SUMOylation of HLS1 is required for its function. A) Apical hook phenotypes of 3-d-old dark-grown siz1-2, siz1-2 hls1-1, HLS1pro:HA-HLS1 hls1-1, and HLS1pro:HA-HLS1^6KR hls1-1 seedlings. Scale bars, 1 mm. B) Quantification of apical hook curvature from seedlings shown in A). Data represent means ± Se from 3 replicates, with each replicate consisting of at least 12 seedlings. Different lowercase letters indicate significant differences (P < 0.05) based on 1-way ANOVA followed by Fisher's Lsd test. C) Subcellular localization of HLS1-GFP and HLS1^6KR-GFP. HLS1-GFP and HLS1^6KR-GFP constructs were transiently transfected into Arabidopsis protoplasts prepared from Col-0. The cell nucleus was labeled by DAPI staining. Scale bars, 20 μm. D) Chemical crosslinking assays showing the oligomerization status of HLS1 in HLS1OE hls1-1, HLS1^6KROE hls1-1, and HLS1OE siz1-2 seedlings. Seedlings were grown on half-strength MS medium in darkness for 3 d and then transferred to light (D to L) for 4 h. Seedlings were harvested and submerged in 0.5% (w/v) PFA solution for 30 min. An anti-HA antibody was used for immunoblotting. The oligomer/monomer ratio was determined using ImageJ, with oligomeric and monomeric HLS1 protein abundance in HLS1OE hls1-1 set to 1. Data represent means ± Sd from 3 independent experiments. Different lowercase letters indicate significant differences (P < 0.05) based on 1-way ANOVA followed by Fisher's Lsd test.