Blue-light-dependent interaction of cryptochrome 1 with SPA1 defines a dynamic signaling mechanism

Abstract

Cryptochromes (CRYs) are blue-light photoreceptors that mediate various light responses in plants and animals. The signaling mechanism by which CRYs regulate light responses involves their physical interactions with COP1. Here, we report that CRY1 interacts physically with SPA1 in a blue-light-dependent manner. SPA acts genetically downstream from CRYs to regulate light-controlled development. Blue-light activation of CRY1 attenuates the association of COP1 with SPA1 in both yeast and plant cells. These results indicate that the blue-light-triggered CRY1-SPA1 interaction may negatively regulate COP1, at least in part, by promoting the dissociation of COP1 from SPA1. This interaction and consequent dissociation define a dynamic photosensory signaling mechanism.

Figure 1.

CRY interacts with SPA in a blue-light-dependent manner in yeast cells. (A) Yeast two-hybrid bait constructs. All proteins are fused with the LexA DNA-binding domain (LexA). (SNT1) N-terminal domain of SPA1; (SCT) C-terminal domain of SPA. (B) Yeast two-hybrid prey constructs. All proteins are fused with the B42 activation domain (AD). (C) Yeast two-hybrid analyses of the CRY–SPA interactions and the interactions of GUS-CCT1 with SPA under blue light (BL) and in darkness (DK). (D,E) Quantitative yeast two-hybrid analyses of the CRY–SPA1 interactions under the indicated fluence rates of blue light (D) and 20 μmol/m²/sec blue light for the indicated exposure time (E). Data are mean ± SD (n = 12).

Figure 2.

Colocalization of CRY1 and CRY2 with SPA1 in onion epidermal cells. Onion peels were cobombarded with the DNA constructs indicated. CRY1 and CRY2 localize together with SPA1 to the NBs in onion epidermal cells. (Dic) Differential interference contrast in light microscope mode. Bars, 10 μm.

Figure 3.

CRY1 interacts with SPA1 in a blue-light-dependent manner in vivo. (A–C) Co-IP using anti-CCT1 antiserum in the extracts from Myc-SPA1/spa1 seedlings grown under continuous white light for 5 d and then transferred to darkness for another 3 d (A,B), or seedlings grown in continuous darkness for 6 d (C) before being exposed to the indicated light conditions for 10 h (A), the indicated fluence rates of blue light (B), or 10 μmol/m²/sec blue light for the indicated exposure time (C). (D) Darkness; (B) blue light (30 μmol/m²/sec); (R) red light (30 μmol/m²/sec); (FR) far-red light (5 μmol/m²/sec). The immunoprecipitates were probed with anti-CCT1 and anti-Myc antibodies, respectively.

Figure 4.

SPA acts genetically downstream from CRY to regulate light-controlled development. (A) Wild-type (WT), cry1, spa1 spa2 spa3 spa4 (spa1234), and cry1 spa1 spa2 spa3 spa4 (cry1spa1234) mutant seedlings grown in blue light (30 μmol/m²/sec) for 6 d. Bars, 2 mm. (B) Wild-type (WT), cry2, spa1 spa2 spa3 (spa123), cry2 spa1 spa2 spa3 (cry2spa123), spa1 spa3 spa4 (spa134), and cry2 spa1 spa3 spa4 (cry2spa134) mutant seedlings grown in blue light (3 μmol/m²/sec) for 6 d. Bars, 2 mm. (C,D) Measurements of hypocotyl lengths of the genotypes of plants shown in A and B, respectively. Data are mean ± SD (n = 30). (E) Differential interference contrast (Dic) images of the abaxial cotyledon epidermis of 7-d-old seedlings with indicated genotypes grown in continuous blue light (10 μmol/m²/sec). Stomatal index (percentages) are listed in each panel. Data are mean ± SD (n = 10). Bars, 20 μm. (F) The flowering phenotype of the plants with indicated genotypes in long days (LDs; 16 h light/8 h dark). Bars, 2 cm. Flowering time was measured as days to flowering and the number of leaves at bolting. Data are mean ± SD (n ≥ 30).

Figure 5.

CRY1 interferes with the COP1–SPA1 interaction under blue light in both yeast and plant cells. (A) Yeast three-hybrid constructs expressing both the bait and the bridge proteins. (BD) DNA-binding domain of GAL4; (P_Met25) inducible promoter driving the expression of the bridge protein; (CC1) coiled-coil domain of COP1; (SCC1) coiled-coil domain of SPA1; (mCRY1) mock CRY1 with a frameshift in CRY1 that does not express CRY1. (B–D) Quantitative yeast three-hybrid analyses showing the effects of CRY1 on the interactions of COP1–SPA1 (B), CC1–SPA1 (C), and SCC1–COP1 (D) under the indicated fluence rates of blue light. The interaction values (Miller units) in darkness were set to 100%, and the relative interaction under blue light was expressed as the percentage of the dark value. Data are mean ± SD (n = 4). (E,F) Co-IP using anti-Myc agarose beads in the extracts from Myc-SPA1/spa1 seedlings (CRY1 present) (E) and Myc-SPA1/cry1 seedlings (CRY1 absent) (F) grown in continuous darkness for 6 d before being exposed to 50 μmol/m²/sec blue light for the indicated time. The immunoprecipitates were probed with anti-COP1, anti-Myc, and anti-CUL1 antibodies, respectively. Relative band intensities were normalized for each panel and are shown below each lane. (G) Mode of CRY1 action. Photoactivation of CRY1 by blue-light irradiation may lead to a change in the conformation of SPA1 and COP1 through physical interactions, as a result of which COP1 dissociates from SPA1, its E3 ligase activity is attenuated, and HY5 is able to accumulate and promote photomorphogenesis.