Plant Molecular Biology

, 68:633

Transcriptome analysis approaches for the isolation of trichome-specific genes from the medicinal plant Cistus creticus subsp. creticus

  • Vasiliki Falara
  • Vasileios Fotopoulos
  • Thanasis Margaritis
  • Thalia Anastasaki
  • Irene Pateraki
  • Artemios M. Bosabalidis
  • Dimitris Kafetzopoulos
  • Costas Demetzos
  • Eran Pichersky
  • Angelos K. Kanellis
Article

DOI: 10.1007/s11103-008-9399-0

Cite this article as:
Falara, V., Fotopoulos, V., Margaritis, T. et al. Plant Mol Biol (2008) 68: 633. doi:10.1007/s11103-008-9399-0

Abstract

Cistus creticus subsp. creticus is a plant of intrinsic scientific interest due to the distinctive pharmaceutical properties of its resin. Labdane-type diterpenes, the main constituents of the resin, exhibit considerable antibacterial and cytotoxic activities. In this study chemical analysis of isolated trichomes from different developmental stages revealed that young leaves of 1–2 cm length displayed the highest content of labdane-type diterpenes (80 mg/g fresh weight) whereas trichomes from older leaves (2–3 or 3–4 cm) exhibited gradual decreased concentrations. A cDNA library was constructed enriched in transcripts from trichomes isolated from young leaves, which are characterized by high levels of labdane-type diterpenes. Functional annotation of 2,022 expressed sequence tags (ESTs) from the trichome cDNA library based on homology to A. thaliana genes suggested that 8% of the putative identified sequences were secondary metabolism-related and involved primarily in flavonoid and terpenoid biosynthesis. A significant proportion of the ESTs (38%) displayed no significant similarity to any other DNA deposited in databases, indicating a yet unknown function. Custom DNA microarrays constructed with 1,248 individual clones from the cDNA library facilitated transcriptome comparisons between trichomes and trichome-free tissues. In addition, gene expression studies in various Cistus tissues and organs for one of the genes highlighted as the most differentially expressed by the microarray experiments revealed a putative sesquiterpene synthase with a trichome-specific expression pattern. Full length cDNA isolation and heterologous expression in E. coli followed by biochemical analysis, led to the characterization of the produced protein as germacrene B synthase.

Keywords

Cistus creticus subsp. creticus DNA microarrays EST analysis Gene expression Germacrene B synthase Trichomes 

Abbreviations

ABA

Abscisic acid

aRNA

Amplified RNA

CcEF1α

Cistus creticus elongation factor 1α

CcGer

Cistus creticus germacrene B synthase

CER1

ECERIFERUM1 sterol desaturase

DTT

1,4-Dithiothreitol

EST

Expression sequence tag

FDP

Farnesyl diphosphate

GC/MS

Gas chromatography/mass spectrometry

GGDPS

Geranyl-geraniol diphosphate synthase

MIPS

Munich Information Center for Protein Sequences

NIST

National Institute of Standards and Technology

PDMS

Polydimethylsiloxane

SCID

Severe combined immunodeficiency disease

SEM

Scanning electron microscopy

Copyright information

© Springer Science+Business Media B.V. 2008

Authors and Affiliations

  • Vasiliki Falara
    • 1
  • Vasileios Fotopoulos
    • 1
    • 2
  • Thanasis Margaritis
    • 3
    • 4
  • Thalia Anastasaki
    • 5
  • Irene Pateraki
    • 1
    • 6
  • Artemios M. Bosabalidis
    • 7
  • Dimitris Kafetzopoulos
    • 3
    • 4
  • Costas Demetzos
    • 5
  • Eran Pichersky
    • 8
  • Angelos K. Kanellis
    • 1
  1. 1.Group of Biotechnology of Pharmaceutical Plants, Laboratory of Pharmacognosy, Department of Pharmaceutical SciencesAristotle University of ThessalonikiThessalonikiGreece
  2. 2.Department of Agricultural Sciences, Biotechnology and Food ScienceCyprus University of TechnologyLimassolCyprus
  3. 3.Institute of Molecular Biology and BiotechnologyFoundation of Research and TechnologyHeraklionGreece
  4. 4.Department of BiologyUniversity of CreteHeraklionGreece
  5. 5.School of PharmacyNational and Kapodistrian University of AthensAthensGreece
  6. 6.Departament de Bioquímica i Biologia Molecular, Facultat de BiologiaUniversitat de BarcelonaBarcelonaSpain
  7. 7.School of BiologyAristotle University of ThessalonikiThessalonikiGreece
  8. 8.Department of Molecular, Cellular, and Developmental BiologyUniversity of MichiganAnn ArborUSA