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.