Integration of auxin and brassinosteroid pathways by Auxin Response Factor 2

Abstract

Plant form is shaped by a complex network of intrinsic and extrinsic signals. Light-directed growth of seedlings (photomorphogenesis) depends on the coordination of several hormone signals, including brassinosteroids (BRs) and auxin. Although the close relationship between BRs and auxin has been widely reported, the molecular mechanism for combinatorial control of shared target genes has remained elusive. Here we demonstrate that BRs synergistically increase seedling sensitivity to auxin and show that combined treatment with both hormones can increase the magnitude and duration of gene expression. Moreover, we describe a direct connection between the BR-regulated BIN2 kinase and ARF2, a member of the Auxin Response Factor family of transcriptional regulators. Phosphorylation by BIN2 results in loss of ARF2 DNA binding and repression activities. arf2 mutants are less sensitive to changes in endogenous BR levels, whereas a large proportion of genes affected in an arf2 background are returned to near wild-type levels by altering BR biosynthesis. Together, these data suggest a model where BIN2 increases expression of auxin-induced genes by directly inactivating repressor ARFs, leading to synergistic increases in transcription.

Fig. 1.

Auxin response is greatly enhanced by and dependent on BRs. (A) BR treatment increases sensitivity to exogenous auxin and synergistically enhances auxin-induced hypocotyl elongation. (B) Reducing BR synthesis with BRZ causes a dramatic reduction in auxin response. Average hypocotyl lengths of 10–15 light-grown 4-day-old seedlings are shown in A and B. (C) Quantitative RT-PCR experiments were performed by using RNA extracted from 5-day-old light-grown seedlings treated for 1, 3, or 6 h with mock (m), BR (b), IAA (a), or combined hormone (ab) treatments. Representative examples of observed patterns are shown. Many genes showed an increase in magnitude and duration of expression when both hormones were present (as shown here for At1g62440); other genes were insensitive to BR effects (as shown here for At4g32280). Error bars indicate standard error.

Fig. 2.

BIN2 interacts with and phosphorylates ARF2. (A) BIN2 interacts with ARF2 in yeast. Yeast was transformed with a bait construct and a prey construct. The bait constructs contain GAL4-DNA binding domain fused with BIN2, and the prey constructs contain GAL4-activation domain fused with ARF2. Interactions between each pair of test proteins were determined by selection for growth on −Ade −His medium containing 5-bromo-4-chloro-3-indolyl-α-d-galactoside (X-α-Gal). (B) In vitro kinase assays using GST-BIN2 and MBP or MBP-ARF2. The autophosphorylation of GST-BIN2 serves as a loading control. Arrows show theoretical mobility of MBP, phosphorylated MBP-ARF2, and autophosphorylated BIN2 kinase.

Fig. 3.

Phosphorylation alleviates ARF2 DNA binding and repressor activity. (A) DNA:protein pull-down experiments with biotinylated DNA fragments from SAUR-15 promoter with ARF2 translated in vitro. ARF2 protein was incubated with BIN2 or kinase-dead [K69R] BIN2 before pull-down experiments with DNA-bound streptavidin beads. Two representative examples from seven similar experiments are shown. BES1 does not interfere with ARF2 binding to DNA. Purified recombinant MBP-BES1 was added where indicated. (B) Yeast repression assay. Repression of the transcription of a LexAop-CYC1::LacZ reporter by a ARF2-LexA fusion in the presence or absence of BIN2 was assessed by β-galactosidase activity. Results are presented as mean of two separate assays of three independent transformants. Error bars indicate standard error.

Fig. 4.

ARF2 is a negative regulator of BR responses. Hypocotyl elongation in response to BRZ (A) and BL (B) is shown. Four-day-old dark-grown wild-type (filled bars) and arf2 (open bars) seedlings were grown on plates with different concentrations of BRZ or BL. Results are presented as mean of two independent experiments ± SD (n = 25).