SIZ1 facilitates the SUMOylation of HLS1. A) In vitro SUMOylation assays showing the SUMOylation of HLS1. SUMOylated HLS1 was detected with anti-MBP and anti-SUMO1 antibodies. SUMO1^GG and SUMO1^AA represent the mature form of SUMO1 and the conjugation-deficient mutant of SUMO1, respectively. B) In vivo SUMOylation assays showing SIZ1-mediated HLS1 SUMOylation in vivo. HA-HLS1 and MYC-SUMO1 constructs were transiently transfected into Arabidopsis protoplasts prepared from Col-0 or siz1-2. Total proteins were extracted and then incubated with HA agarose beads. Anti-HA and anti-MYC antibodies were used to examine HLS1 SUMOylation. C) SUMOylation of HLS1 in HLS1OE, HLS1OE siz1-2, HLS1^K186ROE, and HLS1^6KROE. Total proteins were extracted from 3-d-old dark-grown seedlings and then incubated with HA agarose beads. Anti-HA and anti-SUMO1 antibodies were used to examine HLS1 SUMOylation. D) In vitro SUMOylation assays showing the effects of point mutations of predicted SUMOylation sites on the SUMOylation of HLS1. HA-HLS1 and MYC-SUMO1 constructs were transiently transfected into Arabidopsis protoplasts prepared from Col-0. The SUMOylated HLS1(HLS1-S1)/HLS1 ratio was determined using ImageJ, with wild-type HLS1-S1 and HLS1 abundance 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. E) In vivo SUMOylation assays showing the effects of HLS1 point mutations on the SUMOylation of HLS1. HA-HLS1 and MYC-SUMO1 constructs were transiently transfected into Arabidopsis protoplasts prepared from Col-0. Total proteins were extracted and then incubated with HA agarose beads. Anti-HA and anti-MYC antibodies were used to examine HLS1 SUMOylation. F) In vitro SUMOylation assays showing the effects of HLS1 multiple mutations on the SUMOylation of HLS1. N.A., not available. G) In vivo SUMOylation assays showing the effects of HLS1 multiple mutations on the SUMOylation of HLS1. The SUMOylated HLS1(HLS1-S1)/HLS1 ratio was determined using ImageJ, with wild-type HLS1-S1 and HLS1 abundance 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.