Abstract
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 1β-oxidation and 9,11-desaturation of GAs, but was lacking the 3-oxidation activity. CsGA1ox/ds overexpression in Arabidopsis plants resulted in severely dwarfed plants that could be rescued by the exogenous application of bioactive GA4, confirming that CsGA1ox/ds catabolizes GAs. Substitution of three amino acids in CsGA1ox/ds, Phe93, Pro106, and Ser202, with those typically conserved among GA 3-oxidases, Tyr93, Met106, and Thr202, 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 in Arabidopsis accelerated plant growth and development, indicating that this enzyme variant can produce bioactive GAs in 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 GA9 to GA4, highlighting the importance of these three conserved amino acids for GA 3-oxidase activity.
Highlights
Bioactive gibberellins (GAs) are central regulators of plant growth and development, including seed development
Our results indicate that the GA 3-oxidase conserved amino acids Tyr and Met are important for GA 3-oxidase activity, with Thr having a minor function
In addition to the previously described endogenous GAs from mature seeds of Cucumis sativus (12), we identified several 2-hydroxylated gibberellins in mature seeds; this correlates with the GA 2-oxidase expression at the late stages of seed development (Fig. S6)
Summary
Bioactive gibberellins (GAs) are central regulators of plant growth and development, including seed development. CsGA1ox/ds overexpression in Arabidopsis plants resulted in severely dwarfed plants that could be rescued by the exogenous application of bioactive GA4, 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. 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 Overexpression of this amino acid–modified GA1ox/ds variant in Arabidopsis accelerated plant growth and development, indicating that this enzyme variant can produce bioactive GAs in planta. Our results indicate that these three conserved amino acids among GA
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