Work in the Montgomery lab on phytochrome responses in plants is supported by the National Science Foundation (grant no. MCB-0919100 to B.L.M.) and the Chemical Sciences, Geosciences and Biosciences Division, Office of Basic Energy Sciences, Office of Science, U.S. Department of Energy (grant no. DE FG02 91ER20021 to B.L.M.). We thank Melissa Whitaker for technical assistance during filming and critically reading the manuscript, Stephanie Costigan for experimental assistance, Dr. Louis King for assistance with developing and optimizing Fluorescence-Activated Cell Sorting protocols for Arabidopsis protoplast sorting and Dr. Melinda Frame for assistance with confocal microscopy. We thank Marlene Cameron for graphical design assistance and Karen Bird for editorial assistance.

1. Plant Growth
<ol>
 <li>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. </li>
 <li>Plants are grown for 5 weeks on soil under white illumination of 100 &mu;molm-2s-1 at 22 &deg;C and 70% humidity.</li>
</ol>
2. Leaf Protoplast Isolation (adapted from Denecke and Vitale9)
<ol>
 <li>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, 2.56 mM), 0.75 g calcium chloride dihydrate (CaCl2.2H2O, 6.75 mM), 0.25 g ammonium nitrate (NH4NO3, 3.12 mM), 136.9 g sucrose (0.4M).Dissolve components completely in ~900 ml of deionized, distilled water (ddH2O) and bring the pH to 5.7 with 1 M KOH. Bring final volume to 1 liter and filter sterilize with a 0.2 &mu;m bottle-top filter connected to a vacuum pump.</li>
 <li>Collect green, healthy leaves from plants (~250 ml of leaves loosely packed in a beaker) and rinse with~ 40 ml ddH2O 4 times, followed by rinsing twice with sterile ddH2O. Using a #20 scalpel, cut leaves into thin strips and divide tissue equally into two 50 ml sterile plastic tubes. Prepare 1x leaf digestion mixture by adding 5 ml aliquot of 10x leaf digestion stock solution (10x leaf digestion stock solution: dissolve 2% w/v Macerozyme R-10, 4% w/v Cellulase "Onozuka" R-10 in TEX buffer, filter sterilize with 0.2 &mu;m filter and freeze 5 ml aliquots at -80 &deg;C) to 45 ml fresh TEX buffer. Add ~25 ml of 1x leaf digestion mix per 50 ml tube to cover all leaf tissue.</li>
 <li>Vacuum infiltrate leaf tissue in digestion mixture in open tubes for 1 hr at room temperature using a vacuum desiccator connected to a water pump followed by 3-hr incubation at room temperature on a rocker with gentle shaking. Capped tubes with leaf tissue in 1x leaf digestion mix are kept wrapped in aluminum foil during this process to prevent exposure to light.</li>
 <li>After 3 hours, increase the rocker speed for ~2 minutes to release protoplasts. Filter the crude protoplast suspension through two layers of sterile cheese cloth to remove debris and collect the filtrate in a sterile glass beaker. Filter the filtrate through a sterile 100-&mu;m nylon mesh into a sterile Petri dish. Collect the flow through and transfer it to a new sterile 50 ml tube. Use about 15-20 ml of fresh TEX buffer to wash the sterile Petri dish and collect any protoplasts adhering to the surface. </li>
 <li>Centrifuge the flow through using a swing bucket rotor at 100xg at 10 &deg;C for 15 min (Acceleration 6, Deceleration 0).</li>
 <li>Remove ~25 - 30 ml of the liquid below the floating protoplast layer, which contains residual and pelleted debris, with a sterile 9" glass Pasteur pipette connected to a peristaltic pump without disturbing the floating protoplast layer and leaving ~10 - 15 ml volume.</li>
 <li>Add fresh TEX buffer to a final volume of 40 ml while gently resuspending the protoplasts.</li>
 <li>Repeat steps 2.5 to 2.7 two times to remove as much cellular debris as possible. The centrifugation time is reduced to 10 minutes in the first repetition and to 5 minutes in the final repetition.</li>
 <li>Aspirate floating protoplasts with a cut, sterile 1 ml transfer pipette into a new 15 ml tube. Proceed directly to sorting or store in dark for up to 2 hours at 4 &deg;C until sorting.</li>
</ol>
3. Protoplast Sorting by Fluorescence-Activated Cell Sorting (FACS)
<ol>
	<li>Before sorting, examine protoplasts by Confocal Laser Scanning Microscopy (CLSM) using a 488-nm laser for excitation to confirm protoplast integrity and the presence of GFP fluorescence in the protoplast pool. Minimal debris is necessary to avoid clogging the FACS sorting nozzle.</li>
	<li>Sort isolated protoplasts in TEX buffer via FACS (BD FACSVantage SE, BD Biosciences) using a 200-&mu;m nozzle on a macro sort head at event rates between 6,000 and 15,000, with a system pressure of around 9 p.s.i. following an adapted protocol10.</li>
	<li>Wild-type non-GFP protoplasts are used to determine the autofluorescence thresholds.</li>
 <li>To collect GFP-positive protoplasts, sort cells using an air-cooled argon laser (Spectra Physics Model 177, Newport Corporation, Irvine, CA) operated at 100 mW on a 488-nm argon line to identify GFP fluorescence using a 530/30 band pass filter.</li>
	<li>Following the collection of GFP-positive protoplasts, examine sorted protoplasts by CLSM as detailed in step 3.1.</li>
	<li>Extract total RNA from sorted protoplasts using a Qiagen RNeasy Plant Mini Kit. cDNA can then be prepared for hybridization as described in Affymetrix GeneChip Expression Analysis Technical Manual and then hybridized to AtH1 Arabidopsis Affymetrix whole genome arrays.</li>
</ol>
Representative Results
We detected a significant number of GFP-positive cells in the GFP channel by FACS (Fig. 2). In optimization assays using GFP enhancer-trap parents, the constitutively GFP-expressing line, J0571, displayed ~ 17% to 24% GFP-positive protoplasts, whereas the line with vascular and dermal expression, J1071, had ~1.4% GFP-positive protoplasts (Table 1). Sorting of 3 x 500 &mu;l (~ 1.5 ml total of protoplast suspension) of J0571 protoplasts for 1 h gave ~ 100,000 GFP-positive protoplasts. Sorting of 3 x 500 &mu;l (~ 1.5 ml total protoplast suspension) of J1071 protoplasts for 1.5 h gave ~ 3,000 GFP-positive protoplasts. Confocal images indicated a very high yield of protoplasts for both J0571 and J1071 samples before sorting was carried out (Fig. 3C and 3E), and the sorted fractions contained only bright GFP-fluorescent protoplasts (Fig. 3G and data not shown). This finding confirms that intact GFP-positive protoplasts can be sorted via FACS. The non-GFP protoplasts can also be sorted and collected in a separate channel. The non-GFP protoplasts serve as an ideal negative control for subsequent microarray analyses to detect the specific changes in gene expression upon reduction of holophytochrome levels. RNA extraction from isolated protoplasts (1 ml) yields RNA of sufficient quantity for detection by fluorospectrometry (Fig. 4). Isolated RNA was assessed using a NanoDrop instrument (NanoDrop 1000, Thermo Scientific) and quantified by NanoDrop 3.7.1 RNA quantification software. RNA yields from pre-sorted C24 wild-type protoplasts or FACS-sorted, GFP-positive enhancer trap protoplasts exceeded the minimum 20 ng needed for use in RNA-labeling assays for microarray (Table 2). 
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Enhancer Trap Line</td>
 <td>% of GFP-positive protoplasts</td>
 <td>Number of sorted GFP protoplasts</td>
 </tr>
 <tr>
 <td>J0571</td>
 <td>17.18 % ~ 24.06 %</td>
 <td>26,400 ~ 36,000</td>
 </tr>
 <tr>
 <td>J1071</td>
 <td>~ 1.43 %</td>
 <td>1,000 ~ 1,300</td>
 </tr>
</tbody>
</table>
Table 1. Percentage of GFP-positive protoplasts from two enhancer trap lines before sorting and the number of GFP-positive protoplasts collected by running 500 &mu;L of protoplast suspension through the Fluorescence Activated Cell Sorter (FACSVantage, BD). 
<table border="1" cellspacing="0" cellpadding="0">
<tbody>
 <tr>
 <td>Plant Line</td>
 <td>RNA yield (ng)</td>
 </tr>
 <tr>
 <td>C24 WT</td>
 <td>12486.5</td>
 </tr>
 <tr>
 <td>J0571</td>
 <td>60</td>
 </tr>
 <tr>
 <td>J1071</td>
 <td>71.5</td>
 </tr>
</tbody>
</table>
Table 2. Yield from RNA isolation from protoplasts. RNA was isolated from pre-sorted C24 wild-type or sorted GFP-positive protoplasts from two enhancer trap lines and quantified.
<img src="/files/ftp_upload/1925/1925fig1.jpg" alt="Figure 1" /> Figure 1. GAL4 enhancer-trap-based induction of Biliverdin Reductase (BVR) expression in transgenic Arabidopsis thaliana plants. (A). An individual selected from a library of GAL4-based enhancer trap lines, which contain a GAL4-responsive GFP marker gene, can be crossed with a line containing a GAL4-responsive target gene to induce expression of the target gene in GAL4-containing cells marked by GFP fluorescence. Based on a figure from Dr. Jim Haseloff (http://www.plantsci.cam.ac.uk/Haseloff/geneControl/GAL4Frame.html). (B) Production of phytochrome chromophore, phytochromobilin (P&Phi;B) and holophytochrome in parent lines. (C) Left, Reduction of biliverdin IX&#945; (BV IX&#945;) and P&Phi;B by biliverdin reductase (BVR) activity to BR and P&Phi;R, respectively. BVR activity results in depletion of P&Phi;B and leads to a reduction in the production of photoactive holophytochrome. Right, the reaction catalyzed by BVR is shown.
<img src="/files/ftp_upload/1925/1925fig2.jpg" alt="Figure 2" /> Figure 2. Fluorescence Activated Cell Sorting (FACS) acquisition dot plots. Comparison of protoplast cell sorting for C24 wild type (A), J0571 (B) and J1071 (C). (A) Acquisition dot plot of non-GFP fluorescent C24 wild-type protoplasts excited by a 488-nm argon laser and used to determine autofluorescence threshold. (B) and (C) acquisition dot plots show proportions of protoplasts that are GFP positive in response to excitation by a 488-nm laser. R3 sorting gates in B and C delimit the GFP-positive targets that were sorted by Fluorescence-Activated Cell Sorter (FACSVantage, BD) and collected. Red channel indicates values for chlorophyll autofluorescence from protoplasts and green channel indicates values for GFP fluorescence.
<img src="/files/ftp_upload/1925/1925fig3.jpg" alt="Figure 3" /> Figure 3. Confocal microscopy of plant protoplasts used for cell sorting. Confocal laser scanned images of protoplasts before (A, C, E) and after (G) sorting via Fluorescence-Activated Cell Sorter (FACS). B, D, F and H are DIC images. Protoplasts of C24 wild type (A, B), J1071 (C, D) and J0571 (E, F, G, H) are shown. Images C, E and G are merged images of GFP fluorescence (BP 505 nm - 575 nm) and Autofluorescence (LP 650 nm) obtained from excitation with a 488-nm laser. A through H are average of 4 scans under 63x oil. Bar = 10 &mu;m.
<img src="/files/ftp_upload/1925/1925fig4.jpg" alt="Figure 4" /> Figure 4. Quantification of RNA isolated from protoplasts by fluorospectrometry. RNA extracted from (A) C24 wild type, (B) J0571, and (C) J1071. A indicates RNA for pre-sorted wild-type protoplasts. B and C indicate RNA from GFP-positive protoplasts sorted by Fluorescence-Activated Cell Sorting (FACS). RNA quantification software, NanoDrop 3.7.1, (NanoDrop 1000, Thermo Scientific).

<ol>
	<li>Franklin, K. A., Quail, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phytochrome+functions+in+Arabidopsis+development.">Phytochrome functions in Arabidopsis development.</a> J. Exp. Bot. 61, 11-24 (2010).</li><li>Montgomery, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Right+place,+right+time:+Spatiotemporal+light+regulation+of+plant+growth+and+development.">Right place, right time: Spatiotemporal light regulation of plant growth and development.</a> Plant Signal Behav. 3, 1053-1060 (2008).</li><li>Laplaze, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GAL4-GFP+enhancer+trap+lines+for+genetic+manipulation+of+lateral+root+development+in+Arabidopsis+thaliana.">GAL4-GFP enhancer trap lines for genetic manipulation of lateral root development in Arabidopsis thaliana.</a> J. Exp. Bot. 56, 2433-2442 (2005).</li><li>Costigan, S., Warnasooriya, S. N., Montgomery, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Root-localized+phytochrome+chromophore+synthesis+is+required+for+tissue-specific+photoregulation+of+root+elongation+and+impacts+sensitivity+to+jasmonic+acid+in+Arabidopsis+thaliana.">Root-localized phytochrome chromophore synthesis is required for tissue-specific photoregulation of root elongation and impacts sensitivity to jasmonic acid in Arabidopsis thaliana.</a> , .</li><li>Lagarias, D. M., Crepeau, M. W., Maines, M. D., Lagarias, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+photomorphogenesis+by+expression+of+mammalian+biliverdin+reductase+in+transgenic+Arabidopsis+plants.">Regulation of photomorphogenesis by expression of mammalian biliverdin reductase in transgenic Arabidopsis plants.</a> Plant Cell. , 675-688 (1997).</li><li>Montgomery, B. L., Yeh, K. C., Crepeau, M. W., Lagarias, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+distinct+aspects+of+photomorphogenesis+via+targeted+expression+of+mammalian+biliverdin+reductase+in+transgenic+Arabidopsis+plants.">Modification of distinct aspects of photomorphogenesis via targeted expression of mammalian biliverdin reductase in transgenic Arabidopsis plants.</a> Plant Physiol. 121, 629-639 (1999).</li><li>Warnasooriya, S. N., Montgomery, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+spatial-specific+phytochrome+responses+using+targeted+expression+of+biliverdin+reductase+in+Arabidopsis.">Detection of spatial-specific phytochrome responses using targeted expression of biliverdin reductase in Arabidopsis.</a> Plant Physiol. 149, 424-433 (2009).</li><li>Warnasooriya, S. N., Porter, K. J., Montgomery, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-dependent+anthocyanin+accumulation+and+phytochromes+in+Arabidopsis+thaliana.">Light-dependent anthocyanin accumulation and phytochromes in Arabidopsis thaliana.</a> , .</li><li>Denecke, J., Vitale, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+protoplasts+to+study+protein+synthesis+and+transport+by+the+plant+endomembrane+system.">The use of protoplasts to study protein synthesis and transport by the plant endomembrane system.</a> Methods Cell Biol. 50, 335-348 (1995).</li><li>Birnbaum, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+expression+profiling+in+plants+via+cell+sorting+of+protoplasts+from+fluorescent+reporter+lines.">Cell type-specific expression profiling in plants via cell sorting of protoplasts from fluorescent reporter lines.</a> Nat. Methods. 2, 615-619 (2005).</li><li>Ma, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+control+of+Arabidopsis+development+entails+coordinated+regulation+of+genome+expression+and+cellular+pathways.">Light control of Arabidopsis development entails coordinated regulation of genome expression and cellular pathways.</a> Plant Cell. 13, 2589-2607 (2001).</li><li>Chen, M., Chory, J., Fankhauser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light+signal+transduction+in+higher+plants.">Light signal transduction in higher plants.</a> Annu. Rev. Genet. 38, 87-117 (2004).</li><li>Ulm, R., &amp;amp, N. a. g. y., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signalling+and+gene+regulation+in+response+to+ultraviolet+light.">Signalling and gene regulation in response to ultraviolet light.</a> Curr. Opin. Plant Biol. 8, 477-482 (2005).</li><li>Ma, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ-specific+expression+of+Arabidopsis+genome+during+development.">Organ-specific expression of Arabidopsis genome during development.</a> Plant Physiol. 138, 80-91 (2005).</li><li>Neff, M. M., Fankhauser, C., &amp;amp, C. h. o. r. y., J, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light:+an+indicator+of+time+and+place.">Light: an indicator of time and place.</a> Genes Dev. 14, 257-271 (2000).</li><li>Birnbaum, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+expression+map+of+the+Arabidopsis+root.">A gene expression map of the Arabidopsis root.</a> Science. 302, 1956-1960 (2003).</li></ol>

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 cascade12, 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...

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Anti-BVR antibody</td>
<td>&nbsp;</td>
<td>QED Bioscience Inc.</td>
<td>56257-100</td>
<td>&nbsp;</td>
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<td>Cellulase &#x201C;Onozuka&#x201D; R-10</td>
<td>&nbsp;</td>
<td>SERVA Electrophoresis GmbH, Crescent Chemical Company</td>
<td>MSPC 0930</td>
<td>&nbsp;</td>
<tr>
<td>Gamborg&#x2019;s B5 basal salt mixture</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>G5768</td>
<td>&nbsp;</td>
<tr>
<td>Macerozyme R-10</td>
<td>&nbsp;</td>
<td>SERVA Electrophoresis GmbH, Crescent Chemical Company</td>
<td>PTC 001</td>
<td>&nbsp;</td>
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<td>MES, low moisture content</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M3671</td>
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<td>Murashige and Skoog salts</td>
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<td>Caisson Laboratories</td>
<td>74904</td>
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<td>Phytablend</td>
<td>&nbsp;</td>
<td>Caisson Laboratories</td>
<td>28302</td>
<td>&nbsp;</td>
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<td>RNeasy Plant Minikit</td>
<td>&nbsp;</td>
<td>Qiagen</td>
<td>16419</td>
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investigating tissue- and organ-specific phytochrome responses using facs-assisted cell-type specific expression profiling in arabidopsis thaliana

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.

Watch this Scientific Journal Video about Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana at JoVE.com

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

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.

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

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

investigating-tissue-organ-specific-phytochrome-responses-using-facs

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15.7K Views.  Michigan State University (MSU). 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.

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

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&amp;#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Heterokaryon Technique for Analysis of Cell Type-specific Localization

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling. These proteins display a diverse array of cellular functions including nuclear import/export of RNA and protein, transcriptional regulation, and apoptosis. Interestingly, major cellular reorganizations including cell division, differentiation and transformation, often involve such activities3,4,8,10. The detailed study of these proteins and their respective regulatory mechanisms can be challenging as the stimulation for these localization changes can be elusive, and the movements themselves can be quite dynamic and difficult to track. Studies involving cellular oncogenesis, for example, continue to benefit from understanding pathways and protein activities that differ between normal primary cells and transformed cells6,7,11,12. As many proteins show altered localization during transformation or as a result of transformation, methods to efficiently characterize these proteins and the pathways in which they participate stand to improve the understanding of oncogenesis and open new areas for drug targeting.
Here we present a method for the analysis of protein trafficking and shuttling activity between primary and transformed mammalian cells. This method combines the generation of heterokaryon fusions with fluorescence microscopy to provide a flexible protocol that can be used to detect steady-state or dynamic protein localizations. As shown in Figure 1, two separate cell types are transiently transfected with plasmid constructs bearing a fluoroprotein gene attached to the gene of interest. After expression, the cells are fused using polyethylene glycol, and protein localizations may then be imaged using a variety of methods. The protocol presented here is a fundamental approach to which specialized techniques may be added.

A flexible and efficient method for the characterization of cell type-specific protein localization and nucleocytoplasmic shuttling is described. This heterokaryon approach uses fluorescently-labeled fusion proteins to image protein localizations after cell fusion. The protocol is amenable to steady-state localizations or more dynamic determinations based on live cell imaging.

A significant number of proteins are regulated by subcellular trafficking or nucleocytolasmic shuttling.  These proteins display a diverse array of ...

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as phosphatidylcholine or phosphatidylethanolamine, form bilayer membranes which act as both boundaries and interfaces for chemical exchange between cells and their surroundings. Unlike animals, plant cells have a special organelle for photosynthesis, the chloroplast. The intricate membrane system of the chloroplast contains unique glycerolipids, namely glycolipids lacking phosphorus: monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG)4. The roles of these lipids are beyond simply structural. These glycolipids and other glycerolipids were found in the crystal structures of photosystem I and II indicating the involvement of glycerolipids in photosynthesis8,11. During phosphate starvation, DGDG is transferred to extraplastidic membranes to compensate the loss of phospholipids9,12.
Much of our knowledge of the biosynthesis and function of these lipids has been derived from a combination of genetic and biochemical studies with Arabidopsis thaliana14. During these studies, a simple procedure for the analysis of polar lipids has been essential for the screening and analysis of lipid mutants and will be outlined in detail. A leaf lipid extract is first separated by thin layer chromatography (TLC) and glycerolipids are stained reversibly with iodine vapor. The individual lipids are scraped from the TLC plate and converted to fatty acyl methylesters (FAMEs), which are analyzed by gas-liquid chromatography coupled with flame ionization detection (FID-GLC) (Figure 1). This method has been proven to be a reliable tool for mutant screening. For example, the tgd1,2,3,4 endoplasmic reticulum-to-plastid lipid trafficking mutants were discovered based on the accumulation of an abnormal galactoglycerolipid: trigalactosyldiacylglycerol (TGDG) and a decrease in the relative amount of 18:3 (carbons : double bonds) fatty acyl groups in membrane lipids 3,13,18,20. This method is also applicable for determining enzymatic activities of proteins using lipids as substrate6.

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Composition of polar lipid extracts and the fatty acid composition of individual glycerolipids are determined in a simple and robust lipid profiling experiment. For this purpose, glycerolipids are isolated by thin layer chromatography and subjected to transmethylation of their acyl groups. Fatty acyl methylesters are quantified by gas-liquid chromatography.

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify ...

Isolate Cell-Type-Specific RNAs from Snap-Frozen Heterogeneous Tissue Samples without Cell Sorting

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids potential pitfalls caused by the heterogeneity of tissues and unleashes the powerful transcriptome analysis. The protocol described here demonstrates how to use the Translating Ribosome Affinity Purification (TRAP) method to isolate ribosome-bound RNAs from a small amount of EGFP-expressing cells in a complex tissue without cell sorting. This protocol is suitable for isolating cell-type-specific RNAs using the recently available NuTRAP mouse model and could also be used to isolate RNAs from any EGFP-expressing cells.

This protocol aims to isolate cell-type-specific translating ribosomal mRNAs using the NuTRAP mouse model.

Cellular heterogeneity poses challenges to understanding the function of complex tissues at a transcriptome level. Using cell-type-specific RNAs avoids ...

An Improved Method to Measure Pollen Hydration Profiles in &lt;em&gt;Arabidopsis thaliana&lt;/em&gt;

A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is established between the interacting partners. Studies across a range of species have revealed that a series of molecular checkpoints regulate the pollen-stigma interaction to ensure that only compatible, generally intraspecific pollen is successful in effecting fertilization. In species that possess a 'dry stigma', such as the model plant Arabidopsis thaliana, the first post-pollination, prezygotic compatibility checkpoint is the establishment of pollen hydration. 
This phase of pollination is tightly regulated, whereby signals from the pollen grain elicit the release of water from the stigma, thus permitting pollen hydration. The ability to accurately measure and track pollen hydration over time is key to the design of experiments directed at understanding the regulation of this critical step in reproduction. Published protocols frequently utilize flowers that have been excised from the parent plant, maintained on liquid or solid media, and bulk pollinated. 
This paper describes a noninvasive, in vivo pollination bioassay that permits minute-by-minute hydration tracking of individual A. thaliana pollen grains at high resolution. The assay is highly reproducible, able to detect very subtle variations of pollen hydration profiles, and thus is suitable for the analysis of mutants that affect pathways regulating pollination. Although the protocol is lengthier than those described for bulk pollinations, the precision and reproducibility it provides, along with its in vivo nature, make it ideal for the detailed dissection of pollination phenotypes.

Author Spotlight: A High-Resolution, Single-Grain, In Vivo Pollen Hydration Bioassay for Arabidopsis thaliana  

An improved method to measure pollen hydration profiles in Arabidopsis thaliana is described here. The new method offers higher resolution, is noninvasive, and is highly reproducible. The protocol represents a new tool for a finer dissection of the processes that regulate the early stages of pollination.

Sexual reproduction in flowering plants requires initial interaction between the pollen grain and the stigmatic surface, where a molecular dialog is ...

Pollen Hydration Assay in Arabidopsis thaliana: Raw Data Acquisition and its Measurements

Pollen Hydration Assay in Arabidopsis thaliana: Raw Data Acquisition and its Measurements  

Begin the assay by laying the labeled A.thaliana pollen recipient plant on its side, positioning the emasculated flower on the stage of an inverted microscope to image the stigma. To fix the position of the emasculated recipient flower, immobilize the stem using strips of masking tape. From the pollen donor plant, remove a healthy and freshly-opened flower and place the pollen donor plant under a dissecting microscope.Gather pollen grains using fine-tipped forceps. Remove excess pollen grains by lightly touching the forceps against the donor petals until the pollen grain's monolayer is formed. Return to the pollen recipient plant and use a low power objective lens to focus the inverted microscope on the stigma.Holding the forceps along the opening between the arms of the forceps, carefully approach and unpollinated stigmatic papilla cell. Continue approaching the unpollinated stigmatic papilla cell until the suitably positioned pollen grain on the forceps makes light contact with its surface. Once pollen attachment is confirmed, slowly withdraw the forceps.Orient the pollen grain with its equatorial axis visible and in sharp focus, and immediately switch to a higher power objective lens, like 20x. Capture the first pollen grain image as T-0, and continue capturing images at one-minute intervals for 10 minutes. Adjust the focus to accommodate small movements in the pollen grain or stigma.Record the room's ambient temperature and relative humidity every two minutes, allowing future comparisons between experimental replicates. Save the images in a lossless format, such as ND2, before repeating the pollination and imaging steps for additional pollen grains to acquire data for control and experimental pollinations. Begin the data measurements for pollen hydration assay using image analysis software.Use similar parameters of digital zoom and the approach defining the pollen boundary for all measurements in the datasets. Record the semiminor axis values for all time points in the dataset. Once the measurements are complete for a dataset, export the raw semiminor axis values of each individual time point to a spreadsheet.Present the data in columns per image stack and calculate the percentage semiminor axis change. Repeat the steps and obtain data from at least 15 hydrated pollen grains for each plant line. A few pollen grains may fail to hydrate or may hydrate significantly slower than expected.They're known as dud grains. Exclude these from the dataset. Calculate the mean values for each time point per plant using the specialized software package GraphPad Prism.Analyze the hydration data from wild-type and mutant lines at each time point using unpaired T-test and one-way ANOVA to compare the means of the specific time point of interest. To compare the means across all the time points, perform multiple T-tests between wild-type and mutant lines across multiple time points. The pollen hydration time series data for wild-type plants collected on different days showed that the minimum and maximum values for the means between replicates for all time points were within 3%This representative data for wild-type pollinations demonstrated a high degree of consistency for relatively low sample numbers and across different days.