The Class III Gibberellin 2-Oxidases AtGA2ox9 and AtGA2ox10 Contribute to Cold Stress Tolerance and Fertility.

Many developmental processes in plants are regulated by GA hormones. GA homeostasis is achieved via complex biosynthetic and catabolic pathways. GA catabolic enzymes include GA 2-oxidases that are classified into three classes. Members of class III GA 2-oxidases typically act on GA precursors containing a C20-skeleton. Here, we identified two further members of this class of GA 2-oxidases, namely AtGA2ox9 and AtGA2ox10, in the Arabidopsis (Arabidopsis thaliana) genome. Both genes encode enzymes that have functional similarities to AtGA2ox7 and AtGA2ox8, which are class III GA 2-oxidases that 2ß-hydroxylate C20-GAs. Previously unknown for GA 2-oxidases, AtGA2ox9 performs 2α-hydroxylation of C19-GAs and harbors putative desaturating activity of C20-GAs. Additionally, AtGA2ox9 and AtGA2ox10 exhibit GA 20-oxidase activity. AtGA2ox9 oxidizes carbon-20 to form tricarboxylic acid C20-GAs, whereas AtGA2ox10 produces C19-GA9 AtGA2ox9 transcript levels increase after cold treatment and AtGA2ox10 is expressed mainly in the siliques of Arabidopsis plants. Atga2ox9 loss-of-function mutants are more sensitive to freezing temperatures, whereas Atga2ox10 loss-of-function mutants produce considerably more seeds per silique than wild-type plants. We conclude that in Arabidopsis, AtGA2ox9 contributes to freezing tolerance and AtGA2ox10 regulates seed production.

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 1ß-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.

The Multifunctional Dioxygenases of Gibberellin Synthesis.

Gibberellin (GA) hormones regulate the development of plants and their responses to environmental signals. The final part of GA biosynthesis is catalyzed by multifunctional 2-oxoglutarate-dependent dioxygenases, which are encoded by multigene families. According to their enzymatic properties and physiological functions, GA-oxidases are classified as anabolic or catabolic enzymes. Together they allow complex regulation of the GA biosynthetic pathway, which adapts the specific hormonal needs of a plant during development and interaction with its environment. In this review, we combine recent advances in enzymatic characterization of the multifunctional GA-oxidases, in particular, from cucumber and Arabidopsis that have been most comprehensively investigated.

Ovary-derived precursor gibberellin A9 is essential for female flower development in cucumber.

Gibberellins (GAs) are hormones that control many aspects of plant development, including flowering. It is well known that stamen is the source of GAs that regulate male and bisexual flower development. However, little is known about the role of GAs in female flower development. In cucumber, high levels of GA precursors are present in ovaries and high levels of bioactive GA4 are identified in sepals/petals, reflecting the expression of GA 20-oxidase and 3-oxidase in these organs, respectively. Here, we show that the biologically inactive precursor GA9 moves from ovaries to sepal/petal tissues where it is converted to the bioactive GA4 necessary for female flower development. Transient expression of a catabolic GA 2-oxidase from pumpkin in cucumber ovaries decreases GA9 and GA4 levels and arrests the development of female flowers, and this can be restored by application of GA9 to petals thus confirming its function. Given that bioactive GAs can promote sex reversion of female flowers, movement of biologically inactive precursors, instead of the hormone itself, might help to maintain floral organ identity, ensuring fruit and seed production.

Functional characterization of gibberellin oxidases from cucumber, Cucumis sativus L.

Cucurbits have been used widely to elucidate gibberellin (GA) biosynthesis. With the recent availability of the genome sequence for the economically important cucurbit Cucumis sativus, sequence data became available for all genes potentially involved in GA biosynthesis for this species. Sixteen cDNAs were cloned from root and shoot of 3-d to 7-d old seedlings and from mature seeds of C. sativus. Two cDNAs code for GA 7-oxidases (CsGA7ox1, and -2), five for GA 20-oxidases (CsGA20ox1, -2, -3, -4, and -5), four for GA 3-oxidases (CsGA3ox1, -2, -3, and -4), and another five for GA 2-oxidases (CsGA2ox1, -2, -3, -4, and -5). Their enzymatic activities were investigated by heterologous expression of the cDNAs in Escherichia coli and incubation of the cell lysates with (14)C-labelled, D2-labelled, or unlabelled GA-substrates. The two GA 7-oxidases converted GA12-aldehyde to GA12 efficiently. CsGA7ox1 converted GA12 to GA14, to 15α-hydroxyGA12, and further to 15α-hydroxyGA14. CsGA7ox2 converted GA12 to its 12α-hydroxylated analogue GA111. All five GA 20-oxidases converted GA12 to GA9 as a major product, and to GA25 as a minor product. The four GA 3-oxidases oxidized the C19-GA GA9 to GA4 as the only product. In addition, three of them (CsGA3ox2, -3, and -4) converted the C20-GA GA12 to GA14. The GA 2-oxidases CsGA2ox1, -2, -3, and -4 oxidized the C19-GAs GA9 and GA4 to GA34 and GA51, respectively. CsGA2ox2, -3, and -4 converted GA51 and GA34 further to respective GA-catabolites. In addition to C19-GAs, CsGA2ox4 also converted the C20-GA GA12 to GA110. In contrast, CsGA2ox5 oxidized only the C20 GA12 to GA110 as the sole product. As shown for CsGA20ox1 and CsGA3ox1, similar reactions were catalysed with 13-hydroxlyated GAs as substrates. It is likely that these enzymes are also responsible for the biosynthesis of 13-hydroxylated GAs in vivo that occur at low levels in cucumber.

Stamen-derived bioactive gibberellin is essential for male flower development of Cucurbita maxima L.

Gibberellin (GA) signalling during pumpkin male flower development is highly regulated, including biosynthetic, perception, and transduction pathways. GA 20-oxidases, 3-oxidases, and 2-oxidases catalyse the final part of GA synthesis. Additionally, 7-oxidase initiates this part of the pathway in some cucurbits including Cucurbita maxima L. (pumpkin). Expression patterns for these GA-oxidase-encoding genes were examined by competitive reverse transcription-PCR (RT-PCR) and endogenous GA levels were determined during pumpkin male flower development. In young flowers, GA20ox3 transcript levels are high in stamens, followed by high levels of the GA precursor GA(9). Later, just before flower opening, transcript levels for GA3ox3 and GA3ox4 increase in the hypanthium and stamens, respectively. In the stamen, following GA3ox4 expression, bioactive GA(4) levels rise dramatically. Accordingly, catabolic GA2ox2 and GA2ox3 transcript levels are low in developing flowers, and increase in mature flowers. Putative GA receptor GID1b and DELLA repressor GAIPb transcript levels do not change in developing flowers, but increase sharply in mature flowers. Emasculation arrests floral development completely and leads to abscission of premature flowers. Application of GA(4) (but not of its precursors GA(12)-aldehyde or GA(9)) restores normal growth of emasculated flowers. These results indicate that de novo GA(4) synthesis in the stamen is under control of GA20ox3 and GA3ox4 genes just before the rapid flower growth phase. Stamen-derived bioactive GA is essential and sufficient for male flower development, including the petal and the pedicel growth.