Metabolite profiling
Metabolite extraction was performed essentially by following an established gas chromatography-mass spectrometry (GC-MS)-based metabolite profiling protocol of Lisec et al. (2006) modified. Approximately 50 mg of homogenized plant materials were aliquoted in tubes and extracted in 100% methanol and internal standard (0.2 mg ribitol mL-1 water). 2.0 mL tubes were shaken for 15 min at 70°C and next centrifuged at 11000g for 10 min. The supernatant was transferred to new tubes and, afterwards, 100% chloroform and distilled water were added. Tubes were centrifuged at 2200 gfor 15 min. Finally, 150 μL of the upper phase of each sample were transferred to new 1.5 mL tubes and left to dry overnight in a vacuum concentrator. Sample derivatization was carried out as previously described (Lisec et al., 2006). A pooled reference sample was made by combining 5 µl aliquots from all samples. Sample injection and subsequent peak annotation were performed as described in Dahtt et al. (2019), using the Fiehn GC/MS Metabolomics RTL Library (G1676AA; Agilent). After blank subtraction, the peak area of each metabolite was normalized to the internal standard (i.e., ribitol) in each sample, as well as fresh weight.

Expression analysis by RT-PCR

Total RNA was isolated using TRIzol reagent (Ambion, Life Technology) according to the manufacturer’s recommendations. The total RNA was treated with DNAse I (RQ1 RNase free DNase I, Promega, Madison, WI, USA). The integrity of the RNA was checked on 1% (w/v) agarose gels, and the concentration was measured using a Nanodrop spectrophotometer. Finally, 2 μg of total RNA were reverse transcribed with Superscript II Rnase H2 reverse transcriptase (Invitrogen) and oligo (dT) primer according to the manufacturer’s recommendations. Real-time PCR reactions were performed in a 96-well microplate (Applied Biosystems Applera, Darmstadt, Germany), using Power SYBR Green PCR Master Mix. The primers used here were designed using the open-source program QuantPrime-qPCR primer designed tool (Arvidsson et al., 2008) and are described in Table S2. ACTIN (AT2G37620) was used as internal standard. The relative levels of mRNAs were determined using the 2-ΔCt method (Livak and Schmittgen, 2001). Three biological replicates were processed for each experimental condition.