asia and thrives well under disturbed soils, can grow in hot climates and survives severe water deficit and osmotic stress [36]. Comparative analysis for drought stress tolerance among different crop species showed that finger millet was highly drought tolerant with nearly 90% survivability similar to drought tolerant species peanut and horse gram. However, sunflower, beans and tomato were found to be highly susceptible to drought with only 20% survival rate [37]. Further, extensive screening of finger millet germplasms under drought conditions showed that GPU-28 belonged to tolerant group with a low drought susceptibility index (DSI) of 0.9–1 compared to susceptible genotype of DSI of 1.9. Also, in a study using induction response technique (34), GPU-28 had better acquired tolerance, hence this genotype was chosen for the present study. Due to this extreme hardiness, genes from finger millet would be a valuable resource to improve stress tolerance. From this
PLOS ONE | DOI:10.1371/journal.pone.0137098 September 14, 2015
2 / 21
EcbHLH57 Improves Abiotic Stress Tolerance in Tobacco
context, a drought stress cDNA library of finger millet cv.GPU28 was constructed and several stress responsive transcription factors were identified [35, 38]. In the finger millet stress cDNA library a few bHLH ESTs were identified and the emphasis in this study was on cloning and characterization of stress responsive EcbHLH57 from finger millet, a homolog of OsbHLH057 and AtILR3 in model plant tobacco. Overexpression of EcbHLH57 in tobacco resulted in improved tolerance to diverse abiotic stresses.
Materials and Methods Plant material and stress treatments Finger millet genotype GPU-28 used in this study belongs to tolerant group of genotypes with a low drought susceptibility index (DSI) of 0.9 compared to susceptible genotypes with DSI of 1.9–2.0. Finger millet seedlings (var. GPU-28) were grown in small pots for one month and subjected to drought stress imposed gravimetrically [37]. The leaf material was collected at 100, 80, 60 and 40% field capacities (FC). For seedling level stress treatments, GPU-28 seeds were germinated in petridishes and 7-day-old seedlings were treated with 10 μM ABA, -12 bars polyethylene glycol (PEG)-6000, 300 mM NaCl and 10 μM MV. The treated seedlings were harvested at 12 and 24 h for expression analysis.
Isolation of full-length sequence of EcbHLH57 from finger millet The partial length cDNA of EcbHLH57 was isolated from finger millet stress cDNA library. The 5’ end was amplified using RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) kit (Invitrogen Corporation, Carlsbad, CA, USA) according to manufacturer’s instructions. The full length sequence of 1053 bp was amplified using gene specific primers and the reaction was carried out under standard PCR conditions. Amplified product was cloned into T/A vector (pTZ57/R, MBI Fermentas) and sequenced. The protein sequence was analysed for potential motifs/domains using PLANTsP software tool (http://plantsp.genomics.purdue. edu/index.html). The protein sequences were multiple aligned with close homolog from different species using CLUSTALW multiple alignment tool (http://www.genome.jp/tools/clustalw/) and phylogenetic tree was deduced. These aligned sequences were submitted to SMS2 (http:// www.bioinformatics.org/sms2/) to arrive at the conserved regions within the close homologs. The interaction of EcbHLH57 ortholog AtILR3 with other genes was studied using GeneMANIA tool (www.genemania.org) Generation of transgenic tobacco overexpressing EcbHLH57. The pTZ57/R vector was digested with XbaI and BamHI to release EcbHLH57 cDNA fragment and subcloned into cloning vector IM1.1RBCS. Subsequently PRBCS::EcbHLH57:Trbcs fragment was released by digesting IM1.1RBCS: EcbHLH57 with AscI and PacI and cloned into pBINplus vector. The plasmid was then mobilized into Agrobacterium tumefaciens strain EHA105 by electroporation. Tobacco var. KST-19 was transformed by using Agrobacterium mediated tranformation method [39]. The transformants were selected on Murashige and Skoog (MS) agar medium [40] containing 100 mg/L kanamycin, 350 mg/L cefotaxime. The seeds obtained from T0 transformants were germinated on MS agar medium supplemented with 100 μg/mL kanamycin. Based on the phenotype of the seedlings under the selection pressure for 15-days, segregation ratio was calculated (S2 Table).
Gene expression analysis Total RNA was extracted according to the protocol described by Datta et al. [41] and first strand cDNA was synthesized by oligo (dT) primers using Molony Murine Leukaemia Virus
PLOS ONE | DOI:10.1371/journal.pone.0137098 September 14, 2015
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EcbHLH57 Improves Abiotic Stress Tolerance in Tobacco
reverse transcriptase (MMLV-RT; MBI Fermentas, Hanover, MD, USA) according to manufacturer’s instructions. The cDNA pool was used as a template to perform RT- PCR analysis. PCR conditions were 94°C for 2 min, 25 cycles of 94°C for 45 s, 52–58°C for 30 s, 72°C for 30 s and a final extension of 72°C for 10 min. The quantitative real-time RT-PCR was performed in a qPCR machine (RT-PCR; Opticon2, MJ research, USA) with the fluorescent dye SYBR-green (TAKARA SYBR-green qPCR Kit) following the manufacturer’s protocol. The conditions for the PCR were as follows: 95°C for 2 min, 25 cycles of denaturation at 94°C for 45 s, annealing for 30 sec (56°C and 58°C for genes and elongation factor, respectively), polymerization for 45 s (72°C) followed by plate reading at 72°C for 5 min, estimation of melting curve from 50°C to 95°C and incubation at 72°C for 4 min. The relative expression levels of the selected genes under a given stress condition was calculated using comparative threshold method. Elongation factor was used as internal control for tobacco samples and actin was used as internal control for finger millet samples to normalize qRT- PCR (For primer list See S1 Table). The reaction efficiency for each gene was calculated using software LinRegPCR [42] (S3 Table).
Drought and salinity stress tolerance assays For drought stress imposition, tobacco transgenic and wild type plants were grown in pots and when the plants reached 90 days, soil water status was gradually reduced by gravimetric approach. Gradual moisture stress was imposed by weighing the pots and the loss of water in the pots was replenished with required amount of water to arrive at the desired field capacity (FC) of soil. At the end of stress period when pots reached the required FC (60 and 40%), they were maintained in that particular FC for about 3 days [37]. One set of plants was maintained at 100% FC. Gas exchange parameters such as photosynthetic rate (μmolm−2s−1) and stomatal conductance (mmol−2s−1) were measured using a portable photosynthetic system (LICOR 6400, USA). The plants were assessed for dry matter accumulation by measuring the stem, leaves and root biomass. 30-day-old transgenic and wild type plants were subjected to drought stress by withholding water for 1 week and analysed for relative water content, electrolyte leakage, chlorophyll content and MDA levels. Salinity stress was imposed on 30-day-old transgenic and wild type plants grown hydroponically by supplementing 300 mM NaCl for 7 days. At the end of stress period, the shoot and root fresh weight of plants, electrolyte leakage and chlorophyll content was recorded. For longterm salinity stress, 90-day-old pot grown plants were treated with 100 mM NaCl for 25 days and later grown upto maturity and yield parameters were recorded.
Seedling level stress imposition The germinated seeds of transgenic and wild type were placed on half MS agar medium supplemented with 100 mM NaCl. The seedling growth was observed 7 days after stress treatment. The fresh weight, root length and cotyledon greening of seedlings were recorded. Photographs were taken at the end of the stress period.
Estimation of total chlorophyll content Chlorophyll was extracted from 200 mg of leaf tissue in acetone:DMSO (1:1) and the supernatant was made up to a known volume. The absorbance was recorded at 663 nm and 645 nm using UV-visible spectrophotometer (UV 2450, Shimadzu Corporation, Kyoto, Japan). Total
PLOS ONE | DOI:10.1371/journal.pone.0137098 September 14, 2015
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EcbHLH57 Improves Abiotic Stress Tolerance in Tobacco
chlorophyll was estimated by using the following formula and expressed as mg/g FW [43]. CHLa ¼ ð12:7ðA663 Þ 2:69ðA645 Þ VÞ=ðW 1000Þ CHLb ¼ ð22:9ðA645 Þ 4:68ðA663 Þ VÞ=ðW 1000Þ TCHLðmg=gFWÞ ¼ CHLa þ CHLb Where, CHL = chlorophyll, V = volume, W = weight and TCHL = Total chlorophyll
Electrolyte leakage assay Leaf discs from 30-day-old transgenic and wild type plants subjected to NaCl treatment and drought stress were analyzed for membrane damage by electrolyte leakage assay using conductivity meter (Elico-India, CM183, EC-TDS analyser) [37]. The leaf discs were transferred to deionised water and incubated for 2 h and initial electrical conductivity (EC) was recorded. The samples were then boiled for 15 min and final EC of the resultant solution was recorded. The electrolyte leakage was calculated using the following formula: EC (%) = (IEC/FEC) x 100 where, IEC = initial EC, FEC = final EC
Estimation of relative water content Leaf discs from 30-day-old transgenic and wild type plants subjected to drought stress were analysed for relative water content. Leaf discs were initially weighed to record the fresh weight. Later they were floated in deionized water and incubated for 5 h. The discs were blotted on filter paper to remove excess water and recorded the turgid weight. The leaf discs were ovendried and recorded the dry weight. The relative water content was estimated using the formula: RWC (%) = ((FW−DW) / (TW−DW)) x 100 where FW = fresh weight, DW = dry weight, TW = Turgid weight.
Excised leaf disc assays Leaf discs were made from identical leaves of 3-month-old transgenic and wild type plants were placed on half MS medium containing -6 bars PEG 6000, 200 mM NaCl, and 2 μM Methyl viologen (MV) and incubated for 72 h. After stress period, leaf discs were analysed for total chlorophyll content as described previously [43]
Estimation of H2O2 levels The leaf discs treated with MV was extracted in phosphate buffer of pH 7.5 and 25 μl supernatant was taken and mixed with 275 μl of xylenol orange reagent. The reaction mix was incubated for 30 min at room temperature and absorbance was measured at 560 nm against blank (xylenol orange reagent) [44]. The xylenol orange reagent was prepared in 50 ml of distilled water containing 1ml of 50 mM ferrous ammonium sulphate in 2.5 M H2SO4 and 62.5 μl of 125 μM xylenol orange along with 0.9019 g sorbitol.
Estimation of malondialdehyde (MDA) content MDA content was estimated by using TBARS assay. About 0.5–1.0 g of tissue was homogenized in 5 ml of 5% (w/v) trichloroacetic acid and the homogenate was centrifuged at 12,000 g for 15 min at room temperature. The supernatant was mixed with an equal volume of thiobarbituric acid (0.5% in 20% (w/v) trichloroacetic acid), and the mixture was boiled for 25 min at 100°C, followed by centrifugation for 5 min at 7,500 g to clarify the solution. Absorbance of the
PLOS ONE | DOI:10.1371/journal.pone.0137098 September 14, 2015
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EcbHLH57 Improves Abiotic Stress Tolerance in Tobacco
supernatant was measured at 532 nm and corrected for nonspecific turbidity by subtracting the A600. MDA content in leaf tissue was calculated using standard graph [45].
Analysis of stomatal characteristics In order to observe the stomatal characters, leaves from same aged plants and from same relative position was selected. The epidermal impressions were taken by applying a thin layer of thermocole paste in xylene on abaxial and adaxial surface of leaves. The imprint was removed and placed on glass slide and observed under light microscope with 40X magnification. The stomatal number and epidermal cell number was counted and calculated the stomatal index. The stomatal index was calculated by dividing number of stomata with total number of cells (i, e epidermal cells and stomatal cells) and multiplied by 100.
Statistical analysis The data obtained in different experimental results was analysed using two-way analysis of variance (ANOVA) as per the procedure given by Fischer [46]. Data points with different lowercase letters indicate significant differences (P
