SKIP Interacts with the Paf1 Complex to Regulate Flowering via the Activation of FLC Transcription in Arabidopsis  Ying Cao, Liguo Wen, Zheng Wang, Ligeng Ma  Molecular Plant  Volume 8, Issue 12, Pages 1816-1819 (December 2015) DOI: 10.1016/j.molp.2015.09.004 Copyright © 2015 The Author Terms and Conditions

Figure 1 SKIP Interacts Directly with ELF7 to Promote FLC/MAFs Transcription and Repress the Floral Transition in Arabidopsis. (A) The skip-1 mutant exhibited an early flowering phenotype under different photoperiods (left panel) and at different temperatures (LD conditions, 16 h of light/8 h of dark; right panel). The rosette leaf number in at least 16 plants at bolting was used as an indicator of flowering time. WT (Col-0), wild-type Columbia-0 plants; CL, constant light at 22°C; LD (16L/8D), 16 h of light at 22°C/8 h of dark at 18°C; SD (10L/14D), 10 h of light at 22°C/14 h of dark at 18°C; SD (8L/16D), 8 h of light at 22°C/16 h of dark at 18°C. RLN, rosette leaf number. (B) Relative expression levels of FLC variants and COOLAIR variants in wild-type and skip-1 plants as determined by qRT–PCR. The positions of the primers used are shown in Supplemental Figure 3. The levels of the FLC variants were normalized to that of FLC.1 in wild-type plants. (C) Relative expression levels of unspliced sense and anti-sense FLC transcripts in wild-type and skip-1 plants as determined by qRT–PCR. The positions of the primers used are shown in Supplemental Figure 3. (D) The expression pattern of FLM-β and FLM-δ following growth at 16°C, 22°C, and 27°C under LD conditions in wild-type and skip-1 plants. The levels of FLM-β and FLM-δ were normalized to that of FLM-β at 16°C and FLM-δ at 27°C, respectively, in both wild-type and skip-1 plants. (E) Relative FLC expression levels under different photoperiods (left panel) and at different temperatures (right panel) in wild-type and skip-1 plants as determined by quantitative reverse transcription polymerase chain reaction (qRT–PCR). (F) Demonstration of a physical interaction between SKIP and ELF7 by yeast two-hybrid assay. Positive control, pGADT7-T/pGBKT7-53; negative control, pGADT7-T/pGBKT7-Lam. The indicated combinations of plasmids were co-transformed into the yeast reporter strain AH109, and the interaction of SKIP with ELF7 was assessed by growth on plates lacking leucine, tryptophan, histidine, and adenine. AD, activation domain; BD, binding domain. (G) Demonstration of the in vivo interaction of SKIP with ELF7 by an acceptor photobleaching fluorescence resonance energy transfer (FRET) assay in tobacco cells. Images from the GFP and RFP channels before and after SKIP-RFP photobleaching are shown; different colors indicate the fluorescence intensity. The fluorescence intensities of the donor and acceptor in pre- and post-bleach images were calculated. The FRET efficiency was calculated as EFRET = 1 − IDB/IDA. (H) Co-immunoprecipitation (Co-IP) of SKIP and ELF7 in Arabidopsis. Total protein samples from 9-day-old wild-type and FLAG-ELF7res (a rescue line expressing FLAG-EFL7 under the control of the endogenous promoter in elf7) plants were subjected to immunoprecipitation using anti-FLAG antibodies. The immunoprecipitate was analyzed by western blotting using anti-FLAG or anti-SKIP antibodies as indicated in the right part of the figure. (I) The early flowering phenotypes of skip-1 and elf7-3 single and double mutants under LD conditions at 22°C. The rosette leaf number in at least 14 plants at bolting was used as an indicator of flowering time. RLN, rosette leaf number. (J) Relative expression levels of FLC clade genes in skip-1 and elf7-3 plants as determined by qRT–PCR. ACTIN was used as an internal control. (K) H2B monoubiquitination level of the chromatin of FLC clade genes in skip-1 and elf7-3 plants as determined by ChIP. The immunoprecipitated chromatin was normalized to the input chromatin. (L) The relative levels of H3K4me3 of the chromatin of FLC clade genes in skip-1 and elf7-3 plants as determined by ChIP. The immunoprecipitated chromatin was normalized to that of ACTIN. The values in (B–E) and (J–L) are the mean ± standard deviation of at least three biological replicates. Molecular Plant 2015 8, 1816-1819DOI: (10.1016/j.molp.2015.09.004) Copyright © 2015 The Author Terms and Conditions