Ionic Homeostasis and Redox Metabolism Upregulated by 24-Epibrassinolide are Crucial for Mitigating Nickel Excess in Soybean Plants, Enhancing Photosystem II Efficiency and Biomass

doi:10.21203/rs.3.rs-418596/v1

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Research Article

Ionic Homeostasis and Redox Metabolism Upregulated by 24-Epibrassinolide are Crucial for Mitigating Nickel Excess in Soybean Plants, Enhancing Photosystem II Efficiency and Biomass

https://doi.org/10.21203/rs.3.rs-418596/v1

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posted 10 May, 2021

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Nickel (Ni) excess often generates oxidative stress in chloroplasts, causing redox imbalance, membrane damage and negative impacts on biomass. 24-Epibrassinolide (EBR) is a plant growth regulator of great interest in the scientific community because it is a natural molecule extracted from plants that is biodegradable and environmentally friendly. This study aimed to determine whether EBR can induce benefits on ionic homeostasis and antioxidant enzymes and convey possible repercussions on photosystem II efficiency and biomass, more specifically evaluating nutritional, physiological, biochemical and morphological responses in soybean plants subjected to Ni excess. The experiment was randomized with four treatments, including two Ni concentrations (0 and 200 µM Ni, described as – Ni²⁺ and + Ni²⁺, respectively) and two concentrations of 24-epibrassinolide (0 and 100 nM EBR, described as – EBR and + EBR, respectively). In general, Ni caused deleterious modulatory effects on chlorophyll fluorescence and gas exchange. In contrast, EBR enhanced the effective quantum yield of PSII photochemistry (15%) and electron transport rate (19%) due to upregulation of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase. Exogenous EBR application promoted significant increases in biomass, and these results were explained by the benefits on nutrient contents and ionic homeostasis, demonstrated by increased Ca²⁺/Ni²⁺, Mg²⁺/Ni^+ 2 and Mn²⁺/Ni²⁺ ratios.

Environmental Engineering

Environmental Policy

brassinosteroids

chlorophyll fluorescence

Glycine max

growth

heavy metal

Soybean [Glycine max (L.) Merr.] is a leguminous species that is highly relevant to the Brazilian economy and other producer countries because grains are rich in proteins and oils (Bamji and Corbitt, 2017) and are considered a commodity with several possibilities, primarily human nutrition and animal feed (Majumdar et al., 2019). However, environmental problems connected to heavy metals have been verified in cultivated areas with soybean crops, reducing plant performance and subsequent yield (Küpper and Andresen, 2016; Reis et al., 2017).

Soil contamination by heavy metals often occurs due to inadequate crop management and intensively applied pesticides, fertilizers, and petroleum products (Ayangbenro and Babalola, 2017; Mir et al., 2018), in which these compounds are applied indiscriminately and can cause deleterious effects on plants (Aprile and De Bellis, 2020). Nickel (Ni) excess in agronomic crops is a theme of great importance for food security, attracting the attention of researchers worldwide due to representing a recurrent problem in modern agriculture (M. Yusuf et al., 2011), in which this element is found in contaminated environments as Ni²⁺ (Ameen et al., 2019).

Ni excess often impacts biomass, which is correlated with inadequate uptake, transport and distribution of macro- and micronutrients (Matraszek et al., 2016), including strong limitations on the absorption of Mg, Mn, Zn and Fe (Palacios et al. 1998; Torres et al. 2016). Phytotoxicity linked to Ni negatively modulates photochemical efficiency (Ribeiro et al., 2020), gas exchange (Nazir et al., 2019), water relations and protein biosynthesis (Azeem, 2018). These deleterious effects are occasioned by the overproduction of reactive oxygen species (ROS), such as hydrogen peroxide (H₂O₂), superoxide (O₂⁻) and hydroxyl radicals (-OH) (Amari et al., 2017; Yan et al., 2010). Oxidative stress generated in chloroplasts causes redox imbalance and membrane damage (Gajewska et al., 2006; Israr et al., 2011; Pietrini et al., 2015; Rizwan et al., 2018; Sreekanth et al., 2013).

Brassinosteroids are plant growth regulators that act by stimulating biochemical reactions and physiological responses and modulating cellular functions (Jan et al., 2018; Rahman et al., 2017). Among steroids, there are more than 50 natural and synthetic forms, of which 24-epibrassinolide (EBR) is of great interest to the scientific community because it is a natural molecule extracted from plants that is both biodegradable and environmentally friendly. This molecule plays multiple roles, stimulating chloroplast pigments (Parmoon et al., 2018), light capture (Lima and Lobato, 2017; Yusuf et al., 2017) and CO₂ fixation (Kohli et al., 2017; Sharma et al., 2016), biosynthesis of nucleic acids (Bajguz, 2000; Tanveer et al., 2018) and tissue structures (Fonseca et al., 2020; Rajewska et al., 2016; Ribeiro et al., 2019). Focusing on plant nutrition, Lima et al. (2018) verified that EBR increased the content of essential elements, improving the nutritional balance (Oliveira et al., 2018) and the activities of H+-ATPase enzymes in the root system (Song et al., 2016). In parallel, the literature has verified that EBR positively modulates the antioxidant system (Ahanger et al., 2020; Rodrigues et al., 2020), more specifically controlling ROS overaccumulation (Anjum et al., 2017; Ashraf et al., 2015).

The hypothesis of this research was developed on the deleterious effects provoked by Ni on photochemical efficiency (Drążkiewicz and Baszyński, 2010; Pietrini et al., 2015) and growth (Parida et al., 2003; Rahman et al., 2005) verified in plants grown in environments contaminated by this heavy metal. However, relevant roles linked to EBR in nutritional status (Lima et al., 2018) and the antioxidant system (Santos et al., 2020) suggest that this growth regulator may represent an interesting option to mitigate the negative impacts induced by Ni. This study aimed to determine whether EBR induces benefits for ionic homeostasis and antioxidant enzymes and to reveal possible repercussions on photosystem II efficiency and biomass, more specifically evaluating nutritional, physiological, biochemical and morphological responses in soybean plants subjected to Ni excess.

Location and growth conditions

This experiment was performed at the Campus of Paragominas of the Universidade Federal Rural da Amazônia, Paragominas, Brazil (2°55’ S, 47°34’ W). This study was conducted in a greenhouse with controlled temperature and humidity. The minimum, maximum, and median temperatures were 23.4, 29.8 and 26.3°C, respectively. The relative humidity during the experimental period varied between 60% and 80%.

Plants, containers and acclimation

Seeds of Glycine max (L.) Merr. var. M8644RR Monsoy™ were germinated and grown in 1.2-L pots filled with a mixed substrate of sand and vermiculite at a ratio of 3:1. The plants were cultivated under semi-hydroponic conditions containing 500 mL of distilled water for four days. A nutritive solution described by Pereira et al. (2019) was used for plant nutrition, with ionic strength beginning at 50% (4th day) and later modified to 100% after two days (6th day). After this period, the nutritive solution remained at total ionic strength.

Experimental design

The experiment was randomized with four treatments, including two Ni concentrations (0 and 200 µM Ni, described as – Ni²⁺ and + Ni²⁺, respectively) and two concentrations of 24-epibrassinolide (0 and 100 nM EBR, described as – EBR and + EBR, respectively). Five replicates for each of the four treatments were conducted, yielding a total of 20 experimental units, with one plant per unit.

24-Epibrassinolide (EBR) preparation and application

Ten-day-old plants were sprayed with 24-epibrassinolide (EBR) or Milli-Q water (containing a proportion of ethanol that was equal to that used to prepare the EBR solution) at 5-d intervals until day 30. EBR (0 and 100 nM, Sigma-Aldrich, USA) solutions were prepared by dissolving the solute in ethanol followed by dilution with Milli-Q water [ethanol:water (v/v) = 1:10,000] (Ahammed et al., 2013).

Plant conduction and Ni treatment

Plants received the following macro- and micronutrients contained in the nutrient solution in agreement with Pereira et al. (2019). To simulate high Ni concentration. NiCl₂ was used at concentrations of 0 and 200 µM Ni, which was applied over 8 days (days 22–30 after the start of the experiment). During the study, the nutrient solutions were changed at 07:00 h at 3-day intervals, with the pH adjusted to 5.5 using HCl or NaOH. On day 30 of the experiment, physiological and morphological parameters were measured for all plants, and leaf tissues were harvested for biochemical and nutritional analyses.

Determination of Ni and nutrients

Milled samples (100 mg) of root, stem and leaf tissues were pre-digested in conical tubes (50 mL) with 2 ml of sub boiled HNO₃. Subsequently, 8 ml of a solution containing 4 ml of H₂O₂ (30% v/v) and 4 ml of ultra-pure water were added and transferred to a Teflon digestion vessel in agreement with Paniz et al. (2018). Determination of Ni, P, Ca, Mg, Mn, Zn and Fe was performed using an inductively coupled plasma mass spectrometer (model ICP-MS 7900; Agilent).

Measurement of chlorophyll fluorescence and gas exchange

Chlorophyll fluorescence was measured in fully expanded leaves under light using a modulated chlorophyll fluorometer (model OS5p; Opti-Sciences). Preliminary tests determined the location of the leaf, the part of the leaf and the time required to obtain the greatest F_v/F_m ratio; therefore, the acropetal third of the leaves, which was the middle third of the plant and was adapted to the dark for 30 min, was used in the evaluation. The intensity and duration of the saturation light pulse were 7,500 µmol m^− 2 s^− 1 and 0.7 s, respectively. Gas exchange was evaluated in all plants and measured in the expanded leaves in the middle region of the plant using an infrared gas analyser (model LCPro⁺; ADC BioScientific) in a chamber under constant CO₂, photosynthetically active radiation, air-flow rate and temperature conditions at 360 µmol mol^− 1 CO₂, 800 µmol photons m^− 2 s^− 1, 300 µmol s^− 1 and 28°C, respectively, between 10:00 and 12:00 h.

Determination of antioxidant enzymes, superoxide and soluble proteins

Antioxidant enzymes (SOD, CAT, APX, and POX), superoxide, and soluble proteins were extracted from leaf tissues according to the method of Badawi et al. (2004). Total soluble proteins were quantified using the methodology described by Bradford (1976). SOD activity was measured at 560 nm (Giannopolitis and Ries, 1977), and SOD activity is expressed in mg^− 1 protein. The CAT assay was detected at 240 nm (Havir and McHale, 1987), and the CAT activity is expressed in µmol H₂O₂ mg^− 1 protein min^− 1. The APX assay was measured at 290 nm (Nakano and Asada, 1981), and APX activity is expressed in µmol AsA mg^− 1 protein min^− 1. The POX assay was detected at 470 nm (Cakmak and Marschner, 1992), and activity is expressed in µmol tetraguaiacol mg^− 1 protein min^− 1. O₂⁻ was measured at 530 nm (Elstner and Heupel, 1976).

Quantification of hydrogen peroxide, malondialdehyde and electrolyte leakage

Stress indicators (H₂O₂ and MDA) were extracted using the methodology described by Wu et al. (2006). H₂O₂ was measured using the procedures described by Velikova et al. (2000). MDA was determined by the method of Cakmak and Horst (1991) using an extinction coefficient of 155 mM^− 1 cm^− 1. EL was measured according to Gong et al. (1998) and was calculated by the formula EL (%) = (EC₁/EC₂) × 100.

Determination of photosynthetic pigments and biomass

Chlorophyll and carotenoid determinations were performed using a spectrophotometer (model UV-M51; Bel Photonics) according to the methodology of Lichtenthaler and Buschmann (2001). The biomass of roots and shoots was measured based on constant dry weights (g) after drying in a forced-air ventilation oven at 65°C.

Data analysis

Data were subjected to an analysis of variance, and significant differences between the means were determined using the Scott-Knott test at a probability level of 5% (Steel et al., 2006). Standard deviations were calculated for each treatment.

Plants pretreated with EBR exhibited reduced Ni²⁺ content in tissues

Ni content was significantly increased in the root, stem and leaf tissues (Table 1). However, EBR spray on plants exposed to Ni reduced (P < 0.05) its content in the root, stem and leaf by 38%, 16% and 25%, respectively, compared to plants that received Ni without EBR pretreatment.

Steroids mitigate the stress caused by Ni on nutritional status

Ni excess caused reductions in nutrient content (Table 2). In contrast, treatment with EBR and Ni²⁺ resulted in increased P, Ca, Mg, Mn, Zn and Fe by 14%, 9%, 13%, 21%, 24% and 19% in root tissue, respectively; 18%, 11%, 81, 16%, 18% and 8% in stem, respectively; and 11%, 12%, 39%, 13%, 31% and 17% in leaf, respectively, compared to plants that received Ni²⁺ without EBR. For ionic ratios (Table 3), plants treated with Ni²⁺ excess and sprayed with EBR resulted in increments in Ca^+ 2/Ni^+ 2, Mg^+ 2/Ni^+ 2 and Mn^+ 2/Ni^+ 2 ratios of 77%, 84% and 100% in root, 33%, 115% and 42% in stem and 49%, 86% and 55% in leaf, respectively, compared to equal treatment without EBR.

Pretreatment with EBR minimizes the impacts of Ni on photosynthetic machinery

Ni caused negative changes in chlorophyll fluorescence (Fig. 1). However, EBR spray in plants exposed to Ni²⁺ produced significant increases of 4%, 5% and 1% in the values of F_m, F_v and F_v/F_m, respectively, compared to the same treatment without EBR. For chlorophyll fluorescence (Table 4), treatment with Ni²⁺ + EBR, the variables Φ_PSII, q_P and ETR exhibited increments (P < 0.05) of 15%, 32% and 19%, respectively. Plants pretreated with EBR and submitted to Ni²⁺ exhibited reductions of 21% and 5% in NPQ and EXC, respectively, compared to equal treatment in the absence of EBR. Ni generated deleterious effects on gas exchange (Table 4). However, pre-treatment with EBR in plants stressed with Ni resulted in significant increases in P_N, g_s, WUE, and P_N/C_i, corresponding to 20%, 19%, 16%, and 23%, respectively. Additionally, increased E (3%) and reduced C_i (4%) were verified compared to plants that received the same treatment without EBR. With respect to photosynthetic pigments, a high Ni supply occasioned damage to chlorophyll and carotenoids (Table 4). On the other hand, the EBR and Ni²⁺ treatment significantly increased Chl a, Chl b, total Chl and Car by 37%, 40%, 37% and 31%, respectively, as well as an increase in total Chl/Car (4%) and a decrease in Chl a/Chl b (2%) compared to the same treatment without EBR application.

Antioxidant responses are upregulated in plants treated with EBR and exposed to Ni

Adverse effects promoted by Ni increased the activities of antioxidant enzymes (Fig. 2). However, plants treated with both EBR and Ni presented significant increases in SOD, CAT, APX and POX of 50%, 27%, 40% and 19%, respectively, compared to treatment without EBR. Ni stress induced increases in stress indicators (Fig. 3), but EBR treatment in plants under Ni excess promoted significant reductions in O₂⁻, H₂O₂, MDA and EL of 20%, 5%, 9% and 10%, respectively, compared to the Ni^{2 +} + 0 nM EBR treatment.

EBR suppresses the negative impacts on biomass caused by Ni excess

Toxic action promoted by Ni provoked a decrease in biomass (Fig. 4), but EBR treatment in plants under Ni stress mitigated these effects, with increases of 7%, 12%, 25% and 7% in LDM, RDM, SDM and TDM, respectively, compared to the same treatment without EBR.

Soybean plants pretreated with EBR exhibited reductions in Ni content in the roots, stems and leaves. These results indicate that this steroid likely maximizes the endogenous levels of phytoquelatins, acting on immobilization and detoxification of excess Ni²⁺ ions into the plant cells (Rajewska et al., 2016). Concomitantly, there was a decrease in the negative effects of this heavy metal on Fe, Zn, Mn and Mg contents, improving the absorption and accumulation of these metals, corroborated by the results obtained in this research. Ahmad et al. (2018b) studied the effects of EBR application (10^− 6 M) in Cicer arietinum seedlings subjected to Hg toxicity (15 µM and 30 µM), observing significant reductions in Hg contents in roots and leaves, in addition to increases in Mg, Mn and Ca contents. Surgun et al. (2016) evaluated B toxicity in Arabidopsis thaliana-treated EBR and observed a reduction in B contents in the tissues of the leaf, root and inflorescence, and these results were explained by the authors as due to the better selectivity of the membrane enzymes. Sharma et al. (2011) studied the mechanisms of action connected to EBR in Raphanus sativus seedlings under Ni stress and found increases in root length and activities of antioxidant enzymes in shoot.

Steroid mitigated the stress caused by Ni on the contents of macro- (P, Ca, Mg) and micronutrients (Mn, Fe, Zn), improving nutritional status. These results can be explained by the systemic role promoted by the EBR, stimulating root structures intrinsically related to selectivity and protection of the root tissue against Ni (Ranathunge et al., 2003; Saraiva et al., 2021), as well as protection against biotic and abiotic stress (Barberon et al., 2016; Cui et al., 2016). Matraszek et al. (2017) studied EBR’s effects on the nutritional status of Sinapis alba exposed to four Ni concentrations (0, 0.0004, 0.04 and 0.08 mM Ni) and observed reductions in P, Ca and Mg contents. Yuan et al. (2015) evaluated the roles of EBR in nutrient accumulation in Cucumis sativus plants under Ca(NO₃)₂ stress and described increases in K, P, Mg, Fe and Mn contents in shoot and root tissues. Jan et al. (2018) reported increases in macronutrient contents, more specifically Mg, Ca and P, in Pisum sativum pretreated with EBR (individual or combined with silicon) under cadmium (Cd) stress.

Ca²⁺/Ni²⁺, Mg²⁺/Ni^+ 2 and Mn²⁺/Ni²⁺ ratios were increased after EBR spray in leaves, stems and roots. These results reveal multiple benefits of this steroid on homeostasis, increasing ionic ratios and decreasing the stress generated by Ni (Reis et al., 2017; Ribeiro et al., 2020), confirmed by increases in Ca, Mg and Mn contents and other elements evaluated in this research. Hu et al. (2016) studied the EBR effects in Solanum tuberosum plants under salt stress conditions and described positive effects of this steroid on homeostasis connected to the K⁺/Na⁺ ratio, combined with higher root efficiency and improvement in antioxidant capacity in shoots.

Pretreatment with EBR minimized the impacts of Ni on F_v, F_m and F_v/F_m. Our results demonstrated that this steroid provided protection for photosynthetic machinery, including benefits on the absorption of light energy by chloroplasts. EBR clearly alleviated oxidative damage due to increases in the activities of antioxidant enzymes (SOD, CAT, APX and POX) measured in this research, resulting in reduced concentrations of oxidative compounds, such as superoxide (O₂⁻) and hydrogen peroxide (H₂O₂), revealing a protective role for EBR on chloroplast ultrastructure (Sadeghi and Shekafandeh, 2014). According to Wani et al. (2017), BRs protect PSII against excessive excitation in abiotic stress conditions, preventing possible damage to thylakoid membranes. The positive effects of EBR also induced increases in Φ_PSII, ETR and q_P, which were related to the benefits of F₀ and F_m, as verified in this study. This result demonstrates better absorption and photon capture and maintenance of Q_A oxidation, improving the flow of electrons through PSII. Additionally, EBR reduced EXC, ETR/P_N and NPQ, demonstrating higher efficiency in the use of light and decreased use in secondary processes. Palliotti et al. (2015) studied Vitis vinífera genotypes under conditions of water restriction and reported an increase in ETR/PN; this result was associated with a possible imbalance in the production of electrons during water photolysis and use in photosynthetic machinery, suggesting an increase in alternative drains, including photorespiration. The stress generated by Ni reduced F_v/F_m and NPQ, indicating inhibition of light absorption and energy accumulation in the antenna complex and developing favourable conditions for the overproduction of reactive oxygen species (ROS), which in contact with the membrane cause severe damage to thylakoid structures and pigments (Anjum et al., 2016). Bukhari et al. (2016) described that EBR application attenuated the stress generated by Cr and increased the F_v/F_m values in Nicotiana tabacum seedlings. Pietrini et al. (2015) studied the deleterious effects caused by Ni on chlorophyll fluorescence in plants of Amaranthus paniculatus and detected reductions in the values of Φ_PSII, q_P and NPQ, compromising the functioning of PSII. Research conducted by Santos et al. (2018) using Vigna unguiculata plants sprayed with EBR and under Cd toxicity obtained significant improvements in the values of EXC, ETR, ETR/P_N and NPQ.

EBR mitigated the negative effects caused by Ni on gas exchange. Increases in P_N and WUE promoted by EBR can be explained by the positive effects on PPT and SPT observed in this study. These tissues have a large amount of chloroplasts and contribute to the formation of intercellular spaces that accumulate CO₂ essential for the photosynthetic process (Sorin et al., 2015). Increments observed in E and g_s after EBR application are related to increases in SD and SI detected in this research, suggesting higher efficiency in gas exchange, including the transpiration process and CO₂ assimilation. Our results also indicate that the increase in P_N/C_i values and reduction in C_i occurred due to EBR actions on possible increases in ribulose-1,5-bisphosphate carboxylase/oxygenase activity (reduction in C_i) and by CO₂ fixation (increase in P_N) during the photosynthetic process (Farooq et al., 2009; Shu et al., 2016; X.-J. Xia et al., 2009). Ni interference impaired gas exchange, reducing P_N, g_s, and E, which was related to stomatal limitations, confirmed by reductions in SF and SI, and the non-stomatal implications, corroborated by the overproduction of O₂⁻ and H₂O₂, were verified in this research. Shah et al. (2019) investigated photosynthetic responses and the antioxidant system in Cucumis sativus plants treated with EBR (5 µM) that were subjected to Cd stress (2.5 mM) and confirmed that steroids attenuated (P < 0.05) the effects of heavy metals on P_N, C_i, g_s, and E. Santos et al. (2020) evaluated gas exchange and anatomical structures in Glycine max plants exposed to Zn stress and treated with EBR, obtaining increased P_N, E, g_s, WUE and P_N/C_i and reduced C_i. Khan et al. (2017) measured the deleterious effects provoked by soil contamination with Ni (50, 100 and 200 mg Ni kg^− 1 soil) on gas exchange in Vinca rosea plants and described reductions in g_s, E and P_N. Khaliq et al. (2016) studied alterations caused by Ni toxicity (50 and 100 µM) on carbon fixation in Gossypium hirsutum plants and verified significant reductions in E and P_N.

Ni excess was partially suppressed by exogenous application of EBR, with increases in SOD, CAT, APX and POX activities. EBR clearly improved the performance of the antioxidant system, resulting in more efficient elimination of reactive oxygen species (ROS) due to the positive regulation exerted by this steroid on gene expression connected to these enzymes and subsequent quantitative activation of the antioxidant system (Mohammad Yusuf et al., 2011), reducing damage to the structure of chloroplast cells (Sharma et al., 2017). Increases linked to these enzymes are intrinsically related to the maintenance of photosynthetic pigments (Chl a, Chl b and Car), and significant reductions in stress indicators (H₂O₂ and O₂⁻) were verified in our research. Cao et al. (2005) investigated the biochemical and molecular responses in Arabidopsis thaliana with loss of function for the DET2 gene and interestingly described that the mutation of this gene increased the transcripts linked to the antioxidant system, reducing simulated oxidative stress. Xia et al. (2009b) observed higher tolerance to stress in Cucumis sativus leaves treated with EBR, positively regulating the gene expression and activities of enzymes related to antioxidant metabolism. Oliveira et al. (2019) described that pretreatment with 100 nM EBR in Na⁺ stressed Eucalyptus urophylla plants resulted in increased SOD, CAT, APX and POX, in which the authors found that this steroid minimized deleterious effects on photosynthetic machinery.

In the literature, there are several studies describing the benefits of this steroid, more specifically potentiating antioxidant enzymes, such as Hussain et al. (2019), who evaluated the antioxidant system in Triticum aestivum under EBR application and Mn stress, reporting increases in SOD, CAT and POX enzymes. These authors suggested that EBR likely stimulated the expression of regulatory genes involved in antioxidant defence. Fariduddin et al. (2015) studied the responses of Brassica juncea seedlings sprayed with EBR under Mn toxicity to photosynthetic attributes and redox metabolism, revealing evidence that this natural steroid acts as an efficient stress alleviator.

Exogenous EBR induced decreases in oxidative compounds and cellular damage generated by Ni. These findings reveal that EBR improved the performance of the antioxidant system, controlled H₂O₂ and O₂⁻ overproduction, and subsequently mitigated oxidative damage, as confirmed by the lower MDA and EL values described in this study. H₂O₂ and O₂⁻ are omnipresent in cellular compartments; however, these toxic compounds accumulate during adverse environmental stresses, including Ni excess (Ahmad et al., 2010; Gill and Tuteja, 2010; Gupta et al., 2016). Chandrakar et al. (2017) evaluated Glycine max seedlings treated with EBR (0.5 µM) exposed to As stress and observed decreases in O₂⁻, H₂O₂ and MDA, and these authors suggested that EBR promoted tolerance to oxidative stress by accumulating osmolytes and activating the antioxidant defence system in stressed plants. Dalyan et al. (2018) studied the roles of EBR under ROS overproduction in seedlings of Brassica juncea seedlings under Pb stress and identified reductions in H₂O₂ and MDA values, suggesting a protective role triggered by steroids. Sreekanth et al. (2013) described that Ni at high concentrations can negatively interfere with the balance between detoxification and the generation of ROS. Sirhindi et al. (2016) and (Mir et al., 2018), working with Glycine max plants exposed to Ni, found increases in stress indicators (H₂O₂, O₂⁻, MDA and EL). These authors described that ROS accumulation and the extent of oxidative stress are often correlated with inefficient antioxidant systems in plants under environmental stress conditions.

Steroid positively act on pigments of soybean plants exposed to Ni^+ 2 excess. Maintenance of these photopigments after pretreatment with EBR can be explained by the alleviation of oxidative damage and subsequent positive repercussions on chlorophyll fluorescence, as evidenced in this study. In other words, reductions in O₂⁻ and H₂O₂, combined with less deleterious effects on membranes (MDA and EL), occurred, resulting in better structural and functional integrity of these pigments associated with increments linked to light absorption, confirmed by the increases in ETR and Φ_PSII. Simultaneously, increases observed in Chl a, Chl b and Car are likely associated with a positive modulation induced by EBR in the metabolic pathway linked to the biosynthesis of these pigments (Soares et al., 2016) and with increased contents of essential elements, specifically Mg, which compose the structure of the chlorophyll molecule (Jan et al., 2018). There are studies in the literature revealing the direct relationship between Ni excess and ROS overproduction (Dourado et al., 2015; Muhammad et al., 2013) and its potential deleterious effects, such as peroxidation and degradation of membranes in chloroplast pigments (Ahmad et al., 2010; Gajewska and Skłodowska, 2008; Gill and Tuteja, 2010). Results corroborating our research were described by Ahmad et al. (2018a) when studying the effects of EBR application in Solanum lycopersicon plants stressed with NaCl, demonstrated increases in photosynthetic pigments (total Chl and Car), which were attributed by the authors to changes in enzymatic and nonenzymatic antioxidants, osmolytes and metabolites. Dong et al. (2019) evaluated the mechanisms of action triggered by EBR (0.1 µM) and nitric oxide on gas exchange in Arachis hypogaea plants under Cd toxicity and observed significant increases in Chl a, Chl b, total Chl and Car.

Pretreatment with EBR spray on soybean plants subjected to Ni stress resulted in significant increases in biomass (LDM, RDM, SDM and TDM), suppressing the negative impacts caused by Ni excess. These results confirm the multiple roles of EBR in plant metabolism, specifically increasing essential nutrient contents, reducing ROS levels, improving gas exchange (P_N, E, g_s and WUE) and attenuating the negative effects connected to Ni on chloroplast pigments (Chl a, Chl b and Car). Liu et al. (2019) evaluated the effects induced by brassinosteroid mimetics (EBR, bikinin and brazide) in Zea mays exposed to nicosulfuron toxicity and found beneficial responses linked to steroids on P_N, increments in chlorophyll and reduction in H₂O₂, improving biomass. Zhong et al. (2020) investigated Festuca arundinacea plants sprayed with three EBR concentrations (0.05, 0.10 and 0.20 mg L^− 1) and stressed with Pb (100 mg kg^− 1 soil) and detected increments in biomass (shoot and root), similar to the results of this research.

This research demonstrated the positive effects induced by EBR on antioxidant metabolism and ionic homeostasis in soybean plants subjected to Ni excess. This steroid improved the performance of the photosynthetic machinery, more specifically, the effective quantum yield of PSII photochemistry and electron transport rate due to upregulation of antioxidant enzymes (superoxide dismutase, catalase, ascorbate peroxidase and peroxidase), controlling the overproduction of superoxide and hydrogen peroxide, and reducing oxidative damage to chloroplasts. Exogenous EBR application promoted significant increases in biomass (leaves, roots and stems), and these results were explained by the benefits in nutrient content and ionic homeostasis, demonstrated by increases in Ca²⁺/Ni²⁺, Mg²⁺/Ni^+ 2 and Mn²⁺/Ni²⁺ ratios. Therefore, our results demonstrated that EBR attenuates oxidative damage caused by Ni in soybean plants.

BRs Brassinosteroids

Ca Calcium

CAR Carotenoids

Chl a Chlorophyll a

Chl b Chlorophyll b

C_ⁱ Intercellular CO2 concentration

CO₂ Carbon dioxide

E Transpiration rate

EBR 24-epibrassinolide

EL Electrolyte leakage

ETR Electron transport rate

ETR/P_N Ratio between the apparent electron transport rate and net photosynthetic rate

EXC Relative energy excess at the PSII level

F₀ Minimal fluorescence yield of the dark-adapted state

F_e Iron

F_m Maximal fluorescence yield of the dark-adapted state

F_v Variable fluorescence

F_v/F_m Maximal quantum yield of PSII photochemistry

g_s Stomatal conductance

H₂O₂ Hydrogen peroxide

LDM Leaf dry matter

MDA Malondialdehyde

Mg Magnesium

Mn Manganese

Ni Nickel

NPQ Nonphotochemical quenching

O₂- Superoxide

P Phosphorus

P_N Net photosynthetic rate

P_N/C_i Instantaneous carboxylation efficiency

PSII Photosystem II

q_P Photochemical quenching

RDM Root dry matter

SDM Stem dry matter

TDM Total dry matter

Total Chl Total Chlorophyll

WUE Water-use efficiency

Z_n Zinc

Φ_PSII Effective quantum yield of PSII photochemistry

Author contribution statement

AKSL was the advisor of this project, planning all phases of the research and critically revised the manuscript. MPS and CFM conducted the experiment and performed physiological, biochemical and morphological determinations, as well as wrote and edited the manuscript. BLB carried out the nutritional determinations and critically revised the manuscript. All authors read and approved the final version of manuscript.

Data availability statement

Data are available upon request to the corresponding author.

Conflict of interest

The authors declare that they have no competing interests.

Ethical Approval

Not applicable.

Consent to Participate

Not applicable.

Consent to Publish

Not applicable.

Funding

This research had financial supports from Fundação Amazônia de Amparo a Estudos e Pesquisas (FAPESPA/Brazil), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq/Brazil) and Universidade Federal Rural da Amazônia (UFRA/Brazil) to AKSL.

Ahammed GJ, Choudhary SP, Chen S, Xia X, Shi K, Zhou Y, Yu J (2013) Role of brassinosteroids in alleviation of phenanthrene–cadmium co-contamination-induced photosynthetic inhibition and oxidative stress in tomato. J Exp Bot 64:199–213. https://doi.org/10.1093/jxb/ers323
Ahanger MA, Mir RA, Alyemeni MN, Ahmad P (2020) Combined effects of brassinosteroid and kinetin mitigates salinity stress in tomato through the modulation of antioxidant and osmolyte metabolism. Plant Physiol Biochem 147:31–42. https://doi.org/10.1016/j.plaphy.2019.12.007
Ahmad P, Abd_Allah EF, Alyemeni MN, Wijaya L, Alam P, Bhardwaj R, Siddique KHM (2018a) Exogenous application of calcium to 24-epibrassinosteroid pre-treated tomato seedlings mitigates NaCl toxicity by modifying ascorbate–glutathione cycle and secondary metabolites. Sci Rep 8:13515. https://doi.org/10.1038/s41598-018-31917-1
Ahmad P, Ahanger MA, Egamberdieva D, Alam P, Alyemeni MN, Ashraf M (2018b) Modification of osmolytes and antioxidant enzymes by 24-epibrassinolide in chickpea seedlings under mercury (Hg) toxicity. J. Plant Growth Regul 37:309–322. https://doi.org/10.1007/s00344-017-9730-6
Ahmad P, Jaleel CA, Salem MA, Nabi G, Sharma S (2010) Roles of enzymatic and nonenzymatic antioxidants in plants during abiotic stress. Crit Rev Biotechnol 30:161–175. https://doi.org/10.3109/07388550903524243
Amari T, Ghnaya T, Abdelly C (2017) Nickel, cadmium and lead phytotoxicity and potential of halophytic plants in heavy metal extraction. South African J Bot 111:99–110. https://doi.org/10.1016/j.sajb.2017.03.011
Ameen N, Amjad M, Murtaza B, Abbas G, Shahid M, Imran M, Naeem MA, Niazi NK (2019) Biogeochemical behavior of nickel under different abiotic stresses: toxicity and detoxification mechanisms in plants. Environ Sci Pollut Res 26:10496–10514. https://doi.org/10.1007/s11356-019-04540-4
Anjum SA, Ashraf U, Tanveer M, Khan I, Hussain S, Shahzad B, Zohaib A, Abbas F, Saleem MF, Ali I, Wang LC (2017) Drought induced changes in growth, osmolyte accumulation and antioxidant metabolism of three maize hybrids. Front Plant Sci 08:1–12. https://doi.org/10.3389/fpls.2017.00069
Anjum SA, Tanveer M, Ashraf U, Hussain S, Shahzad B, Khan I, Wang L (2016) Effect of progressive drought stress on growth, leaf gas exchange, and antioxidant production in two maize cultivars. Environ Sci Pollut Res 23:17132–17141. https://doi.org/10.1007/s11356-016-6894-8
Aprile A, De Bellis L (2020) Editorial for Special Issue “Heavy Metals Accumulation, Toxicity, and Detoxification in Plants. Int J Mol Sci 21:4103. https://doi.org/10.3390/ijms21114103
Ashraf U, Kanu AS, Mo Z, Hussain S, Anjum SA, Khan I, Abbas RN, Tang X (2015) Lead toxicity in rice: effects, mechanisms, and mitigation strategies—a mini review. Environ Sci Pollut Res 22:18318–18332. https://doi.org/10.1007/s11356-015-5463-x
Ayangbenro A, Babalola O (2017) A new strategy for heavy metal polluted environments: A review of microbial biosorbents. Int J Environ Res Public Health 14:94. https://doi.org/10.3390/ijerph14010094
Azeem U (2018) Ameliorating nickel stress by jasmonic acid treatment in Zea mays L. Russ Agric Sci 44:209–215. https://doi.org/10.3103/S1068367418030035
Badawi GH, Yamauchi Y, Shimada E, Sasaki R, Kawano N, Tanaka, Kunisuke, Tanaka K (2004) Enhanced tolerance to salt stress and water deficit by overexpressing superoxide dismutase in tobacco (Nicotiana tabacum) chloroplasts. Plant Sci 166:919–928. https://doi.org/10.1016/j.plantsci.2003.12.007
Bajguz A (2000) Effect of brassinosteroids on nucleic acids and protein content in cultured cells of Chlorella vulgaris. Plant Physiol Biochem 38:209–215. https://doi.org/10.1016/S0981-9428(00)00733-6
Bamji SF, Corbitt C (2017) Glyceollins: Soybean phytoalexins that exhibit a wide range of health-promoting effects. J Funct Foods 34:98–105. https://doi.org/10.1016/j.jff.2017.04.020
Barberon M, Vermeer JEM, De Bellis D, Wang P, Naseer S, Andersen TG, Humbel BM, Nawrath C, Takano J, Salt DE, Geldner N (2016) Adaptation of root function by nutrient-induced plasticity of endodermal differentiation. Cell 164:447–459. https://doi.org/10.1016/j.cell.2015.12.021
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. https://doi.org/10.1016/0003-2697(76)90527-3
Bukhari SAH, Wang R, Wang W, Ahmed IM, Zheng W, Cao F (2016) Genotype-dependent effect of exogenous 24-epibrassinolide on chromium-induced changes in ultrastructure and physicochemical traits in tobacco seedlings. Environ Sci Pollut Res 23:18229–18238. https://doi.org/10.1007/s11356-016-7017-2
Cakmak I, Horst WJ (1991) Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max). Physiol Plant 83:463–468. https://doi.org/10.1111/j.1399-3054.1991.tb00121.x
Cakmak I, Marschner H (1992) Magnesium deficiency and high light intensity enhance activities of superoxide dismutase, ascorbate peroxidase, and glutathione reductase in bean leaves. Plant Physiol 98:1222–1227. https://doi.org/10.1104/pp.98.4.1222
Cao S, Xu Q, Cao Y, Qian K, An K, Zhu Y, Binzeng H, Zhao H, Kuai B (2005) Loss-of-function mutations in DET2 gene lead to an enhanced resistance to oxidative stress in Arabidopsis. Physiol Plant 123:57–66. https://doi.org/10.1111/j.1399-3054.2004.00432.x
Chandrakar V, Yadu B, Meena RK, Dubey A, Keshavkant S (2017) Arsenic-induced genotoxic responses and their amelioration by diphenylene iodonium, 24-epibrassinolide and proline in Glycine max L. Plant Physiol Biochem 112:74–86. https://doi.org/10.1016/j.plaphy.2016.12.023
Cui L, Zou Z, Zhang J, Zhao Y, Yan F (2016) 24-Epibrassinoslide enhances plant tolerance to stress from low temperatures and poor light intensities in tomato (Lycopersicon esculentum Mill.). Funct Integr Genomics 16:29–35. https://doi.org/10.1007/s10142-015-0464-x
Dalyan E, Yüzbaşıoğlu E, Akpınar I (2018) Effect of 24-epibrassinolide on antioxidative defence system against lead-induced oxidative stress in the roots of Brassica juncea L. seedlings. Russ J Plant Physiol 65:570–578. https://doi.org/10.1134/S1021443718040118
Dong Y, Chen W, Bai X, Liu F, Wan Y (2019) Effects of exogenous nitric oxide and 24-epibrassinolide on the physiological characteristics of peanut seedlings under cadmium stress. Pedosphere 29:45–59. https://doi.org/10.1016/S1002-0160(17)60376-X
Dourado MN, Franco MR, Peters LP, Martins PF, Souza LA, Piotto FA, Azevedo RA (2015) Antioxidant enzymes activities of Burkholderia spp. strains—oxidative responses to Ni toxicity. Environ Sci Pollut Res 22:19922–19932. https://doi.org/10.1007/s11356-015-5204-1
Drążkiewicz M, Baszyński T (2010) Interference of nickel with the photosynthetic apparatus of Zea mays. Ecotoxicol Environ Saf 73:982–986. https://doi.org/10.1016/j.ecoenv.2010.02.001
Elstner EF, Heupel A (1976) Inhibition of nitrite formation from hydroxylammoniumchloride: A simple assay for superoxide dismutase. Anal Biochem 70:616–620. https://doi.org/10.1016/0003-2697(76)90488-7
Fariduddin Q, Ahmed M, Mir BA, Yusuf M, Khan TA (2015) 24-epibrassinolide mitigates the adverse effects of manganese induced toxicity through improved antioxidant system and photosynthetic attributes in Brassica juncea. Environ Sci Pollut Res 22:11349–11359. https://doi.org/10.1007/s11356-015-4339-4
Farooq M, Wahid A, Basra SMA, Islam-ud-Din (2009) Improving water relations and gas exchange with brassinosteroids in rice under drought stress. J Agron Crop Sci 195:262–269. https://doi.org/10.1111/j.1439-037X.2009.00368.x
Fonseca SS, da Silva BRS, Lobato, AK da S (2020) 24-Epibrassinolide positively modulate leaf structures, antioxidant system and photosynthetic machinery in rice under simulated acid rain. J Plant Growth Regul. https://doi.org/10.1007/s00344-020-10167-4
Gajewska E, Skłodowska M (2008) Differential biochemical responses of wheat shoots and roots to nickel stress: antioxidative reactions and proline accumulation. Plant Growth Regul 54:179–188. https://doi.org/10.1007/s10725-007-9240-9
Gajewska E, Sklodowska M, Slaba M, Mazur J (2006) Effect of nickel on antioxidative enzyme activities, proline and chlorophyll contents in wheat shoots. Biol Plant 50:653–659. https://doi.org/10.1007/s10535-006-0102-5
Giannopolitis CN, Ries SK (1977) Superoxide dismutases: I. occurrence in higher plants. Plant Physiol 59:309–314
Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Biochem PPB 48:909–930. https://doi.org/10.1016/j.plaphy.2010.08.016
Gong M, Li Y-J, Chen S-Z (1998) Abscisic acid-induced thermotolerance in maize seedlings is mediated by calcium and associated with antioxidant systems. J Plant Physiol 153:488–496. https://doi.org/10.1016/S0176-1617(98)80179-X
Gupta P, Srivastava S, Seth CS (2016) 24-epibrassinolide and sodium nitroprusside alleviate the salinity stress in Brassica juncea L. cv. varuna through cross talk among proline, nitrogen metabolism and abscisic acid. Plant Soil 411:483–498. https://doi.org/10.1007/s11104-016-3043-6
Havir EA, McHale NA (1987) Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves. Plant Physiol 84:450–455. https://doi.org/10.1104/pp.84.2.450
Hu Y, Xia S, Su Y, Wang H, Luo W, Su S, Xiao L (2016) Brassinolide increases potato root growth in vitro in a dose-dependent way and alleviates salinity stress. Biomed Res. Int. 2016, 1–11. https://doi.org/10.1155/2016/8231873
Hussain A, Nazir F, Fariduddin Q (2019) 24-epibrassinolide and spermidine alleviate Mn stress via the modulation of root morphology, stomatal behavior, photosynthetic attributes and antioxidant defense in Brassica juncea. Physiol Mol Biol Plants 25:905–919. https://doi.org/10.1007/s12298-019-00672-6
Israr M, Jewell A, Kumar D, Sahi SV (2011) Interactive effects of lead, copper, nickel and zinc on growth, metal uptake and antioxidative metabolism of Sesbania drummondii. J Hazard Mater 186:1520–1526. https://doi.org/10.1016/j.jhazmat.2010.12.021
Jan S, Alyemeni MN, Wijaya L, Alam P, Siddique KH, Ahmad P (2018) Interactive effect of 24-epibrassinolide and silicon alleviates cadmium stress via the modulation of antioxidant defense and glyoxalase systems and macronutrient content in Pisum sativum L. seedlings. BMC Plant Biol 18:146. https://doi.org/10.1186/s12870-018-1359-5
Khaliq A, Ali S, Hameed A, Farooq MA, Farid M, Shakoor MB, Mahmood K, Ishaque W, Rizwan M (2016) Silicon alleviates nickel toxicity in cotton seedlings through enhancing growth, photosynthesis, and suppressing Ni uptake and oxidative stress. Arch Agron Soil Sci 62:633–647. https://doi.org/10.1080/03650340.2015.1073263
Khan WU, Ahmad SR, Yasin NA, Ali A, Ahmad A, Akram W (2017) Application of Bacillus megaterium MCR-8 improved phytoextraction and stress alleviation of nickel in Vinca rosea. Int J Phytoremediation 19:813–824. https://doi.org/10.1080/15226514.2017.1290580
Kohli SK, Handa N, Sharma A, Kumar V, Kaur P, Bhardwaj R (2017) Synergistic effect of 24-epibrassinolide and salicylic acid on photosynthetic efficiency and gene expression in brassica juncea L. Under Pb stress. Turkish J Biol 41:943–953. https://doi.org/10.3906/biy-1707-15
Küpper H, Andresen E (2016) Mechanisms of metal toxicity in plants. Metallomics 8:269–285. https://doi.org/10.1039/C5MT00244C
Lichtenthaler HK, Buschmann C (2001) Chlorophylls and carotenoids: Measurement and characterization by UV-VIS spectroscopy. In: Current Protocols in Food Analytical Chemistry. John Wiley & Sons, Inc., Hoboken, pp 431–438. https://doi.org/10.1002/0471709085.ch21
Lima JV, Lobato AKS (2017) Brassinosteroids improve photosystem II efficiency, gas exchange, antioxidant enzymes and growth of cowpea plants exposed to water deficit. Physiol Mol Biol Plants 23:59–72. https://doi.org/10.1007/s12298-016-0410-y
Lima MDR, Barros Junior UO, Batista BL, Lobato AKS (2018) Brassinosteroids mitigate iron deficiency improving nutritional status and photochemical efficiency in Eucalyptus urophylla plants. Trees - Struct Funct 32:1681–1694. https://doi.org/10.1007/s00468-018-1743-7
Liu S, He Y, Tian H, Yu C, Tan W, Li Z, Duan L (2019) Application of brassinosteroid mimetics improves growth and tolerance of maize to nicosulfuron toxicity. J Plant Growth Regul 38:701–712. https://doi.org/10.1007/s00344-018-9883-y
Majumdar S, Pagano L, Wohlschlegel JA, Villani M, Zappettini A, White JC, Keller AA (2019) Proteomic, gene and metabolite characterization reveal the uptake and toxicity mechanisms of cadmium sulfide quantum dots in soybean plants. Environ Sci Nano 6:3010–3026. https://doi.org/10.1039/C9EN00599D
Matraszek R, Hawrylak-Nowak B, Chwil S, Chwil M (2017) Macronutrient balance of nickel-stressed lettuce plants grown under different sulfur levels. Commun Soil Sci Plant Anal 48:665–682. https://doi.org/10.1080/00103624.2017.1298779
Matraszek R, Hawrylak-Nowak B, Chwil S, Chwil M (2016) Macronutrient composition of nickel-treated wheat under different sulfur concentrations in the nutrient solution. Environ Sci Pollut Res 23:5902–5914. https://doi.org/10.1007/s11356-015-5823-6
Mir MA, Sirhindi G, Alyemeni MN, Alam P, Ahmad P (2018) Jasmonic acid improves growth performance of soybean under nickel toxicity by regulating nickel uptake, redox balance, and oxidative stress metabolism. J Plant Growth Regul 37:1195–1209. https://doi.org/10.1007/s00344-018-9814-y
Muhammad BH, Shafaqat A, Aqeel A, Saadia H, Muhammad AF, Basharat A, Saima AB, Muhammad BG (2013) Morphological, physiological and biochemical responses of plants to nickel stress: A review. African J Agric Res 8:1596–1602. https://doi.org/10.5897/AJAR12.407
Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867–880
Nazir F, Hussain A, Fariduddin Q (2019) Interactive role of epibrassinolide and hydrogen peroxide in regulating stomatal physiology, root morphology, photosynthetic and growth traits in Solanum lycopersicum L. under nickel stress. Environ Exp Bot 162:479–495. https://doi.org/10.1016/j.envexpbot.2019.03.021
Oliveira VP, Lima MDR, Silva BRS, Batista BL, Lobato AKS (2018) Brassinosteroids confer tolerance to salt stress in Eucalyptus urophylla plants enhancing homeostasis, antioxidant metabolism and leaf anatomy. J Plant Growth Regul 38:557–573. https://doi.org/10.1007/s00344-018-9870-3
Palacios G, Gómez I, Carbonell-Barrachina A, Pedreño JN, Mataix J (1998) Effect of nickel concentration on tomato plant nutrition and dry matter yield. J Plant Nutr 21:2179–2191. https://doi.org/10.1080/01904169809365553
Palliotti A, Tombesi S, Frioni T, Silvestroni O, Lanari V, D’Onofrio C, Matarese F, Bellincontro A, Poni S (2015) Physiological parameters and protective energy dissipation mechanisms expressed in the leaves of two Vitis vinifera L. genotypes under multiple summer stresses. J Plant Physiol 185:84–92. https://doi.org/10.1016/j.jplph.2015.07.007
Paniz FP, Pedron T, Freire BM, Torres DP, Silva FF, Batista BL (2018) Effective procedures for the determination of As, Cd, Cu, Fe, Hg, Mg, Mn, Ni, Pb, Se, Th, Zn, U and rare earth elements in plants and foodstuffs. Anal Methods 10:4094–4103. https://doi.org/10.1039/c8ay01295d
Parida B, Chhibba I, Nayyar V (2003) Influence of nickel-contaminated soils on fenugreek (Trigonella corniculata L.) growth and mineral composition. Sci Hortic (Amsterdam) 98:113–119. https://doi.org/10.1016/S0304-4238(02)00208-X
Parmoon G, Ebadi A, Jahanbakhsh S, Hashemi M, Moosavi SA (2018) Effect of exogenous application of several plant growth regulators on photosynthetic pigments of fennel plants. Not Sci Biol 10:508–515. https://doi.org/10.15835/nsb10410356
Pereira YC, Rodrigues WS, Lima EJA, Santos LR, Silva MHL, Lobato AKS (2019) Brassinosteroids increase electron transport and photosynthesis in soybean plants under water deficit. Photosynthetica 57:1–11
Pietrini F, Iori V, Cheremisina A, Shevyakova NI, Radyukina N, Kuznetsov VV, Zacchini M (2015) Evaluation of nickel tolerance in Amaranthus paniculatus L. plants by measuring photosynthesis, oxidative status, antioxidative response and metal-binding molecule content. Environ Sci Pollut Res 22:482–494. https://doi.org/10.1007/s11356-014-3349-y
Rahman H, Sabreen S, Alam S, Kawai S (2005) Effects of nickel on growth and composition of metal micronutrients in barley plants grown in nutrient solution. J Plant Nutr 28:393–404. https://doi.org/10.1081/PLN-200049149
Rahman MF, Ghosal A, Alam MF, Kabir AH (2017) Remediation of cadmium toxicity in field peas (Pisum sativum L.) through exogenous silicon. Ecotoxicol Environ Saf 135:165–172. https://doi.org/10.1016/j.ecoenv.2016.09.019
Rajewska I, Talarek M, Bajguz A (2016) Brassinosteroids and response of plants to heavy metals action. Front Plant Sci 7:1–5. https://doi.org/10.3389/fpls.2016.00629
Ranathunge K, Steudle E, Lafitte R (2003) Control of water uptake by rice (Oryza sativa L.): role of the outer part of the root. Planta 217:193–205. https://doi.org/10.1007/s00425-003-0984-9
Reis AR, dos, de Q, Barcelos JP, de Souza Osório CRW, Santos EF, Lisboa LAM, Santini JMK, dos Santos MJD, Furlani Junior E, Campos M, de Figueiredo PAM, Lavres J, Gratão PL (2017) A glimpse into the physiological, biochemical and nutritional status of soybean plants under Ni-stress conditions. Environ Exp Bot 144:76–87. https://doi.org/10.1016/j.envexpbot.2017.10.006
Ribeiro AT, Oliveira VP, Oliveira Barros Junior U, Silva BRS, Batista BL, Lobato AKS (2020) 24-Epibrassinolide mitigates nickel toxicity in young Eucalyptus urophylla S.T. Blake plants: Nutritional, physiological, biochemical, anatomical and morphological responses. Ann For Sci 77:1–19. https://doi.org/10.1007/s13595-019-0909-9
Ribeiro DGS, Silva BRS, Lobato AKS (2019) Brassinosteroids induce tolerance to water deficit in soybean seedlings: contributions linked to root anatomy and antioxidant enzymes. Acta Physiol Plant 41:1–11. https://doi.org/10.1007/s11738-019-2873-2
Rizwan M, Mostofa MG, Ahmad MZ, Imtiaz M, Mehmood S, Adeel M, Dai Z, Li Z, Aziz O, Zhang Y, Tu S (2018) Nitric oxide induces rice tolerance to excessive nickel by regulating nickel uptake, reactive oxygen species detoxification and defense-related gene expression. Chemosphere 191:23–35. https://doi.org/10.1016/j.chemosphere.2017.09.068
Rodrigues WdaS, Pereira YC, de Souza ALM, Batista BL, Lobato, AK da S (2020) Alleviation of oxidative stress induced by 24-epibrassinolide in soybean plants exposed to different manganese supplies: UpRegulation of antioxidant enzymes and maintenance of photosynthetic pigments. J Plant Growth Regul. https://doi.org/10.1007/s00344-020-10091-7
Sadeghi F, Shekafandeh A (2014) Effect of 24-epibrassinolide on salinity-induced changes in loquat (Eriobotrya japonica Lindl). J Appl Bot Food Qual 87:182–189. https://doi.org/10.5073/JABFQ.2014.087.026
Santos LR, Batista BL, Lobato AKS (2018) Brassinosteroids mitigate cadmium toxicity in cowpea plants. Photosynthetica 56:591–605. https://doi.org/10.1007/s11099-017-0700-9
Santos LR, da Silva BRS, Pedron T, Batista BL, Lobato, AK da S (2020) 24-Epibrassinolide improves root anatomy and antioxidant enzymes in soybean plants subjected to zinc stress. J Soil Sci Plant Nutr 20:105–124. https://doi.org/10.1007/s42729-019-00105-z
Saraiva MP, Maia CF, Silva BRS, Batista BL, Lobato, AK da S (2021) 24-Epibrassinolide induces protection against nickel excess in soybean plants: anatomical evidences. Brazilian J Bot 44:197–205. https://doi.org/10.1007/s40415-021-00701-3
Shah AA, Ahmed S, Yasin NA (2019) 24-epibrassinolide triggers cadmium stress mitigation in Cucumis sativus through intonation of antioxidant system. South African J Bot 127:349–360. https://doi.org/10.1016/j.sajb.2019.11.003
Sharma A, Kumar V, Singh R, Thukral AK, Bhardwaj R (2016) Effect of seed pre-soaking with 24-epibrassinolide on growth and photosynthetic parameters of Brassica juncea L. in imidacloprid soil. Ecotoxicol Environ Saf 133:195–201. https://doi.org/10.1016/j.ecoenv.2016.07.008
Sharma A, Thakur S, Kumar V, Kesavan AK, Thukral AK, Bhardwaj R (2017) 24-epibrassinolide stimulates imidacloprid detoxification by modulating the gene expression of Brassica juncea L. BMC Plant Biol 17:56. https://doi.org/10.1186/s12870-017-1003-9
Sharma I, Pati PK, Bhardwaj R (2011) Effect of 24-epibrassinolide on oxidative stress markers induced by nickel-ion in Raphanus sativus L. Acta Physiol Plant 33:1723–1735. https://doi.org/10.1007/s11738-010-0709-1
Shu S, Tang Y, Yuan Y, Sun J, Zhong M, Guo S (2016) The role of 24-epibrassinolide in the regulation of photosynthetic characteristics and nitrogen metabolism of tomato seedlings under a combined low temperature and weak light stress. Plant Physiol Biochem 107:344–353. https://doi.org/10.1016/j.plaphy.2016.06.021
Sirhindi G, Mir MA, Abd-Allah EF, Ahmad P, Gucel S (2016) Jasmonic acid modulates the physio-biochemical attributes, antioxidant enzyme activity, and gene expression in Glycine max under nickel toxicity. Front Plant Sci 7:1–12. https://doi.org/10.3389/fpls.2016.00591
Soares C, de Sousa A, Pinto A, Azenha M, Teixeira J, Azevedo RA, Fidalgo F (2016) Effect of 24-epibrassinolide on ROS content, antioxidant system, lipid peroxidation and Ni uptake in Solanum nigrum L. under Ni stress. Environ Exp Bot 122:115–125. https://doi.org/10.1016/j.envexpbot.2015.09.010
Song YL, Dong YJ, Tian XY, Kong J, Bai XY, Xu LL, He ZL (2016) Role of foliar application of 24-epibrassinolide in response of peanut seedlings to iron deficiency. Biol Plant 60:329–342. https://doi.org/10.1007/s10535-016-0596-4
Sorin C, Musse M, Mariette F, Bouchereau A, Leport L (2015) Assessment of nutrient remobilization through structural changes of palisade and spongy parenchyma in oilseed rape leaves during senescence. Planta 241:333–346. https://doi.org/10.1007/s00425-014-2182-3
Sreekanth TVM, Nagajyothi PC, Lee KD, Prasad TNVKV (2013) Occurrence, physiological responses and toxicity of nickel in plants. Int J Environ Sci Technol 10:1129–1140. https://doi.org/10.1007/s13762-013-0245-9
Steel RG, Torrie JH, Dickey DA (2006) Principles and procedures of statistics: a biometrical approach, 3rd edn. Academic Internet Publishers, Moorpark
Surgun Y, Çöl B, Bürün B (2016) 24-Epibrassinolide ameliorates the effects of boron toxicity on Arabidopsis thaliana (L.) Heynh by activating an antioxidant system and decreasing boron accumulation. Acta Physiol Plant 38:71. https://doi.org/10.1007/s11738-016-2088-8
Tanveer M, Shahzad B, Sharma A, Biju S, Bhardwaj R (2018) 24-Epibrassinolide; an active brassinolide and its role in salt stress tolerance in plants: A review. Plant Physiol Biochem 130:69–79. https://doi.org/10.1016/j.plaphy.2018.06.035
Torres GN, Camargos SL, Weber OLDS, Maas KDB, Scaramuzza WLMP (2016) Growth and micronutrient concentration in maize plants under nickel and lime applications. Rev Caatinga 29:796–804. https://doi.org/10.1590/1983-21252016v29n403rc
Velikova V, Yordanov I, Edreva A (2000) Oxidative stress and some antioxidant systems in acid rain-treated bean plants protective role of exogenous polyamines. Plant Sci 151:59–66. https://doi.org/10.1016/S0168-9452(99)00197-1
Wani AS, Tahir I, Ahmad SS, Dar RA, Nisar S (2017) Efficacy of 24-epibrassinolide in improving the nitrogen metabolism and antioxidant system in chickpea cultivars under cadmium and/or NaCl stress. Sci Hortic (Amsterdam) 225:48–55. https://doi.org/10.1016/j.scienta.2017.06.063
Wu Q-S, Xia R-X, Zou Y-N (2006) Reactive oxygen metabolism in mycorrhizal and non-mycorrhizal citrus (Poncirus trifoliata) seedlings subjected to water stress. J Plant Physiol 163:1101–1110. https://doi.org/10.1016/j.jplph.2005.09.001
Xia X-J, Huang L-F, Zhou Y-H, Mao W-H, Shi K, Wu J-X, Asami T, Chen Z, Yu J-Q (2009) Brassinosteroids promote photosynthesis and growth by enhancing activation of Rubisco and expression of photosynthetic genes in Cucumis sativus. Planta 230:1185–1196. https://doi.org/10.1007/s00425-009-1016-1
Xia XJ, Zhang Y, Wu JX, Wang JT, Zhou YH, Shi K, Yu YL, Yu JQ (2009) Brassinosteroids promote metabolism of pesticides in cucumber. J Agric Food Chem 57:8406–8413. https://doi.org/10.1021/jf901915a
Yan ZZ, Ke L, Tam NFY (2010) Lead stress in seedlings of Avicennia marina, a common mangrove species in South China, with and without cotyledons. Aquat Bot 92:112–118. https://doi.org/10.1016/j.aquabot.2009.10.014
Yuan L, Zhu S, Shu S, Sun J, Guo S (2015) Regulation of 2,4-epibrassinolide on mineral nutrient uptake and ion distribution in Ca(NO3)2 stressed cucumber plants. J Plant Physiol 188:29–36. https://doi.org/10.1016/j.jplph.2015.06.010
Yusuf M, Fariduddin Q, Ahmad A (2011) 28-Homobrassinolide mitigates boron induced toxicity through enhanced antioxidant system in Vigna radiata plants. Chemosphere 85:1574–1584. https://doi.org/10.1016/j.chemosphere.2011.08.004
Yusuf M, Fariduddin Q, Hayat S, Ahmad A (2011) Nickel: An overview of uptake, essentiality and toxicity in plants. Bull Environ Contam Toxicol 86:1–17. https://doi.org/10.1007/s00128-010-0171-1
Yusuf M, Fariduddin Q, Khan TA, Hayat S (2017) Epibrassinolide reverses the stress generated by combination of excess aluminum and salt in two wheat cultivars through altered proline metabolism and antioxidants. South African J Bot 112:391–398. https://doi.org/10.1016/j.sajb.2017.06.034
Zhong W, Xie C, Hu D, Pu S, Xiong X, Ma J, Sun L, Huang Z, Jiang M, Li X (2020) Effect of 24-epibrassinolide on reactive oxygen species and antioxidative defense systems in tall fescue plants under lead stress. Ecotoxicol Environ Saf 187:109831. https://doi.org/10.1016/j.ecoenv.2019.109831

Table 1. Ni contents in soybean plants sprayed with EBR and exposed to high Ni concentration.

Ni²⁺	EBR	Ni in root (µg g DM^-1)	Ni in stem (µg g DM^-1)	Ni in leaf (µg g DM^-1)
̶	̶	2.29 ± 0.12c	0.11 ± 0.01c	0.12 ± 0.02c
̶	+	1.07 ± 0.08d	0.07 ± 0.01d	0.12 ± 0.01c
+	̶	344.55 ± 6.39a	10.87 ± 0.07a	20.81 ± 1.56a
+	+	213.64 ± 16.14b	9.12 ± 0.47b	15.53 ± 0.27b

Ni = Nickel. Columns with different letters indicate significant differences from the Scott-Knott test (P<0.05). Values described correspond to means from five repetitions with standard deviations.

Table 2. Nutrient contents in soybean plants sprayed with EBR and exposed to high Ni concentration.

Ni²⁺	EBR	P (mg g DM^-1)	Ca (mg g DM^-1)	Mg (mg g DM^-1)	Mn (µg g DM^-1)	Fe (µg g DM^-1)	Zn (µg g DM^-1)
Contents in root
̶	̶	14.28 ± 0.57b	13.83 ± 0.53b	14.31 ± 0.15b	293.94 ± 16.47b	2539.06 ± 67.09b	30.57 ± 0.83b
̶	+	16.17 ± 0.39a	15.89 ± 0.47a	14.92 ± 0.06a	354.79 ± 13.45a	2906.85 ± 65.67a	36.47 ± 0.72a
+	̶	11.16 ± 0.09d	12.24 ± 0.18c	12.29 ± 0.29d	213.41 ± 13.99d	2150.27 ± 72.83c	25.53 ± 0.76c
+	+	12.71 ± 0.91c	13.32 ± 0.87b	13.94 ± 0.18c	258.89 ± 11.91c	2554.58 ± 97.14b	31.55 ± 0.52b
Contents in stem
̶	̶	7.08 ± 0.24b	13.21 ± 0.32b	2.07 ± 0.30c	15.71 ± 0.48b	40.13 ± 0.59b	11.62 ± 0.27b
̶	+	8.45 ± 0.24a	14.23 ± 0.16a	3.23 ± 0.16a	17.68 ± 0.49a	46.71 ± 0.63a	12.29 ± 0.29a
+	̶	6.06 ± 0.02c	11.95 ± 0.22c	1.54 ± 0.20d	13.40 ± 0.48c	32.92 ± 0.69d	7.47 ± 0.29d
+	+	7.18 ± 0.60b	13.32 ± 0.43b	2.78 ± 0.16b	15.55 ± 0.68b	35.43 ± 0.90c	8.81 ± 0.21c
Contents in leaf
̶	̶	9.24 ± 0.12b	13.67 ± 0.50b	3.32 ± 0.14b	46.18 ± 1.99b	92.39 ± 1.65b	21.84 ± 0.70b
̶	+	9.97 ± 0.37a	15.49 ± 0.14a	4.98 ± 0.42a	50.28 ± 0.61a	117.94 ± 0.96a	25.90 ± 0.66a
+	̶	8.06 ± 0.05c	12.26 ± 0.23c	3.39 ± 0.19b	41.30 ± 0.82c	72.24 ± 0.60d	12.61 ± 0.43d
+	+	8.92 ± 0.05b	13.71 ± 0.22b	4.71 ± 0.33a	46.67 ± 0.28b	84.85 ± 0.85c	16.55 ± 0.19c

P = Phosphorus; Ca = Calcium; Mg = Magnesium; Mn = Manganese; Fe = Iron; Zn = Zinc. Columns with different letters indicate significant differences from the Scott-Knott test (P<0.05). Values described correspond to means from five repetitions with standard deviations.

Table 3. Ionic ratios in soybean plants sprayed with EBR and exposed to high Ni concentration.

Ni²⁺		EBR	Root	Stem	Leaf
	Ca²⁺/Ni²⁺ ratio
̶		̶	6089.1 ± 470.0b	123359.6 ± 10882.3b	109973.7 ± 12054.5b
̶		+	15009.3 ± 1519.7a	210916.2 ± 24930.8a	126519.1 ± 6810.6a
+		̶	35.5 ± 0.8d	1099.5 ± 22.10d	592.1 ± 48.1d
+		+	62.8 ± 7.4c	1460.0 ± 56.12c	883.0 ± 9.2c
	Mg²⁺/Ni²⁺ ratio
̶		̶	6298.3 ± 396.8b	19267.0 ± 3382.5b	26802.4 ± 3763.7b
̶		+	14067.4 ± 1011.0a	47970.1 ± 6402.1a	40688.5 ± 4174.2a
+		̶	35.7 ± 1.2d	141.7 ± 19.2d	163.5 ± 13.8d
+		+	65.6 ± 5.0c	305.2 ± 28.2c	303.7 ± 25.7c
	Mn²⁺//Ni²⁺ ratio
̶		̶	129.3 ± 13.7b	146.8 ± 15.2b	371.6 ± 14.9b
̶		+	334.9 ± 32.2a	261.8 ± 28.8a	410.1 ± 10.4a
+		̶	0.6 ± 0.1d	1.2 ± 0.1d	2.0 ± 0.2d
+		+	1.2 ± 0.1c	1.7 ± 0.1c	3.1 ± 0.1c

Ca²⁺/Ni²⁺ = Calcium and nickel ratio; Mg²⁺/Ni²⁺ = Magnesium and nickel ratio and Mn²⁺/Ni²⁺ = Manganese and nickel ratio. Columns with different letters indicate significant differences from the Scott-Knott test (P<0.05). Values described correspond to means from five repetitions with standard deviations.

Table 4. Chlorophyll fluorescence, gas exchange and photosynthetic pigments in soybean plants sprayed with EBR and exposed to high Ni concentration.

Ni²⁺	EBR	Φ_PSII	q_P	NPQ	ETR (µmol m^-2 s^-1)	EXC (µmol m^-2 s^-1)	ETR/P_N
̶	̶	0.24 ± 0.01b	0.43 ± 0.05b	0.18 ± 0.01c	36.2 ± 0.7b	0.70 ± 0.01b	2.09 ± 0.04b
̶	+	0.27 ± 0.01a	0.56 ± 0.03a	0.12 ± 0.01d	39.1 ± 1.0a	0.67 ± 0.01c	2.08 ± 0.04b
+	̶	0.20 ± 0.01c	0.25 ± 0.02d	0.42 ± 0.02a	29.1 ± 1.4d	0.75 ± 0.01a	2.22 ± 0.07a
+	+	0.23 ± 0.01b	0.33 ± 0.03c	0.33 ± 0.01b	34.5 ± 1.0c	0.71 ± 0.01b	2.19 ± 0.03a
Ni²⁺	EBR	P_N(µmol m^-2 s^-1)	E (mmol m^-2 s^-1)	g_s (mol m^-2 s^-1)	C_i (µmol mol^-1)	WUE (µmol mmol^–¹)	P_N/C_i (µmol m^-2 s^-1Pa^-1)
̶	̶	17.29 ± 0.22b	3.11 ± 0.07a	0.39 ± 0.008a	267 ± 7b	5.56 ± 0.15b	0.065 ± 0.002b
̶	+	18.80 ± 0.18a	3.14 ± 0.06a	0.40 ± 0.013a	264 ± 8b	6.00 ± 0.14a	0.071 ± 0.002a
+	̶	13.09 ± 0.64d	2.79 ± 0.26b	0.27 ± 0.009c	281 ± 5a	4.71 ± 0.29c	0.047 ± 0.003d
+	+	15.72 ± 0.31c	2.87 ± 0.10b	0.32 ± 0.011b	271 ± 4b	5.48 ± 0.25b	0.058 ± 0.001c
Ni²⁺	EBR	Chl a (mg g^–1 FM)	Chl b (mg g^–1 FM)	Total Chl (mg g^–1 FM)	Car (mg g^–1 FM)	Ratio Chl a/Chl b	Ratio Total Chl/Car
̶	̶	11.65 ± 0.19b	2.67 ± 0.08b	14.32 ± 0.21b	0.59 ± 0.02b	4.37 ± 0.16a	24.29 ± 1.36a
̶	+	12.78 ± 0.21a	3.27 ± 0.21a	16.05 ± 0.39a	0.63 ± 0.01a	3.91 ± 0.20b	25.71 ± 2.09a
+	̶	6.06 ± 0.37d	1.32 ± 0.07d	7.38 ± 0.42d	0.36 ± 0.08d	4.58 ± 0.25a	20.76 ± 0.92b
+	+	8.27 ± 0.13c	1.85 ± 0.09c	10.11 ± 0.17c	0.47 ± 0.02c	4.49 ± 0.20a	21.51 ± 1.20b

Φ_PSII= Effective quantum yield of PSII photochemistry; q_P = Photochemical quenching coefficient; NPQ = Nonphotochemical quenching; ETR = Electron transport rate; EXC = Relative energy excess at the PSII level; ETR/P_N = Ratio between the electron transport rate and net photosynthetic rate; P_N = Net photosynthetic rate; E = Transpiration rate; g_s = Stomatal conductance; C_i = Intercellular CO₂ concentration; WUE = Water-use efficiency; P_N/C_i = Carboxylation instantaneous efficiency; Chl a = Chlorophyll a; Chl b = Chlorophyll b; Total Chl = Total chlorophyll; Car = Carotenoids. Columns with different letters indicate significant differences from the Scott-Knott test (P<0.05). Values described correspond to means from five repetitions with standard deviations.

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Ionic Homeostasis and Redox Metabolism Upregulated by 24-Epibrassinolide are Crucial for Mitigating Nickel Excess in Soybean Plants, Enhancing Photosystem II Efficiency and Biomass

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Location and growth conditions

Plants, containers and acclimation

Experimental design

24-Epibrassinolide (EBR) preparation and application

Plant conduction and Ni treatment

Determination of Ni and nutrients

Measurement of chlorophyll fluorescence and gas exchange

Determination of antioxidant enzymes, superoxide and soluble proteins

Quantification of hydrogen peroxide, malondialdehyde and electrolyte leakage

Determination of photosynthetic pigments and biomass

Data analysis

Results

Plants pretreated with EBR exhibited reduced Ni²⁺ content in tissues

Steroids mitigate the stress caused by Ni on nutritional status

Pretreatment with EBR minimizes the impacts of Ni on photosynthetic machinery

Antioxidant responses are upregulated in plants treated with EBR and exposed to Ni

EBR suppresses the negative impacts on biomass caused by Ni excess

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Status:

Version 1

Ionic Homeostasis and Redox Metabolism Upregulated by 24-Epibrassinolide are Crucial for Mitigating Nickel Excess in Soybean Plants, Enhancing Photosystem II Efficiency and Biomass

Status:

Version 1

Abstract

Figures

Introduction

Materials And Methods

Location and growth conditions

Plants, containers and acclimation

Experimental design

24-Epibrassinolide (EBR) preparation and application

Plant conduction and Ni treatment

Determination of Ni and nutrients

Measurement of chlorophyll fluorescence and gas exchange

Determination of antioxidant enzymes, superoxide and soluble proteins

Quantification of hydrogen peroxide, malondialdehyde and electrolyte leakage

Determination of photosynthetic pigments and biomass

Data analysis

Results

Plants pretreated with EBR exhibited reduced Ni2+ content in tissues

Steroids mitigate the stress caused by Ni on nutritional status

Pretreatment with EBR minimizes the impacts of Ni on photosynthetic machinery

Antioxidant responses are upregulated in plants treated with EBR and exposed to Ni

EBR suppresses the negative impacts on biomass caused by Ni excess

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Status:

Version 1

Plants pretreated with EBR exhibited reduced Ni²⁺ content in tissues