Gene expression analysis
Plants were grown hydroponically, as described above. Four-week-old
plants were treated with half-strength modified Hoagland´s media with or
without 10 mM MgSO4 for 24 h, after which root and leaf
tissue was collected and immediately frozen in liquid nitrogen. RNA was
isolated using the Spectrum Plant Total RNA Kit (Sigma-Aldrich), DNA was
removed using the Turbo DNA-free Kit (Applied Biosystems), and cDNA
synthesis was done with the Transcriptor High Fidelity cDNA Synthesis
Kit (Roche). Reverse transcription was done with anchored-oligo(DT)18
primers and 1 µg RNA. Quantitative PCR was done with a LightCycler 480
system (Roche) using the LightCycler 480 SYBR Green I Master mix
(Roche). The primers used are listed in Table S1. SAND family protein
(At2g28390) was used to normalize target gene transcription values.