Ravi, B. G. | bioRxiv Science

bioRxiv2021

Cardenolide Increase in Foxglove During Stresses Corresponds to Cholesterol and Phytosterol Biosynthesis

Cardenolides are a group of steroidal metabolites found in Digitalis lanata with potent cardioactive effects on animals. In plants, cardenolides are involved in various stress responses. However, the molecular mechanism of cardenolide increase during stresses is mostly unknown. Here we show that cardenolides were increased upon methyl jasmonate (MJ), sorbitol, potassium (KCl) chloride, and salicylic acid analog (BTH: 2,1,3-benzothiadiazole) treatments. However, the expression of three known genes for cardenolide biosynthesis did not correlate well with these increases. Specifically, the expression of progesterone-5{beta}-reductases (P5{beta}R and P5{beta}R2) did not correlate with cardenolide increase. The expression of 3{beta}-hydroxysteroid dehydrogenase (3{beta}HSD) correlated with cardenolide levels only during the BTH treatment. Mining the D. lanata transcriptome identified genes involved in cholesterol and phytosterol biosynthesis: SSR1 (sterol sidechain reductase), SMO1, and SMO3 (sterol methyl oxidase). Surprisingly, the expression of all three genes correlated well with the cardenolide increase after BTH treatment. Phylogenetic analysis showed that SSR1 is likely involved in both cholesterol and phytosterol biosynthesis. In addition, SMO1 is likely specific to phytosterol biosynthesis, and SMO3 is specific to cholesterol biosynthesis. These results suggest that both cholesterol and phytosterol are involved in cardenolide biosynthesis. In summary, this work shows that cardenolides are important for stress responses in D. lanata and revealed a novel relationship between phytosterol and cardenolide biosynthesis.

plant biology↗

bioRxiv2019

Profiling and structural analysis of cardenolides in two species of Digitalis using liquid chromatography coupled with high-resolution mass spectrometry

Plants of the Digitalis genus contain a cocktail of cardenolides commonly prescribed to treat heart failure. Cardenolides in Digitalis extracts have been conventionally quantified by high-performance liquid chromatography yet the lack of structural information compounded with possible co-eluents renders this method insufficient for analyzing cardenolides in plants. The goal of this work is to structurally characterize cardiac glycosides in fresh-leaf extracts using liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) that provides exact masses. Fragmentation of cardenolides is featured by sequential loss of sugar units while the steroid aglycon moieties undergo stepwise elimination of hydroxyl groups, which distinguishes different aglycones. The sequence of elution follows diginatigenin[->]digoxigenin[->]gitoxigenin[->]gitaloxigenin[->]digitoxigenin for cardenolides with the same sugar units but different aglycones using a reverse-phase column. A linear range of 0.8-500 ng g-1 has been achieved for digoxigenin, {beta}-acetyldigoxin, and digitoxigenin with limits of detection ranging from 0.09 to 0.45 ng g-1. A total of 17 cardenolides have been detected with lanatoside A, C, and E as major cardenolides in Digitalis lanata while 7 have been found in Digitalis purpurea including purpurea glycoside A, B, and E. Surprisingly, glucodigifucoside in D. lanata and verodoxin and digitoxigenin fucoside in D. purpurea have also been found as major cardenolides. As the first MS/MS-based method developed for analyzing cardenolides in plant extracts, this method serves as a foundation for complete identification and accurate quantification of cardiac glycosides, a necessary step towards understanding the biosynthesis of cardenolide in plants.

biochemistry↗