The bHLH Transcription Factors MYC2, MYC3, and MYC4 Are Required for Jasmonate- Mediated Inhibition of Flowering in Arabidopsis  Houping Wang, Yang Li, Jinjing Pan, Dengji Lou, Yanru Hu, Diqiu Yu  Molecular Plant  Volume 10, Issue 11, Pages 1461-1464 (November 2017) DOI: 10.1016/j.molp.2017.08.007 Copyright © 2017 The Author Terms and Conditions

Figure 1 The MYC2/3/4-Mediated JA Pathway Regulates Flowering Time through Inhibiting FT Transcription. (A) Flowering phenotype of the myc2 mutant, myc2/3, myc2/4, myc3/4 double mutants, and myc2/3/4 triple mutant under LD conditions. The picture was taken 23 days after germination. (B) RT–PCR analysis of FT expression in wild-type and myc2/3/4 mutant. 10-day-old plants grown under LD conditions were harvested at given times after dawn (zeitgeber time [ZT] 0). Error bars indicate the SD from three independent biological replicates. *p < 0.01, Student's t-test. (C) Schematic of the FT locus. Bars indicate the G-box-related motifs (CANNTG or core of G-box ACGT). The arrow indicates the putative promoter. Black boxes indicates exons; white boxes indicate the introns. The solid lines or dashed lines indicate the sequences detected or not detected by ChIP assays. (D) Semiquantitative PCR to determine the enrichment of DNA fragments. DNA fragments (∼250 bp) were prepared from 10-day-old 35Spro:MYC2-cMYC transgenic seedings at ZT 15 grown under LD conditions and were immunoprecipitated with anti-cMYC antibodies or anti-IGG antibodies. Input indicates the samples before immunoprecipitation. (E) qPCR of anti-cMYC ChIP in FT chromatin regions. Error bars indicate SD from three experiments quantified by normalization of the cMYC-IP with the corresponding input. The ACTIN2 3′-untranslated sequence was used as an endogenous control. (F) The early flowering phenotype of myc2/3/4 was suppressed in an ft-10 mutant background. Flowering time of the wild-type plants and various mutants were assessed by rosette leaf number under LD conditions. Data are means of at least 20 plants. (G) MeJA response of wild-type plants and myc2/3/4 mutant. Flowering phenotype of the wild-type plants and various mutants treated with MeJA were assessed by rosette leaf number under LD conditions. Error bars indicate the SD from 20 independent plants; Student's t-test; *p < 0.01, **p < 0.001. (H) Exogenous JA regulates FT transcription. GUS staining represents the cotyledon (left) and the first set of rosette leaves (right) of 10-day-old plants at ZT 16 grown under LD conditions. Scale bars, 0.5 mm. (I) qRT–PCR analysis of FT expression in response to exogenous JA. Total RNA was extracted from MeJA or mock-treated wild-type plants and myc2/3/4 mutants at 8 h after dawn. Error bars indicate the SD from three independent biological replicates. *p < 0.05, **p < 0.01, Student's t-test. (J) Schematic of the reporter and effectors used in the transient transactivation assays. (K) Transient dual-luciferase reporter assays show that the expression of FT is affected by the MYC2-mediated JA signaling pathway. Error bars indicate the SD from three independent biological replicates; *p < 0.05, Student's t-test. Molecular Plant 2017 10, 1461-1464DOI: (10.1016/j.molp.2017.08.007) Copyright © 2017 The Author Terms and Conditions