Exogenous Application Of Brassinolide Research Articles

A mutation in the brassinosteroid biosynthesis gene CpDWF5 disrupts vegetative and reproductive development and the salt stress response in squash (Cucurbita pepo).

A Cucurbita pepo mutant with multiple defects in growth and development has been identified and characterized. The mutant dwfcp displayed a dwarf phenotype with dark green and shrinking leaves, shortened internodes and petioles, shorter but thicker roots and greater root biomass, and reduced fertility. The causal mutation of the phenotype was found to disrupt gene Cp4.1LG17g04540, the squash orthologue of the Arabidopsis brassinosteroid (BR) biosynthesis gene DWF5, encoding for 7-dehydrocholesterol reductase. A single nucleotide transition (G > A) causes a splicing defect in intron 6 that leads to a premature stop codon and a truncated CpDWF5 protein. The mutation co-segregated with the dwarf phenotype in a large BC1S1 segregating population. The reduced expression of CpDWF5 and brassinolide (BL) content in most mutant organs, and partial rescue of the mutant phenotype by exogenous application of BL, showed that the primary cause of the dwarfism in dwfcp is a BR deficiency. The results showed that in C. pepo, CpDWF5 is not only a positive growth regulator of different plant organs but also a negative regulator of salt tolerance. During germination and the early stages of seedling development, the dwarf mutant was less affected by salt stress than the wild type, concomitantly with a greater upregulation of genes associated with salt tolerance, including those involved in abscisic acid (ABA) biosynthesis, ABA and Ca2+ signaling, and those coding for cation exchangers and transporters.

Adverse effects of shading on the tea yield and the restorative effects of exogenously applied brassinolide

Shading during cultivation leads to increased tea quality, but its effect on tea yield remains unclear. In this study, shading decreased the tea plant yield because it resulted in the production of thin leaves and shortened internodes as well as a decreased photosynthetic rate. The exogenous application of brassinolide (BL), which is a novel phytohormone, recovered some of the lost yield caused by shading by increasing the leaf area, promoting internode elongation, and increasing the photosynthetic rate. Moreover, BL did not affect the enhancement effect of shading on tea quality. The exogenous application of BL could increase the content of total amino acid (AA), geraniol, chlorophyll and carotenoid, and reduce polyphenols-to-amino acids ratio (P/A) in green tea processed from shaded tea leaves. Our findings have clarified the effects of shading on tea plants and explored measures to address shading-induced yield losses without affecting the enhancement of quality, which have potential implications for limiting shading-induced yield losses in tea plants.

Overexpression of GmMYB14 improves high-density yield and drought tolerance of soybean through regulating plant architecture mediated by the brassinosteroid pathway.

SummaryMYB transcription factors (TFs) have been reported to regulate the biosynthesis of secondary metabolites, as well as to mediate plant adaption to abiotic stresses, including drought. However, the roles of MYB TFs in regulating plant architecture and yield potential remain poorly understood. Here, we studied the roles of the dehydration‐inducible GmMYB14 gene in regulating plant architecture, high‐density yield and drought tolerance through the brassinosteroid (BR) pathway in soybean. GmMYB14 was shown to localize to nucleus and has a transactivation activity. Stable GmMYB14‐overexpressing (GmMYB14‐OX) transgenic soybean plants displayed a semi‐dwarfism and compact plant architecture associated with decreased cell size, resulting in a decrease in plant height, internode length, leaf area, leaf petiole length and leaf petiole angle, and improved yield in high density under field conditions. Results of the transcriptome sequencing suggested the involvement of BRs in regulating GmMYB14‐OX plant architecture. Indeed, GmMYB14‐OX plants showed reduced endogenous BR contents, while exogenous application of brassinolide could partly rescue the phenotype of GmMYB14‐OX plants. Furthermore, GmMYB14 was shown to directly bind to the promoter of GmBEN1 and up‐regulate its expression, leading to reduced BR content in GmMYB14‐OX plants. GmMYB14‐OX plants also displayed improved drought tolerance under field conditions. GmBEN1 expression was also up‐regulated in the leaves of GmMYB14‐OX plants under polyethylene glycol treatment, indicating that the GmBEN1‐mediated reduction in BR level under stress also contributed to drought/osmotic stress tolerance of the transgenic plants. Our findings provided a strategy for stably increasing high‐density yield and drought tolerance in soybean using a single TF‐encoding gene.

Exogenous Brassinolide Enhances the Growth and Cold Resistance of Maize (Zea mays L.) Seedlings under Chilling Stress

This paper aims to elucidate the effects of exogenous brassinolide (BL) on maize germination and seedling growth under chilling stress. The cold-resistant maize hybrid Tiannong 9 and the cold-sensitive hybrid Tianhe 1 were soaked at the germination stage (6 °C) and leaves were sprayed at seedling stage (4 °C), with BL at concentrations of 0, 0.01, 0.1, and 1 mg/L. The germination rate of the maize seeds and the changes in seedling biomass, antioxidant, photosynthetic, and plant endogenous hormone systems and chloroplast ultrastructures were determined. The results showed that the optimum concentration of BL to alleviate chilling stress in maize seedlings was 0.1 mg/L. This rate effectively increased the germination rate and plant biomass of maize and significantly increased the superoxide dismutase (SOD) peroxidase (POD) and catalase (CAT) activities, the net photosynthetic rate (Pn), stomatal conductance (gs) and transpiration rate (Tr), and seedling auxin (IAA), gibberellin (GA3) and trans zeatin nucleoside (t-ZR) contents under chilling stress. In addition, BL significantly reduced the malondialdehyde (MDA) content, abscisic acid (ABA) content, and intercellular carbon dioxide concentration (Ci). In the comparison of mesophyll cells, the chloroplast membrane of the treatment group was tightly attached to the stroma, and some of the plasma membranes were dissolved, but the overall structure of the chloroplast was relatively complete, and the osmiophilic granules were relatively few. The exogenous application of BL can effectively alleviate the damage caused by a low temperature in maize, maintain the normal characteristics of seedlings in chilling environments, and ensure the development and growth of plant tissue in the later stage.

Alteration in morpho-physiological attributes of Leymus chinensis (Trin.) Tzvelev by exogenous application of brassinolide under varying levels of drought stress

Alteration in morpho-physiological attributes of Leymus chinensis (Trin.) Tzvelev by exogenous application of brassinolide under varying levels of drought stress

The Cytochrome P450 Gene CsCYP85A1 Is a Putative Candidate for Super Compact-1 (Scp-1) Plant Architecture Mutation in Cucumber (Cucumis sativus L.).

The dwarf or compact plant architecture is an important trait in plant breeding. A number of genes controlling plant height have been cloned and functionally characterized which often involve in biosynthesis or signaling of plant hormones such as brassinosteroids (BRs). No genes for plant height or vine length have been cloned in cucurbit crops (family Cucurbitaceae). From an EMS-induced mutagenesis population, we identified a super compact (SCP) mutant C257 which was extremely dwarf due to practically no internode elongation. Under dark growing condition, C257 did not undergo skotomorphogenesis and its mutant phenotype could be rescued with exogenous application of brassinolide (BL), suggesting SCP might be a BR-deficient mutant. Segregation analysis revealed a single recessive gene scp-1 that was responsible for the SCP mutation. Map-based cloning combined with a modified MutMap identified CsCYP85A1, a member of the plant cytochrome P450 monooxygenase gene family, as the most possible candidate gene for scp-1, which encodes a BR-C6-oxidase in the BR biosynthesis pathway. We show that a SNP within the second exon of scp-1 candidate gene caused the SCP phenotype. Three copies of the CsCYP85A gene are present in the cucumber genome, but only the scp-1/CsCYP85A1 gene seemed active. The expression of CsCYP85A1 was higher in flowers than in the leaves and stem; its expression in the wild type (WT) was feedback regulated by BL application. Its expression was reduced in C257 as compared with the WT. This was the first report of map-based cloning of a plant height gene in cucurbit crops. The research highlighted the combined use of linkage mapping, an improved MutMap method and allelic diversity analysis in natural populations in quick cloning of simply inherited genes in cucumber. The roles of CsCYP85A1 in regulation of internode elongation in cucumber was discussed.

Exogenous application of brassinolide can alter morphological and physiological traits of Leymus chinensis (Trin.) Tzvelev under room and high temperatures

Plant growth regulating substances are involved in the physiological and metabolic processes of plants and enable them to cope with numerous environmental stresses. The effect of exogenously applied brassinolide (BR) with various concentrations (0.01, 0.1, and 1.0 mg L -1 ) was studied on morphological and physiological traits of Leymus chinensis (Trin.) Tzvelev under room and high temperatures in pots. The experimental results revealed that high temperature stress substantially perturbed growth, photosynthetic pigments, and root activity of L. chinensis; however, the deleterious effects of high temperature were partially ameliorated by the foliar application of BR. Compared to room temperature, high temperature stress decreased the plant height, leaf area, plant fresh and dry weight, chlorophyll a and b content, chlorophyll a/b ratio as well as root activity, while exacerbated the membrane damage as indicated by enhanced production of malondialdehyde (MDA). Accumulation of proline content, soluble protein and sugar content in L. chinensis improved by heat stress, compared with normal temperature; application of BR further improved their production thus aiding in the attainment of tolerance against heat stress. Elevated levels of antioxidant enzymes such as superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), ascorbate peroxidase (APX), and glutathione reductase (GR) were observed under heat stress compared to room temperature, however, application of BR further proved beneficial in this regard. Our results indicated that BR could improve the growth and development of L. chinensis by enhancing the biosynthesis of photosynthetic pigments, osmolytes and antioxidant enzymes system in plants under both room and high temperature.

Phenotypic analysis and molecular characterization of an allelic mutant of the D61 gene in rice

Brassinosteroids (BRs) are a class of plant-specific steroidal hormones that play important roles in multiple biological processes. In this paper, a classic rice mutant gsor300084, showing erect leaves and semi-dwarf stature, was characterized. Morphological analysis in darkness showed that the mesocotyl of the gsor300084 mutant did not elongate when grown in darkness. Coleoptile elongation and root growth were less affected by the exogenous application of brassinolide (BL), the most active form of BR, in gsor300084 than in the wild-type rice variety Matsumae. Lamina joint bending analysis also showed that gsor300084 was less sensitive to exogenous BL than Matsumae. These results suggested that the gsor300084 mutant is defective in BR sensitivity. Map-based cloning indicated that gsor300084 is a novel allelic mutant of the DWARF61 (D61) gene, which encodes the putative BR receptor OsBRI1. A single-base mutation appears in the LRR domain of OsBRI1, changing the 444th amino acid from tryptophan (W) to arginine (R). Subcellular localization analysis suggested that both the wild-type and mutant OsBRI1 protein are localized at the cytoplasmic membrane. Structure modeling revealed that the W444R substitution may affect the perception of BRs by the LRR domain.

Proteomic and Virus-induced Gene Silencing (VIGS) Analyses Reveal That Gossypol, Brassinosteroids, and Jasmonic acid Contribute to the Resistance of Cotton to Verticillium dahliae

Verticillium wilt causes massive annual losses of cotton yield, but the mechanism of cotton resistance to Verticillium dahliae is complex and poorly understood. In this study, a comparative proteomic analysis was performed in resistant cotton (Gossypium barbadense cv7124) on infection with V. dahliae. A total of 188 differentially expressed proteins were identified by mass spectrometry (MALDI-TOF/TOF) analysis and could be classified into 17 biological processes based on Gene Ontology annotation. Most of these proteins were implicated in stimulus response, cellular processes and metabolic processes. Based on the proteomic analysis, several genes involved in secondary metabolism, reactive oxygen burst and phytohormone signaling pathways were identified for further physiological and molecular analysis. The roles of the corresponding genes were further characterized by employing virus-induced gene silencing (VIGS). Based on the results, we suggest that the production of gossypol is sufficient to affect the cotton resistance to V. dahliae. Silencing of GbCAD1, a key enzyme involving in gossypol biosynthesis, compromised cotton resistance to V. dahliae. Reactive oxygen species and salicylic acid signaling may be also implicated as regulators in cotton responsive to V. dahliae according to the analysis of GbSSI2, an important regulator in the crosstalk between salicylic acid and jasmonic acid signal pathways. Moreover, brassinosteroids and jasmonic acid signaling may play essential roles in the cotton disease resistance to V. dahliae. The brassinosteroids signaling was activated in cotton on inoculation with V. dahliae and the disease resistance of cotton was enhanced after exogenous application of brassinolide. Meanwhile, jasmonic acid signaling was also activated in cotton after inoculation with V. dahliae and brassinolide application. These data provide highlights in the molecular basis of cotton resistance to V. dahliae.

Brd1 Gene in Maize Encodes a Brassinosteroid C-6 Oxidase

The role of brassinosteroids in plant growth and development has been well-characterized in a number of plant species. However, very little is known about the role of brassinosteroids in maize. Map-based cloning of a severe dwarf mutant in maize revealed a nonsense mutation in an ortholog of a brassinosteroid C-6 oxidase, termed brd1, the gene encoding the enzyme that catalyzes the final steps of brassinosteroid synthesis. Homozygous brd1–m1 maize plants have essentially no internode elongation and exhibit no etiolation response when germinated in the dark. These phenotypes could be rescued by exogenous application of brassinolide, confirming the molecular defect in the maize brd1-m1 mutant. The brd1-m1 mutant plants also display alterations in leaf and floral morphology. The meristem is not altered in size but there is evidence for differences in the cellular structure of several tissues. The isolation of a maize mutant defective in brassinosteroid synthesis will provide opportunities for the analysis of the role of brassinosteroids in this important crop system.

CYP724B2 and CYP90B3 Function in the Early C-22 Hydroxylation Steps of Brassinosteroid Biosynthetic Pathway in Tomato

We characterized a new cytochrome P450 monooxygenase (P450), CYP724B2, from tomato (Lycopersicon esculentum). CYP724B2 showed 42% and 62% amino acid sequence identity with Arabidopsis DWARF4/CYP90B1 and rice DWARF11/CYP724B1 respectively. Functional assay of CYP724B2 heterologously expressed in insect cells revealed that CYP724B2 catalyzes C-22 hydroxylation of campesterol, indicating that CYP724B2 is a C-22 hydroxylase. We also isolated a tomato CYP90B homolog (CYP90B3) and found that CYP90B3 is a C-22 hydroxylase as well. CYP724B2 and CYP90B3 showed substrate specificities similar to each other toward the biosynthetic intermediate compounds from campesterol to campestanol. Campesterol was the best substrate, and (24R)-ergost-4-en-3-one was also metabolized to the C-22 hydroxylated product to some extent. On the other hand, the P450s catalyzed C-22 hydroxylation of (24R)-5alpha-ergostan-3-one and campestanol at a trace level, indicating that the compounds after C-5alpha reduction are poor substrates of CYP724B2 and CYP90B3. In addition, cholesterol (C27 sterol) and sitosterol (C29 sterol) were also converted to C-22 hydroxylated products by the P450s. Furthermore, CYP724B2 and CYP90B3 genes were ubiquitously expressed, and their transcript levels were down-regulated by the exogenous application of brassinolide. These findings strongly suggest that CYP724B2 and CYP90B3 function in the early C-22 hydroxylation steps of brassinosteroid biosynthetic pathway in tomato.

Arabidopsis cyp51 mutant shows postembryonic seedling lethality associated with lack of membrane integrity.

CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14alpha-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes, CYP51A1 and CYP51A2. Our studies revealed that CYP51A1 should be considered an expressed pseudogene. To study the functional importance of the CYP51A2 gene in plant growth and development, we isolated T-DNA knockout alleles for CYP51A2. Loss-of-function mutants for CYP51A2 showed multiple defects, such as stunted hypocotyls, short roots, reduced cell elongation, and seedling lethality. In contrast to other sterol mutants, such as fk/hydra2 and hydra1, the cyp51A2 mutant has only minor defects in early embryogenesis. Measurements of endogenous sterol levels in the cyp51A2 mutant revealed that it accumulates obtusifoliol, the substrate of CYP51, and a high proportion of 14alpha-methyl-delta8-sterols, at the expense of campesterol and sitosterol. The cyp51A2 mutants have defects in membrane integrity and hypocotyl elongation. The defect in hypocotyl elongation was not rescued by the exogenous application of brassinolide, although the brassinosteroid-signaling cascade is apparently not affected in the mutants. Developmental defects in the cyp51A2 mutant were completely rescued by the ectopic expression of CYP51A2. Taken together, our results demonstrate that the Arabidopsis CYP51A2 gene encodes a functional obtusifoliol 14alpha-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway.

Brassinosteroid/Sterol synthesis and plant growth as affected by lka and lkb mutations of Pea

The dwarf pea (Pisum sativum) mutants lka and lkb are brassinosteroid (BR) insensitive and deficient, respectively. The dwarf phenotype of the lkb mutant was rescued to wild type by exogenous application of brassinolide and its biosynthetic precursors. Gas chromatography-mass spectrometry analysis of the endogenous sterols in this mutant revealed that it accumulates 24-methylenecholesterol and isofucosterol but is deficient in their hydrogenated products, campesterol and sitosterol. Feeding experiments using 2H-labeled 24-methylenecholesterol indicated that the lkb mutant is unable to isomerize and/or reduce the Delta24(28) double bond. Dwarfism of the lkb mutant is, therefore, due to BR deficiency caused by blocked synthesis of campesterol from 24-methylenecholesterol. The lkb mutation also disrupted sterol composition of the membranes, which, in contrast to those of the wild type, contained isofucosterol as the major sterol and lacked stigmasterol. The lka mutant was not BR deficient, because it accumulated castasterone. Like some gibberellin-insensitive dwarf mutants, overproduction of castasterone in the lka mutant may be ascribed to the lack of a feedback control mechanism due to impaired perception/signal transduction of BRs. The possibility that castasterone is a biologically active BR is discussed.