Paul Galland

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.

THE ROLE OF PTERINS IN THE PHOTORECEPTION AND METABOLISM OF PLANTS

Most of the biochemistry of pterins is known from work with bacteria, insects and vertebrates. Great progress has been made in the elucidation of the chemistry and function of pterins in these organisms (for review see: Nichol et al., 1985; Ziegler, 1987). The conspicuous absence of plant material in the 1986 Proceedings of the Eighth International Symposium on Pterines (Cooper and Whitehead, 1986) demonstrates that the interest of plant physiologists and plant biochemists in this area has been rather modest in comparison. The decision to review nonetheless the role of pterins in plants has been largely motivated by our conviction that pterins are relevant to the problem of blue-light reception in plants and microorganisms including fungi. While the manyfold effects of near-UV-blue light (30&500 nm) on phototropism, phototaxis, photomorphogenesis and the metabolism of plants and microorganisms are well known, the nature of the photoreceptor(s) mediating these responses is still a matter of dispute. Flavoproteins are presently considered the most likely candidates (Galland and Senger, 1988; Schmidt, 1980; Senger and Briggs, 1981; Senger and Schmidt, 1986). however, carotenoproteins are still being discussed as possible bluelight receptor pigments (Shropshire, 1980). Little attention has so far been paid to pterins, although their physiochemical properties d o meet the requirements for a receptor pigment acting in the nearultraviolet. Pterins share with flavins properties such as radical formation, participation in redox chains, photosensitizing capacity and absorption of nearultraviolet light. These features make them suitable candidates as receptor or coreceptor pigments in the near-ultra-violet. They could also function as redox partners in flavin-sensitized electron transport chains. In this contribution we review the literature pertinent to the possible role of pterins in photoreception and extend our discussion to their role in metabolism, because of the interrelations between the two functions.

Action spectrum for cryptochrome-dependent hypocotyl growth inhibition in Arabidopsis

Cryptochrome blue-light photoreceptors are found in both plants and animals and have been implicated in numerous developmental and circadian signaling pathways. Nevertheless, no action spectrum for a physiological response shown to be entirely under the control of cryptochrome has been reported. In this work, an action spectrum was determined in vivo for a cryptochrome-mediated high-irradiance response, the blue-light-dependent inhibition of hypocotyl elongation in Arabidopsis. Comparison of growth of wild-type,cry1cry2 cryptochrome-deficient double mutants, and cryptochrome-overexpressing seedlings demonstrated that responsivity to monochromatic light sources within the range of 390 to 530 nm results from the activity of cryptochrome with no other photoreceptor having a significant primary role at the fluence range tested. In both green- and norflurazon-treated (chlorophyll-deficient) seedlings, cryptochrome activity is fairly uniform throughout its range of maximal response (390–480 nm), with no sharply defined peak at 450 nm; however, activity at longer wavelengths was disproportionately enhanced in CRY1-overexpressing seedlings as compared with wild type. The action spectrum does not correlate well with the absorption spectra either of purified recombinant cryptochrome photoreceptor or to that of a second class of blue-light photoreceptor, phototropin (PHOT1 and PHOT2). Photoreceptor concentration as determined by western-blot analysis showed a greater stability of CRY2 protein under the monochromatic light conditions used in this study as compared with broad band blue light, suggesting a complex mechanism of photoreceptor activation. The possible role of additional photoreceptors (in particular phytochrome A) in cryptochrome responses is discussed.

New trends in photobiology the role of flavins as photoreceptors

Abstract Almost every facet in the life of plants and fungi can be controlled by blue light. There are numerous blue-light responses with action spectra resembling the absorption spectra of flavoproteins. The photochemical properties of flavoproteins make them well suited for photoreception between 300 and 500 nm and potentially also beyond 500 nm. Evidence that flavoproteins act as blue-light photoreceptors comes in part from receptor substitution experiments with flavin analogues. Attempts to distinguish flavoproteins with photoreceptor function from bulk flavoproteins are also made by fluorescence lifetime measurements and analysis of light-induced absorbance changes that indicate cytochrome- b reduction. The only well-characterized and isolated flavin-type photoreceptors to date are the DNA photolyases of E. coli , yeast and Streptomyces .

MODIFIED ACTION SPECTRA OF PHOTOGEOTROPIC EQUILIBRIUM IN Phycomyces blakesleeanus MUTANTS WITH DEFECTS IN GENES madA, madB, madC, and madH

Abstract— Action spectra of photogeotropic equilibrium were measured for behavioral mutants of Phycomyces blakesleeanus with defects in the genes madB, madC and madH as well as for a double mutant defective in the genes madA and madC. The action spectra of strains C109 (madB), LI (madC) and L52 (madA madC) all lack the broad near‐ultraviolet peak which extends from 347 to 386 nm in the wild type; the peaks at 414 and 491 nm are also missing in these mutants. The double mutant L52 (madA madC) shows a novel broad peak at 477 nm; the relative quantum effectiveness of L52 at 477 nm is 10 times higher than in LI (madC119). These properties of the double mutant L52 (madA madC) suggest steric interaction of the madA and the madC gene products in the photoreceptor complex. For the hypertropic mutant L84 (madH) the action spectrum and absolute sensitivity are similar to those for wild type. These results confirm and extend previous findings that multiple photoreceptors are mediating phototropism in P. blakesleeanus.

ACTION SPECTRA OF PHOTOGEOTROPIC EQUILIBRIUM IN Phycomyces WILD TYPE AND THREE BEHAVIORAL MUTANTS

Photogeotropic equilibrium action spectra in the range from 301 to 740 nm were made for Phycomyces wild type and the three behavioral mutants C47 (madA35), C109 (madBlOl) and LI (madCIIQ), all of which have a raised phototropic threshold. In addition to two broad peaks at 365 and 455 nm, typical for flavins, the wild type action spectrum shows three novel peaks, which have not been observed previously. These peaks are located at 414, 491 and 650 nm. The 650 nm peak has a relative quantum efficiency of 3 × 10−8 compared to the peak at 414 nm. The wavelength dependent shapes of the fluence‐response curves of the bending angle and the aiming error angle indicate more than one receptor pigment for phototropism. The shape of the action spectrum of C47 is basically unaltered in comparison to wild type. C109 and LI show substantial differences from the wild type. In the near UV two small peaks at 334 and 365 nm appear; the 414 and 491 nm peaks present in wild type and C47 are missing and two new peaks at 529 nm (not well resolved in C109) and 567 nm are found. None of the three mad mutations affects the 650 nm peak. A model of the sensory transduction chain is presented, which incorporates these and other known features.

Microspectrophotometry ofEuglena gracilis

The paraflagellar bodies (PFBs) of isolated flagella ofEuglena gracilis were investigated microspectrophotometrically using a visible- and infrared-light microscope with image analyzer and microspectrophotometer. Flagella with attached PFBs were separated from the cell bodies by a short exposure to near-UV light. Fluorescence-emission spectra (excitation at 365 nm) of single PFBs had maxima near 470 and 520 nm, indicating the presence of pterins and flavins. No fluorescence was associated with the flagella themselves. Pterin- and flavin-like fluorescence emission was also found in blue-fluorescing vesicles distributed throughout the entire cell body ofEuglena. Their characterization by microfluorimetry was greatly aided by the use of chlorophyll-free mutants in which the signal-to-noise ratio was distinctly enhanced because of the lack of chlorophyll fluorescence. Our finding of flavin-like fluorescence associated with PFB strengthens similar earlier reports in the literature. The occurrence of pterin-like fluorescence in the PFB lends further support to our earlier proposal that pterins as well as flavins may function as photoreceptor pigments for near-UV and blue light.

ACTION SPECTRA FOR PHOTOTROPIC BALANCE IN Phycomyces blakesleeanus: DEPENDENCE ON REFERENCE WAVELENGTH and INTENSITY RANGE

Abstract— Action spectra for phototropic balance of Phycomyces blakesleeanus sporangiophores were measured for various reference wavelengths and intensity ranges. Balance action spectra were made at fluence rates of 10‐4 W m‐2 with reference wavelengths of 450 nm, 394 nm, 507 nm, and broadband blue light. For broad‐blue light and 450 nm light as references, typical flavin‐like action spectra were found with a ma jor peak at 455 nm, a secondary peak at 477 nm, and a minor peak at 383 nm; these peaks are wider for broad blue than for 450 nm light. With the 394 nm reference, there is a major peak at 455 nm, a secondary peak at 477 nm and a minor peak at 394 nm. An action spectrum with 507 nm reference has a major peak at 455 nm and a minor peak at 383 nm, but no peak at 477 nm. A balance action spectrum was made with 450 nm reference light near threshold intensity (2 times 10‐8 W m‐2); there, the 386 nm peak is greatly reduced, while the 455 nm peak is enhanced. The intensity dependence of the 386 nm peak was studied in detail for reference light of 450 nm. We found that the relative quantum efficiency of the 386 nm light increases with the logarithm of the 450 nm fluence rate; in the high intensity range (0.3 W m‐2) the relative quantum efficiency of the 386 nm light is 1.3 and approaches zero at 10‐9 W m‐2. These findings indicate that P. blakesleeanus phototropism is mediated by multiple interacting pigments or by a photochromic photoreceptor.

PHOTOINITIATION OF SPORANGIOPHORES IN PHYCOMYCES MUTANTS DEFICIENT IN PHOTOTROPISM AND IN MUTANTS LACKING β‐CAROTENE

Abstract—The formation of sporangiophores from mature Phycomyces mycelium is inhibited in a closed system. Irradiation of the mycelium with blue light reverses the inhibition of spordngiophore formation. Dose response curves for this reaction are established for wild type. β‐caroteneless mutants and for mutants that are deficient in phototropism.

FORTY YEARS OF BLUE‐LIGHT RESEARCH AND NO ANNIVERSARY

The status of near‐ UV/blue‐light research is described and critically assessed. It is argued that the generally accepted proposition of flavins acting as near‐ UV/blue‐light photoreceptors is reasonable, but that also other pigments, specifically pteridines and pterins, should be considered. In view of the fact that a receptor assay, applicable to any organism and near‐UV/blue‐light reaction, is not yet at hand, caution appears to be indicated as to what one can accept as a reasonable “criterion” for the involvement of any proposed photoreceptor pigment.