*2.5. PpaPrx19 Associates with Lignin Biosynthesis Enzymes and Cell Wall-Related Proteins*

Finally, we searched for protein associations by means of STRING (string-db.org). This program provides a network of predicted associations for a particular group of proteins based on high-throughput experimental data, literature and database mining [40]. In the case of PpaPrx19, it is located in the center of a network comprising 10 proteins (Figure S2), which are listed as having unknown functions in the poorly annotated *P. patens* genome. We then searched, for each predicted *P. patens* protein, its closest homolog in *Arabidopsis*. The results are shown in Table 6. The proteins with the highest score are three cinnamyl alcohol dehydrogenases (CADs) and one O-methyl-transferase (OMT). CAD participates in the lignin biosynthetic pathway, catalyzing the conversion of cinnamyl aldehydes into their corresponding alcohols. Unfortunately, the CAD proteins identified by STRING have not been characterized biochemically, although they are known to be expressed in lignifying tissues [41]. The associated OMT has been reported to have high affinity (in the μM range) for a plethora of phenylpropanoids, such as coniferyl alcohol and aldehyde, as well as quercetin [42].

We also performed an analysis in Phytozome (https://phytozome.jgi.doe.gov/pz/ portal.html; accessed on 8 July 2021) and searched for coexpression patterns with PpaPrx19. The list (Table S1) comprises phenylalanine ammonia-lyase (PAL), the first enzyme of phenylpropanoid metabolism, which includes the branch that leads to lignin formation [3]. Other enzyme-encoding genes are also coexpressed with PpaPrx19, such as β-1,3-glucanase-related and exostosin heparin sulfate glycosyltransferase-related, both associated with remodeling of the cell wall [43]. Moreover, the WRKY transcription factors have been reported to be involved in the regulation of lignin deposition [44]. These associations support the putative involvement of PpaPrx19 in the formation of lignin or lignin-like compounds.


**Table 6.** List of proteins PpaPrx19 (PP1S306\_37V6.1) has interactions with, based on STRING.

## **3. Discussion**

The cell wall is characteristic of all plant cells, although its composition varies depending on the cell type, the lineage and environmental conditions. Therefore, the plant cell wall is essential for cell development and in responses to stress, being able to plastically adapt to the cell's needs. Several innovations that arose during plant evolution, such as lignin and suberin, help to promote this plasticity. Lignin is thought to have emerged with vascular plants 450 million years ago, but lignin-like or pre-lignin compounds have been detected in bryophytes and algae [5,45]. The last step of lignin biosynthesis, the oxidation of hydroxycinnamyl alcohols, is catalyzed by class III peroxidases. In this work, we report the purification of a *P. patens* peroxidase, PpaPx19, with the ability to oxidize hydroxycinnamyl alcohols (Tables 4 and 5). This characteristic is surprising not just because *P. patens* does not lignify, but also because of the atypical kinetic properties shown by this peroxidase. In our experiments, PpaPrx19 showed *K*<sup>M</sup> values for hydroxycinnamyl alcohols similar to other peroxidases involved in lignification, from flowering and non-flowering plants. While PpaPrx19 showed *K*<sup>M</sup> values of 16.7 and 20.8 μM for coniferyl and sinapyl alcohols, respectively (Table 5), the gymnosperm *Picea abies* contains two basic peroxidases involved in lignification with reported *K*<sup>M</sup> values for coniferyl alcohol of 16.7 and 23.2 μM [46]. In angiosperms, *K*<sup>M</sup> values have been reported for zinnia (83 μM for coniferyl alcohol) and tomato (11.4 μM for syringaldazine, a chemical analog of sinapyl alcohol) [47,48].

The use of catalytic efficiency (*Kcat/K*M) is preferable in order to compare diverse enzymes and substrates, although very few peroxidase reports calculate this parameter to evaluate enzyme kinetics. In the lycophyte *Selaginella,* two basic peroxidases show values of 3.55 and 28.63 μM−1s−<sup>1</sup> with coniferyl alcohol [20]. GbPrx09 from *Ginkgo biloba*, a gymnosperm, showed values of 4.91 μM−1s−<sup>1</sup> for coniferyl alcohol [17]. In dicots, ZePrx from *Z. elegans* showed a *Kcat/K*<sup>M</sup> ratio for coniferyl alcohol of 1.20 μM−1s−<sup>1</sup> [48] and TPX1 (from tomato) showed a *Kcat/K*<sup>M</sup> for syringaldazine of 1.50 μM−1s−<sup>1</sup> [47]. In monocots, PviPRX9 from *Panicum virgatum* showed a *Kcat/K*<sup>M</sup> ratio for coniferyl alcohol of 1.60 μM<sup>−</sup>1s−<sup>1</sup> [49].

The *Kcat/K*<sup>M</sup> value obtained for PpaPrx19 using coniferyl alcohol as a substrate is not only higher than for peroxidases involved in lignification [48], but also for other enzymes involved in phenylpropanoid metabolism, such as PAL (the first enzyme of the route, [50]), CCR (the first committed enzyme of lignin biosynthesis, [51]) and CAld5H, involved in last steps of lignin biosynthesis [52].

Moreover, our results indicate that PpaPrx19 is able to efficiently use sinapyl alcohol as a substrate. While coniferyl alcohol is easily oxidized by most peroxidases, the capacity of these enzymes to oxidize sinapyl alcohol is not such a common fact and defined a new subgroup named syringyl peroxidases [39]. PpaPrx19 has most of the structural determinants of this new subgroup [39] in the protein primary structure (Figure 3, shaded in red). These structural motifs determine the syringyl oxidase activity shown by peroxidases, but are absent in the two paradigmatic G peroxidases, ATP A2 and HRP A2 from *Arabidopsis*

and horseradish, respectively. These structural motifs agree with the experimental capacity of PpaPrx19 of oxidizing sinapyl alcohol in vitro.

All these data strongly suggest that the peroxidase PpaPrx19 may have been involved in lignin biosynthesis, if such a pathway was present in *P. patens*, i.e., PpaPrx19 fulfills the kinetic and structural requirements to oxidize coniferyl alcohol. The presence of an enzyme involved in the biosynthetic route of a compound that appeared later in an evolutionary context is not surprising. Thermospermine emerges as one example of a metabolite typical of vascular plants recently described in non-vascular plants. The only reported function for thermospermine is the regulation of xylem cell maturation, which makes the function it may have in non-vascular plants unclear [53]. It is widely accepted that promiscuous enzymes with several putative substrates are more likely to be recruited to novel metabolic routes [54]. Therefore, a peroxidase with multiple substrates but with particular affinity for coniferyl alcohol would be a good candidate to participate in what eventually would constitute the pathway leading to lignin formation.

Several reports [55–57] indicate that the first appearance of the entire lignin biosynthesis pathway enzymes (excluding the pathway that leads to syringyl lignin formation), from the catalysis of phenylalanine to coniferyl alcohol formation, took place in mosses (*P. patens*), regardless the fact that *P. patens* does not accumulate lignin in the cell wall. Thus, the *P. patens* genome has all the genes necessary for the biosynthesis of lignin, and according to the results presented in this paper, at least some of the enzymes are expressed and functional, but the route does not take place and lignin is not polymerized. Nonetheless, a pre-lignin pathway has been recently suggested, revealing a role of caffeate units for the formation of the *P. patens* cuticle, coupled with ascorbate metabolism [6]. This finding suggests that the biosynthesis of lignin-like or pre-lignin compounds may not originate from the precursors described for canonical lignin and that the enzymes involved in its synthesis have broader specificity than the enzymes participating in true lignin from vascular plants, making lignin evolution an exciting field to explore.

PpaPrx19 may have a function that in vascular plants was later derived for involvement in lignification. This hypothesis is supported by its structural and kinetic homology to peroxidases with an already described role in lignin biosynthesis, such as ZePrx [39,48], and association with other enzymes of the lignin biosynthetic route. The appearance of a primitive water-conducting system, together with stomata and cuticle, were innovations developed by plants during the transition from water to land. Renault [6] already proved the existence of a pre-lignin pathway involved in the formation of the *P. patens* cuticle. Given all this, although the actual function of PpaPrx19 in *P. patens* physiology remains unclear and should be further studied, it is likely involved in the remodeling of the cell wall in response to environmental stress, based on this peroxidase ability of oxidizing phenolic compounds and its upregulation upon several conditions related to water deficiency, the paradigmatic stress for poikilohydric plants lacking a true vascular system.
