Qbase 2

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).