All mutants used in this study are in the Arabidopsis thaliana Columbia ecotype background. The cry1cry2 double mutant were described as previously32 (link). The lrb1lrb2-1lrb3 (lrb1, Salk_145146; lrb2-1, Salk_001013; lrb3, Salk_082868) and lrb1lrb2-2lrb3 (lrb2-2, Salk_044446) triple mutants were gifts from Dr. Peter Quail and as described previously30 (link),31 (link). cop1-4 and cop1-6 are weak mutant alleles of COP1 as previously described50 (link). cry1cry2lrb1lrb2-2lrb3 and lrb1lrb2-2lrb3cop1-4 mutants were generated by crossing. The genotypes of lrb123 mutants were verified by PCR using the primers listed in Supplementary Table 2 . cop1-4 mutant was verified by PCR using the primers listed in Supplementary Table 2 , followed by Sanger sequencing to confirm the point mutation of COP1 gene. cry1cry2 mutant was verified by western blots using antibodies against CRY1 and CRY2 proteins.
All transgenic lines were generated via Agrobacterium tumefaciens–mediated floral-dip method51 (link). The wild-type plants used for transformation in this study are rdr6-11, which suppresses gene silencing52 (link). For in vivo ubiquitination study, FGFP-CRY2 was introduced into rdr6-11, lrb1lrb2-1lrb3, lrb1lrb2-2lrb3, cop1-4 and cop1-6 background. The FGFP-CRY2/Myc-LRB1 double-overexpression lines were prepared by introducing FGFP-CRY2 into Myc-LRB1/rdr6-11 plants. The transgenic T1 populations were screened on MS plates containing 25 mg/L Glufosinate-ammonium (cat # CP6420, Bomei Biotechnology) and 25 mg/L hygromycin (cat # 10843555001, Roche), and western blots were performed to confirm the expression of both proteins. The same method was used for generating FGFP-CRY2/Myc-LRB2 and FGFP-CRY2/Myc-COP1 double-overexpression lines. For experiments comparing the hypocotyl phenotype and protein degradation kinetics of FGFP-CRY2 and FGFP-CRY2P532L, FGFP-CRY2 and FGFP-CRY2P532L were introduced into cry1cry2rdr6 background. The cry1cry2rdr6 were generated by crossing cry1-30432 (link), cry2-112 (link), and rdr6-1152 (link). The transgenic lines were screened on MS plates with 25 mg/L Glufosinate-ammonium, and lines with similar protein expression level of FGFP-CRY2 and FGFP-CRY2P532L were used for analysis. For lrb123 mutant blue-light hypersensitivity phenotype rescue experiments, FGFP-LRB2 and Myc-LRB2 were transformed into lrb1lrb2-2lrb3 background.
For routine maintenance, Arabidopsis thaliana were grown under long day conditions (16 h light / 8 h dark) at 22 °C. For hypocotyl phenotype analysis, seedlings were grown on MS plates with 3% sucrose at 20–22 °C for 6 days under different light conditions. Light-emitting diode (LED) was used to obtain monochromatic light (blue light, peak 465 nm, half-bandwidth of 25 nm; red light, peak 660 nm, half-bandwidth of 20 nm; far-red light, peak 735 nm, half-bandwidth of 21 nm). For endogenous CRY2 degradation analysis in WT, lrb123, cop1 and lrb123cop1 mutants, seedlings were grown in darkness on MS plates with 3% sucrose for 7 days, then subjected to 30 μmol m−2 s−1 blue light for the indicated time. For FGFP-CRY2 and FGFP-CRY2P532L degradation analysis, seedlings were grown in darkness on MS plates with 3% sucrose for 7 days, then subjected to 100 μmol m−2 s−1 blue light for the indicated time. For immunoprecipitation of polyubiquitinated proteins, 7-day-old etiolated seedlings grown on MS medium containing 3% sucrose were treated with 50 μM MG132 (Cat # S2619, Selleck) in liquid MS in the dark overnight with gentle shaking, and then moved to 30 μmol m−2 s−1 blue light for 5, 10, and 15 min before harvest.
All transgenic lines were generated via Agrobacterium tumefaciens–mediated floral-dip method51 (link). The wild-type plants used for transformation in this study are rdr6-11, which suppresses gene silencing52 (link). For in vivo ubiquitination study, FGFP-CRY2 was introduced into rdr6-11, lrb1lrb2-1lrb3, lrb1lrb2-2lrb3, cop1-4 and cop1-6 background. The FGFP-CRY2/Myc-LRB1 double-overexpression lines were prepared by introducing FGFP-CRY2 into Myc-LRB1/rdr6-11 plants. The transgenic T1 populations were screened on MS plates containing 25 mg/L Glufosinate-ammonium (cat # CP6420, Bomei Biotechnology) and 25 mg/L hygromycin (cat # 10843555001, Roche), and western blots were performed to confirm the expression of both proteins. The same method was used for generating FGFP-CRY2/Myc-LRB2 and FGFP-CRY2/Myc-COP1 double-overexpression lines. For experiments comparing the hypocotyl phenotype and protein degradation kinetics of FGFP-CRY2 and FGFP-CRY2P532L, FGFP-CRY2 and FGFP-CRY2P532L were introduced into cry1cry2rdr6 background. The cry1cry2rdr6 were generated by crossing cry1-30432 (link), cry2-112 (link), and rdr6-1152 (link). The transgenic lines were screened on MS plates with 25 mg/L Glufosinate-ammonium, and lines with similar protein expression level of FGFP-CRY2 and FGFP-CRY2P532L were used for analysis. For lrb123 mutant blue-light hypersensitivity phenotype rescue experiments, FGFP-LRB2 and Myc-LRB2 were transformed into lrb1lrb2-2lrb3 background.
For routine maintenance, Arabidopsis thaliana were grown under long day conditions (16 h light / 8 h dark) at 22 °C. For hypocotyl phenotype analysis, seedlings were grown on MS plates with 3% sucrose at 20–22 °C for 6 days under different light conditions. Light-emitting diode (LED) was used to obtain monochromatic light (blue light, peak 465 nm, half-bandwidth of 25 nm; red light, peak 660 nm, half-bandwidth of 20 nm; far-red light, peak 735 nm, half-bandwidth of 21 nm). For endogenous CRY2 degradation analysis in WT, lrb123, cop1 and lrb123cop1 mutants, seedlings were grown in darkness on MS plates with 3% sucrose for 7 days, then subjected to 30 μmol m−2 s−1 blue light for the indicated time. For FGFP-CRY2 and FGFP-CRY2P532L degradation analysis, seedlings were grown in darkness on MS plates with 3% sucrose for 7 days, then subjected to 100 μmol m−2 s−1 blue light for the indicated time. For immunoprecipitation of polyubiquitinated proteins, 7-day-old etiolated seedlings grown on MS medium containing 3% sucrose were treated with 50 μM MG132 (Cat # S2619, Selleck) in liquid MS in the dark overnight with gentle shaking, and then moved to 30 μmol m−2 s−1 blue light for 5, 10, and 15 min before harvest.
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