Arabidopsis DELLA and JAZ proteins bind the WD-repeat/bHLH/MYB complex to modulate gibberellin and jasmonate signaling synergy

Abstract

Integration of diverse environmental and endogenous signals to coordinately regulate growth, development, and defense is essential for plants to survive in their natural habitat. The hormonal signals gibberellin (GA) and jasmonate (JA) antagonistically and synergistically regulate diverse aspects of plant growth, development, and defense. GA and JA synergistically induce initiation of trichomes, which assist seed dispersal and act as barriers to protect plants against insect attack, pathogen infection, excessive water loss, and UV irradiation. However, the molecular mechanism underlying such synergism between GA and JA signaling remains unclear. In this study, we revealed a mechanism for GA and JA signaling synergy and identified a signaling complex of the GA pathway in regulation of trichome initiation. Molecular, biochemical, and genetic evidence showed that the WD-repeat/bHLH/MYB complex acts as a direct target of DELLAs in the GA pathway and that both DELLAs and JAZs interacted with the WD-repeat/bHLH/MYB complex to mediate synergism between GA and JA signaling in regulating trichome development. GA and JA induce degradation of DELLAs and JASMONATE ZIM-domain proteins to coordinately activate the WD-repeat/bHLH/MYB complex and synergistically and mutually dependently induce trichome initiation. This study provides deep insights into the molecular mechanisms for integration of different hormonal signals to synergistically regulate plant development.

Figure 1.

GA Promotes Trichome Formation in a WD-Repeat/bHLH/MYB Complex–Dependent Manner. (A) Trichome images of wild-type (WT) Landsberg erecta (Ler), ga1-3, penta (ga1-3 rga gai rgl1 rgl2), and della (rga gai rgl1 rgl2 rgl3) plants treated without (Mock) or with GA₃ (GA). The fifth true leaves were selected to count trichome numbers using an environmental scanning electron microscope. (B) Statistical analysis of total trichome numbers in the fifth true leaves of Ler (WT), ga1-3, Q1 (ga1-3 gai rgl1 rgl2), Q2 (ga1-3 rga rgl1 rgl2), Q3 (ga1-3 rga gai rgl1), Q4 (ga1-3 rga gai rgl2), penta, and della plants treated without (Mock) or with GA₃ (GA). Eight leaves for each genotype were used for trichome number measurement in each biological experiment. Data are means (±se) of three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05). (C) Trichome images of Arabidopsis gl3 egl3, gl1, and ttg1 mutants treated without (Mock) or with GA₃ (GA). The fifth true leaves were selected to count trichome numbers. No trichome was observed in gl3 egl3, gl1, and ttg1 mutants.

Figure 2.

Interactions of DELLAs with the WD-Repeat/bHLH/MYB Complex. (A) Schematic diagrams show domain constructs of RGA and RGL2. The diagrams display the conserved DELLA domain. The numbers indicate positions of the first and the last amino acid of the domain constructs. (B) Y2H assays to test the interactions of DELLAs with GL1, EGL3, GL3, and TTG1. The RGA-R (202 to 587 amino acids) and RGL2-R (161 to 547 amino acids) were fused with the LexA DNA BD. GL1, EGL3, GL3, and TTG1 were fused with the activation domain (AD). (C) Expression of GL1, EGL3, GL3, TTG1, and RGA-R in the yeast strains shown in the top panel of (B). GL1, EGL3, GL3, and TTG1 were detected with anti-HA antibody (top). RGA-R was detected with anti-LexA antibody (bottom). (D) and (E) In vitro pull-down assay to verify the interactions of RGA (D) or RGL2 (E) with GL1, EGL3, and GL3. Purified MBP, MBP-GL1, MBP-EGL3, and MBP-GL3 fusion proteins were incubated with the total proteins extracted from the Arabidopsis seedlings transgenic for TAP-RGA (D) or TAP-RGL2 (E). Bound proteins were washed, separated on SDS-PAGE, and immunoblotted with the anti-c-myc antibody (a-myc; top panel). The input lane shows the expression level of myc-RGA (D) or myc-RGL2 (E) in the transgenic plant. The positions of purified MBP, MBP-GL1, MBP-EGL3, and MBP-GL3 separated on SDS-PAGE are indicated with an asterisk (bottom panel; stained by Coomassie blue). (F) BiFC assay to detect the interactions of RGA and RGL2 (fused with C-terminal fragment of YFP) with GL1 and EGL3 (fused with N-terminal fragment of YFP). Construct pairs indicated were coinfiltrated into leaves of N. benthamiana. YFP fluorescence was detected 50 h after infiltration. The nuclei are indicated by 4′,6-diamidino-2-phenylindole (DAPI) staining. (G) and (H) Y2H assays to detect the interactions of RGA-R and RGL2-R with different domains of GL1 (G) and GL3 (H). The schematic diagram showed the AD domain–fused GL1NT and GL1CT, the conserved R2R3 domain and CT domain of GL1 (G), the AD domain–fused GL3NT and GL3CT, and the conserved NT domain and bHLH domain of GL3 (H). The numbers indicate the positions of amino acids. (I) Expression of GL1, GL1NT, GL1CT, and RGA-R in the yeast strains used in (G). GL1, GL1NT, GL1CT, and AD were detected with anti-HA antibody (top). RGA-R was detected with anti-LexA antibody (bottom). (J) Expression of GL3, GL3NT, GL3CT, and RGA-R in yeast strains used in (H). GL3, GL3NT, GL3CT, and AD were detected with anti-HA antibody (top). RGA-R was detected with anti-LexA antibody (bottom).

Figure 3.

DELLA Proteins Inhibit Transcriptional Function of the WD-Repeat/bHLH/MYB Complex. (A) The schematic diagram shows the constructs used in the transient expression assays of (B) to (D). (B) Transient expression assay shows that RGA and RGL2 inhibit transcriptional function of GL1. The GUS reporter and the internal control LUC were cotransformed with the indicated constructs. Data are means (±se) of three biological replicates. Asterisks represent Student’s t test significance compared with GL1 (**P < 0.01). (C) Transient expression assay shows that RGA and RGL2 inhibit transcriptional function of EGL3. Data are means (±se) of three biological replicates. Asterisks represent Student’s t test significance compared with EGL3 (**P < 0.01). (D) Transient expression assay shows that RGA and RGL2 inhibit transcriptional function of GL3. Data are means (±se) of three biological replicates. Asterisks represent Student’s t test significance compared with GL3 (**P < 0.01). (E) The schematic diagram shows the constructs used in the transient expression assays of (F). (F) Transient expression assay shows that activation of GL2 promoter by GL3/GL1 is repressed by RGA. The P_GL2-LUC reporter was cotransformed with the indicated constructs. Data are means (±se) of three biological replicates. Asterisks represent Student’s t test significance compared with GL1+GL3 (**P < 0.01). (G) and (H) Real-time PCR analysis for GL2 (G) and MYB23 (H) in Arabidopsis Ler wild-type (WT), ga1-3, and penta plants. ACTIN8 was used as the internal control. Data are means (±se) of three biological replicates. Asterisks indicate significant differences compared with the wild type by one-way ANOVA analysis with SAS software (**P < 0.01).

Figure 4.

Double Mutations in GL3 and EGL3 Block Trichome Initiation in the penta Mutant. (A) Images of the fifth true leaves of penta and penta gl3 egl3 heptuple mutant treated without (Mock) or with GA₃ (GA). (B) Statistical analysis of total trichome numbers per leaf indicated in (A). Eight leaves for each genotype were used for trichome number measurement in each biological experiment. Data are means (±se) of three biological replicates. (C) and (D) Real-time PCR analysis for GL2 (C) and MYB23 (D) in penta and penta gl3 egl3 mutants treated without (Mock) or with GA₃ (GA). ACTIN8 was used as the internal control. Data are means (±se) of three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05). [See online article for color version of this figure.]

Figure 5.

Overexpression of EGL3 or GL1 Rescues Trichome Initiation in ga1-3 Mutant. (A) Plant images of ga1-3 and EGL3 overexpression transgenic plant in ga1-3 background (ga1-3 EGL3OE) supplied without (Mock) or with GA₃ (GA). (B) Statistical analysis of total trichome numbers in the fifth true leaves of indicated plants in (A). Eight leaves for each genotype were used for trichome number measurement in each biological experiment. Data are means (±se) of three biological replicates. Asterisks indicate significant differences by one-way ANOVA analysis with SAS software (**P < 0.05). (C) Real-time PCR analysis for GL2, MYB23, and GL3 in Arabidopsis wild-type Ler (WT), ga1-3, and ga1-3 EGL3OE plants plated on MS medium. ACTIN8 was used as the internal control. Data are means (±se) of three biological replicates. Asterisks indicate significant differences compared with ga1-3 EGL3OE by one-way ANOVA analysis with SAS software (**P < 0.01). (D) Plant images of 3-month-old ga1-3 and ga1-3 GL1OE transgenic plants. GL1 overexpression obviously increased trichome formation in ga1-3 background. (E) Real-time PCR analysis for GL2 and MYB23 in ga1-3 and ga1-3 GL1OE plants in (D). ACTIN8 was used as the internal control. Data are means (±se) of three biological replicates. Asterisks indicate significant differences compared with ga1-3 by one-way ANOVA analysis with SAS software (**P < 0.01).

Figure 6.

RGA and JAZ1 Coordinately Inhibit Transcriptional Function of EGL3 and GL1. (A) Schematic diagram of the constructs used in transient expression assays in (B) and (C). (B) and (C) Transient expression assay showed that both RGA and JAZ1 proteins could significantly repress transcriptional function of EGL3 (B) and GL1 (C). The GUS/LUC ratios were measured in Arabidopsis leaf protoplasts after transiently transfected with reporter, internal control, and indicated effectors. Data are means (±se) of three biological replicates. Asterisks represent Student’s t test significance between pairs indicated with brackets (*P < 0.05; **P < 0.01).

Figure 7.

GA and JA Function Synergistically to Promote Trichome Initiation. (A) Trichome images of the fifth true leaves from wild-type Columbia-0 (Col-0) treated without (Mock) or with MeJA (JA), GA₃ (GA), GA₃ plus MeJA (GA+JA), paclobutrazol (PAC), or paclobutrazol plus MeJA (PAC+JA). (B) Trichome images of the fifth true leave from ga1-3 treated without (Mock) or with MeJA (JA). (C) Trichome images of the fifth true leave from coi1-1 treated without (Mock) or with GA₃ (GA). (D) Statistical analysis of total trichome numbers in the fifth true leaves of Col-0 with the indicated treatments in (A). Eight leaves for each genotype were used for trichome number measurement in each biological experiment. Data are means (±se) of three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05). (E) and (F) Statistical analysis of total trichome numbers in the fifth true leaves of the indicated genotype with the indicated treatments. Eight leaves for each genotype were used for trichome number measurement in each biological experiment. Data are means (±se) of three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05). (G) Real-time PCR analysis for GL2 and MYB23 in Col-0 plants indicated in (A). ACTIN8 was used as the internal control. Data are means (±se) of three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05). (H) and (I) Real-time PCR analysis for GL2 (H) and MYB23 (I) in the indicated genotype with the indicated treatment in (B) and (C), respectively. ACTIN8 was used as the internal control. Data are means (±se) of three biological replicates. Lowercase letters indicate significant differences by one-way ANOVA analysis with SAS software (P < 0.05).

Figure 8.

A Simplified Model for the Crosstalk between GA and JA. (A) Both DELLAs and JAZs interact with and attenuate WD-repeat/bHLH/MYB complex to inactivate downstream genes and repress trichome formation. GA and JA signal induce degradation of DELLAs and JAZs, respectively, to derepress WD-repeat/bHLH/MYB complex and synergistically and mutually dependently modulate trichome development in Arabidopsis. (B) A previous study showed that the Arabidopsis DELLAs interact with and repress JAZs to release MYC2 that mediates JA-regulated root growth (Hou et al., 2010). (C) A previous study showed that JAZs interact with and repress DELLAs to release PIF3 that regulates GA-mediated hypocotyl elongation in Arabidopsis (Yang et al., 2012). [See online article for color version of this figure.]