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

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 scientic 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 benets on ionic homeostasis and antioxidant enzymes and convey possible repercussions on photosystem II eciency and biomass, more specically 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 – Ni 2+ and + Ni 2+ , 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 uorescence 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 signicant increases in biomass, and these results were explained by the benets on nutrient contents and ionic homeostasis, demonstrated by increased Ca 2+ /Ni 2+ , Mg 2+ /Ni + 2 and Mn 2+ /Ni 2+ ratios.


ement and int
nsively 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 Ni 2+ (Ameen et l., 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 e ciency (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 (H 2 O 2 ), superoxide (O 2 − ) 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


Materials And Methods


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 o

Glycine max (L.) Mer
.var.M8644RR Monsoy™ were germinated and grown in 1.2-L pots lled with a mixed substrate of sand and vermiculite at a ratio of 3:1.The plants were cultivated under semihydroponic 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 modi e

to 100% after two days (6th day).After this period, t
e 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 -Ni 2+ and + Ni 2+ , respectively) and two concentrations of 24-epi

assinolide (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 e

anol that was
qual 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

00] (Aha
med et al., 2013).


Plant conduction and Ni treatment

Plants receive the following macro-and micronutrients contained in the nutrient solution in agreement with Pereira et al. (2019).To simulate high Ni concentration.NiCl 2 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).Du

ng the study, the nutrient solutions were changed at 07:00 h at
-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 HNO 3 .Subsequently, 8 ml of a solution containing 4 ml of H 2 O 2 (30% v/v) and 4 ml of ultra-pure water were added and transferred to a Te on 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 uorescence and gas exchange

Chlorophyll uorescence was measured in fully expanded leaves under light using a modulated chlorophyll uorometer (model OS5p; Opti-Sciences).Preliminary tests determined the location of he 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

ant and was adapted to the dark for 30 min, was used in the evaluat
on.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

ectively, be
ween 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 quanti ed 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 H 2 O 2 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 .O 2 − was measured at 530 nm (Elstner and Heupel, 1976).


Quanti cation of hydrogen peroxide, malondialdehyde and electrolyte leakage

Stress indicators (H 2 O 2 and MDA) were extracted using the methodology described by Wu et al. (2006).

H 2 O 2 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 coe cient of 155 mM − 1 cm − 1 .EL was measured according to Gong et al. (1998) and was calculated by the formula EL (%) = (EC 1 /EC 2 ) × 100.


Determination of photosynthetic pigments and b