The GA 20-oxidase enzymes represent a potentially important rate-limiting step in GA biosynthesis, as evidenced by the increased concentrations of bioactive GA produced by (and GA-overdosed phenotypes of) transgenic Arabidopsis plants overexpressing GA20ox genes (Huang et al., 1998; Coles et al., 1999, see section 1.2.1). A family of five GA20ox genes have been identified in the Arabidopsis genome through sequence similarity (Figure 3.1a, Hedden et al., 2002). The importance of two of these paralogues (AtGA20ox1 and -2) to vegetative growth and development has been directly demonstrated through knockout mutations in the Col-0 ecotype (Rieu et al., 2008). These two paralgoues, plus a third,
AtGA20ox3, have been shown to have GA20ox activity in vitro (Phillips et al., 1995; Xu et al.,
1995). GA20ox enzymes catalyse successive oxidative reactions at Carbon-20 of intermediate GA substrates (Hedden, 1997; Figure 3.1b), resulting in the eventual loss of C-20 and the production of C19-GA species that can be recognised by GA3ox enzymes and processed into biologically active GA.
Loss of AtGA20ox1 and -2 results in a semi-dwarf phenotype, and tissues from these plants contain reduced levels of bioactive GA (Rieu et al., 2008), demonstrating the contribution of GA20ox activity to GA biosynthesis. However, the phenotype of the ga20ox1 ga20ox2 mutant is far less severe than mutants such as ga1-3, in which GA biosynthesis is completely blocked (see section 1.2), suggesting that one or more of the remaining AtGA20ox genes has biological functions during Arabidopsis growth and development. Alternatively, the possibility must be borne in mind that other, unrelated enzymes may perform the same functions as the GA20ox family (or even that other GA biosynthesis pathways exist that circumvent GA20ox activity). There are no published accounts of mutations in known
GA20ox genes causing a severe dwarf phenotype in any plant species. The most probable
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Figure 3.1: The AtGA20ox gene family.
(a) Genomic distribution of the AtGA20ox gene family.
(b) Schematic of GA 20-oxidase activity on non 13-hydroxylated substrates, converting GA12
to GA9. Numerals in italics denote carbon position. Sequential oxidative reactions at carbon
position 20 (blue) eventually result in the loss of this carbon and its replacement by a lactone group derived from carbon 19 (yellow).
(c) Expression profile of AtGA20ox paralogues during Arabidopsis development. Reproduced with permission from Rieu et al. (2008).
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The members of the AtGA20ox gene family demonstrate differential expression patterns during development, as shown through both quantitative Real-Time PCR (qPCR, Rieu et al., 2008) and Northern blot techniques (Phillips et al., 1995). Beyond this, detailed tissue- specific expression patterns of the individual AtGA20ox paralogues have not yet been published, with the exception of an AtGA20ox1::GUS translational fusion reporter gene expressed around the SAM during vegetative growth (Hay et al., 2002). A similar
translational fusion reporter line has been established for AtGA20ox2, but attempts to replicate this approach for AtGA20ox3, -4 and -5 did not demonstrate any GUS staining (Phillips, A., unpublished data).
AtGA20ox1 and -2 are the two most abundant and broadly-expressed GA20ox paralogues
across Arabidopsis development, but expression data and mutant phenotypes suggest that functional specificity exists between them. AtGA20ox1 is practically the only GA20ox paralogue expressed in stem tissues (Phillips et al., 1995; Rieu et al., 2008), a finding
supported by the semi-dwarf phenotype of ga20ox1 loss-of-function mutants (Xu et al., 1995; Rieu et al., 2008). However, despite the absence of AtGA20ox2 from wild-type stem tissue the ga20ox1 ga20ox2 double mutant shows less stem elongation than ga20ox1 (Rieu et al., 2008), demonstrating that functional redundancy also occurs between AtGA20ox paralogues.
AtGA20ox2 stem expression is in fact strongly up-regulated in the ga20ox1 mutant (Rieu et
al., 2008), presumably through homeostatic regulation of the GA biosynthesis pathway (see section 1.4). This highlights the potential for complex relationships between AtGA20ox paralogues. AtGA20ox1, -2 and -3 all show reduced expression under exogenous GA treatment (Phillips et al., 1995, Rieu et al., 2008), indicating that they are all responsive to feedback regulation. In contrast, expression of AtGA20ox4 or -5 did not respond to GA treatment (Rieu et al., 2008). AtGA20ox3 expression has been shown to be significantly up- regulated in the ga20ox1 ga20ox2 mutant background in all tissues tested (leaf, internode and inflorescence, Rieu et al., 2008), suggesting that expression of AtGA20ox1, -2 and -3 is governed by a complex relationship. In wild-type tissues AtGA20ox3 is most strongly expressed in developing siliques and during germination.
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In addition to plant stature, floral development also differs between ga20ox1 ga20ox2 and
ga1-3. ga20ox1 ga20ox2 pistils, stamens and pistils reach a larger final size than those of ga1- 3 (Figure 1.8), the stamens producing viable pollen (Rieu et al., 2008), whilst ga1-3 pollen
development is arrested at the unicellular stage (Cheng et al., 2004). Similarly, ga20ox1
ga20ox2 flowers are female fertile whilst ga1-3 flowers are not (Koornneef & Van der Veen,
1980). However, successful silique-set is reduced across the first 10 flowers in ga20ox1
ga20ox2 compared to wild type Col-0, apparently due to reduced stamen growth and delayed
anther dehiscence (pollen release) creating a mechanical barrier to pollination (Figure 1.8, Rieu et al., 2008). Similar fertility phenotypes are also seen in ga20ox1 (Rieu et al., 2008), and the ga3ox1 ga3ox mutant (Hu et al., 2008), though this is less severe in ga20ox1. Seed- set in these mutants subsequently recovers, which has been associated with restored growth of stamens relative to the pistil in the case of ga3ox1 ga3ox3 (Hu et al., 2008). The mechanism regulating this process is unknown.
Genetic evidence presented in this chapter details the characterisation of AtGA20ox3 function during plant development through the creation and phenotypic analysis of new ga20ox combinatorial mutant lines. The results indicate that AtGA20ox1, -2 and -3 are the three dominant GA20ox paralogues throughout Arabidopsis development, with loss of all three producing a severely dwarfed, infertile phenotype very similar to ga1-3. This is the first instance in which the GA20ox paralogues responsible for the majority of GA biosynthesis have been identified in any plant species, and confirms that the GA20ox pathway comprises the primary route through GA biosynthesis. AtGA20ox3 functions almost entirely redundantly with either AtGA20ox1, -2 or both at all stages of development, dependent on the phenotypic character examined. Loss of AtGA20ox1, -2 and -3 induces an apparent block in stamen development, exhibited by the failure of the tapetum cell layer to degrade in a timely fashion. However, evidence is also presented describing a previously undiscovered mechanism that enhances floral organ growth and development in later flowers of GA-deficient mutants in a manner that is apparently independent of GA biosynthesis.