Cucumber gibberellin 1-oxidase/desaturase initiates novel gibberellin catabolic pathways

Bioactive gibberellins (GAs) are central regulators of plant growth and development, including seed development. GA homeostasis is achieved via complex biosynthetic and catabolic pathways, whose exact activities remain to be elucidated. Here, we isolated two cDNAs from mature or imbibed cucumber seeds with high sequence similarity to known GA 3-oxidases. We found that one enzyme (designated here CsGA3ox5) has GA 3-oxidation activity. However, the second enzyme (designated CsGA1ox/ds) performed multiple reactions, including 1?-oxidation and 9,11-desaturation of GAs, but was lacking the 3-oxidation activity. CsGA1ox/ds overexpression inArabidopsisplants resulted in severely dwarfed plants that could be rescued by the exogenous application of bioactive GA(4), confirming that CsGA1ox/ds catabolizes GAs. Substitution of three amino acids in CsGA1ox/ds, Phe(93), Pro(106), and Ser(202), with those typically conserved among GA 3-oxidases, Tyr(93), Met(106), and Thr(202), respectively, conferred GA 3-oxidase activity to CsGA1ox/ds and thereby augmented its potential to form bioactive GAs in addition to catabolic products. Accordingly, overexpression of this amino acid?modified GA1ox/ds variant inArabidopsisaccelerated plant growth and development, indicating that this enzyme variant can produce bioactive GAsin planta. Furthermore, a genetically modified GA3ox5 variant in which these three canonical GA 3-oxidase amino acids were changed to the ones present in CsGA1ox/ds was unable to convert GA(9)to GA(4), highlighting the importance of these three conserved amino acids for GA 3-oxidase activity.