JXB Advance Access published online on February 13, 2008
Journal of Experimental Botany, doi:10.1093/jxb/erm355
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RESEARCH PAPER |
Molecular cloning and characterization of a salinity stress-induced gene encoding DEAD-box helicase from the halophyte Apocynum venetum
1State Key Laboratory of Crop Biology, College of Agronomy, Shandong Agricultural University, Taian, Shandong 271018, PR China
2Cotton Research Institute, Chinese Academy of Agricultural Sciences, Anyang, Henan, 455100, PR China
* To whom correspondence should be addressed. E-mail: ffshen{at}sdau.edu.cn
Received 19 October 2007; Revised 3 December 2007 Accepted 14 December 2007
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Abstract |
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The genes encoding DEAD-box helicases play a key role in various abiotic stresses, including temperature, light, oxygen, and salt stress. A salt-responsive gene, designated AvDH1, was isolated from the halophyte dogbane (Apocynum venetum) by using suppression subtractive hybridization and RACE (rapid amplification of cDNA ends) PCR. The deduced amino acid sequence has nine conserved helicase motifs of the DEAD-box protein family. The AvDH1 gene is present as a single copy in the dogbane genome. This gene is expressed in response to NaCl and not polyethlene glycol (PEG) nor abscisic acid, and its expression increases with time. The transcription of AvDH1 is also induced by low temperature (4 °C), but its accumulation first increases then decreases with time. The purified recombinant protein contains ATP-dependent DNA helicase activity, ATP-independent RNA helicase activity, and DNA- or RNA-dependent ATPase activity. The ATPase activity of AvDH1 is stimulated more by single-stranded DNA than by double-stranded DNA or RNA. These results suggested that AvDH1 belonging to the DEAD-box helicase family is induced by salinity, functions as a typical helicase to unwind DNA and RNA, and may play an important role in salinity tolerance.
Key words: Apocynum venetum, ATPase, ATP-dependent DNA helicase, ATP-independent RNA helicase, AvDH1, DEAD-box family, salt stress
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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Soil salinity, by inhibiting growth and crop yield, is a severe and increasing constraint on agricultural productivity. The study of plant salt tolerance, with a view to identify and eventually to manipulate the genes involved in salt perception and responses, seems to be a promising approach (Mahajan and Tuteja, 2005; Munns, 2005). To survive salt stress, plants respond and adapt with complex mechanisms, including developmental, morphological, physiological, and biochemical strategies. Due to the large number of genes involved in the response to salt stress, microarrays are increasingly being used to monitor global gene expression changes in Arabidopsis (Kreps et al., 2002), rice (Kawasaki et al., 2001; Chao et al., 2005; Kim et al., 2007), and other systems (Oztur et al., 2002; Wang et al., 2003; Rensink et al., 2005). The products of these salt-responsive genes may provide salt tolerance either directly or indirectly, and ultimately lead to plant adaptation and help the plant to survive and overcome the salt stress condition. Therefore, it is essential to analyse the function of stress-induced genes not only to understand the mechanism of stress tolerance but also to improve the stress tolerance of crops by manipulating such genes (Munns, 2005; Yamaguchi and Blumwald, 2005).
Although the general response to salt stress is similar in all plants, there is a group of plants known as ‘halophytes’ that have evolved unique mechanisms or regulatory pathways that are not found in glycophytes (Wong et al., 2006). The dogbane (Apocynum venetum L.) is native to saline soil of northern China, and is adapted to growth in coastal seawater and the salinity fluctuation resulting from water evaporation and tidal inundation. It grows optimally at 0.2–0.4 M NaCl and requires Na+ for growth (Chen et al., 2007). Although dogbane plants are salt tolerant and can grow in 0.6 M NaCl medium, they do not have salt glands or other morphological alterations either before or after salt adaptation. This suggested that the salt tolerance in dogbane results from mechanisms that are similar to those operating in glycophytes. To elucidate the regulation of salt tolerance in halophyte plants, a large number of NaCl-induced genes from dogbane were analysed by using suppression subtractive hybridization (SSH). The most up-regulated clone S26 of length 976 bp was separated from the subtractive cDNA library. Sequence analysis of the clone revealed that its encoded protein had significant similarity to the DEAD-box helicase.
Helicases are motor proteins that can transiently catalyse the unwinding of energetically stable duplex DNA or RNA molecules by using ATP hydrolysis as the source of energy (Tuteja and Tuteja, 2004a). Biochemical studies and computer analyses have revealed that many DNA and RNA helicases share a core region (
400 amino acids) of highly conserved sequence motifs, and belong to the rapidly growing DEAD-box protein family (Schmid and Linder, 1992; Pause et al., 1993). The important helicase motifs are A/GXXGXGKT, DEAD, SAT, and HRIGRXXR, which have been shown to be responsible, respectively, for initial ATP binding; hydrolysis of ATP; RNA or DNA unwinding; and ATP hydrolysis-dependent RNA or DNA binding (Tuteja and Tuteja, 2004b; Tuteja, 2003; Cordin et al., 2006; Jankowsky and Fairman, 2007). DEAD-box proteins are mainly known as RNA helicases or putative helicases, but some members are also known as DNA helicases (Tuteja and Tuteja, 2004b; Cordin et al., 2006). RNA helicases unfold the secondary structures in RNA and are involved in ribosome biogenesis, RNA splicing, transport, turnover, transcription, translation initiation, RNA interference, RNA editing, and mRNA stabilization and degradation (Cordin et al., 2006), whereas DNA helicases unwind duplex DNA and are involved in replication, repair, recombination, transcription, pre-rRNA processing, and translation initiation (Tuteja and Tuteja, 2004b).
Reports have emerged recently indicating that DEAD-box helicase expression or activity is regulated not only with respect to participation in housekeeping processes such as those mentioned above, but also in response to changes in specific environmental variables, including temperature, light, oxygen, and salt stress (Mahajan and Tuteja, 2005; Owttrim, 2006; Vashisht and Tuteja, 2006). The cyanobacterial, CrhC (Yu and Owttrim, 2000), and fission yeast DEAD-box protein, Ded1 (Liu et al., 2002), respond to cold and oxidative stress, respectively. There is also evidence for the involvement of the DeaD/DeaH-box protein in Desulfovibrio vulgaris Hildenborough surviving in the presence of high concentrations of NaCl (Mukhopadhyay et al., 2006). Increased resistance to several weak organic acids is conferred on Escherichia coli by overexpression of the ATP-dependent helicase RecG from genomic libraries of the acetate-resistant species Acetobacter aceti (Steiner and Sauer, 2003). In sorghum, a salt-responsive transcript HVD1 (Hordeum vulgare DEAD-box protein), encoding a putative ATP-dependent DEAD-box RNA helicase, is induced under salt and cold stress (Nakamura et al., 2004). Two chilling and freezing stress-inducible DEAD-box helicase genes have been identified using Arabidopsis mutants that are impaired in the cold-regulated expression of CBF and their downstream target genes (Gong et al., 2005). Using cDNA microarray analysis of 1300 Arabidopsis genes, a cold stress-inducible DEAD-box helicase gene was identified, but its functions has not yet been defined (Kreps et al., 2002). Pea DNA helicase 45 (PDH45) was found to be induced in pea seedlings in response to high salt (NaCl), dehydration, wounding, and low temperature (Pham et al., 2000). When this gene was transformed to tobacco, it provided high salinity tolerance without affecting yield (Sanan-Mishra et al., 2005). Another gene from Pisum sativum (PDH47, pea DNA helicase 47) was reported to be induced in response to cold and salinity stress in shoots and roots, and heat and abscisic acid (ABA) treatment in roots (Vashisht et al., 2005). These reports suggested that plant helicases might also play an important role in stress tolerance.
Though the involvement of DEAD-box helicase genes in response to stress was reported from plants, only a few stress-induced helicase proteins had been biochemically characterized (Pham et al., 2000; Tuteja and Tuteja, 2004b; Vashisht and Tuteja, 2005, 2006; Vashisht et al., 2005). The exact role of plant DEAD-box helicases largely remains to be elucidated. In the present study, a novel type of salt-responsive gene, termed AvDH1, was cloned from a halophyte dogbane plant. The AvDH1 gene is expressed in a salt-dependent manner and the encoded protein shows striking homology with the DEAD-box helicase. The AvDH1 gene was overexpressed in bacteria and was purified as a 54 kDa protein. Biochemical characterization indicated that AvDH1 exhibits ATP-dependent DNA helicase activity, ATP-independent RNA helicase activity, and DNA- or RNA-dependent ATPase activity. These results showing AvDH1 as a DEAD-box helicase involved in salt stress acclimation provide a unique opportunity to further our understanding of the mechanism by which halophytes respond to salt stress.
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Materials and methods |
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Plant material, growth conditions, and treatments
Seeds of the dogbane (A. venetum L.) (collected from the Yellow River delta, Shandong Province, PR China) were soaked in water at 30 °C for 1 d, for germination. The germinated seeds were transplanted into pots filled with sand and grown in a growth chamber for 21 d with 250 µmol m2 s–1 light intensity and day:night temperatures of 27/19 °C. The seedlings were irrigated at 1 week intervals using Hoagland nutrient solution diluted 1000-fold with water. Cold treatment was conducted by exposure of uniformly developed seedlings at 4 °C for 12 h. For other treatments, uniformly developed seedlings were irrigated with solutions containing the indicated concentrations of NaCl, ABA, and polyethylene glycol (25% PEG was used to simulate low water potential –1.6 MPa) for given time periods (Fig. 3). The plant seedlings were given an entirely new medium without treatments as the control. The plant tissue of treated or control seedlings was harvested by rapid freezing in liquid nitrogen and stored at –8 °C until use.
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PCR-based suppression subtractive hybridization and RACE PCR
Total RNA was extracted using the RNeasy Plant Mini Kit (Qiagen, Fremont, CA, USA). Poly(A)+ RNA was purified from total RNA using the Oligotex-dT 30 super kit (Roche). SSH was carried out using a PCR-Select cDNA Subtraction Kit (Clontech) following the manufacturer's instructions. Aliquots (2.5 µg) of mRNA from control dogbane plants and those treated with 0.6 M NaCl (for 24 h) were prepared as driver and tester RNA samples, respectively. After SSH, the products of PCR were ligated to pGEM-T vector (Promega) for E. coli DH5 competent cell transformation. Positive colonies were picked randomly for plasmid extraction. Northern blot hybridization was carried out to confirm a positive clone for salt induction. Recombinant plasmids were isolated for DNA sequencing using an ABI PRISM 337 DNA Sequencer (Bioasia Co., Shanghai, PR China).
The up-regulated clone S26 of length 976 bp was separated from the subtractive cDNA library. To obtain the 5' and 3' termini of the AvDH1 gene, the 5'- and 3'-rapid amplification of cDNA ends (RACE) was performed using the SMART RACE cDNA Amplification kit (Clontech) following the manufacturer's instructions. An mRNA sample (3 µg) from aerial parts of dogbane treated for 24 h was converted into first-strand cDNA. The gene-specific primer AVPR1 for the antisense strand was designed for 5'-RACE, and the gene-specific primer AVPR2 for the sense strand was used for 3'-RACE (see Supplementary data at JXB online). The sequence of the universal primer for 5'-RACE and 3'-RACE was given in the user manual of the kit. Two gene-specific primers [designed from the 5' and 3'-untranslated region (UTR) of AvDH1 cDNA], AVPR3 (forward), and AVPR4 (reverse), were used to obtain the full-length AvDH1 cDNA. All PCRs were performed using the Advantage 2 PCR enzyme system (TaKaRa, Dalian, PR China) in a TaKaRa PCR Thermal Cycler. The PCR product was subcloned into a pGEM-T Easy vector and sequenced by an ABI PRISM 377 DNA sequencer (Bioasia Co., Shanghai, PR China). A homology search was performed using FASTA, and multiple sequence alignment was done using CLUSTAL W alignment programs.
Southern blot and northern blot analysis
Genomic DNA was isolated from leaves of a 3-week-old dogbane plant. About 20 µg of genomic DNA was digested with EcoRI, HindIII, and SacI, resolved by electrophoresis in a 0.8% agarose gel, and transferred onto a nylon membrane (Amersham-Pharmacia). The filter was probed with the 32P-labelled 3' end portion of AvDH1 cDNA (235 bp long, from 1432 to 1666) or the 32P-labelled conserved region of AvDH1 cDNA (677 bp long, from 336 to 1010). The hybridized membrane was washed with 0.2x SSC (pH 7.2) and 0.1% (w/v) SDS at 60 °C. The hybridization signals were detected by exposing them to film (RX-U, Fuji Film, Tokyo).
Total RNA (30 µg) was run in 1.5% formaldehyde–agarose gels, and blotted onto nylon membranes (Amersham). A 32P-labelled probe of full-length AvDH1 cDNA was prepared using the Megaprime DNA labelling system (Amersham) according to the manufacturer's instructions. The membrane was hybridized with the probe in 50% (w/v) formamide, 5x SSC, 50 mM sodium phosphate (pH 7.0), 0.1% (w/v) SDS, 50 µg ml–1 salmon sperm DNA, and 1x Denhardt's at 45 °C for 12 h. The blots were washed at high stringency and exposed to X-ray film under an intensifying screen at 80 °C. RNA samples for each experiment were analysed in at least two independent blots.
Construction, overexpression, and purification of AvDH1 protein
The coding region of the AvDH1 gene was amplified by PCR using the following two primers. AVPR7 (sense) contains a BamHI restriction endonuclease site (underlined) immediately upstream of the ATG translation initiation codon, and AVPR8 (antisense) contains a HindIII restriction endonuclease site (underlined) and 54 bp downstream of the stop codon of the AvDH1 open reading frame (ORF). PCR amplification was performed using Taq DNA polymerase and the purified product was cloned into plasmid pGEM-T (Promega) according to the protocols provided by the supplier. The cDNA of AvDH1 was verified by sequencing and was cloned into pET28a, resulting in the pET28a-AvDH1 plasmid containing the 6xHis-Tag. The pET28a-AvDH1 plasmid was transformed into E. coli BL21 (DE3) pLysS cells. Several clones were selected, and the authenticity of the clones was verified initially by restriction endonuclease mapping based on sites predicted to be present within the sequence and finally by automated DNA sequencing. The transformed cells were cultured at 37 °C in liquid Luria–Bertani (LB) medium (1% Bacto-tryptone, 0.5% yeast extract, and 1% NaCl, pH 7.5) containing 50 µg ml–1 kanamycin, with vigorous shaking. Isopropyl-1-thio-β-D-galactoside (IPTG) was added to the culture to a final concentration of 0.4 mM when the cell density reached an optical density at 600 nm (OD600) of 0.6–0.8. After 4 h of additional incubation at 37 °C, the cells were harvested by centrifuging at 5000 g for 20 min at 4 °C. The pelleted cells were suspended in a binding buffer (50 mM TRIS-HCl, 0.5 mM NaCl, and 1% Triton X-100, pH 8.0). The suspension was sonicated and the lysate was centrifuged at 10 000 g for 10 min at 4 °C. The supernatant was loaded on an Ni-charged His-Bind column according to the instructions provided by Novagen (Madison, WI, USA). The fractions eluted from the column containing the recombinant AvDH1 protein were collected and loaded on a HiLoad 16/60 Superdex 200 column (GE Healthcare, Pollards Wood, UK) equilibrated with the buffer containing 20 mM TRIS-HCl, 150 mM NaCl, pH 7.8. The resulting peak fractions were analysed by SDS–PAGE and used for all analyses. The protein concentration was determined by the Bradford method using bovine serum albumin (BSA; Sigma) as standard.
ATPase assays
ATPase assays were performed by thin-layer chromatographic detection of 
-32P release from [-32P]ATP (Rodriguez and Carrasco, 1993). The standard reaction mixture (20 µl) contained 15 ng of recombinant AvDH1 proteins in a reaction buffer containing 20 mM HEPES–KOH, pH 8.0, 5 mM MgCl2, 200 mM KCl, 1 mM dithiothreitol (DTT), 40 µM ATP, and 2.6 kBq of [-32P]ATP, and 100 ng of yeast RNA, M13 single-stranded DNA, or yeast genome DNA when necessary. The mixture was incubated at 30 °C for 30 min and quenched by addition of 4 µl of 0.1 M EDTA (pH 8.0). An aliquot (2 µl) was spotted onto PEI–cellulose TLC plates (Fisher Scientific) and developed in 0.5 M LiCl/1.0 M formic acid. The sheets were dried and exposed on X-ray film. When needed, the ATP conversion rate was determined by quantifying 32P release using a phosphorimager.
DNA helicase assay
Four oligonucleotides were synthesized and used for the preparation of the helicase substrates. Oligonucleotides were purified by extraction from a 10% denaturing polyacrylamide gel and labelled in a 50 µl reaction mixture containing 10 pmol of oligonucleotide, 1x polynucleotide kinase buffer, 1.85 MBq of [
-32P]ATP, and 10 U of T4 kinase for 30 min at 37 °C. Labelled oligonucleotides were separated from free nucleotides using a G-25 spin column (Amersham). The substrate used in the standard DNA helicase reaction was prepared by annealing the 5' end-labelled 48mer oligonucleotide (DSPR1) to M13mp19 single-stranded DNA. The single-stranded overhang substrates were prepared by annealing either the 30mer labelled oligonucleotide (DSPR2) for the 5' overhang substrate, or the 30mer labelled DSPR3 for the 3' overhang substrate, to the 79mer oligonucleotide (DSPR4). The blunt DNA substrate was prepared by annealing the labelled 30mer oligonucleotide (DSPR3) used for the 3' overhang substrate to the 30mer complementary oligonucleotide (DSPR5). For each substrate, annealing was performed as follows: 3 pmol of labelled oligonucleotide were mixed with 1.5 pmol of non-labelled oligonucleotide or ssM13mp19 in a 30 µl reaction mixture containing 100 mM NaCl, 20 mM HEPES, pH 7.5, and 15 mM MgCl2. Reactions were placed in a heat block at 100 °C and then slowly cooled to room temperature. The annealed substrate was separated from free oligonucleotides using a G-25 spin column (Amersham).
The standard reaction mixture (15 µl) consisted of 20 mM TRIS-HCl (pH 8.0), 8 mM DTT, 2.0 mM MgCl2, 60 mM KCl, 3.0 mM ATP, 4% sucrose, 80 µg µl–1 BSA (Sigma), 1 ng of 32P-labelled helicase substrate (40 pM), and 15 ng of recombinant AvDH1 protein. The reaction mixture was incubated at 37 °C for 30 min and stopped by the addition of 10 µl of a helicase reaction stop buffer (0.3% SDS, 10 mM EDTA, 5% glycerol, and 0.03% bromphenol blue). After further incubation at 37 °C for 5 min, the substrate and product were separated by electrophoresis on a 12% native polyacrylamide gel (mini gel size, 8x10 cm) in TBE buffer [89 mM Tris borate (pH 8.2) and 2 mM EDTA (pH 8.0)]. As controls, a sample was prepared with a DNA substrate that was heat-denatured by incubating for 10 min at 99 °C and without the addition of AvDH1. After electrophoresis, the gel was exposed to an imaging plate for 2 h, and the bands were detected by a BAS2000 bioimaging analyzer (Fujix, Tokyo, Japan). The DNA unwinding was quantitated by excising the radioactive bands from the gel and counting the radioactivity in a liquid scintillation counter (GMI, USA).
RNA helicase assay
A synthetic double-stranded DNA fragment containing a promoter sequence for T7 RNA polymerase was prepared by annealing 5'-phosphorylated oligonucleotides, RSPR1 and RSPR2, and inserting the duplex between the HindIII and SmaI sites of pUC19 to create pUC-T7p. Similarly, two oligonucleotides, RSPR3 and RSPR4, were phosphorylated, annealed, and inserted between the BamHI and EcoRI sites of pUC19 to create pUC-SP6p, which carries a promoter for SP6 RNA polymerase. pUC-T7p and pUC-SP6p were linearized with EcoRI and HindIII, respectively, and transcribed with T7 or SP6 RNA polymerase (TaKaRa) according to the supplier's instructions. When labelling, 20 mM [-32P]UTP (3700 GBq mmol–1) was included in the reaction. After removal of free nucleotides by passing twice through a G-25 spin column (Amersham), transcripts from pUC-T7p and pUC-SP6p were mixed in 10 mM TRIS-HCl (pH 7.5) and 100 mM NaCl, and incubated sequentially at 80 °C for 10 min and at 45 °C for 3 h to obtain partially double-stranded RNA.
RNA helicase assays were performed in 20 µl reaction mixtures containing 20 mM HEPES–KOH, pH 8.0, 2 mM MgCl2, 1 mM DTT, 1 mM ATP, 10 U of RNasin (Promega), 200 µg ml–1 BSA, 50 fmol of partially duplexed RNA substrate, and 15 ng of recombinant AvDH1 protein, unless otherwise stated. Reactions were quenched, after incubation at 30 °C for 10 min, by addition of 5 µl of a mixture containing 0.1 M TRIS-HCl, pH 7.4, 20 mM EDTA, 0.5% SDS, 0.1% Nonidet P-40, 0.1% xylene cyanol, 50% glycerol, and 0.2 mg ml–1 proteinase K. As controls, a sample was prepared with an RNA substrate that was heat-denatured by incubating for 5 min at 99 °C and without the addition of AvDH1. Reaction products (10 µl) were loaded onto an 8% SDS–polyacrylamide gel and electrophoresed at 100 V. The gel was packed into a hybridization bag and exposed on an imaging plate.
The oligonucleotides used in this experiment are listed in the Supplementary data at JXB online.
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DNA and amino acid sequences of AvDH1
In order to investigate the mechanism of adaptation to salt stress at the molecular level, SSH was carried out using leaves from salt-stressed and untreated control dogbane. The 1864 clones were subjected to reverse northern blotting with labelled tester cDNA (salinity stress). A random selection of 662 positive clones for salt induction was sequenced. An up-regulated clone S26 of length 976 bp was separated from the subtractive cDNA library. Nucleotide sequence analysis of the up-regulated clone revealed that its deduced amino acid sequence shares high sequence homology (73% identity) with DEAD-box RNA helicase cDNAs of Arabidopsis (Aubourg et al., 1999). With RACE PCR techniques, a full-length cDNA, S26 (GenBank accession no. EU145588), was cloned from salt-stressed leaves and renamed as AvDH1 (Apocynum venetum DEAD-box Helicase 1). The sequence analysis shows that it encodes a full-length cDNA of 1738 bp with an ORF of 1341 bp, a 5'-UTR of 76 bp, and a 3'-UTR of 322 bp, including a 21 bp poly(A) tail. A polyadenylation signal sequence (AATAA) of AvDH1 is located 108 bp upstream of the poly(A) tail. The reading frame in this cDNA encodes a 447 amino acid polypeptide having a predicted molecular mass of 50 478 Da and a pI of 7.61.
As shown in Fig. 1, nine conserved amino acid motifs normally found in members of the DEAD-box protein family are all present in the middle region of AvDH1 (amino acids 33–365). In the SAT (Ser-Ala-Thr) motif, however, the first serine residue is changed for threonine in AvDH1. The resulting sequence Thr-Ala-Thr is the same as the consensus motif located at the corresponding position in the DEAD/H family proteins (Koonin, 1991). In the C-terminal region (amino acids 365–447) there are two RGG boxes, a motif involved in RNA binding (Kiledjian and Dreyfuss, 1992), and a nuclear localization signal, KKSRKEKK.
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Comparison of the AvDH1 amino acid sequence with known proteins demonstrates a homology with the DEAD-box family (Fig. 1). The amino acid sequence alignment of AvDH1 revealed that it has the highest sequence identity (74%) with rice DEAD-box ATP-dependent RNA helicase (accession no. NM_001057041), Arabidopsis thaliana RNA helicase (accession no. AJ010456), and Vitis vinifera hypothetical protein (accession no. AM483425). The AvDH1 protein shows a sequence identity of 72% to Nicotiana plumbaginifolia (accession no. P41380 [GenBank] ), 71% to Zea mays (accession no. DQ327709) and Pisum sativum (accession no. Y17186 [GenBank] ), and 65% to Homo sapiens (accession no. NM_014740.2).
Genomic hybridization and copy number of the AvDH1 gene
To estimate the number of AvDH1-related genes in the dogbane genome, genomic DNA was completely digested with EcoRI, HindIII, and SacI, and hybridized with the 32P-labelled 3' end portion of the AvDH1 cDNA sequence generated by RT-PCR under conditions of low (not shown) and high stringency. The probe hybridized to a 6.2 kb HindIII fragment, a 3.4 kb EcoRI fragment, and a 9.8 kb SacI fragment (Fig. 2). There are no recognition sites for EcoRI, HindIII, and SacI in the AvDH1 cDNA from nucleotide sequence analysis. The 3' end portion of the AvDH1 cDNA probe hybridized singly, suggesting that AvDH1 may comprise a single copy number in the dogbane genome. However, extra bands were detected using the conserved (core) region of the AvDH1 cDNA sequence generated by RT-PCR as a probe (Fig. 2), suggesting that somewhat divergent genes exist in the dogbane genome. These results indicate that AvDH1 represents one member of a DEAD-box gene family present in the dogbane genome.
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Tissue distribution and gene expression of AvDH1 under abiotic stresses
Northern blot analysis was performed by using the ORF of AvDH1 cDNA as a hybridization probe to determine in which tissues and organs it is expressed. The results (Fig. 3A) show that a single, strongly hybridizing band of a transcript is expressed in dogbane root, stem, and leaf organs under salinity stress, but not under control conditions after washing the blot at both low stringency (data not shown) and high stringency. These results suggest that the transcription of AvDH1 is salt dependent. Since almost the same amount of the mRNA relative to the total RNA is detected in all the tissues examined, expression of the AvDH1 mRNA is constitutive and the AvDH1 protein seems to play a role in the basic activity of cells under salinity.
To address a potential role for salinity in the regulation of AvDH1 transcription, total RNAs were extracted from aerial parts of 21-d-old seedlings grown in the absence and presence of salinity, and hybridized with the ORF of AvDH1 cDNA probes. As shown in Fig. 3B, the AvDH1 mRNA increases with increasing salinity, but maximal levels were detected in seedlings grown at 0.4 M NaCl; no further increase in AvDH1 transcripts was observed when the NaCl concentration was varied from 0.1 M to 0.6 M NaCl. The rise in AvDH1 mRNA occurred within 1 h, reached a maximum at 8 h, and stayed at a higher transcript level when dogbane seedlings were transferred from control culture medium to 0.4 M NaCl (Fig. 3C).
To determine if expression of the AvDH1 gene is regulated in response to other environmental stresses, 21-d-old dogbane seedlings were exposed to low temperature (4 °C), PEG, and ABA. Control plants were treated in the same manner with water (Fig. 3A). The AvDH1 expression is strongly up-regulated by low temperature, but there is no change in response to water stress due to PEG, in aerial parts of seedlings, compared with non-stressed plants. At low temperature (4 °C), the change in mRNA level occurred within 1 h, reached a maximum at 4 h, declined to a low at 8 h, and then disappeared gradually after 8 h (Fig. 3C). The transcript level is hardly affected after treatment with ABA, suggesting that AvDH1 follows in ABA-independent salt stress signalling networks.
Expression and purification of recombinant AvDH1
To facilitate the biochemical characterization of AvDH1 protein, the AvDH1 ORF was cloned as a translational fusion into E. coli, creating pET28a-AvDH1. Soluble recombinant AvDH1 protein was obtained by IPTG (0.4 mM) induction at 37 °C for 4 h (Fig. 4A). Soluble extract of the cells is able to unwind stable duplex DNA or RNA molecules. No activity was obtained from protein extracts using cells without IPTG induction or cells transformed with the empty vector (data not shown). SDS–PAGE analysis showed a highly expressed 54 kDa additional polypeptide that is induced by IPTG in E. coli transformed with pET28a-AVDH1 (Fig. 4A, lanes 2 and 3), as compared with uninduced cells (Fig. 4A, lane 1). The 54 kDa His-tagged recombinant protein was affinity purified by two chromatographic steps on an Ni-charged His-Bind column (Fig. 4A, lane 3) and gel filtration on a Superdex 200 column (Fig. 4A, lane 4). The protein migrates as a single band on SDS–polyacrylamide gels with an apparent molecular mass of 54 kDa, which agrees with the theoretical molecular mass of 50.5 kDa plus the 3.5 kDa His tag fused with AvDH1.
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Characterization of ATPase activity in the presence of different kinds of nucleic acid species
The ability of AvDH1 to hydrolyse ATP was assayed by incubating purified recombinant AvDH1 with [
-32P]ATP and measuring the release of [-32P]phosphate by TLC. The results indicated that His-tagged AvDH1 protein exhibits a low level of ATPase activity in the absence of RNA or DNA (Fig. 4B, lane 2). The ATPase activity is also Mg2+ dependent. If Mg2+ was omitted from the reaction, the enzyme showed no ATPase activity (Fig. 4B, lane 6). ATPase activity is most active at 29 °C (Fig. 4C), has a pH optimum for activity of 8.0 (Fig. 4D) and a salt optimum of 200 mM KCl (Fig. 4E). To check the effect of different kinds of nucleic acid species on the ATPase activity of AvDH1, these species were included in a standard reaction. The amount of these species used was 100 ng in each case, which allows maximal stimulation of ATPase activity. Under the tested conditions, ATPase activity of AvDH1 is significantly stimulated by the presence of M13 ssDNA (Fig. 4B, lane 5), and shows slight stimulation of ATPase activity in the presence of the same amount of yeast genomic DNA (Fig. 4B, lane 4) or total yeast RNA (Fig. 4B, lane 3), as compared with ssDNA. The ATP hydrolysis activity requires a DNA or RNA cofactor, and the activity is stimulated more by ssDNA compared with dsDNA and RNA. Therefore, AvDH1 shows DNA- or RNA-dependent and Mg2+-dependent ATPase activity.
Characterization of DNA-unwinding activity of AvDH1
The DNA-unwinding activity of AvDH1 was characterized by assaying the displacement of 32P-labelled DNA from a partial duplex DNA substrate. The substrate used for the standard DNA helicase contains hanging tails on both the 5' and 3' ends, as shown in Fig. 5. The purified protein shows DNA-unwinding activity (Fig. 5). The enzyme is heat labile and loses its activity upon heating at 60 °C for 1 min (data not shown). Significant unwinding activity was observed over a broad pH range (pH 6.0–9.0) (Fig. 5A, lanes 3–9) with an optimum near pH 8.0 (lane 7). For this reason, all unwinding assays were performed at pH 8.0. DNA helicase activity is totally dependent upon ATP, as there is no activity in the absence of ATP (Fig. 5B, lane 3). The ATP concentration shows its maximum at 3.0 mM (Fig. 5B, lane 7), while the activity is inhibited at >8 mM (lane 9). The enzyme shows an absolute requirement for Mg2+, as it does not show activity in the absence of Mg2+ (Fig. 5C, lane 3). The optimum concentration of MgCl2 required for the helicase reaction is 2.0 mM (Fig. 5C, lane 6); however, at 10 mM MgCl2, the activity is totally inhibited (Fig. 5C, lane 9). The enzyme shows optimum activity at 60 mm KCl (Fig. 5D, lane 6). At a greater concentration (200 mM) of KCl the activity is inhibited (Fig. 5D, lane 9).
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Next, different DNA templates were tested in order to determine the polarity of translocation along DNA. A 79mer oligonucleotide was synthesized and annealed to two different 5'-radiolabelled oligonucleotides: the first one corresponds to a 30mer oligonucleotide complementary to the 3' part of the 79mer that leads to a DNA substrate with a 49 nucleotide 5' overhang; and the second one corresponds to a 30mer oligonucleotide complementary to the 5' part of the 79mer leading to a DNA substrate with a 49 nucleotide 3' overhang. For the two substrates, equal length complementary oligonucleotides with the same GC content were used in order to get equivalent stability at 37 °C. As shown in Fig. 5E, the AvDH1 protein is able to unwind the two DNA templates. In the presence of a 5' tail, the helicase activity is stimulated and shows more unwinding compared with a 3' tail substrate. The enzyme also fails to unwind blunt-ended duplex DNA (Fig. 5E).
Characterization of RNA-unwinding activity of AvDH1
To determine whether AvDH1 can unwind RNA, an RNA-unwinding activity was analysed using a partially double-stranded RNA as substrate (Fig. 6A). The substrate RNA is stable at 30 °C during the reaction (Fig. 6, lane 1) and heat denaturation produces a faster migrating, single-stranded RNA (Fig. 6, lane 2). The enzyme shows good activity over a broad pH range from 5 to 10 (Fig. 6B, lanes 3–9), with an optimum activity at pH 8 (lane 6). This single-stranded RNA was observed in the presence or absence of ATP (Fig. 6C, lanes 3–8), indicating that RNA-unwinding activity performed by AvDH1 is ATP independent. This RNA unwinding is Mg2+ dependent (Fig. 6D, lanes 3–9), as the protein does not show unwinding activity without Mg2+ (lane 3). The RNA-unwinding activity is optimum at a concentration of 2 mM MgCl2 (lane 6).
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Soil salinity is a major abiotic factor that limits crop productivity in many areas around the world. Salt-inducible genes have been used to increase the salt tolerance of plants by gene transfer (Munns, 2005; Yamaguchi and Blumwald, 2005; Waditee et al., 2007). It has also been suggested that salt could trigger the expression of genes encoding DEAD-box helicases, which play a key role in various abiotic stresses, on the basis of the observation that ectopic expression of PDH45 enhances salt tolerance of transgenic plants (Sanan-Mishra et al., 2005). In this study, a salt-responsive gene cloned from the halophyte dogbane is found to be a DEAD-box protein. Its deduced amino acid sequence has the nine consensus motifs normally found in members of the DEAD-box protein family. The AvDH1 gene is present as a single copy in the dogbane genome. It is a unique helicase specifically up-regulated in response to salt. Its accumulation is also induced by low temperature (4 °C), but there is no change in response to water stress. The present work demonstrated that the purified recombinant protein contains ATP-dependent DNA helicase activity, ATP-independent RNA helicase activity, and DNA- or RNA-dependent ATPase activity. Therefore, AvDH1 is the DEAD-box helicase protein shown actively to unwind DNA and RNA in ‘halophytes’. This gene could be a potential candidate for developing salt stress-tolerant transgenic plants.
AvDH1 is a unique member of the DEAD-box protein family because it contains a striking structural motif TAT instead of SAT, two repeats of ‘RGG’ motifs, and a nuclear localization signal, KKSRKEKK. The serine to threonine modification can be accounted for by a T-to-A transition, from a TCC serine codon to an ACC threonine codon. The resulting sequence Thr-Ala-Thr is the same as the consensus motif located at the corresponding position in the DEAD/H family proteins (Koonin, 1991). In vitro and in vivo mutational analyses of mammalian and yeast eIF-4A have implicated the SAT box in coupling the ATPase and RNA helicase activities (Pause and Sonenberg, 1992; Schmid and Linder, 1992). Crystallographic analysis of the hepatitis C virus RNA helicase suggested that the serine residue of the SAT box is involved in hydrogen bond switching which links the ATPase and helicase activities (Cho et al., 1998; Jankowsky and Fairman, 2007). It had been reported that DEAD-box proteins also contain a modified SAT box, a TAT motif (Fuller-Pace, 1994), a YAT box (Brander et al., 1995), a FAT box (Yu and Owttrim, 2000), and an SRT box (Pham et al., 2000). The replacement of serine with threonine in the SAT motif is conservative, as both amino acids are polar and uncharged. Therefore, the TAT motif instead of SAT does not adversely affect any biochemical activity performed by AvDH1. Structurally, the identified RNA helicases catalysing RNA annealing appear to comprise a unique subclass of SF2 RNA helicases which contain RG-rich C-terminal regions (Yang and Jankowsky, 2005). The involvement of the RGG box in non-sequence-specific RNA binding had been demonstrated for the hnRNP U protein (Kiledjian and Dreyfuss, 1992). The RGG repeat of a spinach DEAD-box protein, PRH75, has also been shown to be capable of binding to RNA (Lorkovic et al., 1997). Five repeats of ‘RGG’ motifs of salt-induced HVD1, a putative ATP-dependent DEAD-box RNA helicase, had also been found in its C-terminal region (Nakamura et al., 2004). Therefore, the specialized functions of AvDH1 may include discriminatory additional RNA-binding motifs that confer substrate specificity. The nuclear localization signal of AvDH1, KKSRKEKK, may direct the DEAD-box protein to a selective subcellular localization.
The involvement of DEAD-box helicase genes in response to abiotic stress has been reported in plants (Mahajan and Tuteja, 2005; Owttrim, 2006). In barley, a salt-responsive transcript HVD1 is induced under salt stress, cold stress, and ABA treatment (Nakamura et al., 2004). PDH45 was found to be induced in pea seedlings in response to high salt, dehydration, wounding, and low temperature (Pham et al., 2000). Another, PDH47, had been reported to be induced in response to cold and salinity stresses, and no significant change in PDH47 mRNA level was observed in response to drought stress (Vashisht and Tuteja, 2005; Vashisht et al., 2005). Obviously, the levels of transcripts of these DEAD-box helicases of glycophytes are regulated by different abiotic stresses, and their expression is part of a general stress response system. In contrast to DEAD-box helicase genes from glycophytes, the AvDH1 transcript is strongly dependent on the salinity of the medium, and increases with time (Fig. 3C). At 0.1 M NaCl, levels of AvDH1 transcript are close to the detection limit. At 0.2 M NaCl, levels increase dramatically, but maximal AvDH1 transcript concentrations are observed at 0.4 M NaCl (Fig. 3B). It should be mentioned that the optimal growth rate is in the same range at NaCl concentrations between 0.2 M and 0.4 M (Chen et al., 2007). In addition, the transcript of AvDH1 is also induced by low temperature (4 °C), but its accumulation first increases then decreases with time (Fig. 3C). The expression of AvDH1 does not change in response to both drought stress and ABA treatment. These results indicate that AvDH1 is not a general stress-induced gene, and induction of the AvDH1 transcript may not be mediated by ABA. Under salt conditions, a single species of AvDH1 mRNA was detected abundantly and constitutively in all the tissues examined (Fig. 3A). It is suggested that AvDH1 plays a role in a basic activity of cells under salt stress. The results further strengthen the fact that AvDH1 is different from other plant stress-induced helicases.
To date, only a few stress-induced helicase proteins have been biochemically characterized in plants (Tuteja and Tuteja, 2004b). The failure to detect helicase activity might be ascribed to the lack of other helper components or a suitable substrate in the reaction. That purified AvDH1 exhibited a strong helicase activity indicates that this protein can unwind duplex DNA or RNA by itself. The AvDH1 protein contains ATP-dependent DNA helicase activity, ATP-independen RNA helicase activity, and DNA- or RNA-dependent ATPase activity, which indicates that this is a novel enzyme functioning specifically in salt stress. This study confirms that AvDH1 is a dual helicase containing both DNA and RNA helicase activities. PDH45 (Pham et al., 2000) and PDH47 (Vashisht et al., 2005) are the best characterized stress-induced helicases containing both DNA and RNA helicase activities. In contrast to the ATP-dependent RNA helicase activities of PDH45 and PDH47, the purified AvDH1 protein possesses an RNA-unwinding activity without ATP. This result indicated that AvDH1 has ATPase-independent RNA helix-destabilizing activity and apparently does not need to interact with other proteins to display RNA-unwinding activity like that of E. coli CsdA (Jones et al., 1996) and yeast Dbp9p (Kikuma et al., 2004). The AvDH1 catalytically requires a fork-like structure for its optimum activity as it has a preference for fork-like structures of the DNA substrate. A 5-tailed fork structure is more stimulatory than a 3-tailed structure (Fig. 5E). Similar results were reported for the pea chloroplast DNA helicase II (Tuteja and Phan, 1998). In contrast, pea chloroplast DNA helicase I (Tuteja et al., 1996), pea nuclear DNA helicases PDH120 (Phan et al., 2003) and PDH45 (Pham et al., 2000), and soybean helicase (Cannon and Heinhorst, 1990) do not require a fork-like structure for optimum activity. The observation that the 3-tailed DNA is a relatively poor substrate compared with the 5-tailed substrate suggested that AvDH1 translocates in the 3' to 5' direction. This property is similar to that of PDH45 (Pham et al., 2000), PDH65 (Tuteja et al., 2001), PDH120 (Phan et al., 2003), and pea chloroplast DNA helicases I and II (Tuteja et al., 1996; Tuteja and Phan, 1998). In contrast, PDH47 contains both the 3'–5' and 5'–3' directional helicase activities (Vashisht and Tuteja, 2005; Vashisht et al., 2005). The observation that AvDH1 exhibits the capacity to hydrolyse ATP is not surprising, as many proteins that possess a helicase core domain previously have been shown to be ATPases (Tuteja and Tuteja, 2004b; Cordin et al., 2006). ATP hydrolysis of AvDH1 is strictly dependent on the presence of RNA or DNA, which is also true for many other DEAD-box proteins characterized so far (Iost et al., 1999; Diges and Uhlenbeck, 2001; Tuteja and Tuteja, 2004b).
What role does AvDH1 perform during adaptation to salt stress in the halophyte A. venetum? The specificity of expression of AvDH1 as a result of salt stress, and the fact that the AvDH1 protein has DNA- and RNA-unwinding activities indicate that AvDH1 may actively unwind secondary structures at the 5' end of target RNAs which will tend to form a double-stranded helix or stem–loop structures under high salt concentrations (Jacobson, 1976). It is RNA secondary structures, such as double-stranded RNA, which inhibit protein synthesis (Baglioni et al., 1978), and degradation of RNAs by RNase (Edy et al., 1976). It was shown that in yeast, factors involved in translation initiation, eIF-4A (Montero-Lomeli et al., 2002) and eIF-1A (Rausell et al., 2003), improved tolerance to lithium and salinity stress, respectively. Increased tolerance to high salinity by overexpressing a helicase gene (PDH45) without affecting yield in tobacco plants suggested that the RNA helicase activity of PDH45 could facilitate transcription by altering the structure of nascent RNA (Sanan-Mishra et al., 2005). The AvDH1 gene is induced by salinity, and purified recombinant protein was found to unwind partial duplex RNA in the absence of ATP. Therefore, its RNA helicase activity probably helps to unwind inhibitory structures in the 5'-UTR of mRNA for efficient translation to take place under salt stress, in turn helping in the synthesis of molecules providing salt tolerance.
In conclusion, a novel DEAD-box helicase gene (AvDH1) has been isolated and several of its important characteristics have been defined, including (i) it is a unique member of the DEAD-box protein family, containing a TAT motif instead of SAT, two repeats of ‘RGG’ motifs, and a nuclear localiztion signal KKSRKEKK; (ii) the gene is expressed in response to NaCl and not PEG or ABA, and its expression increases with time; (iii) AvDH1 contains both ATP-dependent DNA and ATP-independent RNA helicase activities; and (iv) its ATPase activity is DNA or RNA dependent, and stimulated more by ssDNA compared with dsDNA and RNA.
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The supplementary data available at JXB online show the oligonucleotides used in this study.
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This research was supported by the China Key Development Project for Basic Research (973) (grant no. 2007CB116208), and the China Special Program for Research and Industrialization of Transgenic Plant (grant no. JY03-B-05).
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