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First published online March 19, 2008; 10.1104/pp.107.115246 Plant Physiology 147:30-40 (2008) © 2008 American Society of Plant Biologists OPEN ACCESS ARTICLE
Global Characterization of Cell-Specific Gene Expression through Fluorescence-Activated Sorting of Nuclei1,[W],[OA]Department of Plant Sciences (C.Z., R.A.B., G.M.L., D.W.G.), and BIO5 Institute for Collaborative Bioresearch (R.A.B., G.M.L., D.W.G.), The University of Arizona, Tucson, Arizona 85721
We describe a simple and highly effective means for global identification of genes that are expressed within specific cell types within complex tissues. It involves transgenic expression of nuclear-targeted green fluorescent protein in a cell-type-specific manner. The fluorescent nuclei are then purified from homogenates by fluorescence-activated sorting, and the RNAs employed as targets for microarray hybridization. We demonstrate the validity of the approach through the identification of 12 genes that are selectively expressed in phloem.
Multicellular eukaryotes exist in the form of complex tissues and organs, within which different cell types can be recognized and distinguished (Scheres et al., 2004  ). Considerable interest exists in establishing an inventory of these different cell types and furthermore in characterizing the global patterns of gene expression that underlie their establishment, function, and maintenance (Galbraith and Birnbaum, 2006). Complicating this characterization is the requirement to separate the different cell types in purified form prior to gene expression measurements. For both animal and plant species, this is most typically done utilizing hydrolytic enzymes to degrade the extracellular structures linking different cells together within tissues and organs, followed by imposition of some cell separation and enrichment method that is specific for the cell type of interest. Particularly productive has been the use of fluorescent proteins (FPs) as transgenic cell-type markers, employing appropriate regulatory sequences to highlight specific cells, followed by the production of protoplasts and the use of fluorescence-activated cell sorting to purify the FP-tagged protoplasts (Birnbaum et al., 2003, 2005; Nawy et al., 2005; Galbraith and Birnbaum, 2006; Brady et al., 2007).
The problems with methods involving tissue and organ dissociation relate, first, to the potential impact that these procedures may have on cellular function, as reflected by gene expression and, second, to the requirement that the methods be applicable to the organ and cell type of interest. This latter problem, common to all multicellular eukaryotes, is exacerbated particularly in higher plants because subtle changes in cell wall structure can render tissues and organs completely recalcitrant to enzymatic dissolution and release of protoplasts (Galbraith, 2007
A conceptually attractive alternative is to focus not on the cell, but on the transcriptional center of the cell—the nucleus—as the object of selective fluorescent labeling, and then employ gentle tissue homogenization (Galbraith et al., 1983
We have explored this strategy using the model plant Arabidopsis (Arabidopsis thaliana). Utilizing transgenic plants expressing a histone2A-GFP translational fusion within the companion cells of the phloem (Zhang et al., 2005
Cell-Type-Specific Analysis of Gene Expression: Platform Description and Implementation
We have previously shown that gentle disruption of plant tissues allows the release and facile analysis of intact nuclei using flow cytometry (Galbraith et al., 1983
For these experiments, we chose transgenic Arabidopsis plants in which expression of a nuclear-targeted version of GFP was under the control of the SULPHATE TRANSPORTER2;1 (SULTR2;1) promoter (Zhang et al., 2005 In transgenic plants expressing GFP under the control of the SULTR2;1 promoter, nuclear accumulation of GFP is restricted to elongated cells within the stele that fall within the phloem regions of the diarch structure. This is consistent with their identification as companion cells (Fig. 1A ; Supplemental Fig. S2); although we cannot formally exclude that they might also include other subsets of the cells of the stele, evidently they comprise a small minority. Biparametric flow cytometric analysis of homogenates, produced from these plants and counterstained using 4',6-diamidino-phenylindole (DAPI), resolved populations of nuclei with and without GFP fluorescence (Fig. 1, B and D). These were purified by flow sorting, and the RNA was extracted, amplified, and employed for hybridization to whole-genome 70-mer oligonucleotide microarrays.
Preliminary comparisons of transcript levels within the nuclear compartment and those of the total RNA of control (nontransgenic plants) revealed general concordance in global gene expression (Supplemental Fig. S3), suggesting that isolated nuclei can be employed as representative sources of transcriptional information concerning cellular states. For the first series of experiments, examination of differences of transcript levels between GFP-positive PCC nuclei and GFP-negative nuclei, utilizing two biological replications, indicated 0.2% were up-regulated and 1% down-regulated by factors of 2-fold or more, at an adjusted P value of less than 0.01 (Supplemental Table S1). A 5-fold enrichment (adjusted P value = 0.045) of the marker gene, the endogenous SULTR2;1, was also observed.
From the list of genes whose transcripts were found to be enriched within GFP-positive nuclei in the first series of experiments, we selected the first 12, for convenience entitled NUCLEAR-ENRICHED PHLOEM COMPANION CELL (NPCC) genes NPCC1 to NPCC12, corresponding to those having the greatest enrichment. Promoter regions, covering approximately 2,000 bp of genomic sequence 5' to the translational start site (or the entire intergenic region, if shorter than this), were employed to regulate transgenic expression of histone2B-GFP or histone2B-GFP-GFP fusion protein markers (Supplemental Table S2). In all cases, GFP fluorescence was located within the vascular core of transgenic roots, being predominantly associated with the positions of the two phloem sieve tubes and the associated PCCs, confirming the cell-type specificity of expression of the transcripts identified through flow sorting, as well as mapping the basis of this specific regulation to properties of the 5'-cis regions (Figs. 1E and 2; Supplemental Figs. S4–S15). Sieve tube-companion cell localization was strongly implied for NPCC2, NPCC4, and NPCC5 (Fig. 2 ; Supplemental Figs. S4–S6) given the observation that nonnuclear GFP fluorescence was distributed within two parallel tubes running along the axis of the root, with various degrees of organized nuclear fluorescence being found relatively infrequently along this axis (compare with NPCC4). In two cases (NPCC3 and NPCC6) a spacing of nuclear fluorescence similar to that of NPCC4 was seen but with an absence of nonnuclear GFP fluorescence (Fig. 2; Supplemental Fig. S7). This would be consistent with the hypothesis that GFP is localized solely within the nuclei of companion cells. In other cases, nuclear GFP accumulation was observed within a greater proportion of the cells of the stele, including those of the pericycle (NPCC7, NPCC8, NPCC10–NPCC12), and of the lateral root primordia (NPCC7, NPCC11, and NPCC12), which originate from the pericycle. The differences in GFP accumulation, notably the distribution of GFP within the interior of phloem sieve-tubes (NPCC2, NPCC4, and NPCC5), and within the general vascular core tissues (NPCC7, NPCC8, NPCC10, NPCC11, and NPCC12) suggest that some promoters may be active at different stages of phloem development, and hint at the possibility of selective transport and/or translation of the transgene mRNA in the context of the companion cell/sieve tube interface. As a final point, when these data were obtained, none of the 12 genes had been previously described as exhibiting phloem-specific expression, and none had a well-characterized function, indicating the general utility of the method for gene discovery.
Comprehensive Classification of Genes Having Transcripts That Are Enriched in Companion Cell Nuclei
For a comprehensive analysis of transcripts enriched in PCC nuclei, we combined the results of the first two sets of experiments with a third biological replicate to permit application of stringent statistical criteria for significance. This analysis, performed using Pointillist (Hwang et al., 2005a
In terms of genes of established function that are also known to be specifically expressed in the phloem, the entries listed in Supplemental Table S3 include At5g10180, which encodes a sulfate transporter and is the source of the promoter used to drive nuclear expression of GFP within the original transgenic plants employed for flow sorting, as well as At4g14680 and At3g22890, which encode ATP-sulfurylases involved in the initial steps of sulfur assimilation within roots (Lappartient et al., 1999
Comparison, to the genome at large, of the distribution of bioprocess annotational headings for genes whose transcripts were enriched in amounts within the PCC nuclei, indicated overrepresentation of only one classification—that of amino acid transporters (Table I
). This is in accord with the well-known physiological function of the phloem in amino acid transport and, by implication, represents a key molecular function for the PCCs. Within this particular classification, the acidic amino acid transporters At5g49630 and At5g09220 participate in the transport of Glu and other amino acids most important to nitrogen metabolism (Kwart et al., 1993
The  - and β-subunits of Glu dehydrogenase (At5g07440 and At5g18170) have been reported to be expressed uniquely in the companion cells of aerial portions of the plant, and are likely key to amine assimilation and recycling (Kichey et al., 2005; Fontaine et al., 2006). The transcripts for this enzyme and for Gln synthetase (At1g66200) do not appear in our list of NPCC transcripts. Within the root, the transport of Glu is central to the distribution of nitrogen metabolites, which suggests either that root PCCs do not participate directly in Glu/Gln metabolism or that such activity is not characterized by nuclear accumulation of associated transcripts.
Comparison of molecular annotational headings for the NPCC transcripts to those of the whole genome revealed overrepresentation for the terms sulfurylase, ceramidase, and carboxylase (Table I). Notable in the first category are the ATP-sulfurylases, At4g14680 and At3g22890, and coregulation of these genes is of course also consistent with the presence of the sulfate transporter, At5g10180, in the list of NPCC transcripts. The ceramidases, representing the second category, may play important roles in phloem ontology and function given the established involvement of sphingolipids in the regulation of programmed cell death (Liang et al., 2003
Interestingly, the transcript from locus At5g57190 is also enriched within the companion cell nuclei. This transcript encodes a tonoplast-localized phosphatidyl-Ser (PS) decarboxylase (Nerlich et al., 2007 The presence of the various amino acid transporters and permeases within the NPCC transcript list (Supplemental Table S3; Fig. 3 ) is consistent with the key role the phloem plays in the transport and distribution of amino acids and amines within plants. Along this line, another transcript enriched within the PCC nuclei, from At1g59740, is annotated by TIGR (http://www.tigr.org/tdb/at/atgenome/atgenome.html) as a proton-dependent oligopeptide transporter, and therefore may also participate in amine recycling from peptides derived from protein degradation within senescent tissue.
A final, general scan of the annotations associated with the NPCC transcripts revealed a large number of entries (15) described as transcription factors (Fig. 3). A further eight are described simply as zinc finger proteins, and may function either as transcription factors or, perhaps, as RNA-binding proteins associated with regulation of phloem function (Lunde et al., 2007 ). Fourteen of the NPCC transcripts are assigned to the signal transduction category (Fig. 3), implying regulatory roles. Thus, many of the transcripts associated specifically with accumulation in companion cell nuclei have functions consistent with their role in being part of the vasculature, whether that role is in transport or in the sensing of the physiological state of the plant and regulating appropriate responses.
To further explore the regulatory mechanisms associated with cell-type-specific nuclear transcript accumulation, the NPCC gene list (Supplemental Table S3) was divided into quartiles based on the mean raw fluorescent intensity signals recorded for each of the probes representing NPCC transcripts. The groups of genes within each quartile were separately analyzed using Promomer (http://bar.utoronto.ca/ntools/cgi-bin/BAR_Promomer.cgi) for the presence of shared 5-mer sequence motifs within their promoter regions, these regions being empirically defined as the 1,000 bp of sequence upstream of the start of transcription. Table II presents those 5-mer motifs that were significantly overrepresented in the promoter regions for 95% of the genes within that quartile, as compared to the genome at large.
These 5-mer motifs were then compared to plant promoter motifs contained in the PLACE database (http://www.dna.affrc.go.jp/PLACE/signalscan.html). One motif, ROOTMOTIFTAPOX1, identified by the 5-mer ATATT, forms part of the rolD promoter, and is expressed in the elongation zone and vasculature of the root (Elmayan and Tepfer, 1995 ). ROOTMOTIFTAPOX1 also is present in the upstream regions of >95% of the genes listed in the first quartile. Several of the 5-mer motifs (AAAGT, AAAAG, and AAAGA) overlap with DOFCOREZM (AAAG), a plant motif found in the promoters of the genes encoding phosphoenolpyruvate carboxylase and orthophosphate dikinase in maize (Zea mays; Yanagisawa and Schmidt, 1999). In nonphotosynthetic tissues, phosphoenolpyruvate carboxylase performs the anaplerotic function of maintaining the levels of tricarboxylic acid cycle intermediates (Hartwell et al., 1999), and is involved in regulating pH (Fontaine et al., 2002). Interestingly, PPCK1 (At1g08650) is found in our list of NPCC transcripts (Supplemental Table S3). The DOFCOREZM motif was also overrepresented within the genes of the first, second, and fourth quartiles, and is also overrepresented in the promoter regions for all the genes when analyzed together. Individual probes on microarrays are not thought to produce hybridization signals that can be directly compared in terms of transcript abundance. However, the assumptions inherent to microarray normalization are based on general concordance of this relationship across large numbers of genes. It will therefore be interesting to explore, in further experiments, the potential association of promoter motifs with genes according to gene expression level.
While this work was being prepared for publication, a report appeared that describes a comprehensive analysis of global gene expression patterns in the Arabidopsis root (Brady et al., 2007
Of the list of 12 genes (NPCC1–NPCC12) that we selected for confirmation of cell-type specificity tissue promoter tests (Fig. 2; Supplemental Tables S1 and S2), two, NPCC3 (At3g58720) and NPCC11 (At2g18196), are not represented in the probe sets of the Affymetrix ATH-1 GeneChip. Of the remainder, six are found in the phloem-related gene lists as described by Brady et al. (2007)
Given that the overall comparison is across platforms, and that the two approaches interrogate very different points within the process of gene expression, this general level of concordance (60%) is both impressive and encouraging. Because NPCC1 to NPCC12 represent genes producing transcripts showing the highest degree of differential accumulation within the nuclei of PCC cells, they may represent a subset of genes most readily identifiable for, and characteristic of, the phloem. Nevertheless, when comparing the complete gene list of NPCC genes (Supplemental Table S4) to those genes defined as being differentially expressed in APL-, S17-, S32-, and SUC2-GFP fusions, considerably less overlap is seen (Fig. 4
). The degree of overlap within the results for the APL, S17, S32, and SUC2 gene sets (Brady et al., 2007
We are extending this approach to additional cell types within Arabidopsis, including those found in its aerial organs, as well as to different plant species. We have also established that the approach is applicable to mammalian cells and tissues, through demonstration of general concordance between nuclear and cellular transcript levels (Barthelson et al., 2007 ). The approach therefore should be of considerable general interest and utility to investigators addressing the molecular basis of cell-type specificity within complex tissues of any eukaryotic species. Finally, given that identification of cell types is achieved simply using promoter sequences of defined specificities, one can envisage extending this analysis to novel cell types identified in transgenics through the expression of multiple combinations of FPs, but otherwise lacking recognizable (morphological or other) phenotypes, thereby leading to a more sophisticated and comprehensive understanding of organogenesis, microanatomy, and the regulation of cell-type specificity of gene expression.
Biological Materials Transgenic plants, expressing a translational fusion between GFP and histone H2A (HTA6; At5g59870) under the transcriptional control of the promoter of the SULTR2;1 gene (At5g10180), were used for this study. The genetic background of transgenic Arabidopsis (Arabidopsis thaliana) plants is ecotype Columbia (Col-0). The seeds of 10 to 12 independent transgenic T2 lines were combined and treated as one biological replicate to reduce potential influence of mutations caused by T-DNA insertions. Seeds were sterilized and planted on Murashige and Skoog (MS) agar plates supplemented with 2% Suc, 30 mg/L hygromycin, and 1.2% agar. The plates were maintained at 4°C for 2 d before transfer to a Conviron growth chamber under a 16-h day/8-h night illumination regime, with an incident light flux of 150 to 175 µm m–2 s–1, and temperature of 22°C (day) and 20°C (night). The plates were kept in a vertical orientation so that the roots grew on the surface of, rather than within, the agar medium. The roots of 2-week-old seedlings were sampled during the day, between 9 AM and 1 PM (Mountain Standard Time). Three biological replicates were prepared, 1 to 2 weeks apart.
Roots were homogenized by chopping (Zhang et al., 2005 Sorting was done using a Dako-Cytomation (now Beckman-Coulter) MoFlo flow cytometer/cell sorter equipped with a 70-µm flow tip and operated at a sheath pressure of 40 psi. Droplets were generated at 60,000 Hz using a piezo drive voltage of 17, and were sorted with a drop-delay setting of 24. Events were triggered on 90° side scatter, and were thresholded to provide an event rate of 30,000/s, with a sort rate for GFP-positive nuclei of 100/s, and 1,000/s for GFP-negative nuclei. The laser illumination power was set to 40 mW for DAPI excitation at 395 nm, and to 200 mW for GFP excitation at 488 nm. The barrier filters were 450/65 nm for DAPI fluorescence, and 530/40 nm for GFP fluorescence.
Total cellular RNA from whole roots was isolated using RNeasy Plant Mini Kits (QIAGEN) according to the manufacturer's recommendation. Total RNA was extracted from sorted nuclei using the TRIZOL method, following slight modifications of the manufacturer's recommendations (Invitrogen) as follows: nuclei were flow sorted (a final volume of approximately 120–200 µL) directly into 500 µL of TRIZOL reagent. The mixture was shaken vigorously, and stored at room temperature for 5 min. Chloroform (120 µL) was added and the tube was shaken by hand vigorously for 15 s. The tube was centrifuged at 11,900g for 15 min at 4°C. The upper phase was transferred to a new tube and an equal volume of isopropyl alcohol was added. The sample was mixed and kept overnight at –20°C. RNA was sedimented by centrifugation at 11,900g for 15 min at 4°C. After removing the supernatant, 1 mL of 75% ethanol was added to wash the RNA pellet (invisible). The tube was centrifuged at 11,900g for 10 min at 4°C, and the supernatant was completely removed. The tube was then left open for about 5 min to permit evaporation of residual ethanol. RNAase-free H2O (10 µL) was added to redissolve the RNA. The quantity and quality of the RNA was determined using an Agilent 2100 Bioanalyzer (Agilent). For the first set of experiments, two consecutive rounds of amplification were done on 5-µL (approximately 2–20 ng RNA) samples, using ExpressArt mRNA amplification kits (Nano plus Version; Artus GmbH) according to the manufacturer's instructions. Aminoallyl-modified UTP was incorporated into the amplified antisense RNA produced during the second round of in vitro transcription. For the second set of experiments, RNA amplification was done using the Ambion MessageAmp kit.
The amplified antisense RNA samples were labeled with Cy3-NHS or Cy5-NHS fluorescent dyes (Amersham Biosciences) according to the manufacturer's instructions. Labeled targets were purified using RNeasy MiniElute kits (QIAGEN). Arabidopsis whole genome long oligonucleotide microarrays (http://cals.arizona.edu/microarray/deconvolutionver3.0.html) were employed for hybridization, following standard procedures in the Galbraith laboratory (http://cals.arizona.edu/microarray/methods.html), and as previously described (Zanetti et al., 2005
For the second set of experiments, a data set was prepared from one additional hybridized slide. For the combined data sets from the first and second sets of experiments, the data were submitted to analysis with the program Pointillist (Hwang et al., 2005a
Cluster analysis was performed on the log-transformed intensity values for the list of PCC-enriched genes, using the program Genesis (http://genome.tugraz.at/genesisclient/genesisclient_description.shtml). The resulting clusters of genes were further analyzed with Promomer (http://bbc.botany.utoronto.ca/ntools/cgi-bin/BAR_Promomer.cgi) for the presence of 5-mer motifs that are significantly more common in 95% or greater of the 1,000-bp upstream sequences for the gene clusters compared to the Arabidopsis genome in general. Promomer provides a P value for the comparison of cluster genes to the whole genome, and those 5 mers with a P value of 0.05 or smaller are given in Figure 4. Further analysis was done with the aid of the PLACE database (http://www.dna.affrc.go.jp/PLACE/signalscan.html; Prestridge, 1991
To determine the expression patterns of the candidate genes identified through microarray analysis, plasmid constructs were made comprising nuclear-localized versions of GFP coding sequences placed under control of the upstream regulatory sequences. Genomic sequences about 2 kb upstream and up to 24 bp downstream of translational start (ATG) were amplified using PfuUltra high-fidelity DNA polymerase (Stratagene). The primers and related information are listed in Supplemental Table S2. The amplified sequences were ligated into plasmid vectors pBIn1GFP or pBIn2GFP (provided by Dr. Ramin Yadegari, University of Arizona), which express fusion proteins of HTB2-GFP and HTB2-GFP-GFP, respectively. Plasmids carrying the above constructs were introduced into Agrobacterium tumefaciens strain GV3101. Arabidopsis Col-0 was transformed using the floral dip method (Clough and Bent, 1998
Roots from T1 or T2 seedlings were counterstained with 10 µg mL–1 of propidium iodide (Sigma) for 2 min, and were placed on slides carrying a drop of water for observation. GFP fluorescence was imaged by confocal microscopy using a MRC 1024MP (Bio-Rad) confocal scanner attached to an Olympus BX-50 upright microscope, equipped with UPlanFl 4x/0.13, UPlanFl 10x/0.30, and UPlanApo 20x/0.70 objective lenses. LaserSharp2000 (Bio-Rad) was employed for image collection and color merging. For each construct, at least seven independent T1 seedlings were observed. Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number DQ370422.
The following materials are available in the online version of this article.
We thank R. Yadegari (University of Arizona) for his support and helpful suggestions. Received December 19, 2007; accepted March 13, 2008; published March 19, 2008.
1 This work was supported by the National Science Foundation Plant Genome Research Program (grant DBI–0211857 to D.W.G.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: David W. Galbraith (galbraith{at}arizona.edu).
[W] The online version of this article contains Web-only data.
[OA] Open Access article can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.115246 * Corresponding author; e-mail galbraith{at}arizona.edu.
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ABSTRACT
RESULTS AND DISCUSSION
