Plant material - Arabidopsis thaliana
Experiments on Arabidopsis were performed using the wild type (WT) ecotype Col-0 and the two T-DNA insertion lines GABI_437B11 (Atpip1;1 ) (Rosso et al., 2003) and SALK_051107 (Atpip1;3 ) (Alonso et al., 2003) obtained from the Nottingham Arabidopsis Stock Centre (Nottingham, United Kingdom). Homozygous mutants were selected by PCR with specific primers listed in Supplementary Table S1. Homozygous lines were selected based on the absence of a wild type amplicon and the presence of the T-DNA insertion (Supplementary Fig. S1). For the cultivation of plants on soil, ‘Fruhstorfer Nullerde’ (white-peat volcanic clay mixture) was used. The soil substrate was prepared as described previously (Pommerrenig et al., 2018) with some modifications. The pH of a white-peat volcanic clay mixture was first adjusted to around pH 5.5 with CaO (3 g kg-1) and CaCO3 (2 g kg-1). After 1 week, the substrate was fertilized with 250 ml kg-1 of a modified Hoagland solution containing: 60 mM NH4NO3, 23.5 mM KH2PO4, 2.8 mM K2SO4, 13.3 mM MgSO4, 420 µM CuSO4, 360 µM ZnSO4, 2.5 mM MnCl2, 3.3µM NaMoO4, 152 µM NaFeEDTA and 226 µM H3BO3. N deficiency was achieved by reducing the NH4NO3 concentration to 30 mM NH4NO3. Leaves (8 plant replicates per condition, three independent repetitions of the experiment) were harvested after 5 weeks of growth, snap-frozen in liquid N2 and stored at -80 °C. The N and C contents of the leaves were measured using a Euro EA elemental analyser (HEKAtech, Wegberg, Germany). Statistical analysis was performed using SigmaPlot v13 (Systat Software, Erkrath, Germany) utilizing a two way analysis of variance (ANOVA) and the Holm-Sidak post hoc test. Additionally for the qRT-PCR analysis plants were grown using in vitro based conditions as described previously (Schlesier, Breton, & Mock, 2003). To impose N deficiency, the medium’s N content was reduced to 2 mM.