The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the flavor volatiles β-ionone, pseudoionone, and geranylacetone

Authors

  • Andrew J. Simkin,

    1. Horticultural Sciences, Plant Molecular and Cellular Biology Program, PO Box 110690, University of Florida, Gainesville, FL 32611-0690, USA
    Search for more papers by this author
  • Steven H. Schwartz,

    1. Department of Energy-Plant Research Laboratory and Department of Plant Biology, Michigan State University, East Lansing, MI 48824, USA
    Search for more papers by this author
  • Michele Auldridge,

    1. Horticultural Sciences, Plant Molecular and Cellular Biology Program, PO Box 110690, University of Florida, Gainesville, FL 32611-0690, USA
    Search for more papers by this author
  • Mark G. Taylor,

    1. Horticultural Sciences, Plant Molecular and Cellular Biology Program, PO Box 110690, University of Florida, Gainesville, FL 32611-0690, USA
    Search for more papers by this author
  • Harry J. Klee

    Corresponding author
    1. Horticultural Sciences, Plant Molecular and Cellular Biology Program, PO Box 110690, University of Florida, Gainesville, FL 32611-0690, USA
      (fax +001 352 846 2063; e-mail hjklee@ifas.ufl.edu).
    Search for more papers by this author

(fax +001 352 846 2063; e-mail hjklee@ifas.ufl.edu).

Summary

Volatile terpenoid compounds, potentially derived from carotenoids, are important components of flavor and aroma in many fruits, vegetables and ornamentals. Despite their importance, little is known about the enzymes that generate these volatiles. The tomato genome contains two closely related genes potentially encoding carotenoid cleavage dioxygenases, LeCCD1A and LeCCD1B. A quantitative reverse transcriptase-polymerase chain reaction analysis revealed that one of these two genes, LeCCD1B, is highly expressed in ripening fruit (4 days post-breaker), where it constitutes 0.11% of total RNA. Unlike the related neoxanthin cleavage dioxygenases, import assays using pea chloroplasts showed that the LeCCD1 proteins are not plastid-localized. The biochemical functions of the LeCCD1 proteins were determined by bacterial expression and in vitro assays, where it was shown that they symmetrically cleave multiple carotenoid substrates at the 9,10 (9′,10′) positions to produce a C14 dialdehyde and two C13 cyclohexones that vary depending on the substrate. The potential roles of the LeCCD1 genes in vivo were assessed in transgenic tomato plants constitutively expressing the LeCCD1B gene in reverse orientation. This over-expression of the antisense transcript led to 87–93% reductions in mRNA levels of both LeCCD1A and LeCCD1B in the leaves and fruits of selected lines. Transgenic plants exhibited no obvious morphological alterations. High-performance liquid chromatography analysis showed no significant modification in the carotenoid content of fruit tissue. However, volatile analysis showed a ≥50% decrease in β-ionone (a β-carotene-derived C13 cyclohexone) and a ≥60% decrease in geranylacetone (a C13 acyclic product likely derived from a lycopene precursor) in selected lines, implicating the LeCCD1 genes in the formation of these important flavor volatiles in vivo.

Ancillary