Growth phenotype analysis
Seeds were sterilized with 30% bleach for 10 min, followed by washing 5
times with sterile water. Plants were grown for 5 d on half-strength MS
media, after which they were transferred to agar plates with indicated
nutrients for 7 d. They were grown in a growth chamber at 22°C during
the day, 21°C at night, under long day (16 h of light) conditions at 100
μmol m−2 s−1. Plates were scanned,
after which primary root length (primary root growth after the 7 d
treatment) was measured from the resulting images in ImageJ (version
1.50b). Plants of the same genotype from one plate (3-6 plants) were
pooled and weighed to determine seedlings fresh weight. All experiments
were performed with three biological replicates. For the growth response
of adult plants to high Mg2+ concentration in growth
medium, wt and pldα1-1 were grown hydroponically for 3 weeks in
modified half-strength Hoagland´s media (Hoagland & Arnon, 1950)
followed by 10 d in media with or without 10 mM MgSO4.
Nutrient concentrations were as follows:
NH4H2PO4,
KNO3,
Ca(NO3)2.4H2O,
MgSO4.7H2O, 24.5 µM ferric citrate, 0.45
µM KI, 4.85 µM H3BO3, 5.92 µM
MnSO4.4H2O, 0.7 µM
ZnSO4.7H2O, 0.1 µM
Na2MoO4.2H2O, 0.01 µM
CuSO4.5H2O, 0.01 µM
CoCl2.6H2O, 10.02 µM
Na2EDTA, 10 µM
FeSO4.7H2O, 55.51 µM myo-inositol, 0.81
µM nicotinic acid, 0.49 µM pyridoxin, and 2.97 µM thiamin. Aeration of
the hydroponic media was performed every 3 h for 15 min using an
aquarium air pump. Nutrient solution was replaced once weekly. Plants
were grown in a growth chamber at 22°C during the day, 21 °C at night,
under short-day conditions (10 h light per day).