Interaction between BZR1 and PIF4 integrates brassinosteroid and environmental responses

Abstract

Plant growth is coordinately regulated by environmental and hormonal signals. Brassinosteroid (BR) plays essential roles in growth regulation by light and temperature, but the interactions between BR and these environmental signals remain poorly understood at the molecular level. Here, we show that direct interaction between the dark- and heat-activated transcription factor phytochrome-interacting factor 4 (PIF4) and the BR-activated transcription factor BZR1 integrates the hormonal and environmental signals. BZR1 and PIF4 interact with each other in vitro and in vivo, bind to nearly 2,000 common target genes, and synergistically regulate many of these target genes, including the PRE family helix-loop-helix factors required for promoting cell elongation. Genetic analysis indicates that BZR1 and PIFs are interdependent in promoting cell elongation in response to BR, darkness or heat. These results show that the BZR1-PIF4 interaction controls a core transcription network, enabling plant growth co-regulation by the steroid and environmental signals.

Figure 1. BZR1 interacts with PIF4

(a) BiFC assay shows PIFs interaction with BZR1 in tobacco leaf cells. Images show overlay of fluorescence and light view. (b) Co-immunoprecipitation assay of BZR1 with PIF4. Plants expressing BZR1-CFP from native BZR1 promoter and PIF4-myc from native PIF4 promoter or plants expressing only PIF4-myc were incubated at 28°C for 8 hr to accumulate PIF4 protein and treated with 100 nM BL for 1.5 hr. BZR1-CFP was immunoprecipitated using anti-GFP antibody and immunoblotted using anti-myc or anti-GFP antibody. (c) PIFs directly interact with BZR1 in vitro. GST-PIF1 and GST-PIF4 were pulled down by MBP-BZR1 immobilized on maltose agarose beads and eluted and analyzed by immunoblotting using anti-GST antibody. (d) PIF4 interacts with both BZR1 and BZR2 in vitro. MBP-BZR1 and MBP-BZR2 were pulled down by GST-PIF4 immobilized on glutathione-agarose beads and then eluted and analyzed by immunoblotting using anti-MBP antibody. (e) N-terminal DNA binding domain of BZR1 interacts with PIF4 in vitro. Various fragments of BZR1 fused to MBP were pulled down by GST-PIF4 and then eluted, analyzed by immunoblotting using anti-MBP antibody. (f) Both N-terminal and bHLH domain of PIF4 interact with BZR1 in vitro. Various fragments of PIF4 fused to GST were pulled down by MBP-BZR1N and eluted, analyzed by immunoblotting using anti-GST antibody. (g,h) Box diagrams of the various fragments of BZR1 (g) and PIF4 (h) used in the pull-down assays in panels e and f. The black boxes indicate the DNA binding domains. The numbers indicate the amino acids.

Figure 2. BZR1 and PIFs act interdependently in promoting hypocotyl elongation

(a) The pifq mutant is hyposensitive to BL compared to wild type (Col-0). Seedlings were grown on various concentrations of BL under white light for 7 days before hypocotyl lengths were measured. Error bars indicate s.d. (n=10 plants) and ** : P < 0.01. (b) The pifq mutant, but not pif4, is hypersensitive to BRZ. Seedlings were grown on various concentrations of BRZ in the dark for 5 days. Error bars indicate s.d. (n=12 plants) and ** : P < 0.01. (c) bzr1-1D cannot promote etiolation in the dark in the pifq mutant. Seedlings were grown on medium either with (+BRZ) or without (−BRZ) 2 µM BRZ in the dark for 5 days. Scale bar, 5 mm. (d) Quantification data of panel (c). Numbers indicate ratio of average hypocotyl length of bzr1-1D to that of control. Error bars indicate s.d. (n=10 plants) and ** : P < 0.01. (e) PIF4 is required for BZR1 promotion of hypocotyl elongation under light. Seedlings were grown on the mock (M) or 2 µM BRZ (BRZ) medium under red light for 5 days. Numbers indicate ratios of hypocotyl length of bzr1-1D to that of control. Error bars indicate s.d. (n=10 plants) and ** : P < 0.01. (f) Both PIF-4OX and bzr1-1D are required to promote hypocotyl elongation in the bri1-116 (bri1) mutant background under light. Seedlings were grown in the dark or under red light for 5 days. Scale bar, 5 mm. (g) Quantification data of panel (f) (light). Numbers indicate ratio of average hypocotyl length of multiple mutant including bri1-116 to that of bri1-116. Error bars indicate s.d. (n=10 plants) and ** : P < 0.01.

Figure 3. BZR1 and PIF4 share a large number of genomic targets

(a) Total PIF4 binding peaks identified by ChIP-Seq analyses using two statistical methods. Numbers indicate percentage of binding peaks associated with at least one PIF-regulated gene. (b) Venn diagram shows significant overlap between PIF4-binding target genes and PIF-regulated genes identified by microarray analysis of pif4;pif5 or pifq and RNA-Seq analysis of pifq. (c) Distribution of PIF4 binding peaks (Frequency) relative to gene structure (−5 kb to +1 kb downstream of 3’ end). (d) Frequency of shown cis-elements around the PIF4 binding sites. Sequence logo shows the most enriched motifs in the PIF4 binding regions. (e) Venn diagrams of PIF4 target genes, BZR1 target genes identified by previous ChIP-chip assay and all light-regulated genes from the light-treatment microarray assays. (f) Spatial distribution of PIF4 and BZR1 binding peaks along the promoter (−5000 bp to start), coding (start-end, gray), and 3’ (end to +1000 bp) regions of common target genes. Frequency of peak pairs is represented with a color scale. Counts are numbers of peak pairs in certain area. (g) Representative PIF4 and BZR1 binding peaks in the promoters of common target genes (IAA19 and SAUR15). (h) ChIP-reChIP analysis shows that BZR1 and PIF4 co-occupy common targets. The chromatin of 35s-BZR1-myc;35s-PIF4-YFP double transgenic plants was first immunoprecipitated using anti-myc antibody, and then using anti-GFP antibody, and the precipitated DNA was quantified by qPCR. Enrichment of DNA was calculated as ratio between BZR1-myc;PIF4-YFP and wild type control, normalized to that of PP2A coding region as an internal control. Error bars indicate s.d. of three independent experiments (n=3).

Figure 4. BZR1 and PIF4 regulate different genes interdependently and independently

(a) Venn diagram shows significant overlap between BZR1-regulated and PIF-regulated and red light-regulated genes. (b) Heat map of 682 BZR1, PIF and light co-regulated genes. Scale bar shows fold changes (log2 value). (c) Gene ontology (GO) analyses of BZR1 and PIF4 targets, and BZR1-regulated, PIF4-regulated genes. Numbers indicate percentage of genes belonging to each GO category. Total: Arabidopsis total genes. (d) Comparison of BZR1-regulated genes (bzr1-1D vs Col-0 and pifq;bzr1-1D vs pifq) and PIF-regulated genes (bzr1-1D vs pifq;bzr1-1D) identified by RNA-Seq analysis. Differentially expressed genes were defined by 2-fold difference between samples with p-value < 0.01. (e) Scatter plot of log2 fold change values in bzr1-1D/Col-0 or bzr1-1D/pifq;bzr1-1D RNA-Seq data for 1279 BZR1 and PIF co-regulated genes. (f) Scatter plot of log2 fold change values of 2151 BZR1-regulated genes in the wild type vs pifq mutant background. (g, h) qRT-PCR analysis of the expression levels of BZR1 and PIF co-regulated genes and BR biosynthesis genes. Seedlings were grown on 2 µM BRZ medium in the dark (g) or under red light (h) for 5 days. (i) PIF4 activation of target gene expression requires BR signalling and BZR1. qRT-PCR analysis of seedlings grown on media without or with 2 µM BRZ (BRZ) under red light for 5 days. All gene expression levels were normalized to that of PP2A and are shown relative to the expression levels in wild type (Col-0). All error bars indicate s.d. of three independent experiments (n=3).

Figure 5. PREs promote hypocotyl elongation downstream of BZR1 and PIF4

(a) Transient gene expression assays of co-regulation of PRE5 expression by BZR1 and PIF4. The PRE5 promoter (2 kb) fused to firefly luciferase reporter gene was co-transfected with 35S::BZR1, 35S::PIF4, or both, into Arabidopsis mesophyll protoplast. The firefly luciferase activities were normalized by renilla luciferase as an internal control. Error bars indicate s.d. of three independent experiments (n=3). (b) Expression levels of PRE1 to PRE6 in the pre-amiR plants. PRE4 was not detected in this condition. Similar results were obtained in two independent experiments. (c) The pre-amiR plants show dwarfism. Four-week-old plants were photographed. (d, e) The pre-amiR plants had reduced sensitivity to BL. Seedlings were grown on various concentration of BL medium under light for 5days. Representative seedlings grown on either mock (M) or 100 nM BL (BL) media are shown in panel (d). Error bars indicate s.d. (n=10 plants) and ** : P < 0.01. (f) The pre-amiR plants are hypersensitive to BRZ. Seedlings were grown either on mock (M) or 2 µM BRZ in the dark for 5 days. Error bars indicate s.d. (n=10 plants). (g) The pre-amiR plants are hypersensitive to light. Seedlings were grown in various intensities of red light for 5 days. Error bars indicate s.d. (n=10 plants) and ** : P < 0.01. (h) PRE1-OX suppresses the short-hypocotyl phenotype of the pifq mutant. Seedlings were grown under red light for 5 days. Error bars indicate s.d. (n=15 plants).

Figure 6. High temperature promotion of hypocotyl elongation requires both BZR1 and PIF4

(a, b) Both BZR1 and PIF4 are required for high temperature promotion of hypocotyl elongation. Seedlings were grown either on mock (−PPZ) or 2 µM PPZ (+PPZ) at 20°C or 28°C for 7 days. Numbers indicate ratios of hypocotyl lengths of seedlings at 28°C to 20°C. Error bars indicate s.d. (n=10 plants). (c) PIF4 protein accumulates at high temperature. Seedlings expressing PIF4-myc from native PIF4 promoter were grown at 20°C for 5 days and transferred to 28°C for 1, 4 and 24 hr. (d) BZR1 accumulation and phosphorylation status are not significantly affected at high temperature. Seedlings expressing BZR1-CFP from native BZR1 promoter were grown at 20°C for 5 days and transferred to 28°C for 1, 4 and 24 hr. (e) Both BZR1 and PIF4 are required for high temperature-induced gene expressions. Seedlings were grown at 20°C for 4 days and transferred to 28°C for 24 hr (28) or kept at 20°C (20). Relative gene expression levels were normalized to that of PP2A. Similar results were obtained in two independent experiments. (f) The pre-amiR plants are defective in the high temperature promotion of hypocotyl elongation. Seedlings were grown at 20°C or 28°C for 7 days. Error bars indicate s.d. (n=10 plants).