Guard cells surround stomatal pores in the epidermis of plant leaves and stems. Stomatal pore opening is essential for CO2 influx into leaves for photosynthetic carbon fixation. In exchange, plants lose over 95% of their water via transpiration to the atmosphere. Signal transduction mechanisms in guard cells integrate hormonal stimuli, light signals, water status, CO2, temperature, and other environmental conditions to modulate stomatal apertures for regulation of gas exchange and plant survival under diverse conditions. Stomatal guard cells have become a highly developed model system for characterizing early signal transduction mechanisms in plants and for elucidating how individual signaling mechanisms can interact within a network in a single cell. In this review we focus on recent advances in understanding signal transduction mechanisms in guard cells.

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.

Here we describe the development of a microarray-based mapping strategy to rapidly isolate deletion mutant genes. The presented approach is particularly useful for mapping mutant genes that are difficult to phenotype. This strategy uses masking bulk segregant analysis to mask unrelated deletions, thus allowing identification of target deletions by microarray hybridization of pooled genomic DNA from both WT and mutant F 2 populations. Elemental profiling has proven to be a powerful tool for isolation of nutrient and toxic metal accumulation mutants in Arabidopsis. Using microarray mapping, a sodium overaccumulation mutant FN1148 was identified as having a 523-bp genomic deletion within the second exon and intron of the AtHKT1 gene. Further cosegregation, complementation, and comparative analyses among different salt-sensitive mutants confirmed that the deletion within the AtHKT1 gene is responsible for the sodium overaccumulation in shoots and leaf sodium sensitivity of the FN1148 mutant. These results demonstrate that microarray-based cloning is an efficient and powerful tool to rapidly clone ion accumulation or other genetic deletion mutants that are otherwise difficult to phenotype for mapping, such as metabolic or cell signaling mutants.

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