EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana

Authors: Keun Pyo Lee, Dr. Chanhong Kim

Head of Group: Prof. Dr. Klaus Apel

 

GEO accretion number for microarray data: GSE10509

 

Project Summary

Shortly after the release of singlet oxygen(¹O₂), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsisthat reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of ¹O₂ generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of ¹O₂-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of ¹O₂-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid.

Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all ¹O₂-responsive genes. Retrograde control of ¹O₂-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.

 

Experimental Design

Plant Material: The EX1 (At4g33630) T-DNA insertion line SALK_002088 and EX2 (At1g27510) T-DNA insertion line SALK_012127 were obtained from the European Arabidopsis Stock Centre (NASC). Homozygous mutant lines were identified by PCR analysis by using T-DNA-, EX1- and EX2-specific primers. Both T-DNA-lines were crossed with a flu Col-0 line that had been obtained by 5 backcrosses of flu1-1 in Landsberg erecta with wild-type Columbia. The ex1/flu and ex2/flu mutant lines were crossed, and within the segregating F2 population triple mutants were identified by PCR-based genotyping. For the cultivation of mature plants, seeds of wild type, flu, ex1/flu, ex2/flu, and ex1/ex2/flu, all in Col-0 ecotype, were sown on soil and plants were grown under continuous light (100 µmol·m²·s¹).

 

Microarray Hybridization and Analysis

Two individual biological replicates, each containing material of five mature plants of wildtype, flu, ex1/flu, ex2/flu, and ex1/ex2/flu, respectively, were used for the microarray analysis. Plants were germinated on soil and kept under continuous light until the beginning of bolting and then transferred to the dark for 8 h. Dark-incubated mature plants were reilluminated for 30 min and subsequently harvested for RNA extraction.

cRNA Preparation

Total RNAs from the different samples were extracted. The quality of the isolated RNA was determined with a NanoDrop ND 1000 (NanoDrop Technologies, Wilmington) and a Bioanalyzer 2100 (Agilent, Waldbronn, Germany). Only those samples with a 260/280 nm ratio between 1.8-2.1 and a 28S/18S ratio within 1.5-2 were further processed. Total RNA samples (5 mg) were reverse-transcribed into doublestranded cDNA with a One-Cycle cDNA synthesis kit (P/N 900431; Affymetrix, Santa Clara, CA). The doublestranded cDNA was purified using a Sample Cleanup Module (P/N 900371; Affymetrix). The purified doublestranded cDNAs were in vitro-transcribed in the presence of biotin-labeled nucleotides using a IVT labeling kit (P/N 900449; Affymetrix). The biotinylated cRNA was purified by using a Sample Cleanup Module (P/N 900371; Affymetrix), and its quality and quantity were determined with a NanoDrop ND 1000 and Bioanalyzer 2100

Array Hybridization

Biotin-labeled cRNA samples (15 mg) were fragmented randomly to 35-200 bp at 94°C in fragmentation buffer (P/N 900371; Affymetrix) and were mixed in 300 ml of hybridization buffer containing a hybridization control cRNA and control oligo B2 control (P/N 900454; Affymetrix), 0.1 mg/ml herring sperm DNA, and 0.5 mg/ml acetylated BSA in 2-(4-morpholino)-ethane sulfonic acid (Mes) buffer, pH 6.7, before hybridization to GeneChip Arabidopsis ATH1 genome arrays for 16 h at 45°C. Arrays were then washed using an Affymetrix Fluidics Station 450 EukGE-WS2v4_450 protocol. An Affymetrix GeneChip Scanner 3000 was used to measure the fluorescence intensity emitted by the labeled target.

Statistical Analysis

Raw data processing was performed by using the Affymetrix GCOS 1.2 software. After hybridization and scanning, probe cell intensities were calculated and summarized for the respective probe sets by means of the MAS5 algorithm (1). To compare the expression values of the genes from chip to chip, global scaling was performed, which resulted in the normalization of the trimmed mean of each chip to a target intensity (TGT value) of 500 as detailed by Affymetrix. Quality control measures were considered before performing the statistical analysis. These included adequate scaling factors (between 1 and 3 for all samples), and appropriate numbers of present calls were calculated by application of a signedrank call algorithm (2). The efficiency of the labeling reaction and the hybridization performance were controlled with the following parameters: Present calls and optimal 3'/5' hybridization ratios (»1) for the housekeeping genes (GAPDH and ACO7), for the poly(A) spike in controls and the prokaryotic control (BIOB, BIOC, CREX, BIODN). After normalization, "baseline" samples were compared (i.e., wild type after a dark/light shift versus either flu or ex1/flu or ex2/flu or ex1/ex2/flu) and for the comparative analysis of changes in gene expression between wild type and others, only the genes that met the following criteria were considered: (i) The genes should show a reliable level of RNA giving a detection call of P (present in the Affymetrix nomenclature), and (ii) the signals should be changed by at least 2-fold or greater ("difference" call) relative to the baseline sample. Based on these criteria, the remaining genes were selected for analysis using Genespring 7.2 package. The microarray analyses were performed in duplicate using independent samples for wild type, flu, ex1/flu, ex2/flu, and ex1/ex2/flu plants.

 

Reference

EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana

Keun Pyo Lee, Chanhong Kim, Frank Landgraf, and Klaus Apel

10270–10275 PNAS June 12, 2007, vol. 104, no. 24