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.
1. Plant Growth
2. Leaf Protoplast Isolation (adapted from Denecke and Vitale9)
3. Protoplast Sorting by Fluorescence-Activated Cell Sorting (FACS)
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 μl (~ 1.5 ml total of protoplast suspension) of J0571 protoplasts for 1 h gave ~ 100,000 GFP-positive protoplasts. Sorting of 3 x 500 μl (~ 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).
Enhancer Trap Line | % of GFP-positive protoplasts | Number of sorted GFP protoplasts |
J0571 | 17.18 % ~ 24.06 % | 26,400 ~ 36,000 |
J1071 | ~ 1.43 % | 1,000 ~ 1,300 |
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 μL of protoplast suspension through the Fluorescence Activated Cell Sorter (FACSVantage, BD).
Plant Line | RNA yield (ng) |
C24 WT | 12486.5 |
J0571 | 60 |
J1071 | 71.5 |
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.
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 (PΦB) and holophytochrome in parent lines. (C) Left, Reduction of biliverdin IXα (BV IXα) and PΦB by biliverdin reductase (BVR) activity to BR and PΦR, respectively. BVR activity results in depletion of PΦB and leads to a reduction in the production of photoactive holophytochrome. Right, the reaction catalyzed by BVR is shown.
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.
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 μm.
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).
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 downstream signaling components interact with phytochromes in a cell- and tissue-specific manner2, 14, 15.
The expression of downstream candidate genes can be up- or down-regulated upon targeted BVR expression. Through comparative analysis of gene expression changes in UAS-BVR X GAL4-GFP progeny and parental lines, we can identify distinct changes in gene expression where such changes result from localized phytochrome deficiencies. Based on gene expression profiles, we can identify genes that encode negative and positive factors in phytochrome-mediated cell-to-cell signaling. Recent data suggest that several hundred transcripts are induced by protoplasting of plant tissue16. Thus, the ideal negative control for microarray analysis is RNA isolated from non-GFP protoplasts collected during sorting. Transcripts that are induced by protoplasting of plant tissue can be excluded from data analysis using these controls, resulting in the identification of target genes specifically involved in tissue- and organ-specific phytochrome signaling and/or responses. To confirm steady-state changes in expression of genes identified via cell-type-specific expression profiling, qRT-PCR can be carried out on Poly (A) RNA from the sorted GFP-positive and GFP-negative protoplasts.
Genes whose expression is changed significantly in microarray analyses and confirmed by qRT-PCR are identified as candidates involved in discrete phytochrome-mediated intercellular signaling. If T-DNA insertion mutants are already available in candidate genes, they can be analyzed for light-impaired phenotypes. By comparing the phenotypes of mutants carrying mutations in candidate genes and known phytochrome mutants to wild type, we can identify specific responses in which candidate genes have regulatory roles. If the mutants with mutations in candidate genes display phytochrome-deficient phenotypes, this will confirm that the identified downstream target genes are involved in mediating distinct phytochrome-regulated responses.
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.
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 |