Brassinolide induces IAA5, IAA19, and DR5, a synthetic auxin response element in Arabidopsis, implying a cross talk point of brassinosteroid and auxin signaling

Abstract

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 (beta-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.

Figure 1.

The induction kinetics of IAA5 and IAA19 genes after treatment with auxin or BR. A and B, Seven-day-old det2 mutant seedlings treated with 10 nm BL. C and D, Wild-type (WT) seedlings treated with 1 μm, 100 nm, or 10 nm IAA. E and F, WT seedlings treated with 1 μm, 100 nm, or 10 nm BL. The transcript abundance of IAA5 (A, C, and E) and IAA19 (B, D, and F) was analyzed using Taq-Man RTQ RT-PCR. Transcript levels are presented as values relative to those at 0 h, defined as 1, after normalization to 18S ribosomal RNA levels. The data are the means ± se of the results of three independent hormone treatment experiments.

Figure 2.

IAA5 and IAA19 transcripts in WT, det2, and bri1 seedlings. A, Transcript abundance of IAA5 and IAA19 in WT (Col) and det2 seedlings. B, Seven-day-old bri1 or WT (Wassilewskija) mutant seedlings were treated with 10 nm BL for 6 h. The transcript abundance was analyzed using Taq-Man RTQ RT-PCR. Transcript levels are presented as values relative to those of the WT, defined as 1, after normalization to 18S ribosomal RNA levels. The data are the means ± se of the results of three independent hormone treatment experiments.

Figure 3.

Induction kinetics of the DR5-GUS gene after auxin and BL treatment. A, Time course of the DR5-GUS gene response to 10 nm BL or 1 μm IAA treatment. Seven-day-old transgenic Arabidopsis seedlings containing the DR5-GUS gene were treated with BL or IAA for up to 24 h. B, Dose-dependent induction of the DR5-GUS gene in response to IAA treatment. Seven-day-old transgenic Arabidopsis seedlings containing the DR5-GUS gene were treated with 10 nm, 100 nm, or 1 μm IAA for 1 h. C, Dose-dependent induction of the DR5-GUS gene in response to BL. Seven-day-old transgenic Arabidopsis seedlings containing the DR5-GUS gene were treated with BL for 12 h. Levels of GUS gene transcripts were analyzed using Taq-Man RTQ RT-PCR. Transcript levels are presented as values relative to those at 0 h (A) or mock treatment (B and C), both defined as 1, after normalization to 18S ribosomal RNA levels. The data are the means ± se of the results of three independent hormone treatment experiments. The experiments in each panel were performed independently; therefore, the results differ, even under the same conditions.

Figure 4.

Induction of GUS activity in DR5-GUS transgenic seedlings in response to treatment with various plant hormones. Seven-day-old transgenic Arabidopsis seedlings harboring the DR5-GUS gene were treated or mock treated with plant hormones for 24 h; 10 μm solutions of the hormones ABA, 1-aminocyclopropane-1-carboxylate (ACC), zeatin, methyl jasmonate (MeJA), and GA₃ were used. BL and IAA were used at concentrations of 10 nm to 1 μm. GUS activities were determined and presented as values relative to mock-treated plants after normalization to the protein content. The data are the means ± se of the results of three independent hormone treatment experiments, except for IAA treatment at 1 μm (six experiments).

Figure 5.

Histochemical staining of GUS activity in DR5-GUS transgenic Arabidopsis seedlings in response to IAA or BL treatment. Seven-day-old DR5-GUS transgenic seedlings were mock treated (A), treated with 10 nm BL for 12 h (B), treated with 1 μm IAA (C), or treated with 50 nm IAA (D) for 12 h. Seedlings were stained for 24 h for GUS activity in staining buffer containing 1 mm 5-bromo-4-chloro-3-indolyl-β-d-glucuronide.

Figure 6.

Organ-specific expression of the DR5-GUS and IAA genes. Seven-day-old WT seedlings transgenic for a DR5-GUS gene were dissected into shoots (S) and roots (R) before (control) and after treatment with 10 nm BL for 6 h or 1 μm IAA for 1 h. The abundance of GUS (A), IAA5 (B), and IAA19 (C) transcripts was analyzed using Taq-Man RTQ RT-PCR. Transcript levels are presented as values relative to those for control shoots, defined as 1, after normalization to the 18S ribosomal RNA levels. The data are the means ± se of the results of three independent hormone treatment experiments.