Introduction

Plants are often challenged by various environmental stresses which tend to inhibit their growth and development. Amongst these unfavourable environmental cues, drought and salinity are known to impose serious threat to plant growth, survival and productivity. Consequently, plants have established intricate defence mechanism to facilitate their survival and adaptation under these unfavourable conditions. Drought stress is known to reduce the crop productivity by 70% (Kaur et al. 2008; Akram et al. 2013; Lum et al. 2014), and this reduced productivity remains a major concern for wheat grown in both arid and semi-arid areas. Similarly, soil salinity is also one of the important agricultural problems in both arid and semi-arid regions in different parts of the world. Salinity is reported to affect almost every aspect of the plant physiology and biochemistry (Murphy and Durako 2003; Cuartero et al. 2006).These adverse conditions are known to induce the expression of wide range of genes including Zinc Finger Proteins (ZFPs) (Xiong et al. 2002; Bartels and Sunkar 2005; Islam et al. 2009).

Zinc Finger Proteins (ZFPs) are one of the best studied transcription factor families reported to participate in various growth and developmental processes and also participate in both biotic and abiotic responses (Cheung et al. 2007; Kam et al. 2007; Jung et al. 2013). The zinc-binding motifs of ZFPs vary extensively in their structure as well as in functions like DNA or RNA binding to protein–protein interactions (Laity et al. 2001). The C3HC4-type RING finger genes form a large gene family and have been well studied in both Arabidopsis and rice (Wu et al. 2014). According to previous studies, 1400 E3 ubiquitin ligases have been reported and 469 of these proteins have 477 RING domains, thus forming the third-largest gene family in Arabidopsis. Moreover, out of these 477 RING domains, 186 RING-HC type domains were recognized based on the occurrence of typical RING domain structure (Stone et al. 2005). RING-HC proteins in Arabidopsis have been reported to function in various processes like root development, photomorphogenesis, secretory pathways, chromatin remodelling in seed dormancy and expression of various drought stress responsive genes (Matsuda et al. 2001; Hardtke et al. 2002; Wang et al. 2006; Liu et al. 2007; Qin et al. 2008).

Wheat is the most important cereal crop as it is a staple diet for more than one-third of the world population (Abd-El-Haleem et al. 2009). In the present study, we attempt to characterize our previously identified heat induced C3HC4 type TaZnF (Traes_4BS_37569EA13.1; hereafter referred to as TaZnF) under dehydration and salinity stress (Agarwal and Khurana 2017).

Materials and methods

Plant material, growth conditions and stress treatments

Genotypes of Triticum aestivum L. cv. PBW343 and Arabidopsis thaliana ecotype Col0 were used in the current study. For wheat seedlings, seeds were sown in plastic trays and kept in growth chamber (Conviron®, Canada) maintained at 20 ± 1 °C; 16 h light: 8 h dark photoperiodic cycle. While for Arabidopsis, seeds were grown on half MS media and kept in growth room maintained at 22 ± 1 °C and 16 h light: 8 h dark cycle. For expression analysis of TaZnF in response to salt (250 mM) and air dehydration stress, 10-day-old seedlings of PBW343 were given stress exposures at different time points (0, 1, 2, 4, 6 and 8 h). To measure the germination rate of wild type and Arabidopsis transgenics under salt stress, seeds of both wild type and transgenics were grown on half MS media supplemented with 150 mM of NaCl. To further study the effect of salt stress on overall growth of the seedlings, 2 weeks old seedlings of both Arabidopsis transgenics and wild type were subjected to gradual salt stress treatment (200 mM NaCl for 5 days followed by 300 mM NaCl for next 16 days). Moreover, these plants under stress conditions were also used for expression profiling of salt stress related marker genes. To analyse the response under dehydration stress, seeds of both transgenics and wild type were grown on half MS media supplemented with different concentrations of PEG (Polyethylene glycol 2%, 3% and 4%) and also with different concentration of mannitol (100 mM, 200 mM and 300 mM). To monitor survival and overall growth under drought stress, 22-day-old seedlings of both wild type and Arabidopsis transgenics were grown in soilrite and then subjected to drought stress by not watering the plants for 14 days, and then followed by re-watering for the next 5 days. To further monitor the overall growth and yield under severe drought stress, seeds of both wild type and transgenics were grown for one month under normal conditions and were then subjected to drought stress by not watering the plants for one month and thereafter analysis was performed. These plants under stress conditions were also used for expression profiling of drought stress marker genes. A representation of plant stage, kind of stress, level of stress and parameters studied is provided in Table 1.

Table 1 Representation of kind of stress treatment, plant stage, level of stress and parameters analysed
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Expression analysis by Real-time PCR

Total RNA was isolated from 10-day-old seedling of PBW343 and Arabidopsis plants using the RNeasy plant mini kit (Qiagen, Germany). On-column DNaseI treatment was done to remove genomic DNA contamination. Two µg of total RNA was used to synthesize cDNA using the High Capacity cDNA Archive kit (Applied Biosystems, USA) and this was mixed with 200 nM of each primer and SYBR Green PCR Master Mix (Applied Biosystems). The primers were designed using Primer Express version 2.0 (PE Applied Biosystems, USA) using default parameters. Real-time PCR analysis was done using the ABI Prism 7000 Sequence Detection System and Software (PE Applied Biosystems) under the following set of conditions including melt curve and dissociation curve: 95 °C for 10 min, 95 °C for 30 s, 60 °C for 1 min, 72 °C for 1 s; 95 °C for 1 min, 60 °C for 30 s, 95 °C for 30 s and 40 cycles. Two biological replicate and three technical replicate was used for analysis. Actin (KX533928.1) was used as internal control. The relative expression was estimated using 2-ΔΔCT method. Primers used are listed in Supplementary Table S1.

Cloning and plant transformation

For cloning, a 756 bp fragment (cDNA) of TaZnF was amplified from pGEM-T Easy::TaZnF plasmid using Topo::TaZnF-F and TaZnF-R primers (Table S1). This amplicon was then inserted into pENTR/D-TOPO vector (Invitrogen) for making entry clone. This cassette was then moved into pMDC32 destination vector by LR-clonase reaction. Overexpression transgenics of Arabidopsis thaliana Col0 were raised by floral dip transformation (Clough and Bent 1998). For analysis, three independent transgenic lines from T3 homozygous seeds were used and the results here represent at least three replicates.

Estimation of chlorophyll fluorescence

PSII activity was calculated as per Krause and Weis (1991). Pulse amplitude modulation fluorometer (Junior-PAM chlorophyll fluorometer, H. Walz, Germany) was used to measure the modulated chlorophyll fluorescence emission from the upper surface of the leaf. For measuring the induction of fluorescence, leaves were initially kept in dark for 20 min. Measurements of different PSII parameters like maximum photosynthetic efficiency (Fv/Fm), effective photosynthetic efficiency (YII) and Electron Transport Rate (ETR) were measured from rosette leaves from ten plants per transgenic line for both transgenics and wild type seedlings.

Estimation of total chlorophyll

Chlorophyll amount was estimated according to protocol mentioned by Hiscox and Israelstam (1979). Leaf weighing approximately 0.05 g was taken from both wild type and transgenic plants in three different replicates followed by incubation in 5 ml of DMSO for 4 h at 65 °C in dark. Absorbance was then measured at 645 nm and 663 nm using spectrophotometer (Beckman DUTM 640B, Beckman Instruments Inc., USA). Chlorophyll contents were calculated by the given formula.

$${\text{Ch a}} = \left[ {\left( {12.3{\text{A}}_{663} - 0.86{\text{A}}_{645} } \right){\text{xV}}} \right]/{\text{X}} \times 1000 \times {\text{W}}$$
$${\text{Ch b}} = \left[ {\left( {19.3{\text{A}}_{665} - 3.6{\text{A}}_{643} } \right){\text{xV}}} \right]/{\text{X}} \times 1000 \times {\text{W}}$$

where V is the volume of DMSO (mL), X represents path length (1 cm) and W indicates fresh weight (gm).

Measurement of membrane stability

Cell membrane stability was measured according to Bajji et al. (2002). For this, seedlings from both wild type and transgenics were submerged in 5 ml of distilled water in test tubes and then were kept at 30 °C for 30 min. After incubation, electrical conductivity (C1) was measured and then these seedlings were further autoclaved (temperature of 121 °C and pressure 15 psi) for 15 min and C2 was measured. Cell injury was calculated using the equation: CMS = [1-(C1-C2)].

Proline estimation

Proline level was measured according to Bates et al. (1973). For this, tissue weighing approximately 100 mg was grinded in 1 ml of 3% sulphosalicyclic acid followed by centrifugation at 15,000 rpm for 15 min. Supernatant obtained was divided and 400 µl of glacial acetic and 400 µl of ninhydrin was added to it followed by incubation at 100 °C for 1 h. After incubation, reaction was stopped by keeping the reaction mix in ice. 800 µl of toluene was added to the sample and followed by vigorous vortexing. Supernatant thus obtained was transferred to new MCT and absorbance was recorded at 520 nm in a UV–visible spectrophotometer (U-2810 Spectrophotometer, Hitachi, Japan). Proline content was calculated using the given equation:

$$\upmu{\text{moles of proline/gm fresh wt}} = \left( {\upmu{\text{g protein/ml}} \times {\text{ml toluene }}5} \right)/115.5 \upmu{\text{g}}{/}\upmu {\text{moles}} \times {\text{gm sample}}$$

Statistical analysis

Statistical analysis was performed by calculating the mean value and standard error for all replicates. Student’s t test was performed to find out the significant difference between wild type and transgenic lines and P value of ≤ 0.05 was considered to be significant and represented by asterisk *.

Results and discussions

Expression profiling of TaZnF under dehydration and salt stress in wheat

To understand the role of TaZnF under abiotic stress, expression profiling of TaZnF was done by real-time PCR analysis. For this, 10-day-old seedlings of Triticum aestivum L. cv. PBW343 were subjected to air dehydration and salinity treatment for various time intervals. Similar expression pattern was observed in both root and shoot tissues under dehydration stress. The expression level was initially found to decrease followed by gradual increase in the expression level with increase in the duration of stress Fig. 1a, b. The expression level of TaZnF was almost similar in both the tissues till 6 h of stress. At 8 h of stress, the expression was found to be higher in shoots (> 15 fold change) in comparison to roots (> 3 fold change). These results correlate with the expression pattern of CCCH zinc finger (BraA10g002330) in seedlings of B. rapa in response to mannitol stress (Pi et al. 2018). The expression level was found to decrease initially at 1 h of stress followed by further increase with the increase in duration of stress (3-9 h). Moreover, the expression of zf-C3HC4 in rice was also found to be strongly affected in leaf over root tissue of rice seedlings under drought stress (Minh-Thu et al. 2013).

Fig. 1
figure 1

Expression profiling of TaZnF transcription factor under dehydration and salt stress at different time points. Change in transcript abundance of TaZnF in response to dehydration and salt stress in a shoot and b in root tissue with increase in duration of stress. The expression level of control samples i.e. 0 h of treatment was normalized as 1.0. The results shown are the means ± SDs of at least three independent experiments

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The expression level was analysed in roots and shoot tissue under salt stress treatment (Fig. 1a, b). In roots the expression level was found to increase with increase in duration of stress and showed greater than 14 fold changes in expression level at 8 h of stress treatment. However in shoot tissue, the expression level increased gradually till 2 h followed by decrease at 4 h and 6 h and again increased at 8 h of stress. Such differential expression pattern has been reported for CCCH zinc finger proteins (BraA10g016510 and BraA08g018640) in seedlings of B. rapa under NaCl stress. Similarly RR-TZF (Arginine-rich motif-tandem CCCH Zinc Finger) of B. rapa like BraA09g046730 and BraA10g002330 showed increased expression within 3–6 h of stress, after which their expression was found to decline under salt stress (Pi et al. 2018). Another C3HC4 type zinc finger protein (BrRZFP1) from B. rapa was also found to be induced by salt treatment. The transcript level increased several fold after 30 min of stress treatment, reaching to a peak level after 8 h and then returning to its normal (Jung et al. 2013). Recent studies too have revealed that C2H2 zinc finger proteins act as key regulator of salinity and drought stress response (Wang et al. 2018). Another parallel observation was made for AdZFP1 from Artemisia desertorum Spreng (induced in root, stem and leaf under drought stress as well as by salinity), Arabidopsis Zinc finger protein, AZF2 and STZ (strongly induced in response to dehydration and high-salt stress) and AlSAP protein from A. littoralis (induced by salt, osmotic, heat and cold stress) (Yang et al. 2008; Sakamoto et al. 2004; Ben Saad et al. 2010). Thus, together these studies clearly indicate the probable role of TaZnF in both drought and salt stress as each of these processes were found to regulate the expression of TaZnF.

Overexpression of TaZnF confers enhanced tolerance under drought stress to transgenics

To find out the role of TaZnF under drought stress, seeds of both wild type and overexpression transgenics were grown on half MS media supplemented with different concentration of PEG (2%, 3% and 4%). PEG is often used to imitate desiccation in various dehydration based experiments (Premachandra and Shimada 1987). All the transgenic lines were found to grow well on all the different concentration of PEG than wild type plants. They displayed relatively longer root length and increased fresh weight (Fig. 2a–c). Earlier studies have documented that stress conditions results in overproduction of proline in plants which in turn imparts stress tolerance by maintaining the osmotic balance, stabilizing the membrane and also by preventing oxidative burst (Hayat et al. 2012). Therefore proline content was measured and it was found to be significantly higher in transgenics in comparison to wild type (Fig. 2e). Other parameters like membrane stability and chlorophyll content were also studied. Cell membranes are one of the foremost targets of many plant stresses and maintenance of its integrity and stability under water stress; this act as a key factor for enhanced tolerance under drought stress. Transgenics showed better membrane stability as estimated by the electrolyte leakage from cells (Fig. 2d). Under stress conditions, there is decrease in chlorophyll content due to damage done to the chloroplasts by active oxygen species (Hasegawa et al. 2000). Transgenics showed relatively higher chlorophyll content than wild type plants (Fig. 2f). Thus, the transgenics performed better under stress conditions as evident from their higher membrane stability, increased proline and chlorophyll content; an indicator for improved tolerance mechanism. Similar observation was made when transgenics were grown on half MS media supplemented with different concentration of mannitol (100 mM, 200 mM and 300 mM). The transgenics performed well in terms of their increased root length, more lateral branching, and higher fresh weight; and were found to be healthier in comparison to the wild type (Fig. S1a–c). They also showed higher accumulation of proline (Fig. S1d). Thus, these studies clearly indicate the possible role of TaZnF under dehydration stress. Since transgenics were found to perform better under dehydration stress, they were further analyzed for their overall growth and survival under drought stress. Both wild type and transgenic lines were subjected to drought stress for 14 days (refer to material and method section). The overexpression transgenics were found to be more tolerant as indicated by their survival rate (13% for C3.3, 13.33% for C3.4 and 11.67% C3.7), robust and healthier growth while the wild type plants perished under similar conditions (Fig. S3). Even after rewatering, the wild type did not recover while transgenics recovered well and showed profuse and healthier growth (Fig. 3). Further assays were done to examine the effect of overexpression on overall yield of the transgenic plants. For this one month old seedlings of both transgenics and wild type were subjected to drought stress for one month as mentioned in material and method sections. The transgenics were found to grow well and showed early bolting and flowering. The siliques of transgenics were larger in size and more in number; thus eventually leading to higher yield (Fig. 4a, b, Table 2). The leaves of transgenics were also found to be smaller in size, thus indicating the mobilization of carbon resource for seed setting, which further correlates with the early flowering and seed formation phenotype of transgenics (Fig. 4c). This is one of the most common mechanisms of the plants to escape and overcome stress conditions. The transgenics also showed higher proline and chlorophyll content (Table 2). Thus transgenics were found to be more tolerant under severe drought stress (one month) in comparison to wild type plants. These observations correlate with the function of other zinc finger proteins like BrRZFP1 whose overexpression conferred increased tolerance to Nicotiana tabacum transgenics under dehydration stress (Jung et al. 2013). Similar observation was reported for tobacco transgenics overexpressing AdZFP1 gene (tolerance to drought stress) and DST gene (increased tolerance in rice in response to drought and salinity stress) (Yang et al. 2008; Huang et al. 2009). In another analysis by Ben Saad et al. (2012) the expression of AlSAP in rice cv. Nipponbare, was found to enhance the tolerance to drought and salt stress. Further the expression of AlSAP did not show any loss in yield and allowed seed production even after exposure to severe drought stress at the vegetative stage. Collectively, these observations show that overexpression of TaZnF in Arabidopsis enhances the tolerance of transgenics under drought stress and thus it can be said that TaZnF functions as a positive regulator of drought stress response.

Fig. 2
figure 2

TaZnF overexpressing Arabidopsis transgenics showed increased tolerance to dehydration stress. a Effect of dehydration stress on Arabidopsis transgenics and wild type (WT) plants was analyzed by growing seeds of both transgenics and WT on half MS media supplemented with various concentration of PEG (2%, 3% and 4%) and phenotype was observed after 20 days, b root length, c fresh weight, d membrane stability index, e proline content and f chlorophyll content. Asterisk* represent the t-test, P value of ≤ 0.05. Experiment represents the average value of three biological replicates

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Fig. 3
figure 3

TaZnF overexpressing Arabidopsis transgenics showed increased tolerance to drought stress. Effect of drought stress was analyzed by subjecting 22-day-old seedlings of both wild type (WT) and transgenics to drought treatment for 14 days and followed by recovery i.e. rewatering the plants for 5 days

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Fig. 4
figure 4

TaZnF overexpressing Arabidopsis transgenics showed increased tolerance to drought stress. Effect of drought stress on one month old seedlings of transgenics and wild (WT) plants subjected to drought stress for one month. a Phenotype of wild type and transgenics observed after stress treatment, b size of siliques and c leaf morphology under drought stress

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Table 2 Morphological and physiological parameters of TaZnF overexpressing Arabidopsis transgenic plants subjected to drought stress for one month
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Overexpression of TaZnF gene confers increased tolerance under salt stress

To find out the possible role of TaZnF in response to salt stress, seeds of both wild type and overexpression transgenics were grown on half MS supplemented with 150 mM of NaCl. The transgenics showed higher germination rate, longer root length and higher fresh weight (Table 3, Fig. S2a–b). Further studies were conducted to examine the overall growth and survival of transgenics under salt stress conditions. For this, transgenics and wild type plants were subjected to gradual salt stress treatment (refer to material and method section). The transgenics displayed profuse vegetative growth with larger leaves and rosette size in comparison to wild type plants which resulted in increased fresh weight of transgenics (Fig. 5, Table 3). According to previous studies, under salt stress plants collect a large number of compatible solutes like proline, polyols, trehalsose and betaine in the cytosol (Rhodes et al. 2002). Therefore, the effect of salt stress on proline accumulation was analysed and the transgenic plants were found to retain higher proline content (Table 3). These transgenics also showed higher membrane stability and more chlorophyll level than wild type under stress conditions (Table 3). These observations correspond with the previous study by Jung et al. (2013) where BrRZFP1 overexpressing lines of tobacco have been reported to confer increased tolerance to salt stress as noted by their better growth with respect to their fresh weight, shoot and root length. Further, these transgenics also showed high retention of chlorophyll levels. Moreover, the overexpression TaZNF resulted in increased tolerance of Arabidopsis transgenic under salt stress conditions (Ma et al. 2016). Similarly, constitutive expression of TaSAP17-D in Arabidopsis conferred enhanced tolerance to transgenics under salt stress (Xu et al. 2018). In addition, SpRing gene belonging to RING finger E3 ligase has been also reported to be involved in salt stress signalling process in Solanum piminellifolium. The Arabidopsis transgenic overexpressing SpRing gene displayed increased tolerance to salt stress during germination and early stages of seed development (Qi et al. 2016). Transgenic tobacco expressing AlSAP gene also showed an improved tolerance to salinity and drought stress (Ben Saad et al. 2010). Overexpression of ZAT6 also resulted in increased tolerance to rice transgenics under salt stress (Tang and Luo 2018). Thus, these observations together clearly indicate that overexpression of TaZnF confer increased tolerance to transgenics as they display better growth and robustness by retaining chlorophyll, accumulation of proline and increased membrane stability. Hence, these findings clearly indicate that TaZnF is an important determinant and plays a crucial role in several stress-related processes and its overexpression can increase the tolerance or resistance of transgenic plants to salt stress.

Table 3 Morphological and physiological parameters of TaZnF overexpressing Arabidopsis transgenic plants subjected to salt stress
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Fig. 5
figure 5

TaZnF overexpressing Arabidopsis plants showed increased tolerance to salt stress. Effect of salt stress on 2 weeks old TaZnF overexpression Arabidopsis transgenic lines subjected to various concentration of salt stress i.e. 200 mM NaCl for 5 days followed by 300 mM NaCl for 16 days

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Expression profiling of stress marker genes

To get an insight into the fact as to how the overexpression is leading to enhanced tolerance to drought and salt stress, expression profiling of various drought and salt stress marker genes were analysed in both wild and transgenic plants. The expression of these marker genes like RD26, RD20, RD29A, RFD29B, DREB1A, DREB2A, ERD6, ATF1, SOS1, SOS2 and SOS3 were found to be up-regulated in transgenics in comparison to wild type under stress conditions (Fig. 6). The C3.4 transgenic line showed higher level of expression in comparison to other two transgenic lines. The expression level of RD29A, DREB1A and DREB2A was found to be significantly higher (> 100 folds) in transgenics in comparison to the wild type under drought stress. The expression level of salt stress marker genes like ATF1, SOS1, SOS2 and SOS3 was also found to be higher (> 2 fold) in transgenics than the wild type under salt stress. Thus, the increased expression level of stress related genes closely correlates with the tolerant phenotype displayed by TaZnF overexpression transgenic plants under drought and salinity stress. Together these results demonstrate that TaZnF acts as a positive regulator of drought and salt stress via regulation of stress responsive genes in transgenic plants. These observations correspond with the previous report where the expression of salt stress related marker genes was found to be significantly high in Arabidopsis transgenics showing constitutive expression of TaSAP17-D in comparison to the wild type plants (Xu et al. 2018). Another report by Ben Saad et al. (2010) has shown that in tobacco transgenics expressing the AlSAP gene, the expression level of stress related marker genes was higher than wild type plants. Similarly rice transgenics expressing AlSAP also showed higher transcript level of stress-related genes than wild type plants under both stress and control conditions (Ben Saad et al. 2012). Thus together these studies suggests that TaZnF is effective in providing tolerance upon overexpression and may be suitable for engineering in crop plants for corroborating suboptimal growth conditions and overcoming abiotic stresses.

Fig. 6
figure 6

Expression profiling of marker genes in wild type and transgenic lines of Arabidopsis plants overexpressing TaZnF. The expression level of wild type (WT) was normalized as 1.0 and the result shown are the means ± SDs of at least three independent experiments

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Conclusions

To summarize TaZnF acts as a positive regulator for both drought and salt stress response. The overexpression of TaZnF resulted in increased tolerance to both drought and salt stress by regulating the expression of various stress related genes. Thus, these results indicate that TaZnF transcription factor is an important determinant of stress response in plants and changes in its expression level in plant might increase the tolerance level to various abiotic stresses. This further indicates that this gene can be used in genetic engineering for improving abiotic stress tolerance in crop plants.