Abstract
Plants constantly adjust their growth, development and metabolism to the ambient light environment. Blue light is sensed by the Arabidopsis photoreceptors CRY1 and CRY2 which subsequently initiate light signal transduction by repressing the COP1/SPA E3 ubiquitin ligase. While the interaction between cryptochromes and SPA is blue light-dependent, it was proposed that CRY1 interacts with COP1 constitutively, i.e. also in darkness. Here, our in vivo co-immunoprecipitation experiments suggest that CRY1 and CRY2 form a complex with COP1 only after seedlings were exposed to blue light. No association between COP1 and CRY1 or CRY2 was observed in dark-grown seedlings. Thus, our results suggest that cryptochromes bind the COP1/SPA complex after photoactivation by blue light. In a spa quadruple mutant that is devoid of all four SPA proteins, CRY1 and COP1 did not interact in vivo, neither in dark-grown nor in blue light-grown seedlings. Hence, SPA proteins are required for the high-affinity interaction between CRY1 and COP1 in blue light. Yeast three-hybrid experiments also show that SPA1 enhances the CRY1-COP1 interaction. The coiled-coil domain of SPA1 which is responsible for COP1-binding was necessary to mediate a CRY1-SPA1 interaction in vivo, implying that—in turn—COP1 may be necessary for a CRY1-SPA1 complex formation. Hence, SPA1 and COP1 may act cooperatively in recognizing and binding photoactivated CRY1. In contrast, the blue light-induced association between CRY2 and COP1 was not dependent on SPA proteins in vivo. Similarly, ΔCC-SPA1 interacted with CRY2, though with a much lower affinity than wild-type SPA1. In total, our results demonstrate that CRY1 and CRY2 strongly differ in their blue light-induced interaction with the COP1/SPA complex.
Highlights
Plants constantly adjust their growth and development to the ambient light environment
Blue light activates two cryptochrome photoreceptors which subsequently inactivate an E3 ubiquitin ligase consisting of CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) and SUPPRESSOR OF PHYA-105 (SPA) proteins
To investigate whether cryptochromes associate with COP1 in a B-dependent manner in planta, we carried out co-immunoprecipitation studies using transgenic Arabidopsis seedlings expressing YFP-COP1 (35S::YFP-COP1) [32]
Summary
Plants constantly adjust their growth and development to the ambient light environment. Plants have evolved several sets of photoreceptors which include the blue lightabsorbing cryptochromes, phototropins and the ZEITLUPE family, the red/far-red responsive phytochromes and the UV-B photoreceptor UVR8. These photoreceptors regulate growth and development throughout the plant life cycle, such as seed germination, seedling deetiolation, shade avoidance and the induction of flowering. The CRY1 and CRY2 photoreceptors carry two chromophores that are responsible for light absorption, the primary chromophore FAD and a pterin as a second chromophore These chromophores are attached to the highly conserved N-terminal domain of the cryptochrome apoproteins, the Photolyase Homologous Region (PHR). The CRY C-terminal Extensions (CCE) in CRY1 and CRY2 are only loosely conserved but important for cryptochrome signaling activities [4,5,6]
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