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Trp triad-dependent rapid photoreduction is not required for the function of Arabidopsis CRY1
Thursday, 2015/07/23 | 07:53:50
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Jie Gao, Xu Wang, Meng Zhang, Mingdi Bian, Weixian Deng, Zecheng Zuo, Zhenming Yang, Dongping Zhong, and Chentao Lin SignificanceThe Trp triad-dependent photoreduction of the flavin chromophore has been widely accepted as the photoexcitation mechanism of cryptochrome photoreceptors. However, the experimental evidence supporting this hypothesis derived primarily from the biophysical studies in vitro, except for one genetics study of Arabidopsis cryptochrome 1 (CRY1). In contrast to the previous report, we found that all Trp-triad mutations of Arabidopsis CRY1 remained physiologically active in plants, and this result cannot be readily explained by the ATP-dependent enhancement of Trp triad-dependent photoreduction. Our results challenge the widely accepted Trp-triad hypothesis and call for further investigation of alternative electron transport mechanisms to explain cryptochrome photoexcitation. AbstractCryptochromes in different evolutionary lineages act as either photoreceptors or light-independent transcription repressors. The flavin cofactor of both types of cryptochromes can be photoreduced in vitro by electron transportation via three evolutionarily conserved tryptophan residues known as the “Trp triad.” It was hypothesized that Trp triad-dependent photoreduction leads directly to photoexcitation of cryptochrome photoreceptors. We tested this hypothesis by analyzing mutations of Arabidopsis cryptochrome 1 (CRY1) altered in each of the three Trp-triad tryptophan residues (W324, W377, and W400). Surprisingly, in contrast to a previous report all photoreduction-deficient Trp-triad mutations of CRY1 remained physiologically and biochemically active in Arabidopsis plants. ATP did not enhance rapid photoreduction of the wild-type CRY1, nor did it rescue the defective photoreduction of the CRY1W324A and CRY1W400F mutants that are photophysiologically active in vivo. The lack of correlation between rapid flavin photoreduction or the effect of ATP on the rapid flavin photoreduction and the in vivo photophysiological activities of plant cryptochromes argues that the Trp triad-dependent photoreduction is not required for the function of cryptochromes and that further efforts are needed to elucidate the photoexcitation mechanism of cryptochrome photoreceptors.
See: http://www.pnas.org/content/112/29/9135.abstract.html?etoc PNAS July 21, 2015 vol. 112 no. 29 9135-9140
Fig. 4. The Trp-triad mutants of CRY1 are biochemically active. (A–D) Mobility upshift assay showing blue light-dependent phosphorylation of the endogenous CRY1 (Col-4), the GFP-CRY1 control (CRY1), and the indicated mutants of CRY1 and light-independent phosphorylation of the W400A mutant of CRY1. Seven-day-old seedlings grown on MS medium in darkness (0) were exposed to blue light (30 μmol·m−2·s−1) for 10, 30, or 60 min. Samples were extracted and fractionated in a 6% SDS/PAGE gel, blotted, probed with anti-CRY1 antibody, stripped, and reprobed with anti-HSP90 antibody. An aliquot of the W400A mutant from seedlings exposed to blue light for 30 min was treated with Lambda Protein Phosphatase [30(+PPase)]. White dotted lines are drawn to help distinguish the upshifted (i.e., phosphorylated) bands. Levels of proteins from different immunoblots are not directly comparable. (E and F) Coimmunoprecipitation assay showing the blue light-dependent CRY1–SPA1 interaction and the constitutive W400A–SPA1 interaction. CRY1 and SPA1 or W400A and SPA1 were coexpressed in HEK293 cells. The transfected cells were cultured in darkness before exposure to blue light (40 μmol·m−2·s−1) for 2 h or were kept in darkness. The total protein extracts (Input) and immunoprecipitation product (IP-Flag) were fractionated by a SDS/PAGE gel, blotted, probed with the anti-Flag antibody, stripped, and reprobed with the anti-CRY1 antibody. (G) Results of BiFC assays showing the interaction of the CRY1 or mutant proteins with SPA1CT509 in N. benthamiana plants. Three-week-old tobacco plants grown in long-day conditions (16 h light/8 h dark) were cotransformed with Agrobacteria harboring the plasmids encoding nYFP-CRY1W-to-A mutants and cCFP-SPA1CT509. The transformed plants were incubated for 12 h in the dark and then were transferred to white light for 48–72 h. The infected leaf spots were excised and examined under a fluorescence microscope. Images were taken at 10× magnification and are shown in SI Appendix, Fig. S14. The percentage of cells that exhibit BiFC fluorescence signals were calculated by the formula [(number of YFP fluorescent nuclei/number of DAPI-stained nuclei)%]. Data are shown as means and SD (n = 3). |
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