The membrane tethered transcription factor EcbZIP17 from finger millet promotes plant growth and enhances tolerance to abiotic stresses

The occurrence of various stresses, as the outcome of global climate change, results in the yield losses of crop plants. Prospecting of genes in stress tolerant plant species may help to protect and improve their agronomic performance. Finger millet (Eleusine coracana L.) is a valuable source of superior genes and alleles for stress tolerance. In this study, we isolated a novel endoplasmic reticulum (ER) membrane tethered bZIP transcription factor from finger millet, EcbZIP17. Transgenic tobacco plants overexpressing this gene showed better vegetative growth and seed yield compared with wild type (WT) plants under optimal growth conditions and confirmed upregulation of brassinosteroid signalling genes. Under various abiotic stresses, such as 250 mM NaCl, 10% PEG6000, 400 mM mannitol, water withdrawal, and heat stress, the transgenic plants showed higher germination rate, biomass, primary and secondary root formation, and recovery rate, compared with WT plants. The transgenic plants exposed to an ER stress inducer resulted in greater leaf diameter and plant height as well as higher expression of the ER stress-responsive genes BiP, PDIL, and CRT1. Overall, our results indicated that EcbZIP17 improves plant growth at optimal conditions through brassinosteroid signalling and provide tolerance to various environmental stresses via ER signalling pathways.


Results
Isolation and sequence analysis of EcbZIP17. The partial EST sequence of EcbZIP17 obtained from the finger millet EST library was 533 bp in length, lacking a 5′ region. Therefore, Rapid amplification of cDNA ends (5′ RACE) was carried out to obtain full-length cDNA with complete ORF. Sequencing analysis revealed that the ORF of EcbZIP17 was 1,722 bp in length ( Supplementary Fig. 1), translated into a product of 573 aa with a molecular weight of 61 kDa. EcbZIP17 was predicted as a nuclear protein with a leucine rich export signal of 75-83 aa ( Supplementary Fig. 2a,b), a BRLZ domain of 120-184 aa, a TM domain of 249-271 aa, a nuclear localisation signal (NLS) of 223-245 aa, and a canonical S1P protease site of 279-282 aa ( Supplementary Fig. 3a). Homology analysis of EcbZIP17 (AHZ30615.1) revealed 76% identity with a Sorghum bicolor hypothetical protein (Sb02g041070), 75% identity with the maize ZmbZIP17 (BT040011), 48% identity with the rice OsbZIP39 (Os05g34050), and 40% identity with the Arabidopsis AtbZIP17 (NM_129659.2), mainly at the BRLZ domain, TM domain, NLS, and S1P protease site, as well as similarity at the evolutionary and common motif level. However some more homologous proteins (OsbZIP60 (Os07g44950), ZmbZIP28 (BT067808), ZmbZIP60 (BT086464), AtbZIP60 (NM_103458), AtbZIP28 (NM_001202616), and AtbZIP49 (At3g56660)) were used to predict the exact evolutionary relationship in phylogenetic tree construction ( Supplementary Fig. 3b-d). The homologous proteins are known to play a key role in the ER stress response or the UPR. Thus, an NLS at the N-terminal end facing the cytosol and a TM domain at the C-terminal end facing the ER lumen, which is a characteristic feature of the homologous proteins, indicated the putative localisation of EcbZIP17 in the ER membrane.
Expression of EcbZIP17 in finger millet. The expression of EcbZIP17 was observed in all the vegetative parts of the plant, including the leaf, shoot, root, panicle, and germinated seedling; however, the highest expression was noted in the shoot (Fig. 1a), whereas the transcript levels were similar in the panicle and the root. The expression of EcbZIP17 was upregulated under all stress treatments. Under heat, dehydration, ABA, and H 2 O 2 stresses, the expression levels of EcbZIP17 markedly increased after 24 h of treatment (Fig. 1b,c,f,g), whereas under NaCl stress, the expression level increased gradually and then, decreased after 8 h of treatment (Fig. 1d). Under mannitol treatment, the expression level initially increased and then, decreased after 2 h of treatment (Fig. 1e). Apart from the abiotic and oxidative stresses, EcbZIP17 was also upregulated under DTT stress, and the highest expression was observed after 24 h of treatment (Fig. 1h).

Development of transgenic tobacco plants overexpressing EcbZIP17.
A total of 24 putative transgenic lines were generated based on the preliminary screening on kanamycin selection medium. Of these, 13 lines were confirmed by PCR using EcbZIP17 and nptII specific primers ( Supplementary Fig. 4a,b). Southern blot hybridisation revealed that all transgenic lines were independent events with estimated integrations, ranging from one to nine copies of T-DNA. The transgenic lines EcbZIP17E, EcbZIP17O, and EcbZIP17S showed single integrations, whereas the transgenic line EcbZIP17W showed double integrations ( Supplementary Fig. 4c) of EcbZIP17 was higher in EcbZIP17E, EcbZIP17O, EcbZIP17S, and EcbZIP17W compared with the other nine transgenic lines ( Supplementary Fig. 4d). Therefore, the transgenic lines, EcbZIP17E and EcbZIP17S with a single T-DNA insertion, and EcbZIP17W with a double insertion and higher EcbZIP17 expression levels were used for all the experiments in this study, whereas the transgenic lines, EcbZIP17Y and EcbZIP17Z were also used for mannitol stress and drought recovery experiments, respectively, in addition to the other three lines.
Growth of EcbZIP17 transgenic plants under optimal growth conditions. The vegetative growth (i.e. plant height, number of leaves per plant, and leaf area) of transgenic and WT plants was almost similar until the initiation of flowering, whereas significant differences were identified during the transition from the vegetative to the reproductive stage (Fig. 2a). In transgenic plants, we observed an increase in the height by 12-20% and in the girth of the stem by 40-44% within a week. Additionally, a significant increase was observed in the size of the leaves per plant (15-23%), the internode distance (12-16%), the number of pods per plant (28-44%), and the total weight of seeds per plant (14-81%) in transgenic plants (Fig. 2b). Since the transgenic plants performed better under optimal growth conditions than the WT plants, we investigated the expression level of growth related genes of the brassinosteroid signalling pathway, including (EXP10, NtIAA14, NtSEB1, NtBZR1, NtNTR1, NtPP2c4) at the seedling and flowering stages. All these genes are showing better expression at both seedling and flowering stage in transgenic plants compared to WT (Fig. 2c).
Tolerance of EcbZIP17 transgenic plants to salt stress. At germination under NaCl stress, the leaf colour of WT plants showed a severe bleaching, whereas that of all transgenic plants remained green (Fig. 3a). The appearance of transgenic plants that were non-transformants was similar to that of WT plants due to the absence of T-DNA. The fresh weight of transgenic plants was significantly higher than that of WT plants (Fig. 3b). Under salt stress experiment of 30 d old plants, the leaf architecture and chlorophyll content of transgenic and WT plants were similar before and after the stress treatment. However, after one-month of salt stress, the leaf architecture of WT plants markedly changed compared with that of transgenic plants. After 40 d of recovery from the stress treatment, the WT plants were completely bleached, whereas the transgenic plants were fully recovered (Fig. 3c). The chlorophyll content of transgenic and WT plants was lower under salt stress conditions compared with that under optimal growth conditions; however, transgenic plants showed significantly higher chlorophyll content compared with that of WT plants after 40 d of recovery (Fig. 3d). Additionally, transgenic plants also showed better MSI during stress ( Supplementary Fig. 5). β-Tubulin was used as the internal reference gene to normalize the data. Data represents mean ± SE of three technical replicates. Different letters above the error bars indicate significant difference between 0hr and indicated time periods of stress treatment, based on one-way analysis of variance (P < 0.05).
Tolerance of EcbZIP17 transgenic plants to mannitol stress. After 45 d of mannitol stress, the primary root length, number of secondary roots, fresh weight, dry weight, number of leaves per plant, diameter of leaves, and plant height were significantly higher in transgenic plants than in WT plants ( Fig. 4a-d; Supplementary Fig. 6). Tolerance of EcbZIP17 transgenic plants to PEG. Under PEG 6000, the vegetative growth of transgenic plants was significantly higher than that of WT plants ( Supplementary Fig. 7a). Transgenic plants showed a maximum increase of 73% in fresh weight and 76% in dry weight compared with WT plants ( Supplementary  Fig. 7b). However, no significant differences were observed in the chlorophyll content of transgenic and WT plants ( Supplementary Fig. 8).

Plant height and yield of EcbZIP17 transgenic plants after drought recovery. Wilting was noticed
in both transgenic and WT plants, after 11 d of drought stress. Further, 30 d of recovery by watering, all plants were visibly recovered. However, during the flowering stage, the plant height and pod weight of transgenic plants were significantly higher than those of WT plants (Fig. 5a,b).

Tolerance of EcbZIP17 transgenic plants to heat stress.
Under heat stress of whole seedlings, the leaf colour of WT plants became watery brown, and plants started dying after 3 d, whereas the leaves of transgenic plants retained their green colour, and the plants survived (Fig. 6a). The fresh weight of transgenic seedlings was significantly increased after 3 d of treatment (Fig. 6c). In leaf disc assay, after the heat treatment, discs of transgenic plants remained green in colour, whereas those of WT plants turned watery brown (Fig. 6b). A significant retention of the chlorophyll content was observed in transgenic plants compared with WT plants (Fig. 6d).  transgenic plants was larger compared with that of WT plants ( Supplementary Fig. 9a,b). Under 3 mM DTT stress, the vegetative growth of EcbZIP17S was less affected compared with that of WT plants ( Supplementary  Fig. 9c), whereas under 4 mM DTT stress, all WT plants lost their chlorophyll content and became bleached after 30 d of treatment, whereas transgenic plants retained their chlorophyll content ( Supplementary Fig. 9d). The findings of the DTT experiment suggested the significant role of EcbZIP17 in UPR stress tolerance. To further confirm the role of EcbZIP17 in ER stress at the molecular level, the UPR stress responsive genes BiP, PDIL, CNX, and CRT1 were used for expression analysis. Under optimal growth conditions, all the genes showed almost similar expression in transgenic and WT plants, whereas after 6 h of 2.5 mM DTT stress, all the genes were upregulated in transgenic plants compared with WT plants (Fig. 7c), showing that the UPR pathway in transgenic plants was activated under stress conditions.

Discussion
Plants are exposed to different biotic and abiotic stresses during their lifespan and hence, have developed mechanisms to cope with the adverse environmental conditions. Plant species native to harsh environmental conditions and the wild relatives of crop plants are considered rich reservoirs of stress tolerance genes. However, crop plants lost many of the stress tolerant alleles due to domestication and breeding. To improve crop growth and yield under stress conditions, many genes have been characterised at the molecular level, but most of them only provide tolerance to one or a few stresses. In the present study, we identified and characterised a gene that can provide tolerance to multiple abiotic stresses and can perform better under optimal growth conditions.
EcbZIP17 was isolated from finger millet, a crop plant that is tolerant to harsh environmental conditions 50 . This gene encodes a type II transmembrane protein category and thus, contains NLS, TMD, S1P, and S2P sites, which are characteristic of MTTFs. In Arabidopsis, there are four type II MTTF members, AtbZIP17, AtbZIP28, AtbZIP49, and AtbZIP60, which are ER stress signal transducers 17,25,26,51 . Of these, AtbZIP17 and AtbZIP28 are activated by regulated intramembrane proteolysis (RIP). EcbZIP17 was isolated from a cDNA library of stressed finger millet plants and found to be upregulated under different abiotic stresses. Additionally, we characterised EcbZIP17 under optimal growth conditions at different stages of plant development in order to better understand the role of inactivated bZIP17 in the ER membrane to answer the questions raised by Howell 24 . In this regard, transgenic tobacco plants overexpressing EcbZIP17 were evaluated both under optimal growth conditions and different stress conditions.
Under optimal conditions, EcbZIP17 transgenic plants showed better growth, including increased plant height, number of leaves, shoot girth, internode distance, and higher pod and seed yield, than WT plants, suggesting the role of EcbZIP17 in vegetative growth and maturation. Previous studies revealed that overexpression of all MTTFs, AtbZIP28Δ, AtbZIP60, and AtbZIP17Δ from Arabidopsis and EcbZIP60 from finger millet, negatively affected plant growth under optimal conditions, but improved tolerance under stress conditions 25,26,47 . Arabidopsis plants overexpressing ZmbZIP17 showed normal growth under optimal conditions, similar to that of WT plants 52 . In the present study, transgenic plants showed a better vegetative growth under optimal conditions compared with WT plants, revealing the positive effect of overexpressing EcbZIP17. Although, the expression of Under all abiotic stresses EcbZIP17 was upregulated in finger millet and also provided tolerance to drought, salt, heat, PEG 6000, and mannitol stress as well as improved growth in transgenic tobacco plants overexpressing the gene. Under different abiotic stress conditions, the growth parameters of transgenic plants, including biomass, leaf size, primary and secondary root length, and recovery after stress treatment, were significantly improved compared with those of WT plants. These results showed that EcbZIP17 plays a key role in improving tolerance to multiple abiotic stresses. Previous studies reported that the overexpression of AtbZIP60 and AtbZIP28 in Arabidopsis provided tolerance to salt stress and heat stress, respectively 53,54 , whereas the overexpression of  EcbZIP60 in tobacco provided tolerance to both salt and drought stress 47 . Many other members of bZIP family TFs also have been reported to provide tolerance to multiple stresses.
EcbZIP17 transgenic plants showed improved vegetative growth and leaf diameter under DTT stress. DTT is a reducing agent that disrupts the redox environment in the ER, which is required for the disulphide bridge formation in proteins, and results in the accumulation of unfolded proteins, triggering the ER stress response 55 . Abiotic stresses are known to interrupt protein folding and assembly in the ER and activate UPR 56 . The accumulation of unfolded proteins in the ER activates MTTFs, which are processed in the Golgi machinery and then, translocated to the nucleus, increasing the expression of UPR pathway genes such as BiP, PDIL, CNX, and CRT1 24,56 . In Arabidopsis plants transformed with chimeric constructs of AtbZIP28Δ and AtbZIP28 with GFP, proteins were observed in ER structures; however, under tunicamycin and DTT stress, chimeric proteins were observed in the nucleus and triggered the activation of molecular chaperons that facilitated protein folding in the ER by preventing the disaggregation of proteins in the ER lumen 24,57 . Among the ER resident chaperons, BiP is the most well characterised 34,58 . The truncated form of AtbZIP28ΔC was shown to activate the promoter regions of BiP1 and BiP3, which were responsible for the ER stress response through the cis-elements P-UPRE and ERSE 59 . The overexpression of the truncated form of OsbZIP39ΔC and the ectopic expression of the ER stress transducer ZmbZIP17 in Arabidopsis plants also significantly upregulated UPR pathway genes such as BiP, PDIL, CNX and CRT1 under 2 mM DTT stress 52,60 . All these genes were also upregulated in EcbZIP17 transgenic plants under DTT stress, confirming that stress tolerance was due to the same UPR pathway.
Under environmental stress conditions, EcbZIP17 was mobilised from the ER, processed by S1P and S2P in the Golgi apparatus, transported to the nucleus, and eventually activated UPR pathway genes. The DNA binding component of EcbZIP17 translocated to the nucleus after S2P intramembrane cleavage and functioned as a transcription factor, providing stress tolerance. Even in the absence of major stresses, small amounts of bZIPs translocate through the Golgi to the nucleus as plants are constantly subjected to various stresses of low intensity, such as fluctuations in temperature and irrigation times, which activate the brassinosteroid signalling pathway 61 . We also observed upregulation of brassinosteroid signalling pathway genes EXP10, NtIAA14, NtSEB1, NtBZR1, NtNTR1 and NtPP2c4 in the transgenic lines at optimal growth conditions. Previous studies have shown that moderate water stress can stimulate primary root elongation and increase the rate of cell production 62 , whereas high temperature fluctuation can stimulate hypocotyl elongation 63 . These results might explain the improved growth of EcbZIP17 transgenic plants under optimal growth conditions.

Methods
Growth conditions of finger millet plants. Seeds  Isolation and sequence analysis of EcbZIP17. We generated EST data from heat-stressed finger millet seedlings, and selected EcbZIP17 for further characterisation due to its relative higher expression. To obtain the full-length sequence 5′ RACE was performed using heat-stressed RNA sample. A cDNA library was prepared using SMART RACE cDNA amplification kit (Clontech, USA), and a series of nested PCR reactions was performed. Amplicons obtained from nested PCRs were cloned using the pGEMT easy vector (Promega, USA), and at least four colonies were sequenced and submitted to NCBI (Accession no. KF245640.1). The full-length EcbZIP17 cDNA was cloned incorporating KpnI and SalI restriction sites in the forward and reverse primers, respectively.

Expression analysis of EcbZIP17 in different plant tissues and under different stress conditions.
Finger millet seedlings at the three-leaf growth stage were subjected to different abiotic stressors, including heat at 42 °C, dehydration by removing seedlings from the medium and keeping them on 3-mm Whatman sheet, sodium chloride (NaCl; 250 mM), mannitol (300 mM), abscisic acid (ABA; 100 µM), hydrogen peroxide (H 2 O 2 ; 25 mM), and DTT (2.5 mM) 47,64,65 . Seedling samples were collected after 2 h, 4 h, 6 h, 8 h, and 24 h of treatment. Total RNA was isolated from whole seedlings as well from leaf, shoot, root, and panicle samples of matured plants using Spectrum plant total RNA isolation kit (Sigma, USA) to study the expression pattern of EcbZIP17. 1 μg of total RNA was reverse transcribed to single-stranded cDNA using Affynityscript qRT-PCR cDNA synthesis kit (Stratagene, USA). A 1:5 dilution of cDNA pool was used as a template for qRT-PCR expression analysis with gene-specific primers. The qRT-PCR conditions were as follows: 95 °C for 5 min, followed by 38 cycles of 95 °C for 5s, 58 °C for 10s and 72 °C for 25s. The housekeeping gene β-tubulin was used for qRT-PCR normalisation 66 Binary vector construction. A pGEMT-EcbZIP17 plasmid confirmed by sequencing was restricted with KpnI and SalI restriction enzymes. The pCAMBIA2300 binary vector with CAMV35S enhancer-promoter and NptII selection marker was linearized by same enzymes. The restricted ORF of EcbZIP17 was ligated to the linearized pCAMBIA2300 vector at 4 °C overnight using T4 DNA ligase (Fermentas, USA). The resulting construct was mobilised into Agrobacterium tumefaciens EHA105 competent cells by the freeze-thaw method 68 .

Development of transgenic tobacco plants.
Leaves of 20-d-old healthy tobacco (Nicotiana tabacum L. 'Petit Havana') plants were used for Agrobacterium-mediated genetic transformation. Leaf discs were cut and incubated in Murashige and Skoog (MS) pre-culture medium supplemented with 2.5 mg L −1 6-benzylaminopurine (BAP) and 0.1 mg L −1 1-naphthaleneacetic acid (NAA). After 2 d, leaf discs were infected by A. tumefaciens with the pCAMBIA2300-EcbZIP17 vector for 20 min, placed on filter paper to optimise the density of A. tumefaciens cells, and cultured in MS co-culture medium supplemented with 2.5 mg L −1 BAP and 0.1 mg L −1 NAA under dark conditions for 3 d. Next, the leaf disks were transferred to MS selection medium supplemented with 2.5 mg L −1 BAP, 0.1 mg L −1 NAA, 200 mg L −1 kanamycin, and 500 mg L −1 cefotaxime and cultured under light conditions. After 15 d, leaf discs were transferred to fresh selection medium. During this process, calli developed from the infected areas of leaf discs, and the newly developed shoots from calli were sub-cultured to fresh medium in bottles. The elongated shoots were shifted to MS rooting medium supplemented with 200 mg L −1 kanamycin and 500 mg L −1 cefotaxime 69 . The putative transgenic plants were then transferred to small pots, containing soilrite, and later acclimatised to a glasshouse.

Confirmation of putative transgenic plants.
Genomic DNA from transgenic and wild type (WT) plants was isolated using the CTAB (N-cetyl-N,N,N-trimethyl ammonium bromide) method, and the quality and quantity were checked using Nanodrop (Thermo Scientific, USA) and 0.8% agarose gel electrophoresis. The presence of T-DNA was carried out by PCR with gene specific and NptII specific primers and by Southern blot hybridisation.
Southern blot hybridisation. Southern blot hybridisation was carried out to identify the copy number and stable integration of nptII in the genome of EcbZIP17 transgenic plants. A total of 20 μg of genomic DNA from EcbZIP17 transgenic and WT plants was digested by HindIII enzyme. The restricted genomic fragments were resolved by 0.8% agarose gel electrophoresis and transferred to a nitrocellulose membrane (Hybond-N+; Amersham Pharmacia, UK) via the capillary method. The probe preparation was performed using nptII. A 750-bp fragment of the gene was PCR amplified, gel purified, and α[32 P]-dCTP was labelled using the mega prime DNA labelling system (Amersham Biosciences, UK). Further hybridisation and washing were carried out at 65 °C. The hybridised membrane was exposed to an X-ray film in an intensifying cassette under dark room conditions. The cassette was placed at -80 °C for 3 d and developed to visualise the bands 65 .
Expression analysis of EcbZIP17 transgenic plants. qRT-PCR was performed to check the levels of mRNA in transgenic plants overexpressing EcbZIP17. Total RNA was isolated from transgenic and WT plants, and cDNA was prepared using 1.0 μg of total RNA. The expression analysis of EcbZIP17 was performed by qRT-PCR, considering the Ct value of the lowest expressing plant as 1-fold for comparison. The housekeeping gene L25 was used for qRT-PCR normalisation 70 .

Analysis of EcbZIP17 transgenic plants under stress treatments.
Confirmed T1 seedlings obtained from independent T0 plants were analysed under various abiotic stresses. All the experiments were carried out in three replicates. After sterilisation, EcbZIP17-T1 transgenic plants were germinated on MS medium supplemented with 100 mg L −1 kanamycin, whereas WT seeds were germinated on MS medium without kanamycin. At 10d after germination, seedlings were placed on MS medium supplemented with different abiotic stressors, including 10% PEG 6000, 400 mM mannitol, and MS medium supplemented with endoplasmic reticulum (ER) stressor 52,71 (2.5 mM, 3 mM, and 4 mM DTT). To analyse the salinity tolerance at germination level, seeds of EcbZIP17 transgenic plants and WT were germinated on MS agar medium supplemented with 250 mM NaCl 2 and to analyse salt tolerance at mature plant level, 30-d-old transgenic and WT plants were saturated in 200 mM NaCl solution at 3-d intervals continuously for one month. To evaluate response to drought stress at mature plant level, transgenic and WT plants were subjected to water withdrawal for 11 d. Further for recovery study from drought stress, the plants were saturated with water 65 . For the heat stress treatment, 20-d old transgenic plants were grown on MS medium without kanamycin at 42 °C for 3d. Additionally, a leaf disc assay was carried out using mature leaves, keeping 18 leaf discs of transgenic and WT plants in petri dishes, containing autoclaved water at 42 °C for 3 d.
The MSI of transgenic and WT plants was estimated as described by Sairam et al. using an electrolyte leakage conductivity meter 73 . To measure the electrical conductivity E1, samples were placed in test tubes containing 10 ml of deionised water at 42 °C for 30 min, whereas to measure the electrical conductivity E2, the same samples were placed in test tubes containing 10 ml of deionised water at 100 °C for 10 min. The MSI was calculated as follows:

Statistical analysis.
All experiments were carried out in three replicates. One-way analysis of variance (ANOVA) was performed to determine significant differences between the means within each treatment at p < 0.05. GraphPad Prism 5.0 software (Graph Pad, USA) was used for statistical analysis 74 . Lower case letters were used to indicate the significant difference above the error bars.