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Arabidopsis CYP72C1 is an atypical cytochrome P450 that inactivates brassinosteroids

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Abstract

Cytochrome P450 monooxygenases (P450s) are a diverse family of proteins that have specialized roles in secondary metabolism and in normal cell development. Two P450s in particular, CYP734A1 and CYP72C1, have been identified as brassinosteroid-inactivating enzymes important for steroid-mediated signal transduction in Arabidopsis thaliana. Genetic analyses have demonstrated that these P450s modulate growth throughout plant development. While members of the CYP734A subfamily inactivate brassinosteroids through C-26 hydroxylation, the biochemical activity of CYP72C1 is unknown. Because CYP734A1 and CYP72C1 in Arabidopsis diverge more than brassinosteroid inactivating P450s in other plants, this study examines the structure and biochemistry of each enzyme. Three-dimensional models were generated to examine the substrate binding site structures and determine how they might affect the function of each P450. These models have indicated that the active site of CYP72C1 does not contain several conserved amino acids typically needed for substrate hydroxylation. Heterologous expression of these P450s followed by substrate binding analyses have indicated that CYP734A1 binds active brassinosteroids, brassinolide and castasterone, as well as their upstream precursors whereas CYP72C1 binds precursors more effectively. Seedling growth assays have demonstrated that the genetic state of CYP734A1, but not CYP72C1, affected responsiveness to high levels of exogenous brassinolide supporting our observations that CYP72C1 acts on brassinolide precursors. Although there may be some overlap in their physiological function, the distinct biochemical functions of these proteins in Arabidopsis has significant potential to fine-tune the levels of different brassinosteroid hormones throughout plant growth and development.

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Abbreviations

P450:

Cytochrome P450

BR:

Brassinosteroid

SRS:

Substrate recognition site

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Acknowledgments

We thank Dr. Zhimou Wen for technical input on the baculovirus expression and type-I binding experiments. We also thank Dr. Shozo Fujioka for his gift of castasterone precursors for use as binding substrates and Dr. Edward Turk for cloning the Arabidopsis CYP734A1 and CYP72C1 cDNAs. We thank Drs. Christopher Mau and Rodney Croteau for their help in establishing the E. coli expression system. This research was supported by the United States Department of Agriculture 2005-35318-16214 (L.E.T.), the National Science Foundation 0758411 (M.M.N.) and MCB0115068 (M.A.S.). We are also grateful for support from the Department of Energy DE-FG02-08ER15927 (M.M.N.).

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Authors and Affiliations

  1. Department of Biology, The College of New Jersey, 2000 Pennington Rd., Ewing, NJ, 08628, USA

    Leeann E. Thornton

  2. Department of Cell and Developmental Biology, University of Illinois, Urbana, IL, 61801, USA

    Sanjeewa G. Rupasinghe & Mary A. Schuler

  3. Department of Crop and Soil Sciences, Washington State University, Pullman, WA, 99164, USA

    Hao Peng & Michael M. Neff

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  1. Leeann E. Thornton
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  2. Sanjeewa G. Rupasinghe
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Correspondence to Leeann E. Thornton.

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Thornton, L.E., Rupasinghe, S.G., Peng, H. et al. Arabidopsis CYP72C1 is an atypical cytochrome P450 that inactivates brassinosteroids. Plant Mol Biol 74, 167–181 (2010). https://doi.org/10.1007/s11103-010-9663-y

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