Search

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.)

Brassinosteroids (BRs) can regulate various processes in plant development and defense against environmental stress. In this study, the contribution of BRs in the degradation of isoproturon (IPU) in rice has been established. IPU has a significant effect on rice growth, chlorophyll content, and membrane permeability. When treated with 1.0 μmol/L 24-epibrassinolide (EBR), a BR analogue, the associated symptoms of rice poisoning were alleviated as the IPU levels in the rice and growth media were decreased. In the presence of EBR, the activities of several IPU-related detoxification enzymes were enhanced to cope with the stress due to IPU. An RNA-sequencing (RNA-Seq) has been performed to determine the variation of transcriptomes and metabolic mechanisms in rice treated with EBR, IPU, or IPU+EBR. Some of the differentially expressed genes (DEGs) were Phase I-III reaction components of plants, such as cytochrome P450 (CYP450), glutathione S-transferase (GST), glycosyltransferases (GTs), and the ATP-binding cassette transporter (ABC transporter). The expression of some signal transduction genes was significantly up-regulated. The relative content of low-toxicity IPU metabolites increased due to the presence of EBR as determined by UPLC/Q-TOF-MS/MS. The IPU metabolic pathways include enzyme-catalyzed demethylation, hydroxylation, hydrolysis, glycosylation, and amino acid conjugation processes. The results suggest that EBR plays a key role in the degradation and detoxification of IPU. This study has provided evidence that BRs regulate the metabolism and detoxification of IPU in rice, and offers a new approach to ensuring cleaner crops by eliminating pesticide residues in the environment., 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 Inc.)

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).)

Low temperature (LT) greatly restricts grain filling in maize (Zea mays L.), but the relevant molecular mechanisms are not fully understood. To better understand the effect of LT on grain development, 17 hybrids were subjected to LT stress in field trials over 3 years, and two hybrids of them with contrasting LT responses were exposed to 30/20°C and 20/10°C for 7 days during grain filling in a greenhouse. At LT, thousand-kernel weight declined, especially in LT-sensitive hybrid FM985, while grain-filling rate was on average about 48% higher in LT-tolerant hybrid DK159 than FM985. LT reduced starch synthesis in kernel mainly by suppression of transcript levels and enzyme activities for sucrose synthase and hexokinase. Brassinolide (BR) was abundant in DK159 kernel, and genes involved in BR and cytokinin signals were inducible by stress. LT downregulated the genes in light-harvesting complex and photosystem I/II subunits, accompanied by reduced photosynthetic rate and Fv/Fm in ear leaf. The LT-tolerant hybrid could maintain a high soluble sugar content and fast interconversion between sucrose and hexose in the stem internode and cob, improving assimilate allocation to kernel at LT stress and paving the way for simultaneous growth and LT stress responses., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

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.)

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.)

Main Conclusion: Trace amounts of epibrassinolide (EpiBL) could partially rescue wheat root length inhibition in salt-stressed situation by scavenging ROS, and ectopic expression of TaDWF4 or TaBAK1 enhances root salt tolerance in Arabidopsis by balancing ROS level. Salt stress often leads to ion toxicity and oxidative stress, causing cell structure damage and root development inhibition in plants. While prior research indicated the involvement of exogenous brassinosteroid (BR) in plant responses to salt stress, the precise cytological role and the function of BR in wheat root development under salt stress remain elusive. Our study demonstrates that 100 mM NaCl solution inhibits wheat root development, but 5 nM EpiBL partially rescues root length inhibition by decreasing H 2 O 2 content, oxygen free radical (OFR) content, along with increasing the peroxidase (POD) and catalase (CAT) activities in salt-stressed roots. The qRT-PCR experiment also shows that expression of the ROS-scavenging genes (GPX2 and CAT2) increased in roots after applying BR, especially during salt stress situation. Transcriptional analysis reveals decreased expression of BR synthesis and root meristem development genes under salt stress in wheat roots. Differential expression gene (DEG) enrichment analysis highlights the significant impact of salt stress on various biological processes, particularly "hydrogen peroxide catabolic process" and "response to oxidative stress". Additionally, the BR biosynthesis pathway is enriched under salt stress conditions. Therefore, we investigated the involvement of wheat BR synthesis gene TaDWF4 and BR signaling gene TaBAK1 in salt stress responses in roots. Our results demonstrate that ectopic expression of TaDWF4 or TaBAK1 enhances salt tolerance in Arabidopsis by balancing ROS (Reactive oxygen species) levels in roots., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Microplastic (MP), as a new pollutant, not only affects the growth and development of plants but also may affect the secondary metabolites of plants. The anti-tumor role of Pinellia ternata is related to secondary metabolites. The role of brassinolide (BR) in regulating plant resistance is currently one of the research hotspots. The paper mainly explores the regulation of BR on growth and physiology of Pinellia ternata under MP stress. The experimental design includes two levels of MP (0, 1%) and two levels of BR (0, 0.1 mg/L). MP led to a marked reduction in plant height (15.0%), Fv/Fm (3.2%), SOD and APX activity (15.0%, 5.1%), whereas induced an evident raise in the rate of O 2 · - production (29.6%) and GSH content (4.4%), as well as flavonoids (6.8%), alkaloids (75%), and β-sitosterol (26.5%) contents. Under MP addition, BR supply significantly increased plant height (15.7%), aboveground and underground biomass (16.1%, 10.3%), carotenoid and GSH content (11.8%, 4.2%), Fv/Fm (2.9%), and activities of SOD, GR, and MDHAR (32.2%, 21.08%, 20.9%). These results indicate that MP suppresses the growth of P. ternata, although it promotes secondary metabolism. BR can alleviate the inhibitory effect of MP on growth by improving photosynthesis, redox homeostasis, and the AsA-GSH cycle., 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.)

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.

Brassinosteroids (BRs) play vital roles in the plant life cycle and synthetic BRs are widely used to increase crop yield and plant stress tolerance. Among them are 24 R -methyl-epibrassinolide (24-EBL) and 24 S -ethyl-28-homobrassinolide (28-HBL), which differ from brassinolide (BL, the most active BR) at the C-24 position. Although it is well known that 24-EBL is 10% active as BL, there is no consensus on the bioactivity of 28-HBL. A recent outpouring of research interest in 28-HBL on major crops accompanied with a surge of industrial-scale synthesis that produces mixtures of active (22 R ,23 R )-28-HBL and inactive (22 S ,23 S )-28HBL, demands a standardized assay system capable of analyzing different synthetic "28-HBL" products. In this study, the relative bioactivity of 28-HBL to BL and 24-EBL, including its capacity to induce the well-established BR responses at molecular, biochemical, and physiological levels, was systematically analyzed using the whole seedlings of the wild-type and BR-deficient mutant of Arabidopsis thaliana . These multi-level bioassays consistently showed that 28-HBL exhibits a much stronger bioactivity than 24-EBL and is almost as active as BL in rescuing the short hypocotyl phenotype of the dark-grown det2 mutant. These results are consistent with the previously established structure-activity relationship of BRs, proving that this multi-level whole seedling bioassay system could be used to analyze different batches of industrially produced 28-HBL or other BL analogs to ensure the full potential of BRs in modern agriculture.

To identify new steroidal agents with potential biological activities, we synthesized hybrid steroids containing thiazole, pyrazole, isoxazole, thiophene or phthalazine moiety. Epi-androsterone 1 reacted with phenylthiosemicarbazide to afford the corresponding androstane-4-phenyl-3-thiosemicarbazone derivative 2. The latter product was used in the synthesis of a series of annulated steroid derivatives. Also, Epi-androsterone 1 reacted with the thienopyridazine derivative 16 to afford the thieno[3,4-d]pyridazino-N-ylidenoandrostane derivative 17. Compound 17 reacted readily with electron-poor olefins to yield the corresponding phthalazine steroid derivatives. Detailed experimental and spectroscopic evidences for the structures of the newly synthesized compounds are explained. Compounds 3, 7, 8a, 12a, 14, 17 and 21a, were investigated individually as anticancer agents on different panel of human malignant cell lines. Moreover, a computer modelling investigation was performed to speculate the macromolecular targets for the most promising candidate. The results revealed a concentration-dependent reduction in the number of viable cells in all cancer cell lines. Most notably, compound 7 was the most effective compound against all tested cancer cell lines, especially against HepG2 cell line; therefore, the mode of action of this compound against HCC was investigated. Compound 7 was able to induce cell cycle arrest, and DNA fragmentation in HepG2 cells. Moreover, compound 7 induced apoptosis via upregulating the expression of caspase-3, -8, -9, P53, Bax and inhibiting the expression of BCL2, and CDK2 genes. Our results highlighted compound 7 as a promising anti-hepatocellular carcinoma agent, with theoretical, and practical potential binding affinity with CDK2; therefore, more investigations are required to elucidate its chemotherapeutic value as anti-HCC agent., 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.)

Steroidal heterocyclic compounds constitute interesting and promising scaffolds for drug discovery as they have displayed diverse chemical reactivity and several types of biological activities. This study is a concise report on the most recent advancements in the chemistry of the steroid skeleton, including reactions at the A, B, and D ring systems. The modern synthetic methods for the steroidal nitrogen-containing six-membered heterocyclic derivatives from 3-keto-, 6-keto-, 17-keto-, and 20-keto-steroids, as well as 2-Aldo-, 4-Aldo-, 6-Aldo-, and 16-Aldo-steroids, are discussed. However, some other methods for the synthesis of steroidal N-containing 6-membered heterocyclic derivatives are also included. These compounds have shown therapeutic potential as cytotoxic agents against various cell lines and have also shown antiproliferative, anti-inflammatory, and antioxidant activities. Therefore, they could be used as prospective candidates for the development of various medications. This paper not only describes synthetic details involved in creating N-containing 6-membered heterocyclic steroid derivatives, but also provides a brief overview of the medicinal applications of these compounds. This information will be highly useful for the medicinal chemists conducting research in this field., 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 © 2022 Elsevier Inc. All rights reserved.)

Brassinosteroids are plant hormones whose main function is to stimulate plant growth. However, they have been studied for their biological applications in humans. Brassinosteroid compounds have displayed an important role in the study of cancer pathology and show potential for developing novel anticancer drugs. In this review we describe the relationship of brassinosteroids with cancer with focus on the last decade, the mechanisms of cytotoxic activity described to date, and a structure-activity relationship based on the available information., 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 © 2022 Elsevier Inc. All rights reserved.)

Key Message: Exogenous brassinolide promotes Fe absorption through mechanism I strategy, thus improving the tolerance of Malus hupehensis seedlings to Fe deficiency stress. Iron (Fe) deficiency is a common nutritional disorder that results in decreased yield and poor fruit quality in apple production. As a highly active synthetic analog of brassinosteroids, brassinolide (BL) plays numerous roles in plant responses to abiotic stresses. However, its role in Fe deficiency stress in apple plants has never been reported. Herein, we found that the exogenous application of 0.2 mg L -1 BL could significantly enhance the tolerance of apple seedlings to Fe deficiency stress and result in a low etiolation rate and a high photosynthetic rate. The functional mechanisms of this effect were also explored. We found that first, exogenous BL could improve Fe absorption through the mechanism I strategy. BL induced the activity of H + -ATPase and the expression of MhAHA family genes, resulting in rhizosphere acidification. Moreover, BL could enhance the activity of Fe chelate reductase and absorb Fe through direct binding with the E-box of the MhIRT1 or MhFRO2 promoter via the transcription factors MhBZR1 and MhBZR2. Second, exogenous BL alleviated osmotic stress by increasing the contents of osmolytes (proline, solution proteins, and solution sugar) and scavenged reactive oxygen species by improving the activities of antioxidant enzymes. Lastly, exogenous BL could cooperate with other endogenous plant hormones, such as indole-3-acetic acid, isopentenyl adenosine, and gibberellic acid 4, that respond to Fe deficiency stress indirectly. This work provided a theoretical basis for the application of exogenous BL to alleviate Fe deficiency stress in apple plants., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Brassinolide (BL) is a possible plant growth regulator in agriculture, but the presence of a steroid skeleton hampers the field application of BL in agriculture because of its high synthetic cost. We discovered NSBR1 as the first nonsteroidal BL-like compound using in silico technology. Searching for more potent BL-like compounds, we modified the structure of NSBR1 with respect to 2 benzene rings and the piperazine ring. The activity of synthesized compounds was measured in Arabidopsis hypocotyl elongation. The propyl group of butyryl moiety of NSBR1 was changed to various alkyl groups, such as straight, branched, and cyclic alkyl chains. Another substituent, F, at the ortho position of the B ring toward the piperazine ring was changed to other substituents. A methyl group was introduced to the piperazine ring. Most of the newly synthesized compounds with the 3,4-(OH)2 group at the A ring significantly elongated the hypocotyl of Arabidopsis., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Peanut is one of the most important oil crops in the world, the growth and productivity of which are severely affected by salt stress. 24-epibrassinolide (EBL) plays an important role in stress resistances. However, the roles of exogenous EBL on the salt tolerance of peanut remain unclear. In this study, peanut seedlings treated with 150 mM NaCl and with or without EBL spray were performed to investigate the roles of EBL on salt resistance. Under 150 mM NaCl conditions, foliar application of 0.1 µM EBL increased the activity of catalase and thereby could eliminate reactive oxygen species (ROS). Similarly, EBL application promoted the accumulation of proline and soluble sugar, thus maintaining osmotic balance. Furthermore, foliar EBL spray enhanced the total chlorophyll content and high photosynthesis capacity. Transcriptome analysis showed that under NaCl stress, EBL treatment up-regulated expression levels of genes encoding peroxisomal nicotinamide adenine dinucleotide carrier ( PMP34 ), probable sucrose-phosphate synthase 2 ( SPS2 ) beta-fructofuranosidase ( BFRUCT1 ) and Na + /H + antiporters ( NHX7 and NHX8) , while down-regulated proline dehydrogenase 2 ( PRODH ). These findings provide valuable resources for salt resistance study in peanut and lay the foundation for using BR to enhance salt tolerance during peanut production.

We compared the defensive response of rape plants to treatment with 24-epibrassinoldide (10 nM, EBL) before the onset of salt stress (preadaptation stage) and under conditions of chloride salination (150 mM NaCl). It is shown that salt stress inhibits some growth parameters by 30-35%. EBL, regardless of the plant treatment method, showed a pronounced protective effect, first of all, at the level of the assimilating surface, the main photosynthetic pigments, and the photochemical activity of photosystem II. It was established for the first time that the pretreatment of plants with EBL followed by salt stress is accompanied by suppression of NaCl-induced accumulation of proline and an increase in superoxide dismutase activity, whereas the addition of a hormone under salt stress increases the content of carotenoids, which leads to a decrease in the level of lipid peroxidation., (© 2022. Pleiades Publishing, Ltd.)

Postharvest crop yellowing is a major concern in the broccoli industry. The effect and underlying mechanisms of 24-epibrassinolide (EBR) treatment on yellowing in postharvest broccoli were investigated. Treatment with 2 µM EBR markedly inhibited the increase of the yellowing index and L* values, causing higher retention of the metric hue angle and chlorophyll content compared to the control. Treatment also alleviated oxidative damage by preventing the accumulation of malondialdehyde and superoxide anion (O 2 •- ). The ascorbic acid content of broccoli reached its lowest value at the end of its shelf life, whereas that of the treated sample was obviously higher than the control. Moreover, treated broccoli exhibited higher superoxide dismutase, ascorbate peroxidase, and phenylalanine ammonia-lyase activities. Multivariate statistical analysis further demonstrated the effective enhancement of EBR treatment on antioxidant enzymes. These results indicate that exogenous application of EBR ameliorates postharvest yellowing by improving the antioxidant capacity of broccoli., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Previous embryonic fish data have shown caffeine to induce potential teratogenic and long-term neurodevelopmental outcomes through oxidative stress-mediated apoptosis. In this context, antioxidants may have the potential to counteract the caffeine-induced effects. Therefore, the present study aimed to investigate the potential protective role of 24-epibrassinolide (24-EPI), a natural brassinosteroid with proven antioxidant properties, against caffeine-induced teratogenic effects during early zebrafish development. Embryos (~2 h post-fertilization - hpf) were exposed to 0.5 mM caffeine, co-exposed to 24-EPI (0.01, 0.1 and 1 μM) and to 24-EPI alone (1 μM) for 96 h. During exposure, lethal and sublethal developmental parameters were evaluated. At the end of the exposure, biochemical evaluations were made, and 24 h after, different behavioural paradigms were assessed. An increased number of animals showing oedema and malformations were observed after caffeine exposure, while these were reduced after co-exposure to 24-EPI concentration, namely the tail curvature. The results showed oxidative stress and related parameters similar among treatments. Yet, caffeine exposure resulted in locomotor deficits (decreased speed and distance) and disrupted anxiety-like and avoidance responses. The co-exposure to caffeine and to the highest 24-EPI concentrations resulted in less pronounced behavioural deficits. Overall, there was an absence of effects in the embryo/larvae exposed solely to 24-EPI, while caffeine caused developmental and neurotoxic effects. Although further studies are needed, the results showed promising protective effects of the highest 24-EPI concentration tested against the toxicity induced by caffeine in zebrafish., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Heterostyly is a breeding system that promotes outbreeding through a combination of morphological and physiological floral traits. In Turnera these traits are governed by a single, hemizygous S -locus containing just three genes. We report that the S -locus gene, BAHD , is mutated and encodes a severely truncated protein in a self-compatible long homostyle species. Further, a self-compatible long homostyle mutant possesses a T. krapovickasii   BAHD allele with a point mutation in a highly conserved domain of BAHD acyl transferases. Wild type and mutant TkBAHD alleles were expressed in Arabidopsis to assay for brassinosteroid (BR) inactivating activity. The wild type but not mutant allele caused dwarfism, consistent with the wild type possessing, but the mutant allele having lost, BR inactivating activity. To investigate whether BRs act directly in self-incompatibility, BRs were added to in vitro pollen cultures of the two mating types. A small morph specific stimulatory effect on pollen tube growth was found with 5 µM brassinolide, but no genotype specific inhibition was observed. These results suggest that BAHD acts pleiotropically to mediate pistil length and physiological mating type through BR inactivation, and that in regard to self-incompatibility, BR acts by differentially regulating gene expression in pistils, rather than directly on pollen.

The force of gravity is a constant environmental factor. Plant shoots respond to gravity through negative gravitropism and gravity resistance. These responses are essential for plants to direct the growth of aerial organs away from the soil surface after germination and to keep an upright posture above ground. We took advantage of the effect of brassinosteroids (BRs) on the two types of graviresponses in Arabidopsis thaliana hypocotyls to disentangle functions of cell wall polymers during etiolated shoot growth. The ability of etiolated Arabidopsis seedlings to grow upward was suppressed in the presence of 24-epibrassinolide (EBL) but enhanced in the presence of brassinazole (BRZ), an inhibitor of BR biosynthesis. These effects were accompanied by changes in cell wall mechanics and composition. Cell wall biochemical analyses, confocal microscopy of the cellulose-specific pontamine S4B dye and cellular growth analyses revealed that the EBL and BRZ treatments correlated with changes in cellulose fibre organization, cell expansion at the hypocotyl base and mannan content. Indeed, a longitudinal reorientation of cellulose fibres and growth inhibition at the base of hypocotyls supported their upright posture whereas the presence of mannans reduced gravitropic bending. The negative effect of mannans on gravitropism is a new function for this class of hemicelluloses. We also found that EBL interferes with upright growth of hypocotyls through their uneven thickening at the base., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

The postharvest senescence accompanied by yellowing limited the shelf-life of broccoli. In this study, we developed a novel W/O/W double emulsion co-delivering brassinolide and cinnamon essential oil and applied it to broccoli for preservation. Results showed that double emulsion prepared by whey protein concentrate-high methoxyl pectin (1:3) exhibited best storage stability with largest particle size (581.30 nm), lowest PDI (0.23) and zeta potential (-40.31 mV). This double emulsion also exhibited highest encapsulation efficiency of brassinolide (92%) and cinnamon essential oil (88%). The broccoli coated with double emulsion maintained higher chlorophyll contents and activities of chlorophyllase and magnesium-dechelatase were reduced by 9% and 24%, respectively. The energy metabolic enzymes (SDH, CCO, H + -ATPase, Ca 2+ -ATPase) were also activated, inducing higher level of ATP and energy charge. These results demonstrated W/O/W double emulsion co-delivering brassinolide and cinnamon essential delayed the senescence of broccoli via regulating chlorophyll degradation and energy metabolism., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

As an essential element, zinc (Zn) can improve or inhibit the growth of plants depending on its concentrations. In this study, the effects of 24-Epibrassinolide (EBR), one well-known steroid phytohormone regulating plant growth and alleviating abiotic stress damage, on morphological parameters and antioxidant capacities of Sedum lineare were investigated under different Zn doses. Compared to plants only exposed to Zn, simultaneously foliar application of 0.75 μM EBR significantly improved multiple morphological characteristics and such growth-improving effects were more significant at high Zn concentrations. At a detrimental 800 μM Zn, EBR benefitted plant growth most prominently, as shown by that the stem length, fresh weight and internode length were increased by 111%, 85% and 157%, respectively; than Zn solely treated plants. EBR spray also enhanced both the activities of antioxidant enzymes such as peroxidase (POD), ascorbate peroxidase (APX), glutathione reductase (GR), and the contents of antioxidative agents including ascorbic acid (AsA) and glutathione (GSH), which in turn decreased the accumulation of reactive oxygen species (ROS) and alleviated the lipid peroxidation in plants. Thus, by demonstrating that EBR could help S. lineare resist high-zinc stress through strengthening the antioxidant system, this work provided a new idea for expanding the planting range of Crassulaceae plants in heavy metal contaminated soil for phytoremediation purpose in the future., Competing Interests: The authors have declared that no competing interests exist.

Glycogen synthase kinase 3β (GSK3β) is considered an important element of glycogen metabolism; however, it has many other regulatory roles. Changes in the GSK3β signaling mechanism have been associated with various disorders, such as Alzheimer's disease (AD), type II diabetes, and cancer. Although the effects of GSK3β inhibitors on reducing the pathological effects of AD have been described, an effective inhibitor has not yet been developed. Epibrassinolide (EBR), a brassinosteroid (BR), is structurally similar to mammalian steroid hormones. Our studies have shown that EBR has an inhibitory effect on GSK3β in different cell lines. Roscovitine (ROSC), a cyclin-dependent kinase (CDK) inhibitor, has also been identified as a potential GSK3 inhibitor. Within the scope of this study, we propose that EBR and/or ROSC might have mechanistic action in AD models. To test this hypothesis, we used in vitro models and Caenorhabditis elegans (C. elegans) AD strains. Finally, EBR treatment successfully protected cells from apoptosis and increased the inhibitory phosphorylation of GSK3β. In addition, EBR and/or ROSC treatment had a positive effect on the survival rates of C. elegans strains. More interestingly, the paralysis phenotype of the C. elegans AD model due to Aβ42 toxicity was prevented by EBR and/or ROSC. Our findings suggest that EBR and ROSC administration have neuroprotective effects on both in vitro and C. elegans models via inhibitory GSK3β phosphorylation at Ser9., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Cadmium (Cd2+) soil pollution is a global environmental problem caused by the high toxicity of Cd. 24-Epibrassinolide (EBR) is a biodegradable plant steroid involved in response modulation to biotic and abiotic stresses. The aim of this study was to evaluate if the leaf-application of EBR improves the gas exchange and possible repercussions on leaf anatomy in young Eucalyptus urophylla plants exposed to Cd toxicity. The experiment involved six treatments, which included three Cd concentrations (0, 450, and 900 μM) and two EBR concentrations (0 and 100 nM, described as - EBR and + EBR, respectively). Plants exposed to Cd toxicity suffered decreases in leaf anatomical and gas exchange parameters. However, the plants treated with EBR + 900 μM Cd showed an increase of 46%, 40%, and 54% in the net photosynthetic rate, water-use efficiency, and instantaneous carboxylation efficiency, respectively. The EBR application-induced improvements in gas exchange parameters, causing beneficial effects on the photosynthetic apparatus, mainly the effective quantum yield of photosystem II (PSII) photochemistry and electron transport rate. Furthermore, this steroid mitigated the effect of Cd toxicity on leaf anatomical variables, more specifically palisade and spongy parenchyma, which are intrinsically related to stomatal density, and stimulated the net photosynthetic rate of plants., (© 2020 Scandinavian Plant Physiology Society.)

This study explored the effects of brassinolide (BR) soaking, preharvest ultraviolet-B (UV-B) radiation, and their combined treatments on physiological characteristics, chlorophyll fluorescence, and quality of small black bean sprouts during storage. Results indicated that the combined treatments significantly enhanced contents of flavone, free amino acid, and photosynthetic pigment, and activities of phenylalanine ammonia lyase (PAL) and 2-diphenyl-1-picrylhydrazyl(DPPH) radical scavenging in sprouts stored for 5 days compared with BR treatment alone. The combined treatments significantly increased total phenols content and PAL activity, and reduced malonaldehyde content in sprouts compared with UV-B radiation alone. The inhibitory effect of BR or UV-B on fluorescence of photosystem II was weakened by their combined treatments. Comprehensive analysis indicated that the combined treatments could be used to maintain postharvest small black bean sprouts with high levels of nutritional ingredients by probably keeping high photosynthetic capacity, PAL activity, and DPPH radical scavenging rate in sprouts., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Background: Boscalid is often used to extend the storage time of postharvest cherry tomato. Pesticide residue has become an issue of food safety. This study sought to investigate the spatial distribution of boscalid residue in cherry tomato fruits and to determine the effect of 24-epibrassinolide (EBR) in promoting boscalid degradation., Results: Boscalid could quickly penetrate into cherry tomatoes, but mainly remained in the peel. The migration of boscalid from the peel into the core was a time-consuming and complex process during storage. After 72 h, boscalid residues in the pulp and the core began to accumulate gradually. The exogenous application of EBR activated peroxidase, glutathione reductase and glutathione S-transferase, and effectively promoted the degradation of boscalid by a maximum decrease of 44.8% in peel, 54.0% in pulp and 71.2% in core., Conclusion: As one of the common pesticides, boscalid had a strong ability to enter the cherry tomato and thus become a potential risk for public consumption. Therefore, rational use of pesticides is recommended. The results of this study indicate that the possible risk of boscalid residue could be alleviated by EBR pretreatment through activating detoxification enzymes. © 2020 Society of Chemical Industry., (© 2020 Society of Chemical Industry.)

B-BOX proteins are zinc finger transcription factors that play important roles in plant growth, development, and abiotic stress responses. In this study, we identified 15 PavBBX genes in the genome database of sweet cherry. We systematically analyzed the gene structures, clustering characteristics, and expression patterns of these genes during fruit development and in response to light and various hormones. The PavBBX genes were divided into five subgroups. The promoter regions of the PavBBX genes contain cis -acting elements related to plant development, hormones, and stress. qRT-PCR revealed five upregulated and eight downregulated PavBBX genes during fruit development. In addition, PavBBX6 , PavBBX9 , and PavBBX11 were upregulated in response to light induction. We also found that ABA, BR, and GA 3 contents significantly increased in response to light induction. Furthermore, the expression of several PavBBX genes was highly correlated with the expression of anthocyanin biosynthesis genes, light-responsive genes, and genes that function in multiple hormone signaling pathways. Some PavBBX genes were strongly induced by ABA, GA, and BR treatment. Notably, PavBBX6 and PavBBX9 responded to all three hormones. Taken together, BBX proteins likely play major roles in regulating anthocyanin biosynthesis in sweet cherry fruit by integrating light, ABA, GA, and BR signaling pathways.

In plants, brassinosteroids (BRs) are a class of steroidal hormones that are involved in numerous physiological responses. However, the function of BRs in cold tolerance in cotton has not been explored. In this study, cotton seedlings were treated with five concentrations (0, 0.05, 0.1, 0.2, 0.5 and 1.0 mg/L) of 24-Epibrassinolide (EBR) at 4°C. We measured the electrolyte leakage, malondialdehyde (MDA) content, proline content, and net photosynthesis rate (Pn) of the seedlings, which showed that EBR treatment increased cold tolerance in cotton in a dose-dependent manner, and that 0.2 mg/L is an optimum concentration for enhancing cold tolerance. The function of EBR in cotton cotyledons was investigated in the control 0 mg/L (Cold+water) and 0.2 mg/L (Cold+EBR) treatments using RNA-Seq. A total of 4,001 differentially expressed genes (DEGs), including 2,591 up-regulated genes and 1,409 down-regulated genes were identified. Gene Ontology (GO) and biochemical pathway enrichment analyses showed that EBR is involved in the genetic information process, secondary metabolism, and also inhibits abscisic acid (ABA) and ethylene (ETH) signal transduction. In this study, physiological experiments showed that EBR can increase cold tolerance in cotton seedlings, and the comprehensive RNA-seq data shed light on the mechanisms through which EBR increases cold tolerance in cotton seedlings., Competing Interests: The authors have declared that no competing interests exist.

Brassinosteroids are promising agents for alleviating the negative effects of salinity on plants, but the mechanism of their protective action is far from being understood. We investigated the effect of pretreatment with 24-epibrassinolide (24-EBL) on the photosynthetic and physiological parameters of potato plants under progressive salinity stress caused by root application of 100 mM NaCl. Salinity clearly inhibited primary photosynthetic processes in potato plants by reducing the contents of photosynthetic pigments, photosynthetic electron transport and photosystem II (PSII) maximal and effective quantum yields. These negative effects of salinity on primary photosynthetic processes were mainly due to toxic ionic effects on the plant's ability to oxidize the plastoquinone pool. Pretreatment with 24-EBL alleviated this stress effect and allowed the maintenance of plastoquinone pool oxidation and the efficiency of photosystem II photochemistry to be at the same levels as those in unstressed plants; however, the pretreatment did not affect the photosynthetic pigment content. 24-EBL pretreatment clearly alleviated the decrease in leaf osmotic potential under salinity stress. The stress-induced increases in lipid peroxidation and proline contents were not changed under brassinosteroid pretreatment. However, 24-EBL pretreatment increased the peroxidase activity and improved the K + /Na + ratio in potato leaves, which were likely responsible for the protective 24-EBL action under salt stress.

Plants exhibit several restrictions under waterlogging conditions, including stomatal limitations, negative impacts on gas exchange, lower nutrient uptake and reduced growth. 24-epibrassinolide (EBR) is a polyhydroxylated steroid, with the advantages to be a natural and biodegradable molecule, presenting beneficial roles in metabolic and physiological processes. The aim of this research is to investigate whether EBR can protect soybean plants against damage caused by waterlogging and evaluate the responses associated with the root and leaf anatomy, photosynthetic machinery and biomass. This study used a completely randomized factorial design with two water conditions (control and waterlogging) and three concentrations of 24-epibrassinolide (0, 5 and 10 nM EBR). This steroid stimulated the activities of enzymes linked to the antioxidant system and resulted in minor damage to the chloroplast membranes. EBR maximized the efficiency of photosystem II and improved the gas exchange, which was explained by the higher density and index of the stomata in addition to the increased chlorophyll content and electron transport rate. In root structures, EBR mitigated the impact of waterlogging on vascular cylinder and metaxilem, suggesting maintenance and functions of these structures in plants stressed.

Embryonic studies have demonstrated the neurotoxic, teratogenic, and neurobehavioral toxicity of ethanol (EtOH). Although multiple mechanisms may contribute to these effects, oxidative stress has been described as the major damage pathway. In this regard, natural antioxidants have the potential to counteract oxidative stress-induced cellular damage. Therefore, the present study aimed to investigate the potential protective role of 24-epibrassinolide (24-EPI), a natural brassinosteroid with proved antioxidant properties, in EtOH-induced teratogenic effects during early zebrafish development. Embryos (~2 h post-fertilization - hpf) were exposed to 1 % EtOH, co-exposed to 24-EPI (0.01, 0.1 and 1 μM) and to 24-EPI alone (1 μM) for 24 h. Following exposure, biochemical evaluations were made at 26 hpf, developmental analysis was made throughout the embryo-larval period, and behavioural responses were evaluated at 120 hpf. Exposure to 1 % EtOH caused an increase in the number of malformations, which were diminished by 24-EPI. In addition, EtOH induced an accumulation of GSSG and consequent reduction of GSH:GSSG ratio, indicating the involvement of oxidative mechanisms in the EtOH-induced effects. These were reverted by 24-EPI as proved by the GSSG levels and GSH:GSSG ratio that returned to control values. Furthermore, exposure to EtOH resulted in behavioural deficits at 120 hpf as observed by the disrupted response to an aversive stimulus, suggesting the involvement of neurotoxic mechanisms. 24-EPI restored the behavioural deficits observed in a dose-dependent manner. The absence of effects in the embryos exposed solely to 24-EPI showed its safety during the exposure period. In conclusion, EtOH caused developmental teratogenicity and behavioural toxicity by inducing glutathione changes, which were prevented by 24-EPI., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Brassinosteroids (BRs) are known to improve salt tolerance of plants, but not in all situations. Here, we show that a certain concentration of 24-epibrassinolide (EBL), an active BR, can promote the tolerance of canola under high-salt stress, but the same concentration is disadvantageous under low-salt stress. We define this phenomenon as hormonal stress-level-dependent biphasic (SLDB) effects. The SLDB effects of EBL on salt tolerance in canola are closely related to H 2 O 2 accumulation, which is regulated by polyamine metabolism, especially putrescine (Put) oxidation. The inhibition of EBL on canola under low-salt stress can be ameliorated by repressing Put biosynthesis or diamine oxidase activity to reduce H 2 O 2 production. Genetic and phenotypic results of bri1-9, bak1, bes1-D, and bzr1-1D mutants and overexpression lines of BRI1 and BAK1 in Arabidopsis indicate that a proper enhancement of BR signaling benefits plants in countering salt stress, whereas excessive enhancement is just as harmful as a deficiency. These results highlight the involvement of crosstalk between BR signaling and Put metabolism in H 2 O 2 accumulation, which underlies the dual role of BR in plant salt tolerance., (© 2020 John Wiley & Sons Ltd.)

Involvement of nitrate reductase (NR) and nitric oxide synthase (NOS)-like enzyme in 24-epibrassinolide (EB)-triggered nitric oxide (NO) synthesis to improve iron deficiency (ID) tolerance in strawberry plants was studied. EB was sprayed to strawberry plants every two days for two weeks. Then, the EB-treated plants were pre-treated with inhibitors of NR, tungstate, or NOS, L-NAME for 3 h. During the first three weeks, Fe was supplied as 100 μM EDTA-Fe or FeSO 4 to Fe-sufficient or Fe-deficient plants, respectively. Thereafter, plants were subjected for further three weeks to control (100 μM EDTA-Fe) and Fe deficiency (ID; without Fe). ID reduced biomass, chlorophyll, and chlorophyll fluorescence, while increased oxidative stress parameters, ascorbate (AsA), glutathione (GSH), endogenous NO, and the activities of NR, NOS, and antioxidant enzymes. Pre-treatments with EB and EB + SNP improved ID tolerance of strawberry by improving leaf Fe 2+ , plant growth, and antioxidant enzyme activities, and causing a further elevation in AsA, GSH, NO, NR and NOS. L-NAME application reversed NOS activity, but it did not eliminate NO, however, tungstate application reversed both NR activity and NO synthesis in plants exposed to ID + EB, suggesting that NR is the main contributor of EB-induced NO synthesis to improve ID tolerance in strawberry plants., Competing Interests: Declaration of competing interest The authors declare no conflict of interest among them., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

Lead (Pb) is a highly toxic heavy metal to plants, animals, and human beings. The use of growth regulators has reversed the effects of heavy metal stress on germination and early plant development. The aim of this study was to evaluate the effect of brassinosteroids on seed germination and seedling growth of Brassica juncea (L.) Czern. & Coss. under Pb stress conditions. Two forms of application of 24-epibrassinolide (EBL) were evaluated, application on seeds in pre-soaking and on germination paper, using EBL concentrations of 0, 10 -10 , 10 -8 , and 10 -6  M. Germination and seedling growth parameters were evaluated during the germination test. The activity of the enzymes superoxide dismutase, catalase, peroxidase, and ascorbate peroxidase were determined, as well as the lead content in the seeds and seedlings. The EBL applied at the 10 -8  M concentration was the most effective in overcoming Pb stress in both forms of application. The antioxidant enzyme defense system was compromised by Pb exposure. However, 10 -8  M EBL increased the activity of antioxidant enzymes such as catalase and peroxidase to overcome the toxic effects caused by Pb. In addition, EBL at the concentration of 10 -8  M increased Pb content in seedlings without affecting seedling growth., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Background: Brassinolide (BR), as a new type of plant hormones, is involved in the processes of plant growth and stress response. Previous studies have reported the roles of BR in regulating plant developmental processes and also response tolerance to abiotic stresses in plants. The main purpose of our study was to explore whether nitric oxide (NO) plays a role in the process of BR-induced adventitious root formation in cucumber (Cucumis sativus L.)., Results: Exogenous application of 1 μM BR significantly promoted adventitious rooting, while high concentrations of BR (2-8 μM) effectively inhibited adventitious rooting. NO donor (S-nitroso-N-acerylpenicillamine, SNAP) promoted the occurrence of adventitious roots. Simultaneously, BR and SNAP applied together significantly promoted adventitious rooting and the combined effect was superior to the application of BR or SNAP alone. Moreover, NO scavenger (c-PTIO) and inhibitors (L-NAME and Tungstate) inhibited the positive effects of BR on adventitious rooting. BR at 1 μM also increased endogenous NO content, NO synthase (NOS-like) and Nitrate reductase (NR) activities, while BRz (a specific BR biosynthesis inhibitor) decreased these effects. In addition, the relative expression level of NR was up-regulated by BR and SNAP, whereas BRz down-regulated it. The application of NO inhibitor (Tungstate) in BR also inhibited the up-regulation of NR., Conclusion: BR promoted the formation of adventitious roots by inducing the production of endogenous NO in cucumber.

Lupinus angustifolius L. is a legume culture known as a source of valuable feed protein and the N 2 -fixator for improving soil fertility. However, its low ecological resistance does not allow for a stable yield of the crop. Earlier, we have shown that steroid phytohormone 24-epibrassinolide (EBR) increases the tolerance of lupine to chlorine ions by activating the protective proteins in ripening seeds (such as proteinase inhibitors that prevent protein breakdown) and lectins. Here we investigated the effect of EBR on the functional status of the N 2 -fixing system in root nodules, protein synthesis in ripening seeds and the resistance of lupine plants to various pathogens. It was found that EBR enhanced the nodulation process, N 2 -fixing activity of nitrogenase and the accumulation of poly-β-hydroxybutirate in the bacteroides, increased the leghemoglobin content in the nodules as well as the metabolic activity of bacteroides. According to data on the inclusion of 14 C-leucine in maturing seed proteins, EBR increased the accumulation of protein in them against the background of a short-term decrease in protein synthesis and its subsequent regeneration to the control level. Gradual inhibition of protein synthesis, characteristic of other legumes, was observed in control variants of lupine. EBR increased lupine resistance to phytopathogenic fungi of Colletotrichum genus and insects of Noctuidae and Scarabaeidae families. We concluded that a more complete implementation of the potential productivity and sustainability of lupine under the action of EBR was achieved due to the anabolic/anti-catabolic effect on the N 2 fixation system in root nodules, as well as on protein synthesis in ripening seeds., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Lead is one of the most hazardous pollutants to both the environment as well as human beings. As one of the approaches to enhance phytoremediation, brassinosteroids are predicted as a potential candidate phytohormone for assisted phytoremediation. Few studies have focused on the physiological regulations of tall fescue plants (Festuca arundinacea Schreb.), a potential phytoremediation species, for its responses to applications of brassinosteroids under lead stress. Therefore, the objectives of this study were to investigate the effects of foliar application of 24-epibrassinolide, a brassinosteroids analogue, on reactive oxygen species accumulation and antioxidative defense systems of tall fescue when exposed to lead, and ultimately its potential to be used in phytoremediation. When exposed to lead (1000 mg/kg) for 80 d, decreases in shoot and root biomass of tall fescue biomass as well as chlorophyll and carotenoid productions were found. Foliar application of 24-epibrassinolide at three rates and five applications every 7 d improved the biomass of both shoots and roots, and increased the photosynthetic pigments. The improved lead tolerance in tall fescue plants after 24-epibrassinolide applications was associated with reduced H 2 O 2 and O 2 .- accumulations and increased antioxidative enzyme activities including superoxide dismutase, catalase, and guaiacol peroxidase. Additionally, osmoprotectants increased and lipid peroxidation decreased. Ultimately, foliar applications of 24-epibrassinolide enhanced the lead recovery rate of tall fescue plants, proving its potential role in phytoremediation for soil contaminated with heavy metals such as lead., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Arabidopsis PIN2 protein directs transport of the phytohormone auxin from the root tip into the root elongation zone. Variation in hormone transport, which depends on a delicate interplay between PIN2 sorting to and from polar plasma membrane domains, determines root growth. By employing a constitutively degraded version of PIN2, we identify brassinolides as antagonists of PIN2 endocytosis. This response does not require de novo protein synthesis, but involves early events in canonical brassinolide signaling. Brassinolide-controlled adjustments in PIN2 sorting and intracellular distribution governs formation of a lateral PIN2 gradient in gravistimulated roots, coinciding with adjustments in auxin signaling and directional root growth. Strikingly, simulations indicate that PIN2 gradient formation is no prerequisite for root bending but rather dampens asymmetric auxin flow and signaling. Crosstalk between brassinolide signaling and endocytic PIN2 sorting, thus, appears essential for determining the rate of gravity-induced root curvature via attenuation of differential cell elongation.

Plant steroid hormones brassinosteroids (BRs) regulate plant growth and development at many different levels. Recent research has revealed that stress-responsive NAC (petunia NAM and Arabidopsis ATAF1, ATAF2, and CUC2) transcription factor RD26 is regulated by BR signaling and antagonizes BES1 in the interaction between growth and drought stress signaling. However, the upstream signaling transduction components that activate RD26 during drought are still unknown. Here, we demonstrate that the function of RD26 is modulated by GSK3-like kinase BIN2 and protein phosphatase 2C ABI1. We show that ABI1, a negative regulator in abscisic acid (ABA) signaling, dephosphorylates and destabilizes BIN2 to inhibit BIN2 kinase activity. RD26 protein is stabilized by ABA and dehydration in a BIN2-dependent manner. BIN2 directly interacts and phosphorylates RD26 in vitro and in vivo. BIN2 phosphorylation of RD26 is required for RD26 transcriptional activation on drought-responsive genes. RD26 overexpression suppressed the brassinazole (BRZ)  insensitivity of BIN2 triple mutant bin2 bil1 bil2, and BIN2 function is required for the drought tolerance of RD26 overexpression plants. Taken together, our data suggest a drought signaling mechanism in which drought stress relieves ABI1 inhibition of BIN2, allowing BIN2 activation. Sequentially, BIN2 phosphorylates and stabilizes RD26 to promote drought stress response., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Background: Brassinosteroids (BRs) have a positive effect on many processes during plant growth and development, and in response to various abiotic stressors. Low-temperature (LT) stress constricts the geographic distribution, growth, and development of wucai (Brassica campestris L. ssp. chinensis var. rosularis Tsen). However, there is little information on the global gene expression of BRs under LT stress in wucai. In this study, the molecular roles of 24-epibrassinolide (EBR) after exogenously application, were explored by RNA sequencing under LT conditions., Results: According to the Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses, photosynthesis was significantly enriched after spraying EBR under LT. The transcripts encoding the photosystem II (PSII) oxygen-evolving enhancer protein, photosystem I (PSI) subunit, light-harvesting chlorophyll protein complexes I and II, and ferredoxin were up-regulated after the application of EBR. Transcripts encoding several key enzymes involved in chlorophyll biosynthesis were also up-regulated, accompanied by significant differences in the contents of 5-aminolevulinic acid (ALA), porphobilinogen (PBG), protoporphyrin IX (Proto IX), Mg-protoporphyrin IX (Mg-proto IX), protochlorophyllide (Pchl), and photosynthetic pigments. Notably, transcriptional and physiological analyses revealed that under LT stress, plant responses to EBR involved a major reorientation of photosynthesis, as well as porphyrin and chlorophyll metabolism., Conclusion: This study explored the role of EBR as an LT stress tolerance mechanism in wucai. At the transcription level, LT tolerance manifests as an enhancement of photosynthesis, and the amelioration of porphyrin and chlorophyll metabolism.

: The present research was performed to assess the effect of 24-epibrassinolide (EBR) on salt-stressed soybean plants. Salt stress suppressed growth, biomass yield, gas exchange parameters, pigment content, and chlorophyll fluorescence, but all these parameters were up-regulated by EBR supply. Moreover, salt stress increased hydrogen peroxide, malondialdehyde, and electrolyte leakage. EBR supplementation reduced the accumulation of oxidative stress biomarkers. The activities of superoxide dismutase and catalase, and the accumulation of proline, glycinebetaine, total phenols, and total flavonoids increased with NaCl stress, but these attributes further increased with EBR supplementation. The activities of enzymes and the levels of non-enzymatic antioxidants involved in the Asc-Glu cycle also increased with NaCl stress, and further enhancement in these attributes was recorded by EBR supplementation. Salinity elevated the methylglyoxal content, but it was decreased by the EBR supplementation accompanying with up-regulation of the glyoxalase cycle (GlyI and GlyII). Salinity enhanced the Na + uptake in root and shoot coupled with a decrease in uptake of Ca 2+ , K + , and P. However, EBR supplementation declined Na + accumulation and promoted the uptake of the aforementioned nutrients. Overall, EBR supplementation regulated the salt tolerance mechanism in soybean plants by modulating osmolytes, activities of key enzymes, and the levels of non-enzymatic antioxidants., Competing Interests: The authors declare no conflict of interest.

The aim of current investigation was to perform proteomics and physio-chemical studies to dissect the changes in contrasting varieties (S-22 and PKM-1) of Lycopersicon esculentum under low-temperature stress. Plant grown under variable low-temperature stress were analysed for their growth biomarkers, antioxidant enzyme activities, and other physiological parameters, which headed toward the determination of protein species responding to low-temperature and 24-epibrassinolide (EBL) concentrations. The plants grown under temperatures, 20/14, 12/7, and 10/3 °C recorded significantly lower growth biomarkers, SPAD chlorophyll, net photosynthetic rate and carbonic anhydrase activity in S-22 and PKM-1. Moreover, the combined effect of EBL and hydrogen peroxide (H 2 O 2 ) significantly improved the parameters mentioned above and consecutively upgraded the different antioxidant enzymes (CAT and SOD) with higher accumulation of proline under stress and stress-free environments. Furthermore, proteomics study revealed that the maximum number of differentially expressed proteins were detected in S-22 (EBL + H 2 O 2 ); while treatment with EBL + H 2 O 2  + low temperature lost expression of 20 proteins. Overall, three proteins (O80577, Q9FJQ8, and Q9SKL2) took a substantial part in the biosynthesis of citrate cycle pathway and enhanced the growth and photosynthetic efficiency of tomato plants under low-temperature stress., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Oryza sativa BRASSINAZOLE RESISTANT 1 (OsBZR1) is the closest rice homolog of the Arabidopsis BZR1 and bri1-EMS-SUPPRESSOR 1 (BES1)/BZR2 transcription factors. OsBZR1 plays a central role in the rice brassinosteroid signaling pathway. Despite its functional importance, the control mechanism by which the cellular stability of OsBZR1 is regulated has not yet been fully elucidated. Here, we report that a rice U-box E3 ubiquitin (Ub) ligase OsPUB24 acts as a negative regulator in the BR signaling pathway via the 26S proteasome-dependent degradation of OsBZR1. The ospub24 T-DNA knock-out mutant and Ubi:RNAi-OsPUB24 knock-down rice plants displayed enhanced seedling growth, increased lamina joint bending, and hypersensitivity to brassinolide (BL). The expressions of the BR biosynthetic genes suppressed by BR in a negative feedback loop were lower in the mutant progeny than in the wild-type rice plants, which indicated increased BR responses in the mutant line. OsPUB24 ubiquitinated OsBZR1, resulting in the proteasomal degradation of OsBZR1. In addition, the stability of OsPUB24 was downregulated by BL and bikinin, an inhibitor of Oryza sativa Shaggy/GSK3-like kinase 22 (OsSK22). OsSK22, the homolog of Arabidopsis BRASSINOSTEROID INSENSITIVE 2 (BIN2) protein kinase, phosphorylated OsPUB24 and elevated the cellular stability of OsPUB24. Our findings suggest that OsPUB24 participates in OsBZR1 turnover, and that the regulatory networks of OsPUB24, OsSK22 and OsBZR1 are crucial for fine-tuning the BR response in rice., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

In this study, the effect of 24-Epibrassinolide (EBL) on antioxidant system in Arabidopsis thaliana were investigated under arsenate [As(V)] stress. The enzyme activity of superoxide dismutase (SOD) and catalase (CAT), total antioxidant status, malondialdehyde (MDA) level and free proline content, as well as the expression levels of SOD isoforms (Cu-ZnSODs, FeSODs and MnSOD), CAT isoforms (CAT1, CAT2 and CAT3), some heat shock proteins (Hsp70-4 and Hsp90-1) and proline biosynthesis (P5CS1 and P5CS2) genes were determined in rosette leaves of eight-week old plants under exposure of 100 and 200 μM As(V) and/or 1 μM EBL treatments for 24 h. Total SOD and CAT enzyme activities increased as a result of 100 μM As(V) + EBL treatments compared to 100 μM As(V) treatment. Total antioxidant and proline levels increased in plants subjected to As(V), and the treatment of EBL together with stress caused further increase. As the MDA level increased in As-treated plants, 100 μM As(V) + EBL treatment decreased MDA level. Transcript levels of CSD1, CSD2, FSD1, FSD2, MSD1 and CAT2 genes increased as a result of combined treatment of EBL and As(V) compared to control and alone stress treatments (except CSD1 gene). Expression level of CSD3, CAT1 and CAT3 genes were downregulated in response to As(V) and/or EBL treatments. EBL application alone and in combination with As(V) elevated the expression level of P5CS1 gene dramatically. Treatment with 100 μM As(V) and EBL increased the transcript level of Hsp70-4 and Hsp90-1 genes in leaves compared to 100 μM As(V) treatment. To our best knowledge, this is the first detailed study to evaluate the improving effect of EBL on antioxidant defense system at biochemical and transcriptional level in A. thaliana plants under As(V) stress., (Copyright © 2019 Elsevier GmbH. All rights reserved.)