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Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cells

  • Original Article
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Lipids

Abstract

Tobacco BY-2 cell suspensions are our preferred model for studying isoprenoid biosynthesis pathways, due to their easy genetic transformation and the efficient absorption of metabolic precursors, intermediates, and/or inhibitors. Using this model system, we have analyzed the effects of chemical and genetic blockage of cycloartenol synthase (CAS, EC 5.4.99.8), an oxidosqualene cyclase that catalyzes the first committed step in the sterol pathway of plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Short-term treatments (24 h) resulted in accumulation of oxidosqualene with no changes in the final sterol products. Interestingly, long-term treatments (6 days) induced down-regulation in gene expression not only of CAS but also of the SMT2 gene coding sterol methyltransferase 2 (EC 2.1.1.41). This explains some of the increase in 24-methyl sterols at the expense of the 24-ethyl sterols stigmasterol and sitosterol. In our alternative strategy, CAS gene expression was partially blocked by using an inducible artificial microRNA. The limited effectiveness of this approach might be explained by some dependence of the machinery for RNAi formation on an operating MVA/sterol pathway. For comparison we checked the effect of RO 48-8071 on a green cell suspension of Arabidopsis and on seedlings, containing a small spectrum of triterpenes besides phytosterols. Triterpenes remained essentially unaffected, but phytosterol accumulation was clearly diminished.

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Abbreviations

CAS:

Cycloartenol synthase

NtCAS:

Nicotiana tabacum CAS

RO 48-8071:

(4-Bromophenyl)[2-fluoro-4-[[6-(methyl-2- propenylamino)hexyl]oxy]phenyl]-methanone

SMT:

Sterol methyltransferase

amiRNA:

Artificial micro interfering RNA

RNAi:

Interfering RNA

microRNA:

Small interfering RNA

MVA:

Mevalonic acid

BY-2 cells:

(Tobacco) Bright Yellow-2 cells

LAS:

Lanosterol synthase

MEP:

Methyl erythritol phosphate

PFTE:

Polytetrafluoroethylene

GC-FID:

Gas chromatography with flame ionization detector

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Acknowledgments

This work was financially supported by the Centre National de la Recherche Scientifique (CNRS), by the Université de Strasbourg and by the Agence Nationale de la Recherche (ANR grants “Terpene”, NT05-3-45695 and “Biosis”, BLAN06-2_135891). We wish to thank Prof. Nam-Hai Chua (Rockefeller University) for providing us with the pER8 estradiol inducible vector. We thank Dr. Marie-Claire Criqui (IBMP Strasbourg) for providing the MM2D cells. We are indebted to Prof. Katrina Cornish (Department of Horticulture and Crop Science, The Ohio State University, Wooster OH, 44691, USA) for critically reading the English manuscript.

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  1. Elisabet Gas-Pascual

    Present address: Department of Horticulture and Crop Science, The Ohio State University, Wooster, OH, 44691, USA

Authors and Affiliations

  1. Institut de Biologie Moléculaire des Plantes, CNRS UPR 2357, Université de Strasbourg, 28, rue Goethe, 67083, Strasbourg, France

    Biljana Simonovik, Hubert Schaller & Thomas J. Bach

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  1. Elisabet Gas-Pascual
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  2. Biljana Simonovik
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Correspondence to Thomas J. Bach.

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G. J. Schroepfer, Jr. Memorial Sterol Symposium.

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Gas-Pascual, E., Simonovik, B., Schaller, H. et al. Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cells. Lipids 50, 761–772 (2015). https://doi.org/10.1007/s11745-015-4036-6

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