ऊतक और अंग विशेष Phytochrome प्रतिक्रियाएँ में FACS की सहायता सेल प्रकार विशिष्ट अभिव्यक्ति रूपरेखा का उपयोग की जांच Arabidopsis thaliana</em
Summary
स्थानिक विशेष phytochrome प्रतिक्रियाओं का आणविक आधार ट्रांसजेनिक पौधों है कि एक्ज़िबिट ऊतक और अंग विशेष phytochrome कमियों का उपयोग कर जांच की जा रही है. विशिष्ट कोशिकाओं के अलगाव प्रतिदीप्ति सक्रिय सेल माइक्रोएरे विश्लेषण द्वारा पीछा छंटनी स्थानिक विशेष phytochrome प्रतिक्रियाओं में शामिल जीनों की पहचान करने के लिए उपयोग किया जा रहा है द्वारा प्रेरित phytochrome क्रोमोफोर रिक्तीकरण का प्रदर्शन.
Abstract
Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.
Protocol
1. संयंत्र विकास पुष्टि यूएएस – बी.वी.आर. एक्स GAL4 GFP बढ़ाने जाल अलग लाइन के रूप में 4 वर्णित (सारांश के लिए चित्र देखें 1.) और जंगली प्रकार या माता – पिता की लाइनों की धरती पर बोया जाता है, यानी ~ प्रति पंक्?…
Discussion
जीन प्रोटीन के माध्यम से अभिव्यक्ति की रूपरेखा (1) ने संकेत दिया है कि Arabidopsis seedlings में जीनों के 30% से अधिक प्रकाश विनियमित 11 कर रहे हैं और (2) प्रकाश संकेत transduction phytochrome संकेतन झरना 12, 13 में शामिल प्रोटीन एन्क?…
Acknowledgements
मोंटगोमरी प्रयोगशाला में पौधों में phytochrome प्रतिक्रियाओं पर कार्य राष्ट्रीय विज्ञान फाउंडेशन (अनुदान नहीं. BLM MCB-0,919,100) और रसायन विज्ञान, Geosciences और बायोसाइंसेज प्रभाग, मूल ऊर्जा विज्ञान के कार्यालय, विज्ञान के कार्यालय, अमेरिका विभाग द्वारा समर्थित है ऊर्जा (अनुदान नहीं. FG02 डे BLM 91ER20021). हम शूटिंग के दौरान तकनीकी सहायता के लिए मेलिस्सा Whitaker धन्यवाद और समीक्षकों पांडुलिपि पढ़ने, प्रयोगात्मक सहायता, विकास और प्रतिदीप्ति सक्रिय सेल अनुकूलन सहायता के लिए confocal साथ Arabidopsis मूलतत्त्व छँटाई और डॉ. मेलिंडा फ़्रेम के लिए प्रोटोकॉल छंटनी के साथ सहायता के लिए डा. लुई राजा के लिए स्टेफ़नी Costigan माइक्रोस्कोपी. हम ग्राफिकल डिजाइन सहायता और संपादकीय सहायता के लिए करेन बर्ड के लिए मार्लिन कैमरून धन्यवाद.
Materials
| Material Name | Type | Company | Catalogue Number | Comment |
|---|---|---|---|---|
| Anti-BVR antibody | QED Bioscience Inc. | 56257-100 | ||
| Cellulase “Onozuka” R-10 | SERVA Electrophoresis GmbH, Crescent Chemical Company | MSPC 0930 | ||
| Gamborg’s B5 basal salt mixture | Sigma | G5768 | ||
| Macerozyme R-10 | SERVA Electrophoresis GmbH, Crescent Chemical Company | PTC 001 | ||
| MES, low moisture content | Sigma | M3671 | ||
| Murashige and Skoog salts | Caisson Laboratories | 74904 | ||
| Phytablend | Caisson Laboratories | 28302 | ||
| RNeasy Plant Minikit | Qiagen | 16419 |
References
- Franklin, K. A., Quail, P. H. Phytochrome functions in Arabidopsis development. J. Exp. Bot. 61, 11-24 (2010).
- Montgomery, B. L. Right place, right time: Spatiotemporal light regulation of plant growth and development. Plant Signal Behav. 3, 1053-1060 (2008).
- Laplaze, L. GAL4-GFP enhancer trap lines for genetic manipulation of lateral root development in Arabidopsis thaliana. J. Exp. Bot. 56, 2433-2442 (2005).
- Costigan, S., Warnasooriya, S. N., Montgomery, B. L. Root-localized phytochrome chromophore synthesis is required for tissue-specific photoregulation of root elongation and impacts sensitivity to jasmonic acid in Arabidopsis thaliana. , .
- Lagarias, D. M., Crepeau, M. W., Maines, M. D., Lagarias, J. C. Regulation of photomorphogenesis by expression of mammalian biliverdin reductase in transgenic Arabidopsis plants. Plant Cell. , 675-688 (1997).
- Montgomery, B. L., Yeh, K. C., Crepeau, M. W., Lagarias, J. C. Modification of distinct aspects of photomorphogenesis via targeted expression of mammalian biliverdin reductase in transgenic Arabidopsis plants. Plant Physiol. 121, 629-639 (1999).
- Warnasooriya, S. N., Montgomery, B. L. Detection of spatial-specific phytochrome responses using targeted expression of biliverdin reductase in Arabidopsis. Plant Physiol. 149, 424-433 (2009).
- Warnasooriya, S. N., Porter, K. J., Montgomery, B. L. Light-dependent anthocyanin accumulation and phytochromes in Arabidopsis thaliana. , .
- Denecke, J., Vitale, A. The use of protoplasts to study protein synthesis and transport by the plant endomembrane system. Methods Cell Biol. 50, 335-348 (1995).
- Birnbaum, K. Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines. Nat. Methods. 2, 615-619 (2005).
- Ma, L. Light control of Arabidopsis development entails coordinated regulation of genome expression and cellular pathways. Plant Cell. 13, 2589-2607 (2001).
- Chen, M., Chory, J., Fankhauser, C. Light signal transduction in higher plants. Annu. Rev. Genet. 38, 87-117 (2004).
- Ulm, R., &, N. a. g. y., F, . Signalling and gene regulation in response to ultraviolet light. Curr. Opin. Plant Biol. 8, 477-482 (2005).
- Ma, L. Organ-specific expression of Arabidopsis genome during development. Plant Physiol. 138, 80-91 (2005).
- Neff, M. M., Fankhauser, C., &, C. h. o. r. y., J, . Light: an indicator of time and place. Genes Dev. 14, 257-271 (2000).
- Birnbaum, K. A gene expression map of the Arabidopsis root. Science. 302, 1956-1960 (2003).
Citer Cet Article
Warnasooriya, S. N., Montgomery, B. L. Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana. J. Vis. Exp. (39), e1925, doi:10.3791/1925 (2010).