Strigolactone Signaling in Arabidopsis Regulates Shoot Development by Targeting D53-Like SMXL Repressor Proteins for Ubiquitination and Degradation

Abstract

Strigolactones (SLs) are carotenoid-derived phytohormones that control many aspects of plant development, including shoot branching, leaf shape, stem secondary thickening, and lateral root growth. In rice (Oryza sativa), SL signaling requires the degradation of DWARF53 (D53), mediated by a complex including D14 and D3, but in Arabidopsis thaliana, the components and mechanism of SL signaling involving the D3 ortholog MORE AXILLARY GROWTH2 (MAX2) are unknown. Here, we show that SL-dependent regulation of shoot branching in Arabidopsis requires three D53-like proteins, SUPPRESSOR OF MORE AXILLARY GROWTH2-LIKE6 (SMXL6), SMXL7, and SMXL8. The smxl6 smxl7 smxl8 triple mutant suppresses the highly branched phenotypes of max2 and the SL-deficient mutant max3. Overexpression of a mutant form of SMXL6 that is resistant to SL-induced ubiquitination and degradation enhances shoot branching. Exogenous application of the SL analog rac-GR24 causes ubiquitination and degradation of SMXL6, 7, and 8; this requires D14 and MAX2. D53-like SMXLs form complexes with MAX2 and TOPLESS-RELATED PROTEIN2 (TPR2) and interact with D14 in a GR24-responsive manner. Furthermore, D53-like SMXLs exhibit TPR2-dependent transcriptional repression activity and repress the expression of BRANCHED1. Our findings reveal that in Arabidopsis, D53-like SMXLs act with TPR2 to repress transcription and so allow lateral bud outgrowth but that SL-induced degradation of D53-like proteins activates transcription to inhibit outgrowth.

Figure 1.

Shoot Architecture of smxl Mutants and Suppression of Shoot Branching in the max3-9 Mutant. (A) Shoots of representative plants after 7 weeks of growth in a 16-h-light/8-h-dark photoperiod. All mutations are in the Col-0 background, and genotypes of mutants are as indicated. Bar = 5 cm. (B) Number of primary rosette (RI) branches of at least 0.5 cm recorded in plants shown in (A). Values are means ± se (n = 14); significant differences revealed by Tukey's multiple comparison test are indicated by letters above bars (P < 0.05). (C) Number of secondary cauline (CII) branches of at least 0.5 cm recorded in plants shown in (A). Values are means ± se (n = 14); significant differences revealed by Tukey's multiple comparison test are indicated by letters above bars (P < 0.05).

Figure 2.

Expression of MAX4 Is Repressed in smxl6/7/8 and Is Unresponsive to GR24^5DS. Expression of MAX4 in 10-d-old seedlings of Col-0 and the indicated mutants, each treated with 5 µM GR24^5DS in 0.5× MS liquid medium for 4 h. Values are means ± se (n = 3); ns, no significant difference; **P < 0.01 indicated by Student’s t test.

Figure 3.

Overexpression of SMXL6D-GFP Enhances the Outgrowth of Axillary Buds and MAX4 Expression. (A) Shoots of representative wild-type (Col-0) and transgenic plants expressing 35S:SMXL6-GFP or 35S:SMXL6D-GFP genes after 7 weeks of growth in a 16-h-light/8-h-dark photoperiod. Bar = 5 cm. (B) Number of primary rosette (RI) branches of at least 0.5 cm recorded in plants shown in (A). Values are means ± se (n = 15); significant differences revealed by Tukey's multiple comparison test are indicated by different letters above bars (P < 0.05). (C) Number of secondary cauline (CII) branches of at least 0.5 cm recorded in plants shown in (A). Values are means ± se (n = 15); Tukey's multiple comparison test revealed no significant differences (P < 0.05). (D) RNA levels of MAX4 relative to ACTIN2 in 10-d-old seedlings of wild-type (Col-0) and transgenic plants expressing 35S:SMXL6-GFP or 35S:SMXL6D-GFP. Seedlings were treated with 5 µM rac-GR24 in 0.5× MS liquid medium for 4 h before isolation of RNA for RT-qPCR. Values are means ± se (n = 3); ns, no significant difference; asterisks indicate significant difference (**P < 0.01) revealed by Student’s t test.

Figure 4.

Regulation of Leaf Shape by SMXL6, 7, and 8 in Arabidopsis. (A) Rosettes and the fifth leaves of 3-week-old wild type (Col-0) and the indicated mutants. Bars = 1 cm. (B) Quantitative analysis on the ratio of leaf length to leaf width for the fifth leaves shown in (A). Values are represented as mean ± se (n = 12); **P < 0.01 indicated by Student’s t test. (C) Rosettes and the fifth leaves of 3-week-old wild-type (Col-0) and transgenic plants expressing 35S:SMXL6-GFP and 35S:SMXL6D-GFP. Bars = 1 cm. (D) Quantitative analysis on the ratio of leaf length to leaf width for the fifth leaves shown in (C). Values are represented as mean ± se (n = 12); **P < 0.01 indicated by Student’s t test. (E) Hypocotyl length of 7-d-old light-grown seedlings in the wild type (Col-0) and the indicated mutants. Values are represented as mean ± se (n = 16); **P < 0.01 indicated by Student’s t test.

Figure 5.

GFP-SMXL6, 7, and 8 Fusion Proteins Are Polyubiquitinated and Degraded in Response to rac-GR24 Treatment in Arabidopsis. (A) Ubiquitination of GFP-SMXL6, GFP-SMXL7, and GFP-SMXL8 in protoplasts. Arabidopsis (Col-0) protoplasts were transformed with plasmids encoding each fusion protein, incubated for a 12-h period of protein synthesis, then pretreated with 50 µM MG132 for 1 h and treated with 40 µM rac-GR24 or 40 µM KAR₁ for 1 h. Proteins were isolated for immunoprecipitation with agarose-conjugated anti-GFP monoclonal antibody followed by immunoblotting with antiubiquitin antibody (upper panel) or anti-GFP (lower panel). (B) Levels of GFP-SMXL6, GFP-SMXL7, and GFP-SMXL8 proteins in wild-type protoplasts after plasmid transformation and treatment with 40 µM rac-GR24 or 40 µM KAR₁ for the times indicated. Proteins were detected by immunoblotting with anti-GFP monoclonal antibody. Relative amounts of proteins were determined by densitometry and normalized to loadings determined by Ponceau staining (red) and expressed relative to the value at zero time. (C) Ubiquitination of SMXL6-GFP and SMXL6D-GFP in wild-type (Col-0) Arabidopsis containing 35S:SMXL6-GFP and 35S:SMXL6D-GFP transgenes. Seedlings were treated after 10 d of growth with 50 µM MG132 for 1 h, then with 2 µM rac-GR24 in 0.5× MS liquid medium for 10 min. Proteins were detected as in (A). (D) Levels of SMXL6-GFP and SMXL6D-GFP proteins in wild-type (Col-0) Arabidopsis containing 35S:SMXL6-GFP and 35S:SMXL6D-GFP transgenes. Seedlings were treated after 10 d of growth with 2 µM rac-GR24 in 0.5× MS liquid medium for the times indicated. Proteins were detected as in (B). (E) Ubiquitination of SMXL6-GFP in wild-type (Col-0), max2-1, and d14-1 transgenic plants each expressing 35S:SMXL6-GFP. Seedlings were treated after 10 d of growth with 50 µM MG132 for 1 h, then with 2 µM rac-GR24 in 0.5× MS liquid medium for 10 min. Proteins were detected as in (A). (F) Level of SMXL6-GFP in wild-type (Col-0), max2-1, and d14-1 transgenic plants each expressing 35S:SMXL6-GFP. Seedlings were treated after 10 d of growth with 2 µM rac-GR24 in 0.5× MS liquid medium for the times indicated. Proteins were detected as in (B).

Figure 6.

Interactions between SMXL6 and MAX2. (A) Subcellular localizations of GFP, GFP-SMXL6, GFP-SMXL7, and GFP-SMXL8 in Arabidopsis wild-type (Col-0) protoplasts. A plasmid construct expressing 35S:SV40NLS-mCherry was cotransformed to label the nucleus. BF, bright-field. Bar = 10 μm. (B) In vivo interaction between Flag-SMXL6 and GFP-MAX2 revealed by co-IP assay in protoplasts prepared from the wild type (Col-0). After transformation and incubation for 11 h, protoplasts were pretreated with 40 µM rac-GR24 for 1 h, then cells were broken and immunoprecipitation (IP) with agarose-conjugated anti-GFP monoclonal antibody was performed under 40 µM rac-GR24 treatment, following which the SMXL6 recombinant protein was detected with an anti-Flag monoclonal antibody, while GFP-MAX2 fusion protein and GFP were detected with an anti-GFP monoclonal antibody. Input means total protein lysate without immunoprecipitation. (C) In vivo interaction between Flag-SMXL6 and GFP-MAX2 revealed by the co-IP assay in protoplasts prepared from the wild type (Col-0) and d14-1. Following transformation and incubation for 12 h, cells were broken and immunoprecipitation and immunoblot were conducted as in (B).

Figure 7.

Interactions between D53-Like SMXLs and Arabidopsis D14. (A) SMXL6 and SMXL7 interact with D14 in yeast cells upon rac-GR24 treatment. Yeast cells were cotransformed with constructs encoding the binding domain (BD) fused to D14 and the activation domain (AD) fused to each SMXL. Cells were plated on selective media in the absence (left panel) and presence (right panel) of 10 µM rac-GR24. (B) The interaction between SMXL6 and D14 responds to rac-GR24 in a dose-dependent manner. Yeast cells were cotransformed with constructs encoding BD-D14 and AD-SMXL6. Cells were plated on selective media in the presence of increasing amount of rac-GR24. (C) In vivo interaction between Flag-SMXL6 and GFP-D14 revealed by the co-IP assay in protoplasts made from the wild type and max2-1. After transformation and incubation for 12 h, cells were broken and then immunoprecipitation (IP) with agarose-conjugated anti-GFP monoclonal antibody was performed, following which the SMXL6 recombinant protein was detected with an anti-Flag monoclonal antibody, while GFP-D14 fusion protein and GFP were detected with an anti-GFP monoclonal antibody. Input represents total protein lysate without immunoprecipitation. (D) In vivo interaction between HA-D14 and GFP-SMXL6 revealed by the co-IP assay in max2-1 protoplasts in the absence or presence of rac-GR24. After transformation and incubation for 11 h, protoplasts were pretreated with 100 µM rac-GR24 for 1 h and then cells were broken and immunoprecipitation (IP) with agarose-conjugated anti-GFP monoclonal antibody was performed under 100 µM rac-GR24 treatment, following which the HA-D14 recombinant protein was detected with an anti-HA monoclonal antibody, while GFP-SMXL6 fusion protein and GFP were detected with an anti-GFP monoclonal antibody.

Figure 8.

SMXL6, 7, and 8 Interact with TPR2. (A) Interactions of TPR2 with SMXL6, 7, and 8 in yeast cells. Yeast cells were cotransformed with constructs encoding the binding domain (BD) fused to each SMXL and the activation domain (AD) fused to TPR2 and plated on selective media. (B) Interactions in Arabidopsis protoplasts of SMXL6, SMXL7, or SMXL8 with TPR2. Protoplasts were prepared from wild type (Col-0) and cotransformed with genes encoding Flag-TPR2 and either GFP-SMXL or GFP-SMXLΔEAR (both for each of SMXL6, 7, and 8). After incubation for 12 h, cells were broken and immunoprecipitation (IP) with agarose-conjugated anti-GFP monoclonal antibody was performed, following which the Flag-TPR2 was detected with an anti-Flag monoclonal antibody, while GFP-SMXL and GFP-SMXLΔEAR proteins were detected with an anti-GFP monoclonal antibody.

Figure 9.

SMXL6, 7, and 8 Cooperate with TPR2 to Repress the Expression of SL-Responsive Genes. (A) SMXL6, 7, and 8 show transcriptional repression activities in transient expression assays in Arabidopsis. Protoplasts were cotransformed with two plasmids (Supplemental Figure 8), one comprising a luciferase reporter with upstream enhancer sequence and the other encoding GAL4, a GAL4-SMXL fusion, or GAL4-SMXLΔEAR fusion. After 12 h of incubation, luciferase was assayed. Values are means ± se (n = 4); ns, no significant difference; **P < 0.01 determined by Student’s t test. (B) The effects of TPR2 on transcriptional repression activities of SMXL6, 7, and 8 in Arabidopsis. Protoplasts were cotransformed with three plasmids (Supplemental Figure 8): one comprising the luciferase reporter, another encoding either GAL4 or GAL4-SMXL fusion, and the third either GFP (as a control) or TPR2. After 12 h of incubation, luciferase was assayed. Values are means ± se (n = 4); ns, no significant difference; **P < 0.01 determined by Student’s t test. (C) and (D) Expression of BRC1 in nonelongated axillary buds of primary rosette (RI) branches (C) and secondary cauline (CII) branches (D) of Col-0 and the mutants indicated. Values are means ± se (n = 3 or 4); **P < 0.01 determined by Student’s t test. (E) A model of the SL signaling complex in Arabidopsis that includes SL-dependent interaction of Arabidopsis D14 with both MAX2 and SMXL proteins, although the sequence in which these interactions occur is not known. It is not known if BRC1 is a direct or indirect target of this SL signaling mechanism. ASK, CUL1, RBX, and E2 are components of the ubiquitination complex. U, Ubiquitin; TFs, transcription factors (unidentified).