overexpression of maize zmdbp3 enhances tolerance to drought and cold stress in transgenic...
TRANSCRIPT
Biologia 64/6: 1108—1114, 2009Section Cellular and Molecular BiologyDOI: 10.2478/s11756-009-0198-0
Overexpression of maize ZmDBP3 enhances tolerance to droughtand cold stress in transgenic Arabidopsis plants
Chang-TaoWang & Yin-Mao Dong*
Beijing Key Lab of Plant Resources Research and Development, Beijing Technology and Business University, Beijing 100037,People’s Republic of China; e-mail: [email protected]
Abstract: C-repeat/dehydration-responsive element binding factors (CBF/DREBs), belonging to the AP2/ERF super-family, play a vital regulatory role in abiotic stress responses in plants. The ZmDBP3 gene, a member of the A-1 subgroupof the CBF/DREB subfamily, was isolated from maize seedlings. The predicted ZmDBP3 protein contained a putativenuclear localization signal and an activation region. As a trans-acting factor, the ZmDBP3 protein accumulated in thenucleus in a subcellular localization assay, and activated CRT/DRE-containing genes under normal growth conditions intransgenic Arabidopsis plants. ZmDBP3 transcription was highly activated by cold and moderately by salt. Overexpressionof ZmDBP3 improved drought and cold stress tolerance in transgenic Arabidopsis plants. These results suggested thatZmDBP3 produces a CRT/DRE-binding transcription factor and may have an important role in improving drought andcold tolerance in plants.
Key words: transcription factor; CBF/DREB; abiotic stress; induction kinetics; transgenic plants; maize.
Abbreviations: ABA, abscisic acid; COR/RD, cold-regulated/responsive to dehydration; CBF/DREBs, C-repeat/dehydration-responsive element binding factors; CRT/DREs, C-repeat/dehydration-responsive elements; DREB, DRE binding protein;EST, expressed sequence tag; NLS, nuclear localization signal; RACE, rapid amplification of cDNA ends.
Introduction
Environmental stresses, including drought, salt andcold, influence the growth of plants and the productiv-ity of crops. Plants are thought to have common mech-anisms for adaptation to these stresses. On exposure tocontinuously changing stress surroundings, plants pro-duce various biochemical and physiological responses toacquire stress tolerance (Yamaguchi-Shinozaki & Shi-nozaki 2006). Transcription factors interact with cis-elements present in the promoter region of variousstress-related genes and thus up-regulate the expres-sion of many genes resulting in imparting tolerance tostresses (Xu et al. 2008a). Thus, transcription factorsare powerful tools for genetic engineering as their over-expression can lead to the up-regulation of a whole ar-ray of genes under their control (Agarwal et al. 2006).CBF/DREBs are key regulatory factors that func-
tion primarily in stress tolerance by activating a batteryof target genes with C-repeat/dehydration-responsiveelements (CRT/DREs) in their promoter regions, suchas rd29A, cor15A, cor6.6 and cor47 (Stockinger etal. 1997; Liu et al. 1998; Oh et al. 2007). Thefirst isolated cDNAs encoding DRE-binding proteinswere CBF1 (Stockinger et al. 1997), DREB1A andDREB2A (Liu et al. 1998) from Arabidopsis. Since
then, a variety of CBF/DREBs genes were success-fully identified and investigated in species. Arabidop-sis CBF1/DREB1B was induced by low temperatureand its overexpression was found to activate down-stream cold-responsive genes and improved freezing tol-erance in plants (Jaglo-Ottosen et al. 1998). Arabidop-sis DREB1A/CBF3 and rice OsDREB1A conferreddrought and freezing tolerance in transgenic Arabidop-sis plants (Dubouzet et al. 2003). Recently, it was re-ported that overexpression of maize ZmDREB2A andwheat TaAIDFa resulted in increased thermotoleranceand/or drought tolerance in plants (Qin et al. 2007;Xu et al. 2008b). Similar responses in tolerance tohigh salinity were observed in transgenic plants over-expressing Physcomitrella patens PpDBF1, chickpeaCAP2 and barley HvDREB1 (Shukla et al. 2006; Liuet al. 2007; Xu et al. 2009). These results suggest thatCBF/DREBs play a significant role in the stress toler-ance of plants.In maize (Zea mays), an important food and feed
crop in the world, only a few CBF/DREB transcrip-tion factors have been described (Jiang et al. 2004; Qinet al. 2004, 2007). In this paper, we report the cloningand characterization of the ZmDBP3 gene. The expres-sion pattern and subcellular localization of ZmDBP3were investigated. Importantly, overexpression of the
* Corresponding author
c©2009 Institute of Molecular Biology, Slovak Academy of Sciences
Characterization of maize ZmDBP3 gene 1109
ZmDBP3 gene improved drought and cold tolerance intransgenic Arabidopsis plants.
Material and methods
Plant materials and stress treatmentsSeedlings of maize (X178) grown at 25◦C for 10 days weresubjected to various abiotic stresses. Seedlings were exposedto air on filter paper for induction of rapid drought condi-tions, or placed in a 4◦C chamber for cold stress. To mimicsalinity and for abscisic acid (ABA) treatments, seedlingswere transferred into solutions containing 2% NaCl and200 µM ABA, respectively. Materials were collected at 0,1, 2, 5, 12, 24 or 48 h after treatments. Harvested leaveswere dropped immediately into liquid nitrogen and storedat −80◦C for RNA extraction.
Isolation of entire cDNA and genomic sequenceIn order to isolate the genes encoding DREB from maize, theArabidopsis DREB1A/CBF3 sequence (Liu et al. 1998) wasused as a query to search the expressed sequence tag (EST)database of maize (http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/Blast/index.cgi). EST sequences containingAP2 domains were obtained and further systematic phylo-genetic analyses of those sequences were carried out on thebasis of homology of AP2 domains. Using the rapid ampli-fication of cDNA ends (RACE) method, several full-lengthcDNA sequences were isolated from total RNA of maizecv. X178. In order to study the characteristics and func-tions of a member belonging to the A-1 subgroup of theDREB subfamily, ZmDBP3 was chosen for further anal-yses (GenBank Acc. No. FJ805751). RACE was carriedout as described in the Instruction Manual (Rapid Am-plification of cDNA Ends System, Gibco-BRL, Rockville,MD, USA). The full-length cDNA sequence or genomic se-quences of ZmDBP3 was amplified using the specific primerset of (5’-CAAGATGTGTCCGACCAAGAAG-3’) and (5’-CTAGTAGCTCCACAGTGGCACATC-3’).
RT-PCR and quantitative-RT-PCRTotal RNA was extracted from leaves using Trizol Reagents(TianGen, Beijing, China). One-step RT-PCR was per-formed using total RNA as the template according to theinstructions of the manufacturer (TianGen, Beijing, China).RT-PCR of actin was run at the same time to normalize theamount of added template. The amplified products were runon a 1.5% agarose gel and visualized with the Gel Doc EQSystem (Bio-Rad, Richmond, CA, USA).
The quantitative RT-PCR were conducted using theABI Prism 7000 system (Applied Biosystems, USA). Theactin gene was used as an internal reference. The transcriptanalysis of the ZmDBP3 gene was performed using gene-specific primers (5’-CATGAGCTGGGATCTATACTAC-3’and 5’-CAAGGTATCAACGTCCTCA-3’), which were lo-cated in the 3’-untranslated region of the gene. Validationexperiments were performed to demonstrate that amplifica-tion efficiency of the ZmDBP3 specific primers were approx-imately equal to the amplification efficiency of the endoge-nous reference primers. Quantification of the target geneexpression was carried out with comparative CT method(Livak & Schmittgen 2001). Average CT values for the tar-get gene from at least three PCRs were normalized to aver-age CT values for actin from the same cDNA preparationsand analysed using Microsoft Excel.
Subcellular localization analysisThe full-length cDNA fragment containing the coding regionof ZmDBP3 was amplified, and fused to the N-terminus ofthe hGFP gene under control of the 2×CaMV35S promoter.Subcellular localization of the ZmDBP3::hGFP fusion pro-tein and hGFP control in onion epidermal cells was moni-tored using a confocal microscope (Leica Microsystem, Hei-delberg, Germany) 48 h after particle bombardment trans-formation.
Arabidopsis transformation and stress treatmentTo construct an expression vector for Arabidopsis, the full-length ZmDBP3 cDNA was ligated into the modified vec-tor pBI121 under the control of the CaMV35S promoter.Columbia (Col-0) ecotype Arabidopsis plants were trans-formed using the vacuum infiltration method. Transfor-mants were selected on MS medium containing 50 µg/mLkanamycin. T2 generation plants were used for further anal-ysis.
Sixty Arabidopsis plants of two independent linesgrown for 3 weeks at 22◦C were subjected to stress treat-ment. For drought stress treatment, plants were grown at22◦C without watering for 12 days; and then re-water atnormal growing conditions. For cold stress treatment, trans-genic and wild-type Arabidopsis were exposed to −6◦C for12 h, then returned to 22◦C for 2 weeks.
GenBankThe nucleotide sequences reported in this paper was submit-ted to the GenBank database (Benson et al. 2009) under theaccession number FJ805751 (ZmDBP3).
1 gcaaaataccactcgagcacaattcaagcagcagcgaaggtagccacaacatccactctactactcgagagctaagaaatcgttcaacaggtgatccagaacaag106 ATGTGTCCGACCAAGAAGGAGATGAGTGCCGAGTCGTCGGGCTCGGCGAGCAGCTGGACTTCGGCCTCGGCCTCGGCCTCGACCTCGACCTCGCCGGAGCACCAG1 M C P T K K E M S A E S S G S A S S W T S A S A S A S T S T S P E H Q 211 ACCGTGTGGACGTCGCCGCCGAAGCGGCCCGCGGGGCGGACCAAGTTCCGCGAGACGCGGCACCCCGTGTTCCGTGGCGTCCGGCGCCGCGGCAGCGCCGGGCGG36 T V W T S P P K R P A G R T K F R E T R H P V F R G V R R R G S A G R 316 TGGGTGTGCGAGGTGCGCGTGCCCGGGAGGCGCGGGTGCAGGCTCTGGCTCGGCACGTTCGACGCCGCCGAGGCCGCCGCGCGCGCGCACGACGCCGCCATGCTC71 W V C E V R V P G R R G C R L W L G T F D A A E A A A R A H D A A M L 421 GCCATCGCCGGCGCGAGCGCGTGCCTCAACTTCGCCGACTCCGCGTGGCTGCTCGCGGTCCCCGCCTCGTACGCCAGCCTTGCCGAGGTCCGCCGTGCGGTCGCC106 A I A G A S A C L N F A D S A W L L A V P A S Y A S L A E V R R A V A 526 GAGGCCGTGGAGGACTTTCAGCGCCGCGAGGCGGCCGCCGGGGACGACGCGCGCTCGGCCACATCGCCGACGCCGTCCACCTCGGGCACCGACGACGATGCTGCC141 E A V E D F Q R R E A A A G D D A R S A T S P T P S T S G T D D D A A 631 ACTGACGGCGAGGAGTCGTCACCGGCCACGGAGGTCTCGTCGTTCCAGCTCGACGTGTTCGATAACATGAGCTGGGATCTATACTACGCGAGCATGGCGCAGGGA176 T D G E E S S P A T E V S S F Q L D V F D N M S W D L Y Y A S M A Q G 736 ATGCTGATGGAGCTGCCGTCCGCAGTTCCGGCGTTCGGAGACGACGGCTACACCAATGTCGCCGATGTGCCACTGTGGAGCTACTAGaattgaggacgttgatac211 M L M E L P S A V P A F G D D G Y T N V A D V P L W S Y * 841 cttgaatttctcttctttttccagttggctccccatgccaaattttgggactgtagtggtaatgaaaacagagcaagtttggaaaaggaaaacaaaaaaaa
Fig. 1. Nucleotide sequence of the ZmDBP3 gene the deduced amino acid sequence. The AP2/ERF domain is single underlined;the basic amino acids that potentially act as NLS in the N-terminal are shown in boxes; The acidic amino acids that may act as atranscriptional activation domain are shown in italics; and one potential N-linked glycosylation site is signified by broken underline.
1110 C.-T.Wang et al.
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Fig. 2. Sequence analysis of ZmDBP3. (a) Alignment of ZmDBP3 with other closely related CBF/DREB proteins. The conservedAP2/ERF domain is underlined and the N-terminal basic amino acid stretch that may function as a putative NLS is indicatedby dots. Asterisks indicate the conserved V14 and V19/E19. Consensus sequences are exhibited in black. (b) Phylogenetic tree ofZmDBP3 with CBF/DREBs from other plants. Phylogenetic tree was constructed by the MEGA program. The accession number ofeach appended protein is as follows: ZmDREB1A: AF450481; ZmCBF3: AAY33832; HvCBF3: AF298230; ZmDREB2A: NP 001105876;TaAIDFa: AY781361; HvDRF1: AY223807; HvCBF1: AF418204; HvCBF2: AF442489; FaDREB2A: AJ717396; TaDREB1: DQ195068;TaCBF1: AF376136; OsDREB1A: AF300970; ZmDBF1: AAM80486; ZmDBF2: AF493799; ZmABI4: AY125490; GhDBP1: AY174160;GhDREB1L: DQ409060; AtDREB1A: AB007787; AtDREB1B: AB007788; AtDREB1C: AB007789; AtDREB2A: AB007790; Gm-DREBa: AAT12423; GmDREBb: AAQ57226; GmDREBc: AAP83131; PgDREB2A: AAV90624; ScCBF: AAL35759; VrCBF1a:AAR28671; OsDREB2A: AF300971; CaDREBLP1: AY496155; TaDREB6: AAX13289; PeDREB2: ABY19375; ABI4: A0MES8; Gm-DREB2: ABB36645; RAP2.10: Q9SW63; RAP2.1: Q8LC30; AtRAP2: AAP04063; RAP2.4: NP 177931; GhDBP2: AAT39542; Os-DREB3:NP 001048142; GhDBP1L: ABD65473; TINY: Q39127.
Results
Isolation and characterization of ZmDBP3 cDNAIt has been reported that overexpression of the Ara-bidopsis DREB1A/CBF3 gene under the control of the
CaMV35S promoter resulted in strong expression ofdownstream stress-inducible genes and the transgenicplants had acquired higher tolerance to drought, highsalt and freezing stresses (Liu et al. 1998; Kasuga et al.1999; Gilmour et al. 2000; Maruyama et al. 2004; Oh et
Characterization of maize ZmDBP3 gene 1111
ZmDBP3:hGFP
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Fig. 3. Subcellular localization of the ZmDBP3 protein in onion epidermis cells. The fusion construct for ZmDBP3::hGFP and thecontrol plasmid hGFP were introduced into onion epidermis cells by particle bombardment, respectively. The transformed cells werecultured on MS medium at 25◦C for 16 h and observed under a confocal microscope. Photographs were taken in the dark field forgreen fluorescence (a) and (d), in bright light for morphology of the cells (b) and (e), and in combination (c) and (f).
al. 2005). In order to isolate the homologous genes inmaize, the Arabidopsis DREB1A/CBF3 sequence (Liuet al. 1998) was used as a query to search the ESTdatabase of maize. Several assembled sequences wereobtained. Using the RACE method, several full-lengthcDNA sequences were isolated from maize. Of these,ZmDBP3, a homologous gene ofDREB1A/CBF3, waschosen for further analyses.The cDNA sequences ofZmDBP3 comprised a 717
bp ORF encoding a 25.3 kDa protein with pI 4.81(Fig. 1). The deduced amino acid sequence of ZmDBP3contains a highly conserved AP2/ERF domain withtwo conserved amino acids, 14th valine and the 19th
glutamate residues that characterize CBF/DREB pro-teins (Sakuma et al. 2002) (Figs 1 and 2a). Also,ZmDBP3 contain a putative basic amino acid regionthat potentially acts as a nuclear localization sig-nal (NLS), an acidic amino acid region that mightact as a transcriptional activation domain (AD) inthe C-terminal region, and a potential N-linked gly-cosylation site (NXS/T, where X denotes any aminoacid residue) (Fig. 1). Phylogenic analysis further con-firmed that ZmDBP3 belongs to the A-1 subgroup ofthe CBF/DREB subfamily, with the signature motifs(PKK/RPAGRxKFxETRHP and DSAWR/L) directlyflanking the AP2 domain (Jaglo et al. 2001). Amongmembers, ZmDBP3 shared 75, 53 and 41% identitywith OsDREB1A, ZmDREB1A and DREB1A/CBF3proteins, respectively (Fig. 2).
Subcellular localization of ZmDBP3 proteinThe ZmDBP3 protein, with a putative NLS in its N-terminal region, was expected to localize to the nu-cleus (Fig. 1b). To investigate the biological activityof putative NLSs, ZmDBP3 gene was fused to the N-terminus of the hGFP gene, and transferred into onionepidermal cells to investigate intra-cellular localization.The ZmDBP3::hGFP fusion protein was observed tobe mainly localized to the nucleus, whereas the controlhGFP was uniformly distributed throughout the onionepidermal cell. These results suggest that ZmDBP3 is
a nuclear protein, possibly serving as a transcriptionfactor (Fig. 3).
ZmDBP3 is induced by cold and salt stressesTotal RNA was extracted from maize seedlings sub-jected to various abiotic stress conditions, such asdrought, salt, cold and exogenous ABA. Quantita-tive RT-PCR showed thatZmDBP3 transcripts accu-mulated greatly in leaves in response to cold and salt(Fig. 4). Under cold conditions, ZmDBP3 mRNA be-gan to accumulate at 1 h and reached its maximum at12 h after treatment, and last out to 48 h (Fig. 4c). Un-der salt treatment, the expression pattern of ZmDBP3was similar to that with cold treatment, but the initialtime of salt-induced transcription was later (5 h) thanwith cold treatment (Fig. 4b). In contrast to salt andcold, ZmDBP3 transcript did not respond to droughtand exogenous ABA treatments (Fig. 4a,d).
Improved survival rate under drought and cold stressesPrevious studies showed that overexpression ofDREB1A/CBF3 and OsDREB1A genes led to droughtand cold stress tolerance (Liu et al. 1998; Kasuga etal. 1999; Gilmour et al. 2000; Dubouzet et al. 2003;Maruyama et al. 2004; Oh et al. 2005). The ZmDBP3gene under the control of CaMV35S was transformedinto Arabidopsis plants to identify its functions. Five in-dependent transgenic lines were obtained by kanamycinselection. Two lines (TL-1 and TL-2) with high expres-sion levels of ZmDBP3 were chosen for further analy-sis. Overexpression of ZmDBP3 resulted in the accumu-lation of cor15a mRNA with the CRT/DRE elementsin promoter in the transgenic Arabidopsis plants undernormal growth conditions (Fig. 5a).Subsequently, we carried out drought and cold
stress tolerance tests on seedlings of 35S::ZmDBP3transgenic and wild-type Arabidopsis grown for 3 weeksat 22◦C. Under water-free conditions, almost all of wild-type plants were dead after 12 days, whereas 30–33.3%of the transgenic plants survived. Similarly, after expo-sure to −6◦C for 12 h for cold treatment, most of wild-
1112 C.-T.Wang et al.
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Fig. 4.
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Expression patterns of maize ZmDBP3 under different abiotic stress conditions, including drought (a), NaCl (b), cold (c) andABA (d). Total RNA was isolated from leaves of maize seedlings. The actin gene was used as an internal reference.
type plants were dead; on the contrary, 71.6–73.3% ofthe transgenic plants survived (Fig. 5b). These experi-ments indicated that ZmDBP3 bound to CRT/DRE el-ements and activated downstream genes, and enhanceddrought and cold tolerance in Arabidopsis plants.
Discussion
CBF/DREBs were shown to regulate many key func-tions, especially abiotic stress responses in plants. Overthe past decade, A-1 group genes of CBF/DREB fam-ily were extensively investigated and characterized inAradopsis and rice. The expression of AtDREB1A andAtDREB1B is induced by cold, but not by dehydra-tion, or high salt stress (Liu et al. 1998; Shinwari etal. 1998). Similarly, CBF genes also exhibited high ex-pression in response to low temperature (Medina et al.1999). In rice, OsDREB1A and OsDREB1B were in-duced rapidly after cold exposure and did not respondto ABA treatment. In this paper, ZmDBP3 transcrip-tion was highly activated by cold and moderately bysalt (Fig. 4). OsDREB1C showed constitutive expres-sion and the expression ofOsDREB1D was not detectedwith or without any stress (Dubouzet et al. 2003). Hotpepper Ca-DREBLP1, a member of A-1 group genes,was rapidly induced by dehydration, high salinity and,to a lesser extent, by mechanical wounding but not bycold stress (Hong & Kim 2005). This expression patternis quite similar to that of DREB2A. Clearly, A-1 groupgenes exhibit variable expression patterns in differentplant species.
In addition, genetic variation may result from dif-ferences in the regulatory motifs present in promotersin different cultivars (Xu et al. 2008b). Kume et al(2005) found that the A-1 group gene, WCBF2, dis-played differential expression during long-term cold ac-climation in two wheat cultivars in a manner similarto TaDREB1, a DBF2-type gene (Shen et al. 2003).Therefore, more A-1 group genes need to be isolated inorder to assess the CBF/DREB regulation.Overexpression of AtDREB1A and OsDREB1A
in transgenic Arabidopsis resulted in an enhancedfreezing and dehydration tolerance, but showed severegrowth retardation under normal growth conditions(Liu et al. 1998; Kasuga et al. 1999; Dubouzet et al.2003). cDNA microarray analysis of 35S::AtDREB1Aand 35S::OsDREB1A transgenic plants revealed sev-eral genes had two-fold higher expression level thanin the wild-type. These gene products may functionin stress tolerance in plants, including cor15a, rd29A,rd17, kin1, kin2 and erd10 (Liu et al. 1998; Kasuga etal. 1999; Dubouzet et al. 2003). Shen et al (2003) in-dicated that a monocot gene transferred to dicots maynot function effectively as it did in the monocot. How-ever, intact Arabidopsis DREB2A and rice OsDREB2Afrom A-2 group does not activate downstream genes.It was considered to require post-translational modifi-cation for activation (Liu et al. 1998; Dubouzet et al.2003; Sakuma et al. 2006a). Overexpression of ZmDBP3conferred better survival rate to transgenic Arabidop-sis plants under stress conditions as compared to thewild-type plants (Fig. 5). Therefore, the ZmDBP3 gene
Characterization of maize ZmDBP3 gene 1113
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Fig. 5. Effect of ZmDBP3 expression on drought and cold tol-erance in transgenic Arabidopsis plants. (a) Expression pat-terns of ZmDBP3, cor15a and cor6.6 in wild-type (WT) and35S::ZmDBP3 transgenic Arabidopsis plants (lanes TL-1 and TL-2) under normal growing conditions. Parallel reactions using actinin primers were carried out to normalize the amounts of addedtemplate. (b) Survival rates of WT and CaMV35S::ZmDBP3transgenic Arabidopsis lines (TL-1 and TL-2) were estimated af-ter cold stress treatment. Stress treatments were applied to trans-genic Arabidopsis and WT 3-week-old plants under common con-ditions. Results are averages of three replicates ± S.D.
is likely to be important component for abiotic signaltransduction pathways, and encodes a regulator underabiotic stress conditions.
Acknowledgements
This research was financially supported by the Beijing NewStar Project on Science & Technology of China (2008B08).
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Received March 18, 2009Accepted September 15, 2009