Introduction

As a toxic metalloid, arsenic (As) is a potential hazard to ecological systems due to anthropogenic impacts and environmental activities such as dumping of industrial and municipal wastes, the use of different fertilizers, pesticides, mining, and fossil fuel consumption (Mehmood et al. 2017; Ramezani et al. 2021). Humans may be exposed to As toxicity in several ways, such as the plant-soil–water system and by drinking As-contaminated water (Khalid et al. 2017). Arsenic in irrigation water and soil enters the food chain through absorption by the plant. High levels of As in soil and irrigation water not only limit the growth and productivity of crops but can also be a dangerous threat to human health by entering the food chain (Abbas et al. 2018; Ghorbani et al. 2020). Toxic levels of As induce dysfunction and oxidation of bio-molecules in plant cells through the excessive accumulation of reactive oxygen species, including singlet oxygen, hydroxyl radical, and hydrogen peroxide (H2O2) (Kohli et al. 2019; Ghorbani et al. 2021). To counteract the oxidative stress induced by As toxicity, the antioxidant defense system is naturally activated in plants, which includes the accumulation of non-enzymatic compounds such as glutathione (GSH), as well as improving the activity of antioxidant enzymes and the glyoxalase system (Afzal et al. 2018; Ghorbani et al. 2020). The glyoxalase system plays a critical role in diminishing abiotic stress-induced oxidative stress by detoxifying methylglyoxal (MG) (Hossain et al. 2012). Chelation of toxic metals in the cytoplasm by phytochelatins (PCs) and their sequestration in vacuoles is recognized an important defense mechanism for plant tolerance to heavy metal (HM) toxicity (Park et al. 2013). Therefore, stimulation of plant defense systems can effectively alleviate the negative effects induced by As stress in plants. In this regard, methyl jasmonate (MJ), as an effective bio-stimulant involved in the plant defense mechanism under biotic and abiotic stresses (Bari and Jones 2009; del Amor and Cuadra-Crespo 2011), could play an outstanding role in improving plant tolerance under As toxicity.

Jasmonates are important phytohormones in higher plants that are derived from lipids and mainly include methyl jasmonate (MJ) and jasmonic acid. Jasmonates are known to act as signal agents or elicitors in many biochemical, physiological, and molecular processes, including leaf senescence, phenolic production, fruit ripening, root growth, germination, and defence responses (Reyes-Díaz et al. 2016; Asghari 2019; Mousavi et al. 2020). Furthermore, the exogenous application of MJ plays an important role in diminishing the phytotoxic effects of HM, e.g., As (Mousavi et al. 2020), cadmium (Per et al. 2016), copper (Poonam et al. 2013), and lead (Salavati et al. 2021). However, the defense mechanisms induced by MJ in improving As toxicity tolerance need to be examined more deeply with regard to the role of nitrate reductase (NR)-mediated nitric oxide (NO).

NR enzymes are one of the main enzymes that synthesize NO in plants exposed to stressful conditions (Fu et al. 2018). NO as a signaling molecule has been reported to play a positive role in modulating the defense mechanisms of plants under various stresses such as cadmium stress (Ahmad et al. 2018; Gerami et al. 2018), As stress (Singh et al. 2017), drought (Montilla-Bascón et al. 2017), cold stress (Fan et al. 2015), and salinity (Ahmad et al., 2016). Apart from the well-known protective properties of MJ and NO in plants' tolerance to various stresses, the role of MJ and NO interaction in the response of rice plants under As toxicity has not been well studied. Therefore, the present experiment was designed to investigate the possible functions of NR-triggered NO in MJ-mediated As tolerance with respect to the content of photosynthetic pigments, the activity of some antioxidant enzymes and the glyoxalase cycle, as well as the expression level of genes responsible for As sequestration in vacuoles using sodium tungstate (ST) as an NR inhibitor. The application of sodium tungstate (ST) as an NR inhibitor abolished the positive impacts of MJ on the induction of As tolerance in rice by diminishing NR-mediated endogenous NO.

Materials and Methods

Plant Materials and Treatments

The experiments were done on rice (Oryza sativa L. cv. IR64) in greenhouse conditions with 70–75% humidity. After treating the seeds with a NaOCl (1%) solution for surface sterilization, seed germination was performed in peat moss (autoclaved twice). A MJ treatment (0.5 mM) was applied to 12-day-old seedlings by foliar spray for 3 days (once a day). A MJ solution was prepared using MJ (C13H20O3) with 0.1% ethanol and 0.01% tween-20 in distilled water. Control plants were sprayed with an equal volume of distilled water containing 0.1% ethanol and 0.01% tween-20. Before transplanting the seedlings, the roots were immersed in a 0.1 mM sodium tungstate (ST) solution for 3 h as ST treatments (Kaya 2021). Fifteen-day-old seedlings were transferred to plastic containers filled with 1/2-strength Hoagland solution containing 50 µM As (NaAsO2) or without As, which were replaced with fresh solution every 3 days. After transplanting rice seedlings, a foliar spray of 0.1 mM sodium nitroprusside (SNP) was applied twice (once every week). Temperature, light period and light intensity of the greenhouse were maintained at 25 ± 3 °C, 16 h light and 300–350 μmol m−2 s−1, respectively (Ghorbani et al. 2011). Samples were collected 2 weeks after beginning As treatment. After recording the plant height, the total dry weight was estimated by incubating the samples at 75 °C for 48 h (Ghorbani et al. 2009). After harvesting, fresh samples were rapidly frozen in liquid nitrogen and kept at -80 °C for biochemical and molecular attributes.

Chlorophyll Florescence and Photosynthetic Pigments

The PAM fluorometer (PAM 2500, Walz) was employed to appraise the chlorophyll fluorescence value of rice leaves after 30 min dark adaptation (Ghorbani et al. 2018b). Fresh leaves were applied to assess the content of photosynthetic pigments using 80% acetone. After centrifugation and reading of the supernatants at 460, 645, and 663 nm, the contents of chlorophyll a, b, and carotenoids were calculated by the method outlined by Lichtenthaler (1987).

Nitric Oxide (NO) Content

By determining nitrite (NO2−) content using the Griess reagent, NO content was obtained according to Zhou et al. (2005). Extraction buffer (cold acetic acid (50 mM, pH 3.6) including 4% zinc diacetate) was used for the homogenization of fresh roots and leaves. After centrifugation at 12,000 g for 15 min at 4 °C, the supernatants were mixed with charcoal and incubated at room temperature for 30 min. Then, the Griess reagent was added to the samples and read at 540 nm.

Arsenic Content

By digesting the dried root and shoot tissues in HNO3:H2O2 (1:4 ratio), the root and shoot concentrations of As were obtained using an ICP-MS (Agilent 7500 cx).

Glutathione (GSH) and Phytochelatins (PCs)

Glutathione extraction was achieved using meta-phosphoric acid (6%, pH 2.8) containing EDTA (1 mM). After centrifugation, polyvinylpolypyrrolidone was added to the supernatants and centrifuged at 12,000 × g for 20 min (Yu et al. 2003). Supernatants were combined with yeast GSH reductase (GR, 20 IU mL–1), 5,5′-dithiobis(2-nitrobenzoic acid (2.4 mg mL–1), NADPH (1.9 mg mL–1), and potassium phosphate buffer (0.1 M, pH 7.0) containing EDTA (5 mM). After reading at 412 nm, the total GSH content was expressed as µmol g–1 FW following the method of Adams and Liyanage (1991). After determining non-protein thiols (Howe and Merchant, 1992), the content of PCs was obtained by subtracting the GSH content from the content of non-protein thiols (De Vos et al. 1992).

Nitrate Reductase (NR) Activity

An extraction buffer containing HEPES–KOH (0.1 M, pH 7.5), 1,4-dithiothreitol (5 mM), polyvinylpyrrolidone (1%), Triton X-100 (0.1%), glycerol (10%), flavin adenine dinucleotide (20 μM), EDTA (1 mM) and phenylmethylsulfonyl fluoride (0.5 mM) was applied to quantify the leaf activity of NR. After centrifugation, the supernatants were utilized to quantify NR activity by the method of Sun et al. (2014). Assay buffer containing HEPES–KOH (50 mM, pH 7.5), MgCl2 (10 mM), 1,4-dithiothreitol (1 mM), KNO3 (2 mM) and NADH (0.2 mM) was mixed with the supernatants and incubated at 30 °C for 30 min. After adding Zn-acetate (0.5 M), N-(1-naphthyl) ethylenediamine (0.02%) in HCl (0.2 M) and sulfanilamide (1%) in HCl (3 M), the produced nitrate was determined by reading at 520 nm.

Electrolyte Leakage (EL)

After washing the leaves with distilled water, the leaf discs were incubated in distilled water at room temperature on a shaker, and after 24 h, electrical conductivity was recorded (EC1). Then, to determine EC2, the samples were autoclaved at 120 °C for 20 min (Dionisio-Sese and Tobita (1998). After recording EC2, EL was calculated as: EL (%) = (EC1/EC2) × 100.

Hydrogen Peroxide (H2O2), Methylglyoxal (MG) and Malondialdehyde (MDA) Contents

The method of Loreto and Velikova (2001) was used to quantify the leaf H2O2 content. Trichloroacetic acid (TCA, 1%) was employed to extract fresh leaves. After centrifugation of the extracts, K buffer (10 mM) and potassium iodide (1 M) were added to the supernatants and read at 390 nm.

Homogenization of fresh leaves with perchloric acid (5%) was performed to quantify MG using the procedure of Wild et al. (2012). After centrifugation, charcoal and saturated potassium carbonate were utilized to decolorize and neutralize the supernatants. After adding N-acetyl-L-cysteine and NaH2PO4, the absorbance of the mixtures was recorded at 288 nm.

The method of Weisany et al. (2012) was applied to measure leaf MDA levels. Trichloroacetic acid (1%, w:v) was used to homogenize fresh leaves, and after centrifugation, thiobarbituric acid (0.5%) and TCA (20%) were mixed with the supernatants. The mixtures were placed in a water bath (90 °C) for 30 min, and after cooling, the absorbance of the samples was read at 532 nm.

Extraction and Assay of Enzymes

Extraction buffers including potassium phosphate buffer (50 mM, pH 7.0), glycerol (10%), KCl (100 mM), ascorbic acid (1 mM) and β-mercaptoethanol (5 mM) were employed to homogenize fresh leaves. After centrifugation of the extracts, supernatants were used to estimate enzyme activity and protein content (Bradford 1976).

The reaction mixtures containing enzyme extract, K-P buffer (50 mM, pH 7.0) and H2O2 (5.9 mM) were employed to quantify the activity of catalase (CAT) (Chance and Maehly 1955). CAT activity was quantified by recording a decline in the OD of 240 nm following the decomposition of H2O2 for 1 min. One unit of the CAT enzyme represents the amount needed to decompose H2O2 per min.

The reaction solution containing enzyme extract, phosphate buffer (50 mM, pH 7.8), EDTA (0.1 mM), nitroblue tetrazolium (NBT, 0.75 µM), riboflavin (2 mM), Na2CO3 (50 mM) and methionine (13.33 mM) was utilized to assess the activity of superoxide dismutase (SOD) based on the inhibition of photochemical reduction of NBT (Dhindsa et al. 1981).

The protocol of Nakano and Asada (1981) was utilized to assess the activity of the ascorbate peroxidase (APX) enzyme by recording a change in the absorbance of the reaction solution containing K–P buffer (50 mM, pH 7.0), enzyme extracts, EDTA (0.1 mM), ascorbic acid (0.5 mM) and H2O2 (0.1 mM) at 290 nm. The amount needed to oxidize ascorbate per min was defined as one unit of the APX enzyme.

By regarding the alteration in the absorbance of the mixture including enzymatic extracts, K-P buffer (0.1 mM, pH 7.0), NADPH (0.2 mM), EDTA (1 mM) and oxidized glutathione (1 mM) at 340 nm, the activity of the glutathione reductase (GR) was obtained following the method optimized by Hasanuzzaman et al. (2011). The amount required to catalyze the reduction of oxidized glutathione per min was defined as one unit of GR enzyme.

The procedure of Hasanuzzaman et al. (2012) was applied to quantify the activity of glyoxalase (Gly) I enzyme by reading the reaction mixture (the enzyme assay solution, K–P buffer (100 mM, pH 7.0), MG (3.5 mM), glutathione (1.7 mM) and MgSO4 (15 mM)) at 240 nm.

A reaction mixture of enzyme extract, Tris − HCl buffer (100 mM, pH), 5,5′-dithio-bis(2-nitrobenzoic acid) (0.2 mM) and SD-lactoylglutathione (1 mM) was employed to determine the activity of Gly II enzyme following Hossain et al. (2010) method by recording the absorbance changes at 240 nm.

Gene Expression

After extraction of the total RNA from roots and leaves using TRIzol reagent (Invitrogen, USA), first-strand cDNA synthesis was performed by the RevertAid™ Reverse Transcriptase kit (Fermentase, Germany) (Ghasemi-Omran et al. 2021). qPCR reactions were performed using Maxima SYBR Green/ROX qPCR Master Mix (Thermo Scientific). The specific primers (Table S1) used for GSH1, PCS, ABCC1 and Actin (internal control) genes were designed by an online program (Primer3). The 2−ΔΔCT method was utilized to analyze qPCR data (Ghorbani et al. 2019a).

Statistical Analysis

Five biological replications were used for morphological and biochemical traits (at least 3 technical repeats for each replication), while gene expression was analyzed based on three biological replications (at least 3 technical repeats for each replication). All data were analyzed by SAS 9.1 software (Ghorbani et al. 2019b). In the tables and figures, the means (± standard division) were compared at a 0.05% level of confidence by the LSD test.

Results

Morphological and Photosynthetic Attributes

Arsenic treatment (50 μM) significantly diminished height and total dry weight by 22.6 and 25.8%, respectively, compared to untreated plants. In As-stressed plants, MJ, MJ + SNP, and MJ + ST + SNP treatments enhanced plant height by 14.2, 20.3 and 3.4% and total dry weight by 16, 25.8 and 14.3%, respectively, however, MJ + ST treatment diminished height and total dry weight by 11.7 and 4.9%, respectively, over As-exposed plants alone. Furthermore, all treatments (MJ, MJ + ST, MJ + SNP and MJ + ST + SNP) did not induce a significant effect on height and total dry weight in control plants (CP) (Table 1).

Table 1 Morphological traits, photosynthetic pigments and Fv/Fm value in rice plants sprayed with methyl jasmonate (MJ, 0.5 mM) under arsenic (As, 50 µM) treatment combined with sodium tungstate (ST, 0.1 mM) and sodium nitroprusside (SNP, 0.1 mM)
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In CP, the application of MJ, MJ + ST, MJ + SNP, and MJ + ST + SNP treatments did not induce significant differences in the content of chlorophyll a, b, carotenoids or Fv/Fm values. The addition of 50 μM As lessened the contents of chlorophyll a, b, carotenoids, and Fv/Fm by 54.6, 60.7, 28, and 37.2%, respectively, over CP. In As-exposed plants, the application of MJ, MJ + SNP and MJ + ST + SNP treatments significantly improved the photosynthetic pigments and Fv/Fm value in comparison with As-treated plants alone, which recorded the highest increase under MJ + SNP treatment. However, MJ + ST treatment did not cause a significant difference in the photosynthetic pigments or Fv/Fm value in As-stressed plants (Table 1).

The Contents of NO and As

A significant rise in root and leaf contents of NO was recorded by 58 and 68.5%, respectively, under As stress over CP. In plants treated with 50 μM As, the exogenous application of MJ, MJ + SNP, and MJ + ST + SNP treatments caused a significant increase in root and leaf NO content, and the highest improvement was observed in plants treated with MJ + SNP. However, MJ + ST treatment lessened root and leaf NO levels in As-stressed plants (Table 2).

Table 2 Endogenous NO levels and arsenic accumulation in rice plants sprayed with methyl jasmonate (MJ, 0.5 mM) under arsenic (As, 50 µM) treatment combined with sodium tungstate (ST, 0.1 mM) and sodium nitroprusside (SNP, 0.1 mM)
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The addition of 50 μM As caused the accumulation of 462.7, 88.57, and 551.2 μg/gDW of As in the roots, shoots and whole plant, respectively. However, MJ, MJ + SNP and MJ + ST + SNP treatments diminished As accumulation in roots (50.5, 57.5 and 21.3%), shoots (68.2, 71.2 and 45.5%) and whole plants (53.3, 59.7 and 25.2%) in As-treated plants. MJ + ST treatment increased the concentration of As in the roots and the whole plant by 4 and 4%, respectively, in As-treated plants (Table 2).

Oxidative Stress Markers

The application of MJ, MJ + ST, MJ + SNP, and MJ + ST + SNP treatments did not induce significant differences in the leaf levels of H2O2, MG, MDA, and EL in CP; however, the application of As significantly increased the leaf levels of H2O2, MG, MDA, and EL by 4.3, 2, 3.1, and 2.4-fold, respectively, over CP. In plants treated with As, the application of MJ and MJ + SNP significantly diminished the levels of H2O2, MG, MDA, and EL over As-treated plants alone, and the lowest levels of these traits were observed under MJ + SNP treatment. The use of MJ + ST significantly enhanced the levels of MG and MDA, while it did not induce significant differences in H2O2 and EL levels in As-stressed plants. MJ + ST + SNP treatment declined the levels of H2O2 and MDA, and increased the level of MG in the leaves of As-treated plants (Fig. 1A, B, C and D).

Fig. 1
figure 1

Levels of hydrogen peroxide (H2O2, A), methylglyoxal (MG, B), malondialdehyde (MDA, C) and electrolyte leakage (EL, D) in rice leaves sprayed with methyl jasmonate (MJ, 0.5 mM) under arsenic (As, 50 µM) treatment combined with sodium tungstate (ST, 0.1 mM) and sodium nitroprusside (SNP, 0.1 mM). Different letters in each column show significant differences at the p < 0.05 based on the LSD test (means ± SD, n = 5)

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Antioxidant Enzymes and the Glyoxalase Pathway

The results showed that MJ, MJ + ST, MJ + SNP, and MJ + ST + SNP treatments did not induce significant differences in the activity of CAT, SOD, APX, and GR enzymes in CP. However, As treatment increased the activity of CAT, SOD, APX, and GR in the leaves of rice plants by 57.4, 37.4, 64.2, and 51.5%, respectively over CP. In As-treated plants, MJ and MJ + SNP treatments upregulated the activity of these enzymes compared to As-treated plants alone. Although MJ + ST did not have a significant effect on SOD activity, it decreased the activity of CAT, APX, and GR in As-treated plants. MJ + ST + SNP treatment enhanced the activity of SOD and GR enzymes in As-treated plants, while it did not cause a significant difference in CAT and APX (Fig. 2A, B, C and D).

Fig. 2
figure 2

The activities of catalase (CAT, A), superoxide dismutase (SOD, B), ascorbate peroxidase (APX, C) and glutathione reductase (GR, D) in rice leaves sprayed with methyl jasmonate (MJ, 0.5 mM) under arsenic (As, 50 µM) treatment combined with sodium tungstate (ST, 0.1 mM) and sodium nitroprusside (SNP, 0.1 mM). Different letters in each column show significant differences at the p < 0.05 based on the LSD test (means ± SD, n = 5)

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When rice plants were treated with 50 μM As, the activity of Gly I and II enzymes was elevated by 35.8 and 47.6%, respectively, over CP. When As-treated plants were supplied with MJ and MJ + SNP, a further increase in the activity of these enzymes was recorded. However, MJ + ST treatment significantly declined Gly I and II activity in As-treated plants. MJ + ST + SNP treatment did not induce a significant difference in Gly I activity but decreased Gly II activity in As-exposed plants (Fig. 3A and B).

Fig. 3
figure 3

The activities of glyoxalase I (Gly I, A) and Gly II (B) in rice leaves sprayed with methyl jasmonate (MJ, 0.5 mM) under arsenic (As, 50 µM) treatment combined with sodium tungstate (ST, 0.1 mM) and sodium nitroprusside (SNP, 0.1 mM). Different letters in each column show significant differences at the p < 0.05 based on the LSD test (means ± SD, n = 5)

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The Activity of NR Enzyme

In CP, the application of MJ and MJ + SNP treatments did not cause significant differences in NR activity. However, MJ + ST and MJ + ST + SNP treatments lessened NR activity by 56 and 56.2%, respectively, over CP. The application of As significantly enhanced NR activity over CP. The application of MJ and MJ + SNP further enhanced NR activity in As-stressed plants. However, MJ + ST and MJ + ST + SNP treatments lowered NR activity in As-stressed plants compared to plants treated with As alone (Fig. 4).

Fig. 4
figure 4

The activity of nitrate reductase in rice leaves sprayed with methyl jasmonate (MJ, 0.5 mM) under arsenic (As, 50 µM) treatment combined with sodium tungstate (ST, 0.1 mM) and sodium nitroprusside (SNP, 0.1 mM). Different letters in each column show significant differences at the p < 0.05 based on the LSD test (means ± SD, n = 5)

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Root and Leaf Contents of PCs and GSH

The application of MJ, MJ + ST, MJ + SNP, and MJ + ST + SNP treatments did not cause a significant difference in the root and leaf contents of PCs in CP. Arsenic treatment elevated the root and leaf contents of PCs by 52.4 and 74%, respectively over CP. In As-treated plants, MJ, MJ + SNP, and MJ + ST + SNP applications significantly enhanced the root accumulation of PCs by 38.8, 51.4, and 17.5%, respectively, while MJ + ST treatment did not induce significant effects on PCs content in roots compared to As-exposed plants alone. In the leaves of As-treated plants, MJ + ST treatment lessened the content of PCs by 9.4% and MJ + ST + SNP treatment increased the content of PCs by 8.7%. However, MJ and MJ + SNP treatments did not induce significant effects on the leaf content of PCs in As-exposed plants (Fig. 5A and B).

Fig. 5
figure 5

The root and leaf contents of phytochelatins (PCs, A and B) and glutathione (GSH, C and D) in rice plants sprayed with methyl jasmonate (MJ, 0.5 mM) under arsenic (As, 50 µM) treatment combined with sodium tungstate (ST, 0.1 mM) and sodium nitroprusside (SNP, 0.1 mM). Different letters in each column show significant differences at the p < 0.05 based on the LSD test (means ± SD, n = 5)

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Significant increases in root and leaf content of GSH were observed under As treatment by 20.1 and 31.1%, respectively over CP. In As-treated plants, applications of MJ, MJ + SNP, and MJ + ST + SNP significantly enhanced root and leaf accumulation of GSH over As-exposed plants alone. The MJ + ST treatment did not induce a significant difference in GSH accumulation in the roots, however, it significantly declined GSH content in the leaves in As-exposed plants (Fig. 5C and D).

Root and Leaf Expression of GH1, PCS and ABCC1 Genes

The results of gene expression revealed that the application of MJ, MJ + ST, MJ + SNP, and MJ + ST + SNP treatments did not induce significant effects on root and leaf expression of the GSH1 gene in CP. However, a significant upregulation in GSH1 expression was found in the roots and leaves of As-treated rice plants over CP. In As-treated plants, the application of MJ, MJ + SNP, and MJ + ST + SNP treatments upregulated GSH1 expression in the roots and leaves over As-treated plants alone, and the highest increase was found under MJ + SNP treatment. When As-treated plants were exposed to MJ + ST treatment, GSH1 expression in the roots and leaves revealed a significant downregulation over As-treated plants alone (Fig. 6A and B).

Fig. 6
figure 6

The relative expression of GSH1 (A and B), PCS (C and D) and ABCC1 (E and F) in the roots and leaves of rice plants sprayed with methyl jasmonate (MJ, 0.5 mM) under arsenic (As, 50 µM) treatment combined with sodium tungstate (ST, 0.1 mM) and sodium nitroprusside (SNP, 0.1 mM). Different letters in each column show significant differences at the p < 0.05 based on the LSD test (means ± SD, n = 5)

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When rice plants were treated with 50 μM As, PCS expression in roots and leaves was upregulated by 2.7- and 2.1-fold, respectively over CP. In As-treated plants, MJ, MJ + SNP, and MJ + ST + SNP treatments significantly upregulated PCS expression in the roots and the highest increase was recorded in plants treated with MJ + SNP. However, MJ + ST treatment lessened PCS expression in the roots of As-treated plants. MJ, MJ + SNP, and MJ + ST + SNP treatments did not induce significant differences in PCS expression in the leaves of As-exposed plants, while MJ + ST treatment declined PCS expression (Fig. 6C and D).

A significant upregulation in ABCC1 expression was found in the roots and leaves of rice plants treated with 50 μM As over CP. In the roots, MJ, MJ + SNP, and MJ + ST + SNP treatments increased ABC1 expression in As-exposed plants, with the highest rise observed under MJ + SNP treatment, while MJ + ST treatment did not induce a significant difference in ABCC1 expression. In the leaves, MJ + ST + SNP treatment upregulated ABCC1 expression and MJ + ST treatment downregulated ABCC1 expression in As-treated plants. However, MJ and MJ + SNP treatments did not induce significant effects on ABCC1 expression (Fig. 6E and F).

Discussion

Here, the possible role of the NR enzyme in As tolerance induced by MJ in rice plants was evaluated. Although the positive impacts of MJ on improving the tolerance of plants under As stress have been confirmed (Mousavi et al. 2020; Verma et al. 2020), no report of the possible role of NR in MJ-mediated As tolerance has been recorded. The results revealed that 50 μM As treatment diminished the growth and biomass of the rice plant, as previously recorded on rice (Ghorbani et al. 2020), oilseed rape (Farooq et al. 2016) and maize (Kaya et al. 2020). Arsenic stress has been shown to reduce plant growth by disrupting ionic homeostasis, inducing oxidative stress, and dysfunction of the photosynthetic apparatus (Hasanuzzaman and Fujita 2013; Ghorbani et al. 2020; Ahmad et al. 2020). However, the application of MJ significantly improved the growth and biomass of rice plants under As stress, which is consistent with the results obtained on rice (Mousavi et al. 2020; Verma et al. 2020) and oilseed rape (Farooq et al. 2016). These results confirm that MJ, as a growth regulator, can effectively improve the tolerance of plants to As toxicity. The exogenous application of MJ has been shown to reduce As uptake and diminish oxidative stress by modulating the expression of As transporters and augmenting the antioxidant defense system, thereby improving plant growth under As phytotoxicity (Mousavi et al. 2020; Verma et al. 2020). To investigate the possible role of NR enzyme in improving plant tolerance to As stress induced by MJ, ST was used as an inhibitor of NR activity in this study to prevent the synthesis or accumulation of NO during MJ application. The results showed that ST application prevented the positive effects induced by MJ, indicating that MJ improves the tolerance of rice plants under As toxicity by inducing NR activity. Kaya et al. (2020) affirmed that inhibition of NR activity abolished salicylic acid-induced As stress tolerance, indicating the important role of NR-synthesized NO in plants' tolerance to As stress. In the current study, SNP was used as a NO donor to confirm the participation of NR-synthesized NO in MJ-induced As tolerance. As previously indicated by Singh et al. (2016) and Hasanuzzaman and Fujita (2013), the application of SNP restored the ameliorative effects of MJ in MJ + ST-treated plants and enhanced the positive effects of MJ in MJ-treated plants under As stress, indicating the role of NO in improving plant tolerance to As stress. Thus, these results reveal that NR-synthesized NO as a signal molecule is activated by MJ and plays an important role in inducing MJ-induced As tolerance in rice plants.

Arsenic treatment adversely affected the chlorophylls and carotenoids contents and Fv/Fm value in rice, which indicates the destructive effect of As toxicity on the efficiency of photosynthetic apparatus. Arsenic stress has been reported to diminish photosynthetic pigments and disrupt photosynthetic centres by inducing chlorophyll-degrading enzymes and reducing the activity of enzymes involved in chlorophyll synthesis, as well as increasing the accumulation of toxic radicals and inducing oxidative stress (Zemanová et al. 2020; Ghorbani et al. 2020). The application of MJ alleviated the negative effects of As stress on photosynthetic attributes, indicating the protective effects of MJ on the photosynthetic pigments. The protective effects of MJ on the photosynthetic pigments of rice under As stress can be due to a reduction in the levels of reactive oxygen species (ROS) and an increase in the activity of antioxidant enzymes, as previously affirmed by Coelho et al. (2020) and Mousavi et al. (2020). ST treatment reversed the protective effects of MJ on photosynthetic pigments, indicating the participation of NR and NO in MJ-induced protective responses on the photosynthetic pigments of rice under As stress. The use of SNP abolished the inhibitory effects of ST on the protective role of MJ on the photosynthetic apparatus, which confirms the role of NO in MJ-induced defense responses. Increased photosynthetic pigments and protection of the photosynthetic apparatus by NO under As stress have been previously reported (Singh et al. 2016; Ahmad et al. 2020). Thus, MJ protects the photosynthetic pigments of rice plants under As toxicity by activating NR and increasing the endogenous level of NO.

The results indicated that MJ lessened the uptake of As by the roots and its translocation to the shoots and, consequently, diminished the accumulation of As in the whole plant, which could play an important role in mitigating the toxic effects of As on rice. The MJ-mediated decline in As content has been announced in Brassica napus (Farooq et al. 2016) and rice (Verma et al. 2020). MJ has been demonstrated to diminish the uptake and translocation of As in As-stressed rice plants by modulating the expression of As transporters (Mousavi et al. 2020). However, the application of ST completely reversed the MJ-mediated decrease in As accumulation, suggesting that NR-synthesized NO may be involved in modulating the expression of As transporters induced by MJ. Kaya et al. (2020) indicated that NR-mediated NO is implicated in reducing brassinosteroid-induced cadmium accumulation in pepper plants under cadmium toxicity. As a result, SNP application eliminated the inhibitory effects of ST and, as a result, reduced the accumulation of As in MJ + ST-treated plants under As toxicity, which affirms the role of MJ-induced endogenous NO in reducing As uptake in As-stressed rice. Similar results of an exogenous NO-induced decrease in As accumulation in Vicia faba (Ahmad et al. 2020) and rice (Singh et al. 2016) under As stress have been previously published.

Arsenic treatment enhanced the accumulation of H2O2 and MG, resulting in damage to bio-membranes and induction of EL, indicating the toxic effects of As on rice, as reported by Ghorbani et al. (2020) and Mousavi et al. (2020). However, MJ diminished the levels of H2O2 and MG by increasing the activity of antioxidant enzymes (CAT, SOD, GR, and APX) and the glyoxalase system (Gly I and II), thereby protecting bio-membranes (reducing MDA and EL). Farooq et al. (2016) revealed that MJ raised the tolerance of Brassica napus under As stress by upregulating the expression of CAT, SOD, APX, and peroxidase enzymes. ST treatment eliminated the upregulation in the activity of antioxidant enzymes and the glyoxalase cycle mediated by MJ, which increased H2O2, MG, MDA and EL in the leaves of rice plants under As stress. Kaya et al. (2020) indicated that NR and NO are involved in increasing the activity of antioxidant enzymes and reducing oxidative stress by brassinosteroid in cadmium-stressed pepper plants, indicating the essential role of NR-mediated NO in the defense responses of plants under HM stress. To confirm the role of exogenous NO in the induction of the defense mechanism triggered by MJ, SNP was used to replace ST-eliminated endogenous NO. The results revealed that the use of SNP reversed the mitigating effects of MJ on As-induced oxidative stress in MJ + ST-treated rice plants, indicating the significant role of NR-synthesized endogenous NO in inducing the MJ-mediated antioxidant defense mechanism. The participation of NR-synthesized NO in the induction of salicylic acid-mediated antioxidant defense systems under drought stress has also been reported by Kaya (2021) in pepper plants. It has previously been shown that the external application of NO augmented the antioxidant defense system and the glyoxalase cycle, thereby improving plant tolerance to As stress (Hasanuzzaman and Fujita 2013; Singh et al. 2016). However, our results suggest for the first time in the literature that MJ, by activating NR and enhancing the internal level of NO, augments the antioxidant defense system and the glyoxalase cycle, thereby improving the tolerance of rice plants to As phytotoxicity.

Phytochelatins are important defense compounds synthesized from GSH that, by chelating toxic metal ions, protect metabolic organelles against HM toxicity (Hall 2002; Ghorbani et al. 2018a). The results indicated that MJ enhanced the root and leaf expression of GSH1 and the root expression of PCS and ABCC1 in As-treated rice plants, which is consistent with increasing the root contents of PCs and GSH and the leaf content of GSH. It has been shown that increasing the expression of genes implicated in the sequestration of toxic metal ions can play an important role in enhancing plant tolerance under HM toxicity (Hasan et al. 2015). Thus, MJ protects metabolic organelles against toxic As by increasing the accumulation of GSH and PCs and the sequestration of As in vacuoles, which can play an important role in increasing the tolerance of rice plants under As stress. However, the positive effects of MJ on the induction of the As sequestration mechanism was abolished by ST treatment, suggesting the role of NR-synthesized NO as a signaling molecule downstream of MJ. The hindering effects of ST on the MJ-induced sequestration mechanism were reversed by SNP, which confirms the role of NR and NO in the induction of MJ-mediated sequestration mechanism. The role of NR-synthesized NO downstream of the phytohormones brassinosteroid and salicylic acid under cadmium and drought stress, respectively, has been previously announced. Thus, our findings show for the first time that MJ, by activating NR and increasing the endogenous level of NO, upregulated the expression of GSH1, PCS and ABCC1 and increased the accumulation of GSH and PCs compounds to promote plant tolerance under As toxicity.

Conclusion

The results revealed that MJ, by activating NR and increasing endogenous NO, increased rice plant tolerance to As toxicity, suggesting that NO may be involved as a signaling molecule downstream of the MJ-induced defense mechanism in As-stressed rice plants. To confirm the role of NR and NO in the induction of MJ-mediated defense mechanism, ST was used as an NR inhibitor, which revealed that ST reversed the positive effects of MJ on the antioxidant defense system, the glyoxalase cycle and As sequestration mechanism in As-stressed rice plants. To provide further evidence, SNP as a NO donor abolished the inhibitory effects of ST on the defense mechanism induced by MJ, which confirms the contribution of MJ-induced internal NO in improving the tolerance of rice plants under As stress. Thus, by inducing NR enzyme and increasing the internal level of NO, MJ augmented the antioxidant defense system and the glyoxalase cycle, as well as modulating As sequestration mechanism, which effectively reduced As accumulation and mitigated oxidative stress, protected the photosynthetic apparatus and bio-membranes, and thus promoted the growth and biomass of rice plants under As stress. In the future, the association of other signaling molecules or enzymes in inducing phytohormones-mediated abiotic stresses tolerance requires to be investigated.