title: A dwarf mutant strain of Pharbitis nil, Uzukobito (kobito), has defective brassinosteroid biosynthesis.
abstract: Japanese morning glory (Pharbitis nil) is a model plant characterized by a large stock of spontaneous mutants. The recessive mutant Uzukobito shows strong dwarfism with dark-green rugose leaves. The phenotype was rescued by the application of brassinolide, a bioactive brassinosteroid (BR), indicating that Uzukobito was a BR-deficient mutant. A detailed analysis of the endogenous BR levels in Uzukobito and its parental wild-type plant showed that Uzukobito had a lower level of BRs downstream of (24R)-24-methyl-5alpha-cholestan-3-one and (22S, 24R)-22-hydroxy-24-methyl-5alpha-cholestan-3-one than those in wild-type plants, while their immediate precursors (24R)-24-methylcholest-4-en-3-one and (22S, 24R)-22-hydroxy-24-methylcholest-4-en-3-one accumulated relatively more in Uzukobito. These results indicate that Uzukobito had a defect in the conversion of (24R)-24-methylcholest-4-en-3-one and (22S, 24R)-22-hydroxy-24-methylcholest-4-en-3-one to their 5alpha-reduced forms, which is catalyzed by de-etiolated2 (DET2) in Arabidopsis. The P. nil ortholog of the DET2 gene (PnDET2) was cloned and shown to have the greatest similarity to DET2 among all the putative genes in Arabidopsis. Uzukobito had one amino acid substitution from Glu62 to Val62 in the deduced amino acid sequence of PnDET2. Recombinant PnDET2 expressed in COS-7 cells was found to be a functional steroid 5alpha-reductase (S5alphaR) converting (24R)-24-methylcholest-4-en-3-one to (24R)-24-methyl-5alpha-cholestan-3-one, while PnDET2 with the mutation did not show any catalytic activity. This shows that a plant S5alphaR can convert an intrinsic substrate. All these results clearly demonstrate that the Uzukobito phenotype resulted from a mutation on PnDET2, and a morphological mutant has been characterized at the molecular level among a large stock of P. nil mutants.
authors: Department of Applied Biological Chemistry, The University of Tokyo, Bunkyo-ku, Tokyo 113-8657, Japan. ayoshi@pgr1.ch.a.u-tokyo.ac.jp
authors: Yoshihito
authors: Y
authors: Suzuki
authors: Kazuyuki
authors: K
authors: Saso
authors: Shozo
authors: S
authors: Fujioka
authors: Shigeo
authors: S
authors: Yoshida
authors: Eiji
authors: E
authors: Nitasaka
authors: Shinji
authors: S
authors: Nagata
authors: Hiromichi
authors: H
authors: Nagasawa
authors: Suguru
authors: S
authors: Takatsuto
authors: Isomaro
authors: I
authors: Yamaguchi
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002522:Chlorocebus aethiops; D017124:Conserved Sequence; D017343:Genes, Plant; D005808:Genes, Recessive; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D010802:Phylogeny; D010937:Plant Growth Regulators / Q000096:biosynthesis / Q000235:genetics*; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019657:Solanaceae / Q000145:classification / Q000235:genetics* / Q000254:growth & development; D013256:Steroids / Q000378:metabolism*; D014162:Transfection
chemical_list: D010937:Plant Growth Regulators; D010940:Plant Proteins; D011994:Recombinant Proteins; D013256:Steroids