Arabidopsis Arogenate Dehydratases: Influence on Aromatic Amino Acid and Downstream Phenylpropanoid Biosynthesis
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Arogenate dehydratases (ADTs) perform the final step of phenylalanine (Phe) biosynthesis in plants. As one of twenty protein-coding amino acids, Phe is essential in all living organisms, and in plants it is also a precursor to thousands of different phenylpropanoid that have wide-ranging functions in planta. Undoubtedly, the most important of these (in terms of vascular plant evolution and survival), is the polymer lignin, which provides additional strength to cell walls, enabling plants to grow upright, and retain water in a desiccating environment. The goal of this study was to understand the role of ADTs in the chloroplast-localized pathway upstream of Phe, particularly regarding possible influences on downstream cytosol/cell wall localized phenylpropanoid biosynthesis. All six ADT isoenzymes from Arabidopsis thaliana were first individually biochemically characterized; this confirmed Arabidopsis ADTs preferentially utilized arogenate as a substrate. Subsequent studies focused on characterization of ADTs in planta. Accordingly, Arabidopsis knockout (KO) lines were obtained for five/six ADTs and were crossed to obtain double, triple and quadruple KO lines. These KOs were assessed for potential reductions in free Phe levels and lignin contents over the course of growth and development. Results of these analyses identified vast reductions in lignin levels in certain ADT KO lines (up to 70% in a quadruple KO compared to WT). Amino acid analyses identified diminished Phe levels in the same lines with reduced lignin contents, but apparently only in lignified tissue during times when lignin biosynthesis and deposition are most active. Together, these analyses revealed that the isoenzyme ADT5 is prominently involved in the synthesis of Phe as a precursor for lignin. Finally, an analysis of ADT expression/localization using the â-glucuronidase (GUS) reporter gene, found that ADT5 is mainly expressed in the lignifying regions of the stem, further supporting the above findings. Together, these studies showed that individual ADTs in Arabidopsis have distinct roles to accommodate the many different requirements for Phe in different cell types and tissues.