Arabidopsis cryptochrome mediates responses to magnetic fields that have been applied in the absence of light, consistent with flavin reoxidation as the primary detection mechanism. Cryptochromes are highly conserved blue-light-absorbing flavoproteins which have been linked to the perception of electromagnetic stimuli in numerous organisms. These include sensing the direction of the earth's magnetic field in migratory birds and the intensity of magnetic fields in insects and plants. When exposed to light, cryptochromes undergo flavin reduction/reoxidation redox cycles leading to biological activation which generate radical pairs thought to be the basis for magnetic sensitivity. However, the nature of the magnetically sensitive radical pairs and the steps at which they act during the cryptochrome redox cycle are currently a matter of debate. Here, we investigate the response of Arabidopsis cryptochrome-1 in vivo to a static magnetic field of 500 μT (10 × earth's field) using both plant growth and light-dependent phosphorylation as an assay. Cryptochrome responses to light were enhanced by the magnetic field, as indicated by increased inhibition of hypocotyl elongation and increased cryptochrome phosphorylation. However, when light and dark intervals were given intermittently, a plant response to the magnetic field was observed even when the magnetic field was given exclusively during the dark intervals between light exposures. This indicates that the magnetically sensitive reaction step in the cryptochrome photocycle must occur during flavin reoxidation, and likely involves the formation of reactive oxygen species.

Cryptochromes are evolutionarily conserved blue light receptors with many roles throughout plant growth and development. They undergo conformational changes in response to light enabling interaction with multiple downstream signaling partners. Recently, it has been shown that cryptochromes also synthesize reactive oxygen species (ROS) in response to light, suggesting the possibility of an alternate signaling mechanism. Here we show by fluorescence imaging and microscopy that H 2 0 2 and ROS accumulate in the plant nucleus after cryptochrome activation. They induce ROSregulated transcripts including for genes implicated in pathogen defense, biotic and abiotic stress. Mutant cryptochrome alleles that are non-functional in photomorphogenesis retain the capacity to induce ROS-responsive phenotypes. We conclude that nuclear biosynthesis of ROS by cryptochromes represents a new signaling paradigm that complements currently known mechanisms. This may lead to novel applications using blue light induced oxidative bursts to prime crop plants against the deleterious effects of environmental stresses and toxins.Cryptochromes are blue light sensing receptors that regulate multiple processes of plant growth and development, including photomorphogenesis, de-etiolation, flowering initiation, stress response, and hormone signaling 1-3 . They are localized in the nucleus and function both directly and indirectly as core regulators of more than 20% of total cellular transcripts 4-6 . Structurally, cryptochromes are flavoproteins consisting of a conserved N-terminal light sensing domain (designated the PHR domain) and a less well-conserved C-terminal domain (designated CCE) of variable length that is important for nuclear localization and signaling 7 . Upon illumination, Arabidopsis cryptochromes interact with protein partners including phytochromes (PhyA and PhyB), the WD-repeat protein SPA1, the E3 ubiquitin ligase COP1, and bHLH transcription factors CIB 1,2 , PIF3 and PIF4 8,9 which have a signaling role in photomorphogenesis or flowering initiation. Most of these partners attach to the PHR domain and release the CCE domain from the protein surface 10,11 . The ensuing suppression of COP1/SPA1-regulated degradation of transcription factors, such as HFR1 and HY5/HYH, results in photomorphogenesis and interaction with a large network of phytochrome, hormone, and stress signaling pathways [12][13][14][15] .Recently, it has been reported that plant cryptochromes release reactive oxygen species (ROS) including hydrogen peroxide (H 2 O 2 ) and superoxide (O 2• −) subsequent to illumination 16,17 . This follows from reoxidation of the bound flavin cofactor from the light activated radical (FADH°) or reduced (FADH-) redox state back to the oxidized (FADox) resting dark-adapted state 18 . ROS are damaging byproducts of metabolism originating in the chloroplasts, peroxisomes or mitochondria. They are also induced by several environmental stresses and function as core regulators of cellular processes including stress tolerance, root growth...

We report here our systematic studies of the heme dynamics and induced protein conformational relaxations in two redox states of ferric and ferrous cytochrome c upon femtosecond excitation. With a wide range of probing wavelengths from the visible to the UV and a site-directed mutation we unambiguously determined that the protein dynamics in the two states are drastically different. For the ferrous state the heme transforms from 6-fold to 5-fold coordination with ultrafast ligand dissociation in less than 100 fs, followed by vibrational cooling within several picoseconds, but then recombining back to its original 6-fold coordination in 7 ps. Such impulsive bond breaking and late rebinding generate proteinquakes and strongly perturb the local heme site and shake global protein conformation, which were found to completely recover in 13 and 42 ps, respectively. For the ferric state the heme however maintains its 6-fold coordination. The dynamics mainly occur at the local site, including ultrafast internal conversion in hundreds of femtoseconds, vibrational cooling on the similar picosecond time scale, and complete ground-state recovery in 10 ps, and no global conformation relaxation was observed.

We report here our systematic characterization of resonance energy transfer between intrinsic tryptophan and the prosthetic heme group in myoglobin in order to develop a novel energy-transfer pair as a molecular ruler in heme proteins to study local conformation fluctuations. With site-directed mutagenesis, we designed four tryptophan mutants along the A-helix of myoglobin and each mutant contains only a single tryptophan-heme energy-transfer pair. With femtosecond resolution, we observed, even at separation distances of 15-25 A, ultrafast energy transfer in tens to hundreds of picoseconds. On these time scales, the donor and acceptor cannot be randomized and the orientation factor in Forster energy transfer is highly restricted. Thus, direct measurement of the orientation-factor changes at different mutation sites reveals relative local structure flexibility and conformation fluctuations as particularly demonstrated here for positions of tryptophan 7 and 14. More importantly, the local environment relaxation occurs on the similar time scales of the energy transfer dynamics, resulting in a nonequilibrium dynamic process. With femtosecond- and wavelength-resolved fluorescence dynamics, we are able to determine the time scales of such nonequilibrium energy-transfer dynamics and elucidate the mechanism of the nonexponential energy-transfer dynamics caused by local dynamic heterogeneity and/or local environment relaxation.

Cryptochromes are flavoprotein photoreceptors with multiple signaling roles during plant de-etiolation and development. Arabidopsis cryptochromes (cry1 and cry2) absorb light through an oxidized flavin (FADox) cofactor which undergoes reduction to both FADH° and FADH− redox states. Since the FADH° redox state has been linked to biological activity, it is important to estimate its concentration formed upon illumination in vivo. Here we model the photocycle of isolated cry1 and cry2 proteins with a three-state kinetic model. Our model fits the experimental data for flavin photoconversion in vitro for both cry1 and cry2, providing calculated quantum yields which are significantly lower in cry1 than for cry2. The model was applied to the cryptochrome photocycle in vivo using biological activity in plants as a readout for FADH° concentration. The fit to the in vivo data provided quantum yields for cry1 and cry2 flavin reduction similar to those obtained in vitro, with decreased cry1 quantum yield as compared to cry2. These results validate our assumption that FADH° concentration correlates with biological activity. This is the first reported attempt at kinetic modeling of the cryptochrome photocycle in relation to macroscopic signaling events in vivo, and thereby provides a theoretical framework to the components of the photocycle that are necessary for cryptochrome response to environmental signals.