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First published online November 6, 2003; 10.1104/pp.103.030031 Plant Physiology 133:1843-1853 (2003) © 2003 American Society of Plant Biologists Brassinolide Induces IAA5, IAA19, and DR5, a Synthetic Auxin Response Element in Arabidopsis, Implying a Cross Talk Point of Brassinosteroid and Auxin SignalingPlant Science Center, RIKEN, Suehirocho, Tsurumi, Yokohama, Kanagawa 230-0045, Japan (A.N., H.G., Y.S., S.Y.); Department of Biological Sciences, Graduate School of Science, Tokyo Metropolitan University, Hachioji, Tokyo 192-0397, Japan (K.H., T.K.); Plant Functions Lab., RIKEN, Hirosawa, Wako, Saitama 351-0198, Japan (M.T.F., S.Y.); Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-0033, Japan (S.S.); and Graduate School of Science and Engineering, Saitama University, Saitama, Saitama 338-8570, Japan (A.N., S.Y.)
Despite numerous physiological studies addressing the interactions between brassinosteroids (BRs) and auxins, little is known about the underlying molecular mechanisms. We studied the expression of IAA5 and IAA19 in response to treatment with indole acetic acid (IAA) or brassinolide (BL), the most active BR. Exogenous IAA induced these genes quickly and transiently, whereas exogenous BL induced them gradually and continuously. We also found that a fusion of DR5, a synthetic auxin response element, with the GUS (  -glucuronidase) gene was induced with similar kinetics to those of the IAA5 and IAA19 genes in response to both IAA and BL treatment of transgenic plants. These results suggest that the IAA genes are induced by BL, at least in part, via the activation of the auxin response element. Endogenous IAA levels per gram fresh weight did not increase when seedlings of Arabidopsis wild type (WT) or the BR-deficient mutant det2 were treated with BL. Furthermore, the levels of IAA transcripts were lower in the det2 mutant than in the WT, even though endogenous IAA levels per gram fresh weight were higher in the det2 mutant than in the WT. In conclusion, the lack of evidence for auxin-mediated activation of early auxin-inducible genes in response to BL suggests that the BR and auxin signaling pathways independently activate the transcriptional system of the IAA and DR5-GUS genes.
Exogenous application of brassinosteroids (BRs) to plants at nanomolar to micromolar concentrations produces a wide spectrum of physiological effects. These include promotion of cell elongation and division, enhancement of tracheary element differentiation, delaying of abscission, enhancement of gravity-induced bending, promotion of ethylene biosynthesis, and enhancement of stress resistance, as reviewed by Clouse and Sasse (1998  ) and Sasse (1999). A number of BR-deficient mutants have been identified in Arabidopsis, pea (Pisum sativum), and tomato (Lycopersicon esculentum; for review, see Clouse and Feldmann, 1999; Clouse, 2002; Fujioka and Yokota, 2003). These mutants exhibit dwarfism when grown in either light or dark conditions. Many of these mutants also have dark-green leaves, reduced fertility, a prolonged life span, and abnormal skotomorphogenesis. BR-insensitive mutants have been identified in Arabidopsis, pea, tomato, and rice (Oryza sativa; for review, see Clouse and Feldmann, 1999; Clouse, 2002; Fujioka and Yokota, 2003). The molecular mechanisms of BR action, however, remain unclear.
It has been suggested that the actions of BRs are related to auxin action (Mandava, 1988
Recently, we found that a number of early auxin-inducible genes, belonging to the GH3, SAUR (Hagen and Guilfoyle, 2002
In our experimental system, BL significantly promoted the growth of Arabidopsis seedlings in both the WT and deetiolated2 (det2) mutant, a BR-deficient mutant blocked early in BR biosynthesis (Fujioka et al., 1997
Induction of IAA5 and IAA19 Genes by BL and IAA Treatment
We studied expression of the IAA5 and IAA19 genes in response to treatment with exogenous auxin and BR. Seven-day-old det2 seedlings were treated with 10 nM BL, and the induction kinetics of the two genes were analyzed with real-time quantitative (RTQ) reverse transcriptase (RT)-PCR (Fig. 1, A and B). The IAA5 and IAA19 genes were induced by BL treatment with kinetics similar to the responses of IAA3, SAUR-AC1, and BRU6/GH3-2 to BL, as well as similarly to genes implicated in cell wall organization, as described previously (Goda et al., 2002
To compare the induction kinetics of the IAA genes by BL treatment with those in response to IAA, WT (Columbia [Col-0]) seedlings were treated with 10 nM, 100 nM, or 1 µM BL or IAA for up to 24 h. Both IAA genes were quickly induced by IAA treatment and showed stronger responses with higher IAA concentrations (Fig. 1, C and D). The IAA5 and IAA19 genes were induced by over 80- and nearly 40-fold, respectively, and transcript levels peaked 1 h after IAA treatment. In contrast, BL induced both IAA genes with much different kinetics than did IAA. The maximum induction of these genes by BL in WT plants ranged from 3- to 5-fold (Fig. 1, E and F). The point of maximum transcript accumulation of these IAA genes after BL treatment occurred later than did the maximum point after auxin induction. The BL induction kinetics in the WT were similar to the BL induction kinetics of the det2 mutant. The timing and the level of peak induction by BL were independent of BL concentration.
The levels of the IAA5 and IAA19 transcripts were compared between WT plants and det2 mutant plants. The levels were higher in WT plants than in det2 mutant plants (Fig. 2A), indicating that these genes are down-regulated in the det2 mutant, possibly because this mutant has a lower endogenous level of BR (Fujioka et al., 1997
AuxREs, which consist of a TGTCTC sequence and an adjacent or overlapping coupling element, were defined based on the auxin-responsive promoter of the soybean GH3 gene (Liu et al., 1994 To gain insight into the mechanisms of BL induction of primary auxin-responsive genes, we studied the involvement of the DR5 AuxRE in BL-induced gene expression. Transgenic Arabidopsis seedlings containing the DR5-GUS reporter gene were treated with either 1 µM IAA or 10 nM BL, and the abundance of GUS transcripts was analyzed by RTQ RTPCR (Fig. 3A). IAA quickly induced GUS transcript accumulation, with similar kinetics to the induction of the IAA5 and IAA19 genes by IAA. GUS transcript abundance peaked 1 h after IAA treatment and returned to the basal level by 12 h after treatment, with a maximum induction of about 100-fold. In contrast, BL significantly induced the DR5-GUS gene at 12 h (Student's t test, P < 0.05) with peak induction of approximately 10-fold. These observations indicated that both BL and IAA induce the DR5-GUS reporter gene at the transcriptional level. We also tested the dose dependence of DR5-GUS induction by both IAA and BL at the time of the maximum induction in Figure 3A (1 h for IAA induction and 12 h for BL induction). IAA induced the DR5-GUS gene dose dependently between 10 nM and 1 µM (Fig. 3B). We also tested BL dose response of the DR5-GUS gene between 10 pM and 1 µM (Fig. 3C). At lower BL concentrations, i.e. between 1 pM and 10 nM, BL induction of the DR5-GUS gene was dose dependent. At 10 nM BL, we sometimes observed saturated responses and sometimes did not (individual data not shown). Therefore, 10 nM BL was close to the saturation level. In contrast, the BL induction was less dose dependent between 100 nM and 1 µM (Fig. 3C). Based on these observations, the time- and dose-dependent induction kinetics of the DR5-GUS gene seems to be similar to those of the IAA5 and IAA19 genes in response to IAA or BL, although maximum induction of the DR5-GUS was higher than that of the IAA5 and IAA19 genes. These observations suggest that exogenously applied BL induces primary auxin-responsive genes, at least in part, by activating the same cis-element as auxin, the AuxRE. The above observations suggest that exogenous BL increases endogenous auxin levels, or exogenous BL increases the sensitivity of seedlings for auxin, or the AuxRE is a shared cis-element that functions in both the auxin and BR signaling pathways. The lag period for BL-induced gene expression may be due to the time needed to induce auxin biosynthesis, to activate the auxin signaling pathway, or to directly modulate the transcriptional system of AuxRE without involvement of auxin. It may be difficult to clearly distinguish the latter two cases.
The above findings prompted us to determine the endogenous free IAA levels in response to treatment with exogenous BL. We used det2 mutant seedlings and WT seedlings not only because det2 shows clearer IAA gene induction in response to BL treatment (Fig. 1) but also because effects of endogenous BRs can be studied. Seven-day-old det2 and WT seedlings were treated with 10 nM BL for 12 h and then analyzed for fresh weight and endogenous free IAA levels (Table I). The fresh weight per seedling of BL-treated det2 or BL-treated WT seedlings was 150% or 115%, respectively, that of mock-treated seedlings, indicating that exogenous BL strongly promotes seedling growth. The amount of IAA per plant increased as the fresh weight increased. However, IAA levels per gram fresh weight did not change in response to BL treatment of either det2 or WT seedlings. This result indicates that exogenous BL induces the DR5-GUS reporter gene without detectable increases in IAA levels per gram fresh weight. It also indicates that lower endogenous BR levels in the det2 mutant do not result in reduced IAA levels but instead result in higher IAA levels.
Natural AuxRE D1-4 is stimulated specifically by auxin and not by other plant hormones (cytokinin, GA3, ethylene, abscisic acid [ABA], and jasmonate) in carrot (Daucus carota) protoplasts (Ulmasov et al., 1995
To analyze the organ specificity of the DR5-GUS reporter activity induced by BL or IAA treatment, 7-d-old DR5-GUS transgenic seedlings were treated with either of these hormones for 12 h and then stained for GUS activity (Fig. 5). In the control plants, GUS activity was detectable only at the edges of cotyledons and root tips (Fig. 5A) under our staining conditions. When seedlings were treated with 1 µM IAA, both shoots and roots stained (Fig. 5C), similar to the staining after 50 µM NAA treatment observed by Ulmasov et al. (1997
The organ-specific induction of the IAA and DR5-GUS genes was analyzed by RTQ RT-PCR. Seven-day-old DR5-GUS transgenic seedlings were treated with either 10 nM BL for 6 h or 1 µM IAA for 1 h; then, the seedlings were divided into shoots and roots. All three genes were expressed more strongly in roots than in shoots (Fig. 6, A-C, insets). The induction of these three genes in response to BL treatment was stronger in shoots, whereas their induction was limited in roots (Fig. 6, A-C, insets). This result was consistent with the histochemical staining of the DR5-GUS reporter (compare Fig. 5, B with A). Conversely, IAA induced these genes in both shoots and roots, although the degree of the induction in shoots and roots varied with each gene (Fig. 6). The maximum induction shown in Figure 6 is higher than in the previous figures. This may be because the transcript levels shown in Figure 6 were normalized to those in shoots of control seedlings, in which the maximum level of DR5-GUS transcripts was lower than in roots.
The respective fresh weight per seedling of the BL-treated det2 mutant and the WT reached 150% and 115% that of mock-treated seedlings after a 12-h BL treatment (Table I). During the growth promotion induced by BL treatment, the IAA5 gene was induced as a primary BL-responsive gene, similar to the SAUR-AC1 gene. Because the IAA5 and IAA19 genes are induced by BL as quickly as the genes involved in cell elongation and cell wall organization (Goda et al., 2002 ), it is possible that these IAA genes are involved in growth acceleration in response to BL. A precise dose-dependent kinetics study of the IAA5 and IAA19 genes after IAA treatment (Fig. 1) revealed that induction of the IAA genes was as rapid as that observed in previous studies (Conner et al., 1990; Abel et al., 1995). In contrast, BL induced IAA genes more moderately and more continually than IAA treatment in both det2 and the WT (Fig. 1). The timing of maximum transcript accumulation after BL induction was independent of BL concentration between 10 nM and 1 µM and later than that after IAA induction. The magnitude of maximum transcript accumulation was also independent of BL concentration at these concentrations. From these observations, it is apparent that IAA and BL induce the two IAA genes with different kinetics and different dose dependency.
Xu et al. (1995
In this study, the IAA5 and IAA19 genes (Fig. 1) and the DR5-GUS reporter gene (Figs. 2 and 3) were induced within 30 min and remained activated up to 24 h posttreatment. A 12-h BL treatment accelerated seedling growth (Table I). However, the endogenous free IAA levels per gram fresh weight did not change in either det2 or WT seedlings (Table I). It is possible that endogenous IAA was increased in response to BL treatment, but the increase was below the limits of detection. For example, it was increased mainly in cotyledons as a result of transport from other organs. Alternatively, endogenous IAA might not increase in response to BL treatment. If this is the case, the AuxRE is not specific to auxin but responds to both auxins and BRs. Of note, endogenous IAA levels were higher in the BR-deficient mutant det2 than in the WT (Table I). This indicates that the lower endogenous BRs in the det2 mutant do not result in lower endogenous IAA levels. Moreover, the abundance of IAA5 and IAA19 transcripts was lower in det2 seedlings than in WT seedlings (Fig. 2), suggesting that decreased BRs down-regulate IAA5 and IAA19 gene expression in the det2 mutant not via decreased endogenous IAA levels but with increased IAA levels. Müssig et al. (2002
Although both IAA and BL activate the IAA5, IAA19, and DR5-GUS genes, a number of characteristics distinguish BL and IAA induction. IAA induction is steep and transient, consistent with the proposed hypothesis that IAA proteins function as self-repressors (Ulmasov et al., 1997
Because IAA induction of the IAA5, IAA19, and DR5-GUS genes did not show saturation, the expression of these genes might be related to a response to higher IAA levels. The most remarkable physiological response to treatment with higher IAA concentrations is growth retardation, which is most significant in roots. This appears consistent with the tissue-specific induction of the DR5-GUS reporter; IAA induces DR5-GUS more strongly in roots than in shoots (Fig. 5). In contrast, the application of approximately 1 µM BL promotes shoot growth (e.g. Li et al., 1996
The early auxin-inducible genes, IAA5, IAA19, and DR5-GUS, are induced by BL in Arabidopsis seedlings, without increases in IAA levels per gram fresh weight. Endogenous IAA levels were higher in the det2 mutant than in the WT, whereas the levels of IAA5 and IAA19 transcripts were lower in the det2 mutant than in the WT. The lack of evidence for auxin-mediated activation of early auxin-inducible genes in response to BL suggests that the BR and auxin signaling pathways activate the transcriptional system of the IAA and DR5-GUS genes independently. Very recently, we demonstrated that AXR1 is involved in BR-mediated elongation and SAUR-AC1 gene expression in Arabidopsis (Nakamura et al., 2003
Plant Materials and Growth Conditions
Arabidopsis ecotype Col-0 was used as the WT in this study unless otherwise noted. The Arabidopsis mutant det2-1 (Chory et al., 1991
Isolation of RNAs and RTQ RT-PCR were performed essentially as described previously (Shimada et al., 2001
The DR5-GUS construct and transgenic plants were described by Ulmasov et al. (1997
GUS activities were measured by using the GUS activity kit (Sigma, St. Louis) according to the manufacturer's instructions. Plants were ground in liquid nitrogen, then homogenized in 500 µL of extraction buffer containing 50 mM sodium phosphate (pH 7.0), 10 mM EDTA, 10 mM
For GC single-ion monitoring MS analyses of free IAA, fresh plant material was carefully weighed, frozen in liquid nitrogen, and stored at -80°C. The material was then ground in liquid nitrogen using a mortar and pestle. After addition of [13C6]IAA at a rate of 30 pg/1 mg fresh weight (Cambridge Isotope Lab, Andover, MA) as an internal standard, the material was extracted in 80% (v/v) acetone with 0.1 mg mL-1 2,6-di-tert-butyl-4-methylphenol (BHT) for 60 min. After centrifugation, the supernatant was collected. The pellet was re-extracted for 90 min, and the supernatant was brought to a water phase in a rotary evaporator. After adjustment of the pH to 2.0, the aqueous phase was then partitioned twice against ether containing 0.01 mg mL-1 BHT. The combined ether phase, which contained ether-soluble acidic and neutral substances, was concentrated to about 0.6 mL under a nitrogen stream. After adjustment of the pH to 10.0 with 2% (w/v) NaHCO3, vortexing for 1 min, and centrifugation, the organic phase was discarded. The pH was adjusted to 2.0, an equal volume of ether containing 0.01 g mL-1 BHT was added, and the material was dried under a nitrogen stream and dissolved in methanol. IAA was purified by HPLC using a Nucleosil N(CH3)2 column (Senshu, Tokyo) and a mobile phase of methanol with 0.03% (w/v) acetic acid. The purified IAA fraction was dried under a nitrogen stream and trimethylsilylated with N-methyl-N-trimethylsilyltrifluoroacetamide at 60°C for 15 min. Splitless injections were made into a GC-single-ion monitoring-MS system (QP5050A, Shimadzu, Kyoto) equipped with a capillary column (DB-1, 0.25-mm x 30 m i.d., 0.25-µm film thickness; J&W Scientific, Folsom, CA). A linear temperature gradient was applied from 80°C to 280°C with an increase of 20°C min-1. The injection temperature of the GC was 250°C, the ion source temperature of the MS was 250°C, a helium flow of 1.2 mL min-1 was applied, the ionization potential was 70 eV, and the scan time was 0.2 s. The percentages of IAA molecules labeled with 13C were calculated from the relative intensities of m/z 202 to 208 and 319 to 325 ions after subtraction of background.
We thank Dr. Tom J. Guilfoyle for providing the DR5-GUS system and Mr. Narumasa Miyauchi for technical assistance with molecular techniques. Received July 10, 2003; returned for revision August 18, 2003; accepted September 7, 2003.
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.030031. * Corresponding author; e-mail shimada{at}postman.riken.go.jp; fax 81-45-503-9492.
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