Isolation and characterization of a buffalograss (Buchloe dactyloides) dehydration responsive element binding transcription factor, BdDREB2

Gene 536 (2014) 123–128

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Isolation and characterization of a buffalograss (Buchloe dactyloides) dehydration responsive element binding transcription factor, BdDREB2 Pan Zhang, Peizhi Yang, Zhiqiang Zhang, Bo Han, Weidong Wang, Yafang Wang, Yuman Cao, Tianming Hu ⁎ Department of Grassland Science, College of Animal Science and Technology, Northwest A&F University, Yangling, Shaanxi 712100, China

a r t i c l e

i n f o

Article history: Accepted 27 November 2013 Available online 12 December 2013 Keywords: Buffalograss BdDREB2 RACE Expression analysis Tobacco transformation Drought stress

a b s t r a c t Dehydration responsive element binding (DREB) transcription factors play an important role in the regulation of stress-related genes. These factors contribute to resistance to different abiotic stresses. In the present study, a novel DREB transcription factor, BdDREB2, isolated from Buchloe dactyloides, was cloned and characterized. The BdDREB2 protein was estimated to have a molecular weight of 28.36 kDa, a pI of 5.53 and a typical AP2/ERF domain. The expression of BdDREB2 was involved in responses to drought and salt stresses. Overexpression of BdDREB2 in tobacco showed higher relative water and proline content, and was associated with lower MDA content under drought stress, suggesting that the transgenic tobacco may tolerate drought stress better. Results demonstrate that BdDREB2 may play an important role in the regulation of abiotic stress responses, and mediate many physiological pathways that enhance stress tolerance in plants. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Abiotic stresses such as salinity and drought limit crop growth and production worldwide. Knowledge about alleviation of this loss is still limited due to the complexity of both the stress condition and plant responses (Lucas et al., 2011). Plants respond to abiotic stresses through complex tolerance mechanisms, which are activated by the expression of many genes. Transcription factors play central roles in the downstream regulation of genes contributing to stress tolerance (Seki et al., 2002; Yamaguchi-Shinozaki and Shinozaki, 2006; Zhu, 2002). The dehydration responsive element binding (DREB) transcription factor, which binds to dehydration-responsive element/C-repeat (DRE/CRT) cisacting elements in gene promoters and active transcription of downstream genes, plays an important role in regulating stress-related genes (Yamaguchi-Shinozaki and Shinozaki, 1994). DREB transcription factor regulates many stress inducible genes that contain DRE binding sites in their promoters (Khedr et al., 2011; Kizis et al., 2001). The Abbreviations: BdDREB2, Buchloe dactyloides dehydration responsive element binding transcription factor 2; DREB, dehydration responsive element binding transcription factor; DRE/CRT, dehydration-responsive element/C-repeat; AP2/EREBP, APETLA2/ethylene responsive element binding protein; AP2/ERF, APETLA2/ethylene responsive element binding factor; NaClO, sodium hypochlorite; PEG 6000, polyethylene glycol 6000; NaCl, sodium chloride; RT-PCR, reverse transcription-polymerase chain reaction; RACE, rapid amplification of cDNA end; GSP, gene specific primers; ORF, open reading frame; CaMV 35S, cauliflower mosaic virus 35S; WT, wild type; RWC, relative water content; MDA, malonyldialdehyde; TBA, thiobarbituric acid; ANOVA, analysis of variance; LSD, least significant difference. ⁎ Corresponding author. Tel.: +86 13709124728; fax: +86 29 87092355. E-mail addresses: [email protected] (P. Zhang), [email protected] (P. Yang), [email protected] (Z. Zhang), [email protected] (B. Han), [email protected] (W. Wang), [email protected] (Y. Wang), [email protected] (Y. Cao), [email protected] (T. Hu). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.11.060

DREB transcription factor belongs to the APETLA2/ethylene responsive element binding protein (AP2/EREBP) family, which is characterized by a conserved APETLA2/ethylene responsive element binding factor (AP2/ERF) domain and is induced under abiotic stress conditions. The amino acid sequence of the AP2/ERF domain is highly conserved and the 14th and 19th amino acids distinguish the DREB (valine and glutamic acid, respectively) from the ERF (alanine and aspartic acid, respectively) gene classes (Latini et al., 2007; Sakuma et al., 2002). These putative DREB proteins consist of two subclasses, specifically DREB1 and DREB2, and perform distinct functions in plants (Agarwal et al., 2006). The DREB1 genes play a critical role in cold-responsive gene expression while DREB2 genes are expressed under dehydration and high-salt stresses (Nayak et al., 2009; Xiong and Fei, 2006). In Arabidopsis, AtDREB1A responds to low temperatures and AtDREB2A is regulated by drought and salt stresses (Nakashima et al., 2006). DREB genes have been identified in many plant species under various abiotic stresses (Lee et al., 2004). DREB2 transcription factor could be a promising candidate gene involved in drought and salt tolerance (Chen et al., 2007, 2009). DREB genes from different species have been overexpressed in many plants like Arabidopsis thaliana, Oryza sativa, Brassica juncea, and Triticum aestivum (Gutha and Reddy, 2008; Tsutsui et al., 2009; Wang et al., 2006; Zhao et al., 2007). Overexpression of the DREB genes led to physiological variation and improved stress tolerance in oxidative, osmotic, and freezing stresses in transgenic plants (Ban et al., 2011; J.Q. Chen et al., 2008; Dubouzet et al., 2003; Gutha and Reddy, 2008; Ni et al., 2010). Drought responsive turfgrass cultivars are becoming increasingly important as water becomes a more limited resource and environmental issues become a greater concern to the public (Koski et al., 2001). In recent years, buffalograss (Buchloe dactyloides) has gained popularity as a turfgrass for its low maintenance requirements, low nutrient

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requirements, drought and heat tolerance, and limited susceptibility to pests and disease (Budak et al., 2005; Frank et al., 2004; Springer et al., 2005; Steinke et al., 2011; Sun et al., 2011). Buffalograss is a perennial, warm-season (C4), stoloniferous, open-pollinated, dioecious grass. It is native to semi-arid regions of the Great Plains of North America and distributed from Montana to northern Mexico (Frank et al., 2004; Gulsen et al., 2004). Since its introduction, it has become the most widely cultivated turfgrass in northern China and is usually planted on riversides and roadsides to help prevent soil erosion. It is also used for lawns, parks, cemeteries, airfields, athletic fields, golf courses, and pastures (Zhou et al., 2011). Although there are numerous genetically diverse buffalograss germplasm resources available for use with turfgrass, forage, and conservation, current knowledge of resistance genes and their use in genetic improvement in buffalograss lag behind most crop plants. The roles of DREB genes from Arabidopsis and O. sativa in abiotic stress tolerance, including salt, drought, oxidation, and freezing are well characterized (Dubouzet et al., 2003; Nakashima et al., 2006; Yamaguchi-Shinozaki and Shinozaki, 1994; Zhu, 2002). However, there are no report on cloning and characterization of DREB genes from buffalograss. Isolation of DREB genes from buffalograss can help to identify the function and mechanism of DREB proteins, and provide potential improvements in buffalograss abiotic stress tolerance. In the present study, we isolated a novel DREB gene (BdDREB2) from B. dactyloides. The expression pattern in response to abiotic stress and function in transgenic tobacco was investigated. To our knowledge, this is the first report of isolation of DREB-like genes from buffalograss. Our results indicated that BdDREB2, a novel transcription factor, may be involved in abiotic stress response and provided insight into the role of BdDREB2. 2. Materials and methods 2.1. Plant culture and stress treatment Unbudded buffalograss (B. dactyloides (Nutt.) Engelm) seeds were obtained from the Animal Husbandry Institute of Chinese Academy of Agricultural Sciences. Seeds were surface sterilized in 10% sodium hypochlorite (NaClO) solution for 10 min, washed with distilled water four to five times, soaked for germination, and sowed in a tray containing quartz sand. Seedlings were then placed in a light incubator (30 °C, 16 h light/8 h dark daily). A time-course experiment was carried out to investigate the responses to various stress conditions (Latini et al., 2013). Two-week-old seedlings were exposed to drought, cold and salt stresses. For drought treatment, seedlings were watered with 20% (w/v) polyethylene glycol 6000 (PEG 6000) and were sampled at 0 (control), 0.5, 1, 3, 6, 12, and 24 h. For cold treatment, seedlings were moved to a 4 °C cold room, and harvested at 6 h. For salt treatment, seedlings were irrigated with 3% (w/v) NaCl solution, and harvested at 6 h. All samples were placed in liquid nitrogen immediately and stored at −80 °C before proceeding to RNA extraction. 2.2. Isolation and sequence analysis of BdDREB2 To isolate the DREB2 gene in buffalograss, degenerate primers were designed based on the conserved amino acid sequences of DREBs from Cynodon dactylon, Poa pratensis, Schedonorus arundinaceus, Hordeum brevisubulatum, Helianthus annuus, T. aestivum, Pennisetum glaucum and A. thaliana. The primer sequences were as follows: forward primer, 5′-CCB GCC AAR GGK TCS AAG AAR GG-3′; reverse primer, 5′-TAC ATT GCY CTD GCM GCY TCR TC-3′, where B denotes C, G or T, D denotes A, G or T, K denotes G or T, M denotes A or C, R denotes A or G, S denotes C or G, and Y denotes C or T. Total RNA was extracted from buffalograss leaves that had been drought-induced for 6 h using a Trizol reagent (TaKaRa, Japan) according to the manufacturer's protocol. Genomic DNA was removed via

digestion with RNase-free DNase I (TaKaRa, Japan). The full-length cDNA sequence of the BdDREB2 gene was cloned using reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA end (RACE) method with the use of a SMART™ RACE cDNA Amplification Kit in accordance with the manufacturer's protocol (Clontech, US). Gene specific primers (GSP) were: 5GSP 5′-GAG CAG CCT CCA GAG CAG TAG GGA ACG AG-3′ and 3GSP 5′-CTC GTT CCC TAC TAC TGC TCT GG-3′. Full-length BdDREB2 cDNA was amplified using the primers 5′-GAT GGA GCG GGT GGA GGT GC-3′ and 5′-GGG TTG AAG CGT TTC CTA ACA AG-3′. The amplification conditions were: predenaturation at 95 °C for 3 min, 40 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 60 s, followed by a final extension step of 72 °C for 5 min. All of the PCR products were cloned into the pMD18-T vector, transformed into Escherichia coli DH5α and sequenced at Shanghai Sangon Biological Engineering Technology & Services Co. Ltd. The predicted amino acid and nucleotide sequences were analyzed using DNAMAN (Lynnon Biosoft). Multiple sequence alignments and phylogenetic trees were generated through an Internet Blast search (http://www.ncbi.nlm.nih.gov/BLAST) and DNAMAN.

2.3. Expression analysis of BdDREB2 under stress treatments Semiquantitative RT-PCR was used to determine the expression patterns of BdDREB2 under stress treatments. Total RNA was extracted from leaves and roots of buffalograss seedlings using a Trizol reagent (TaKaRa, Japan) according to the manufacturer's protocol. Genomic DNA was removed via digestion with RNase-free DNase I (TaKaRa, Japan). For stress treatments, total RNA was extracted from buffalograss seedlings under drought, cold and salt stresses for 6 h. For time-course expression, buffalograss seedlings were harvested at 0 (control), 0.5, 1, 3, 6, 12, and 24 h under drought stress. For tissue-specific expression, roots and leaves of buffalograss were sampled at 6 h of drought treatment. Then 18S rRNA universal primers (forward/reverse: 5′-AGT ATG GTC GCA AGG CTG AA-3′ and 5′-CAT TCA ATC GGT AGG AGC GA-3′) were used as an internal standard. Specific primers (5′-CCT GAC TCC ATC GCT GAG ACA AT-3′ and 5′-GTG CTG ACG TGC AAC CTG AG-3′) were used to amplify the BdDREB2 gene. The PCR conditions were: initial denaturation at 95 °C for 3 min, followed by 36 cycles of denaturation at 95 °C for 30 s, annealing at 50 °C for 30 s and extension at 72 °C for 60 s. Three replicates from different RNA extracts were used.

2.4. Overexpression vector construction and generation of transgenic tobacco An overexpression vector was constructed on the base of the pBI121 vector (TaKaRa, Dalian, China). The open reading frame (ORF) of the BdDREB2 gene was amplified with specific primers modified to include Xba1 and BamHI restriction sites (underlined), BdDREBXba1: 5′-GCT CTA GAA TGG AGC GGG TGG AGG TGC-3′ and BdDREBBamHI: 5′-CGG GAT CCT GAG AAG AGA CTG AAC-3′. The amplification products were inserted into a pBI121 vector downstream from a cauliflower mosaic virus 35S (CaMV 35S) promoter. The BdDREB2 binary vector was mobilized into Agrobacterium tumefaciens (LBA4404) by a CaCl2 freezing method (Xue and Loveridge, 2004). The Agrobacterium-mediated transform method was used to transform tobacco by the leaf disc co-cultivation method (Latini et al., 2007). Putative transgenic plants were selected on MS solid medium containing 500 μg/ml cefathiamidine and 100 μg/ml kanamycin. The expression level of BdDREB2 in transgenic tobacco was measured by semi-quantitative RT-PCR using BdDREB2 gene specific primers (5′-CCT GAC TCC ATC GCT GAG ACA AT-3′ and 5′-GTG CTG ACG TGC AAC CTG AG-3′) and using 18S rRNA universal primers (forward: 5′-AGT ATG GTC GCA AGG CTG AA-3′ and reverse: 5′-CAT TCA ATC GGT AGG AGC GA-3′) as an internal standard.

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Fig. 1. Compare the deduced amino acid sequences of BdDREB2 with other DREB-like proteins. The amino acid sequences are shown below: BdDREB2 (EF512460), Buchloe dactyloides; CdDREB1 (AAS46284) and CdDREB2 (AAS46285), Cynodon dactylon; FaDREB2 (AAR11157), Festuca arundinacea; HbDREB2 (AAU29412), Hordeum brevisub; PpDREB2 (AAS59530), Poa pratensis; SbDREB2 (ABD66654), Sorghum bicolor; ZmDREB (AAN76733), Zea mays; OsDREB (AAO39764), Oryza sativa. The conserved amino acids are shaded. The valine (V) and glutamic acid (E) residues at the 14th and 19th positions are shown by a box.

2.5. Analysis of drought tolerance in transgenic tobacco Five-week-old tobacco seedlings (wild type (WT) and three randomly selected transgenic lines) were used for further analysis. Well-watered plants were treated without irrigation for 0 (control) and 15 days, then the leaves of plants were harvested for physiological parameter assays.

Relative water content (RWC) was determined according to Chen et al. (2008) with little modification. Malonyldialdehyde (MDA) content was measured using a modified thiobarbituric acid (TBA) method (Puckette et al., 2007; Yang et al., 2013). Proline content was determined spectrophotometrically following the method of Bates et al. (1973). At least three plants (plant clones from the transgenic plants) from each line

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were used in each experiment. Each experiment was performed at least three times. Data were analyzed for mean difference by analysis of variance (ANOVA) in SPSS 20.0 with a least significant difference (LSD) test (P b 0.05).

3. Results 3.1. Cloning and sequence analysis of the buffalograss BdDREB2 gene The full-length cDNA of BdDREB2 from buffalograss was cloned by RT-PCR and RACE (GenBank accession no. EF512460). BdDREB2 was 1050 bp in length, and contained an ORF of 254 amino acids. The BdDREB2 protein was estimated to have a molecular weight of 28.36 kDa and a pI of 5.53. Multiple sequence alignment showed that BdDREB2 was very similar to other DREB2-related proteins throughout the AP2/ERF domain and the N-terminal region (Fig. 1). BdDREB2 protein possesses a typical AP2/ERF domain of 57 amino acids with a DRE cis-acting element. Valine (V) and glutamic acid (E) residues were found at the 14th and 19th positions. Results indicated that BdDREB2 was a new member of the AP2/ERF transcription factor family in bufflograss. A phylogenetic tree was constructed from the deduced amino acid sequences of BdDREB2 and other DREBs released in GeneBank and other databases. Results suggested that BdDREB2 was attributable to the DREB2 subgroups by comparing to DREB1 transcription factors. Moreover, phylogenetic analysis indicated that BdDREB2 had the highest homology in amino acid sequence with C. dactylon CdDREB2 (Fig. 2). Putative amino acid sequences also exhibited close identity with P. pratensis, T. aestivum, and Festuca arundinacea. 100% 90% 80% 70% 60% 50% 40%

BUCHLOE_DACTYLOIDES

30%

3.2. Expression patterns of BdDREB2 in buffalograss under abiotic stresses To investigate the expression patterns of BdDREB2 in buffalograss under various abiotic stresses, expression levels of BdDREB2 were determined using semi-quantitative RT-PCR. After cold (4 °C for 6 h), salt (3% NaCl for 6 h) and drought (20% PEG for 6 h) treatment, BdDREB2 was up-regulated by salt and drought stresses (Fig. 3A). However, the expression level of BdDREB2 under cold stress showed no significant difference compared to control (buffalograss cultivated under normal condition). In buffalograss seedlings, the expression level of BdDREB2 under drought stress increased with time and reached the maximum at 6 h, then remained at 12 and 24 h (Fig. 3B). Under control conditions, we found the expression of BdDREB2 in buffalograss leaves and roots, and the expression levels significantly increased in both after 6 h of drought treatment. However, the increased level in roots was obviously higher than that in leaves (Fig. 3C). 3.3. Effect of overexpression of BdDREB2 on drought tolerance To determine the function of the BdDREB2 gene, we overexpressed the ORF of the BdDREB2 gene, driven by a CaMV 35S promoter in tobacco. A total of 10 independent transgenic lines were obtained by cefathiamidine and kanamycin resistance selection. Four homozygous transgenic lines were chosen randomly to measure BdDREB2 transcription level. Semi-quantitative RT-PCR analysis showed that the expression level of BdDREB2 was similar in the transgenic lines (Fig. 4A). Growth of transgenic plants was slower than wild type plants under normal conditions (0 day) and WT plants were more wilted than transgenic lines under drought stress for 15 days (Fig. 4B). RWC, MDA content and proline content were measured and compared with transgenic and WT plants before and after drought stress. No significant difference in RWC (Fig. 4C), MDA content (Fig. 4D) and proline content (Fig. 4E) between

88% 82%

CYNODON_DACTYLON SORGHUM_BICOLOR

79%

POA_PRATENSIS 96% FESTUCA_ARUNDINACEA

97% 88% 54%

SCHEDONORUS_ARUNDINACEUS HORDEUM_BREVISUBULATUM PENNISETUM_GLAUCUM

50% 63%

TRITICUM_DURUM

49%

ARABIDOPSIS_THALIANA 42%

HELIANTHUS_ANNUUS TRITICUM_AESTIVUM

46% ZEA_MAYS

39%

67%

ORYZA_SATIVA ASPARAGUS_OFFICINALIS Fig. 2. Phylogenetic analysis of BdDREB2 with other DREBs released in GeneBank and other databases. Genes and corresponding accession numbers are as follows: Buchloe dactyloides BdDREB2 (EF512460); Cynodon dactylon CdDREB2 (AY462118), Sorghum bicolor SbDREB2 (DQ403725), Poa pratensis PpDREB2 (AY553331), Festuca arundinacea FaDREB2 (AY436639), Schedonorus arundinacea SaDREB2 (AJ786400), Hordeum brevisub HbDREB2 (AY728807), Pennisetum glaucum PgDREB2 (AY829439), Helianthus annuus HaDREB2 (AY508007), Arabidopsis thaaliana AtDREB2A (NM_001036760), Triticum aestivum TaDREB2 (AY781345), Triticum durum TdDRF1 (JN571427), Asparagus officinalis AoDREB3 (DQ211835), Zea mays ZmDREB (AF448789), and Oryza sativa OsDREB (AY196209).

Fig. 3. Expression patterns of BdDREB2 in buffalograss in response to abiotic stresses. The amplified fragment was 358 bp in length. A: Expression of BdDREB2 in response to control (normal condition), cold (4 °C for 6 h), salt (3% NaCl for 6 h), and drought (20% PEG for 6 h) stresses. B: Expression of BdDREB2 after 20% PEG treatment for 0, 0.5, 1, 3, 6, 12, and 24 h. C: Expression of BdDREB2 in buffalograss leaves and roots after 0 (control) and 6 h of 20% PEG treatment. M: The molecular marker (DL2000).

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Fig. 4. Function characterization of BdDREB2 expressed in transgenic tobacco lines. A: Semi-quantitative RT-PCR analysis of BdDREB2 expression in representative transgenic lines. WT, wild type; 1, 2, 3 and 4, transgenic tobacco line 1, line 2, line 3 and line 4; M, molecular marker. B: Phenotype of wild type (WT) and transgenic line (line 1 and line 2) seedlings under 0 and 15 days of drought treatment. The two transgenic plants displayed growth retardation as evidenced by reduced plant height and delayed flowering. Under drought stress, WT was more wilted than transgenic lines. C: Changes in relative water content (RWC) in leaves of wild type and transgenic tobacco seedlings under 0 and 15 days of drought treatment. D: Changes in the content of malonyldialdehyde (MDA) in leaves of WT and transgenic tobacco seedlings under 0 and 15 days of drought treatment. E: Changes in the content of proline in leaves of WT and transgenic tobacco seedlings under 0 and 15 days of drought treatment. Five-week-old plants were subjected to drought stress, and leaves were harvested before drought stress (0 days) and 15 days after drought stress. Data are shown as mean ± SE. Different letters indicate significant difference (P b 0.05).

WT and transgenic plants was detected before drought stress (0 day). However, RWC and proline content in all transgenic plants were higher (P b 0.05) than WT plants. MDA content in all transgenic plants was lower (P b 0.05) than WT plants after drought stress. 4. Discussion Studying the regulation of stress-inducible genes may improve our understanding of the mechanisms by which plants maintain growth and thrive under abiotic stress conditions. Identifying the transcription factors that mediate responses to abiotic stress is an important prerequisite for use of stress-inducible genes in crop improvement (Rae et al., 2011). DREB transcription factor is one of the most promising candidate genes conferring drought tolerance in several crops (YamaguchiShinozaki and Shinozaki, 2005, 2006). However, there have been no reports on B. dactyloides DREB genes and the physiological processes regulated by DREBs under drought stress. In the present study, we identified a BdDREB2 gene from B. dactyloides and demonstrated that it has a typical AP2/ERF domain. The conserved region between V and E was found to be AEIR (Fig. 1) rather than C/SEV/LR demonstrating that BdDREB2 was consistent with the characteristics of DREB2 subclass (Riechmann and Meyerowitz, 1998; Sakuma et al., 2002). In addition, phylogenetic tree analysis suggested that BdDREB2 was attributable to the DREB2 subgroups by comparing to DREB1 transcription factors. These data demonstrated that BdDREB2 was one new member of the DREB2 subclass and belonged to the AP2/EREBP family. Different DREBs may play various roles in plants, be involved in several pathways and participate in crosstalk among pathways during the response to abiotic stress (Peng et al., 2011). Semi-quantitative RTPCR analysis showed that the BdDREB2 gene was up-regulated by high salt and drought stresses, while low temperature had no effect (Fig. 3A). The expression level of the BdDREB2 gene increased after drought treatment and the increased level in the root was higher than

that in leaves compared with the expressions under control conditions. This result confirmed that BdDREB2 is an abiotic stress response gene and may contribute to plant stress tolerance (Gupta et al., 2010; Lucas et al., 2011; Yang et al., 2009). Compared with wild type plants, the drought tolerance of transgenic tobacco increased under drought stress (Fig. 4). Drought-induced expression of the AtDREB1A gene in transgenic wheat delayed water stress symptoms under greenhouse conditions (Pellegrineschi et al., 2004). Overexpression of the BdDREB2 gene could cause a delayed growth retardation phenotype in transgenic tobacco. Plant metabolism is dependent on leaf water status and RWC has been proposed as a selection criterion for drought tolerance in many crops (Kizis et al., 2001). RWC has been proposed as an important indicator of plant cell health (Chen et al., 2012). Our study found that the RWC in transgenic plants was higher than in WT plants under drought stress, suggesting that they were in a healthier state than non-transgenic tobacco plants. MDA is an end product of lipid peroxidation and has been used extensively as an indicator for free radical-caused membrane injury under various abiotic stress conditions (Alexieva et al., 2001). Our result showed that the MDA content in leaves of transgenic plants was lower than WT plants under drought stress, suggesting that the degree of damage to a cell membrane was lower in transgenic plants. Accumulation of proline is an important parameter of drought tolerance in plants and proline may act as a radical scavenger or cellular osmotic regulator (Manivannan et al., 2007; Paleg and Aspinall, 1981). In this study, proline content in the transgenic lines was higher than in WT plants under drought condition. Previous studies have shown that overexpression of DREB family genes could elevate proline content (Ban et al., 2011; Zhao et al., 2007). Therefore, the elevation of proline content may have contributed to drought tolerance in transgenic plants. These results may reflect an increase in osmotic adjustment and decreased damage to plasma membranes under drought stress, suggesting that transgenic tobacco may have tolerated drought stress better.

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In summary, our results showed that BdDREB2 is likely involved in the responses to drought and salt stresses. Furthermore, overexpression of BdDREB2 in tobacco showed higher RWC and proline content, which was associated with lower MDA content under drought stress. These results suggest that BdDREB2 can mediate many physiological pathways to enhance stress tolerance in plants. Thus, with the character of BdDREB2 established, various physiological, molecular, and genetic studies can be undertaken to provide a better understanding of how the DREB gene is regulated in buffalograss under various stress conditions. Conflict of Interest The author declare that there was no conflict of interest. Acknowledgments We would like to thank Professor Roger Gates for his advices on English writing. This work was supported by the National Science Foundation of China (30901050, 31272490), the National Key Technology R&D Program in the 12th Five-Year Plan of China (2011BAD17B05), the Northwest A&F Univesity Cyrus Tang Breeding Foundation and the major project for Tibetan forage industry (2011-2015). Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2013.11.060. References Agarwal, P.K., Agarwal, P., Reddy, M.K., Sopory, S.K., 2006. Role of DREB transcription factors in abiotic and biotic stress tolerance in plants. Plant Cell Rep. 25, 1263–1274. Alexieva, V., Sergiev, I., Mapelli, S., Karanov, E., 2001. The effect of drought and ultraviolet radiation on growth and stress markers in pea and wheat. Plant Cell Environ. 24, 1337–1344. Ban, Q.Y., Liu, G.F., Wang, Y.C., 2011. A DREB gene from Limonium bicolor mediates molecular and physiological responses to copper stress in transgenic tobacco. J. Plant Physiol. 168, 449–458. Bates, L.S., Waldren, R.P., Teare, I.D., 1973. Rapid determination of free proline for waterstress studies. Plant Soil 39, 205–207. Budak, H., Shearman, R.C., Gulsen, O., Dweikat, I., 2005. Understanding ploidy complex and geographic origin of the Buchloe dactyloides genome using cytoplasmic and nuclear marker systems. Theor. Appl. Genet. 111, 1545–1552. Chen, M., et al., 2007. GmDREB2, a soybean DRE-binding transcription factor, conferred drought and high-salt tolerance in transgenic plants. Biochem. Biophys. Res. Commun. 353, 299–305. Chen, D., et al., 2008a. Identification of dehydration responsive genes from two nonnodulated alfalfa cultivars using Medicago truncatula microarrays. Acta Physiol. Plant. 30, 183–199. Chen, J.Q., Meng, X.P., Zhang, Y., Xia, M., Wang, X.P., 2008b. Over-expression of OsDREB genes lead to enhanced drought tolerance in rice. Biotechnol. Lett. 30, 2191–2198. Chen, J., Xia, X., Yin, W., 2009. Expression profiling and functional characterization of a DREB2-type gene from Populus euphratica. Biochem. Biophys. Res. Commun. 378, 483–487. Chen, T., Yang, Q., Zhang, X., Ding, W., Gruber, M., 2012. An alfalfa (Medicago sativa L.) ethylene response factor gene, MsERF11, enhances salt tolerance in transgenic Arabidopsis. Plant Cell Rep. 1–10. Dubouzet, J.G., et al., 2003. OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression. Plant J. 33, 751–763. Frank, K.W., et al., 2004. Nitrogen rate and mowing height effects on turf-type buffalograss. Crop Sci. 44, 1615–1621. Gulsen, O., Heng-Moss, T., Shearman, R., Baenziger, P.S., Lee, D., Baxendale, F.P., 2004. Buffalograss germplasm resistance to Blissus occiduus (Hemiptera: Lygaeidae). J. Econ. Entomol. 97, 2101–2105. Gupta, K., Agarwal, P.K., Reddy, M.K., Jha, B., 2010. SbDREB2A, an A-2 type DREB transcription factor from extreme halophyte Salicornia brachiata confers abiotic stress tolerance in Escherichia coli. Plant Cell Rep. 29, 1131–1137. Gutha, L.R., Reddy, A.R., 2008. Rice DREB1B promoter shows distinct stress-specific responses, and the overexpression of cDNA in tobacco confers improved abiotic and biotic stress tolerance. Plant Mol. Biol. 68, 533–555. Khedr, A.H.A., et al., 2011. A DREB gene from the xero-halophyte Atriplex halimus is induced by osmotic but not ionic stress and shows distinct differences from glycophytic homologues. Plant Cell Tissue Organ Cult. 106, 191–206.

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