Analysis and manipulation of specialized metabolism in solanaceae

Abstract

Plants produce a wide range of specialized metabolites. These compounds act in a myriad of ways producing attractive colors and aromas for pollinators and seed dispersal, flavor volatiles, and defensive compounds. Steroidal glycoalkaloids (SGAs) are biosynthetically produced from a cholesterol backbone. Most plants produce phytosterols, including β-sitosterol, stigmasterol, and campesterol, from a single sterol pathway, while Solanum has a branch off of the main sterol biosynthetic pathway that generates cholesterol and SGAs. Squalene is a terpene precursor of all phytosterols. The cholesterol branch shares several enzymes while several key steps are performed by enzymes that are duplicated from genes in the main branch. Cholesterol is modified, nitrogen is incorporated, and the resulting SGAs are glycosylated. Jasmonic acid responsive ethylene response factor 4 (JRE4) is a major regulator of the cholesterol branch and SGA biosynthesis. SGAs can impart bitter flavors and also provide defense against insect herbivory and fungal infection. The duplicated genes are DWARF5 (in general phytosterol biosynthesis) and 7DHCR (in the cholesterogenesis pathway). Arabidopsis dwf5 mutants have a short stature and smaller dark green leaves due to deficiencies in brassinosteroid production. CS72 carries a single nucleotide polymorphism in DWF5, dwf5-4. 7DHCR was cloned from MTX-851 and transformed into a dwf5-4 line. Relative sterol content was measured using UHPLC-HRAMS, and a Zorbax SB-CN column with an APCI source. Squalene content increased in the dwf5-4 line compared to the 7DHCR complementation line, while β-sitosterol and campesterol content decreased. These results suggest 7DHCR has a broader substrate specificity than previously understood. Several species in the genus Jaltomata, a close relative of tomato, produce a red nectar. Jaltomata bohsiana produces a clear nectar and lacks a protein found in red-nectared species. To better understand this trait, an Agrobacterium-mediated transformation protocol was developed. J. bohsiana seeds were germinated in sterile Magenta boxes to produce explants from the cotyledons and hypocotyl. After 48 hours, these were co-cultured with AGL1 carrying a binary vector based on pDIRECT21_C. TAIL-PCR showed the insert was present with plant DNA from most closely matches data from fully sequenced Solanum relatives as one would expect given the lack of available Jaltomata bohsiana genomic sequence information.