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First published online August 11, 2006; 10.1104/pp.106.084871 Plant Physiology 142:553-563 (2006) © 2006 American Society of Plant Biologists Transcriptional Regulation of Gibberellin Metabolism Genes by Auxin Signaling in Arabidopsis1,[W]Instituto de Biología Molecular y Celular de Plantas, Universidad Politécnica de Valencia, Consejo Superior de Investigaciones Científicas, 46022 Valencia, Spain (M.F., D.A., L.G.-C., M.A.B.); and Rothamsted Research, Harpenden, Herts AL5 2JQ, United Kingdom (J.P.-G., A.L.P., P.H.)
Auxin and gibberellins (GAs) overlap in the regulation of multiple aspects of plant development, such as root growth and organ expansion. This coincidence raises questions about whether these two hormones interact to regulate common targets and what type of interaction occurs in each case. Auxins induce GA biosynthesis in a range of plant species. We have undertaken a detailed analysis of the auxin regulation of expression of Arabidopsis (Arabidopsis thaliana) genes encoding GA 20-oxidases and GA 3-oxidases involved in GA biosynthesis, and GA 2-oxidases involved in GA inactivation. Our results show that auxin differentially up-regulates the expression of various genes involved in GA metabolism, in particular several AtGA20ox and AtGA2ox genes. Up-regulation occurred very quickly after auxin application; the response was mimicked by incubations with the protein synthesis inhibitor cycloheximide and was blocked by treatments with the proteasome inhibitor MG132. The effects of auxin treatment reflect endogenous regulation because equivalent changes in gene expression were observed in the auxin overproducer mutant yucca. The results suggest direct regulation of the expression of GA metabolism genes by Aux/IAA and ARF proteins. The physiological relevance of this regulation is supported by the observation that the phenotype of certain gain-of-function Aux/IAA alleles could be alleviated by GA application, which suggests that changes in GA metabolism mediate part of auxin action during development.
The plant hormones GA and auxin overlap in the regulation of many developmental programs, most of them related to cell expansion and differentiation. For instance, both hormones seem to participate in the signals that trigger early fruit development in response to fertilization in many different species, including pea (Pisum sativum) and Arabidopsis (Arabidopsis thaliana; García-Martínez et al., 1991  ; Reinecke et al., 1995; Rodrigo et al., 1997; Vivian-Smith and Koltunow, 1999), as well as in the promotion of organ expansion, such as the Arabidopsis hypocotyl (Smalle et al., 1997; Cowling and Harberd, 1999; Collett et al., 2000) and pea stems (Ross and O'Neill, 2001), or in the promotion of Arabidopsis root growth (Fu and Harberd, 2003). Although it is possible that auxin and GAs exert their action through independent mechanisms, an interaction between these two hormones (and other convergent signals) has been demonstrated. This is the case of pericarp growth in pea fruits, which is synergistically induced by GAs and auxin (Ozga and Reinecke, 1999), or hypocotyl and stomata development, for which GAs are absolutely required, but their action is modulated by auxins and ethylene (Saibo et al., 2003).
The interaction between GAs and auxin has been intensively investigated recently, revealing at least two mechanisms. In roots, auxin signaling has been shown to induce the degradation of the negative GA-signaling element RGA, thereby promoting GA signaling and root growth (Fu and Harberd, 2003
The regulation of GA biosynthesis by auxin has been attributed to changes in the expression level of the genes encoding GA biosynthetic or deactivating enzymes. The most important regulatory steps that determine the concentration of the active GA species (GA1 or GA4) are the final biosynthetic reactions, catalyzed by GA 20-oxidases (GA20ox) and GA 3-oxidases (GA3ox), and deactivation reactions catalyzed by GA 2-oxidases (GA2ox; Hedden and Phillips, 2000
The mechanism of the auxin-signaling pathway allows the plant cell to transform very rapidly changes in auxin concentration into changes in gene expression. Upon auxin binding, the F-box-containing auxin receptors accelerate the 26S proteasome-dependent degradation of short-lived transcriptional repressors, Aux/IAA, which in turn allows ARF transcription factors to modulate gene expression by binding to the promoters of auxin-regulated genes (Dharmasiri et al., 2005a A clearer picture of the molecular mechanism by which auxin regulates GA biosynthesis and inactivation may be obtained by answering the following questions. (1) Given that the genes encoding GA biosynthesis are regulated by both auxin and GA, is there a functional connection between these two processes? (2) Is auxin regulation of GA metabolism genes mediated directly by Aux/IAA-ARF auxin signaling elements? We have addressed these issues by systematically analyzing the expression of GA metabolism genes in Arabidopsis seedlings in response to auxin. We found that the magnitude and kinetics of gene activation by auxin are different, depending on the particular GA metabolism gene under study, and that activation by auxin also occurs in the absence of GA-signaling elements that modify the expression of these genes in the GA homeostatic mechanism. These results indicate that regulation of GA metabolism by auxin and GAs occurs through independent pathways. The fast induction by auxin occurs in the absence of de novo protein synthesis and is reduced by the proteasome inhibitor MG132, suggesting that it is dependent on the rapid turnover of short-lived Aux/IAA repressors of auxin signaling. In fact, several mutants harboring dominant versions of these repressors are defective in the auxin up-regulation of the GA metabolism genes, indicating a direct involvement of these signaling elements in this control. Furthermore, certain phenotypes of these mutants could be rescued by GA treatment.
Auxin Up-Regulates GA Metabolism Genes
Control of expression of GA metabolism genes by auxin has been described for several species. To investigate whether this control also occurs in Arabidopsis, we analyzed changes in transcript levels of GA metabolism genes by quantitative reverse transcription (RT)-PCR in light-grown seedlings subject to exogenous auxin treatment. In Arabidopsis, the later regulatory enzymes in the GA pathway are encoded by small gene families: GA20ox are encoded by at least five genes (AtGA20ox1–AtGA20ox5); GA3ox are encoded by at least four genes (AtGA3ox1–AtGA3ox4); and GA2ox are encoded by at least seven genes (AtGA2ox1–AtGA2ox8, with AtGA2ox5 a pseudogene; Hedden et al., 2002
Auxin Effect on GA Biosynthesis Genes Does Not Require the DELLA Proteins GAI and RGA
GA metabolism is tightly controlled by GA through negative feedback regulation of GA20ox and GA3ox expression and positive feed-forward regulation of GA2ox expression (Hedden and Phillips, 2000
Significantly, expression of the GA biosynthesis genes AtGA20ox1 and AtGA20ox2 is up-regulated in response to at least two inputs: reduced GA levels and increased auxin levels. This raises the question as to whether these two inputs use the same or independent mechanisms to control the expression of these two target genes. Although the lack of correlation between up-regulation by auxin and feedback regulation by GAs for certain GA metabolism genes suggests that these mechanisms are independent, interaction may still occur specifically for those two genes that are up-regulated by both signals. This model for GA signaling proposes that the feedback mechanism operates through the activity of DELLA proteins (Fleet and Sun, 2005
Our expression analysis shows that GAI and RGA mediate, to different extents, feedback regulation of AtGA20ox1, AtGA20ox2, AtGA20ox4, and AtGA3ox1 because the elevated mRNA levels of these genes in the GA-deficient ga1-3 were reduced in ga1-3 gai-t6 rga-24 triple-mutant seedlings (Fig. 2
), which lack GAI and RGA activity (Dill and Sun, 2001
Auxin Signaling Directly Controls Expression of Several GA Metabolism Genes
Auxin has a broad effect on gene expression (Goda et al., 2004
The fast up-regulation of these genes resembles the behavior of direct targets of auxin signaling. Based on this model for auxin signaling, we would predict that one or more of the short-lived Aux/IAA proteins would be involved in repressing, through physical interaction with ARF transcription factors, the ARF-mediated auxin induction of these genes (Dharmasiri et al., 2005a ; Kepinski and Leyser, 2005). Preventing de novo protein synthesis must cause rapid depletion of Aux/IAA repressors in the cell, thus activating ARF-regulated genes. As shown in Figure 4
, cycloheximide treatment resulted in high mRNA levels of the GA metabolism genes regulated by auxin. This result suggests that Aux/IAA proteins present in the cell before treatment might mediate the regulation of GA metabolism genes by auxin, and this is supported by the observation that NAA potentiated the effect of cycloheximide (Fig. 4). The transcript levels of the non-auxin-regulated genes AtGA3ox1, AtGA3ox2, and AtGA2ox1 slightly increased in response to cycloheximide treatment (data not shown), suggesting the activity of a labile repressor in the control of their basal expression, which may also contribute to the cycloheximide effect seen on GA metabolism genes regulated by auxin. In addition, impairment of Aux/IAA degradation by inhibition of the 26S proteasome with MG132 (Gray et al., 2001; Ouellet et al., 2001) prevented induction of GA metabolism genes by auxin (Supplemental Fig. S3). Treatment with MG132 in the absence of exogenous auxin did not alter expression levels of AtGA20ox1, AtGA20ox2, AtGA2ox2, and AtGA2ox6, suggesting that any effect on DELLA stability, which is also controlled by the proteasome pathway (Fu et al., 2002), is not sufficient to alter basal expression levels of these genes, and therefore the effect of the inhibitor on expression of GA metabolism genes is likely to be via the auxin pathway. Only in the case of AtGA2ox3 could the increase in expression after MG132 treatment be caused by stabilization of DELLA proteins because this gene was also up-regulated by paclobutrazol (Supplemental Fig. S2). The relatively different magnitude of the effect caused by MG132 on the induction of these genes may reflect different responsiveness of the promoters to the activity of the Aux/IAA proteins. Taken together, these results strongly suggest that some GA metabolism genes may represent primary targets in response to auxin, and that this induction may be regulated by Aux/IAA-ARF dimers.
Expression of GA Metabolism Genes Is Regulated by Aux/IAA-ARF Auxin-Signaling Elements
To confirm the involvement of Aux/IAA proteins in the control of GA metabolism genes by auxin, we analyzed the response of these genes to NAA treatments in mutant lines harboring gain-of-function alleles of some Aux/IAA genes: axr2-1/iaa7 (Nagpal et al., 2000
The involvement of ARF transcription factors was tested for NPH4/ARF7 by analyzing NAA induction in nph4-1 null mutant seedlings (Harper et al., 2000 ; Fig. 5). NPH4/ARF7 seems to be involved in the regulation of all the auxin-inducible genes, in some cases controlling NAA induction and in other cases regulating basal transcript levels. It has been shown that nph4-1 null mutants resemble msg2-1 dominant mutants regarding auxin regulation of differential hypocotyl growth; moreover, both proteins interact physically, indicating that they act together in this control (Tatematsu et al., 2004). Although the behavior of AtGA20ox1 and AtGA2ox2 was similar in nph4-1 and msg2-1 seedlings—indicating that MSG2/IAA19 and NPH4/ARF7 may act together in the regulation of these two genes by auxin—their effects on the expression of the other genes were both qualitatively and quantitatively different, suggesting that other Aux/IAA and ARFs must also be involved.
Consistent with the hypothesis that Aux/IAA-ARF pairs direct the regulation of GA metabolism genes by auxin, loss of function of TIR1, a member of the small family of F-box proteins that act as auxin receptors (Dharmasiri et al., 2005a In summary, these results suggest that auxin controls GA metabolism gene expression through a highly branched signaling network involving different Aux/IAA-ARF elements.
A connection has been clearly established between auxin and GA metabolism genes in the context of transient, exogenous auxin applications. To investigate whether this regulation reflects a mechanism that operates in response to changes in endogenous auxin concentration, we studied steady-state mRNA levels of GA metabolism genes in the auxin-overproducing mutant yucca (Zhao et al., 2001
Auxin Regulation of GA Metabolism Genes Is Tissue Specific
To gain insight into the actual localization of the auxin regulation of GA metabolism gene expression, we investigated the spatial regulation of some GA metabolism genes in response to auxin treatment by using reporter-gene fusions (Fig. 7
). AtGA20ox2 and AtGA2ox2 reporter genes showed complementary expression patterns in cotyledons of control seedlings; AtGA2ox2 was also expressed in the shoot apical region (Jasinsky et al., 2005
GAs Partially Mediate the Action of Auxin in Seedlings
The observation that the expression of GA metabolism genes responds to changes in endogenous auxin concentration and that the regulation is tissue specific prompted the question of whether this regulation is physiologically relevant. Indeed, the exaggerated hypocotyl elongation provoked by auxin overproduction in the yucca mutant was completely dependent on GA biosynthesis, as observed in the dose-response curve of hypocotyl growth relative to increasing concentrations of paclobutrazol (Fig. 8A
); the paclobutrazol effect was completely reverted by simultaneous application of GA3. In a similar way, the enhanced hypocotyl growth favored by the stimulation of auxin synthesis at higher temperatures (Gray et al., 1998
On the other hand, the short hypocotyl and the de-etiolation phenotypes shown by several gain-of-function mutants in Aux/IAA genes when grown in darkness (Leyser et al., 1996 ; Kim et al., 1998; Nagpal et al., 2000) are similar to the phenotype of dark-grown GA-deficient mutants (Alabadí et al., 2004). Exogenous application of GA3 to dark-grown axr2-1, axr3-1, and shy2-2 mutants partially compensated for the defect in hypocotyl elongation, but did not compensate for cotyledon opening (data not shown). Additionally, GA3 treatment partially rescued the molecular phenotype of CAB2 and RbcS observed in these mutants when grown in darkness (Fig. 8, B and C). These results suggest that at least part of the phenotype caused by auxin insensitivity in these mutants is mediated by a deficiency in GA biosynthesis. To identify other potential targets for auxin regulation via GA, we reviewed microarray expression data available in Genevestigator (https://www.genevestigator.ethz.ch/at) and searched for genes up-regulated by auxin and GA. Among the genes selected, we examined by quantitative RT-PCR the expression of one of them (At4g37770) and, as shown in Supplemental Figure S4, auxin-induced up-regulation was dramatically reduced in the presence of paclobutrazol. In contrast, the expression of IAA32 (At2g01200), which is only induced by auxin, showed no alteration in the response to auxin even in the presence of paclobutrazol. This result confirms that auxin can indeed exert its action through GA or in an autonomous manner, depending on the particular target examined.
Interaction between hormone pathways appears as an important strategy used by plants to integrate environmental and developmental cues. These interactions may occur through shared signaling elements, as illustrated by the SPINDLY protein, which participates in the transduction of signals triggered by GAs and cytokinins in Arabidopsis (Greenboim-Wainberg et al., 2005 ), or the DELLA protein RGA that integrates signals from ethylene, auxin, and GAs in growth processes (Achard et al., 2003; Fu and Harberd, 2003; Vriezen et al., 2004). An additional strategy developed by plants is to modulate metabolism of one hormone by a second hormone. This is the case, for example, for the homeostatic control between auxin and cytokinin in Arabidopsis, in which auxin exerts negative control on cytokinin pools by acting on its biosynthetic pathway (Nordström et al., 2004). In this work, we show that auxins control the expression of genes encoding enzymes involved in GA metabolism in Arabidopsis seedlings. Auxins act on the GA biosynthesis pathway by regulating specifically the expression of two genes encoding enzymes that participate in rate-limiting steps, AtGA20ox1 and AtGA20ox2, supporting the standing model that auxin generally promotes GA biosynthesis. This step in GA biosynthesis would then represent the key entry point for auxin control of GA biosynthesis in Arabidopsis seedlings. This situation is comparable to the auxin promotion of GA biosynthesis in tobacco (Wolbang and Ross, 2001), whereas auxin induces GA biosynthesis in pea through the control of a different step, the one catalyzed by PsGA3ox1 (O'Neill and Ross, 2002). Nevertheless, PsGA3ox1 induction by auxin seems to proceed through a different mechanism, given that cycloheximide is able to block this induction, contrary to the case for AtGA20ox1 and AtGA20ox2 genes in Arabidopsis. Another important difference is that, unlike pea and tobacco, certain GA2ox genes are also up-regulated by auxin treatments in Arabidopsis seedlings. Moreover, this induction is likely direct (see below) and not the consequence of positive feed-forward regulation by GAs, as proposed for PsGA2ox2 in pea (O'Neill and Ross, 2002). AtGA20ox1 expression has also been shown to be up-regulated in response to auxin transport inhibitors and in the tir3-1 mutant, which also alters auxin transport, and in both cases this up-regulation has been related to reduced growth caused by the altered auxin distribution (Desgagné-Penix et al., 2005).
To interpret the physiological consequences of the regulation by auxin of GA metabolism genes, we have to consider at least one additional level of regulation: tissue specificity for the expression of the different GA metabolism genes. Our analysis resolves the actual localization of the regulation by auxin in seedlings for AtGA20ox2 and AtGA2ox2. The expression patterns of these genes do not overlap so that localized auxin accumulation would promote either GA biosynthesis or deactivation, depending on the tissue, leading to different responses. For example, auxin might participate in maintaining shoot apical meristem activity by enhancing GA deactivation through specific regulation of AtGA2ox2 gene expression at the boundary of the meristem (Jasinsky et al., 2005
Auxin-induced degradation of the DELLA proteins GAI and RGA mediates several growth responses in Arabidopsis (Achard et al., 2003
Although induction by auxin of GA metabolism genes fits this model of auxin signaling, it is difficult to assign roles to individual Aux/IAA proteins in this particular auxin response based solely on the analysis of the effects of aux/iaa-dominant mutations (Fig. 5), given both the nature of the mutations and also that, in many instances, gain of function of one particular Aux/IAA gene alters the expression of other members in the family (Tian et al., 2002
Studies on the auxin control of primary response genes led to the discovery of a sequence element in their promoters, TGTCTC, important for this response and that allowed the identification of ARF1, an Arabidopsis transcription factor that binds to this sequence element (Ulmasov et al., 1995 In conclusion, our results indicate that auxin signaling affects GA metabolism mainly via direct up-regulation of expression of several AtGA20ox and AtGA2ox genes. This regulation operates through a mechanism that is independent of the feedback regulation mediated by the DELLA proteins GAI and RGA, and involves one or more Aux/IAA and ARF-signaling elements.
Plant Materials and Growth Conditions Arabidopsis (Arabidopsis thaliana) accessions Columbia-0 (Col-0) and Landsberg erecta (Ler) were used as wild type. Seeds of the GA-deficient ga1-3 mutant were obtained from the Arabidopsis Biological Resource Center (ABRC), and ga1-3 rga-24 gai-t6 were provided by Dr. Tai-ping Sun (Duke University), both in Ler background. Seeds of the auxin-signaling mutants tir1-1, axr2-1, and axr3-1 were obtained from the ABRC; nph4-1 seeds were provided by Dr. Emmanuel Liscum (University of Missouri); msg2-1 by Dr. Kotaro Yamamoto (Hokkaido University); yucca by Dr. Yunde Zhao (University of California) and Dr. Joanne Chory (Salk Institute), all in Col-0 background; and shy2-2 was provided by Dr. Jason Reed (University of North Carolina) in Ler background. All seeds were surface sterilized for 15 min in 70% (v/v) ethanol and 0.01% (v/v) Triton X-100, followed by 10 min in 96% (v/v) ethanol. Seeds were stratified in sterile water at 4°C for 4 d in darkness and then germinated in 50 mL of 0.5x Murashige and Skoog liquid medium, supplemented with 1% (w/v) Suc. Stratification of ga1-3 and ga1-3 rga-24 gai-t6 seeds was done in water containing 10 µM GA3 and the seeds were then rinsed several times with water before sowing. Seedlings were grown at 20°C under continuous fluorescent white light (fluence rate of 90–100 µmol m–2 s–1) for 6 d. For the experiments shown in Figure 8, seeds were sterilized as above, sown on 0.5x Murashige and Skoog agar plates supplemented with 1% (w/v) Suc, and stratified at 4°C for 4 d in the dark. For light-grown seedlings, plates were kept at 20°C under continuous fluorescent white light (fluence rate of 90–100 µmol m–2 s–1 for the first 2 d and 10–15 µmol m–2 s–1 for the next 5 d). For dark-grown seedlings, plates were placed in the light for 8 to 9 h, wrapped in several layers of aluminum foil, and kept at 20°C for a total of 7 d. Seedlings grown in liquid media were treated with 50 µM NAA (Sigma), 100 µM GA3 (Duchefa), or mock treated with ethanol 0.14% (v/v, final concentration) for the indicated times. Paclobutrazol (Duchefa) was added to a final concentration of 0.5 µM, whereas control seedlings were mock treated with acetone 0.005% (v/v, final concentration). Inhibitors of protein synthesis (cycloheximide, 10 µM final concentration; Sigma) and 26S proteasome (MG132, 50 µM final concentration; Sigma) were added to the media for the indicated times; control seedlings for the MG132 treatment were mock treated with DMSO 0.1% (v/v, final concentration). In agar plates, GA3 (10 µM final concentration) and paclobutrazol (0.01–1 µM, final concentration) were added to the media after autoclaving. Control plates for GA3 and paclobutrazol treatments contained 0.014% ethanol (v/v, final concentration) and 0.01% acetone (v/v, final concentration), respectively.
Total RNA was extracted from frozen, whole seedlings using the RNeasy plant mini kit (Qiagen). Genomic DNA was eliminated during RNA purification with the RNase-free DNase set (Qiagen). Two micrograms of total RNA were used to synthesize first-strand cDNA using the SuperScript first-strand synthesis system for RT-PCR (Invitrogen). cDNA samples were diluted in a total volume of 40 µL.
One microliter of cDNA was used for quantitative RT-PCR using SYBR Green PCR master mix (Applied Biosystems) following the manufacturer's recommendations and an ABI Prism 7000 sequence detection system (Applied Biosystems). Each sample was assayed in triplicate. Expression levels were calculated relative to EF1-
Two micrograms of total RNA were used for northern analysis. Northern blotting, hybridization with 32P-labeled CAB2 and RbcS probes, and washes were carried out as described previously (Alabadí et al., 2004
The AtGA20ox2::
For histochemical analyses, whole seedlings were prefixed for 20 min in cold 90% (v/v) acetone at room temperature, washed once in cold staining buffer (50 mM sodium phosphate buffer, pH 7, 0.2% [v/v] Triton X-100, 2 mM potassium ferricyanide, 2 mM potassium ferrocyanide), and incubated in 2 mM X-GlcA (in staining buffer) first on ice for 1 h, then at 37°C for 8 h. Tissue was dehydrated in ethanol series (20% [v/v], 35% [v/v], 50% [v/v], 30 min each at room temperature), then postfixed 30 min at room temperature in 50% (v/v) ethanol, 5% (v/v) formaldehyde, 10% (v/v) acetic acid. Tissue was further dehydrated in 70% (v/v) ethanol and photographed with Nomarski optics.
Seedlings were placed on an acetate sheet and scanned at a resolution of 800 dots per inch. Hypocotyl lengths were measured as described previously (Alabadí et al., 2004
The following materials are available in the online version of this article.
We thank Dr. Joanne Chory (Salk Institute, La Jolla, CA), Dr. Emmanuel Liscum (University of Missouri, Columbia, MO), Dr. Jason Reed (University of North Carolina, Chapel Hill, NC), Dr. Tai-ping Sun (Duke University, Raleigh, NC), Dr. Kotaro Yamamoto (Hokkaido University, Sapporo, Japan), and Dr. Yunde Zhao (University of California, La Jolla, CA) for seeds; Dr. G. Vachon (Université Joseph Fourier, Grenoble, France) for advice on the quantitative RT-PCR determination of GA metabolism gene expression; and Dr. J. Carbonell, Dr. J.L. García-Martínez, Dr. O. Nilsson, and Dr. M.A. Pérez-Amador for discussions and useful comments on the manuscript. Received June 8, 2006; accepted August 3, 2006; published August 11, 2006.
1 This work was supported by the Spanish Ministry of Education and Science (MEC; grant nos. BIO2001–1558 and BIO2004–02355); the European Molecular Biology Organization Young Investigator Programme (grant to M.A.B.); a Ramón y Cajal contract with the Spanish MEC (to D.A.); a Spanish FPI fellowship (to M.F.); the European Union Research Training Network INTEGA (J.P.-G.); and a Core Strategic Grant from the Biotechnology and Biological Sciences Research Council to Rothamsted Research (to A.L.P. and P.H.). 
2 These authors contributed equally to the paper.
3 Present address: Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX1 3RB, UK. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Miguel A. Blázquez (mblazquez{at}ibmcp.upv.es).
[W] The online version of this article contains Web-only data. www.plantphysiol.org/cgi/doi/10.1104/pp.106.084871 * Corresponding author; e-mail mblazquez{at}ibmcp.upv.es; fax 34–963877859.
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ABSTRACT
RESULTS
. Transcript level in the control sample for each gene was set to 1, and level in the treated sample was calculated relative to the corresponding control value. Error bars represent SD.
-Glucuronidase Reporter Fusion and Plant Transformation
EMBL3 genomic clone containing the At20ox2 gene (J.P. Coles and A.L. Phillips, unpublished data). Construction of the AtGA20ox2 promoter::GUS fusion and Agrobacterium-mediated transformation of Arabidopsis were carried out as described for AtGA20ox1::GUS (Desgagné-Penix et al., 2005