Исследование тканей и органов конкретных Фитохром ответы с помощью FACS-помощь камерного типа Конкретные профилирования выражения в Arabidopsis thaliana</em
Summary
Молекулярные основы пространственно-конкретных ответов фитохрома в настоящее время исследованы с помощью трансгенных растений, которые обладают тканей и органов конкретных фитохрома недостатков. Выделение специфических клеток выставке индуцированных фитохрома истощения хромофора по флуоресценции, активированных сортировки клеток следуют анализ микрочипов в настоящее время используется для идентификации генов, вовлеченных в пространственно-конкретных ответных фитохрома.
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. Роста растений Подтвержденные UAS-BVR X GAL4-GFP усилитель ловушку линии выделяли, как описано 4 (для резюме см. рис. 1) и дикого типа или родительских линий и посеянное на почве, т.е. ~ 2000 стерилизованных семян на линии. Растения выращиваются в течение 5 недель на почве под белой …
Discussion
Экспрессия генов профилирования через микрочипы (1) показал, что более 30% генов Arabidopsis сеянцы света регулируется 11 и (2) выявила обширная группа генов, кодирующих светового сигнала трансдукции белков, участвующих в фитохрома сигнальный каскад 12, 13 . Такие эксперименты позволя?…
Acknowledgements
Работа в Монтгомери лаборатории на фитохрома ответы в растениях при поддержке Национального научного фонда (грант №. MCB-0919100 с БЛМ) и химических наук, наук о Земле и биологических наук отдела Управления основной энергии наук, Управление по науке департамента США Энергия (грант №. DE FG02 91ER20021 для BLM). Мы благодарим Мелисса Уитакер за техническую помощь во время съемок и критически чтения рукописи, Стефани Костиган для экспериментальных помощи, доктор Louis King за помощь в разработке и оптимизации флуоресценции, активированных сортировки клеток Arabidopsis протоколов для сортировки протопластов и д-р Мелинда Рамка для помощи конфокальной микроскопии. Мы благодарим Марлен Камерон для графического дизайна и помощь Карен птиц за помощь в редактировании.
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).
Cite This 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).