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. 2004 Mar;16(3):596-615.
doi: 10.1105/tpc.019000. Epub 2004 Feb 18.

Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant

Nathalie Leonhardt  1 , June M Kwak, Nadia Robert, David Waner, Guillaume Leonhardt, Julian I Schroeder
Affiliations

Microarray expression analyses of Arabidopsis guard cells and isolation of a recessive abscisic acid hypersensitive protein phosphatase 2C mutant

Nathalie Leonhardt et al. Plant Cell. 2004 Mar.

Erratum in

  • Plant Cell. 2005 Apr;17(4):1330-3

Abstract

Oligomer-based DNA Affymetrix GeneChips representing about one-third of Arabidopsis (Arabidopsis thaliana) genes were used to profile global gene expression in a single cell type, guard cells, identifying 1309 guard cell-expressed genes. Highly pure preparations of guard cells and mesophyll cells were isolated in the presence of transcription inhibitors that prevented induction of stress-inducible genes during cell isolation procedures. Guard cell expression profiles were compared with those of mesophyll cells, resulting in identification of 64 transcripts expressed preferentially in guard cells. Many large gene families and gene duplications are known to exist in the Arabidopsis genome, giving rise to redundancies that greatly hamper conventional genetic and functional genomic analyses. The presented genomic scale analysis identifies redundant expression of specific isoforms belonging to large gene families at the single cell level, which provides a powerful tool for functional genomic characterization of the many signaling pathways that function in guard cells. Reverse transcription-PCR of 29 genes confirmed the reliability of GeneChip results. Statistical analyses of promoter regions of abscisic acid (ABA)-regulated genes reveal an overrepresented ABA responsive motif, which is the known ABA response element. Interestingly, expression profiling reveals ABA modulation of many known guard cell ABA signaling components at the transcript level. We further identified a highly ABA-induced protein phosphatase 2C transcript, AtP2C-HA, in guard cells. A T-DNA disruption mutation in AtP2C-HA confers ABA-hypersensitive regulation of stomatal closing and seed germination. The presented data provide a basis for cell type-specific genomic scale analyses of gene function.

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Figures

Figure 1.
Figure 1.
Transcription Inhibitors Suppress Stress-Induced PAL Gene Expression during Guard Cell Isolation. RT-PCR analyses of PAL in guard cells extracted in the absence (GC) or presence (GC + Inh) of the transcription inhibitors actinomycin D (33 mg/L) and cordycepin (100 mg/L). The Actin2 gene was amplified as a control.
Figure 2.
Figure 2.
GeneChip Hybridizations Show Genomic Scale Reproducibility. Scatter plots comparing the raw signal intensities of two independent experiments from guard cells (A), guard cells treated with ABA (B), mesophyll cells (C), and mesophyll cells treated with ABA (D). Each gene is represented by one dot. For each gene, the raw RNA expression level in one experiment is given on the x axis, and the expression level for the same gene in the other experiment is plotted on the y axis. The solid diagonal lines indicate a difference by a factor of 2 between the two hybridizations for visual reference. Significantly expressed genes detected as Present or Marginal in two or three samples (see Methods) are represented by black dots, whereas genes for which expression levels were not significant in two or three samples are shown as gray dots.
Figure 3.
Figure 3.
Comparison of Predicted Functional Distribution of Guard Cell– and Mesophyll Cell–Expressed Genes Shows a Higher Relative Portion of Photosynthesis Gene Expression in Mesophyll Cells. Distribution of guard cell and mesophyll cell profiles among 11 major classes was performed using the MIPS database (http://mips.gsf.de/proj/thal/db/tables/tables_func_frame.html).
Figure 4.
Figure 4.
Comparison of Guard Cell–Expressed Genes versus Mesophyll Cell–Expressed Genes. Scatter plot of the normalized signal intensity values from guard cell versus mesophyll cell comparison (average of two and three replicates for mesophyll cells and guard cells, respectively) shows that many genes are expressed in both guard cells and mesophyll cells, albeit many at substantially different levels.
Figure 5.
Figure 5.
Cluster Analyses of Six Distinguishable ABA-Dependent Expression Responses of Guard Cell– and Mesophyll Cell–Expressed Genes. (A) Six clusters showing distinctive ABA gene regulation patterns. Note that in the presented y axis scales, a value of 2 does not refer to a twofold increase in expression level (see Methods for log-derived values). Group I cluster contains ABA-induced genes in both guard cells and mesophyll cells. Group II cluster contains ABA-induced mRNAs only in guard cells. Group III cluster contains ABA-induced mRNAs only in mesophyll cells. Group IV cluster contains ABA-repressed mRNAs both in guard cells and mesophyll cells. Group V cluster contains ABA-repressed mRNAs only in guard cells. Group VI cluster contains ABA-repressed mRNAs only in mesophyll cells. Colors represent relative expression level of a gene after ABA treatment. Red indicates increased expression, and blue indicates reduced expression. (B) Venn diagram presentation shows that 69 mRNAs are ABA induced only in guard cells (GC), 100 only in mesophyll cells (MC), and 21 in both cell types. (C) Venn diagram presentation shows that 64 mRNA levels are repressed by ABA only in guard cells (GC), 51 only in mesophyll cells (MC), and three in both cell types.
Figure 6.
Figure 6.
RT-PCR Analyses Independently Confirm Results Obtained from Chip Hybridization Experiments. RT-PCR was performed using guard cell and mesophyll cell RNA with primers for selected genes from guard cell preferential genes showing no ABA modulation (CER2 and calcium-dependent protein kinase [At3g50530]) and from Figure 5–derived group I (dehydrin [At3g50970], PP2C [At3g11410], and COR47), group II (trehalose-6-phosphate synthase and LEA) and group V (KAT1). Results are from 24 and 27 RT-PCR cycles. Actin2 gene was used as control. Results from 8 of 29 tested genes are illustrated. PCR was repeated at least twice. GC; guard cells; GC + ABA, guard cells treated with ABA; MC, mesophyll cells; MC + ABA, mesophyll cells treated with ABA.
Figure 7.
Figure 7.
Schematic Representation of ABA-Regulated Guard Cell–Expressed Genes in Current Working Model for ABA Signal Transduction Shows That the Transcript Levels of ABA Signal Transducers Are Regulated by ABA in Guard Cells. Negative regulators and effectors are shown in red for clarity. Colors in boxes represent relative expression level of a gene before and after ABA treatment (+ABA). Low message level genes are included for this model, and genes not present on the chip are excluded in the model.
Figure 8.
Figure 8.
atp2C-HA-1 Mutant Shows ABA Hypersensitivity in Stomatal Response and ABA Inhibition of Seed Germination. (A) Genomic organization of AtP2C-HA gene. Exons are shown as boxes, whereas introns are shown as lines. The insertion site and orientation of the T-DNA in the atp2C-HA-1 mutant are indicated. (B) RNA gel blot confirms disruption of the AtP2C-HA mRNA in atp2C-HA-1 mutant and ABA induction in the wild-type plants. (C) Stomatal aperture measurements show that ABA-induced stomatal closing is ABA hypersensitive in the atp2C-HA-1 T-DNA disruption mutant (open bars) compared with the wild type (black bars) and that the AtP2C-HA cDNA complemented this phenotype (hatched bars, line 1). Stomatal apertures were measured 3 h after addition of 0.01 or 0.1 μM ABA. (D) The atp2C-HA-1 (atp2C-HA) mutation causes ABA hypersensitivity in ABA inhibition of seed germination, which is complemented by the AtP2C-HA cDNA. (E) RNA gel blots (left) and RT-PCR (right) show that At2PC-HA expression is restored in the atp2C-HA-1 complemented lines. Bottom left, total RNA control; bottom right, actin 2 control. Error bars represent SE of n = 6 independent experiments with 240 stomata each analyzed for the wild type and atp2C-HA-1 (atp2C-HA) and of two independent experiments with 80 stomata each analyzed for a complemented atp2C-HA-1 (line 1) in (C) and >50 seeds at each data point in (D).
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References

    1. Allen, G.J., Chu, S.P., Harrington, C.L., Schumacher, K., Hoffmann, T., Tang, Y.Y., Grill, E., and Schroeder, J.I. (2001). A defined range of guard cell calcium oscillation parameters encodes stomatal movements. Nature 411, 1053–1057. - PubMed
    1. Allen, G.J., Murata, Y., Chu, S.P., Nafisi, M., and Schroeder, J.I. (2002). Hypersensitivity of abscisic acid-induced cytosolic calcium increases in the Arabidopsis farnesyltransferase mutant era1–2. Plant Cell 14, 1649–1662. - PMC - PubMed
    1. Anderson, J.A., Huprikar, S.S., Kochian, L.V., Lucas, W.J., and Gaber, R.F. (1992). Functional expression of a probable Arabidopsis thaliana potassium channel in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 89, 3736–3740. - PMC - PubMed
    1. Bartholomew, D.M., Bartley, G.E., and Scolnik, P.A. (1991). Abscisic acid control of rbcs and cab transcription in tomato leaves. Plant Physiol. 96, 291–296. - PMC - PubMed
    1. Becker, J.D., Boavida, L.C., Carneiro, J., Haury, M., and Feijo, J.A. (2003). Transcriptional profiling of Arabidopsis tissues reveals the unique characteristics of the pollen transcriptome. Plant Physiol. 133, 713–725. - PMC - PubMed

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