Evolution of phenolic conjugate incorporation in plant sporopollenin

Sporopollenin, a structurally complex and chemically recalcitrant biopolymer, forms the outer exine layer of plant spores and pollen, protecting male gametes from environmental stresses. Varying levels of phenolic constituents are incorporated as building blocks into sporopollenin in many species, but how this process evolved remains unclear. Using an optimized alkaline hydrolysis method, along with NMR spectroscopy and GC-MS, we determined that plants have evolved the ability to incorporate phenolic compounds into sporopollenin in a phylogenetically ordered manner. Covalently linked phenolic constituents, including p-coumarate (p-CA), ferulate, p-hydroxybenzoate, naringenin, the canonical monolignol p-hydroxyphenyl unit and guaiacyl unit, occur in sporopollenin of vascular but not nonvascular plants. Evolutionary analyses showed that the metabolic scaffold for phenolic precursors evolved before the integration of phenolics into sporopollenin in vascular plants. The conserved multicopper oxidase SCULP1, which incorporates p-CA into sporopollenin, co-occurred with p-coumaroylation of sporopollenin in vascular plants, likely contributing to the prevalence of p-coumaroylated sporopollenin. These findings provide an evolutionary framework for understanding genetic associations with sporopollenin chemical diversification and plant adaptation.