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Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint

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Abstract

Biosynthesis of the p-menthane monoterpenes in peppermint occurs in the secretory cells of the peltate glandular trichomes and results in the accumulation of primarily menthone and menthol. cDNAs and recombinant enzymes are well characterized for eight of the nine enzymatic steps leading from the 5-carbon precursors to menthol, and subcellular localization of several key enzymes suggests a complex network of substrate and product movement is required during oil biosynthesis. In addition, studies concerning the regulation of oil biosynthesis have demonstrated a temporal partition of the pathway into an early, biosynthetic program that results in the accumulation of menthone and a later, oil maturation program that leads to menthone reduction and concomitant menthol accumulation. The menthone reductase responsible for the ultimate pathway reduction step, menthone-menthol reductase (MMR), has been characterized and found to share significant sequence similarity with its counterpart reductase, a menthone-neomenthol reductase, which catalyzes a minor enzymatic reaction associated with oil maturation. Further, the menthone reductases share significant sequence similarity with the temporally separate and mechanistically different isopiperitenone reductase (IPR). Here we present immunocytochemical localizations for these reductases using a polyclonal antibody raised against menthone-menthol reductase. The polyclonal antibody used for this study showed little specificity between these three reductases, but by using it for immunostaining of tissues of different ages we were able to provisionally separate staining of an early biosynthetic enzyme, IPR, found in young, immature leaves from that of the oil maturation enzyme, MMR, found in older, mature leaves. Both reductases were localized to the cytoplasm and nucleoplasm of the secretory cells of peltate glandular trichomes, and were absent from all other cell types examined.

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Notes

  1. Numbering of p-menthane monoterpene carbons is based on the limonene carbon skeleton in which the methyl group (C7) is attached to C1 and the isopropyl group (C8, C9 and C10) is attached to C4 of the cyclohexanyl ring.

Abbreviations

GPP:

Geranyl diphosphate

IPD:

Isopiperitenol dehydrogenase

IPR:

Isopiperitenone reductase

MEP:

Methyl erythritol phosphate

MR:

Menthone reductase

MMR:

Menthone-menthol reductase

MNR:

Menthone-neomenthol reductase

PR:

Pulegone reductase

NADPH:

Nicotinamide adenine dinucleotide phosphate

EDTA:

Ethylenediaminetetraacetic acid

IgG:

Immunoglobulin

TBST:

Tris-buffered saline-tween

BSA:

Bovine serum albumin

NBT:

Nitro-blue tetrazolium

BCIP:

5-Bromo-4-chloro-3-indolyl phosphate

ER:

Endoplasmic reticulum

TEM:

Transmission electron microscopy

anti-MMR:

Polyclonal antiserum raised against MMR

HPF-FS:

High-pressure freezing and freeze substitution

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Acknowledgments

We thank the staff of the Franceschi Microscopy and Imaging Center at Washington State University for technical assistance and use of equipment. We thank Dr. Kent McDonald of the Electron Microscope Laboratory at the University of California at Berkeley for preparing freeze-substituted samples of peppermint leaves. We thank Dr. Christopher Mau and Dr. Yujia Wu for helpful discussions, and we thank Juliana Gothard for growing the plants.

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  1. Institute of Biological Chemistry, Washington State University, Pullman, WA, 99164-6340, USA

    Glenn W. Turner, Edward M. Davis & Rodney B. Croteau

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  1. Glenn W. Turner
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  2. Edward M. Davis
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  3. Rodney B. Croteau
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Correspondence to Glenn W. Turner or Rodney B. Croteau.

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Turner, G.W., Davis, E.M. & Croteau, R.B. Immunocytochemical localization of short-chain family reductases involved in menthol biosynthesis in peppermint. Planta 235, 1185–1195 (2012). https://doi.org/10.1007/s00425-011-1567-9

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