Appl Biochem Biotechnol DOI 10.1007/s12010-015-1738-4
Expression of Finger Millet EcDehydrin7 in Transgenic Tobacco Confers Tolerance to Drought Stress Rajiv Kumar Singh 1 & Vivek Kumar Singh 1 & Sanagala Raghavendrarao 1 & Mullapudi Lakshmi Venkata Phanindra 1 & K. Venkat Raman 1 & Amolkumar U. Solanke 1 & Polumetla Ananda Kumar 1,2 & Tilak Raj Sharma 1
Received: 5 August 2014 / Accepted: 29 June 2015 # Springer Science+Business Media New York 2015
Abstract One of the critical alarming constraints for agriculture is water scarcity. In the current scenario, global warming due to climate change and unpredictable rainfall, drought is going to be a master player and possess a big threat to stagnating gene pool of staple food crops. So it is necessary to understand the mechanisms that enable the plants to cope with drought stress. In this study, effort was made to prospect the role of EcDehydrin7 protein from normalized cDNA library of drought tolerance finger millet in transgenic tobacco. Biochemical and molecular analyses of T0 transgenic plants were done for stress tolerance. Leaf disc assay, seed germination test, dehydration assay, and chlorophyll estimation showed EcDehydrin7 protein directly link to drought tolerance. Northern and qRT PCR analyses shows relatively high expression of EcDehydrin7 protein compare to wild type. T0 transgenic lines EcDehydrin7(11) and EcDehydrin7(15) shows superior expression among all lines under study. In summary, all results suggest that EcDehydrin7 protein has a remarkable role in drought tolerance and may be used for sustainable crop breeding program in other food crops. Keywords Finger millet . Drought . EcDehydrin7 . Transgenics and qRT PCR
* Polumetla Ananda Kumar [email protected] * Tilak Raj Sharma [email protected] Amolkumar U. Solanke [email protected] 1
National Research Centre on Plant Biotechnology, LBS Building, Pusa Campus, New Delhi 110012, India
2
Present address: Institute of Biotechnology, Acharya N.G. Ranga Agricultural University, Hyderabad 500030, India
Appl Biochem Biotechnol
Introduction Finger millet (Eleusine coracana (L.) Gaertn.) is a hardy cereal known for its great level of tolerance against drought, salinity, and diseases. The seed of finger millet contains valuable amino acid called methionine, which is lacking in the diets of hundreds of millions of the poor who live on starchy staples such as cassava. It is also enriched in calcium, iron, and phosphate. Finger millet is also a popular food among diabetic patients because of its slow digestion. Crop has major role in food, nutrition, and financial security to resource poor farmers and consumers [1–3]. In current situation, climate change has become an alarming issue. Current climate prediction report indicates that average surface temperature will rise by 3–5 °C in the near future, drastically affecting global agricultural systems [4]. This repercussion will lead to increase frequency of drought, flood, and heat waves [5, 6]. Plants being sessile in nature have to face the environmental stress like drought, salinity, high and low temperature, intense light, and excessive ozone or CO2, and in response, it activates a diverse set of physiological, metabolic, and defense systems to adapt and survive the periods of drought stress. The mechanism of tolerance and susceptibility to abiotic stresses is very complex. Abiotic stress possesses great threat to crop loss worldwide, causing average yield losses of more than 50 % for major crops [7–9]. Being outstanding properties, Finger millet crop is tagged as orphan crop because there is meager genetic and genomic resources available till date. This is due to negligence of research, preference of particular food, less interest in scientific community, green revolution’s adverse effect, and government policies. So far, very few numbers of genes/TFs from finger millet have been cloned and characterized. Therefore, with background stated, functional characterization of EcDehydrin7 protein in transgenic tobacco plant was undertaken to characterize its role in drought stress. This gene was identified in our earlier study and was found to be drought and heat responsive [10].
Materials and Methods Plant Sample and Growth Condition Finger millet (Eleusine coracana) cv. MR1 was chosen as an experimental material for analysis. Seeds were sterilized with 5 % teepol detergent for 30 min with gentle shaking, followed by washing under the running tap water. Seeds were then surface sterilized with 4 % sodium hypochlorite solution for 12 min with intermittent shaking, followed by four to six washes with sterile water and transfer of four to five seeds on MS medium. Culture bottles were kept in culture room maintained at 28 ± 1 °C with a 16 h/8 h light and dark cycles for seed germination. Fluorescent tubes of 40W/54 (Philips India Limited, India) were used as source of light, providing light at a fluence rate of 50–100 μmol m−2 s−1.
RACE for Open Reading Frame of Selected Genes In order to get full-length open reading frame (ORF) of selected genes, RACE was performed by SMARTer RACE cDNA Amplification Kit (Clontech USA). Full-length ORF was obtained with the help of gene finding online tool FGENESH (softberry.com). Finally, EcDehydrin7 gene was selected for characterization and validation in tobacco.
Appl Biochem Biotechnol
Sequence Analysis of EcDehydrin7 Gene Nucleotide composition was analyzed using online BioEdit program. The percentage of A, C, G, and T nucleotides of EcDehydrin7 nucleotide sequence was calculated. The amino acid composition, molecular weight, and isoelectric point (PI) of EcDehydrin7 protein were determined with online program provided by ExPASy (http://web.expasy.org/compute_pi/). To understand the evolutionary history of EcDehydrin7, we identified homologs of EcDehydrin7 gene at the genome-wide level and performed evolutionary analysis across distantly related plant. We performed BLASTP search among sequenced genomes of land plants in plant database (www.plantgdb.org) using EcDehydrin7 as queries. Based on homology in the range of 69 to 82 %, 12 genes were selected for investigation. Phylogenetic tree was generated based on protein sequences using the neighbor-joining algorithm of MEGA software version 6, with method p-distance, bootstrap replicates of 1500, and pairwise deletion missing data [11].
Vector Construction and Molecular Cloning Entire coding sequence of EcDehydrin7 protein was isolated from drought tolerance finger millet (Eleucine coracana cv. MR1) by using the primers of 5′-CGGGATCCCACGGAACCA CCGGAACCGG-3′ (BamHI site underlined) and 5′-ACGGAGCTCTTAGTGCTGGCCGG GGAGCT-3′ (SacI site underlined), and the amplified gene fragment was ligated in the corresponding sites of pBI121 binary vector (Clontech Laboratories, USA) by replacing the GUS reporter gene under the control of constitutive CaMV35S promoter. The resulted vector construct was named as pBI121-35S:: EcDehydrin7. Ligated vector was mobilized in Escherichia coli DH5α competent cells via heat shock method at 42 °C for 90 s. Positive clones were selected using kanamycin 100 mg l−1 marker. Positive clones were confirmed by PCR method using gene-specific primers and nptII primers. PBI121-EcDehydrin7 binary construct was mobilized in to Agrobacterium tumefacience (LBA4404) via electroporation (Bio-Rad, USA). Positive clones were selected on rifampicin 10 mg l−1 + kanamycin 50 mg l−1. Clones with PBI121-EcDehydrin7 were confirmed by restriction digestion and PCR using gene-specific primers and nptII primers.
Plant Transformation Transformation of tobacco was performed by Agrobacterium-mediated transformation using leaf discs as explants [12]. Positive transformants, which survive on MS medium containing 300 mg l−1 kanamycin, were further transferred to MS medium containing 200 mg l−1 kanamycin. Bottles were kept in tissue culture growth chamber for hardening. Healthy plants were transferred to pots, and further biochemical and molecular analyses of T0 transgenic lines were done.
Analysis of Transgenic Plants for Drought Tolerance Leaf Disc Assay Five-week-old healthy WT and T0 transgenic tobacco lines (2, 4, 11, 13, and 15) with expanded leaves were taken for leaf discs (1.0 cm2) preparation with the help cork borer. Leaf
Appl Biochem Biotechnol
discs were kept in Petri plates containing solution of 200 mM mannitol for dehydration stress. Three replicates were kept. Petri plates were incubated in tissue culture room for 72 h. Observation was carried out for number of greenish and yellowish leaf disc. Finally, graph was plotted.
Seed Germination Assay For germination test assay, WT and T0 transgenic tobacco seeds were taken. Seeds were sterilized with alcohol (70 %) and sodium hypochlorite (1 %) and germinated on 90-mm Petri dishes containing 1/2 MS medium added with 200 mM mannitol. Germination assay was done in two replicates. Germination, root length, and vigor were judged with WT seeds.
Chlorophyll Estimation of Transgenic Lines Control and T0 transgenic plants were taken in soil pots, and water-deficit experiment was done in glass house on 5-week-old tobacco plants. The leaf total chlorophyll content was monitored after 15 days subjecting the plants to water stress [13]. The chlorophyll content was measured by extraction from leaf tissue in 95 % alcohol. Leaf slices weighing about 2 g were placed in 10 ml of alcohol in 25-ml tubes protected from light overnight. Absorbance (OD) of the extract was obtained at 665 and 649 nm, respectively. Total chlorophyll content (mg/g fresh wt) = [(13 × 95 × OD665 − 6 × 88 × OD649) + (24 × 96 × OD649 − 7 × 32 × OD665) × V]/ (1000 × W), with V being the volume of the extract and W being the fresh weight of leaf tissue in gram. Assay was done in three replicates per line per treatment, and graph was plotted.
Expression Profile of EcDehydrin7 in T1 Transgenic Tobacco Lines Total RNA was isolated from T1 transgenic and WT tobacco plants using spectrum Plant Total RNA Kit (Sigma) as per manufacturer’s instructions. Fifteen micrograms of total RNA was resolved on a 1.2 % formaldehyde denaturing gel and transferred to the nitrocellulose membrane (Hybond-N+, Amersham Biosciences, UK) using diethylpyrocarbonate (DEPC) treated 20× SSC. Pre-hybridization and hybridization were performed with ULTRAhyb hybridization buffer (Ambion, USA) according to the manufacturer’s instructions. The blot was hybridized at 42 °C with α [32P]dCTP labeled EcDehydrin7 417-bp fragment by mega prime DNA labeling system (Amersham Biosciences, UK). The hybridized membrane was washed at 42 °C, and the membrane was exposed to X-ray film. The cassette was incubated at −70 °C for 7 days. Finally, film was developed. Real-time amplification reactions were performed using SYBR Green detection chemistry and run on 96-well plates with the MX3000 Real-time PCR (Stratagene). Three biological replicates for each of the eight samples of transgenic tobacco were used for real-time PCR analysis, and three technical replicates were analyzed for each biological replicate. A no-template control (NTC) was also included in each run for each gene. Each reaction contained 1 μl fourfold diluted cDNA template, 0.4 μm of each primer, and 1× SYBR Green Master Mix (USB Stratagene) in a final volume of 25 μl was subjected to the following conditions: 50 °C for 3 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s in 96-well optical reaction plates (Bio-Rad, USA). The melting curves were analyzed at 60–95 °C after 40 cycles. The close range of efficiencies between the targets and controls allowed for a ΔΔCT analysis using Actin 1a as calibrator. Error was calculated as standard error of the mean (SEM).
Appl Biochem Biotechnol
Results In the present study, effort was made to analyze the possible role of EcDehydrin7 gene in water-deficit stress tolerance. The isolated and characterized EcDehydrin7 from cDNA macroarray was amplified by the 5′ RACE PCR technique using degenerate primers to sequence 5′ region to get the complete gene sequence. The RACE PCR product was cloned in pGEMT-Easy vector and sequenced. The coding region of the gene was identified by FGEN ESH of softberry online software using aligned sequence. The cDNA of EcDehydrin7 gene contained a 417-bp open reading frame (ORF) encoding a protein of 138 amino acids with a calculated molecular weight of about 14.02 kDa and an isoelectric point of 9.40. The fulllength cDNA sequence of EcDehydrin7 was submitted to NCBI GenBank (Accession No. KM096446).
Sequence Analysis of EcDehydrin7 Gene Nucleotide composition was analyzed using BioEdit program. The percentages of A, C, G, and T nucleotides are 25.90, 30.70, 33.33, and 10.07, respectively, with 64.03 % of GC content in EcDehydrin7 nucleotide sequence, whereas the protein sequence has a high content of glycine (27.54 %) followed by threonine (15.94), lysine (9.42), and methionine (6.52). In order to find evolutionary relationship of EcDehydrin7, BLASTP program was employed to identify the homologs of EcDehydrin7 protein sequences with that of other plant species. Phylogenetic tree result showed two clusters, one large (A) and other small (B). Within the large cluster, there are two sub-clusters (clusters 1 and 2). The results showed bootstrap value (70) for EcDehydrin7 is statistically significant and is close to Triticum turgidum protein followed by Setaria italica protein and dehydrin1 and RAB17 proteins of Zea mays. Phylogenetic tree showed that most of the sequences are aligned with dehydrin or RAB17 protein sequences of different crop plants within two sub-clusters. Further ClustalW2 multiple sequence alignment of EcDehydrin7 with other species was carried out with homologous sequences of other species.
Cloning of EcDehydrin7 for Plant Transformation The coding region (ORF of 417 bp) was amplified using specific primers having restriction sites and cloned in pGEMT-Easy vector and sequenced. Further, the ORF of EcDehydrin7 was cloned in a binary vector pBI121 under the control of constitutive Cauliflower Mosaic Virus 35S promoter. The recombinant binary vector was named pBI121-35S:: EcDehydrin7 (Fig. 1). Recombinant clones were confirmed with restriction digestion and mobilized to Agrobacterium strain LBA4404 by electroporation. Recombinant clones in Agrobacterium were also confirmed by PCR and restriction digestion prior to plant transformation. Fifteen transgenic tobacco lines were generated and confirmed by gene-specific PCR, and five lines showing expression in northern analysis were used for physiological analysis.
RNA Expression Profiling of Transgenic Lines Northern blot analysis showed the transgenic lines containing EcDehydrin7 gene are transcribed. It was observed that expression level among transgenic lines were different (Fig. 2a). The EcDehydrin7(15) line showed higher expression followed by EcDehydrin7(11), EcDehydrin7(2), EcDehydrin7(4), and EcDehydrin7(13). There was
Appl Biochem Biotechnol
EcDHN7
pBI121 EcDehydrin7 13,288 bp
Fig. 1 Map of Binary vector pBI121-35S:: EcDehydrin7
B
EcDehydrin7(15)
EcDehydrin7(14)
EcDehydrin7(13)
EcDehydrin7(11)
EcDehydrin7(4)
EcDehydrin7(3)
A
EcDehydrin7(2)
no mRNA transcript accumulation of EcDehydrin7 gene in case of transgenic EcDehydrin7(3) and EcDehydrin7(14) lines. This may be due to gene rearrangement or more copy number (gene silencing) or would have fallen in heterochromatic region in the host genome as these lines are PCR positive for the EcDehydrin7 gene. qRT PCR result also showed relatively similar expression in transgenic lines compared to control plant (Fig. 2b).
12 10 8 6 4 2 0
Fig. 2 Expresssion profile of EcDehydrin7 in transgenic lines. a Northern blot analysis with gene-specific probe. b qRT PCR analysis of selected transgenic lines; Actin1a was used as a normalizer
Appl Biochem Biotechnol
Abiotic Stress Tolerance Analysis of Transgenic Tobacco Lines To find the effect of overexpression of EcDehydrin7 in tobacco, the EcDehydrin7 cDNA was fused to Cauliflower Mosaic Virus 35S promoter and transferred to tobacco by Agrobacteriummediated transformation. After selection on kanamycin-containing medium, putative transgenic tobacco plants were further analyzed by PCR. No growth inhibition or phenotypic alterations were observed in transgenic plants compared to the WT. The transgenic tobacco plants developed normally and did not exhibit any growth retardation. Flowering and seed setting was also normal in transgenic lines.
Chlorophyll Estimation of Transgenic Plants Total chlorophyll content was determined for transgenic lines. Results showed total chlorophyll content in the range of 1.8 to 2.5 mg/g. It was found that there are more amount of chlorophyll in the transgenic lines EcDehydrin7(15) 2.5 mg/g, followed by EcDehydrin7(11), EcDehydrin7(2), EcDehydrin7(4), and EcDehydrin7(13) compared to that in WT plant (Fig. 3). We observed the total chlorophyll content was low in WT, whereas it was relatively more in the transgenics after 15 days of water-deficit stress. This result tells that the overexpression of EcDehydrin7 can impart tolerance to drought stress in transgenic tobacco.
Leaf Disc Assay and Seed Germination Test Assay In order to find tolerance capacity, the WT and transgenic plants were exposed to dehydration and osmotic stresses, and their phenotypic response was examined by leaf disc senescence assay in mannitol. The maximum percentage of green leaf discs was observed in line EcDehydrin7(15), followed by EcDehydrin7(11), EcDehydrin7(2), EcDehydrin7(4), and EcDehydrin7(4) after 72 h of treatment with 200 mM mannitol (Fig. 4a, b). Seed germination test assay was conducted, and response was observed to see the root length and vigor of transgenic lines at 200 mM mannitol. Results showed root length and vigorness of transgenic lines; EcDehydrin7(15) was more followed by EcDehydrin7(11), EcDehydrin7(2), EcDehydrin7(4), and EcDehydrin7(13) compared to control plant (Fig. 4c). 3
(mg/g)
2.5 2 1.5 1 0.5 0
Fig. 3 Chlorophyll estimation of transgenic plants expression EcDehydrin7. Graph showing the chlorophyll content of transgenic lines in milligram per gram
Appl Biochem Biotechnol
A
Percentage
(%)
B
30 25
% of green leaf disc
20 15 10
% of yellow leaf disc
5 0
C
Fig. 4 Physiological analysis of transgenic plants. a Leaf disc senescence assay of WT and T0 transgenic plants in 200 mM mannitol. b Bars showing percentage of response to 200 mM mannitol. c WT and T1 transgenic seed germination on MS media containing 200 mM mannitol and their phenotyping after 20 days
Discussion The dehydrin or RAB (responsive to ABA) genes express during desiccation stages after embryo maturation of seeds in all angiosperms; and in case of vegetative tissue, it express by osmotic stress while imposing abiotic stress like cold, salt, or drought [14, 15]. Dehydrin genes were further identified and compared from barley and corn [16]. In earlier study, we have isolated and characterized drought responsive Dehydrin7 gene from Finger millet, a drought tolerant crop [10]. Here, we generated transgenic plants overexpressing EcDehydrin7 gene and characterized them for stress tolerance. Our results showed more expression of this gene in drought and heat stress compared to control plants. The RNA blot analysis revealed that the
Appl Biochem Biotechnol
EcDehydrin7 gene was transcribed in the transgenic tobacco lines. It confirmed the presence of EcDehydrin7 transcript in transgenic tobacco lines. EcDehydrin7(11) and EcDehydrin7(15) lines showed more expression compare to other lines. The expression variation of EcDehydrin7 gene in transgenic lines may be due to gene copy number and their locus in the host genome. Northern blot analysis result was also supported by qRT PCR assay. Our result showed the transgenic tobacco plant leaves contain more chlorophyll than wild-type plant. We observed that the transgenic plants exhibited higher growth on mannitol than the wild type. Even leaf disc assay showed more green leaf disc under manniol. These findings suggest that transgenic plants overexpression EcDehydrin7 provides tolerance under drought stress conditions.
Conclusion EcDehydrin7 gene showed upregulated expression in heat, drought + heat, and salt stresses. Biochemical and molecular analyses showed EcDehydrin7 gene directly involved in abiotic stress tolerance. Here, our results suggested that EcDehydrin7 gene has active role in imparting the drought stress tolerance. This is the first report of EcDehydrin7 gene from finger millet for abiotic stress tolerance using transgenic approach, and further, it can be used to improve the drought tolerance in other monocot plants. Acknowledgments The authors are thankful to the Department of Biotechnology (DBT), Govt. of India, and Indian Council of Agricultural Research (ICAR) for financial assistance; and AICP on small millets, University of Agricultural Sciences, Bangalore, for providing seeds of finger millet.
References 1. Tenywa, J. S., Nyende, P., Kidoido, M., Kasenge, V., Oryokot, J., & Mbowa, S. (1999). Prospects and constraints of finger millet production in Eastern Uganda. African Crop Science Journal, 7, 569–583. 2. Tadele, Z. (2007). Role of orphan crops in enhancing and diversifying food production in Africa. African Technology Development Forum - Journal, 6, 9–15. 3. Rahman, H., Jagadeeshselvam, N., Valarmathi, R., Sachin, B., Sasikala, R., Senthil, N., Sudhakar, D., Robin, S., & Muthurajan, R. (2014). Transcriptome analysis of salinity responsiveness in contrasting genotypes of finger millet (Eleusine coracana L.) through RNA-sequencing. Plant Molecular Biology, 85, 485–503. 4. Solomon, S. (ed). (2007). Climate change 2007—the physical science basis: Working group I contribution to the fourth assessment report of the IPCC (Vol. 4). Cambridge University Press. 5. IPCC special report on carbon dioxide capture and storage, Chap. 5. http://www.ipcc.ch/pdf/special-reports/ srccs/srccs_wholereport.pdf. Accessed 14 October 2008. 6. Mittler, R., & Blumwald, E. (2010). Genetic engineering for modern agriculture: challenges and perspectives. Annual Review of Plant Biology, 61, 443–462. 7. Boyer, J. S. (1982). Plant productivity and environment. Science, 218, 443–448. 8. Bray, E. A., Bailey-Serres, J., & Weretilnyk, E. (2000). Responses to abiotic stress. In B. B. Buchanan, W. Gruissem, & R. L. Jones (Eds.), Biochemistry and Molecular Biology of Plants (pp. 1158–1203). Waldorf: American Society of Plant Biologists. 9. de Carvalho, M. H. C. (2008). Drought stress and reactive oxygen species. Plant Signal Behavior, 3, 156– 165. 10. Singh, R. K., Phanindra, M. L. V., Singh, V. K., Sonam, R. S., Solanke, A. U., & Kumar, P. A. (2014). Isolation and characterization of drought responsive EcDehydrin7 gene from finger millet (Eleusine coracana (L.) Gaertn.). Indian Journal of Genetics and Plant Breeding, 74, 456–462. 11. Tamura, K., Stecher, G., Peterson, D., Filipski, A., & Kumar, S. (2013). MEGA6: molecular evolutionary genetics analysis version 6.0. Molecular Biology and Evolution, 30, 2725–2729.
Appl Biochem Biotechnol 12. Horsch, R. B., Fry, J. E., Hoffmann, N. L., Eichholtz, D., Rogers, S. A., & Fraley, R. T. (1985). A simple and general method for transferring genes into plants. Science, 227, 1229–1231. 13. Arnon, D. I. (1949). Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiology, 24, 1–15. 14. Mundy, J., & Chua, N. H. (1988). Abscisic acid and water-stress induce the expression of a novel rice gene. EMBO Journal, 7, 2279–2286. 15. Dure, L. S. (1993). Structural motifs in LEA proteins. In E. A. Bray, & T. J. Close (Eds.), Plant responses to cellular dehydration during environmental stress (pp. 91–103). Rockville: The American Society of Plant Physiologist. 16. Close, T. J., Kortt, A. A., & Chandler, P. M. (1989). A cDNA-based comparison of dehydration-induced proteins (dehydrins) in barley and corn. Plant Molecular Biology, 13, 95–108.
Expression of Finger Millet EcDehydrin7 in Transgenic Tobacco Confers Tolerance to Drought Stress Rajiv Kumar Singh 1 & Vivek Kumar Singh 1 & Sanagala Raghavendrarao 1 & Mullapudi Lakshmi Venkata Phanindra 1 & K. Venkat Raman 1 & Amolkumar U. Solanke 1 & Polumetla Ananda Kumar 1,2 & Tilak Raj Sharma 1
Received: 5 August 2014 / Accepted: 29 June 2015 # Springer Science+Business Media New York 2015
Abstract One of the critical alarming constraints for agriculture is water scarcity. In the current scenario, global warming due to climate change and unpredictable rainfall, drought is going to be a master player and possess a big threat to stagnating gene pool of staple food crops. So it is necessary to understand the mechanisms that enable the plants to cope with drought stress. In this study, effort was made to prospect the role of EcDehydrin7 protein from normalized cDNA library of drought tolerance finger millet in transgenic tobacco. Biochemical and molecular analyses of T0 transgenic plants were done for stress tolerance. Leaf disc assay, seed germination test, dehydration assay, and chlorophyll estimation showed EcDehydrin7 protein directly link to drought tolerance. Northern and qRT PCR analyses shows relatively high expression of EcDehydrin7 protein compare to wild type. T0 transgenic lines EcDehydrin7(11) and EcDehydrin7(15) shows superior expression among all lines under study. In summary, all results suggest that EcDehydrin7 protein has a remarkable role in drought tolerance and may be used for sustainable crop breeding program in other food crops. Keywords Finger millet . Drought . EcDehydrin7 . Transgenics and qRT PCR
* Polumetla Ananda Kumar [email protected] * Tilak Raj Sharma [email protected] Amolkumar U. Solanke [email protected] 1
National Research Centre on Plant Biotechnology, LBS Building, Pusa Campus, New Delhi 110012, India
2
Present address: Institute of Biotechnology, Acharya N.G. Ranga Agricultural University, Hyderabad 500030, India
Appl Biochem Biotechnol
Introduction Finger millet (Eleusine coracana (L.) Gaertn.) is a hardy cereal known for its great level of tolerance against drought, salinity, and diseases. The seed of finger millet contains valuable amino acid called methionine, which is lacking in the diets of hundreds of millions of the poor who live on starchy staples such as cassava. It is also enriched in calcium, iron, and phosphate. Finger millet is also a popular food among diabetic patients because of its slow digestion. Crop has major role in food, nutrition, and financial security to resource poor farmers and consumers [1–3]. In current situation, climate change has become an alarming issue. Current climate prediction report indicates that average surface temperature will rise by 3–5 °C in the near future, drastically affecting global agricultural systems [4]. This repercussion will lead to increase frequency of drought, flood, and heat waves [5, 6]. Plants being sessile in nature have to face the environmental stress like drought, salinity, high and low temperature, intense light, and excessive ozone or CO2, and in response, it activates a diverse set of physiological, metabolic, and defense systems to adapt and survive the periods of drought stress. The mechanism of tolerance and susceptibility to abiotic stresses is very complex. Abiotic stress possesses great threat to crop loss worldwide, causing average yield losses of more than 50 % for major crops [7–9]. Being outstanding properties, Finger millet crop is tagged as orphan crop because there is meager genetic and genomic resources available till date. This is due to negligence of research, preference of particular food, less interest in scientific community, green revolution’s adverse effect, and government policies. So far, very few numbers of genes/TFs from finger millet have been cloned and characterized. Therefore, with background stated, functional characterization of EcDehydrin7 protein in transgenic tobacco plant was undertaken to characterize its role in drought stress. This gene was identified in our earlier study and was found to be drought and heat responsive [10].
Materials and Methods Plant Sample and Growth Condition Finger millet (Eleusine coracana) cv. MR1 was chosen as an experimental material for analysis. Seeds were sterilized with 5 % teepol detergent for 30 min with gentle shaking, followed by washing under the running tap water. Seeds were then surface sterilized with 4 % sodium hypochlorite solution for 12 min with intermittent shaking, followed by four to six washes with sterile water and transfer of four to five seeds on MS medium. Culture bottles were kept in culture room maintained at 28 ± 1 °C with a 16 h/8 h light and dark cycles for seed germination. Fluorescent tubes of 40W/54 (Philips India Limited, India) were used as source of light, providing light at a fluence rate of 50–100 μmol m−2 s−1.
RACE for Open Reading Frame of Selected Genes In order to get full-length open reading frame (ORF) of selected genes, RACE was performed by SMARTer RACE cDNA Amplification Kit (Clontech USA). Full-length ORF was obtained with the help of gene finding online tool FGENESH (softberry.com). Finally, EcDehydrin7 gene was selected for characterization and validation in tobacco.
Appl Biochem Biotechnol
Sequence Analysis of EcDehydrin7 Gene Nucleotide composition was analyzed using online BioEdit program. The percentage of A, C, G, and T nucleotides of EcDehydrin7 nucleotide sequence was calculated. The amino acid composition, molecular weight, and isoelectric point (PI) of EcDehydrin7 protein were determined with online program provided by ExPASy (http://web.expasy.org/compute_pi/). To understand the evolutionary history of EcDehydrin7, we identified homologs of EcDehydrin7 gene at the genome-wide level and performed evolutionary analysis across distantly related plant. We performed BLASTP search among sequenced genomes of land plants in plant database (www.plantgdb.org) using EcDehydrin7 as queries. Based on homology in the range of 69 to 82 %, 12 genes were selected for investigation. Phylogenetic tree was generated based on protein sequences using the neighbor-joining algorithm of MEGA software version 6, with method p-distance, bootstrap replicates of 1500, and pairwise deletion missing data [11].
Vector Construction and Molecular Cloning Entire coding sequence of EcDehydrin7 protein was isolated from drought tolerance finger millet (Eleucine coracana cv. MR1) by using the primers of 5′-CGGGATCCCACGGAACCA CCGGAACCGG-3′ (BamHI site underlined) and 5′-ACGGAGCTCTTAGTGCTGGCCGG GGAGCT-3′ (SacI site underlined), and the amplified gene fragment was ligated in the corresponding sites of pBI121 binary vector (Clontech Laboratories, USA) by replacing the GUS reporter gene under the control of constitutive CaMV35S promoter. The resulted vector construct was named as pBI121-35S:: EcDehydrin7. Ligated vector was mobilized in Escherichia coli DH5α competent cells via heat shock method at 42 °C for 90 s. Positive clones were selected using kanamycin 100 mg l−1 marker. Positive clones were confirmed by PCR method using gene-specific primers and nptII primers. PBI121-EcDehydrin7 binary construct was mobilized in to Agrobacterium tumefacience (LBA4404) via electroporation (Bio-Rad, USA). Positive clones were selected on rifampicin 10 mg l−1 + kanamycin 50 mg l−1. Clones with PBI121-EcDehydrin7 were confirmed by restriction digestion and PCR using gene-specific primers and nptII primers.
Plant Transformation Transformation of tobacco was performed by Agrobacterium-mediated transformation using leaf discs as explants [12]. Positive transformants, which survive on MS medium containing 300 mg l−1 kanamycin, were further transferred to MS medium containing 200 mg l−1 kanamycin. Bottles were kept in tissue culture growth chamber for hardening. Healthy plants were transferred to pots, and further biochemical and molecular analyses of T0 transgenic lines were done.
Analysis of Transgenic Plants for Drought Tolerance Leaf Disc Assay Five-week-old healthy WT and T0 transgenic tobacco lines (2, 4, 11, 13, and 15) with expanded leaves were taken for leaf discs (1.0 cm2) preparation with the help cork borer. Leaf
Appl Biochem Biotechnol
discs were kept in Petri plates containing solution of 200 mM mannitol for dehydration stress. Three replicates were kept. Petri plates were incubated in tissue culture room for 72 h. Observation was carried out for number of greenish and yellowish leaf disc. Finally, graph was plotted.
Seed Germination Assay For germination test assay, WT and T0 transgenic tobacco seeds were taken. Seeds were sterilized with alcohol (70 %) and sodium hypochlorite (1 %) and germinated on 90-mm Petri dishes containing 1/2 MS medium added with 200 mM mannitol. Germination assay was done in two replicates. Germination, root length, and vigor were judged with WT seeds.
Chlorophyll Estimation of Transgenic Lines Control and T0 transgenic plants were taken in soil pots, and water-deficit experiment was done in glass house on 5-week-old tobacco plants. The leaf total chlorophyll content was monitored after 15 days subjecting the plants to water stress [13]. The chlorophyll content was measured by extraction from leaf tissue in 95 % alcohol. Leaf slices weighing about 2 g were placed in 10 ml of alcohol in 25-ml tubes protected from light overnight. Absorbance (OD) of the extract was obtained at 665 and 649 nm, respectively. Total chlorophyll content (mg/g fresh wt) = [(13 × 95 × OD665 − 6 × 88 × OD649) + (24 × 96 × OD649 − 7 × 32 × OD665) × V]/ (1000 × W), with V being the volume of the extract and W being the fresh weight of leaf tissue in gram. Assay was done in three replicates per line per treatment, and graph was plotted.
Expression Profile of EcDehydrin7 in T1 Transgenic Tobacco Lines Total RNA was isolated from T1 transgenic and WT tobacco plants using spectrum Plant Total RNA Kit (Sigma) as per manufacturer’s instructions. Fifteen micrograms of total RNA was resolved on a 1.2 % formaldehyde denaturing gel and transferred to the nitrocellulose membrane (Hybond-N+, Amersham Biosciences, UK) using diethylpyrocarbonate (DEPC) treated 20× SSC. Pre-hybridization and hybridization were performed with ULTRAhyb hybridization buffer (Ambion, USA) according to the manufacturer’s instructions. The blot was hybridized at 42 °C with α [32P]dCTP labeled EcDehydrin7 417-bp fragment by mega prime DNA labeling system (Amersham Biosciences, UK). The hybridized membrane was washed at 42 °C, and the membrane was exposed to X-ray film. The cassette was incubated at −70 °C for 7 days. Finally, film was developed. Real-time amplification reactions were performed using SYBR Green detection chemistry and run on 96-well plates with the MX3000 Real-time PCR (Stratagene). Three biological replicates for each of the eight samples of transgenic tobacco were used for real-time PCR analysis, and three technical replicates were analyzed for each biological replicate. A no-template control (NTC) was also included in each run for each gene. Each reaction contained 1 μl fourfold diluted cDNA template, 0.4 μm of each primer, and 1× SYBR Green Master Mix (USB Stratagene) in a final volume of 25 μl was subjected to the following conditions: 50 °C for 3 min, 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s, 60 °C for 30 s, and 72 °C for 30 s in 96-well optical reaction plates (Bio-Rad, USA). The melting curves were analyzed at 60–95 °C after 40 cycles. The close range of efficiencies between the targets and controls allowed for a ΔΔCT analysis using Actin 1a as calibrator. Error was calculated as standard error of the mean (SEM).
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Results In the present study, effort was made to analyze the possible role of EcDehydrin7 gene in water-deficit stress tolerance. The isolated and characterized EcDehydrin7 from cDNA macroarray was amplified by the 5′ RACE PCR technique using degenerate primers to sequence 5′ region to get the complete gene sequence. The RACE PCR product was cloned in pGEMT-Easy vector and sequenced. The coding region of the gene was identified by FGEN ESH of softberry online software using aligned sequence. The cDNA of EcDehydrin7 gene contained a 417-bp open reading frame (ORF) encoding a protein of 138 amino acids with a calculated molecular weight of about 14.02 kDa and an isoelectric point of 9.40. The fulllength cDNA sequence of EcDehydrin7 was submitted to NCBI GenBank (Accession No. KM096446).
Sequence Analysis of EcDehydrin7 Gene Nucleotide composition was analyzed using BioEdit program. The percentages of A, C, G, and T nucleotides are 25.90, 30.70, 33.33, and 10.07, respectively, with 64.03 % of GC content in EcDehydrin7 nucleotide sequence, whereas the protein sequence has a high content of glycine (27.54 %) followed by threonine (15.94), lysine (9.42), and methionine (6.52). In order to find evolutionary relationship of EcDehydrin7, BLASTP program was employed to identify the homologs of EcDehydrin7 protein sequences with that of other plant species. Phylogenetic tree result showed two clusters, one large (A) and other small (B). Within the large cluster, there are two sub-clusters (clusters 1 and 2). The results showed bootstrap value (70) for EcDehydrin7 is statistically significant and is close to Triticum turgidum protein followed by Setaria italica protein and dehydrin1 and RAB17 proteins of Zea mays. Phylogenetic tree showed that most of the sequences are aligned with dehydrin or RAB17 protein sequences of different crop plants within two sub-clusters. Further ClustalW2 multiple sequence alignment of EcDehydrin7 with other species was carried out with homologous sequences of other species.
Cloning of EcDehydrin7 for Plant Transformation The coding region (ORF of 417 bp) was amplified using specific primers having restriction sites and cloned in pGEMT-Easy vector and sequenced. Further, the ORF of EcDehydrin7 was cloned in a binary vector pBI121 under the control of constitutive Cauliflower Mosaic Virus 35S promoter. The recombinant binary vector was named pBI121-35S:: EcDehydrin7 (Fig. 1). Recombinant clones were confirmed with restriction digestion and mobilized to Agrobacterium strain LBA4404 by electroporation. Recombinant clones in Agrobacterium were also confirmed by PCR and restriction digestion prior to plant transformation. Fifteen transgenic tobacco lines were generated and confirmed by gene-specific PCR, and five lines showing expression in northern analysis were used for physiological analysis.
RNA Expression Profiling of Transgenic Lines Northern blot analysis showed the transgenic lines containing EcDehydrin7 gene are transcribed. It was observed that expression level among transgenic lines were different (Fig. 2a). The EcDehydrin7(15) line showed higher expression followed by EcDehydrin7(11), EcDehydrin7(2), EcDehydrin7(4), and EcDehydrin7(13). There was
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pBI121 EcDehydrin7 13,288 bp
Fig. 1 Map of Binary vector pBI121-35S:: EcDehydrin7
B
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no mRNA transcript accumulation of EcDehydrin7 gene in case of transgenic EcDehydrin7(3) and EcDehydrin7(14) lines. This may be due to gene rearrangement or more copy number (gene silencing) or would have fallen in heterochromatic region in the host genome as these lines are PCR positive for the EcDehydrin7 gene. qRT PCR result also showed relatively similar expression in transgenic lines compared to control plant (Fig. 2b).
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Fig. 2 Expresssion profile of EcDehydrin7 in transgenic lines. a Northern blot analysis with gene-specific probe. b qRT PCR analysis of selected transgenic lines; Actin1a was used as a normalizer
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Abiotic Stress Tolerance Analysis of Transgenic Tobacco Lines To find the effect of overexpression of EcDehydrin7 in tobacco, the EcDehydrin7 cDNA was fused to Cauliflower Mosaic Virus 35S promoter and transferred to tobacco by Agrobacteriummediated transformation. After selection on kanamycin-containing medium, putative transgenic tobacco plants were further analyzed by PCR. No growth inhibition or phenotypic alterations were observed in transgenic plants compared to the WT. The transgenic tobacco plants developed normally and did not exhibit any growth retardation. Flowering and seed setting was also normal in transgenic lines.
Chlorophyll Estimation of Transgenic Plants Total chlorophyll content was determined for transgenic lines. Results showed total chlorophyll content in the range of 1.8 to 2.5 mg/g. It was found that there are more amount of chlorophyll in the transgenic lines EcDehydrin7(15) 2.5 mg/g, followed by EcDehydrin7(11), EcDehydrin7(2), EcDehydrin7(4), and EcDehydrin7(13) compared to that in WT plant (Fig. 3). We observed the total chlorophyll content was low in WT, whereas it was relatively more in the transgenics after 15 days of water-deficit stress. This result tells that the overexpression of EcDehydrin7 can impart tolerance to drought stress in transgenic tobacco.
Leaf Disc Assay and Seed Germination Test Assay In order to find tolerance capacity, the WT and transgenic plants were exposed to dehydration and osmotic stresses, and their phenotypic response was examined by leaf disc senescence assay in mannitol. The maximum percentage of green leaf discs was observed in line EcDehydrin7(15), followed by EcDehydrin7(11), EcDehydrin7(2), EcDehydrin7(4), and EcDehydrin7(4) after 72 h of treatment with 200 mM mannitol (Fig. 4a, b). Seed germination test assay was conducted, and response was observed to see the root length and vigor of transgenic lines at 200 mM mannitol. Results showed root length and vigorness of transgenic lines; EcDehydrin7(15) was more followed by EcDehydrin7(11), EcDehydrin7(2), EcDehydrin7(4), and EcDehydrin7(13) compared to control plant (Fig. 4c). 3
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Fig. 3 Chlorophyll estimation of transgenic plants expression EcDehydrin7. Graph showing the chlorophyll content of transgenic lines in milligram per gram
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Fig. 4 Physiological analysis of transgenic plants. a Leaf disc senescence assay of WT and T0 transgenic plants in 200 mM mannitol. b Bars showing percentage of response to 200 mM mannitol. c WT and T1 transgenic seed germination on MS media containing 200 mM mannitol and their phenotyping after 20 days
Discussion The dehydrin or RAB (responsive to ABA) genes express during desiccation stages after embryo maturation of seeds in all angiosperms; and in case of vegetative tissue, it express by osmotic stress while imposing abiotic stress like cold, salt, or drought [14, 15]. Dehydrin genes were further identified and compared from barley and corn [16]. In earlier study, we have isolated and characterized drought responsive Dehydrin7 gene from Finger millet, a drought tolerant crop [10]. Here, we generated transgenic plants overexpressing EcDehydrin7 gene and characterized them for stress tolerance. Our results showed more expression of this gene in drought and heat stress compared to control plants. The RNA blot analysis revealed that the
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EcDehydrin7 gene was transcribed in the transgenic tobacco lines. It confirmed the presence of EcDehydrin7 transcript in transgenic tobacco lines. EcDehydrin7(11) and EcDehydrin7(15) lines showed more expression compare to other lines. The expression variation of EcDehydrin7 gene in transgenic lines may be due to gene copy number and their locus in the host genome. Northern blot analysis result was also supported by qRT PCR assay. Our result showed the transgenic tobacco plant leaves contain more chlorophyll than wild-type plant. We observed that the transgenic plants exhibited higher growth on mannitol than the wild type. Even leaf disc assay showed more green leaf disc under manniol. These findings suggest that transgenic plants overexpression EcDehydrin7 provides tolerance under drought stress conditions.
Conclusion EcDehydrin7 gene showed upregulated expression in heat, drought + heat, and salt stresses. Biochemical and molecular analyses showed EcDehydrin7 gene directly involved in abiotic stress tolerance. Here, our results suggested that EcDehydrin7 gene has active role in imparting the drought stress tolerance. This is the first report of EcDehydrin7 gene from finger millet for abiotic stress tolerance using transgenic approach, and further, it can be used to improve the drought tolerance in other monocot plants. Acknowledgments The authors are thankful to the Department of Biotechnology (DBT), Govt. of India, and Indian Council of Agricultural Research (ICAR) for financial assistance; and AICP on small millets, University of Agricultural Sciences, Bangalore, for providing seeds of finger millet.
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