David Kaufholdt

This review article deals with the pathways of cellular and global molybdate distribution in plants, especially with a full overview for the model plant Arabidopsis thaliana. In its oxidized state as bioavailable molybdate, molybdenum can be absorbed from the environment. Especially in higher plants, molybdenum is indispensable as part of the molybdenum cofactor (Moco), which is responsible for functionality as a prosthetic group in a variety of essential enzymes like nitrate reductase and sulfite oxidase. Therefore, plants need mechanisms for molybdate import and transport within the organism, which are accomplished via high-affinity molybdate transporter (MOT) localized in different cells and membranes. Two different MOT families were identified. Legumes like Glycine max or Medicago truncatula have an especially increased number of MOT1 family members for supplying their symbionts with molybdate for nitrogenase activity. In Arabidopsis thaliana especially, the complete pathway followed by molybdate through the plant is traceable. Not only the uptake from soil by MOT1.1 and its distribution to leaves, flowers, and seeds by MOT2-family members was identified, but also that inside the cell. the transport trough the cytoplasm and the vacuolar storage mechanisms depending on glutathione were described. Finally, supplying the Moco biosynthesis complex by MOT1.2 and MOT2.1 was demonstrated.

Polyprenylated xanthones are natural products with a multitude of biological and pharmacological activities. However, their biosynthetic pathway is not completely understood. In this study, metabolic profiling revealed the presence of 4-prenylated 1,3,5,6-tetrahydroxyxanthone derivatives in St. John's wort (Hypericum perforatum) root extracts. Transcriptomic data mining led to the detection of 5 variants of xanthone 4-prenyltransferase (HpPT4px) comprising 4 long variants (HpPT4px-v1 to HpPT4px-v4) and 1 short variant (HpPT4px-sh). The full-length sequences of all 5 variants were cloned and heterologously expressed in yeast (Saccharomyces cerevisiae). Microsomes containing HpPT4px-v2, HpPT4px-v4, and HpPT4px-sh catalyzed the addition of a prenyl group at the C-4 position of 1,3,5,6-tetrahydroxyxanthone; 1,3,5-trihydroxyxanthone; and 1,3,7-trihydroxyxanthone, whereas microsomes harboring HpPT4px-v1 and HpPT4px-v3 additionally accepted 1,3,6,7-tetrahydroxyxanthone. HpPT4px-v1 produced in Nicotiana benthamiana displayed the same activity as in yeast, while HpPT4px-sh was inactive. The kinetic parameters of HpPT4px-v1 and HpPT4px-sh chosen as representative variants indicated 1,3,5,6-tetrahydroxyxanthone as the preferred acceptor substrate, rationalizing that HpPT4px catalyzes the first prenylation step in the biosynthesis of polyprenylated xanthones in H. perforatum. Dimethylallyl pyrophosphate was the exclusive prenyl donor. Expression of the HpPT4px transcripts was highest in roots and leaves, raising the question of product translocation. C-terminal yellow fluorescent protein fusion of HpPT4px-v1 localized to the envelope of chloroplasts in N. benthamiana leaves, whereas short, truncated, and masked signal peptides led to the disruption of plastidial localization. These findings pave the way for a better understanding of the prenylation of xanthones in plants and the identification of additional xanthone-specific prenyltransferases.