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Heavy metals like cadmium (Cd) contamination occur in conjunction with the rising CO 2 threatening food security and safety. Foliar application of 24-Epibrassinolide (EBR) was found to ameliorate Cd stress and improve nutrient availability in crops. However, its role under elevated CO 2 is currently unknown. Accordingly, a pot experiment was conducted in open-top chambers (CO 2 at 400 and 600 μmol mol -1 ) to determine the protective effect of EBR on wheat plants under different Cd concentrations (0, 2, and 4 mg kg -1 ) in soil. The foliar application of EBR significantly improved growth, biomass, photosynthesis, proline, total phenol, and total soluble protein in Cd stress treatments under elevated CO 2 . Simultaneously, it significantly (p ≤ 0.05) increased catalase (42.89 %), superoxide dismutase (26.53 %), peroxidase (28.10 %), and ascorbate peroxidase (61.70 %) while reduced malondialdehyde (35.53 %), hydrogen peroxide (19.94 %), and electrolyte leakage (23.55 %) under elevated CO 2 compared to ambient CO 2 conditions. Furthermore, EBR and elevated CO 2 interactively showed a maximum reduction in Cd concentrations and accumulation in the wheat roots (39.74,41.63 %), shoots (46.83,44.87 %), and grains (27.52,29.06 %) respectively. Elevated CO 2 and Cd stress interactively showed a significant reduction in nutrient content. Conversely, the EBR application recovered and significantly increased calcium, magnesium, iron, zinc, and copper content in wheat roots, shoots, and grains. Our findings inferred that EBR foliar application reduced Cd toxicity and improved plant growth and nutritional quality under elevated CO 2 ., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Plants often experience variations in light intensity, referred to as light stress, that negatively impact important aspects of plant growth and development, including photosynthesis and antioxidant system. The photosynthetic machinery is susceptible to these disturbances, especially photosystem II and its reaction centers. We aimed to evaluate the role of brassinosteriod in plants under both high and low light conditions by examining various physiological parameters such as photosynthetic efficiency, pigment levels, and enzymatic activity of various antioxidant enzymes in one month old tomato plants. We investigated various chlorophyll fluorescence parameters under low light (LL) and high light (HL) conditions and the associated gene expression related to photosynthesis, including plastocyanin, ferredoxin, and photosystem II oxygen-evolving enhancer protein 3 (PsbQ). Our results indicate that exogenous brassinosteroid application considerably increased tolerance to both high and low light stress in 4-week-old tomato as treated plants displayed enhanced photosynthesis, reduced oxidative damage, and increased antioxidant enzyme activity in comparison to control plants. Furthermore, brassinosteroid treatment enhanced the expression of genes associated with antioxidant pathways, which significantly contributed to the recovery of chlorophyll fluorescence parameters crucial for plant growth and development. Our results provide valuable insights into how brassinosteroid reduces light-induced stress in tomato plants.

Silica nanoparticles (SiNPs) and brassinolide (BR) have been used as nano-fertilizer and growth regulator, respectively to enhance crop tolerance to abiotic stress. However, it is unclear whether a combination of the two (BR + SiNPs) is more beneficial than single application of BR or SiNPs to improve the growth of deficit-irrigated sugar beets. In this study, a two-year (2022-2023) field experiment was conducted to investigate the effects of foliar spraying of water (CK), SiNPs, BR, and BR + SiNPs on the antioxidant defense, photosynthetic capacity, dry matter accumulation, nutrient uptake, and yield of sugar beets under full irrigation (100% of crop evapotranspiration (ETc), W1) and deficit irrigation (60% ETc, W2). The results showed that compared with the application of BR or SiNPs, the application of BR + SiNPs could enhance the antioxidant defense, osmoregulation, and photosynthesis of the full-irrigated and deficit-irrigated sugar beet leaves, and ultimately improved the water status, growth, and yield of sugar beet plants. There was no significant difference in the net revenue (NR) between BR + SiNPs treatment and CK under W1 conditions. However, the NR of the BR + SiNPs treatment increased by 27.0% (p < 0.05) compared with that of CK under W2 conditions, and there was no significant difference in NR between BR + SiNPs and SiNPs treatments. A comprehensive evaluation using entropy weight combined with technique for order preference by similarity to ideal solution method found that under deficit irrigation condition, spraying SiNPs could improve the growth of sugar beet, increase the TY, NR, and water use efficiency, and reduce costs compared with spraying BR + SiNPs. Therefore, foliar spraying of SiNP on deficit-irrigated sugar beets can be used to improve sugar beet growth and reduce the potential economic losses caused by deficit irrigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Gamma irradiation-based mutant creation is one of the most important methods for rice plant mutagenesis breeding and molecular biology research. Although median lethal dose irradiation severely damages rice seedlings, applying brassinolide (BR) can increase the survival rate of irradiated seedlings. In this study, we investigated the effects of soaking seeds in solutions containing different BR concentrations (0.001, 0.01, 0.1, 1.0, and 5.0 μmol/L) and then spraying the resulting seedlings twice with 0.1 μmol/L BR. The combined BR treatments markedly decreased the superoxide anion (O 2 •- ), hydrogen peroxide (H 2 O 2 ), and malondialdehyde contents but increased the chlorophyll content. An appropriate BR treatment of gamma-irradiated samples substantially increased the activities of the antioxidant enzymes superoxide dismutase, peroxidase, and ascorbate peroxidase as well as the proline, ascorbic acid, and glutathione contents in rice seedling shoots. The BR treatment also promoted the growth of seedlings derived from irradiated seeds and increased the shoot and root fresh and dry weights. Most notably, soaking seeds in 0.01 or 0.1 μmol/L BR solutions and then spraying seedlings twice with 0.1 μmol/L BR significantly increased the final seedling survival rate and decreased mutant loss. The study results suggest that exogenous BR treatments can protect rice seedlings from gamma irradiation stress by enhancing antioxidant metabolism.

Bitter melon (Momordica charantia L.) is well-known for its high protein, steroid, alkaloid, mineral, lipid, triterpene, and phenolic compound content, as well as its medicinal properties, particularly its anti-diabetic effects. To investigate the impact of elicitors on the morphology and phytochemical characteristics of bitter melon (Jounpouri cultivar) over two consecutive years (2018 and 2019), we conducted a field experiment. The study aimed to determine the effects of Ethrel, brassinosteroids (BRs), and k-carrageenan on yield and the production of anti-diabetic agents in M. charantia farm crops. The elicitors included ten levels, ranging from a control group to Ethrel (100, 300, and 600 mg l - 1 ), brassinosteroids (BRs) (0.1, 0.5, and 1 mg l - 1 ), and k-carrageenan (200, 400, and 600 mg l - 1 ). These characteristics included leaf area, leaf length, leaf width, fruit parameters, carbohydrate content, total phenols and flavonoid accumulation, antioxidant activity, total acid, ascorbic acid, momordicine, and charantin. Across both years, we observed the highest flavonoid accumulation and antioxidant activity in the Ethrel treatment group. Specifically, applying 0.5 mg l - 1 BRs and 300 mg l - 1 Ethrel led to an 18.8% and 14.8% increase in momordicine content, respectively. All elicitor treatments, particularly at 0.1 mg l - 1 BRs, significantly increased leaf area, leaf length, and leaf width compared to the control group in both cropping years. Additionally, the application of all elicitors resulted in increased fruit weight, dimensions, and yield over the two consecutive years. Notably, in 2018, 600 mg l - 1 Ethrel contributed to enhanced fruit weight and yield, while in 2019, 0.5 mg l - 1 BRs exhibited the same effect. Metabolic and physiological changes in bitter squash induced by employed elicitors over two different years (2018-2019) are strongly dependent on a variety of environmental factors such as temperature and rainfall. In conclusion, using BRs as an elicitor has the potential to optimize the health benefits of bitter melon by increasing the content of two bioactive molecules, momordicine and charantin., (© 2024. The Author(s).)

Bassinosteroids (BRs) can induce plant defence responses and promote plant growth. In this work, we evaluated the effect of a natural (EP24) and a synthetic (BB16) brassinosteroid on strawberry (Fragaria vesca ) plants exposed to saline stress. Treated plants showed higher shoot dry weight and root growth compared to untreated control plants. In BR-treated plants, crown diameters increased 66% and 40%, leaf area 148% and 112%, relative water content in leaves 84% and 61%, and SPAD values 24% and 26%, in response to BB16 and EP24, respectively. A marked stomatal closure, increased leaflet lignification, and a decrease in cortex thickness, root diameter and stele radius were also observed in treated plants. Treatments also reduces stress-induced damage, as plants showed a 34% decrease in malondialdehyde content and a lower proline content compared to control plants. A 22% and 15% increase in ascorbate peroxidase and total phenolic compound activities was observed in response to BB16, and a 24% increase in total flavonoid compound in response to both BRs, under stress conditions. These results allow us to propose the use of BRs as an environmentally safe crop management strategy to overcome salinity situations that severely affect crop yield.

There has been a growing concern over soil cadmium (Cd) pollution, underscoring the importance of finding effective remediation strategies. Willow trees have emerged as promising candidates for phytoremediation of Cd-contaminated soils. Nevertheless, the specific potential of a novel willow genotype, NJU513, in remediating Cd-polluted soil remains unexplored. Hence, the primary objectives of this study were twofold: firstly, to ascertain the suitability of the willow genotype NJU513 for remediating Cd-contaminated soil; and secondly, to elevate its remediation efficciency with the application of epibrassinolide (Brs). In the pot-culture experiment without Brs, its leaf and stem Cd concentrations were 203 mg kg -1 and 65.1 mg kg -1 , with a bioaccumulation factor (BCF) of 20.8 and 6.68, respectively. In the pot-culture experiment with Brs, the corresponding Cd concentrations were 226 mg kg -1 and 59.2 mg kg -1 , with a BCF of 23.1 and 6.06, respectively. In addition, the extracted Cd contents were higher in the Brs treatments (1.11-1.37 mg plant -1 ) than in the no-Brs treatments (0.78-0.96 mg plant -1 ) because Brs increased the plant biomass and leaf BCF. The mechanism underlying the Cd accumulation of NJU513 leaves with and without Brs was revealed by a transcriptome analysis. The expression levels of genes related to metal ion binding, channel activity, and transporters in leaves were up-regulated, which contributed to the high Cd accumulation and stress tolerance. Analyses of soil metabolites and bacteria in the presence and absence of Brs spraying on willow leaves indicated that soil organic compounds with carboxyl and amino groups may induce Cd activation and passivation, respectively. This study provides valuable insights for developing woody plant varieties that can be used for remediating Cd-contaminated soil., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

The introduction of arsenic, a hazardous metalloid, into the soil system due to heavy industrialization has negatively affected agricultural productivity, resulting in limited crop yields. A recent breakthrough in stress-responsive hormones, specifically brassinosteroids, has extensively covered the role of antioxidant enzyme defense systems in heavy metal stress mitigation. Considering the antioxidant properties and metal complex formation abilities of polyphenols, our study focuses on examining their role in arsenate toxicity amelioration by 24-epibrassinolide. We demonstrate enhanced growth parameters of sodium arsenate-stressed seedlings upon application of 24-epibrassinolide, with increased root and shoot polyphenol levels analyzed by high-performance liquid chromatography. Specifically, the concentration of catechin, sinapic acid, 4-hydroxy benzoic acid, protocatechuic acid, 4-coumaric acid, and myricetin were elevated, indicating induction of phenylpropanoid signaling pathway. Further, we also report a decrease in the generation of superoxide anions and hydrogen peroxide validated the antioxidant effects of these metabolites through the nitrobluetetrazolium and diaminobenzidine staining method. In addition, evaluation of transcript level of genes encoding for specific enzymes of the phenylpropanoid pathway in shoot and root showed a significant upregulation in mRNA expression of phenylalanine ammonia-lyase-1, cinnamate-4-hydroxylase, and caffeic acid o-methyltransferase-1 upon exogenous application of 24-epibrassinolide in arsenate stressed Oryza sativa., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Grape fruit are harvested in the late summer or early fall and need to be stored at low temperatures to prevent enfeeblement and prolong their shelf-life. This study aimed to determine the effects of abscisic acid (ABA), brassinolide (BR) and ABA + BR (ABR) treatment on the berry quality of 'Shine Muscat' under low temperatures. ABA and BR maintained fruit appearance, cellular structure, weight, firmness. ABR treatments reduced the loss of fruit aroma. Furthermore, the transcriptome and metabolome analysis revealed that ABA, BR, and ABR treatments maintained the quality of fruits during the low temperatures period by influencing chlorophyll metabolism, carotenoid metabolism, flavonoid metabolism, unsaturated fatty acid, and terpene metabolism. These findings identify key genes and metabolites for ABA and BR-induced maintenance of grape fruit quality during cold storage, expanding our understanding of postharvest storage quality maintenance of grape fruit at the transcript and metabolic levels., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Background: Carotenoids have various physiological functions, such as immune regulation and cancer prevention. Germination could further improve the content of carotenoids in maize seeds. In this study, yellow maize seeds (Suyu 29) were soaked and germinated with different concentrations of 24-epibrassinolide. The changes of germination percentage, sprout length, bioactive components, antioxidant capacity and carotenoid content of the maize seeds were analyzed. Additionally, the relative expression of key genes in the carotenoid synthesis pathway was investigated., Results: The results showed that the sprout length, germination percentage, soluble protein, free amino acids, proline, endogenous abscisic acid, vitamin C, total phenolics and carotenoids displayed a significant increasing trend compared with the control group (P < 0.05). The activity of superoxide dismutase and peroxidase increased by 55.1% and 58.5% versus the control group, and the antioxidant capacity of 2,2-diphenyl-1-picrylhydrazyl, 2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) and ferric reducing antioxidant power was 19.8%, 13.4% and 44.1% higher than that of the control group (P < 0.05). Compared with the control group, the expression of genes was significantly up-regulated (P < 0.05). Under the treatment of 0.1 mg L -1 of 24-epibrassinolide, carotenoid content reached the highest value. The carotenoids showed a positive correspondence with antioxidant enzyme activity, antioxidant capacity and total phenolics content (P < 0.05)., Conclusion: This study showed that 0.1 mg L -1 of exogenous 24-epibrassinolide promoted the accumulation of carotenoids and improved the antioxidant capacity and the quality of germinated maize seeds. It could provide a method for the development of germinated maize products enriched in carotenoids. © 2024 Society of Chemical Industry., (© 2024 Society of Chemical Industry.)

Exogenous brassinolide (BR) and strigolactones (SLs) play an important role in alleviating salt stress in maize. We studied the morphological and physiological responses of the salt-sensitive genotype PH4CV and salt-tolerant genotype Zheng58 to BR (1.65 nM), SL (1 µM), and BS (1.65 nM BR + 1 µM SL) under salt stress. Phenotypic analysis showed that salt stress significantly inhibited the growth of maize seedlings and significantly increased the content of Na + in the roots. Exogenous hormones increased oxidase activity and decreased Na + content in the roots and mitigated salt stress. Transcriptome analysis showed that the interaction of BR and SL is involved in photosynthesis-antenna proteins, the TCA cycle, and plant hormone signal transduction pathways. This interaction influences the expression of chlorophyll a/b-binding protein and glucose-6-phosphate isomerase 1 chloroplastic, and aconitase genes are affected. Furthermore, the application of exogenous hormones regulates the expression of genes associated with the signaling pathways of cytokinin (CK), gibberellins (GA), auxin (IAA), brassinosteroid (BR), abscisic acid (ABA), and jasmonic acid (JA). Additionally, exogenous hormones inhibit the expression of the AKT2/3 genes, which are responsible for regulating ion transduction and potassium ion influx. Four candidate genes that may regulate the seedling length of maize were screened out through WGCNA. Respective KOG notes concerned inorganic ion transport and metabolism, signal transduction mechanisms, energy production and conversion, and amino acid transport and metabolism. The findings of this study provide a foundation for the proposition that BR and SL can be employed to regulate salt stress alleviation in maize.

Plants have complex detoxification and metabolic systems that enable them to deal with environmental pollutants. We report accumulation of the pesticide isoproturon (IPU) in a BR signaling pathway for mutant bzr4- 3/5 rice to be significantly higher than in wild-type (WT) rice controls and for exogenous 24-epibrassinolide to reverse toxic symptoms in WT rice but not in mutants. A genome-wide RNA sequencing study of WT/ bzr4 rice is performed to identify transcriptomic changes and metabolic mechanisms under IPU exposure. Three differentially expressed genes in yeast cells increase the degradation rate of IPU in a growth medium by factors of 1.61, 1.51, and 1.29 after 72 h. Using UPLC/Q-TOF-MS/MS, five phase I metabolites and five phase II conjugates are characterized in rice grains, with concentrations generally decreasing in bzr4 rice grains. OsBZR4, a regulator of IPU degradation in rice, may eliminate IPU from edible parts of food crops by regulating downstream metabolic genes.

Increased anthropogenic activities over the last decades have led to a gradual increase in cadmium content in the soil, which, due to its high mobility in soil, makes Cd accumulation in plants a serious threat to the health of animals and humans. Plant hormones including melatonin (Mel) and brassinosteroids (BR) are known to provide tolerance against various abiotic stresses. In this work, the role of combined and separate exogenous application of Mel and BR on Cd stress in cherry tomato plants was examined. Cd stress significantly reduced tomato growth by inducing oxidative stress and reduced K + uptake in roots and shoots. Combined application of Mel and BR reduced detrimental effects of Cd in tomato by (i) reducing Cd accumulation in the shoot; (ii) increasing the activities of different antioxidants (SOD, CAT, APX, GR); (iii) triggering higher expression of genes relating to Cd vacuolar sequestration (Na + /H + EXCHANGER, SlNHX1; NATURAL RESISTANCE-ASSOCIATED MACROPHAGE PROTEIN 6, SlNRAMP6), and Cd transport and detoxification (HEAVY-METAL-ASSOCIATED 3, SlHMA3; PLANT CADMIUM RESISTANT 2, SlPCR2); and (iv) improving plant K + homeostasis and contents in root and shoot. The latter trait was associated with the reduced gene expression of K + -permeable outward rectifying channel (SlGORK3), and transcriptional upregulation of high affinity potassium transporter 5 (SIHAK5) under Cd stress. A separate application of Mel and BR showed tissue-specific regulation of tomato growth and Cd tolerance by regulating antioxidant activities, K + uptake, Cd uptake, and translocation from root to shoot and their endogenous contents. Melatonin per se was more effective in improving Cd tolerance in shoot while beneficial BR effects were more pronounced in roots, and their combined application was effective in both tissues. Taken together, reported results show tissue-specific regulation of Cd tolerance by Mel and BR in cherry tomato plants and demonstrate the efficiency of combined Mel + BR treatment as a practical tool to reduce Cd accumulation and mitigate its negative effects on plant growth., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Cadmium (Cd) pollution is a serious threat to food safety and human health. Minimization of Cd uptake and enhancing Cd tolerance in plants are vital to improve crop yield and reduce hazardous effects to humans. In this study, we investigate the effect of a synergistic system with phytohormone (24-Epibrassinolide, EBL) and silicon (Si) on Cd toxicity and accumulation of rice plants. The results revealed that Si, EBL and their combination rescued Cd-induced growth inhibition, as evidenced by the increased dry weight of root and shoot. The chlorophyll content and photosynthetic performance were improved. The activity of antioxidant enzymes (SOD, POD and CAT) was increased and oxidative stress was alleviated. More importantly, Cd content in root was decreased by 20.25%, 17.72% and 27.84%, while Cd content in shoot decreased by 21.17%, 16.47% and 25.88%, respectively. Moreover, Si, EBL and Si + EBL treatment enriched cell wall-bound Cd and reduced Cd toxicity to functional organelles. Meanwhile, the residual form of Cd was enriched and the highly toxic forms of Cd (inorganic and water-soluble Cd) were decreased. The joint application showed better effects than applying Si and EBL alone. Collectively, this study provides an effective way for Cd toxicity mitigation in rice plants.

Low-temperature (LT) is one of the major abiotic stresses that restrict the growth and development of maize seedlings. Brassinolides (BRs) have been shown to enhance LT tolerance in several plant species; the physiological and molecular mechanisms by which BRs enhance maize tolerance are still unclear. Here, we characterized changes in the physiology and transcriptome of N192 and Ji853 seedlings at the three-leaf stage with or without 2 μM 2,4-epibrassinolide (EBR) application at 25 and 15 °C environments via high-performance liquid chromatography and RNA-Sequencing. Physiological analyses revealed that EBR increased the antioxidant enzyme activities, enhanced the cell membrane stability, decreased the malondialdehyde formation, and inhibited the reactive oxygen species (ROS) accumulation in maize seedlings under 15 °C stress; meanwhile, EBR also maintained hormone balance by increasing indole-3-acetic acid and gibberellin 3 contents and decreasing the abscisic acid level under stress. Transcriptome analysis revealed 332 differentially expressed genes (DEGs) enriched in ROS homeostasis, plant hormone signal transduction, and the mitogen-activated protein kinase (MAPK) cascade. These DEGs exhibited synergistic and antagonistic interactions, forming a complex LT tolerance network in maize. Additionally, weighted gene co-expression network analysis (WGCNA) revealed that 109 hub genes involved in LT stress regulation pathways were discovered from the four modules with the highest correlation with target traits. In conclusion, our findings provide new insights into the molecular mechanisms of exogenous BRs in enhancing LT tolerance of maize at the seedling stage, thus opening up possibilities for a breeding program of maize tolerance to LT stress.

Key Message: Exogenous application of 24-epibrassinolide can alleviate oxidative damage, improve photosynthetic capacity, and regulate carbon and nitrogen assimilation, thus improving the tolerance of grapevine (Vitis vinifera L.) to drought stress. Brassinosteroids (BRs) are a group of plant steroid hormones in plants and are involved in regulating plant tolerance to drought stress. This study aimed to investigate the regulation effects of BRs on the carbon and nitrogen metabolism in grapevine under drought stress. The results indicated that drought stress led to the accumulation of superoxide radicals and hydrogen peroxide and an increase in lipid peroxidation. A reduction in oxidative damage was observed in EBR-pretreated plants, which was probably due to the improved antioxidant concentration. Moreover, exogenous EBR improved the photosynthetic capacity and sucrose phosphate synthase activity, and decreased the sucrose synthase, acid invertase, and neutral invertase, resulting in improved sucrose (190%) and starch (17%) concentrations. Furthermore, EBR pretreatment strengthened nitrate reduction and ammonium assimilation. A 57% increase in nitrate reductase activity and a 13% increase in glutamine synthetase activity were observed in EBR pretreated grapevines. Meanwhile, EBR pretreated plants accumulated a greater amount of proline, which contributed to osmotic adjustment and ROS scavenging. In summary, exogenous EBR enhanced drought tolerance in grapevines by alleviating oxidative damage and regulating carbon and nitrogen metabolism., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Water deficiency stress reduces yield in grain legumes, primarily due to a decrease in the pods number. Melatonin (ML) and 24-epibrassinolide (EBL) are recognized for their hormone-like properties that improve plant tolerance to abiotic stresses. This study aimed to assess the impact of different concentrations of ML (0, 100, and 200 µM) and EBL (0, 3, and 6 µM) on the growth, biochemical, and physiological characteristics of chickpea plants under water-stressed conditions., Results: The study's findings indicated that under water-stressed conditions, a decrease in seed (30%) and pod numbers (31%), 100-seed weight (17%), total chlorophyll content (46%), stomatal conductance (33%), as well as an increase in H 2 O 2 (62%), malondialdehyde content (40%), and electrolyte leakage index (40%), resulted in a 40% reduction in chickpea plants grain yield. Our findings confirmed that under water-stressed conditions, seed oil, seed oil yield, and seed protein yield dropped by 20%, 55%, and 36%, respectively. The concurrent exogenous application of ML and EBL significantly reduces oxidative stress, plasma membrane damage, and reactive oxygen species (ROS) content. This treatment also leads to increased yield and its components, higher pigment content, enhanced oil and protein yield, and improved enzymatic and non-enzymatic antioxidant activities such as catalase, superoxide dismutase, polyphenol oxidase, ascorbate peroxidase, guaiacol peroxidase, flavonoid, and carotenoid. Furthermore, it promotes the accumulation of osmoprotectants such as proline, total soluble protein, and sugars., Conclusions: Our study found that ML and EBL act synergistically to regulate plant growth, photosynthesis, osmoprotectants accumulation, antioxidant defense systems, and maintain ROS homeostasis, thereby mitigating the adverse effects of water deficit conditions. ML and EBL are key regulatory network components in stressful conditions, with significant potential for future research and practical applications. The regulation metabolic pathways of ML and EBL in water-stressed remains unknown. As a result, future research should aim to elucidate the molecular mechanisms by employing genome editing, RNA sequencing, microarray, transcriptomic, proteomic, and metabolomic analyses to identify the mechanisms involved in plant responses to exogenous ML and EBL under water deficit conditions. Furthermore, the economical applications of synthetic ML and EBL could be an interesting strategy for improving plant tolerance., (© 2024. The Author(s).)

Nickel (Ni) and copper (Cu) contamination have become major threats to plant survival worldwide. 24-epibrassinolide (24-EBR) and melatonin (MT) have emerged as valuable treatments to alleviate heavy metal-induced phytotoxicity. However, plants have not fully demonstrated the potential mechanisms by which these two hormones act under Ni and Cu stress. Herein, this study investigated the impact of individual and combined application of 24-EBR and MT on the growth and physiological traits of Primula forbesii Franch. subjected to stress (200 μmol L -1 Ni and Cu). The experiments compared the effects of different mitigation treatments on heavy metal (HM) stress and the scientific basis and practical reference for using these exogenous substances to improve HM resistance of P. forbesii in polluted environments. Nickel and Cu stress significantly hindered leaf photosynthesis and nutrient uptake, reducing plant growth and gas exchange. However, 24-EBR, MT, and 24-EBR + MT treatments alleviated the growth inhibition caused by Ni and Cu stress, improved the growth indexes of P. forbesii, and increased the gas exchange parameters. Exogenous MT effectively alleviated Ni stress, and 24-EBR + MT significantly alleviated the toxic effects of Cu stress. Unlike HM stress, MT and 24-EBR + MT activated the antioxidant enzyme activity (by increasing superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT)), significantly reduced reactive oxygen species (ROS) accumulation, and regulated ascorbate and glutathione cycle (AsA-GSH) efficiency. Besides, the treatments enhanced the ability of P. forbesii to accumulate HMs, shielding plants from harm. These findings conclusively illustrate the capability of 24-EBR and MT to significantly bolster the tolerance of P. forbesii to Ni and Cu stress., (© 2024. The Author(s).)

Cold injury is a major environmental stress affecting the growth and yield of crops. Brassinosteroids (BRs) and salicylic acid (SA) play important roles in plant cold tolerance. However, whether or how BR signaling interacts with the SA signaling pathway in response to cold stress is still unknown. Here, we identified an SA methyltransferase, TaSAMT1 that converts SA to methyl SA (MeSA) and confers freezing tolerance in wheat (Triticum aestivum). TaSAMT1 overexpression greatly enhanced wheat freezing tolerance, with plants accumulating more MeSA and less SA, whereas Tasamt1 knockout lines were sensitive to freezing stress and accumulated less MeSA and more SA. Spraying plants with MeSA conferred freezing tolerance to Tasamt1 mutants, but SA did not. We revealed that BRASSINAZOLE-RESISTANT 1 (TaBZR1) directly binds to the TaSAMT1 promoter and induces its transcription. Moreover, TaBZR1 interacts with the histone acetyltransferase TaHAG1, which potentiates TaSAMT1 expression via increased histone acetylation and modulates the SA pathway during freezing stress. Additionally, overexpression of TaBZR1 or TaHAG1 altered TaSAMT1 expression and improved freezing tolerance. Our results demonstrate a key regulatory node that connects the BR and SA pathways in the plant cold stress response. The regulatory factors or genes identified could be effective targets for the genetic improvement of freezing tolerance in crops., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Main Conclusion: In this study, six ZaBZRs were identified in Zanthoxylum armatum DC, and all the ZaBZRs were upregulated by abscisic acid (ABA) and drought. Overexpression of ZaBZR1 enhanced the drought tolerance of transgenic Nicotiana benthamian. Brassinosteroids (BRs) are a pivotal class of sterol hormones in plants that play a crucial role in plant growth and development. BZR (brassinazole resistant) is a crucial transcription factor in the signal transduction pathway of BRs. However, the BZR gene family members have not yet been identified in Zanthoxylum armatum DC. In this study, six members of the ZaBZR family were identified by bioinformatic methods. All six ZaBZRs exhibited multiple phosphorylation sites. Phylogenetic and collinearity analyses revealed a closest relationship between ZaBZRs and ZbBZRs located on the B subgenomes. Expression analysis revealed tissue-specific expression patterns of ZaBZRs in Z. armatum, and their promoter regions contained cis-acting elements associated with hormone response and stress induction. Additionally, all six ZaBZRs showed upregulation upon treatment after abscisic acid (ABA) and polyethylene glycol (PEG), indicating their participation in drought response. Subsequently, we conducted an extensive investigation of ZaBZR1. ZaBZR1 showed the highest expression in the root, followed by the stem and terminal bud. Subcellular localization analysis revealed that ZaBZR1 is present in the cytoplasm and nucleus. Overexpression of ZaBZR1 in transgenic Nicotiana benthamiana improved seed germination rate and root growth under drought conditions, reducing water loss rates compared to wild-type plants. Furthermore, ZaBZR1 increased proline content (PRO) and decreased malondialdehyde content (MDA), indicating improved tolerance to drought-induced oxidative stress. The transgenic plants also showed a reduced accumulation of reactive oxygen species. Importantly, ZaBZR1 up-regulated the expression of drought-related genes such as NbP5CS1, NbDREB2A, and NbWRKY44. These findings highlight the potential of ZaBZR1 as a candidate gene for enhancing drought resistance in transgenic N. benthamiana and provide insight into the function of ZaBZRs in Z. armatum., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Salt stress is a critical limiting factor for fruit yield and quality of apples. Brassinosteroids (BRs) play an important role in response to abiotic stresses. In the present study, application of 2,4- Epicastasterone on seedlings of Malus 'M9T337' and Malus domestica 'Gala3' alleviated the physiological effects, such as growth inhibition and leaf yellowing, induced by salt stress. Further analysis revealed that treatment with NaCl induced expression of genes involved in BR biosynthesis in 'M9T337' and 'Gala3'. Among which, the expression of BR biosynthetic gene MdBR6OX2 showed a three-fold upregulation upon salt treatment, suggesting its potential role in response to salt stress in apple. MdBR6OX2, belonging to the CYP450 family, contains a signal peptide region and a P450 domain. Expression patterns analysis showed that the expression of MdBR6OX2 can be significantly induced by different abiotic stresses. Overexpressing MdBR6OX2 enhanced the tolerance of apple callis to salt stress, and the contents of endogenous BR-related compounds, such as Typhastero (TY), Castasterone (CS) and Brassinolide (BL) were significantly increased in transgenic calli compared with that of wild-type. Extopic expression of MdBR6OX2 enhanced tolerance to salt stress in Arabidopsis. Genes associated with salt stress were significantly up-regulated, and the contents of BR-related compounds were significantly elevated under salt stress. Our data revealed that BR-biosynthetic gene MdBR6OX2 positively regulates salt stress tolerance in both apple calli and Arabidopsis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

Heavy metals (HMs) at a concentration above the threshold level act as environmental pollutants and very often threaten the agricultural productivity globally. Finding affordable and environmentally sustainable deliverables to address this issue is therefore a top focus. Phytohormones alleviate the HMs-induced toxicity and positively influence the plant growth. Considering the importance of phytohormones, the present study aimed to assess the effect of 24-epibrassinolide (24-EBL; 10 µM) as seed soaking treatment on growth performance of Zea mays (L.) contaminated separately with increasing concentrations (50-400 mg.kg -1 ) of lead (Pb) and cadmium (Cd). With increasing metal concentrations, growth and plant biometric criteria were reduced. For instance, Cd at 400 mg.kg -1 soil reduced the germination efficiency (56%), root (77%) and shoot (69%) dry weight, total chlorophyll (64%), and carotenoid content (45%). Contrarily, both HMs caused increase in stress biomarkers and antioxidant enzymes in seedling. However, exogenous administration of 24-EBL significantly enhanced the growth attributes, photosynthetic pigments, proline, MDA, and antioxidant enzyme activity while reducing the harmful effects of HMs stress on Z. mays. For instance, 24-EBL (10 µM) improved the germination percentage, root biomass, chl a, chl b, total chlorophyll, and carotenoid content by 16, 21, 17, 34, 18, and 15%, respectively, in 50 mg.Pb.kg -1 soil-treated Z. mays plants. Furthermore, the amounts of proline, MDA, and antioxidant enzymes in foliage of Z. mays were interestingly and dramatically lowered by 24-EBL application. Uptake of metals in plant organs was significantly reduced when 24-EBL was applied to Pb- and Cd-treated Z. mays. The recent findings help us better understand how 24-EBL regulates growth and development of Z. mays as well as how it boosts HMs' resilience, which could increase the possibility of employing 24-EBL to increase Z. mays productivity. Thus, the present findings confirmed the potentiality of pre-soaking the seed in 24-EBL solution that neutralizes the toxic effects of heavy metals in Z. mays plants. Therefore, it is suggested that applying phytohormones including 24-EBL in removal of heavy metal stress in plants is the best possible solution in sustainable agriculture., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Broomrape (Orobanche cumana) negatively affects sunflower, causing severe yield losses, and thus, there is a need to control O. cumana infestation. Brassinosteroids (BRs) play key roles in plant growth and provide resilience to weed infection. This study aims to evaluate the mechanisms by which BRs ameliorate O. cumana infection in sunflower (Helianthus annuus). Seeds were pretreated with BRs (1, 10, and 100 nM) and O. cumana inoculation for 4 weeks under soil conditions. O. cumana infection significantly reduced plant growth traits, photosynthesis, endogenous BRs and regulated the plant defence (POX, GST), BRs signalling (BAK1, BSK1 to BSK4) and synthesis (BRI1, BR6OX2) genes. O. cumana also elevated the levels of malondialdehyde (MDA), hydroxyl radical (OH - ), hydrogen peroxide (H 2 O 2 ) and superoxide (O 2 •- ) in leaves/roots by 77/112, 63/103, 56/97 and 54/89%, as well as caused ultrastructural cellular damages in both leaves and roots. In response, plants activated a few enzymes, superoxide dismutase (SOD), peroxidase (POD) and reduced glutathione but were unable to stimulate the activity of ascorbate peroxidase (APX) and catalase (CAT) enzymes. The addition of BRs (especially at 10 nM) notably recovered the ultrastructural cellular damages, lowered the production of oxidative stress, activated the key enzymatic antioxidants and induced the phenolic and lignin contents. The downregulation in the particular genes by BRs is attributed to the increased resilience of sunflower via a susceptible reaction. In a nutshell, BRs notably enhanced the sunflower resistance to O. cumana infection by escalating the plant immunity responses, inducing systemic acquired resistance, reducing oxidative or cellular damages, and modulating the expression of BR synthesis or signalling genes., (© 2024 Scandinavian Plant Physiology Society.)

As the core of Brassinosteroids (BR) signaling pathway, BR-resistant (BZR) transcription factor regulates thousands of targeted genes mediating photomophogenesis, pollen sterility, cell expansion and stress response. Pecan (Carya illinoinensis) is a famous trees species of Carya, and its nut has high nutritional and economic values. However, there has no report on BZR genes family in pecan yet. Herein, totals of seven CiBZR members were identified in pecan genome, which were predicted to be hydrophilic unstable proteins and located in the nucleus. CiBZR genes had close evolutionary relationships with CcBZRs and JrBZRs in both Carya cathayensis and Juglans regia. These seven CiBZR genes were located independently on 7 chromosomes without doubling or tandem duplication. Based on the analysis of conserved motifs and gene structures, CiBZR genes were divided into three categories. More than 40 cis-acting elements were found in the 2 kb promoter regions of CiBZRs, which were mainly involved in hormone, light, and stress response, and plant growth and development. Notably, some of these CiBZR proteins were mainly located in the nucleus, had the self-activation ability and interaction relationship with BIN2 kinase, and negatively regulated the expression of CiCPD and CiDWF4. Gene expressions analysis further showed that CiBZR genes could express in many tissues and shared similar expression trends during embryo development. Moreover, most CiBZR genes responded to BR, Gibberellin (GA), Strigolactone (SL), salt, acid and osmotic stress. This study provides theoretical basis for the subsequent study on the role of CiBZR family genes in plant growth, development and stress responses., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Brassinosteroids (BR) play crucial roles in plant development and abiotic stresses in plants. Exogenous application of BR can significantly enhance cold tolerance in rice. However, the regulatory relationship between cold tolerance and the BR signaling pathway in rice remains largely unknown. Here, we characterized functions of the BR receptor OsBRI1 in response to cold tolerance in rice using its loss-of-function mutant (d61-1). Our results showed that mutant d61-1 was less tolerant to cold stress than wild-type (WT). Besides, d61-1 had lower levels than WT for some physiological parameters, including catalase activity (CAT), superoxide dismutase activity (SOD), peroxidase activity (POD), peroxidase activity (PRO), soluble protein, and soluble sugar content, while malondialdehyde content (MDA) and relative electrical conductivity (REC) levels in d61-1 were higher than those in WT plants. These results indicated that the loss of OsBRI1 function resulted in decreased cold tolerance in rice. In addition, we performed RNA sequencing (RNA-seq) of WT and d61-1 mutant under cold stress. Numerous common and unique differentially expressed genes (DEGs) with up- and down-regulation were observed in WT and d61-1 mutant. Some DEGs were expressed to various degrees, even opposite, between CK1 vs. T1 (WT) and CK2 vs. T2 (d61-1). Among these specific DEGs, some typical genes are involved in plant tolerance to cold stress. Through weighted correlation network analysis (WGCNA), 50 hub genes were screened in the turquoise and blue module. Many genes were involved in cold stress and plant hormone, such as Os01g0279800 (BRI1-associated receptor kinase 1 precursor), Os10g0513200 (Dwarf and tiller-enhancing 1, DTE1), Os02g0706400 (MYB-related transcription factor, OsRL3), etc. Differential expression levels of some genes were verified in WT and d61-1 under cold stress using qRT-PCR. These valuable findings and gene resources will be critical for understanding the regulatory relationships between cold stress tolerance and the BR signaling pathways in rice., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Bud dormancy is an important physiological process during winter. Its release requires a certain period of chilling. In pear (Pyrus pyrifolia), the abscisic acid (ABA)-induced expression of DORMANCY-ASSOCIATED MADS-box (DAM) genes represses bud break, whereas exogenous gibberellin (GA) promotes dormancy release. However, with the exception of ABA and GA, the regulatory effects of phytohormones on dormancy remain largely uncharacterized. In this study, we confirmed brassinosteroids (BRs) and jasmonic acid (JA) contribute to pear bud dormancy release. If chilling accumulation is insufficient, both 24-epibrassinolide (EBR) and methyl jasmonic acid (MeJA) can promote pear bud break, implying that they positively regulate dormancy release. BRASSINAZOLE RESISTANT 2 (BZR2), which is a BR-responsive transcription factor, inhibited PpyDAM3 expression and accelerated pear bud break. The transient overexpression of PpyBZR2 increased endogenous GA, JA, and JA-Ile levels. In addition, the direct interaction between PpyBZR2 and MYELOCYTOMATOSIS 2 (PpyMYC2) enhanced the PpyMYC2-mediated activation of Gibberellin 20-oxidase genes PpyGA20OX1L1 and PpyGA20OX2L2 transcription, thereby increasing GA3 contents and accelerating pear bud dormancy release. Interestingly, treatment with 5 μm MeJA increased the bud break rate, while also enhancing PpyMYC2-activated PpyGA20OX expression and increasing GA3,4 contents. The 100 μm MeJA treatment decreased the PpyMYC2-mediated activation of the PpyGA20OX1L1 and PpyGA20OX2L2 promoters and suppressed the inhibitory effect of PpyBZR2 on PpyDAM3 transcription, ultimately inhibiting pear bud break. In summary, our data provide insights into the crosstalk between the BR and JA signaling pathways that regulate the BZR2/MYC2-mediated pathway in the pear dormancy release process., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2023. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The bulbil expansion of P. ternata is a key period for its yield formation, and the process of bulbil expansion is often subjected to short-term heavy precipitation. It is not clear whether the short-term waterlogging can affect bulbil expansion. Brassinolide (BR) is widely believed to enhance plant tolerance to abiotic stress. The study investigated the effects of normal water (C), waterlogging (W), waterlogging + BR (W + B), waterlogging + propiconazole (W + P) on P. ternata at the bulbil expansion period in order to assess P. ternata's ability to cope with waterlogging during the bulbil expansion stage and the regulation effects of BR on the process. The biomass of P. ternata was significantly increased after waterlogging. W treatment significantly reduced the H 2 O 2 and MDA contents, the rate of O 2 ⋅ - production and the activities of antioxidant enzymes compared with the C group. AsA and GSH contents were significantly reduced by W treatment. However, the ratios of AsA/DHA and GSH/GSSG were slightly affected by W treatment. The rate of O 2 ∙ - production and H 2 O 2 content in W + B group were significantly lower than those in W group. The POD, APX, and GR activities, and GSH content in W + B group were evidently increased compared with the W group. Soluble sugar and active ingredients contents were significantly increased after waterlogging, and the enhancement was reinforced by BR. In conclusion, waterlogging reduced oxidative stress in P. ternata under the experimental conditions. BR treatment under waterlogging had a positive effect on P. ternata by enhancing antioxidant capacity and promoting the accumulation of soluble sugars and active ingredients., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

Brassinosteroid (BR) has been shown to modulate plant tolerance to various stresses. S-nitrosoglutathione reductase (GSNOR) is involved in the plant response to environment stress by fine-turning the level of nitric oxide (NO). However, whether GSNOR is involved in BR-regulated Na + /K + homeostasis to improve the salt tolerance in halophyte is unknown. Here, we firstly reported that high salinity increases the expression of BR-biosynthesis genes and the endogenous levels of BR in mangrove Kandelia obovata. Then, salt-induced BR triggers the activities and gene expressions of GSNOR and antioxidant enzymes, thereafter decrease the levels of malondialdehyde, hydrogen peroxide. Subsequently, BR-mediated GSNOR negatively regulates NO contributions to the reduction of reactive oxygen species generation and induction of the gene expression related to Na + and K + transport, leading to the decrease of Na + /K + ratio in the roots of K. obovata. Finally, the applications of exogenous BR, NO scavenger, BR biosynthetic inhibitor and GSNOR inhibitor further confirm the function of BR. Taken together, our result provides insight into the mechanism of BR in the response of mangrove K. obovata to high salinity via GSNOR and NO signaling pathway by reducing oxidative damage and modulating Na + /K + homeostasis., (© 2023 John Wiley & Sons Ltd.)

Brassinosteroids (BRs) are a group of polyhydroxylated steroids for plant growth and development, regulating numerous physiological and biochemical processes and participating in multi-pathway signaling in plants. 24-Epibrassinolide (EBR) is the most commonly used BR for the investigation of the effects of exogenous steroidal phytohormones on plant physiology. Although SlSERK3B is considered a gene involved in the brassinosteroid (BR) signaling pathway, its specific role in plant growth and development has not been reported in detail. In this study, tomato ( Solanum lycopersicum L.) seedlings treated with 0.05 μmol L -1 EBR showed a significant increase in plant height, stem diameter, and fresh weight, demonstrating that BR promotes the growth of tomato seedlings. EBR treatment increased the expression of the BR receptor gene SlBRI1 , the co-receptor gene SlSERK3A and its homologs SlSERK3B , and SlBZR1 . The SlSERK3B gene was silenced by TRV-mediated virus-induced gene silencing (VIGS) technology. The results showed that both brassinolide (BL) content and BR synthesis genes were significantly up-regulated in TRV-SlSERK3B-infected seedlings compared to the control seedlings. In contrast, plant height, stem diameter, fresh weight, leaf area and total root length were significantly reduced in silenced plants. These results suggest that silencing SlSERK3B may affect BR synthesis and signaling, thereby affecting the growth of tomato seedlings. Furthermore, the photosynthetic capacity of TRV- SlSERK3B -infected tomato seedlings was reduced, accompanied by decreased photosynthetic pigment content chlorophyll fluorescence, and photosynthesis parameters. The expression levels of chlorophyll-degrading genes were significantly up-regulated, and carotenoid-synthesising genes were significantly down-regulated in TRV- SlSERK3B -infected seedlings. In conclusion, silencing of SlSERK3B inhibited BR signaling and reduced photosynthesis in tomato seedlings, and this correlation suggests that SlSERK3B may be related to BR signaling and photosynthesis enhancement.

Brassinosteroids (BRs) are a class of plant steroid hormones that are essential for plant growth and development. BRs control important agronomic traits and responses to abiotic stresses. Through the signaling pathway, BRs control the expression of thousands of genes, resulting in a variety of biological responses. The key effectors of the BR pathway are two transcription factors (TFs): BRASSINAZOLE RESISTANT 1 (BZR1) and BRI1-EMSSUPPRESSOR 1 (BES1). Both TFs are phosphorylated and inactivated by the Glycogen synthase kinase 3 BRASSINOSTEROID INSENSITIVE2 (BIN2), which acts as a negative regulator of the BR pathway. In our study, we describe the functional characteristics of HvGSK1.1 , which is one of the GSK3/SHAGGY-like orthologs in barley. We generated mutant lines of HvGSK1.1 using CRISPR/Cas9 genome editing technology. Next Generation Sequencing (NGS) of the edited region of the HvGSK1.1 showed a wide variety of mutations. Most of the changes (frameshift, premature stop codon, and translation termination) resulted in the knock-out of the target gene. The molecular and phenotypic characteristics of the mutant lines showed that the knock-out mutation of HvGSK1.1 improved plant growth performance under salt stress conditions and increased the thousand kernel weight of the plants grown under normal conditions. The inactivation of HvGSK1.1 enhanced BR-dependent signaling, as indicated by the results of the leaf inclination assay in the edited lines. The plant traits under investigation are consistent with those known to be regulated by BRs. These results, together with studies of other GSK3 gene members in other plant species, suggest that targeted editing of these genes may be useful in creating plants with improved agricultural traits.

Applying brassinolide (BL, a phytohormone) in combination with pyraclostrobin (Pyr, a fungicide) has shown effective disease control in field trials. However, the mechanism by which BL + Pyr control disease remains uncertain. This work compared the disease control and defense responses of three pretreatments (BL, Pyr, and BL + Pyr) in Arabidopsis thaliana . We found that BL + Pyr improved control against Pyr-sensitive Hyaloperonospora arabidopsidis and Botrytis cinerea by 19 and 17% over Pyr, respectively, and achieved 29% control against Pyr-resistant B. cinerea . Furthermore, BL + Pyr outperformed BL or Pyr in boosting transient H 2 O 2 accumulation, and the activities of POD, APX, GST, and GPX. RNA-seq analysis revealed a more potent activation of defense genes elicited by BL + Pyr than by BL or Pyr. Overall, BL + Pyr controlled disease by integrating the elicitation of plant innate disease resistance with the fungicidal activity of Pyr.

Brassinosteroids (BRs) play many pivotal roles in plant growth and development, especially in cell elongation and vascular development. Although its biosynthetic and signal transduction pathway have been well characterized in model plants, their biological roles in Eucalyptus grandis , a major hardwood tree providing fiber and energy worldwide, remain unclear. Here, we treated E. grandis plantlets with 24-epibrassinolide (EBL), the most active BR and/or BR biosynthesis inhibitor brassinazole. We recorded the plant growth and analyzed the cell structure of the root and stem with histochemical methods; then, we performed a secondary growth, BR synthesis, and signaling-related gene expression analysis. The results showed that the BRs dramatically increased the shoot length and diameter, and the exogenous BR increased the xylem area of the stem and root. In this process, EgrBRI1, EgrBZR1, and EgrBZR2 expression were induced by the BR treatment, and the expressions of HD-ZIPIII and cellulose synthase genes were also altered. To further verify the effect of BRs in secondary xylem development in Eucalyptus, we used six-month-old plants as the material and directly applied EBL to the xylem and cambium of the vertical stems. The xylem area, fiber cell length, and cell numbers showed considerable increases. Several key BR-signaling genes, secondary xylem development-related transcription factor genes, and cellulose and lignin biosynthetic genes were also considerably altered. Thus, BR had regulatory roles in secondary xylem development and differentiation via the BR-signaling pathway in this woody plant., Competing Interests: The authors declare there are no competing interests., (©2024 Zhou et al.)

Plant growth regulators are routinely added to in vitro culture media to foster the growth and differentiation of the cells, tissues, and organs. However, while the literature on usage of the more common auxins, cytokinins, gibberellins, abscisic acid, and ethylene is vast, other compounds that also have shown a growth-regulating activity have not been studied as frequently. Such substances are also capable of modulating the responses of plant cells and tissues in vitro by regulating their growth, differentiation, and regeneration competence, but also by enhancing their responses toward biotic and abiotic stress agents and improving the production of secondary metabolites of interest. This chapter will discuss the in vitro effects of several of such less frequently added plant growth regulators, including brassinosteroids (BRS), strigolactones (SLs), phytosulfokines (PSKs), methyl jasmonate, salicylic acid (SA), sodium nitroprusside (SNP), hydrogen sulfite, various plant growth retardants and inhibitors (e.g., ancymidol, uniconazole, flurprimidol, paclobutrazol), and polyamines., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Gibberellins (GAs) play a crucial role in regulating secondary growth in angiosperms, but their effects on the secondary growth of gymnosperms are rarely reported. In this study, we administered exogenous GA 3 to two-year-old P. massoniana seedlings, and examined its effects on anatomical structure, physiological and biochemical changes, and gene expression in stems. The results showed that exogenous GA 3 could enhance xylem development in P. massoniana by promoting cell division. The content of endogenous hormone (including auxins, brassinosteroids, and gibberellins) were changed and the genes related to phytohormone biosynthesis and signaling pathway, such as GID1, DELLA, TIR1, ARF, SAUR, CPD, BR6ox1, and CYCD3, were differentially expressed under GA 3 treatment. Furthermore, GA 3 and BR (brassinosteroid) might act synergistically in promoting secondary growth in P. massoniana. Additionally, lignin content was significantly increased after GA 3 treatment accompanied by the express of lignin biosynthesis related genes. PmCAD (TRINITY&#95;DN142116&#95;c0&#95;g1), a crucial gene involved in the lignin biosynthesis, was cloned and overexpressed in Nicotiana benthamiana, significantly promoting the xylem development and enhancing stem lignification. It was regarded as a key candidate gene for improving stem growth of P. massoniana. The findings of this study have demonstrated the impact of GA 3 treatment on secondary growth of stems in P. massoniana, providing a foundation for understanding the molecular regulatory mechanism of stem secondary growth in Pinaceae seedlings and offering theoretical guidance for cultivating new germplasm with enhanced growth and yield., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Exogenous application of phytohormones is getting promising results in alleviating abiotic stresses, particularly heavy metal (HMs). Jasmonate (JA) and brassinosteroid (BR) have crosstalk in bamboo plants, reflecting a burgeoning area of investigation. Lead (Pb) is the most common pollutant in the environment, adversely affecting plants and human health. The current study focused on the foliar application of 10 µM JA and 10 µM BR in both single and combination forms on bamboo plants grown under Pb stress (0, 50, 100, 150 µM) with a completely randomized design by four replications. The study found that applying 10 µM JA and 10 µM BR significantly improves growth and tolerance by reducing oxidative stress, reactive oxygen species including hydrogen peroxide (H 2 O 2 , 32.91%), superoxide radicals (O 2 -• , 33.9%), methylglyoxal (MG, 19%), membrane lipoperoxidation (25.66%), and electrolyte leakage (41.5%) while increasing antioxidant (SOD (18%), POD (13%), CAT (20%), APX (12%), and GR (19%)), non-antioxidant (total phenolics (7%), flavonols (12.3%), and tocopherols (13.8%)), and glyoxylate activity (GLy I (13%), GLy II (19%)), proline content (19%), plant metal chelating capacity (17.3%), photosynthetic pigments (16%), plant growth (10%), and biomass (12%). We found that JA and BR, in concert, boost bamboo species' Pb tolerance by enhancing antioxidant and glyoxalase cycles, ion chelation, and reducing metal translocation and accumulation. This conclusively demonstrates that utilizing a BR-JA combination form at 10 µM dose may have the potential to yield optimal efficiency in mitigating oxidative stress in bamboo plants., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cold stress (CS) significantly limits cucumber yield. However, it remains unclear whether and how sodium nitrophenolate (CSN) regulates plant responses to cold stress. Here, H 2 O, CSN, 24-epibrassinolide (EBR), and CSN + EBR were sprayed on cucumber seedlings before or after CS, and on control plants. We found that CSN, EBR, or EBR + CSN pre-treatment improved seedling growth under normal conditions (control condition) and cold tolerance under CS conditions. EBR pre-treatment promoted the expression of approximately half of the genes involved in BR synthesis and signaling and CsICE-CsCBF-CsCOR under CS. However, CSN pre-treatment promoted almost all the expression of BR synthesis and signaling genes, and CsICE-CsCBF-CsCOR genes, which showed the highest expression in early CS, remarkably improving the cold tolerance of cucumber. Interestingly, EBR and CSN had a superimposed effect on the expression of BR synthesis and signaling and CsICE-CsCBF-CsCOR genes, which rapidly increased their expression under normal temperature. Spraying EBR after CS accelerated seedling recovery, whereas CSN had the opposite effect. However, spraying CSN combined with EBR accelerated the recovery of CS-injured seedlings and was better than spraying EBR alone. Although CS-injured seedlings were negatively influenced by CSN, pre-treatment with CSN accelerated seedling growth and increased cold tolerance, suggesting that the effect of CSN was related to whether the seedlings were damaged by CS. In conclusion, we firstly found that CSN enhanced cold tolerance by activating BR signaling, contributing to the gene expression of ICE-CBF-COR and that CSN + EBR contributed to cold tolerance and CS-injured seedling recovery in cucumber., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2023 Elsevier Masson SAS. All rights reserved.)

Zinc (Zn) is an important element in plants, but over-accumulation of Zn is harmful. The phytohormone brassinosteroids (BRs) play a key role in regulating plant growth, development, and response to stress. However, the role of BRs in watermelon ( Citrullus lanatus L.) under Zn stress, one of the most important horticultural crops, remains largely unknown. In this study, we revealed that 24-epibrassinolide (EBR), a bioactive BR enhanced Zn tolerance in watermelon plants, which was related to the EBR-induced increase in the fresh weight, chlorophyll content, and net photosynthetic rate (Pn) and decrease in the content of hydrogen peroxide (H 2 O 2 ), malondialdehyde (MDA), and Zn in watermelon leaves. Through RNA deep sequencing (RNA-seq), 350 different expressed genes (DEG) were found to be involved in the response to Zn stress after EBR treatment, including 175 up-regulated DEGs and 175 down-regulated DEGs. The up-regulated DEGs were significantly enriched in 'phenylpropanoid biosynthesis' pathway (map00940) using KEGG enrichment analysis. The gene expression levels of PAL , 4CL , CCR , and CCoAOMT , key genes involved in phenylpropanoid pathway, were significantly induced after EBR treatment. In addition, compared with Zn stress alone, EBR treatment significantly promoted the activities of PAL, 4CL, and POD by 30.90%, 20.69%, and 47.28%, respectively, and increased the content of total phenolic compounds, total flavonoids, and lignin by 23.02%, 40.37%, and 29.26%, respectively. The present research indicates that EBR plays an active role in strengthening Zn tolerance, thus providing new insights into the mechanism of BRs enhancing heavy metal tolerance.

The synthesis and biological evaluation of brassinosteroids (BRs) analogs with chemical modification in the side alkyl chain is a matter of current interest. Recently, a series of BR analogs with phenyl or benzoate groups in the alkyl chain have been reported. The effect of substitution in the aromatic ring on the biological activities of these new analogs has been evaluated, and the results suggest that the bioactivity is enhanced by substitution with an F atom. In this context, we have synthesized, characterized, and evaluated a series of new analogs of 23,24-bisnorcholenic type in which the benzoate group at the C-22 position is substituted with an F atom at " ortho or para " positions. Plant growth-promoting activities were evaluated by using the rice lamina inclination test and bean second internode biotest. The results obtained with both bioassays indicate that the compound with an F atom in the para position on the aromatic ring is the most active BR analog and in some cases is even more active than brassinolide. The docking study confirmed that compounds with an F atom adopt an orientation similar to that predicted for brassinolide, and the F atom in the " para " position generates an extra hydrogen bond in the predicted binding position.

Background: Brassinolide, known as the seventh plant hormone, can improve the photosynthetic capacity of plants, promote plant growth and development, promote the formation of horticultural crop yield, improve the quality of horticultural crops, and also improve the ability of plants to resist biological and abiotic stresses., Results: The effects of different concentrations of exogenously sprayed 2,4-epibrassinolide (EBR) on growth, physiological and photosynthetic characteristics of 'All-round large leaf coriander' were studied in substrate culture. The results showed that 0.05, 0.1, and 0.5 mg.L - 1 EBR promoted the growth of coriander and increased the aboveground fresh and dry weights, with 0.5 mg.L - 1 EBR having the most significant effect. Spraying 0.1 mg.L - 1 EBR increased the content of soluble sugars and protein of coriander leaves. Spraying 0.1 and 0.5 mg.L - 1 EBR significantly increased the chlorophyll content and photosynthetic parameters of coriander leaves, and 0.5 mg.L - 1 EBR also significantly increased the chlorophyll fluorescence parameters of coriander leaves. Spraying 0.5 mg.L - 1 EBR upregulated the expression of CsRbcS, CsFBPase, and CsAld. Correlation analysis showed that aboveground fresh weight under exogenous EBR treatment was significantly positively correlated with aboveground dry weight, plant height, P n , G s , C i , and CsAld (P < 0.05), and soluble sugar content was significantly positively correlated with the number of leaves, Y (II) , qP, and CsRbcS. The results of the principal component analysis (PCA) showed that there was a significant separation between the treatment and the control groups. Spraying 0.5 mg.L - 1 EBR can promote the growth of coriander, improve the quality of coriander leaves, and strengthen coriander leaf photosynthetic capacity. This study provides new insights into the promotion of coriander growth and development following the application of exogenous EBR., Conclusion: Exogenous EBR treatment increased coriander plant height, leaf growth and aboveground dry weight, and enhanced photosynthesis. Exogenous spraying of 0.5 mg.L - 1 EBR had the most significant effect., (© 2023. The Author(s).)

Brassinosteroids (BRs) play pivotal roles in improving plant stress tolerance. To investigate the mechanism of BR regulation of salt tolerance in kiwifruit, we used 'Hongyang' kiwifruit as the test material. We exposed the plants to 150 mmol/L NaCl stress and irrigated them with exogenous BR (2,4-epibrassinolide). The phenotypic analysis showed that salt stress significantly inhibited photosynthesis in kiwifruit, leading to a significant increase in the H 2 O 2 content of leaves and roots and a significant increase in Na + /K + , resulting in oxidative damage and an ion imbalance. BR treatment resulted in enhanced photosynthesis, reduced H 2 O 2 content, and reduced Na + /K + in leaves, alleviating the salt stress injury. Furthermore, transcriptome enrichment analysis showed that the differentially expressed genes (DEGs) related to BR treatment are involved in pathways such as starch and sucrose metabolism, pentose and glucuronate interconversions, and plant hormone signal transduction, among others. Among the DEGs involved in plant hormone signal transduction, those with the highest expression were involved in abscisic acid signal transduction. Moreover, there was a significant increase in the expression of the AcHKT1 gene, which regulates ion transduction, and the antioxidant enzyme AcFSD2 gene, which is a key gene for improving salt tolerance. The data suggest that BRs can improve salt tolerance by regulating ion homeostasis and reducing oxidative stress.

Excessive use of pesticides against pests has contaminated agricultural crops and raised global concerns about food safety. This research investigates the alleviation effects of 24-epibrassinolide (EBL) seed priming on diazinon (DZ) pesticide toxicity. The experiment was conducted with eight groups including control, DZ, EBL (10 µM), EBL (0.1 µM), EBL (0.01 µM), EBL (10 µM) + DZ, EBL (0.1 µM) + DZ, and EBL (0.01 µM) + DZ. Plants grown with the lowest concentration of EBL (0.01 µM) exhibited an upward increase in the activity of SOD, CAT, POD, APX, GR, and GST enzymes under DZ toxicity stress. In contrast, higher concentrations of EBL showed some inhibitory effects on the activity of antioxidant enzymes. In addition, low concentrations of EBL elevated the free radical scavenging capacity (DPPH), iron-reducing antioxidant power (FRAP), photosynthesis rate (Pn), stomatal conductance (Gs) and proline, and protein contents. EBL also reduced lipid peroxidation (MDA levels) in the DZ-exposed plants, leading to membrane integrity. The favorable effects of EBL were more evident when plants were exposed to pesticides than normal growth conditions. The results indicated that EBL seed priming intensifies the antioxidant enzymes system activity, and helps maize plants against toxic effects of DZ under proper concentration., (© 2023. The Author(s).)

Brassinosteroids (BRs), the sixth major phytohormone, can regulate plant salt tolerance. Many studies have been conducted to investigate the effects of BRs on plant salt tolerance, generating a large amount of research data. However, a meta-analysis on regulating plant salt tolerance by BRs has not been reported. Therefore, this study conducted a meta-analysis of 132 studies to elucidate the most critical physiological mechanisms by which BRs regulate salt tolerance in plants from a higher dimension and analyze the best ways to apply BRs. The results showed that exogenous BRs significantly increased germination, plant height, root length, and biomass (total dry weight was the largest) of plants under salt stress. There was no significant difference between seed soaking and foliar spraying. However, the medium method (germination stage) and stem application (seedling stage) may be more effective in improving plant salt tolerance. BRs only inhibit germination in Solanaceae. BRs (2 μM), seed soaking for 12 h, and simultaneous treatment with salt stress had the highest germination rate. At the seedling stage, the activity of Brassinolide (C 28 H 48 O 6 ) was higher than that of Homobrassinolide (C 29 H 50 O 6 ), and post-treatment, BRs (0.02 μM) was the best solution. BRs are unsuitable for use in the germination stage when Sodium chloride is below 100 mM, and the effect is also weakest in the seedling stage. Exogenous BRs promoted photosynthesis, and antioxidant enzyme activity increased the accumulation of osmoregulatory and antioxidant substances and reduced the content of harmful substances and Na + , thus reducing cell damage and improving plant salt tolerance. BRs induced the most soluble protein, chlorophyll a, stomatal conductance, net photosynthetic rate, Glutathione peroxidase, and root-Ca 2+ , with BRs causing Ca 2+ signals in roots probably constituting the most important reason for improving salt tolerance. BRs first promoted the accumulation of Ca 2+ in roots, which increased the content of the above vital substances and enzyme activities through the Ca 2+ signaling pathway, improving plant salt tolerance.

The generation of adventitious roots (ARs) is the key to the success of cuttings. The appropriate environment for AR differentiation in tea plants is acidic. However, the mechanism is unclear. In this study, pH 4.5 was suitable condition for the differentiation of AR in tea plants. At the base of cuttings, the root primordia differentiated ARs more rapidly at pH 4.5 than pH 7.0, and nine AR differentiation-related genes were found to be differentially expressed in 30 days, the result was also validated by qRT-PCR. The promoter regions of these genes contained auxin and brassinosteroid response elements. The expression levels of several genes which were involved in auxin and brassinosteroid synthesis as well as signaling at pH 4.5 compared to pH 7.0 occurred differential expression. Brassinolide (BL) and indole-3-acetic acid (IAA) could affect the differentiation of ARs under pH 4.5 and pH 7.0. By qRT-PCR analysis of genes during ARs generation, BL and IAA inhibited and promoted the expression of CsIAA14 gene, respectively, to regulate auxin signal transduction. Meanwhile, the expression levels of CsKNAT4, CsNAC2, CsNAC100, CsWRKY30 and CsLBD18 genes were up-regulated upon auxin treatment and were positively correlated with ARs differentiation.This study showed that pH 4.5 was the most suitable environment for the root primordia differentiation of AR in tea plant. Proper acidic pH conditions promoted auxin synthesis and signal transduction. The auxin initiated the expression of AR differentiation-related genes, and promoted its differentiated. BL was involved in ARs formation and elongation by regulating auxin signal transduction., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The steroidal hormone brassinosteroid (BR) has been shown to positively regulate cell expansion in plants. However, the specific mechanism by which BR controls this process has not been fully understood. In this study, RNA-seq and DAP-seq analysis of GhBES1.4 (a core transcription factor in BR signaling) were used to identify a cotton cell cycle-dependent kinase inhibitor called GhKRP6. The study found that GhKRP6 was significantly induced by the BR hormone and that GhBES1.4 directly promoted the expression of GhKRP6 by binding to the CACGTG motif in its promoter region. GhKRP6-silenced cotton plants had smaller leaves with more cells and reduced cell size. Furthermore, endoreduplication was inhibited, which affected cell expansion and ultimately decreased fiber length and seed size in GhKRP6-silenced plants compared with the control. The KEGG enrichment results of control and VIGS-GhKRP6 plants revealed differential expression of genes related to cell wall biosynthesis, MAPK, and plant hormone transduction pathways - all of which are related to cell expansion. Additionally, some cyclin-dependent kinase (CDK) genes were upregulated in the plants with silenced GhKRP6. Our study also found that GhKRP6 could interact directly with a cell cycle-dependent kinase called GhCDKG. Taken together, these results suggest that BR signaling influences cell expansion by directly modulating the expression of cell cycle-dependent kinase inhibitor GhKRP6 via GhBES1.4., (© 2023 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd.)

Peppermint has gained a promising status due to the presence of a high proportion of bioactive compounds, especially menthol. Due to its pharmacological efficacy, the demand for its plant-based bioactive compounds necessitates its cultivation worldwide. Brassinosteroids are polyhydroxylated sterol derivatives that regulate diverse processes and control many agronomic traits during plant growth and development. A factorial randomised pot experiment was performed in the net house to investigate the effect of 24-Epibrassinolide (EBL) on the growth, physiology, essential oil content, stomatal behaviour and trichome development of the three cultivars of peppermint. Four levels of foliage-applied EBL, viz. 0, 10 -5 , 10 -6 and 10 -7  M were applied to the three cultivars of peppermint (Kukrail, Pranjal and Tushar). Among the different treatments of EBL, the application of 10 -6  M increased shoot length by 38.84, 37.59 and 36.91%, root length by 36.73, 29.44 and 33.47%, chlorophyll content by 24.20, 22.48 and 23.32%, P N by 32.88, 32.61 and 33.61%, EO content by 32.72, 30.00 and 28.84%, EO yield per plant by 66.66, 77.77 and 73.33% and menthol yield per plant by 127.27, 110 and 118.18% in Kukrail, Tushar and Pranjal respectively, compared with their respective control plants. Further, the 10 -6  M EBL exhibited improved trichome size and density, cellular viability and menthol content of the oil analysed from scanning electron microscopy, confocal laser scanning microscopy and GC-MS respectively as compared to the control. In conclusion, out of different levels of EBL, two sprays of 10 -6  M EBL proved effective in enhancing the morphophysiological features and productivity of mint plants, particularly for cultivar Kukrail., (© 2023. Springer Nature Limited.)

The endogenous brassinosteroid (BS) profile was for the first time shown to change in response to salt stress in potato plants. A group of 6-keto-BSs was identified and found to significantly increase in content during salinization in contrast to other groups of hormones examined. A tenfold reduction in the level of endogenous BSs in mutant Arabidopsis thaliana plants with impaired biosynthesis (det2) (or reception (bri1)) of phytosteroids decreased their salt resistance, as evidenced by a lower efficiency of photochemical processes of photosystem II (PSII) and growth inhibition. The results confirmed the idea that endogenous BSs are involved in the formation of salt resistance in plants., (© 2023. Pleiades Publishing, Ltd.)

Inorganic phosphate (Pi) is a necessary macronutrient for basic biological processes. Plants modulate their root system architecture (RSA) and cellular processes to adapt to Pi deprivation albeit with a growth penalty. Excess application of Pi fertilizer, on the contrary, leads to eutrophication and has a negative environmental impact. We compared RSA, root hair elongation, acid phosphatase activity, metal ion accumulation, and brassinosteroid hormone levels of Solanum lycopersicum (tomato) and Solanum pennellii, which is a wild relative of tomato, under Pi sufficiency and deficiency conditions to understand the molecular mechanism of Pi deprivation response in tomato. We showed that S. pennellii is partially insensitive to phosphate deprivation. Furthermore, it mounts a constitutive response under phosphate sufficiency. We demonstrate that activated brassinosteroid signaling through a tomato BZR1 ortholog gives rise to the same constitutive phosphate deficiency response, which is dependent on zinc overaccumulation. Collectively, these results reveal an additional strategy by which plants can adapt to phosphate starvation., (© 2023 The Authors. New Phytologist © 2023 New Phytologist Foundation.)

Five new brassinosteroid analogues were synthetized from 3β-acetoxy-23,24-dinorchol-4-en-22-oic acid. All the obtained compound showed significant activity in the Rice Lamina Inclination Test. Interestingly the effects of the methyl ester of 3β-hydroxy-6-oxo-23,24-dinorcholan-22-oic acid (14) at concentrations of 1 × 10 -7 and 1 × 10 -6 M proved to be higher than those produced by brassinolide. In silico Molecular Docking and Induced fit docking (IFD) simulations for the compounds with the highest biological activity data were carried out to investigate the binding mode interactions into the brassinolide-binding groove which revealed that the compound 14 had high binding energy values and a good affinity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

The molecular processes behind Parkinson's disease (PD) remain under debate although mitochondrial oxidative stress generation has been proposed as a fundamental contributor. In this context, different brassinosteroids have shown neuroprotective action hampering oxidative stress. This study determined the effects of 24-Epibrassinolide (24-EPI) against 6-hydroxydopamine- (6-OHDA-) induced toxicity using the zebrafish embryonic model. Embryos were exposed to 250 μM 6-OHDA or co-exposed to 24-EPI (0.01, 0.1, and 1 μM) for 3 days, starting at 48 h post-fertilization (hpf). During the experimental period, developmental parameters were assessed. At 120 hpf, larvae were tested for behavioural phenotypes with different biochemical biomarkers and tyrosine hydroxylase- (TH-) reactive neurons being also assessed. Exposure to 6-OHDA induced a decrease in body length while no other morphological phenotypes were noticed. A significant decrease in TH-neurons immunofluorescence, a decreased locomotion (speed and distance moved), and an increased absolute angle were found in 6-OHDA-exposed embryos. These outcomes were rescuable by the co-exposure with 24-EPI. Surprisingly, the direct effects of 6-OHDA on reactive oxygen species (ROS) were not observed in the present study supporting the involvement of other molecular pathways in the 6-OHDA-induced effects during embryonic development. Overall, the results obtained confirm PD-like symptoms induced by 6-OHDA during embryonic development which were reverted by 24-EPI. Although antioxidative signalling pathways deserve further scrutiny, the findings support the further investigation of 24-EPI neuroprotective effects., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)