Abstract
This is a protocol for isolation of guard cell enriched samples from Arabidopsis thaliana plants for RNA extraction. Leaves are blended in ice-water and filtered through nylon mesh to obtain guard cell enriched fragments. With guard cell enriched samples, gene expression analysis can be done, e.g., comparing different gene expression levels in guard cells versus whole leaf to determine if a gene of interest is predominantly expressed in guard cells. It can also be used to study the effect of treatments or different genetic backgrounds in the regulation of the guard cell expressed genes.
Keywords: Arabidopsis thaliana, Guard cell isolation, RNA
Background
Isolation of guard cells for RNA extraction has traditionally relied on guard cell protoplast extraction (Leonhardt et al., 2004) or epidermal peels (Pandey et al., 2010). Manual dissection of guard cells from freeze-dried leaves has also been used (Bates et al., 2012). The protoplast procedure may introduce unwanted changes in gene expression from wounding effects and other methods are time consuming, require special equipment or expensive reagents (e.g., transcriptional inhibitors). Hence there is a need for a fast and simple protocol to isolate guard cells for gene expression analysis. Here we further describe a quick ice blender method for isolation of guard cell enriched tissue (Bauer et al., 2013), in the form of epidermal fragments without mesophyll and other vascular cells. This method uses a nylon mesh to collect epidermis from blended leaf tissue. The adequately large pore size of the nylon mesh allows mesophyll and other vascular cells to pass through while retaining the epidermal fragments. We propose that the critical factor in this protocol is the type of blender used to process the samples.
Materials and Reagents
Equipment
Procedure
Collect leaves from 4-7 week old Arabidopsis plants grown in soil at 12 h/12 h photoperiod, use 17-18 plants and 4-5 leaves per plant. If plants are smaller in size, use more leaves per plant.
Data analysis
Isolated guard cell enriched fragments can be visualized with a stereo microscope (Zeiss SteREO Discovery.v20), and no mesophyll fragments (green tissue) should be present (Figure 4A). While testing different blenders, successful isolation of guard cell enriched fragments was achieved with the Braun (800 W) blender but not with a blender with lower effect (Figure 4). Figure 4. Isolated guard cell enriched fragments under a microscope. A. Sample isolated with the Braun blender; B. Sample isolated with a small 220 W blender. To control the quality of guard cell enrichment, real-time quantitative PCR can be done with guard cell expressed genes like HT1 and GORK, by comparing their expression level in guard cell enriched sample versus a whole leaf sample (Figure 5; for details about the qPCR conditions see Jalakas et al., 2017). Figure 5. Gene expression in Col-0 guard cell enriched sample compared to leaf sample. The mean of three biological replicates are shown; error bars depict ± SEM.
Acknowledgments
This protocol was adapted from the research article Bauer et al. (2013). This work was funded by the Estonian Ministry of Science and Education (IUT2-21 to H.K.), the European Regional Development Fund (Center of Excellence in Molecular Cell Engineering CEMCE to H.K.), the Academy of Finland (grant number #307335, Center of Excellence in Primary Producers 2014-2019 to M.B.).
References
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