| Cryptochromes are blue light receptors that regulate various photomophogenic responses in plant, including inhibition of hypocotyl elongation, stimulation of cotyledon expansion, and regulation of flowering time. It is not clear about how cryptochromes mediate blue light regulating photomophogenic responses.To understand the underling mechanism, the wild-type(col-4) and some blue light receptor mutants(cry1-304,cry1cry2) of Arabidopsis were used in this study, and they all are in the col background. Two-dimensional electrophoresis (2-DE), matrix assisted laser desorption/ionization time of flight mass spectrometry (MALDI- TOF-TOF /MS), high compacity trap mass spectrometry and bioinformatics technique were used in this presentation. In order to understand the underling mechanism of how cryptochromes mediate blue light to regulate photomophogenic responses and to understand the light responses in Arabidopsis, the total or nucleus proteins extracted from seedlings of cry1-304 and col-4, which were grown under different light. The results of this study were listed as follows:(1)Extract the ptoteins of cry1-304 and col-4of Arabidopsis thaliana 7d seedling which were grown under the white light, blue light and dark. To study the difference of protein expression level between cry1-304 and col-4, a proteome approach based on two-dimensional gel electrophoresis (2-DE) was applied. Image acquisition and analysis, 44 protein spots were differently expressed in cry1-304 and col-4, 21 different protein spots were identified by the Matrix Assisted Laser Desorption/ Ionization-time of Flight Mass Spectrometry-Mass Spectrometry (MALDI-TOF-TOF- MS). In white light, 15 protein spots Up-regulated in cry1-304 and 6 protein spots down-regulated in cry1-304.In blue light 17 protein spots Up-regulated in cry1-304 and 4 protein spots down-regulated in cry1-304. In dark, 7 protein spots Up-regulated in cry1-304, 9 protein spots down-regulated in cry1-304,9 protein spots were no alteration between cry1-304 and col-4, 2 protein spots were not appear in dark cry1-304 and col-4. 2 spots of protein were identified to be unclassified proteins, and the other proteins were categorized into several functional groups including 3 protein spots of them were metabolism, 4 protein spots were energy, 3 protein spots were defense,4 protein spots were transcription, 2 protein spots were protein synthesis, 1 protein spot is RNA processing, 1 protein spot is protein fate and 1 protein spot is Cellular Transport and Transport Mechanisms. The expression of genes corresponding to four protein spots was analyzed by semi quantitative RT-PCR. One of them the mRNA and protein levels was correlated well confirmed, but the other three in some instance, the level of mRNAs and the expression level of the related proteins were inconsistent. This difference could be affected by post-translational modifications. Protein profiles are classified by K-means clustering function in the statistics toolbox in Matlab 2006, and found that the differentially expressed proteins formed six clusters reflecting coregulation. It seems likely that CRY1 is the photoreceptor responsible for mediating the blue light effect on gene expression. These data indicate that light controls Arabidopsis development through coordinately regulating many gene expressions. This research provided a new way to study of cry1 mediating blue light-dependent regulation of gene expression. The results indicated that light control of Arabidopsis development was co-regulated by many other related genes.(2)To study the difference at the protein expression level of the cry1cry2 and col-4, we used a proteome approach based on 2-D gel electrophoresis (2-DE) to compare the protein patterns of Arabidopsis species cry1cry2 and col-4. The 75 different protein spots were identified by the Matrix Assisted Laser Desorption/ Ionization-time of Flight Mass Spectrometry-Mass Spectrometry (MALDI-TOF-TOF- MS). Between cry1cry2 and wild type: In dark there are 18 of 67 protein spots are decreased and 49 of 67 are increased; In blue light 44 protein spots are decreased and 24 protein spots are increased; In red light, 39 protein spots are decreased and 25 protein spots are increased. The protein expression profile of these 40 or 46 protein spots showed more than 2-fold changes between etiolated and blue light-grown wild type seedlings or etiolated and red light-grown wild type seedlings,whereas 49 or 40 protein expression was changed at least 2-fold in the cry1cry2 mutant. These proteins are involved in carbohydrate, energy metabolism, Cellular Structural Organization, Defence Stress and Detoxification, Protein fate, structural cell walls and so on. Most of them are chloroplasts or mitochondria proteins. We identified 5 new light reflect proteins, they are Ribulose-phosphate 3-epimerase, Glyceraldehyde-3-phosphate dehydrogenase A, Fructose-bisphosphate aldolase, Malate dehydrogenase and Glyceraldehyde-3-phosphate dehydrogenase B, this five proteins all down- relaguted in cry1cry2. 7 spots were involved in hypocotyl elongation and cotyledon expansion, they are Quercetin 3-O-methyltransferase, Alpha-xylosidase precursor, Subtilisin-like protease precursor, glycine dehydrogenase, Glycine-rich protein and Glycine hydroxymethyltransferase. To dissect the proteomic responses to each light, a clustering analysis was performed on the 75 protein spots which had been identified and were found to be regulated differentially. In WT or cry1cry2, the definitively classified protein spots formed six clusters, In WT and cry1cry2 the definitively classified protein spots formed nine clusters, and the blue light and red light of cry1cry2 are more closely related than dark response. In the present study, through clustering analysis of mutants and wild type, We demonstrated that the protein expression profile may study the relationship of light response under different lights in the different mutants for the first time.(3)From above research, we reported a comparative proteomic study of protein expressions in the wild type Arabidopsis and the cry1cry2 mutant seedlings grown under continuous blue light or red light. As expected, the cry1cry2 mutation altered protein expression profile in response to blue light. Surprisingly, we also found significant differences of the protein expression profiles between wild type and the cry1cry2 mutant seedlings grown in red light. To further test whether cryptochromes may regulate mRNA expression in both blue-light-dependent and blue-light- independent manner to cause changes of protein abundances, we examined mRNA expression of the wild type and the cry1cry2 mutant seedlings grown in dark, red light, or blue light, using both conventional RT-PCR and real-time Q-PCR methods. RNA expression analyses showed that the cry1cry2 mutation affects not only blue-light-dependent but also blue-light-independent mRNA expression changes. These results indicated that cryptochromes may act in a blue light-independent manner to affect phytochrome regulation of gene expression and development. Furthermore, cryptochromes also play roles in the control of gene expression mediated by the red/far-red light receptors phytochromes.(4)We use 2D-gel, MALDI-TOF-TOF and high compacity trap mass spectrometry identified col-4, cry1-304 and cry1cry2 nuclear protein, three proteins cellular localizated in nuclear were identified in two mutants.The protein of 14-3-3-like protein GF14 lambda is increased in the two mutants compare to col-4. This protein has protein banging function, localiazed in nuclear membrane, We used fluorescence localiazation analysis this protein, It is expressed in nuclear. These results provide a good basis for the further study of the functions of CRY in the nucleus in the future. Our observation may suggest that protein expression is significantly influencedby inactivation of the gene CRY, and blue light acting through CRY regulates the expression of many proteins. This is the first time that cryptochrome regulation of protein and mRNA expressions in response to both blue light and red light has been reported. Our study may provide a useful overview of how cryptochromes affect patterns of protein expression, and the basis for further analysis the function of CRY.
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