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Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cell Suspensions: A Proteomic Analysis

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  • Published:
Lipids

Abstract

The effect of an inhibitor of cycloartenol synthase (CAS, EC 5.4.99.8) on the proteome of tobacco BY-2 cells has been examined. CAS catalyzes the first committed step in phytosterol synthesis in plants. BY-2 cells were treated with RO 48-8071, a potent inhibitor of oxidosqualene cyclization. Proteins were separated by two-dimensional electrophoresis and spots, that clearly looked differentially accumulated after visual inspection, were cut, in-gel trypsin digested, and peptides were analyzed by nano-HPLC–MS/MS. Distinct peptides were compared to sequences in the data banks and attributed to corresponding proteins and genes. Inhibition of CAS induced proteins that appear to mitigate the negative effects of the chemical exposure. However, as all enzymes that are directly involved in phytosterol biosynthesis are low-abundant proteins, significant changes in their levels could not be observed. Differences could be seen with enzymes involved in primary metabolism (glycolysis, pentose phosphate pathway etc.), in proteins of the chaperonin family, and those, like actin, that participate in formation and strengthening of the cytoskeleton and have some impact on cell growth and division.

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Fig. 1

Abbreviations

BY-2 cells:

Bright Yellow cells

CAS:

Cycloartenol synthase

CHAPS:

3-[(3-Cholamidopropyl)dimethylammonio]-1-propanesulfonate

EC:

Enzyme catalogue

EMBL:

European Molecular Biology Laboratory

ER:

Endoplasmic reticulum

GAP:

Glyceraldehyde 3-phosphate

IEF:

Isoelectric focusing

IPG:

Immobilized pH gradient

HMG-CoA:

3-Hydroxy-3-methylglutaryl-coenzyme A

HMGR:

HMG-CoA reductase

HSP:

Heat shock protein

LAS:

Lanosterol synthase

2D PAGE:

Two-dimensional polyacrylamide gel electrophoresis

PBS:

Phosphate buffer saline

PDB:

Protein data bank

RuBisCO:

Ribulose bisphosphate carboxylase/oxidase

SAM:

S-Adenosyl-methionine

SMT:

Sterol methyltransferase

SDS:

Sodium dodecyl sulfate

SSRP1:

Structure-specific recognition protein 1

TCA:

Trichloroacetic acid

TOF:

Time of flight

Tris:

Trishydroxymethylaminomethane, or 2-amino-2-hydroxymethyl-1,3-propanediol

VIGS:

Virus-induced gene silencing

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Author notes

  1. Elisabet Gas-Pascual

    Present address: Department of Horticulture and Crop Science, The Ohio State University, 208 Williams Hall, 1680 Madison Avenue, Wooster, OH, 44691, USA

Authors and Affiliations

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

    Elisabet Gas-Pascual, Biljana Simonovik, Dimitri Heintz, Marc Bergdoll, 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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11745_2015_4041_MOESM1_ESM.tif

Fig. S1. Protein category and enriched cellular processes. Identified proteins have been distributed into 7 major categories (and 5 minor categories), depending on the cellular processes to which are more likely to be involved. Values represent  the percentage of proteins in each category vs the total number of proteins identified.(TIFF 51 kb)

11745_2015_4041_MOESM2_ESM.xlsx

Table S1: Identified proteins, selected peptide sequences, protein coverage and spectral information based on Mascot MS/MS dataset. Information provided in this table complements Table 1. Up to 25 peptides were identified from each spot. Predicted proteins represented by less than 3 peptides were discarded. Only the first peptide for each identified protein is shown. (XLSX 25 kb)

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Gas-Pascual, E., Simonovik, B., Heintz, D. et al. Inhibition of Cycloartenol Synthase (CAS) Function in Tobacco BY-2 Cell Suspensions: A Proteomic Analysis. Lipids 50, 773–784 (2015). https://doi.org/10.1007/s11745-015-4041-9

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