Abstract
In the present work, lignin-like fractions were isolated from several ancestral plants –including moss (Hypnum cupressiforme and Polytrichum commune), lycophyte (Selaginella kraussiana), horsetail (Equisetum palustre), fern (Nephrolepis cordifolia and Pteridium aquilinum), cycad (Cycas revoluta), and gnetophyte (Ephedra fragilis) species– and structurally characterized by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. Py-GC/MS yielded marker compounds characteristic of lignin units, except in the H. cupressiforme, P. commune and E. palustre “lignins,” where they were practically absent. Additional structural information on the other five samples was obtained from 2D-NMR experiments displaying intense correlations signals of guaiacyl (G) units in the fern and cycad lignins, along with smaller amounts of p-hydroxyphenyl (H) units. Interestingly, the lignins from the lycophyte S. kraussiana and the gnetophyte E. fragilis were not only composed of G- and H-lignin units but they also incorporated significant amounts of the syringyl (S) units characteristic of angiosperms, which appeared much later in plant evolution, most probably due to convergent evolution. The latter finding is also supported by the abundance of syringol derivatives after the Py-GC/MS analyses of these two samples. Regarding lignin structure, β−O−4′ alkyl-aryl ethers were the most abundant substructures, followed by condensed β−5′ phenylcoumarans and β−β′ resinols (and dibenzodioxocins in the fern and cycad lignins). The highest percentages of alkyl-aryl ether structures correlated with the higher S/G ratio in the S. Kraussiana and E. fragilis lignin-like fractions. More interestingly, apart from the typical monolignol-derived lignin units (H, G and S), other structures, assigned to flavonoid compounds never reported before in natural lignins (such as amentoflavone, apigenin, hypnogenol B, kaempferol, and naringenin), could also be identified in the HSQC spectra of all the lignin-like fractions analyzed. With this purpose, in vitro synthesized coniferyl-naringenin and coniferyl-apigenin dehydrogenation polymers were used as standards. These flavonoids were abundant in H. cupressiforme appearing as the only constituents of the moss lignin-like fraction (including 84% of dimeric hypnogenol B) and their abundance decreased in those of S. Kraussiana (with amentoflavone and naringenin representing 14% of the total aromatic units), and the two ancient gymnosperms (0.4–1.2%) and ferns (0–0.7%).
Highlights
The first land plants to colonize Earth, which evolved from freshwater green algae (Charophyceae), appeared around 450 Mya as confirmed by the fossil record (Kenrick and Crane, 1997)
Especially thanks to the great advances in two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy during the last two decades, the presence of flavonoids incorporated into the lignin polymer has been discovered, acting as natural lignin monomers together with other phenolic precursors biosynthesized outside the monolignol pathway
Lignins from precursors synthesized through the classical monolignol biosynthetic route were not significantly detected in the two moss species, as the more ancestral plants analyzed
Summary
The first land plants to colonize Earth, which evolved from freshwater green algae (Charophyceae), appeared around 450 Mya as confirmed by the fossil record (Kenrick and Crane, 1997). Before land plants developed the ability to synthesize lignin, it is believed that flavonoid compounds were responsible for protecting them from UV radiation (Clayton et al, 2018; Davies et al, 2020). Flavonoids are a large family of secondary metabolites ubiquitously found in land plants (Grotewold, 2006). These phenolic compounds, with a C6-C3-C6 carbon framework, are synthesized from phenylalanine and malonyl-CoA through the phenylpropanoid pathway. Especially thanks to the great advances in two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy during the last two decades, the presence of flavonoids incorporated into the lignin polymer has been discovered, acting as natural lignin monomers together with other phenolic precursors biosynthesized outside the monolignol pathway (del Río et al, 2020, 2021). All flavonoids with a free 4 -OH in the B-ring are potentially susceptible of being enzymatically oxidized, forming radicals compatible with lignification in cross-coupling reactions with monolignols
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