Journal of Plant Biology Research 2016 5(2) :48-57
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REGULARARTICLE
Molecular cloning and characterization of a novel SiDREB2L gene encoding DRE-binding transcription factor2 protein from Foxtail millet (Setaria italica. L., Cv. Prasad).
Merum Pandurangaiah, Gunupuru LR, Veeranagamallaiah G, Kiranmai K, Venkatesh B, Ranganayakulu G and Chinta Sudhakar* Plant molecular Biology Laboratory, Department of Botany, Sri Krishnadevaraya University, Anantapuram, Andhra Pradesh, INDIA.
ABSTRACT Dehydration–responsive element biding transcription factor plays important roles in stress signaling responses and in imparting stress endurance in plants. In this study, a DREB2-like transcription factor, SiDREB2L gene has been isolated from the stress tolerant foxtail millet (Setaria italica L) and SiDREB2L ORF is 663bp in length and codes for 221aminoacid length polypeptide with molecular mass of 16.86kDa and pI of 9.74. On the basis of the gene annotation studies, multiple sequence alignment and phylogenetic analysis, the SiDREB2L was classified as an A-2 subfamily member of the DREB family. The deduced amino acid sequence of the SiDREB2L gene showed high sequence similarity with other AtDREB2A (BAA33794.1), OsDREB2A (AFB77198.1), ZmDREB2A (AB218832), SiDREB2 (HQ132744.1), SoDREB2A (JQ736812.1), HcDREB2 (KC203598.1), SbDREB2 (EU500653.1), HvAP2/ERRBP (HQ647359.1), CdDREB2 (AY462118.1) and BdDREB2 (EF512460.1) proteins and also shared sequence similarity with the conserved ERF/AP2 DNA binding domain region of other AP2 DNA domain regions. The SiDREB2L gene expression was significantly increased in leaf tissues due to drought, dehydration, salinity and cold stress conditions. The results indicate that SiDREB2L gene functions as a novel transcription factor and involved in the responses of abiotic stresses. The SiDREB2L gene is one of the potential candidate gene involved in improving stress tolerance of crop plants. Keywords: DREB2 type transcription factors, fox tail millet, In-silico, Gene expression, Setaria italica L., Transcription activation Assay and Abiotic stresses.
INTRODUCTION Many intensive reports of molecular and genomic analyses have proven that there are several transcriptional regulatory pathways involved in stress-responsive gene expression. The cis-acting elements and trans-acting factors are involved in the transcriptional regulation of a signalling network of genes controlling by various biological processes, including abiotic and biotic stress responses [1]. * Corresponding author :
[email protected];
[email protected]
Drought/dehydration responsive element binding (DREB) proteins, one of the subgroups of AP2/EREBP transcription factor family, which plays important roles in plant response and adaptation to abiotic stresses [2, 3]. Dehydration responsive element (DRE) with the core sequence A/GCCGAC and similar elements CRT (C-repeat) were identified as important cis-acting elements in regulating gene expression under abiotic stresses such as
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drought, salinity, and low temperatures in Arabidopsis [4]. In Arabidopsis, 146 AP2/EREBP-related transcription factors reported so far, were classified into five subfamilies, viz., DREB, APETALA2, RAV (related to ABI3/VP1), ERF (ethyleneresponsive transcription factor) and a very specific gene, AL079349, based on the similarities of their DNA-binding domain (AP2 domain) [5]. The DREB subfamily was further divided into 6 subgroups (A-1 to A-6) [2]. Subgroups A-1 and A-2, harbouring the DREB1-type and DREB2-type genes, respectively, are the largest ones that are involved in two ABA-independent pathways [6, 7]. The DREB1-type genes (AtDREB1A, AtDREB1B, and AtDREB1C) were thought to be involved in cold-responsive pathway, whereas DREB2-type genes (AtDREB2A and AtDREB2B) were mainly involved in osmoticresponsive pathway [1]. Foxtail millet (Setaria italica L. Cv. prasad) is one of the important stress tolerant millets [8, 9]. It serves as staple food for majority of the populations in arid and semi-arid regions like Rayalaseema region of Andhra Pradesh, India. In spite of its importance as food crop, understanding of the molecular aspects of this plant’s response to adverse abiotic stresses factors (viz., drought and salt stress) is relatively rudimentary. Although several DREB proteins have been isolated from a wide variety of plants however, only a few reports are available from dry land millets [10, 11, 12, 13]. Here, we report a novel DREB2L homolog from a salt tolerant foxtail millets cultivar Prasad and an endeavour was made to analyse the expression profile of SiDREB2L gene under different stress conditions and its DNA-binding ability were investigated. MATERIALS & METHODS Plant material and growth conditions: Seeds of foxtail millet (Setaria italica. L Cv. Prasad) obtained from Andhra Pradesh Agriculture Experimental Station, Nandhyal, Andhra Pradesh, India. Seeds were surface
sterilized with 0.1% (W/V) sodium hypochlorite solution for 1 min, rinsed three times with autoclaved distilled water and were germinated in plastic pots containing 4kg of soil: manure (3:1) mixture and maintained by watering daily under a natural photoperiod (1012 hours; temperature 28±4°C) in the botanical garden for three weeks. Three week old plants were subjected to water stress by withholding water supply for 5 days. Leaf samples were collected and flash frozen in liquid nitrogen for RNA extraction. cDNA from the stressed samples were used for PCR amplification of the DREB2L gene. For quantitative RT-PCR analysis, three week old foxtail millet seedlings were exposed to four abiotic stressors which included drought, dehydration, salt and cold. For salt stress treatment, the plants were up-rooted carefully and placed in a 250mM salt (NaCl) solution with external aeration. Dehydration stress was imposed by up-rooting the plants from pots and placing them on filter paper under room conditions (air-dry) for different time intervals. For drought treatment, plants were subjected to water stress conditions by withholding water supply for 5 days. Afterwards, the plants were supplied with water to reach soil moisture levels (SML) to 25%. SML was regularly measured by standardised gravimetric method until drought symptoms appeared on the plants [14]. For cold treatment, plants were kept at 10°C in a growth cabinet and leaf samples were collected at different time intervals. Samples were collected after 0, 3, 6, 12 and 24 hours post stress treatments. The leaf samples were harvested, flash frozen in liquid N2 and stored at -80°C prior to RNA extraction. In parallel, a control set of plants were also maintained for all the experiments. RNA extraction and cDNA synthesis Total RNA from leaf samples was extracted using the RNeasy plant kit (Qiagen, USA) according to the manufacturer’s instructions. DNase treatment of extracted total RNA was
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performed using the TURBO DNA-free TM kit (Ambion Inc., USA). Concentration of total RNA was quantified with UV-vis spectrophotometer (Shimadzu Japan) and visually assessed via gel electrophoresis (1.2% (w/v) agarose gel). First strand cDNA was synthesized in 12.5 µL reaction volume from 1 μg of total RNA, oligo-dT primer (Invitrogen, USA), MMLV reverse transcriptase (Invitrogen, USA) according to the manufacturer’s protocol. The cDNA was diluted to 50µL and stored in -20°C until further use. Cloning of SiDREB2L gene: Degenerative primers to amplify SiDREB2L gene were designed from the consensus amino acids sequences of full length homologous protein sequences available from NCBI database. Putative homologous consensus region of SiDREB2L from foxtail millet was PCR amplified from cDNA. PCR was performed using Invitrogen Taq™ (Invitrogen, USA), and the reaction conditions were as follows: 30 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 45 s, and extension at 72°C for 1 min. An initial denaturation step of 5 min at 95°C and a final elongation step at 72°C for 10 min were also performed. The amplified product was checked on 1% (w/v) agarose gel. Amplicon was purified using QIA quick Gel Extraction Kit (Qiagen, USA) and ligated in pTZ57R/T (Thermo scientific, USA). Positive colonies were sequenced at Eurofins Genomics, Bangalore, India. Gene expression analysis by qRT-PCR in response to various abiotic stress: Quantitative RT-PCR analysis was used to analyse SiDREB2L gene expression under various abiotic stress conditions. Real time primers were designed based on the obtained full length sequence using IDT primer designing tool. The cDNA from various stress conditions was used as template for qRT-PCR analysis using SiDREB2L gene specific primers (IDT Primer designing tool) forward 5’AAGGCACGTGTCAACTTTCC-3’, reverse 5’CACCCCAGTCTCCACAGATT- 3’, and SiActin7 as housekeeping gene, forward 5’GAACCCCAAGGCTAACAG-3’, reverse 5’CAGTGGTGGTGAAGGAGTA -3’. qRT-PCR was carried in StepOne RT-PCR machine (Applied Biosystems, USA) with following thermal cycling parameters :step 1: 95°C, 5 min, 1 cycle; step 2:
95°C, 1 min; 57°C, 45 sec; 72°C, 2 min for 40 cycles. The final extension was carried at 72°C for 10 min, 1 cycle. After 40 cycles, the specificity of the amplifications was checked by heating from 60°C to 95°C with a ramp speed of 1.9°C minute-1, resulting in melting curves. Triplicate (three biological replica) measurements were carried out to determine the mRNA abundance of each gene in each sample. Data analysis was performed using SDS 2.2.1 software (Applied Biosystems, USA). All quantitative RT-PCR analyses were conducted in triplicates. The threshold cycle (CT) values obtained by real-time RT-PCR were used to calculate the relative gene expression using the formula 2^-(CT target gene – CT housekeeping gene), as described [15] previously. Subcellular localization of SiDREB2L protein:
To determine the subcellular localization of SiDREB2L, the DREB2L coding region was fused to YFP under the control of 35S promoter [16]. The fusion plasmid (pAM-PAT-35sSiDREB2L-YFP) and the control vector (pAMPAT-35s-YFP) were introduced into Agrobacterium tumefaciens stain AGL-2 by electroporation. The Agrobacterium tumefaciens carrying YFP or SiDREB2L-YFP were transiently transformed into leaves of 4week-old Nicotiana benthamiana by syringe infiltration. Epidermal cells of N. benthamiana leaves expressing transgenic vectors were assayed for fluorescence at 24 hours post infiltration. YFP excitation was performed at 515 nm and emission detected in the range of 525-600 nm using an Olympus fluoview FV1000 fluorescent microscope. Transactivation analysis of SiDREB2L using yeast one-hybrid analysis: The binding specificity and transactivation activity of the SiDREB2L protein was investigated in yeast (Saccharomyces cerevisiae) strain AH109. Full length SiDREB2L was amplified with primers having EcoRI and BamHI sites. The product was cloned into yeast expression vector pGBKT7 (pBD) which having the reporter genes, His-tag and LacZ. pGBKT7-DREB2L and pBD were transformed to yeast strain AH109, following methods in clontech yeast protocol hand book. The transformed colonies were verified by Colony PCR for positive colonies. Independent colonies for empty vector (pBD) and
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pBD-DREB2L were taken and used to study transcription activity assay. The yeast strains were streaked on SD/-Trp and SD/-His plates containing 5 mM 3-amino-1, 2, 4-triazole (3-AT) for transcription activity assay and on SD/-Trp for X- ἀ -D-Galactoside (X-ἀ-D-Gal) assay to check the reporter gene LacZ activation. The colony lift filter assay was performed with following methods in clontech yeast protocol hand book. The plates were incubated at 30°C for 3 days. Statistical analysis
All the data analyses were conducted using MINITAB 16 (Minitab Ltd., Coventry, UK).Normal distribution of data sets was determined using the Ryan Joiner test [17] within Minitab. Non-normally distributed data sets were transformed to fit a normal distribution using the Johnson transformation [17] within Minitab and the statistical significance of differences was assessed using one-way analysis of variance incorporating Tukey’s test (P < 0.05). RESULTS Characterization of SiDREB2L from Setaria italica Foxtail millet SiDREB2L gene was PCR amplified from a three week old Setaria italica seedlings subjected to drought for 12hrs. The ORF of Setaria italica is 660bp in length (Fig.1). The ORF codes for a polypeptide of 220 amino acids in length with a predicted molecular mass of 23.7 KDa and predicted iso-electrical point (pI) of 9.69. Protein blast analysis shows the encoded protein aligned with AP2/ERF DNA binding domain against various AP2/ERF proteins, suggesting the isolated gene encodes a protein which is a DREB2 type protein in poaceae family. The isolated cDNA product is designated as SiDREB2L based on the sequence similarity with other DREB2 homologous genes. Structural and phylogenetic analysis of the SiDREB2L The alignment of the AP2/ERF DNA binding domain against various AP2/ERF proteins suggested that this cDNA encoded DREB2 type proteins in poaceae members. Therefore, the isolated cDNA clone was designated as SiDREB2L. Besides the highly conserved AP2 DNA binding domain,
SiDREB2L contained a typical DREB2-type nuclear localization signal (NLS) consensus from various
Figure 1: SiDREB2L gene was amplified from drought stressed leaf samples. The amplified product was run on 1% agarose gel and visualized by ethidium bromide stain. M: DNA ladder (1kb, Thermo scientific). 1: SiDREB2L PCR product (660bp).
crop species. Multiple sequence alignment and phylogenetic analysis showed that SiDREB2L shared high similarity with other DREB2-related proteins such as AtDREB2A (BAA33794.1), OsDREB2A (AFB77198.1), ZmDREB2A (AB218832), SiDREB2 (HQ132744.1), SoDREB2A (JQ736812.1), HcDREB2 (KC203598.1), SbDREB2 (EU500653.1), HvAP2/ERF (HQ647359.1), CdDREB2 (AY462118.1) and BdDREB2 (EF512460.1) in public databases (Fig. 2). Phylogenetic analysis indicated that SiDREB2L belongs to the A-2 group, according to the classification of AP2/ERF transcription factors in Arabidopsis (Fig. 3). Analysis of SiDREB2L expression by qRT-PCR in stressed seedlings
Quantitative real time-PCR analysis showed that SiDREB2L was highly expressed in stressed leaf samples, with the highest gene upregulation in drought and dehydration stress conditions. In general, the SiDREB2L expression was up-regulated in leaves by all the four stresses tested (Fig. 4). The SiDREB2L transcripts highly accumulated and reached its maximum level about 9.8 fold by drought and 7.6 fold by dehydration stress. Salt stress and cold stress resulted an increase the transcript levels of SiDREB2L by 5.7 fold and 3.9 fold respectively. These qRT-PCR results strongly
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Figure. 2 Multiple sequence alignment of SiDREB2L protein with other DREB2 group proteins.
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AP2 domain
AP5 domain AP3 domain AP6 domain AP4 domain
AP1 domain
Figure. 3 Phylogenetic relationship of SiDREB2L protein. Phylogenetic tree was derived for SiDREB2L and AP2/EREBP group family protein sequences using the programme ClustalW2 tool. The SiDREB2L shows high homology with A1domain proteins are AtDREB1A (NP_567720.1),OsDREB1A(AEW67332.1),OsDREB1B(AAN02488.1),FaDREB1A(CAG30550.1),NtDREB1A (ABD65969.1),CiDREB1A(ABM21468.1),CbDREB1A(ABM21468.1),AtDREB1B(NP_567721.1),CiDREB1B( AHI59151.1),BjDREB1B(ABX00639.1),CmDREB1B(ABD90467.1),AtDREB1C(NP_567719.1),LiDREB1C(A ET87124.1);A2domainproteins:AtDREB2A(BAA33794.1),EaDREB2A(AAS58438.1),SbDREB2A(ADE35085. 1),NbDREB2A(CBI83762.1),PsDREB2A(ADL27988.1),VuDREB2A(AEY75222.1),CaDREB2A(ABB05044.1) ,MtDREB2A(ABJ88942.1),OsDREB2A(AFB77198.1),CvDREB2A(ABR23508.1),AtDREB2B(BAA33795.1), MnDREB2B(AHJ25989.1);AP3domainproteins:AtABI4(NP_181551.1), ZmABI4(AAM95247.1), PpABI4 (AFN25691.1); AP4 domain proteins: AtTINY (CAA64359.1), GmTINY (ACP40513.1), AvTINY (ACI95260.1), ZmTINY (NP_001147298.1), MtTINY (XP_003630740.1), AhTINY (CAW30742.1), RcTINY (XP_002516502.1); AP5 domain proteins: AtRAP2 (AAP04063.1), OsRAP2 (NP_001173158.1), (AHA84148.1), GmRAP2 (XP_003526587.1), CsRAP2 (XP_006472629.1), CaRAP2 (XP_004514265.1); AP6 domain proteins: AtERF1 (AAD03545.1), NtERF1 (BAA07321.1), CaERF (ADZ55304.1), AdERF10 (ADJ67439.1), MnERF1B (EXB54035.1), CaERF1B (XP_004497839.1), MtERF (XP_003590048.1) and cluster with other DREB2s.
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Drought stress
Dehydration stress
Salt stress
Cold stress
Figure. 4 Gene expression of SiDREB2L under various abiotic stresses
indicate that SiDREB2L gene is differentially regulated under various abiotic stress conditions. Transactivation analysis and subcellular localization of SiDREB2L protein In order to determine whether the SiDREB2L gene encodes a protein which has a potential transcription activator function, we used yeast expression system (Saccharomyces cerevisiae) to study its role. The ORF of SiDREB2L was fused to the DNA binding domain of the yeast transcriptional activator GAL4 (GAL4-BD) in the vector pGBKT7 to identify transcription activation activity. pBD-DREB and pBD (negative control) were transferred to yeast strain AH109. The yeast cells transformed with pBD-DREB grew well in SD/-Trp and SD/-His plates containing 5 mM 3-amino-1, 2, 4-triazole (3AT) but the yeast cells transformed with pBD could survive in SD/-Trp medium only. This result indicates that the His reporter gene was activated with the presence of SiDREB2L protein. The colony lift assay results indicate the activation of Lac-Z reporter gene which turns X-ἀ-D-Gal blue in colour in the presence of SiDREB2L protein (Fig. 5). These results strongly suggest that SiDREB2L acts as a potential transcription activator. In plants transcription factor family members of AP2/ERF have been shown to localise in the nucleus of rice
protoplasts [18] and in onion epidermal cells [19]. Subcellular localisation of SiDREB2L protein was analysed by transient expression of YFP fusion protein expressed in tobacco leaves (Fig. 6). The control tobacco leaves expressing YFP protein was distributed in both the nucleus and cytoplasm of tobacco cells, the tobacco cells which are expressing YFP- SiDREB2L were only detected in the nucleus (Fig. 6).
DISCUSSION Plant transcription factor genes play an important role in translating the environmental stress signals into changes in gene expression levels of plants under stressful conditions. So far, huge work on plant transcription factor family genes regulating abiotic stress responses has taken place but mainly focused on single TFs and their isolated functions as hubs, which have many partner proteins, in dynamic networks and share cross-talk between different pathways [2, 3]. Dehydration-responsive element binding gene products are found to be involved in gene expression regulation of many stress-related genes besides the desiccation and low temperature responsive genes. More than 40 genes downstream of CBF/DREB have been identified through the use of both cDNA and GeneChip microarrays [3] and many of their protein products,
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SD/Trp
SD/-His
pBD-DREB
pBD
pBD-DREB
pBD
pBD-DREB
pBD
Figure. 5 Transactivation activity of SiDREB2L protein in Yeast. Transactivation activity analysis of SiDREB2L was performed using yeast strain AH109. The transformants pBD-DREB and pBD (pGBKT7) were streaked on SD/-Trp and SD/-His plates containing 5 mM 3-amino-1, 2, 4-triazole (3-AT). Colony lift filter assay suggests the activation of LacZ gene.
Fluorescence
Bright field
Merge
A SiDREB2L -YFP
B YFP
Figure. 6 Subcellular localization analysis of SiDREB2L protein. A] YFP and SiDREB2L, B] YFP fusion protein were transiently expressed under the control of the CaMV 35S promoter in N. benthamiana leaves and observed with a confocal microscope. The images were presented fluorescence, bright field and merge of bright field and fluorescence. The bars represents 10µm.
include RNA-binding proteins, sugar transport proteins, carbohydrate metabolism-related proteins, LEA proteins, KIN (cold-inducible) proteins, osmoprotectant biosynthesis proteins, and protease inhibitors, function against stresses and are probably responsible for the stress tolerance of plants [20]. In this research paper, a SiDREB2L, encoding a DREB2-type transcription factor from a stress tolerant foxtail millet cultivar under
desiccation stress conditions was isolated to elucidate its role in drought tolerance mechanism. Gene annotation results demonstrated that the SiDREB2L gene shares identity with the DRE-ciselement in a sequence specific manner (Fig. 2). Similar results were also observed for AtDREB2A, ZmDREB2A and OsDREB2A proteins [2, 13, 21]. These results clearly state that some A-2 group members are DRE-binding proteins, and to further
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support the hypothesis that all 6 sub-groups of the AP2/ERF family may bind to DRE-related sequences, as proposed by Sukuma et al 2002 [2]. Using the substitution method Sukuma and colleagues constructed artificial mutant proteins with residue substitution as V14 and E19 in the AP2/ERF domain of DREB1/2 proteins and demonstrated that valine at position 14 is necessary for the interaction of DREB type proteins with DRE-cis-element, while the amino acid in position 19 could vary slightly [2]. The amino acids in positions 14 and 19 of the AP2/ERF domains of SiDREB2L with other A-2 group proteins are valine (V) and alanine (A), respectively (Figure 3). Finally, these results demonstrated that this SiDREB2L protein can bind to the DRE element (Figure 5). The results for the direct plant proteins are consistent with those obtained for the artificial mutant DREB proteins. Phylogenetic analysis results suggests that, despite sharing high sequence homology and some similar functions with AtDREB2A, ZmDREB2A, SiDREB2, SoDREB2A, HcDREB2, SbDREB2, HvAP2/ERF, CdDREB2 and BdDREB2 indicates a role distinctive from other such proteins. The results of transactivation and nucleus localization studies also showed that SiDREB2L gene sequence specifically bound to AP2/ERF domains, and suggests that the acidic N-terminus is crucial for the ability of SiDREB2L to act as potential transcriptional activator (Figure 6). Therefore these results support the hypothesis that V14 is more important than E19 for determining the DNA-binding specificity of DREB type transcription factors [2, 21]. The qRT-PCR results revealed that the SiDREB2L gene expression is induced by drought, dehydration, salt and cold treatments (Figure 4), and this results are similar to AtDREB2A, ZmDREB2A, HvDREB1 and OsDREB2A, an A-2 group member in plants, which were also induced by drought, dehydration, salt and cold treatments in plant seedlings [2, 3, 13, 21, 22, 23, 24]. These findings indicate that SiDREB2L is a one member of DREB2 sub family in millets like Setaria italica. As SiDREB2L showed a close similarity with other DREB2 genes and highly responsive to drought, dehydration, Salt and cold stress conditions, therefore this gene could be an important member of the DREB2 protein family and acts as a transcription activator that may play a
distinct role in plant responses to environmental stresses.
CONCLUSION ACKNOWLEDGENT This research work was supported in a form of research grant (SR/SO/PS/0001/2011) to CS by Department of Science & Technology, SERB, Govt of India, New Delhi. MP thanks to CSIR EMR-1 for providing Research Associate fellowship (grant no: 09/383/0053/2013) Govt. of India, New Delhi, which is being greatly acknowledged.
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