Selaginella moellendorffii subsp. oxylipin Hieron. 1901

2.5. S. moellendorffii oxylipin profiling

The aerial parts of S. moellendorffii plants possessed complex oxylipins patterns (Fig. 5) similar to those described before for S. martensii (Ogorodnikova et al., 2015). The most prominent oxylipins were the products of DES and AOS activities. These were the divinyl ethers (DE1 – DE6, Fig. 5) and the cyclopentenones, cis - and trans -12-oxo-PDA, as well as the iso -12-oxo-PDA, 15(Z)-12-oxo-9(13),15-phytodienoic acid. The spectra for the detected divinyl ethers and cyclopentenones corresponded to those described before (Ogorodnikova et al., 2015). The oxylipin profile of S. moellendorffii, besides the mentioned products, contained several epoxyalcohols. These were 11-hydroxy-12,13-epoxy-9-octadecenoic (5), 11-hydroxy-12,13-epoxy-9,15-octadecadienoic (5a), 9-hydroxy-12,13-epoxy-10-octadecenoic acid (12) and 9-hydroxy-12,13-epoxy-10,15-octadecadienoic acid (12a) acids (EAS products). The mass spectra of epoxyalcohols 5 and 5a are described in Supplementary Information. The mass spectra of epoxyalcohols 12 and 12a have been described earlier (Toporkova et al., 2018a).

The genome of S. moellendorffii contains at least 10 CYP74 genes (Banks et al., 2011) belonging to four subfamilies CYP74J, CYP74K, CYP74L and CYP 74M. All these sequences possess significant peculiarities of catalytically essential domains, primarily the “F/L toggle” at the SRS-1 near the N-end and the I-helix groove domain (including two amino acids behind it). Normally in AOS and EAS sequences the “F/L toggle” contains phenylalanine, whereas in HPL and DES, it contains leucine. Phe/Leu substitution and vice versa regularly leads to alteration in the catalytic mechanism (Lee et al., 2008; Toporkova et al., 2018b, 2020b). Among S. moellendorffii CYP 74s the “F/L toggle” contains leucine only in two sequences, CYP74M1 and CYP74M3 (Fig. 6), which are DESs. The absence of other CYP74s, except SmDES1 (CYP74M1) and SmDES3 (CYP74M3), having a Leu at this site suggested the absence of HPLs in S. moellendorffii. However, SmHPL/AOS (CYP74L1) possesses significant HPL activity, especially towards 13-hydroperoxides of linoleic and α- linolenic acids. At the same time, its “F/L toggle” contains phenylalanine. To date, this is the only CYP74 HPL containing phenylalanine at this site.

The I-helix groove domain and its environment in the CYP74L sequences also look unusual. Their alignment (Fig. 6) with other CYP74s shows that all three CYP74Ls have a substitution of the glycine in the next position after the I-helix groove domain. AOSs and HPLs of flowering plants normally have a conserved Gly at this site. In DESs, Gly is substituted with another residue (Fig. 6). DESs have either Ala (CYP74D DESs) or bulkier residues like Glu (CYP74B16, LuDES), or Thr (CYP74Q1, RaDES), or Leu (CYP74H1, AsDES). The importance of this site for DES activity was confirmed by site-directed mutagenesis experiments; the E292G mutant form of LuDES (CYP74B16) exhibited AOS activity (Toporkova et al., 2013). In CYP74L sequences this glycine is substituted with another amino acid – leucine (CYP74L1) or phenylalanine (CYP74L2 and CYP74L3). However, the CYP74 L enzymes possessed mainly AOS or HPL activities. No DES activity was detected.

Position #3 of HBD is occupied with a conserved Phe in AOSs and HPLs (Fig. 6). All known DESs except the RaDES (CYP74Q1) have another residue instead of Phe at this position (Fig. 6). Mutation at this site may alter the CYP 74 type of catalysis. So, the F295I mutant form of LeAOS3 (CYP74C3) possessed HPL activity (Toporkova et al., 2008). CYP74 L enzymes have a substitution of this conservative phenylalanine with other residue – valine (CYP74L1) or methionine (CYP74L2 and CYP74L3).

Data obtained indicated that the recombinant CYP74L1, CYP74L2 and CYP74L3, despite belonging to the same subfamily and sharing high homology, possess different catalytic activities. The CYP74L2 specifically exhibited AOS activity. In contrast, CYP74L1 possessed mainly HPL activity towards its preferred substrates, 13-hydroperoxides. Conversely, the 9-HPOD and 9-HPOT, relatively poor substrates for CYP74L1, were converted largely to AOS products. The AOS products of both CYP74L1 and CYP74L2 were represented mainly by α ketols which are products of spontaneous hydrolysis of corresponding allene oxides, short-living primary AOS products. Besides, the conversion of 13-HPOT led to a substantial yield of the cyclopentenone cis -12-OPDA. Unlike CYP74L1 and CYP74L2, CYP74L3 exhibited quite low HPL/EAS activity towards 13-HPOD, 9-HPOT, and 9-HPOD (Supplementary Fig. S1) and was fully inactive towards 13-HPOT. Low activity of CYP74L3 may be caused by the deletion of sixteen amino acids in the middle of the sequence (Fig. 1).

The CYP74Ls expand the list of characterized enzymes of oxylipin biosynthesis in spikemoss. The S. moellendorffii CYPome includes at least two DESs, i.e. SmDES1 (CYP74M1) and SmDES2 (CYP74M3) (Gorina et al., 2016), producing divinyl ethers DE1 – DE6 (Fig. 5). Divinyl ethers oxylipins play self-defensive and antipathogenic roles in plants (Weber et al., 1999; Grechkin, 2002; Gran´er et al., 2003; Cowley and Walters, 2005; Prost et al., 2005; Toporkova et al., 2018a; Deboever et al., 2020). Moreover, expression of DES genes is increased in response to pathogenic microorganisms (Weber et al., 1999; Stumpe et al., 2001; Fammartino et al., 2007), viral attack (Nelson, 2011) or elicitor treatment (Gobel ¨et al., 2001). The epoxyalcohols 5, 5a, 12, and 12a can be synthesized by SmEAS (CYP74M2, Toporkova et al., 2018a). Epoxyalcohols and products of their hydrolysis, trihydroxy acids, were shown to participate in the defence responses against phytopathogens (Kato et al., 1985; Prost et al., 2005). Cis - and trans -isomers of 12-oxo-PDA, as well as iso -12-oxo-PDA, can be synthesized by SmAOS1 (CYP74L2) described in the present report or other putative AOSs, i.e. CYP74J1, CYP74K1, CYP74K2, or the before characterized SmAOS2 (CYP74K3) (Pratiwi et al., 2017). 12-oxo-PDA appears to play a significant role in mediating resistance to pathogens and pests such as Botrytis cinerea (Scalschi et al., 2015), beet armyworm (Spodoptera exigua larvae) (Bosch et al., 2014 a, 2014b), brown planthopper (Nilaparvata lugens) (Guo et al., 2014), and corn leaf aphid (Rhopalosiphum maidis) (Varsani et al., 2019; Grover et al., 2020). The absence of HPL products in the S. moellendorffii oxylipin profile (Fig. 5) suggests the absence of constitutive expression of SmHPL1 (CYP74L1) gene. Presumably, it might be expressed under stress conditions. HPL-synthesized hexenals are also involved in herbivore resistance. In addition, the HPL branch yields the wound phytohormone traumatin and compounds with bactericidal, fungicidal or antioxidant properties (Pietryczuk and Czerpak, 2011).

The SmHPL1 (CYP74L1) is the first CYP74 enzyme of S. moellendorffii possessing hydroperoxide lyase activity. Earlier it was suggested that CYPome of S. moellendorffii does not contain any HPLs (Gorina et al., 2016). This assumption was based on the peculiarities of catalytically essential domains of CYP74s. Moreover, SmHPL1 (CYP74L1) presents one more example of a CYP74 enzyme exhibiting a different catalytic behaviour depending on substrate. Recently the dual function CYP74C HPL/EASs have been described (Toporkova et al., 2018b). LuDES (CYP74B16) was identified as dual function DES/HPL (with minor EAS activity) depending on substrate (Toporkova et al., 2020a). Additional EAS activity was exhibited by CYP74B HPLs (Toporkova et al., 2020b), as well as by carrot allene oxide synthase DcAOS (CYP74B33) (Gorina et al., 2019b). A common intermediate of all reactions controlled by CYP74s is the epoxyallylic radical (Fig. 7). Depending on the substrate of the SmHPL1, the epoxyallylic radical undergoes either (i) a single electron oxidation followed by proton loss to form the allene oxide (AOS pathway), or (ii) recombination with a hydroxyl radical to the epoxyalcohol (EAS pathway), or (iii) isomerization to the vinyloxycarbinyl radical which recombines with a hydroxyl radical, leading to the hemiacetal (HPL pathway), see Fig. 7.

Like other CYP74 subfamilies, the CYP74L subfamily consists of different enzymes, namely AOSs and HPLs. The CYP74A subfamily includes 13-specific AOSs and 9/13-specific EASs. The CYP74B subfamily comprises 13-specific HPLs and DESs, as well as at least one 9-specific AOS. The CYP74C subfamily includes 9/13-specific AOSs and double function HPL/EASs, and the CYP74M subfamily is composed of 13-specific DESs and EASs (Fig. 8).

Expanding knowledge on proteins (including P450s) in S. moellendorffii, one of the oldest vascular plants, is essential for understanding molecular evolution (Weng et al., 2008a,b, 2011; Chen et al., 2011; Anderberg et al., 2012; Cheon et al., 2013; Yokota et al., 2017; Alber et al., 2019; Ferrari et al., 2020). Spikemosses are phylogenetically situated between bryophytes (mosses, liverworts, and hornworts) and euphyllophytes, which include flowering plants. The lipoxygenase pathway of S. moellendorffii, S. martensii (Ogorodnikova et al., 2015), P. patens and M. polymorpha (Mukhtarova et al., 2020) possess some peculiarities compared to flowering plants. For instance, S. moellendorffii (present work), S. martensii (Ogorodnikova et al., 2015), P. patens and M. polymorpha (Mukhtarova et al., 2020) possess a prominent iso -12-OPDA, which is relatively uncommon in flowering plants. On the other hand, jasmonates, widespread in flowering plants, have been detected neither in S. moellendorffii (present work) nor in mosses, including P. patens and M. polymorpha (Bowman et al., 2017; Ponce de Leon et al., 2015; Mukhtarova et al., 2020). Pratiwi et al. (2017) reported the detection of jasmonate in S. moellendorffii. However, the only evidence presented was the SIM LC-MS analysis, while no spectrometry data was provided. We observed no signs of jasmonate presence in S. moellendorffii (present work), nor in S. martensii (Ogorodnikova et al., 2015). The lack of jasmonates was attributed before to the absence of 12-oxo-PDA reductase OPR 3 in non-flowering plants (Bowman et al., 2017; Ponce de Leon et al., 2015). The OPDA system is more ancient a stress signalling pathway than the jasmonate one. However, the OPDA system already includes almost all the components of the jasmonate system (Schluttenhofer, 2020). In flowering plants, the emergence of jasmonates as more specific ligands than OPDA for signal receptors improved the efficiency of functioning of this signalling system.

The OPDA system is an older a stress signalling pathway than the jasmonate system. However, the OPDA system included almost all components of the jasmonate system (Schluttenhofer, 2020). In flowering plants, the emergence of jasmonates as more specific ligands for signaling receptors than OPDA seems to have increased the efficiency of functioning of this signalling system.

Diversity of CYP74 genes in S. moellendorffii, including DESs (CYP74M1 and CYP74M3), EAS (CYP74M2), HPL (CYP74L1), AOSs (including the characterized AOSs CYP74K3 and CYP74L2, as well as the putative AOSs CYP74J1, CYP74K1, and CYP74K2), establishes the complexity of the oxylipin profile in spikemoss tissues. Recent findings on the enzymes of lipoxygenase pathway in the green alga K. flaccidum, the liverwort M. polymorpha (Koeduka et al., 2015), the moss P. patens (Mukhtarova et al., 2020), the spikemoss Selaginella (Ogorodnikova et al., 2015; Gorina et al., 2016; Pratiwi et al., 2017; Toporkova et al., 2018a), as well as the results of present work, shed light onto the enzymatic apparatus of oxylipin biosynthesis of non-flowering plants and its changes in the course of early land plant evolution.