Molecular Evolution of Pyrrolizidine Alkaloid Biosynthesis in the Tribe Crotalarieae (Fabaceae)
Final Report Abstract
We report about a project that was embedded in our research on the evolution of pathways in plant secondary metabolism. Pyrrolizidine alkaloids (PAs), our model system, occur in the tribe Crotalarieae within the Fabaceae only in the genera Crotalaria and Lotononis, while most other genera of this clade are characterized by quinolizidine alkaloids (QAs). In Crotalaria, PA biosynthesis is linked to nodulation. It was shown that homospermidine synthase (HSS), the first enzyme of PA biosynthesis, is expressed exclusively in root nodules. Therefore, we tested the hypothesis that also QA biosynthesis might be regulated by nodulation. We used Lupinus arboreus as model plant that was cultivated and infected with a species-specific rhizobial strain under controlled conditions in vitro. Data on QA content and of transcripts encoding ornithine decarboxylase (ODC) and its paralog lysine decarboxylase (LDC), the first specific enzyme of QA biosynthesis, do not support any link between QA biosynthesis and nodulation. However, biochemical characterization of heterologously expressed ODC and LDC in E. coli showed that ODC has a clear preference for the decarboxylation of ornithine to putrescine, while LDC strongly prefers lysine as substrate for the decarboxylation to cadaverine. This functional divergence of the gene duplicates is more pronounced that described previously for the enzyme pair of Lupinus angustifolius. Using polyclonal antibodies, we succeeded to label HSS in the root nodules of Crotalaria spectabilis. The enzyme is localized in the parenchyma of the nodules underneath the cortex. No label is detected in the inner part of the nodules with the symbionts. First analyses using MS imaging co-localized monocrotaline N-oxide, the only PA produced by C. spectabilis in significant amounts, with HSS. Efforts to establish a hairy root system as an easy-to-use system to generate transgenic plant lines to study putative promotor regions of the HSS-encoding gene suffered from bad reproducibility. Instead, we developed a protocol for stable transformation of C. spectabilis plants with Agrobacterium tumefaciens that results in transgenic plants that nodulate after infection with a Bradyrhizobium strain. This is a valuable tool for further research on the regulation and evolution of specific genes of PA biosynthesis. Identification of sequences encoding HSS and its paralog deoxyhypusine synthase of various species of the genistoid clade allowed the identification of two independent duplications of the DHS-encoding gene. This first duplication resulted in a paralog that was lost in some lineages, but that was recruited as HSS in the genus Crotalaria. The consequences of the second duplication event are still under study.
