Arabidopsis cryptochrome 2

Here we present the design and evaluation of four strategies for light-inducible activation of TrkA in the absence of NGF. Our strategies involve the light-sensitive protein Arabidopsis cryptochrome 2 and its binding partner CIB1.

These results demonstrate a CRY-BIC negative-feedback circuitry that regulates the activity of each other.

CRY2 and CIB1, Arabidopsis proteins that interact upon light illumination

Molecular basis for blue light-dependent phosphorylation of Arabidopsis cryptochrome 2

The nuclear clearing phenotype was dependent on the presence of a dimerization domain contained within the CRY2-fused transcriptional activators.

by suppressing the activity of CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1), resulting in activation of the transcription activator ELONGATED HYPOCOTYL 5 (HY5) that is associated with chromatins of the BIC promoters

While adopting this approach to regulate transcription in mammalian cells, we observed light-dependent redistribution and clearing of CRY2-tethered proteins within the nucleus.

suggesting a novel photoreceptor co-action mechanism to sustain blue light sensitivity of plants under the broad spectra of solar radiation in nature.

two negative regulators of Arabidopsis cryptochromes, Blue light Inhibitors of Cryptochromes 1 and 2 (BIC1 and BIC2), inhibit cryptochrome function by blocking blue light-dependent cryptochrome dimerization

Here we show that cryptochromes mediate light activation of transcription of the BIC genes

We found that Arabidopsis cryptochrome 2 (CRY2) undergoes blue light-dependent homodimerization to become physiologically active.

We hypothesize that regulated dimerization governs homeostasis of the active cryptochromes in plants and other evolutionary lineages.

We identified BIC1 (blue-light inhibitor of cryptochromes 1) as an inhibitor of plant cryptochromes that binds to CRY2 to suppress the blue light-dependent dimerization, photobody formation, phosphorylation, degradation, and physiological activities of CRY2.

enabled through the optical regulation of target receptor clustering and downstream signalling using noncovalent interactions with engineered Arabidopsis Cryptochrome 2 (Cry2)

Thus, the signaling state lifetimes of plant cryptochromes are not, or are only moderately, stabilized in planta.

we estimate the in vivo half-lives of the signaling states of cry1 and cry2 to be in the range of 5 and 16 min, respectively

We found that all trp-triad mutations of CRY2 tested lost photoreduction activity in vitro but retained the physiological and biochemical activities in vivo.

the trp-triad residues are evolutionarily conserved in the photolyase/cryptochrome superfamily for reasons of structural integrity rather than for photochemistry per se

Some of the trp-triad mutations of CRY2 remained responsive to blue light; others, such as CRY2(W374A), became constitutively active.

These results support the hypothesis that cryptochromes mediate blue-light responses via a photochemistry distinct from trp-triad-dependent photoreduction

In contrast to wild-type CRY2, which undergoes blue-light-dependent interaction with the CRY2-signaling proteins SUPPRESSOR OF PHYA 1 (SPA1) and cryptochrome-interaction basic helix-loop-helix 1 (CIB1), the constitutively active CRY2(W374A) interacts with SPA1 and CIB1 constitutively.

These results demonstrate that the active form of Cry2 contains FADH(.) (whereas catalytically active photolyase requires fully reduced flavin (FADH(-))).

suggest that cryptochromes could represent photoreceptors using flavin redox states for signaling differently from DNA-photolyase for photorepair

Our results showed that an 80-residue motif, referred to as NC80, was sufficient to confer the physiological function of CRY2.

Green light irradiation of Cry2 causes a change in the equilibrium of flavin oxidation states and attenuates Cry2-controlled responses such as flowering.

suggesting that the blue light-induced CRY2 phosphorylation causes a conformational change to derepress the NC80 motif

Here we have shown that Arabidopsis Cry2 undergoes a photocycle in which semireduced flavin (FADH(.)) accumulates upon blue light irradiation.

the CRY2 C-terminal tail was found to be required for the blue light-induced CRY2 phosphorylation but not for the CRY2 activity

We propose that the PHR domain and the C-terminal tail of the unphosphorylated CRY2 form a "closed" conformation to suppress the NC80 motif in the absence of light. In response to blue light, the C-terminal tail of CRY2 is phosphorylated and electrostatically repelled from the surface of the PHR domain to form an "open" conformation, resulting in derepression of the NC80 motif and signal transduction to trigger photomorphogenic responses.