Transcriptomic and Physiological Studies Unveil that Brassinolide Maintains the Balance of Maize's Multiple Metabolisms under Low-Temperature Stress.

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.

PmLBD3 links auxin and brassinosteroid signalling pathways on dwarfism in Prunus mume.

BACKGROUND: Grafting with dwarf rootstock is an efficient method to control plant height in fruit production. However, the molecular mechanism remains unclear. Our previous study showed that plants with Prunus mume (mume) rootstock exhibited a considerable reduction in plant height, internode length, and number of nodes compared with Prunus persica (peach) rootstock. The present study aimed to investigate the mechanism behind the regulation of plant height by mume rootstocks through transcriptomic and metabolomic analyses with two grafting combinations, 'Longyan/Mume' and 'Longyan/Peach'. RESULTS: There was a significant decrease in brassinolide levels in plants that were grafted onto mume rootstocks. Plant hormone signal transduction and brassinolide production metabolism gene expression also changed significantly. Flavonoid levels, amino acid and fatty acid metabolites, and energy metabolism in dwarf plants decreased. There was a notable upregulation of PmLBD3 gene expression in plant specimens that were subjected to grafting onto mume rootstocks. Auxin signalling cues promoted PmARF3 transcription, which directly controlled this upregulation. Through its binding to PmBAS1 and PmSAUR36a gene promoters, PmLBD3 promoted endogenous brassinolide inactivation and inhibited cell proliferation. CONCLUSIONS: Auxin signalling and brassinolide levels are linked by PmLBD3. Our findings showed that PmLBD3 is a key transcription factor that regulates the balance of hormones through the auxin and brassinolide signalling pathways and causes dwarf plants in stone fruits.

24-epibrassinolide enhances drought tolerance in grapevine (Vitis vinifera L.) by regulating carbon and nitrogen metabolism.

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.

24-Epibrassinolide mitigates arsenate stress in seedlings of Oryza sativa (IR-20) via the induction of phenylpropanoid pathway.

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.

Exploring phytoremediation potential of willow NJU513 for cadmium-contaminated soil with and without epibrassinolide treatment.

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.

A cotton endoreduplication gene, GaTOP6B, regulates trichome branching development.

Trichomes are specialized epidermal structures that protect plants from biotic and abiotic stresses by synthesizing, storing, and secreting defensive compounds. This study investigates the role of the Gossypium arboreum DNA topoisomerase VI subunit B gene (GaTOP6B) in trichome development and branching. Sequence alignment revealed a high similarity between GaTOP6B and AtTOP6B, suggesting a conserved function in trichome regulation. Although AtTOP6B acts as a positive regulator of trichome development, functional analyses showed contrasting effects: Virus-induced gene silencing (VIGS) of GaTOP6B in cotton increased trichome density, while its overexpression in Arabidopsis decreased trichome density but enhanced branching. This demonstrates that GaTOP6B negatively regulates trichome number, indicating species-specific roles in trichome initiation and branching between cotton and Arabidopsis. Overexpression of the GaTOP6B promotes jasmonic acid synthesis, which in turn inhibits the G1/S or G2/M transitions, stalling the cell cycle. On the other hand, it suppresses brassinolide synthesis and signaling while promoting cytokinin degradation, further inhibiting mitosis. These hormonal interactions facilitate the transition of cells from the mitotic cycle to the endoreduplication cycle. As the level of endoreduplication increases, trichomes develop an increased number of branches. These findings highlight GaTOP6B's critical role as a regulator of trichome development, providing new genetic targets for improving cotton varieties in terms of enhanced adaptability and resilience.

Mitigating Ni and Cu ecotoxicity in the ecological restoration material and ornamental Primula forbesii Franch. with exogenous 24-epibrassinolide and melatonin.

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.

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.

24-epibrassinolide as a multidimensional regulator of rice (Oryza sativa) physiological and molecular responses under isoproturon stress.

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.

BR regulates wheat root salt tolerance by maintaining ROS homeostasis.

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 H2O2 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.

Molecular mechanisms underlying low temperature inhibition of grain filling in maize (Zea mays L.): coordination of growth and cold responses.

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.

Physiological and biochemical effects of 24-Epibrassinolide on drought stress adaptation in maize (Zea mays L.).

Maize production and productivity are affected by drought stress in tropical and subtropical ecologies, as the majority of the area under maize cultivation in these ecologies is rain-fed. The present investigation was conducted to study the physiological and biochemical effects of 24-Epibrassinolide (EBR) as a plant hormone on drought tolerance in maize. Two maize hybrids, Vivek hybrid 9 and Bio 9637, were grown under three different conditions: (i) irrigated, (ii) drought, and (iii) drought+EBR. A total of 2 weeks before the anthesis, irrigation was discontinued to produce a drought-like condition. In the drought+EBR treatment group, irrigation was also stopped, and in addition, EBR was applied as a foliar spray on the same day in the drought plots. It was observed that drought had a major influence on the photosynthesis rate, membrane stability index, leaf area index, relative water content, and leaf water potential; this effect was more pronounced in Bio 9637. Conversely, the activities of antioxidant enzymes such as catalase (CAT), ascorbate peroxidase (APX), and superoxide dismutase (SOD) increased in both hybrids under drought conditions. Specifically, Vivek hybrid 9 showed 74% higher CAT activity under drought conditions as compared to the control. Additionally, EBR application further enhanced the activity of this enzyme by 23% compared to plants under drought conditions. Both hybrids experienced a significant reduction in plant girth due to drought stress. However, it was found that exogenously applying EBR reduced the detrimental effects of drought stress on the plant, and this effect was more pronounced in Bio 9637. In fact, Bio 9637 treated with EBR showed an 86% increase in proline content and a 70% increase in glycine betaine content compared to untreated plants under drought conditions. Taken together, our results suggested EBR enhanced tolerance to drought in maize hybrids. Hence, pre-anthesis foliar application of EBR might partly overcome the adverse effects of flowering stage drought in maize.

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.

Brassinolide as potential rescue agent for Pinellia ternata grown under microplastic condition: Insights into their modulatory role on photosynthesis, redox homeostasis, and AsA-GSH cycling.

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 O2·- production (29.6%) and GSH content (4.4%), as well as flavonoids (6.8%), alkaloids (75%), and ß-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.

Sodium nitrophenolate mediates brassinosteroids signaling to enhance cold tolerance of cucumber seedling.

Cold stress (CS) significantly limits cucumber yield. However, it remains unclear whether and how sodium nitrophenolate (CSN) regulates plant responses to cold stress. Here, H2O, 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.

Waterlogging faced by bulbil expansion improved the growth of Pinellia ternata and its effect reinforced by brassinolide.

The bulbil expansion of P. ternata is a key period for its yield formation, and the process of bulbil expansion is often subjected to short-term heavy precipitation. It is not clear whether the short-term waterlogging can affect bulbil expansion. Brassinolide (BR) is widely believed to enhance plant tolerance to abiotic stress. The study investigated the effects of normal water (C), waterlogging (W), waterlogging + BR (W + B), waterlogging + propiconazole (W + P) on P. ternata at the bulbil expansion period in order to assess P. ternata's ability to cope with waterlogging during the bulbil expansion stage and the regulation effects of BR on the process. The biomass of P. ternata was significantly increased after waterlogging. W treatment significantly reduced the H2O2 and MDA contents, the rate of O2⋅- production and the activities of antioxidant enzymes compared with the C group. AsA and GSH contents were significantly reduced by W treatment. However, the ratios of AsA/DHA and GSH/GSSG were slightly affected by W treatment. The rate of O2∙- production and H2O2 content in W + B group were significantly lower than those in W group. The POD, APX, and GR activities, and GSH content in W + B group were evidently increased compared with the W group. Soluble sugar and active ingredients contents were significantly increased after waterlogging, and the enhancement was reinforced by BR. In conclusion, waterlogging reduced oxidative stress in P. ternata under the experimental conditions. BR treatment under waterlogging had a positive effect on P. ternata by enhancing antioxidant capacity and promoting the accumulation of soluble sugars and active ingredients.

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 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.

Anti-cancer activity, and molecular docking of novel hybrid heterocyclic steroids revealed promising anti-hepatocellular carcinoma agent: Implication of cyclin dependent kinase-2 pathway.

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.

Exogenous 24-epibrassinolide alleviates chilling injury in peach fruit through modulating PpGATA12-mediated sucrose and energy metabolisms.

24-Epibrassinolide (EBR) may act as a modulator for chilling injury in peach fruit during cold storage. In this study, we screened a EBR-induced GATA-type zinc finger protein PpGATA12. The objective of this study was to investigate the potential roles of EBR treatment and transcriptional regulation of PpGATA12 in regulating chilling resistance of peaches. In the current study, we found that EBR treatment promoted the activities and transcriptions of energy and sucrose metabolism-related enzymes, maintained higher ATP content and energy status, improved the accumulation of sucrose and hexose. Furthermore, molecular biology assays suggested that PpGATA12 up-regulated transcriptions of sucrose metabolism-related genes including PpSS and PpNI, and energy metabolism-related genes including PpCCO, PpSDH and PpH+-ATPase. These results provided a new insight that the enhancement of chilling resistance in peach fruit by EBR treatment might be closely related to the regulatory role of PpGATA12 on sucrose and energy metabolisms.