Regulation of the WD-repeat/bHLH/MYB complex by gibberellin and jasmonate

Abstract

The phytohormones gibberellin (GA) and jasmonate (JA) regulate various aspects of plant development, growth and defense. Previous studies showed that both DELLA repressors in GA pathway and JA-ZIM domain (JAZ) proteins in JA pathway interact with and repress the WD-repeat/bHLH/MYB transcriptional complex to inhibit trichome initiation, and GA and JA respectively induce DELLAs and JAZs degradation to synergistically enhance trichome formation. In this study, we showed that the DELLA protein RGA and JAZ1 competitively bind to ENHANCER OF GLABRA3 (EGL3), a bHLH component of the WD-repeat/bHLH/MYB complex. GA and JA differently affect the expression and protein stability of the components of the WD-repeat/bHLH/MYB complex, and EGL3 and GL3 repress the expression of JAZ genes as a feedback. The novel findings help to understand the mechanism of the WD-repeat/bHLH/MYB complex in GA/JA-regulated trichome formation.

Keywords: Gibberellin; WD-repeat/bHLH/MYB complex; jasmonate.

Figure 1.

RGA and JAZ1 Competitively Bind to EGL3. In vitro pull-down assay showed that the interaction between MBP-EGL3 and GST-JAZ1 is attenuated by myc-RGA in a dosage-dependent manner. GST and GST-JAZ1 proteins were purified from E. coli using GST resin beads, and MBP-EGL3 protein was purified from E. coli using amylose resin beads. Total protein was extracted from 5 g of myc-RGA transgenic plants. Immobilized EGL3 was incubated with GST, GST-JAZ1 or myc-RGA as shown. The immunoprecipitated proteins were separated on SDS-PAGE and detected by anti-GST or anti-myc antibodies. The plus sign indicates the amount of myc-RGA. The total input of MBP-EGL3 was detected by anti-MBP antibody.

Figure 2.

Regulation of the Expression of WD-repeat/bHLH/MYB Complex by Gibberellin. (A) Real-time PCR analysis for GL1, GL3, EGL3 and TTG1 in Col-0 wild-type treated without (Mock) or with Gibberellin (GA). ACTIN8 was used as the internal control. Error bars represent SE. (B) Real-time PCR analysis for GL1, GL3, EGL3 and TTG1 in the Landsberg erecta (Ler) wild-type, ga1-3, Q1 (ga1-3 gai-t6 rgl1-1 rgl2-1, wild-type for RGA and RGL3), Q3 (ga1-3 gai-t6 rgl1-1 rga-t2, wild-type for RGL2 and RGL3), penta (ga1-3 gai-t6 rga-t2 rgl1-1 rgl2-1, wild-type for RGL3) and della (gai-t6 rga-t2 rgl1-1 rgl2-1 rgl3-1) plants grown on MS medium. ACTIN8 was used as the internal control. Error bars represent SE.

Figure 3.

Effects of Jasmonate and Gibberellin on the Protein Stability of the WD-repeat/bHLH/MYB Complex. The Agrobacterium strain containing myc-fused GL1 (myc-GL1), GL3 (myc-GL3), or EGL3 (myc-EGL3) was infiltrated into N. benthamiana leaves. After 50 h, total protein was extracted from N. benthamiana leaves, incubated without (Mock) or with jasmonate (JA), gibberellin (GA), or MG132 for the indicated time (hours), and myc-GL1 (A), myc-GL3 (B), or myc-EGL3 (C) was detected by immunoblot using anti-myc antibody. The PVDF membrane was stained with Memstain to serve as loading control.

Figure 4.

Expression of JAZ Genes in wide-type and the gl3 egl3 Double Mutant. Real-time PCR analysis for JAZ1, JAZ2, JAZ5, JAZ6, JAZ7, JAZ8, JAZ9 and JAZ10 in the 2 week-old seedlings of wild type (WT) and gl3 egl3 treated with 100 µM Methyl-jasmonate for 0, 1 or 2 hours. ACTIN8 was used as the internal control. Error bars represent SE.