Digiprep graphite block digestion system

Shoot tissues were cleaned with milliQ water, while root tissues were desorbed as described (Talke et al., 2006 (link)) and dried at 60 °C for 3 d. Shoot samples (10–50 mg of tissues) were then acid-digested in DigiPrep tubes with 3 ml of ≥65% HNO₃ (Sigma-Aldrich) on a DigiPrep Graphite Block Digestion System (SCP Science) as follows: 15 min at 45 °C, 15 min at 65 °C, and 90 min at 105 °C. After cooling, sample volumes were adjusted to 10 ml with milliQ water, and 200 µl ≥65% HNO₃ was added. Metal concentrations were determined using ICP-AES with a Vista-AX instrument (Varian, Melbourne, Australia) as described (Nouet et al., 2015 (link)).

Col-0 (wild-type) and hma2hma4 mutant plants as well as the complemented mutants (four to five independent T3 homozygous lines per promoter construct) were analysed. Eighteen days after germination on modified Hoagland medium in short days, the seedlings were transferred to hydroponic trays (Araponics, Tocquin et al., 2003 (link)) with Hoagland medium containing 1 μM ZnSO₄ and grown in short days for 2 weeks before initiating the treatments. Plants were then cultivated on either 0.2 μM ZnSO₄ or 0.05 μM CdSO₄ for 4 weeks under long days. Root and rosette tissues of two to three plants were harvested separately and desorbed as described (Talke et al., 2006 (link)). Plant tissues were dried at 60°C for 3 days. Tissues (10–50mg) were then acid-digested in a DigiPrep tube with 3ml 65% HNO₃ (Sigma-Aldrich) on a DigiPrep Graphite Block Digestion System (SCP Science) as follows: 15min at 45°C, 15min at 65°C, and 90min at 105°C. After cooling, sample volumes were adjusted to 10ml with milliQ water, and 200 µl >65% HNO₃ was added. Metal concentration was measured using inductively coupled plasma atomic emission spectroscopy with a Vista-AX instrument (Varian, Melbourne, Australia).

Root and leaf tissues were harvested separately. Shoot tissues were rinsed in Milli-Q water, whereas root tissues were desorbed and washed as described previously (Talke et al., 2006 (link)). Dried tissue samples were acid- digested in DigiPrep tubes with 3ml of ≥65% (w/w) HNO₃ (Sigma-Aldrich) on a DigiPrep Graphite Block Digestion System (SCP Science) as follows: 15min at 45 °C, 15min at 65 °C, and 90min at 105 °C. After cooling, sample volumes were adjusted to 10ml with Milli-Q water and 200 µl of ≥65% HNO₃. Element concentrations were determined by inductively coupled plasma-atomic emission spectroscopy (Vista AX, Varian).