Synergistic effects of melatonin and 24-epibrassinolide on chickpea water deficit tolerance.

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 H2O2 (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.

Cold Acclimation and Deacclimation of Winter Oilseed Rape, with Special Attention Being Paid to the Role of Brassinosteroids.

Winter plants acclimate to frost mainly during the autumn months, through the process of cold acclimation. Global climate change is causing changes in weather patterns such as the occurrence of warmer periods during late autumn or in winter. An increase in temperature after cold acclimation can decrease frost tolerance, which is particularly dangerous for winter crops. The aim of this study was to investigate the role of brassinosteroids (BRs) and BR analogues as protective agents against the negative results of deacclimation. Plants were cold-acclimated (3 weeks, 4 °C) and deacclimated (1 week, 16/9 °C d/n). Deacclimation generally reversed the cold-induced changes in the level of the putative brassinosteroid receptor protein (BRI1), the expression of BR-induced COR, and the expression of SERK1, which is involved in BR signal transduction. The deacclimation-induced decrease in frost tolerance in oilseed rape could to some extent be limited by applying steroid regulators. The deacclimation in plants could be detected using non-invasive measurements such as leaf reflectance, chlorophyll a fluorescence, and gas exchange monitoring.

Exogenous 24-epibrassinolide (EBL) facilitates cell growth of Chlorella pyrenoidosa under high temperatures by enhancing the photosynthetic energy utilization and alleviating oxidative damage.

The microalga Chlorella pyrenoidosa is cultivated extensively for its constituents, which are of significant economic worth. Large-scale growth of C. pyrenoidosa in outdoor environments is subject to various stressors such as elevated temperature. The purpose of this study was to assess the protective effects of exogenous 24-epibrassinolide (EBL) on C. pyrenoidosa under high-temperature conditions. Compared to a temperature of 30°C, increasing the temperature to 43°C reduced the enzymatic capacity for carbon assimilation and resulted in the buildup of reactive oxygen species (ROS), thus reducing photosynthesis and proliferation. It was observed that exogenous EBL protected C. pyrenoidosa cells against high temperatures, with an optimal EBL concentration of 100 nM, resulting in enhanced capacity for photosynthetic carbon assimilation with a notable reduction in the imbalance between the absorption of light and energy used under high-temperature conditions. The addition of 100 nM EBL resulted in a 25.4% increase in cell density when exposed to elevated temperatures for 7 days. In addition, exogenous EBL reduced ROS production and increased the activities of critical antioxidant enzymes. This, in turn, mitigated heat-induced oxidative damage, resulting in advantageous outcomes in terms of cellular development and maintenance.

HvGSK1.1 Controls Salt Tolerance and Yield through the Brassinosteroid Signaling Pathway in Barley.

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.

RNAi-Mediated Suppression of OsBBTI5 Promotes Salt Stress Tolerance in Rice.

This study explores the impact of RNAi in terms of selectively inhibiting the expression of the OsBBTI5 gene, with the primary objective of uncovering its involvement in the molecular mechanisms associated with salt tolerance in rice. OsBBTI5, belonging to the Bowman-Birk inhibitor (BBI) family gene, is known for its involvement in plant stress responses. The gene was successfully cloned from rice, exhibiting transcriptional self-activation in yeast. A yeast two-hybrid assay confirmed its specific binding to OsAPX2 (an ascorbate peroxidase gene). Transgenic OsBBTI5-RNAi plants displayed insensitivity to varying concentrations of 24-epibrassinolide in the brassinosteroid sensitivity assay. However, they showed reduced root and plant height at high concentrations (10 and 100 µM) of GA3 immersion. Enzyme activity assays revealed increased peroxidase (POD) and superoxide dismutase (SOD) activities and decreased malondialdehyde (MDA) content under 40-60 mM NaCl. Transcriptomic analysis indicated a significant upregulation of photosynthesis-related genes in transgenic plants under salt stress compared to the wild type. Notably, this study provides novel insights, suggesting that the BBI gene is part of the BR signaling pathway, and that OsBBTI5 potentially enhances stress tolerance in transgenic plants through interaction with the salt stress-related gene OsAPX2.

Physiological Characteristics and Transcriptome Analysis of Exogenous Brassinosteroid-Treated Kiwifruit.

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 H2O2 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 H2O2 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.

Effects of Plant Regulators on the Seed Germination and Antioxidant Enzyme Activity of Cotton under Compound Salt Stress.

Salinity stress significantly hampers cotton seed germination and seedling growth. Employing plant growth regulators stands out as an effective strategy to mitigate salt stress. In this study, we assessed the impact of varying concentrations of natural composite salt conditions (0%, 0.6%, and 1.2%) on cotton seed germination, seedling growth, and physiology. Additionally, we explored the effects of compound sodium nitrophenolate (CSN: 2 mg·L-1 and 10 mg·L-1), 24-epibrassinolide (EBR: 0.02 mg·L-1 and 0.1 mg·L-1), and gibberellic acid (GA: 60 mg·L-1 and 300 mg·L-1), against a control (CK: distilled water) group. The results indicate that with an increase in the composite salt concentration, the germination potential (GP) and germination rate (GR) of cotton seeds gradually decrease. Simultaneously, the fresh weight and root vitality of seedlings also correspondingly decrease, while the degree of membrane lipid peroxidation increases. Under high-salt (1.2%) conditions, soaking treatments with CSN and EBR significantly enhance both GP (45-59% and 55-64%) and GR (30-33% and 39-36%) compared to the CK. However, the GA treatment does not increase the GP and GR of cotton. Moreover, under high-salt (1.2%) conditions, CSN and EBR treatments result in the increased activities of superoxide dismutase (56-66% and 71-80%), peroxidase (20-24% and 37-51%), and catalase (26-32% and 35-46%). Consequently, cotton exhibits a relatively good tolerance to natural composite salts. Soaking treatments with CSN and EBR effectively improve cotton germination by enhancing antioxidant enzyme activities, thereby strengthening cotton's tolerance to salt stress. These findings offer new insights for enhancing the salt tolerance of cotton.

Assessing the effects of 24-epibrassinolide and yeast extract at various levels on cowpea's morphophysiological and biochemical responses under water deficit stress.

BACKGROUND: Due to the factor of water deficit, which has placed human food security at risk by causing a 20% annual reduction in agricultural products, addressing this growing peril necessitates the adoption of inventive strategies aimed at enhancing plant tolerance. One such promising approach is employing elicitors such as 24-epibrassinolide (EBR) and yeast extract, which are potent agents capable of triggering robust defense responses in plants. By employing these elicitors, crops can develop enhanced adaptive mechanisms to combat water deficit and improve their ability to withstand drought condition. This study investigates the impact of different levels of EBR (0, 5, 10 µm) and yeast extract (0 and 12 g/l) on enhancing the tolerance of cowpea to water deficit stress over two growing seasons. RESULTS: The findings of this study demonstrate that, the combined application of EBR (especially 10 µm) and yeast extract (12 g/l) can increase seed yield (18%), 20-pod weight (16%), the number of pods per plant (18%), total chlorophyll content (90%), and decrease malondialdehyde content (45%) in cowpea, compared to plants grown under water deficit stress without these treatments. Upon implementing these treatments, impressive results were obtained, with the highest recorded values observed for the seed yield (1867.55 kg/ha), 20-pod weight (16.29 g), pods number per plant (9), and total chlorophyll content (19.88 mg g-1 FW). The correlation analysis indicated a significant relationship between the seed yield, and total chlorophyll (0.74**), carotenoids (0.82**), weight of 20 seeds (0.67**), and number of pods (0.90**). These traits should be prioritized in cowpea breeding programs focusing on water deficit stress. CONCLUSIONS: The comprehensive exploration of the effects of EBR and yeast extract across various levels on cowpea plants facing water deficit stress presents a pivotal contribution to the agricultural domain. This research illuminates a promising trajectory for future agricultural practices and users seeking sustainable solutions to enhance crops tolerance. Overall, the implications drawn from this study contribute significantly towards advancing our understanding of plant responses to water deficit stress while providing actionable recommendations for optimizing crop production under challenging environmental conditions.

The Exogenous Application of 24-Epibrassinolide (24-EBL) Increases the Cd and Pb Resilience in Zea mays (L.) by Regulating the Growth and Physiological Mechanism.

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.

Exogenous Application of 24-Epibrassinolide Confers Saline Stress and Improves Photosynthetic Capacity, Antioxidant Defense, Mineral Uptake, and Yield in Maize.

Salinity is one of the major environmental stresses threatening crop production, the natural ecosystem, global food security, and the socioeconomic health of humans. Thus, the development of eco-friendly strategies to mitigate saline stress and/or enhance crop tolerance is an important issue worldwide. Therefore, this study was conducted during the summer of 2022 to investigate the potential of 24-Epibrassinolide (EBL) for mitigating saline stress and improving photosynthetic capacity, antioxidant defense systems, mineral uptake, and yield in maize (Zea mays L.) grown under a controlled hydroponic system. Three saline stress levels-S1 (control/no added NaCl), S2 (60 mM NaCl), and S3 (120 mM NaCl)-were continuously applied with nutrient solution, whereas exogenous EBL (i.e., control, 0.1 µM and 0.2 µM) was applied as exogenous application three times (i.e., 40, 55, 70 days after sowing). The experiment was designed as a split-plot in a randomized complete block design (RCBD) in which saline stress was the main factor and EBL treatment was the sub-factor. Results showed that saline stress significantly affected plant growth, physiological performance, biochemistry, antioxidant activity, and yield attributes. However, the exogenous application of EBL at 0.2 µM significantly mitigated the salt stress and thus improved plant performance even under 120 mM NaCl saline stress. For instance, as compared to untreated plants (control), 0.2 µM EBL application improved plant height (+18%), biomass (+19%), SPAD (+32%), Fv/Fm (+28%), rate of photosynthesis (+11%), carboxylation efficiency (+6%), superoxide dismutase (SOD +14%), catalase (CAT +18%), ascorbate peroxidase (APX +20%), K+ (+24%), 100-grain weight (+11%), and grain yield (+47%) of maize grown under salt stress. Additionally, it resulted in a 23% reduction in Na+ accumulation in leaves and a 25% reduction in for Na+/K+ ratio under saline stress as compared to control. Furthermore, the Pearson's correlation and principal component analysis (PCA) highlighted the significance of exogenous EBL as saline stress mitigator in maize. Overall, our results indicated the protective effects of EBL application to the alleviation of saline stress in crop plants. However, further exploration of its mechanism of action and crop-specific response is suggested prior to commercial use in agriculture.

Preharvest 24-epibrassinolide treatment prolongs harvest duration and shelf life in sweet corn.

Sweet corn is perishable and have limited harvest duration and shelf life due to their quality deterioration. Reactive oxygen species (ROS) are one of the most predominant factors for maintaining quality of sweet corn during and after harvest. Brassinosteroids (BRs) can enhance the activity of antioxidant enzymes and decrease the ROS level in plants. In this study, we found that a bioactive BR (24-epibrassinolide, EBR) treatment before harvest markedly inhibited change of quality indicators (MDA content, weight loss rate, and soluble sugar content) during and after harvest. Further analysis revealed that EBR promoted the activity and transcriptions of antioxidant enzymes, maintaining lower ROS level in kernels. Meanwhile, exogenous EBR increased the expression level of genes controlling sucrose transport in sweet corn kernels. Bioinformatics and binding analysis identified that BR transcription factor ZmBES1/ZmBZR1-10 might potentially bind to and upregulate transcriptions of antioxidant enzyme genes including SOD and POD genes, and sucrose transport-related genes including SUT and SWEET genes. These results indicated that exogenous application of EBR ameliorates quality during and after harvest by improving the antioxidant capacity and photosynthetic assimilates accumulation rate of sweet corn, thus prolonging harvest duration and shelf life in sweet corn.

24-Epibrassinolide Facilitates Adventitious Root Formation by Coordinating Cell-Wall Polyamine Oxidase- and Plasma Membrane Respiratory Burst Oxidase Homologue-Derived Reactive Oxygen Species in Capsicum annuum L.

Adventitious root (AR) formation is a critical process in cutting propagation of horticultural plants. Brassinosteroids (BRs) have been shown to regulate AR formation in several plant species; however, little is known about their exact effects on pepper AR formation, and the downstream signaling of BRs also remains elusive. In this study, we showed that treatment of 24-Epibrassinolide (EBL, an active BR) at the concentrations of 20-100 nM promoted AR formation in pepper (Capsicum annuum). Furthermore, we investigated the roles of apoplastic reactive oxygen species (ROS), including hydrogen peroxide (H2O2) and superoxide radical (O2•-), in EBL-promoted AR formation, by using physiological, histochemical, bioinformatic, and biochemical approaches. EBL promoted AR formation by modulating cell-wall-located polyamine oxidase (PAO)-dependent H2O2 production and respiratory burst oxidase homologue (RBOH)-dependent O2•- production, respectively. Screening of CaPAO and CaRBOH gene families combined with gene expression analysis suggested that EBL-promoted AR formation correlated with the upregulation of CaPAO1, CaRBOH2, CaRBOH5, and CaRBOH6 in the AR zone. Transient expression analysis confirmed that CaPAO1 was able to produce H2O2, and CaRBOH2, CaRBOH5, and CaRBOH6 were capable of producing O2•-. The silencing of CaPAO1, CaRBOH2, CaRBOH5, and CaRBOH6 in pepper decreased the ROS accumulation and abolished the EBL-induced AR formation. Overall, these results uncover one of the regulatory pathways for BR-regulated AR formation, and extend our knowledge of the functions of BRs and of the BRs-ROS crosstalk in plant development.

Assessment of Biological Activity of 28-Homobrassinolide via a Multi-Level Comparative Analysis.

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 24R-methyl-epibrassinolide (24-EBL) and 24S-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 (22R,23R)-28-HBL and inactive (22S,23S)-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.

Exogenous 24-epibrassinolide promoted growth and nitrogen absorption and assimilation efficiency of apple seedlings under salt stress.

Introduction: High salinity significantly hampers global agricultural productivity. Plants typically undergo lower nitrogen utilization efficiency (NUE) under salt stress. As an active byproduct from brassinolide biosynthesis, 24-epibrassinolide (EBR) is involved in regulating the stress-treated plant N absorption and assimilation. However, the exogenous EBR application effects' on N absorption and assimilation in apple exposed to the salt-stressed condition remains unclear. Methods: We sprayed exogenous EBR (0.2 mg L-1) on apple dwarf rootstock (M9T337) seedlings (growing hydroponically) under salt (NaCl) stress in a growth chamber. We analyzed the seedling development, photosynthesis and its-mediated C fixation, N ( NO 3 - ) absorption and assimilation in reponse to exogenous EBR application under salt stress. Results: The findings demonstrated that NaCl stress greatly hampered seedlings' root growth and that exogenous EBR application obviously alleviated this growth suppression. Exogenous EBR-treated plants under NaCl stress displayed the more ideal root morphology and root activity, stronger salt stress tolerance and photosynthetic capacity as well as higher C- and N-assimilation enzyme activities, NO 3 - ion flow rate and nitrate transporter gene expression level than did untreated plants. Furthermore, the results of isotope labeling noted that exogenous EBR application also enhanced 13C-photoassimilate transport from leaves to roots and 15 NO 3 - transport from roots to leaves under NaCl stress. Conclusion: Our findings imply that exogenous EBR application, through strengthening photosynthesis, C- and N-assimilation enzyme activities, nitrate absorption and transport as well as synchronized optimizing the distribution of seedlings' C and N, has a fundamental role in improving NUE in apple rootstock seedlings under salt stress.

24-Epibrassinolide confers zinc stress tolerance in watermelon seedlings through modulating antioxidative capacities and lignin accumulation.

Zinc (Zn) is an important element in plants, but over-accumulation of Zn is harmful. It is well-known that brassinolide (BR) plays a key role in the regulation of abiotic stress responses in plants. However, the effects of brassinolide on alleviating Zn phytotoxicity in watermelon (Citrullus lanatus L.) seedlings are not clear. The purpose of this study was to study the effect of 24-epibrassinolide (EBR, one of the bioactive BRs) on Zn tolerance of watermelon seedlings and its potential resistance mechanism. Exposure to excessive Zn significantly inhibited shoot and root fresh weight of watermelon, but this could be significantly alleviated by the optimum 0.05 µM EBR. Exogenous spraying EBR increased the pigments and alleviated oxidative damage caused by Zn through reducing Zn accumulation and the levels of reactive oxygen species (ROS) and malonaldehyde (MDA) and increasing the activities of antioxidant enzymes and contents of ascorbic acid (AsA) and glutathione (GSH). Importantly, the relative mRNA levels of antioxidant genesincluding Cu/Zn-superoxidedismutase (Cu-Zn SOD), catalase (CAT), ascorbic acid peroxidase (APX), and glutathione reductase (GR) were significantly induced after EBR treatment. In addition, EBR pre-treatment induced lignin accumulation under Zn stress, and the activity of phenylalanine ammonia-lyase (PAL) and 4-coumaric ligase (4CL), two key enzymes regulating lignin synthesis, also tended to be consistent. Collectively, the present research proves the beneficial effects of EBR in response to Zn stress through enhancing antioxidant defense and lignin accumulation and provides a new insight into the mechanism of BR-enhancing heavy metal tolerance.

Protective effects of 24-epibrassinolide against the 6-OHDA zebrafish model of Parkinson's disease.

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.

Exogenous Brassinosteroid Enhances Zinc tolerance by activating the Phenylpropanoid Biosynthesis pathway in Citrullus lanatus L.

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 (H2O2), 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.

Epi-Brassinolide Regulates ZmC4 NADP-ME Expression through the Transcription Factors ZmbHLH157 and ZmNF-YC2.

Maize is a main food and feed crop with great production potential and high economic benefits. Improving its photosynthesis efficiency is crucial for increasing yield. Maize photosynthesis occurs mainly through the C4 pathway, and NADP-ME (NADP-malic enzyme) is a key enzyme in the photosynthetic carbon assimilation pathway of C4 plants. ZmC4-NADP-ME catalyzes the release of CO2 from oxaloacetate into the Calvin cycle in the maize bundle sheath. Brassinosteroid (BL) can improve photosynthesis; however, its molecular mechanism of action remains unclear. In this study, transcriptome sequencing of maize seedlings treated with epi-brassinolide (EBL) showed that differentially expressed genes (DEGs) were significantly enriched in photosynthetic antenna proteins, porphyrin and chlorophyll metabolism, and photosynthesis pathways. The DEGs of C4-NADP-ME and pyruvate phosphate dikinase in the C4 pathway were significantly enriched in EBL treatment. Co-expression analysis showed that the transcription level of ZmNF-YC2 and ZmbHLH157 transcription factors was increased under EBL treatment and moderately positively correlated with ZmC4-NADP-ME. Transient overexpression of protoplasts revealed that ZmNF-YC2 and ZmbHLH157 activate C4-NADP-ME promoters. Further experiments showed ZmNF-YC2 and ZmbHLH157 transcription factor binding sites on the -1616 bp and -1118 bp ZmC4 NADP-ME promoter. ZmNF-YC2 and ZmbHLH157 were screened as candidate transcription factors mediating brassinosteroid hormone regulation of the ZmC4 NADP-ME gene. The results provide a theoretical basis for improving maize yield using BR hormones.

Seed Priming with Nanoparticles and 24-Epibrassinolide Improved Seed Germination and Enzymatic Performance of Zea mays L. in Salt-Stressed Soil.

Saline stress is one of the most critical abiotic stress factors that can lessen crops' productivity. However, emerging nanotechnology, nano-fertilizers, and developing knowledge of phytochromes can potentially mitigate the negative effects of saline stress on seed germination. Therefore, the aim of this study was to investigate the effects of seed priming either with zinc oxide nanoparticles (ZnO-NPs; 50 and 100 mg L-1) or 24-epibrassinolide (EBL; 0.2 and 0.4 µM) and their combinations on maize (Zea mays L.) grains sown in salt-stressed soil (50 and 100 mM NaCl). Saline stress treatments significantly affected all germination traits and chemical analysis of seeds as well as α-amylase activity. Compared to un-primed seeds, seed priming with ZnO-NPs or EBL and their combinations significantly increased the cumulative germination percentage, germination energy, imbibition rate, increase in grain weight, K+ content, and α-amylase activity, and significantly reduced germination time, days to 50% emergence, Na+ uptake, and Na+/K+ ratio of maize sown in salt-stressed-soil (50 or 100 mM NaCl). The combination of 100 mg ZnO-NPs L-1 + 0.2 µM EBL resulted in the highest improvements for most of the studied traits of maize seeds sown in salt-stressed soil in comparison to all other individual and combined treatments.

Effects of the timing of epibrassinolide spraying on yield and nitrogen use efficiency of wide-belt sowing wheat.

In this study, we investigated the effects of epibrassinolide spraying at different growth stages on grain yield and nitrogen use efficiency (NUE), and uptake efficiency (UPE) of wide-belt sowing wheat. The results showed that epibrassinolide spraying enhanced wheat grain yield by increasing the number of kernels per spike and (or) 1000-kernel weight, and improved NUE by promoting aboveground nitrogen accumulation and improving UPE. However, the magnitudes of such enhancements in yield and NUE differed among spraying times. Spraying epibrassinolide at the erecting and filling stages, jointing and filling stages, erecting, jointing, and filling stages, as well as erecting, flowering, and filling stages, produced the greatest increase in the number of kernels per spike and 1000-kernel weight, which led to substantial yield increases (12.8%-14.0%), and the greatest increase in aboveground nitrogen accumulation, which improved UPE by 16.4%-18.8%, and resulted in a significant improvement in NUE. Therefore, spraying epibrassinolide at the erecting and filling stage or jointing and filling stages could achieve high yield and NUE in wide-belt sowing wheat.