1 Introduction

Soybean (Glycine max L.) is the most widely cultivated legume around the world due to its high protein and oil content (Singh et al. 2008; Nisa et al. 2016; Baig et al. 2018). Its world production reached 338 million tons in the 2017/2018 harvest, with the United States, Brazil and Argentina being the main producers (FAO 2018). In field conditions, it has been frequently observed that the growth and development of this species can be affected by abiotic stresses induced by nutritional imbalances (Wang et al. 2015; Santos et al. 2017), metal toxicity (Balasaraswathi et al. 2017; Reis et al. 2018), water deficiency (Kunert et al. 2016; Wijewardana et al. 2019), salinity (Shu et al. 2017) and high temperatures (Allen Jr. et al. 2018).

Zinc is the second most necessary micronutrient for plants (Jain et al. 2010), being the deficiency of this element caused by the weathering process in tropical soils (Suhr et al. 2018). On the other hand, the zinc toxicity in plants is mainly determined by the anthropic activity associated to deposition of pollutants rich in heavy metals (Nagajyoti et al. 2010). Many plants contain 3 to 100 μg Zn g−1 dry matter, which is considered sufficient to promote adequate plant growth rates, while concentrations above 300 μg Zn g−1 are generally considered toxic (Noulas et al. 2018). The Zn content in soil is variable depending on its physical and chemical characteristics, but concentrations higher than 100 μg g−1 in soil are unusual (Rezapour et al. 2014; Antoniadis et al. 2018). However, plants frequently exhibit symptoms of Zn deficiency in shoots with concentrations lower than 2 μg Zn g−1 dry matter (Sinclair and Krämer 2012).

Zinc is essential for plant growth (Sadeghzadeh 2013; Hafeez et al. 2013) and plays important roles in essential processes, such as membrane biosynthesis, photosynthetic machinery, hormonal regulation, metabolism of lipids and nucleic acids, gene expression, and protein synthesis (Hänsch and Mendel 2009; Noulas et al. 2018; Manaf et al. 2019). Additionally, Zn is the single metal required in all six classes of the enzymes (oxidoreductases, transferases, hydrolases, lyases, isomerases and ligases) essential during photosynthesis processes and subsequent starch accumulation (Palmer and Guerinot 2009; Tripathi et al. 2015).

Deficiency linked to zinc frequently results in lower biomass and yield (Hidoto et al. 2017), reduces chlorophyll levels (Kosesakal and Unal 2009; Samreen et al. 2017) and minor efficiency linked to antioxidant system, more specifically related to superoxide dismutase (SOD) enzyme (Singh et al. 2019). Chloroplast ultrastructure is affected, resulting in abnormalities in leaf structure leading to leaf chlorosis (Kim and Wetzstein 2003; Fu et al. 2015). In relation to the photosynthetic machinery, decreases in the photochemical efficiency and the activities of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and carbonic anhydrase (CA) enzymes have been reported (Salama et al. 2006; Tavallali et al. 2009; Hajiboland and Amirazad 2010). However, excess levels of Zn also promote deleterious effects on crop yield (Tripathi et al. 2015) because Zn toxicity negatively affects CO2 assimilation and stomatal mechanisms (Azzarello et al. 2012), thus decreasing the transpiration rates and water content in the leaf (Sagardoy et al. 2009) and resulting in a lower biomass (Marques et al. 2017).

The root is a vital organ of the plant and has specialized tissues with important functions connected to influx of water and nutrients (Barberon et al. 2016). The exodermis and endodermis are tissues that act in regard to protection and selectivity of the root, thus contributing to the symplastic immobilization of excesses of Zn in the vacuoles of the root cells (Enstone et al. 2003; Arrivault et al. 2006; Sinclair and Krämer 2012). The cortex is a tissue with a storage capacity for water and nutrients in the root (Hameed et al. 2009). However, under conditions of oxidative stress, reduction and disintegration of cortical cells can occur (Singh et al. 2007; Talukdar 2013), negatively impacting the respiration and nutrient content of the root tissue (Schneider et al. 2017). In plants exposed to low/high availability of nutrients, the cortical tissue can be replaced by the cortical aerenchyma of the root, which allows for a higher allocation of the nutrients to other plant functions, such as growth and reproduction (Fan et al. 2003; Lynch 2007; Postma and Lynch 2011; Saengwilai et al. 2014).

The exogenous application of 24-epibrassinolide (EBR) can be a possible solution to mitigate the damage caused by deficiencies and excess Zn in plants because EBR is one of the most bioactive forms of brassinosteroids (BRs); it is extracted from plants and is biodegradable (Azhar et al. 2017). This steroid presents a broad spectrum of systemic action on plant metabolism (Oh et al. 2012), including CO2 (Li et al. 2016b), gas exchange (Swamy and Rao 2009), photochemical efficiency (Thussagunpanit et al. 2015), antioxidant metabolism (Xia et al. 2009) and growth rate (Abdullahi et al. 2003). In addition, BRs activate proton pumps, stimulate the synthesis of proteins and nucleic acids (Bajguz 2000) and modulate cellular expansion and division (Zhiponova et al. 2013).

This study has focused on the gap in the literature in relation to EBR’s hypothetical effects in regard to Zn. Zn is the second most common micronutrient required by plants; however, deficiencies and excesses of Zn promote deleterious effects on soybean plants. Interestingly, EBR can be a possible solution to mitigate the damage caused by deficiencies and excesses of Zn in plants because this steroid presents a spectrum of actions linked to increments in nutrient content (Lima et al. 2018), reactive oxygen species scavenging (Oliveira et al. 2019) and stimulation of biomass (Maia et al. 2018). Therefore, the aim of this research was to determine whether EBR can mitigate oxidative stress in soybean plants subjected to different Zn supplies and to evaluate its possible repercussions on anatomical, nutritional, biochemical, physiological and morphological behaviours.

2 Materials and Methods

2.1 Location and Growth Conditions

The experiment was performed at the Campus of Paragominas of the Universidade Federal Rural da Amazônia, Paragominas, Brazil (2°55’ S, 47°34’ W). The study was conducted in a greenhouse with the temperature and humidity controlled. The minimum, maximum, and median temperatures were 21, 31 and 25.2 °C, respectively. The relative humidity during the experimental period varied between 60% and 80%.

2.2 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 eight days. A modified Hoagland and Arnon (1950) solution was used for nutrients, with the ionic strength beginning at 50% (6th day) and later modified to 100% after two days (8th day). After this period, the nutritive solution remained at total ionic strength.

2.3 Experimental Design

The experiment followed a completely randomized factorial design with two concentrations of 24-epibrassinolide (0 and 100 nM EBR, described as - EBR and + EBR, respectively) and three Zn supplies (0.2, 20 and 2000 μM Zn, described as low, control and high supply of Zn). With five replicates for each of six treatments, a total of 30 experimental units were used in the experiment, with one plant in each unit.

2.4 24-Epibrassinolide (EBR) Preparation and Application

Ten-day-old seedlings 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 35. The 0 and 100 nM EBR (Sigma-Aldrich, USA) solutions were prepared in agreement with Ahammed et al. (2013). Based on preliminary studies and literature available (Lima and Lobato 2017; Maia et al. 2018; Pereira et al. 2019; Oliveira et al. 2019), the EBR is more efficient in plants pretreated (10th day). On the other hand, Zn was applied only on 20th day after experimental implementation due to need of leaf area and plant tissue sufficient to make all analyses involved in this research.

2.5 Plant Conduction and Zn Supplies

The plants received the following macro- and micronutrients contained in the nutrient solution: 8.75 mM KNO3, 7.5 mM Ca(NO3)2·4H2O, 3.25 mM NH4H2PO4, 1.5 mM MgSO4·7 H2O, 62.50 μM KCl, 31.25 μM H3BO3, 2.50 μM MnSO4·H2O, 0.63 μM CuSO4·5H2O, 0.63 μM NaMoO4·5H2O and 250 μM NaEDTAFe·3H2O, with Zn concentrations adjusted to each treatment. For Zn treatments, ZnCl2 was used at concentrations of 0.2 μM (low) and 20 μM (control) and 2000 μM (high) applied over 15 days (days 20–35 after the start of the experiment). Plants were maintained from 8th to 20th day under equal Zn concentration (20 μM Zn), considered as control treatment. One plant per pot was used to examine the plant parameters. On day 35 of the experiment, all plants were harvested and analysed.

2.6 Measurement of Chlorophyll Fluorescence and Gas Exchange

The 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 Fv/fm ratio; therefore, the acropetal third of the leaves, which was the middle third of the plant and adapted to the dark for 30 min, was used in the evaluation. The intensity and duration of the saturation light pulse were 7500 μmol m−2 s−1 and 0.7 s, respectively. The gas exchange was evaluated in all plants, measuring expanded leaves in middle region of the plant under constant conditions of a CO2 concentration, using an infrared gas analyser (model LCPro+; ADC BioScientific), photosynthetically active radiation, air-flow rate and temperature in a chamber 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. Previous tests using equal soybean variety and greenhouse were conducted to configure the equipment and determinate the work conditions. The water-use efficiency (WUE) was estimated according to Ma et al. (2004), and the instantaneous carboxylation efficiency (PN/Ci) was calculated using the formula that was described by Aragão et al. (2012).

2.7 Quantifications Linked to Anatomical Variables

Samples were collected from the middle region of the leaf limb of fully expanded leaves and roots 5 cm from the root apex, being used five samples to examine the anatomical variables. Subsequently, all collected botanical material was fixed in FAA 70 for 24 h, dehydrated in ethanol and embedded in historesin Leica™ (Leica, Nussloch, Germany). Transverse sections with a thickness of 5 μm were obtained with a rotating microtome (model Leica RM 2245, Leica Biosystems) and were stained with toluidine blue (O’Brien et al. 1964). For stomatal characterization, the epidermal impression method was used according to Segatto et al. (2004). The slides were observed and photomicrographed under an optical microscope (Motic BA 310, Motic Group Co. LTD.) coupled to a digital camera (Motic 2500, Motic Group Co., LTD.). The images were analysed with Motic plus 2.0, which was previously calibrated with a micrometre slide supplied by the manufacturer. The anatomical parameters evaluated were polar diameter of the stomata (PDS), equatorial diameter of the stomata (EDS), epidermis thickness from adaxial leaf side (ETAd), epidermis thickness from abaxial leaf side (ETAb), palisade parenchyma thickness (PPT), spongy parenchyma thickness (SPT), and the ratio PPT/SPT. In both leaf faces, the stomatal density (SD) was calculated as the number of stomata per unit area and the stomatal functionality (SF) as the ratio PDS/EDS according to Castro et al. (2009). The stomatal index (SI %) was calculated as the percentage of stomata in relation to total epidermal cells by area. In root samples, the root epidermis thickness (RET), root endodermis thickness (RDT), root cortex diameter (RCD), vascular cylinder diameter (VCD) and root metaxylem diameter (RMD) were measured.

2.8 Extraction 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). The extraction mixture was prepared by homogenizing 500 mg of fresh plant material in 5 ml of extraction buffer, which consisted of 50 mM phosphate buffer (pH 7.6), 1.0 mM ascorbate and 1.0 mM EDTA. Samples were centrifuged at 14,000×g for 4 min at 3 °C, and the supernatant was collected. Quantification of the total soluble proteins was performed using the method described by (Bradford 1976). Absorbance was measured at 595 nm, using bovine albumin as a standard.

2.9 Superoxide Dismutase Assay

For the SOD assay (EC 1.15.1.1), 2.8 ml of a reaction mixture containing 50 mM phosphate buffer (pH 7.6), 0.1 mM EDTA, 13 mM methionine (pH 7.6), 75 μM NBT, and 4 μM riboflavin was mixed with 0.2 ml of supernatant. The absorbance was then measured at 560 nm (Giannopolitis and Ries 1977).One SOD unit was defined as the amount of enzyme required to inhibit 50% of the NBT photoreduction. The SOD activity was expressed in unit mg−1 protein.

2.10 Catalase Assay

For the CAT assay (EC 1.11.1.6), 0.2 ml of supernatant and 1.8 ml of a reaction mixture containing 50 mM phosphate buffer (pH 7.0) and 12.5 mM hydrogen peroxide were mixed, and the absorbance was measured at 240 nm (Havir and McHale 1987). The CAT activity was expressed in μmol H2O2 mg−1 protein min−1.

2.11 Ascorbate Peroxidase Assay

For the APX assay (EC 1.11.1.11), 1.8 ml of a reaction mixture containing 50 mM phosphate buffer (pH 7.0), 0.5 mM ascorbate, 0.1 mM EDTA, and 1.0 mM hydrogen peroxide was mixed with 0.2 ml of supernatant, and the absorbance was measured at 290 nm (Nakano and Asada 1981). The APX activity was expressed in μmol AsA mg−1 protein min−1.

2.12 Peroxidase Assay

For the POX assay (EC 1.11.1.7), 1.78 ml of a reaction mixture containing 50 mM phosphate buffer (pH 7.0) and 0.05% guaiacol was mixed with 0.2 ml of supernatant, followed by addition of 20 μL of 10 mM hydrogen peroxide. The absorbance was then measured at 470 nm (Cakmak and Marschner 1992). The POX activity was expressed in μmol tetraguaiacol mg−1 protein min−1.

2.13 Determination of Superoxide Concentration

To determine O2−, 1 ml of extract was incubated with 30 mM phosphate buffer [pH 7.6] and 0.51 mM hydroxylamine hydrochloride for 20 min at 25 °C. Then, 17 mM sulphanilamide and 7 mM α-naphthylamine were added to the incubation mixture for 20 min at 25 °C. After the reaction, ethyl ether was added in the identical volume and centrifuged at 3000×g for 5 min. The absorbance was measured at 530 nm (Elstner and Heupel 1976).

2.14 Extraction of Nonenzymatic Compounds

Nonenzymatic compounds (H2O2 and MDA) were extracted as described by Wu et al. (2006). Briefly, a mixture for extraction of H2O2 and MDA was prepared by homogenizing 500 mg of fresh leaf materials in 5 mL of 5% (w/v) trichloroacetic acid. Then, the samples were centrifuged at 15,000 x g for 15 min at 3 °C to collect the supernatant.

2.15 Determination of Hydrogen Peroxide Concentration

To measure H2O2, 200 μL of supernatant and 1800 μL of reaction mixture (2.5 mM potassium phosphate buffer [pH 7.0] and 500 mM potassium iodide) were mixed, and the absorbance was measured at 390 nm (Velikova et al. 2000).

2.16 Quantification of Malondialdehyde Concentration

MDA was determined by mixing 500 μL of supernatant with 1000 μL of the reaction mixture, which contained 0.5% (w/v) thiobarbituric acid in 20% trichloroacetic acid. The mixture was incubated in boiling water at 95 °C for 20 min, with the reaction terminated by placing the reaction container in an ice bath. The samples were centrifuged at 10,000×g for 10 min, and the absorbance was measured at 532 nm. The nonspecific absorption at 600 nm was subtracted from the absorbance data. The MDA–TBA complex (red pigment) amount was calculated based on the method of Cakmak and Horst (1991), with minor modifications and using an extinction coefficient of 155 mM−1 cm−1.

2.17 Determination of Electrolyte Leakage

Electrolyte leakage was measured according to the method of Gong et al. (1998) with minor modifications. Fresh tissue (200 mg) was cut into pieces 1 cm in length and placed in containers with 8 mL of distilled deionized water. The containers were incubated in a water bath at 40 °C for 30 min, and the initial electrical conductivity of the medium (EC1) was measured. Then, the samples were boiled at 95 °C for 20 min to release the electrolytes. After cooling, the final electrical conductivity (EC2) was measured. The percentage of electrolyte leakage was calculated using the formula EL (%) = (EC1/EC2) × 100.

2.18 Determination of Photosynthetic Pigments

The chlorophyll and carotenoid determinations were performed with 40 mg of leaf tissue, being used five samples per treatment. The samples were homogenized in the dark with 8 mL of 90% methanol (Nuclear). The homogenate was centrifuged at 6000×g for 10 min at 5 °C. The supernatant was removed, and chlorophyll a (Chl a) and b (Chl b), carotenoid (Car) and total chlorophyll (total Chl) contents were quantified using a spectrophotometer (model UV-M51; Bel Photonics), according to the methodology of Lichtenthaler and Buschmann (2001).

2.19 Determination of Nutrients

Samples with 100 mg of milled samples were weighed in 50-mL conical tubes (FalconR, Corning, Mexico) and pre-digested (48 h) with 2 ml of sub boiled HNO3 (DST 1000, Savillex, USA). After, 8 ml of a solution containing 4 ml of H2O2 (30% v/v, Synth, Brasil) and 4 ml of ultra-pure water (Milli-Q System, Millipore, USA) were added, and the mixture was transferred to a Teflon digestion vessel, closed and heated in a block digester (EasyDigest®, Analab, France) according to the following program: i) 100 °C for 30 min; ii) 150 °C for 30 min; iii) 130 °C for 10 min; iv) 100 °C for 30 min and; and v) left to cool. The volume was made to 50 mL with ultra-pure water, and iridium was used as an internal standard at 10 μg l−1. The determinations of Zn, P, K, Mg, Fe, Cu and Mo were carried out using an inductively coupled plasma mass spectrometer (ICP-MS 7900, Agilent, USA). Certified reference materials (NIST 1570a and NIST 1577c) were run in each batch for quality control purposes. All found values were in agreement with certified values.

2.20 Measurements of Morphological Parameters

The growth of roots, stems and leaves was measured based on constant dry weights (g) after drying in a forced-air ventilation oven at 65 °C.

2.21 Data Analysis

The data were submitted to ANOVA and applied Scott–Knott test at a probability level of 5% (Steel et al. 2006). All statistical procedures used the Assistat software.

3 Results

3.1 Zn Contents in Plants after EBR and Zn Treatments

The low and high Zn supplies promoted changes in the contents of this element in the root, stem and leaf tissues of soybean plants (Table 1). Plants sprayed with EBR and exposed to low Zn presented increases in Zn concentrations of 48% (root), 42% (stem) and 41% (leaf) when compared to the same treatment without EBR. However, the control + EBR treatment exhibited increases of 44%, 50% and 12% in root, stem and leaf, respectively. In relation to the high Zn with EBR, significant decreases were detected in the Zn contents in the stem and leaf by 21% and 10%, respectively, but there was an increase in the root tissue of 7%.

Table 1 Zn contents in soybean plants sprayed with EBR and exposed to different Zn supplies
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3.2 Root Structures Were Positively Modulated by EBR

The low and high Zn supplies resulted in negative changes in root anatomy (Fig. 1). However, the application of EBR in the plants submitted to the low Zn treatment promoted increases for RET, RDT, RCD, VCD and RMD of 16%, 3%, 14%, 33% and 74% (Table 2), respectively, when compared to the same treatment without EBR, while the control + EBR treatment had increases of 10%, 5%, 10%, 38% and 5%, respectively. Plants exposed to high Zn + EBR had increases of 25%, 7%, 9% 29% and 69%, respectively.

Fig. 1
figure 1

Root cross sections in soybean plants sprayed with EBR and exposed to different Zn supplies. Low Zn without EBR (A), Low Zn with EBR (B), Control Zn without EBR (C), Control Zn with EBR (D), High Zn without EBR (E), High Zn with EBR (F). RE = Root epidermis; RC = Root cortex; RD = Root endodermis; VC = Vascular cylinder; RM = Root metaxylem. Bars: 300 μm

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Table 2 Root anatomy in soybean plants sprayed with EBR and exposed to different Zn supplies
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3.3 EBR Maximized the Nutrient Contents

Soybean plants exposed to low and high concentrations of Zn had reductions (P < 0.05) in nutrient contents in their tissues (Table 3). However, plants subjected to a low Zn supply and sprayed with EBR had increases in the values of K, P, Mg, Fe, Cu and Mo at 14%, 15%, 9%, 29%, 23% and 42% (root); 4%, 16%, 15%, 16%, 30% and 12% (stem); 25%, 13%, 12%, 10%, 7% and 55% (leaf), respectively, compared with the same treatment without EBR (Table 3). In the high Zn treatment with EBR, we also observed increases in K, P, Mg, Fe, Cu and Mo of 29%, 13%, 24%, 19%, 37% and 10% in roots; 7%, 12%, 50%, 9%, 9% and 4% in stems; and 6%, 28%, 15%, 17%, 17% and 50% in leaves compared with the equal treatment without EBR.

Table 3 Nutrient contents in soybean plants sprayed with EBR and exposed to different Zn supplies
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The steroid provoked benefits for the photosynthetic machinery of plants under Zn stress.

Plants with low and high Zn supplies exhibited reductions in Fm, Fv and Fv/fm, but increase in F0, in relation control treatment (Fig. 2). In Fm, the EBR application resulted in increases of 2% and 2% in the low and high supplies, respectively, when related to the same treatment without EBR. For Fv, we detected increases of 3% and 3% in plants under low and high Zn supplies with EBR, respectively. In Fv/fm, a low Zn with EBR had an increase of 1%, while the control + EBR showed an increment of 2% in relation to the same treatment without EBR. Decreases in ΦPSII, qP and ETR and increases in NPQ, EXC and ETR/PN were verified under low and high Zn in soybean plants (Table 4). However, plants treated with 100 nM EBR and exposed to low Zn had increases of 4%, 4%, and 5% for ΦPSII, qP and ETR, respectively, and reductions in NPQ (8%) and EXC (1%) compared to the low Zn without EBR. In relation to the high Zn with EBR, there were increases of 16%, 12%, and 14% for ΦPSII, qP and ETR, respectively, and decreases in NPQ (8%) and EXC (6%) and ETR/PN (5%) compared with the same treatment in the absence of EBR.

Fig. 2
figure 2

Minimal fluorescence yield of the dark-adapted state (F0), maximal fluorescence yield of the dark-adapted state (Fm), variable fluorescence (Fv) and maximal quantum yield of PSII photochemistry (Fv/fm) in soybean plants sprayed with EBR and exposed to different Zn supplies. Columns with different letters indicate significant differences from the Scott-Knott test (P < 0.05). Columns corresponding to means from five repetitions and standard deviations

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Table 4 Chlorophyll fluorescence in soybean plants sprayed with EBR and exposed to different Zn supplies
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Fig. 3
figure 3

Leaf cross sections in soybean plants sprayed with EBR and exposed to different Zn supplies. Low Zn without EBR (A), Low Zn with EBR (B), Control Zn without EBR (C), Control Zn with EBR (D), High Zn without EBR (E), High Zn with EBR (F). EAd = adaxial epidermis; EAb = Abaxial epidermis; PP = Palisade parenchyma; SP = Spongy parenchyma. Bars: 200 μm

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3.4 Exogenous EBR Improved the Gas Exchange

The low and high Zn supplies had negative effects on gas exchange (Table 5). However, the application of EBR in plants with a low Zn supply resulted in increases of PN, gs, WUE and PN/Ci of 5%, 14%, 10% and 32%, respectively, and decreases of 20% for Ci when compared to the same treatment without EBR. The high Zn + EBR had incremental changes in PN, E, gs, WUE and PN/Ci of 20%, 9%, 13%, 10% and 59%, respectively, and a reduction of 19% in Ci.

Table 5 Gas exchange in soybean plants sprayed with EBR and exposed to different Zn supplies
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EBR action enhanced the stomatal performance in plants exposed to different Zn supplies.

The stomatal characteristics showed decreases in SD, SF and SI, as well as increases in PDS and EDS on the adaxial and abaxial faces of soybean leaves exposed to the low and high Zn concentrations (Table 6). The action of EBR on the adaxial face of leaves in both treatments (low and high Zn) caused increases in SD (26% and 76%, respectively), SF (5% and 4%, respectively) and SI (24% and 57%, respectively) and reductions in PDS (4% and 8%, respectively) and EDS (9% and 12%, respectively). For the abaxial face, the low and high Zn supplies with 100 nM EBR spray promoted increases in SD (13% and 30%, respectively), SF (4% and 2%, respectively) and SI (6% and 8%, respectively) and decreases in values of PDS (5% and 8%, respectively) and EDS (10% and 11%, respectively) when compared to the same treatment in the absence of EBR.

Table 6 Stomatal characteristics in soybean plants sprayed with EBR and exposed to different Zn supplies
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Beneficial repercussions on leaf anatomy promoted by the steroids in plants under Zn stress.

The low and high concentrations of Zn promoted negative changes in the leaf anatomy (Fig. 3). However, plants under low Zn and EBR had increases in ETAd (21%), ETAb (25%), PPT (11%) and SPT (12%) and a reduction in PPT/SPT (2%) compared with the same treatment without EBR (Table 7). For the high Zn with EBR, we observed significant increases in ETAd (19%), ETAb (14%), PPT (10%) and SPT (16%) and a decrease in PPT/SPT (6%).

Table 7 Leaf anatomy in soybean plants sprayed with EBR and exposed to different Zn supplies
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Antioxidant enzymes were stimulated after EBR spray in plants treated with different Zn concentrations.

Soybean plants exposed to low and high Zn supplies had increases (P < 0.05) in SOD, CAT, APX and POX values (Fig. 4). The application of 100 nM EBR in plants under a low Zn supply provoked significant increases of 26% 18%, 66% and 25%, respectively, when compared to the low supplement Zn with 0 nM EBR. The high Zn + EBR resulted in significant increases in the activities of SOD (29%), CAT (24%), APX (72%) and POX (44%) compared with the same treatment in the absence of EBR (Fig. 4).

Fig. 4
figure 4

Activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and peroxidase (POX) in soybean plants sprayed with EBR and exposed to different Zn supplies. Columns with different letters indicate significant differences from the Scott-Knott test (P < 0.05). Columns corresponding to means from five repetitions and standard deviations

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Oxidative stress induced by different Zn supplies was alleviated after treatment with the steroid.

The oxidant compounds (O2− and H2O2) and indicators of cell damage (MDA and EL) in plants exposed to low and high Zn supplies showed increases (Fig. 5). However, plants with a low supply of Zn and 100 nM EBR had reductions in O2− (46%), H2O2 (6%), MDA (17%) and EL (10%) levels compared to the low Zn and 0 nM EBR plants. In relation to the high Zn with EBR, decreases were verified in O2− (29%), H2O2 (2%), MDA (15%) and EL (6%) in comparison with the same treatment in the absence of EBR.

Fig. 5
figure 5

Superoxide (O2−), hydrogen peroxide (H2O2), malondialdehyde (MDA) and electrolyte leakage (EL) in soybean plants sprayed with EBR and exposed to different Zn supplies. Columns with different letters indicate significant differences from the Scott-Knott test (P < 0.05). Columns corresponding to means from five repetitions and standard deviations

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EBR prevented the degradation of photosynthetic pigments in plants under Zn stress.

In both treatments (low and high Zn), a concentration of 100 nM EBR promoted maximization of the photosynthetic pigments (Table 8), increasing the levels of Chl a (29% and 31%, respectively), Chl b (95% and 65%, respectively), Chl total (38% and 35%, respectively) and Car (38% and 45%, respectively) when compared with equal treatment without EBR (0 nM). In addition, there were reductions in the Chl a/Chl b ratio of 31% and 14% and in the Chl/Car ratio of 4% and 5%, respectively.

Table 8 Photosynthetic pigments in soybean plants sprayed with EBR and exposed to different Zn supplies
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Effects deleterious on the biomass were mitigated in plants treated with EBR and subjected to Zn stress.

Plants under low and high Zn supplies presented improvements in growth when receiving EBR application, for the low Zn + EBR increases of 25%, 32%, 5% and 22% of LDM, RDM, SDM and TDM, respectively, compared to low Zn + 0 nM EBR (Fig. 6). In the high Zn with EBR, we also detected increases in the values of LDM, RDM, SDM and TDM of 14%, 5%, 12% and 10%, respectively.

Fig. 6
figure 6

Leaf dry matter (LDM), root dry matter (RDM), stem dry matter (SDM) and total dry matter (TDM) in soybean plants sprayed with EBR and exposed to different Zn supplies. Columns with different letters indicate significant differences from the Scott-Knott test (P < 0.05). Columns corresponding to means from five repetitions and standard deviations

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4 Discussion

Plants exposed to low and control Zn + 100 nM EBR had increases in Zn content, suggesting that this steroid improved the absorption, transport and accumulation of Zn in the evaluated tissues. This result can be associated with the intense interaction between Zn2+ ions and organic acids, such as histidine, to form soluble Zn complexes, favouring the absorption and accumulation of this metal in the cytosol of the root cells (Khodamoradi et al. 2015). Histidine is an amino acid that plays a central role in the homeostasis of Zn2+ ions, facilitating the mobility of this element in the xylem sap via symplastic transport (Kozhevnikova et al. 2014; Khodamoradi et al. 2015). On the other hand, exogenous EBR also minimized the toxic effects of Zn, reducing the Zn content in the tissues exposed to a high Zn supply. This reduction is related to higher synthesis of phytochelatins (PC) in the root cells (Anwar et al. 2018). PC contributes to detoxification mechanisms of heavy metals (Rajewska et al. 2016), chelating the metal ions and forming complexes, with consequent immobilization of this metal in the cytoplasm of root cells (Bajguz and Hayat 2009; Bajguz 2010). Tadayon and Moafpourian (2019) verified that foliar application of 0.4 mg L−1 EBR increased the efficiency of foliar application of Zn and B, affecting the chemical and reproductive characteristics of Vitis vinifera plants.

EBR revealed beneficial effects on root tissues (RET, RDT, RCD, VCD and RMD). Increases in the expression of RET, RDT and RCD demonstrated that EBR modulated growth linked to the root meristem through cellular expansion and differentiation, conferring higher protection to this organ (Wei and Li 2016). The epidermis, endoderm and cortex are tissues that are associated with the mechanism of protection and selectivity in the roots, and the increases detected in these tissues contribute to forming a barrier against biotic and abiotic stresses (Cui 2015; Barberon et al. 2016). EBR has positive effects on VCD and RMD, suggesting that the higher densities of these tissues must facilitate transport of water and nutrients via the symplast (Meyer et al. 2011). Reductions in RET and RCD promoted by the high Zn supply (500 μM ZnSO4) were verified by Bazihizina et al. (2014) after studying the impacts of this metal on the cellular structure of Nicotiana tabacum roots. Maia et al. (2018) observed in a study with Solanum lycopersicum plants that a spray with 100 nM EBR promoted increases in RET (9%), RDT (14%), RCD (12%), VCD (7%) and RMD (17%).

Plants treated with low and high concentrations of Zn and sprayed with 100 nM EBR presented increases in the contents of macronutrients (K, P, and Mg) and micronutrients (Fe, Cu and Mo). The increments induced by the EBR on Zn contents (mainly under low and control Zn supplies) can be explained by the increases in RDM, suggesting higher amounts of root hairs, because this tissue have large contact surface exposed to substrate, facilitating the uptake and mobility of the Zn in plant tissues (Tanaka et al. 2014). These results revealed that the EBR mitigated the negative impacts of Zn on the ionic homeostasis of these essential elements in the absorption channels, optimizing the transport and assimilation process of H2PO4−, Ca2+, Mg2+, Mn2+ Fe3+, Cu2+ and Zn2+ ions by the roots (Karlidag et al. 2011). The exogenous steroid increased the uptake of the Mg2+ ion in the root and improved the transport of this element from root to shoot, increasing the chlorophyll levels and improving the photosynthetic characteristics (Fiedor et al. 2008; Yuan et al. 2015). In addition, increases in Fe, Cu, Zn and Mn contribute to a better response related to the antioxidant system because they are metal cofactors of the three main forms of SOD (Fe-SOD, Cu / Zn-SOD and Mn-SOD) (Hänsch and Mendel 2009; Abreu and Cabelli 2010). A study conducted by Samreen et al. (2017) evaluating the effect of Zn stress (0, 1 and 2 μM Zn) on the growth, chlorophyll content and mineral content of Vigna radiata plants verified that Zn toxicity had deleterious effects on P, K, and Fe contents in plants. Billard et al. (2015) investigated the impacts of Zn deficiency on nutritional status and protein modifications in Brassica napus plants and found that Zn-deficient plants exhibited a lower absorption of elements (K, Mg and Fe).

The exogenous application of 100 nM EBR mitigated the negative effects of low and high Zn supplies on F0, Fm, Fv and Fv/fm, indicating that EBR reduced photoinhibition and improved photochemical efficiency. Reductions in F0 and increases in Fm suggested that EBR enhanced the electron transfer from the primary plastoquinone acceptor (QA) to the secondary plastoquinone acceptor (QB) on the acceptor side of PSII, reflecting positively on Fv/fm (Shu et al. 2016). Andrejić et al. (2018), studying the impact of Zn excess (250, 500 and 1000 mg Zn kg-1) on gas exchange and chlorophyll fluorescence in plants Miscanthus × giganteus, verified reductions in Fm, Fv and Fv/fm, while Xia et al. (2009) confirmed in a study with Cucumis sativus that 0.1 μM EBR increased the Fv/fm values, optimizing the activity of PSII.

The highest values of ΦPSII, qp and ETR are intrinsically related to the increase in Fv/fm, as previously described in this study. These results confirm that exogenous EBR maximized the energy capture efficiency by the PSII open-reaction centres in Zn-stressed plants (Zhang et al. 2013; Jia et al. 2015). In addition, the increase in ETR, as indicated by higher values of ΦPSII, corroborates that EBR increased the capacity of the photosynthetic apparatus to maintain the QA in the oxidized state, optimizing the transport of electrons through PSII (Dobrikova et al. 2014). Siddiqui et al. (2018), investigating the chlorophyll fluorescence of Brassica juncea plants pretreated with two BRs, detected increases promoted by EBR (10−8 M) in ΦPSII (19%), qp (17%) and ETR (19%), while foliar spray of HBL (10−8 M) promoted increases of 17%, 16% and 18% for ΦPSII, qp and ETR, respectively.

The decrease induced by EBR in the NPQ, EXC and ETR/PN of plants exposed to the low and high Zn supplies revealed that the application of this steroid resulted in less excitation energy dissipation in the form of heat, avoiding the damage by photoinhibition in the centres of reaction (Ogweno et al. 2008; Zhang et al. 2015). Additionally, reductions of the expression of EXC and ETR/PN indicated that the excess electrons were used less often for secondary processes, such as photorespiration, and thus were potentially available for primary processes, such as reductions of NADP+ during the biochemical fixation of CO2 (Silva et al. 2012). Lima et al. (2018) found that the application of 100 nM EBR in Eucalyptus urophylla under Fe deficiency significantly reduced the values of NPQ (19%), EXC (14%) and ETR/PN (16%), promoting protection of PSII against possible damages caused by the excess of excitation and improving the use of electrons during the photochemical activity.

EBR minimized the damage caused by Zn concentrations (low and high) on gas exchange. Increases in PN and E are positively related to the improvements expressed in gs, and these effects are explained by the positive impact of EBR on the enzymatic activities of CA (Hayat et al. 2011) and RuBisCO (Yu et al. 2004), which are key enzymes in the initial process of photosynthesis. The high CA activity increases the carboxylation state of RuBisCO in the Calvin cycle, consequently decreasing Ci and inducing PN maximization (Hasan et al. 2011; Alyemeni and Al-Quwaiz 2016). The increase in WUE is associated with the benefits promoted by EBR on PN. In addition, PN/Ci values were increased in EBR-treated plants due to increased PN and a simultaneous reduction in Ci. Fei et al. (2016) detected reductions in PN, gs and E, as well as increases in Ci promoted by the low Zn supplement in Citrus sinensis plants. Ouni et al. (2016), analysing the effects of Zn concentrations (100 and 300 ppm) on the gas exchange of Polypogon monspeliensis, observed reductions in PN, gs and WUE values under the highest concentration of Zn (300 ppm). However, Jiang et al. (2012) demonstrated that the effects of EBR foliar spraying (0.1 μM) improved the gas exchange (PN, gs and Ci) in Cucumis sativus plants.

Exogenous EBR (100 nM) had positive effects on stomatal characteristics (SD, PDS, EDS, SF and SI). The increases of SD, SF and SI revealed that the EBR improved stomatal performance, corroborated by higher values detected for gs. This steroid regulates stomatal development, activating specific proteins that act on the stomatal intracellular signalling pathway (Kim et al. 2012; Casson and Hetherington 2012), maximizing the gas exchange and increasing the opportunity for CO2 uptake by the mesophyll cells (PPT and SPT) (Flexas et al. 2008, 2012). Additionally, the reductions observed in PDS and EDS reveal beneficial interferences of the EBR in the stomata form, inducing stomata to be more elliptic and providing increases in SF (Martins et al. 2015). Subba et al. (2014), investigating physiological and biochemical changes induced by nine concentrations of Zn (0–20 mM Zn) in Citrus reticulata seedlings, observed reductions in SD in the leaves of plants exposed to deficiency (0, 1, 2, 3 and 4 mM Zn) and excess (10, 15 and 20 mM Zn) when compared to a sufficient concentration (5 mM Zn).

Plants treated with EBR (100 nM) and exposed to Zn supplies (low and high) had beneficial effects on leaf anatomy (ETAd, ETAb, PPT and SPT). The increases in PPT and SPT are connected to increments shown in PN and PN/Ci because the gas exchange has an influence on the mesophyll, facilitating CO2 diffusion from the environment to the carboxylation sites in the chloroplasts (Ennajeh et al. 2010). The high values of ETAd and ETAb in plants sprayed with EBR can be explained by the higher values of E and WUE, in which the epidermis is a coating tissue, clearly contributing to the use of water and avoiding excessive loss of water during the transpiration process (Javelle et al. 2011). Kim and Wetzstein (2003) investigated Carya illinoinensis plants subjected to Zn deficiency and reported decreases in PPT and SPT and found a reduction in the number of cells of the palisade parenchyma per length in leaf. Mattiello et al. (2015) examined the impacts of Zn deficiency on physiological and anatomical characteristics of Zea mays leaves during 0, 2, 6, 10, 14, 18 and 22 days after the Zn omission; they reported intense reductions in their size, composed of 44% mesophyll and 8% intercellular space. In addition, the epidermal area on the adaxial and abaxial surfaces corresponded to 15% and 10%, respectively.

The application of EBR (100 nM) contributed to an increase in the activities of the SOD, CAT, APX and POX enzymes of the plants exposed to the low and high Zn supplies, revealing the intrinsic action of this substance on antioxidant metabolism. These changes contribute to a higher photochemical efficiency, as evidenced by the increases in Fv/fm and ETR. A study conducted by He et al. (2016) evaluating the enzymatic responses and growth of Solanum melongena seedlings subjected to Zn toxicity (10% Zn) + 0.1 μM EBR detected increases in the activities of SOD (20%), CAT (25%), APX (11%) and POX (17%). Li et al. (2016a), investigating the exogenous effects of EBR on Solanum lycopersicum seedlings, also found benefits on the antioxidant system, in which 5 nM EBR notably increased the activities of the SOD, CAT and APX enzymes under Zn stress.

Exogenous EBR (100 nM) promoted reductions in ROS levels (O2− and H2O2) and mitigated the membrane damage (MDA and EL) in Glycine max plants exposed to Zn stress (low and high), and these results were attributed to higher activity of the antioxidant enzymes (SOD, CAT, APX and POX) as previously detected in this study. In cells, the SOD enzyme rapidly converts O2− to H2O2, while the CAT and APX enzymes act to dissociate H2O2, with consequent formation of H2O and O2, reducing the concentrations of oxidizing compounds (Li et al. 2016a). On the other hand, high concentrations of O2− and H2O2 often promote lipid peroxidation (MDA), inducing electrolyte leakage (EL) and negatively impacting the membrane function (Kumari et al. 2010; Gallego et al. 2012). Ramakrishna and Rao (2012) evaluated Raphanus sativus seedlings subjected to three concentrations of EBR (0.5, 1.0 and 2 μM) and exposed to Zn stress and verified significant reductions in O2− (57%), H2O2 (27%) and EL.

Plants under Zn stress (low and high) and sprayed with EBR had increases in the levels of Chl a, Chl b, Chl total and Car, and these effects were related to lower accumulation of ROS (O2− and H2O2) in leaf tissue, reducing the oxidative damage to the structures and functions of the thylakoid membranes (Ramakrishna and Rao 2012). This result was confirmed by the decreases in the MDA and EL levels previously described in this study. In addition, EBR promoted an increase in Mg content, which is a structural element of the chlorophyll molecule (Fiedor et al. 2008). These benefits induced by EBR enhanced pigment biosynthesis and promoted a positive impact on the photosynthetic apparatus. Mateos-Naranjo et al. (2018) found reductions in Chl a, Chl b and Car levels in Juncus acutus plants exposed to Zn toxicity (100 mM Zn). However, Ramakrishna and Rao (2015) demonstrated that foliar application of EBL and HBL at concentrations of 0.5, 1.0 and 2.0 μM effectively alleviated the deleterious effects of Zn toxicity on Raphanus sativus, protecting mainly the chloroplast membranes and increasing Chl a, Chl b and Car.

The EBR application reduced the deleterious effects on plant biomass (LDM, RDM, SDM and TDM) caused by low and high Zn supplementation. These results suggest that EBR stimulated cell division and elongation in roots, stems and leaves, increasing the rate of growth and development (Müssig 2005; Que. et al. 2018). The increase in biomass can be explained by the benefits to root anatomy, gas exchange, antioxidant enzymes (SOD, CAT, APX and POX) and nutrient contents demonstrated in this study (Shahbaz et al. 2008; Hayat et al. 2012; Santos et al. 2018). Pascual et al. (2016) reported significant reductions in the LDM and RDM values of Glycine max plants subjected to Zn deficiency. Research conducted by Marques et al. (2017) studying Jatropha curcas plants subjected to different concentrations of Zn (100, 200, 300, 400 and 600 μM) observed a decrease in plant biomass (leaf, stem and root) after exposure to a higher concentration of Zn (600 μM).

5 Conclusion

Our study proved that 24-epibrassinolide mitigated the oxidative stress induced by different zinc supplies in soybean plants. In other hand, plants exposed to high zinc supply without 24-epibrassinolide application presented deleterious effects more intense. The steroid spray alleviated the impact produced by zinc stress on nutritional status because these results were intrinsically linked to improvements on vascular cylinder and metaxylem, improving the magnesium, phosphorus, potassium, iron, copper and molybdenum contents. In relation to the photosynthetic machinery of plants treated with 24-epibrassinolide and exposed to high and low zinc supplies, antioxidant enzymes play crucial roles, dismutating superoxide and hydrogen peroxide, and protecting the chloroplast membranes, with clear positive repercussions on chlorophylls, effective quantum yield of photosystem II photochemistry and electron transport rate. The stimulation induced by this substance on gas exchange can be explained by the favourable conditions detected for stomatal density, stomatal index, palisade parenchyma and spongy parenchyma, enhancing the carbon dioxide diffusion in the chloroplasts. Finally, an interesting result found in this research is related to 24-epibrassinolide application on leaves promoting beneficial effects on root anatomy, validating the systemic action of this steroid.