Arogenate dehydratases (ADTs) are enzymes found within the aromatic amino acid pathway. They are responsible for catalyzing the final step in phenylalanine (Phe) biosynthesis in vascular plants. While being essential for protein production in all living systems, Phe additionally is the starting precursor to a multitude of secondary metabolites produced in the phenylpropanoid pathway. Our group discovered that by knocking out ADT isoenzymes in Arabidopsis thaliana , measurable reductions in lignin levels can be achieved in stem tissue. This finding provides the opportunity to study potential mechanisms related to lignin biosynthesis and could have implications in bioengineering applications where alterations in lignin level might be desired.

Any alteration to a gene family, as important as that of the ADTs, imparts plant-wide biomolecular changes and because of this, it is not only important to know that lignin is reduced but that optimal plant function is maintained or to understand how it has been changed in order to mediate any undesirable effects. Here we utilized a multitude of analytical platforms and data analysis techniques on both ADT knock-outs (KOs) and over-expression (OE) lines. By using both KO and OE lines we could provide validation to our findings, as KO and OE mutants of the same enzyme/s typically show converse biomolecular abundance changes. As a systems level understanding was desired, we utilized a multi-omics strategy (metabolomics, transcriptomics and proteomics).

Identified metabolites showed which metabolite and metabolite classes were most affected. Major KEGG defined pathway changes were identified at the transcript and protein enzyme family level. Integration of all omics data revealed which enzymatic reactions were most correlated to observed metabolite abundance changes. Network and clustering algorithms identified patterns of molecular change between metabolites, transcripts and proteins and these patterns were further correlated to reveal possible post-transcriptional regulatory processes involved in lignin biosynthesis.

Taken altogether, these data informed us of how ADT alterations affect the entire biomolecular system of Arabidopsis and also revealed targets for future studies aimed at elucidating further how lignin biosynthesis is regulated at the post-transcriptional and translational levels.