Submitted to: Plant Journal
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/6/2004
Publication Date: 11/10/2004
Citation: Basset, G., Ravanel, S., Quinlivan, E., White, R.A., Giovannoni, J.J., Rebeille, F., Nichols, B., Shinozaki, K., Gregory, J., Hanson, A. 2004. Folate synthesis in plants: the last step of the p-aminobenzoate branch is catalyzed by a plastidial aminodeoxychorismate lyase. Plant Journal. 40:453-461. Interpretive Summary: Tetrahydrofolate and its derivatives, commonly grouped under the generic name of folates, belong to the B vitamin group and are vital cofactors for enzymes that mediate one-carbon transfer reactions. As major carriers and donors of one-carbon units, folates are involved in a wide range of key metabolic functions including the biosynthesis of methionine, purines, thymidylate and, specifically in plants, in photorespiration. While bacteria, fungi and plants synthesize folates de novo, vertebrates do not, and consequently are totally dependent on a dietary supply. As plant foods are major sources of folates for humans and folate deficiency is a worldwide health problem, engineering plants with enhanced folate content is an appealing way to address folate malnutrition. The undertaking of this engineering requires that plant folate synthesis first be understood. In this study, we identified Arabidopsis and tomato cDNAs encoding functional ADC lyases, and showed that the plant ADC lyase is targeted to plastids where it is involved in folate synthesis. Because tomato fruit is our model for folate engineering and evidence suggests that the expression of other folate biosynthesis genes declines steeply in ripening fruits, we also analyzed the expression of ADC lyase during tomato fruit development. We have outlined a strategy for folate engineering in maturing fruit tissues based on our results.
Technical Abstract: In plants, the last step in the synthesis of the p-aminobenzoate (PABA) moiety of folate remains to be elucidated. In Escherichia coli, this step is catalyzed by the PabC protein, a b-lyase that converts 4-amino-4-deoxychorismate (ADC) - the reaction product of the PabA and PabB enzymes - to PABA and pyruvate. So far, the only known plant enzyme involved in PABA synthesis is ADC synthase, which has fused domains homologous to E. coli PabA and PabB and is located in plastids. ADC synthase has no lyase activity, implying that plants have a separate ADC lyase. No such lyase is known in any eukaryote. Genomic and phylogenetic approaches identified Arabidopsis and tomato cDNAs encoding PabC homologs with putative chloroplast targeting peptides. These cDNAs were shown to encode functional enzymes by complementation of an E. coli pabC mutant, and by demonstrating that the partially purified recombinant proteins convert ADC to PABA. Plant ADC lyase is active as dimer and is not feedback inhibited by physiological concentrations of PABA, its glucose ester, or folates. The full-length Arabidopsis ADC lyase polypeptide was translocated into isolated pea chloroplasts and, when fused to green fluorescent protein, directed the passenger protein to Arabidopsis chloroplasts in transient expression experiments. These data indicate that ADC lyase, like ADC synthase, is present in plastids. As shown previously for the ADC synthase transcript, the level of ADC lyase mRNA in the pericarp of tomato fruit falls sharply as ripening advances, suggesting that the expression of these two enzymes is coregulated.