Guard cell purification and RNA isolation suitable for high-throughput transcriptional analysis of cell-type responses to biotic stresses.

Stomata, micro-pores on the leaf surface, are formed by a pair of guard cells. In addition to controlling water loss and gas exchange between the plant and the environment, these cells act as immunity gates to prevent pathogen invasion of the plant apoplast. Here, we report a brief procedure to obtain highly pure guard cell preparations using conditions that preserve the guard cell transcriptome as much as possible for a robust high-throughput RNA sequence analysis. The advantages of this procedure included i) substantial shortening of the time required for obtaining high yield of >97% pure guard cell protoplasts (GCP), ii) extraction of enough high quality RNA for direct sequencing, and iii) limited RNA decay during sample manipulation. Gene expression analysis by reverse transcription quantitative polymerase chain reaction revealed that wound-related genes were not induced during release of guard cells from leaves. To validate our approach, we performed a high-throughput deep-sequencing of guard cell transcriptome (RNA-seq). A total of 18,994 nuclear-encoded transcripts were detected, which expanded the transcriptome by 70%. The optimized GCP isolation and RNA extraction protocols are simple, reproducible, and fast, allowing the discovery of genes and regulatory networks inherent to the guard cells under various stresses.