Direct Interaction of Arabidopsis Cryptochromes with COP1 in Mediation of Photomorphogenic Development Haiyang Wang, Li-Geng Ma, Jin-Ming Li, Hong-Yu Zhao, Xing Wang Deng

Supplementary Material

Supplemental Figure 1. A working model of cryptochrome signaling. CRY (either CRY1 or CRY2) contains a conserved N-terminal chromophore (flavin) binding domain and a distinct C-terminal extension (CCT). In the model, it is proposed that each CCT can independently interact with the C-terminal WD-40 repeat domain (G) of COP1. In the dark, COP1 is in an active conformation and promotes the degradation of HY5 and other substrates via a 26S proteasome-mediated process. Blue light activates CRY, possibly through a light-driven intramolecular redox reaction (electron transfer from the flavin occurs within the cryptochrome molecule itself), and causes a change in the redox state and/or the conformation of the CCT. The activated CCT (presumably similar to that in the GUS-CCT fusion protein) interacts with COP1 in a different configuration, and rapidly inactivate the E3 ubiquitin ligase activity of COP1, thus abrogating the COP1-dependent degradation of HY5 and other substrates. Abbreviations: F: flavin; N: N-terminus of COP1; C: C-terminus of COP1; Zn: Ring-finger domain; Coil: Coiled-coil domain; G: WD-40 repeat domain.

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Methods

Yeast two-hybrid assay. The CCT1 and CCT2 coding regions were obtained via PCR and cloned into the yeast two-hybrid vectors pEG202 and pJG4-5 (1) to generate LexA-CCT1, LexA-CCT2, AD-CCT1 and AD-CCT2. The LexA- and AD- GUS-CCTs fusion constructs were generated by adding the GUS gene coding region inframe. The AD-COP1 and its various deletion forms were described in (2). The various yeast two-hybrid assay clones for subunits of the COP9 signalosome, the procedures for yeast transformation, mating and liquid assay were described in (3).

Experimental procedure for in vivo co-immunoprecipitation. Dark-grown (three days old) Arabidopsis seedlings were briefly treated with blue light (16.2 mol m^-2s^-1) and then processed for co-immunoprecipitation assay in a buffer containing 50 mM Tris, pH7.5, 150 mM NaCl, 10 mM MgCl₂, 0.1% NP-40, 1 mM PMSF and 1X complete protease inhibitors (Roche). A CRY2 antibody (provided by C. Lin, Univ. California, Los Angeles) and protein A-agarose beads (Sigma) were used to precipitate the immunoprotein complex. SDS-PAGE gel and western blot analysis were performed according to standard procedures.

Transient gene expression in onion epidermal cells. The GFP-CCT1 expression plasmid was generated by cloning the CCT1 coding region into the vector pRTL2-S65TGFP (2). The expression plasmids for GFP-COP1 and the nontagged COP1, as well as the experimental procedures for transient expression in onion epidermal cells were described in (2).

Generation of the GUS-CCTs transgenic plants. The CCT1 and CCT2 coding regions were cloned into the vector pRTL2-GUS/NIa (4) to replace the NIa coding region to generate pRTL2-GUS-CCT1 and pRTL2-GUS-CCT2. The expression cassettes GUS-CCT1 and GUS-CCT2 driven by the CaMV 35S promoter were excised from pRTL2-GUS-CCT1 and pRTL2-GUS-CCT2 and cloned into the plant transformation binary vector pPZP222 (5). Stable transformation of Arabidopsis was conducted as described in (6). Transgenic seedlings were selected with Gentamycin (100ug/ml; Sigma). Twelve and fourteen independent lines were generated for GUS-CCT1 and GUS-CCT2, respectively. T2 seedlings display the cop-like phenotype were chosen and used in this study.

Microarray. We constructed a microarray containing 9216 Arabidopsis ESTs, representing about 6120 unique genes (7). To minimize the inherent variability of the microarray assay and to ensure the reproducibility of our results, at least two microarray slides (four replicates) were used to analyze the mRNA abundance of each sample pair. The first slide was probed with cDNAs labeled with Cy-3 dUTP and Cy-5 dUTP. The labeling of the sample pair was reversed on the second slide to overcome potential artifacts due to the dye-related differences, different laser settings, and nonlinearities of photomultiplier tubes in the scanner. The hybridization signals from each replicated ESTs were averaged and used for analysis. The correlation coefficient of the ratios from the replicates is higher than 0.95, suggesting an excellent reproducibility among individual arrays in the same experiment. For the details of microarray slide preparation, RNA isolation, probe labeling, hybridization, washing, scanning, and the programs for data analysis, see (7) and http://info.med.yale.edu/wmkeck/dna_arrays.htm.

References and Notes

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7. L. Ma, et al., Plant Cell (2001, In Press).