Oxylipin biosynthesis in spikemoss Selaginella moellendorffii: Identification of allene oxide synthase (CYP 74 L 2) and hydroperoxide lyase (CYP 74 L 1) Author Toporkova, Yana Y. Author Askarova, Elena K. Author Gorina, Svetlana S. Author Mukhtarova, Lucia S. Author Grechkin, Alexander N. text Phytochemistry 2022 113051 2022-03-31 195 1 12 http://dx.doi.org/10.1016/j.phytochem.2021.113051 journal article 10.1016/j.phytochem.2021.113051 1873-3700 8235350 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 ). 2.6. Alignment of amino acid sequences of CYP 74Ls with other CYP 74s The genome of S. moellendorffii contains at least 10 CYP 74 genes (Banks et al., 2011) belonging to four subfamilies CYP 74J, CYP 74K, CYP 74 L and CYP 74 M . 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, CYP 74 M 1 and CYP 74 M 3 ( Fig. 6 ), which are DESs. The absence of other CYP 74s, except SmDES1 ( CYP 74 M 1) and SmDES3 ( CYP 74 M 3), having a Leu at this site suggested the absence of HPLs in S. moellendorffii . However, SmHPL/ AOS ( CYP 74 L 1) 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 CYP 74 HPL containing phenylalanine at this site. The I-helix groove domain and its environment in the CYP 74 L sequences also look unusual. Their alignment ( Fig. 6 ) with other CYP 74s shows that all three CYP 74Ls 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 ( CYP 74D DESs) or bulkier residues like Glu ( CYP 74 B 16, LuDES), or Thr ( CYP 74 Q 1, RaDES), or Leu ( CYP 74H1, AsDES). The importance of this site for DES activity was confirmed by site-directed mutagenesis experiments; the E292G mutant form of LuDES ( CYP 74 B 16) exhibited AOS activity ( Toporkova et al., 2013 ). In CYP 74 L sequences this glycine is substituted with another amino acid – leucine ( CYP 74 L 1) or phenylalanine ( CYP 74 L 2 and CYP 74 L 3). However, the CYP 74 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 ( CYP 74 Q 1) 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 F 295I mutant form of LeAOS3 ( CYP 74 C 3) possessed HPL activity ( Toporkova et al., 2008 ). CYP 74 L enzymes have a substitution of this conservative phenylalanine with other residue – valine ( CYP 74 L 1) or methionine ( CYP 74 L 2 and CYP 74 L 3). 3. Discussion Data obtained indicated that the recombinant CYP 74 L 1, CYP 74 L 2 and CYP 74 L 3, despite belonging to the same subfamily and sharing high homology, possess different catalytic activities. The CYP 74 L 2 specifically exhibited AOS activity. In contrast, CYP 74 L 1 possessed mainly HPL activity towards its preferred substrates, 13-hydroperoxides. Conversely, the 9- HPOD and 9- HPOT , relatively poor substrates for CYP 74 L 1, were converted largely to AOS products. The AOS products of both CYP 74 L 1 and CYP 74 L 2 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 CYP 74 L 1 and CYP 74 L 2, CYP 74 L 3 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 CYP 74 L 3 may be caused by the deletion of sixteen amino acids in the middle of the sequence ( Fig. 1 ). The CYP 74Ls expand the list of characterized enzymes of oxylipin biosynthesis in spikemoss. The S. moellendorffii CYPome includes at least two DESs, i.e. SmDES1 ( CYP 74 M 1) and SmDES2 ( CYP 74 M 3) ( Gorina et al., 2016 ), producing divinyl ethers DE 1 – DE 6 ( 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 ( CYP 74 M 2, 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 ( CYP 74 L 2) described in the present report or other putative AOSs, i.e. CYP 74J1, CYP 74K1, CYP 74K2, or the before characterized SmAOS2 ( CYP 74K3) (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 ( CYP 74 L 1 ) 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 ). Fig. 5. The TIC chromatogram of oxylipins (Me/TMS) from the aerial parts of S. moellendorffii plants. DE1 , (ω5 Z )-etheroleic acid; DE2 , etheroleic acid; DE3 , (11 Z )- etheroleic acid; DE4 , etherolenic acid; DE5 , (ω5 Z )-etherolenic acid; DE6 , (11 Z )-etherolenic acid; 5 , 11-hydroxy-12,13-epoxy-9-octadecenoic acid; 5a , 11-hydroxy- 12,13-epoxy-9,15-octadecadienoic acid; 10 , eicosanoic acid; 11 , 13-ketooctadecadienoic acid; 12 , epimeric 9-hydroxy-12,13-epoxy-10-octadecenoic acid; 12a , epimeric 9-hydroxy-12,13-epoxy-10,15-octadecadienoic acid. Additionally, 13-HOD, as well as cis and trans isomers of 12-OPDA were detected. Fig. 6. The multiple alignment of the CYP74L1, CYP74L2, and CYP74L3 sequences of S. moellendorffii with other CYP74s described earlier. Conservative structures are marked as follows: the I-helix groove region (SRS-4) is numbered 1–6, two positions after the I-helix groove region are marked by ◆ symbol, the F/L toggle is marked by ▾ symbol. Fig. 7. The scheme of catalytic mechanisms of the CYP74 L enzymes . The SmHPL1 ( CYP 74 L 1) is the first CYP 74 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 CYP 74s. Moreover, SmHPL1 ( CYP 74 L 1) presents one more example of a CYP 74 enzyme exhibiting a different catalytic behaviour depending on substrate. Recently the dual function CYP 74 C HPL/EASs have been described ( Toporkova et al., 2018b ). LuDES ( CYP 74 B 16) was identified as dual function DES /HPL (with minor EAS activity) depending on substrate ( Toporkova et al., 2020a ). Additional EAS activity was exhibited by CYP 74 B HPLs ( Toporkova et al., 2020b ), as well as by carrot allene oxide synthase DcAOS ( CYP 74 B 33) ( Gorina et al., 2019b ). A common intermediate of all reactions controlled by CYP 74s 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 CYP 74 subfamilies, the CYP 74 L subfamily consists of different enzymes, namely AOSs and HPLs. The CYP 74A subfamily includes 13-specific AOSs and 9/13-specific EASs. The CYP 74 B subfamily comprises 13-specific HPLs and DESs, as well as at least one 9-specific AOS . The CYP 74 C subfamily includes 9/13-specific AOSs and double function HPL/EASs, and the CYP 74 M subfamily is composed of 13-specific DESs and EASs ( Fig. 8 ). Fig. 8. The unrooted phylogenetic tree of the CYP74 clan. Classified CYP74 subfamilies are marked with their letter designation (A, B, C etc.). Subfamilies consisting of more than one member are outlined with unclosed curves (semi-ellipses). Plant CYP74s: As, Allium sativum ; AsDES, CYP74H1, GI:83414021; At, Arabidopsis thaliana ; AtAOS, CYP74A1, GI:15239032; AtHPL, CYP74B2, GI:3822403; Ca, Capsicum annuum ; CaHPL, CYP74B1, GI:1272340; Cm, Cucumis melo ; CmHPL/EAS, CYP74C2, GI:14134199; Cs, C. sativus ; CsHPL/EAS, CYP74C1, GI:101211324; CsHPL/EAS/AOS, CYP74C31 GI:101211574; Dc, Daucus carota ; DcAOS, CYP74B33, GI:10821971; Gm, Glycine max ; GmHPL/EAS (CYP74C13_Gm), KRH29541.1; Hv, Hordeum vulgare ; HvAOS2, CYP74A3, GI:7452981; HvHPL, CYP74F3, GI: 22265998; Kf, K. flaccidum (green alga); KfAOS, SI:LC032459; Le, Solanum lycopersicum ; LeAOS1, CYP74A1, GI:7581989; LeAOS2, CYP74A2, GI:7677376; LeAOS3, CYP74C3, GI:25991603; LeHPL, CYP74B3, GI:7677378; LeDES (CYP74D1), NP001234527.1; Lu, Linum usitatissimum ; LuAOS, CYP74A1, GI:1352186; LuDES, CYP74B16, GI:379048766; Mp, M. polymorpha ; MpAOS1, SI:LC032457.1, MpAOS2, SI:LC032458.1; Mt, Medicago truncatula ; MtHPL3, CYP74B5, GI:63081244; MtHPL1/EAS, CYP74C13, GI:33504430; Nt, Nicotiana tabacum ; NtDES, CYP74D3; GI: 107799697; Os, Oryza sativa ; OsAOS, CYP74A4, GI:115455571; OsHPL1, CYP74E2, GI:115445057; OsHPL2, CYP74E1, GI:125538638; Pa, Parthenium argentatum ; PaAOS, CYP74A1, GI:218511958; Pd, Prunus dulcis ; PdHPL, CYP74C5, GI:33300600; Pg, Psidium guajava ; PgHPL, CYP74B5, GI:13183137; Pi, Petunia inflata ; PiCYP74C9, GI:85720841; Pp, P. patens ; PpAOS1, CYP74A1, GI:22217985; PpAOS2, CYP74A8, GI:168014176; PpHPL, CYP74G1, GI:76057841; Ra, Ranunculus acris ; RaDES, CYP74Q1, GI:768564485; Rj, Ranunculus japonicus ; RjEAS, CYP74A88, SI:MK061531; Sm, S. moellendorffii ; SmDES1, CYP74M1, GI:9660714; SmEAS, CYP74M2, GI:9637471 SmDES2, CYP74M3, GI:9654395; SmAOS2, CYP74K3, EFJ20163.1; CYP74L1, XP002969700.1; CYP74L2, XP002972651.1; CYP74L3, EFJ25870.1; St, Solanum tuberosum ; StAOS2, CYP74A6, GI:86769479; StAOS3, CYP74C10, GI:56605358; StHPL/EAS, CYP74C4, GI:102588560; StDES, CYP74D2, GI:12667099; Zm, Zea mays ; ZmAOS, CYP74A19, GI:223947589; ZmHPL, CYP74F2, GI:162462890. CYP74 clan members: Es, E. siliculosus (brown alga); EsEAS, CYP5164B1, GI:1109557544; Mn, M. nodulans (proteobacteria); MnHPL, SI:WP_015932840.1; Msp, Methylobacterium sp. 4 –46; MspCYP74, SI:WP_012335549.1. Ap, A. palmata (Metazoa) ; ApAOS, GI:187948710; Bf, B. floridae (Metazoa) ; BfEAS, CYP440A1, GI:189312561; Nv, N. vectensis (Metazoa) ; NvEAS, CYP443D1, GI:5516222; NvHPL/EAS CYP443C1 (GenBank QJI54761.1). 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 CYP 74 genes in S. moellendorffii , including DESs ( CYP 74 M 1 and CYP 74 M 3), EAS ( CYP 74 M 2), HPL ( CYP 74 L 1), AOSs (including the characterized AOSs CYP 74K3 and CYP 74 L 2, as well as the putative AOSs CYP 74J1, CYP 74K1, and CYP 74K2), 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.