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  • Plant Materials and Growth Conditions: All the mutants were in the Columbia-0 (Col-0) background and Col-0 was the control for all experiments. Double and triple mutants were in the cat2 (SALK_076998) background as previously described [24]. The dnd1 double mutants were previously described [27]. Sterilized seeds were placed on Murashige and Skoog (MS) medium (1/2 MS salts, and 0.7% agar), stratified for three days and transferred to a growth chamber (Sanio Electric Co) at 21°C/19°C under a 12 h day/12 h night regime, light intensity 120 μmol m-2 s-1, and 70% relative humidity. One week old plants were transplanted into pots with 1:1 peat:vermiculate and grown on soil for four weeks in controlled growth rooms. Three experimental repeats were used for gene expression analysis. Plant material was collected from five weeks old plants. Three groups of leaves at different developmental stages were selected based on leaf age: old (leaves with visual lesions, leaf position 5–7), mature (fully developed leaves without lesions, leaf position 9–12), young (developing leaves, no lesions, leaf position 12–14). Eight leaves from each age class were pooled, frozen in liquid nitrogen and stored at -80°C. Total RNA was extracted using the Spectrum Total RNA extraction kit (Sigma Aldrich). The expression of marker genes involved in PCD regulation was measured with real time quantitative PCR (qPCR). Three biological repeats were used for gene expression analysis with qPCR. RNA was treated with DNAseI and reverse transcription was performed using 2 μg of RNA with the RevertAid Premium Reverse Transcriptase (RT) and Ribolock Rnase inhibitor according to manufacturer’s instructions (Thermo Fisher Scientific). After reverse transcription the reaction was diluted to the final volume of 100 μl. 1 μl was used for PCR with EvaGreen ROX (Solis Biodyne). The cycle conditions in the ABI 7900HT Fast RT PCR System (Applied Biosystems) were: 95°C 10 min, 40 cycles with 95°C 15 s, 60°C 30 s, 72°C 30 s and ending with melting curve analysis. Normalization of the data was performed in qBase 2.0 (Biogazelle), with three reference genes TIP41, YLS8 and SAND. Primer amplification efficiencies were determined in qBase from a cDNA dilution series. Primer sequences and amplification efficiencies can be found in S1 Table. Data normality was tested and subsequently 2-base logarithmed for statistical analyses. Factorial ANOVA posthoc analyses Fisher LSD was used to evaluate significant differences between mutant and leaf age, and One-Way ANOVA to changes in gene expression with leaf age (Statistica 7.1, Stat Soft Inc). Previous analysis of gene expression in cat2 using genes from the gene ontology category “cell death” in [24] identified several genes with strongly increased expression in various LMMs, and in response to SA and pathogen treatment. 26 genes of these genes were tested at different leaf age classes (old, mature, young) in Col-0 and cat2. Five marker genes; FMO1, PLA2A, WRKY75, WRKY40 and GLTP were chosen as qPCR marker genes based on significant differences in expression between different leaf ages. Analysis of Marker Gene Expression in Public Gene Expression Data: The selected marker genes used for qPCR were analyzed with the Condition Search tool “Perturbations”in Genevestigator https://genevestigator.com/gv/doc/intro_plant.jsp [31]. Marker gene expression is shown in response to hormone and pathogen treatment and in LMMs.
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