Introduction

Faba bean (Vicia faba L.), a legume family member, is one of the most major feed and food legumes ranking fourth worldwide after lentil (Lens culinaris Medik.), chickpea (Cicer arietinum L.) and pea (Pisum sativum L.) (http://faostat.fao.org). In Asia, Africa and several developing countries, faba bean is a valuable crop which is a rich source of dietary protein used for human consumption, animal nutrition, and farming systems. In the Mediterranean region, especially countries in North Africa including Tunisia, field faba bean is the most produced grain legume. In 2018, the national production yield was 64,508 tons cultivated on a surface of 54,907 hectares (FAOSTAT 2018). Indeed, Tunisia's average faba bean yield is generally low, at about 1.12 t/ha compared to the worldwide one (2.26 t/ha) (FAOSTAT 2018). Moreover, the yield and production of faba beans in Tunisia, are dependent on seasonal conditions, characterized by wide fluctuations and thus vary significantly from year to year. This unstable productivity is apparently due to a lack of adequate genetic resources. Currently, the crop is cultivated in semi-arid areas where receive average annual precipitation of more than 400 mm per year. Several studies found that drought at various stages of plant development as well as heat during the reproductive growth and pod filling stages are the major problem of this legume (De Costa et al. 1997; Alghamdi 2007; Al-Suhaibani 2009). Furthermore, several studies have shown that faba bean is more susceptible to water deficit compared to common bean, pea, and chickpea (Amede and Schubert 2003). Similar results were also reported by Khan et al. (2010) and Torres et al. (2010). These authors revealed that faba bean is more susceptible to water deficit than other grain legumes mainly due to its relatively shallow rooted and its disability to mediating osmotic adjustment under water-limited conditions. Ouji et al. (2016) mentioned that water deficit is the major constraint to pulse crops production in cultivated fields of semi-arid environments of Tunisia characterized by terminal drought stress.

Drought stress is a major environmental stress that affects numerous metabolic, physiological, and biochemical processes, limiting plant growth and crop yield worldwide (Pandey et al. 2017). Ghassemi-Golezani et al. (2009) revealed that water deficit reduced plant growth, grain filling, grain weight, grains per plant and consequently considerably reduced grain yield in faba bean cultivars. Similar data were reported by Abid et al. (2017) who showed that water limitation reduced growth (leaf area, root fresh weight, shoot fresh and plant height) and physiological (net CO2 assimilation rate and relative water content) parameters, the activity of antioxidant enzymes and reduced mean grain yield in 11 studied faba bean genotypes. Plant responses to water deficit are complex, involving multiple levels of regulation (Sourour et al. 2017). It is worthy to mention that understanding these responses will help the screening for genotypes tolerant to water-limited conditions. To cope with environmental stresses, plant activates multiple responses involving cascades of gene networks and molecular signaling controlling abiotic stress response in plants including biosynthesis of plant hormones and activation of process of reactive oxygen species (ROS) detoxification in plant cells (Krannich et al. 2015).

High-throughput next-generation sequencing (NGS) technologies including RNA-Seq (RNA sequencing) (Song et al. 2017), DNA microarray (Khan et al. 2017), and in silico analysis (Muthuramalingam et al. 2017) help in the recognition of drought stress-related genes and in enlightening of metabolic pathways affected by drought stress and consequently leading to an acceleration in faba bean crop improvement through genetic and genomic tools.

In silico analysis approach has been successfully used in many species like Glycine max (soybean), Medicago truncatula (barrel medic), Cajanus cajan (pigeon pea), Phaseolus vulgaris (common bean) and Oryza sativa (rice) (Soares-Cavalcanti et al. 2012; Zhang et al. 2013; Sinha et al. 2016; Büyük and Aras 2017; Muthuramalingam et al. 2017, respectively) for the analysis of global patterns of gene expression in response to various abiotic stress. Indeed, identification and molecular characterization of osmotic stress-responsive genes known from the model legume Medicago truncatula in faba bean using in silico approach could contribute to a better comprehension of the early mechanisms at the molecular level and pathways involved in osmotic stress response and adaptation in this leguminous crop.

Drought is a potential constraint which alters plant metabolism and consequently leads to decreases of plant growth and yields by influencing various biological processes of plants like photosynthesis, photosynthetic pigments biosynthesis, plant water relations, hormone biosynthesis, ion uptake and translocation, nutrient metabolism and respiration (Nadeem et al. 2019). During the processes, generating signals in various cell organelles caused by perturbations due to stress were perceived. These signals activate the differential expression of various proteins which were found to be involved in signal transduction including transcription factors comprising members of ABA signaling pathways and other regulatory proteins (transcription factors like ZFP, bZIP, WRKY and NAC), protein kinases and/or phosphatases, redox regulatory proteins, drought-related, proteins proteins related to sugar metabolism and osmotic adjustment leading to plant protection against drought stress (Li et al. 2016; Yang et al. 2019). The main objective of this study is to investigate molecular pathways contributing to osmotic stress responses in faba bean. For this aim, in silico and transcriptomic analysis were employed to identify and study the expression of candidate genes by qRT-PCR in two faba bean genotypes with contrasting osmotic stress response. The findings are valuable and can serve as a basis for developing of faba bean varieties resistant to osmotic stress.

Materials and methods

Plant material

Two genotypes (Giza 3 and Bachar) were used in the present study for comparative expression analysis to identify putative osmotic stress-response genes in faba bean (Vicia faba L. var. minor). Giza 3 genotype acknowledged susceptible to drought stress (Abdellatif et al. 2012) was obtained from ICARDA (Aleppo, Syrian Arab Republic) and the one at Bachar, tolerant to drought stress (Maazaoui et al. 2016) from Regional Field Crop Research Center of Beja (CRRGC, Tunisia). The present work was conducted during the period of 2017–2018 in a glasshouse compartment under controlled environmental conditions with 23 ± 2 °C temperature, 55/65% relative humidity, 270 μmol of photons/m2/s PAR and 16/8 h photoperiod day/night regime. Seeds were surface sterilized by immersing in sodium hypochlorite (NaOCl at 5%) for 5 min and then washed in sterile distilled water several times. After, seeds were soaking for 12 h in distilled water before placed on perlite for germination as reported by Mhamdi et al. (1999). Two weeks old, seedlings were pulled and the roots were washed 5 times with distilled water. Afterwards, a total of 90 seedlings from each cultivar were grown hydroponically in a 40 × 17 × 10 cm (length, width and height, respectively) nutrient reservoir containing 5 L of Hoagland solution (Hoagland and Arnon 1950) in a randomized complete block design with three replicates. The experimental boxes were continuously aerated with aeration tubes and covered with black papers to avoid the algal growth.

NaCl (sodium chloride), ABA (abscisic acid) and PEG (polyethylene glycol) treatment

Salinity and osmotic stresses were applied by using NaCl (200 mM) and PEG-6000 (21%), respectively. After one week of the establishment of the seedlings in Hoagland solution, the nutrient medium was replaced with Hoagland solution (5L/box) containing PEG-6000 and NaCl. Moreover, non-treated faba bean seedlings were used as a control. Leaves and roots were collected at 0 (control), 3, 6, 12, 24 and 48 h time points after each treatment. Besides, ABA treatment (100 µM) was carried out by adding it into the Hoagland culture solution and spraying on the leaves of the 3-week-old seedlings. Leaves samples were harvested at 0, 1, 3, 6 and 12 h after treatment and all control plants were treated with sterile distilled water. Plant materials collected at each sampling time were frozen in liquid nitrogen and stored at − 80 °C until further use. Each treatment contained three biological replications and repeated three times.

Relative water content (RWC)

The RWC was evaluated during 48 h of stress treatments as described by Downey and Miller (1971). To obtain the fresh mass (FM), five fully expanded leaves were randomly taken from the control or osmotic/salt-treated groups of each genotype at the same time point during the stress period and weighted. Then, samples were instantly floated on distilled water in the dark for 24 h at 4 °C to estimate the turgid weight (TW). Finally, samples were dried at 70 ± 2 °C for 3 days and weighed (dry mass, DM).

RWC was determined according to the following equation:

$$ {\text{RWC }}\;(\% ) = \left[ {\left( {{\text{FM}}{-}{\text{DM}}} \right)/\left( {{\text{TW}}{-}{\text{DM}}} \right)} \right] \, \times { 1}00. $$

Malondialdehyde content (MDA)

Lipid peroxidation level was measured by determining malondialdehyde (MDA) content in plant tissues according to Hodges et al. (1999). Samples (0.5 g) of leaf tissues were homogenized in 10 ml of 0.1% (w/v) TCA (trichloroacetic acid). The mixture was centrifuged at 12,000 rpm for 15 min. A 2 ml aliquot from the supernatant was added to 2 ml of 10% TCA contained 0.5% TBA (thiobarbituric acid). Samples were placed in a water bath at 100 °C for 30 min and centrifuged at 12,000 rpm for 15 min after cooled by incubation on ice. Then the absorbance was measured at 440, 532 and 600 nm in the spectrophotometer (Spectro UV–Vis Dual Beam PC, UV-S-2007; LABOMED, INC.). An extinction coefficient of 155/(mM/cm) was used to determine MDA content (Heath and Packer 1968).

Hydrogen peroxide content (H 2 O 2 )

H2O2 level in tissues was estimated colorimetrically following the method of Mukherjee and Choudhuri (1983). Briefly, 0.5 g of frozen leaf powder was mixed with 3 ml of refrigerated acetone (10%) and centrifuged at 12,000 rpm for 15 min. A 2 ml of extracted solution was homogenized with 1 ml of titanium sulfate (1%) and 0.5 ml of 30% ammonium hydroxide solution. After the precipitate was formed, the homogenate was centrifuged at 4 °C for 10 min at 12,000 rpm. The resulting precipitate was dissolved in 10 ml of 2 M H2SO4 after washing three times with acetone and the absorbance was recorded at 415 nm. The H2O2 content was calculated by referring to a H2O2 standard curve. Each measurement was repeated at least three times.

Free proline content

Proline was estimated following the method of Bates et al. (1973). A 0.1 g of dry leaf tissues were extracted in 10 ml of 3% (w/v) sulfosalicylic acid over-night. The extract was centrifuged for 10 min at 5000 rpm. Two ml of extracts were homogenized with an equal volume of fresh ninhydrin solution and glacial acetic acid before heading for 1 h in boiling water. Subsequently, free proline was extracted by adding 4 ml toluene. Proline content was estimated by read absorbance at 520 nm using l-proline as a standard. The analyses were performed in triplicate.

Total soluble sugars content

The soluble sugar was determined spectrophotometrically according to the method of Dubois et al. (1956). A 0.1 g of dry leaf tissues was extracted by heating for 15 min in a 95 °C water bath by adding 5 ml of 95% ethanol (v/v) solution. After centrifugation at 3000 rpm for 10 min, the supernatants of the alcoholic extracts were kept in the refrigerator at 4 °C for the determination of total soluble sugars content. Indeed, 200 µl of extract were homogenized with 1.8 ml of sterile deionized water and the samples were rigorously mixed. After adding 2 ml of phenol (5%) and 10 ml of sulphuric acid to each sample, the homogenate was kept at 25 °C for 1 h. Total soluble sugar contents were recorded at 490 nm using glucose as a sugar standard.

Database searches

Li et al. (2009) performed a DNA microarray in salt-stressed roots of Medicago truncatula treated with 180 mM NaCl for 6, 24, and 48 h in order to identify salt stress-response genes and regulatory pathways associated with salt stress tolerance. Data of the gene expression quantification was deposited at Gene Expression Omnibus (GEO) under series number GSE13921. According to these authors, the log2 fold change [logFC] values were used to identify the differentially expressed genes (DEGs) in respective stress. Indeed, up-regulated or down-regulated genes were defined that its fold change was positively higher or negatively lower than two times of the standard deviation in any of the three time points. Publicly available EST database of faba bean were downloaded from dbEST of NCBI. The identified Medicago truncatula sequences were used for data mining the NCBI in search for faba bean homologues using an E value cutoff of 1 × 10−4 (Table 1). BLAST algorithm and NCBI databases (EST, nucleotide and protein) were used for homology searches.

Table 1 Summary of faba bean (Vicia faba) selected genes showing homology with osmotic stress related Medicago truncatula genes
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Extraction of total RNA and cDNA synthesis

Total RNA was extracted from leaves and roots tissue according to the CTAB method as reported previously by Chang et al. (1993). The quality and concentration of total RNA was measured spectrophotmetrically using UV-2700 (Shimadzu, Tokyo, Japan) by determining the ratio of absorbance at 260, 280 and 230 nm and its integrity were checked by agarose gel electrophoresis (1.2%). The remaining genomic DNA traces from RNA samples was removed by treatment with 1 μl of DNase I (Biomatik; Wilmington, Delaware, USA); then 2 µg were used for first-strand cDNA synthesis using 200 U MMLV reverse transcriptase (Biomatik; Wilmington, Delaware, USA) according to the manufacturers’ instructions. The cDNA products were stored at − 20 °C until used for PCR analysis.

Semi-quantitative RT-PCR

Organ-specific gene expression in roots, leaves, stems, and cotyledons was determined in Bachar 3-week-old seedlings while in flowers (0 days after pollination) at 8-week-old Bachar plants. In seeds gene expression was measured 9 days after pollination. Total RNA was extracted according to the CTAB method and first-strand cDNA synthesis was carried out as described over. Primers were designed using Primer3 program (Rozen and Skaletsky, 2000). PCR reactions were performed in a 20 µl final volume as follows: 95 °C for 5 min; 25–35 cycles of 95 °C for 30 s, 55–60 °C for 30 s and 72 °C for 30 s. A final elongation step was performed at 72 °C for 5 min. Faba bean VfEF1α was used as an internal standard for data normalization. Reactions which replicated three times were amplified on a DNA thermal cycler (Applied Biosystems® 2720, Foster City, CA, USA). An aliquot of 15 µl of obtained products was electrophoresed on a 1.2% agarose gels and DNA bands were stained with ethidium bromide before exposed at 254 nm for visualizing.

Quantitative RT-PCR (qRT-PCR)

For qRT-PCR, reactions were run in a 7300 Real-Time PCR thermocycler (Applied Biosystems, Foster City, USA) and were performed in a final volume of 25 µl containing 200 µM primers (Table 2), 12.5 μl Maxima SYBR Green Master Mix, 2 µl cDNA and 9 μl H2O. The amplifications of all genes were technically replicated 3 times using the following program: 95 °C for 10 min, 40 cycles at 95 °C for 30 s, 60 °C for 1 min. Melting curve analysis was performed by heating from 60 to 95 °C, 0.5 °C per 5 s increments. Results were analyzed by StepOne™ Software v2.2.2 (Applied Biosystems, USA). Relative gene expression analysis was accomplished with 2−ΔΔCt method (Schmittgen and Livak 2008). The R package was used to generate heatmaps of differential gene expression.

Table 2 Primers used for RT-PCR analysis
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Statistical analyses

Data were obtained from at least three replicates and represent mean ± standard deviation (SD). One-way analysis (P < 0.001) was applied to determine the significance of the results between different treatments and then Turkey’s multiple comparison tests at 5% (P < 0.05) were performed. ANOVA analysis was conducted using statistical software SPSS 13.0.

Results

Physiological analyses

Characterization of the genotypes differing in their response to osmotic and salt stress was performed by measurement of physiological parameters such as RWC. The RWC was not significantly different between studied genotypes under control condition (Fig. 1a, b). However, underwater stress (3 h, 6 h and 12 h), Giza 3 exhibited slightly, not significant, lower RWC than Bachar. However, at 24 h and 48 h, Giza 3 showed a slightly higher but not significant RWC value compared to Bachar. A similar pattern was obtained under salt stress at 3 and 6 h, whereas at 12, 24 and 48 h Bachar showed higher and significant RWC value compared to Giza 3. In general, under salt stress conditions Giza 3 showed lower RWC value than Bachar. Thus, under equivalent osmotic potential, the effects of NaCl were more harmful than PEG on RWC of Bachar and Giza 3.

Fig.1
figure 1

Effects of PEG and NaCl treatments on relative water content (a, b, respectively), proline (µg/mg DW) (c, d, respectively), soluble sugars (µg/mg DW) (e and f respectively), H2O2 (µM/g FW) (g, h, respectively) and MDA (nmol/g FW) content (i, j, respectively) in leaves of Bachar and Giza 3. The values are means (± SD) of four replicates. Letters above the bars represent significant differences at P < 0.05 (Tukey’s pairwise comparison)

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Proline accumulation was used as a drought-tolerance selection criterion. In this study, leaves of both studied genotypes showed an increase of proline accumulation under osmotic and salt stress conditions (Fig. 1c, d). Under osmotic stress, the increase varied from 31-fold at 48 h, 1.90-fold at 3 h, 1.60-fold at 6 h and 0.80-fold at 12 h for Bachar and only 5.35-fold at 48 h for Giza 3. Thus, 48 h after osmotic stress induction, Bachar registered the biggest proline content, being followed by Giza 3, and the lowest content was registered at 24 h in both studied genotypes. Moreover, leaves of Bachar showed a high proline content under salt stress during the experimental period. The increase varied from 4.50-fold at 48 h, threefold at 3 h, 1.90-fold at 6 h, 1.05-fold at 12 h and 0.75-fold at 24 h. In Giza 3 an increase by 0.60-fold only at 48 h was observed. Overall, the behavior of Bachar and Giza 3 under osmotic and salt stress showed genotypic variability in the accumulation of proline.

In the present study, data in Fig. 1e, f showed that the initial level of soluble sugar of Bachar and Giza 3 plants was not significantly different under control condition. On the other hand, total soluble sugars content in Bachar and Giza 3 was significantly higher under osmotic and salt stress application compared to the respective controls. Moreover, Bachar had the highest soluble sugars content during osmotic stress; while the lowest soluble sugars content was observed in Giza 3 (Fig. 1e). In Bachar, soluble sugars content increased after 3, 6, 12, 24 and 48 h, approximately up to 9, 9.6, 11, 10.5 and 5.8 folds, respectively. Similar pattern in soluble sugars accumulation was also observed under salt stress and control (Fig. 1f). Osmotic and salinity treatments significantly increased soluble sugars concentration in Bachar compared to Giza 3.

Data shown in Fig. 1g, h revealed that under osmotic and salt pressure, the H2O2 content increased significantly in both genotypes compared to the control plants. The responses of both genotypes to osmotic and salt stress were different. For all time points analyzed, osmotic challenged Giza 3 plants exhibited the highest H2O2 content. At 3, 6, 12, 24 and 48 h, the H2O2 level increased to about 31%, 44%, 76%, 96% and 138%, respectively in Bachar and to about 52%, 76%, 76%, 110% and 147%, respectively in Giza 3 in comparison to the controls (Fig. 1g). Similarly, following treatment with NaCl, the Bachar and Giza 3 leaves (Fig. 1h) displayed an increase in H2O2 concentration at each time point of the time course. Although a higher H2O2 induction in Giza 3 leaves prior to the beginning of the NaCl treatment was observed (Fig. 1h), at 3 h after treatment Bachar and Giza 3 revealed a similar H2O2 content. However, a greater augmentation in H2O2 was observed in Giza 3 compared to Bachar, when subjected to salt stress at 6, 12 and 24 h.

MDA content, as an indicator of lipid peroxidation, was induced during the NaCl and PEG treatments in both studied genotypes (Fig. 1i, j). Giza 3 osmotic stressed plants showed higher levels of MDA than Bachar after 0, 3 and 6 h of treatment. However, no significant changes in MDA concentration were observed between Giza 3 and Bachar after 12, 24 and 48 h of osmotic stress induced by PEG (Fig. 1i).

Similar to the changes in H2O2 content, after exposure to osmotic stress, MDA increase was significantly higher in Giza 3 than in Bachar. Indeed, MDA accumulation in Giza 3 significantly increased by 40%, 25%, 36% and 23% at 3, 6, 24, and 48 h, respectively, compared to control. NaCl treatment showed changes very similar to PEG which resulted in a significant increase in MDA content at 3, 6, 24 and 48 h. In contrast, there was a remarkable decrease in MDA accumulation at 12 h in both genotypes (Fig. 1j). Compared to control plants, MDA accumulation significantly increased by 53%, 118%, 33% and 51% at 3, 6, 24 and 48 h respectively in Giza 3; while MDA accumulation significantly increased by 53%, 30%, 5% and 6%, at 3, 6, 24 and 48 h respectively in Bachar.

Expression profiles of selected genes in different faba bean tissues

To highlight the roles of the 25 selected osmotic stress-responsive genes (Table 1) in plant development, PCR was conducted to determine their tissue specificity patterns in leaves, cotyledons, roots, stems, flowers and seeds (Fig. 2). Faba bean elongation factor 1-alpha (VfELF1A) was used as an internal control and it was ubiquitously expressed in all tested organs. The expression of all studied genes showed in at least one tissue. Moreover, most of the selected genes (19 among 25, 76%) were differentially expressed in the different analyzed tissue. For instance, VfCOMT and VfDELLA were not found in flowers, whereas VfWRKY and VfLEA were slightly expressed. In the other hand, VfNod19 mRNA was not found in seeds and stems tissue, however a preferential accumulation of this transcript was revealed in root tissue. VfLEA6, VfWRKY, VfPS/TP, VfGST, VfLEA, VfDELLA, VfLEA3, VfHMT and VfPOX are highly expressed in leaves and lower in other tissues.

Fig. 2
figure 2

Tissue-specific expression using semiquantitative RT-PCR analysis of selected genes. RNA was isolated from vegetative tissues, such as leaves (L), cotyledons (C), roots (R), stems (St), as well as reproductive tissues, like flowers (F), and seeds (Se). Transcripts of faba bean elongation factor 1-alpha (VfELF1A) were used as a control

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Expression patterns of the selected genes under osmotic and salt stress

In order to determine early and genes differentially and strongly induced by osmotic and salt stress treatments, real-time PCR was carried out to analyze gene expression patterns of selected genes by using fold-change values transformed to Log2 of the fold difference in expression in leaves and roots stressed samples as compared to the corresponding control at 0 h time point. The hierarchical clustering of the genes is represented in heat maps which were performed to shown the expression patterns of selected genes in response to osmotic and salt stress. The white rectangles in the heatmap represent the gene showing maximum expression at a particular time point. Differential regulation of the majority of selected genes was shown in Fig. 3 under osmotic and salt stress in leaves tissue during the time-course. Obtained data indicate significant differences between gene expression patterns at every time point. Overall, significant differences in gene expression level were observed after 3 h of osmotic and salt stress treatments, suggesting a quick transcriptional regulation in both studied genotypes to the abiotic stress response. Most candidate genes showed high expression in osmotic and salt-stressed plants (Bachar and Giza 3), although some significant differences could also be observed. For the osmotic treatment (Fig. 3a), studied genes could be broadly divided into four clusters. The first cluster contains 8 (32%) members (VfERF, VfPOX, VfRD22, VfDELLA, VfLEA3, VfLEA, VfNAC and VfCIPK) of selected abiotic response genes, which were widely upregulated after 3 h by osmotic treatment, and the maximum induction being recorded at 6 h of PEG treatment. Cluster 2 has 10 (40%) members (VfMATE, VfHMT, VfDHN, VfPS/TP, VfUDP, VfGST, VfLEA6, VfMEM, VfERF-B3 and VfERD). Indeed, VfMATE, VfHMT, VfDHN, VfPS/TP and VfERD showed upregulated response at 3 or 6 h, but downregulated at 12, 24 and 48 h after osmotic treatment. However, VfUDP, VfGST, VfLEA6, VfMEM and VfERF-B3 showed a weakly upregulated expression at 3 or 6 h of stress, while at the other time spans, their expression remained unchanged. Cluster 3 contains 3 (12%) genes (VfTK, VfWRKY and VfNod19), which were mainly upregulated with the increased level of osmotic treatment after 3 h, and the VfTK in particular was highly induced by PEG treatment in Bachar and Giza 3 at 6 and 24 h, respectively. However, VfWRKY exhibited high expression level at 3 and 6 h respectively. In the other hand VfNod19 showed high level of expression at 12 and 24 h in Bachar and Giza 3 respectively. Cluster 4 mainly consists of 4 (16%) genes (VfMYB-related, VfAP2LP, VfCOMT and VfMYB), which were widely downregulated by osmotic treatment (Fig. 3a).

Fig. 3
figure 3

Heat map representation of the differential expression of the candidate genes in response to PEG-6000 (a) and NaCl (b) in the leaves of Giza 3 and Bachar at 0 (control), 3, 6, 12, 24 and 48 h after stress induction. White and red indicate higher and lower expression values, respectively. The intensity of the colors is proportional to the absolute value of log2 of the fold difference in gene expression

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For the salt stress treatment, the heatmap displayed five clusters of genes (Fig. 3b). Most (9, 36%) members (VfMATE, VfDHN, VfPS/TP, VfGST, VfLEA3, VfCIPK, VfNAC, VfWRKY, and VfPOX) of cluster 1 were mainly upregulated after the salt treatment and exhibited the highest expression at 12 and 48 h in Bachar and at 6 h in Giza 3. The 7 (28%) members (VfLEA, VfLEA6, VfMEM, VfHMT, VfUDP, VfERF-B3 and VfAP2LP) of cluster 2 were weakly upregulated or showed a relatively stable expression pattern under salt stress. Cluster 3 contained the smallest (2, 8%) members (VfTK and VfNod19) which were downregulated in continuation of salt stress for 48 h. The 4 (16%) members (VfERF, VfRD22, VfDELLA, and VfERD) of cluster 4 were mainly upregulated at 3 and 6 h of salt treatment but were downregulated after 12 h in Bachar. A similar expression of the members of cluster 4 in Giza 3 was observed, but the differences that they were upregulated at 6 and 12 h of salt stress and then downregulated at 24 h and 48 h of salt stress. Cluster 5 has 3 (12%) members (VfMYB-related, VfCOMT, and VfMYB) of the 25 studied genes, which were widely downregulated at 3, 6, and 24 h in leaves tissue of Bachar and at 3, 12, 24 and 48 h in Giza 3.

The expression of the selected genes in root tissues of Bachar and Giza 3 showed also different trends related to the duration of osmotic and salt stress (Fig. 4). Based on a hierarchical clustering analysis, the 25 studied genes were mainly clustered into three clusters when roots of both studied genotypes were subjected to osmotic stress, as shown in Fig. 4a. The first cluster represented 9 (36%) genes (VfTK, VfRD22, VfPS/TP, VfLEA3, VfMEM, VfDHN, VfAP2LP, VfUDP and VfNod19) which were found to be upregulated under osmotic challenge. The maximum accumulation of the first 6 members of the cluster was recorded after osmotic stress for 3, 6 and 12 h in both Bachar and Giza 3, while VfAP2LP, VfUDP and VfNod19 showed a striking induction at 24 and 48 h. The induction of VfTK, VfRD22, VfPS/TP, VfLEA3, VfMEM and VfDHN was noted especially in Bachar, while the induction in VfAP2LP, VfUDP and VfNod19 was recorded especially in Giza 3. The second cluster gathered together 14 (56%) genes (VfERF, VfWRKY, VfNAC, VfERD, VfGST, VfMATE, VfPOX, VfMYB, VfHMT, VfCOMT, VfLEA, VfCIPK, VfERF-B3 and VfLEA6) with a striking induction under osmotic stress at 6 and 12 h; they were found to be downregulated at 24 and 48 h, though there are some exceptions in the expressions of VfLEA, VfCIPK, VfERF-B3 and VfLEA6 which were obviously weakly upregulated or unchanged under osmotic stress. The third cluster was composed of 2 (8%) genes (VfDELLA and VfMYB-related) which were widely downregulated after PEG treatment.

Fig. 4
figure 4

Heat map representation of the differential expression of the candidate genes in response to PEG-6000 (a) and NaCl (b) in the roots of Giza 3 and Bachar at 0 (control), 3, 6, 12, 24 and 48 h after stress induction. White and red indicate higher and lower expression values, respectively. Intensity of the colors is proportional to the absolute value of log2 of the fold difference in gene expression

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As shown in Fig. 4b, the expression of all the selected genes in root tissue could be induced or repressed under NaCl stress treatment. The heatmap disclosed the genes distributed in five clusters. Cluster 1 and 2 (11 genes) showed most of the upregulated genes. Under salt conditions, all the 5 (20%) genes (VfLEA3, VfTK, VfNAC, VfMEM and VfPS/TP) of cluster 1 were found to be significantly upregulated at 3, 6 or 12 h in the root tissues of Bachar and at 3 or 48 h in Giza 3. The 6 members (VfDHN, VfPOX, VfERF, VfMATE, VfERD and VfGST) of cluster 2 were mainly upregulated after the NaCl treatment and exhibited the highest expression at 6 and 12 h in the root tissues of Bachar and at 3 and 48 h in Giza 3. However, we found that in Bachar members of cluster 2 were not significantly regulated under salt stress at 3, 24 and 48 h and in Giza 3 at 6, 12 and 24 h, as their expression patterns when compared to the control ones remained unaffected or downregulated in roots tissue. Seven (28%) members (VfRD22, VfLEA, VfERF-B3, VfUDP, VfLEA6, VfAP2LP, and VfCIPK) of cluster 3 showed no change or downregulated expression at each time point of the time course in root tissues of Bachar and Giza 3 with respect to the control conditions. The 5 (20%) members (VfMYB, VfWRKY, VfHMT, VfNod19, and VfCOMT) of cluster 4 were mainly upregulated after 3 h under NaCl treatment but were downregulated especially at 48 h in Bachar and Giza 3. The 2 (8%) members (VfDELLA and VfMYB-related) of cluster 5 had a similar expression with cluster 4, but the difference was the both VfDELLA and VfMYB-related were found to be dramatically downregulated at most time points.

Effects of exogenous ABA (abscisic acid) application on the expression patterns of 17 selected genes

Various phytohormones control multiple biological processes and act as regulators of plant response to abiotic stresses. ABA is a central regulator of environmental constraints responses in plants. To assess the differential expression of VfRD22, VfLEA, VfLEA6, VfAP2LP, VfCIPK, VfMYB, VfWRKY, VfMYB-related, VfPOX, VfDHN, VfERF, VfUDP, VfMATE, VfERD, VfLEA3, VfNAC, and VfGST under treatment with exogenous ABA, their expression profiles were investigated in the leaves (Fig. 5a) and roots (Fig. 5b) tissue of Bachar and Giza 3 using qRT-PCR analyses during 0 (control), 1, 3, 6 and 12 h durations of ABA treatment. Based on hierarchical clustering analysis, the 17 selected genes were mainly clustered into 4 clusters, as shown in Fig. 5a. All the studied genes were responsive to ABA treatment in the two studied genotypes. Interestingly, most of the studied genes revealed a similar expression pattern in Bachar and Giza 3, although some significant differences could also be observed. Cluster 1 contains 4 (23.5%) members (VfCIPK, VfLEA, VfERF, and VfPOX) which were widely upregulated after 1 h by ABA treatment, and up to the highest expression level at 12 h (Fig. 5a). Expression of the 4 (23.5%) members (VfDHN, VfMATE, VfLEA3 and VfWRKY) of cluster 2 was upregulated after 1 h by ABA treatment compared with control but downregulated or nearly unchanged at 12 h. Similar to cluster 1, cluster 3 consists of 4 (23.5%) genes (VFNAC, VfRD22, VfUDP and VfMYB-related) showing upregulated expression in response to the same treatment across all time points, although expressed at low levels. Five members (29.5%) of cluster 4 showed a weakly upregulated at 1, 3 and 6 h, though the expression level was more pronounced at 3 h but downregulated or nearly unchanged at 12 h after ABA treatment (Fig. 5a).

Fig. 5
figure 5

Heat map representation of the differential expression of 17 selected candidate genes in response to exogenous ABA treatment (100 µM) in leaves (a) and roots (b) of Giza 3 and Bachar at 0 (control), 1, 3, 6, and 12 h. White and red indicate higher and lower expression values, respectively. Intensity of the colors is proportional to the absolute value of log2 of the fold difference in gene expression

Full size image

For the ABA treatment of roots, these genes could be divided also into 4 clusters (Fig. 5b). With ABA treatment, the 5 (29.5%) genes (VfCIPK, VfERF, VfLEA, VfNAC and VfPOX) of cluster 1 showed significantly enhanced transcript accumulations in roots of Bachar and Giza 3, though the expression level was more pronounced at 6 h, where the expression was constitutive (Fig. 5b). The inducibility by ABA was clear from the gene expression pattern of cluster 2 in Bachar and Giza 3, the 5 (29.5%) genes (VfLEA3, VfDHN, VfMATE, VfERD, and VfWRKY) were widely upregulated after 1 h, then downregulated to reached control levels at 12 h. The highest expression level was shown in VfWRKY, which in particular was highly induced at 1 h after ABA treatment in Bachar and at 3 h in Giza 3. Cluster 3, representing a total of 6 (35%) genes (VfUDP, VfLEA6, VfGST, VfMYB, VfRD22 and VfAP2LP), were slightly upregulated at 1 and 3 h by ABA treatment in both genotypes but downregulated or nearly unchanged in Bachar at 6 and 12 h. However, expression was nearly unchanged at 6 h but downregulated at 12 h in Giza 3. Moreover, VfMYB-related which contains cluster 4 was downregulated by ABA treatment at an early stage (1 and 3 h after treatment) but was weakly upregulated at 6 and 12 h.

Discussion

In this study, physiological and biochemical stress indicators in response to environmental challenges showed that Bachar and Giza 3 displayed a variability of responses to osmotic and salt stress (Fig. 1).

Data revealed that osmotic and salt stress induced by PEG and NaCl, respectively, significantly affected RWC in Bachar and Giza 3. Under osmotic stress at 0, 3, 6 and 12 h, the RWC was recorded slightly highest in Bachar compared to Giza 3. In the other hand, similar results were obtained under salt application, although some significant differences could be observed. Indeed, at 12, 24 and 48 h, the RWC was higher in Bachar, than in Giza 3. Earlier significant decline and greater extent decrease in RWC were found in Giza 3 than in Bachar, which suggested a better osmotic and salt adaptation in Bachar. The results obtained here for Vicia faba were almost similar to previous studies (Siddiqui et al. 2015; Abid et al. 2017). Ghuge et al. (2010) also reported that osmotic stress induced by NaCl and PEG significantly reduced the relative water content of the potato plant. Results of this work are in agreement with previous studies in Vicia faba (Alghamdi et al. 2015) and other plant species like rice (Swapna and Shylaraj 2017) reporting that osmotic tolerant genotypes showed higher RWC rather than osmotic sensitive genotypes.

Proline is the most widely accumulated osmolyte under abiotic stress and which was correlated with tolerance to environmental constraints. Moreover, its acts as an antioxidant, and stress-related signaling molecule (Hayat et al. 2012). According to these authors, rapid and high amounts of free proline accumulation are a typical plant response to abiotic stress. Data showed that amount of free proline in leaves of Bachar and Giza 3 increased significantly under osmotic and salt stress. Under osmotic stress, proline significantly and particularly accumulated in Bachar at 48 h in comparison with Giza 3. Moreover, under salt stress Bachar plants increased proline content more than Giza 3. This increase was very significant at all time points. Augmentation of proline content in leaf tissues of faba bean due to abiotic stress has been noted in other researches (Siddiqui et al. 2015; Migdadi et al. 2016; Abid et al. 2017). Similar results were reported in several crops such as wheat (Mwadzingeni et al. 2016) and chickpea (Awasthi et al. 2017) which accumulate high level of proline in drought tolerant genotypes than sensitive. Data of the present investigation suggested that prominent accumulation of proline in Bachar constitutes a protective mechanism to decrease oxidative damage reducing osmotic stress in this genotype. According to Hare and Cress (1997), the level of proline accumulated in stressed-plants relatively correlated with the amount of carbohydrates. Indeed, the amount of soluble sugars was positively associated with abiotic stress tolerance in various plant species like faba bean (Siddiqui et al. 2015; Abid et al. 2017), rice (Redillas et al. 2012) and sugar beet (Wu et al. 2014). Current studies validated these findings with enhanced soluble sugars levels in osmotic/salt stressed Bachar and Giza 3 plants in comparison to control. Interestingly, these enhancements were more pronounced in the cultivar Bachar than in the Giza 3 throughout all time points. The amount of soluble sugar accumulated in Bachar and Giza 3 stressed-plants suggesting a potential role in osmotic adjustment, with a better performance of Bachar, particularly in maintaining a better water balance under osmotic stress (Farooq et al. 2009). Indeed, significant variations in free proline and soluble sugars appeared at earlier osmotic and salt stages in Bachar than in Giza 3.

Environmental challenges, contribute to formation of ROS (reactive oxygen species) in the cells. Among the various ROS, H2O2 is one of the most abundant in aerobic biological systems, being highly reactive and toxic. H2O2 causes perturbation of basic metabolic pathways, damages the membrane lipids, induces protein denaturation and may also inactivate enzymes, induces DNA mutations and at high level, it could lead to cell death (Sharma et al. 2012). As for indicators of oxidative stress, MDA and H2O2 contents were determined in leaves of Bachar and Giza 3. In this study, the H2O2 contents significantly increased with osmotic and salt stress progressed in the Bachar and Giza 3 cultivars but in contrast with proline and soluble sugars parameters, H2O2 has negative effects on osmotic tolerance. Differences were found in H2O2 contents between Bachar and Giza 3 seedlings when exposed to osmotic and salt pressure. At all time points Giza 3 exhibited the highest H2O2 content under PEG and NaCl treatments and compared to Bachar, it showed higher increased rate in contents of H2O2. Results of this study indicated significant differences among Bachar and Giza 3 for H2O2 accumulation and scavenging. These data were similar to the one obtained by Abid et al. (2017) in Vicia faba.

Among the lipid peroxidation products, malondialdehyde (MDA) is the most indicator of oxidative damage and used as potential marker to screen the tolerant and susceptible genotypes (Xu et al. 2008). In olive tree and turfgrass, low MDA content was correlated to abiotic stress tolerance (Bacelar et al. 2007; Xu et al. 2013). Leaf MDA content increased significantly in Bachar and Giza 3 osmotic and salt-stressed plant showing that the salinization and dehydration of faba bean is associated with lipid peroxidation mechanisms. Giza 3 had the highest MDA content at 3 and 6 h after PEG treatment, while the highest MDA content under salt stress appeared at 6 h after NaCl treatment. Results of this study suggested less severe lipid membrane damage in Bachar than Giza 3. Thus, lower level in leaves of Bachar suggests better protection of this cultivar against the peroxidation of the membrane under abiotic stress. The results are in good agreement with previous work, in which higher MDA have been reported in sensitive faba bean genotypes compared to the tolerant ones (Siddiqui et al. 2015; Abid et al. 2017). Similar results were reported by Chugh et al. (2013) in maize. Compared with Bachar, Giza 3 possessed a higher increased rate in contents of MDA and H2O2 and earlier significant changes were found in Giza 3, which suggested that Bachar possessed better osmotic tolerance and stronger osmotic adaptation.

The results indicated that Bachar and Giza 3 respond differentially to osmotic and salt exposure induced by PEG and NaCl. Compared with Giza 3, Bachar had the lowest MDA and H2O2 content and higher RWC. In the other hand, proline and soluble sugars content was the highest in this cultivar which suggest that Bachar was less affected than Giza 3 under these stress conditions. These data suggest that Bachar is more resistant to NaCl and PEG-induced osmotic stress than Giza 3. These observations are in agreement with some reports demonstrating that osmotic response of faba bean is greatly affected by genetic factors (Siddiqui et al. 2015; Migdadi et al. 2016; Abid et al. 2017).

In a context of unprecedented climate change, the development of drought and salt-tolerant cultivars is one of the most significant challenges of traditional Vicia faba breeding programs in the last decade and critical to sustain this grain legume production, particularly in the Mediterranean ecosystem. The traditional breeding approach limited the development of faba bean cultivars for abiotic stress tolerance. Indeed, plant response to abiotic stress is a complex trait involving several genes related with various cellular signaling pathways. Therefore, it is imperative to first identify genes involved in osmotic stress tolerance for understand the mechanism of abiotic stress tolerance. This may be lead to create new crop varieties with increased tolerance to environmental constraints. In recent years, several transcriptomic approaches including RNA deep sequencing and microarray (Hossain et al. 2016; Lu et al. 2017) have been employed to elucidate the differential expression of RNA transcripts involved in osmotic response which leads to increased knowledge about osmotic signaling pathways. Interestingly, for plants whose genomes have not been completely or partially sequenced, computational approaches such as in silico analysis provide a good way for discovering genes involved in plant stress tolerance (Ram and Sharma 2013).

Based on microarray data in salt-stressed Medicago truncatula plants (Li et al. 2009), an in silico analysis was performed in order to identify gene networks involved in faba bean abiotic stress tolerance. A total of 123 and 104 faba bean genes showing homology with upregulated and downregulated salt stress response genes in Medicago truncatula, respectively were determined. Among the identified genes, 25 (Table 1) were selected. These genes showed different functional categories including ABA-responsive. Their expression patterns were analyzed by real time PCR in leaves and roots tissue of Bachar and Giza 3. Some of the key genes are classified as transcription factors like WRKY, NAC, ERF, AP2LP and MYB. Others consist of functional proteins related to abiotic stress tolerance including LEA and CIPK or ROS detoxify enzymes like GST and POX. Most of the selected genes differentially expressed in response to abiotic stress, suggesting a potential role in the modulation of drought and salt stress tolerance. Moreover, transgenic lines over-expressing some of those genes, such as Tamarix hispida expressing GST (Yang et al. 2014), wheat plants overexpressing DHN-5 (Brini et al. 2007) or ERF (Rong et al. 2014), transgenic lines of Arabidopsis expressing TaWRKY1 and TaWRKY33 (Mao et al. 2011) or TaMYB2A (He et al. 2016) and transgenic rice plants expressing NAC transcription factor (Hong et al. 2016) showed increased tolerance to drought and salt stress. Indeed, expression patterns of these genes have been carried out in several plants under drought and salt constraint but have not been previously studied in Vicia faba. PCR data showed that most selected genes were expressed in various organs (leaf, root, flower, stem, cotyledon, and seed) of faba bean, but showed different spatial transcript accumulation pattern. This indicates that they may participate in growth and development and each member might play a particular physiological function.

Most selected genes showed significant differences in the relative mRNA levels between Bachar and Giza 3 under PEG, NaCl and ABA treatments. Indeed, under salt and osmotic stress, gene encoding VfPOX, VfLEA3, VfNAC, VfCIPK, VfLEA, VfWRKY, VfERF, VfRD22, VfDELLA, VfNod19, and VfTK showed constitutive upregulation in the leaves tissue at all examined time points, while VfMYB-related, VfMYB, VfCOMT and VfAP2LP showed constitutive downregulation. The remaining genes (VfERD, VfMATE, VfGST, VfDHN, VfPS/TP, VfLEA6, VfTMEM and VfERF-B3) were upregulated or downregulated at only specific time points, indicating a temporal control of gene transcription under osmotic stress. In general, the expression of these genes was upregulated in the leaves of the tolerant cultivar Bachar. In the root tissues of PEG and NaCl-stressed Bachar and Giza 3 plants, expressions for 7 genes (VfPOX, VfLEA3, VfNAC, VfWRKY, VfERF, VfRD22, and VfTK) out of 11 induced genes in leaves showed upregulated in a constitutive manner. Moreover, VfDHN, VfPS/TP, VfHMT and VfMYB were also found to show upregulation under all the examined conditions, whereas VfMYB-related, VfCIPK and VfDELLA showed downregulation. The rest of the genes are either expressed or turned off under specific conditions. Current evidence links changes in ABA levels and the expression of specific ABA-reponsive genes related to environmental stress response and tolerance. ABA application induced the expression of 16 out of 17 (94%) of the studied genes in the leaves and roots tissue of Bachar and Giza 3 compared to control, whereas the expression of the remaining gene (VfMYB-related) significantly downexpressed. Results of this study were in accordance with previous reports, which found that most of the studied genes are involved directly or indirectly in abiotic stresses responses in plants and many of them have been reported as key regulators of plant response to osmotic stress. The WRKY transcription factors play crucial roles in growth, development and abiotic stresse response in plant. Indeed, some WRKY genes like Arabidopsis WRKY8 (Hu et al. 2013), Moso bamboo PeWRKY83 (Wu et al. 2017), cotton GhWRKY68 (Jia et al. 2015) and soybean GmWRKY21/54 (Zhou et al. 2008) were upregulated by drought, salt or ABA treatment. Interestingly, gain-of-function of these WRKY genes enhanced transgenic lines' tolerance to osmotic stress. Exposed to salinity, 10 GSTs and one POX genes encoding antioxidant enzymes were upregulated in root tissues of citrus plants and played important protective roles in coping with excessive accumulation of ROS (Xie et al. 2018). Expression profiles of VfGST and VfPOX suggest their role in stress adaptation and tolerance towards various abiotic stresses.

In plants, the MYB (myeloblastosis) proteins constitute one of the largest transcription factors family which play a number of roles in regulating plant growth, metabolism and various stress responses. Dubos et al. (2010) classified MYB proteins into 4 classes (1R-MYB, R2R3-MYB, 3R-MYB and 4R-MYB) based to the number of conserved MYB DNA-binding domain. Overall, 155 and 197 MYB genes were identified in Oryza sativa and Arabidopsis thaliana and among them; several members are upregulated by various abiotic stresses. Overall, 155 and 197 MYB genes were identified in rice and Arabidopsis and among them; several members are upregulated by various abiotic stresses. Indeed, using real time PCR, the expression of 28 OsMYB genes among 60 showed significantly increased under drought stress in rice. However, among the 21 MYB genes analyzed in Arabidopsis 7 AtMYB were upregulated and 7 AtMYB were downregulated (Katiyar et al. 2012). In watermelon, exogenous ABA application induced expression of 19 MYB genes, whereas the 4 rest genes showed repressed expression in comparison with the control (Xu et al. 2017). The MYB gain-of-function mutants in rice (Yang et al. 2012) and Arabidopsis (Ding et al. 2008) showed increased resistance to abiotic stress. These results indicated that a large number of MYB genes could play a role against abiotic stress in rice and Arabidopsis. VfMYB exhibited significantly reduced expression in leaves tissue under PEG and NaCl treatment. However, VfMYB-related showed significantly reduced transcript levels under PEG, NaCl and ABA treatment in leaves and roots of Bachar and Giza 3. This could suggest the involvement of VfMYB-related in the crosstalk of different signaling and metabolic pathways. This was in line with early results found in watermelon reported by Xu et al. (2017). These authors revealed a remarkably reduction in the expression of ClaMYB1R7 gene encoding MYB-related class under salinity and exogenous application of ABA.

The AP2/ERF family is a large gene family transcription factors which is unique to plant and they play major roles not only in plant growth, development and metabolite biosynthesis but also are considered as central regulators in cold, drought, salt and heat stress responses (Gu et al. 2017). According to the classification adopted by Licausi et al. (2010) based on the difference of the common DNA binding domain copy numbers, the superfamily AP2/ERF includes five subfamilies: AP2 (APETALA2), ERF (ethyleneresponsive-element-binding-factor) which could be classified into six groups (B1–B6), DREB (dehydration responsive element binding), RAV (related to ABI3/VP) and other proteins (Soloist). Generally, AP2/ERF members are stimulated by ABA signals and can be induced in plants by different abiotic stresses (Gu et al. 2017). Rice, tomato and tobacco transgenic lines overexpressing ERF members showed enhanced resistance to drought stress and salinity (Guo et al. 2004; Lu et al. 2010; Zhang et al. 2010). Moreover, mutants tomato and wheat overexpressed SlERF5 (Pan et al. 2012) and TaERF3 (Rong et al. 2014) exhibited significantly enhanced tolerance to abiotic stress. Obtained data showed that faba bean AP2/ERF members differentially expressed suggesting a crucial role in abiotic stress and ABA response. Indeed, VfERF and VfERF-B3 were upregulated, while VfAP2LP was significantly downregulated particularly in leaves tissue under osmotic and ABA treatments. Recently, Dossa et al. (2016) reported similar results in sesame regarding the transcripts accumulation of AP2/ERF under water deficit. Indeed, this environmental stress has decreased the transcript abundance of AP2si115, AP2si47, AP2si103 and AP2si11.

VfNAC transcription factor was also found to be strongly and differentially induced in leaves and roots of Bachar and Giza 3 in response to different treatments. This suggested a potential usefulness of VfNAC in improving abiotic stress resistance of faba bean. Various NAC transcription factors have been documented as central regulators during plant stress response (Puranik et al. 2012). Moreover, several NAC genes such as OsNAC6 were induced by ABA, cold, salt and drought stresses (Ohnishi et al. 2005). Overexpressing of rice OsNAC6 gene conferred dehydration and enhanced resistance to salinity (Nakashima et al. 2007). More recently, in transgenic rice overexpression of ONAC022 also caused an increase in drought and salt stress tolerance (Hong et al. 2016).

In plants, late embryogenesis abundant (LEA) proteins form a large family associated with plant responses and adaption to environmental stress, that includes seven different groups (LEA1–LEA7) based on their sequence similarities, repeated motifs and amino acid composition (Battaglia et al. 2008). In different plant species, genes called dehydrins (DHNs), responsive to dehydration (RD), early responsive to dehydration (ERD), cold-inducible (KIN), cold regulated (COR) and responsive to ABA (RAB) encodes for LEA type proteins have been reported previously (Zhu 2002). It is well known that LEA proteins are potentially expressed under different environmental stresses like salinity, drought, cold and heat as well as ABA treatment. Moreover, overexpression of LEA proteins from various groups showed improved tolerance to abiotic stress. Indeed, rice plants overexpressed OsLEA4 gene exhibited high tolerance to various stresses such as drought, salinity and heavy metals (Hua et al. 2016).

In the other hand, overexpression of dehydrine gene (OsDhn1) in rice improved its tolerance to drought and salt stress by effectively scavenging ROS (Kumar et al. 2014). In this work, VfLEA, VfLEA3, VfLEA6, VfDHN and VfERD genes were induced under PEG, NaCl, and ABA treatments, but showed completely different expression patterns among Bachar and Giza 3 suggesting their potential role in salt and osmotic stresses response in faba bean. Multiple genes encoding calcium sensors in plants decode Ca2+ signals including calcium-dependent protein kinases (CDPKs), calmodulin-dependent protein kinases (CaMKs), calmodulin-like proteins (CML), calmodulin (CaM) and calcineurin B-like [CBL] protein interaction protein kinase (CIPK) have been shown to be involved in the regulation of abiotic stress response in plants (Das and Pandey 2010). According to Kim et al. (2003), ABA, salinity, drought and cold treatment resulted in the higher accumulation of CIPK3 transcript in Arabidopsis. Moreover, drought, ABA, PEG, cold, salt and heat treatment significantly induced the expression of ZmCIPK16 in maize (Zhao et al. 2009). Interestingly, rice plants overexpressed OsCIPK03, OsCIPK12 and OsCIPK15 gene exhibited a higher cold, drought and salt tolerance (Xiang et al. 2007). Overall, transcripts accumulation of VfCIPK gene showed increased after treatment with PEG, NaCl and ABA and showed different trends related to the duration of stress exposure in Bachar and Giza 3, indicating their possible function in Vicia faba abiotic stress response.

Conclusion

From this study, we can conclude that PEG (21%) and NaCl (200 mM) treatments affected significantly physiological and biochemical parameters, with lesser effects on Bachar compared to Giza 3. Indeed, Bachar showed greater accumulation of proline and soluble sugars and less accumulation of MDA and H2O2 under osmotic and salt stress, suggested that this cultivar exhibited higher osmotic and salt tolerance than Giza 3 through the accumulation of osmoprotectants and a better antioxidant machinery performance under osmotic stress. Analysis of expression profiles showed a differential transcriptional regulation of the selected genes in leaves and roots tissue of Bachar and Giza 3. The latter is an aspect of the varietal differences in osmotic stress tolerance, suggesting also that these genes might function collaboratively and play crucial roles to modulate faba bean plant response and tolerance to different environmental stresses. These results may contribute to decipher the transcriptional regulation signaling networks in response to osmotic and salt stress in faba bean.

Author contribution statement

GA: performing the experiments, conducted research, and writing of the manuscript. MNS, RNO and YM: planning of the analysis and correcting of the manuscript. SHJ, EG, KS, J-PB, MEA, SE, FB and MJ: helped in the analysis of the results and editing the manuscript. All the authors have read and approved the final manuscript.