**2. Results**

#### *2.1. Abiotic Stress Strongly Modulates the Expression of a Basic Peroxidase in P. patens*

*P. patens* is a moss that is highly resistant to abiotic stress, compared to other model plants such as *Arabidopsis thaliana*, and is especially tolerant to desiccation [15], in line with its phylogenetic position as a moss, and whose ancestors were early colonists of land around 500 million years ago. This bryophyte is thus a useful tool to study responses to abiotic stress. Peroxidases are enzymes known to change their expression pattern in response to different types of stress [16,17]. Here, we selected different abiotic stresses and monitored in a time course both peroxidase activity and isoform pattern from protein extracts of *P. patens* gametophores grown in liquid medium. The results (Figure 1A) show that H2O2 caused an early increase in peroxidase activity, peaking 1h after treatment. The addition of ascorbic acid, a known H2O2 scavenger, returned peroxidase activity to control levels. NaCl and salicylic acid (SA) also enhanced peroxidase activity 24 h after treatment, going back to control levels afterwards. Mannitol slightly changed peroxidase activity throughout the treatment. Although both osmotic and salt stress can be abscisic acid (ABA) dependent, *P. patens* peroxidase activity in response to ABA treatment did not mirror mannitol or salt stress responses, but strongly decreased from 8 h after the hormone addition. Moreover, SA and mannitol led to the disappearance of a strongly basic peroxidase isoform, which was instead induced by H2O2 and NaCl (Figure 1B, arrow). We quantified free phenolics as a proxy to measure the stress caused by the different treatments. H2O2, salt and mannitol treatments significantly enhanced the amount of total phenols in *P. patens* gametophores (Figure 1C). Based on these results, we pursued the purification and further characterization of a strongly basic peroxidase that was induced by H2O2 and salt, two major stresses faced by the first plants that colonized land.

**Figure 1.** Salt and oxidative stress upregulate the expression of a basic peroxidase from *P. patens*. (**A**) Time-course determination of peroxidase activity extracted from *P. patens* gametophores that were subjected to different stress treatments: H2O2 with or without ascorbic acid (Asc), salt, mannitol (Man), abscisic acid (ABA) and salicylic acid (SA). TMB was used as substrate and peroxidase activity was normalized to control conditions at each time point (shown as a dashed line). (**B**) Effect at 24 h of different treatments on peroxidase isoenzyme pattern revealed by IEF stained with 4-MN. C, control; pI, isoelectric point. (**C**) Effect of stress treatments at 96 h on soluble phenolic content in *P. patens* cultures. Data presented are average values ± SD of *n* = 3 experiments. Statistical analysis was carried out with Duncan's test, asterisks represent statistical differences from control (*p* < 0.05).
