Physcomitrella patens subsp. gametophores, (Hedw.) Bruch & Schimp. (Hedw.) Bruch & Schimp.

Mukhtarova, Lucia S., Lantsova, Natalia V., Khairutdinov, Bulat I. & Grechkin, Alexander N., 2020, Lipoxygenase pathway in model bryophytes: 12 - oxo- 9 (13), 15 - phytodienoic acid is a predominant oxylipin in Physcomitrella patens, Phytochemistry (112533) 180, pp. 1-7 : 2-3

2.1. Oxylipins of P. patens gametophores

The oxylipin profile of P. patens gametophores is presented in Fig. 1A .

The predominant endogenous oxylipin was product 1 (retention time 16.2 min, equivalent chain length (ECL) 20.61). For further characterization of the lipoxygenase pathway in P. patens and structural identification of product 1, the homogenate of gametophores was incubated with α linolenic acid. The GC-MS chromatogram of products (Me/ TMSi) of α linolenate conversion is presented in Fig. 1B . The second product (by abundance) was the peak of 13-hydroxy-9,11,15-octadecatrienoic acid (Me/TMSi). Minorities were represented by peaks of cis -12- OPDA (Me) and α ketol, 12-oxo-13-hydroxy-9,15-octadecadienoic acid (Me/TMSi), as seen from Fig. 1B . The mass spectrum of cis -12-OPDA (Me) and the corresponding mass fragmentation scheme are presented in Supplementary Fig. 1 .

The mass spectrum of predominant product 1 possessed M + at m/z 306 (16%), [M – MeO] + at m/z 275 (12), m/z 217 (14), [M – C1/C6] + at m/z 177 (78), [M – C1/C8] + at m/z 149 (100), m/z 135 (38), and m/z 91 (65). The spectrum matched that of iso -12-OPDA, i.e. 12-oxo-9(13),15- phytodienoic acid (Me) ( Vick et al., 1979). Catalytic hydrogenation converted compound 1 (Me) to product, whose mass spectrum (Supplementary Fig. 2 ) possessed M + at m/z 310 (0.1%), [M – MeO] + at m/z 279 (1%), [M – C14/C18 +H] + at m/z 240 (4), [M – C1/C8] + at m/z 153 (15), and m/z 83 (100). These spectral patterns fully corresponded to that of the cyclopentanone 12-oxophytonoic acid (Me), which is also formed upon the hydrogenation of 12-OPDA ( Grechkin et al., 2002). The sequential NaBH 4 reduction, methylation, hydrogenation over PtO 2, and trimethylsilylation converted compound 1 to products, appearing as a group of three closely eluting peaks during the GC-MS analysis and having the identical mass spectral patterns (Supplementary Fig. 3 ): [M – Me] + at m/z 369 (4%), [M – Me – MeO] + at m/z 337 (3), m/z 224 (3), m/z 159 (6), [ CH 2 –– CH – CH ––O + –SiMe 3] at m/z 129 (100), [SiMe 3] at m/z 73 (70). The spectral patterns matched that of cyclopentanol 12-hydroxyphytonoic acid (Me/TMSi) ( Grechkin et al., 2002). Its appearance as a few peaks is explained by the formation of enantiomers and geometric isomers of cyclopentanol. Overall, the described data are fully consistent with the identification of compound 1 as the Me ester of 12-oxo-9(13),15-phytodienoic acid (iso -12-OPDA). For final structural approval, compound 1 was purified, and the NMR data were recorded.

The NMR spectral data for compound 1 are presented in Table 1 . The attribution of signals of protons at C2/C8 and C14/C18 was confirmed by the 1 H– 1 H–COSY correlations. The 1 H-NMR spectrum possessed only two olefinic protons H15 and H16. At the same time, the 13 C chemical shifts ( Table 1 ) estimated from the HSQC and HMBC spectral data, demonstrated the presence of double bond between C9 and C13, the cyclopentenone carbons bearing the side chains. Position of C9 and C13 towards the neighbouring functional groups (particularly, H8 and H14) is unequivocally confirmed by the heteronuclear multiple bond correlations (1 H, 13 C-HMBC data, Table 1 ). Other chemical shifts were unambiguously attributed thanks the 1 H– 1 H–COSY, HSQC and HMBC spectra, for instance, the homonuclear and heteronuclear multiple bond correlations (estimated from the HMBC spectral data). To summarize, the data fully confirmed the identification of compound 1 as 12-oxo-9 (13),15-phytodienoic acid (Me), i.e., the iso -12-OPDA.

The less abundant product 2, possessing similar product patterns to those of compound 1, had shorter retention time (13.2 min) and the equivalent chain length (ECL) 18.53. The mass spectrum of product 2 ( Fig. 2B ) possessed M + at m/z 278 (25%), [M – Et] + at m/z 249 (10), [M – MeO] + at m/z 247 (5), [M – C1/C4] + at m/z 177 (100), [M – C1/C6] + at m/z 149 (97), m/z 135 (36), m/z 122 (40), m/z 105 (34), m/z 91 (77), m/z 79 (79), and m/z 67 (41). The spectral patterns of product 2 indicated the structure of 2,3-dinor analogue of compound 1, i.e. 2,3-dinor-12-oxo-9(13)-PDA (Me) (dinor- iso -12-OPDA). The sequential NaBH 4 reduction, methylation, hydrogenation over PtO 2, and trimethylsilylation turned product 2 to compound 2a, whose mass spectrum (Supplementary Fig. 4) possessed [M – Me] + at m/z 341 (7%), [M – Me – MeO] + at m/z 309 (1), [M – TMSiOH] + at m/z 266 (5), m/z 234 (4%), m/z 164 (7), [ CH 2 –– CH – CH ––O + –SiMe 3] at m/z 129 (100), [SiMe 3] at m/z 73 (60). Fragment CH 2 –– CH – CH ––O + –SiMe 3 representing a base peak at m/z 129 is distinguishing for TMSi-cyclopentanol structures. Overall, the described mass spectral data allow ascribing the structures of 1- hydroxy-2-(2’(Z)-pentenyl)-cyclopentane-3-hexanoic acid (Me/TMSi) and 2,3-dinor-12-oxo-9(13)-PDA (Me) (dinor- iso -12-OPDA) to compounds 2a and 2, respectively.

The hydroxy fatty acids have also been detected in P. patens . The hydroxy acids 13-hydroxy-9,11,15-octadecatrienoic (13-HOT) and 13- hydroxy-9,11-octadecadienoic acids (13-HOD) (Me/TMSi) derived from the 13-lipoxygenase activity were detected (their spectral data are not presented). Upon the sequential reduction with sodium borohydride, hydrogenation over PtO 2, methylation with diazomethane, and trimethylsilylation these products turned to the 13-hydroxystearic acid (Me/ TMSi), thus confirming the identification of 13-HOT and 13-HOD. Besides, there was a minor product, eluting close before the methyl stearate, whose mass spectrum possessed [M – Me] + at m/z 337 (0.6%), [M – MeO] + at m/z 321 (0.4%), [M – MeCH 2 CH ––CHCH 2] + at m/z 283 (62), [M – ( CH 2) 5 COOMe] + at m/z 223 (1), m/z 161 (23), m/z 133 (48), m/z 119 (26), m/z 105 (15), m/z 91 (24), [SiMe 3] + at m/z 73 (100). The main characteristic fragment at m/z 283 allowed the preliminary identification of the product as the 11-hydroxy-7,9,13-hexadecatrienoic acid (11-HHT, Me/TMSi). For further structural confirmation, the product was subjected to the sequential reduction with sodium borohydride, hydrogenation over PtO 2, methylation with diazomethane, and trimethylsilylation. The mass spectrum of the resulting saturated analogue possessed [M – Me] + at m/z 343 (0.3%), [M – MeO] + at m/z 327 (0.4%), [M – Me – MeO] + at m/z 311 (3%), [M – Pe] + at m/z 287 (21), m/z 258 (6), [M – ( CH 2) 9 COOMe] + at m/z 173 (62), m/z 159 (6), m/z 129 (6), [ CH 2 ––O + –SiMe 3] at m/z 103 (12), m/z 75 (41), [SiMe 3] + at m/z 73 (100). The fragmentation patterns matched that for 11-hydroxypalmitic acid (Me/TMSi) ( Osipova et al., 2010), thus confirming the identification of the unreduced product as the 11-HHT (Me/TMSi). The ratio of 11-HHT to 13-HOT was ca. 1:10, as estimated by integration of the total ion current chromatograms. The hydroxy derivatives of arachidonic and eicosapentaenoic acids were present only at trace levels (results not presented).

The following minor oxylipins were detected (by order of elution) except the products mentioned above. First, dodecane-1,12-dioic acid (Me). Second, 12-hydroxydodecanoic acid (Me/TMSi). Both compounds were detected upon the NaBH 4 reduction of products and their catalytic hydrogenation over PtO 2. Besides, the 9-oxononanoic acid (Me), azelaic acid (Me), (2 E)-4-hydroxy-2-nonenoic acid (Me/TMSi) were detected. All these products are biosynthesized via the HPL pathway, occurring in P. patens ( Stumpe et al., 2006) . The spectra of mentioned chain cleavage products (not shown) match those for the same non-volatile HPL products published before ( Mukhtarova et al., 2011).

Figures

Fig. 1. The GC-MS oxylipin profiles of P. patens (A, B) and M. polymorpha (C, D). A and C, the profiles of endogenous oxylipins (Me/TMSi), the mixed ion current chromatograms (m/z 149, 177, 210, 225, 238, 278, 306, 308, 311). B and D, profiles of oxylipins (Me/TMSi) biosynthesized upon the in vitro preincubations of P. patens and M. polymorpha with α linolenic acid. The experimental details are described under “Experimental”.

Fig. 2. The electron impact mass spectra and fragmentation schemes (insets) for products 1 (A), 2 (B), and 3 (C).

Fig. 3. The proposed pathways of biosynthesis of major cyclopentenones in P. patens and M. polymorpha. β oxn, β oxidation.