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.

Exogenous supplementation of Sulfur (S) and Reduced Glutathione (GSH) Alleviates Arsenic Toxicity in Shoots of Isatis cappadocica Desv and Erysimum allionii L.

The current study was conducted to investigate the role of sulfur (S) and reduced glutathione (GSH) in mitigating arsenic (As) toxicity in Isatis cappadocica and Erysimum allionii. These plants were exposed for 3 weeks to different concentrations (0, 400 and 800 µM) of As to measure fresh weight, total chlorophyll, proline and hydrogen peroxide (H2O2) content, As and S accumulation, and guaiacol peroxidase (POD) and glutathione S-transferase (GST) along with the supplementation of 20 mg L-1 of S and 500 µM of GSH. Results revealed the significant reduction of fresh weight (especially in E. allionii), activities of POD and GST enzymes and proline content as compare to control. However, the application of S and GSH enhanced the fresh weight. Inhibition in H2O2 accumulation and improvement in antioxidant responses were measured with the application of S and GSH. Hence, the supplementation of S and GSH enhanced fresh weight and total chlorophyll in both I. cappadocica and E. allionii by alleviating the adverse effects of As stress via decreased H2O2 content and restricted As uptake.

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.

Interaction of plant growth regulators and reactive oxygen species to regulate petal senescence in wallflowers (Erysimum linifolium).

BACKGROUND: In many species floral senescence is coordinated by ethylene. Endogenous levels rise, and exogenous application accelerates senescence. Furthermore, floral senescence is often associated with increased reactive oxygen species, and is delayed by exogenously applied cytokinin. However, how these processes are linked remains largely unresolved. Erysimum linifolium (wallflower) provides an excellent model for understanding these interactions due to its easily staged flowers and close taxonomic relationship to Arabidopsis. This has facilitated microarray analysis of gene expression during petal senescence and provided gene markers for following the effects of treatments on different regulatory pathways. RESULTS: In detached Erysimum linifolium (wallflower) flowers ethylene production peaks in open flowers. Furthermore senescence is delayed by treatments with the ethylene signalling inhibitor silver thiosulphate, and accelerated with ethylene released by 2-chloroethylphosphonic acid. Both treatments with exogenous cytokinin, or 6-methyl purine (which is an inhibitor of cytokinin oxidase), delay petal senescence. However, treatment with cytokinin also increases ethylene biosynthesis. Despite the similar effects on senescence, transcript abundance of gene markers is affected differentially by the treatments. A significant rise in transcript abundance of WLS73 (a putative aminocyclopropanecarboxylate oxidase) was abolished by cytokinin or 6-methyl purine treatments. In contrast, WFSAG12 transcript (a senescence marker) continued to accumulate significantly, albeit at a reduced rate. Silver thiosulphate suppressed the increase in transcript abundance both of WFSAG12 and WLS73. Activity of reactive oxygen species scavenging enzymes changed during senescence. Treatments that increased cytokinin levels, or inhibited ethylene action, reduced accumulation of hydrogen peroxide. Furthermore, although auxin levels rose with senescence, treatments that delayed early senescence did not affect transcript abundance of WPS46, an auxin-induced gene. CONCLUSIONS: A model for the interaction between cytokinins, ethylene, reactive oxygen species and auxin in the regulation of floral senescence in wallflowers is proposed. The combined increase in ethylene and reduction in cytokinin triggers the initiation of senescence and these two plant growth regulators directly or indirectly result in increased reactive oxygen species levels. A fall in conjugated auxin and/or the total auxin pool eventually triggers abscission.

Identification and stress-induced expression of three 3ß-hydroxysteroid dehydrogenases from Erysimum crepidifolium Rchb. and their putative role in cardenolide biosynthesis.

3ß-Hydroxysteroid dehydrogenases (3ßHSD) are supposed to be involved in cardenolide biosynthesis in plants. Erysimum crepidifolium Rchb., a member of the Brassicaceae accumulating cardenolides, is a close relative to Arabidopsis thaliana. Full length cDNAs encoding for three individual 3ßHSDs (EcHSD1, EcHSD2, EcHSD3) were isolated from E. crepidifolium leaves. EcHSD1 and EcHSD2 encode proteins assembled from 257 amino acids whereas EcHSD3 encodes a protein assembled from 260 amino acids. All three proteins qualify as members of the short-chain dehydrogenases/reductases family of proteins (SDRs). EcHSD1 and EcHSD2 shared a high amino acid sequence identity of about 86% and 91% with putative 3ßHSDs of A. thaliana (AT2G47140 and AT2G47130). EcHSD3 showed high homology to the A. thaliana SDRs AT2G47150 (74%) and AT2G47120 (81%). All three EcHSD genes were expressed in Escherichia coli and the recombinant enzymes were characterized biochemically. All three recombinant EcHSDs catalyzed the dehydrogenation of pregnenolone and the 3-reduction of 5α/ß-pregnane-3,20-dione when NAD and NADH were used as cosubstrates, respectively. After exposure to different stress conditions, no increased transcription was seen for EcHSD1 whereas EcHSD2 was expressed four times higher under osmotic stress than under control conditions. EcHSD3 expression was 10 times and 6 times higher after osmotic stress and MeJA treatment, respectively, than in controls.

Progesterone 5ß-reductase of Erysimum crepidifolium: cDNA cloning, expression in Escherichia coli, and reduction of enones with the recombinant protein.

Erysimum is a genus of the Brassicaceae family closely related to the genus Arabidopsis. Several Erysimum species accumulate 5ß-cardenolides. Progesterone 5ß-reductases (P5ßRs) first described in Digitalis species are thought to be involved in 5ß-cardenolide biosynthesis. P5ßRs belong to the dehydrogenase/reductase super-family of proteins. A full length cDNA clone encoding a P5ßR was isolated from Erysimum crepidifolium leaves by 5'/3' RACE-PCR (termed EcP5ßR). Subsequently, the P5ßR cDNAs of another nine Erysimum species were amplified by RT-PCR using 5' and 3' end primers deduced from the EcP5ßR cDNA. The EcP5ßR cDNA is 1170bp long and encodes for 389 amino acids. The EcP5ßR cDNA was ligated into the vector pQE 30 UA and the recombinant His-tagged protein (termed rEcP5ßR) was over-expressed in Escherichia coli and purified by Ni-chelate affinity chromatography. Kinetic constants were determined for progesterone, 2-cyclohexen-1-one, isophorone, and NADPH. The by far highest specificity constant (k(cat)K(M)⁻¹) was estimated for 2-cyclohexen-1-one indicating that this monocyclic enone may be more related to the natural substrate of the enzyme than progesterone. The atomic structure of rEcP5ßR was modelled using the crystal structure of P5ßR from Digitalis lanata 2V6G as the template. All sequence motifs specific for SDRs as well as the NFYYxxED motif typical for P5ßR-like enzymes were present and the protein sequence fitted into the template smoothly.