Abstract:

Arabidopsis cryptochromes are blue/UV-A light receptors that mediate blue light inhibition of hypocotyl elongation and photoperiodic promotion of floral initiation. Sequence analyses show that the majority of plant cryptochromes are composed of two domains, the N-terminal photolyase-related domain (PHR) and the C-terminal domain. The PHR domain is the site of chromophore binding, while the C-terminal functions in nuclear/cytoplasmic localization and protein-protein interaction. In this work we demonstrate that blue light-dependent cryptochrome 1 (CRY1) phosphorylation is closely associated with the function or regulation of the photoreceptor and that the overall structure of CRY1 is critical to its phosphorylation. An analysis of in-vivo behaviors of the full-length cryptochrome 2 CRY2 fused to GFP at either the N-terminal (GFP-CRY2) or the C-terminal (CRY2-GFP) showed that while GFP-CRY2 exerts light-dependent biochemical and physiological activities, CRY2-GFP is constitutively active. CRY2-GFP is constitutively phosphorylated, it promotes de-etiolation in dark and light conditions and it promotes floral initiation. It readily forms nuclear bodies in response to light while formation of nuclear foci in GFP-CRY2 could be promoted only in the presence of a 26S proteasome inhibitor, suggesting that the accumulation of photo-excited CRY2 in nuclear speckles is associated with light-induced degradation of the photoreceptor. An assessment of CRY2 nuclear bodies formation patterns suggests that these foci are a diverse and highly responsive population that changes in form, number and size as conditions change. Nuclear bodies evolve throughout time, with a suggested trend of cycling that can be observed after prolonged time exposure. The association of CRY2 nuclear bodies with degradation via the 26S proteasome complex seems to be legitimate after transcription and translation inhibitors produced no effect on the formation of nuclear domains and their pattern of distribution. Three of the examined lysine-to-arginine ubiquitin-conjugate mutants, showed interesting phenotypes with suggested modification of CRY2 activity, especially in K612R where an apparent reduction in activity, compared to the parental CRY2-GFP line, can be observed.