In Arabidopsis thaliana, the blue light photoreceptor cryptochromes (CRY) act to promote photomorphogenic development and the transition from vegetative to floral development in long days (LDs). We previously proposed that one of the mechanisms by which CRY regulates light responses is via its physical interaction with COP1, a RING motif-containing E3 ligase. Under LDs, the transcription of FLOWERING LOCUS T (FT) is activated by CONSTANS (CO) in leaf, and the FT protein moves to the shoot apex to induce flowering. CO protein is degraded in darkness, whereas it is stabilized by the CRYmediated signal. However, the mechanism underlying this process is unknown. We show in this report that CO acts genetically downstream of COP1 and CRY to regulate flowering time. In addition, COP1 physically interacts with CO and functions as an E3 ligase, ubiquitinating CO in vitro and reducing CO levels in vivo. These results suggest that COP1 acts as a repressor of flowering by promoting the ubiquitin-mediated proteolysis of CO in darkness and that CRY-mediated signal may negatively regulate COP1, thereby stabilizing CO, activating FT transcription, and inducing flowering.

blue light photoreceptor ͉ phototropin ͉ water evaporation ͉ photosynthesis T he stomatal pores of higher plants act as ports that tightly regulate the uptake of CO 2 for photosynthesis and the evaporation of water for transpiration. Situated in the epidermis, they are surrounded by a pair of guard cells, which regulate their opening in response to environmental and internal signals, including light, humidity, CO 2 , phytohormones, calcium, and reactive oxygen species (1-5). Stomata are closed in darkness but open in response to blue light.Blue light responses are primarily mediated by four blue light photoreceptors in Arabidopsis: cryptochrome (CRY)1, CRY2, phototropin (PHOT)1, and PHOT2. Major blue light responses mediated by CRY1 and CRY2 include inhibition of hypocotyl elongation (6-8), enhancement of cotyledon expansion (9), anthocyanin accumulation (8, 10, 11), and regulation of flowering time (12-15). CRY1 and CRY2, together with the red͞ far-red light receptor phytochromes, also serve to entrain the circadian clock (16). There is now evidence for a third CRY (CRY3) in Arabidopsis, the role of which is presently unknown (17). PHOT1 and PHOT2 work together to mediate phototropism, blue light-induced chloroplast migration, and blue lightdependent regulation of stomatal opening (18)(19)(20)(21)(22). Recent studies have shown that CRY and PHOT perform overlapping roles. For examples, PHOT functions at early stages to regulate photomorphogenic development, including rapid inhibition of hypocotyl elongation (23) and enhancement of cotyledon expansion (24), and CRY and PHOT function together to enhance phototropism under low fluence rate blue light (25).Insight into the signaling mechanism of Arabidopsis CRY was obtained through the demonstration that transgenic plants expressing the C-terminal domain of either CRY1 (CCT1) or CRY2 (CCT2) fused to ␤-glucuronidase (GUS) display a constitutive photomorphogenic (COP) phenotype (11), which is similar to that of mutants of both COP1 and COP9 signalosome, the negative regulators of photomorphogenesis (26,27). Both CCT1 and CCT2 were shown to bind to COP1 (28,29), indicating that the signaling mechanism of Arabidopsis CRY1 and CRY2 is mediated through negative regulation of COP1 by direct CRY-COP1 interaction. It is now demonstrated that Arabidopsis CRY1 N-terminal domain mediates homodimerization, which is required for light activation of CCT1 (30).The purpose of the present study was to determine the role of the Arabidopsis CRY and COP1 signaling system in the regulation of stomatal opening. Through molecular, genetic, and physiological analyses, we demonstrate that CRY acts additively with PHOT to mediate blue light-induced stomatal opening and that COP1 is a repressor of stomatal opening and likely acts downstream of CRY and PHOT signaling pathways. Materials and MethodsExperimental procedures of construction of expression cassettes and transformation, antibody production, PCR, Western blot, and construction of the various double, triple, and quadruple mutants can b...