Fig. 2

The thermomorphogenesis-related gene YUCCA2 is directly co-regulated by APOLO and VIM1. A Histochemical localization of GUS activity in 4-day-old seedlings containing the proYUCCA2:GUS construct, grown at 23 °C or 29 °C. Scale bars, 0.1 cm. B Boxplots showing hypocotyl length quantification ratio at 29 °C over 23 °C of 4-day-old yuc2 seedlings and their associated wild-type (WT). Values are represented by colored points. Representative morphological phenotypes are shown on the right. Scale bars, 1 cm. CYUCCA2 (YUC2) transcript levels in 4-day-old WT, OE APOLO-1, and vim1-3 seedlings treated or not with heat (29 °C) for 6 h. Asterisks indicate Student’s t test ≤ 0.05; n = 3 between each corresponding genotype and WT. D Epigenetic profile at the YUC2 locus. Tracks 1 to 3 [52]: APOLO recognition by chromatin isolation by RNA purification (ChIRP) sequencing, using ODD (Track 1) and EVEN (Track 2) sets of probes against APOLO. ChIRP negative control using LacZ probes is shown in Track 3. Tracks 4 to 7 [61]: R-loop formation by DNA:RNA immunoprecipitation (DRIP) sequencing, on Watson (Track 4), Crick strand (Track 5), or unstranded sequencing (Track 6). DRIP negative control after RNAseH treatment is shown in Track 7. Gene annotation is shown at the bottom. On the YUCCA2 schematic representation in the bottom, red dots indicate the presence of six GAAGAA/TTCTTC boxes which may mediate APOLO recognition according to Ariel et al. [52]. EAPOLO association to DNA of the YUC2 locus by ChIRP-qPCR in WT, RNAi APOLO-1, and CRISPR-APOLO plants. The background level was determined using a set of probes against LacZ RNA. F RNA-DNA hybrid (R-loop) formation at the YUC2 locus by DRIP-qPCR in WT and RNAi APOLO-1 plants. G R-loop formation at the YUC2 locus by DRIP-qPCR in WT and OE APOLO-1 plants at 23 °C or 29 °C. Asterisks indicate a significant reduction (Student’s t test ≤ 0.05; n = 3) between R-loop levels at 23 °C or 29 °C in WT plants. H Chromatin immunoprecipitation (ChIP)-qPCR analysis of VIM1 binding at the YUC2 promoter in 4-day-old VIM1 over-expression (OE VIM1-1) seedlings treated or not with heat (29 °C) for 6 h. I Methylated DNA immunoprecipitation (MeDIP)-qPCR analysis at the YUC2 promoter in 4-day-old APOLO over-expression (OE APOLO-1) or vim1-3 mutant seedlings treated or not with heat (29 °C) for 6 h. In A, one representative picture out of ten stained seedlings is shown. In B, results are the mean of three biological replicates and letters indicate significant differences compared to WT, based on a Mann-Whitney test (α = 0.05; n ≥ 110). In C, transcript levels are normalized relatively to the untreated control to show fold changes. Bars represent average ± SD (n = 3 biological replicates). In E, F, bars represent average ± SD (n = 3 biological replicates). In G, H, results are expressed as a percentage of the INPUT fraction. Anti-IgG antibodies were used as a negative control. Bars represent SD (n = 3 biological replicates), except for H (n = 2 closest biological replicates out of 3 performed, all showing the same trend) and the asterisk indicates the Student’s t test ≤ 0.05, between 23 and 29 °C. In E and G, asterisks indicate the Student’s t test ≤ 0.05; n = 3, between WT and the corresponding genotype. In I asterisks indicate the Student’s t test ≤ 0.05; n = 3, between 23 and 29 °C for each genotype