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Plant Science 166 (2004) 1167–1175

Isolation and characterization of a dehydrin-like gene from drought-tolerant Boea crassifolia
Ye Shen1 , Ming-Juan Tang, Yuan-Lei Hu, Zhong-Ping Lin∗
College of Life Science, National Key Laboratory of Protein Engineering and Plant Genetic Engineering, Peking University, Beijing 100871, China Received 11 October 2003; received in revised form 7 December 2003; accepted 19 December 2003

Abstract A novel dehydrin-like gene, designated BcDh2, was isolated from Boea crassifolia (Hemsley) by using mRNA differential display and rapid amplification of cDNA ends (RACE). The full length of BcDh2 cDNA consists of 700 nucleotides and has an open reading frame of 399 bp. The deduced amino acid sequence of BcDh2 has 133 amino acid residues which forms a 15 kDa polypeptide with a calculated pI of 7.95. The putative polypeptide composed of one consensus Y-segment, a stretch of serine residues and two Lysine-rich repeats which presented a typical Yn SK2 structure of dehydrins. The transcripts of BcDh2 highly accumulated when the plants were treated with drought, salinity, exogenous abscisic acid (ABA) and moderated heat shock, while accumulated slightly in response to low temperature stress. BcDh2 also accumulated slightly in response to wounding signals such as MeJA and low concentration of salicylic acid. For further investigation, the promoter region of BcDh2 gene isolated was fused to the -glucuronidase reporter gene (GUS) and transferred into tobacco (W38). The assays of GUS activity in transgenic plants treated by drought, cold, heat, exogenous ABA and MeJA were in accord with Northern analysis of BcDh2 mRNA in B. crassifolia. © 2004 Elsevier Ireland Ltd. All rights reserved.
Keywords: Boea crassifolia; mRNA differential display; Dehydrins; Abiotic stress; Wounding stress

1. Introduction Dehydrins, also known as late embryogenesis abundant (LEA) D-II protein family, have been the most commonly observed proteins that accumulate in plants in response to drought, low temperature, exogenous abscisic acid (ABA), or wounding signals, such as MeJA and SA. A majority of dehydrins, both in angiosperm and gymnosperm, are composed of several typical domains joined together in a few characteristic patterns with numerous minor permutations [1,2]. There are usually 1–11 consensus Lys-rich amino acid sequence EKKGIMDKIKEKIPG (K-segment) at the C-terminus of the dehydrins studied, which forms a putative amphipathic -helix. Many dehydrins contain a tract of contiguous serine residues (the S-segment) in the central of the

Corresponding author. Tel.: +86-10-6275-3062; fax: +86-10-6275-9652. E-mail address: linzp@pku.edu.cn (Z.-P. Lin). 1 Present address: Photosynthesis Research Center, Institute of Botany, Chinese Academy of Sciences, No. 20, Nanxincun, Xiangshan, Beijing 100093, China. Tel.: +86-10-6259-1431; fax: +86-10-6259-4105. E-mail address: niffay shen@yahoo.com

protein that may be phosphorylated and related to nuclear transport by binding to nuclear localization signal peptides. At the N-terminal in a majority of dehydrins, there is one or two copies of a short amino acid repeat, T/VDEYGNP (Y-segment), which is homologous to a portion of the nucleotide binding site of chaperones. According to these characteristics of dehydrins described above, a “YSK” shorthand is used to facilitate comparison and classification of different types of dehydrins. So far, the function of the dehydrins has not yet been demonstrated. Many studies, however, revealed that they were associated with macromolecules, such as nucleoprotein complexes in the nucleus and endomembrane in the cytoplasm, which suggested that the dehydrins might be surfactants that could inhibit the coagulation of macromolecules and preserve their structural integrity under the condition of cellular dehydration or low temperature [2]. Boea crassifolia (Hemsley) is a kind of drought-tolerant plant belonging to the family Gesneriaceae. This perennial stemless herb mainly distributes on stony stiffs with an elevation ranging from 700 to 3200 m above sea level in southwest of China [3,4], and can survive drought environment during dry seasons. mRNA differential display was carried out to analysis the expression patterns of drought-induced

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APR18 and APR20). the plants were put at 32 ◦ C for 8 h. crassifolia. (AP1. the DNA fragments were blotted onto Hybond-N+ filters (Amersham Pharmarcia. After separation on 1% agarose gel. respectively. 37 ◦ C or 42 ◦ C for 6 h. 2. containing 15% PEG. For heat treatment. the promoter of BcBh2 was obtained from the genomic DNA of B. [8]. and then the PCR amplification was carried out using GSP2 and AAP supplied with the kit. the seedlings saturated with deionized water were exposed in the air at room temperature. For the low temperature treatment. the swab contained no MeJA but only ethanol solution. 2. For further investigation. the filters were washed twice with 1 × SSC plus 0. The cDNA fragments were electrophoretically separated on a 5. After hybridization. The material for this study were sterilized by 0. [9]. Two gene-specific primers were designed according to the partial sequence of the BcDh2 gene: GSP1 (5 -ATGCTCGGACACACACACCT-3 ) and GSP2 (5 -TTCGTGATGTGGCTGGTTGT-3 ). Six microlitres of each reamplification mixture were dotted on Hybond-N+ membrane filter (Amersham Pharmarcia. Southern blot analysis Genomic DNA was extracted from leaves as described by Ausubel et al. plants were placed in a glass containing a cotton swab to which 500 l of a 10% MeJA solution prepared in ethanol was added [5].1168 Y. and dehydrated to 70. China. Reverse Northern blotting.4. 65. Germany). signal detection was performed as described in the manufacturer’s instruction (Roche. The expression pattern of BcDh2 under various stresses were also studied. For its control. Materials and methods 2. The salinity and exogenous ABA treatment were carried out by incubating plants on MS medium containing 150 mM NaCl and 75 mM ABA at 22 ◦ C for 8 and 24 h. [7] to reduce false positives arose from differential display. crassifolia leaves by using the acid-Guanidinium Thiocyanate method. 2. USA). crassifolia by mRNA differential display and RACE. and 2. USA). USA) according to manufacture’s recommendation. For cold treatment. crassifolia. For MeJA treatment. plants were incubated in sealed flasks at 30 ◦ C. as described in the Protocols and Applications Guide of Promega. After prehybridization and hybridization. For heat treatment. 5 -RACE and RT-PCR The 5 -end of the cDNA was obtained using 5 -RACE system for rapid amplification of cDNA ends (GibcoBRL. the plants were incubated in 30 ml MS liquid medium with 75 M ABA. Full-length cDNA was amplified by using RT-PCR and cloned into pGEM-T easy vector.5. Plant material and stress treatments B.1% HgCl and grow on hormone-free Murashige-Skoog medium at 22 ◦ C with a photoperiod of 18 h light/6 h dark.1%SDS at room temperature for 5 min. Total RNA from drought-treated plants was used as a template for the reverse transcription reaction with GSP1. Total cDNA probes were synthesized by reverse transcription of 5 g total RNA for each sample from drought-treated and untreated B. twice with 0. Sequence analysis of the promoter region and plant transformation with chimeric constructs confirmed the BcDh2 to be a stress-inducible gene. Three anchored primers . / Plant Science 166 (2004) 1167–1175 gene in B.6% denatured-polyacrylamide gel. ABA was dissolved in methanol. Six recombinant colonies issued from each DD band were chosen and subjected to a second-round reverse Northern blotting for verification. filters were washed according to Sambrook et al. 58 and 43% of its original fresh weight. The differential display was performed with the fluroDD kit (Genomyx.1. For MeJA treatment. 0. and subjected to X-ray film exposure. Promega). AP6 and AP10) were used in combination with four 10 mers arbitrary primers (APR7. crassifolia by adapter-mediated PCR (AMPCR) chromosome walking. the plants were incubated at 4 ◦ C for 14 h. Its possible roles in stress and defense response were discussed. crassifolia was collected in August of 1998 from Guizhou Province.1% SDS at 65 ◦ C for 15 min. cloning and sequencing Reverse Northern blotting was carried out as described by Zegzonti et al. respectively. In this paper we reported the isolation of a novel dehydrin-like gene BcDh2 from B. EcoRI. For exogenous ABA treatment. for 24 h at 22 ◦ C. The cDNA fragments of the interested were cloned into pGEM-T Easy vectors according to the manufacturer’s protocol (Promega. Transgenic tobacco were treated with the following conditions: Drought treatment were carried out by incubating the plantlets in 30 ml of MS liquid medium.25 and 0.5 mM SA at 22 ◦ C for 24 h [6]. USA). 2. APR9. Shen et al.5% methanol was used as the control for ABA treatment. 2. respectively. The interested cDNA fragment clones were sequenced and subjected to BLAST on NCBI database.1. The BcDh2 cDNA was labeled as a probe with [α-32 P]-dCTP by randomly primed labeling method (Prime-a-Gene Labeling System. For drought treatment.2. respectively. plants were incubated on MS containing 0. USA) according to manufacturer’s instruction. EcoRV and HindIII). DNA was digested with restriction endonucleases (BglII.3. For SA treatment. the seedlings were placed in liquid MS supplemented with 5% MeJA for 8 h. plants were incubated at 4 ◦ C for 8 and 24 h. RNA isolation and mRNA differential display Total RNA was extracted from both drought-treated and untreated B.1×SSC plus 0.

The deduced amino acid sequence of BcDh2 gene consists of 133 amino acids with a predicted molecular mass of 15 kDa and a pI of 7. 3. to yield to pT-pBcDh2. These characteristics of the predicted polypeptide demonstrated that BcDh2 cDNA encodes a typical Yn SK2 dehydrins. GUS activities were measured in seven to nine independent transgenic lines transformed with the same construct. Sequence analysis of this fragment revealed its homology to the dehydrin-like gene cloned in many other plant species. BglII. AMPCR was performed according to Mueller and Wold [10] and Liu (a Ph. Alignments of amino acid sequence show that the BcDh2 shares high homology with Elaeis guineensis (African oil palm).95 as expected.1 were obtained as described previously and used as a control. 2. A 40-cycle touch-down PCR (TD-PCR) consisted of six cycles/◦ C declining at 2 ◦ C increments from 72 to 64 ◦ C followed by 10 cycles at 62 ◦ C. Extracts were standardized based on protein concentration. and then subjected to PCR amplification. was ligated with the Sau3AI adapter.6. 2. Transgenic plants carrying the CaMV 35S promoter fused to GUS in pCAMBIA 1305. EcoRV and HindIII-digested genomic DNA from B. The probe labeling. The PCR fragment descried above was cloned into the pGEM-T Easy.5% formaldehyde agarose gel electrophoresis with 20 g each lane [8] and blotted onto Hybond-N+ filters.1. which was loaded with the BglII. the sense primer (5 -GGATCCGTATGGACATTTCTATACT-3 ) and the antisense primer (5 -ACCATGGCAGCCTTCGTACCTTCTCGTTTC-3 ). Pro 9. In the 3 -flanking region. designed according to the known sequence of the BcDh2 promoter. The tobacco (W38) was transformed with Agrobacterium tumefaciens strain LBA4404 harboring the BcDh2 promoter–GUS fusion constructs by leaf-disk method. adaptermediated PCR (AMPCR) was used for chromosome walking. were used to amply the whole promoter region. crassifolia and its control. BcDh2 promoter–GUS fusions were generated by inserting a 1023 bp fragment (+84 to −939 bp relative to the transcription start site) of the plasmid pT-PBcDh2 into the EcoRI/NcoI site of the binary vector pCAMBIA 1305. hybridized to the BcDh2 gene probe . Shen et al. Two gene-specific primers were designed according to the sequence of the BcDh2 cDNA: GSP1 (5 -GGCTCCAACATGAG GTCCGCCGTATT-3 ) and GSP2: (5 -GTCTTCCTCAATTGGTCACCAATTTGGT-3 ).2.8. Results 3.1 by removing the CaMV 35S-promoter from the original plasmid. was selected for further investigation.8% and lacks of Cys. A slightly modification was presented in the 15 amino acid consensus sequence. Adapter-mediated PCR chromosome walking To isolate the promoter region of BcDh2 gene. and two repeats of Lys-rich consensus 15 amino acid sequence EKKGIMDKIKEKIPG (K-segment) at the C-terminal region. 1996) with some modifications. crassifolia digested with BamHI. as determined by the method of Bradford [12]. GUS activity is expressed as nmol 4-methylumbelliferyl. 3. prehybridization and hybridization were performed as Southern blot analysis described above. Phe and Trp residues. GUS activity measurements Crude extracts prepared from leaves of transgenic tobacco plants were subjected to GUS activity measurement as described by Jefferson et al. Isolation of the dehydrin-like gene BcDh2 by using differential display Differential display performed with 12 primer combinations resulted in the isolation of 12 fragments that presented different expressions between drought-treated B. BclI and XhoII. [11].5%. Construction of chimeric gene and plant transformation The two primers. The full-length cDNA. No typical Kozak’s consensus sequence was found around the translation initiation codon. Southern blot analysis of BcDh2 A single band in each lane. Lys 9. BcDh2 (GenBank accession AY243045).D. a 357 bp fragment that showed significant positive signal in reverse Northern blot. 2.9.-d-glucuronide per minute per mg protein. / Plant Science 166 (2004) 1167–1175 1169 2.Y. Hydrophilicity analysis indicated that the BcDh2 was highly hydrophilic polypeptides. TDEYGNP (Y-segment) in one copy at the N-terminal region. Craterostigma plantagineum and Lycopersicon esculentum (tomato) (Fig. His 10. Its amino acid composition presents a compositional bias for Gly18%.7. 3. a stretch of seven serine residues (S-segment) in the central part. EcoRI. respectively. Characterization of the BcDh2 cDNA and its polypeptide The BcDh2 gene has an open reading frame of 399 bp.3.8%. The BcDh2 contains a short amino acid sequence. Homology comparison on NCBI GenBank database indicated that BcDh2 is a dehydrin-like gene but a new number in dehydrins family. crassifolia. was obtained by using 5 -RACE succeeded with RT-PCR. The genomic DNA of B. dissertation in Peking University. Of these fragments. one polyadenylation signal (AATAAA) was found 100 bp downstream of the translation stop codon [13]. Northern blot analysis Total RNA was separated by 1. 1B).

The translation start codon is indicated in bold. WUN-motif. ELI-box3 and box I are boxed. and an asterisk marks the stop codon. CE3 is shaded. 1. MBS. The numbers indicate the relative positions of the amino acids. . Shen et al. C-repeat. HSE.1170 Y. ERE. P-box. Craterostigma plantagineum (GenBank accession P22239) and Lycopersicon esculentum (GenBank accession P22240). Shaded box indicate identical amino acids and highly conservative replacements. (B) Comparison of deduced amino acid sequence of BcDh2 (GenBank accession AY243045) with dehydrin from Elaeis guineensis (GenBank accession AF236067). (A) The nucleotide sequence of the BcDh2 gene and predicted amino acid sequence. An intron is in small case and found within the coding region. Structure of the BcDh2 gene. The transcription start site is labeled +1. The putative TATA and poly (A) are underlined. The ABRE response elements. / Plant Science 166 (2004) 1167–1175 Fig.

. 3A). Northern analysis of plant treated by abiotic stresses. 8 h (2) and 24 h (3). Fig. As a result. respectively. Expression of BcDh2 gene in response to different stresses 3. EcoRI (B). Southern analysis of genomic DNA from B. equal amounts of total RNA isolated from the materials dehydrated to 70. 58 and 43% of original fresh weight were subjected to Northern blot analysis. 65% (3). crassifolia.Y. 3. crassifolia genome.1. 2. (C) Plants treated were with 50 M ABA for 0 h (1). (A) Plants saturated with deionized water was dehydrated to 70% (2). / Plant Science 166 (2004) 1167–1175 1171 Fig. 2). (Continued ). and control was treated with 2. The expression of BcDh2 gene could also be induced with salinity treatment. while no accumulation of BcDh2 transcripts was detected in control (Fig.5% methanol. (B) Plants were treated with 150 M NaCl for 0 h (1). respectively. 1.4. (Fig.4. Southern blot under high-stringency washing condition indicated that the BcDh2 gene is probably present in a single copy gene in B. 8 h (2) and 24 h (3). When treated with 150 mM NaCl. Total DNA was digested with BglII (A). 58% (4) and 43% (5) of original fresh weight. EcoRV (C) and HindIII (D). the Fig. the BcDh2 transcripts increased within water-stress time course. Shen et al. 3. Drought and salinity stress In order to examine the accumulation of BcDh2 transcripts during water deficit. 65.

0. 3. Low temperature stress and heat treatment The expression of BcDh2 gene in response to cold treatment was relatively weak.1172 Y. there were also two MYB binding sites (MBS) [20]. 30 ◦ C (2). crassifolia were treated with 10% MeJA for 8 h (2) and 24 h (3). 5A shows that the accumulation of BcDh2 gene was observed at 8 h MeJA treatment.3. The transcription start point determined by primer extension was assumed to be 82 bp upstream of the putative translation start codon (data not shown).25 mM or 0. so we wanted to know if the BcDh2 gene responded to wounding signal. Exogenous ABA treatment Fig. 5B shows that the BcDh2 transcript was detected at low concentration of SA (0. crassifolia were treated by low temperature for 0 h (1). −36 bp.5. and control was treated with 2. a CE3 [18] element (GACGcttgtc) at position −75 bp. −128 and −172 bp with substantial homology to ABA responsive elements (ABREs. and were identified by PCR amplification. 1A). Northern blotting analysis of plants treated by extreme temperature stress. can be induced by wounding and jasmonate-treatment.2 kb fragment was produced from restriction fragments generated by digestions with XhoII. however. Northern blotting analysis of plants treated with wounding and defense-response signal molecules. Fig. TACCGACAT) [19] at position −82 bp. Each treatment included 7–9 independent transgenic . The sequence of the BcDh2 promoter has been deposited in GenBank databases under the accession number AY243044. Some cis-acting elements related to abiotic stress response were predicted in the promoter region according to the PLACE [15] and PlantCARE [16] database. a 1. 37 ◦ C (3) and 42 ◦ C (4). WUN-motif (wound-responsive element). for example. 3. 8 h (2) and 24 h (3). PgDhn1 [14]. ERE-element (ethylene responsive element). Besides.6. Our results demonstrated that BcDh2 transcripts accumulated on earlier MeJA treatment.4. Fig. and an AP2-like binding site was also presented.4. Northern analysis was also performed to investigate whether the BcDh2 mRNA was accumulated in response to SA. / Plant Science 166 (2004) 1167–1175 Fig. but no signal was detected at 24 h. MeJA and SA treatment Some dehydrin-like genes. Activity of the BcDh2 promoter in transgenic tobacco Tobacco plants were transformed with the p1305-Pde carrying 954 bp of the BcDh2 promoter region coupled with GUS reporter gene. Since MeJA and SA was a couple of antagonists in the wounding signal pathway. box I (gibberellin-responsive element. while a typical CAAT box was missing (Fig. Fig. (A) B.5% EtOH (1). The accumulation of BcDh2 transcripts was sensitive to different temperature: it increased significantly at 30 ◦ C. A little expression of BcDh2 gene was detected in control because of the induction of 2. respectively. while declined sharply at 37 ◦ C. which were moderate and extreme heat treatment. For the control.5% methanol served as a solvent for ABA in the medium. 4. heat shock element (HSE. TCA-element (salicylic acid responsive element). (B) Plants were treated at 22 ◦ C (1). Shen et al. accumulation of BcDh2 transcripts increased significantly after 8 h.1 mM) rather than at higher concentration (0. Therefore. and got the lowest level at 42 ◦ C among these three treatments (Fig.5 mM (4). plants were grown at a normal condition of 22 ◦ C. plants were grown at 30. There were three sequence motifs (with ACGT as core sequence) at positions −107. 3B). and increased a little after long exposure in low temperature up to 24 h (Fig. For heat treatment. Its accumulation could be detected after 8-h cold treatment in 4 ◦ C.25 mM (3) and 0. ELI-box3 (elicitor-responsive element). 4B). Several motifs corresponding to known regulatory signals of eucaryotic gene expression presented further 5 of transcription start site: Two TATA box motifs were found at −30. Cloning and sequence analysis of the BcDh2 promoter The adaptor-mediated PCR method was applied to obtain the promoter of BcDh2.1 mM (2). 37 and 42 ◦ C for 6 h. It could be clearly seen that the transcription level had an intention of decrease within the time course of treatment. respectively. the steady-state level of BcDh2 transcription was up-regulated by the moderate heat treatment but a little at extreme heat temperature. 3C shows the accumulation of BcDh2 transcripts when treated with 50 M exogenous ABA at 8 and 24 h.4. declined after 24 h (Fig. (B) Plants were treated with different concentration of salicylic acid for 24 h: control (1).2.5 mM). 4A). 3. TATC) and P-box (gibberellin-responsive element) predicted in the BcDh2 promoter region. 3. PyACGTGGC) [17].4. 5. (A) B. After the secondary amplification. several poly (A) boxes [21]. aA/T/GAAAt/a/ctta). 3. 0. a DRE/C-repeat (drought-responsive element.

while the acidic dehydrins are mainly cold-responsive proteins [22. The sporamine gene in leaves and stems of sweet potato may be activated by MeJA and JA. thaliana revealed that more than fifty defense-related genes are co-induced by SA and JA. 6. the BcDh2 gene is induced strongly by drought stress rather than cold stress. / Plant Science 166 (2004) 1167–1175 1173 Fig. transcripts for JA-responsible and biosynthesis genes were prominent. Being a kind of basic protein. JA and ABA play a key role in separate signal transduction pathways [36]. because the concentration of NaCl used was 150 mM. Moreover. SA. suggesting that the two signals coordinately regulate these genes [43]. but not during cold treatment. but 37 ◦ C or 42 ◦ C is extreme temperature. The existence of more than two ABREs at appropriated positions in promoter regions is sufficient for ABA-inducible transcription. Assay of GUS activity in transgenic tobacco plants containing the whole promoter of BcDh2 under exogenous ABA. which are known to be involved in wounding and defense response. which have been observed in the promoter regions of some drought. Other explanation for BcDh2 gene’s slight response to cold is that osmotic signals are membrane proteins. nuclear run-on transcription experiments showed that a number of cold-regulated genes were controlled primarily at the post-transcriptional level [25]. such as Dc3 [34] and PgDhn1 [14]. Microarray analysis in defense-inducing treated A. tobacco plants that were subjected to GUS activity measurement. which mediates rapid physiological response by causing closure of stomata. the transcripts accumulation of BcDh2 respond to MeJA and SA was relatively low. In conclusion. especially to SA. and in addition.23]. cold. the GUS activities were significantly above the background of untransformed plants. On the other hand. ABA and MeJA treatment. [27] that the amount of the expression of gene at 6 h is higher than at that 24 h in the microarray analysis of drought. Some results indicated that ABA usually does not strongly induce systemic wound-response genes. and acts as one of the endogenous wound-induced signals involved in regulating the expressions of several defense genes. the ABA-induced gene’s transcripts were found to be up-regulated [27]. basic dehydrins are highly expressed during dehydration. The expression of BcDh2 gene didn’t increase any more during salinity treatment time course. the expression of BcDh2 was induced faintly by cold stress according to the GUS activity assay and Northern blot analysis. Similar results have been demonstrated by Ozturk et al. an inhibitor of the octadecanoid signal transduction pathway can down-regulated those wound-responsible genes [35]. Fig. osmotic signals were impaired.30]. The number of Lysine-rich repeats is linearly related to the response to low temperature [26]. These results implied that BcDh2 might not be a major factor in wounding-defense in B. Temperature 30 ◦ C is a moderate heat stress temperature. For all these treatments. while the BcDh2 mRNA accumulated strongly at 30 ◦ C. at the 10 h time point. JA rapidly accumulates in leaves and stems via the octaderanoid pathway [37–39]. cold. and BcDh2 has only two of such repeats. [28] thought that higher efficiency of osmotic stress gene was the result of increased enzymatic activity of a rate-limiting signal protein. which plays an important role in vivo of signal transduction pathways in response to abiotic stress [29. Discussion In the BcDh2 promoter region. compared to other primary wounding signals. heat and MeJA treatment. At extreme temperatures. some results indicate that SA and JA act synergistically to induce tobacco PR16 expression [42].or salinity-treated barley. which did not severely affect the plants. Some ABA-inducible genes are also sensitive to JA and MeJA. drought. Shen et al. Xiong et al. and also induces dehydrins transcription [31]. our results indicate that BcDh2 is not only responsive to stress signals. However. which was similar to PgDhn1 [14]. but also to defense signals.33]. . such as systemin and JA [40]. The expression of BcDh2 reached a higher level after 8 h of treatment. Some dehydrin-like genes are not induced by heat shock.Y. an ABA-responsive complex is also important for high level of ABA induction [32. heat stress. Thus. The endogenous ABA content increases when plants perceive dehydration stress. Although several predicted WUN-motif and SA-responsive elements presented in the promoter region of BcDh2 gene. crassifolia. 4. In spite of the existence of several DRE and ABRE elements in the BcDh2 promoter.and ABA-responsive genes. After wounding. Most of the stress-responsive genes have also been found to be induced by exogenous application of ABA. These results confirmed that the BcDh2 promoter could be induced by drought. several putative cis-acting elements are present. and plays a vital role in both systemic acquired resistance signaling and disease resistance. but be down-regulated by SA [41]. Among the dehydrins family. which could be impaired directly by low temperature or indirectly by the physical and chemical changes in the membrane lipids that occur at low temperature [24]. 6 shows the statistical data of GUS activities under various stresses. The microarray analysis of drought-treated barley showed that at the 6 h time point.

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