Regulation of Arabidopsis cryptochrome 2 by blue-light-dependent phosphorylation

125

Cryptochromes are sensory blue light receptors mediating various responses in plants and animals. Studies on the mechanism of plant cryptochromes have been focused on the flowering plant Arabidopsis. In the genome of the unicellular green alga Chlamydomonas reinhardtii, a single plant cryptochrome, Chlamydomonas photolyase homologue 1 (CPH1), has been identified. The N-terminal 500 amino acids comprise the lightsensitive domain of CPH1 linked to a C-terminal extension of similar size. We have expressed the light-sensitive domain heterologously in Escherichia coli in high yield and purity. The 59-kDa protein bears exclusively flavin adenine dinucleotide in its oxidized state. Illumination with blue light induces formation of a neutral flavin radical with absorption maxima at 540 and 580 nm. The reaction proceeds aerobically even in the absence of an exogenous electron donor, which suggests that it reflects a physiological response. The process is completely reversible in the dark and exhibits a decay time constant of 200 s in the presence of oxygen. Binding of ATP strongly stabilizes the radical state after illumination and impedes the dark recovery. Thus, ATP binding has functional significance for plant cryptochromes and does not merely result from structural homology to DNA photolyase. The light-sensitive domain responds to illumination by an increase in phosphorylation. The autophosphorylation takes place although the protein is lacking its native C-terminal extension. This finding indicates that the extension is dispensable for autophosphorylation, despite the role it has been assigned in mediating signal transduction in Arabidopsis.Blue light governs many responses of organisms to environmental conditions. Cryptochromes have been shown to act as sensory blue light photoreceptors in plants and animals, with their action being as diverse as their origin (1). From sequence analysis, cryptochromes have been divided into three subgroups: animal, plant, and DASH cryptochromes (2). Animal cryptochrome synchronizes the circadian clock of Drosophila to the 24-h rhythm (3). In mammals, cryptochrome has been suggested to be involved in circadian entrainment (4), and it functions independent of light as a main component of the biological clock. Arabidopsis cryptochromes 1 and 2 (AtCRY1 2 and AtCRY2) mediate de-etiolation responses, entrain the circadian clock, trigger programmed cell death induced by singlet oxygen, and regulate flowering time, stomatal opening, and production of anthocyanin (5-8). DASH cryptochromes are mostly found in bacteria but also in Neurospora crassa, aquatic vertebrates (9), and Arabidopsis (AtCRY3) (10). Their putative role as sensory receptors has recently been challenged by showing that they act as repair enzymes for single-stranded DNA (11).The 500 N-terminal amino acids constitute the photolyase homology region (PHR) with reference to the DNA repairing enzyme. In this domain all cryptochromes characterized so far carry flavin adenine dinucleotide (FAD) as light-sensitive chromophore (2, 1...

Section: Discussionmentioning

confidence: 98%

Blue Light Induces Radical Formation and Autophosphorylation in the Light-sensitive Domain of Chlamydomonas Cryptochrome

Immeln

Schlesinger

Heberle

et al. 2007

125

“…It seems that cryptochromes have conserved the photoactivation part of the photolyase photochemistry but accumulate the flavin radical instead of fully reduced flavin and use this process for signaling, most likely by conformational changes in their C-terminal output domains that are sufficient for Cry signaling (47,48). This work, together with studies on light-driven electron transfer (17), phosphorylation (34,49,50), and dimerization (48) of cryptochromes provides a foundation for analyzing in detail the early events in the light activation and signal transduction of plant cryptochromes.…”

Section: Discussionmentioning

confidence: 99%

“…Cry2 is degraded in vivo when irradiated with blue light, most likely resulting from direct absorption of light by Cry2 followed by proteasomal degradation (28,34). To determine whether the above described photoreversible responses were also reflected in the stability of Cry2, we analyzed Cry2 protein levels under the same conditions as for the quantification of FT transcripts.…”

Section: Resultsmentioning

confidence: 99%

The Signaling State of Arabidopsis Cryptochrome 2 Contains Flavin Semiquinone

Banerjee

Schleicher

Meier

et al. 2007

242

315

Cryptochrome (Cry) photoreceptors share high sequence and structural similarity with DNA repair enzyme DNA-photolyase and carry the same flavin cofactor. Accordingly, DNA-photolyase was considered a model system for the light activation process of cryptochromes. In line with this view were recent spectroscopic studies on cryptochromes of the CryDASH subfamily that showed photoreduction of the flavin adenine dinucleotide (FAD) cofactor to its fully reduced form. However, CryDASH members were recently shown to have photolyase activity for cyclobutane pyrimidine dimers in single-stranded DNA, which is absent for other members of the cryptochrome/photolyase family. Thus, CryDASH may have functions different from cryptochromes. The photocycle of other members of the cryptochrome family, such as Arabidopsis Cry1 and Cry2, which lack DNA repair activity but control photomorphogenesis and flowering time, remained elusive. Here we have shown that Arabidopsis Cry2 undergoes a photocycle in which semireduced flavin (FADH ⅐ ) accumulates upon blue light irradiation. Green light irradiation of Cry2 causes a change in the equilibrium of flavin oxidation states and attenuates Cry2-controlled responses such as flowering. These results demonstrate that the active form of Cry2 contains FADH ⅐ (whereas catalytically active photolyase requires fully reduced flavin (FADH ؊ )) and suggest that cryptochromes could represent photoreceptors using flavin redox states for signaling differently from DNA-photolyase for photorepair.Blue light signaling has been historically of great interest in plants, because distinct blue light receptors are involved in such key processes as photomorphogenesis, phototropism, and initiation of flowering (1). Cryptochromes (Crys) 2 are UV-A/blue light photoreceptors (also found in animals and bacteria (2)) and are implicated in several of these responses. Because of their high sequence and structural similarity (3-6) and identical flavin cofactor content as DNA-photolyase (7, 8), it was hypothesized that cryptochromes might use the same mechanism for signaling as does photolyase for catalysis, which is light-driven electron transfer from the fully reduced flavin FADH Ϫ (2, 4, 7-9). Indeed, spectroscopic studies on Vibrio cholerae Cry1 (10) and Arabidopsis Cry3 (11) have shown that blue light illumination results in the formation of fully reduced flavin similar to the photoactivation process of DNA-photolyase (2). However, CryDASH members were recently shown to have photolyase activity for cyclobutane pyrimidine dimers in single-stranded DNA (12), which is absent for other members of the cryptochrome/photolyase family.The photoreduction of flavin in DNA-photolyase involves several aromatic amino acid residues (mostly tryptophans) that transfer electrons from the protein surface to the flavin adenine dinucleotide (FAD), which is buried in a U-shaped conformation in the ␣-helical domain of the protein (13-16). The cryptochrome structures solved so far, namely Synechocystis Cry-DASH (3), the photolyase-related d...

“…Quite often, the phosphorylation state of a given protein alone determines the "on/ off" state of a given signaling process (51-54). In this regard, it is intriguing to note that the three major morphogenic photoreceptor types in plants (cryptochrome, phytochrome, and phototropin) exhibit protein kinase activity (4,(55)(56)(57)(58)(59)(60)(61) and that protein phosphorylation/ dephosphorylation events are associated with their signaling pathways (5,(62)(63)(64)(65)(66). In this study, we have demonstrated that the phot1-interacting protein NPH3 is a phosphoprotein in etiolated seedlings and that the BL-induced dephosphorylation of this protein may be necessary for phot1-dependent phototropic signaling.…”

Section: Discussionmentioning

confidence: 99%

Regulation of Phototropic Signaling in Arabidopsis via Phosphorylation State Changes in the Phototropin 1-interacting Protein NPH3

Pedmale

Liscum

2007

141

187

Phototropism, or the directional growth (curvature) of various organs toward or away from incident light, represents a ubiquitous adaptive response within the plant kingdom. This response is initiated through the sensing of directional blue light (BL) by a small family of photoreceptors known as the phototropins. Of the two phototropins present in the model plant Arabidopsis thaliana, phot1 (phototropin 1) is the dominant receptor controlling phototropism. Absorption of BL by the sensory portion of phot1 leads, as in other plant phototropins, to activation of a C-terminal serine/threonine protein kinase domain, which is tightly coupled with phototropic responsiveness. Of the five phot1-interacting proteins identified to date, only one, NPH3 (non-phototropic hypocotyl 3), is essential for all phot1-dependent phototropic responses, yet little is known about how phot1 signals through NPH3. Here, we show that, in dark-grown seedlings, NPH3 exists as a phosphorylated protein and that BL stimulates its dephosphorylation. phot1 is necessary for this response and appears to regulate the activity of a type 1 protein phosphatase that catalyzes the reaction. The abrogation of both BL-dependent dephosphorylation of NPH3 and development of phototropic curvatures by protein phosphatase inhibitors further suggests that this post-translational modification represents a crucial event in phot1-dependent phototropism. Given that NPH3 may represent a core component of a CUL3-based ubiquitin-protein ligase (E3), we hypothesize that the phosphorylation state of NPH3 determines the functional status of such an E3 and that differential regulation of this E3 is required for normal phototropic responsiveness.