ELONGATED HYPOCOTYL5 ( HY5 ) and HY5 HOMOLOGUE ( HYH ) maintain shade avoidance suppression in UV‐B

SUMMARY Reductions in red to far‐red ratio (R:FR) provide plants with an unambiguous signal of vegetational shade and are monitored by phytochrome photoreceptors. Plants integrate this information with other environmental cues to determine the proximity and density of encroaching vegetation. Shade‐sensitive species respond to reductions in R:FR by initiating a suite of developmental adaptations termed shade avoidance. These include the elongation of stems to facilitate light foraging. Hypocotyl elongation is driven by increased auxin biosynthesis promoted by PHYTOCHROME INTERACTING FACTORs (PIF) 4, 5 and 7. UV‐B perceived by the UV RESISTANCE LOCUS 8 (UVR8) photoreceptor rapidly inhibits shade avoidance, in part by suppressing PIF4/5 transcript accumulation and destabilising PIF4/5 protein. Here, we show that longer‐term inhibition of shade avoidance is sustained by ELONGATED HYPOCOTYL 5 (HY5) and HY5 HOMOLOGUE (HYH), which regulate transcriptional reprogramming of genes involved in hormone signalling and cell wall modification. HY5 and HYH are elevated in UV‐B and suppress the expression of XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH) genes involved in cell wall loosening. They additionally increase expression GA2‐OXIDASE1 (GA2ox1) and GA2ox2, encoding gibberellin catabolism enzymes that act redundantly to stabilise the PIF‐inhibiting DELLA proteins. UVR8 therefore regulates temporally distinct signalling pathways to first rapidly inhibit and subsequently maintain suppression of shade avoidance following UV‐B exposure.

(WL) or 0.06 (+FR).UV-B was provided at 1 μmolm -2 s -1 .hy5/hyh mutants (Ws) grown in WL were used as a negative control.A transgenic uvr8-1/GFP-UVR8 line (Ler) grown in WL was used as a positive control.120 µg protein was loaded for Ws samples and 50 µg for the positive control.Ponceau staining of the Rubisco large subunit (rbcL) was used as loading control.Each blot represents an independent biological repeat.
Table S9.Primer sequences used for qPCR.F = forward primer, R = reverse primer.

Figure S1 .
Figure S1.UV-B increases HY5 abundance in Ws seedlings.Western blots of HY5 in Ws seedlings grown for 5 d in 16 h light/ 8 h dark cycles at 20 o C before transfer at dawn to WL, WL+UV-B (UV-B), WL+FR (FR) or WL+FR+UV-B (FRUV-B) for 4 h.R:FR values were 2.5 (WL) or 0.06 (+FR).UV-B was provided at 1 μmolm -2 s -1 .hy5/hyh mutants (Ws) grown in WL were used as a negative control.A transgenic uvr8-1/GFP-UVR8 line (Ler) grown in WL was used as a positive control.120 µg protein was loaded for Ws samples and 50 µg for the positive control.Ponceau staining of the Rubisco large subunit (rbcL) was used as loading control.Each blot represents an independent biological repeat.

FR
Figure S2.Volcano plots of differentially expressed transcripts in Ws and hy5/hyh seedlings treated with (a) WL, (b) WL+UV-B, (c) WL+ FR and (d) WL+ FR+ UV-B.Seedlings were grown for 7 d in WL at 20 o C before transfer at dawn to different light conditions (WL, +FR, +UV-B or +FR+UV-B) for 4 h.R:FR values were 8 (WL) or 0.06 (+FR).UV-B was provided at 1 μmolm -2 s -1 .The x-axis represents log2 of the fold change in transcript abundance and the y-axis represents log10 of the pvalue.Genes displaying significant fold changes > 2 (FDR corrected p value of <0.05) are shown in red.Transcripts induced by HY5/HYH (and therefore down-regulated in the hy5/hyh mutant) are shown on the left of each plot.Transcripts repressed by HY5/HYH (and therefore up-regulated in the hy5/hyh mutant) are shown on the right of each plot.
Figure S3.UV-B-mediated suppression of XTH transcript abundance requires UVR8.Relative abundance of XTH8, XTH17 and XTH19 transcript in Ler and uvr8-1 (a, c, e) and Ws and hy5/hyh (b, d, f) seedlings in different light conditions, measured by qPCR.Plants were grown for 10 d in 16 h light/ 8 h dark cycles at 20 o C before before transfer at dawn to WL, WL+UV-B (UV-B), WL+FR (FR) or WL+FR+UV-B (FRUV-B) for 4 h.R:FR values were 2.5 (WL) or 0.06 (+FR).UV-B was provided at 1 μmolm -2 s -1 .Data represent the mean of 2 (a, c, e) or 3 (b, d, f) independent biological repeats ± SE.
Figure S4.XTH enzymes are likely to act redundantly to control petiole elongation during shade avoidance.Plants were grown for 10 d in 16 h light/ 8 h dark cycles of WL before transfer to different light conditions: WL, WL+UV-B (UV-B), WL+FR (FR) or WL+FR+UV-B (FRUV-B) for 9 d.R:FR values were 2.5 (WL) or 0.06 (FR).UV-B was provided at 1 μmolm -2 s -1 .The largest rosette leaf was used for petiole measurements.Boxes represent 25th to 75th percentile.Bars show the median petiole length of at least 7 plants in each treatment.Boxes represent 25 th to 75 th percentile, whiskers represent spread of data within 1.5 * interquartile range.(n ≥ 12).Different letters represent statistically different mean values (P< 0.05) using a 2-way ANOVA with Tukey multiple comparison test.