Introduction

Chromium (Cr) is one of the most abundant natural elements in the Earth’s crust and a dangerous heavy metal that is toxic to both flora and fauna (Emsley 2001; Velma et al. 2009; Wang et al. 2013). Chromium exists naturally in both trivalent (CrIII) and hexavalent (CrVI) states and shows variability in mobility, bioavailability, and toxicity (Hayat et al. 2012). Hexavalent Cr is water soluble and more toxic than CrIII. Besides occurring naturally, both forms of this metal accumulate in the environment from anthropogenic activities (Chary et al. 2008; Ali et al. 2011; Kováčik et al. 2013). The release of Cr from tanneries, electroplating factories, chemical plants, and refractory steel industries represents major sources of Cr contamination in the environment (Shanker et al. 2005; Ganesh et al. 2009; Homa et al. 2016; Gorny et al. 2016). In the last few decades, Cr pollution has drastically increased. As a result, Cr toxicity is now a major threat to agricultural land and water bodies (Choudhary et al. 2012). Excess Cr contamination in soil and water results in accumulation of this toxic element in plants and subsequently into the food chain (Wang et al. 2013). Chromium is potently mutagenic and carcinogenic to biological systems (Emsley 2001), even at low concentrations. In plants, Cr inhibits seed germination and plant growth, alters plant metabolism, reduces nutrient uptake, and inhibits photosynthesis by negatively affecting chlorophyll (chl) formation and chloroplast ultrastructure (Shanker et al. 2005; Gbaruko and Friday 2007). Like other toxic metals, Cr can cause oxidative stress in plants by reducing CO2 fixation and photosynthesis through inhibiting electron transport and inactivating the Calvin cycle enzymes and ultimately affects growth and development (Shanker et al. 2005; Sundaramoorthy et al. 2010; Zhang et al. 2010). Chromium induced oxidative stress oxidizes cellular components of plants such as lipids, proteins, and nucleic acids through excess generation of reactive oxygen species (ROS) including singlet oxygen (1O2), superoxide anion (O2 •−), hydrogen peroxide (H2O2), and the hydroxyl radical (OH•) (Apel and Hirt 2004; Hasanuzzaman and Fujita 2012; Trinh et al. 2014). To alleviate the harmful effects of heavy metals including Cr, plants avoid metal binding to the cell wall, decrease transport across the cell membrane, and increase compartmentalization and metal chelation or exclusion (Li et al. 2002; Hall 2002; Gratão et al. 2005; Hasanuzzaman et al. 2012a). Furthermore, to minimize oxidative stress, plant cells have non-enzymatic antioxidants (ascorbic acid, AsA; glutathione, GSH; phenolic compounds; alkaloids; α-tocopherol and non-protein amino acids) and antioxidant enzymes (superoxide dismutase, SOD; catalase, CAT; ascorbate peroxidase, APX; glutathione reductase, GR; monodehydroascorbate reductase, MDHAR; dehydroascorbate reductase, DHAR; glutathione peroxidase, GPX; and glutathione S-transferase, GST) within the antioxidant defense system (Gill and Tuteja 2010; Hasanuzzaman et al. 2012a). Methylglyoxal (MG) is another cytotoxic reactive oxidative compound that is spontaneously produced in plants. However, under abiotic stresses, MG production increases several fold, resulting in damage to the cellular ultra-structural apparatus and causing mutation and even cell death (Yadav et al. 2005, 2008; Hasanuzzaman et al. 2017a). Plants have an MG detoxification system consisting of two essential enzymes, glyoxalase I (Gly I) and glyoxalase II (Gly II), which maintain the balance of MG production with the help of GSH (Yadav et al. 2008; Nahar et al. 2016a). However, the effectiveness of this system differs to a great extent with plant genotypes, dose, and duration of heavy metal stress.

Using exogenous protectants such as plant hormones, organic acids, signaling molecules, and trace elements is now common in research and is expected to enhance abiotic stress tolerance including that of metal toxicity. γ-aminobutyric acid (GABA) is a four-carbon non-protein amino acid that is present in living systems from bacteria to plants, and even in vertebrates (Bouché and Fromm 2004). GABA is involved in an array of metabolic processes in plants, and it is considered to be an internal signal molecule that can swiftly congregate and respond to environmental stimuli (Roberts 2007). Recently, exogenous GABA has been found to enhance plant tolerance to oxidative damage from chilling stress in tomato and wheat (Malekzadeh et al. 2012, 2014), heat stress in rice (Nayyar et al. 2014), drought stress in black pepper (Vijayakumari and Puthur 2016), and aluminum (Al) toxicity in barley (Song et al. 2010). However, GABA’s role in plant response to Cr stress is still unknown. Also, the combined performance of the antioxidant defense and glyoxalase systems under Cr stress has not yet been investigated. On the other hand, different genera of the Brassicaceae family are known as hyperaccumulators of heavy metals, meaning they can easily accumulate high amount of heavy metals (Prasad and Freitas 2003). Therefore, the purpose of this study was to investigate the physiological effect of GABA on metal accumulation, the biochemical and physiological parameters including the antioxidant defense and glyoxalase systems, as well as to determine the effectiveness of counteracting Cr stress in Brassica juncea L. As far as we know, this study is the first to report such an investigation on role of GABA in mustard seedlings under Cr toxicity.

Materials and methods

Plant materials and stress treatments

Uniform seeds of mustard (Brassica juncea L. cv. BARI Sharisha 11) were selected and surface sterilized with 70% ethanol followed by washing several times with sterilized distilled water. After that, the sterilized seeds were sown in Petri dishes (9 cm) lined with six layers of filter paper moistened with 10 mL of distilled water for germination and kept for 2 days in a germinator. Each Petri dish contained 60 germinated seedlings, which were grown under controlled conditions (light, 350 µmol photon m−2 s−1; temperature, 25 ± 2 °C; relative humidity, 65–70%) in a growth chamber; 5000-fold diluted Hyponex solution (Hyponex, Japan) was applied as nutrient every day as necessary. Eight-day-old mustard seedlings were exposed to GABA (125 ​μM) and Cr (0.15 and 0.3 mM K2CrO4) separately and in combination. The concentrations of 0.15 and 0.3 mM K2CrO4 were considered as mild stress and severe stress, respectively. The control seedlings were grown in Hyponex solution only. After 5 days of Cr treatment, the leaves and roots were harvested and used to study various morphological and physiological parameters. The experiment was conducted following a completely randomized design (CRD) with six treatments and was repeated three times under the same conditions.

Measurement of growth parameters

Plant height was measured for each treatment and expressed as centimeters (cm). Ten randomly selected fresh seedlings from each treatment were weighed, recorded, and considered as fresh weight (FW). Dry weight (DW) was determined after drying the seedlings at 80 °C in an oven for 48 h. Both DW and FW were expressed in milligrams (mg).

Measurement of relative water content

Relative water content (RWC) was measured according to Barrs and Weatherly (1962). Whole leaf discs were weighed as FW and then floated on distilled water in Petri dishes and kept in a dark place. After 8 h, the leaf discs were weighed again after removing excess surface water, and considered as turgid weight (TW). Finally, DW was measured after drying at 80 °C for 48 h. Leaf RWC was calculated using the following formula:

$${\rm{RWC (\% ) = }}\left[ {\left( {{\rm{FW - DW}}} \right) - \left( {{\rm{TW - DW}}} \right)} \right] \times 100$$

Measurement of Cr content, biological concentration factor (BCF), biological accumulation coefficient (BAC) and translocation factor (TF)

Chromium content was determined by using an atomic absorption spectrophotometer (Hitachi Z-5000, Japan). The plant samples were oven dried at 80 °C for 72 h. The dried samples from the roots and shoots (0.1 g) were ground and digested separately with acid mixture (HNO3:HClO4 = 5:1v/v) for 48 h at 80 °C. The absorbance of the samples was then recorded from the atomic absorption spectrophotometer, and the Cr content of the shoots and roots was calculated using a standard curve of known concentration.

BCF, BAC, and TF value were measured according to Malik et al. (2010).

BCF = Metal (roots)/ metal (growing media)

BAC = Metal (shoots)/ metal (growing media)

TF = Metal (shoots)/ metal (roots)

Measurement of phytochelatin content

Phytochelatin (PC) content was determined by subtracting the amount of GSH from the amount of total non-protein thiols. To examine non-protein thiols, the leaves were homogenized in 3% (w/v) sulfosalicylic acid. Using Ellman’s reaction mixture, non-protein thiol content was measured at 412 nm (Ellman 1959).

Measurement of chlorophyll content

Chlorophyll content was measured following the method of Arnon (1949). The leaves (0.5 g) were extracted with 10 mL 80% v/v acetone and supernatant was obtained by centrifuging at 2000 × g for 10 min. After diluting the supernatant, the absorbance was measured with a UV-visible spectrophotometer at 663 and 645 nm for chl a and chl b content, respectively.

Measurement of proline content

Proline (Pro) content was measured according to Bates et al. (1973). Fresh leaf tissue (0.5 g) was homogenized in 5 mL of 3% sulfosalicylic acid in an ice-cold condition, and the homogenate was centrifuged at 11,500 × g for 15 min. The supernatant (1 mL) was mixed with 1 mL of acid ninhydrin and 1 mL of glacial acetic acid, and the mixture was placed in a water bath (100 °C) for 1 h. The mixture was then transferred to a test tube and kept on ice for cooling. Toluene (2 mL) was added to the cooled mixture and mixed thoroughly using a vortex machine. After few minutes, chromophore-containing toluene was read spectrophotometrically at 520 nm. The proline content of the sample was determined by comparing with a standard curve of known concentration of Pro.

Histochemical detection of hydrogen peroxide and superoxide

Hydrogen peroxide (H2O2) and superoxide (O2 •−) were localized histochemically by staining the Brassica leaves according to the method described in Chen et al. (2010). Leaves were stained in 0.1% 3-diaminobenzidine (DAB) and 0.1% nitrobluetetrazolium chloride (NBT) solution for 24 h under a dark condition for H2O2 and O2 •−, respectively. Incubated leaves were then blenched by immersing in boiling ethanol. After that, brown spots appeared resulting from the reaction of DAB with H2O2 and dark blue spots appeared resulting from the reaction of NBT with O2 •−. Photographs were then taken by placing the leaves on glass.

Measurement of lipid peroxidation

The level of lipid peroxidation was measured by estimating malondealdehyde (MDA) content according to Heath and Packer (1968) with slight modification by Hasanuzzaman et al. (2012b). Leaf samples (0.5 g) were homogenized in 3 mL 5% (w/v) trichloroacetic acid (TCA), and the homogenate was centrifuged at 11,500 × g for 15 min. The supernatant (1 mL) was mixed with 4 mL of thiobarbituric acid (TBA) reagent (0.5% of TBA in 20% TCA). The reaction mixture was heated at 95 °C for 30 min in a water bath and then quickly cooled in an ice bath and centrifuged again at 11,500 × g for 10 min. The absorbance of the colored supernatant was measured at 532 nm and was corrected for non-specific absorbance at 600 nm. MDA content was calculated by using extinction coefficient 155 mM−1 cm−1 and expressed as n mol of MDA g−1 FW.

Determination of hydrogen peroxide content

Hydrogen peroxide was determined according to the method of Yu et al. (2003). Leaf tissue (0.5 g) was homogenized with 3 mL of 50 mM potassium–phosphate (K–P) buffer (pH 6.5) at 4 °C. The homogenate was centrifuged at 11,500 × g for 15 min. The supernatant (2 mL) was mixed with 666.4 μL of 0.1% TiCl4 in 20% H2SO4 (v/v) and was kept at room temperature for 10 min. After that, the mixture was again centrifuged at 11,500 × g for 12 min. The supernatant was then measured spectrophotometrically at 410 nm to determine H2O2 content using extinction coefficient 0.28 μM−1 cm−1 and was expressed as n mol g−1 FW.

Measurement of methylglyoxal level

Methylglyoxal was measured following the method of Wild et al. (2012). Leaves were homogenized in 5% perchloric acid and centrifuged at 4 °C for 10 min at 11,000 × g. The supernatant was decolorized by adding charcoal. The decolorized supernatant was neutralized by adding a saturated solution of sodium carbonate at room temperature. The neutralized supernatant was used to estimate MG by adding sodium dihydrogen phosphate and N-acetyl-l-cysteine to a final volume of 1 mL. Formation of the product N-α-acetyl-S-(1-hydroxy-2-oxoprop-1-yl) cysteine was recorded after 10 min at a wavelength of 288 nm, and the MG content was calculated using a standard curve of known concentration.

Extraction and measurement of ascorbate and glutathione

Fresh leaves (0.5 g) were homogenized in 3 mL ice-cold 5% meta-phosphoric acid containing 1 mM ethylenediaminetetraacetic acid (EDTA) using a mortar and pestle. The homogenate was centrifuged at 11,500 × g for 12 min at 4 °C, and the supernatant was collected to analyze for AsA and GSH. Ascorbate content was determined following the method of Huang et al. (2005) with some modifications. The supernatant was neutralized with 0.5 M K-P buffer (pH 7.0), and the oxidized fraction was reduced by 0.1 M dithiothretitol. Total and reduced AsA was assayed spectrophotometrically at 265 nm in 100 mM K-P buffer (pH 7.0) with 0.5 units of ascorbate oxidase (AO). A specific standard curve of AsA was used for quantification. Oxidized AsA (DHA) was calculated by subtracting AsA from total AsA. The GSH pool was assayed according to a previously described method (Yu et al. 2003) with modifications as described by Paradiso et al. (2008). Aliquots (0.2 mL) of supernatant were neutralized with 0.3 mL of 0.5 M K-P buffer (pH 7.0). Based on enzymatic recycling, GSH is oxidized by 5,5-dithio-bis (2-nitrobenzoic acid) (DTNB) and reduced by nicotinamide adenine dinucleotide phosphate (NADPH) in the presence of GR, and GSH content was evaluated by the rate of absorption changes at 412 nm of 2-nitro-5-thiobenzoic acid (NTB) generated from the reduction of DTNB. Oxidized glutathione (GSSG) was determined after removing GSH by 2-vinylpyridine derivatization. Standard curves with known concentrations of GSH and GSSG were used. The content of GSH was calculated by subtracting GSSG from total GSH.

Determination of protein

The protein concentration of each sample was determined according to Bradford (1976) using BSA as a protein standard.

Enzyme extraction and assays

Leaf tissue (0.5 g) was homogenized in 1 mL of 50 mM ice-cold K-P buffer (pH 7.0) containing 100 mM KCl, 1 mM AsA, 5 mM β-mercaptoethanol, and 10% (w/v) glycerol using a pre-cooled mortar and pestle. The homogenates were centrifuged at 11,500 × g for 10 min and the supernatants were used to determine enzyme activity. A temperature of 0–4 °C was maintained for all the activities.

Lipoxygenase (LOX; EC 1.13.11.12) activity was determined following the method of Doderer et al. (1992) by monitoring the increase in absorbance at 234 nm using linoleic acid as a substrate, and the activity was calculated using extinction coefficient 25 mM−1 cm−1.

Superoxide dismutase (SOD; EC 1.15.1.1) activity was measured based on the xanthine-xanthine oxidase system following the method of El-Shabrawi et al. (2010). The reaction mixture contained K-P buffer (50 mM), 2.24 mM NBT, catalase (0.1 units), xanthine oxidase (0.1 units), xanthine (2.36 mM), and enzyme extract. A change in absorbance was read at 560 nm. Superoxide dismutase activity was expressed as units (amount of enzyme required to inhibit NBT reduction by 50%) min−1 mg−1 protein.

Catalase (CAT; EC: 1.11.1.6) activity was assayed following the method of Hasanuzzaman et al. (2012b) by monitoring the decrease in absorbance at 240 nm for 1 min caused by the decomposition of H2O2. The reaction mixture contained 50 mM K-P buffer (pH 7.0), 15 mM H2O2, and enzyme solution in a final volume of 700 μL. The reaction was initiated with the enzyme extract and activity was calculated using extinction coefficient 39.4 M−1 cm−1.

Ascorbate peroxidase (APX; EC: 1.11.1.11) activity was determined according to the method of Nakano and Asada (1981) with a solution mixture of 50 mM K-P buffer (pH 7.0), 0.5 mM AsA, 0.1 mM H2O2, 0.1 mM EDTA, and enzyme extract in a final volume of 700 μL. The activity was measured by observing the decrease in absorbance at 290 nm for 1 min using extinction coefficient 2.8 mM−1 cm−1.

Monodehydroascorbate reductase (MDHAR, EC: 1.6.5.4) activity was measured following the method described in Hossain et al. (1984). The reaction mixture contained 50 mM Tris-HCl buffer (pH 7.5), 0.2 mM NADPH, 2.5 mM AsA, 0.5 units of AO, and enzyme solution in a final volume of 700 μL. The absorbance was measured at 340 nm for 1 min and activity was calculated using extinction coefficient 6.2 mM−1 cm−1.

Dehydroascorbate reductase (DHAR; EC: 1.8.5.1) activity was assayed according to the method of Nakano and Asada (1981). The reaction buffer contained 50 mM K-P buffer (pH 7.0), 2.5 mM glutathione (GSH), 0.1 mM EDTA, and 0.1 mM dehydroascorbate (DHA). The activity was measured from the change in absorbance at 265 nm for 1 min and calculated using extinction coefficient 14 mM−1 cm−1.

Glutathione reductase (GR; EC: 1.6.4.2) activity was assayed according to the method of Hasanuzzaman et al. (2012b) by monitoring the decrease in absorbance at 340 nm for 1 min. The reaction mixture contained 0.1 M K-P buffer (pH 7.0), 1 mM EDTA, 1 mM GSSG, 0.2 mM NADPH, and enzyme extract. The activity was calculated using extinction coefficient 6.2 mM−1 cm−1.

Glutathione peroxidase (GPX; EC: 1.11.1.9) activity was assayed using the method of Elia et al. (2003). The reaction mixture consisted of 100 mM K-P buffer (pH 7.0), 1 mM EDTA, 1 mM sodium azide (NaN3), 0.12 mM NADPH, 2 mM GSH, 1 unit GR, 0.6 mM H2O2 (as a substrate), and 20 μL of sample solution. The oxidation of NADPH was recorded at 340 nm for 1 min and the activity was calculated using extinction coefficient 6.62 mM−1 cm−1.

Glyoxalase I (Gly I; EC: 4.4.1.5) activity was determined following the method of Hasanuzzaman et al. (2012b). The assay mixture contained 100 mM K–P buffer (pH 7.0), 15 mM magnesium sulphate, 1.7 mM GSH, and 3.5 mM MG in a final volume of 700 μL. The increase in absorbance was recorded at 240 nm for 1 min. The activity was calculated using extinction coefficient 3.37 mM−1 cm−1.

Glyoxalase II (Gly II; EC: 3.1.2.6) activity was determined according to Principato et al. (1987). The reaction mixture contained 100 mM Tris-HCl buffer (pH 7.2), 0.2 mM DTNB, and 1 mM S-D-lactoylglutathione (SLG). The change in absorbance was recorded at 412 nm, and the activity was calculated using extinction coefficient 13.6 mM−1 cm−1.

Statistical analysis

The data were subjected to analysis of variance (ANOVA), and the mean differences were compared by Fischer's Least Significant Difference (LSD) test using XLSTAT v.2016 software (Addinsoft 2016) from three replicates. Differences at P  ≤ 0.05 were considered significant.

Results

Growth and biomass

In our study, the Cr-stressed seedlings showed reduced growth compared with the control seedlings including plant height, FW and DW (Table 1). Plant height decreased by 20 and 25%, FW decreased by 20 and 28%, and DW decreased by 9 and 18% under mild (0.15 mM K2CrO4) and severe (0.3 mM K2CrO4) stress, respectively, compared with control. Adding GABA to the Cr-stressed plants alleviated the growth inhibitory effects and increased plant height, FW, and DW significantly (in contrast to Cr treatment without GABA) (Table 1).

Table 1 Growth, leaf relative water content (RWC), and proline (Pro) content of mustard seedlings under chromium (Cr) stress
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Plant water status and Pro content

Leaf RWC decreased significantly under both levels of Cr stress (Table 1). As a result, plants suffered from water deficit stress. Increased Pro level in the Cr-stressed seedlings further confirmed the water deficit condition. Proline content was increased by 113 and 186% under mild and severe stress, respectively. However, exogenous application of GABA to the Cr-stressed plants increased leaf RWC and decreased Pro content (Table 1).

Photosynthetic pigments

Chlorophyll (a+b) as well as total chl content decreased gradually with the increase in Cr stress levels. Chlorophyll a content decreased by 54 and 69%, and chl b content decreased by 44 and 59% under mild and severe stress, respectively, resulting in reduced total chl [chl (a + b)] content in contrast to control (Fig. 1). Compared with Cr stress alone, adding GABA with Cr was effective in restoring the content of chl a, chl b, and total chl in the Cr-treated seedlings (Fig. 1). Adding GABA to the mild and severe Cr-stressed plants increased chl (a+b) content by 32 and 38%, respectively, in contrast to Cr stress only.

Fig. 1
figure 1

Chlorophyll (chl) a (a), chl b (b), and total chl (c) content of mustard seedlings under chromium (Cr) stress. Cr1, Cr2, and GABA indicate 0.15 mM K2CrO4, 0.3 mM K2CrO4, and 125 µM γ-aminobutyric acid, respectively. Means (±SD) were calculated from three replications (n = 3) for each treatment. Bars with different letters are significantly different at P  ≤ 0.05 applying Fisher’s LSD test

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Histochemical detection of H2O2 and O2 •−generation

Leaves were subjected to staining by DAB and NBT to see the generation of ROS: DAB staining resulted in brown patches of H2O2 and NBT staining resulted in dark blue spots of O2 •−. Chromium stress increased H2O2 and O2 •− generation in the leaves compared with control (Fig. 2). Applying GABA to the Cr-stressed plants decreased the spots on the leaves produced by H2O2 and O2 •−, indicating a reduction in ROS generation compared with Cr stress alone (Fig. 2).

Fig. 2
figure 2

Histochemical detection of hydrogen peroxide (H2O2) (a) and superoxide (O2 •ˉ) (b) in the leaves of mustard seedlings under chromium (Cr) stress. Cr1, Cr2, and GABA indicate 0.15 mM K2CrO4, 0.3 mM K2CrO4, and 125 µM γ-aminobutyric acid, respectively

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Oxidative stress indicators

Compared with control, lipid peroxidation (MDA content) increased by 34% under mild stress and by 58% under severe stress (Fig. 3a). The content of H2O2 increased by 20 and 43% and that of LOX by 39 and 71% under mild and severe stress, respectively (Fig. 3b, c). The rise in H2O2 and LOX activity was responsible for the increase in lipid peroxidation under Cr stress. Conversely, applying GABA reduced the H2O2 content and LOX activity, which decreased lipid peroxidation in the mustard seedlings under Cr stress (in contrast to the Cr-affected plants without GABA) (Fig. 3).

Fig. 3
figure 3

Malondialdehyde (MDA) content (a), hydrogen peroxide (H2O2) content (b), and lipoxygenase (LOX) activity (c) of mustard seedlings under chromium (Cr) stress. Cr1, Cr2, and GABA indicate 0.15 mM K2CrO4, 0.3 mM K2CrO4, and 125 µM γ-aminobutyric acid, respectively. Means (±SD) were calculated from three replications (n = 3) for each treatment. Bars with different letters are significantly different at P  ≤ 0.05 applying Fisher’s LSD test

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AsA-GSH pool

Chromium stress decreased the AsA content and increased the DHA content, which ultimately decreased the AsA/DHA ratio in the plants (Fig. 4a–c). Compared with control, the AsA/DHA ratio was decreased by 70 and 83% resulting from mild and severe stress, respectively. Adding GABA to the Cr-stressed plants decreased the DHA content, increased the AsA content, and finally the AsA/DHA ratio increased by 81 and 51% under mild and severe stress, respectively (compared with Cr stress only) (Fig. 4a–c). Compared with control, the GSH and GSSG content increased at both levels of Cr stress resulting in 16 and 36% reduction in the GSH/GSSG ratio under mild and severe stress, respectively (Fig. 4d–f). Adding GABA to the Cr-stressed plants increased the GSH/GSSG ratio by 25 and 35%, respectively (compared with stress-affected seedlings without GABA) (Fig. 4f).

Fig. 4
figure 4

Ascorbate (AsA) content (a), dehydroascorbate (DHA) content (b), AsA/DHA ratio (c), glutathione (GSH) content (d), oxidized glutathione (GSSG) content (e), and GSH/GSSG ratio (f) of mustard seedlings under chromium (Cr) stress. Cr1, Cr2, and GABA indicate 0.15 mM K2CrO4, 0.3 mM K2CrO4, and 125 µM γ-aminobutyric acid, respectively. Means (±SD) were calculated from three replications (n = 3) for each treatment. Bars with different letters are significantly different at P  ≤ 0.05 applying Fisher’s LSD test

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Antioxidant enzymes

Compared with control, APX activity was increased by 21 and 25% under mild and severe stress, respectively (Fig. 5a). Adding GABA to the plants under both levels of Cr stress further increased APX activity. MDHAR activity decreased by 29 and 38% after exposing the mustard seedlings to mild and severe stress, respectively (compared with control) but increased after adding GABA (by 26% under mild stress and 35% under severe stress, compared with Cr stress alone) (Fig. 5b). Compared with the control plants, mild and severe stress decreased DHAR activity by 33 and 53%, respectively. However, GABA treatment increased DHAR activity by 36 and 51% under mild and severe stress, respectively; compared with Cr stress alone (Fig. 5c). GR activity increased significantly under mild stress and slightly under severe stress in contrast to the control plants. Applying GABA to the Cr-treated plants increased GR activity by 18% under mild stress and 16% under severe stress, compared with Cr stress alone (Fig. 5d). Superoxide dismutase activity was increased by 15 and 14% after exposing the seedlings to mild and severe stress, respectively. Exogenous addition of GABA to the Cr-treated plants further increased SOD activity (by 14% under mild stress and 23% under severe stress, compared with Cr stress alone) (Fig. 6a). On the other hand, CAT activity decreased by 32 and 40% under mild and severe stress, respectively, in contrast to the control plants. Adding GABA to the Cr-treated plants increased CAT activity by 25 and 36% under mild and severe stress, respectively, compared with Cr stress alone (Fig. 6b). Mild stress increased GPX activity by 19%, but severe stress decreased GPX activity by 18%, in contrast to the control plants. On the other hand, GABA treatment increased GPX activity by 11 and 29% under mild and severe stress, respectively; in contrast to Cr stress alone (Fig. 6c).

Fig. 5
figure 5

Ascorbate peroxidase (APX) (a), monodehydroascorbate reductase (MDHAR) (b), dehydroascorbate reductase (DHAR) (c), and glutathione reductase (GR) (d) activities of mustard seedlings under chromium (Cr) stress. Cr1, Cr2, and GABA indicate 0.15 mM K2CrO4, 0.3 mM K2CrO4, and 125 µM γ-aminobutyric acid, respectively. Means (±SD) were calculated from three replications (n = 3) for each treatment. Bars with different letters are significantly different at P  ≤ 0.05 applying Fisher’s LSD test

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Fig. 6
figure 6

Superoxide dismutase (SOD) (a), catalase (CAT) (b), and glutathione peroxidase (GPX) (c) activities of mustard seedlings under chromium (Cr) stress. Cr1, Cr2, and GABA indicate 0.15 mM K2CrO4, 0.3 mM K2CrO4, and 125 µM γ-aminobutyric acid, respectively. Means (±SD) were calculated from three replications (n = 3) for each treatment. Bars with different letters are significantly different at P  ≤ 0.05 applying Fisher’s LSD test

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MG content and glyoxalase system

Glyoxalase I activity increased by 20 and 16% under mild and severe stress, respectively, but Gly II activity decreased in the Cr-treated seedlings in a dose-dependent manner, compared with the control plants (Fig. 7a, b). Supplementing the Cr-treated plants with GABA increased Gly I activity (slightly) and Gly II activity (in contrast to Cr stress alone). On the other hand, in contrast to control, MG content increased by 57 and 99% under mild and severe stress, respectively (Fig. 7c). Adding GABA to the Cr-stressed seedlings decreased the MG content, which may be the result of upregulation of the activities of Gly I and Gly II (Fig. 7).

Fig. 7
figure 7

Glyoxalase I (Gly I) activity (a), Glyoxalase II (Gly II) activity (b), and methylglyoxal (MG) content (c) of mustard seedlings under chromium (Cr) stress. Cr1, Cr2, and GABA indicate 0.15 mM K2CrO4, 0.3 mM K2CrO4, and 125 µM γ-aminobutyric acid, respectively. Means (±SD) were calculated from three replications (n = 3) for each treatment. Bars with different letters are significantly different at P  ≤ 0.05 applying Fisher’s LSD test

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Cr uptake and detoxification mechanism

Accumulation of Cr in the seedlings followed a dose-dependent manner where higher Cr content was observed in the roots, compared with the shoots (Fig. 8a, b). Adding GABA with Cr to the plant growing media decreased Cr content in the roots and shoots. Compared with Cr stress alone, shoot Cr content was decreased by 100% under mild stress and 73% under severe stress after adding GABA. Similarly, root Cr content decreased by 13% under mild stress and 11% under severe stress after adding GABA (Fig. 8a, b). The uptake and translocation of Cr in the plants were reflected in the values of BCF, BAC, and TF (Fig. 8c–e). High BCF, BAC, and TF values were observed at both levels of Cr in the seedlings, and adding GABA decreased these values slightly or considerably. Phytochelatin content increased to a great extent at both levels of Cr stress, with increases of 69 and 102% in the mustard seedlings subjected to mild and severe stress, respectively (compared with control). However, supplementation with GABA reduced the PC content compared with Cr stress alone (Fig. 8f).

Fig. 8
figure 8

Shoot chromium (Cr) (a), root Cr (b), biological concentration factor (BCF) (c), biological accumulation coefficient (BAC) (d), translocation factor (TF) (e), and phytochelatin (PC) content (f) of mustard seedlings under chromium (Cr) stress. Cr1, Cr2, and GABA indicate 0.15 mM K2CrO4, 0.3 mM K2CrO4, and 125 µM γ-aminobutyric acid, respectively. Means (±SD) were calculated from three replications (n = 3) for each treatment. Bars with different letters are significantly different at P  ≤ 0.05 applying Fisher’s LSD test. ND - Not detected

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Discussion

Growth and development are necessary for the continuing existence and multiplication of any plant species. However, increasing Cr concentration in growing media gradually decreases the FW and DW of shoots and roots as well as plant growth (Sundaramoorthy et al. 2010). In the present study, Cr toxicity in the mustard plants resulted in growth reduction as indicated by decreased plant height, FW, and DW. Similar growth reduction was recorded in Glycine max L. (Ganesh et al. 2009) and paddy (Sundaramoorthy et al. 2010) under Cr stress. The soil and plant water relationship, nutrient uptake, photosynthesis, and so on are altered under Cr stress, which finally reduces plant growth (Panda and Choudhury 2005). Exogenous application of GABA considerably restores plant growth and development under abiotic stress by improving water status, photosynthetic activity, and upregulating antioxidants (Nayyar et al. 2014; Song et al. 2010). Similarly, in our study, GABA suppressed the growth inhibitory effects and increased the plant height, FW, and DW of the mustard seedlings. Song et al. (2010) reported that in contrast to stress treatment, GABA increased the fresh weight and root length of barley seedlings under Al stress.

Heavy metals in growing media can decrease water uptake by modulating osmotic potential, which is also an approach for avoiding water uptake as well as entry of toxic ions into the plant cells. Root damage by heavy metals is another reason for hampering water uptake and decreasing tissue water content (Fahr et al. 2013). In our present study, leaf RWC significantly decreased under Cr stress. In addition, Pro content increased considerably because of Cr toxicity. A higher Pro level in the Cr-stressed seedlings confirmed the water deficit condition. Free Pro works as an osmoprotectant and antioxidant, which creates the proper environment for various metabolic activities and increases stress tolerance (Yadav 2010; Nahar et al. 2016a, b). Exogenous application of GABA increased leaf RWC and decreased the Pro content in the tested mustard plants. As a result, plants gained relief from dehydration. This result corroborates the findings of Li et al. (2016), Vijayakumari and Puthur (2016), and Nayyar et al. (2014) who reported that exogenous application of GABA increased RWC under different abiotic stress conditions. Furthermore, Malekzadeh et al. (2014) documented that Pro content decreased under chilling stress after applying GABA.

Leaf chlorosis is a common injurious consequence of Cr stress. Choudhury and Panda (2004) reported that a high dose of Cr (1 mM) altered the chloroplastic membrane completely with severe divergence of thylakoids in moss. On the other hand, applying GABA increases the chl content and photochemical efficiency of plants under stress conditions (Nayyar et al. 2014; Li et al. 2017). Similarly, the decrease in chl content in the present study is a common deleterious effect of Cr stress, which resulted in chlorosis of the mustard leaves. The severity of the deleterious effects increased in a dose-dependent manner. However, applying GABA recovered the chl content of the Cr-stressed seedlings.

Reactive oxygen species destroy cell biomolecules causing lipid peroxidation and fatty acid oxidation (Hasanuzzaman et al. 2012a; Nahar et al. 2016a, b). Lipid peroxidation destroys the integrity and function of cell membranes as well as results in cell death (Panda and Choudhury 2005). Chromium-induced oxidative stress in the present study increased in a dose-dependent manner that is reflected in the overproduction of H2O2, O2 •−, and MDA in the mustard seedlings, which is also clear from the results of histochemical detection in the leaves and their contents at the cellular level. LOX activity also increased under Cr stress in a similar fashion, which increased lipid peroxidation. Similar findings were reported in other studies of Cr stress in different crops (Choudhary et al. 2012; Kováčik et al. 2013). Exogenous application of GABA enhanced stress tolerance in creeping bentgrass (Li et al. 2016), tomato (Malekzadeh et al. 2014), and pepper (Li et al. 2017) by upregulating the activity of antioxidant enzymes. In our study, applying GABA reduced lipid peroxidation in the Cr-treated plants compared with Cr stress alone. The resulting lower ROS production and LOX activity is due to the action of GABA in upregulating the antioxidant defense system of the mustard seedlings.

Ascorbate is an important water-soluble antioxidant that scavenges a range of ROS including 1O2, O2 •−, H2O2, and OH•. Moreover, AsA is involved in synthesizing zeaxanthin, which is used in photosynthesis and reduces oxidative damage (Conklin et al. 1996). In plant cells, AsA is regenerated by the antioxidant enzymes, MDHAR and DHAR. Therefore, the upregulation or downregulation of these two vital enzymes maintains the content of AsA at the cellular level (Gill and Tuteja 2010, Hasanuzzaman et al. 2012a; Nahar et al. 2016a, b). The Cr-stressed mustard plants had a lower AsA level and higher DHA level because of high production of ROS, viz. H2O2, under stress conditions. Reduced AsA content is also involved with increased APX activity and the diminished activities of DHAR and MDHAR (enzymes recycle DHA to AsA) under Cr stress. Previous many findings reported high DHA content, excess ROS production, and oxidative stress in toxic metal affected seedlings (Rahman et al. 2016; Hasanuzzaman et al. 2017b). Under stress conditions, GABA increases the activity of a number of vital enzymes of the AsA-GSH cycle, which finally enhances the AsA and GSH levels (Li et al. 2016, 2017). In our present study, adding exogenous GABA to the Cr-affected seedlings increased the MDHAR and DHAR activities as well as AsA content, decreased the DHA content, and finally increased the AsA/DHA ratio to some extent. Glutathione is also a prospective non-enzymatic antioxidant that works as a substrate of GPX and reduces ROS by preventing protein thiol oxidation and protein denaturation (Noctor et al. 2002; Hasanuzzaman et al. 2012c; Hasanuzzaman and Fujita 2013). GSH content increases significantly under mild stress and slightly under severe stress, which is a common feature in plants exposed to heavy metals (Hasanuzzaman and Fujita 2012). Higher GR activity is desirable to recycle GSH and increase its content, which our findings corroborate. In the present study, the GR activity of the mustard seedlings significantly increased under mild stress but not under severe stress. The level of GSSG also increased significantly under Cr stress. Moreover, the mustard plants showed a decreased GSH/GSSG ratio under Cr stress, which is consistent with excess ROS production and oxidative stress (Szollosi et al. 2009; Nahar et al. 2016b). The GSH/GSSG ratio as part of the redox signaling pathway is important for plant growth and development including the cell cycle, gene expression, and protein activity under both normal and stress conditions (Szalai et al. 2009; Fotopoulos et al. 2010; Nahar et al. 2016a, b). Applying exogenous GABA to the Cr-stressed seedlings increased the GSH/GSSG ratio because of the higher GSH and lower GSSG contents. Adding GABA to the Cr-treated plants also increased the enzyme activity of the AsA-GSH cycle including APX, MDHAR, DHAR, and GR. As a result, it can be said that GABA played a positive role in upregulating the AsA-GSH cycle and thereby provided tolerance against Cr toxicity.

Among the antioxidants, SOD is considered a main enzyme that provides first line defense against ROS because it catalyzes the dismutation process of O2 •− to H2O2 (Hasanuzzaman et al. 2012a; Gill et al. 2015). Catalase and APX then remove H2O2 by converting it to O2 and H2O (Miller et al. 2008). Chromium stress in the mustard seedlings did not significantly increase SOD activity but decreased CAT activity. Diminished CAT activity was due to higher generation of H2O2 in the mustard seedlings, compared with the control plants. Glutathione peroxidase has a vital function in defending cell components against oxidative damage by diminishing the activities of peroxides and different electrophiles (Gill and Tuteja 2010; Hasanuzzaman et al. 2012a). Glutathione peroxidase activity increased in the mustard seedlings under mild stress but decreased under severe stress. Panda and Choudhury (2005) reported that enzymatic antioxidants (SOD, CAT, etc.) are highly vulnerable under Cr stress resulting in a decline in their catalytic actions. Generally, plant cells have intracellular equilibrium between ROS production and scavenging through the actions of different antioxidant enzymes (Gill and Tuteja 2010; Song et al. 2010; Hasanuzzaman et al. 2012a). This redox homeostasis needs the well-organized synchronization of reactions in diverse cell compartments and is managed by multifarious signal transduction pathways (Dutilleul et al. 2003). The function of GABA has been shown in diverse plant species under different stresses by increasing the activity of different antioxidant enzymes (Nayyar et al. 2014; Li et al. 2016). In the present study, GABA upregulated the activities of SOD, CAT, and GPX under Cr stress in contrast to Cr stress alone. As a result, the mustard plants gained protection from oxidative damage caused by Cr toxicity.

Methylglyoxal is a cytotoxic compound, normally present in a lower amount in plant cells, but increases several fold under stress conditions depending on stress intensity and duration (Yadav et al. 2005, 2008; Rahman et al. 2016; Hasanuzzaman et al. 2017a). Therefore, upregulation of the glyoxalase system is crucial for plants to build up stress tolerance against toxic MG-induced oxidative stress (Yadav et al. 2005, 2008). The glyoxalase system consists of two vital enzymes, Gly I and Gly II, which can detoxify MG in a two-step reaction. In the first step, the Gly I enzyme converts MG to SLG, where GSH acts as a co-factor. In the second step, Gly II transforms SLG to d-lactate, where GSH is recycled (Yadav et al. 2005; Mustafiz et al. 2010; Hasanuzzaman et al. 2017a). Chromium stress in the present study noticeably increased MG content in a dose-dependent fashion, which clearly linked with the enhanced Gly I and diminished Gly II activity. Similar differential modulation of the activity of the glyoxalase system enzymes and excess MG level were recorded in previous studies with other heavy metal stresses. No previous studies have determined the effect of GABA in the glyoxalase system under abiotic stress, but in our study, we examined the glyoxalase system with MG content and found that adding GABA to the Cr-affected plants increased the activity of the glyoxalase enzymes as well as GSH content, which were strongly involved in detoxifying MG. Therefore, GABA might have a role in detoxifying MG by upregulating the glyoxalase system enzymes and GSH content at the cellular level.

In our study, Cr uptake by the shoots and roots of the mustard seedlings increased in a dose-dependent manner, where the roots accumulated more Cr than the shoots. Our results corroborated with the findings of Sundaramoorthy et al. (2010) and Wang et al. (2013). However, applying GABA in combination with Cr stress reduced Cr uptake in both the shoots and roots, even no Cr was observed in mild stressed plant. The Cr-affected mustard seedlings showed increased BCF, BAC, and TF, but GABA reduced their values considerably or to some extent.

Phytochelatins are oligomers of the γ-Glu-Cys unit produced from GSH. The PC synthase enzyme is responsible for PC production (Jozefczak et al. 2012) that utilizes GSH with a blocked thiol group for PC synthesis, and heavy metal ions that bind to GSH cause the enzyme to work faster (Vatamaniuk et al. 2000). Under metal stress conditions, PC binds to metal and transfer it to the cell vacuole to reduce metal toxicity (Zagorchev et al. 2013). In the present study, Cr toxicity increased PC and GSH content, which corroborates the findings of previous studies on Cd stress (Nahar et al. 2016a). Phytochelatin content rises when cells require more PC to stay alive in an environment with elevated concentrations of metal ions (Vatamaniuk et al. 2000). Applying GABA to the Cr-stressed plants reduced the Cr uptake in the shoots and roots of the mustard seedlings as well as decreased the PC content, which indicates alleviation of metal toxicity.

Conclusion

In summary, morphological, physiological, and metabolic analyses of mustard seedlings treated with Cr stress alone or in combination with GABA confirmed that Cr stress indirectly reduces plant growth by causing oxidative stress. Adding GABA to the Cr-stressed plants improved Cr tolerance by reducing metal uptake and increasing water status and chl content through upregulating the enzymes of the antioxidant defense and glyoxalase systems. However, the underlying physiological mechanism needs to be illuminated. For that reason, further study is required to elucidate the mode of action of GABA in plants exposed to Cr stress.