A triple helix-loop-helix/basic helix-loop-helix cascade controls cell elongation downstream of multiple hormonal and environmental signaling pathways in Arabidopsis

Abstract

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.

Figure 1.

PRE1 and IBH1 Antagonistically Regulate Cell Elongation Through Overlapping Transcriptomes. (A) PRE1 suppresses the effects of IBH1. Seedlings of Col, IBH1-Ox, PRE1-Ox, and IBH1-Ox/PRE1-Ox were grown on medium under light for 7 d. (B) Venn diagram shows the overlap of genes regulated by IBH1-Ox and PRE1-Ox. (C) Hierarchical cluster analysis of the genes differentially expressed in IBH1-Ox versus Col and PRE1-Ox/IBH1-Ox versus IBH1-Ox. The numerical values for the yellow-to-blue gradient bar represent log₂-fold change relative to the control sample.

Figure 2.

The Basic Domain of IBH1 Is Not Required for Its Function in Inhibition of Cell Elongation. (A) Various degrees of dwarf phenotype observed among T1 plants overexpressing wild-type IBH1. (B) Number and percentage of transgenic plants showing each category of phenotype severity observed among populations of transgenic plants overexpressing wild-type IBH1 or mutant IBH1 containing deletion of the basic domain (IBH1ΔB) or of the AS domain (IBH1ΔAS). [See online article for color version of this figure.]

Figure 3.

IBH1 Interacts with HBI1 in Vitro and in Vivo. (A) Yeast two-hybrid assays of interactions between indicated IBH1 proteins and PRE1, BEE2, or HBI1. The AS domain is required for the IBH1 interaction with HBI1 but not for interaction with PRE1. (B) A gel blot of MBP and MBP-IBH1 was probed with GST-HBI1 followed by horseradish peroxidase–labeled anti-GST antibody or stained with Ponceau S (Stain). (C) Coimmunoprecipitation assays show IBH1 interacts with HBI1 in plant cells. 35S:HBI1-YFP was transformed into the protoplasts prepared from the 35S:IBH1-myc transgenic plants. Immunoprecipitation was performed using anti-YFP antibody, and immunoblots were probed with anti-myc or anti-YFP antibodies. [See online article for color version of this figure.]

Figure 4.

HBI1 Is a Positive Regulator of Cell Elongation. (A) Representative plants of the wild type and HBI1 cosuppression (CS) and overexpression (#1 and 2#) lines were grown in soil for 4 weeks. (B) Quantitative RT-PCR analyses of HBI1 expression in plants represented in (A) using PP2A as the internal control. Error bars indicate sd. (C) Seedling phenotypes of HBI1-CS and HBI1-Ox transgenic plants grown on half-strength MS medium under dim light for 14 d. The top images show the smaller cotyledon of HBI1-CS lines. (D) Quantification hypocotyl lengths of wild-type (WT) and HBI1 transgenic plants. Error bars represent sd. (E) Phenotype of HBI1-SRDX plants grown in soil for 4 weeks. (F) Quantitative RT-PCR analyses of expression of EXP1 and EXP8 in wild-type, HBI1-CS, HBI1-SRDX, and HBI1-Ox plants. PP2A was used as the internal control. Error bars indicate sd from three biologic repeats.

Figure 5.

HBI1 Positively Regulates BR Responses. (A) and (B) Overexpression of HBI1 partly suppresses the bri1-5 phenotype. (A) The bri1-5 and HBI1-Ox/bri1-5 plants were grown in soil for 4 weeks. The bottom panels show immunoblots probed with anti-YFP antibodies and Ponceau S staining for loading control. (B) The average hypocotyl lengths of the wild type (WS), bri1-5, and HBI1-Ox/bri1-5 grown on half-strength MS medium under light for 6 d. Error bars show sd. (C) and (D) Overexpression of HBI1 reduces sensitivity to the BR biosynthesis inhibitor PPZ. Wild-type and HBI1-Ox plants were grown on half-strength MS medium containing 100 nM (+), 2 μM (++) PPZ (C), or with indicated concentrations of PPZ (D). (D) The hypocotyl lengths were measured from at least 15 plants. Error bars represent sd. (E) and (F) The HBI1-Ox plants show longer hypocotyls than the wild type in the presence of the GA biosynthesis inhibitor PAC. Wild-type and HBI1-Ox plants were grown on half-strength MS medium containing 10 nM (+), 1 μM (++) PAC (E), or with indicated concentrations of PAC (F) under constant light. The average hypocotyl lengths were measured from at least 15 plants. Error bars represent sd.

Figure 6.

IBH1 and PRE1 Antagonistically Regulate the Activity of HBI1. (A) and (B) Overexpression of IBH1 or knockdown the expression of PREs suppresses the phenotype of HBI1-Ox. Plants (A) or detached leaves (B) were photographed after growth in soil for 4 weeks. (C) In vitro DNA pull-down assays of HBI1 DNA binding activity. The indicated MBP or MBP fusion proteins purified from Escherichia coli were incubated with a biotinylated DNA fragment of the EXP1 promoter immobilized on streptavidin beads. The DNA-bound proteins were immunoblotted using anti-MBP antibody. (D) ChIP-qPCR analysis of HBI1 binding to the EXP1 and EXP8 promoters and the effects of IBH1-Ox and pre-amiR on HBI1 DNA binding in vivo. Heterozygous transgenic 35S:HBI1-YFP/Col-0, 35S:HBI1-YFP/35S:IBH1-myc, and 35S:HBI1-YFP/35S:pre-amiR F1 plants grown in a greenhouse for 4 weeks were used for the ChIP-qPCR analysis. Error bars indicate sd of three biological repeats. (E) ChIP-qPCR analysis of the effects of BR, GA, and light on HBI1 DNA binding in vivo. ChIP-qPCR was performed using 35S:HBI1-YFP and 35S:YFP grown in half-strength MS liquid medium with or without 2 µM PPZ or 1 µM PAC for 5 d under constant light or in the dark. The plants grown on 2 µM PPZ were treated with mock solution (BL−) or 100 nM BL (BL+) for 6 h; plants grown on 1 µM PAC were treated with mock solution (GA−) or 10 µM GA₃ (GA+) for 6 h; plants grown in the dark were treated with white light (35 µM/m²/s) or kept in the dark for 6 h. Error bars indicate sd of three biological repeats. (F) Transient assays show HBI1 activation of the pEXP1:LUC reporter gene. Arabidopsis protoplasts were transformed with the dual luciferase reporter construct containing pEXP1:LUC (luciferase) and 35S:REN (renilla luciferase) and constructs overexpressing the indicated effecters. The LUC activity was normalized to REN. Error bars indicate sd of three biological repeats.

Figure 7.

PREs, IBH1, and HBI1 Mediate Hypocotyl Elongation Responses to BR, GA, Temperature, and Light. (A) and (B) Wild-type (WT) and transgenic 35S:pre-amiR, 35S:IBH1-myc, and 35S:HBI1-SRDX plants were grown under constant light for 7 d in half-strength MS medium with (+) or without (−) indicated hormones: 100 nM BR (A), 1 μM GA3, and 100 nM PAC (B). Right panels: The hypocotyl lengths were measured from at least 15 plants. Error bars represent sd. (C) Wild-type and transgenic 35S:pre-amiR, 35S:IBH1-myc, and 35S:HBI1-SRDX plants were grown at 20 or 29°C for 7 d. The hypocotyl lengths were measured from at least 15 plants. Error bars represent sd. (D) Wild-type and transgenic 35S:pre-amiR, 35S:IBH1-myc, and 35S:HBI1-SRDX plants were grown under different light conditions for 7 d. The hypocotyl lengths and relative lengths (percentage of length in the dark) were calculated from at least 10 plants. Error bars represent sd.

Figure 8.

Diagrams of the Signaling Network Mediating Cell Elongation Regulation by Multiple Environmental and Hormonal Signals. The diagram on the right side summarizes conceptually the detailed diagram on the left, where the primary and secondary transcription factors (TFs) are grouped by colored boxes, arrows show activation and bar-ended lines show inhibition, blue lines show regulation by protein–protein interactions, and red lines show transcriptional regulation. Dashed lines show unknown or speculated mechanisms. The component and mechanisms discovered in this study are marked by bold text and thicker lines, respectively.