Introduction

Cadmium (Cd) is a common toxic heavy metal (HM) in agricultural soils posing adverse effects on growth, development and yield of crops (Ahmad et al. 2017a, b; Kaur et al. 2017; Yang et al. 2016; Rizwan et al. 2017a, b). Cd is known to hamper vital physiological activities, including photosynthesis, respiration, chlorophyll biosynthesis, assimilation of mineral nutrients and modulation of the signalling cascades of antioxidant systems (Anjum et al. 2014; Ahmad et al. 2015; Kovacik et al. 2017, Su et al. 2017). Cd stress imposes osmotic, ionic, as well as oxidative stress in different plants resulting in the dysfunction of plant metabolism (Kaur et al. 2017; Cuypers et al. 2010; Rizwan et al. 2017a, b). Cd stress is responsible for the overproduction of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), the hydroxyl radical (OH·), singlet oxygen (1O2) and the superoxide radical (O2•−), which are strong oxidants that cause protein oxidation, lipid peroxidation and DNA and RNA damage (Per et al. 2016; Sewelam et al. 2016).

Plants can cope with the Cd-induced oxidative stress through an efficient signalling network, which involves mineral nutrient homeostasis, production of enzymatic and non-enzymatic antioxidants and accumulation of comparative cytosolutes (Li et al. 2017; Silva et al. 2016; Mnasri et al. 2015). Stress physiologists are involved in developing strategies that underpin Cd-stress tolerance; however, the expanding field is emerging. Of the various adopted practices to reverse HM stress effects, application of plant growth regulators (PGRs) and mineral nutrients to achieve HM stress tolerance remains least explored and discussed.

PGRs crucially mediate plants’ response, metabolism and survival rate under both normal and abiotic environmental pressures. Methyl jasmonate (MeJ) is a methyl ester of jasmonic acid and is a lipid-derived naturally occurring PGR, which has a profound effect on various morphological, physiological, biochemical and antioxidant responses of plants against different stresses (Wasternack 2014; Hanaka et al. 2015; Sharma and Laxmi 2016; Ahmad et al. 2016a, b; Huang et al. 2017). The stimulatory effect of MeJ was found to be dose dependent and concentrations from 10−7 to 10−5 M have enhanced photosynthetic pigments and activity of photosystem II (PSII), which was severely down-regulated by Cd (Kovacik et al. 2011; Maksymiec and Krupa 2002) and Cu stress (Hanaka et al. 2015). It has been observed that MeJ can alleviate the stress imposed by Cd (Ahmad et al. 2017a, b), lead (Pb) (Piotrowska et al. 2009), arsenic (As) (Farooq et al. 2016, 2018), copper (Cu) (Poonam et al. 2013) boron (B) (Aftab et al. 2011) and nickel (Ni) (Sirhindi et al. 2016). On the other hand, studies performed on various plants have established that the addition of N can alleviate a range of toxicities in plants, such as water logging (Roy Chowdhury et al. 2017), UV-B radiation (Correia et al. 2005), freezing (Liu et al. 2017), elevated carbon dioxide (Ruiz-Vera et al. 2017) and metal stress (Giansoldati et al. 2012). Lin et al. (2011) working on rice showed that the deficiency of N under Cd stress significantly reduced chlorophyll, protein and nitrate contents.

Mentha arvensis, also known as mentholmint, belongs to the Lamiaceae family and is a perennial herbaceous plant distributed widely in the temperate regions of Europe, western and central Asia, eastern region of Siberia and North America and east to the Himalayan plane (Lawrence 2007). The plant has been widely cultivated for extraction of useful products which have diaphoretic, antiviral, anti-spasmodic, choleretic, stomachic, carminative, antifungal, antibacterial and vermifuge properties (Naeem et al. 2017), However, the regulatory effect of MeJ and N in reducing toxic effects of Cd stress in mentholmint (M. arvensis) has not been reported.

Nitrogen (N) is an important common limiting mineral nutrient element, which is the main constituent of all amino acids, proteins and a number of N-containing compounds that play a profound role in plant abiotic stress tolerance (Cetner et al. 2017; Mohanty et al. 2018; Siddiqui et al. 2008a, b; Khan et al. 2017). The metabolism of N is one of the fundamental physiological processes in plants in which N-mediated metabolites and related enzymes play a significant role against metal stress (Ren et al. 2017; Ai et al. 2017). The N-assimilation rate has been shown to be up-regulated in plants exposed to elevated metal stress (Khan et al. 2016a, b). N can also act as an important signalling agent, which triggers expression of genes, N-assimilation processes (Thind et al. 2018), metabolism of carbohydrates (Marschner 2011), rate of net photosynthesis (Singh et al. 2016a, b), antioxidant defence and osmoprotectant systems (Siddiqui et al. 2008a, b), PGRs, vitamins and nucleic acids (Beevers and Hageman 1969), chlorophyll biosynthesis (Zhang et al. 2014), and can maintain nutrient homeostasis (Xiao et al. 2017).

Considering the importance, the goals of the present research were to determine the interactive effect of foliar MeJ in the presence or absence of N in countering the detrimental effects of Cd stress in menthol mint.

Materials and Methods

Experimental Methodology

Healthy and uniform suckers of M. arvensis Linn. ‘Kushal’ were sterilized with NaOCl (5%) for 4 min followed by a washing with deionized and double distilled water (DDW) and then transplanted in earthen pots. Each experimental pot (30 × 45 cm) contains 7.5 kg mixture of soil and farmyard manure (6:1). The physicochemical characteristics of the soil were sandy loam texture, pH (1:2) 7.5, EC (1:3) 0.52 m mhos cm−1 and 92.6, 8.5 and 135.4 mg available of critical mineral nutrients, namely N, P and K per kg of soil, respectively. After germination, thinning was manually conducted and 3–4 healthy seedlings were maintained in each pot. The 150 mg kg−1 Cd stress was given basally in the form of cadmium chloride (CdCl2) through the soil at 30 days of transplantation (DAT). Methyl jasmonate (MeJ) and basal nitrogen (N) were given thrice 15 days apart beginning 15 days after Cd stress at 45, 60 and 75 DAT. The concentration of 4 g N per kg soil and MeJ was based on our earlier findings (Mohammad et al. 1998; Khan et al. 2009). The source of N was urea. Each pot was given DDW (200 mL pot−1) on every alternate day to keep the soil moist. The experiment was conducted in net house of Botany Department, Aligarh Muslim University Aligarh, India, with average day/night temperature of 25 ± 3/14 ± 2 °C; relative humidity, 65 ± 5%; photosynthetically active radiation (PAR), 810 ± 20 µmol m−2 s−1; critical photoperiod, 10–12 h. Each experiment was conducted in five replications. The plants were harvested at 90 DAT and biochemical, antioxidant enzyme and osmolyte content were determined in upper young leaves. The Cd, MeJ and N treatments were as follows:

  • 0 mg kg−1 Cd + 0 M MeJ + 0 g N; T0 (control);

  • 0 mg kg−1 Cd + 1 µM MeJ + 0 g N; T1

  • 0 mg kg−1 Cd + 0 M MeJ + 4 g N; T2

  • 0 mg kg−1 Cd + 1 µM MeJ + 4 g N; T3

  • 150 mg kg−1 Cd + 0 M MeJ + 0 g N; T4

  • 150 mg kg−1 Cd + 1 µM MeJ M + 0 g N; T5

  • 150 mg kg−1 Cd + 0 M MeJ + 4 g N; T6

  • 150 mg kg−1 Cd + 1 µM MeJ + 4 g N; T7

Determination of Growth and Yield Parameters

Shoot and root length were measured using a scale. Samples were kept in an oven and dried and then dry weight (DW) was recorded.

Estimation of Leaf Relative Water Content (LRWC) and Leaf Area

The standard method of Yamasaki and Dillenburg (1999) based on recording fresh, turgid and dry weight of leaf discs was adopted to estimate LRWC. LRWC was calculated using the following formula:

$${\text{LRWC}}=\frac{{{\text{Fresh weight}} - {\text{dry weight}}}}{{{\text{Turgid weight}} - {\text{dry weight}}}} \times 100$$

Leaf area (LA) was calculated by using graph sheets.

Determination of Photosynthetic Attributes

Chlorophyll contents were analysed using a portable SPAD chlorophyll meter (SPAD-502; Konica, Minolta Sensing, Inc., Japan).

Leaf gas exchange measurements viz-net photosynthetic rate (PN), stomatal conductance (gs) and carbon dioxide assimilation rate (Ci) were measured on the uppermost fully expanded leaves in full and bright sunlight between 10:00 and 12:00 h using IRGA (Li-COR 6400, Li-COR, Lincoln, NE, USA).

Chlorophyll fluorescence parameters were recorded with a junior PAM chlorophyll fluorometer (Heinz Mess und Regeltechnik, Heinz Walz Gmbh D-91090, Germany). The fully expanded horizontal Mentha leaves were dark-adapted for 30 min before measuring Fv/Fm (Li et al. 2007).

Determination of Rubisco Content

The Usuda (1985) standard protocol was employed to determine the activity of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco; EC4.1.1.39) by monitoring the oxidation of NADH at 28 °C and 342 nm.

Determination of Osmoprotectants

The content of glycine betaine (GB) was determined according to the method of Grieve and Grattan (1983). The acid ninhydrin based method of Bates et al. (1973) was used to estimate proline content.

Determination of Oxidative Stress Biomarkers

Hydrogen peroxide (H2O2) production, lipid peroxidation in the form of malondialdehyde (MDA) and electrolyte leakage (EL) was estimated using the methods of Velikova et al. (2000), Madhava Rao and Sresty (2000) and Dionisio-Sese and Tobita (1998) respectively. Electrolyte leakage was calculated using the following formula:

$${\text{Electrolyte leakage}}=\frac{{EC1 - EC0}}{{EC2 - EC0}} \times 100$$

The content of superoxide (O2−) was quantified by the estimating the production of nitrite as a result of reaction of hydroxylamine with superoxide anions by employing the method of Wang and Luo (1990). The absorbance of reaction mixture was taken at 530 nm and O2− generation rate was calculated from the standard curve of sodium nitrite and expressed as µg g−1 (FW).

Estimation of Antioxidant Activities

Preparation of Enzyme Extract and Assay

Leaf material was homogenized in a pre-chilled pestle and mortar in 2 mL 100 mM potassium phosphate buffer (pH 7.0) having 1% of polyvinyl pyrrolidone and centrifuged at 12,000×g for 30 min at 4 °C; the supernatant was used to determine different enzyme activities. Superoxide dismutase activity (SOD, EC1.15.1.1) was measured using the nitroblue tetrazolium (NBT) reduction method of Van Rossum et al. (1997). Catalase (CAT: 1.11.1.6) activity was determined by monitoring the decomposition of H2O2 for 2 min at 240 nm (Aebi 1986). Ascorbate peroxidase (APX, 1.11.1.11) activity was assayed according to Nakano and Asada (1981). Glutathione reductase (GR, 1.6.4.2) activity was measured according to the method of Foster and Hess (1980). The activities of these enzymes were expressed in EU mg protein−1. All spectrophotometric analyses used in the present study was conducted using a portable spectrophotometer (ELICO SL 171 MINI SPEC).

Determination of Ascorbate, Carotenoids

Ascorbate contents were measured according to the method of Huang et al. (2005). Carotenoid content in leaves was estimated using the method of Lichtenthaler and Buschmann (2001). The freshly collected leaf samples were homogenized with a mortar and pestle in 100% acetone and then the optical density (OD) was read at 470 nm to determine carotenoid contents.

Determination of Nitrate Reductase (NR) Activity and Nitrogen Use Efficiency

NR activity was determined by adopting the method proposed by Jaworski (1971). A fresh leaf sample (500 mg) was crushed in phosphate buffer (pH 7.5) to which a mixture of 5% isopropanol and 0.2 M potassium nitrate were added, and the mixture was incubated at 30 °C for 120 min. After incubation of the reaction mixture, 0.02% of N-1-naphthyl ethylenediamine dihydrochloride and 1% sulphanilamide was added. The absorption of the coloured solution was recorded at 540 nm. Nitrogen use efficiency (NUE) was calculated asthe ratio of net photosynthesis to N content per unit LA.

Analysis of Root and Leaf Cd

Samples were dried in oven at 85 °C for 24 h and were then digested in a tri-acid (H2SO4, HNO3, HCLO4) mixture at the ratio of 5:1:1v/v at 80 °C on a dry block heater. HNO3 and H2O2 were added to make a transparent solution. The contents were determined by atomic absorption spectrophotometer (GBC, 932 plus; GBC Scientific Instruments, Braeside, Australia) and expressed as µg/g (DW).

Determination of Leaf Mineral Nutrients

Leaf N content was determined by the acid-peroxide digested method of Lindner (1944). The optical density of the reaction medium was read at 525 nm. The method of Fiske and Subbarow (1925) was adopted for P determination. The intensity of the blue colour was recorded at 620 nm.

Determination of Leaf K Content by Flame Emission Spectroscopy

The acid-peroxidase digested aliquot was also used for determining the content of K by flame photometer (Model, ELICO, CL22D, India), which was made to operate at 10 psi pressure (Hald 1947). All the critical nutrient concentrations were expressed in mg g−1 DW.

Scanning Electron Microscopy

Fresh leaf samples were fixed first with 2.5% glutaraldehyde plus 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.0) for 4 h. The leaves were again fixed with 1% osmium tetroxide in phosphate buffer (pH 7.0) for 50 min and washed with phosphate buffer for 10 min. After fixing, alcohol dehydration (50%, 70%, 80%, 90%, 95% and 100%) was carried out. Finally, leaves were dehydrated in a Carl Zeiss EVO 40 (Germany) scanning electron microscope critical point dryer with liquid CO2. The leaf samples were coated with gold–palladium and observed under a Carl Zeiss EVO 40 (Germany) scanning electron microscope (SEM) at high voltage (15 kV) and magnification of × 1500.

Statistical Analysis

Data were analysed following one-way analysis of variance (ANOVA) using SPSS software version 17. The values represented are means ± SE (n = 5). Different letters indicate significant differences between treatments at P ≤ 0.05. The principal component analysis was performed by Minitab Mtb EXE (2) software.

Results

Effects of MeJ and N on Morphological and Yield Parameters Under Cd Stress

The shoot length decreased significantly (P ≤ 0.05) by 14.53% in T4 (Cd) in comparison to that of the control (T0). Treatment with MeJ to Cd-stressed plants in T5 (Cd + MeJ) showed an increase of 7.70% over Cd-stressed plants. A similar increase of 5.39% was recorded for N application in T6 (Cd + N) over Cd-stressed plants. However, the combined application of MeJ + N in the presence of Cd stress in T7 (Cd + MeJ + N) resulted a more conspicuous and significant increase of 23.24% relative to their individual treatment (Fig. 1a).

Fig. 1
figure 1

a Length of shoot and root, b shoot fresh and dry weight, c leaf relative water content, d leaf area, e maximum PSII efficiency, f photochemical and non-photochemical quenching, g maximum capture efficiency and actual quantum yield, h Rubisco activity of cornmint cultivar ‘Kushal’ grown with/without 150 mg kg−1 Cd in precence of either alone MeJ or N or both. Data are presented as treatments mean ± SE (n = 5). Data followed by same letters are not significantly different by DMRT test at (P ≤ 0.05). 0 mg kg−1 Cd + 0 M MeJ + 0 g N (T0) (control); 0 mg kg−1 Cd + 1 µM MeJ + 0 gN (T1); 0 mg kg−1 Cd + 0 M MeJ + 4 g N (T2); 0 mg kg−1 Cd + 1 µM MeJ + 4 g N (T3); 150 mg kg−1 Cd + 0 M MeJ + 0 g N (T4); 150 mg kg−1 Cd + 1 µM MeJ + 0 g N (T5); 150 mg kg−1 Cd + 0 M MeJ + 4 g N (T6); 150 mg kg−1 Cd + 1 µM MeJ + 4 g N (T7)

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The length of root was also negatively influenced by Cd treatment in T4 and it decreased by 26.60% relative to that of the control. Application of MeJ to Cd-stressed plants increased the root length by 36.84% compared to that of the T4 treatment (Fig. 1a). Nitrogen supply in T6 resulted in a significant root length increase of 23.79% over Cd-stressed plants.

A retardation in shoot fresh and dry mass by 22.36% and 46.73%, respectively, was also observed after exposing plants to Cd stress in T4 with respect to that of the control. However, the cumulative treatment of MeJ + N in T7 (Cd + MeJ + N) had a more significant effect and an increase of 35.17% in shoot fresh weight and 52.88% in shoot dry weight with respect to Cd plants in T4 was noticed (Fig. 1b).

MeJ and N Restore LRWC and LA

A significant (P ≤ 0.05) decrease of 30.81% was recorded in LRWC in T4 with respect to that of the T0 control treatment. MeJ in T5 significantly increased LRWC by 26.99% and N in T6 by 24.41%; however, in T7 treatment, LRWC was enhanced by 28.77% with the combined treatment of MeJ and N with respect to Cd-stressed plants (Fig. 1c).

Cd stress decreased LA by 14.22% in T4 relative to that of the control plants. The LA of non-stressed plants in T1 and T2 increased by 13.33% and 10.40%, respectively, in comparison to that of the T0 control plants. The combined effects of MeJ and N in T3 (MeJ + N) was more effective in the non-stressed plants and LA showed a significant increase of 18.18% compared to that of the T0 control plants (Fig. 1d). Regarding Cd stress, MeJ in T5 increased LA by 8.56% and N in T6 increased LA by 5.13%, but MeJ + N in T7 increased LA by 10.11% (Fig. 1d).

Influence of MeJ and N on Photosynthetic Attributes

The SPAD value declined by 31.37% in Cd-treated plants relative to that of the control (Table 1). Supplementing Cd-stressed plants with MeJ or N improved SPAD content by 24.04% and 17.34%, respectively, as compared to that of Cd-stressed plants. The combined application of MeJ + N (T7) to Cd-treated plants further increased total Chl content by 29.97% as compared to Cd alone.

Table 1 SPAD value, net photosynthesis PN (µmol CO2 m−2 s−1), stomatal conductance gs (mmol m−2 s−1) and intercellular CO2 concentration Ci (µmol mol−1) responses of cornmint cultivar ‘Kushal’ grown with/without 150 mg kg−1 Cd in precence of either alone MeJ or N or both
Full size table

The PN decrease was 44.26% in Cd-stressed plants (T4) relative to that of the control (T0) (Table 1). Supplementing Cd-stressed plants with MeJ or N improved PN by 34.91% and 12.85% in the T5 and T6 treatments, respectively. The combined supply of MeJ and N in T7 further increased the rate of PN by 48.79% as compared to that of Cd-stressed plants alone.

The decrease in gs and carbon dioxide assimilation efficiency by Cd was nullified completely by MeJ in plants receiving N input in T7 (Cd + MeJ + N) and the values were 4.92 and 8.51% relative to Cd-stressed plants in T4 (Table 1).

The chlorophyll fluorescence parameters showed a varied trend at the respective treatments. Cd stress in T4 decreased the maximal PSII (Fv/Fm) by 39.13% (Fig. 1e), photochemical quenching (qP) by 16.86% (Fig. 1f), actual quantum yield (f PSII) by − 21% (Fig. 1g) and maximum capture efficiency (f exc) by 36.11% (Fig. 1g), with respect to that of the control T0. Non-photochemical quenching (NPQ) increased by 38.02% (Fig. 1f). Treatment of Cd-stressed plants with MeJ in T5 increased Fv/Fm by 31.14%, qP by 12.65%, f PSII by 23.91% and f exc by 19.29%, but reduced NPQ by 12.67% relative to that of Cd-stressed plants alone. The combined dose of MeJ and N in T7 increased Fv/Fm, qP, f PSII and f exc by 44%, 14.81%, 40.67% and 48.88%, respectively, and decreased NPQ by 22.53% with respect to that of the Cd-stressed plants alone.

The activity of Rubisco showed a significant decrease of 28.66% in the T4 treatment relative to that of the control plants (Fig. 1h). A non-significant increase by N supply in T2 was noted relative to that of the control plants. The combined treatment of MeJ and N in T7 (MeJ + N) enhanced Rubisco activity by 42.21% over that of Cd-stressed plants.

Effects of MeJ and N on Osmoprotectants

Cd at the T4 concentration enhanced the accumulation of proline and GB by 1.52- and 2.90-fold, respectively, in comparison to that of the control (Fig. 2a). Furthermore, the highest proline and GB contents were found in treatment T7 (MeJ + N) with the combined supply of MeJ and N, as compared to N and MeJ alone under Cd stress. Treatment with Cd + MeJ + N in T7 exhibited a 1.69-fold higher GB content than Cd-fed plants in T4 (Fig. 2a).

Fig. 2
figure 2

a Contents of proline and glycine betaine, b superoxide ion (O2−), c electrolyte leakage and d TRABS and H2O2 of cornmint cultivar ‘Kushal’ grown with/without 150 mg kg−1 Cd in precence of either alone MeJ or N or both. Data are presented as treatments mean ± SE (n = 5). Data followed by same small and capital letters are not significantly different by DMRT test at (P ≤ 0.05). 0 mg kg−1 Cd + 0 M MeJ + 0 g N (T0) (control); 0 mg kg−1 Cd + 1 µM MeJ + 0 gN (T1); 0 mg kg−1 Cd + 0 M MeJ + 4 g N (T2); 0 mg kg−1 Cd + 1 µM MeJ + 4 g N (T3); 150 mg kg−1 Cd + 0 M MeJ + 0 g N (T4); 150 mg kg−1 Cd + 1 µM MeJ + 0 g N (T5); 150 mg kg−1 Cd + 0 M MeJ + 4 g N (T6); 150 mg kg−1 Cd + 1 µM MeJ + 4 g N (T7)

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Effects of MeJ and N on TRABS Content, EL, H2O2 and O2 − Production

Cd caused a significant increase in TRABS content, EL and H2O2 accumulation by 53.97%, 67.26% and 56.66%, respectively, in T4 as compared to that of the T0 control treatment (Fig. 2c, d). Application of MeJ to plants receiving Cd and N, reduced the TRABS content by 47.12%, EL by 55.68% and H2O2 accumulation by 42.66% in T7 relative to that of Cd-only (T4) treated plants alone.

Cd stress leads to a significant (P ≤ 0.05) increase in O2− content by 52.72% as compared to T0 control plants. Both MeJ and N reduced O2− production in Cd-treated plants with respect to Cd-treated plants, however, decrease was more significant (P ≤ 0.05) when Cd-treated plants received MeJ in presence on N at T7 by 34.62% as compared to only Cd contaminated plants T4 (Fig. 2b).

MeJ and N Enhanced the Activity of Antioxidant Enzymes

Cd stress in T4 enhanced activities of SOD, CAT, APX and GR by 83.72%, 6.02%, 40.49% and 32.11%, respectively, with respect to that of the T0 control plants. Moreover, application of MeJ and N to plants in the T7 treatment increased the SOD by 64.65%, CAT by 8.77%, APX by 34.83% and GR by 56.47% as compared to that of the T4 treatment (Fig. 3a–d). However, no significant increase was noted in SOD, APX or GR activities in the T1and T2 treatments as compared to that of the T0 plants.

Fig. 3
figure 3

Activities of a SOD, b CAT, c APX, d GR, e AsA and f carotenoid content of cornmint cultivar ‘Kushal’ grown with/without 150 mg kg−1 Cd in precence of either alone MeJ or N or both. Data are presented as treatments mean ± SE (n = 5). Data followed by same letters are not significantly different by DMRT test at (P ≤ 0.05). 0 mg kg−1 Cd + 0 M MeJ + 0 g N (T0) (control); 0 mg kg−1 Cd + 1 µM MeJ + 0 gN (T1); 0 mg kg−1 Cd + 0 M MeJ + 4 g N (T2); 0 mg kg−1 Cd + 1 µM MeJ + 4 g N (T3); 150 mg kg−1 Cd + 0 M MeJ + 0 g N (T4); 150 mg kg−1 Cd + 1 µM MeJ + 0 g N (T5); 150 mg kg−1 Cd + 0 M MeJ + 4 g N (T6); 150 mg kg−1 Cd + 1 µM MeJ + 4 g N (T7)

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Enhancement of Ascorbate and Carotenoids with Supplementation with MeJ and N

Cd stress caused a significant (P ≤ 0.05) decrease of 42.88% in AsA in the T4 treatment with respect to that of the T0 control. The Cd-induced decrease in AsA was significantly overcomed by supplementing with MeJ and N in combination in T7 rather than with individual treatment of MeJ or N relative to that of the Cd-stressed plants in T4 treatment (Fig. 3e).

The carotenoid content showed a non-significant increase when control plants were given MeJ or N in T1 and T2, respectively. Cd stress increased the carotenoid content by 50.84% with respect to that of the T0 (control) plants. However, treatment T7 enhanced the carotenoid content by 52.30% compared to the Cd-only treatment (Fig. 3f).

Nitrate Reductase Activity and Nitrogen Use Efficiency

Plants treated with Cd in T4 showed a significant decrease in NR activity by 55.00% with respect to that of the T0 control plants (Fig. 4a). Plant supplementation with a combined dose of MeJ + N in T3 increased the activity of NR by 35.48% over that of the control plants. Similarly, the combined dose of MeJ + N to Cd-stressed pants in T7 significantly (P ≤ 0.05) increased the activity of NR by 57.14% as compared to that of Cd-only stressed plants.

Fig. 4
figure 4

Activity of a NR and b nitrogen use efficiency of cornmint cultivar ‘Kushal’ grown with/without 150 mg kg−1 Cd in precence of either alone MeJ or N or both. Data are presented as treatments mean ± SE (n = 5). Data followed by same letters are not significantly different by DMRT test at (P ≤ 0.05). 0 mg kg−1 Cd + 0 M MeJ + 0 g N (T0) (control); 0 mg kg−1 Cd + 1 µM MeJ + 0 gN (T1); 0 mg kg−1 Cd + 0 M MeJ + 4 g N (T2); 0 mg kg−1 Cd + 1 µM MeJ + 4 g N (T3); 150 mg kg−1 Cd + 0 M MeJ + 0 g N (T4); 150 mg kg−1 Cd + 1 µM MeJ + 0 g N (T5); 150 mg kg−1 Cd + 0 M MeJ + 4 g N (T6); 150 mg kg−1 Cd + 1 µM MeJ + 4 g N (T7)

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Photosynthetic-NUE exhibited a decreasing trend with the Cd application in T4 and the decrease was 1.64-fold with respect to that of the control plants. However, the combined treatment of MeJ and N in T7 alleviated the decrease in NUE and the increase was 1.47-fold with respect to that of the Cd-stressed plants in T4 (Fig. 4b).

Stomatal Response Studied Under Electron Microscopy

In the T0 control plants, normal stomatal aperture along with characteristic guard cells were observed, but with Cd stress, closed stomata were observed in T4. However, plants receiving MeJ in presence of N decreased the Cd effects on stomata and MeJ along with N prevented the stomatal closure in T7 (Fig. 5a–c).

Fig. 5
figure 5

Leaf stomatal behaviour of mentholmint (Mentha arvensis L.) under control a: Cd b and Cd + MeJ + N c. The stomatal opening and closing were studied under the scanning electron microscopes at × 1500 (a–c). A single representative of three treatments is shown

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Root and Leaf Cd Content

The plants accumulated higher Cd in roots (~ 11.92 µg−1 DW) than in leaves (~ 3.04 µg−1 DW). Application of N or MeJ reduced Cd content both in roots and leaves, but the higher reduction occurred in plants supplied with the combined treatment of N and MeJ in T7, in which significant (P ≤ 0.05) reductions of 73.68% and 61.74% in leaves and roots, respectively, were observed relative to that of Cd-treated plants alone (Fig. 6).

Fig. 6
figure 6

Content of Cd in root and leaf of cornmint cultivar ‘Kushal’ grown with/without 150 mg kg−1 Cd in precence of either alone MeJ or N or both. Data are presented as treatments mean ± SE (n = 5). Data followed by same letters are not significantly different by DMRT test at (P ≤ 0.05). 0 mg kg−1 Cd + 0 M MeJ + 0 g N (T0) (control); 0 mg kg−1 Cd + 1 µM MeJ + 0 gN (T1); 0 mg kg−1 Cd + 0 M MeJ + 4 g N (T2); 0 mg kg−1 Cd + 1 µM MeJ + 4 g N (T3); 150 mg kg−1 Cd + 0 M MeJ + 0 g N (T4); 150 mg kg−1 Cd + 1 µM MeJ + 0 g N (T5); 150 mg kg−1 Cd + 0 M MeJ + 4 g N (T6); 150 mg kg−1 Cd + 1 µM MeJ + 4 g N (T7)

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Mineral Nutrient Contents

Under Cd stress, the contents of mineral nutrients (N, P, K) decreased by 49.62%, 29.41% and 14.58%, respectively, in T4 in comparison to that of the T0 (control) plants (Table 2). However, in the control plants supplemented with MeJ + N, N content increased significantly (P ≤ 0.05) by 31.79% relative to that of the control plants. In T7, the dual application of MeJ and N to Cd-stressed plants significantly increased the N content by 41.73% over Cd-stressed plants in T4. The P content increased with both MeJ and N in T5 and T6, respectively, but the increase was non-significant with respect to Cd-stressed plants in T4. K content increased by 36.68% with the combined dose of MeJ and N in T7 with respect that of the Cd-stressed plants in T4 (Table 2).

Table 2 Concentrations of nitrogen, phosphorous and potassium of corn mint cultivar ‘Kushal’ grown with/without 150 mg kg−1 Cd in presence of either alone MeJ or N or both
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Principal Component Analysis (PCA)

PCA was used to assess the relation between Cd stress, MeJ and N application on various physiological and biochemical properties in mentholmint plants. According to PCA, growth and photosynthetic characters were in opposite direction with root and leaf Cd content, H2O2, O2•−, EL and TRABS content. In contrast, mineral mutrient contents were in positive direction with growth and photosynthetic parameters. Plant growth and photosynthesis under Cd stress increased with the application of MeJ and N via increase of mineral nurient contents and photosynthesis and by regulation of antioxidants and osmolytes. This was proved by PCA that antioxidants and osmolytes characters lie between plant growth and photosynthesis and oxidative stress biomarkers (Fig. 7).

Fig. 7
figure 7

Principal component analysis (PCA) of various parameters of mentholmint plants under cadmium (Cd) and methyl jasmonate (MeJ) or nitrogen (N). SL shoot length, RL root length, SFM shoot fresh mass, SDM shoot dry mass, LRWC leaf relative water content, LA leaf area, PN net photosynthesis, gs stomatal conductance, Ci intercellular CO2 concentration, N nitrogen, P phosphorous, K potassium, GB glycine betaine, superoxide ion (O2−), EL electrolyte leakage, TRABS thiobarbituric acid reactive substances, H2O2 hydrogen peroxide, SOD superoxide dismutase activity, CAT catalase, APX ascorbate peroxidise, GR glutathione reductase, AsA ascorbate, Fv/Fm maximum PSII efficiency, Qp photochemical, NPq non-photochemical quenching, NR nitrate reductase, NUE nitrogen use efficiency

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Discussion

Cd is a non-essential toxic biometal that alters the physiology and metabolism of plants (Kaur et al. 2017; Anetor et al. 2016; Ahmad et al. 2015). In the present study, we observed significant repression of growth parameters because of Cd stress and our results are in agreement with the earlier results of Ahmad et al. (2017a, b) for Vicia faba, Per et al. (2016) for Brassica juncea, Shen et al. (2017) for Brassica campestris and Rizwan et al. (2017a, b) for Oryza sativa. Restricted plant growth because of Cd results from its cumulative effects on the uptake of water and minerals, metabolism of sugars, photosynthetic efficiency, cell division and elongation and enzyme activity (Shen et al. 2017; Ahmad et al. 2015). N supplementation protected M. arvensis from effects of Cd stress because application of N in the present study increased the mineral nutrient content, particularly N, and thus many N-containing metabolites and compounds that benefit enzyme activities associated with plant growth (Shah et al. 2017). N supplementation also promotes cell division and elongation of the mesophyll cells resulting in enhanced growth of leaf blades (Mac Adam et al. 1989), reflecting the significant enhancement in the photosynthetic efficiency and production of assimilates. Application of N and MeJ prevented the toxic effects of Cd on the uptake of essential elements, such as N, P and K, and improved the photosynthetic N-use efficiency, therefore resulting in greater allocation of N to Rubisco proteins. Similar to our results, Per et al. (2016) observed MeJ-mediated alleviation of Cd stress through increased nutrient assimilation. Yan et al. (2013) demonstrated significant alleviation of Cd toxicity in Capsicum after the application of MeJ. In the present study, application of N + MeJ exhibited maximal alleviation of Cd-induced deleterious effects on growth, suggesting the possible existence of crosstalk between N and MeJ.

Cd treatment reduced LRWC in the present study and similar decrease in LRWC has been reported in mustard and faba bean under Cd stress (Ahmad et al. 2011, 2017a, b). It was interesting to observe that application of N and MeJ caused a significant increase in the LRWC under normal and stress conditions probably because of the greater uptake of mineral nutrients, such as K, and the production of proline and GB. Li et al. (2007) have demonstrated that N supplementation increases root development in Cd-stressed plants and hence optimum water absorption capacity is maintained to a considerable extent. Cd stress significantly reduced LA and this might be due to reduced supply and incorporation of compounds to be utilized in plant growth in addition to restricted LRWC and cell division. Supply of N and MeJ restores the metabolic activity of leaf tissues and increases nutrient assimilation dynamics in plants (Fan et al. 2016; Iqbal et al. 2015; Gomez et al. 2010). Aplication of optimum N nutrition has the capacity to alleviate abiotic stress in crops plants by sustaining various metabolic activities even at reduced tissue water potential (Saud et al. 2017). In the present study, N application increase LRWC and this might be due to optimum water tissue balance, similar N-mediated increase in LRWC in wheat plants under N management has been reported (Akram et al. 2014). MeJ also alleviates decrease in LRWC probably by maintaining optimum water balance in mentholmint plants.

Cd significantly decreased the SPAD value and leaf gas exchange parameters in the present study. Cd triggers degradation of chlorophyll proteins by enhancing chlorophyllase activity and affecting the activity of their bio-synthesizing enzymes (Assche and Clijsters 1990; Alyemini et al. 2018). A reduction in chlorophyll content because of Cd treatment has been previously reported by Masood et al. (2012a, b), Khan et al. (2016a, b) in mustard and Abd-Allah et al. (2015) in sunflower plants. In agreement with our results, Singh et al. (2016a, b) and Per et al. (2016) also demonstrated individually the N and MeJ-induced protection of photosynthesis under NaCl- and Cd-stressed in mustard plants. N supply has been observed to alleviate abiotic stress-triggered deleterious effects on photosynthetic potential by increasing N-assimilation, activity of Rubisco and photosynthetic-NUE (Akram et al. 2014; Singh et al. 2016a, b; Iqbal et al. 2015). Reports describing the combined effect of N and MeJ are rare; however, in the present study, application of MeJ along with N improved N-assimilation (N content and NR activity) and resulted in the maintenance of Rubisco functioning and hence reduced the cyclic electron around PSII, thereby restricting the generation of toxic O2− (Makino 2003) which is agreement with our results. In addition, MeJ and N regulated the stomatal functioning under Cd stress (Fig. 5a–c) leading to continuous gas flow exchange and the associated gas exchange parameters. Similar effects of added N on increasing photosynthesis in B. juncea plants was reported by Mohammad et al. (1998) and Siddiqui et al. (2008), Malus prunifolia (Huang et al. 2018). In the present study, both N and MeJ mediated modulation of photosynthesis (increased PN) which may have resulted from the increased diffusion rate of CO2 from the atmosphere (increased A) into the intercellular spaces via the open stomatal aperture under Cd stress (Fig. 5c). Regulation and reversal of Cd-induced inhibition of PSII activity by the exogenous application of MeJ (Per et al. 2016) has been reported; however, reports regarding photosynthetic regulation involving the combined effect of N and MeJ on PSII activity under Cd stress are not available. Moreover, higher proline and GB content in plants because of N and MeJ either individually or jointly protected the carboxylase activity of Rubisco and the activity of PSII in the present study by quickly eliminating ROS and maintaining the tissue water content.

Proline and GB are common compatible organic osmoprotectants having regulatory roles in imparting tolerance to plants against abiotic stresses (Per et al. 2017; Sirhindi et al. 2016; Chen and Murata 2009). There is direct relationship of proline and N as available N in plants is directly stored as proline (Rhodes et al. 1999). Proline biosynthesis relies on an N source, as well as the application method (Neuberg et al. 2010) and in the present study, application of N along with MeJ increased the synthesis of proline under normal conditions and was maintained to highest level in Cd-treated plants with maximal accumulation observed in N + MeJ supplemented Cd seedlings. Accumulation of proline under Cd stress is a prevalent phenomenon in plants. Greater accumulation of proline and GB in N + MeJ treated Cd-stressed conditions could stabilize the process of protein synthesis and protein structure, thus preventing stress-mediated protein denaturation and maintaining enzyme activities (Sharmila and Saradhi 2002), and could lead to rapid detoxification of Cd-triggered ROS. Zakery-Asl et al. (2014) also reported that N application in Phaseolus vulgaris plants results in an increment of proline content in root and foliar organs via increasing the activities proline bio-synthesizing enzymes. Enhanced osmolyte accumulation during metal stress serves (i) to balance the water potential of plants (Sirhindi et al. 2016), (ii) as a metal chelator (Sharma et al. 1998), (iii) as free radical neutralization (Zhao 2011) and (iv) as an energy source for growth revival after stress release (Kavi Kishor and Sreenivasulu 2014). MeJ is also known to induce enzymes related to proline biosynthesis during stress conditions (Chen and Kao 1993) and greater accumulation of osmolytes results from the up-regulation and down-regulation of biosynthetic and catabolic genes of osmolyte generating pathways, respectively (Ahanger et al. 2017). Gao et al. (2004) found that the exogenously applied MeJ induced significant increases in GB biosynthesis in pear plants subjected to water stress conditions.

Cd stress triggered excessive generation of ROS causing increased peroxidation of membranes and leakage of essential cellular components and protein modification (Srivastava et al. 2014; Guo et al. 2016). Previously, increased ROS after Cd treatment has been reported in crops such as mustards, (Masood et al. 2012a, b) chickpea (Ahmad et al. 2016a, b) and tomato (Singh et al. 2018). Reduced ROS accumulation in MeJ and N supplemented seedlings may be attributed to the maintenance of efficient ROS-scavenging. In agreement with this observation, Per et al. (2016) have also demonstrated significant reduction in ROS generation, EL and TBARS under application of MeJ. MeJ has earlier been evidenced to change the fatty acid composition of membranes which makes them less prone to the damages done by ROS (Wang 1999). Moreover, optimizing ROS concentration can maintain the intimate crosstalk of ROS with other stress signalling components, such as transcription factors, leading to better elicitation of stress responses (Ahanger et al. 2017). N application to Cd-treated soybean plants resulted in a decrease of Cd stress, which was correlated with a reduction in Cd accumulation (Konotop et al. 2012) which is in aggrement with the present results. Further strengthening and protection against Cd was mediated by MeJ application, which was observed as significantly reduced endogenous Cd ion concentrations (Fig. 6) and such dilution in endogenous Cd after N and MeJ application may be attributed to greater biomass production. Similar ameliorative effects of exogenous MeJ against metal stress have been reported in rice (Singh and Shah 2014) and oil seed rape (Farooq et al. 2018) seedlings.

N-mediated alleviation of Cd stress via increased antioxidant enzyme activities has been reported in poplar plants by up-regulation and improved gene expression of antioxidant enzymes because of N supply has been reported in Populus plants under Cd stress (Zhang et al. 2014). Similarly, N-mediated alleviation of Cd stress via increased antioxidant enzyme activities has been reported in poplar plants by Zhang et al. (2017). Sirhindi et al. (2016) demonstrated that MeJ application protected Glycine max from the damaging effects of Ni by increasing the gene expression of antioxidant enzymes leading to improved metabolic and photosynthetic efficiency. Similar to these reports, application of MeJ and N in the present study significantly reduced the Cd-mediated excess accumulation of ROS by stimulating the activities of SOD, CAT, APX and GR. The higher activity of antioxidant enzymes in response to MeJ may be attributed to the direct interaction of MeJ with ROS (Chen et al. 2014). Increased CAT, APX and GR activity because of MeJ application in Cd-stressed plants has been reported by others as well (Per et al. 2016; Yan et al. 2015; Keramat et al. 2009). Increased APX and GR activity in N and MeJ supplemented plants resulted in rapid elimination of H2O2 via the AsA-GSH pathway. Application of MeJ + N further elevated the APX and GR activity, and increased GR activity by MeJ treatment in Arabidopsis thaliana plants has been reported to impart metal-stress tolerance at the post-translational level (Xiang 1998). Strengthening of the antioxidant system because of MeJ + N application apparently reduced ROS production and their consequent effects leading to alleviation of Cd-induced oxidative stress in M. arvensis. Greater GR activity and the content of AsA, and carotenoids in MeJ and N treated plants protected the photosynthetic electron transport by maintaining the optimal concentration of NADP and the redox homeostasis for maintaining the activity of key enzymes and levels of ROS. A similar ameliorative role of MeJ via increased antioxidant enzyme activities during Cd stress has been reported by Per et al. (2016) in B. juncea, Yan et al. (2015) in Solanum nigrum, Keramat et al. (2009) in Glycine max and Chen et al. (2014) in Kandelia obovata.

In the present study, contents of carotenoids increased under Cd stress. The contents of AsA decreased with Cd stress, which is in agreement with the finding of Chao et al. (2010) for rice plants who observed a decline in AsA content in response to Cd toxicity. AsA is a potent antioxidant that scavenges excess ROS in plants (Akram et al. 2017). Supplementation of MeJ + N further enhanced the AsA contents in the Cd-stressed plants, which could further protect the metabolism of M. arvensis. Increased AsA contents because of MeJ in Cd-treated K. obovata was also reported by Chen et al. (2014) and MeJ-induced up-regulated expression of genes encoding enzymes for AsA biosynthesis has been reported in A. thaliana (Wolucka et al. 2005). Greater production of AsA and carotenoids after the supplementation of N and MeJ may have improved the potentiality of M. arvensis plants to withstand the Cd stress by modulating the antioxidant defence pathways, such as AsA-GSH.

Supplementation of N and MeJ positively regulated the uptake and assimilation of N via improvement of NR activity. Greater uptake and assimilation of N sources, such as nitrates, mediates increased incorporation of available N into amino acids (Goel and Singh 2015). Similar decreases in N-assimilation by Cd stress have been reported by Gill et al. (2012) in Lepidium sativum. Enhanced N-assimilation after proper mineral application can be beneficial in arbitrating the demand for the key precursor molecules required for the synthesis of stress responsive metabolites, such as proline (Pandey and Agarwal 1998). Increased NUE because of N supplementation has been reported in Chile peppers (Huez Lopez et al. 2011). Hampered uptake capacity of nutrients because of Cd stress can be one of the possible reasons for restricted N-assimilation and external application of N and MeJ may have increased the expression of proteins required for enhanced mineral uptake at the root level, making them available for enzymes, such as NR, and hence increase NUE. Availability of nutrients and plant growth regulators increases N-assimilation under adverse environmental conditions (Iqbal et al. 2011; Singh et al. 2016a). Therefore, it can be suggested that increased N-assimilation because of N and MeJ supplementation might increase the synthesis of metabolites, optimizing the antioxidant defence and osmoprotectant systems in Cd-stressed plants and leading to protection of photosynthesis.

Supplementation of MeJ and N reduced the translocation of Cd from root-to-shoot leading to significant declines in oxidative damage. Similar effects of MeJ in the reduction of Cd toxicity have been reported by Per et al. (2016) in B. juncea and Yan et al. (2013) in Capsicum frutescens. Exogenous phytohormone application has also been reported to modulate metal toxicity by reducing the root-to-shoot metal translocation rate (Singh et al. 2015).

Cd exhibits direct competition with important mineral nutrients (Nazar et al. 2012; Asgher et al. 2014). Our results showed that Cd accumulation considerably reduced the uptake of essential mineral nutrients, which was mitigated by application of N and MeJ (Table 2). N and MeJ restore the mechanism of root-to-shoot translocation of key mineral elements and these observations are in agreement with Kováčik et al. (2011) for Scenedesmus quadricauda and Gomez et al. (2010) for Solanum lycopersicum. Rossato (2002) while working on Brassica napus observed the positive regulatory role of N and MeJ in increasing uptake and assimilation of mineral elements. Positive effects of N and MeJ were obvious when applied jointly and proved to be of considerable importance in the mitigating Cd stress. Increased uptake of mineral nutrients by Bothriochloa ischaemum was observed because of application of N (Ai et al. 2017). Plants showing enhanced uptake and assimilation of key mineral elements exhibited greater accumulation of free amino acids and tolerance to abiotic stresses (Kavi Kishor and Sreenivasulu 2014; Singh and Shah 2014).

Conclusion

Cadmium stress induced growth and photosynthetic inhibition, and enhanced ROS biosynthesis. Uptake and assimilation of mineral nutrients, stomatal movements and photosynthetic pigments declined in M. arvensis because of Cd; however, exogenous supplementation of MeJ and N optimized ROS metabolism reversing the negative effects of Cd-induced oxidative damage on physio-morphological attributes by protecting chlorophyll, Rubisco and stomatal functioning, therefore leading to growth maintenance. Such Cd stress mitigating roles of N and MeJ can be attributed to their cumulative effects on antioxidant metabolism, nutrient assimilation and osmolyte synthesis. More importantly, the maintenance of redox components, such as AsA, and carotenoids, because of N and MeJ application may have led to significant protection of the photosynthetic system. Decreased root-to-shoot translocation of Cd in N and MeJ treated M. arvensis seedlings could be exploited for its improved growth and productivity on metal and metalloid effected soils. A schematic representation drawn from the present work that how MeJ and N mediates Cd changes to improve growth and photosynthesis in given in Fig. 8.

Fig. 8
figure 8

A schematic representation showing the potential mechanism of Cd stress alleviation by combined treatment of exogenous MeJ and N. Cd stress causes oxidative stress in plants by orchestrating the generation of ROS and stomatal inhibition. Exogenous application of MeJ and N in the present study enhances the N-assimilation by directly stimulating the activity of NR and N content or by signalling pathways. Additionally, MeJ directly enhanced osmolytes, nutrient contents and activity of various antioxidants. The redox status of antioxidant defence system optimizes the ROS balance, which was altered by Cd stress. Efficient ROS metabolism maintained by MeJ and N improved growth and photosynthesis in Cd persistence in Mentha arvensis plants. Cd cadmium, N nitrogen, MeJ methyl jasmonate, ROS reactive oxygen species. Pointed arrows show promoting and blunt arrows show inhibitory interactions respectively

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