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Keywords:

  • chilling tolerance;
  • GhDREB1;
  • gibberellic acid (GA3);
  • promoter;
  • transcription factor;
  • transgenic tobacco

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    The transcription factors C-repeat binding factors/dehydration-responsive element binding proteins (CBFs/DREBs) control the expression of many stress-inducible genes in Arabidopsis.
  • • 
    A cDNA clone, designated GhDREB1, was isolated from cotton (Gossypium hirsutum) by cDNA library screening.
  • • 
    Northern blot analysis indicated that mRNA accumulation of GhDREB1 was induced by low temperatures and salt stress, but was not induced by abscisic acid (ABA) or drought stress in cotton seedlings. Transgenic tobacco (Nicotiana tabacum) plants overexpressing GhDREB1 displayed stronger chilling tolerance than wild-type plants. Their leaf chlorophyll fluorescence, net photosynthetic rate and proline concentrations were higher than those of control plants during low-temperature treatment. However, under normal growth conditions, the transgenic tobacco plants exhibited retarded growth and delayed flowering. Interestingly, GhDREB1 transcripts in cotton seedlings were negatively regulated by gibberellic acid (GA3) treatment. Analysis of the promoter of the GhDREB1 gene revealed the presence of one low-temperature and four gibberellin-responsive elements. Green fluorescent protein (GFP) signal intensity or β-glucuronidase (GUS) activity driven by the GhDREB1 promoter was clearly enhanced by low temperature but repressed by GA3.
  • • 
    These results suggest that GhDREB1 functions as a transcription factor and plays an important role in improving cold tolerance, and also affects plant growth and development via GA3.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Low temperature, drought and high salinity are critical environmental factors that limit agricultural production world-wide. When plants are exposed to environmental stress, they undergo physiological and biochemical adaptations (Choi et al., 2002; Xiong et al., 2002). Plants acclimate to environmental stresses by activating cascades or network events starting with stress perception and ending with the expression of many effector genes (Shinozaki et al., 2003; Shou et al., 2004). Extensive studies have elucidated the regulatory mechanisms of stress-responsive gene expression, and the manipulation of stress-responsive genes for transcription factors should prove useful for improving plant stress tolerance (Jaglo-Ottosen et al., 1998; Kasuga et al., 1999; Cheong et al., 2003; Dubouzet et al., 2003; Mukhopadhyay et al., 2004; Agarwal et al., 2006).

Low-temperature stress includes two different processes, chilling and freezing. Chilling stress (0–10°C) causes membrane leakiness as a result of an inability to increase membrane fluidity and inhibition of photosythetic processes, whereas freezing stress (below 0°C) leads to cellular dehydration caused by the formation of ice crystals in the extracellular space (Zhang et al., 2004; Ensminger et al., 2006; Verslues et al., 2006). During the past decade, a family of transcription factors known as dehydration-responsive element binding proteins (DREBs) has been identified in Arabidopsis, and a number of DREB-like proteins have been isolated from other plant species (Liu et al., 1998; Guo et al., 2002; Shen et al., 2003). These factors interact with cold- and dehydration-responsive elements (DREs) and enhance tolerance to freezing, drought and high salinity in plants (Jaglo et al., 2001; Kizis et al., 2001; Kasuga et al., 2004). DRE elements contain the conserved CCGAC core sequence, which is sufficient to induce gene transcription under cold stress and is present in the promoters of many cold-inducible genes (Stockinger et al., 1997; Kim et al., 2002; Narusaka et al., 2003). Expression of DREB1 genes is strongly induced by low-temperature stress, whereas expression of DREB2 genes is induced by dehydration, indicating that two independent families of DREBs function as trans-acting factors in two separate signal-transduction pathways under low-temperature and dehydration conditions (Liu et al., 1998; Medina et al., 1999; Shinozaki et al., 2003; Sakuma et al., 2006). A number of transgenic lines overexpressing the DREB1 gene showed significantly improved cold tolerance but a marked decrease of plant height and delayed flowering time compared with wild-type plants (Liu et al., 1998; Gilmour et al., 2000; Dubouzet et al., 2003; Kasuga et al., 2004).

Cotton (Gossypium hirsutum) is one of the oldest and most important fiber and oil crops. Its growth and yield are severely inhibited at low temperature, especially at germination and emergence stages (Ashraf, 2002). Our interest has therefore focused on identifying genes whose expression is correlated with low-temperature induction in seedlings of cotton. Towards this goal, a cDNA library was constructed using mRNA isolated from low-temperature-induced seedlings of a chilling-tolerant cotton cultivar (ZM19), and screened using differential hybridization cDNAs encoding specific proteins whose activity may contribute to cold tolerance. In this paper, a cDNA clone, GhDREB1, encoding a DREB-like transcription factor, was isolated and characterized. Our results indicated that the expression of GhDREB1 was strongly induced by low-temperature exposure (0°C) and repressed by gibberellic acid (GA3). Transgenic tobacco (Nicotiana tabacum) plants overexpressing GhDREB1 showed higher chilling tolerance than wild-type plants. The 5′ upstream promoter region of GhDREB1 conferred enhanced expression of the green fluorescent protein (GFP) reporter gene under low-temperature conditions in the transgenic tobacco cells. We suggest that the product of the GhDREB1 gene functions as a transcription factor and plays an important role in improving chilling tolerance, and also affects plant growth and development via GA3.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Plant material, growth conditions and treatments

Seeds of cotton (Gossypium hirsutum L.) cv. ZM19 were provided by the Chinese Academy of Agricultural Sciences. Seedlings were grown in a growth chamber for 20 d with 300 µmol m−2 s−1 light intensity and day:night temperatures of 28 : 20°C. For the low-temperature treatment, uniformly developed seedlings were transferred to a temperature of 0°C for given time periods. For other treatments, uniformly developed seedlings were cultured in solutions containing the indicated concentrations of NaCl, abscisic acid (ABA) or polyethylene glycol (PEG) for given time periods (Fig. 2a). The leaves, roots and stems were harvested directly into liquid nitrogen and store at –80°C for later use.

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Figure 2. Northern blot analysis of Gossypium hirsutum dehydration-responsive element binding protein 1 (GhDREB1) gene expression induced by low-temperature and salt stresses in cotton (G. hirsutum). Total RNA was extracted from cultivar ZM19. About 20 µg of total RNA was analyzed by RNA gel blotting. The blot was hybridized with a 5′-cDNA fragment of GhDREB1. The ethidium bromide-stained rRNA is shown as a loading control. (a) Expression of GhDREB1 induced by 0°C, 250 mm NaCl, 100 µm abscisic acid (ABA) or drought (25% PEG6000) for 2 h. (b) GhDREB1 gene expression induced by low temperature at different times. (c) Tissue-specific expression of GhDREB1 in cotton seedlings with a treatment of 0°C for 2 h.

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cDNA library construction and screening

Poly(A)+ RNA (0.5 µg) isolated from cotyledons of ZM19 seedlings treated at 0°C for 24 h was used to synthesize first-strand cDNA, which was then amplified by long distance polymerase chain reaction (PCR) according to the manufacturer's protocol (SMART‘ cDNA Library Construction Kit; Clontech, Mountain View, CA, USA). The double-stranded cDNA was digested by the SfiI enzyme, and then fractionated using Chroma Spin-400 (Clontech). Fragments longer than 500 bp were cloned into SfiI-digested dephosphorylated λTripIEx2 arms with T4 DNA ligase. The recombinants were packaged in vitro with Packagene (Promega, Madison, WI, USA).

The cDNA library was screened using differential hybridization (once with the untreated cotyledon cDNA probe and once with the 0°C treated cotyledon cDNA probe). Plaques at a density of 104 plaques per plate (15-cm diameter) were transferred onto the membrane. Prehybridization, hybridization, and washing were performed as described previously (Zheng et al., 1998). Positive clones were plaque-purified by two additional rounds of plaque hybridization with the same probes. Clones exclusively or preferentially hybridized by the 0°C treated cotyledon cDNA probe were selected. Of these, one cDNA clone, GhDREB1, is described in this paper.

Isolation and characterization of the GhDREB1 promoter

The GhDREB1 promoter fragment was isolated by adaptor PCR using a TaKaRa long and accurate PCR in vitro cloning kit (TaKaRa, Dalian, China), according to the manufacturer's protocol. Briefly, c. 5 µg of genomic DNA digested with BamHI was ligated to the dephosphorylated BamHI adaptor. Half of the ligated products were amplified in the first round of PCR using the 5′-adaptor primer C1, 5′-GTACATATTGTCGTTAGAACGCGTAATACGACTCA-3′, and the GhDREB1 3′ specific primer S1, 5′-ATAATCTTGAACTACAAAATCCA-3′. The second round of PCR was performed using the first PCR product as template with the 5′-adaptor primer C2, 5′-CGTTAGAACGCGTAATACGACTCACTATAGGGAGA-3′, and the second 3′ specific primer S2, 5′-CATCGGAAAAATTCACCGGACGAT-3′. The PCR product was cloned into the pGEM-T easy vector (Promega) and sequenced. the PLACE database (Higo et al., 1999) and PlantCARE (Lescot et al., 2002) were used for promoter nucleotide sequence analysis.

To analyze promoter activity, the pBINmGFP5-ER and pBI121 β-glucuronidase (GUS) plasmids were used to construct the transformation vectors containing the GhDREB1 promoter. The fusion constructs (GhDREB1::GFP and GhDREB1::GUS) and control plasmids (35S::GFP and 35S::GUS) were introduced into tobacco Bright Yellow 2 (BY-2) suspension-cultured cells by Agrobacterium tumefaciens-mediated transformation (Koroleva et al., 2004). The GhDREB1::GFP and 35S::GFP transformed cells were cultured on Murashige-Skoog (MS) medium at 25°C for 2 d and GFP signals were observed by fluorescence microscopy (Olympus BX50; Olympus, Tokyo, Japan). The GhDREB1::GUS and 35S::GUS transformed cells were cultured in NT medium (MS medium containing 1 mg l−1 VB1 and 0.6 mg l−1 2,4-D) at 25°C with 125 r.p.m. shaking for 1 d and then treated at 8°C for 12 h. GUS activities were measured using the method of Jefferson as described previously (Xue et al., 2005).

Northern blot analysis

Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Fremont, CA, USA). RNA samples for each experiment were analyzed in at least two independent blots. Hybridization was performed in the same manner as the cDNA library screening. The specific GhDREB1 cDNA fragment was labeled with [α-32P]dCTP by priming a gene labeling system from Promega, and used as a hybridization probe.

Subcellular localization of the GhDREB1 protein

The GhDREB1 cDNA with the termination codon removed was fused in-frame to the GFP reporter gene in the pBINmGFP5-ER vector and verified by DNA sequencing. The fusion construct for GhDREB1-GFP and the GFP control plasmid were introduced into an onion (Allium cepa) epidermis cell by biolistic bombardment transformation as described previously (Kinkema et al., 2000). The transformed cells were cultured on MS medium at 25°C for 2 d (16 h light and 8 h dark) and observed under a fluorescence microscope (Olympus BX50).

Analysis of transgenic tobacco plants under cold stress conditions

Tobacco (Nicotiana tabacum cv. NC89) seedlings were grown on sterile MS medium and were used for leaf-disc transformation. The Agrobacterium tumefaciens strain LBA4404 and the pBI121-based binary vector pHAGSK were used for transformation. The GUS gene of the vector was replaced with GhDREB1 at the BamHI and SacI restriction sites. The Agrobacterium-mediated transformation and regeneration procedures were as previously described (Kano-Murakami et al., 1993).

T0 transgenic tobacco plants were identified by PCR to amplify the neomycin phosphotransferase II gene with specific primers (5′-CGCATGATTGAACAAGATGG-3′ and 5′-TCCCGCTCAGAAGAACTCGTC-3′). The corresponding T1 transgenic seedlings segregating at a ratio of ~3 : 1 (resistant:sensitive) were selected to propagate the T2 generation, which was used for further analysis.

Chlorophyll fluorescence values and net photosynthetic rate (Pn) were measured using a FMS-2 pulse-activated modulation fluorometer (Hansatech, Cambridge, UK) and CIRAS-2 portable system (PP-Systems, Hitchen, UK) according to the method described by Genty et al. (1990), respectively.

Approximately 100 seeds each from the wild type and 16 transgenic T2 lines were planted on MS medium and incubated at 0°C after germination. This experiment was performed three times. The seedlings were photographed after 10 d followed recovery at 25°C for 2 d.

Proline analysis

Fresh leaf material (500 mg) was extracted with 5 ml of 3% sulfosalicylic acid at 100°C for 10 min with shaking. The extracts were filtered through glass wool and analyzed for proline content using the acid ninhydrin method. Briefly, 2 ml of the aqueous extract was mixed with 2 ml of glacial acetic acid and 2 ml of acid ninhydrin reagent (1.25 g of ninhydrin, 30 ml of glacial acetic acid and 20 ml of 6 m orthophosphoric acid) and heated at 100°C for 30 min. After cooling, the reaction mix was partitioned against toluene (4 ml) and the absorbance of the organic phase was determined at 520 nm. The resulting values were compared with a standard curve constructed using known amounts of proline (Sigma, St Louis, MO, USA).

Target gene analysis of GhDREB1 in transgenic tobacco plants

Total RNA isolation from transgenic and control tobacco plants and RNA-blot hybridization were performed as described above in ‘Northern blot analysis’. The probes were produced by reverse transcriptase (RT)–PCR using the primers 5′-TATGGGAACCCAGTCCATCACACTGGA-3′ and 5′-CTTTAAAGGGATTTTATTGTTTGGCAG-3′ for NtERD10B, and 5′-AGGTTGAAGAGGGTAGCGCAAACG-3′ and 5′-GCCACTTCCTCTGTCTTCTTTTGC-3′ for NtERD10C. Equal RNA loading in the experiments was monitored by rRNA staining.

Semiquantitative RT-PCR was employed using total RNAs treated with RNase-free DNase I (Promega). DNA-free total RNA (1 µg) was reverse-transcribed with reverse transcriptase (Promega), according to the manufacturer's protocol. The reverse transcription products were then amplified by PCR using the GhDREB1 primers (5′-ATGGATTTTGTAGTTCAAGATTAT-3′ and 5′-TTAAATAGAATAACTCCATAAAGG-3′) and histone primers (5′-ATGGATTTTGTAGTTCAAGATTAT-3′ and 5′-TTAAATAGAATAACTCCATAAAGG-3′).

Quantification of endogenous gibberellins by enzyme-linked immunosorbent assay (ELISA)

ELISA was performed according to the procedure of Yang et al. (2001). Sixteen uniform transgenic tobacco seedlings from independent lines and 16 wild-type seedlings were used. Briefly, samples were homogenized in liquid nitrogen and extracted in cold 80% (volume/volume (v/v)) methanol with butylated hydroxytoluene (1 mmol l−1) overnight at 4°C. After centrifugation at 10 000g (4°C) for 20 min, the extracts were passed through a C18 Sep-Pak cartridge (Waters, Milford, MA, USA) and dried in N2. The residues were dissolved in phosphate-buffered saline (PBS) (0.01 mol l−1; pH 7.4). Ovalbumin solution (10 mg ml−1) was added to each well to block nonspecific binding after the 96-well microtitration plates (Nunc, Roskilde, Denmark) had been coated with synthetic GA1-ovalbumin conjugates in NaHCO3 buffer (50 mmol l−1; pH 9.6) overnight at 37°C. Then horseradish peroxidase-labeled goat anti-rabbit immunoglobulins were added to each well and incubated for 1 h at 37°C. The enzyme reaction was carried out in the dark at 37°C for 15 min after the substrate (orthophenylenediamino) had been added, and then terminated using 3 mol l−1 H2SO4. Calculations using the ELISA data were performed for absorbance recorded at 490 nm. The cross-reactivity of antibodies raised against GA1-ovalbumin to GA3-ovalbumin was 32% under the analytical conditions used.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Characterization of the G. hirsutum GhDREB1 cDNA clone

A cDNA clone, GhDREB1, was isolated from a G. hirsutum cotyledon cDNA library by differential hybridization screening to identify genes involved in cold tolerance. The full sequence of the GhDREB1 cDNA consisted of 958 nucleotides with an open reading frame of 648 nucleotides, starting at nucleotide position 70, where a potential translation initiation codon was flanked by eukaryotic translation initiation consensus motifs (Kizak, 1981). A termination codon was located at position 718, and a potential polyadenylation signal at position 928 (Joshi, 1987).

The deduced amino acid sequence of the cDNA showed that it encoded a polypeptide containing 216 amino acids which shared a DNA-binding domain with other DREB transcription factors previously characterized in higher plants. The sequence identities to Arabidopsis AtDREB1A/B/C are 54, 54 and 53%, respectively, but it shares less similarity (only 23%) to AtDREB2A. Consistent with other higher plant DREB proteins, the GhDREB1 protein is predicted by the Chou–Fasman approach to contain one α-helix and three β-sheets, forming a DNA-binding domain (Fig. 1). Furthermore, GhDREB1 contains a four-amino-acid sequence (DSAW) at the end of its DNA-binding domain that is highly conserved in the DREB1-type proteins. GhDREB1 also has a basic amino acid stretch (PKRRAGRKKFR) near its N-terminal region that has been suggested to function as a nuclear localization signal (NLS) in DREB1-related proteins (Dubouzet et al., 2003). Overall, the GhDREB1 cDNA we isolated from cotton might encode an AtDREB1-like transcription factor.

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Figure 1. Comparison of the deduced amino acid sequence of Gossypium hirsutum dehydration-responsive element binding protein 1 (GhDREB1) with its homologs. Gaps have been introduced to optimize the alignment. Identical or conserved amino acids are shaded in black or gray, respectively. The N-terminal basic amino acid stretch (PKRRAGRKKFR) which may function as a putative nuclear localization signal (NLS) is indicated by dots. The conserved DNA-binding domains are underlined (double line) and the four-amino-acid signature sequence (DSAW) at the end of the domain is indicated by asterisks. The accession numbers in GenBank and sources of the proteins are as follows: GhDREB1 (AY422828), Gossypium hirsutum; CaDREBLP1 (AY496155), Capsicum annuum; LeCBF1 (AY034473), Lycopersicon esculentum; HvCBF1 (AF418204), Hordeum vulgare; AtDREB1A (AB007787), Arabidopsis thaliana; BnCBF7 (AF499032), Brassica napus.

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To determine whether GhDREB1 represents a single locus in the G. hirsutum genome or whether it is a member of a multigene family, a Southern blot analysis was performed using the GhDREB1 cDNA as a probe. The results showed that GhDREB1 hybridized to between two and four genomic restriction fragments, indicating the presence of a small gene family of GhDREB1 in cotton (data not shown).

The expression of GhDREB1 is up-regulated by low temperature

To determine which type of stress could induce GhDREB1 expression, total RNA was extracted from 9-d-old cotton seedlings treated with low temperature (0°C), 250 mm NaCl, 10 mm ABA or 25% PEG for northern blot analysis. The results showed that GhDREB1 gene expression was induced by low temperature and high-salinity stress but not by ABA or drought (Fig. 2a). Interestingly, GhDREB1 mRNA accumulation appeared to reach a peak within 2 h of treatment at 0°C (Fig. 2b). In order to further determine the pattern of tissue-specific expression of GhDREB1 under low-temperature conditions, different organs of 9-d-old seedlings treated at 0°C for 2 h were harvested for RNA extraction. GhDREB1 gene expression was detected in all organs, with the highest expression found in leaves (Fig. 2c).

Overexpression of GhDREB1 in tobacco plants improves tolerance to chilling stress

To confirm the in vivo functions of the GhDREB1 gene during low-temperature stress in plants, we ectopically expressed the GhDREB1 gene in tobacco. A total of 23 transgenic tobacco plants were obtained. Thirty-two kanamycin-resistant T2 plantlets (from 16 lines) were selected for low-temperature and salt tolerance assays. Sixteen uniformly developed transgenic tobacco seedlings and 16 wild-type tobacco seedlings were treated with 250 mm NaCl for 30 d or 0°C for 6 d, respectively. Overexpression of the GhDREB1 gene did not confer elevated tolerance to salt stress in transgenic tobacco (data not shown). As shown in Fig. 3(a), however, the transgenic plants exhibited enhanced chilling tolerance compared with wild-type tobacco plants. All transgenic tobacco plants were able to survive at 0°C for 6 d followed recovery at 25°C, whereas all leaves of the wild-type plants became wilted and curled and eventually 82% of wild-type plants died.

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Figure 3. Low-temperature tolerance of transgenic and wild-type tobacco (Nicotiana tabacum) plants. (a) Phenotype of transgenic and wild-type tobacco seedlings growing in soil in pots treated at 0°C for 6 d followed by recovery at 25°C for 2 d. (b) Phenotype of transgenic and wild-type tobacco seedlings after seed germination treated at 0°C for 10 d followed by recovery at 25°C for 2 d. (c) Northern blot analysis of Gossypium hirsutum dehydration-responsive element binding protein 1 (GhDREB1) in transgenic and wild-type (WT) tobacco plants. T1, T2 and T3 are three independent transgenic lines. (d) Proline concentrations in 4-wk-old transgenic and wild-type tobacco plants before and after low-temperature treatment (0°C for 24 h). Sixteen independent transgenic lines were used and data are mean ± standard error. (e) Net photosynthetic rate (Pn) of transgenic (open circles) and wild-type (closed circles) tobacco during the 0°C treatment. (f) Chlorophyll fluorescence measurements for transgenic lines (open circles) and wild type (closed circles) during the 0°C treatment. Data are mean ± standard error of four replicates. FW, fresh weight.

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To test whether the cold resistance induced by overexpression of GhDREB1 is also effective at the seed germination stage, the response of young transgenic seedlings to cold stress was analyzed. After germination, the young seedlings were incubated at 0°C in the dark for 10 d. As shown in Fig. 3(b), wild-type seedlings almost died, whereas all transgenic seedlings survived and developed green cotyledons under such conditions. These results indicate that the improved cold resistance conferred by overexpression of GhDREB1 was found in both early seedling and later seedling stages.

The enhanced chilling tolerance of transgenic tobacco plants was further verified by measuring chlorophyll fluorescence (variable fluorescence (Fv)/maximum fluorescence (Fm)) and net photosynthetic rate (Pn). After 2 d at 0°C, reductions in the maximum photochemical efficiency of photosystem II (PSII) in the dark-adapted state (Fv/Fm) and Pn were considerably larger in wild-type plants than in 35S::GhDREB1-transformed plants throughout the time course (Fig. 3e,f). Moreover, northern blot analysis showed that the GhDREB1 gene was expressed in all the transgenic tobacco plants that had improved chilling tolerance (Fig. 3c), indicating that the cold-resistance changes in 35S::GhDREB1 plants were the result of the transgene expression.

Proline is required for protein biosynthesis and in many organisms also acts as a protective agent for cells under osmotic stress. To determine whether the overexpression of GhDREB1 might result in elevated concentrations of proline in transgenic tobacco plants, concentrations of free proline were analysed. Under normal growth conditions, free proline concentrations in the GhDREB1-overexpressing plants were approximately 2-fold higher than those in the control plants (Fig. 3d), suggesting that some downstream genes activated by GhDREB1 are involved in proline synthesis, conferring higher plant tolerance to chilling stress.

GhDREB1 targets the nucleus and acts as a transcriptional activator to enhance transcription of downstream genes

To determine the subcellular localization of GhDREB1 in plant cells, a reporter gene encoding GFP in the binary vector pBINmGFP5-ER was fused to GhDREB1, and subjected to a transient assay using onion epidermis cells (Fig. 4). The individual cells transformed with CaMV35S::GhDREB1-GFP exhibited green fluorescence predominantly at the nucleus (Fig. 4b), whereas the cells transformed with the CaMV35S::GFP control construct showed GFP signals in both the cytoplasm and the nucleus. The results suggest that GhDREB1 is a nuclear protein.

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Figure 4. Subcellular localization of Gossypium hirsutum dehydration-responsive element binding protein 1 (GhDREB1). The fusion construct for GhDREB1–green fluorescent protein (GFP) and the GFP control plasmid were introduced into onion (Allium cepa) epidermis cells by biolistic bombardment transformation. The transformed cells were cultured on Murashige-Skoog (MS) medium at 28°C for 2 d and observed under a microscope. The photographs were taken in brightlight for the morphology of the cell (a) and in dark field for green fluorescence (b) and 35S::GFP control (c, d). (e) Expression of the GhDREB1 target genes (NtERD10B and NtERD10C; ERD, early response to dehydration) in 35S:GhDREB1 transgenic tobacco plants (Nicotiana tabacum; T1 and T2) and in wild-type plants (WT). Each lane was loaded with total RNA (20 µg) isolated from 4-wk-old seedlings under a low-temperature treatment (0°C for 24 h) and control conditions (25°C). Equal amounts of RNA were present in each sample, as confirmed by ethidium bromide staining of rRNAs.

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In order to better understand the role of GhDREB1 in cold tolerance in transgenic tobacco, we examined the expression of NtERD10B and NtERD10C (ERD, early response to dehydration), which have been described as C-repeat binding factor (CBF)/DREB1 and C. arietnum AP2 (CAP2) target genes in transgenic tobacco plants (Kasuga et al., 2004; Shukla et al., 2006). NtERD10B and NtERD10C transcripts were undetectable in the absence of cold stress in control plants, but in transgenics they were greatly elevated even under normal growth conditions (Fig. 4c). This indicates that GhDREB1 acts as a transcriptional activator of NtERD10B and NtERD10C and that a correlative link exists between their expression and chilling tolerance.

Tobacco plants overexpressing GhDREB1 exhibited growth retardation and the flowering delay phenotype

When the phenotypes of the transgenic tobacco plants were compared with those of the wide-type plants, growth retardation of the transgenic tobacco plants was observed. All transgenic plants grew slowly and delayed flowering for c. 20 d under normal growth conditions (Fig. 5a), suggesting that GhDREB1 also influences plant growth and development.

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Figure 5. Delayed flowering phenotype of 35S:GhDREB1 (DREB1, dehydration-responsive element binding protein 1) transgenic tobacco (Nicotiana tabacum) plants under normal growth conditions. (a) The phenotype of 4-month-old 35S:GhDREB1 transgenic and wide-type tobacco plants grown in the glasshouse under normal growth conditions. (b) Gibberellic acid (GA1+3) contents in 35S:GhDREB1 transgenic and wide-type tobacco plants. Each column represents the mean ± standard error for 16 plants. FW, fresh weight.

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Because gibberellins (GAs) are known to play an important role in many aspects of plant growth and development, including stem growth and flowering (Richards et al., 2001), we were interested to determine whether there was a correlation between GA content and phenotype in the transgenic tobacco plants. By ELISA, we quantified the endogenous amount of GA1 and GA3, the major active GAs in tobacco plants. The endogenous amount of GA1+3 in the 35S::GhDREB1 plants was only 47% of that in the control plants (Fig. 5b), suggesting that the growth retardation and flowering delay phenotype of transgenic plants might be involved in decreasing the amount of GAs present.

The GhDREB1 promoter contains low-temperature and GA responsive elements

To clarify the mechanism responsible for the inducible expression of GhDREB1 by low-temperature stress, an 807-bp GhDREB1 promoter fragment was isolated by adaptor PCR with reference to the transcriptional start site determined by the GhDREB1 cDNA sequence. Sequence analysis revealed that the GhDREB1 promoter contains CAAT and TATA motifs located at nucleotides –66 and –35 relative to the transcriptional start site, respectively (Fig. 6). Each of these motifs is characteristic of eukaryotic gene promoters.

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Figure 6. Nucleotide sequences of the 5′ flanking region (GenBank accession number AY920495) and N-terminal coding region of the cotton (Gossypium hirsutum) dehydration-responsive element binding protein 1 (GhDREB1) gene. Numbering is relative to the transcription start (shown by shading and labeled ‘+1’) as determined by forecast. The GhDREB1 coding region starting from an ATG is shown in bold letters. P-box, gibberellin-responsive element; TATC-box, gibberellin-responsive element; LTR, low-temperature responsive element; myeloblastosis (MYB)15-like, MYB15-like binding site; ICE1-like, inducer of C-repeat binding factor (CBF) expression 1-like binding site.

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Using computer prediction, interestingly, we found that the 807-bp promoter region contains several motifs probably related to low-temperature and gibberellin signals, such as a P-box (a gibberellin-responsive element), a TATC-box (a cis-acting element involved in gibberellin responsiveness), a low-temperature responsive element (LTR) (a cis-acting element involved in low-temperature responsiveness), an myeloblastosis (MYB)15-like binding site and an inducer of CBF expression 1 (ICE1)-like binding site, suggesting that the low-temperature inducible expression of GhDREB1 might be regulated at the level of transcription (Baker et al., 1994; Chinnusamy et al., 2003; Duan & Schuler, 2005; Agarwal et al., 2006; Zhiponova et al., 2006).

To determine whether the GhDREB1 promoter responds to low-temperature signals and GA, the CaMV35S promoter in the binary vectors pBINmGFP5-ER and pBI121GUS was substituted by the 807-bp GhDREB1 promoter. Transient GFP and GUS assays were conducted by introducing the chimeric gene into tobacco BY-2 suspension-cultured cells using Agrobacterium tumefaciens-mediated transformation. Fluorescence microscope observations showed that the GFP signal intensity driven by the GhDREB1 promoter in the transfected cells was clearly enhanced after 12 h of treatment at 8°C, whereas the green fluorescence driven by the CaMV35S promoter was not changed, indicating that the 807-bp GhDREB1 promoter could respond to cold stress signals (Fig. 7a). Furthermore, high GUS activities in the GhDREB1::GUS transfected cells were detected after treatment at 8°C (there was undetectable GUS activity at 4°C and lower induction at 12°C). Interestingly, the induced GUS activity was highly repressed by supplying GA3 (Fig. 7b), whereas GUS activity in CaMV35S::GUS-transfected cells was not affected by GA3 (data not shown), confirming the presence of a correlation of GhDREB1 expression, the endogenous amount of GAs and the growth retardation and flowering phenotype in transgenic tobacco plants.

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Figure 7. Fluorescent analysis of green fluorescent protein (GFP) and β-glucuronidase (GUS) driven by the Gossypium hirsutum dehydration-responsive element binding protein 1 (GhDREB1) promoter in transgenic BY-2 tobacco (Nicotiana tabacum) suspension cells. (a) Fluorescent and corresponding phase-contrast images. The transformed BY-2 cells were treated at 8°C for 12 h and at 25°C as a control. GhDREB1::GFP and 35S::GFP constructs were used to transform BY-2 cells. (b) GUS activities of the transformed cells treated at 8°C for 12 h and at 25°C as a control. GhDREB1::GUS and 35S::GUS constructs were used to transform BY-2 cells. The transformed cells were treated with (+) or without (–) 80 µm gibberellic acid (GA3).

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Exogenous GA3 does repress GhDREB1 expression in cotton seedlings

To further confirm the effect of GA3 on GhDREB1 expression in cotton seedlings, we performed northern blot hybridization and relative quantitative reverse transcription–PCR (RQRT-PCR) analyses. Under normal growth conditions, the transcript of GhDREB1 was undetectable in both GA-treated and control plants. After exposing cotton seedlings to 0°C for 24 h, however, the accumulation of GhDREB1 mRNA in GA-treated plants was significantly less than that in control plants (Fig. 8a). Because we could not easily detect GhDREB1 transcripts from the seedlings grown in normal conditions by standard northern blot analysis, we conducted RQRT-PCR analyses for the quantitative measurement of GhDREB1 transcripts. As shown in Fig. 8(b), GhDREB1 expression was repressed in GA3-treated plants. These results strongly suggest that the transcriptional suppression of the GhDREB1 gene is a consequence of supplying GA3 to the cotton seedlings.

image

Figure 8. Gossypium hirsutum dehydration-responsive element binding protein 1 (GhDREB1) expression negatively regulated by exogenous gibberellic acid (GA3) in cotton seedlings. One-week-old cotton seedlings were treated with (+) or without (–) 80 µm GA3 for 3 h and then treated at 0°C or 25°C for 2 h. Total RNA was extracted from the treated seedlings. (a) Total RNA (20 µg in each lane) was analyzed by RNA gel blotting and hybridized with a GhDREB1-specific probe. The ethidium bromide-stained rRNA band in the agarose gel is shown as a loading control. (b) The GhDREB1-specific band amplified from total RNA by relative quantitative reverse transcription–polymerase chain reaction (RQRT-PCR). The RQRT-PCR conditions were verified by a linear amplification of a histone-specific band. The picture represents one of three independent experiments that gave similar results.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Low temperature is a major abiotic factor that limits crop productivity in many areas around the world. A number of studies have demonstrated that overexpression of cold-induced genes can confer cold tolerance to transgenic plants (Puhakainen et al., 2004; Shou et al., 2004; Zhu et al., 2004). It has also been suggested that low temperature could trigger the expression of DREB1 transcription factors, which play a key role in cold tolerance, on the basis of the observation that ectopic expression of AtDREB1A and AtDREB1B induces the transcription of genes containing the DRE promoter element and enhances cold tolerance of transgenic plants (Jaglo-Ottosen et al., 1998; Liu et al., 1998; Gilmour et al., 2000; Hsieh et al., 2002). In this study, a cotton DREB1-like transcription factor, GhDREB1, was isolated from a G. hirsutum cotyledon cDNA library. Our in vivo targeting experiment using a GhDREB1-fused GFP as a florescent marker demonstrated that the fusion protein was localized to the nucleus of onion epidermis cells (Fig. 4b), suggesting that GhDREB1 is a nuclear protein and functions as a transcription factor.

We examined the expression patterns of GhDREB1 in relation to various environmental stresses. The results showed that GhDREB1 in cotton seedlings was significantly induced by treatment at 0°C and weakly induced by treatment with 250 mm NaCl. However, it was not regulated by drought stress or exogenous ABA (Fig. 2a), suggesting that the GhDREB1 gene functions in an ABA-independent pathway, similar to BnCBF7 in Brassica napus and OsDREB1A in rice (Oryza sativa), but unlike AtDREB1 in Arabidopsis (Jaglo et al., 2001; Dubouzet et al., 2003; Knight et al., 2004).

Because GhDREB1 transcripts are not usually detected in RNA-blot hybridizations under nonstress conditions, it is generally presumed that the GhDREB1 gene is not transcribed, or is transcribed at lower levels, at normal growth temperatures. To investigate the mechanism whereby the expression of GhDREB1 is regulated by low temperature, we isolated the promoter region of the GhDREB1 gene from the cotton genome. Sequence analysis of the GhDREB1 promoter revealed the existence of one putative low-temperature responsive element, two ICE1-like binding sites and an MYB15-like binding site (Fig. 6), suggesting that induction of the GhDREB1 gene in response to low temperature is involved in the regulation of transcription factors, such as ICE1 and MYB15 in Arabidopsis (Chinnusamy et al., 2003; Agarwal et al., 2006).

To confirm in vivo functions of the GhDREB1 gene during low-temperature stress in plants, we ectopically expressed the GhDREB1 gene in tobacco plants. All transgenic plants exhibited enhanced chilling tolerance compared with wild-type tobacco plants (Fig. 3a). Further determination of photosynthetic efficiency revealed that the Fv/Fm ratio and Pn were less affected by low-temperature treatment in transgenic plants (Fig. 3e,f). Proline is a compatible organic molecule that accumulates in plants when they are exposed to environmental stress, and plays multiple roles in plant adaptation to stress, including chilling (Sung et al., 2003). A significant correlation between freezing tolerance and an increase in proline concentration in shoot and bud tissue of grapevines (Vitis vinfera) after exposure to low temperatures was found (Delauney & Verma, 1993). Under cold stress at normal growth temperatures, free proline concentrations in the GhDREB1-overexpressing plants were approximately 2-fold higher than those in the control plants (Fig. 3d), suggesting that some downstream genes activated by GhDREB1 are involved in proline synthesis, conferring higher plant tolerance to cold stress and protecting the photosynthetic apparatus of plant cells through osmotic regulation. Moreover, we examined the expression of NtERD10B and NtERD10C, which encode group 2 late embryogenesis abundant proteins and function as target genes of CBF/DREB1 in transgenic tobacco (Kasuga et al., 2004). Their transcripts were undetectable in the absence of cold stress in control plants, while in transgenics they were greatly elevated even under normal growth conditions (Fig. 4c), indicating that GhDREB1 acts as a transcriptional activator in transgenic tobacco.

However, constitutive overexpression of GhDREB1 in transgenic tobacco plants did not increase freezing tolerance. When treated at –2°C for 2 d, none of the wild-type and transgenic tobacco plants were able to recover (data not shown). A similar phenotype was observed in transgenic tobacco overexpressing AtDREB1A (Kasuga et al., 2004). In previous work, we found that freezing tolerance was increased in all GhDREB1 transgenic Arabidopsis plants (unpublished data). Zhang et al. (2004) demonstrated that tomato (Lycopersicon esculentum) LeCBF1 in a heterologous system could activate the Arabidopsis cold related (COR) genes involved in increasing freezing tolerance, but that LeCBF1 in tomato plants did not up-regulate equivalent genes. Microarray analysis revealed that the targets of LeCBF1 in tomato were not homologs of the freezing tolerance genes up-regulated by AtDREB1 in Arabidopsis, but the target genes have a potential role in tolerance of chilling conditions. Taken together, these observations indicated that chilling-sensitive plant species, such as tobacco and tomato, have evolved differently from chilling-resistant plant species, such as Arabidopsis, in their responses to cold stress. It is reasonable to conclude that the functions of DREBs in two types of plant species are conserved but that their target genes are very different.

Interestingly, overexpression of GhDREB1 also delayed development, and the transgenic lines showed a late flowering phenotype (Fig. 5a). Similar growth retardation was also observed in transgenic Arabidopsis and other plant species overexpressing AtDREB1A (Kasuga et al., 1999; Hsieh et al., 2002; Magome et al., 2004). GAs are essential phytohormones that control many aspects of plant development, including flowering (Ogawa et al., 2003). In view of the development-delayed phenotype of transgenic plants, it is reasonable to speculate that overexpression of DREB1A in the transgenics may interfere with GA accumulation. By ELISA, we found that the bioactive GAs in GhDREB1-overexpressing lines were less than half of the wild type (Fig. 5b). The GhDREB1 promoter contains GA responsive elements, and the expression of GhDREB1 is repressed by GA, a positive growth regulator, in two independent systems (Figs 7b, 8). Therefore, GhDREB1 may play an important role in GA signaling, although the exact mechanisms remain largely unknown. However, one can conclude that GhDREB1 plays a negative role in plant growth and development, and that this negative action is normally repressed by GA. Overaccumulation of GhDREB1 antagonizes the GA repression.

On the basis of these observations, we propose that there exist at least two regulatory pathways for GhDREB1, one being typical positive regulation in cold stress signaling and the other novel negative control by bioactive GAs. We can thus begin to understand the molecular basis for interactions among GhDREB1, GA, plant cold tolerance and development delay. As we learn more about DREBs and their regulation, the design of efficient strategies for crop improvement should become possible.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Dr J. Haseloff (MRC Laboratory of Molecular Biology, Cambridge, UK) for the GFP construct pBINmGFP5-ER. This work was supported by the National Basic Research Program (grant no. 2006CB1001006) and the National Natural Science Foundation (grant nos 30570144 and 30500042) in China.

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  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
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