Photoactivation Mechanism Of Blue Light Receptor Cryptochrome 1 In Arabidopsis

Plants were influenced by various environmental factors during their growth and development. In the process of long-term evolution, plants formed a series of sensory system to receive and transduce specific incoming environmental signals. Light is one of key environmental signals for plants and can be sensed by photoreceptors in plants. Photoreceptors absorb a photon and transduce the signal downstream in response to the changes of ambient environment. Thirteen photoreceptors were found by now in Arabidopsis thaliana, which can be further classified into three types: red/far-red light receptors, blue/UV-A light receptors and UV-B light receptor. Cryptochrome, originally discovered in Arabidopsis, is one of blue light receptors.Cryptochromes are flavoproteins, sharing the similar sequence and protein structure with photolyase. The flavin co-factor(FAD) of cryptochromes can be photoreduced in vitro by electron transportation of three evolutionarily conserved tryptophan residues known as trp-triad. It was hypothesized that the trp triad-dependent photoreduction directly lead to photoactivation of cryptochrome photoreceptors. According to this hypothesis, cryptochromes in the resting state contained oxidized FAD are presumed resting state; light triggers a sequential electron transfer from three tryptophan residues and a proton transfer from unknown source to reduce FAD* to a neutral semiquinone(FADH?), FADH? is the presumed signaling state. The photoactived cryptochromes, in the form of an opened protein structure, could interact with their signal proteins, and eventually induce a series of physiological and biochemical changes. The hypothesis, however, is under debate now. Firstly, most of evidences supporting the trp-triad-dependent photoreduction came from the experiments in vitro. Secondly, the light-dependent catalytic activity of photolyase, the presumed ancestor of cryptochrome, does not require the trp-triad-dependent photoreduction. Thirdly, the trp-triad-dependent photoreduction and the photobiochemical and photophysiological activities of Arabidopsis CRY2 are irrelevant. The only genetics study reported so far in support of the hypothesis came from a study of Arabidopsis CRY1. It was reported that mutations in two of the three trp-triad residues of Arabidopsis CRY1 abolished the activities of CRY1 in transgenic plants. However, It has not been reported that wheather there is a fundamental difference in photoactivation mechanism between At CRY1 and At CRY2 or photolyase. To test if trp-triad-dependent photoreduction effect photoactivation of CRY1,and further investigate the photoactivation mechanism of CRY1, in this study, we prepared site-specific mutation of trp-triad(W324, W377 and W400) and other conserved tryptophan(W334 and W356)by individually replacing each of the three tryptophans with either phenylalanine(F) or alanine(A) that is redox inert in protein, using the site-directed mutagenesis method. Then the biochemical and physiological activities of these mutants were analyzed both in vivo and in vitro.1. The photoreduction of CRY1 trp-triad mutants in vitro.Five trp-triad mutant proteins were expressed and purified from insect cell, which were referred to as W324 A, W324 F, W377 A, W377 F and W400 F. The specific absorb spectra of FAD in different redox state were analyzed. The photoreduction of all trp-triad mutated proteins were abolished or dramatically reduced, which confirmed that the trp-triad residues are indeed required for the rapid photoreduction of Arabidopsis CRY1 in vitro.2. The physiological activities of CRY1 trp-triad mutants in vivo.Overexpression transgentic plants were prepared by transforming the mutated genes into cry1 null mutant mediated by agrobacterium. All the plants using for analysis were T3 homozygous lines, referred to as GFP-CRY1、GFP-CRY1W324F、GFP-CRY1W324 A, GFP-CRY1W377 F, GFP-CRY1W377 A, GFP-CRY1W400 and GFP-CRY1W400 A. All trp-triad mutations of CRY1 remained physiological functions of wild-type CRY1, such as CRY1 mediated blue light inhibition of hypocotyl growth, CRY1 mediated blue light stimulation of anthocyanin accumulation and blue light induction of related gene m RNA expression. Two mutants exhibited the activity stronger than that of the wild-type GFP-CRY1 control. GFP-CRY1W400 A exhibited constitutive activity in blue light, red light, far-red light and even darkness, and GFP-CRY1W377 A showed similar but weaker activity to GFP-CRY1W400 A. These results demonstrated that the trp triad-dependent photoreduction is not required for the physiological activities of CRY1.3. The photobiochemical activities of CRY1 trp-triad mutants in vivo.Protein phosphorylation of all mutant proteins were tested. All tested proteins undergo blue light-dependent phosphorylation, except W400 A, who undergo protein phosphorylation in both blue light and darkness. Then we tested the interaction between W400 A and SPA1(CRY1’s signal protein) using the Human Embryonic Kidney co-expression system and co-immunoprecipitation assay. The result showed that W400 A constitutive interacted with SPA1, further proved its constitutive activity. The trp-triad mutations and SPA1CT509 were further co-expressed in Nicotiana benthamiana. Bi FC assay showed that all trp-triad mutants could interact with SPA1CT509 in plant cells. We concluded that the trp-triad-dependent photoreduction is not required for the photobiochemical activities of CRY1.4. The effect of ATP-enhanced photoreduction on the activities of CRY1.1 m M ATP were added while testing the photoreduction of trp-triad mutations in vitro to analyse whether ATP could affect the photoreduction of CRY1 mutants. In contrast with ATP enhanced the photoreduction of CRY2 as reported, we found that ATP did not enhance rapid photoreduction of the wild-type CRY1 or rescue the defective photoreduction of the W324 A and W400 A mutants. ATP did enhance photoreduction of W324 F, W377 A and W377 F, but still weaker compared with wild-type CRY1. However, all these trp-triad mutants maintained activities of CRY1 in vivo. These results demonstrated that a lack of correlation between the ATP-enhanced photoreduction and activities of CRY1.5. The analysis of the other two evolutionarily conserved tryptophan mutants of CRY1.There are two other evolutionarily conserved tryptophans in FAD binding cavity(W334 and W356). Similar mutation assay were proformed, and found out that mutations of these two tryptophan residues exhibited no significant impairment in either photoreduction in vitro or physiological and photobiochemical activities in vivo.The photoactivated CRY1 induce a series of physiological and photobiochemical response. According to our results, all trp-triad mutants of CRY1 maintained the physiological and photobiochemical activities as wild-type CRY1, which means these trp-triad mutants can still be photoactivated. Taken together, we concluded that the trp triad-dependent photoreduction is not the photoactivation mechanism of CRY1.