title: Recombinant yeast as a functional tool for understanding bitterness and cucurbitacin biosynthesis in watermelon (Citrullus spp.).
abstract: Cucurbitacins are a group of bitter-tasting oxygenated tetracyclic triterpenes that are produced in the family Cucurbitaceae and other plant families. The natural roles of cucurbitacins in plants are probably related to defence against pathogens and pests. Cucurbitadienol, a triterpene synthesized from oxidosqualene, is the first committed precursor to cucurbitacins produced by a specialized oxidosqualene cyclase termed cucurbitadienol synthase. We explored cucurbitacin accumulation in watermelon in relation to bitterness. Our findings show that cucurbitacins are accumulated in bitter-tasting watermelon, Citrullus lanatus var. citroides, as well as in their wild ancestor, C. colocynthis, but not in non-bitter commercial cultivars of sweet watermelon (C. lanatus var. lanatus). Molecular analysis of genes expressed in the roots of several watermelon accessions led to the isolation of three sequences (CcCDS1, CcCDS2 and ClCDS1), all displaying high similarity to the pumpkin CpCPQ, encoding a protein previously shown to possess cucurbitadienol synthase activity. We utilized the Saccharomyces cerevisiae strain BY4743, heterozygous for lanosterol synthase, to probe for possible encoded cucurbitadienol synthase activity of the expressed watermelon sequences. Functional expression of the two sequences isolated from C. colocynthis (CcCDS1 and CcCDS2) in yeast revealed that only CcCDS2 possessed cucurbitadienol synthase activity, while CcCDS1 did not display cucurbitadienol synthase activity in recombinant yeast. ClCDS1 isolated from C. lanatus var. lanatus is almost identical to CcCDS1. Our results imply that CcCDS2 plays a role in imparting bitterness to watermelon. Yeast has been an excellent diagnostic tool to determine the first committed step of cucurbitacin biosynthesis in watermelon.
authors.affiliation: Institute of Plant Sciences, Newe Ya'ar Research Centre, Agricultural Research Organization, Ramat Yishay, Israel.
authors.forename: Rachel
authors.initials: R
authors.lastname: Davidovich-Rikanati
authors.forename: Lior
authors.initials: L
authors.lastname: Shalev
authors.forename: Nadine
authors.initials: N
authors.lastname: Baranes
authors.forename: Ayala
authors.initials: A
authors.lastname: Meir
authors.forename: Maxim
authors.initials: M
authors.lastname: Itkin
authors.forename: Shahar
authors.initials: S
authors.lastname: Cohen
authors.forename: Kobi
authors.initials: K
authors.lastname: Zimbler
authors.forename: Vitaly
authors.initials: V
authors.lastname: Portnoy
authors.forename: Yutaka
authors.initials: Y
authors.lastname: Ebizuka
authors.forename: Masaaki
authors.initials: M
authors.lastname: Shibuya
authors.forename: Yosef
authors.initials: Y
authors.lastname: Burger
authors.forename: Nurit
authors.initials: N
authors.lastname: Katzir
authors.forename: Arthur A
authors.initials: AA
authors.lastname: Schaffer
authors.forename: Efraim
authors.initials: E
authors.lastname: Lewinsohn
authors.forename: Ya'akov
authors.initials: Y
authors.lastname: Tadmor
mesh_terms: D000595:Amino Acid Sequence; D029773:Citrullus / Q000737:chemistry / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D054728:Cucurbitacins / Q000096:biosynthesis*; D005421:Flavoring Agents / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013649:Taste
chemical_list: D005421:Flavoring Agents; D010940:Plant Proteins; D054728:Cucurbitacins
keywords: bitterness; cucurbitacins; cucurbitadienol synthase; watermelon