**1. Introduction**

Land colonization by plants and their subsequent diversification was one of the most important events in the history of life. Terrestrialization forced plants to cope with new stresses absent in the aquatic medium, such as UV light and limited water supply. To avoid water loss, plants developed different strategies to accumulate water in their tissues, to supply it or to minimize its loss. These first land plants, such as mosses, were poikilohydric, whose water potential was equilibrated with surrounding water sources [1]. Likewise, the first evolutionary radiation among land plants is related to the diversification of tracheids, which appeared in vascular plants (tracheophytes), about 450 million years ago and they have been defined as single-celled conduits with lignin in their cell wall [2]. Lignin is a polyphenolic polymer that confers structural support and flexural stiffness to the aerial part of the plant and provides water impermeability, including resistance against tensile forces of water columns. Lignins are mainly formed from the oxidative coupling of three *p*-hydroxycinnamyl alcohols: *p*-coumaryl, coniferyl and sinapyl alcohols (monolignols). The cross-coupling reaction of monolignol radicals produces a hydrophobic heteropolymer composed of *p*-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) units [3].

Although lignin has traditionally been linked to vascular plants, polyphenols and lignin-like compounds have been found in species without a true vascular system, such as charophycean green algae [4] and bryophytes [5,6]. Lignin-like compounds are polyphenolic polymers usually detectable with typical methods of lignin determination, such as acetyl bromide or nitrobenzene oxidation, but unlike lignin, they lack β-*O*-4 bonds and aryl-glycerol-β-aryl ether structures. The composition is very variable and in many cases

**Citation:** Martínez-Cortés, T.; Pomar, F.; Novo-Uzal, E. Evolutionary Implications of a Peroxidase with High Affinity for Cinnamyl Alcohols from *Physcomitrium patens*, a Non-Vascular Plant. *Plants* **2021**, *10*, 1476. https://doi.org/10.3390/ plants10071476

Academic Editors: Penélope García-Angulo and Asier Largo-Gosens

Received: 20 June 2021 Accepted: 15 July 2021 Published: 19 July 2021

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unknown but not related to the three *p*-hydroxycinnamyl alcohols that are considered markers for lignins. This finding implies that at least part of the phenylpropanoid pathway that eventually led to lignin biosynthesis was present in algae, and the presence of lignin in tracheids may only have involved the expression of those genes in a different type of cell [7].

The last step of lignin biosynthesis, the oxidation of monolignols, is driven by laccases [8] and peroxidases [9]. Secretory plant peroxidases (class III Prx) are heme-containing glycoproteins that oxidize diverse substrates using hydrogen peroxide as an electron donor. Peroxidases are usually rich in isoenzymes, generated from post-transcriptional and posttranslational modifications [10], with expression patterns usually dependent on development and stress conditions, which make it difficult to assign specific functions to individual peroxidase isoenzymes. Nonetheless, diverse responses to a plethora of stresses or growth conditions have been reported, especially in *Arabidopsis*, indicating specific functions for the different isoforms [9,11].

*Physcomitrium (Physcomitrella) patens* is a bryophyte used as a model organism for evolutionary developmental biology and non-vascular plant studies. *P. patens* shows high tolerance to different environmental cues, such as drought and osmotic and saline stresses, which allows survival in periods when water supply is a limiting factor [12]. RedoxiBase reports 53 class III peroxidases and four pseudogenes in the *P. patens* genome. However, information about *P. patens* peroxidase functions is scarce. The best characterized peroxidase is Prx34 (PpaPrx13 according to RedoxiBase nomenclature), which was reported to play a role upon fungal attack and catalyze ROS production [13,14].

In this paper, we report the purification of one *P. patens* peroxidase, upregulated upon salt and oxidative stresses. This enzyme was further purified and characterized, showing homology to angiosperm peroxidases involved in lignification, and with a catalytic efficiency against coniferyl alcohol, a precursor of lignin, of the same order as angiosperm lignification-related peroxidases, despite the fact that *P. patens* does not contain lignin in its cell walls.
