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Proc Natl Acad Sci U S A. Oct 18, 2005; 102(42): 15253–15258.
Published online Oct 7, 2005. doi:  10.1073/pnas.0504279102
PMCID: PMC1257699
From the Cover
Plant Biology

The UGT73C5 of Arabidopsis thaliana glucosylates brassinosteroids

Abstract

Steroid hormones are essential for development, and the precise control of their homeostasis is a prerequisite for normal growth. UDP-glycosyltransferases (UGTs) are considered to play an important regulatory role in the activity of steroids in mammals and insects. This study provides an indication that a UGT accepting plant steroids as substrates functions in brassinosteroid (BR) homeostasis. The UGT73C5 of Arabidopsis thaliana catalyses 23-O-glucosylation of the BRs brassinolide (BL) and castasterone. Transgenic plants overexpressing UGT73C5 displayed BR-deficient phenotypes and contained reduced amounts of BRs. The phenotype, which was already apparent in seedlings, could be rescued by application of BR. In feeding experiments with BL, wild-type seedlings converted BL to the 23-O-glucoside; in the transgenic lines silenced in UGT73C5 expression, no 23-O-glucoside was detected, implying that this UGT is the only enzyme that catalyzes BL-23-O-glucosylation in seedlings. Plant lines in which UGT73C5 expression was altered also displayed hypocotyl phenotypes previously described for seedlings in which BR inactivation by hydroxylation was changed. These data support the hypothesis that 23-O-glucosylation of BL is a function of UGT73C5 in planta, and that glucosylation regulates BR activity.

Keywords: glucosylation, glycosyltransferase, homeostasis, plant, steroid

Steroid hormones play a conserved role in regulating development in eukaryotic organisms ranging from fungi and plants to insects, vertebrates, and mammals. Brassinosteroids (BRs) are plant-specific polyhydroxylated derivatives of 5α-cholestane, structurally similar to cholesterol-derived animal steroid hormones and ecdysteroids from insects (1). The BRs function in cell elongation and differentiation and have been particularly studied in relation to processes such as germination, development in the light and dark, and senescence (2).

BR biosynthesis has been well characterized, with key steps in plant and animal steroid synthesis functionally conserved (3). For example, the Arabidopsis gene DET2 encoding a steroid 5α-reductase, when expressed in a human cell line, was capable of reducing several mammalian steroids (4). In Arabidopsis, the biologically most active BR is brassinolide (BL). This is the end product of a complex set of pathways in which campesterol, formed through general sterol synthesis, is converted via a series of reductions, hydroxylations, epimerizations, and oxidations to other BRs and ultimately to BL (5). Although it is now considered that there are >50 BRs in plants (5), most research has focused on BL. Cell surface receptors have been identified that recognize BL (6-8) and initiate a complex signaling cascade that controls BR target gene expression and exerts the hormone's effects (9, 10).

In contrast to our detailed understanding of BL synthesis and signal transduction, the mechanisms that control BR homeostasis remain largely unknown. Recent grafting experiments performed with pea plants have shown that BRs do not undergo long-distance transport in this species (11), suggesting they act locally, close to their site(s) of synthesis. Feedback control of the expression of genes encoding biosynthetic enzymes, such as the cytochrome P450 Constitutive Photomorphogenesis and Dwarfism (CPD) (12), from the signaling pathway has been demonstrated (13, 14). However, levels of active hormone may also be regulated in a variety of other ways. For example, both castasterone (CS), the direct precursor of BL, and BL were inactivated by hydroxylation at the C-26 position, a process catalyzed by the cytochrome P450 phyB activation-tagged suppressor 1-dominant (BAS1) (CYP72B1/CYP734A1), in Arabidopsis (15, 16). The closest homologue of BAS1, CYP72C1/SOB7, is also thought to be involved in hydroxylation and inactivation of BRs (17-19). Similarly, C-26 hydroxylation controls ecdysone levels in insects (20).

Conjugates of BRs with fatty acids, glucose, and disaccharides have been identified in explants and cell extracts of plants (5), and, although the effect of conjugation on BR bioactivity in planta is not known, many other phytohormones are inactivated by this mechanism (21). Enzymes involved in the glucosylation of plant hormones such as auxins, cytokinins, and abscisic acid belong to a subset of family 1 UDP-glycosyltransferases (UGTs) defined by the presence of a C-terminal consensus sequence (22). Proteins of this class have also been shown to accept mammalian and insect steroid hormones as substrates. In mammals, the typical donor sugar is UDP-glucuronic acid, and conjugation of androgens and estrogens by members of UGT subfamily 2B is thought to regulate hormone activity (23, 24). Insect viruses, such as baculoviruses, use glucosylation to inactivate ecdysteroids in their hosts and prolong the survival of infected larvae by blocking molting and pupation (25, 26).

This study describes the functional characterization of a UGT that accepts plant steroids as substrates. UGT73C5 specifically catalyzes 23-O-glucosylation of the BRs, BL, and CS in Arabidopsis plants. Overexpression of the UGT led to a BR-deficient phenotype, suggesting that glucosylation reduces the biological activity of BRs. Seedlings in which UGT73C5 expression was silenced lacked detectable BL-23-O-glucosylation activity and, under specific light conditions, displayed a hypocotyl phenotype consistent with an impairment in BR inactivation. It was previously reported that UGT73C5 increases tolerance against the Fusarium mycotoxin deoxynivalenol (DON) when overexpressed in plants (27). The implications of the dual activity of UGT73C5 toward both BRs and exogenous mycotoxins in vivo are discussed.

Materials and Methods

Plant Materials and Growth Conditions. Arabidopsis thaliana ecotype Columbia (Col-0) was used as the wild-type for generation of all plant material described in this study. General plant handling and transformation protocols as well as β-glucuronidase (GUS) staining followed standard procedures (28). A null allele of DET2 (det2-1; ref. 29) was used to analyze UGT73C5 promoter-GUS reporter (UGT73C5p-GUS; ref. 27) activity in a BR-deficient background.

For generation of a translational UGT73C5-GUS fusion (UGT73C5f-GUS), the GUS vector pPZP-GUS.1 (27), was used. The reporter was constructed by amplifying the full-length UGT73C5 promoter + ORF using Pfu polymerase (Stratagene) and the primers 73C5-GUS-fw and 73C5F-GUS-rv (for all primer sequences, see Table 4, which is published as supporting information on the PNAS web site) and cloning the product in frame with the GUS gene in the vector.

For BR growth response and hypocotyl elongation assays, mother plants were cultivated in a controlled environment of 16-h/8-h light-dark cycle (150 μmol/m2·s1 white light) at a temperature of 21°C/17°C (±1). To analyze the BR response of UGT73C5oe plants, seedlings were grown vertically for 7 days in the light (16-h/8-h light-dark cycle, 90-110 μmol/m2·s1 white light, 21°C, ±1) before they were transferred to media containing different concentrations of 24-epibrassinolide (24-epiBL) (OlChemIm, Olomouc, Czech Republic) and to control plates lacking the hormone and 24-epibrassinolide incubated for another 10 days.

Analysis of hypocotyl elongation under different light conditions or in the dark was performed as described by Fankhauser and Casal (30) and is outlined in detail in Supporting Text, which is published as supporting information on the PNAS web site. For all light conditions, a fluence rate of 8-10 μmol/m2·s1 was used.

RNA Interference (RNAi) to Specifically Silence UGT73C5. To specifically knock down UGT73C5 in wild type, we chose to use the 5′ and 3′ untranslated regions of At2g36800. The regions upstream and downstream of the UGT73C5 start and stop codons were PCR-amplified from genomic DNA by using the following pairs of oligonucleotides: 73C5-55′, 73C5-53′, and 73C5-35′, 73C5-33′, respectively. Both fragments were then linked by PCR by using oligonucleotides 73C5-55′ and 73C5-33′ and taking advantage of the overlapping sequences of 73C5-53′ and 73C5-35′. The resulting fragment (73C5-53 UTR) was cloned into pGEM-T (Invitrogen), excised by XhoI/BamHI digestion, and then cloned into pENTR4 (Invitrogen). The RNAi vector was made using LR clonase (Invitrogen) and pentry4-73c5 and phellsgate8 (31).

Transcript Analysis. Semiquantitative RT-PCR was performed as described (27) by using specific primers for UGT73C5 (27), UGT73C6, TCH4, or BEE3. UBQ5 was used as an internal template control. Analysis of the silenced lines was conducted after DON treatment to induce UGT73C5 expression. Ten-day-old seedlings were transferred to liquid Arabidopsis thaliana salts (ATS) media and incubated for 48 h on an orbital shaker (60 rpm) before adding 5 ppm (16.9 μM) of DON (SigmaAldrich) dissolved in 70% EtOH. The plants were harvested after 4 h of treatment, and RT-PCR was performed as described above.

Analysis of BR Levels in Plants Using GC/MS. For BR measurements, plants were grown under continuous light conditions (150 μmol/m2·s1 white light) for 4 weeks before the tissue of aerial plant parts was harvested. Fifty grams (fresh weight) of plant material was lyophilized and extracted twice with 500 ml of MeOH:CHCl3 (4:1), and deuterium-labeled internal standards 1 ng/g fresh weight (fw) were added. BR quantification was performed as described by using GC/MS (32).

Analysis of Metabolism of BL and CS in Plants Using Liquid Chromatography Tandem MS (LC-MS/MS). The synthesis of BR glucosides used in this study will be described elsewhere (H.S., unpublished data).

For metabolic experiments, 7-day-old seedlings (Col-0, UGT73C5 overexpression line, UGT73C5 RNAi lines) were transferred to 200-ml flasks containing 30 ml of liquid half-strength MS medium supplemented with 1% (wt/vol) sucrose and grown at 21°C in the light on an orbital shaker (100 rpm). Five days after transfer, an ethanol solution (1 μg/μl) of castaster one (CS) (14 μl) or brassinolide (BL) (14.4 μl) was added. The seedlings were incubated for another 1 or 3 day(s) (depending on the experiment) and then extracted with methanol. The methanol extract was partitioned between ethyl acetate and water. The ethyl acetate-soluble fraction was purified by silica gel cartridge (Sep-Pak Vac RC Silica, 500 mg; Waters), which was eluted with 10 ml of CHCl3-MeOH (93:7) and 10 ml of CHCl3-MeOH (7:3). The eluate with 30% MeOH was subjected to LC-MS/MS analysis (for mass spectrometer settings, see Supporting Text).

Functional Expression in Yeast. Heterologous expression of UGT73C5 in yeast was performed as described by using yeast strain YZGA515 expressing either UGT73C5 or transformed with an empty vector as a control (27). The yeast was grown to an A600 0.3 in selective medium, harvested and diluted to an A600 of 2.0 in glucose-supplemented yeast peptone dextrose medium (5% glucose). The BRs, BL, and CS, dissolved in DMSO (50 μg/10 μl), were added and after 18 h of incubation the yeast cells were harvested, washed with ice-cold water, and extracted with methanol/water (4:1). Subsequent analysis of BR glucosides was conducted with LC-MS/MS, as described before.

Results

Arabidopsis Plants Constitutively Overexpressing UGT73C5 Show BR-Deficient Phenotypes and an Altered BR Response. Constitutive overexpression of UGT73C5 in Arabidopsis led to a typical BR-deficient phenotype (2). This is illustrated in Fig. 1A, which compares a wild-type Col-0 plant with a high overexpression transgenic line (UGT73C5oe). The molecular analysis of independent homozygous lines overexpressing UGT73C5 has been described in Poppenberger et al. (27); the overexpression lines (1319/2, 1319/3) showing high levels of recombinant protein have been used in this study, and both lines displayed a near-identical appearance, with features highly characteristic of BR deficiency (2).

Fig. 1.
Overexpression of UGT73C5 in Arabidopsis results in BR-deficient phenotypes. (A Left) Four-week-old adult plants grown under continuous white light. Wild-type Col-0 and an Arabidopsis plant homozygous for a UGT73C5 overexpression construct (line 1319/2; ...

The phenotype of the UGT73C5oe lines was already apparent at the seedling stage. The plants were smaller than wild type, with reduced petiole length and leaf elongation (Fig. 1B). Because application of BR restores normal growth in dwarf mutants with reduced levels of BRs (33, 34), the BR 24-epiBL was applied to seedlings of UGT73C5oe to determine whether the phenotype of the transgenic line could be reverted to wild type. The results, illustrated in Fig. 1B, show that application of the BR restored a wild-type morphology; growth was not visibly impaired, even at the very high levels of 5 μM 24-epiBL. In contrast, wild-type seedlings reacted to the phytohormone as expected with elongation of young aerial tissues, with leaf bending and chlorosis. In summary, these data show that the morphological effects of overexpression of UGT73C5 could be rescued by external BR, suggesting that the seedlings were deficient in active phytohormone. Furthermore, the high expression of UGT73C5 in the seedlings reduced the impact of unphysiological levels of epiBR in the media.

To investigate whether the morphological evidence for BR deficiency could be verified at the molecular level, the expression of genes known to be regulated by BL was analyzed. Using semiquantitative RT-PCR, we showed that the expression of TCH4, a xyloglucan endotransglucosylase/hydrolase (35) that is substantially down-regulated in BR-deficient mutants such as det2-1 (36), was strongly reduced in UGT73C5oe plants. The expression of BEE3, a basic helix-loop-helix transcription factor, is known to be slightly reduced in BR mutants such as bri1 (37) and was found to be similarly lower in the transgenic plants compared with wild type (Fig. 1C).

Therefore, at both the morphological and molecular levels, there is evidence that overexpression of UGT73C5 is correlated with BR deficiency.

BR Levels Are Changed in the UGT73C5oe Lines. To determine whether levels of free BRs were changed in plants overexpressing UGT73C5, endogenous BRs were analyzed in aerial tissues of 4-week-old plants in two independent experiments. The summary in Table 1 shows that amounts of a number of BRs were significantly reduced in plants overaccumulating the UGT. These data are illustrated within the context of the BR biosynthetic pathway in Fig. 4, which is published as supporting information on the PNAS web site, and shows typhasterol, 6-deoxocastasterone, and CS were all decreased by ≈40-60% in the two transgenic lines compared with wild type. The level of BL was below the detection limit in both wild-type and the transgenic lines.

Table 1.
Endogenous levels of BRs

Overexpression of UGT73C5 Leads to Accumulation of 23-O Glucosides of CS and BL in Feeding Experiments. Initial experiments indicated that BR glucosides were below the level of detection in wild type and the transgenic overexpression lines. Therefore, an alternative strategy was used in which seedlings were incubated in media containing either BL or CS for 3 days, and the metabolites formed were identified by their retention time and fragmentation pattern in LC-MS/MS. Using authentic chemically synthesized reference standards, it was possible to unequivocally identify the nature of the metabolites formed during the incubation period. The results are shown in Table 2 and in Fig. 5, which is published as supporting information on the PNAS web site. Only one principal glucoside was formed in the feeding experiments, whether in the wild-type plants or in the overexpression lines: the 23-O-glucoside of either BL or CS (Fig. 5 A and B). The levels of the BL-23-O-glucoside and the CS-23-O-glucoside were two orders of magnitude higher in the transgenic lines compared with wild type (Table 2).

Table 2.
Metabolites of BRs

Overall, the results are consistent with the hypothesis that recombinant UGT73C5 recognizes the 23 hydroxyl position of both BL and CS in planta. Heterologous expression of UGT73C5 in yeast also indicated that, on feeding BL or CS, only the 23-O-glucosides were formed (data not shown).

Seedlings Impaired in UGT73C5 Expression Exhibit No Detectable BL C-23 Glucosylation Activity. No insertional mutants were identified in the publicly available Arabidopsis T-DNA collections. Therefore, RNAi was used to knock down UGT73C5 expression. Because the gene has closely related homologues, the less conserved regions of the 5′ and 3′ UTRs of UGT73C5 mRNA were targeted for silencing. Fifteen independent homozygous lines expressing the silencing construct were analyzed for reduced levels of UGT73C5 mRNA using semiquantitative RT-PCR analysis. Given that UGT73C5 expression is low, seedlings of wild-type and the knockdown lines were treated with the mycotoxin DON, previously shown to significantly increase steady-state levels of UGT73C5 mRNA (27). Fig. 2A illustrates the results of five independent lines in which varying degrees of silencing were achieved. Silencing was strongest in lines 37/1 and 24/4, whereas line 1/5 had no significant reduction in transcript level. To investigate the specificity of the silencing, mRNA levels of UGT73C6, the closest homologue to UGT73C5, with 87% identity at the amino acid level were investigated. Transcript levels of UGT73C6 were slightly affected in the silenced lines but not to the same extent as those of the target gene, UGT73C5.

Fig. 2.
Phenotypic characterization of plants in which UGT73C5 expression is silenced. (A) Arabidopsis plants homozygous for a UGT73C5 RNAi construct were analyzed for changes in UGT73C5 and UGT73C6 transcript levels using semiquantitative RT-PCR. Seedlings of ...

The feeding experiments described in the previous section were repeated to compare glucosylation of BRs in wild type with the transgenic lines in which UGT73C5 expression was impaired. Three knockdown lines were analyzed, two in which silencing was complete (24/4, 37/1) and the third (1/5) in which no silencing was detected. The results of feeding BL for a 1-day incubation period are shown in Table 3 and Fig. 2B. Levels of the glucoside were near identical in wild type and line 1/5. The 23-O-glucoside of BL was not detected in the silenced lines. Fig. 2B compares the fragmentation patterns in the mass chromatograms in line 1/5 and one of the silenced lines (24/4).

Table 3.
Metabolism of BL

To summarize, the synthesis of the BL-23-O-glucoside in wild-type seedlings is due to UGT73C5 activity.

Changing UGT73C5 Expression Leads to Changes in Hypocotyl Elongation. As shown in Fig. 1B, seedlings overexpressing UGT73C5, when grown in the light, displayed a clear BR-deficient phenotype. It is known that plants that overaccumulate BRs develop longer hypocotyls (38), whereas BR-deficient mutants have impaired hypocotyl growth (2), suggesting that BRs are involved in the regulation of hypocotyl elongation. Fig. 2C illustrates changes in hypocotyl elongation when seeds were germinated either in the dark or in different light conditions (for a detailed description of the conditions used, see Supporting Text). Seedlings overexpressing UGT73C5 displayed a clear phenotype consistent with BR deficiency under all light conditions tested. Surprisingly, in the dark, hypocotyl elongation of UGT73C5oe plants was not impaired.

When seedlings of the UGT73C5 silenced lines were grown in the dark, hypocotyl elongation was not changed compared with wild type (Fig. 2C). Insertional mutants in BAS1, a cytochrome P450 suggested to be involved in BR inactivation (15), displayed a subtle phenotype under low fluence rates of different spectra of light (16). Similar conditions that revealed the phenotype in the BAS1 knockout were also applied in this study to determine the effects on lines silenced in UGT73C5 expression. Small increases in length were observed in low fluence rates of monochromatic red or blue light, compared with both wild type and the RNAi control-line 1/5 (Fig. 2C).

When a dose-response of hypocotyls to external 24-epiBL was performed for the different lines, UGT73C5oe plants again displayed a clear phenotype (a reduced elongation) but only in light-grown seedlings, not in the dark. The silenced lines did not show differences compared with wild type under the conditions tested (Fig. 6, which is published as supporting information on the PNAS web site).

UGT73C5 Promoter Activity and Protein Levels Increase in Elongating Hypocotyls. The tissue-specific expression of UGT73C5 was analyzed histochemically in transgenic Arabidopsis plants homozygous for UGT73C5-GUS reporter constructs. The generation of a transcriptional UGT73C5 promoter-GUS line (UGT73C5p-GUS) has been described (27). A translational reporter (UGT73C5f-GUS) was constructed by fusing the UGT73C5 promoter and ORF N-terminally to the GUS gene.

To obtain indications on the regulation of UGT73C5 expression in elongating hypocotyls, we analyzed GUS expression in seedlings of the reporter lines 48 h after transferring them to the dark. The promoter activity of UGT73C5, although present in hypocotyls of seedlings grown in the light, was found to increase during hypocotyl elongation in the dark (Fig. 3A). Analysis of the translational UGT73C5-GUS fusion confirmed that levels of the translation product corresponded to promoter activity (Fig. 3B).

Fig. 3.
Induction of UGT73C5 expression correlates with hypocotyl elongation. (A) Two-day-old seedlings of a transcriptional UGT73C5p-GUS reporter line (27) were shifted from the light to the dark and incubated for 48 h. Reporter expression was determined by ...

The UGT73C5p-GUS reporter was crossed into det2-1 (39), a mutant blocked in an early step of BR biosynthesis and known to be BR-deficient as well as impaired in hypocotyl elongation (29). As shown in Fig. 3C, det2-1 plants carrying the reporter construct failed to elongate when the seedlings were transferred to the dark, and UGT73C5 expression did not increase in the hypocotyls of those plants. These data show that an induction of UGT73C5 expression is correlated with hypocotyl elongation.

Discussion

BRs play a major role in regulating the developmental plasticity of plants and their responsiveness to changes in the external environment. It is suggested that the steroid hormones act locally close to their site(s) of production, and that their levels may be controlled within individual cells (11, 40). In principle, BR cellular homeostasis could involve regulation of synthesis, degradation or inactivation, and it is already known that the expression of genes encoding biosynthetic enzymes is feedback-regulated by BR levels (41-43). Little is known, however, of BR degradation or inactivation, with the exception of P450-mediated hydroxylation (15-17) and a suggested route via sulfonation (44).

This study provides an indication that a UGT accepting BRs as substrates may function in BR homeostasis. UGT73C5 glucosylates the 23-OH position of the BRs, BL, and CS. Interestingly, early work on the catabolism of exogenously supplied BL had shown that BL was almost exclusively converted to its 23-O-glucoside in mung bean explants (45). This study now confirms that the principal glucosylation site of BL supplied to seedlings of Arabidopsis is also the 23 hydroxyl position. As yet, we have no data on the amounts of glucoside relative to other BL metabolites that may be formed and therefore no insight about the predominant pathway of BL inactivation.

Although BL is suggested to be the biologically most active of BRs in Arabidopsis, typically the hormone is below the level of detection in wild-type plants (11, 38, 46), and therefore it is perhaps not surprising that endogenous 23-O-glucoside of BL was not detectable in this study. It was necessary to supply BRs to the plants to reveal either an increased or decreased amount of 23-O-glucosides in the overexpression or silenced lines, respectively. Overexpression of the UGT led to a massive 500-fold increase in BL-23-O-glucoside, whereas silencing the gene resulted in undetectable levels of the glucoside. These data support the hypothesis that 23-O-glucosylation of BL in planta is a function of UGT73C5, and that the UGT is the primary enzyme in seedlings that catalyzes this reaction.

Overexpression of UGT73C5 in Arabidopsis grown in the light led to morphological and molecular phenotypes consistent with those described in earlier studies for mutants deficient in active BRs (2). Application of BL to biosynthetic mutants rescued a wild-type phenotype and similarly in this study, the phenotypic effects of overexpression of UGT73C5 were reversed when light-grown seedlings were treated with BL. This again is consistent with a deficiency in active BRs in the transgenic lines, which was confirmed by analysis of endogenous BRs. In our study, a phenotype characteristic for BR deficiency was not revealed in dark-grown seedlings. Although it is well recognized that light and BR signaling pathways interact (9), as yet we do not have an explanation for these data.

Overexpression of the UGT led to decreased amounts of typhasterol, 6-deoxocastasterone and CS. Unlike BL, levels of these BRs can be detected in wild-type plants, but their bioactivity is considered lower, and they are thought to represent biosynthetic precursors of BL (2). Previous studies on BR-deficient mutants have also shown decreases in BRs late in the biosynthetic pathway and have been unable to detect BL in either wild type or the mutants (12, 29, 38). These other studies, in line with the results presented here, also demonstrated that under conditions when the BRs late in biosynthesis decreased, amounts of upstream BRs increased.

None of the glucosides of the three BRs decreased in amounts in UGT73C5oe lines have ever been detected in plants using the currently available methods and, again in this study, it was not possible to correlate the decrease observed in CS levels with an increase in CS-glucoside. The only means of detecting the CS-23-O-glucoside was after the application of CS to plants when overexpression of UGT73C5 led to a 2-order-of-magnitude increase in the metabolite compared with wild type, and again, no glucoside could be detected in the silenced lines.

Although our results are consistent with the hypothesis that 23-O-glucosylation of BRs by UGT73C5 is responsible for their inactivation and this is the cause of the observed BR deficiency, the data do not provide conclusive evidence. However, under conditions in which 23-O-glucosylation did not occur in seedlings, i.e., in plants in which UGT73C5 was silenced, elongation of hypocotyls under low fluence rates of monochromatic red or blue light was increased, albeit to a small extent. A similar subtle phenotype has previously been described to be characteristic for plants in which inactivation of BRs by hydroxylation was compromised (16, 19). Interestingly, a much stronger BR-over-accumulation phenotype was observed in a later study of double knockouts involving BAS1 and its closest homologue, SOB7, when additional phenotypes were revealed, suggesting redundancy of function in the P450 inactivation pathway (19). It is possible that there is redundancy also in the glucosylation pathway, and this may give rise to the lack of strong phenotypes in the line silenced only in UGT73C5. In this context, Arabidopsis family 1 UGTs have been characterized (47), and UGT73C5 belongs to a clade of seven genes, six of which are found in a tandem repeat on chromosome 2. It may prove necessary to modulate the expression of other members of the UGT73C subfamily to reveal additional phenotypes.

It has been shown that the expression of UGT73C5 is developmentally regulated, with promoter activity greatest in the hypocotyls and roots of young seedlings and already decreasing as the seedlings age (27). This study shows that in wild-type plants, as hypocotyl elongation occurs, UGT73C5 expression increases. It is considered that BRs regulate cell elongation in hypocotyls, because in BR-deficient mutants, hypocotyl extension is impaired (12, 29, 38). Our hypothesis is that UGT73C5 is involved in BR homeostasis, and therefore expression of the gene may well increase at those developmental stages and physiological conditions that are controlled by BR levels.

Screening an Arabidopsis cDNA library functionally expressed in yeast led to the identification of UGT73C5 through its ability to glucosylate and detoxify the fungal toxin DON (27). In that study, overexpression of UGT73C5 in Arabidopsis also led seedlings of the transgenic plants to exhibit increased tolerance against DON. There is much current discussion about plant UGTs and the ability of individual enzymes to recognize multiple substrates (48). Many parallels exist between the plant and mammalian UGTs in their recognition of endogenous and exogenous substrates (22). The mammalian enzymes that glucuronylate steroid hormones are also known to function in phase II detoxification pathways (49, 50). The data described in this study establish a foundation to explore the possibility of dual roles of UGTs in planta.

Supplementary Material

Supporting Information:

Acknowledgments

We thank the horticultural staff of the University of York, especially Colin Abbott and Alison Sutcliff, for high-quality plant care. We thank Masayo Sekimoto and Makoto Kobayashi for technical assistance. Tobias Sieberer, Christian Luschnig, and Eng-Kiat Lim are acknowledged for helpful discussions. We also thank Tobias Sieberer for critical comments on the manuscript and Steve Penfield and Barbara Willet for providing equipment used in the light experiments. This work was supported by grants from the United Kingdom Biotechnology and Biological Sciences Research Council (87/EGA16205); the Garfield Western Foundation; the Austrian Federal Ministry for Education, Science and Culture (Austrian Genome Program GEN-AU, GZ 200.051/6-VI/1/2002), and a Grant-in-Aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan [to H.S. (no. 15590034)]. B.P. received fellowships from the Austrian Academy of Sciences (DOC program) and from the Austrian Science Fund Austrian Science Fund/Fonds zur Förderung der Wissenschaffichen Forschung (Erwin Schrödinger Fellowship).

Notes

Author contributions: B.P., F.E.V., and S.Y. designed research; B.P., S.F., K.S., and G.L.G. performed research; S.F., S.H., H.S., and S.T. contributed new reagents/analytic tools; B.P. and S.F. analyzed data; B.P. and D.B. wrote the paper; and S.F., G.A., S.Y., and D.B. provided leadership to junior colleagues.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: BL, brassinolide; BR, brassinosteroid; CS, castasterone; DON, deoxynivalenol; UGT, UDP-glycosyltransferase; RNAi, RNA interference; LC-MS/MS, liquid chromatography tandem MS; GUS, β-glucuronidase; epiBL, epibrassinolide; fw, fresh weight.

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