The Multifunctional Dioxygenases of Gibberellin Synthesis Theo Lange 1,2, * and Maria Jo~ ao Pimenta Lange 1,2, * 1 Institut f€ ur Pflanzenbiologie, Technische Universit € at Braunschweig, Mendelssohnstr. 4, D-38106 Braunschweig, Germany 2 These authors contributed equally to this work. *Corresponding authors: Theo Lange, E-mail, theo.lange@tu-bs.de; Fax, 0049-531-391-8180; Maria Jo~ ao Pimenta Lange, E-mail, m.pimenta@tu-bs.de; Fax, 0049- 531-391-8180 (Received March 7, 2020; Accepted April 14, 2020) 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 proper- ties 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 multi- functional GA-oxidases, in particular, from cucumber and Arabidopsis that have been most comprehensively investigated. Keywords: Anabolism • Catabolism • GA-oxidases • Gibberellin biosynthesis • Multifunctional enzymes • 2-Oxoglutarate-dependent dioxygenases. Introduction Gibberellins (GAs) are tetracyclic diterpenoid compounds with a high structural variation. Today 136 structures have been described, and there are many more to come. Of these, only a few function as plant hormones in higher plants. The various structures implicate multiple biosynthetic pathways that, in higher plants, are normally divided into three parts based on the enzymes involved and their subcellular compartmentation (reviewed by Yamaguchi 2008, Hedden and Phillips 2015). GAs are formed from trans-geranylgeranyl diphosphate, a common C 20 -precursor for diterpenoids. The first part is catalyzed by soluble enzymes and is localized in proplastids of the plant cell, leading to ent-kaurene. This reaction occurs in two steps and is catalyzed by the terpene cyclases ent-copalyl diphos- phate synthase and ent-kaurene synthase. In the second part, ent-kaurene is oxidized to the general GA precursor, GA 12 , in six steps via ent-kaurenol, ent-kaurenal, ent-kaurenoic acid, ent-7α- hydroxykaurenoic acid and GA 12 -aldehyde (Lange 1998). Two membrane-associated, NADPH-dependent cytochrome P-450 monooxygenases, ent-kaurene oxidase (KO) and ent-kaurenoic acid oxidase (KAO), are involved. These enzymes are localized in the endoplasmic reticulum and, in the case of KO, also in the outer envelope of the plastid (Helliwell et al. 2001). In general, the gene families that code for enzymes of these early steps are less complex compared to the ones that encode the final steps of GA biosynthesis. The third and final part of the pathway is catalyzed by GA- oxidases that belong to the 2-oxoglutarate-dependent dioxyge- nases (2-ODDs) family, in the cytosol and the nucleus of the cell (Helliwell et al. 2001, Chen et al. 2014). GA-oxidases appear to be highly regulated, with multigene families that often encode multifunctional enzymes. The complexity of the GA pathways and the apparent redundancy of the involved GA-oxidases per- mit the fine-tuning of GA biosynthesis in a spatiotemporal manner regulating plant development and responses to envir- onmental signals. According to their enzymatic properties, GA-oxidases are main- ly classified into GA 7-oxidases (GA7oxs), GA 20-oxidases (GA20oxs), GA 3-oxidases (GA3oxs) and GA 2-oxidases (GA2oxs). Based on the phylogenetic analysis of 41 plant species, it was proposed the existence of four additional classes of GA- oxidases with unknown catalytic properties (designated as groups A, B, C and D), in addition to the known families (Huang et al. 2015). Arabidopsis and cucumber members of groups A and B in Huang’s study do not show GA-oxidase activity in our hands (un- published results). However, one member of group C was recently identified as a GA-oxidase, GAS2 (Fig. 1A, Liu et al. 2019), and group D contains a previously identi fied GA7ox, CsGA7ox2 (Pimenta Lange et al. 2013, Sun et al. 2018). In this review, we focus on 2-ODDs involved in the final part of the GA biosynthetic pathway. Recent advances in the char- acterization of these GA-oxidases and their multifunctionality will be described for two model plants, cucumber (Cucumis sativus L.) and Arabidopsis thaliana, but we will also include recent breakthroughs achieved with other plant species. In the following sections, the GA-oxidases are assembled into ana- bolic and catabolic enzymes, according to their functions in GA metabolism. A protein alignment obtained for known Arabidopsis and cucumber GA-oxidases shows their phylogen- etic relation in Fig. 1A. Anabolic GA-Oxidases There are four classes of anabolic GA-oxidases: three belong to the established families, GA7oxs, GA20oxs and GA3oxs Plant Cell Physiol. 61(11): 1869–1879 (2020) doi:10.1093/pcp/pcaa051, Advance Access publication on 28 April 2020, available online at https://academic.oup.com/pcp # The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. 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