RFWD2 protein, human

All plasmids used in this study were generated by In-Fusion Cloning methods (https://www.takarabio.com/products/cloning/in-fusion-cloning). The sequences subcloned into plasmids were verified by Sanger sequencing. The mammalian cell expression vectors used in this study were made with pQCMV-Flag-GFP (Supplementary Fig. 4), pQCMV-GFP (Supplementary Fig. 4), and pCMV-Myc^{20 (link)}. pQCMV-Flag-GFP vector was modified from the commercial plasmid pEGFP-N1 (Clontech), which allows the inserted gene expressed under control of the CMV promoter and fused with dual in-frame tags of Flag and GFP. pQCMV-GFP vector was further modified from pQCMV-Flag-GFP by removing the coding sequence of Flag tag. pCMV-Myc vector was described previously^{20 (link)}. To generate plasmids expressing Flag-LRB1, Flag-LRB2, Flag-CRY2, and Flag-CRY2^D387A, the coding sequences (CDS) of all the above genes were amplified either from Arabidopsis cDNA or plasmids made before by PCR, the purified PCR products were then subcloned into SpeI/KpnI-digested pQCMV-Flag-GFP vector through in-fusion. Myc-LRB1, Myc-LRB2, Myc-CRY2, Myc-SPA1, and Myc-COP1 plasmids were prepared by cloning the CDS of the genes into the BamHI site of pCMV-Myc vector. For plasmids expressing HA-PPK1 and HA-PPK1^D267N, the coding sequences of PPK1 and PPK1^D267N were PCR-amplified from plasmids made before by using the primers, of which a 2x HA coding sequence was attached to the 5’ end of forward primer, and introduced into SpeI/KpnI-digested pQCMV-GFP vector by in-fusion. The gene-specific primers for constructs used for expressing recombinant proteins in HEK293T cells are listed in Supplementary Table 2.
The BiFC constructs were built with the vectors described previously^{47 (link)}. The coding sequences of LRB1 (AT2G46260), LRB2 (AT3G61600), CRY2 (AT1G04400), and CRY2^D387A were PCR-amplified and cloned into pDONR/Zeo entry vector through BP reaction (Gateway™ BP Clonase™ II Enzyme mix, Cat. # 11789020, Invitrogen). The entry constructs were then introduced into the destination vector pX-nYFP (N-terminus of YFP fused to C-terminus of genes) or pcCFP-X (C-terminus of CFP fused to N-terminus of genes) by LR reaction (Gateway™ LR Clonase™ II Enzyme mix, Cat. # 11791020, Invitrogen). The gene-specific primers of Gateway cloning for constructs used for BiFC assys are listed in Supplementary Table 2.
The split-LUC constructs were made with the vectors described previously^{48 (link)}. CRY2 were fused to the C-terminus of cLUC (the C-terminus of Luciferase). LRB1ΔBTB were generated by seamlessly connecting two fragments, 1-426 bp and 637-1683 bp of LRB1 coding sequence, with 15 bp overlaps, by infusion cloning method. LRB2ΔBTB were generated with two fragments, 1-432 bp and 751-1683 bp of LRB2 coding sequence through the same method as making LRB1ΔBTB. LRB1ΔBTB and LRB2ΔBTB were then fused to the N-terminus of nLUC (the N-terminus of Luciferase) by in-fusion. The primers for constructs used for Split-LUC assays are listed in Supplementary Table 2.
pFGFP (Supplementary Fig. 5) and pDT1H (Supplementary Fig. 5) binary vectors were used for creating overexpression transgenic lines. The pFGFP binary vector was modified from pCambia3301^{20 (link)}, which allows the interested genes to express under Actin2 promoter and fuse with 2x Flag and GFP tags. The pDT1H binary vector, possessing two independent expression cassettes that allows two genes to assemble into one vector, was modified from previously published vector pDT1^{49 (link)} by replacing the BAR gene with HPT gene to convert basta resistance to hygromycin resistance in plants. The CDS of LRB1, LRB2, CRY2, and CRY2^P532L were cloned into BamHI-digested pFGFP vector to generate FGFP-LRB1, FGFP-LRB2, FGFP-CRY2, FGFP-CRY2^P532L with 2x Flag and GFP tags fused to N-terminus of the genes. The native promoters of LRB1 and LRB2 were cloned into SacI/SpeI-digested FGFP-LRB1 and FGFP-LRB2 to generate ProLRB1::FGFP-LRB1 and ProLRB2::FGFP-LRB2, respectively. The CDS regions of LRB1, LRB2, and COP1 (AT2G32950) were cloned into the BamHI site of pDT1H vector to produce Myc-LRB1, Myc-LRB2, and Myc-COP1 with 4x Myc tags fused to N-terminus of the genes. The primers for constructs used for plant transformation are listed in Supplementary Table 2.

All mutants used in this study are in the Arabidopsis thaliana Columbia ecotype background. The cry1cry2 double mutant were described as previously^{32 (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 previously^{30 (link),31 (link)}. cop1-4 and cop1-6 are weak mutant alleles of COP1 as previously described^{50 (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 method^{51 (link)}. The wild-type plants used for transformation in this study are rdr6-11, which suppresses gene silencing^{52 (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-CRY2^P532L, FGFP-CRY2 and FGFP-CRY2^P532L were introduced into cry1cry2rdr6 background. The cry1cry2rdr6 were generated by crossing cry1-304^{32 (link)}, cry2-1^{12 (link)}, and rdr6-11^{52 (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-CRY2^P532L 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-CRY2^P532L 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.

GST-UBE1, UBcH5b, His-hCOP1, p53-His, Strep-azurin, and MSAC-Ub were over-expressed in E. coli BL21 (DE3) cell cultures and purified as above. The assays were performed in 50 μl reaction buffer containing 25 mM Tris (pH 7.5), 150 mM NaCl, 20 mM MgCl₂, 5 mM ATP, 0.5 μM purified GST-UBE1, 1 μM purified UBCH5b, 0.5 μM purified hCOP1, 2 μM p53-His, 1 μM fluorescently labeled ubiquitin (UbIR488) and 0, 4, 12, 36, 108 μM Strep-azurin, respectively. Ubiquitin was expressed and purified bearing a MSAC overhang at the N-terminus. The cysteine residue in the overhang was targeted FITC (MedMol, 75350-46-8) dye followed the reported method^{40 (link),45 (link)}. Control experiments of the ubiquitination assay were carried out in three conditions, (1) E1/E2/Ub/p53 in the same concentration as above, (2) E1/E2/Ub/hCOP1, (3) E1/E2/Ub/hCOP1 supplied with wild type azurin at 108 μM. Reactions were incubated at 30 °C for 4 h, stopped by adding protein loading buffer, resolved by SDS-PAGE, and analyzed by using FLA5100 (Typhoon, Fuji, Japan). Each experiment was performed at least two individual times.

The photon‐to‐photon upconversion efficiency can be calculated with ηp−p(%)=∫λupIup(λ)hcdλλincPinchc that describes the utilization rate of incoming infrared photons for visible photons.^[21]h and c are the Planck constant and the speed of light in the vacuum. λ_up and I_up(λ) are the upconversion visible wavelength and its corresponding intensity. λ_inc and P_inc are the incident infrared wavelength and the related irradiation power of interest. If enough band bending is satisfied, the leakage current under the dark contributes to the upconversion luminance under the infrared stimulus. However, in principle, the device can no longer distinguish a low‐intensity signal with upconversion luminance comparable to the leakage luminance, i.e., a poor signal‐to‐noise ratio. In this regard, we reported the maximum upconversion efficiency at which the device maintained linear response (LDR region) and excluded the leakage luminance to conform to the upconversion luminance in response to the infrared stimulus. The same practice was adopted on the OUD managing single‐type charge carriers in the Supporting Information for consistency. The photoluminescence spectrum, transmittance spectrum, absorption spectrum, EQE spectrum, specific detectivity spectrum, brightness, and current density were recorded according to the previous practice.^[26] The AVT can be calculated by the equation of AVT(%)=∫T(λ)P(λ)S(λ)dλ∫P(λ)S(λ)dλ where T, P, S, and λ represent the transmittance spectra, the human photopic response, solar photon flux, and the optical wavelength. A detailed description of the ultrafast pump‐probe spectroscopy can be found in Supporting Information. To evaluate the spatial resolution of the device, the system was set up on the operational stage of the optical microscope (Taiwan Microscope Enterprise). The infrared light source (M780L3, Thorlabs) was first collimated by the adapter (COP1‐B Olympus, Thorlabs) and propagated through the photomasks with a line‐shaped pattern (line spacing 3, 5, and 10 µm). The large‐area, semi‐transparent device revealed the infrared images, which were subsequently captured by the CCD camera for image resolution determination. Actual photos for the overall system setup can be found in the Supporting Information (Figure S13, Supporting Information).