Introduction

Abiotic stresses cause significant loss in global agricultural productivity and the yield losses in major crops are up to an extent of 60 % (Boyer 1982). Plants employ diverse stress adaptive mechanisms to survive and reproduce under stress conditions. Some of the whole plant and cellular level response mechanisms to stresses have been unraveled. During the past one decade significant progress has been made in unraveling abiotic stress signaling pathways, identification of signaling components and transcription regulators (Chinnusamy et al. 2010). Most signaling events lead to expression and/or activation of diverse transcription factors (TFs) belonging to the families of dehydration responsive element (DRE) binding factor/c-repeat (CRT) binding factor (CBF), basic Leucine Zipper (bZIP), basic helix-loop-helix (bHLH), calmodulin-binding transcriptional activator (CAMTA), no apical meristem (NAM), ATAF1-2 and cup shaped cotyledon (CUC) (NAC), nuclear factor-Y (NF-Y), WRKY, myelocytomatosis (MYC), myeloblastosis (MYB), and zinc finger homeo domain (ZFHD), which in turn orchestrate the expression of effector genes involved in stress acclimation (Yang et al. 2010). Over-expression of some of these TFs in isolation or in combination improved stress tolerance in different plants (Karaba et al. 2007; Lata and Prasad 2011; Pruthvi et al. 2014).

Amongst wide set of stress responsive TFs, CBFs are considered as an important class of proteins involved in cold acclimation in Arabidopsis. Low temperature induces the expression of CBF3/DREB1, which in turn activates several genes involved in cold acclimation (Liu et al. 1998). An important TF, designated as Inducer of CBF Expression 1 (ICE1) is a bHLH protein that is constitutively expressed and localized in the nucleus (Chinnusamy et al. 2003). ICE1 is a positive regulator of CBF genes. In Arabidopsis, cold stress activates ICE1, and the activated ICE1 binds to the promoter of CBF3 to induce its expression (Chinnusamy et al. 2003, 2010). Under cold stress ICE1 protein stability is negatively regulated by high expression of osmotically responsive gene 1 (HOS1), a RING finger ubiquitin E3 ligases (Dong et al. 2006) and positively regulated by sumoylation mediated by a SUMO E3 ligase, SIZ1 (SAP and Miz1) in Arabidopsis (Miura et al. 2007). Over-expression of wild-type ICE1 protein (Chinnusamy et al. 2003) or its mutant version with high trans-activation stability and low proteolysis enhanced the freezing tolerance in Arabidopsis (Miura et al. 2007). ICE1 also negatively regulates MYB15, a MYB TF that binds to CBF3 promoter and represses the expression (Agarwal et al. 2006). Several studies indicated that the CBF cold response pathway is conserved across plant species and over-expression of CBF3/DREB1 improves tolerance to multiple abiotic stresses in plants (Chinnusamy et al. 2010; Lata and Prasad 2011).

Constitutive over-expression of AtICE1 resulted in enhanced osmolyte accumulation and cold tolerance in cucumber and rice (Liu et al. 2010; Xiang et al. 2008). Abscisic acid (ABA) and salt stress up-regulated the ICE1 expression (Chinnusamy et al. 2003), and ice1 mutant showed ABA-induced expression of P CBF3::LUC reporter (Chinnusamy et al. 2003). Additionally, constitutive or stress inducible expression of the ICE1 target, CBF3, has been shown to impart multiple abiotic stress tolerance in several plant species (Lata and Prasad 2011). The expression of CBFs is induced by ABA, although to a lower level as compared with cold induction (Knight et al. 2004). These studies suggested that abiotic stress induced expression of CBFs may be at least in part under the control of ICE1 regulation (Chinnusamy et al. 2006) and ICE1 over-expression may improve tolerance to other abiotic stresses apart from cold-stress. Oryza rufipogon bHLH2 gene, a homolog of AtICE1, conferred enhanced salt tolerance when constitutively expressed in Arabidopsis (Zhou et al. 2009). In this study we are demonstrating the role of ICE1 in imparting salinity, desiccation and drought stress tolerance using transgenic tobacco constitutively over-expressing OsICE1 and AtICE1. We show that ICE1 has roles in minimizing oxidative stress effects and also delaying leaf senescence.

Materials and methods

Analysis of abiotic stress tolerance of Arabidopsis plants over-expressing AtICE1

Arabidopsis thaliana transgenic seeds over-expressing AtICE1 gene under the control of strong constitutive super promoter generated earlier (Chinnusamy et al. 2003) were used to examine the role of ICE1 in different abiotic stress tolerance. Seeds were surface sterilized and inoculated on basal Murashige and Skoog (MS; Murashige and Skoog 1962) media and stratified at 4o C for two days. To assess the stress tolerance, five days old seedlings of wild-type (WT) and AtICE1 over-expressing transgenic plants were transplanted onto MS media supplemented with NaCl (100 mM) and growth was analyzed after seven days. Total plant biomass was recorded after the stress treatment. Simultaneously, to evaluate oxidative stress tolerance, five days old seedlings of WT and AtICE1 over-expressers were transplanted onto MS media supplemented with methyl viologen (MV; 5 μM). Stress tolerance was assessed by visible phenotype seven days after the imposition of stress.

Construction of AtICE1 and OsICE1 expression vectors and transformation of tobacco

Full-length cDNA clones of OsICE1 (AK102594; Os01g0928000) and AtICE1 (At3g26744) were obtained from Rice Genomic Research Centre (RGRC, www.rgrc.dna.affrc.go.jp) and (RIKEN, www.bgrc.riken.jp) Japan, respectively. Both the inserts were amplified using proof reading Phusion™ DNA polymerase (Finnzymes, USA). Gateway cloning® strategy (Invitrogen, USA) was adopted to construct binary vectors harboring AtICE1 and OsICE1 and the details of the primers used for cloning are provided in the Table S1. The OsICE1 and AtICE1 genes were cloned into pK7WG2.0 gateway binary vector (www.psb.ugent.be) under the control of CaMV35S promoter. These constructs were verified by sequencing and the resulting binary vectors were mobilized into Agrobacterium tumefaciens strain EHA105 by electroporation (Electroporator 2510, Eppendorf®, Germany).

Transgenic tobacco (Nicotiana tabacum cv. KST) over-expressing AtICE1 and OsICE1 were generated by A. tumefaciens mediated transformation approach using leaf discs as explants (Horsch et al. 1985). Leaf discs were co-cultivated with Agrobacterium harbouring recombinant binary vector on MS media supplemented with 6-benzylaminopurine (2.25 mg/L) and naphthalene acetic acid (0.1 mg/L) for regeneration. The regenerated shootlets were rooted on MS media supplemented with indole butyric acid (2 mg/L). The rooted plantlets were hardened, transplanted to pots filled with potting mixture and grown under control conditions in transgenic containment facility. Positive transformants were selected by incubating transgenic (T0 generation) seeds on MS media supplemented with kanamycin (100 mg/L) for a week. The putative transgenic lines with good root growth and better phenotype were selected and progressed to next generation.

Seeds of the T0 transformants were germinated on MS media supplemented with kanamycin (100 mg/L) to select putative transgenic plants. The putative transgenic plants were selected based on PCR and RT-PCR. In T2 generation, three independent transgenic lines over-expressing both AtICE1 (denoted as AtICE1-1, 2, 3) and OsICE1 (denoted as OsICE1-1, 2 and 3) with significant transgene expression and normal phenotype were selected and advanced to T3 generation for all the experiments.

RNA isolation and gene expression analysis

Total RNA was isolated by phenol-choloform method (Sambrook and Russell 2001) and was used to prepare cDNA using oligo(dT) primer and M-MuLV reverse transcriptase (MBI Fermentas) according to the manufacture’s protocol. RT-PCR was carried out under standard PCR conditions using gene specific primers (Table S1). Real-time quantitative RT-PCR (qRT-PCR) was performed in reaction mixture containing SYBR green (DyNAmo SYBR-Green qPCR Kit FiNNZYMES, Finland) in a real-time PCR machine (MJ Research, USA & MJ Bioworks, Inc.). For normalization, expression of a housekeeping gene actin was used. Relative transcript levels were calculated using comparative Ct method (Pfaffl 2001).

For expression profiling of ICE1 target genes in tobacco, leaf samples were collected from the WT and transgenic tobacco plants subjected to dehydration stress for 20 d. Total RNA was extracted and cDNA was synthesized as mentioned above. Using the cDNA as template, RT-PCR was performed under standardized conditions using target gene specific primers. The list of genes along with their locus ID, details of primers used are given in the Table S1.

Characterization of tobacco transgenic plants

Seedling level assay

Transgenic tobacco (T3 generation) plants over-expressing AtICE1 and OsICE1 under constitutive promoter (CaMV35S) were used for assessing the role of ICE1 in abiotic stress tolerance. Seeds were surface sterilized and inoculated on MS media supplemented with NaCl (300 mM) and polyethylene glycol (PEG)-8000 (25 % w/v). The seedlings were allowed to grow for 10 d and stress effect was analyzed by recording root and shoot growth and also estimating chlorophyll stability index (CSI) (Beltagi 2008). To estimate the CSI, leaf material from the seedlings exposed to different abiotic stresses were washed in distilled water and total chlorophyll was extracted from leaves in 80 % (v/v) acetone: Dimethyl sulfoxide (1:1 v/v) mixture. The absorbance of the extract was recorded at 663 and 645 nm using UV-Visible spectrophotometer (UV-Vis, Shimadzu, Japan Model DU800). Total chlorophyll expressed as mg/g of fresh weight (Hiscox and Israelstam 1979) and CSI was calculated using the following formula.

$$ \mathrm{C}\mathrm{S}\mathrm{I}\kern0.32em \left(\%\right)=\left\{\left(\mathrm{total}\kern0.45em \mathrm{chlorophyll}\kern0.45em \mathrm{before}\kern0.45em \mathrm{stress}\hbox{-} \mathrm{total}\kern0.45em \mathrm{chlorophyll}\kern0.45em \mathrm{under}\kern0.45em \mathrm{stress}\right)/\left(\mathrm{total}\kern0.45em \mathrm{chlorophyll}\kern0.45em \mathrm{under}\kern0.45em \mathrm{stress}\right)\right\}\times 100 $$

Imposition of drought at whole plant level

A gravimetric approach was followed to mimic drought stress as described earlier (Karaba et al. 2007). For this, a known amount of soil was filled in pots and drenched with water. The soil was then allowed to attain saturation (100 % field capacity, FC) and selected transgenic and WT seedlings were transplanted in these pots. All the pots were maintained at 100 % FC for 30 d. Later, one set of plants were maintained at 100 % FC (control) while the other sets were allowed to reach 40 % FC gradually (water stressed) by controlled irrigation.

Effects of moisture deficit condition on physiological processes were quantified as cell viability and cell membrane stability (CMS). Cell viability was estimated by 2,3,5-triphenyl-tetrazoliumchloride (TTC) reduction test, as described by Towill and Mazur (1975). Electrolyte leakage that reflects loss of membrane integrity was estimated by recording electrical conductivity (EC) using EC-TDS analyzer (ELICO-CM183). Initial electrolytes leaked were recorded (I), subsequently, leaf segments were boiled for 30 min and allowed to cool down and final reading (F) was recorded. Similarly, the leakage was also measured for leaf discs without any stress treatment (control, initial reading (CI) and final reading (CF)) (Tripathy et al. 2000). The CMS was calculated using the following formula.

$$ CMS=\left\{\left[1-\left(\mathrm{I}/\mathrm{F}\right)\right]/\left[1-\left(CI/CF\right)\right]\right\}\ast 100 $$

Ethylene-induced senescence assay

Leaf discs collected from three weeks old transgenic and WT tobacco plants were surface sterilized and incubated in ethrel solution (1200 ppm) in dark up to 42 h to induce senescence. After 42 h of incubation, total chlorophyll was quantified and CSI was calculated (Beltagi 2008).

Salinity and dehydration stress assay

From three weeks old plants, leaf discs were collected, surface sterilized, and incubated in NaCl (300 mM) or MV (7 μM) for 42 h. Similarly, to induce dehydration stress leaf discs were floated on PEG 8000 (25 % w/v) for 42 h and then CMS was estimated.

Statistical analysis

The data generated in all the physiological assays was analyzed by student’s t-test (two tailed) to compare statistical differences between WT and transgenic plants.

Results

Evaluation of Arabidopsis transgenic plants over-expressing AtICE1

To examine the role of ICE1 in abiotic stress tolerance, Arabidopsis transgenic plants over-expressing ICE1 (Chinnusamy et al. 2003) were used. Exposure to salinity (NaCl, 100 mM) for seven days induced tip necrosis/blackening in WT plants whereas ICE1 transgenic plants were devoid of such symptoms (Fig. 1a). ICE1 overexpressors showed lower levels of chlorophyll degradation (Fig. 1b) with significantly higher biomass than WT (Fig. 1c) after prolonged NaCl stress of 20 d. MV-induced oxidative stress caused albino phenotype in WT seedlings after seven days, whereas such phenotypes were not noticed in ICE1 transgenic plants (Fig. 1d). These results indicated that the ICE1 is involved in salt and oxidative stress tolerance in Arabidopsis.

Fig. 1
figure 1

Response of wild-type (WT) and ICE1 over-expressing Arabidopsis seedlings to salt and oxidative stresses. a and b Phenotypic appearance of seedlings grown on MS agar medium supplemented with 100 mM NaCl for 7 and 20 d respectively. c Biomass of Arabidopsis transgenics expressing ICE1 after 20 d of NaCl induced stress. d Effect of methyl viologen (MV; 5 μM) induced oxidative stress on performance of transgenics and WT after 7 d

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Generation of transgenic tobacco plants expressing AtICE1 and OsICE1 and their evaluation

To further evaluate the role of ICE1, we developed transgenic tobacco constitutively over-expressing AtICE1 and OsICE1 (Fig. S1a). We confirmed the expression of transgenes by semi-quantitative and quantitative-RT-PCR analysis in leaf tissue of tobacco transgenic plants (Fig. S1b and S1c). Seedlings of the selected transgenic lines exposed to either NaCl (300 mM) or PEG-8000 (25 % w/v) stress showed better phenotype than untransformed WT plants 10 d post stress imposition (Figs. 2a and 3a). Transgenic lines showed higher CSI (Figs. 2b and 3b), better root (Figs. 2c and 3c) and shoot growth (Fig. 3d) than WT plants under these conditions, although under NaCl stress there was no significant difference in shoot growth (Fig. 2d). Further, we evaluated drought stress tolerance of plants grown in pots by controlled irrigation. After 20 d of moisture deficit condition, transgenic plants exhibited significantly higher cell viability as reflected by percent TTC reduction (Fig. 4a) and CMS than WT plants (Fig. 4b).

Fig. 2
figure 2

Response of transgenic tobacco plants over-expressing AtICE1 and OsICE1 to salt stress. a Seedling growth, b CSI and c & d percent reduction in root and shoot growth of WT and transgenic seedlings, respectively, grown on MS medium supplemented with NaCl (300 mM) for 10 d are presented. Data shown are mean of three replicates, error bars represents standard error and asterisks indicate the significance between WT and ICE1 over-expressed tobacco plants, as described by Student’s t test: *P < 0.05, **P < 0.005 and ***P < 0.0001

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Fig. 3
figure 3

Response of transgenic tobacco plants over-expressing AtICE1 and OsICE1 to PEG induced dehydration stress. a Seedling growth, b CSI and c & d percent reduction in root and shoot growth of WT and transgenic seedlings, respectively, grown on MS medium supplemented with PEG 8000 (25 %) for 10 d are presented. Data shown are mean of three replicates, error bars represents standard error and asterisks indicate the significance between WT and ICE1 over-expressed tobacco plants, as described by Student’s t test: *P < 0.05, **P < 0.005 and ***P < 0.0001

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Fig. 4
figure 4

Response of transgenic tobacco plants over-expressing AtICE1 and OsICE1 to drought. WT and transgenic plants were grown in pots for 30 d under normal condition and subsequently, irrigation was withheld for 20 d to impose drought. a Percent TTC reduction over control and b CMS during stress was used to assess the drought tolerance of WT and ICE1 transgenics. Data shown are mean of three replicates, error bars represents standard error and asterisks indicate the significance between WT and ICE1 overexpressed tobacco plants, as described by Student’s t test: *P < 0.05, **P < 0.005 and ***P < 0.0001

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ICE1 retards ethylene and stress-induced leaf senescence

Many abiotic stresses such as oxidative stress induces leaf senescence (Sedigheh et al. 2011). To determine whether ICE1 over-expression alters this response, leaf discs of WT and ICE1 transgenics were exposed to ethylene for 42 h. As a result, chlorosis was significant in WT than that of ICE1 expressing transgenic lines, suggesting that ICE1 delays ethylene induced leaf senescence. CSI was also significantly higher in transgenic lines as compared to WT treated with ethrel (Fig. S2a). Further, to examine the role of ICE1 in imparting cellular tolerance under different abiotic stresses, leaf tissue was exposed to NaCl (300 mM), PEG-8000 (25 %) and MV (7 μM). ICE1 over-expressors retained significantly high CMS as compared to WT under these stresses (Fig. S2b, c and d).

ICE1 over-expression enhanced the expression of stress responsive genes

To study the effect of ICE1 over-expression on the expression of senescence associated and stress responsive genes, we examined the expression pattern of a few selected target genes of ICE1 by semi-quantitative RT-PCR. ICE1 over-expression enhanced the expression of stress responsive genes such as HSF1 (TC5803), EREBP3 (TC9256), ERD10 (At1g20450), COR47 (TC4052) and HVA22 (TC5661), where as the expression levels of WRKY6 (TC3961) was relatively less in transgenic lines as compared to WT (Fig. S3).

Discussion

Over-expression of several ICE1 homologs including AtICE1 and AtICE2 (Chinnusamy et al. 2003; Fursova et al. 2009), OsICE1 (Xiang et al. 2008; Liu et al. 2010), TaICE141 and TaICE187 (Badawi et al. 2008), CsICE1 (Wang et al. 2012), MdCIbHLH1 (Feng et al. 2012) and EcaICE1 (Lin et al. 2014) have been shown to enhance cold tolerance in plants. The roles of bHLH and ICE1 homologs have also been extended in other abiotic stresses like drought, osmoticum and salinity. Many ICE1 homologs such as OsbHLH001 and OsbHLH002 (Li et al. 2006), OrbHLH2 (Zhou et al. 2009), CbICE53 (Zhou et al. 2012) and CdICE1 (Chen et al. 2012) can impart salinity and osmotic stress tolerance. OrICE1 has been shown to have role only under salt and osmoticum but not under cold (Zhou et al. 2009), CdICE1 showed tolerance to cold, salinity and drought (Chen et al. 2012). In a recent study, under cold stress, more complex regulation of ICE1 by OPEN STOMATA 1 (OST1) has been reported (Ding et al. 2015). OST1 phosphorylates ICE1, which stabilizes the TF and promotes its transcriptional activity. Moreover, OST1 interferes with the interaction between HOS1 and ICE1, which also contributes to ICE1 stability and suppresses HOS1-mediated ICE1 degradation.

To explore the possible regulation of ICE1 expression in different plants, we analyzed upstream regulatory region (UTR) of AtICE1 and OsICE1 using STIF2db (http://caps.ncbs.res.in/stifdb2). The results indicated that AtICE1 has 4Myb_box5_myb, 1Myb_box1_MYB, 6HSE1_HSF and 1WRKY TF binding regions whereas the OsICE1 has no specific stress TF binding sites predicted in the 1000 bp UTR and upstream region. This suggests that the protein probably, is regulated differentially in different species. From this view, we selected ICE1 gene from both Arabidopsis (AtICE1) and rice (OsICE1) for examining its role in cold sensitive model plant tobacco. Independent transgenic lines expressing these genes exhibited typical phenotype of tolerance to different abiotic stresses as evidenced by excised leaf disc, seedling and whole plant assays.

ICE1 protein has been shown to play an important role in cold acclimation since its discovery (Chinnusamy et al. 2003), by regulating expression of COR genes through CBF3 (Chinnusamy et al. 2006). In Arabidopsis Chinnusamy et al. (2003) showed that ICE1 transcripts are constitutively expressed in all the tissues and the levels were high under different abiotic stresses like cold, NaCl and ABA. However, under dehydration stress, the ICE1 induction was not in high levels as compared to other stresses (Chinnusamy et al. 2003). Further, GFP-ICE1 fusion protein was localized to nucleus independent of stress (cold or warm) conditions. We, in this study, attempted to demonstrate additional role of ICE1 in abiotic stress acclimation. At seedling stage, A. thaliana transgenic lines over-expressing AtICE1 exhibited normal phenotype under NaCl-and MV-induced stresses with significantly higher biomass in prolonged NaCl stress than the WT (Fig. 1). Similarly, ICE1 expressing tobacco transgenic seedlings exposed to NaCl and PEG-8000 induced stresses maintained better growth and development (Figs. 2 and 3). The stress tolerant phenotype of both A. thaliana and tobacco transgenic plants over-expressing ICE1 is probably due to the activation of diverse cellular tolerance pathways.

Significantly higher membrane integrity, chlorophyll stability and cell viability observed under stressful condition (Figs. 4 and S2) in ICE1 over-expressors suggest that the TF acts as upstream regulator coordinating multiple cellular tolerance mechanisms. This is evident from our downstream target gene expression study. We examined the expression of a few ICE1 target genes containing MYB, WRKY and HSE cis elements in their promoters. The downstream targets such as HVA22, ERD10, COR47, HSF1 and WRKY6 were selected using STIFdb analysis (Sundar et al. 2008). The AtICE1 and OsICE1 over-expressers showed up-regulation of HVA22, ERD10 (dehydrin), and COR47 (dehydrin) (Fig. S3). These downstream genes, which were induced by ICE1, have been demonstrated to play a role in abiotic stress acclimation (Zhou et al. 2009). We also observed up-regulation of HSF1 transcripts in transgenic lines, which is known to be essential for stress acclimation (McMillan et al. 1998). Similarly, in ICE1 over-expressers, we noticed reduced expression of WRKY6, a gene having role in senescence-associated processes (Robatzek and Somssich 2002). Further, EREBP3 a negative regulator of senescence (Ma et al. 2009) was found to be up-regulated in ICE1 over-expressers. This altered regulation of WRKY6 and EREBP3 relates the ethylene-induced delayed leaf senescence in transgenic plants. Taken together, the results indicate that constitutive expression of ICE1 can improve cellular tolerance to multiple abiotic stresses through diverse mechanisms. Further work towards identifying downstream targets of ICE1 and their interactions would be useful for targeted crop improvement towards abiotic stress tolerance.