Margaret Ahmad

Light-induced electron transfer in a cryptochrome blue-light photoreceptor.

Cryptochromes are flavoproteins implicated in multiple blue light–dependent signaling pathways regulating, for example, photomorphogenesis in plants or circadian clocks in animals. Using transient absorption spectroscopy, it is demonstrated that the primary light reactions in isolated Arabidopsis thaliana cryptochrome-1 involve intraprotein electron transfer from tryptophan and tyrosine residues to the excited flavin adenine dinucleotide cofactor.*Note: The version of this paper initially published online was incorrectly labeled as an Article; the correct category should be Brief Communication. We apologize for any inconvenience this may have caused. This mistake has been corrected in the HTML and print version of the paper.

Magnetic intensity affects cryptochrome-dependent responses in Arabidopsis thaliana

Cryptochromes are blue-light absorbing photoreceptors found in many organisms where they have been involved in numerous growth, developmental, and circadian responses. In Arabidopsis thaliana, two cryptochromes, CRY1 and CRY2, mediate several blue-light-dependent responses including hypocotyl growth inhibition. Our study shows that an increase in the intensity of the ambient magnetic field from 33–44 to 500xa0μT enhanced growth inhibition in A. thaliana under blue light, when cryptochromes are the mediating photoreceptor, but not under red light when the mediating receptors are phytochromes, or in total darkness. Hypocotyl growth of Arabidopsis mutants lacking cryptochromes was unaffected by the increase in magnetic intensity. Additional cryptochrome-dependent responses, such as blue-light-dependent anthocyanin accumulation and blue-light-dependent degradation of CRY2 protein, were also enhanced at the higher magnetic intensity. These findings show that higher plants are sensitive to the magnetic field in responses that are linked to cryptochrome-dependent signaling pathways. Because cryptochromes form radical pairs after photoexcitation, our results can best be explained by the radical-pair model. Recent evidence indicates that the magnetic compass of birds involves a radical pair mechanism, and cryptochrome is a likely candidate for the avian magnetoreception molecule. Our findings thus suggest intriguing parallels in magnetoreception of animals and plants that appear to be based on common physical properties of photoexcited cryptochromes.

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.

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.

Ethylene-induced Arabidopsis hypocotyl elongation is dependent on but not mediated by gibberellins

Ethylene, or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC), can stimulate hypocotyl elongation in the light. It is questioned whether gibberellins (GAs) play a role in this response. Tests with light of different wavelengths demonstrated that the ethylene response depends on blue light and functional cryptochrome signalling. Levels of bio-active GA(4) were reduced in seedlings showing an ethylene response. Furthermore, ACC treatment of seedlings caused accumulation of the DELLA protein RGA, a repressor of growth. Concurrently, transcript levels of several GA biosynthesis genes were up-regulated and GA inactivation genes down-regulated by ACC. Hypocotyl elongation in response to ACC was strongly reduced in seedlings with a diminished GA signal, while being vigorously stimulated in a quadruple DELLA knock-out mutant with constitutive GA signalling. These data show that ethylene-driven hypocotyl elongation is mainly blue light-dependent and that this ethylene response, although GA dependent, hence needing a basal GA level, is not mediated by GA, but rather acts via a separate pathway.

Multiple interactions between cryptochrome and phototropin blue-light signalling pathways in Arabidopsis thaliana

Higher plants contain two structurally unrelated flavoprotein blue-light photoreceptors, the cryptochromes and the phototropins, which mediate largely distinct response pathways. Cryptochromes regulate plant development and photomorphogenesis whereas phototropins are primarily implicated in photomovement responses such as phototropism and chloroplast relocation. In the present study we identify interactions between cryptochromes and phototropins in several photoresponses of Arabidopsis thaliana. Cryptochromes are shown to exert a positive effect on phototropic curvature under long-term irradiation conditions. Specifically, in a phot1-deficient genetic background (phot1 mutant), curvature is reduced in the absence of cryptochromes, particularly at wavelengths where cryptochromes show preferential absorption. Phototropins in turn exert a small promotive effect on such cryptochrome-mediated responses as hypocotyl elongation and anthocyanin accumulation. These effects are apparent in a cryptochrome-deficient (cry1cry2 mutant) genetic background. In addition to positive interactions between signalling pathways, we demonstrate that the cryptochromes also exert a negative regulatory effect. Levels of phot1 protein decrease in blue light as a function of cryptochrome photoreceptor activation. This negative regulation occurs in part at the level of phot1 transcription but may also involve post-transcriptional mechanisms. These two classes of photoreceptor thereby reciprocally modulate their overall responsivity to blue light through multiple forms of interaction.

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.

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.

Human cryptochrome-1 confers light independent biological activity in transgenic drosophila correlated with flavin radical stability

Cryptochromes are conserved flavoprotein receptors found throughout the biological kingdom with diversified roles in plant development and entrainment of the circadian clock in animals. Light perception is proposed to occur through flavin radical formation that correlates with biological activity in vivo in both plants and Drosophila. By contrast, mammalian (Type II) cryptochromes regulate the circadian clock independently of light, raising the fundamental question of whether mammalian cryptochromes have evolved entirely distinct signaling mechanisms. Here we show by developmental and transcriptome analysis that Homo sapiens cryptochrome - 1 (HsCRY1) confers biological activity in transgenic expressing Drosophila in darkness, that can in some cases be further stimulated by light. In contrast to all other cryptochromes, purified recombinant HsCRY1 protein was stably isolated in the anionic radical flavin state, containing only a small proportion of oxidized flavin which could be reduced by illumination. We conclude that animal Type I and Type II cryptochromes may both have signaling mechanisms involving formation of a flavin radical signaling state, and that light independent activity of Type II cryptochromes is a consequence of dark accumulation of this redox form in vivo rather than of a fundamental difference in signaling mechanism.

Heterologous Expression of Photoactivated Adenylyl Cyclase (PAC) Genes from the Flagellate Euglena gracilis in Insect Cells

Abstract The unicellular, green flagellate wild-type Euglena gracilis (strain Z) possesses two genes of the photoactivated adenylyl cyclase (PAC) family. The corresponding gene products were found to be responsible for step-up (but not step-down) photophobic responses as well as both positive and negative phototaxis. The proteins consist of two PACα (Mr 105 kDa) and two PACβ (90 kDa) subunits. In an effort to produce sufficient amounts of PAC proteins, several routes of overexpression have been tried including homologous expression in Euglena and heterologous expression in Escherichia coli. All these approaches were hampered by low yield or formation of inclusion bodies. Therefore we decided to attempt a heterologous expression in an insect cell line. PACα and PACβ were separately cloned in the transfer vector pBacPAK9 with a His tag attached. The transfer vector was subsequently cotransfected via baculovirus into the insect cells and amplified. For the expression both recombinant viruses (containing PACα and PACβ, respectively) were cotransfected simultaneously into insect cells. The expressed proteins were analyzed in Western blots using PACα and PACβ antibodies. Most of the proteins were found to be in soluble form in high yield. The recombinant PAC proteins were purified via their attached His tag on an anti-His resin. Adenylyl cyclase activity was quantified after blue-light excitation using a cAMP enzyme immunoassay kit.