A phenol-enriched cuticle is ancestral to lignin evolution in land plants

Abstract

Lignin, one of the most abundant biopolymers on Earth, derives from the plant phenolic metabolism. It appeared upon terrestrialization and is thought critical for plant colonization of land. Early diverging land plants do not form lignin, but already have elements of its biosynthetic machinery. Here we delete in a moss the P450 oxygenase that defines the entry point in angiosperm lignin metabolism, and find that its pre-lignin pathway is essential for development. This pathway does not involve biochemical regulation via shikimate coupling, but instead is coupled with ascorbate catabolism, and controls the synthesis of the moss cuticle, which prevents desiccation and organ fusion. These cuticles share common features with lignin, cutin and suberin, and may represent the extant representative of a common ancestor. Our results demonstrate a critical role for the ancestral phenolic metabolism in moss erect growth and cuticle permeability, consistent with importance in plant adaptation to terrestrial conditions.

Figure 1. The hydroxycinnamoyl-shikimate pathway in angiosperms and the P. patens hydroxycinnamoyl-threonate pathway established in this study.

The figure shows p-coumaroyl-2-theonate as PpCYP98 substrate, but p-coumaroyl-4-threonate is also converted by the enzyme. The p-coumaroyl-shikimate molecule is drawn according to the structure experimentally determined by Levsh et al..

Figure 2. PpCYP98 is essential for P. patens gametophore development.

(a–d) GUS staining pattern in PpCYP98:uidA lines indicated a prominent expression in developing gametophores. (a) Bud (emerging gametophore). Scale bar, 50 μm. (b) Young gametophores. Scale bar, 0.5 mm. (c) One-month-old gametophore. Scale bar, 2 mm. (d) Apical leaf from 1-month-old gametophore (joined pictures). No GUS staining is visible in the midrib. Scale bar, 0.5 mm. (e–j) ΔPpCYP98 mutants fail to develop normal gametophores. (e,f) Six-week-old colonies grown on agar plates. Arrowheads indicate gametophores. Scale bars, 5 mm. (g–h) Close-up views of gametophores. Scale bars, 0.5 mm. (i,j) Toluidine blue-stained cross section of gametophores. Arrowheads indicate phyllids in the WT. Scale bars, 0.1 mm.

Figure 3. PpCYP98 is a phenolic ring meta-hydroxylase and uses esters of threonic acid as substrates.

(a) Ultraviolet chromatogram showing the absence of major peaks in the ΔPpCYP98 mutant gametophore crude extract. IS, internal standard (morin). (b) Names and structures of molecules at the indicated retention times (RT). (c) PpCYP98-dependent conversion of p-coumaroyl-2-threonate (pC2T) and p-coumaroyl-4-threonate (pC4T) esters into corresponding caffeoyl threonate esters (C2T and C4T). Control reactions without NADPH were concurrently analysed. Molecules were detected using dedicated multiple reaction monitoring (MRM) methods. Note that two caffeoyl-2-threonate isomers are produced from the two p-coumaroyl-2-threonate isomers present in the synthetic substrate, shown in Supplementary Fig. 6. (d) Acid hydrolysis of crude extracts demonstrates the total absence of caffeate in gametophores of the ΔPpCYP98 mutants. Results are the mean+standard error from three independent biological samples for WT and three independent mutant lines. Asterisk indicates a significant difference between mutants and WT (P-value=0.037; two-tailed Student's t-test for samples of unequal variance).

Figure 4. PpCYP98 produces cutin caffeoyl units and is critical for cutin formation in P. patens.

(a) Toluidine blue permeability staining indicates a cuticle defect in the ΔPpCYP98 mutant gametophore. Scale bars, 0.5 mm. (b) Transmission electron micrographs of the phyllid outer cell surface showing alteration of the ΔPpCYP98 mutant cuticle layer. cut, cuticle; cw, cell wall; pm, plasma membrane. Scale bars, 0.5 μm. (c) Comparative analysis of WT and mutant cutin gametophore composition. Results are the mean+standard error from three independent biological samples for WT and three independent lines for the mutant. n.d., not detected. (d) Exogenous caffeate supply (20 μM) restores growth of the ΔPpCYP98 mutant gametophore. Scale bars, 0.2 mm. (e) Exogenous caffeate (20 μM) supply restores cuticle impermeability to toluidine blue of the mutant phyllids. Scale bar, 0.5 mm.