DELLA Proteins Promote Anthocyanin Biosynthesis via Sequestering MYBL2 and JAZ Suppressors of the MYB/bHLH/WD40 Complex in Arabidopsis thaliana  Ye Xie, Huijuan Tan, Zhaoxue Ma, Jirong Huang  Molecular Plant  Volume 9, Issue 5, Pages 711-721 (May 2016) DOI: 10.1016/j.molp.2016.01.014 Copyright © 2016 The Author Terms and Conditions

Figure 1 The MBW Complex Is Involved in GA-Inhibited Anthocyanin Biosynthesis. (A and B) Effect of PAC on anthocyanin biosynthesis in WT (Col-0 and Ler), MYBs-RNAi, tt8 gl3 egl3, and ttg1-1 seedlings. Mock, without PAC treatment. Scale bar in (A) represents 1 mm. (C) Effect of GA and PAC on anthocyanin content in WT, tt8-1, gl3 egl3, and tt8 gl3 egl3 seedlings. (D) Relative gene expression of the MBW complex subunit in 3-day-old WT seedlings treated with GA for 4, 8, and 12 h. The expression levels of the genes in Mock were set to 1. Error bars represent ±SE (n = 3). Two biological replicates were analyzed with similar results. Error bars in (B) and (C) represent ±SD (n = 3). Asterisks indicate significant differences in anthocyanin content between Mock and PAC (B), or between indicated treatments (C) at *p < 0.05 and **p < 0.01 (Student's t-test), respectively. Molecular Plant 2016 9, 711-721DOI: (10.1016/j.molp.2016.01.014) Copyright © 2016 The Author Terms and Conditions

Figure 2 MYBL2 Is Involved in DELLA-Regulated Anthocyanin Biosynthesis. (A and B) Anthocyanin accumulation in leaf petioles of 5- to 6-week-old WT, ga1-3, ProRGA:RGAd17, and mybl2 plants. Scale bar in (A) represents 2 cm. (C) Anthocyanin accumulation in branch stems of mature WT, ProRGA:RGAd17, and mybl2 plants without or with GA treatment. Two-week-old seedlings were sprayed with 20 μM GA3 once a week. The red squares show the magnified images. (D and E) Anthocyanin content and gene expression in WT (Ler), MYBL2OE (Ler background), Pro35S:TAP-RGAd17 (RGAd17), Pro35S:TAP-GAId17 (GAId17) plants, RGAd17/MYBL2OE, and GAId17/MYBL2OE lines. The expression levels of the genes in WT seedlings were set to 1. Error bars represent ±SE (n = 3). Two biological replicates were analyzed with similar results. (F) Effect of GA on anthocyanin content of WT (Col-0) and MYBL2OE seedlings. (G) Effect of GA on anthocyanin content of WT and mybl2 seedlings. The percentages indicate relative anthocyanin content, which was set to 100% in Mock. Error bars in (B), (D), (F), and (G) represent ±SD (n = 3). Asterisks indicate significant differences in anthocyanin content between different genotypes (**p < 0.01, Student's t-test). Molecular Plant 2016 9, 711-721DOI: (10.1016/j.molp.2016.01.014) Copyright © 2016 The Author Terms and Conditions

Figure 3 RGA Promotes Transcriptional Activity of the MBW Complex by Sequestering MYBL2. (A) Y2H analysis of DELLAs interaction with MYBL2. DELLAs were fused with the activation domain and MYBL2 was fused with the binding domain. Transformed yeast cells were grown on SD-Trp/-Leu (-LT), or SD-Trp/-Leu/-His/-Ade (-LTHA) media. (B) Pull-down assays of RGA interaction with JAZ1, TT8, and MYBL2. His-tagged RGA was incubated with immobilized GST or GST-tagged proteins. Arrows indicate the detected GST-tagged proteins or GST alone. (C) CoIP analysis of RGA interaction with MYBL2. RGAd17-3 × HA and 6 × Myc-MYBL2 were detected with the anti-HA and anti-Myc antibodies, respectively. (D and E) Competitive binding assays of RGA and TT8 to MYBL2. The mixture of MBP-RGA and GST-TT8 was added to immobilized His-MYBL2. The gradient indicates the increasing amount of MBP-RGA. Binding of RGA and TT8 to PAP1 (E) was used as a control. (F and G) Competitive binding assays of RGA and EGL3 to MYBL2. Binding of RGA and EGL3 to PAP1 (G) was used as a control. (H and I) Dual-luciferase assays of MYBL2 and/or RGA on PAP1/TT8 (H) and PAP1/EGL3 (I) activation of the DFR promoter. The ProDFR-LUC reporter was cotransformed with the various constructs into tobacco leaves. Relative LUC activities were normalized to the REN internal control. The LUC/REN ratio in the leaves transformed with the vector was set as 1. Error bars represent ±SD (n = 3). Letters above the error bars indicate significant differences by one-way ANOVA (p < 0.05, Duncan's multiple-range test). Molecular Plant 2016 9, 711-721DOI: (10.1016/j.molp.2016.01.014) Copyright © 2016 The Author Terms and Conditions

Figure 4 DELLA-Regulated Anthocyanin Biosynthesis Is Mediated by JAZs. (A–C) Effects of GA, JA, or GA plus JA on anthocyanin accumulation (A), content (B), and gene expression (C) in WT. mRNA abundance were analyzed in seedlings treated with a hormone or hormones at the indicated times. The percentages (B) indicate relative anthocyanin content between the indicated treatments. Letters above the error bars indicate significant differences by one-way ANOVA (p < 0.01, Duncan's multiple-range test). (D–F) Effects of GA, JA, or PAC on anthocyanin accumulation (D), content (E), and gene expression (F) in WT and coi1-2. mRNA abundance were analyzed in seedlings treated with or without a hormone or PAC. The numbers (E) indicate the fold changes of anthocyanin content in PAC or JA treatment against those in Mock. Letters above the error bars indicate significant differences by two-way ANOVA (p < 0.05, Duncan's multiple-range test). (G–I) Competitive binding assays of RGA and PAP1 (G), TT8 (H), or EGL3 (I) to JAZ1. Scale bars in (A) and (D) represent 1 mm. Error bars in (B) and (E) represent ±SD (n = 3). In (C) and (F), the expression levels of genes in Mock were set to 1. Error bars represent ±SE (n = 3). Two biological replicates were analyzed with similar results. Molecular Plant 2016 9, 711-721DOI: (10.1016/j.molp.2016.01.014) Copyright © 2016 The Author Terms and Conditions

Figure 5 DELLAs Play Important Roles in Abiotic Stress-Induced Anthocyanin Biosynthesis. (A and B) Effects of abiotic stresses including low phosphate (LP), low nitrogen (LN), and low temperature (LT) on anthocyanin content in WT, mutants, and transgenic plants. CK, without stress. Error bars represent ±SD (n = 3). Asterisks indicate significant changes in anthocyanin content at *p < 0.05 and **p < 0.01 (Student's t-test), respectively, between the WT and mutants or transgenic plants. (C) Western blotting analysis of RGA levels under stress conditions. Total proteins were extracted from cotyledons and hypocotyls of 2-week-old Pro35S:TAP-RGA seedlings. (D) A model GA-regulated anthocyanin biosynthesis through DELLA proteins. Under normal conditions or in the presence of GA, DELLAs are degraded to liberate MYBL2/JAZs, which inhibit the formation and activity of MBW complex, thereby switching off the expression of anthocyanin biosynthetic genes. In contrast, under abiotic stresses, DELLAs accumulate and sequester MYBL2/JAZs, leading to MBW complex formation, which then activates anthocyanin biosynthesis and ultimately makes a contribution to stress tolerance. Arrows, positive regulation; blunt ends, negative regulation; dashed arrow and question mark, other potential pathways. Molecular Plant 2016 9, 711-721DOI: (10.1016/j.molp.2016.01.014) Copyright © 2016 The Author Terms and Conditions