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

Abstract

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.

Fig. 1

Assessing the yield and purity of GCP preparations. A, Laser scanning confocal micrographs of guard cell and mesophyll cell protoplasts (I=green channel, II=red channel, III=DIC, IV=merged channels). Note the size difference. B, Purity of GCPs extracted using long and short incubation protocols calculated as percentages of total protoplast extracted (MCP and GCP). C, Number of GCPs isolated in long and short methods. Results are shown as means (n=3) ± standard error.

Fig. 2

Amount of RNA extracted from long and short protocols. A, GCPs were isolated from 50 leaves and GCP suspension was equally divided for total RNA extraction using either the Qiagen column or Trizol reagent, thus yield is expressed in μg per 25 leaves. Transcription inhibitors were not added during guard cell protoplasting. B, Total RNA extracted from GCPs using Qiagen column in presence or absence of the transcription inhibitor antibiotics cordycepin (0.01%) and actinomycin D (0.0033%). Results are shown as means (n=3) ± standard error. Statistical significance between the means (short versus long) was detected with two-tailed Student’s t-test (*** refers to P<0.001, * refers to P <0.05).

Fig. 3

Affect of transcription inhibitor antibiotics (actinomycin and cordycepin) in wound-responsive gene transcription during GCP preparation (2-h procedure). Transcript abundance of the indicated genes relative to the procedure with antibiotics was determined by RT-qPCR analysis. Results are shown as mean (n=6) ± standard error. Statistical significance of the difference between means (with antibiotics versus without antibiotics) was detected with two-tailed Student’s t-test (*** = P<0.001, * = P<0.05).

Fig. 4

Effect of GCP preparation time on transcript abundance. Long procedure takes >6 h whereas the short procedure can be finished in about 2 h. Transcript abundance of the indicated genes relative to the >6 h procedure was determined by RT-qPCR analysis. Results are shown as mean (n=6) ± standard error. Statistical significance of the difference between means (short versus long procedure) was detected with two-tailed Student’s t-test (*** = P<0.001, ** = P<0.01, * = P<0.05).

Fig. 5

Functional categorization of guard cell expressed genes (18,994) according to the three broad Gene Ontology categories cellular component (A), molecular function (B), and biological process (C) using the GO slim tool available at TAIR.