Metabolic Characterization of Arabidopsis thaliana Sirtuins and Lysine Acylation

Sirtuins from the Sir2 (silent information regulator 2) gene family are highly conserved, from prokaryotes to eukaryotes. Sirtuins are NAD⁺–dependent deacetylase enzymes that have recently been described as general deacylases. Sirtuins target diverse protein substrates that regulate a broad range of cellular metabolism especially central carbon metabolism. Lysine acylations are readily reversible post-translational modifications (PTMs) that can alter enzyme activity and protein function. Lysine acylation as a regulator of metabolism is a fairly new discovery and very little is known about sirtuins and lysine acylation in plants beyond histone acetylation. There are two Sir2 family genes in Arabidopsis thaliana, SIRT1 and SIRT2. Here we explore the role sirtuins have on the regulation of acylation in Arabidopsis wildtype, sirtuin single and double knockout mutants. Western blot analyses using an anti-acetyl-lysine antibody to investigate global differences in lysine acetylation in the SIRT mutants compared to wildtype indicate that a large number of proteins are acetylated in aerial and root tissue. Currently, we are also investigating other acylation modifications via Western blot analyses. We have identified lysine propionylation, crontonylation, and succinylation as prevalent PTMs in plant proteins. Lysine acetylation is the only lysine-acyl PTM that has previously been identified in plants. To increase our understanding of the regulation of metabolism by sirtuins a metabolomics comparison of the ecotypes was also conducted using liquid chromatography mass spectrometry. Principal component analysis illustrates a separation of wildtype and the sirtuin mutant metabolic profiles, suggesting sirtuins have a role in regulating plant metabolism.