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 Fv, Fm and Fv/Fm. 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 ΦPSII, ETR and qP, which were related to the benefits of F0 and Fm, as verified in this study. This result demonstrates better absorption and photon capture and maintenance of QA oxidation, improving the flow of electrons through PSII. Additionally, EBR reduced EXC, ETR/PN 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 Fv/Fm 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 Fv/Fm 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 ΦPSII, qP 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/PN and NPQ.
EBR mitigated the negative effects caused by Ni on gas exchange. Increases in PN 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 gs 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 PN/Ci values and reduction in Ci occurred due to EBR actions on possible increases in ribulose-1,5-bisphosphate carboxylase/oxygenase activity (reduction in Ci) and by CO2 fixation (increase in PN) during the photosynthetic process (Farooq et al., 2009; Shu et al., 2016; X.-J. Xia et al., 2009). Ni interference impaired gas exchange, reducing PN, gs, 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 PN, Ci, gs, 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 PN, E, gs, WUE and PN/Ci and reduced Ci. 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 gs, E and PN. 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 PN.
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 ΦPSII. 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 (PN, E, gs 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 PN, 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.