Four enzymes control natural variation in the steroid core of Erysimum cardenolides.

Plants commonly produce families of structurally related metabolites with similar defensive functions. This apparent redundancy raises the question of underlying molecular mechanisms and adaptive benefits of such chemical variation. Cardenolides, a class defensive compounds found in the wallflower genus Erysimum (L., Brassicaceae) and scattered across other plant families, show substantial structural variation, with glycosylation and hydroxylation being common modifications of a steroid core, which itself may vary in terms of stereochemistry and saturation. Through a combination of chemical mutagenesis and analysis of gene coexpression networks, we identified four enzymes involved in cardenolide biosynthesis in Erysimum that work together to determine stereochemistry at carbon 5 of the steroid core: Ec3ßHSD, a 3ß-hydroxysteroid dehydrogenase, Ec3KSI, a ketosteroid isomerase, EcP5ßR2, a progesterone 5ß-reductase, and EcDET2, a steroid 5α-reductase. We biochemically characterized the activity of these enzymes in vitro and generated CRISPR/Cas9 knockout lines to confirm activity in vivo. Cardenolide biosynthesis was not eliminated in any of the knockouts. Instead, mutant plants accumulated cardenolides with altered saturation and stereochemistry of the steroid core. Furthermore, we found variation in carbon 5 configuration among the cardenolides of 44 species of Erysimum, where the occurrence of some 5ß-cardenolides is associated with the expression and sequence of P5ßR2. This may have allowed Erysimum species to fine-tune their defensive profiles to target specific herbivore populations over the course of evolution. SIGNIFICANCE STATEMENT: Plants use an array of toxic compounds to defend themselves from attack against insects and other herbivores. One mechanism through which plants may evolve more toxic compounds is through modifications to the structure of compounds they already produce. In this study, we show how plants in the wallflower genus Erysimum use four enzymes to fine-tune the structure of toxic metabolites called cardenolides. Natural variation in the sequence and expression of a single enzyme called progesterone 5ß-reductase 2 partly explains the variation in cardenolides observed across the Erysimum genus. These alterations to cardenolide structure over the course of evolution suggests that there may be context-dependent benefits to Erysimum to invest in one cardenolide variant over another.

Aphid Resistance Segregates Independently of Cardenolide and Glucosinolate Content in an Erysimum cheiranthoides (Wormseed Wallflower) F2 Population.

Plants in the genus Erysimum produce both glucosinolates and cardenolides as a defense mechanism against herbivory. Two natural isolates of Erysimum cheiranthoides (wormseed wallflower) differed in their glucosinolate content, cardenolide content, and their resistance to Myzus persicae (green peach aphid), a broad generalist herbivore. Both classes of defensive metabolites were produced constitutively and were not further induced by aphid feeding. To investigate the relative importance of glucosinolates and cardenolides in E. cheiranthoides defense, we generated an improved genome assembly, genetic map, and segregating F2 population. The genotypic and phenotypic analysis of the F2 plants identified quantitative trait loci, which affected glucosinolates and cardenolides, but not the aphid resistance. The abundance of most glucosinolates and cardenolides was positively correlated in the F2 population, indicating that similar processes regulate their biosynthesis and accumulation. Aphid reproduction was positively correlated with glucosinolate content. Although the overall cardenolide content had little effect on aphid growth and survival, there was a negative correlation between aphid reproduction and helveticoside abundance. However, this variation in defensive metabolites could not explain the differences in aphid growth on the two parental lines, suggesting that processes other than the abundance of glucosinolates and cardenolides have a predominant effect on aphid resistance in E. cheiranthoides.

Cardiac glycosides protect wormseed wallflower (Erysimum cheiranthoides) against some, but not all, glucosinolate-adapted herbivores.

The chemical arms race between plants and insects is foundational to the generation and maintenance of biological diversity. We asked how the evolution of a novel defensive compound in an already well-defended plant lineage impacts interactions with diverse herbivores. Erysimum cheiranthoides (Brassicaceae), which produces both ancestral glucosinolates and novel cardiac glycosides, served as a model. We analyzed gene expression to identify cardiac glycoside biosynthetic enzymes in E. cheiranthoides and characterized these enzymes via heterologous expression and CRISPR/Cas9 knockout. Using E. cheiranthoides cardiac glycoside-deficient lines, we conducted insect experiments in both the laboratory and field. EcCYP87A126 initiates cardiac glycoside biosynthesis via sterol side-chain cleavage, and EcCYP716A418 has a role in cardiac glycoside hydroxylation. In EcCYP87A126 knockout lines, cardiac glycoside production was eliminated. Laboratory experiments with these lines revealed that cardiac glycosides were highly effective defenses against two species of glucosinolate-tolerant specialist herbivores, but did not protect against all crucifer-feeding specialist herbivores in the field. Cardiac glycosides had lesser to no effect on two broad generalist herbivores. These results begin elucidation of the E. cheiranthoides cardiac glycoside biosynthetic pathway and demonstrate in vivo that cardiac glycoside production allows Erysimum to escape from some, but not all, specialist herbivores.

Aphid resistance segregates independently of cardiac glycoside and glucosinolate content in an Erysimum cheiranthoides (wormseed wallflower) F2 population.

Plants in the genus Erysimum produce both glucosinolates and cardiac glycosides as defense against herbivory. Two natural isolates of Erysimum cheiranthoides (wormseed wallflower) differed in their glucosinolate content, cardiac glycoside content, and resistance to Myzus persicae (green peach aphid), a broad generalist herbivore. Both classes of defensive metabolites were produced constitutively and were not induced further by aphid feeding. To investigate the relative importance of glucosinolates and cardiac glycosides in E. cheiranthoides defense, we generated an improved genome assembly, genetic map, and segregating F2 population. Genotypic and phenotypic analysis of the F2 plants identified quantitative trait loci affecting glucosinolates and cardiac glycosides, but not aphid resistance. The abundance of most glucosinolates and cardiac glycosides was positively correlated in the F2 population, indicating that similar processes regulate their biosynthesis and accumulation. Aphid reproduction was positively correlated with glucosinolate content. Although overall cardiac glycoside content had little effect on aphid growth and survival, there was a negative correlation between aphid reproduction and helveticoside abundance. However, this variation in defensive metabolites could not explain the differences in aphid growth on the two parental lines, suggesting that processes other than the abundance of glucosinolates and cardiac glycosides have a predominant effect on aphid resistance in E. cheiranthoides.

Cardiac glycosides protect wormseed wallflower (Erysimum cheiranthoides) against some, but not all, glucosinolate-adapted herbivores.

The chemical arms race between plants and insects is foundational to the generation and maintenance of biological diversity. We asked how the evolution of a novel defensive compound in an already well-defended plant lineage impacts interactions with diverse herbivores. Erysimum cheiranthoides (Brassicaceae), which produces both ancestral glucosinolates and novel cardiac glycosides, served as a model.We analyzed gene expression to identify cardiac glycoside biosynthetic enzymes in E. cheiranthoides and characterized these enzymes via heterologous expression and CRISPR/Cas9 knockout. Using E. cheiranthoides cardiac glycoside-deficient lines, we conducted insect experiments in both the laboratory and field.EcCYP87A126 initiates cardiac glycoside biosynthesis via sterol side chain cleavage, and EcCYP716A418 has a role in cardiac glycoside hydroxylation. In EcCYP87A126 knockout lines, cardiac glycoside production was eliminated. Laboratory experiments with these lines revealed that cardiac glycosides were highly effective defenses against two species of glucosinolate-tolerant specialist herbivores but did not protect against all crucifer-feeding specialist herbivores in the field. Cardiac glycosides had lesser to no effect on two broad generalist herbivores.These results begin elucidation of the E. cheiranthoides cardiac glycoside biosynthetic pathway and demonstrate in vivo that cardiac glycoside production allows Erysimum to escape from some, but not all, specialist herbivores.

Acropetal and basipetal cardenolide transport in Erysimum cheiranthoides (wormseed wallflower).

Plant specialized metabolites are often subject to within-plant transport and have tissue-specific distribution patterns. Among plants in the Brassicaceae, the genus Erysimum is unique in producing not only glucosinolates but also cardenolides. Ten cardenolides were detected with varying abundance in different tissues of Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower). As is predicted by the optimal defense theory, cardenolides were most abundant in young leaves and reproductive tissues. The lowest concentrations were observed in senescing leaves and roots. Crosses between wildtype E. cheiranthoides and a mutant line with an altered cardenolide profile showed that the seed cardenolide phenotype is determined entirely by the maternal genotype. Prior to the development of the first true leaves, seedling cotyledons also had the maternal cardenolide profile. Hypocotyl grafting experiments showed that the root cardenolide profile is determined entirely by the aboveground plant genotype. In further grafting experiments, there was no evidence of cardenolide transport into the leaves, but a mixed cardenolide profile was observed in the stems and inflorescences of plants that had been grafted at vegetative and flowering growth stages, respectively. Together, these results indicate that E. cheiranthoides leaves are a site of cardenolide biosynthesis.

Less Is More: a Mutation in the Chemical Defense Pathway of Erysimum cheiranthoides (Brassicaceae) Reduces Total Cardenolide Abundance but Increases Resistance to Insect Herbivores.

Erysimum cheiranthoides L (Brassicaceae; wormseed wallflower) accumulates not only glucosinolates, which are characteristic of the Brassicaceae, but also abundant and diverse cardenolides. These steroid toxins, primarily glycosylated forms of digitoxigenin, cannogenol, and strophanthidin, inhibit the function of essential Na+/K+-ATPases in animal cells. We screened a population of 659 ethylmethanesulfonate-mutagenized E. cheiranthoides plants to identify isolates with altered cardenolide profiles. One mutant line exhibited 66% lower cardenolide content, resulting from greatly decreased cannogenol and strophanthidin glycosides, partially compensated for by increases in digitoxigenin glycosides. This phenotype was likely caused by a single-locus recessive mutation, as evidenced by a wildtype phenotype of F1 plants from a backcross, a 3:1 wildtype:mutant segregation in the F2 generation, and genetic mapping of the altered cardenolide phenotype to one position in the genome. The mutation created a more even cardenolide distribution, decreased the average cardenolide polarity, but did not impact most glucosinolates. Growth of generalist herbivores from two feeding guilds, Myzus persicae Sulzer (Hemiptera: Aphididae; green peach aphid) and Trichoplusia ni Hübner (Lepidoptera: Noctuidae; cabbage looper), was decreased on the mutant line compared to wildtype. Both herbivores accumulated cardenolides in proportion to the plant content, with T. ni accumulating higher total concentrations than M. persicae. Helveticoside, a relatively abundant cardenolide in E. cheiranthoides, was not detected in M. persicae feeding on these plants. Our results support the hypothesis that increased digitoxigenin glycosides provide improved protection against M. persicae and T. ni, despite an overall decrease in cardenolide content of the mutant line.