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. 2021 Apr 3;33(7):2258–2272. doi: 10.1093/plcell/koab102

Figure 9.

Figure 9

ELF3 represses the expression of FT in combination with LUX and ELF4 as part of the EC. A, GA promotes FT expression through repression of ELF3 by the GAF1-TPR complex. B, Schematic representation of the reporter and effector. A 3,000-bp fragment of the FT promoter was fused with the LUC gene. The effector plasmid expressed full-length ELF3, ELF4, LUX, GAL4BD-ELF3 (GAL4 DNA-binding domain fused ELF3), GAL4BD-ELF4, and GAL4BD-LUX under the control of the CaMV 35S promoter with a viral translation enhancer (Ω). C, Transient expression assay of ELF3, ELF4, and LUX. The effector, reporter, and internal control constructs were co-transfected into Arabidopsis protoplasts. The transfected cells were incubated for 20 h and then Luc and rLUC activities were measured. The results are shown as LUC/rLUC activity. Error bars indicate sd of three biological replicates (n = 3). Asterisks represent Student’s t test significance compared with mock treated control (*P < 0.05). D, Transient expression assay of GAL4BD-ELF3, GAL4BD-ELF4, and GAL4BD-LUX. The effector, reporter, and internal control constructs were co-transfected into Arabidopsis protoplasts. The transfected cells were incubated for 20 h and then Luc and rLUC activities were measured. The results are shown as LUC/rLUC activity. Error bars indicate sd of three biological replicates (n = 3). Asterisks represent Student’s t test significance compared with mock treated control (*P < 0.05). E, Identification of GAF1-binding regions in the FT promoter in vitro. An EMSA was performed using the recombinant LUX protein. Oligonucleotides containing FTpro (−216 to −196, wild-type; lanes 1–4) or mutated (mt) FTpro (mt; lane5) were used as probes. Red letters indicate mutated bases. Wild-type and mt indicate competition with a 1,000-fold excess of the unlabeled wild-type and mutated probe, respectively. The specific GAF1-DNA complexes are indicated by an arrowhead. +, Addition to the reaction mixtures; –, omission from the reaction mixtures. F, Transient expression assay of ELF3, ELF4, and LUX. The effector, reporter, and internal control constructs were co-transfected into Arabidopsis protoplasts. The transfected cells were incubated for 20 h and then Luc and rLUC activities were measured. The results are shown as LUC/rLUC activity. Error bars indicate sd of three biological replicates (n = 3). Asterisks represent Student’s t test significance compared with mock treated control (*P < 0.05). G, A triangle in the schematic map indicates the LUX-binding site in the FT promoter. The 200-bp region of the FT promoter used in the ChIP assay is indicated on the right. The PRR9 promoter region was used as a positive control and UBQ11 was used as a negative control. LUX binds to a region of the FT promoter in vivo. ChIP assays were performed with pre-immune serum or anti-LUX in Col-0. The level of each co-precipitated DNA fragment was quantified by real-time PCR and normalized to the input DNA. Error bars indicate sd of three technical replicates (n = 3). Asterisks represent Student’s t test significance compared with mock treated or pre-immune control (*P < 0.05). Experiments were repeated twice with independently grown plants, with similar results.