On the evolution of lignin biosynthesis in plants

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Lignin is a defining feature of all vascular plants. This phenolic polymer is incrusted into woody secondary cell walls to provide mechanical stability and hydrophobicity necessary for upright growth and long-distance water transport. Despite common occurrence in all vascular plant lineages, the biosynthetic route to lignin appears to be different in different plant lineages. In flowering plants, the rate limiting step towards the majority of lignin monomers is catalyzed by the enzyme 4-coumaroyl-shikimate-3'-hydroxylase (C3'H / CYP98). However, in lycopods, an early diverging vascular plant lineage, biosynthesis to the same lignin monomers proceeds through a different route. Fittingly, we have recently shown that in lycopods CYP98 is unable to metabolize 4-coumaroyl-shikimate, but utilizes different substrates, presumably to produce defense related secondary metabolites derived from the same pathway. Surprisingly, a fern CYP98 was also unable to use 4-coumaroyl-shikimate, and thus monolignol biosynthesis in ferns also appears to employ distinct intermediates. Even in conifers, CYP98 substrate profiles are distinct from lignin related C3'H enzymes. We thus hypothesize that lignin biosynthesis was modified significantly independently multiple times during land plant evolution. We will test this hypothesis by functionally characterizing the single CYP98s from a conifer and from a fern species. Besides detailing biochemical characterizations, we will need to compare gene expression profiles of these CYP98 genes with genes known to be involved in lignin biosynthesis through analysis of large-scale gene expression data. These will also be mined to identify novel candidate genes involved in lignin biosynthesis in these lineages. Importantly, we need to establish approaches to generate mutants in these non-model species to functionally test a direct involvement of these genes in wood formation. We will generate loss-of-function mutants that will be characterized for changes typically found in flowering plant cyp98 mutants, including changes in lignin composition. Alterations in leaf morphology and metabolite composition will be assessed as these are affected in a non-lignin related moss cyp98 mutant. These experiments will show whether CYP98 is involved in lignin biosynthesis in ferns and conifers or if alternative pathways have evolved. If the latter holds true, candidate genes identified from the gene expression data will be tested for their ability to rescue the cyp98a3 loss of function phenotype in the model plant Arabidopsis and, if warranted, then further characterized biochemically and through reverse genetic approaches. Taken together, this program will test the evolutionary trajectory of lignin biosynthesis and may identify evolutionary optimizations of a biochemical pathway that was crucial for plants to conquer land and that defines wood properties and its uses by humans.