Fig. 7: GCR1/2 directly repress the expression of LFS.
From: Spatiotemporal formation of glands in plants is modulated by MYB-like transcription factors
a Venn diagram showing the overlap of up-regulated transcriptional factors in gland (referred to Supplementary Fig. 1), down-regulated genes in pMTR1: GCR1 and up-regulated genes in cr-gcr1/2. b The relative expression level of LFS and HD9 in pMTR1: GCR1 and cr-gcr1/2 based on the transcriptome data. c LUC assay. GCR1 and GCR2 inhibited the transcriptional activity of LFS promoter and mutation of the MYB domain in GCR1 and GCR2 (GCR1-mMYB, GCR2-mMYB) disrupted the inhibition in tobacco protoplast. d Y1H shows GCR1 and GCR2 bind to the promoter fragments of LFS. The bold black lines represent the promoter sequence of LFS. The red vertical lines indicate motifs (referred to Supplementary Fig. 13). GCR1 binds to LFS promoter fragments pLFS-2, pLFS-4 and pLFS-5 containing motif 1, 6, 7, 8 and 9 (referred to Supplementary Fig. 13). GCR2 interacts with LFS promoter fragments pLFS-4 and pLFS-5. Three cell dilutions for each interaction were presented. e Y1H shows mutation of conserved amino acids in MYB domain of GCR1 and GCR2 abolish the interaction between GCR1/2 and LFS promoter. The black boxes represent the amino acid sequence of GCR1 and GCR2. The yellow boxes show the MYB domains. Three conserved amino acids (Trp, W; Pro, P; Leu, L) in the MYB domain of GCR1 and GCR2 are replaced with serine (S) to construct GCR1mMYB and GCR1mMYB. SD/−2: -Ura/-Trp. Three cell dilutions for each interaction were presented. f The ChIP-qPCR analysis shows the interaction between GCR1 and the promoter of LFS. g Biotin-labelled DNA IP assays show GCR1 interacts with pLFS-2 and pLFS-5 and GCR2 interacts with pLFS-4 and pLFS-5. Mutation motifs of promoter fragments (pLFS-2m, pLFS-4m, pLFS-5m) disrupt the interaction between GCR1/2 and its own promoter fragments. Promoter fragments and mutant fragments labelled with biotin were used as bait and incubated with streptavidin-agarose beads. His-GST protein was used as negative control. Immunoprecipitated proteins were detected by anti-His. Data are shown as mean ± SD (n = 3 (b), 3 ~ 4 (c) and 3 (g) biological replicates). p-values were calculated by unpaired two-sided t-test.
