Origins and Evolution of Cuticle Biosynthetic Machinery in Land Plants

Abstract

The aerial epidermis of land plants is covered with a hydrophobic cuticle that protects the plant against environmental stresses. Although the mechanisms of cuticle biosynthesis have been extensively studied in model plants, particularly in seed plants, the origins and evolution of cuticle biosynthesis are not well understood. In this study, we performed a comparative genomic analysis of core components that mediate cuticle biosynthesis and characterized the chemical compositions and physiological parameters of cuticles from a broad set of embryophytes. Phylogenomic analysis revealed that the cuticle biosynthetic machinery originated in the last common ancestor of embryophytes. Coexpansion and coordinated expression are evident in core genes involved in the biosynthesis of two major cuticle components: the polymer cutin and cuticular waxes. Multispecies analyses of cuticle chemistry and physiology further revealed higher loads of both cutin and cuticular waxes in seed plants than in bryophytes as well as greater proportions of dihydroxy and trihydroxy acids, dicarboxylic acids, very-long-chain alkanes, and >C28 lipophilic compounds. This can be associated with land colonization and the formation of cuticles with enhanced hydrophobicity and moisture retention capacity. These findings provide insights into the evolution of plant cuticle biosynthetic mechanisms.

Figure 1.

Evolutionary analyses of plant cuticle biosynthetic genes. A, Ortholog survey of 32 cuticle biosynthetic genes in 41 genomic data sets from the Archaeplastida. Columns in orange, green, purple, and blue indicate the presence of orthologs of genes involved in cutin biosynthesis, cuticular wax formation, transport, and regulation, respectively. The ortholog numbers corresponding to the various plant species are labeled, and the phylogenetic trees of plants were based on Gao et al. (2018), Puttick et al. (2018), Han et al. (2019), and Jiao et al. (2020). B, Expansion and coevolution analysis of LACS, CUS, and EH1. Gene numbers of LACS, CUS, and EH1 in land plant and algal species are shown as numbers in rectangles and circles respectively, and the numbers of gene gains during evolution are labeled with red plus symbols.

Figure 2.

Coexpression network analysis of cuticle biosynthetic modules in Arabidopsis, S. lycopersicum, O. sativa, Z. mays, P. abies, S. moellendorffii, and P. patens. Plant cuticle biosynthesis gene families are indicated by node colors listed on the left. The coexpression networks were reconstructed using the highest reciprocal rank (HRR) method, where genes with an HRR < 30 were connected with gray lines.

Figure 3.

Comparative analysis of cutin composition in 13 representative land plant species. A, Comparative analysis of amounts of total cutin monomers in A. agrestis, M. polymorpha, P. patens, S. moellendorffii, H. selago, B. lanuginosus, P. virginianum, P. abies, G. biloba, Z. mays, O. sativa, S. lycopersicum, and Arabidopsis. B, Heat map of the normalized amounts of cutin monomers (percentage of total cutin monomers) in 13 representative land plant species. C to E, Comparative analyses of amounts (percentage of total cutin monomers) of dihydroxy and trihydroxy acids (C), dicarboxylic acids (D), and phenolic compounds (E) in 13 representative land plant species. The phylogenetic trees were based on Gao et al. (2018), Puttick et al. (2018), Han et al. (2019), and Jiao et al. (2020). For A and C to E, three independent biological replicates were analyzed, and results are shown as means ± se. Asterisks indicate significant difference by Student’s t test (**P < 0.01).

Figure 4.

Comparative analysis of cuticular wax composition in 13 representative land plant species. A, Comparative analysis of amounts of total cuticular wax in A. agrestis, M. polymorpha, P. patens, S. moellendorffii, H. selago, B. lanuginosus, P. virginianum, P. abies, G. biloba, Z. mays, O. sativa, S. lycopersicum, and Arabidopsis. B, Heat map of the normalized amounts of cuticular wax components (percentage of total cuticular waxes) from 13 representative land plant species. C to E, Comparative analyses of amounts (percentage of total cuticular waxes) of VLC alkanes (C), >C28 lipophilic compounds (D), and VLC fatty acids (E) in 13 representative land plant species. The phylogenetic trees were based on Gao et al. (2018), Puttick et al. (2018), Han et al. (2019), and Jiao et al. (2020). For A and C to E, three independent biological replicates were analyzed, and results are shown as means ± se. Asterisks indicate significant difference by Student’s t test (**P < 0.01).

Figure 5.

Comparative analyses of cuticle hydrophobicity and moisture retention capacity in 13 representative land plant species. A, Hydrophobicity analysis in the phyllids or leaves of A. agrestis, M. polymorpha, P. patens, S. moellendorffii, H. selago, B. lanuginosus, P. virginianum, P. abies, G. biloba, Z. mays, O. sativa, S. lycopersicum, and Arabidopsis. Contact angles from at least 50 water droplets were separately measured, and five independent biological replicates were analyzed. Results are shown as means ± se. Asterisks indicate significant difference by Student’s t test (**P < 0.01). B, Comparative analysis of moisture retention capacity in the phyllids or leaves of A. agrestis, M. polymorpha, P. patens, S. moellendorffii, H. selago, B. lanuginosus, P. virginianum, P. abies, G. biloba, Z. mays, O. sativa, S. lycopersicum, and Arabidopsis. Numbers of hours in a dehydrating environment are shown at the bottom of the graphs. Three independent biological replicates were analyzed, and results are shown as means ± se. Asterisks indicate significant difference by Student’s t test (**P < 0.01). The phylogenetic trees were based on Gao et al. (2018), Puttick et al. (2018), Han et al. (2019), and Jiao et al. (2020).

Figure 6.

A model for the origin and evolution of cuticle biosynthetic machinery during plant land colonization. Although several components mediating cuticle biosynthesis, transport, and even regulation emerged in algae, additional important cuticle biosynthetic machinery originated in the last common ancestor of embryophytes. Gene expansion and emergence occurred afterward, resulting in changes in cuticle chemical composition and contributions to plant physiology. During the evolutionary transition from bryophytes to seed plants, both cutin and cuticular wax loads increased, together with the proportions of dihydroxy and trihydroxy acids, dicarboxylic acids, VLC alkanes, and >C28 lipophilic compounds, while the relative amounts of phenolic compounds and VLC fatty acids decreased, leading to the formation of cuticles in seed plants with enhanced hydrophobicity and moisture retention properties. Ma, Million years ago.