Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Summary

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

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 planta^5-7. 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 photoperception^{4, 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. Plant Growth

1. Confirmed UAS-BVR X GAL4-GFP enhancer trap line isolated as described⁴ (For summary see Fig. 1) and wild-type or parental lines are sown on soil, i.e. ~2000 sterilized seeds per line.
2. Plants are grown for 5 weeks on soil under white illumination of 100 μmolm^-2s^-1 at 22 °C and 70% humidity.

2. Leaf Protoplast Isolation (adapted from Denecke and Vitale⁹)

1. To isolate protoplasts, prepare TEX buffer. For 1 liter TEX buffer, weigh out the following components: 3.1 g Gamborg's B5 salts, 0.5 g 2-(N-morpholino)ethanesulfonic acid (MES,....

Discussion

Gene expression profiling through microarrays (1) has indicated that more than 30% of the genes in Arabidopsis seedlings are light regulated¹¹ and (2) has identified a vast group of genes encoding light signal transduction proteins involved in the phytochrome signaling cascade^{12, 13}. Such experiments suggest that light induces rapid and long-term changes in gene expression. Each pool of phytochromes may control only a subset of developmental and adaptive responses. Moreover, it is likely that downst.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-BVR antibody	QED Bioscience Inc.	56257-100
Cellulase “Onozuka” R-10	SERVA Electrophoresis	MSPC 0930
Gamborg’s B5 basal salt mixture	Sigma-Aldrich	G5768
Macerozyme R-10	SERVA Electrophoresis	PTC 001
MES, low moisture content	Sigma-Aldrich	M3671
Murashige and Skoog salts	Caisson Laboratories	74904
Phytablend	Caisson Laboratories	28302
RNeasy Plant Minikit	Qiagen	16419