Our research reported that plants exposed to SAR suffered intense disturbances in their metabolism, demonstrated in anatomical, biochemical, nutritional, physiological and morphological aspects. However, the exogenous application of EBR attenuated the harmful effects caused by SAR in soybean plants, collaborating directly with the development of leaf structures, proven in the photosynthetic mechanism, resulting in higher biomass accumulation.
Benefits provided by EBR were found in the stomatal characteristics, being directly related to the increments in SD, SF and SI, resulting in higher density and favouring the formation of elliptical structures (PDS and EDS) even in plants affected by SAR. These results can be explained by the functionality of this steroid, improving the stomatal performance (Hussain et al. 2019). The SD, SF, and SI increase intrinsically related to the benefits detected in gs, E, Ci and PN, confirming that EBR improved stomatal performance and alleviated the disturbances caused by SAR (Hafeez et al. 2021). SAR causes damage to stomata, reducing mobility and regulation capacity, resulting in an imbalance between E and PN (Santos et al., 2006). Reductions detected in PDS and EDS in plants exposed to SAR and EBR suggest that EBR maximised water utilisation during the gas exchange, maintaining CO2 influx and H2O efflux, evidenced by the increase in WUE (Liu et al. 2021) and improving SF, characterised by more elliptical stomata, justifying the better ratio of this stomatal pattern, compared to more extensive and cylindrical stomata (Souza et al. 2018). Additionally, the highest number of stomata (SD) is an essential and relevant strategy, as it improves gas exchange without significant CO2 losses during photosynthesis and transpiration (Diatta et al. 2021; Zeng et al. 2022). Sousa et al., (2021) evaluated pre-treatment with EBR in tomato plants under salt stress, where they simulated two saline conditions (0 and 150 mM of NaCl) and two concentrations of 24-epibrassinolide (0 and 100 nM EBR) and detected that the functional and anatomical characteristics of stomata improved with treatment with EBR, resulting in increases in SD, SF and SI values and a decrease in PDS and EDS.
Plants exposed to EBR spray showed increases in TTD and TTS on both leaf faces, revealing that EBR contributed to the leaf protection mechanism against damage caused by SAR. Trichomes act as a defence system; epidermal structures are classified into glandular and non-glandular (Yang et al. 2023). The density and size of trichomes vary in response to abiotic factors that cause stress to the plant (Huebbers et al. 2023), such as extreme temperatures and water loss (Parusnath et al. 2022); these structures positively modulate E and reduce foliar exposure to ultraviolet radiation (Garcia et al. 2022). Guedes et al. (2021) cultivated Oryza sativa subjected to Pb stress, where they observed that EBR (100 nM) attenuated the deleterious effects of Pb and benefited the trichome structures, increasing density and size (TS and TD).
EBR spray significantly increased photosynthetic pigments Chl a, Chl b, total Chl and Car. These results suggest that EBR, directly and indirectly, stimulates the biosynthesis of these pigments (Gupta and Seth 2022; Yanhua et al. 2023), as confirmed by the reductions in ROS and increases in total Chl found in this study. The highest proportion of Car after EBR spraying indicates that the light-gathering and antioxidant pigment was protected against oxidative stress, confirmed with the highest Total Chl concentrations (Zhong et al. 2020). In the photosynthetic process, chlorophylls capture light energy to transform CO2 and water into carbohydrates (biochemical energy) and oxygen (Zuo et al. 2017). Occasionally, SAR destroys the integrity of chloroplasts and thus exponentially decreases the chlorophyll contents (Du et al. 2017). Additionally, increased chloroplast acidity inhibits photosynthetic electron transport into PSII (Debnath et al. 2020). Ma et al., (2019) submitted Cinnamomum camphora to SAR associated with high temperature (40ºC) and found that the association of these factors significantly decreased the contents of photosynthetic pigments and consequently PN. Zhang et al., (2020) studied the effect of SAR on Camellia sinensis plants, where SAR significantly decreased Chl a, Chl b, Car and Fv/Fm values. being also verified that lower pH values cause negative impacts on the photosynthesis process and plant growth. Sun et al. (2012) observed the interactive effects of Cd and acid rain on soybean plants, where the association of these two factors exponentially decreased the chlorophyll contents.
The exogenous application of EBR positively impacted the gas exchange of plants subjected to SAR, resulting in increases in the values of PN, E, gs, WUE and PN/Ci, being connected to beneficial effects on stomatal characteristics (SD, SI, SF) verified in this study. Together, these benefits on the gas exchange can be associated with the actions promoted by the EBR in the photosynthetic apparatus, more precisely in the improvements announced in the efficiency of the PSII (Jia et al. 2023), corroborating with the results found in ΦPSII, qp and ETR. PSII is one of the most sensitive components of the photochemical mechanism in response to stress caused by SAR (Liu et al., 2023). EBR acts in the regulatory function against SAR in photosynthetic processes, improving the efficiency of light capture and the activity of the photosynthetic apparatus, including absorption and availability of CO2 for PN (He et al. 2022). Increases in PN can be explained by the maximisations detected in PPT and SPT, promoted by EBR. PN is dependent on the availability of CO2, solar radiation and available water (Li et al. 2022), and CO2 diffusion depends on the anatomical characteristics of the leaf tissue, including stomatal dimensions and functionality (Pang et al. 2023), measured in SD, SF, SI, PDS and EDS and described in this research, providing positive repercussions on PN/Ci. Wang et al. (2014) combined lanthanum chloride (LaCl3) and SAR, showing negative impacts on the photosynthetic apparatus of rice plants, decreasing PN and gs, but increasing Ci. Xia et al. (2022) associated EBR with the nitric oxide (NO) in kiwi seedlings subjected to drought, resulting in improved gas exchange, inducing increments in PN, gs and E.
Our results demonstrated that EBR treatment attenuated the harmful effects of SAR on plants, significantly increasing Fm, Fv and Fv/Fm and directly contributing to the reduction in F0. Therefore, the EBR actions increased the photochemical efficiency of the reaction centre linked to PSII (Barros Junior et al. 2020). The application of EBR increased the ΦPSII, qp and ETR values, indicating that the steroid attenuated the harmful interference caused by SAR in plants (Debnath et al. 2021). EBR benefited the transport of electrons from the PSII reaction centre through the primary plastoquinone acceptor (QA) to the secondary plastoquinone acceptor (QB), favouring the transfer of excitation energy from the light-harvesting complex (Wang et al., 2015). On the other hand, EBR reduced the values of NPQ, EXC and ETR/PN, confirming that EBR acted to minimise heat dissipation, resulting in higher energy available for the photosynthetic process intrinsically related to the PSII antenna complex (Cunha et al., 2021). Hu et al., (2014) verified decreases in chlorophyll contents, Fv/Fm and an increase in Fo on soybean plants exposed to Pb combined with SAR. Liu et al., (2018) evaluated the impacts of SAR on Cunninghamia lanceolata, describing that chlorophyll fluorescence parameters were negatively affected, resulting in a significant reduction of Fv/Fm.
EBR favoured leaf anatomy (ETAd, ETAb, PPT and SPT) in plants subjected to SAR stress, and these results are related to increases in PN, PN/Ci, E and WUE, previously described in this research. EBR can protect the SPT's structure, increasing its thickness compaction and maximising the accumulation and conductance of CO2 to the chloroplast cells, where carboxylation by RuBisCO will occur (Pereira et al. 2020). On the other hand, EBR amplifies the thickness of the PPT, improving water flow within the tissues, and these results are related to the increases observed in WUE (Hernandez and Park 2022). Concomitantly, EBR amplified ETAd and ETAb values, optimising water use efficiency and reducing excessive water loss during the transpiration process, detected by E (Silva et al. 2020; Tadaiesky et al. 2021). In this context, ETAd and ETAb act as a covering tissue, being the plant's first line of defence, and they have a protective function against abiotic stresses, including SAR, as they regulate water losses, gas exchange, thermoregulation and light absorption in plants (Fonseca et al. 2020). Andrade et al. (2020), investigating Joannesia princeps, observed intense damage to the leaf surface in treatment using SAR at pH 3.0; chlorotic spots, necrosis, and leaf blade curling confirmed the high sensitivity of the species to SAR stress.
EBR maximised the activities of antioxidant enzymes (SOD, CAT, APX and POX) in soybean plants subjected to SAR; these results are intrinsically involved with the ability of this growth regulator to stimulate the antioxidant system, increasing the activities of the main enzymes that act in redox metabolism, suppressing ROS overproduction and reducing damage to cell membranes (Pereira et al. 2020). Plants subjected to SAR can suffer from oxidative stress, and the high concentration of O2 detects this damage− and H2O2 in plant tissues, which increases membrane permeability and lipid peroxidation (EL and MDA) (Fonseca et al. 2020). The antioxidant defence system is triggered by SOD (key enzyme); this enzyme catalyses the reaction of O2−, transforming it into H2O2, then CAT, APX and POX break down the H2O2 molecules converting it into oxygen (O2) and water (H2O), thus protecting plant metabolism from the deleterious effects of ROS, caused by exposure to SAR (Ramakrishna and Rao 2012). A study conducted by Zhang et al. (2020) investigating the physiological and biochemical responses of Camellia sinesis seedlings exposed to SAR at three different pH levels (2.5, 3.5 and 4.5) concluded that this stress induced an increase in the activities of antioxidant enzymes (SOD, CAT, APX and POX), to combat the accumulation of ROS. Shu et al. (2019), studying the ecophysiology of Jatropha curcas seedlings on SAR and different soil types, observed increased SOD, CAT and POX activity at high acidity levels (pH at 2.5, 3.5 and 4.5).
Plants pretreated with EBR presented mitigation of the overproduction of ROS (O2− and H2O2) and reduced damage to the membranes (EL and MDA) in plants subjected to SAR; these results are linked with the antioxidant machinery due to the ability of this molecule to amplify the action detoxification of SOD, CAT APX and POX enzymes, protecting cell membranes and organelles, such as the chloroplast, against loss of structure and function, maintaining the proper functioning of intracellular biochemical and molecular processes essential for plant metabolism (Maia et al. 2022). Naturally, organelles such as mitochondria, chloroplasts and peroxisomes produce ROS due to aerobic metabolic processes, including respiration and photosynthesis (Zhang et al. 2021). However, exposure to SAR can generate high production of ROS, causing oxidative damage and damage to lipid membranes, proteins, photosynthetic pigments, enzyme activity and nucleic acids (Srivastava et al. 2014). Parallel to the results obtained in this study, Zhang et al. (2021), evaluating blackberry seedlings exposed to SAR (pH 3.5, 4.5 and 5.6), observed significant increases in H2O2 and MDA in SAR at pH 5.6. In a study conducted by Wang et al. (2022) found higher levels of lipid peroxidation (MDA) in Zelkova serrata seedlings exposed to SAR (pH 2.5, 4.0 and 5.6).
EBR stimulated the biomass of soybean plants subjected to SAR, protecting against damages on LDM, RDM, SDM and TDM. These results confirm the positive repercussions of this growth regulator in plants exposed to SAR, favouring stomatal characteristics, photosynthetic pigments, chlorophyll fluorescence, gas exchange, antioxidant machinery and leaf anatomy. SAR stress inhibits plant growth and development (Liu et al. 2022), causing phytotoxicity due to acidity found in SAR, degrading photosynthetic pigments essential for the functioning of the photosynthetic machinery of plants and consequently delaying their growth (Zhang et al. 2020a). In Quercus acutissima and Cunninghamia lanceolata plants, SAR (pH 3.5) decreased growth rate, growth rate in basal diameter, growth rate in total root length and total root surface area in both species due to physiological changes and biochemical caused by the high acidity of SAR (Liu et al. 2022). In Camellia sinensis plants exposed to SAR at pH 3.5, reductions in root length (22%) and plant height (6%) were observed when compared to the control treatment (Zhang et al. 2021). In Horsfieldia hainanensis seedlings, SAR caused a decrease in biomass (roots, stems, and leaves) at more acidic (pH 2.5), reducing the biomass (29%) of seedlings exposed to SAR when compared to the control treatment (Huang et al. 2019).