*2.2. PpaPrx19 Is a 36 kDa Basic Peroxidase*

We extracted total protein from *P. patens* gametophores grown in control conditions and we then followed a three-step protocol for purification, including ammonium sulfate precipitation, hydrophobic chromatography on phenyl Sepharose and cationic chromatography on SP Sepharose. A fractionated precipitation with ammonium sulfate did not allow the partial purification of the peroxidases of interest, which led us to consider just one fraction, precipitating the proteins with 95% (NH4)2SO4. The fraction was pooled into phenyl Sepharose chromatography, obtaining two major fractions of peroxidases (Figure 2A). The first eluted fraction (F1) contained only acidic peroxidases, while F2 contained both neutral and basic peroxidases and therefore was selected to continue the purification process (Figure 2). After this step, the specific activity for F2 reached 266 nkat mg−<sup>1</sup> protein (Table 1). The F2 was then loaded into a cationic exchange chromatography, and the peroxidase bound to SP Sepharose matrix was eluted with a linear gradient of 9.5–11.5 pH. Neutral peroxidases were not retained in the matrix and only one peak of peroxidase activity was eluted, at a pH of 10.9 (Figure 2A). The fraction arising from cationic chromatography migrated as

two different bands of 36 and 46 kDa in SDS-PAGE electrophoresis, but an IEF showed only one peroxidase with the pI value determined to be 10.04 (Figure 2B). The peptide mass fingerprinting of the two resultant proteins enabled us to detect that the protein of 46 kDa corresponded to a lipase (accession number XP\_001755452). The 36 kDa protein was identified as a predicted protein (access number XP\_001781554) which corresponded to PpaPrx19 (according to RedoxiBase nomenclature).

**Figure 2.** Purified PpaPrx19 is a strongly basic peroxidase. (**A**) Purification process of PpaPrx19, including adsorption chromatography on phenyl Sepharose (upper panel, peak F2) and cationic exchange chromatography on SP Sepharose (lower panel). Profiles of peroxidase activity and protein are denoted either by a continuous or by a dotted line, respectively. (**B**) Protein fingerprint in SDS-PAGE (left) and peroxidase isoenzyme pattern in IEF (right) of the crude extract (1) and the purified peroxidase (2) SDS-PAGE and IEF were revealed using the silver staining method and 4-MN in the presence of H2O2, respectively. (**C**) Dependence on pH of the purified PpaPrx19 activity. Data presented are average values ± SD of *n* = 3 experiments.

**Table 1.** Purification of basic peroxidase PpaPrx19 from *P. patens*. Peroxidase activity was measured using TMB as substrate.


We confirmed by RT-qPCR that *PpaPrx19* was strongly induced after a treatment with NaCl and that gene expression was modulated in response to other stresses such as hydrogen peroxide and mannitol (Figure S1).

We also evaluated the dependence on pH of PpaPrx19 enzymatic activity, using a different pH (4.0 to 9.0) in the reaction mixture. The purified peroxidase showed the highest activity at pH 5.0, but it rapidly decreased at pH > 6 and showed no activity at pH above 7.0 (Figure 2C). These results do not differ from other peroxidases purified from different sources, with the optimum pH between 4.5 and 6.5 [18–20]. pH is critical for peroxidase activity because pH values outside the optimum prevent the heme from binding to the active site of the enzyme [21].

#### *2.3. PpaPrx19 Is Homologous to Peroxidases with a Role in Lignification*

PpaPrx19 is 332 amino acids long, including a 26 aa N-terminal signal peptide, and it is targeted to the secretory pathway according to analysis with SIGNALP [22] and TAR-GETP [23] programs. The exon–intron pattern of PpaPrx19 is the second most abundant for *P. patens* and classic for class III peroxidases, consisting in three exons and two introns [13]. In a BLAST search, PpaPrx19 showed the highest identity at the protein level with two other *P. patens* peroxidases (PpaPrx18 and PpaPrx09) and the moss *Tortula ruralis* (Table 2). The rest of the listed peroxidases belong to gymnosperms and angiosperms, and show identity values below 50%, emphasizing the evolutionary distance among them and pointing out the unique characteristics of this peroxidase, at the amino acid level.

**Table 2.** Comparison of PpaPrx19 mature protein sequence with other class III peroxidases. The most similar peroxidase sequences based on BLASTP searches against the RedoxiBase database were used.


Given these low identity values, we blasted PpaPrx19 against *Arabidopsis* peroxidases, in order to infer a putative function. Most of the peroxidases with the highest identity level have a reported role in lignification (Table 3). This was a surprising result, given that *P. patens* has an internal water-conducting system constituted by hydroids and living cells with thick walls [24] containing pre-lignin and lignin-like polyphenols but no true lignin (defined as the polymerized compounds found in vascular plants) has been described [5,6].

**Table 3.** Reported function of *Arabidopsis* peroxidases which show highest homology to mature PpaPrx19 protein sequence after a BLAST search with Redoxibase.

