Brassinosteroid biosynthesis

Researchers applied exogenous 24-epibrassinolide (BL) or its biosynthesis inhibitor brassinazole (BRZ) to slanted hybrid poplar trees (Populus deltoids × Populus nigra) and measured the morphology of gravitropic stems, anatomy and chemistry of secondary cell wall. We furthermore analyzed the expression levels of auxin transport and cellulose biosynthetic genes after 24-epibrassinolide (BL) or brassinazole (BRZ) application. The BL-treated seedlings showed no negative gravitropism bending, whereas application of BRZ dramatically enhanced negative gravitropic bending. BL treatment stimulated secondary xylem fiber elongation and G-fiber formation on the upper side of stems but delayed G-fiber maturation. BRZ inhibited xylem fiber elongation but induced the production of more mature G-fibers on the upper side of stems. Wood chemistry analyses and immunolocalization demonstrated that BL and BRZ treatments increased the cellulose content and modified the deposition of cell wall carbohydrates including arabinose, galactose and rhamnose in the secondary xylem. The expression of cellulose biosynthetic genes, especially those related to cellulose microfibril deposition (PtFLA12 and PtCOBL4) was significantly upregulated in BL- and BRZ-treated TW stems compared with control stems. The significant differences of G-fibers development and negative gravitropism bending between 24-epibrassinolide (BL) and brassinazole (BRZ) application suggest that brassinosteroids are important for secondary xylem development during tension wood formation. [1]

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Further, Researchers treated fibers from GhEXL3-OE/Cas9 and WT ovules with 1 μm of BL and 5 μm of BRZ, respectively. Fiber length statistics after 10 days of incubation in darkness show that compared with control, exogenous application of BR significantly promoted fiber elongation, and BRZ inhibited fiber elongation. Compared with BRZ-treated WT fiber, BRZ-treated GhEXL3-OE transgenic cotton showed longer fibers, suggesting that overexpression of GhEXL3 partially restored the suppression of fiber cell elongation by BRZ. In contrast, the application of BR to GhEXL3-Cas9 showed much shorter fibers than BR-treated WT fibers (Figure 3c,d). We utilized qRT-PCR analysis to examine the expression profiles of fiber elongation related genes, including GhTUB1, GhACT1, GhEXPA1, GhKCS10, GhEXPA4, and GhCERP (Figure S4). The results show that the expression of all fiber elongation related genes was significantly downregulated in GhEXL3-Cas9 fibers. However, this downregulation was partially restored after BR treatment. Conversely, treatment of GhEXL3-OE fibers with BRZ resulted in significant suppression of highly expressed fiber elongation related genes. These results suggest that GhEXL3 is involved in BR signaling and affects the expression of genes related to fiber elongation, which regulates fiber elongation [2].

In vitro ovule culture [2]
Ovules obtained from WT and GhEXL3 transgenic cotton plants at 1 DPA were subjected to several treatments. Initially, they were immersed in 75% anhydrous ethanol for 5 min, followed by a brief 2- to 3-sec exposure to 95% anhydrous ethanol, and eventually rinsed five times with sterile water. Under aseptic conditions, the ovules were carefully extracted. Both brassinolide (BL) at concentrations of 0.1, 1, and 5 μm and brassinazole (BRZ) at concentrations of 0.1, 1, and 5 μm were dissolved in anhydrous ethanol and added to BT medium (Qin et al., 2007). Subsequently, ovules of WT and GhEXL3 transgenic cotton were immersed in a BT medium containing different concentrations of BL andbrassinazole (BRZ). They were then incubated at 30°C, shielded from light, for 10 days, after which the fiber length was measured.

[1]. Effects of exogenous 24-epibrassinolide and brassinazole on negative gravitropism and tension wood formation in hybrid poplar (Populus deltoids × Populus nigra). Planta.2019 May;249(5):1449-1463.
[2]. GhEXL3 participates in brassinosteroids regulation of fiber elongation in Gossypium hirsutum. Plant J. 2024 Oct;120(2):491-504.

Brassinazole is a triazole compound of but-2-ol, with its 2, 3, and 4 positions substituted by phenyl, 1H-1,2,4-triazol-1-yl, and p-chlorophenyl, respectively. It is an inhibitor of brassinosterol biosynthesis. Brassinazole inhibits the biosynthesis of brassinosterol. It belongs to the triazole class, tertiary alcohol class, and monochlorobenzene class of compounds. Exogenous 24-epibrassinol (BL) and brassinazole (BRZ) regulate G fiber cell wall development and secondary xylem cell wall carbohydrate biosynthesis during the formation of tensionwood in hybrid poplar. Brassinosterols (BRs) play an important role in regulating geotropism and vascular development. This article reports the effects of brassinosterols on negative geotropism and G fiber cell wall development in the stems of woody angiosperms. We applied exogenous 24-epibrassinolide (BL) or its biosynthesis inhibitor brassinolide (BRZ) to tilted hybrid poplar (Populus triangularis × Populus tomentosa) and measured the morphology, secondary cell wall anatomy, and chemical composition of geotropic stems. Furthermore, we analyzed the expression levels of auxin transport and cellulose biosynthesis genes after application of BL or BRZ. BL-treated seedlings did not exhibit negative geotropism, while BRZ treatment significantly enhanced it. BL treatment stimulated elongation of secondary xylem fibers and G fiber formation on the upper side of the stem but delayed G fiber maturation. BRZ inhibited xylem fiber elongation but induced the production of more mature G fibers on the upper side of the stem. Wood chemical analysis and immunolocalization results indicated that BL and BRZ treatments increased cellulose content in the secondary xylem and altered the deposition of cell wall carbohydrates, including arabinose, galactose, and rhamnose. Compared with the control group, the expression of cellulose biosynthesis genes in tension wood stems treated with BL and BRZ was significantly upregulated, especially genes related to cellulose microfibril deposition (PtFLA12 and PtCOBL4). The significant differences in G fiber development and negative geotropism after treatment with 24-epibrassinolide (BL) and brassinolide (BRZ) suggest that brassinosteroids are crucial for the development of secondary xylem during tension wood formation. Our results provide potential insights into the regulation of G fiber cell wall development by brassinosteroids to achieve negative geotropism during tension wood formation. [1] Cotton fiber (upland cotton, Gossypium hirsutum) is an ideal model for studying the molecular mechanisms of plant cell elongation at the single-cell level. Brassinosteroids (BRs) play a crucial role in regulating plant growth and development. However, the mechanism by which BRs affect cotton fiber elongation remains not fully elucidated. This study identified an exordium-like protein (GhEXL3) by analyzing the transcriptome of cotton fibers overexpressing the BR-deficient mutant pagoda 1 (pag1) and BRI1-EMS-SUPPRESSOR 1 (GhBES1.4, a key transcription factor encoding the BR signaling pathway). CRISPR/Cas9 knockout of GhEXL3 inhibited cotton fiber elongation, while overexpression promoted it, indicating a positive regulatory role for fiber elongation. Furthermore, in vitro ovule culture experiments showed that GhEXL3 overexpression partially counteracted the inhibitory effect of brassinolide (BRZ) on cotton fiber elongation, further confirming GhEXL3's involvement in the BR signaling pathway. Additionally, our results indicate that GhBES1.4 directly binds to the E-box (CACGTG) motif in the GhEXL3 promoter region and enhances its transcription. RNA-seq analysis showed that overexpression of GhEXL3 upregulated the expression of EXPs, XTHs, and other genes related to fiber elongation. In conclusion, our study helps to understand the mechanism by which BR regulates cotton fiber elongation by directly regulating GhEXL3 expression through GhBES1.4. [2]

C18H18CLN3O

327.8080

327.113

C, 65.95; H, 5.53; Cl, 10.81; N, 12.82; O, 4.88

224047-41-0

15477807

White to off-white solid powder

1.2±0.1 g/cm3

533.1±60.0 °C at 760 mmHg

276.2±32.9 °C

0.0±1.5 mmHg at 25°C

1.616

3.28

1

3

5

23

369

0

YULDTPKHZNKFEY-UHFFFAOYSA-N

InChI=1S/C18H18ClN3O/c1-18(23,15-5-3-2-4-6-15)17(22-13-20-12-21-22)11-14-7-9-16(19)10-8-14/h2-10,12-13,17,23H,11H2,1H3

4-(4-chlorophenyl)-2-phenyl-3-(1H-1,2,4-triazol-1-yl)butan-2-ol

Brassinazole; 224047-41-0; 4-(4-chlorophenyl)-2-phenyl-3-(1H-1,2,4-triazol-1-yl)butan-2-ol; (Rac)-Brassinazole; UNII-N9XRW3TF90; N9XRW3TF90; 1-[2-(4-Chlorophenyl)-1-(1-hydroxy-1-phenylethyl)ethyl]-1,2,4-triazole; CHEBI:73177;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)