Reduction of Pyrrolizidine Alkaloid Levels in Comfrey (Symphytum officinale) Hairy Roots by RNAi Silencing of Homospermidine Synthase.

Comfrey is a medicinal plant, extracts of which are traditionally used for the treatment of painful inflammatory muscle and joint problems, because the plant contains allantoin and rosmarinic acid. However, its medicinal use is limited because of its toxic pyrrolizidine alkaloid (PA) content. PAs encompass more than 400 different compounds that have been identified from various plant lineages. To date, only the first pathway-specific enzyme, homospermidine synthase (HSS), has been characterized. HSS catalyzes the formation of homospermidine, which is exclusively incorporated into PAs. HSS has been recruited several times independently in various plant lineages during evolution by duplication of the gene encoding deoxyhypusine synthase (DHS), an enzyme of primary metabolism. Here, we describe the establishment of RNAi knockdown hairy root mutants of HSS in Symphytum officinale. A knockdown of HSS by 60 - 80% resulted in a significant reduction of homospermidine by ~ 86% and of the major PA components 7-acetylintermedine N-oxide and 3-acetylmyoscorpine N-oxide by approximately 60%. The correlation of reduced transcript levels of HSS with reduced levels of homospermidine and PAs provides in planta support for HSS being the central enzyme in PA biosynthesis. Furthermore, the generation of PA-depleted hairy roots might be a cost-efficient way for reducing toxic by-products that limit the medicinal applicability of S. officinale extracts.

Variability of Pyrrolizidine Alkaloid Occurrence in Species of the Grass Subfamily Pooideae (Poaceae).

Pyrrolizidine alkaloids (PAs) are a class of secondary metabolites found in various unrelated angiosperm lineages including cool-season grasses (Poaceae, subfamily Pooideae). Thesinine conjugates, saturated forms of PA that are regarded as non-toxic, have been described to occur in the two grass species Lolium perenne and Festuca arundinacea (Poaceae, subfamily Pooideae). In a wider screen, we tested various species of the Pooideae lineage, grown under controlled conditions, for their ability to produce thesinine conjugates or related structures. Using an LC-MS based targeted metabolomics approach we were able to show that PA biosynthesis in grasses is limited to a group of very closely related Pooideae species that produce a limited diversity of PA structures. High variability in PA levels was observed even between individuals of the same species. These individual accumulation patterns are discussed with respect to a possible function and evolution of this type of alkaloid.

ATPase-dependent role of the atypical kinase Rio2 on the evolving pre-40S ribosomal subunit.

Ribosome synthesis involves dynamic association of ribosome-biogenesis factors with evolving preribosomal particles. Rio2 is an atypical protein kinase required for pre-40S subunit maturation. We report the crystal structure of eukaryotic Rio2-ATP-Mg(2+) complex. The active site contains ADP-Mg(2+) and a phosphoaspartate intermediate typically found in Na(+), K(+) and Ca(2+) ATPases but not protein kinases. Consistent with this finding, ctRio2 exhibits a robust ATPase activity in vitro. In vivo, Rio2 docks on the ribosome, with its active site occluded and its flexible loop positioned to interact with the pre-40S subunit. Moreover, Rio2 catalytic activity is required for its dissociation from the ribosome, a necessary step in pre-40S maturation. We propose that phosphoryl transfer from ATP to Asp257 in Rio2's active site and subsequent hydrolysis of the aspartylphosphate could be a trigger to power late cytoplasmic 40S subunit biogenesis.