Unraveling the molecular circuits controlling plant cryptochrome activity
Final Report Abstract
The mechanisms of activation and inactivation of plant cryptochrome (CRY) blue light photoreceptors were only partially understood at the beginning of this project. With regard to activation, it was known that the flavin (FAD) cofactor of cryptochromes, which is bound to the Photolyase Homology Region (PHR) domain, is present in its fully oxidized form in the ground state and, after light absorption, changes into the semi-reduced form (FADHo), which represents the lit-state of this photoreceptor. The electron and proton transfer pathways required for this were analyzed in detail. It was also known that cryptochromes are phosphorylated in the lit-state by photoregulatory protein kinases (PPK) and subsequently interact with numerous signaling components, of which only COP1, SPAs and CIBs may be mentioned here as examples. Furthermore, a number of studies have shown that cryptochromes oligomerize after light excitation. Oligomerization is presumably the prerequisite for all the above-mentioned signal transduction processes. Furthermore, it was known that the inactivation of cryptochromes involves at least two processes, namely the transfer of the electron from FADHo to molecular oxygen with the formation of ROS (superoxide radical, hydrogen peroxide), as well as the binding of Blue Light Inhibitor of Cryptochrome proteins (BIC1 and BIC2 in Arabidopsis) to CRY, which prevent oligomerization or promote the transition of the oligomer to the monomer. The studies carried out in this project with recombinant proteins and cryoEM showed that the CRY1 oligomer is a tetramer. Interestingly, the oligomerization of CRY1 could be induced not only by light but also by hydrogen peroxide. A previously described hyperactive mutant of CRY1 (cry1L407F) was already an oligomer in the dark state. This underlines the statement that CRY is only biologically active in the oligomeric state. We also found that recombinant BIC1 binds covalently heme to a cysteine (Cys99). However, only a small proportion of BIC1 was occupied by heme. Independently of this, we initially postulated that BIC1 possibly functions as a peroxidase and inactivates the hydrogen peroxide formed during dark reversion of CRY. However, this assumption was not supported by extensive studies conducted by us. Furthermore, a mutant of BIC1 in which all cysteines were replaced by alanine (BIC3CA) was biologically active after overexpression in Arabidopsis plants, suggesting that the heme binding observed in vitro is not required for the biological activity of BIC1.
