Cloning and heterologous expression

Total RNA was extracted from frozen and ground Populus × canescens and Picea abies plant material using the InviTrap Spin Plant RNA Mini Kit (Invitek, Berlin, Germany) and cDNA libraries were prepared using the SuperScript III First-Strand Synthesis SuperMix (Thermo Fisher Scientific, Waltham, MA, USA). For expression of HDR proteins, truncated sequences were used in a gateway cloning system (Thermo Fisher Scientific) using appropriate primers (Supplemental Table S3). Gel-purified genes of interest were introduced into pDONOR207 vectors followed by transformation into One Shot™ TOP10 competent E. coli (Thermo Fisher Scientific). Positive clones were verified by Sanger sequencing, further subcloned into a pDEST15 expression vector (Thermo Fisher Scientific) and transformed into BL21-AI™ One Shot™ competent E. coli (Thermo Fisher Scientific). These were used directly for inoculation of a 12 mL preculture of LB medium, incubated for 72 h at 18 °C and 220 rpm, and further used to inoculate 100 mL LB. The medium was supplemented with 1 mM L-cysteine and ferric ammonium citrate (30 µg × mL⁻¹) to ensure proper iron supply for the formation of the iron-sulfur-cluster of the HDR according to (Graewert et al. 2009). The culture was grown until OD₆₀₀ reached 0.6, induced with 0.2% (w/v) L-arabinose and grown overnight at 18 °C and 220 rpm. The whole procedure was performed with two technical replicates.