complementation of DmCRY mutation by HsCRY1. For the purposes of this study, the biological effects of HsCRY1 are interpreted solely as a ‘readout’ phenotype enabling assessment of light dependent and/or independent biological function of the photoreceptor within a living organism. The acceleration of eclosion (developmental timing) that is mediated by HsCRY1 occurs in the absence of light (complete darkness) consistent with light-independent activity of HsCRY1 in transgenic Drosophila as in mammals. However, the effectiveness of HsCRY1 in mediating this response is enhanced in white light (Fig. 2a) indicating an additional level of light sensitivity of this reaction. Wavelength dependence of the eclosion phenotype shows that in HsCRY1 expressing flies, acceleration of development as compared to wt controls is observed in blue light and is even more pronounced in UV/A (380 nm) as compared to DD. However, in red light, although developmental timing is accelerated in comparison to DD, there is no effect of cryptochrome, either of DmCRY or HsCRY1, on this phenotype. Therefore, sensitivity to light conferred by Type II cryptochromes corresponds to the absorption spectrum of the bound flavin chromophore and is confined to the blue/UV-A spectral region, consistent with the wavelength sensitivity of type I (Drosophila) cryptochrome [8,9]. Transcriptome analysis Transcriptome analysis has supported these developmental observations by providing evidence of both light-dependent and light-independent transcriptional roles of HsCRY1 in developing pupa. The fact that many of the same genes are regulated by both HsCRY1 and DmCRY but in opposing manner (Fig. 3) contributes to the validation of this approach as a test for cryptochrome-dependent function, since the same genes are regulated differently dependent on the cryptochrome context. This also suggests that heterologous HsCRY1 is implicated in some of the same response pathways as DmCRY, possibly by targeting some of the same transcription factors non-productively and thereby sequestering them. In the case of light-dependent transcriptional responses, these are obtained within two hours of blue light illumination and may well result from direct interaction of cryptochromes with transcription factor binding to the regulated promoters. In this respect, identification of recurring motifs in genes regulated by both HsCRY1 and DmCRY (bound by an IRF-like transcription factors in mice and STAT transcription factors in flies) further supports the validity of the approach and suggests possible targets for both HsCRY1 and DmCRY that may have homologs in both flies and mammalian systems. Transcriptome analysis also produced the more unexpected finding that DmCRY can mediate considerable transcriptional responses in the complete absence of light, in contrast to the light- dependent mechanism underlying DmCRY clock function. Although a few isolated prior reports of a light-independent role for cryptochrome in the fly circadian clock exist [10,14,45], these roles involve uniquely clock entrainment in certain peripheral tissues or to temperature cycles. In the present work, by contrast, significant transcriptional activity mediated by DmCRY occurs even in the dark, reminiscent of findings for Arabidopsis cryptochrome, which also showed significant light-independent transcriptional responsivity [46]. Both Type I (Drosophila) and plant cryptochrome-1 show robust light-responsivity and undetectable constitutive activity for a number of responses in the absence of light. The observed light-independent response of DmCRY may therefore be indicative of a small pool of ‘activated’ cryptochrome that accumulates in the dark, possibly involving the radical flavin state in equilibrium with the inactive (oxidized) flavin state (see arguments developed below). Alternatively, there may be more Human Cryptochrome Light Responsivity than one means of cryptochrome function in signaling pathways, one of which can occur in the absence of light. A further unexpected finding from transcriptome analysis is the observation that both HsCRY1 and DmCRY regulate many genes that have been associated previously with abiotic and biotic stress, disease resistance, and responsiveness to oxidative stress [35]. In addition to several Osiris genes that seem to be regulated by cryptochrome (DmCRY and HsCRY1), which may involve immune responsiveness, a link to biotic stress has been proposed involving the Toll-like receptor pathway and the expression of Drosomycin (Dro) genes [37], consistent with our observation that Dro3 is differentially regulated by DmCRY (Fig. 4a). This activity is apparently unrelated to a role in the circadian clock, and may therefore be among the emerging novel functions of DmCRY that have recently been reported [25]. A role for cryptochrome in stress response has not yet been described in Drosophila, however, in mammals recent studies have raised a potential role for CRY in apoptosis and in the response to stress [47,48]. In plants, the role of CRY in programmed cell death (PCD) and in the response to various abiotic and biotic stresses has also been described in several studies [49–52]. Therefore, the present work opens up the prospect of a new field of Drosophila cryptochrome function and its role in stress. Given that similar pathways are regulated by HsCRY1 in this transgenic system, these may furthermore involve conserved pathways and possible homologous molecular targets in mammals and other systems. Implications for animal cryptochrome signaling mechanism In previously proposed mechanisms for light activation of both plant and animal cryptochromes has involved photoreduction from oxidized flavin to form a relatively stable radical interme- diate, which is the presumed signaling state [9,12,13]. Conforma- tional change related to signaling has been shown to require radical formation in Arabidopsis cryptochrome [53] and a form of light-induced conformational change requiring the presence of the radical form is proposed as the basis for signaling also in insect cryptochrome [54]. In all cryptochromes and photolyases investigated to date, flavin photoreduction occurs by electron transfer to excited state flavin from a nearby trp residue in the protein, which in turn is reduced through a chain of at least three conserved trp residues (known as the trp triad) by external reductant at the protein surface (see [1] and references therein). Return to darkness restores oxidized flavin, which resets the system to the default ‘off’ signaling state, which can be reactivated by subsequent further illumination [55]. Recently, this notion has been challenged by the observation that mutations in conserved trp residues of this electron transfer chain that prevent photoreduction of insect cryptochromes in vitro still show biological activity in vivo [25,54]. However, this confusion is resolved by the observation that photoreduction in vivo still occurs in such mutants, most likely by alternative electron transfer pathways within the protein or in the extracellular environment (see discussion in [9]). In the present work, the observed stability of the anionic radical flavin in the isolated purified HsCRY1 photoreceptor is entirely consistent with a signaling mechaism requiring flavin radical formation for cryptochrome activation. Given this property in vitro, a significant proportion of Type II cryptochrome would occur in the anionic flavin state in the reducing intracellular environment in vivo, as has in fact been observed in prior studies [9]. The accumulation of anionic radical activated ‘signaling on’ state would explain the constitutive function of mammalian Type II cryptochromes in the absence of light, whereas the remaining proportion of oxidized flavin form that can be photoreduced by PLoS ONE | www.plosone.org 11 March 2012 | Volume 7 | Issue 3 | e31867