Oxylipins in moss development and defense

Oxylipins are oxygenated fatty acids that participate in plant development and defense against pathogen infection, insects, and wounding. Initial oxygenation of substrate fatty acids is mainly catalyzed by lipoxygenases (LOXs) and α-dioxygenases but can also take place non-enzymatically by autoxidation or singlet oxygen-dependent reactions. The resulting hydroperoxides are further metabolized by secondary enzymes to produce a large variety of compounds, including the hormone jasmonic acid (JA) and short-chain green leaf volatiles. In flowering plants, which lack arachidonic acid, oxylipins are produced mainly from oxidation of polyunsaturated C18 fatty acids, notably linolenic and linoleic acids. Algae and mosses in addition possess polyunsaturated C20 fatty acids including arachidonic and eicosapentaenoic acids, which can also be oxidized by LOXs and transformed into bioactive compounds. Mosses are phylogenetically placed between unicellular green algae and flowering plants, allowing evolutionary studies of the different oxylipin pathways. During the last years the moss Physcomitrella patens has become an attractive model plant for understanding oxylipin biosynthesis and diversity. In addition to the advantageous evolutionary position, functional studies of the different oxylipin-forming enzymes can be performed in this moss by targeted gene disruption or single point mutations by means of homologous recombination. Biochemical characterization of several oxylipin-producing enzymes and oxylipin profiling in P. patens reveal the presence of a wider range of oxylipins compared to flowering plants, including C18 as well as C20-derived oxylipins. Surprisingly, one of the most active oxylipins in plants, JA, is not synthesized in this moss. In this review, we present an overview of oxylipins produced in mosses and discuss the current knowledge related to the involvement of oxylipin-producing enzymes and their products in moss development and defense.

Bryophytes, including mosses, liverworts, and hornworts, are early divergent land plants that are phylogenetically placed between unicellular green algae and flowering plants. Mosses were the first plants to conquest land and have evolved adaptation mechanisms to tolerate extreme conditions such as desiccation and exposure to damaging UV-B radiation and to resist coevolving pathogens and herbivores (Rensing et al., 2008). Mosses use many alternative metabolic pathways, some of which are not present in flowering plants, and probably this has allowed mosses to occupy and function in very different habitats (Rensing et al., 2007). In addition to polyunsaturated C18 fatty acids, mosses have also large amounts of polyunsaturated C20 fatty acids which are rarely present in flowering plants due to the lack of the corresponding biosynthetic enzymes (Gill and Valivety, 1997). In mosses like Physcomitrella patens (P. patens) and Mnium cuspidotum, 20:4 reaches up to 30% of total fatty acids (Anderson et al., 1972;Grimsley et al., 1981;Beike et al., 2014). The enzymes involved in the biosynthesis of 20:4 and eicosapentaenoic acid (20:5) have been identified and characterized in P. patens (Girke et al., 1998;Zank et al., 2002;Kaewsuwan et al., 2006). 20:4 and 20:5 are produced from 18:2 and 18:3, respectively, by a reaction series involving the activities of a ∆6-desaturase, a ∆6-elongase, and a ∆5-desaturase (Kaewsuwan et al., 2006). The high abundance of long and very long chain fatty acids together with the presence of oxylipins derived from 20:4 and 20:5 represent a metabolic difference between mosses and flowering plants that may provide a metabolic advantage to the adaptation capacity of mosses to severe environmental conditions (Mikami and Hartmann, 2004). LOX-derived oxylipins produced from C20 and C18 polyunsaturated fatty acids are also found in multicellular algae, where they play a role in defense responses against an algal pathogen (Bouarab et al., 2004). In unicellular algae, aldehydes derived from C20 fatty acids accumulate after wounding where they may play defensive roles (Pohnert, 2000;Pohnert and Boland, 2002). Thus, like algae, mosses have both octadecanoid and eicosanoid pathways. This review is focused on current knowledge related to oxylipins produced in mosses with a special emphasis on the role played by oxylipinproducing enzymes and their products in moss development and defense.

Oxylipins in Moss Development
Mosses are land plants with a relatively simple developmental pattern with alternating haploid gametophyte and diploid sporophyte generations. The gametophyte consists of two distinct developmental stages; the juvenile filamentous protonema with chloronema and caulonema types of cells, and the adult gametophores which are leafy shoots composed of a non-vascular stem with leaves, the reproductive organs, and filamentous rhizoids (Reski, 1998; Figure 2A). The germination of a haploid spore or the division of a protoplast lead to the formation of chloronema cells with characteristic perpendicular cross walls and a high density of chloroplasts. From chloronemal filaments caulonemal cells arise subsequently with oblique cross walls and low density of chloroplasts. Branching of caulonemal cells result in new chloronemal or caulonemal cells leading to the formation of secondary chloronemal or caulonemal filaments and buds (Cove and Knight, 1993;Cove et al., 2006). Buds develop into leafy gametophores upon which the diploid sporophyte generation is formed leading to new haploid spores (Cove et al., 2006; Figure 2A). In mosses fatty acid compositions vary depending on the type of tissue. While 16:0 and 20:4 content are similar in protonemal filaments and leafy gametophores of different moss species, 18:2 and 18:3 are more abundant in gametophores and protonemal tissues, respectively (Beike et al., 2014). These metabolic differences correlate with differences in expression levels of fatty acid desaturases encoding genes (Beike et al., 2014). In maturing sporophytes of the moss Mnium cuspidotum, 16:0 and 20:4 are the most abundant fatty acids, while 18:2 increases when spores have matured and are ready for dispersal, reaching similar levels as 16:0 (Anderson et al., 1972). The most abundant free hydro(per)oxy fatty acids present in protonemal tissues of P. patens are 13-hydroperoxy linoleic acid and 12-HPETE (Stumpe et al., 2010), which can be further metabolized by the corresponding enzymes producing oxylipins with possible roles in moss development. However, the specific functions of the C18:2, C18.3, and C20:4 pathways in different tissues and during moss development are at present unknown. In P. patens, functional analysis of genes encoding enzymes involved in fatty acid and oxylipin biosynthesis can be performed by targeted gene disruption or single point mutation, due to its high rate of homologous recombination (Schaefer, 2002). The P. patens genes encoding ∆6-desaturase, ∆6-elongase, and ∆5-desaturase have been functionally characterized by targeted gene disruption, confirming their involvement in 20:4 and 20:5 production (Girke et al., 1998;Zank et al., 2002;Kaewsuwan et al., 2006). Although 20:4 and 20:5 decrease drastically in these mutants, protonema and gametophores of the knockout plants do not have a visible altered phenotype, suggesting that 20:4 and 20:5 are not necessary, or that residual amounts of these fatty acids are sufficient for normal development (Girke et al., 1998;Zank et al., 2002;Kaewsuwan et al., 2006). Some P. patens knockout mutants in genes encoding oxylipin-producing enzymes have also been generated. PpHPL, PpAOS1, and PpAOS2 knockout mutant have no developmental alterations (Stumpe et al., 2006b;Scholz et al., 2012), while individual PpAOC1 and PpAOC2 knockout mutants have reduced fertility, aberrant sporophyte morphology, and interrupted sporogenesis (Stumpe et al., 2010). Double PpAOC1 and PpAOC2 knockout mutants were unable to obtain suggesting that depletion of both enzymes is lethal (Stumpe et al., 2010). As mentioned previously, only the PpAOS1 mutant is highly impaired in OPDA synthesis, although the amount produced is sufficient for normal development (Scholz et al., 2012). A. thaliana mutants defective in oxylipin production, including OPDA-and JA-deficient mutants, and mutants in the COI-1 gene are male sterile (Browse, 2009), while a mutation in the tomato COI-1 gene renders female sterile plants (Li et al., 2004). Thus, different oxylipins participate in the development of reproductive structures, both in mosses and flowering plants. Further analysis of the different genes encoding enzymes involved in the LOX pathways are needed to understand their participation in different moss developmental processes. For example, while PpAOC1 and PpAOC2 have similar expression patterns in protonemal and gametophores tissues, PpAOC3 is preferentially expressed in protonemata tissues where it could play a more specific role (Hashimoto et al., 2011). Recent studies performed with P. patens Ppα-DOX-GUS reporter lines have shown that Ppα-DOX is expressed during development in tips of protonemal filaments with maximum expression levels in mitotically active undifferentiated apical chloronemal and caulonemal cells (Machado et al., 2015; Figure 2B). Ppα-DOX-GUS is also highly expressed in other type of mitotically active cells, including apical cells of regenerating protoplasts ( Figure 2B). The role played by Ppα-DOX-derived oxylipins in undifferentiated apical cells, which are self-renewing stem cells, needs further investigation. Interestingly, the mammalian COX-derived oxylipin PGE 2 has sustaining effects on undifferentiation and stimulate self-renewal and proliferation (Goessling et al., 2009;Hoggatt et al., 2009), and Ppα-DOXderived oxylipins could play similar functions. In young buds Ppα-DOX transcripts accumulate in cells leading to rhizoids and axillary hair formation suggesting that local cues present in these types of cells contribute to Ppα-DOX expression. Auxin is a good candidate since in young and adult gametophores, Ppα-DOX is expressed in auxin producing tissues, including rhizoids and axillary hairs primordial, and axillary hairs and rhizoid (Machado et al., 2015; Figure 2B). Gametophytes and sporophytes of Ppα-DOX mutant are similar to wild-type plants indicating that this enzyme is not essential for proper moss development (Machado et al., 2015). However, incubating wild-type tissues with Ppα-DOX-derived oxylipins, or overexpressing Ppα-DOX, alter P. patens development leading to smaller moss colonies with less protonemal tissues (Machado et al., 2015). The Ppα-DOXderived aldehyde, heptadecatrienal, is responsible for the reduced protonemal filament growth ( Figure 3A). Moss colonies are also smaller and have less protonemal tissues when moss tissues are grown in the presence of 13-LOX-derived oxylipins, including OPDA and methyl jasmonate (Ponce de León et al., 2012; Figure 3A). OPDA and jasmonate also reduce rhizoid length (Ponce de León et al., 2012), consistently with the growth arrest of A. thaliana seedlings and roots incubated with these oxylipins (Vellosillo et al., 2007;Mueller et al., 2008 ; Figures 3B,C). Interestingly, the inhibitory effect of OPDA on growth, either by OPDA application or by the generation of overexpressing MpAOC plants which produces high levels of OPDA, was also observed in the liverwort M. polymorpha (Yamamoto et al., 2015), suggesting a conserved response to this oxylipin among bryophytes. In contrast, JA did not affect M. polymorpha growth (Yamamoto et al., 2015), indicating that the growth inhibitory activity of jasmonate is not conserved among mosses and liverworts. One possible explanation is that M. polymorpha does not have the downstream components necessary for sensing the presence of JA. However, putative orthologs of the jasmonate ZIM-domain (JAZ) repressor and the receptor COI have been identified in M. polymorpha (Katsir et al., 2008;Wang et al., 2015). In addition, sequence alignment demonstrates that the binding sites are well conserved between COI orthologs of P. patens and M. polymorpha and A. thaliana COI (Wang et al., 2015). Further studies are needed to understand the differential response to jasmonates between different bryophytes. Mueller et al. (2008) have suggested that in flowering plants the inhibition of growth observed with OPDA is related to the inhibition of cell cycle progression. Studies in A. thaliana have shown that jasmonate reduces both cell number and cell size in roots and leaves (Chen et al., 2011;Noir et al., 2013). Consistently, protonemal tissues grown in the presence of heptadecatrienal have filaments with smaller caulonemal cells and abnormal cell divisions (Machado et al., 2015). Taking together, all these observations indicate that like more complex plants where oxylipins synthesized from different biochemical pathways act as regulators of development, a fine-tuning mechanism operates in order to regulate oxylipins concentrations in moss tissues for proper development. P. patensand A. thaliana-derived oxylipins involved in plant development are schematized in Figure 4.

FIGURE 4 | Regulation of development and defense by plant oxylipins.
Flowering plants like A. thaliana have 9-lipoxygenases and 13-ipoxygenases that catalyze the oxygenation of polyunsaturated fatty acids, mainly linoleic acid (18:2) and linolenic acid (18:3), to form fatty acid hydroperoxides. The resulting hydroperoxides are further metabolized to produce a variety of oxylipins, including hydroxy fatty acids and aldehydes, which play different roles in development and defense. α-Dioxygenase utilizes 18:2 and 18:3 to form 2-hydroperoxy fatty acids that are converted to 2-hydroxy fatty acids and aldehydes, with regulatory functions in plant defense against microbial pathogens and insects. The moss P. patens has besides polyunsaturated C18 fatty acids, polyunsaturated C20 fatty acids and can produce a broad range of oxylipins derived from them. P. patens 13-lipoxygenases and α-dioxygenase utilize mainly 18:2 and 18:3 as substrates producing similar oxylipins as in flowering plants, including (+)-cis-12-oxo-phytodienoic acid (OPDA), 2-hydroxy fatty acids and aldehydes with functions in development and defense against microbial pathogens. However, jasmonates are not synthesized in P. patens and the 9-lipoxygenase pathway seems not to be present. In addition, 12-lipoxygenases produce oxylipins from arachidonic (20:4) and eicosapentaenoic acids (20:5), including 12-oxo-dodecatrienoic acid (12-ODTE) and aldehydes whose functions are at present unknown.
Physcomitrella patens is infected by several broad host range pathogens, including the phytopathogenic bacteria Pectobacterium carotovorum (P. carotovorum) and Pectobacterium wasabiae, the fungus B. cinerea and the oomycetes Pythium irregular and Pythium debaryanum (Ponce de León, 2011;Ponce de León and Montesano, 2013). After pathogen assault, P. patens activates a defense response similar to flowering plants, including accumulation of reactive oxygen species (ROS), reinforcement of the cell wall, localized cell death known as the hypersensitive response, and activation of defense genes (Ponce de León and Montesano, 2013). P. patens respond to biotic stress by increasing the endogenous levels of free unsaturated fatty acid, and inducing the expression of genes encoding different PpLOXs, PpAOS, and an oxophytodienoic acid reductase (Ponce de León et al., 2007Oliver et al., 2009). These studies suggest that both the 13-LOX and the 12-LOX pathways are activated after pathogen assault. PpLOX1 and both PpLOX1 and PpLOX6 transcript levels increase after Pythium and B. cinerea infection, respectively, and OPDA concentrations increase in moss tissues infected with both pathogens (Oliver et al., 2009;Ponce de León et al., 2012). Further studies are needed to evaluate the involvement of the other PpLOXs as well as the oxylipins produced in response to pathogen infection. In flowering plants OPDA is active as a defense signal and regulates defense gene expression (Stintzi et al., 2001;Taki et al., 2005;Browse, 2009). OPDA may play similar roles in mosses since expression levels of the defense gene encoding a phenylalanine ammonia-lyase increase in P. patens tissues treated with this oxylipin (Oliver et al., 2009). Moreover, OPDA is a very active oxylipin with antimicrobial activity against several microbial pathogens (Prost et al., 2005), and can therefore contribute to reduce the pathogen population in moss tissues.
Ppα-DOX transcript levels and activity increase in P. patens tissues in response to P. carotovorum elicitors and B. cinerea (Machado et al., 2015). Ppα-DOX-GUS fused proteins accumulate in leaves and protonemal tissues of elicitors-treated and B. cinerea-inoculated plants. A protective role against invading pathogens has been proposed for Ppα-DOX since Ppα-DOX-GUS accumulates in P. patens cells surrounding B. cinerea infected cells (Machado et al., 2015; Figure 3D); this resembles the expression pattern of Arabidopsis thaliana α-DOX1-GUS in cells surrounding tissues infected with Pseudomonas syringae . Under normal growth conditions, Ppα-DOX is expressed in protonemal filaments, in rhizoids, and in axillary hairs where it can function as a permanent protection system (Machado et al., 2015). Moreover, functional studies suggest that Ppα-DOX-derived oxylipins protect tissues against cell death caused by elicitors. While Ppα-DOX disrupted mutant have no phenotype and respond similar to wild-type plants, overexpressing Ppα-DOX or treating plants with α-DOX-derived oxylipins, increase protection against cellular damage caused by P. carotovorum elicitors (Machado et al., 2015). P. patensand A. thaliana-derived oxylipins involved in plant defense are schematized in Figure 4.

Oxylipins in Other Bryophytes
Compared to mosses, only few studies on oxylipin profiling and enzymes involved in oxylipin formation have been conducted in other bryophytes. In recent years more studies in the liverwort M. polymorpha have been performed due to its phylogenetic position as an earliest diverging clade of land plants and the possibility to generate knockout mutants by homologous recombination (Ishizaki et al., 2013). M. polymorpha emits C 8 volatiles and produces OPDA after mechanical wounding (Kihara et al., 2014;Yamamoto et al., 2015). Arachidonic acid (20:4) and eicosapentaenoic acid (20:5) are essential for C 8 volatiles production in this bryophyte since a fatty acid desaturase knockout mutant with undetectable levels of 20:4 and 20:5 produce only minimal amounts of the C 8 volatiles (Kihara et al., 2014). Volatiles, including C 5 , C 6 , and C 10 oxylipins were also identified in the volatile profile of the liverwort Chiloscyphus pallidus (Toyota and Akakawa, 1994). The enzymatic activities of three LOXs of M. polymorpha have been characterized in vitro, and all of them have 15-LOX activity against 20:4 and 20:5 (Kanamoto et al., 2012). Nine additional putative genes encoding LOX are present in the EST database of M. polymorpha (Kihara et al., 2014). Besides, an AOC encoding gene has been identified in M. polymorpha, which is induced after wounding and OPDA treatment (Yamamoto et al., 2015). This chloroplastic MpAOC is involved in the synthesis of OPDA from the unstable allene oxide 12,13-EOT. The overexpression of MpAOC leads to higher OPDA content and to smaller liverwort plants (Yamamoto et al., 2015). Thus, OPDA acts as a signaling molecule regulating both development and response to wounding in this liverwort.

Conclusion
In addition to having polyunsaturated C18 fatty acids, algae and mosses also have large amounts of polyunsaturated C20 fatty acids from which they can produce a broad range of oxylipins. In contrast, flowering plants have lost the C20 pathway in the course of evolution. Studies on moss C20-derived oxylipins are therefore of great interest, and will further extend our understanding of the involvement of these metabolites in plant adaptation to biotic and abiotic stress. At the present time, only a few studies in the moss P. patens have demonstrated a function for oxylipinproducing enzymes. PpAOC1 and PpAOC2 are needed for spore maturation and dehiscing of the spore capsules, while Ppα-DOXderived oxylipins participate in development and defense against bacteria. Functional studies in P. patens are needed to gain more insights into the diversity of oxylipin metabolic pathways and their involvement in moss development and defense. The evaluation of disease severity and activation of defense mechanisms against pathogens in P. patens mutants, including available mutants in PpHPL, PpAOS1, PpAOS2, PpAOC1, and PpAOC2 will certainly improve our understanding of the role played by oxylipins to cope with pathogens. Studies in other bryophytes like the moss Ceratodon purpureus, and the liverwort M. polymorpha (Brücker et al., 2005;Ishizaki et al., 2013), where homologous recombination based gene disruption is feasible, will also provide valuable information on oxylipin biosynthesis, diversity, and plasticity in early divergent land plants. The lack of JA in P. patens is intriguing since this is an important hormone in flowering plants for adaptation to stress. The high content of long and very long chain fatty acids and the production of a wider range of oxylipins including C20-derived oxylipins in moss may be an effective and alternative source of bioactive compounds that contribute to the capacities of these organisms to adapt to diverse environments. The use of other oxylipins than jasmonates for defense responses could be a more general mechanism in bryophytes since M. polymorpha does not synthesize JA. Besides, JA may have appeared later in the evolution of plants as a hormone to maximize fitness associated to a higher level of complexity. In conclusion, further studies are needed to obtain a more complete scenario with regard to the evolution of the different oxylipinproducing pathways in bryophytes and the role played by the corresponding enzymes in development and adaptation to stress.