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GUS Staining of Guard Cells to Identify Localised Guard Cell Gene Expression   

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Original research article

A brief version of this protocol appeared in:
Plant Physiology
Feb 2017

Abstract

Determination of a gene expression in guard cells is essential for studying stomatal movements. GUS staining is one means of detecting the localization of a gene expression in guard cells. If a gene is specially expressed in guard cells, the whole cotyledons or rosette leaf can be used for GUS staining. However, if a gene is expressed in both mesophyll and guard cells, it is hard to exhibit a clear expression of the gene in guard cells by a GUS staining image from leaf. To gain a clear guard cell GUS image of small G protein ROP7, a gene expressed in both mesophyll and guard cells, we peeled the epidermal strips from the leaf of 3-4 week-old plants. After removing the mesophyll cells, the epidermal strips were used for GUS staining. We compared the GUS staining images from epidermal strips or leaf of small G protein ROP7 and RopGEF4, a gene specifically expressed in guard cells, and found that GUS staining of epidermal strips provided a good method to show the guard cell expression of a gene expressed in both mesophyll and guard cells. This protocol is applicable for any genes that are expressed in guard cells of Arabidopsis, or other plants that epidermal strips can be easily peeled from the leaf.

Keywords: Guard cells, Gene expression, GUS staining, Epidermal strip, Leaf

Background

Stomatal movements regulate the gas exchange between plants and environment, therefore, it is important to reveal the mechanism of the opening or closure of stomata. Determination of the guard cell expression of a gene is essential for studying its role in stomatal movements. There are several ways to identify the expression of a gene in guard cells. One way is to check the RNA expression of a gene in guard cells by RT-PCR (Jeon et al., 2008; Takimiya et al., 2013). To do so, the protoplasts of mesophyll and guard cells need to be separated. Another way is to check the GUS signal in guard cells of the transgenic plant expressing GUS driven by a gene’s native promoter. In some reports, the evidence of both RNA expression and GUS signal in guard cells were provided (Zheng et al., 2002; Jeon et al., 2008). As for the GUS signal in guard cells, if a gene is specifically expressed in guard cells, like OST1, MYB60, ROP11 and RopGEF4, a distinguished GUS signal in guard cells can be obtained from a GUS staining image with whole leaf (Mustilli et al., 2002; Li et al., 2012; Li and Liu, 2012; Rusconi et al., 2013). However, if a gene is expressed in both mesophyll and guard cells, like ROP10 and RopGEF2, the GUS signal in guard cells is hard to be distinguished from the mesophyll background (Zheng et al., 2002; Li and Liu, 2012). After GUS staining procedure, the leaf will become soft, and it is very difficult to peel the epidermal strips. Therefore, just after the leaf was excised from the plants, we peeled the epidermal strips from the leaf, and the strips were used for GUS staining after the mesophyll cells were removed. By this method, we obtained a clear guard cell GUS image of ROP7, a gene expressed in both mesophyll and guard cells.

Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
How to cite:  Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Liu, Z., Wang, W., Zhang, C., Zhao, J. and Chen, Y. (2017). GUS Staining of Guard Cells to Identify Localised Guard Cell Gene Expression. Bio-protocol 7(14): e2446. DOI: 10.21769/BioProtoc.2446.
  2. Wang, W., Liu, Z., Bao, L. J., Zhang, S. S., Zhang, C. G., Li, X., Li, H. X., Zhang, X. L., Bones, A. M., Yang, Z. B. and Chen, Y. L. (2017). The RopGEF2-ROP7/ROP2 Pathway Activated by phyB Suppresses Red Light-Induced Stomatal Opening. Plant Physiol 174(2): 717-731.
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