Introduction

Plants grow and survive in diverse climatic habitats and are often exposed to stressful conditions of both biotic and abiotic nature. Environmental stresses such as salinity, extreme temperatures, drought, chemical toxicity, oxidative stress, and insect infestation are some of the serious threats to agriculture and result in crop losses worldwide, reducing average yields of most crop plants by more than 50 % (Wang et al. 2003; Vinocur and Altman 2005). To overcome these limitations and to improve production, it is necessary to develop crop cultivars that are capable of alleviating stress.

In nature, animals, pests, and pathogens thrive on plants as a source of subsistence. Agricultural productivity losses caused by herbivorous insects have been estimated to be 10–20 % for major crops grown worldwide (Ferry et al. 2004; Kumari et al. 2014). Among these herbivorous insect, H. armigera is the most destructive species. H. armigera is a serious polyphagous pest and has developed resistance to a number of chemical pesticides (Reed and Pawar 1982; Whalon et al. 2004). In India, losses in the yield of tomato crop due to H. armigera alone amount to 22–38 % (Dhandapani et al. 2003) or one thousand crore rupees per annum (Padmanaban and Arora 2002; Arora et al. 2012, 2014).

Temperature and salinity are the other major environmental factors that adversely affect crop productivity around the world. The exposure of plants to multiple stress factors results primarily in cell damage, leading to secondary effects, such as, osmotic or oxidative stress (Wang et al. 2003; Mittler 2006; Li et al. 2014). Disruption of cellular homeostasis induced by exposure to these stresses often causes protein dysfunction. Therefore, cells must employ efficient mechanisms to preserve and maintain proteins in their native conformation and prevent aggregation of denatured proteins. One of the universal cellular responses to abiotic stress is induction of chaperone protein (GroEL) and other heat shock proteins (Hsps). These accumulate in both, prokaryotic and eukaryotic cells in response to exposure to various stress conditions (Forreiter et al. 1997; Lee et al. 1997; Heckathorn et al. 1998; Low et al. 2000; Scharf et al. 2001; Kotak et al. 2007) and help the system to endure the environmental distress. As molecular chaperone, GroEL is a key component contributing to cellular homeostasis under both optimal and adverse growth conditions. It is responsible for protein folding, assembly, translocation, and degradation in a broad array of normal cellular processes; it also functions in the stabilization of proteins and membranes, and can assist in protein refolding under stress conditions (Lindquist 1986; Lindquist and Craig 1988). Studies demonstrating overexpression of Hsps in plants conferring tolerance to various stressful conditions have been reported (Song and Ahn 2011; Mu et al. 2013; Ruibal et al. 2013; Personat et al. 2014).

A GroEL homolog XnGroEL is secreted by X. nematophila, a Gram-negative bacterium residing in the gut of a soil nematode of the genus Steinernema as a symbiont (Akhurst et al. 1993; Forst et al. 1997; Herbert and Goodrich-Blair 2007). The bacteria-nematode association is highly toxic to many insect species and causes rapid larval death. The protein was orally insecticidal to the major crop pest, H. armigera. The XnGroEL protein was found to bind to chitin in the peritrophic membrane and cause cessation of growth and development of the larvae (Joshi et al. 2008). In our earlier report, XnGroEL was shown to confer resistance against H. armigera in transgenic tobacco (Kumari et al. 2014).

In this investigation, we report the development of transgenic tomato plants by Agrobacterium mediated transformation with DNA sequence encoding XnGroEL protein and examine the response of transgenic plants constitutively expressing XnGroEL to various abiotic stresses and the crop pest H. armigera. Our results demonstrate that this technology could be expanded to many other crop species to improve their tolerance against environmental stresses. To the best of our knowledge, this is the first report showing that the overexpression of a GroEL homolog from X. nematophila confers tolerance to salt and heat stress as well as to insect attack in transgenic tomato plants.

Materials and methods

Construction of expression vector and transformation of tomato

The 1.7 kb gene encoding the 58 kDa XnGroEL protein was isolated from X. nematophila genome (Accession no. AY184491) as described in our previous report (Joshi et al. 2008). Our previous study described the cloning of XnGroEL gene into a plant expression vector pBI121 (Kumari et al. 2014). The construct was called pBI121-XnGroEL. The gene construct was mobilized into Agrobacterium tumefaciens strain LBA4404 by the freeze-thaw method (Hofgen and Willmitzer 1988). The tomato seedlings were raised by germinating seeds of tomato (c.v. Pusa Early Dwarf) soaked for 1–2 h in tap water in egg trays containing soil: vermiculite (1:1) under controlled growth conditions. Tomato transformation was performed via Agrobacterium-mediated transformation method using cotyledon explants as described by Madhulatha et al. (2007). After inoculating with A. tumefaciens, the plant cultures were maintained at 25 °C under a 16 h photoperiod. After 6 weeks, regenerated shoots were transferred to rooting medium. The plantlets were watered with Hoagland solution (Hoagland and Arnon 1950) and hardened for 2 weeks before transfer to soil in pots. After hardening, plants were maintained in the glasshouse for flowering and seed set. The temperature of the greenhouse was maintained within a range of 25–30 °C.

DNA isolation, PCR and Southern blot analysis

Tomato genomic DNA was isolated from fresh leaf tissue of transgenic, untransformed control (UC), and vector control (VC) plants using the CTAB method (Rogers and Bendich 1994) and quantitated spectrophotometrically. The presence of the transgene XnGroEL in the T1 transgenic lines was confirmed by PCR analysis using primers: FP 5′-GGATCCATGGCAGCTAAAGA-3′ and RP 5′-GAGCTCTCACATCATGCCG-3′. The amplified products were separated by electrophoresis on a 0.8 % agarose gel.

Southern blot analysis was performed according to the standard procedures (Sambrook et al. 1989). Genomic DNA (20 µg/lane) from UC, VC, and PCR positive plants was digested with EcoRI overnight at 37 °C and fractionated on 0.8 % agarose gel, denatured, neutralized, and blotted onto a nylon membrane (Amersham Pharmacia, USA). The probe for the XnGroEL gene was amplified from a pBI121 vector containing XnGroEL gene by PCR and was labeled with α-[32P] dCTP using a random primer labeling kit (Amersham Pharmacia Biotech, UK) and used for hybridization of the membrane at 54 °C using standard procedures (Sambrook et al. 1989).

RNA extraction and RT-PCR analysis

Total RNA was extracted from the mature leaf tissue of transgenic, UC, and VC tomato plants using the Plant RNeasy extraction kit (Qiagen, Valencia, USA) and RT-PCR amplification was done using an RT-PCR kit (AccuScript, Stratagene, USA) in accordance with the manufacturer’s instructions. Total RNA (1 µg) was used for generation of first-strand cDNA using Moloney Murine Leukaemia Virus-Reverse Transciptase (MMLV reverse transcriptase). The gene-specific primers used for amplification of XnGroEL cDNA were as described above and primers for tomato actin gene (FP 5′-TGGACTCTGGTGATGGTGTC-3′ and RP 5′-CCTCCAATCCAAACACTGTA-3′) were used as internal control. The RT-PCR products were visualized after electrophoresis on 1.2 % agarose gel.

Western blot analysis

Mature leaves (100 mg fresh weight) from transgenic, UC, and VC tomato plants were ground to a fine powder in liquid nitrogen, homogenized in 200 µl protein extraction buffer (20 % v/v glycerol, 62.5 mM Tris-HCl, pH 6.8, 5 % v/v 2-mercaptoethanol, 0.1 % w/v SDS, plant protease inhibitor cocktail; G Biosciences, St Louis, MO, USA) and centrifuged at 13,000 rpm for 15 min at 4 °C. Protein concentration was estimated in the supernatant by the method of Bradford (1976). The samples were fractionated on a 12 % SDS-PAGE (Sambrook et al. 1989) and transferred to nitrocellulose membrane (Towbin et al. 1979) using a semi-dry transfer system (Owl Separation Systems, UK) according to the manufacturer’s instructions. After blocking, the membrane was immunoblotted with rabbit antisera against XnGroEL (1:10,000 dilution) for 3 h (Joshi et al. 2008; Kumari et al. 2014) and secondary goat anti-rabbit IgG conjugated with alkaline phosphatase (1:10,000 dilution; Sigma-Aldrich) for 1 h. The membrane was developed with BCIP/NBT (5-bromo-4-chloro-3-indolylphosphate p-toluidine and nitro-blue tetrazolium chloride). All the dilutions of primary and secondary antibodies were made in TBST containing 1 % BSA.

Insect bioassay of transgenic plants against H. armigera

H. armigera cultures were reared on a semi-synthetic diet (Gupta et al. 2010). Young leaves of three transgenic (Xn2, Xn3, and Xn4), UC and VC plants were selected for the insect bioassays. The detached leaves were briefly washed with sterile distilled water, dried and placed on moist Whatman no.1 filter paper discs in petri dishes. Ten newly hatched larvae (neonates) of H. armigera were released on a leaf of transgenic, UC, and VC plants, respectively. Three replicates for each transgenic line, UC, and VC plants were set up. The plates were sealed with parafilm and maintained for 5 days in a controlled environment growth chamber at 27 ± 2 °C (16 h light) with 75 % relative humidity. Larval mortality, leaf damage, weight scores, and pupation rate were recorded. For leaf damage, every leaf was assessed for percent damage using a scale 1–9 (where 1 equals to 10 % damage, 2 equals 11–20 % damage and so forth). The experiment was carried out three times under identical conditions.

Leaf disc senescence assay and measurement of chlorophyll content

Leaf discs of 1 cm diameter were excised from healthy and fully expanded tomato leaves of similar age from transgenic, UC, and VC plants and kept in half strength Hoagland solution containing NaCl (200, 400, and 600 mM), mannitol (200, 400, and 600 mM), and CdCl2 (10, 20, and 40 mM), respectively, for 3–4 days. For cold stress, leaf discs were exposed to 4 °C for 5 days while heat stress was given for 10 h at 42 °C. Leaf discs kept in half strength Hoagland solution were taken as control. The chlorophyll content was measured spectrophotometrically after extraction in 80 % acetone (Arnon 1949). The experiment was repeated thrice with three different transgenic lines.

Testing transgenic tomato plants under salinity and heat stress

The 2-weeks-old seedlings were transferred to plastic pots and maintained under 16 h light/8 h dark and 25 °C, either in absence or presence of 200 mM NaCl for 2 weeks. The transgenic plants were also exposed to a high temperature of 42 °C for 16 h. Three plants from each transgenic, UC, and VC lines were taken for this analysis.

Biochemical analysis of XnGroEL transgenic lines

Estimation of relative water content (RWC), lipid peroxidation, electrolytic leakage, Fv/Fm ratio, and hydrogen peroxide (H2O2) was carried out as described below. Estimation of various antioxidant enzymes, including superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) was performed following the well-established standard protocols.

Measurement of relative water content (RWC)

The procedure of Barr and Weatherley (1962) was followed for determination of RWC, fresh leaves were detached and weighed immediately to record fresh weight (FW), followed by dipping them in distilled water for 12 h. The leaves were then blotted to wipe off excess water, weighed to record turgid weight (TW) and subjected to oven drying at 70 °C for 24 h to record dry weight (DW). The RWC was determined by the equation:

$${\text{RWC}} = \, \left( {{\text{FW}} - {\text{DW}}} \right)\, \times \,100/\left( {{\text{TW}} - {\text{DW}}} \right)$$

Measurement of MDA content

The malondialdehyde (MDA) content in leaves was measured according to the protocol described by Heath and Packer (1968). The tomato leaves were ground to a fine powder in liquid nitrogen. 3 ml of 10 % trichloroacetic acid was added to 0.2 g of the powder and left at 4 °C overnight. After centrifugation at 1000×g for 20 min, the supernatant was transferred to a new tube for measurements. 2 ml of 0.6 % thiobarbituric acid (TBA) was added to 2 ml of the supernatant. The mixture was vortexed thoroughly, heated in boiling water for 15 min, cooled immediately and centrifuged. Absorbance values of the supernatant were detected at wavelengths of 532 and 450 nm using water as blank. The MDA content was calculated using the following formula:

$${\text{MDA content }}\left( {\upmu{\text{mol/g fresh weight}}} \right) \, = \, 6.45\, \times \,{\text{OD}}_{532} {-} \, 0.56\, \times \,{\text{OD}}_{450}$$

Measurement of electrolyte leakage

Electrolyte leakage was determined according to the method described by Sairam and Srivastava (2002). Leaves were collected and washed with deionized water thrice in order to remove electrolytes adhering to the surface. They were then placed in test tubes and immersed in 10 ml of deionized water and the electrical conductivity (EC1) was measured. After incubation at 55 °C for 30 min, the EC2 was determined again. The samples were heated in boiling water for 1 h before the total conductivity was measured in the solution (EC3). Relative ion leakage was expressed as a percentage of the total conductivity:

$${\text{Relative Electrolyte Leakage}}\, = \,\left[ {\left( {{\text{EC}}2 \, - {\text{ EC}}1} \right)/{\text{EC}}3} \right]\, \times \,100$$

Fv/Fm ratio

Photosynthetic efficiency of tomato plants was determined by measuring the chlorophyll fluorescence. Chlorophyll fluorescence from the adaxial side of the leaf was monitored using a portable chlorophyll fluorometer (Handy-PEA, Hansatech Instruments, Ltd., UK). Photochemical efficiency of leaves, as determined by chlorophyll fluorescence ratio (Fv/Fm), was monitored before and after salt and heat treatments. Measurements were taken during the light cycle on the leaves using the saturation pulse method after 30 min of dark adaptation (white light 8000 µmol/m2 s−1 for 0.8 s).

In planta histochemical detection of O2 − and H2O2

Accumulation of O2 − and H2O2 was examined based on histochemical staining by nitroblue tetrazolium (NBT) and 3,3′-diaminobenzidine (DAB), respectively, as described earlier (Kumar et al. 2013). For detection of O2 −, the leaves from UC, VC, and XnGroEL-expressing plants grown under normal and stress conditions, respectively were picked and floated in 50 mM sodium phosphate (pH 7.5) containing 0.2 % NBT. The dark blue insoluble formazan compound formed by reaction of NBT with O2 − was visualized and photographed. For detecting H2O2, the leaves were immersed overnight in a solution of DAB (1 mg ml−1, pH 3.8). Thereafter, chlorophyll was removed by boiling in ethanol for 10 min and the reddish-brown coloration denoting the H2O2 content was photographed.

Estimation of H2O2 content

The accumulation of H2O2 in leaves was determined using the method of Velikova et al. (2004). Frozen leaf tissue (0.5 g) was ground to a fine powder in the presence of liquid nitrogen and 5 ml of 0.1 % (w/v) TCA was added to it. The homogenate was centrifuged at 12,000×g for 15 min at room temperature. To 5 ml of the supernatant obtained, 0.5 ml of 1 M potassium phosphate buffer (pH 7.0) and 1 ml of potassium iodide (1 M) were added. The absorbance of the mixture was taken at 390 nm after vortexing. The amount of H2O2 was calculated using the equation:

$${\text{H}}_{2} {\text{O}}_{2} \left( {\upmu{\text{mol}}/{\text{g fresh weight}}} \right) = 1 \, + \, 227.8 \times {\text{OD}}_{390}$$

Measurement of the activities of antioxidant enzymes

Two-weeks-old plants were treated with 200 mM NaCl for 2 weeks. For heat stress, 2-weeks-old plants were kept at 42 °C for 16 h after which the leaves were harvested. The specific activities of various antioxidant enzymes in the leaves of UC, VC, and transgenic plants were determined following the well established standard protocols.

Superoxide dismutase (SOD) activity

SOD activity was assayed by measuring the inhibition of photochemical reduction of NBT spectrophotometrically at 560 nm using the procedure described by Gupta et al. (1993). A 3 ml reaction mixture contained 50 mM phosphate buffer (pH 7.8), 13 μM methionine, 63 μM NBT, 1.3 μM riboflavin, and 100 μl of the enzyme extract. Riboflavin was added last and the tubes were shaken and placed 30 cm below two 15-W fluorescent tubes for 10 min. One unit of SOD activity is defined as the amount of enzyme required to cause 50 % inhibition of the reduction of NBT.

Ascorbate peroxidase (APX) activity

APX activity was determined using the method of Chen and Asada (1989). Leaves were homogenized in 100 mM sodium phosphate buffer (pH 7.0) containing 5 mM ascorbate, 10 % glycerol, and 1 mM EDTA. The activity was determined in 1 ml reaction mixture containing 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM ascorbate, 0.3 mM H2O2, and 50 μl of the enzyme extract. The decrease in absorbance of the reaction mixture was recorded at 290 nm. Enzyme activity was expressed as units per milligram of protein. One unit of APX activity is defined as the amount of enzyme required to reduce 1 μmol of H2O2 min−1 under the assay conditions.

Catalase (CAT) activity

The activity of CAT was measured following the procedure of Aebi (1984). The assay mixture contained 3.2 mM H2O2 in 50 mM phosphate buffer (pH 7.0) and 100 μl of enzyme extract in a total volume of 3 ml. The CAT activity was estimated by a decrease in absorbance of H2O2 at 240 nm. One unit of CAT is defined as the amount of enzyme dismuting 1 nmol of H2O2 per minute.

Statistical analysis

All the experiments were performed at least thrice independently. For bioassay and various growth parameters of the UC, VC, and XnGroEL T1 transgenic plants, values are presented as means of three replicates. Here, the ‘mean of three replicates’ represents the ‘mean of three independent plants’. The results were assessed by Student’s t test. Significance was defined as p ≤ 0.05 (*) and p ≤ 0.001 (**).

Results

Development and characterization of tomato plants transformed with XnGroEL

The tomato lines (Xn1, Xn2, Xn3, and Xn4) were produced by incorporating the DNA sequence encoding XnGroEL (Fig. 1a) in the genome. The vector control plants were also regenerated with a vector control construct (Fig. 1b). Phenotypically there were no significant differences amongst the vector control (VC) and the transformed lines (Xn1–Xn4), as compared with the untransformed control (UC) plants. The transformed plants grew normally in the glasshouse where they flowered and produced seeds. Seeds from T0 generation of transformed plants were harvested (Fig. 1c) and presence of the XnGroEL in the T1 transgenic tomato plants was confirmed by PCR using gene-specific primers (Fig. 1d).

Fig. 1
figure 1

Tomato transformation and molecular analyses of T1 transgenic plants. a Schematic representation of the pBI121-XnGroEL construct used for tomato transformation. The location of primers used for PCR and RT-PCR is indicated by arrows. The region used as probe for Southern blot analysis is also indicated. b Schematic representation of the vector control (VC) construct used for tomato transformation. c Different stages of regeneration of transformed tomato explants. d PCR amplification of a 1.7 kb fragment using XnGroEL sequence specific primers, pBI121-XnGroEL construct was used as positive control (+ve). e Southern blot of genomic DNA from independent tomato transformants (Xn1, Xn2, Xn3, and Xn4). f RT-PCR analysis confirming expression of XnGroEL gene in young fully expanded leaves of transgenic tomato plants. Actin was used as an internal control (lower panel). g Western blot of transgenic plants Xn1, Xn2, Xn3, and Xn4 expressing XnGroEL protein. UC and VC plants were used as negative controls. Lower panel shows the coomassie stained SDS-PAGE gel for checking the equal loading of proteins

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Southern blot analysis of genomic DNA from T1 transformed plants confirmed integration of the transgene in the tomato plants (Fig. 1e). In the lines Xn2, Xn3, and Xn4, a single-copy transgene insertion was observed, while in Xn1 line two copies were found to be integrated in the genome. The RT-PCR analysis showed that mRNA of XnGroEL was expressed in the transgenic lines, but not in the UC and VC plants (Fig. 1f). Expression of XnGroEL protein in transgenic tomato plants was demonstrated by western blot analysis using anti-XnGroEL antibody (Fig. 1g). Western blot analysis confirmed the presence of a single polypeptide of predicted molecular weight of 58-kDa protein reacting positively with the antibody. A very faint band corresponding to the XnGroEL protein was detected in the UC and VC plants. We assume that the polyclonal antibodies might have cross-reacted with the endogenous Hsps as they are conserved in nature.

Resistance of transformed tomato plants to H. armigera

Biological activity of the XnGroEL protein was tested in detached leaves of transgenic plants. The effect on survival and development of the larvae on transgenic, UC, and VC plants was monitored throughout the course of the assay (Fig. 2). Mortality of H. armigera neonatal larvae was 96.7, 67, and 80 % on the leaves of transgenic lines Xn2, Xn3, and Xn4, respectively, compared to 6.7 % in UC and VC plants on day 2. The mortality increased to 100, 90, and 100 %, respectively, in the lines Xn2, Xn3, and Xn4 compared to 17.8 % in UC and VC plants on day 4 (Fig. 2a). The increased expression of XnGroEL protein was correlated with reduced insect growth and corresponding reduction in larval induced tissue damage by 90, 70, and 80 %, respectively (Fig. 2b). After 4 days, the surviving larvae present on the transgenic plants (line Xn3) had remained stunted in growth, whereas those fed on the control plants (UC and VC) had developed to the 2nd instar stage, gained more body mass and resulted in healthy pupae. A reduction in larval weight by ~86 % with corresponding reductions in the rate of pupation by ~90 % (Table S1) was observed in larvae feeding on the transgenic plants (line Xn3).

Fig. 2
figure 2

Insect bioassay: evaluation of toxic effect of XnGroEL protein expressed in transgenic tomato plants on H. armigera. a Percent mortality on day 2 and day 4. b Leaf damage rating. c UC and d VC leaves showing damage by H. armigera. e Intact transgenic tomato leaf (Xn2) showing resistance to H. armigera attack. f, g Larvae on leaves of UC and VC plants resulting in development of healthy pupae, respectively. h Poor growth of larvae fed on transgenic leaf (Xn2) and photographed on day 4 after larval infestation. *, ** denote significance at p ≤ 0.05 and p ≤ 0.001, respectively

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Stress tolerance of transgenic tomato plants expressing XnGroEL

Leaf disc senescence assay (see “Materials and methods” section) was used for assessing the tolerance of plants towards various abiotic stresses. This was performed with T1 plants under salinity (200, 400, and 600 mM NaCl), dehydration (200, 400, and 600 mM mannitol), heavy metals (10, 20, and 40 mM CdCl2), cold (4 °C) and high temperature (42 °C) stress. Leaf disc senescence assays showed a clear advantage of transgenic lines over UC and VC plants in overcoming the deleterious effects of NaCl. The control plants showed rapid senescence and extensive bleaching reflecting symptoms of injury due to salinity stress, while the transgenic lines performed better under similar conditions. Chlorosis was also less in transgenic lines under mannitol stress (simulating dehydration stress), CdCl2 stress, and cold stress while the controls were bleached under similar conditions. At high temperature, the transgenic lines showed chlorophyll depletion, but in other respects they were better than the control plants (Fig. 3a).

Fig. 3
figure 3

Assessment of XnGroEL transgenic tomato plants for their tolerance towards various abiotic stresses. a Leaf disc senescence assays for abiotic stress tolerance in transgenic tomato plants. Experiments were performed on three transgenic lines (Xn2, Xn3, and Xn4) subjected to either NaCl (200, 400, and 600 mM) for salinity stress, mannitol (200, 400, and 600 mM) for dehydration stress, CdCl2 (10, 20, and 40 mM) for heavy metal stress, 4 °C for cold stress or 42 °C for heat stress. Leaf discs floated in Hoagland solution served as the experimental control. The chlorophyll content (µg/g fresh weight) retained in corresponding leaf discs shown in a is depicted by histograms, b NaCl, c Mannitol, d CdCl2, e 4 °C cold, and f 42 °C heat. The data represents mean ± SE of three biological replicates. *, ** denote significance at p ≤ 0.05 and p ≤ 0.001, respectively

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The results of leaf disc senescence assays were confirmed biochemically by estimating the chlorophyll content, which in the leaf discs from all the transgenic plants was 1.2- to 1.7-fold more as compared to those from UC and VC plants at elevated NaCl concentrations. There was nearly complete loss of chlorophyll in the control plants, whereas the XnGroEL transformants showed relatively less reduction in total chlorophyll content at the highest NaCl concentration (600 mM) used (Fig. 3b). Similar, observations were recorded in response to other stresses. Figure 3c, shows 1.3- to 1.6-fold higher chlorophyll content in the lines Xn2, Xn3, and Xn4 when compared with UC and VC lines after exposure to mannitol. Chlorophyll content after exposure to heavy metals (CdCl2 stress) was more than two fold higher in the transformed leaves as compared to the leaves from UC and VC plants (Fig. 3d). Furthermore, tolerance to both cold (4 °C) and heat (42 °C), was higher in the leaves from transformed plants as indicated by 1.4- and 1.6-fold higher chlorophyll contents on comparison with leaf dics from UC and VC plants, respectively (Fig. 3e, f). These results were found to be statistically significant. These observations established a positive relationship between the expression of XnGroEL and hardiness to the general stress conditions encountered by plants.

Effect of XnGroEL expression on relative water content, membrane damage, and photosynthetic efficiency of transgenic plants

The performance of plants under extended exposures of salt and heat stress was also tested. One set of plants was irrigated with 200 mM NaCl solution for 2 weeks to induce salt stress. In another set, the plants were exposed to 42 °C for 16 h. In a control set, transgenic, UC, and VC plants were irrigated with water. Growth and development of the transgenic plants were indistinguishable from those of UC and VC plants. The transgenic plants (Xn2) showed better growth under salt and heat stress as compared to the UC and VC plants where the leaves showed curling, yellowing, and dropping characteristics (Fig. 4a). The other two transgenic lines (Xn3 and Xn4) showed similar performance as Xn2 under salt and heat stress (photographed not shown).

Fig. 4
figure 4

Effect of exposure of transgenic and control plants to salinity and heat stress. a Control panel shows the phenotype of UC, VC, and transgenic tomato plants under non-stress condition. Middle panel shows response of transgenic, UC, and VC control plants after exposure to 200 mM NaCl for 2 weeks. Bottom panel shows the response of transgenic, UC, and VC control plants after 16 h of heat stress (42 °C). Biochemical analysis of transgenic and control plants when exposed to salt and heat stress. b Relative water content. c Lipid peroxidation expressed in terms of MDA content. d Percentage electrolytic leakage. e Chlorophyll fluorescence. *, ** denote significance at p ≤ 0.05 and p ≤ 0.001, respectively

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The salt and heat induced changes in relative water content, MDA (lipid peroxidation product), electrolyte leakage, and photosynthetic efficiency were also measured in the transgenic lines (Xn2, Xn3, and Xn4), UC, and VC plants. The water holding capacity of transgenic plants was determined by measuring the relative water content of leaves under salt and heat stress. The XnGroEL transgenic lines maintained the relative water content by ~1.7-fold higher when exposed to salt stress and ~2-fold higher on exposure to heat stress as compared to UC and VC plants (Fig. 4b).

The MDA content and electrolyte leakage were significantly reduced in XnGroEL transgenic lines by ~1.4- and ~2-fold, respectively, during these stress conditions, as compared with UC and VC plants (Fig. 4c, d).

The Fv/Fm ratio is a measure of the proportion of light absorbed by chlorophyll molecules associated with PSII in the photochemical reaction and thus, an indication of actual photosynthetic efficiency (Harbinson et al. 1989; Heckathorn et al. 1998). To examine salt and heat stress tolerance of the transgenic plants, changes in chlorophyll fluorescence was also measured. The initial chlorophyll fluorescence ratio (Fv/Fm), was similar in the transgenics and the control (UC and VC) plants. However, following stress treatment, the Fv/Fm ratio of XnGroEL expressing transgenic plants was higher by ~1.5-fold (p ≤ 0.05) and ~1.7-fold (p ≤ 0.05), compared to control plants when exposed to salt and heat stress, respectively (Fig. 4e).

O2 − and H2O2 accumulation and ROS detoxification in transgenic and control plants

Salt and heat stresses are accompanied by the formation of reactive oxygen species (ROS) such as O2 −, H2O2, and OH− radicals that cause damage to biological membranes and macromolecules (Wang et al. 2003). The visual observation of accumulation of ROS (O2 − and H2O2) was performed by histochemical staining of leaves with nitroblue tetrazolium (NBT) and 3,3′-diaminobenzidine (DAB), respectively. Formation of purple spots indicative of superoxide (O2 −) accumulation increased in the leaves of control plants compared to leaves of transgenic plants (Fig. 5a).

Fig. 5
figure 5

ROS accumulation and antioxidant enzyme machinery in transgenic and control plants. Histochemical assessment of relative a superoxide (O2 −) and b hydrogen peroxide (H2O2) accumulation in transgenic and control plants exposed to salt and heat stress. Plants irrigated with water served as the untreated control. c Changes in the level of H2O2 content in XnGroEL transgenic lines, UC, and VC were estimated. Effects of XnGroEL-expression on antioxidant enzyme activity under stress treatments. Treatment details are described in “Materials and methods” section. d Changes in the activity of superoxide dismutase (SOD) in transgenic, UC, and VC plants. e Changes in ascorbate peroxidase (APX) activity in transgenic, UC, and VC plants. f Catalase (CAT) activity in transgenic, UC, and VC plants. *, ** denote significance at p ≤ 0.05 and p ≤ 0.001, respectively

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Furthermore, the leaves of XnGroEL expressing plants displayed less brown colouration and diffused staining by DAB relative to the control (UC and VC) plants indicating less hydrogen peroxide (H2O2) accumulation in the transgenic lines (Fig. 5b). These results were corroborated by measuring the H2O2 content of the plants. The H2O2 level was reduced by ~2-fold (p ≤ 0.001) and ~1.8-fold (p ≤ 0.001) in the transgenic lines, as compared to UC and VC plants when exposed to salt and heat stress, respectively (Fig. 5c). These results indicate that overexpression of XnGroEL helps in reducing the accumulation of ROS in transgenic plants.

The effect of expression of XnGroEL on the activity of enzymes that help in quenching toxic radicals, e.g. superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) was also measured in plants. The activities of all these enzymes increased by approximately two to three fold in all the transgenic lines compared to controls on exposure to salt and heat stress (Fig. 5d–f). Together, these results suggest that XnGroEL increased tolerance of plants against salt and heat stress by activating antioxidant enzymes and as a consequence there was reduction in ROS accumulation in the transgenic tomato plants.

Discussion

Molecular chaperones are key components contributing to cellular homeostasis under both optimal and adverse conditions. Many molecular chaperones are stress proteins and are classified as Hsps (Lindquist 1986; Lindquist and Craig 1988; Fink 1999). Denaturation of enzymes and proteins is the primary signal of cellular distress. The chaperones/Hsps play a critical role in maintaining proteins in their functional conformation and preventing aggregation of non-native proteins during stress (Vierling 1991; Hendrick and Hartl 1993; Boston et al. 1996; Hartl 1996; Waters et al. 1996; Queitsch et al. 2000; Frydman 2001; Torok et al. 2001; Bosl et al. 2006; Goloubinoff and De Los Rios 2007). We have earlier reported insecticidal properties of XnGroEL, a molecular chaperone of bacterial origin, against H. armigera larvae (Joshi et al. 2008; Kumari et al. 2014). Here, we evaluate efficacy of XnGroEL in providing protection against biotic and abiotic stresses to transgenic tomato plants.

In X. nematophila, GroEL is expressed at a much higher level under normal conditions and transported outside the cell to perform its specific extracellular function. Insecticidal property of XnGroEL protein was shown earlier in in vitro bioassays (Joshi et al. 2008) and later in transgenic tobacco plants (Kumari et al. 2014). It was shown to bind chitin and brush border membrane of the larval gut (Joshi et al. 2008), however, the exact mechanism of action was not established. It was suspected that being a large heptameric molecule, it blocked access of chitinase to chitin in the larval gut peritrophic membrane, a critical step in remodeling of the gut membrane during development. As a consequence, metamorphosis and growth was adversely affected leading to death of the larvae. The detached leaf assay of XnGroEL transgenic tomato lines against H. armigera reaffirms its lethal action in a commercially important crop plant.

GroEL is a global stress protein and has orthologs across the spectrum of living organisms. In the backdrop of chaperoning activity of XnGroEL observed earlier (Joshi 2007; Joshi et al. 2008), we evaluated protective role of the protein in transgenic tomato plants against abiotic stress and ROS, a leading cause of cellular damage. Abiotic stresses like high salinity and high temperature cause oxidative stress due to disturbance in ROS production and scavenging (Zhu 2001; Mittler 2002; Xiong et al. 2002). The ROS are highly reactive and toxic to biological systems and cause membrane damage through lipid peroxidation (Gill and Tuteja 2010; Gill et al. 2012; Huang et al. 2010). They partially reduce or activate derivatives of oxygen and damage DNA, proteins, and other biomolecules, resulting in cell death (Mittler et al. 2004; Ouyang et al. 2010).

The enhanced level of tolerance to multiple stresses is demonstrated through various markers like higher-chlorophyll, and relative water content, and reduction in lipid peroxidation, electrolyte leakage, and H2O2 production. A concomitant increase in the activity of antioxidant enzymes (SOD, APX, and CAT) in the transgenic plants further augments the cellular defence against unfavourable environmental conditions (Yeh et al. 2002; Apel and Hirt 2004; Ogawa et al. 2007; Swindell et al. 2007; Jiang et al. 2009; Song and Ahn 2011; Li et al. 2014). Thus, increased antioxidant activity of the transgenic lines helps to suppress toxic ROS levels and their effect on membrane (lipid peroxidation). A direct consequence of the decreased MDA level (lipid peroxidation product) was a better photosynthetic performance (increased Fv/Fm ratio) of the transgenic tomato lines. Accumulation of MDA beyond a threshold level has been reported to cause irreparable damage to the photosynthetic machinery (Mishra and Singhal 1992). Overall, maintenance of better water balance, photosynthetic efficiency, and higher activities of antioxidant enzymes, the hallmarks of stress tolerance, most likely protect the XnGroEL transgenic lines from deleterious effects of oxidative damage caused by salt and heat stress. Moreover, analogous to GroEL, overexpression of XnGroEL is expected to safeguard general health of the transgenic plants by preventing protein/enzyme denaturation and activating the antioxidant machinery under biotic and abiotic stress conditions (Shi et al. 2001; Hall 2002; Murakami et al. 2004; Wang et al. 2004; Schramm et al. 2006; Jiang et al. 2009; Tripp et al. 2009, Mu et al. 2013).

In conclusion, this study demonstrates that the overexpression of the bacterial protein XnGroEL can lead to alleviation of biotic and abiotic stresses, thus, demonstrating its dual role. However, more comprehensive evaluation needs to be done before this technology could be applied more widely for protection of important agricultural crops against environmental stresses.