Biosynthesis of cuticular alkylresorcinols in selected grass species Brachypodium distachyon and Secale cereale

Alkylresorcinols are phenolic lipids which occur in diverse plant species as well as microorganisms. In plants, alkylresorcinols are usually deposited at or near the surfaces where they are thought to serve as a first line of defense. Earlier work in our lab had shown the surface accumulation of alkylresorcinols in Secale cereale leaves was mainly restricted to the cuticle. However, direct evidence showing the protective role of these bioactive compounds at the surface is still insufficient. The current work was to investigate the biosynthesis of cuticular alkylresorcinols in order to get a better understanding of their biological function. This research focused on S. cereale, since it had previously been shown to contain relatively large amounts of alkylresorcinols, and on Brachypodium distachyon, a closely related genetic model system with completely sequenced genome. First, chemical analyses revealed that the cuticular wax covering leaves of B. distachyon included 5% of alkylresorcinols with alkyl chains varying from C₁₇ to C₂₅. Therefore, it was hypothesized that both species have genes encoding alkylresorcinol synthases (ARSs). A central goal of this work was to clone and characterize potential ARSs. One ARS (BdARS) was cloned from B. distachyon by mining the Brachypodium expressed sequence tag libraries and one ARS (ScARS) was cloned from S. cereale using a homology-based cloning strategy. In vivo biochemical characterization in yeast Saccharomyces cerevisiae demonstrated that both enzymes were capable of using C₁₀ to C₂₂ fatty acyl-CoAs with malonyl-CoA to generate a broad range of alkylresorcinols. Organ-specific expression in leaves but not in roots was observed for both BdARS and ScARS. Additionally, the expression pattern of ScARS matched the time-course of cuticular alkylresorcinol accumulation along the leaf of S. cereale. An investigation into their subcellular localization revealed that both ARSs were likely localized to the endoplasmic reticulum membrane. All these results taken together support the idea that BdARS and ScARS are the enzymes responsible for the biosynthesis of cuticular alkylresorcinols, and that the cuticular alkylresorcinols are indeed biosynthesized for a protective function associated with the wax lining the surface of grass leaves.Science, Faculty ofBotany, Department ofGraduat