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.