Abstract
BackgroundA common limitation in guard cell signaling research is that it is difficult to obtain consistent high expression of transgenes of interest in Arabidopsis guard cells using known guard cell promoters or the constitutive 35S cauliflower mosaic virus promoter. An additional drawback of the 35S promoter is that ectopically expressing a gene throughout the organism could cause pleiotropic effects. To improve available methods for targeted gene expression in guard cells, we isolated strong guard cell promoter candidates based on new guard cell-specific microarray analyses of 23,000 genes that are made available together with this report.ResultsA promoter, pGC1(At1g22690), drove strong and relatively specific reporter gene expression in guard cells including GUS (beta-glucuronidase) and yellow cameleon YC3.60 (GFP-based calcium FRET reporter). Reporter gene expression was weaker in immature guard cells. The expression of YC3.60 was sufficiently strong to image intracellular Ca2+ dynamics in guard cells of intact plants and resolved spontaneous calcium transients in guard cells. The GC1 promoter also mediated strong reporter expression in clustered stomata in the stomatal development mutant too-many-mouths (tmm). Furthermore, the same promoter::reporter constructs also drove guard cell specific reporter expression in tobacco, illustrating the potential of this promoter as a method for high level expression in guard cells. A serial deletion of the promoter defined a guard cell expression promoter region. In addition, anti-sense repression using pGC1 was powerful for reducing specific GFP gene expression in guard cells while expression in leaf epidermal cells was not repressed, demonstrating strong cell-type preferential gene repression.ConclusionThe pGC1 promoter described here drives strong reporter expression in guard cells of Arabidopsis and tobacco plants. It provides a potent research tool for targeted guard cell expression or gene silencing. It is also applicable to reduce specific gene expression in guard cells, providing a method for circumvention of limitations arising from genetic redundancy and lethality. These advances could be very useful for manipulating signaling pathways in guard cells and modifying plant performance under stress conditions. In addition, new guard cell and mesophyll cell-specific 23,000 gene microarray data are made publicly available here.
Highlights
A common limitation in guard cell signaling research is that it is difficult to obtain consistent high expression of transgenes of interest in Arabidopsis guard cells using known guard cell promoters or the constitutive 35S cauliflower mosaic virus promoter
Guard cells and mesophyll cells exposed to abscisic acid (ABA) were analyzed, as ABA synthesis is induced under several stress conditions
The following criteria were used for selection of strong guard cell promoter candidates
Summary
A common limitation in guard cell signaling research is that it is difficult to obtain consistent high expression of transgenes of interest in Arabidopsis guard cells using known guard cell promoters or the constitutive 35S cauliflower mosaic virus promoter. The stomatal aperture is regulated by multiple physiological factors such as light, CO2, and plant hormones including abscisic acid (ABA) [1,2,3,4,5] These stimuli regulate the stomatal aperture by affecting the cellular activities of the two adjacent guard cells, which form the stomata. A dominant mutation or a knock out mutation in an essential gene at the whole plant level might be lethal This problem could be avoided by expressing the mutated gene or silencing the specific gene in guard cells only. Intact plant imaging of single cells requires specific reporter gene expression in target cells with low background in the surrounding cells
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