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Cytochrome P450 710A1/A2 as Brassinosteroid C24-Desaturases to Connect C27- and C28-Brassinosteroids Biosynthesis in Arabidopsis thaliana

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Abstract

MBP-fused Arabidopsis CYP710A1 and CYP710A2 were obtained from E. coli, and their biochemical functions were examined to characterize the C24-desaturase(s) involved in converting 28-norcastaterone (28-norCS) into dolichosterone (DS). When 28-norCS was used as a substrate, GC–MS analysis revealed that 28-norCS was converted into dehydro-28-norCS, most likely due to Δ24-28-norCS exposure to NADPH-CYP reductase. When only MBP-CYP710A1 and MBP-CYP710A2 were used as co-enzyme sources, MBP-fused sterolmethyltransferase 1 (MBP-SMT1) successfully catalyzed the C24-methylation of 28-norCS to DS, verifying that CYP710A1 and CYP710A2 act as C24-desaturases for SMT1-mediated 28-norCS to DS C24-methylation intermediated by Δ24-28-norCS. During seedling growth, primary root lengths in cyp710a1 and cyp710a2 were significantly attenuated than those in the wild-type. The abated primary root growth in cyp710a1 and cyp710a2 was restored by exogenously applied dolichosterone and castasterone, indicating that cyp710a1 and cyp710a2 are brassinosteroid-deficient mutants from the disrupted conversion of 28-norCS to DS and CS in Arabidopsis. In addition, CYP710A1 and CYP710A2 expressions were downregulated in bes1-D and bzr1-D. Chromatin immune precipitation assay revealed that BES1 and BZR1 directly bind to E-box and BRRE sequences on CYP710A1 and CYP710A2 promotor regions, indicating that CYP710A1 and CYP710A2 expressions are feedback regulated by brassinosteroid signaling via BES1 and BZR1 in Arabidopsis. Conclusively, CYP710A1 and CYP710A2 serve as C24-desaturases to connect C27- to C28-brassinosteroids biosynthesis, which is strictly regulated by brassinosteroid signaling to maintain homeostasis for regulating Arabidopsis growth and development.

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Data Availability

All data supporting the findings of this study are available within the paper and within its supplementary data published online.

Abbreviations

MBP:

Maltose binding protein

28-norCS:

28-norcastaterone

DS:

Dolichosterone

MBP-SMT1:

MBP-fused sterolmethyltransferase 1

CYP:

Cytochrome P450

BRs:

Brassinosteroids

BL:

Brassinolide

28-norBRs:

C27-BRs with no alkyl group

CS:

Castasterone

CR:

Campesterol

CN:

Compestanol

CHR:

Cholesterol

CHN:

Cholestanol

6-deoxo-28-norTY:

6-deoxo-28-nortyphasterol

SAM:

S-adenosyl-methionine

SMTs:

Sterol methyltransferases

Col-0:

Columbia-0

En-2:

Enkheim

MS:

Murashige–Skoog

ABRC:

Arabidopsis biological resource center

LB:

Luria–Bertani

O/N:

Overnight

STR:

Sitosterol

EtOAc:

Water-saturated ethyl acetate

STG:

Stigmasterol

DMR:

Desmosterol

SiO2 :

Silica

MSTFA:

N-trimethylsilyl-N-methyl trifluoroacetamide

TMS:

Trimethylsilyl

SIM:

Selected ions monitoring

BMB:

Bismethaneboronate

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Funding

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean Government’s Ministry of Science, ICT, and Future Planning (2021R1A2C1007516 to S.-K. Kim).

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Author notes

  1. Chaiweon Seo and Jinyoung Moon have equally contributed to this study.

Authors and Affiliations

  1. Department of Life Science, Chung-Ang University, Seoul, 06974, Republic of Korea

    Chaiweon Seo, Jinyoung Moon, Hyeon Soong Yeom, Yeongil Bae, Jeehee Roh & Seong-Ki Kim

  2. Department of Plant Biotechnology and Bioinformatics, Ghent University, Ghent, Belgium

    Eunji Kim

  3. VIB-UGent Center for Plant Systems Biology, Ghent, Belgium

    Eunji Kim

Authors
  1. Chaiweon Seo
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Contributions

CS, JM, JR, and S-KK planned the experiments; CS, JM, HSY, YB, and EK performed the experiments; CS, JM, JR, and S-KK analyzed the data; and CS, JM, JR, and S-KK wrote and revised the manuscript for publication.

Corresponding authors

Correspondence to Jeehee Roh or Seong-Ki Kim.

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The authors declare no potential conflicts of interest.

Supplementary Information

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Supplementary file1 (DOCX 22 KB)

12374_2023_9409_MOESM2_ESM.pptx

Supplementary file2 (PPTX 1584 KB) Supplementary Figure S1. MBP-fused CYP710A1, CYP710A2, and SMT1 preparation. CYP710A1, CYP710A2, and SMT1 were introduced into a pMALc2x vector, and the constructs were transformed into a BL21 E. coli strain to express MBP-fused proteins. MBP-fused proteins inducted by IPTG were purified, concentrated, and confirmed through SDS-PAGE. Red triangles represent MBP, MBP-710As (MBP-CYP710A1 and MBP-CYP710A2), and MBP-SMT1. MBP, MBP-SMT1, MBP-CYP710A1, and MBP-CYP710A2 molecular weights were 42.5 kDa, 80.8 kDa, 98.2 kDa, and 98.9 kDa, respectively. Supplementary Figure S2. (A) SALK_014626 (cyp710a1) genotyping results. (B) SALK_134856 (cyp710a2) genotyping results. Each asterisk indicates a sample identified as a homozygous mutant used in the experiment. (C) cyp710a1 x cyp710a2 genotyping results

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Seo, C., Moon, J., Yeom, H.S. et al. Cytochrome P450 710A1/A2 as Brassinosteroid C24-Desaturases to Connect C27- and C28-Brassinosteroids Biosynthesis in Arabidopsis thaliana. J. Plant Biol. 67, 59–70 (2024). https://doi.org/10.1007/s12374-023-09409-1

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