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A dwarf mutant strain of Pharbitis nil , Uzukobito ( kobito ), has defective brassinosteroid biosynthesis
Author(s) -
Suzuki Yoshihito,
Saso Kazuyuki,
Fujioka Shozo,
Yoshida Shigeo,
Nitasaka Eiji,
Nagata Shinji,
Nagasawa Hiromichi,
Takatsuto Suguru,
Yamaguchi Isomaro
Publication year - 2003
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2003.01887.x
Subject(s) - biology , brassinosteroid , mutant , arabidopsis , brassinolide , pharbitis nil , wild type , gene , amino acid , biochemistry , genetics , botany , plant growth
Summary 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 (24 R )‐24‐methyl‐5α‐cholestan‐3‐one and (22 S , 24 R )‐22‐hydroxy‐24‐methyl‐5α‐cholestan‐3‐one than those in wild‐type plants, while their immediate precursors (24 R )‐24‐methylcholest‐4‐en‐3‐one and (22 S , 24 R )‐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 (24 R )‐24‐methylcholest‐4‐en‐3‐one and (22 S , 24 R )‐22‐hydroxy‐24‐methylcholest‐4‐en‐3‐one to their 5α‐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 Glu 62 to Val 62 in the deduced amino acid sequence of PnDET2. Recombinant PnDET2 expressed in COS‐7 cells was found to be a functional steroid 5α‐reductase (S5αR) converting (24 R )‐24‐methylcholest‐4‐en‐3‐one to (24 R )‐24‐methyl‐5α‐cholestan‐3‐one, while PnDET2 with the mutation did not show any catalytic activity. This shows that a plant S5αR 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.