Current Research

Introduction

Cryptochromes are blue light receptors found throughout the biological kingdom. They have marked structural similarity to photolyases, a class of flavoprotein that uses light Energy to repair UV-damaged DNA. Unlike photolyases, cryptochromes do not repair DNA but have instead evolved novel roles in signalling. The first cryptochrome gene, cry1, was identified by gene tagging and isolation of the hy4 mutation of Arabidopsis thaliana, which is deficient in blue light sensing. This was the first flavin – type blue light receptor to be identified in any organism (1).

DOMAIN STRUCTURE OF CRYPTOCHROMES AND PHOTOLYASES

Structurally, both cryptochrome and photolyases are globular proteins that bind two light absorbing chromophores, a catalytic flavin chromophore and an antenna pigment that may be either a flavin or a folate derivative in photolyases. Cryptochromes have a c-terminal domain of variable length, that is not found in photolyases and which may play a role in substrate recognition or accessibility (2).

Interest in cryptochromes has been greatly stimulated by the discovery in many organisms including drosophila and humans of related receptors that were found to have important roles in the circadian clock (3). Further interest in cryptochromes has also been recently stimulated by their implication in magnetic sensitivity in a number of organisms (4). These studies are particularly of importance as migratory birds have long been supposed to orient by a magnetic compass based on a blue light absorbing photoreceptor such as found in the cryptochromes.

References:

  1. HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor.
    Ahmad M and Cashmore AR.
    Nature 366, 162-166 (1993).
  2. Photolyase and cryptochrome blue-light photoreceptors.
    Sancar A.
    Adv Protein Chem. 69, 73-100 (2004)
  3. The cryptochromes.
    Lin C, Todo T.
    Genome Biol. 6, 220 (2005)
  4. Chemical magnetoreception in birds: the radical pair mechanism.
    Rodgers CT, Hore PJ.
    Proc Natl Acad Sci U S A. 106(2):353-60 (2009)

Studies on the photocycle of cryptochrome

How does a protein with a domain structure highly similar to photolyases, a DNA repair enzyme, come to function as a photoreceptor? What is the mechanism of light capture and how is this energy transformed into a biological signal by cryptochrome?

To address this question, we have studied the reactions of purified plant cry protein in response to illumination in vitro using a combination of analytical techniques. These include steady state and time resolved UV/vis absorption spectroscopy (6, 25, 30, 31), fluorescence spectroscopy (30,31,35), FTIR (26) and EPR (30,31). These data showed cry is photoreduced to the flavin radical state in response to light by a process involving both intramolecular electron transfer and proton transfer to the oxidized flavin. The light-dependent reactions identified in vitro were related to activation of cryptochrome in vivo by action spectroscopy (20) and physiological studies of plant cryptochrome responses (25,30,31).

Furthermore, direct interconversion of flavin redox states could be confirmed in living cells using a novel application of epr spectroscopy (30,31). Similar photoreactions were found to occur in animal type cryptochromes (drosophila and human), suggesting a common mechanism of light activation (35). Comparative electrochemical studies of photolyase and cry midpoint potentials are in agreement with the proposed mechanism of radical accumulation for cryptochrome activation (36). Finally, a possible role for folate as light sensing antenna pigment in plant cryptochromes has been demonstrated using fluorescence techniques combined with action spectroscopy in vivo (35).

In sum, our studies have shown both similarities and differences beween cryptochromes and their ancestral photolyases in their responsivity to light and in their primary photoreactions. Both photolyases and cryptochromes undergo light dependent electron transfer reactions and flavin photoreduction under appropriate in vitro conditions. The critical difference appears to lie in the fact that oxidized flavin in cryptochrome is converted by light to the radical state, whereas in photolyases flavin is fully reduced by light. Upon return to darkness, cryptochrome bound flavin rapidly reoxidizes whereas in photolyases flavin remains reduced.

Thus, cryptochromes have evolved a mechanism of activation that involves cycling between flavin redox forms. Oxidized flavin is the resting state in darkness and the radical form represents the signaling state.


A model of cry1 photocycle. cry1 exists in three interconvertible redox forms, FAD, FADH*, and FADH-

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The FAD form is inactive and accumulates to high levels in the dark. Blue light triggers photoreduction of FAD to establish a photoequilibrium that favors FADH• over FAD or FADH–. The flavosemiquinone state is the signaling state of the receptor. Green light is absorbed by the radical and shifts the photoequilibrium to the fully reduced form (FADH–), which is inactive. Reversion to oxidized flavin occurs in the dark, involving electron acceptors that are as yet uncharacterized.
Comparison between radical formation in photolyases and cry1. Purified A. thaliana CPD photolyase and cry1 at the identical protein concentration were subject to photoreduction under identical conditions (20 µmol m–2s–1 blue (400–500 nm) light, 2 mM dithiothreitol) and absorption spectra taken at the indicated intervals. Building and decay kinetics of the FADH• absorption are presented at 550 nm for cry1 (open squares) and at 595 nm for photolyase (open circles). a.u., arbitrary units.Bouly, J.-P. et al. J. Biol. Chem. 2007;282:9383-9391

 

Current research interests focus on linking these light dependent changes in the photoreceptor to the signaling process, in particular to structural or surface changes in the receptor that may be important for substrate recognition or productive binding interactions. We are particularly interested in comparative Studies of plant and animal type cryptochromes, as these appear to have independently evolved similar Mechanisms of light activation and photoreceptor function. Studies are currently under way investigating the photocycle of both insect (drosophila) and vertebrate (human and robin) cryptochomes.


Additional Research Interests

1. Studies on ATP binding and autokinase activity of cryptochrome.

We have demonstrated that purified preparations of both plant (Arabidopsis) and animal (human cryptochrome Hscry1) have ATP binding activity and plant cry1 shows autokinase activity that is regulated by both light and flavin redox state (22). Furthermore mutants of Arabidopsis cry1 that fail to undergo light depedent electron transfer also show reduced stimulation of autokinase activity (25).

Current research interests focus on linking this ATP binding and/or autohosphorylation activity to activation of both plant and animal type cryptochromes.


2. Interaction of plant cryptochromes with additional plant photoreceptors

Plants can sense the visible spectrum using a variety of photosensory receptors. In addition to cryptochromes, these include those absorbing in the red and infra-red (phytochromes) and an additional, unrelated class of flavin typeblue light receptor known as phototropins, which were identified initially as receptors for phototropic curvature. Physiological studies showed considerable synergy between phytochrome and cryptochrome signaling pathways (11) and we have observed direct interaction of purified Arabidopsis cry1 and cry2 with phytochrome A in vitro and also in yeast two hybrid interaction studies (15). Furthermore, both cry1 and cry2 C-terminal domains are substrates for phosphorylation by phyA – dependent kinase activity. Both positive and negative interactions between cryptochromes and phototropin dependent signaling pathways have been observed (14,33), although many of these interactions appear to occur at downstream points far removed from the photoreceptors themselves.

Current research interests focus on assessing whether direct interaction between photoreceptors does indeed occur in vivo and could have functional significance.


3. Cryptochromes and plant hormone signaling pathways

In plants, many of the effects of light mimic those of plant hormones, in particular auxins, cytokinins, brassinosteroids, and giberellic acid. It therefore seems likely that light and plant hormone signaling pathways are closely linked and that points of convergence should be identifiable. We have investigated the process of anthocyanin accumulation in Arabidopsis, which is regulated by both cryptochromes and cytokinins. We have found that the transcription factor hy5, which is a positive regulator of cryptochrome signaling, is indeed directly regulated by the plant hormone cytokinin and therefore represents such a point of convergence (29). We further showed that auxin transport is implicated in transmission of the cryptochrome signal from the shoot to stimulate root elongation growth in response to blue light (27).

Current research interests involve in depth analysis for effects of plant hormones on multiple cryptochrome dependent Responses, with the goal of identifiying further possible downstream substrates for cryptochrome function.


4. Cryptochrome and magnetoreception

Orientation in birds is based on a magnetic compass mechanism which requires blue/green light. Cryptochrome has been proposed as the magnetic receptor based on its ability to form radical pairs that may be sensitive to applied magnetic fields. We have shown that plant growth responses in blue light are enhanced in the presence of a static applied field of 10 times higher than the local (earth) field. Furthermore magnetic responsivity depends on the presence of functional cryptochrome (28).

The effects of magnetic field on plant growth responses were observed independently in several different laboratories including in Frankfurt, GE and Paris, FR (28), Irvine, USA (T. Ritz, personal communication) and Marburg, GE (P.

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Galland,personal communication).

Current research interests involve further defining the parameters for magnetic sensitivity in plants by providing a dose response curve at multiple magnetic field strenghs and under different illumination conditions. Parallell studies with purified cryptochrome proteins from plants and birds (robin) are in progress to define possible magnetosensitive photoreactions which

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could be linked to magnetic orientation.