Chemodiversity in Selaginella: a reference system for parallel and convergent metabolic evolution in terrestrial plants

Abstract

Early plants began colonizing the terrestrial earth approximately 450 million years ago. Their success on land has been partially attributed to the evolution of specialized metabolic systems from core metabolic pathways, the former yielding structurally and functionally diverse chemicals to cope with a myriad of biotic and abiotic ecological pressures. Over the past two decades, functional genomics, primarily focused on flowering plants, has begun cataloging the biosynthetic players underpinning assorted classes of plant specialized metabolites. However, the molecular mechanisms enriching specialized metabolic pathways during land plant evolution remain largely unexplored. Selaginella is an extant lycopodiophyte genus representative of an ancient lineage of tracheophytes. Notably, the lycopodiophytes diverged from euphyllophytes over 400 million years ago. The recent completion of the whole-genome sequence of an extant lycopodiophyte, S. moellendorffii, provides new genomic and biochemical resources for studying metabolic evolution in vascular plants. 400 million years of independent evolution of lycopodiophytes and euphyllophytes resulted in numerous metabolic traits confined to each lineage. Surprisingly, a cadre of specialized metabolites, generally accepted to be restricted to seed plants, have been identified in Selaginella. Initial work suggested that Selaginella lacks obvious catalytic homologs known to be involved in the biosynthesis of well-studied specialized metabolites in seed plants. Therefore, these initial functional analyses suggest that the same chemical phenotypes arose independently more commonly than anticipated from our conventional understanding of the evolution of metabolism. Notably, the emergence of analogous and homologous catalytic machineries through convergent and parallel evolution, respectively, seems to have occurred repeatedly in different plant lineages.

Keywords: Selaginella; chemodiversity; convergent evolution; parallel evolution; specialized metabolism.

FIGURE 1

(A) A simplified cladogram illustrating the phylogeny of the green plant lineage. Selaginella is the only genus under the order Selaginellales (highlighted in red), and represents an ancient lineage of vascular plants, lycopodiophytes. (B) A representative expansion of enzyme families implicated in specialized metabolism during plant evolution. The gene number for each enzyme family counted per haploid genome from five species representative of the green plant lineage is shown. The proportion of the sum of these genes in relation to the haploid gene complement is denoted on top of each bar. Due to our currently limited knowledge of the temporal expression and biochemical function of specialized metabolic enzymes, the enzyme families and numbers of members listed likely underestimate the true biosynthetic potential of any green plant.

FIGURE 2

Examples of divergent, parallel, and convergent evolution in plant specialized metabolism. (A) A cladogram showing the phylogenetic relationship of several land plant type III polyketide synthases (PKSs) involved in specialized metabolism. Whereas the expansion of enzyme families by duplication and selection over time leads to functional divergence among descendants, identical biochemical functions do arise independently from homologous ancestral forms through parallel evolution. 2PS, 2-pyrone synthase; CHS, chalcone synthase; PCS, pentaketide chromone synthase; STS, stilbene synthase. (B) Divergence of three plant type III PKSs, 2PS, CHS, and PCS. Mutational trajectories reshape the volume of the active sites (green surface representation) directly correlated with divergent biochemical functions. (C) An overlay of the active sites of CHS from alfalfa (green) and two STSs from pine (magenta) and peanut (yellow), respectively. Parallel evolution of STSs from CHSs in several plant lineages involves distinct mutational trajectories in otherwise homologous three-dimensional structures. These mutations at disparate positions in the STSs’ primary sequences lead to the same conformational shifts of Thr132 in the active sites (red arrow). These separate alterations of the same structural component dictate a switch from cyclization by Claisen condensation in CHSs to cyclization by aldol condensations in STSs. (D) Convergent evolution of flavone synthases (FNS) in plants. Whereas most flowering plants examined to date contain type II FNSs belonging to the cytochrome P450 enzyme family, plants in the Apiaceae employ type I FNSs belonging to the non-homologous 2-oxoglutarate-dependent dioxygenase family.

FIGURE 3

Flavonoids identified from Selaginella. (A) Common flavones and flavanones: apigenin (1), luteolin (2), chrysoeriol (3), genkwanin (4), 4′,7-dihydroxyflavanone (5), 4′,5,7-trihydroxyflavanone (6). (B) Chalcones: 6′-deoxychalcone (7), 6′-deoxychalcone, 4-O-β-glucoside (8). (C) Flavones with unusual ring substitutions: 6-(2′-hydroxy-5′-carboxyphenyl)-apigenin (9), 5-carboxymethyl-4′,7-dihydroxyflavone (10), 5-carboxymethyl-4′,7-dihydroxyflavone ethyl ester (11), 5-carboxymethyl-4′,7-dihydroxyflavone butyl ester (12), 5-carboxymethyl-4′,7-dihydroxyflavone-7-O-β-D-glucopyranoside (13). (D) Biflavonoids: 2′,8^′′-biapigenin (14), taiwaniaflavone (15), 7,4′-di-O-methylrobustaflavone (16), 7′-O-methylrobustaflavone (17), 4′-O-methylrobustaflavone (18), robustaflavone (19), 7,4′-di-O-methyl-2′,3′-dihydrorobustaflavone (20), 7,4′,7^′′-tri-O-methyl-2′,3′-dihydrorobustaflavone (21), 7,4′,7^′′,4^′′′-tetra-O-methylamentoflavone (22), kayaflavone (23), heveaflavone (24), ginkgetin (25), 7,7′-di-O-methylamentoflavone (26), 4′,7′-di-O-methylamentoflavone (27), isoginkgetin (28), bilobetin (29), podocarpusflavone A (30), sostetsuflavone (31), amentoflavone (32), sumaflavone (33), 2,3-dihydroamentoflavone (34), 2′, 3′-dihydroamentoflavone (35), tetrahydroamentoflavone (36), delicaflavone (37), ochnaflavone (38), cryptomerin B (39), pulvinatabiflavone (40), 7-O-methyl-hinokiflavone (41), isocryptomerin (42), hinokiflavone (43), 2′,3^′′-dihydroisocryptomerin (44), 2,3-dihydrohinokiflavone (45), 2′,3′-dihydrohinokiflavone (46), tetrahydrohinokiflavone (47).

FIGURE 4

Lignans identified from Selaginella. (A) Lignans with the β-β′/ γ-O-α′/ α-O-γ′ linkage: (-)-lirioresinol A (48), (+)-syringaresinol (49), (+)-syringaresinol-4,4′-di-O-β-D-glucopyranoside (50). (B) Lignans with the β-β′/ γ-O-γ′ linkage: matairesinol (51), wikstromol (52), notracheloside (53), matairesinol-4,4′-di-O-β-D-glucopyranoside (54), styraxlignolide D (55). (C) Lignans with the β-β′/α-O-γ′ linkage:lariciresinol (56), lariciresinol-4-O-β-glucopyranoside (57). (D) Neolignans: tamariscinoside B (58), (2R, 3S)-dihydro-2-(3′,5′-dimethoxy-4′-hydroxyphenyl)-7-methoxy-5-acetyl-benzofuran (59), tamariscinoside C (60). (E) Secolignans: 3,4-trans-3-hydroxymethyl-4-[bis(4-hydroxyphenyl)methyl]butyrolactone (61), 2,3-trans-3,4-trans-2-methoxy-3-hydroxymethyl-4-[bis(4-hydroxyphenyl)methyl]tetrahydrofuran (62).

FIGURE 5

Selaginellins identified from Selaginella. Selaginellin (63), Selaginellin A (64), Selaginellin B (65), Selaginellin C (66), Selaginellin D (67), Selaginellin E (68), Selaginellin F (69), Selaginellin I (70), Selaginellin J (71), Selaginellin M (72), Selaginellin N (73), Selaginellin G (74), Selaginellin K (75), Selaginellin L (76), Selaginellin H (77).

FIGURE 6

Other phenolic compounds identified from Selaginella. (A) Simple phenylpropanoids: caffeic acid (78), ferulic acid (79), syringin (80). Benzenoids: 4-hydroxybenzoic acid (81), vanillic acid (82), syringic acid (83), tamariscina ester A (84). (B) Uncommon phenylpropanoid derivatives: neolloydosin (85), 3-hydroxy-1-(4-hydroxy-3-methoxyphenyl)propan-1-one (86), 3-hydroxy-1-(4-hydroxy-3,5-dimethoxyphenyl)propan-1-one (87), arbutin (88), 1-(4′-hydroxy-3′-methoxyphenyl)glycerol (89). (C) Coumarins: umbelliferone (90), 3-(4-hydroxyphenyl)-6,7-dihydroxycoumarin (91), isopimpinellin (92). (D) Anthraquinones and chromones: chrysophanol (93), emodin (94), physcion (95), aloe-emodin (96), chrysophanol-8-O-glucoside (97), emodin-8-O-glucoside (98), physcion-8-O-glucoside (99), uncinoside A (100), uncinoside B (101), and 8-methyleugenitol (102).

FIGURE 7

Alkaloids identified from Selaginella. (A) N-methyltyramine derivatives: hordenine (103), hordenine-O-α-rhamnopyranoside (104), N-methyltyramine-O-α-rhamnopyranoside (105), hordenine-O-[(6-O-cinnamoyl)-O-β-glucopyranosyl]-α-rhamnopyranoside (106), hordenine-O-[(6-O-p-coumaroyl)-O-β-glucopyranosyl]-α-rhamnopyranoside (107). (B) Hydroxycinnamoyl polyamine alkaloids: paucine (108), paucine 3′-β-D-glucopyranoside (109), N¹-cis-p-coumaroylagmatine (110).

FIGURE 8

Terpenoids identified from Selaginella. (A) Monoterpenes: linalool (111), (4Z,6E)-2,7-dimethyl-8-hydroxyocta-4,6-dienoic acid 8-O-β-D-glucopyranoside (112). (B) Sesquiterpenes: cedrol (113), (+)-(3S)-nerolidol (114), (+)-germacrene D (115), (-)-β-elemene (116), β-sesquiphellandrene (117). (C) Diterpenes: gibberellin A4 (118), gibberellin A24 (119), ent-copalyl diphosphate (120), miltiradiene (121), λ-7,13e-dien-15-ol (122). (D) Triterpenes: β-sitosterol (123), β-daucosterin (124), pulvinatadione (125), 3β,16̩-dihydroxy-5〈,17^®-cholestan-21-carboxylic acid (126), 3^®-acetoxy-16〈-hydroxy-5α, 17β-cholestan-21-carboxylic acid (127), 3β-(3-hydroxybutyroxy)-16α-hydroxy-5α, 17β-cholestan-21-carboxylic acid (128), glycyrrhetinic acid (129), friedelin (130).