Jean-Pierre Bouly

Cryptochrome Blue Light Photoreceptors Are Activated through Interconversion of Flavin Redox States

Cryptochromes are blue light-sensing photoreceptors found in plants, animals, and humans. They are known to play key roles in the regulation of the circadian clock and in development. However, despite striking structural similarities to photolyase DNA repair enzymes, cryptochromes do not repair double-stranded DNA, and their mechanism of action is unknown. Recently, a blue light-dependent intramolecular electron transfer to the excited state flavin was characterized and proposed as the primary mechanism of light activation. The resulting formation of a stable neutral flavin semiquinone intermediate enables the photoreceptor to absorb green/yellow light (500–630 nm) in addition to blue light in vitro. Here, we demonstrate that Arabidopsis cryptochrome activation by blue light can be inhibited by green light in vivo consistent with a change of the cofactor redox state. We further characterize light-dependent changes in the cryptochrome1 (cry1) protein in living cells, which match photoreduction of the purified cry1 in vitro. These experiments were performed using fluorescence absorption/emission and EPR on whole cells and thereby represent one of the few examples of the active state of a known photoreceptor being monitored in vivo. These results indicate that cry1 activation via blue light initiates formation of a flavosemiquinone signaling state that can be converted by green light to an inactive form. In summary, cryptochrome activation via flavin photoreduction is a reversible mechanism novel to blue light photoreceptors. This photocycle may have adaptive significance for sensing the quality of the light environment in multiple organisms.

Cryptochrome photoreceptors cry1 and cry2 antagonistically regulate primary root elongation in Arabidopsis thaliana

Cryptochromes are blue-light receptors controlling multiple aspects of plant growth and development. They are flavoproteins with significant homology to photolyases, but instead of repairing DNA they function by transducing blue light energy into a signal that can be recognized by the cellular signaling machinery. Here we report the effect of cry1 and cry2 blue light receptors on primary root growth in Arabidopsis thaliana seedlings, through analysis of both cryptochrome-mutant and cryptochrome-overexpressing lines. Cry1 mutant seedlings show reduced root elongation in blue light while overexpressing seedlings show significantly increased elongation as compared to wild type controls. By contrast, the cry2 mutation has the opposite effect on root elongation growth as does cry1, demonstrating that cry1 and cry2 act antagonistically in this response pathway. The site of cryptochrome signal perception is within the shoot, and the inhibitor of auxin transport, 1-N-naphthylphthalamic acid, abolishes the differential effect of cryptochromes on root growth, suggesting the blue-light signal is transmitted from the shoot to the root by a mechanism that involves auxin. Primary root elongation in blue light may thereby involve interaction between cryptochrome and auxin signaling pathways.

ATP Binding Turns Plant Cryptochrome Into an Efficient Natural Photoswitch

Cryptochromes are flavoproteins that drive diverse developmental light-responses in plants and participate in the circadian clock in animals. Plant cryptochromes have found application as photoswitches in optogenetics. We have studied effects of pH and ATP on the functionally relevant photoreduction of the oxidized FAD cofactor to the semi-reduced FADH· radical in isolated Arabidopsis cryptochrome 1 by transient absorption spectroscopy on nanosecond to millisecond timescales. In the absence of ATP, the yield of light-induced radicals strongly decreased with increasing pH from 6.5 to 8.5. With ATP present, these yields were significantly higher and virtually pH-independent up to pH 9. Analysis of our data in light of the crystallographic structure suggests that ATP-binding shifts the pKa of aspartic acid D396, the putative proton donor to FAD·−, from ~7.4 to >9, and favours a reaction pathway yielding long-lived aspartate D396−. Its negative charge could trigger conformational changes necessary for signal transduction.

Evidence of a light-sensing role for folate in Arabidopsis cryptochrome blue-light receptors.

Arabidopsis cryptochromes cry1 and cry2 are blue-light signalling molecules with significant structural similarity to photolyases--a class of blue-light-sensing DNA repair enzymes. Like photolyases, purified plant cryptochromes have been shown to bind both flavin and pterin chromophores. The flavin functions as a light sensor and undergoes reduction in response to blue light that initiates the signalling cascade. However, the role of the pterin in plant cryptochromes has until now been unknown. Here, we show that the action spectrum for light-dependent degradation of cry2 has a significant peak of activity at 380 nm, consistent with absorption by a pterin cofactor. We further show that cry1 protein expressed in living insect cells responds with greater sensitivity to 380 nm light than to 450 nm, consistent with a light-harvesting antenna pigment that transfers excitation energy to the oxidized flavin of cry1. The pterin biosynthesis inhibitor DHAP selectively reduces cryptochrome responsivity at 380 nm but not 450 nm blue light in these cell cultures, indicating that the antenna pigment is a folate cofactor similar to that of photolyases.

Dealing with light: The widespread and multitasking cryptochrome/photolyase family in photosynthetic organisms ☆

Light is essential for the life of photosynthetic organisms as it is a source of energy and information from the environment. Light excess or limitation can be a cause of stress however. Photosynthetic organisms exhibit sophisticated mechanisms to adjust their physiology and growth to the local environmental light conditions. The cryptochrome/photolyase family (CPF) is composed of flavoproteins with similar structures that display a variety of light-dependent functions. This family encompasses photolyases, blue-light activated enzymes that repair ultraviolet-light induced DNA damage, and cryptochromes, known for their photoreceptor functions in terrestrial plants. For this review, we searched extensively for CPFs in the available genome databases to trace the distribution and evolution of this protein family in photosynthetic organisms. By merging molecular data with current knowledge from the functional characterization of CPFs from terrestrial and aquatic organisms, we discuss their roles in (i) photoperception, (ii) biological rhythm regulation and (iii) light-induced stress responses. We also explore their possible implication in light-related physiological acclimation and their distribution in phototrophs living in different environments. The outcome of this structure-function analysis reconstructs the complex scenarios in which CPFs have evolved, as highlighted by the novel functions and biochemical properties of the most recently described family members in algae.

Conformational change induced by ATP binding correlates with enhanced biological function of Arabidopsis cryptochrome.

Cryptochromes are widely distributed blue light photoreceptors involved in numerous signaling functions in plants and animals. Both plant and animal‐type cryptochromes are found to bind ATP and display intrinsic autokinase activity; however the functional significance of this activity remains a matter of speculation. Here we show in purified preparations of Arabidopsis cry1 that ATP binding induces conformational change independently of light and increases the amount and stability of light‐induced flavin radical formation. Nucleotide binding may thereby provide a mechanism whereby light responsivity in organisms can be regulated through modulation of cryptochrome photoreceptor conformation.

Diatom Phytochromes Reveal the Existence of Far-Red-Light-Based Sensing in the Ocean

Diatom phytochromes (DPH) display high sensitivity to far-red light in the far-red poor aquatic environment, opening new perspectives on signaling mechanisms in the marine realm. The absorption of visible light in aquatic environments has led to the common assumption that aquatic organisms sense and adapt to penetrative blue/green light wavelengths but show little or no response to the more attenuated red/far-red wavelengths. Here, we show that two marine diatom species, Phaeodactylum tricornutum and Thalassiosira pseudonana, possess a bona fide red/far-red light sensing phytochrome (DPH) that uses biliverdin as a chromophore and displays accentuated red-shifted absorbance peaks compared with other characterized plant and algal phytochromes. Exposure to both red and far-red light causes changes in gene expression in P. tricornutum, and the responses to far-red light disappear in DPH knockout cells, demonstrating that P. tricornutum DPH mediates far-red light signaling. The identification of DPH genes in diverse diatom species widely distributed along the water column further emphasizes the ecological significance of far-red light sensing, raising questions about the sources of far-red light. Our analyses indicate that, although far-red wavelengths from sunlight are only detectable at the ocean surface, chlorophyll fluorescence and Raman scattering can generate red/far-red photons in deeper layers. This study opens up novel perspectives on phytochrome-mediated far-red light signaling in the ocean and on the light sensing and adaptive capabilities of marine phototrophs.

AKINβ3, a plant specific SnRK1 protein, is lacking domains present in yeast and mammals non-catalytic β-subunits

The SNF1/AMPK/SnRK1 heterotrimeric kinase complex is involved in the adaptation of cellular metabolism in response to diverse stresses in yeast, mammals and plants. Following a model proposed in yeast, the kinase targets are likely to bind the complex via the non-catalytic β-subunits. These proteins currently identified in yeast, mammals and plants present a common structure with two conserved interacting domains named Kinase Interacting Sequence (KIS) and Association with SNF1 Complex (ASC), and a highly variable N-terminal domain. In this paper we describe the characterisation of AKINβ3, a novel protein related to AKINβ subunits of Arabidopsis thaliana, containing a truncated KIS domain and no N-terminal extension. Interestingly the missing region of the KIS domain corresponds to the glycogen-binding domain (β-GBD) identified in the mammalian AMPKβ1. In spite of its unusual features, AKINβ3 complements the yeast sip1Δsip2Δgal83Δ mutant. Moreover, interactions between AKINβ3 and other AKIN complex subunits from A. thaliana were detected by two-hybrid experiments and in vitro binding assays. Taken together these data demonstrate that AKINβ3 is a β-type subunit. A search for β-type subunits revealed the existence of β3-type proteins in other plant species. Furthermore, we suggest that the AKINβ3-type subunits could be plant specific since no related sequences have been found in any of the other completely sequenced genomes. These data suggest the existence of novel SnRK1 complexes including AKINβ3-type subunits, involved in several functions among which some could be plant specific.

Multisignal control of expression of the LHCX protein family in the marine diatom Phaeodactylum tricornutum

Highlight Multiple stress signalling pathways regulate LHCX family gene expression in the diatom Phaeodactylum tricornutum to attune acclimation responses efficiently in highly variable ocean environments.

Single Amino Acid Substitution Reveals Latent Photolyase Activity in Arabidopsis cry1

Cryptochromes are flavoprotein receptors found throughout the biological kingdom. In vertebrates, cryptochromes function in the circadian clock, are linked to human cancers, and have been proposed as magnetoreceptors in migratory birds. All cryptochromes are characterized by their striking structural similarity to light-activated DNA-repair enzymes, photolyases, despite their widespread occurrence and different signaling roles. Like photolyases, cryptochromes bind a light-absorbing flavin cofactor (FAD) in a hydrophobic pocket and undergo intraprotein electron transfer and photoreduction in response to light. 7] However, unlike photolyases, cryptochromes have known signaling roles in plants and animals and do not repair DNA. The nature of the distinguishing characteristics required for signaling has remained elusive. It has recently been shown that animal and plant cryptochromes accumulate oxidized (OX) flavin in the dark and form the semi-reduced radical form (SR) upon illumination, whereas photolyases under the same conditions accumulate fully reduced anionic flavin (RED) in the dark, which is required for DNA repair. It has also been shown that the SR form of plant and insect cryptochromes is correlated with biological activity. 9,11] Although the functional significance of the flavin oxidation state is still under discussion, a critical difference between cryptochromes with known signaling roles and photolyases that repair DNA is the oxidation state of bound flavin in vivo. Herein, we explore how the flavin redox state may provide a clue as to how plant and animal cryptochromes evolved from ancestral photolyases. Mechanistically, protonation of flavin may result from a conserved amino acid at position 396 of Arabidopsis cry1 (Atcry1), which is a negatively charged aspartic acid (D) residue in all plant cryptochromes, whereas in E. coli and other photolyases this is a neutral asparagine (N) or positively charged lysine (K) residue. In cryptochromes, the D residue at this position has been suggested as a possible proton donor for flavin upon illumination 15] and may explain the difference in redox potentials and hence, the favored flavin redox states. To demonstrate that redox state may indeed be a defining distinction between cryptochromes and photolyases, the mutation D396N was introduced into Atcry1 and the purified recombinant protein isolated from a baculovirus expression system (Figure 1). The purified D396N mutant protein binds OX flavin as is the case for wild-type protein (Figure 1a, panels 1, 2; before illumination t0 = dark). The absorption spectra remain unchanged by the mutation of D to N (Figure 1 a, panels 1, 2), consistent with D396 being protonated in the dark. Upon illumination under aerobic conditions, in the presence of a mild reducing agent (10 mm b-mercaptoethanol (BME)), transition to the SR (FADH8) form of flavin was detected by increased absorbance between 500–600 nm. With further illumination, significant formation of the fully reduced FADH (RED) redox form can be seen because of a continuing decrease in absorbance at 450 nm, without an increase at 500–600 nm (Figure 1a, panels 1, 3). Under these same illumination conditions, wild-type protein was only slightly reduced, failed to accumulate the RED form, and also accumulated far less of the SR form (Figure 1a, panels 2, 4). Thus, illumination of the mutant protein D396N results in formation of the RED flavin, useful for DNA repair, rather than the SR flavin, which is correlated with cryptochrome activity. 9] Another striking difference reported between cryptochromes and photolyases is the marked stability of the RED flavin in photolyases as compared to cryptochromes, where transition to OX occurs rapidly upon the return to darkness after illumination. 14] To determine whether the stability of the RED form is enhanced in the D396N mutant protein, Atcry1 was reduced in the presence of a strong reducing agent (10 mm dithiothreitol (DTT)) to drive SR accumulation under aerobic conditions. Samples were returned to darkness and spectra taken at intervals; the increase in absorbance at [*] Dr. S. Burney, T. Roussel, Dr. N. Hoang, Dr. J.-P. Bouly, Prof. Dr. M. Ahmad PCMP, UR5, Universit Paris VI, Casier 156 4 Place Jussieu, 75005 Paris (France) E-mail: margaret.ahmad@upmc.fr