RNA-Seq of iaa3 + BR replicate 2 Basecalls performed using CASAVA version 1.?Total reads were mapped to the Arabidopsis thaliana genome (TAIR9, www.arabidopsis.org) using TopHat software. Read counts corresponding genes were generated using HTSeq with union mode.Genome_build: TAIR9

RNA-Seq of pifq replicate 1 Total reads were mapped to the Arabidopsis thaliana genome (TAIR9, www.arabidopsis.org) using TopHat software. Read counts corresponding genes were generated using HTSeq with union mode.Genome Build:Q_L1.count.txt: Arabidopsis TAIR9

RNA-Seq of Col-0 replicate 1 Total reads were mapped to the Arabidopsis thaliana genome (TAIR9, www.arabidopsis.org) using TopHat software. Read counts corresponding genes were generated using HTSeq with union mode.Genome Build:C_L1.count.txt: Arabidopsis TAIR9

RNA-Seq of pifq;bzr1-1D replicate 2 Total reads were mapped to the Arabidopsis thaliana genome (TAIR9, www.arabidopsis.org) using TopHat software. Read counts corresponding genes were generated using HTSeq with union mode.Genome Build:QB_L2.count.txt: Arabidopsis TAIR9

RNA-Seq of Col-0 replicate 1 Total reads were mapped to the Arabidopsis thaliana genome (TAIR9, www.arabidopsis.org) using TopHat software. Read counts corresponding genes were generated using HTSeq with union mode.Genome Build:C_L1.count.txt: Arabidopsis TAIR9

Chromatin IP against PIF4 transcription factor Alignment : The raw sequence data were processed using Illumina sequence data analysis pipeline GAPipeline1.3.2. Sequences in Solexa FASTQ format were mapped to the Arabidopsis genome, TAIR9. Only unique mapping reads were used to determine PIF4 binding peaks.Peaks: Peak detection was performed with Plant Research International ChIP-seq Analysis Tool (PRI-CAT) (http://www.ab.wur.nl/pricat/)Genome Build:Col.aln: Arabidopsis genome (TAIR9)

RNA-Seq of pifq replicate 2 Total reads were mapped to the Arabidopsis thaliana genome (TAIR9, www.arabidopsis.org) using TopHat software. Read counts corresponding genes were generated using HTSeq with union mode.Genome Build:Q_L2.count.txt: Arabidopsis TAIR9

Chromatin IP against PIF4 transcription factor Alignment : The raw sequence data were processed using Illumina sequence data analysis pipeline GAPipeline1.3.2. Sequences in Solexa FASTQ format were mapped to the Arabidopsis genome, TAIR9. Only unique mapping reads were used to determine PIF4 binding peaks.Peaks: Peak detection was performed with Plant Research International ChIP-seq Analysis Tool (PRI-CAT) (http://www.ab.wur.nl/pricat/)Genome Build:PIF4.aln: Arabidopsis genome (TAIR9)PIF4_peaks.wig: Arabidopsis genome (TAIR9)

Environmental and endogenous signals, including light, temperature, brassinosteroid (BR), and gibberellin (GA), regulate cell elongation largely by influencing the expression of the paclobutrazol-resistant (PRE) family helix-loop-helix (HLH) factors, which promote cell elongation by interacting antagonistically with another HLH factor, IBH1. However, the molecular mechanism by which PREs and IBH1 regulate gene expression has remained unknown. Here, we show that IBH1 interacts with and inhibits a DNA binding basic helix-loop-helix (bHLH) protein, HBI1, in Arabidopsis thaliana. Overexpression of HBI1 increased hypocotyl and petiole elongation, whereas dominant inactivation of HBI1 and its homologs caused a dwarf phenotype, indicating that HBI1 is a positive regulator of cell elongation. In vitro and in vivo experiments showed that HBI1 directly bound to the promoters and activated two EXPANSIN genes encoding cell wall-loosening enzymes; HBI1's DNA binding and transcriptional activities were inhibited by IBH1, but the inhibitory effects of IBH1 were abolished by PRE1. The results indicate that PREs activate the DNA binding bHLH factor HBI1 by sequestering its inhibitor IBH1. Altering each of the three factors affected plant sensitivities to BR, GA, temperature, and light. Our study demonstrates that PREs, IBH1, and HBI1 form a chain of antagonistic switches that regulates cell elongation downstream of multiple external and endogenous signals.

Brassinosteroid (BR) regulates a wide range of physiological responses through the activation of BRASSINAZOLE RESISTANT1 (BZR1), whose activity is tightly controlled by its phosphorylation status and degradation. Although BZR1 appears to be degraded in distinct ways in response to different hormonal or environmental cues, little is known about how BR signaling regulates its degradation. Here we show that the BR-regulated U-box protein PUB40 mediates the proteasomal degradation of BZR1 in a root-specific manner in Arabidopsis (Arabidopsis thaliana). BZR1 levels were strongly reduced by plant U-box40 (PUB40) overexpression, whereas the pub39 pub40 pub41 mutant accumulated much more BZR1 than wild type in roots. The bzr1-1D gain-of-function mutation reduced the interaction with PUB40, which suppressed PUB40-mediated BZR1 degradation in roots. The cell layer-specific expression of PUB40 in roots helps induce selective BZR1 accumulation in the epidermal layer. Both BR treatment and loss-of-function of PUB40 expanded BZR1 accumulation to most cell layers. In addition, BZR1 accumulation increased the resistance of pub39 pub40 pub41 to low inorganic phosphate availability, as observed in bzrl-1D. BRASSINOSTEROID-INSENSITIVE2-induced phosphorylation of PUB40, which mainly occurs in roots, gives rise to BZR1 degradation through enhanced binding of PUB40 to BZR1 and PUB40's stability. Our results suggest a molecular mechanism of root-specific BZR1 degradation regulated by BR signaling.