title: Single cell subtractive transcriptomics for identification of cell-specifically expressed candidate genes of pyrrolizidine alkaloid biosynthesis.
abstract: Progress has recently been made in the elucidation of pathways of secondary metabolism. However, because of its diversity, genetic information concerning biosynthetic details is still missing for many natural products. This is also the case for the biosynthesis of pyrrolizidine alkaloids. To close this gap, we tested strategies using tissues that express this pathway in comparison to tissues in which this pathway is not expressed. As many pathways of secondary metabolism are known to be induced by jasmonates, the pyrrolizidine alkaloid-producing species Heliotropium indicum, Symphytum officinale, and Cynoglossum officinale of the Boraginales order were treated with methyl jasmonate. An effect on pyrrolizidine alkaloid levels and on transcript levels of homospermidine synthase, the first specific enzyme of pyrrolizidine alkaloid biosynthesis, was not detectable. Therefore, a method was developed by making use of the often observed cell-specific production of secondary compounds. H. indicum produces pyrrolizidine alkaloids exclusively in the shoot. Homospermidine synthase is expressed only in the cells of the lower leaf epidermis and the epidermis of the stem. Suggesting that the whole pathway of pyrrolizidine alkaloid biosynthesis might be localized in these cells, we have isolated single cells of the upper and lower epidermis by laser-capture microdissection. The resulting cDNA preparations have been used in a subtractive transcriptomic approach. Quantitative real-time polymerase chain reaction has shown that the resulting library is significantly enriched for homospermidine-synthase-coding transcripts providing a valuable source for the identification of further genes involved in pyrrolizidine alkaloid biosynthesis.
authors.affiliation: Botanical Institute and Botanical Garden, Christian-Albrechts University Kiel, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Sievert
authors.affiliation: Institute for Pharmaceutical Biology, TU Braunschweig, Mendelssohnstrasse 1, D-38106 Braunschweig, Germany.
authors.forename: Till
authors.initials: T
authors.lastname: Beuerle
authors.affiliation: Botanical Institute and Botanical Garden, Christian-Albrechts University Kiel, Germany.
authors.forename: Julien
authors.initials: J
authors.lastname: Hollmann
authors.affiliation: Botanical Institute and Botanical Garden, Christian-Albrechts University Kiel, Germany. Electronic address: dober@bot.uni-kiel.de.
authors.forename: Dietrich
authors.initials: D
authors.lastname: Ober
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism; D028082:Boraginaceae / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018076:DNA, Complementary / Q000235:genetics; D019143:Evolution, Molecular; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism; D018547:Plant Stems / Q000378:metabolism; D011763:Pyrrolizidine Alkaloids / Q000737:chemistry / Q000378:metabolism*; D060888:Real-Time Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid
chemical_list: D018076:DNA, Complementary; D011763:Pyrrolizidine Alkaloids; D019883:Alkyl and Aryl Transferases; C028376:homospermidine synthetase
keywords: Evolution; Homospermidine synthase; Laser-capture microdissection; Secondary metabolism; Specialized metabolism; Suppression subtractive hybridization