Biosynthesis Of 20-hydroxyecdysone Research Articles

A parasitoid regulates 20E synthesis and antibacterial activity of the host for development by inducing host nitric oxide production.

Parasitoids are important components of the natural enemy guild in the biological control of insect pests. They depend on host resources to complete the development of a specific stage or whole life cycle and thus have evolved towards optimal host exploitation strategies. In the present study, we report a specific survival strategy of a fly parasitoid Exorista sorbillans (Diptera: Tachinidae), which is a potential biological control agent for agricultural pests and a pest in sericulture. We found that the expression levels of nitric oxide synthase (NOS) and nitric oxide (NO) production in host Bombyx mori (Lepidoptera: Bombycidae) were increased after E. sorbillans infection. Reducing NOS expression and NO production with an NOS inhibitor (NG-nitro-L-arginine methyl ester hydrochloride) in infected B. mori significantly impeded the growth of E. sorbillans larvae. Moreover, the biosynthesis of 20-hydroxyecdysone (20E) in infected hosts was elevated with increasing NO production, and inhibiting NOS expression lowered 20E biosynthesis. More importantly, induced NO synthesis was required to eliminate intracellular bacterial pathogens that presumably competed for shared host resources. Inhibiting NOS expression down-regulated the transcription of antimicrobial peptide genes and increased the number of bacteria in parasitized hosts. Collectively, this study revealed a new perspective on the role of NO in host-parasitoid interactions and a novel mechanism for parasitoid regulation of host physiology to support its development.

Comparative Transcriptomic Analysis of Genes in the 20-Hydroxyecdysone Biosynthesis in the Fern Microsorum scolopendria towards Challenges with Foliar Application of Chitosan.

Microsorum scolopendria is an important medicinal plant that belongs to the Polypodiaceae family. In this study, we analyzed the effects of foliar spraying of chitosan on growth promotion and 20-hydroxyecdysone (20E) production in M. scolopendria. Treatment with chitosan at a concentration of 50 mg/L in both young and mature sterile fronds induced the highest increase in the amount of accumulated 20E. Using RNA sequencing, we identified 3552 differentially expressed genes (DEGs) in response to chitosan treatment. The identified DEGs were associated with 236 metabolic pathways. We identified several DEGs involved in the terpenoid and steroid biosynthetic pathways that might be associated with secondary metabolite 20E biosynthesis. Eight upregulated genes involved in cholesterol and phytosterol biosynthetic pathway, five upregulated genes related to the methylerythritol 4-phosphate (MEP) and mevalonate (MVA) pathways, and several DEGs that are members of cytochrome P450s and ABC transporters were identified. Quantitative real-time RT-PCR confirmed the results of RNA-sequencing. Taken together, we showed that chitosan treatment increased plant dry weight and 20E accumulation in M. scolopendria. RNA-sequencing and DEG analyses revealed key enzymes that might be related to the production of the secondary metabolite 20E in M. scolopendria.

Physiological, epigenetic, and proteomic responses in Pfaffia glomerata growth in vitro under salt stress and 5-azacytidine.

Plants adjust their complex molecular, biochemical, and metabolic processes to overcome salt stress. Here, we investigated the proteomic and epigenetic alterations involved in the morphophysiological responses of Pfaffia glomerata, a medicinal plant, to salt stress and the demethylating agent 5-azacytidine (5-azaC). Moreover, we investigated how these changes affected the biosynthesis of 20-hydroxyecdysone (20-E), a pharmacologically important specialized metabolite. Plants were cultivated in vitro for 40days in Murashige and Skoog medium supplemented with NaCl (50mM), 5-azaC (25μM), and NaCl + 5-azaC. Compared with the control (medium only), the treatments reduced growth, photosynthetic rates, and photosynthetic pigment content, with increase in sucrose, total amino acids, and proline contents, but a reduction in starch and protein. Comparative proteomic analysis revealed 282 common differentially accumulated proteins involved in 87 metabolic pathways, most of them related to amino acid and carbohydrate metabolism, and specialized metabolism. 5-azaC and NaCl + 5-azaC lowered global DNA methylation levels and 20-E content, suggesting that 20-E biosynthesis may be regulated by epigenetic mechanisms. Moreover, downregulation of a key protein in jasmonate biosynthesis indicates the fundamental role of this hormone in the 20-E biosynthesis. Taken together, our results highlight possible regulatory proteins and epigenetic changes related to salt stress tolerance and 20-E biosynthesis in P. glomerata, paving the way for future studies of the mechanisms involved in this regulation.

CYP314A1-dependent 20-hydroxyecdysone biosynthesis is involved in regulating the development of pupal diapause and energy metabolism in the Chinese citrus fruit fly, Bactrocera minax.

Diapause is an environmentally preprogrammed period of arrested development, and characterized by metabolic depression that can occur during any development stage of insect. The insect steroid hormone 20-hydroxyecdysone (20E), is converted from ecdysone by the cytochrome P450 enzyme shade (CYP314A1), and it exerts a potent effect on the induction and maintenance of diapause in obligatory diapause insects. However, the regulatory mechanism of 20E in obligatory diapause development remains unclear. In this study, the function of 20E in the pupal diapause of Bactrocera minax was investigated. We determined the expression pattern of Halloween P450 genes from larval to adult B.minax, and found differential expression of CYP314A1 from other P450 genes, with a high level in larvae and a low level in pupae. Dysfunction of CYP314A1 by dsCYP314A1 microinjection in third-instar larvae caused significant larval mortality or abnormal pupae. Compared with dsGFP and DEPC-water, dsCYP314A1-injected larvae had significantly reduced 20E titer and altered energy metabolism, and many individuals failed to pupate. Exogenous 20E microinjected into late third-instar larvae or 20E fed to early third-instar larvae both caused similar energy metabolism changes. The 20E-treated larvae of B.minax had reduced total lipids and increased amounts of trehalose and glycogen. Furthermore, 20E-treated diapause individuals showed rapid pupal development. The 20E biosynthesis was regulated by the expression of CYP314A1, and was involved in the induction and termination phase of obligate diapause by regulating energy metabolism in B.minax. © 2022 Society of Chemical Industry.

Effect of methyl jasmonate on production of 20-hydroxyecdysone and turkesterone in hairy roots of Silene linicola C.C.Gmelin

Methyl jasmonate (MeJ) affects the regulation of secondary metabolism, and it is considered to be a promising elicitor in the culture of cells, tissues and plant organs. High-performance liquid chromatography method was used to identify the composition of ecdysteroids in hairy roots of Silene linicola. MeJ was found to have a stimulating effect on ecdysteroid biosynthesis in this culture. Addition of MeJ at a concentration of 100 μM increased the biosynthesis of 20-hydroxyecdysone by 74% (day 3), and that of turkesterone by 35% (day 6). The share of turkesterone in total ecdysteroid content in the investigated samples was up to 60%, and the content of 20-hydroxyecdysone was up to 30%. The study shows that MeJ is a promising stimulator of ecdysteroid biosynthesis in hairy roots of S. linicola.

Functional characterization of CYP71D443, a cytochrome P450 catalyzing C-22 hydroxylation in the 20-hydroxyecdysone biosynthesis of Ajuga hairy roots

20-Hydroxyecdysone (20HE), a molting hormone of insects, is also distributed among a variety of plant families. 20HE is thought to play a role in protecting plants from insect herbivores. In insects, biosynthesis of 20HE from cholesterol proceeds via 7-dehydrocholesterol and 3β,14α-dihydroxy-5β-cholest-7-en-6-one (5β-ketodiol), the latter being converted to 20HE through sequential hydroxylation catalyzed by four P450 enzymes, which have been cloned and identified. In contrast, little is known about plant 20HE biosynthesis, and no biosynthetic 20HE gene has been reported thus far. We recently proposed involvement of 3β-hydroxy-5β-cholestan-6-one (5β-ketone) in 20HE biosynthesis in the hairy roots of Ajuga reptans var. atropurpurea (Lamiaceae). In this study, an Ajuga EST library was generated from the hairy roots and P450 genes were deduced from the library. Five genes with a high expression level (CYP71D443, CYP76AH19, CYP76AH20, CYP76AH21 and CYP716D27) were screened for a possible involvement in 20HE biosynthesis. As a result, CYP71D443 was shown to have C-22 hydroxylation activity for the 5β-ketone substrate using a yeast expression system. The hydroxylated product, 22-hydroxy-5β-ketone, had a 22R configuration in agreement with that of 20HE. Furthermore, labeling experiments indicated that (22R)-22-hydroxy-5β-ketone was converted to 20HE in Ajuga hairy roots. Based on the present results, a possible 20HE biosynthetic pathway in Ajuga plants involved CYP71D443 is proposed.

Biosynthesis of 20-hydroxyecdysone in plants: 3β-Hydroxy-5β-cholestan-6-one as an intermediate immediately after cholesterol in Ajuga hairy roots

3β-Hydroxy-5β-cholestan-6-one was identified in the EtOAc extract of Ajuga hairy roots by micro-analysis using LC–MS/MS in the multiple reaction mode (MRM). Furthermore, administration of (2,2,4,4,7,7-2H6)- and (2,2,4,4,6,7,7-2H7)-cholesterols to the hairy roots followed by LC–MS/MS analysis of the EtOAc extract of the hairy roots indicated that cholesterol was converted to the 5β-ketone with hydrogen migration from the C-6 to the C-5 position. These findings, in conjunction with the previous observation that the ketone was efficiently converted to 20-hydroxyecdysone, strongly suggest that the 5β-ketone is an intermediate immediately formed after cholesterol during 20-hydroxyecdysone biosynthesis in Ajuga sp. In addition, the mechanism of the 5β-ketone formation from cholesterol is discussed.

Induced accumulation of 20-hydroxyecdysone in cell suspension cultures of Vitex glabrata R.Br.

This study describes the effects of culture medium, culture temperature, sucrose concentration and cholesterol feeding on cell growth and 20-hydroxyecdysone production in suspension cultures of Vitex glabrata , an important medicinal plant in Thailand. Cell growth and 20-hydroxyecdysone production were not significantly different when cells were cultivated on B5 or half-strength MS medium. However, cultivation of V. glabrata cell cultures at 25°C yielded 1.06- and 1.09-fold higher values of cell growth and 20-hydroxyecdysone content, respectively than those at 30°C. Sucrose at 30 and 40 g/L favors the production of 20-hydroxyecdysone in suspension cultures of V. glabrata . Feeding of cholesterol at 5 mg/L, as precursor for biosynthesis of 20-hydroxyecdysone, yielded 1.11-fold higher accumulation of 20-hydroxyecdysone than the control cells. Increasing of cholesterol to 10 mg/L resulted in decreased production of 20-hydroxyecdysone. Key words : Vitex glabrata, 20-hydroxyecdysone, suspension culture, cholesterol.

Phantom encodes the 25-hydroxylase of Drosophila melanogaster and Bombyx mori: a P450 enzyme critical in ecdysone biosynthesis

We have reported recently the identification and characterization of the last three mitochondrial cytochrome P450 enzymes (CYP) controlling the biosynthesis of 20-hydroxyecdysone, the molting hormone of insects. These are encoded by the following genes: disembodied ( dib, Cyp302a1, the 22-hydroxylase); shadow (sad, Cyp315a1, the 2-hydroxylase); and shade ( shd, Cyp314a1, the 20-hydroxylase). Employing similar gene identification and transfection techniques and subsequent biochemical analysis of the expressed enzymatic activity, we report the identity of the Drosophila gene phantom ( phm), located at 17D1 of the X chromosome, as encoding the microsomal 25-hydroxylase ( Cyp306a1). Similar analysis following differential display-based gene identification has also resulted in the characterization of the corresponding 25-hydroxylase gene in Bombyx mori. Confirmation of 2,22,25-trideoxyecdysone (3β,5β-ketodiol) conversion to 2,22-dideoxyecdysone (3β,5β-ketotriol) mediated by either Phm enzyme employed LC, MS and definitive NMR analysis. In situ developmental gene analysis, in addition to northern, western and RT-PCR techniques during Drosophila embryonic, larval and adult development, are consistent with this identification. That is, strong expression of phm is restricted to the prothoracic gland cells of the Drosophila larval ring gland, where it undergoes dramatic changes in expression, and in the adult ovary, but also in the embryonic epidermis. During the last larval–larval transition in Bombyx, a similar expression pattern in the prothoracic gland is observed, but as in Drosophila, slight expression is also present in other tissues, suggesting a possible additional role for the phantom enzyme.

Molecular cloning of NADPH-cytochrome P450 oxidoreductase from silkworm eggs. Its involvement in 20-hydroxyecdysone biosynthesis during embryonic development

Molecular cloning of NADPH-cytochrome P450 oxidoreductase from silkworm eggs. Its involvement in 20-hydroxyecdysone biosynthesis during embryonic development

Molecular cloning of NADPH-cytochrome P450 oxidoreductase from silkworm eggs. Its involvement in 20-hydroxyecdysone biosynthesis during embryonic development.

Using RT-PCR, a cDNA fragment of NADPH-cytochrome P450 oxidoreductase from silkworm, Bombyx mori, was cloned from three-day-old nondiapause eggs. RACE was used to isolate the ends of the DNA. The full-length cDNA obtained was composed of 3471 bp with an open reading frame encoding a protein of 687 amino-acid residues with a relative molecular mass of 77 700. The protein, fused with glutathione S-transferase, was expressed in Escherichia coli and purified to homogeneity. The fused protein not only had NADPH-dependent cytochrome c-reducing activity, but also acted as an electron carrier from NADPH to bovine adrenal 21-hydroxylase P450 in the steroid hydroxylation reaction, confirming that the protein is the silkworm NADPH-cytochrome P450 oxidoreductase. Ecdysone 20-hydroxylase activity in the nondiapause egg microsomes increased until the fourth day after oviposition, and then decreased, little being detected on the ninth day. An antibody raised against the P450 reductase inhibited the ecdysone hydroxylation. Immunoblot analyses of the microsomes indicated that the P450 reductase protein appeared distinctly in the three-day-old nondiapause eggs and, in contrast to the developmental pattern of ecdysone hydroxylase activity, continued to increase as the embryos developed. These results suggest that ecdysone hydroxylation in the early stage of embryogenesis is dependent on the presence of both P450 reductase and ecdysone 20-hydroxylase P450, but its gradual reduction in the later stage may be due to the decrease in the level of ecdysone 20-hydroxylase P450.

Biosynthesis of 20-hydroxyecdysone in Ajuga hairy roots: the possibility of 7-ene introduction at a late stage

Administration of [3α- 2 H ]-3β-hydroxy-5β-cholestan-6-one to hairy roots of Ajuga reptans var. atropurpurea followed by 2 H -NMR spectroscopic analysis of the resulting 20-hydroxyecdysone so formed revealed that the substrate was efficiently incorporated into the latter. Additionally, [5β,7α,7β- 2 H 3 ]-2β,3β-dihydroxy-5β-cholestan-6-one was converted into 20-hydroxyecdysone. These findings clearly indicate that Ajuga hairy roots are capable of introducing a double bond at the 7-position at a late stage of 20-hydroxyecdysone biosynthesis, suggesting the possibility of an alternative biosynthetic pathway which does not involve 7-dehydrocholesterol as an obligatory intermediate.

Biosynthesis of sterols and ecdysteroids in Ajuga hairy roots.

Hairy roots of Ajuga reptans var. atropurpurea produce clerosterol, 22-dehydroclerosterol, and cholesterol as sterol constituents, and 20-hydroxyecdysone, cyasterone, isocyasterone, and 29-norcyasterone as ecdysteroid constituents. To better understand the biosynthesis of these steroidal compounds, we carried out feeding studies of variously 2H- and 13C-labeled sterol substrates with Ajuga hairy roots. In this article, we review our studies in this field. Feeding of labeled desmosterols, 24-methylenecholesterol, and 13C2-acetate established the mechanism of the biosynthesis of the two C29-sterols and a newly accumulated codisterol, including the metabolic correlation of C-26 and C-27 methyl groups. In Ajuga hairy roots, 3alpha-, 4alpha-, and 4beta-hydrogens of cholesterol were all retained at their original positions after conversion into 20-hydroxyecdysone, in contrast to the observations in a fern and an insect. Furthermore, the origin of 5beta-H of 20-hydroxyecdysone was found to be C-6 hydrogen of cholesterol exclusively, which is inconsistent with the results in the fern and the insect. These data strongly support the intermediacy of 7-dehydrocholesterol 5alpha,6alpha-epoxide. Moreover, 7-dehydrocholesterol, 3beta-hydroxy-5beta-cholest-7-en-6-one (5beta-ketol), and 3beta,14alpha-dihydroxy-5beta-cholest-7-en-6-one (5beta-ketodiol) were converted into 20-hydroxyecdysone. Thus, the pathway cholesterol-->7-dehydrocholesterol-->7-dehydrocholesterol 5alpha,6alpha-epoxide-->5beta-ketol-->5beta-k etodiol is proposed for the early stages of 20-hydroxyecdysone biosynthesis. 3beta-Hydroxy-5beta-cholestan-6-one was also incorporated into 20-hydroxyecdysone, suggesting that the introduction of a 7-ene function is not necessarily next to cholesterol. C-25 Hydroxylation during 20-hydroxyecdysone biosynthesis was found to proceed with ca. 70% retention and 30% inversion. Finally, clerosterol was shown to be a precursor of cyasterone and isocyasterone.

Mechanism of C-2 hydroxylation during the biosynthesis of 20-hydroxyecdysone in Ajuga hairy roots.

Feeding synthetic [2beta-2H]- and [2alpha-2H]-cholesterols to the hairy roots of Ajuga reptans var. atropurpurea and 2H-NMR analysis of the biosynthesized 20-hydroxyecdysone revealed that hydroxylation at C-2 proceeds with retention of configuration. Feeding [2alpha,3alpha-2H2]cholesterol followed by 2H-NMR analysis of the 2,3,22-triacetate of the resulting 20-hydroxyecdysone ruled out a mechanism which involves a partial loss of the 2alpha-hydrogen. The steric course of C-2 hydroxylation in Ajuga hairy roots is identical with that reported in the insect, Schistocerca gregaria.

Biosynthesis of 20-hydroxyecdysone in Ajuga hairy roots: fate of 6α- and 6β-hydrogens of lathosterol

The fate of 6α- and 6β-hydrogens of lathosterol during the transformation into 20-hydroxyecdysone was chased by feeding [3α,6β-2H2]- and [3α,6α-2H2]-lathosterols to hairy roots of Ajuga reptans var. atropurpurea. The behavior of 6β-hydrogen, which mostly migrated to the C-5 position of 20-hydroxyecdysone, was in agreement with that of C-6 hydrogen of cholesterol. The results strongly supported the view that cholesterol and lathosterol are first metabolized into 7-dehydrocholesterol, which is then converted into 20-hydroxyecdysone via 7-dehydrocholesterol 5α,6α-epoxide in the hairy roots.

Biosynthesis of 20-hydroxyecdysone in Ajuga hairy roots: Hydrogen migration from C-6 to C-5 during cis-A/B ring formation

Feeding of deuterium labeled cholesterols including [6- 2H]cholesterol and [3α,6- 2H 2]-cholesterol to hairy roots of Ajuga reptans var. atropurpurea followed by 2H-NMR analysis of the biosynthesized 20-hydroxyecdysone revealed that most of the deuterium atom located at C-6 of cholesterol migrated to the C-5 position of 20-hydroxyecdysone.

Biosynthesis of 20-hydroxyecdysone in Ajuga hairy roots: Stereochemistry of C-25 hydroxylation

Feeding of [ 13C 2]acetate to hairy roots of Ajuga reptans var. atropurpurea followed by 13C-NMR analysis of the biosynthesized cholesterol and 20-hydroxyecdysone indicated that C-25 hydroxylation in 20-hydroxyecdysone biosynthesis proceeds both in retention and inversion mechanisms. Feeding studies of [26- 13C]- and [27- 13C]cholesterols established that the ratio of retention and inversion mechanisms is ca. 3:1.

3β-Hydroxy-5β-cholest-7-en-6-one as an intermediate of 20-hydroxyecdysone biosynthesis in a hairy root culture of Ajuga reptans var. atropurpurea

[3α-2H]-, [4α-2H]- and [4β-2H]-Cholesterols and [3α-2H]- and [5-2H]-3β-hydroxy-5β-cholest-7-en-6-ones were converted with a hairy root culture of Ajuga reptans var. atropurpurea into 20-hydroxyecdysone, in which the deuterium atoms retained their original positions, thus strongly suggesting that 3β-hydroxy-5β-cholest-7-en-6-one is an obligatory intermediate in the biosynthesis of ecdysteroids in the plant.