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Keywords:

  • auxin;
  • auxin-response element;
  • auxin-response factor;
  • brassinosteroid;
  • rice;
  • transcriptional regulation

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

The phytohormones auxins and brassinosteroids are both essential regulators of physiological and developmental processes, and it has been suggested that they act inter-dependently and synergistically. In rice (Oryza sativa), auxin co-application improves the brassinosteroid response in the rice lamina inclination bioassay. Here, we showed that auxins stimulate brassinosteroid perception by regulating the level of brassinosteroid receptor. Auxin treatment increased expression of the rice brassinosteroid receptor gene OsBRI1. The promoter of OsBRI1 contains an auxin-response element (AuxRE) that is targeted by auxin-response factor (ARF) transcription factors. An AuxRE mutation abolished the induction of OsBRI1 expression by auxins, and OsBRI1 expression was down-regulated in an arf mutant. The AuxRE motif in the OsBRI1 promoter, and thus the transient up-regulation of OsBRI1 expression caused by treatment with indole-3-acetic acid, is essential for the indole-3-acetic acid-induced increase in sensitivity to brassinosteroids. These findings demonstrate that some ARFs control the degree of brassinosteroid perception required for normal growth and development in rice. Although multi-level interactions between auxins and brassinosteroids have previously been reported, our findings suggest a mechanism by which auxins control cellular sensitivity to brassinosteroids, and further support the notion that interactions between auxins and brassinosteroids are extensive and complex.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Phytohormones govern every aspect of growth and development in plants. Many physiological phenomena are regulated either synergistically or antagonistically by multiple phytohormones, and recent studies have revealed extensive interactions among various phytohormone signaling pathways. Auxins and brassinosteroids have a synergistic effect on hypocotyl elongation in various plant species (Yopp et al., 1981; Katsumi, 1985), including maize (Zea mays), pea (Pisum sativum), adzuki bean (Phaseolus angularis) and cucumber (Cucumis sativus). A similar synergistic response was observed for promotion of an upright leaf angle (leaf lamina inclination) in rice (Oryza sativa), which is caused by differential cell elongation by adaxial and abaxial cells in the lamina joint (Yokota and Mori, 1992). The rice lamina inclination bioassay, which was originally developed to detect auxin activity (Maeda, 1965), is more sensitive to brassinosteroids than to auxins, and detects brassinosteroids in a dose-dependent manner (Wada et al., 1981). This assay has been used for purification and isolation of brassinosteroids from various plant species and for evaluation of the biological potency of natural and synthetic brassinosteroids. Interestingly, auxin treatment increases the sensitivity of rice lamina to brassinosteroids (Takeno and Pharis, 1982; Kim et al., 1990; Fujioka et al., 1998), suggesting that auxins and brassinosteroids synergistically regulate this physiological phenomenon.

Significant progress has been made in elucidating both auxin and brassinosteroid signaling pathways. Binding of auxins to members of the TIR1/AFB family of F-box proteins triggers the degradation of Aux/IAA transcriptional repressors, thereby allowing auxin-response factor (ARF) transcription factors, which show either activator or repressor activity, to regulate the expression of auxin-responsive genes (Gray et al., 2001; Zenser et al., 2001; Guilfoyle and Hagen, 2007). In Arabidopsis thaliana, BIN2 kinase negatively regulates brassinosteroid signaling by phosphorylating the BES1 and BZR1 transcription factors, which bind directly to the promoter of brassinosteroid-responsive genes to regulate their expression (He et al., 2002; Yin et al., 2002; Gendron and Wang, 2007; Li and Jin, 2007; Kim and Wang, 2010). Global auxin- and brassinosteroid-induced gene expression analyses using microarray technology have identified a large number of transcriptional target genes shared by the two phytohormone families (Goda et al., 2004; Nemhauser et al., 2004, 2006).

Here, we show that auxins stimulate brassinosteroid perception in rice by increasing the level of the brassinosteroid receptor OsBRI1. We also found that a member of the ARF family regulates the level of OsBRI1 expression to achieve normal plant growth and development. Brassinosteroids are involved in regulation of a number of growth and developmental processes, many of which are also controlled by auxins. In addition to the fact that a large number of transcriptional target genes are shared by auxins and brassinosteroids, our demonstration of the effect of auxins on brassinosteroid sensitivity indicates a complex relationship between the signaling pathways of these two phytohormones in rice.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Exogenous auxin treatment increases OsBRI1 expression in rice

Treatment of seedlings with indole-3-acetic acid (IAA, a bioactive auxin) induced a transient increase in OsBRI1 expression. The level of OsBRI1 mRNA more than doubled after 1 h, but decreased to the pre-treatment level after 4 h (Figure 1a). However, expression of the OsBRI1 paralogs in rice, OsBRL1 and OsBRL3 (Nakamura et al., 2006), and the brassinosteroid biosynthesis enzyme genes CYP90B2 and CYP724B1 (C-22 hydroxylase; Sakamoto et al., 2006a), CYP90D2 and CYP90D3 (C-23 hydroxylase; Sakamoto et al., 2012), CYP90A3 and CYP90A4 (unknown function; Sakamoto and Matsuoka, 2006) and CYP85A1 (C-6 oxidase; Hong et al., 2002; Mori et al., 2002) did not show a similar IAA-induced transient increase (Figs 1b and S1). The level of OsBRI1 protein also increased significantly for at least 3 h after IAA treatment (Figure 1c), suggesting that auxin treatment increases brassinosteroid sensitivity by triggering production of the brassinosteroid receptor.

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Figure 1. Auxins increase OsBRI1 expression. (a) Quantitative RT-PCR analysis of OsBRI1 expression in rice seedlings treated with various phytohormones. Wild-type seedlings were treated with an auxin (IAA), a gibberellin (GA3), a cytokinin (iP), ethylene (ETP), abscisic acid (ABA), a brassinosteroid (BL) or a jasmonate (MeJA). Numbers under the bars indicate the number of hours after each phytohormone treatment. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of six biological repeats. Bars labeled with asterisks differ significantly (< 0.001, Dunnett's post hoc test) from untreated plants (0 h); N, not significant. (b) Quantitative RT-PCR analysis of the expression of OsBRI1 paralogs OsBRL1 and OsBRL3 and brassinosteroid biosynthesis enzyme genes CYP90B2 (C-22 hydroxylase), CYP90D2 (C-23 hydroxylase), CYP90A3 (unknown function) and CYP85A1 (C-6 oxidase) in rice seedlings treated with IAA. Numbers under the bars indicate the number of hours after IAA treatment. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of six biological repeats. Bars labeled with asterisks differ significantly (< 0.01, Dunnett's post hoc test) from untreated plants (0 h); N, not significant. (c) Accumulation of OsBRI1 protein after IAA treatment. The right panel shows protein gel-blot analyses of OsBRI1 (top) and histone H4 (middle). Numbers indicate the number of hours after IAA treatment. Immunoblotting was performed using 20 μg of crude protein extract from rice seedlings treated with IAA. Coomassie Brilliant Blue-stained Rubisco small subunit protein is shown as a loading control (CBB, bottom). In the left panel, levels of OsBRI1 were normalized against the values obtained for histone H4. Values are the means ± SD of six biological repeats. Bars labeled with asterisks differ significantly (Dunnett's post hoc test) from the untreated plants (0 h): *< 0.05, ***< 0.001. (d) Quantitative RT-PCR analysis of OsBRI1 and OsIAA1 expression in rice seedlings treated with various concentrations of IAA. Wild-type seedlings were harvested 1 h after IAA treatment at the indicated concentrations. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of six biological repeats. Bars labeled with asterisks differ significantly (Dunnett's post hoc test) from the untreated plants (0 m): **< 0.01, ***< 0.001. N, not significant. (e) Quantitative RT-PCR analysis of OsBRI1 and OsIAA1 expression in rice seedlings treated with IAA. Numbers under the bars indicate the time after IAA treatment. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of six biological repeats. Bars labeled with asterisks differ significantly (Dunnett's post hoc test) from the untreated plants (0 min): **< 0.01, ***< 0.001. N, not significant. (f) Quantitative RT-PCR analysis of OsBRI1 expression in IAA-treated rice seedlings pre-treated with cycloheximide 3 h before IAA treatment. Numbers under the bars indicate the time after IAA treatment. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of six biological repeats. Bars labeled with asterisks differ significantly (Dunnett's post hoc test) from the untreated plants (0 min): **< 0.01, ***< 0.001. N, not significant.

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We also examined the effects of other phytohormone treatments including a gibberellin (gibberellin A3, GA3), a cytokinin (isopentenyladenine, iP), ethylene (as ethephon, ETP), abscisic acid (ABA) and a jasmonate (methyl jasmonate, MeJA) on the expression of OsBRI1. Although these phytohormone treatments significantly increased expression of the reporter gene for each phytohormone under our experimental conditions (Figure S2), they did not significantly change OsBRI1 mRNA levels (Figure 1a) or production of the OsBRI1 protein (Figure S3). However, the level of OsBRI1 mRNA decreased significantly within 4 h after treatment with brassinolide (BL, a bioactive brassinosteroid) due to feedback down-regulation by the homeostatic system (Figure 1a).

The magnitude of the IAA-induced response was dose-dependent: exogenous IAA had its maximum effect on OsBRI1 expression at concentrations of 20 μm and higher (Figure 1d). An early auxin-responsive gene, OsIAA1 (Thakur et al., 2001), showed a similar dose response (Figure 1d). It is noteworthy that the significant increase in the level of OsBRI1 mRNA occurred within 10 min after IAA treatment, and that the kinetics of the increase were very similar to those of OsIAA1 mRNA (Figure 1e). This rapid induction of OsBRI1 was observed even when indirect effects were blocked by cycloheximide (Figure 1f), which suggests that OsBRI1 expression is directly stimulated by exogenous application of an auxin.

ARF transcription factors participate in the regulation of OsBRI1 expression

The early response of OsBRI1 expression to auxins suggests that OsBRI1 is a member of the early auxin-response gene family targeted by ARF transcription factors. The ARF proteins are encoded by a multi-gene family in plants, and 25 genes from this family have been identified in the rice genome (Wang et al., 2007). Among them, nine genes encode transcriptional activators and the other 16 genes encode transcriptional repressors (Shen et al., 2010). To confirm the involvement of ARF transcription factors in the regulation of brassinosteroid receptor gene expression, we examined the level of OsBRI1 expression in a rice arf mutant. We used a loss-of-function mutant of an activator ARF, OsARF11, which is an ortholog of Arabidopsis ARF5. osarf11-1 is a loss-of-function mutant of OsARF11 caused by insertion of a Tos17 retrotransposon (Figure S4). Like weak brassinosteroid-related mutants, osarf11-1 showed a slight reduction in plant height (to 83% of the wild-type height, = 10, < 0.001), and a significant reduction in the leaf angle of flag leaves from the wild-type value of 23.3° to 18.5° (= 10, < 0.001) (Figure 2a). This mutant also showed the narrow-leaf phenotype that is typical of auxin-deficient or auxin-insensitive rice mutants (Figure 2b) (Fujino et al., 2008; Qi et al., 2008), and the ratio of blade width to blade length of the flag leaves was significantly reduced (to 0.033, versus 0.050 in the wild-type plants, = 10, < 0.001). These mutant phenotypes were complemented by introduction of a 9.7 kb genomic segment containing the OsARF11 gene (Figure S5). Our finding that the steady-state levels of OsIAA1, OsSAUR13 and OsGH3-1 mRNA in the osarf11-1 mutant plants decreased to 72, 79 and 74%, respectively, of those in the wild-type plants (Figure 2c) confirmed that OsARF11, which has a glutamine-rich middle region, acts as a positive regulator of auxin signaling.

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Figure 2. Auxin-response factor transcription factors participate in the regulation of OsBRI1 expression. (a) Comparison of gross morphology between a wild-type plant (left) and an osarf11-1 mutant plant (right). Scale bar = 20 cm. (b) Leaf morphology of a wild-type plant (left) and an osarf11-1 mutant plant (right). Scale bar = 1 cm. (c) Quantitative RT-PCR analysis of OsIAA1, OsSAUR13 and OsGH3-1 expression in wild-type (W) and osarf11-1 (M) plants. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of six biological repeats. Bars labeled with asterisks differ significantly (< 0.001, Student's t test) from the wild-type plants. (d) Quantitative RT-PCR analysis of OsBRI1 expression in osarf11-1 seedlings treated with IAA. Numbers under the bars indicate the number of hours after IAA treatment. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of six biological repeats. Bars labeled with different letters differ significantly (Tukey–Kramer HSD test, < 0.001).

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In the untreated osarf11-1 mutant, the level of OsBRI1 mRNA was 73% of that in untreated wild-type plants; however, the kinetics of the increase in OsBRI1 mRNA after IAA treatment were similar to that in the wild-type (Figure 2d). At 1 h after IAA treatment, the levels of OsBRI1 mRNA in the wild-type and osarf11-1 were 2.2 and 1.6 times greater, respectively, than in the untreated wild-type control. The ratio of the relative expression levels of OsBRI1 mRNA in IAA-treated osarf11-1 after 1 h (1.6) to that in untreated osarf11-1 (0.73) was approximately 2.2, demonstrating that osarf11-1 retains the ability to respond to IAA. These results suggest that multiple OsARF proteins, including OsARF11, function in transcriptional regulation of OsBRI1 in wild-type rice.

A constitutively active Aux/IAA repressor suppresses OsBRI1 expression

Current models propose that ARFs are repressed by interaction with Aux/IAA repressors, and that auxin-stimulated degradation of Aux/IAA repressors releases the ARFs and restores their function (Guilfoyle and Hagen, 2007). Because the expression of OsBRI1 appears to be controlled by OsARFs, we examined whether OsBRI1 expression depends on the degradation of Aux/IAA repressors. We used OsIAA3(P58L)-GR transgenic rice to produce a constitutively active Aux/IAA repressor (Inukai et al., 2005). In these transformants, a single amino acid substitution from Pro to Leu at position 58, which is located in the auxin-dependent degradation-related domain, increases the stability of OsIAA3(P58L) against degradation by auxins. The OsIAA3(P58L) protein in these transformants was fused to the steroid hormone-binding domain of the glucocorticoid receptor (GR) to enable chemical control of its activity using the steroid hormone dexamethasone (DEX). The fusion protein OsIAA3(P58L)–GR is active only when the plants are treated with DEX. Inukai et al. (2005) generated more than 20 independent transgenic lines carrying OsIAA3(P58L)-GR, and selected several lines in which auxin-deficient phenotypes were successfully induced only by DEX treatment. In these lines, the auxin signal was strongly but not completely suppressed by DEX treatment. In the OsIAA3(P58L)-GR transformants, DEX treatment decreased the level of OsBRI1 expression to 60% of that in the untreated transformants within 1 h (Figs 3a and S6).

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Figure 3. Aux/IAA protein suppresses IAA-induced OsBRI1 expression. (a) Quantitative RT-PCR analysis of OsBRI1 expression in DEX-treated OsIAA3(P58L)-GR transgenic seedlings. Numbers under the bars indicate the time after DEX treatment. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of three biological repeats for each of the three independent transgenic lines. Bars labeled with asterisks differ significantly (< 0.001, Dunnett's post hoc test) from the untreated plants (0 min). N, not significant. (b) Quantitative RT-PCR analysis of OsBRI1 expression in IAA-treated OsIAA3(P58L)-GR transgenic seedlings pre-treated with DEX 1 h before the IAA treatment. Numbers under the bars indicate the time after IAA treatment. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of three biological repeats for each of the three independent transgenic lines. Bars labeled with asterisks differ significantly (< 0.001, Dunnett's post hoc test) from the untreated plants (–DEX). N, not significant.

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We also examined the effect of auxins on the DEX-treated OsIAA3(P58L)-GR transformants. IAA treatment 1 h after DEX treatment increased the expression of OsBRI1 to only 1.2 times that in the DEX-untreated transformants (Figs 3b and S7). The ratio of the relative expression levels of OsBRI1 mRNA in both DEX- and IAA-treated OsIAA3(P58L)-GR transformants (1.2) to that in DEX-treated transformants (0.6) was 2.0, demonstrating that DEX-treated OsIAA3(P58L)-GR transformants retain the ability to respond to IAA. These results suggest that degradation of Aux/IAA repressors is essential for the auxin-dependent increase in OsBRI1 expression, and confirms that some OsARF proteins, including OsARF11, positively regulate the expression of OsBRI1.

An ARF transcription factor binds to the OsBRI1 promoter

The early auxin response of OsBRI1 expression and the suppression of OsBRI1 expression in the osarf11-1 mutant suggest that ARF transcription factors regulate OsBRI1 transcription. To investigate the possibility of a direct interaction between ARF transcription factors and the OsBRI1 sequence, a 9.4 kb segment of OsBRI1, which is sufficient to complement the mutant phenotypes seen in the d61-1 mutant (Yamamuro et al., 2000), was cleaved into 23 fragments (<450 bp) and analyzed by an electrophoresis mobility shift assay (EMSA). The recombinant OsARF11 protein expressed in Escherichia coli cells bound to one of the 23 fragments, resulting in a band with lower mobility than the unbound fragments (Figure S8). This fragment contains an AuxRE motif (TGTCTC; Ulmasov et al., 1995) at positions –1986 to –1981 (taking the translation initiation site as +1; Figure 4a). The AuxRE has been identified in the promoters of some early auxin-responsive genes, and ARFs bind to the AuxRE to regulate transcription of these genes (Hagen and Guilfoyle, 2002). To examine whether OsARF11 interacts with this AuxRE in the OsBRI1 promoter, we performed further EMSAs (Figure 4b). OsARF11 bound to a 60 bp OsBRI1 fragment containing the intact AuxRE (wild-type, WT), but did not bind to a fragment containing the mutated AuxRE (MT). The amount of retarded complex was reduced by addition of increasing concentrations of unlabeled WT fragment as a competitor (‘WT comp.’, Figure 4b). The binding of OsARF11 to the WT fragment was not affected by addition of the unlabeled OsBRI1 fragment containing the mutated AuxRE sequence as a competitor (‘MT comp.’, Figure 4b), demonstrating that the interaction between the WT fragment and OsARF11 depends on the presence of an intact AuxRE sequence in the fragment.

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Figure 4. Auxin-induced OsBRI1 expression depends on the presence of an auxin-response element (AuxRE). (a) Genomic structure of OsBRI1. An AuxRE (TGTCTC) was found in the promoter region of OsBRI1. (b) Core sequence of the AuxRE found in the OsBRI1 (wild-type, WT) and mutant (MT) sequences. Labeled WT or MT fragments were used as probes. Binding affinity was estimated using non-labeled WT or MT fragments as competitors (comp.) at the relative concentrations indicated. OsARF11–DNA complexes are indicated by the arrowhead. (c) Quantitative RT-PCR analysis of GUS gene expression in IAA-treated OsBRI1(WT)::GUS and OsBRI1(MT)::GUS transgenic seedlings. Numbers under the bars indicate the number of hours after IAA treatment. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of three biological repeats for each of three independent transgenic lines. Bars labeled with different letters differ significantly (Tukey–Kramer HSD test, < 0.05).

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Auxin-induced OsBRI1 expression depends on the presence of an AuxRE in the OsBRI1 promoter

Our finding that OsARF11 binds to the AuxRE in vitro suggests that this sequence motif may act as a cis-acting regulatory element controlling OsBRI1 expression by rice ARF proteins in vivo. To evaluate the effect of the AuxRE present in the promoter of OsBRI1, we generated transgenic plants with constructs that replaced the OsBRI1 ORF with the GUS reporter gene in a 9.4 kb OsBRI1 segment containing either the WT or MT AuxRE sequence (OsBRI1(WT)::GUS and OsBRI1(MT)::GUS, respectively). In transformants with these constructs, the kinetics of the increase in endogenous OsBRI1 and OsIAA1 mRNA levels were the same as in wild-type (non-transgenic) plants (Figure S9), suggesting that auxin responses were not affected in these transformants. Without IAA treatment, the level of GUS gene expression was slightly but significantly lower in the OsBRI1(MT)::GUS transformants than in the OsBRI1(WT)::GUS transformants (0 h after IAA treatment in Figs 4c and S10). This result suggests that the AuxRE sequence in the OsBRI1 promoter is necessary for the precise control of OsBRI1 expression, and that endogenous auxins partially regulate the level of OsBRI1 expression in rice. A transient but significant increase in GUS gene expression was observed 1 h after IAA treatment in the OsBRI1(WT)::GUS transformants, whereas no significant change was observed in the OsBRI1(MT)::GUS transformants (Figs 4c and S10). These results clearly indicate that the AuxRE in the OsBRI1 promoter is essential for the transient increase in OsBRI1 expression induced by IAA treatment.

Auxins increase the expression of brassinosteroid-responsive genes

To confirm whether the IAA-induced increase in OsBRI1 expression affects brassinosteroid signaling, we monitored the expression of a brassinosteroid-responsive gene, BU1, that participates in the regulation of rice lamina inclination. BU1 is thought to be a primary brassinosteroid-responsive gene, because brassinosteroids increase BU1 expression in the presence of the protein synthesis inhibitor cycloheximide, indicating that de novo protein synthesis is not required (Tanaka et al., 2009). At 24 h after treatment with IAA, no increase in BU1 expression was detected (Tanaka et al., 2009), but if the IAA-induced increase in OsBRI1 expression and subsequent accumulation of the OsBRI1 protein enhanced brassinosteroid sensitivity, we would expect that IAA treatment would at least transiently increase BU1 expression without the exogenous treatment with brassinosteroids. As expected, IAA treatment transiently increased BU1 expression to 7.0 times that in untreated plants (Figure 5a). However, BU1 expression did not increase significantly until approximately 30 min after IAA treatment, and did not reach a maximum until 1–2 h after IAA treatment. Cycloheximide treatment completely abolished the increase in BU1 mRNA levels that occurred in response to IAA treatment (Figure 5b). Taken together, these data indicate that IAA treatment indirectly induces BU1 expression.

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Figure 5. Auxins increase BU1 expression. (a) Quantitative RT-PCR analysis of BU1 expression in rice seedlings treated with IAA. Expression levels were normalized against the values obtained for histone H3. Numbers under the bars indicate the time after IAA treatment. Values are the means ± SD of six biological repeats. Bars labeled with asterisks differ significantly (Dunnett's post hoc test) from the untreated plants (0 min): *< 0.05, ***< 0.001. N, not significant. (b) Quantitative RT-PCR analysis of BU1 expression in IAA-treated rice seedlings pre-treated with cycloheximide 3 h before IAA treatment. Numbers under the bars indicate the time after IAA treatment. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of six biological repeats. The statistical significance of differences was determined by using Dunnett's post hoc test in comparison with the untreated plants (0 min). No significant differences were observed. (c) Quantitative RT-PCR analysis of BU1 expression in BL-treated rice seedlings pre-treated with IAA 4 h before the BL treatment. Seedlings were washed just before BL treatment to remove the IAA. Numbers under the bars indicate the number of hours after BL treatment. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of six biological repeats. Bars labeled with different letters differ significantly (Tukey–Kramer HSD test, < 0.001). (d) Quantitative RT-PCR analysis of OsBRI1 expression in d61-10 mutant seedlings treated with IAA. Numbers under the bars indicate the time after IAA treatment. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of six biological repeats. Bars labeled with asterisks differ significantly (Dunnett's post hoc test) from the untreated plants (0 min): **< 0.01, ***< 0.001. N, not significant. (e) Quantitative RT-PCR analysis of BU1 expression in d61-10 mutant seedlings treated with IAA. Expression levels were normalized against the values obtained for histone H3. Numbers under the bars indicate the time after IAA treatment. Values are the means ± SD of six biological repeats. The statistical significance of differences was determined by Dunnett's post hoc test in comparison with the untreated plants (0 min). No significant differences were observed.

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We also examined the effect of auxin pre-treatment on brassinosteroid-induced BU1 expression. Without IAA pre-treatment, BU1 expression was gradually increased by BL treatment to 10 times that in untreated plants after 6 h (Figure 5c, –IAA), whereas BU1 expression in IAA pre-treated plants was 19 times the level in untreated plants by 6 h after BL treatment (Figure 5c, +IAA). These results show that IAA treatment increases sensitivity to brassinosteroids and downstream brassinosteroid-responsive gene expression.

To exclude the possibility that auxin-induced BU1 expression was not mediated by the function of OsBRI1, we monitored the expression of BU1 in the d61-10 mutant, which has a loss-of-function mutation in OsBRI1 (Nakamura et al., 2006). The steady-state level of OsBRI1 mRNA in the absence of IAA treatment was slightly higher in d61-10 than in the wild-type due to feed-forward up-regulation by the homeostatic system (compare Figs 1e and 5d). In d61-10, the level of OsBRI1 mRNA increased significantly within 10 min after IAA treatment (Figure 5d), and the kinetics of the increase were similar to those in the wild-type. Because the promoter sequence of the OsBRI1 gene in d61-10 has no mutation compared to that in the wild-type, this result supports our conclusion that the AuxRE in the OsBRI1 promoter is essential for the transient increase in OsBRI1 expression induced by IAA treatment. As expected, the level of BU1 mRNA in the absence of IAA treatment was strongly suppressed in the brassinosteroid-insensitive d61-10 mutant (to approximately 35% of that in wild-type plants), and was not affected by IAA treatment (Figure 5e). These results confirm that auxin-induced BU1 expression depends on the function of the intact OsBRI1 receptor, for which levels are increased by IAA treatment.

Auxin-induced BU1 expression depends on the presence of the AuxRE in the OsBRI1 promoter

To confirm our hypothesis that auxin-induced BU1 expression is mediated by the transient increase in OsBRI1 expression, we generated transgenic plants carrying construct containing the 9.4 kb OsBRI1 segment with either the WT or MT AuxRE sequence (D61 and mpD61 constructs, respectively) by introducing them into d61-10. d61-10 plants showed severe dwarfism (plant height was approximately one-fifth that of the wild-type) and defects in reproductive development; these phenotypes were complemented by the D61 construct, which contained the WT AuxRE (D61-d61-10, Figure 6a–c). In contrast, introduction of the mpD61 construct, which contained the mutated AuxRE (mpD61-d61-10), did not completely rescue the plant height of d61-10: the mean plant height of three plants for each of the three independent transgenic lines was 85% that of wild-type (Figure 6a,d,e). The BU1 expression level was not significantly different between mpD61-d61-10 and the wild-type (Figure S11). Because BU1 RNAi plants did not show a dwarf phenotype (Tanaka et al., 2009), BU1 appears not to function in the control of plant height.

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Figure 6. Auxin-induced BU1 expression depends on the presence of the auxin-response element (AuxRE) in the OsBRI1 promoter. (a) Phenotype of the wild-type plants. Scale bar = 20 cm. (b) Phenotype of the d61-10 mutant plants. (c) Phenotypes of three independent transgenic d61-10 lines carrying an intact OsBRI1 gene (D61-d61-10). (d) Phenotypes of three independent transgenic d61-10 lines carrying the mutated AuxRE-containing OsBRI1 gene (mpD61-d61-10). (e) Plant height of the wild-type plants and the D61-d61-10 and mpD61-d61-10 transformants. Values are the means ± SD of nine plants (wild-type) and three plants for each of the three independent transgenic lines (D61-d61-10 and mpD61-d61-10 transformants). Bars labeled with asterisks differ significantly from wild-type plants (< 0.001, Dunnett's post hoc test). N, not significant. (f) Quantitative RT-PCR analysis of BU1 expression in BL-treated D61-d61-10 and mpD61-d61-10 transgenic seedlings pre-treated with IAA 4 h before the BL treatment. Numbers under the bars indicate the number of hours after BL treatment. Expression levels were normalized against the values obtained for histone H3. Values are the means ± SD of three biological repeats for each of the three independent transgenic lines. Bars labeled with different letters differ significantly (Tukey–Kramer HSD test, < 0.001).

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In d61-10 transgenic plants carrying the D61 construct (D61-d61-10), BU1 expression gradually increased to nine times that in untreated plants by 6 h after BL treatment (–IAA; Figs 6f and S12); in IAA-pre-treated plants, the BU1 expression level reached 17 times that in untreated plants by 6 h after BL treatment (+IAA; Figs 6f and S12). The kinetics of the increase in the endogenous BU1 mRNA level in the transgenic plants were similar to that in the wild-type (non-transgenic) plants (Figure 5c). The slightly lower increase in the transgenic plants may reflect a difference in plant growth conditions: the transgenic seedlings used for the expression analysis were grown on medium containing a high concentration of the antibiotic hygromycin, which appears to have a slight growth retardation effect even in hygromycin-resistant transgenic rice. On the other hand, IAA pre-treatment had no effect on BL-induced BU1 up-regulation in d61-10 transgenic plants carrying the mpD61 construct (mpD61-d61-10; Figs 6f and S12). These results clearly show that the AuxRE motif in the OsBRI1 promoter, and thus the transient up-regulation of OsBRI1 expression caused by IAA treatment, is essential for the IAA-induced increased sensitivity to brassinosteroids in regulation of BU1 expression.

The synergistic effects of auxins on brassinosteroid-induced rice lamina inclination depend on the presence of an AuxRE in the OsBRI1 promoter

We also tested the effect of auxins on the sensitivity to brassinosteroids in a lamina inclination bioassay. As previously mentioned, rice leaves can be used for the quantitative bioassay of brassinosteroids, and simultaneous treatment with auxins increases the sensitivity of rice lamina to brassinosteroids (Takeno and Pharis, 1982; Kim et al., 1990; Fujioka et al., 1998). When wild-type seedlings were used for the lamina inclination bioassay (Figure 7), the leaf blade was slightly bent from the axis of the leaf sheath in the absence of exogenous BL (29.8°; –IAA, 0 m BL), and the leaf angle was increased in the presence of increasing concentrations of BL; the difference was significant even at the lowest level of BL application. Treatment with IAA alone (+IAA, 0 m BL) showed no significant increase in inclination compared with the control (–IAA), whereas simultaneous treatment with IAA and BL significantly increased the leaf angle compared to treatment with BL alone. When D61-d61-10 transgenic seedlings were tested, the leaf angle was also increased by simultaneous treatment with IAA and BL (Figure 7), as in the wild-type seedlings. However, IAA did not significantly increase the leaf angle in the mpD61-d61-10 transgenic seedlings (Figure 7). The results of the lamina inclination bioassay thus confirm that the AuxRE motif in the OsBRI1 promoter, and thus the transient up-regulation of OsBRI1 expression caused by IAA treatment, is essential for the IAA-induced increased sensitivity to brassinosteroids in rice.

image

Figure 7. Effect of auxins on the degree of brassinosteroid-induced rice lamina inclination. Two-week-old wild-type seedlings, D61-d61-10 transgenic rice seedlings and two independent lines of mpD61-d61-10 transgenic rice seedlings were simultaneously treated with IAA (20 μm) and BL (as indicated). The leaf angle of the third leaf was measured 4 weeks after the treatment. Values are the means ± SD of ten plants. Bars labeled with different letters differ significantly (Tukey–Kramer HSD test, < 0.001).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Although an auxin-induced increase in OsBRI1 expression was observed under artificial conditions involving IAA treatment of rice seedlings, our results for the rice arf mutant osarf11-1 and OsIAA3(P58L)-GR transgenic rice clearly showed that some members of the ARF family, which are key auxin signal mediators, regulate OsBRI1 expression at appropriate levels for normal plant growth and development. Nemhauser et al. (2004) suggested that auxins also increased the expression of Arabidopsis BRI1 and therefore influences the brassinosteroid signaling capacity in Arabidopsis. They carefully studied the inter-dependence of brassinosteroid and auxin signaling in Arabidopsis, and demonstrated that increased auxin levels saturate the brassinosteroid-stimulated growth response and greatly reduce brassinosteroid effects on gene expression. These results can be explained by the increased BRI1 level caused by auxin treatment, and therefore support our finding that auxins increase the sensitivity of brassinosteroid perception. A recent study in Arabidopsis showed that auxin enhanced brassinosteroid biosynthesis in part by inhibiting BZR1 feedback repression of the DWARF4 gene, which encodes a C-22 hydroxylase that is involved in a rate-limiting step in brassinosteroid biosynthesis (Chung et al., 2011). However, expression of the DWARF4 ortholog in rice, CYP90B2/OsDWARF4, was not induced by IAA (Figure 1b). Because the other brassinosteroid biosynthetic enzyme genes were also not induced by IAA treatment (Figs 1b and S1), the mechanisms of regulation of brassinosteroid biosynthesis may differ between Arabidopsis and rice, and thus auxins may not regulate brassinosteroid biosynthesis in rice.

In our experiments, we also monitored the expression of a brassinosteroid-responsive gene, BU1. Brassinosteroid-induced BU1 up-regulation was not as rapid as that induced by IAA treatment. In general, brassinosteroid-induced transcription is weak and slow, whereas auxin-induced transcription is relatively rapid and has a much higher amplitude (Abel et al., 1994; Gil et al., 1994; Goda et al., 2002; Müssig et al., 2002; Nemhauser et al., 2004; Hardtke, 2007; Hardtke et al., 2007). These kinetics differences may reflect the high and low complexity of brassinosteroid and auxin signal transduction pathways, respectively (Hardtke et al., 2007), or may reflect the slow transport of exogenously applied brassinosteroids in plants (Yokota et al., 1992; Symons and Reid, 2004; Savaldi-Goldstein et al., 2007; Symons et al., 2008). As for OsBRI1, IAA treatment also increased the expression of BU1, but only after 30 min or more. In comparison, the increase in the levels of OsBRI1 mRNA began within 10 min after IAA treatment (compare Figs 1e and 5a). The delay in the increase in BU1 expression suggests that the transient up-regulation of this gene was not a direct result of IAA treatment, but was due to increased sensitivity to brassinosteroids. This hypothesis is strongly supported by our findings that cycloheximide treatment completely abolished the increase in BU1 expression that occurred in response to IAA treatment (Figure 5b), that auxin-induced BU1 expression depends on the function of the intact OsBRI1 receptor, whose levels are increased by IAA treatment (Figure 5e), that IAA pre-treatment had no effect on BL-induced BU1 up-regulation in d61-10 transgenic plants carrying the mpD61 construct (Figure 6f), and that the AuxRE motif was not present in the 5′ flanking region of BU1 (at least not within –5000 bp, taking the translation initiation site as +1). In Figure 6(f), we compare D61-d61-10 (transgenic plants with an intact AuxRE) and mpD61-d61-10 (transgenic plants with a mutated AuxRE) to evaluate the effect of the AuxRE present in the promoter of OsBRI1. This allowed us to evaluate the effect of the AuxRE without the need to consider other auxin effects on brassinosteroid responses. The intact AuxRE in D61-d61-10 doubled the level of brassinosteroid receptor gene expression in response to auxin treatment (significant difference), whereas the mutant AuxRE in mpD61-d61-10 did not produce a significant increase in expression. Because GUS gene expression driven by an OsBRI1 promoter containing the intact AuxRE doubled in response to auxin treatment, whereas gene expression from constructs containing the mutated AuxRE showed no response (Figure 4c), we believe that these results provide direct evidence that changes in the levels of the OsBRI1 receptor are responsible for changes in the brassinosteroid response.

We hypothesized that the IAA-induced increase in OsBRI1 mRNA and OsBRI1 protein levels, which were twice those in untreated seedlings by 1–2 h after treatment, would affect brassinosteroid signaling. Indeed, IAA pre-treatment doubled the level of BU1 mRNA after BL treatment, but only in the presence of an intact AuxRE in the OsBRI1 promoter. We therefore believe that the auxin-induced increase in brassinosteroid sensitivity is an early event in transcriptional auxin–brassinosteroid synergism. Simultaneous treatment with IAA and BL increases brassinosteroid activity, as measured by the rice lamina inclination bioassay (Takeno and Pharis, 1982; Kim et al., 1990; Fujioka et al., 1998). In our experiments, the intact AuxRE in D61-d61-10 significantly increased the brassinosteroid-induced rice lamina inclination in response to IAA co-application, whereas the mutant AuxRE in mpD61-d61-10 did not. These results confirm that the AuxRE motif in the OsBRI1 promoter, and thus the transient up-regulation of OsBRI1 expression caused by IAA treatment, is essential for the IAA-induced increased sensitivity to brassinosteroids in rice.

Auxins and brassinosteroids are known for their overlapping activities in physiological assays (Halliday, 2004; Mockaitis and Estelle, 2004; Hardtke, 2007; Hardtke et al., 2007; Depuydt and Hardtke, 2011). At the molecular level, auxin- and brassinosteroid-induced gene expression analyses based on microarray technology have identified a large number of transcriptional target genes shared by these two phytohormones (Goda et al., 2002, 2004; Müssig et al., 2002; Yin et al., 2002; Nemhauser et al., 2004, 2006). It is also well known that the prototypical auxin-responsive reporter gene construct consisting of multiple AuxREs, DR5::GUS, is brassinosteroid-responsive (Bao et al., 2004; Nemhauser et al., 2004; Wang et al., 2005; Yin et al., 2005). These studies suggest that auxin–brassinosteroid synergism occurs through combinational regulation of expression of common target genes. In addition, auxin and brassinosteroid transcriptional response pathways are inter-dependent, i.e. changes in gene expression in response to one phytohormone require the function of the other phytohormone (Nemhauser et al., 2004, 2006; Hardtke et al., 2007). These results, together with our findings, suggest complex interactions between auxins and brassinosteroids. IAA-induced accumulation of OsBRI1 appears to occur at the start of a cascade of interactions that increase the brassinosteroid responses induced by IAA; a small but significant increase in OsBRI1 is amplified through interactions between auxin and brassinosteroid signaling to produce changes in the sensitivity of the rice lamina joint to brassinosteroids.

As previously mentioned, BIN2 kinase negatively regulates brassinosteroid signaling by phosphorylating the BES1 and BZR1 transcription factors, and changes in the phosphorylation status of BES1 and BZR1 alter their activity, resulting in altered expression levels of their target brassinosteroid-responsive genes (He et al., 2002; Yin et al., 2002; Gendron and Wang, 2007; Li and Jin, 2007; Kim and Wang, 2010). Auxins increase the expression of Arabidopsis BRI1 (Nemhauser et al., 2004), but accumulation of non-phosphorylated BES1 and BZR1 was not observed after auxin treatment (He et al., 2002; Yin et al., 2002). These findings suggest that auxins induce BRI1 expression in Arabidopsis but do not stimulate brassinosteroid signaling. At present, it is not clear whether the phosphorylation of BES1 and BZR1 orthologs in rice is affected by auxin treatment. Although further analysis is required to understand the mechanism by which auxins increase the brassinosteroid response, the auxin–brassinosteroid synergism may differ between rice and Arabidopsis, i.e. auxins regulate brassinosteroid biosynthesis in Arabidopsis (but not in rice), whereas auxins stimulate brassinosteroid signaling in rice (but not in Arabidopsis). Multi-level interactions between auxins and brassinosteroids have previously been reported (Clouse and Sasse, 1998; Halliday, 2004; Mockaitis and Estelle, 2004; Woodward and Bartel, 2005; Hardtke, 2007; Hardtke et al., 2007; Depuydt and Hardtke, 2011), but our results clearly suggest a mechanism by which auxins control cellular sensitivity to brassinosteroids, and further support the notion that interactions between auxins and brassinosteroids are extensive and complex.

In conclusion, auxins directly stimulate brassinosteroid perception by regulating the expression of a brassinosteroid receptor gene, and this phenomenon is probably conserved between monocots (rice) and dicots (Arabidopsis), a hypothesis that is supported by the results of Nemhauser et al. (2004). These findings contribute to explaining how auxin co-application improves cellular sensitivity to brassinosteroids. We also provide evidence that auxins act as regulators to maintain brassinosteroid perception at appropriate levels for normal plant growth and development. These results indicate that possible changes in cellular sensitivity to brassinosteroids should be considered when the effects of auxins on the regulation of various growth and developmental processes in plants are examined. Our findings suggest that brassinosteroid-stimulated growth responses differ depending on whether or not auxin signaling is active. Future questions to be addressed include identifying where auxin signaling functions in plants, and how the increased sensitivity to brassinosteroids induced by auxins affects the regulation of various growth and developmental processes in plants. We envisage that a detailed analysis of mpD61-d61-10 transformants will help to answer these questions.

Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Plant materials and phytohormone treatments

Seeds of wild-type rice (Oryza sativa L. ‘Nipponbare’), mutants, and transformants were sterilized in 1% NaClO for 30 min and sown on Murashige and Skoog agar medium. The seedlings were grown in a growth chamber (MLR-351H; Sanyo Electric, http://www.sanyo-biomedical.jp/) at 28°C under continuous light (169 μmol m−2 sec−1 photosynthetically active radiation photon flux density, measured using RGB quantum meter 101EG; Nippon Medical & Chemical Instruments, http://www.nihonika.co.jp/) provided by cool-white fluorescent tubes (FL40SSW/37; Toshiba, http://www.toshiba.co.jp) for 2 weeks. Seedlings were selected for uniformity of growth, gently washed to remove Murashige and Skoog agar medium from the roots, and transferred into containers filled with distilled water to which no additional nutrients were added (five plants per 60 ml container). After acclimatization for 2 days, phytohormone treatment was performed using an auxin (IAA; 20 μm, except where indicated), a gibberellin (10 μm GA3), a cytokinin (5 μm isopentenyladenine), ethylene (supplied as 50 μm ethephon), abscisic acid (ABA; 100 μm), a brassinosteroid [brassinolide (BL); 100 nm except where indicated] or a jasmonate (100 μm methyl jasmonate) by adding the phytohormone to the water. These concentrations were determined based on the results of previous research (Sakai et al., 2003; Sakamoto et al., 2006b, 2011; Qi et al., 2011; Seo et al., 2011). For cycloheximide treatment, seedlings were pre-treated with 100 μm cycloheximide for 3 h before treatment with 20 μm IAA. Co-application of IAA and BL for the gene expression analyses was performed by pre-treating the seedlings with 20 μm IAA for 4 h, washing their roots to remove the IAA, and then treating them with 100 nm BL. For the rice lamina inclination bioassays, 2-week-old wild-type and transgenic rice seedlings were transplanted into Murashige and Skoog agar medium containing IAA (20 μm) and BL (as indicated). After 4 weeks, the leaf angle of the third leaf on each plant was measured (ten plants for each line). All phytohormone and cycloheximide treatments were performed in the growth chamber under the growing conditions described above.

Gene expression analysis

Total RNA was extracted from whole seedlings using an RNeasy plant mini kit (Qiagen, http://www.qiagen.com/). Single-strand cDNAs were synthesized using an Advantage RT-for-PCR kit (Clontech, http://www.clontech.com/). Quantitative RT-PCR was performed using an iCycler iQ real-time PCR system (Bio-Rad Laboratories, http://www.bio-rad.com/) as described previously (Sakamoto et al., 2011). Expression levels were normalized against the values obtained for histone H3, which was used as the internal reference gene. OsGA2ox3 (Sakai et al., 2003), OsRR5 (Jain et al., 2006), OsERF3 (Jung et al., 2010), OsBZ8 (Nakagawa et al., 1996) and OsMYC2 (Lorenzo et al., 2004) were used as reporter genes for the gibberellin, cytokinin, ethylene, abscisic acid, brassinosteroid and jasmonate signals, respectively. Primer sequences are listed in Table S1.

Protein expression analysis

A DNA fragment encoding a partial OsBRI1 amino acid sequence (Asn530 to Ser715) was inserted in the sense orientation as a translational fusion into the pET-32a expression vector (Novagen, http://www.novagen.com) and expressed in E. coli BL21 (DE3) cells (Stratagene, http://www.stratagene.com). The over-produced recombinant protein was purified using Talon metal affinity resin (Clontech) and separated by SDS–PAGE. The appropriate band was excised from the SDS–PAGE gel and used directly for production of rabbit polyclonal antibodies. OsBRI1 was immunoblotted using 20 μg of total protein extracted from seedlings harvested at the indicated times after IAA treatment. Total protein was extracted from whole seedlings using CelLytic P reagent (Sigma-Aldrich, http://www.sigma-aldrich.com). Protein samples were separated by 8% SDS-PAGE and transferred to a Hybond enhanced chemiluminescence membrane (Amersham, http://www.gelifesciences.com) by semi-dry blotting. The blots were incubated with anti-OsBRI1 antiserum raised in rabbits and then with goat anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (MBL, https://res.mbl.co.jp). The blots were also incubated with anti-histone H4 polyclonal antibody (Imgenex, http://www.imgenex.com) as an internal reference. Peroxidase activity was detected by using the SuperSignal West Pico Complete rabbit IgG detection kit (Pierce, http://www.piercenet.com) and an LAS-3000 imager (Fujifilm, http://www.fujifilm.com), and was quantified using Multi Gauge software version 3.0 (Fujifilm).

Statistics

For gene and protein expression experiments, we performed six biological repeats for wild-type and mutant plants and nine biological repeats for transgenic plants (three biological repeats for each of three independent transgenic lines). Statistical analysis of the data (ratio of the target value to the internal reference) was performed using the JMP statistical package version 5.1.2 (SAS Institute, http://www.sas.com). Comparisons between pairs of groups were performed using Student's t test. Comparisons between more than two groups were performed using one-way anova followed by Dunnett's test for comparison of treatment groups with a control (wild-type or untreated plants), and the Tukey–Kramer HSD test for comparison between all treatment groups.

Electrophoresis mobility shift assay

Full-length OsARF11 cDNA was inserted in the sense orientation into the pCold TF expression vector (Takara, http://www.takarabio.com). Recombinant OsARF11 protein was expressed in ArcticExpress E. coli cells (Stratagene). The recombinant protein was purified using Talon metal affinity resin (Clontech). OsBRI1 promoter fragments containing the wild-type OsBRI1 AuxRE or the mutated AuxRE were amplified by PCR from rice genomic DNA. The primer sequences were 5′-AACTGCAGGTACCTCTAACATGCGTACACA-3′ and 5′-AACTGCAGATAAGATCTTCCTTGGTATGAC-3′ for the wild-type OsBRI1 AuxRE (WT), and 5′-ATGCGTACACAATTCTTACAGTCTCAGGTCATACCAAGGA-3′ and 5′-CCTTGGTATGACCTGAGACTGTAAGAATTGTGTACGCATG-3′ to generate the AuxRE mutation (MT). Amplified fragments were cloned into pBluescript II SK (Stratagene), and their identities were confirmed by sequence analysis. The PCR-amplified fragments were excised using restriction endonucleases, purified by 10% PAGE, and labeled with biotin using a biotin 3′ end DNA labeling kit (Pierce). The electrophoresis mobility shift assay was performed using a LightShift chemiluminescent EMSA kit (Pierce)

Transgenic rice

For complementation of the osarf11-1 mutation, the wild-type genomic sequence from −3020 to +6652 (taking the translation initiation site as +1) was amplified by PCR. The primer sequences were 5′-GCCTCGAGGCTTGGGTAGTCCTCTCCGT-3′ and 5′-GCGGATCCCTGCCATCTTTCCGTGCTC-3′. Amplified fragments were cloned into pBluescript II SK (Stratagene) and their identities were confirmed by sequence analysis. This 9.7 -kb genomic segment containing the OsARF11 gene was inserted into the pCAMBIA1300 binary vector (Cambia, http://www.cambia.org/). The OsBRI1 fragments were PCR-amplified from rice genomic DNA. The primer sequences were 5′-GCTCTAGACGCCGGCCCGCGCGCCCTCTCT-3′ and 5′-ACCTGTCTAGACTGTGGAGATTGGAGAAGC-3′ for the 9.4 kb segment, 5′-GCTCTAGACGCCGGCCCGCGCGCCCTCTCT-3′ and 5′-TCCCCCGGGATGTACGAGCGAGCTCACTGC-3′ for the promoter with a 5′ untranslated region, and 5′-GCGAGCTCAAACAACAACCACCGACACACAGG-3′ and 5′-GCGTCGACTGTGGAGATTGGAGAAGCTAC-3′ for the 3′ untranslated region. Fragments containing the promoter with a 5′ untranslated region and the 9.4 kb segment of OsBRI1 (from positions −4825 to +4571, taking the translation initiation site as +1) containing the AuxRE mutation were amplified using the primers described in the previous section. Amplified fragments were cloned into the pBluescript II SK vector (Stratagene), and their identities were confirmed by sequence analysis. The OsBRI1 promoter sequences with either the WT AuxRE or the MT AuxRE, the GUS gene fragment obtained from pBI121 (Clontech, Palo Alto, CA, USA), and the 3′ untranslated region of OsBRI1 were inserted into the pBI101-Hm vector (kindly provided by Dr. K. Nakamura, Nagoya University, Japan). The 9.4 kb segment of OsBRI1 with either the WT AuxRE or the MT AuxRE was inserted into the pCAMBIA1300 vector. These gene constructs were introduced into Agrobacterium tumefaciens strain EHA105, and Agrobacterium-mediated transformation of rice was performed as previously described (Hiei et al., 1994). Transgenic plants were selected on Murashige and Skoog agar medium containing 50 mg l−1 hygromycin, and then grown in a greenhouse at 28°C under ambient light conditions.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

We thank Mio Tonouchi, Asako Tokida-Segawa and Yukiko Tanabe for technical assistance, and the GenBank project of the National Institute of Agrobiological Science in Japan for providing osarf11-1 (NC2659) seeds and OsARF11 cDNA (AK103452). T.S. was supported by Grants-in-Aid for Young Scientists (numbers 19688001 and 24780005) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. S.F. was supported by Grants-in-Aid for Scientific Research (B) (numbers 19380069 and 23380066) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References
  9. Supporting Information
FilenameFormatSizeDescription
tpj12071-sup-0001-FigureS1.pdfapplication/PDF118KFigure S1. Effect of IAA treatment on brassinosteroid biosynthetic enzyme gene expression.
tpj12071-sup-0002-FigureS2.pdfapplication/PDF122KFigure S2. Effect of phytohormone treatment on reporter gene expression.
tpj12071-sup-0003-FigureS3.pdfapplication/PDF82KFigure S3. Effect of phytohormone treatment on OsBRI1 level.
tpj12071-sup-0004-FigureS4.pdfapplication/PDF143KFigure S4. Molecular characterization of the Tos17 insertion mutant osarf11-1.
tpj12071-sup-0005-FigureS5.pdfapplication/PDF7111KFigure S5. Phenotypic complementation of osarf11-1 by introduction of the OsARF11 gene.
tpj12071-sup-0006-FigureS6.pdfapplication/PDF117KFigure S6. Effect of DEX treatment on OsBRI1 expression in the OsIAA3(P58L)-GR transformants.
tpj12071-sup-0007-FigureS7.pdfapplication/PDF117KFigure S7. Effect of IAA treatment on OsBRI1 expression in DEX pre-treated OsIAA3(P58L)-GR transformants.
tpj12071-sup-0008-FigureS8.pdfapplication/PDF310KFigure S8. The auxin signal transcription factor OsARF11 binds to the promoter region of OsBRI1.
tpj12071-sup-0009-FigureS9.pdfapplication/PDF117KFigure S9. Relative OsBRI1 and OsIAA1 expression levels in OsBRI1(WT)::GUS and OsBRI1(MT)::GUS transformants after IAA treatment.
tpj12071-sup-0010-FigureS10.pdfapplication/PDF118KFigure S10. Effect of IAA treatment on GUS gene expression in OsBRI1(WT)::GUS and OsBRI1(MT)::GUS transformants.
tpj12071-sup-0011-FigureS11.pdfapplication/PDF114KFigure S11. Relative BU1 expression levels in d61-10 mutant and mpD61-d61-10 transgenic plants.
tpj12071-sup-0012-FigureS12.pdfapplication/PDF119KFigure S12. Effect of BL treatment on BU1 expression in IAA pre-treated D61-d61-10 and mpD61-d61-10 transformants.
tpj12071-sup-0013-TableS1.pdfapplication/PDF67KTable S1. Primer sequences used in gene expression analysis.
tpj12071-sup-0014-Supplemental-Figure-legends.pdfapplication/PDF109K 

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