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.

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.

Formation of nitrogen-containing six-membered heterocycles on steroidal ring system: A review.

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.

Cancer and brassinosteroids: Mechanisms of action, SAR and future perspectives.

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.

Brassinolide alleviates Fe deficiency-induced stress by regulating the Fe absorption mechanism in Malus hupehensis Rehd.

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.

The Mechanisms Underlying Salt Resistance Mediated by Exogenous Application of 24-Epibrassinolide in Peanut.

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.

Structure modification of nonsteroidal brassinolide-like compound, NSBR1.

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.

Comparison of Protective Reactions of Rape Seeds to Chloride Salination at Exposure to Epibrassinolide before or during Salt Stress.

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.

Pistil Mating Type and Morphology Are Mediated by the Brassinosteroid Inactivating Activity of the S-Locus Gene BAHD in Heterostylous Turnera Species.

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.

Epibrassinolide improves the growth performance of Sedum lineare upon Zn stress through boosting antioxidative capacities.

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.

Epibrassinolide prevents tau hyperphosphorylation via GSK3ß inhibition in vitro and improves Caenorhabditis elegans lifespan and motor deficits in combination with roscovitine.

Glycogen synthase kinase 3ß (GSK3ß) is considered an important element of glycogen metabolism; however, it has many other regulatory roles. Changes in the GSK3ß signaling mechanism have been associated with various disorders, such as Alzheimer's disease (AD), type II diabetes, and cancer. Although the effects of GSK3ß 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 GSK3ß 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 GSK3ß. 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 Aß42 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 GSK3ß phosphorylation at Ser9.

24-epibrassinolide alleviates postharvest yellowing of broccoli via improving its antioxidant capacity.

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 (O2•-). 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.

24-Epibrassinolide modulates the neurodevelopmental outcomes of high caffeine exposure in zebrafish (Danio rerio) embryos.

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.

A novel W/O/W double emulsion co-delivering brassinolide and cinnamon essential oil delayed the senescence of broccoli via regulating chlorophyll degradation and energy metabolism.

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, Ca2+-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.

Brassinolide soaking and preharvest UV-B radiation influence the shelf life of small black bean sprouts.

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.

Genome-Wide Identification of the B-BOX Genes that Respond to Multiple Ripening Related Signals in Sweet Cherry Fruit.

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 GA3 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.

Transcriptomic analyses show that 24-epibrassinolide (EBR) promotes cold tolerance in cotton seedlings.

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.