Introduction

Environmental stresses have adverse effects on plant growth. To survive, plants have developed defense mechanisms to perceive signals from their surroundings and appropriately respond to the different stresses by modulating the expression of response genes (Lee et al. 1999; Ramanjulu and Bartels 2002). Transcription factors function pivotally in activating defense gene expression (Shinozaki and Dennis 2003; Chen and Zhu 2004; Xu et al. 2008a). More than 1,500 genes encode transcription factors in the Arabidopsis genome (Riechmann et al. 2000). Among them, dehydration-responsive element-binding proteins (DREBs; also referred to as CBF) have received considerable attention in the past decade. A variety of DREB genes were identified and investigated in various species, including rice (Oryza sativa), maize (Zea mays L.), soybean (Glycine max), and wheat (Triticum aestivum; reviewed in Agarwal et al. 2006; Xu et al. 2008b).

DREBs play an important role in stress signal regulatory pathways by specifically binding to C-repeats/dehydration responsive elements (DREs/CRTs; A/GCCGAC) to control the expression of RD/COR genes, such as rd29A, kin1, rab17, cor6.6, and cor15A. DREB genes were found to be involved in response to abiotic stresses (Stockinger et al. 1997; Liu et al. 1998; Gilmour et al. 1998; Haarke et al. 2002). For example, CBF1 was induced by low temperature and its overexpression activated downstream cold-responsive genes and improved freezing tolerance in plants (Jaglo-Ottosen et al. 1998). Arabidopsis DREB1A/CBF3 and rice OsDREB1A conferred drought and freezing tolerance in transgenic Arabidopsis plants (Dubouzet et al. 2003). Recently, it was reported that overexpression of maize ZmDREB2A and wheat TaAIDFa resulted in increased thermotolerance and/or drought tolerance in plants (Qin et al. 2007; Xu et al. 2008b). Isolation and functional characterization of more DREB genes could facilitate understanding the mechanisms by which plants perceive and transduce adverse environmental stimuli to trigger adaptive responses.

Only few DREB transcription factors have been described in maize (Z. mays), an important food and feed crop in the world (Jiang et al. 2004; Qin et al. 2004, 2007; Wang and Dong 2009). In this paper, we report the cloning and characterization of the ZmDBP2 gene. The expression pattern, subcellular localization, and putative cis-acting elements in the promoter of ZmDBP2 were investigated. Importantly, overexpression of the ZmDBP2 gene improved drought tolerance in transgenic Arabidopsis plants.

Materials and Methods

Plant Materials and Stress Treatments

Seedlings of maize (X178) grown at 25°C for 10 days were subjected to various abiotic stresses. All treatments were applied for 5 h or for times otherwise indicated. Seedlings were exposed to air on filter paper for induction of rapid drought conditions, or placed in a 4°C chamber for cold stress. To mimic salinity and for abscisic acid (ABA) treatments, seedling roots were soaked into solutions containing 2% salt and 200 µM ABA, respectively.

Suppression Subtractive Hybridization

A suppression subtractive hybridization (SSH) cDNA library was constructed using the PCR-SelectTM cDNA Subtraction kit (Clontech, Palo Alto, CA, USA) according to the manufacturer’s instructions. Total RNA was extracted from drought- and salt-treated leaves harvested at 5 h. Poly(A)+ RNA was extracted from total RNA using the Oligotex mRNA Mini Kit (Qiagen, Hilden, Germany). Equal amounts of two mRNA samples were pooled and used to make tester cDNAs. Similarly, driver cDNAs were derived from nontreated maize leaves harvested concomitantly. After two rounds of subtractive hybridization and two rounds of PCR amplification, the second round PCR amplification product was ligated into the pGEM-T Easy vector (Promega, Madison, WI, USA), and ligation mixtures were transformed into Escherichia coli JM109. Cloned products were sequenced and results were compared with GenBank database sequences using the BLAST homology search program.

Isolation of Full-Length cDNA and Genomic Sequences

Several homologous cDNA fragments were obtained using the target sequence as a query to search the maize EST database. Based on the putative sequences, the full-length cDNA sequence or genomic sequences of ZmDBP2 were amplified using the primer set (5′-GCTCCATCCAAGCTGTAGTCCT-3′) and (5′-GCTCATGACAGGATGGAATCC-3′).

First-Strand cDNA Synthesis and Quantitative RT-PCR

Total RNA was extracted from leaves using Trizol Reagents (TianGen, Beijing, China). The real-time quantitative RT-PCR (Q-RT-PCR) were conducted using the ABI Prism 7000 system (Applied Biosystems, USA). The actin gene was used as an internal reference. The transcript analysis of the ZmDBP2 gene was performed using gene-specific primers (5′-GCCCGATGGCATTTTAGACG-3′ and 5′-AACCAGGAGATTAGCACGCA-3′), which were located in the 3′-untranslated region of the gene. Validation experiments were performed to demonstrate that amplification efficiency of the ZmDBP2 specific primers were approximately equal to the amplification efficiency of the endogenous reference primers. Quantification of the target gene expression was carried out by the comparative CT method (Livak and Schmittgen 2001). Average CT values for the target gene from at least three PCRs were normalized to average CT values for actin from the same cDNA preparations and analyzed using Microsoft Excel.

Subcellular Localization Analysis

The full-length cDNA fragment containing the coding region of ZmDBP2 was amplified and fused to the N terminus of the hGFP gene under control of the 2× CaMV35S promoter. Subcellular localization of the ZmDBP2::hGFP fusion protein and hGFP control in onion epidermal cells was performed as described by Xu et al. (2007).

Arabidopsis Transformation

To construct an expression vector for Arabidopsis, the full-length ZmDBP2 cDNA was ligated into the modified vector pBI121 under the control of the CaMV35S promoter. Columbia (Col-0) ecotype Arabidopsis plants were transformed using the vacuum infiltration method. Transformants were selected on MS medium containing 50 μg mL−1 kanamycin. T2 generation plants were used for further analysis.

Drought and Salt Stress Treatment of Transgenic Plants

Sixty Arabidopsis plants of two independent lines grown for 3 weeks at 22°C were subjected to stress treatments. For drought stress treatment, plants were grown at 22°C without watering for 12 days and then rewatered under normal growing conditions. For salt stress, plants were irrigated with 200 mM NaCl under normal growing conditions. Survival rates were calculated 10 days later.

Results

Isolation and In Silico Analysis of ZmDBP2 cDNA

We constructed a maize SSH cDNA library to isolate new genes that mediate drought stress responses. One cDNA fragment of 349 bp (MF245) showed sequence homology to DREB proteins in the BLAST search (BLASTX). BLASTN was performed on the maize EST database using the MF245 sequence. Using sequence assembly and RT-PCR, we obtained the full-length cDNA sequence and designated it as ZmDBP2 (GenBank accession no. FJ805750). The genomic sequence was isolated using PCR primers corresponding to the 5′- and 3′-ends of ZmDBP2 cDNA. Sequence alignment of ZmDBP2 with its genomic sequence indicated that ZmDBP2 has no intron.

ZmDBP2 comprised a 1,032-bp ORF encoding a 36.7-kD protein with pI 6.6 (Fig. 1). The deduced amino acid sequences of ZmDBP2 contain a highly conserved AP2/ERF domain. Two conserved amino acids, 14th Val and 19th Leu, within the AP2/ERF domain are characteristic of DREB proteins (Sakuma et al. 2002; Figs. 1 and 2a). ZmDBP2 contains two acidic amino acid regions that might act as transcriptional activation domains and two potential N-linked glycosylation sites (NXS/T, where X denotes any amino acid), but has no clearly basic amino acid region that potentially acts as a nuclear localization signal (NLS; Fig. 1). Phylogenic analysis confirmed that ZmDBP2 belongs to the A-6 subgroup of the DREB subfamily (Fig. 2b). Among members, ZmDBP2 shares 53% and 47% identity with maize ZmDBF1 and soybean GmDREBb proteins, respectively (Fig. 2a).

Fig. 1
figure 1

Nucleotide and deduced amino acid sequences of the ZmDBP2 gene. The ORF sequence is represented by uppercase letters, and untranslated regions are indicated by lowercase letters. The deduced amino acid sequence is shown below the DNA sequence. The AP2/ERF domain is single underlined; acidic and Ser/Thr-rich regions that may act as transcriptional activation domains are shown in bold italics, and two potential N-linked glycosylation sites are in bold script

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Fig. 2
figure 2

Comparison and phylogenetic relationship of maize ZmDBP2 with other plant DREBs. a Amino acid alignment of DREB proteins. RAP2.4 (NP_177931), OsDBF1 (BAD37688), ZmDBF1 (AAM80486), and GmDREBb (AY296651). The conserved AP2/ERF domain is underlined and asterisks indicate the conserved V14 and L19. Three β-sheets and one amphipathic α-helix are marked over the corresponding sequences. b Phylogenetic tree of ZmDBP2 with DREBs from other plants. The phylogenetic tree was constructed by the MEGA program. The accession number of each appended protein is as follows: OsDBF1: BAD37688, CiDREB1: ABR53728, AoDREB3: ABB89754, PtDREB18: EEE94094, HvDRF1: AY223807, HvCBF1: AF418204, HvCBF2: AF442489, FaDREB2A: AJ717396, HbDREB2: AY728807, TaDREB1: DQ195068, TaCBF1: AF376136, OsDREB1: AY064403, ZmDBF1: AAM80486, ZmDBF2: AF493799, ZmABI4: AY125490, GhDBP1: AY174160, GhDREB1L: DQ409060, AtDREB1A: AB007787, AtDREB1B: AB007788, AtDREB1C: AB007789, AtDREB2A: AB007790, AtDREB2B: AB007791, AtDREB2C: NM_129594, GmDREBa: AAT12423, GmDREBb: AAQ57226, GmDREBc: AAP83131, PgDREB2A: AAV90624, ScCBF: AAL35759, VrCBF1a: AAR28671, OsDREB2A: AF300971, CaDREBLP1: AY496155, TaDREB6: AAX13289, PpDREB2: AAS59530, AsDREB2: ABS11171, PeDREB2: ABY19375, CdDREB2: AAS46285, SbDREB2: ABX45045, ABI4: A0MES8, GmDREB2: ABB36645, RAP2.10: Q9SW63, RAP2.1: Q8LC30, AtRAP2: AAP04063, RAP2.4: NP_177931, GhDBP2: AAT39542, OsDREB3:NP_001048142, GhDBP1L: ABD65473, and TINY: Q39127

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ZmDBP2 Protein Is Not Localized in the Nucleus

Transcription factors are commonly expected to localize to the nucleus. As shown in Fig. 3, ZmDBP2::hGFP fusion protein, like hGFP, was uniformly distributed throughout the onion epidermal cell, suggesting that ZmDBP2 probably has no nuclear localization activity, which agrees with the results of protein subcellular localization prediction (WoLF PSORT; http://wolfpsort.org/).

Fig. 3
figure 3

Subcellular localization of the ZmDBP2 protein in onion epidermis cells. The fusion construct for ZmDBP2::hGFP and the control plasmid hGFP were introduced into onion epidermis cells by particle bombardment. Transformed cells were cultured on MS medium at 25°C for 16 h and observed under a confocal microscope. Photographs were taken in dark field for green fluorescence (a and d), in bright light for morphology of the cells (b and e), and in combination (c and f)

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ZmDBP2 Is Induced by Drought, Salt, and Exogenous ABA

To assess responses to various abiotic stress conditions, we qualitatively surveyed the transcript expressions of ZmDBP2 using Q-RT-PCR. Total RNA was extracted from maize seedlings subjected to drought, salt, cold, and exogenous ABA. As shown in Fig. 4a, ZmDBP2 transcripts accumulated greatly in response to drought and moderately to salt and exogenous ABA, but cold treatment had no effect.

Fig. 4
figure 4

Expression of ZmDBP2 under different abiotic stress conditions. a All treatments were applied for 5 h. In a, CK, D, S, C, and A indicate control, drought, salt, cold, and ABA treatment, respectively. b–d The kinetics of mRNA ZmDBP2 accumulation of under drought, salt, and exogenous ABA over 48 h

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We studied the kinetics of mRNA accumulation in maize seedlings over 48 h. Under drought condition, ZmDBP2 mRNA began to accumulate at 1 h and reached its maximum at 5 h after treatment (Fig. 4b). Under high-salt and exogenous ABA treatment, the expression pattern of ZmDBP2 was similar to that with drought treatments, but the peak time for salt-induced transcription was later than for drought treatment (Fig. 4c, d).

ZmDBP2 Promoter Contains Several Putative Stress-Responsive Elements

To further investigate the mechanism responsible for regulation of ZmDBP2 gene expression, we cloned a 1.62-kb promoter region upstream of the ZmDBP2 ATG start codon from maize genomic DNA. Putative cis-acting elements in the promoter region were identified using PLACE (a database of plant cis-acting elements, http://www.dna.affrc.go.jp/PLACE/). Several distinct regulatory motifs homologous to cis-acting elements involved in responses to abiotic stresses and plant hormones were identified (Fig. 5). These included two DPBF binding core sequences (ACACNNG), two ABRE elements (TACGTGKC, K represents G or T), two RAV binding sequences (CAACA), a ARF site (TGTCTC), a MBS site (CAACTG), and a W-box element (TTGACC). In addition, there were sequences resembling a TC-rich repeat (GTTTTTTTAC), a Py-rich stretch (TTTCTCTTCT), CGTCA motif (CGTCA), and a GCC-box element (GCCGCC).

Fig. 5
figure 5

The promoter sequence of the ZmDBP2 gene. The ATG translation initiation codon is assigned as position 1 in the nucleotide sequence, and the nucleotide positions upstream of position 1 are presented as negative numbers. The putative cis-acting elements are indicated in bold, and the names are given under the elements. DPBF DPBF binding core sequence, ABRE ABRE element, ARF ARF site, MBS MBS site, W-box W-box element, TC-rich TC-rich repeat, Py-rich Py-rich stretch, CGTCA CGTCA motif, GCC-box GCC-box element

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Improved Drought Tolerance in Transformed Arabidopsis Plants

The ZmDBP2 gene under the control of CaMV35S was transformed into Arabidopsis plants to further investigate its functions. The transcription of COR/RD genes with the CRT/DRE element in their promoters probably regulated by ZmDBP2 gene were analyzed. As shown in Fig. 6a, overexpression of ZmDBP2 in the transgenic Arabidopsis plants under normal growth conditions resulted not only in the accumulation of its own mRNA but also of transcripts corresponding to rd29A and rab17. Thus, it is likely that the presence of ZmDBP2 leads to improved stress tolerance in transgenic Arabidopsis plants.

Fig. 6
figure 6

Effect of ZmDBP2 expression on drought and salt tolerance in transgenic Arabidopsis plants. a Expression patterns of ZmDBP2, rd29A, and rab17 in wild-type (WT) and 35S::ZmDBP2 transgenic Arabidopsis plants (lines TL-4 and TL-9) under normal growing conditions. Parallel reactions using actin in primers were carried out to normalize the amounts of added template. b Survival rates of WT and CaMV35S::ZmDBP2 transgenic Arabidopsis lines (TL-4 and TL-9) were estimated after stress treatments. Stress treatments were applied to transgenic Arabidopsis and WT 3-weeks-old plants under common conditions. Results are averages of three replicates ± SD

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Responses to abiotic stresses were analyzed using 35S::ZmDBP2 transgenic Arabidopsis plants. Seedlings of transgenic and wild-type Arabidopsis grown for 3 weeks at 22°C were used for analyses of survival rates under drought and NaCl stresses (Fig. 6b). Under water-free conditions, most (more than 80%) wild-type plants were dead after 12 days, whereas 71.7–83.3% of the transgenic plants survived and continued to grow after rewatering. Ten days after 200 mM NaCl treatment, almost all of the transgenic plants, like the wild-type plants, had died, although the loss of greenness of transgenic plants was slower than that of wild-type plants (data not shown). These experiments indicated that overexpression of ZmDBP2 resulted in enhancement of drought tolerance in Arabidopsis plants.

Discussion

The transcription of DREB genes are strongly and rapidly upregulated in response to abiotic stresses in numerous plant species (reviewed in Agarwal et al. 2006). Therefore, it is important to elucidate the mechanisms underlying the transmission of DREB-mediated abiotic stress signals in order to understand enhanced crop stress tolerance. DREBs, which show variations in some conserved motifs and in biological functions in different species, were divided into six groups (A-1, A-2, A-3, A-4, A-5, and A-6) based on different response profiles after a range of stresses (Dubouzet et al. 2003; Agarwal et al. 2006). For example, expression of A-1 group genes is specifically induced by low-temperature stress in Arabidopsis and rice (Liu et al. 1998; Sakuma et al. 2002; Dubouzet et al. 2003), whereas A-2 group genes respond to dehydration and high-salt stresses (Liu et al. 1998; Dubouzet et al. 2003). However, gene expression in the other groups has been studied in only a limited number of plant species and most of their functions remain undetermined. We therefore isolated an A-6 group transcription factor gene, ZmDBP2, from maize.

The ZmDREB2 protein showed no nuclear localization activity (Fig. 3). Most transcription factors have one or two putative NLSs. Xu et al. (2007) isolated a wheat AP2/ERF factor, TaERF1, containing three NLSs that synergistically controlled its subcellular localization. Interestingly, the AP2/ERF domain of TaERF1 plays dual roles in DNA-binding affinity and nuclear localization. As a transcription factor, ZmDBP2 possibly enters the nucleus but after interaction with other transcription factors.

There are ABA-dependent and ABA-independent regulatory systems for stress-responsive gene expression in plants (Yamaguchi-Shinozaki and Shinozaki 2006). A-2 subgroup members are key regulators in ABA-independent gene expression under abiotic stress conditions (Nakashima et al. 2000; Sakuma et al. 2002; Chinnusamy et al. 2006; Nakashima and Yamaguchi-Shinozaki 2006). Expression pattern analyses revealed that ZmDBP2 transcripts were induced by exogenous ABA (Fig. 4). Putative ABA-responsive motifs, such as DPBF binding core sequences, ARF site, and ABRE elements, were also found in the promoter of ZmDBP2. These results suggest that ZmDBP2 may act in an ABA-mediated signal transduction pathway.

ZmDBP2 transcription was activated by drought and by salt (Fig. 4). Putative drought- and salt-responsive motifs were found in the promoter of ZmDBP2, such as an MBS site (responsive to drought), W-box element (responsive to drought and salt), DPBF binding core sequences, and ABRE elements (responsive to drought as well as ABA). A maize A-2 group gene, ZmDREB2A, was shown to accumulate under heat stress in maize seedlings (Qin et al. 2007). Clearly, DREB genes are involved in multiple stress responses, and more of them need to be isolated and tested in order to gain a more comprehensive understanding of their similarities and differences in response to various stresses.

Overexpression of ZmDBP2 activated CRT/DRE-containing genes under normal growth conditions and conferred drought tolerance in transgenic Arabidopsis plants (Fig. 6). However, overexpression of ZmDBP2 did not dramatically enhance resistance to salt. Although a gene may respond to a stress, such a response does not necessarily imply that the gene will play a positive role in increasing resistance to the stress (Liu et al. 1998; Xiong and Yang 2003). It is possible that some gene products might require posttranslational modifications or interaction with other factors to achieve such a response, or on the other hand, overexpression of some genes might not be enough to enhance resistance to clearly detectable or adequate levels (Xu et al. 2007).