Brassinosteroid, gibberellin and phytochrome impinge on a common transcription module in Arabidopsis

Abstract

Brassinosteroid and gibberellin promote many similar developmental responses in plants; however, their relationship remains unclear. Here we show that BR and GA act interdependently through a direct interaction between the BR-activated BZR1 and GA-inactivated DELLA transcription regulators. GA promotion of cell elongation required BR signalling, whereas BR or active BZR1 suppressed the GA-deficient dwarf phenotype. DELLAs directly interacted with BZR1 and inhibited BZR1-DNA binding both in vitro and in vivo. Genome-wide analysis defined a BZR1-dependent GA-regulated transcriptome, which is enriched with light-regulated genes and genes involved in cell wall synthesis and photosynthesis/chloroplast function. GA promotion of hypocotyl elongation requires both BZR1 and the phytochrome-interacting factors (PIFs), as well as their common downstream targets encoding the PRE-family helix-loop-helix factors. The results demonstrate that GA releases DELLA-mediated inhibition of BZR1, and that the DELLA-BZR1-PIF4 interaction defines a core transcription module that mediates coordinated growth regulation by GA, BR and light signals.

Fig. 1. BR signalling and BZR1 activity are required for GA promotion of hypocotyl elongation

(a) Wild type and BR mutants were grown under the constant light for 7 days on medium with or without 1 μM GA₃ and 1 μM brassinolide (BL, the most active brassinosteroid). (b) BR-deficient and insensitive mutants show a GA-insensitive phenotype, which is suppressed by bzr1-1D. Wild type (Col) and BR mutant seedlings were grown in the dark for 6 days on medium containing 1 μM PAC and different concentrations of GA₃, and with 10 nM BL where indicated (+BL). Error bars, s.e.m. (n=32 plants) (c) bzr1-1D partly suppressed ga1-3 phenotype. (d) BR promotes cell elongation in GA mutants. Wild type (Col), ga1-3, sly1-10 were grown under light for 7 days on medium containing 1 μM PAC or 1 μM BL as indicated. (e) GA enhances the BR sensitivity. Wild type and det2-1 were grown under constant light for 7 days on medium with or without 1 μM GA₃ and different concentrations of BL. Error bars, s.e.m. (n=35 plants). (f, g) Removing DELLAs enhances BR sensitivity. Ler, gai-1 and della seedlings were grown for 7 days under constant light on medium containing 2 μM brassinazole (BRZ, an inhibitor of BR synthesis) and 10 nM (+), 1 μM (++) (f), or different concentrations (g) of BL. Error bars, s.e.m. (n=25 plants).

Fig. 2. RGA interacts with BZR1 and inhibits BZR1 DNA binding activity in vitro and in vivo

(a) Wild type and dominant mutant forms of BZR1 and RGA interact in yeast. (b) A diagram of the structure of RGA. (c) The LHR1 domain is necessary for both RGA homodimerization and the interaction with BZR1; the SAW domain is also required for interaction with BZR1. (d, e) MBP and MBP-fusions with BZR1 protein were incubated with GST-RGA bound to glutathione-agarose beads and then eluted and analyzed by anti-MBP immunoblotting. (f, g) BZR1 and RGA interact in plants. (f) The seedling of Col and pBZR1:BZR1-CFP grown in medium containing 100 nM PAC under light for 7 days were treated with 100 nM BL 1hr, and then co-immunoprecipitation was performed using anti-YFP antibody and immunoblotted using anti-RGA and anti-YFP antibodies. (g) Immunoprecipitation was performed using anti-YFP antibody on transgenic Arabidopsis plants expressing 35S::BZR1-Myc only or co-expressing 35S::BZR1-Myc and 35S::RGA-YFP, and immunoblotted using anti-Myc or anti-YFP antibodies. (h) RGA inhibits BZR1 DNA binding in vitro. MBP-BZR1 pre-incubated with MBP or MBP-RGA was incubated with biotinylated DNA fragments from the IAA19 and SAUR-AC1 promoters immobilized on streptavidin beads. The DNA-bound proteins were immunoblotted using anti-MBP antibody. (i) GA increases BZR1-DNA binding in vivo. ChIP was performed using anti-YFP antibodies followed by qPCR analysis. BZR1 binding was calculated as ratio between BZR1-CFP and 35S::YFP control, normalized to that of the control gene CNX5. Error bars mean s.d. of three biological repeats. Significant differences between GA and mock treatment are marked by asterisk (p < 0.01). (j, k) Transient reporter gene assays show RGA inhibition of BZR1 transcription activity. Arabidopsis protoplasts were transformed with the dual luciferase reporter construct containing pPRE::LUC (luciferase) and 35S::REN (renilla luciferase), and constructs overexpressing the indicated effecters. The LUC activity was normalized to REN. Error bars, s.d. of three biological repeats. a: Significant difference compared with BZR1 and BZR1+RGA samples (p < 0.01). b: Significant difference compared with BZR1 and BZR1+GAI samples. (p < 0.05). #: No Significant difference compared with BZR1 and BZR1+RGL2 samples (p > 0.05).

Fig. 3. GA and BR co-regulate large number of genes through DELLAs and BZR1

(a) Venn diagram shows the overlaps between sets of genes differentially expressed in dark-grown bri1-116 versus wild type (bri1-116 D vs WT D), ga1-3 versus wild type (ga1-3 D vs WT D), and light-grown versus dark-grown wild type (WT L vs D). (b) Scatter plot of log2 fold change values for 419 genes differentially expressed of ga1-3 D versus WT D and bri1-116 D versus WT D. Red and blue colors indicate light-activated and light-repressed genes, respectively; black color indicates the genes are not regulated by light. (c) Hierarchical clusters analysis of the genes differentially expressed in bzr1-1D/bri1-116 versus bri1-116 (bzr1-1D/bri1-116), bri1-116 versus WT (bri1-116), ga1-3 versus Ler (ga1-3), and della/ga1-3 versus ga1-3 (della/ga1-3). The gradient bar represents log2 of the ratio. Genes are listed in table S1. (d-h) RNA-Seq analyses of genes affected by GA treatments or by bzr1-1D in BR-deficient plants (grown on 2 μM PPZ medium) (2 μM is right). (d) Venn diagram shows overlaps between sets of genes affected by GA treatment in wild type (Col) plants grown on medium containing PAC (Col GA+/-) or on medium containing PAC and PPZ (Col_PPZ GA+/-), and genes affected by bzr1-1D in the presence of PAC and PPZ (bzr1-1D_PPZ vs Col_PPZ). (e-g) Hierarchical clusters analysis of the expression data of the genes in class A (e), B (f) and C (g) in panel d. (h) Gene Ontology analysis of cellular functions represented by GA up- and down-regulated genes in each gene class (A to D) shown in panel d. All genes detected in RNA-Seq samples were used as control (random).

Fig. 4. BZR1 and PIF4 are required for the GA promotion of hypocotyl elongation

(a) Venn diagram shows the percentage of GA regulated genes among the gene sets that BZR1 and PIF4 bind to and/or regulate. PIF4 and BZR1 targets were identified by PIF4 ChIP-Seq and BZR1 ChIP-chip, respectively; PIFs- and BZR1-regulated genes were differentially expressed in pifq versus WT and in bzr1-1D/bri1-116 versus bri1-116 grown in the dark. GA-regulated genes were differentially expressed in ga1-3 versus WT in the dark. Numbers show the percentages of each gene set that are GA-regulated genes. (b, c) PIFs are required for BZR1 mediated GA promotion of hypocotyl elongation. Seedlings were grown in the dark for 5 days on medium containing 0.5 μM PAC, 10 μM PPZ with or without 1 μM GA_3. Error bars, s.d. (n=10 plants). Asterisks mark significant differences between GA and mock treatments (p < 0.01). (d, e), Both BZR1 and PIFs are required for GA promotion of hypocotyl elongation in light. Seedlings were grown under red light for 5 days on medium containing 0.1 μM PAC, 2 μM PPZ, and 0 (M) or 1 μM GA₃ (GA). Error bars, s.d. (n=10 plants). Asterisks mark significant differences between GA and mock treatments (p < 0.01).

Fig. 5. GA promotion of cell elongation requires the BZR1 and PIF4 targets PREs

(a, b) Quantitative RT-PCR analyses of gene expression after GA treatment in wild type, bri1-119 and bzr1-1D/bri1-119 (a) and BL treatment in wild type (Col) and gai-1 (b). Total RNAs were extracted from 7-day-old seedlings treated with mock solution, 10 μM GA₃, or 100 nM BL for 3 hr. The PP2A gene was analyzed as an internal control. Error bars are s.d. of three biological replicates. (c) Suppressing PREs (pre-amiR) and overexpression of IBH1 (IBH1-Ox) reduce hypocotyl elongation response to GA. Error bars, s.d. (n=25 plants). Asterisks indicate significant difference between GA and mock treatments (p < 0.01). (d, e) The 35S:PRE1-YFP transgenic plants (PRE1-Ox) show reduced sensitivities to the GA biosynthesis inhibitor PAC (d) and BR biosynthesis inhibitor BRZ (e). Seedlings were grown on medium containing different concentrations of PAC (d) or BRZ (e) for 7 days under light. Relative hypocotyl lengths were measured from at least 30 plants and normalized to the untreated plants. Error bars represent s.d. (n=33) (f) ChIP-reChIP analyses show that PRE1, PRE6, EXP1 and EXP8 are the common targets of BZR1 and PIF4. Chromatin from transgenic Arabidopsis expressing both 35S::BZR1-myc and 35S::PIF4-YFP was immunoprecipitated sequentially using anti-myc and anti-YFP antibodies, and then analyzed by qPCR. BZR1 and PIF4 binding was calculated as ratio between BZR1-myc/PIF4-YFP transgenic plants and Col control, normalized to that of the control gene PP2A. Error bars are s.d. of three biological replicates. Asterisks indicate significant difference to PP2A gene (p < 0,01) (g) The model for the signalling network integrating BR, GA and light signals. BZR1 and PIF4 form a functional complex to regulate a large number of genes that contribute to hypocotyl elongation; these include PREs, which in turn inactivates IBH1, leading to cell elongation. DELLAs interact with BZR1 and PIFs to inhibit their DNA binding ability. Signal transduction activated by BR, GA and light/phytochrome modulates the levels of BZR1, DELLAs, and PIFs, respectively, thereby controlling the activity of BZR1-PIF complex and cell elongation.