Extended Data Fig. 7: Generation and characterization of estradiol-inducible fie-amiR-es and SSTS/AAAA/fie mutants.
From: Glucose-driven TOR–FIE–PRC2 signalling controls plant development
a, Screening of optimal amiRNA in the protoplast system. Empty amiRNA expression vector as a control (Ctrl) or each of the eight amiRNA candidate plasmids was co-transfected with a heat shock promoter-driven FIE-FLAG plasmid. The HBT-GFP-HA plasmid serves as an internal control for all co-transfection experiments. Immunoblot analysis indicated similar GFP-HA protein levels. The most potent amiRNA4 abolished FIE-FLAG expression and was chosen to generate estradiol-inducible fie-amiR-es transgenic plants. b, RT-qPCR analysis of FIE transcripts in 14-day transgenic fie-amiR-es lines without or with 10 μM of estradiol treatment. UBQ10 transcripts served as an internal control for normalization. Data show mean ± s.d. from 3 biological replicates. Data were analysed by unpaired two-sided Student’s t test. c, Protein blot analysis of FIE protein levels in 14-day transgenic fie-amiR-es lines. Three independent fie-amiR-es lines were crossed to the FLAG-GFP-FIE transgenic plant. The FLAG-GFP-FIE protein was eliminated with 10 μM of estradiol treatment. Tubulin was used for the loading control. d, The development phenotype of the fie-amiR-es lines was similar to that of the SSTS/AAAA/fie mutant. Three independent fie-amiR-es lines and the SSTS/AAAA/fie mutant showed small, narrow and curled leaves. The GFP-FIE/fie plant showed normal development. Scale bar, 10 mm. Experiments were conducted in three biological repeats with similar results. e, The H3K27me3 level was greatly decreased in 14-day fie mutant plants. Values are the relative level of H3K27me3 compared with the corresponding H3 control, with immunoblot signals in WT set as 1.0. f, Genome browser view of H3K27me3 ChIP-seq read densities. The Arabidopsis genome region within Chr3:15800000..18000000 is shown. g, Venn diagram of H3K27me3 target genes in Arabidopsis from three independent genome wide analyses. The H3K27me3 targets in our study cover 79.7% of genes from Zhang et al. and 84.5% of genes from Bouyer et al12. Data in a, c, and e are representatives of three biological replicates each.
