*4.2. Protein Extraction and Precipitation*

Gametophore samples (400 g) were homogenized in 50 mM Tris-HCl buffer (pH 7.5) containing 1 mM EDTA, 1 M KCl, and 0.05 g PVPP per g of tissue. The homogenate was filtered through nylon layers and centrifuged at 27,000× *g* for 30 min at 4 ◦C. The

supernatant was dialyzed on cellulose membranes. After protein precipitation with 95% saturation ammonium sulfate, the precipitate was resuspended in 50 mM Tris-HCl pH 7.5 and dialyzed overnight against the same buffer.

## *4.3. Purification of PpaPrx19*

Purification of *P. patens* peroxidase 19 was performed in the AKTA System (GE Healthcare, Barcelona, Spain). The dialyzed sample was concentrated in Amicon® Ultra (Merck Millipore, Barcelona, Spain) and dissolved in 1.5 M (NH4)2SO4. In the first step, hydrophobic chromatography, proteins were separated on a phenyl SepharoseTM 6 Fast Flow (GE Healthcare, Barcelona, Spain) 31.5 × 1.0 cm gel bed column at a flow rate 1.0 mL·min−1, and fractions of 5.0 mL were recovered. The eluent chromatography program was as follows: from 0 to 160 min (100% A, 0% B), from 160 to 360 min (0% to 100% B) and from 360 to 515 min (100% B), where buffer A was 50 mM Tris-HCl (pH 7.5) containing 1.5 M (NH4)2SO4, and buffer B was 50 mM Tris-HCl (pH 7.5).

The second step involved ion-exchange chromatography on SP Sepharose Fast Flow (GE Healthcare). To do so, the peroxidase-enriched fractions obtained from the hydrophobic chromatography were dialyzed against 50 mM CAPS, (pH 9.5), and loaded on a 17.5 × 1.0 cm gel bed column equilibrated with 50 mM CAPS (pH 9.5), at a flow rate of 1.0 mL min−1. Fractions of 1.0 mL were recovered. The eluent chromatography program was as follows: from 0 to 70 min (100% A, 0% B), from 70 to 170 min (0–100% B) and from 170 to 330 min (100% B), where buffer A was 50 mM CAPS (pH 9.5), and buffer B was 50 mM CAPS (pH 11.5).
