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.

Niche differences may explain the geographic distribution of cytotypes in Erysimum mediohispanicum.

Polyploidisation has played an important role in plant diversification, and variation in ploidy level may be found not only between species of the same genus, but also within a single species. Although establishing the adaptive significance of polyploidy to explain the geographic distribution of cytotypes is challenging, the occurrence of different cytotypes in different ecological niches may suggest an adaptive role of genome duplication. We studied the adaptive significance of the geographic distribution of cytotypes across the entire distribution range of the endemic Erysimum mediohispanicum (Brassicaceae). For that, we have used climate variables, population elevation and soil properties to model ecological niches for the different cytotypes. In addition, we analysed the effect that ploidy level has on the floral phenotype. We found a clear geographic pattern in the distribution of cytotypes, with diploid individuals occurring in the southernmost part of the distribution range, while tetraploids were found in the northern area. A contact (mosaic) zone between both cytotypes was identified, but diploids and tetraploids occur in sympatry in only one population (although in a highly unbalanced proportion). Gene flow between different cytotypes seems to be negligible, as evident from an almost complete absence of triploids and other minority cytotypes. Niches occupied by both cytotypes showed subtle, but significant differences, even in the contact zone. Precipitation was higher in regions occupied by tetraploid individuals, which present wider corolla tubes and thinner but taller stalks than diploids. Our findings highlight the potential role of polyploidy in the ecological adaptation of E. mediohispanicum to both abiotic factors and biotic interactions.

Reproductive success through high pollinator visitation rates despite self incompatibility in an endangered wallflower.

PREMISE OF THE STUDY: Self incompatibility (SI) in rare plants presents a unique challenge-SI protects plants from inbreeding depression, but requires a sufficient number of mates and xenogamous pollination. Does SI persist in an endangered polyploid? Is pollinator visitation sufficient to ensure reproductive success? Is there evidence of inbreeding/outbreeding depression? We characterized the mating system, primary pollinators, pollen limitation, and inbreeding/outbreeding depression in Erysimum teretifolium to guide conservation efforts. METHODS: We compared seed production following self pollination and within- and between-population crosses. Pollen tubes were visualized after self pollinations and between-population pollinations. Pollen limitation was tested in the field. Pollinator observations were quantified using digital video. Inbreeding/outbreeding depression was assessed in progeny from self and outcross pollinations at early and later developmental stages. KEY RESULTS: Self-pollination reduced seed set by 6.5× and quadrupled reproductive failure compared with outcross pollination. Pollen tubes of some self pollinations were arrested at the stigmatic surface. Seed-set data indicated strong SI, and fruit-set data suggested partial SI. Pollinator diversity and visitation rates were high, and there was no evidence of pollen limitation. Inbreeding depression (δ) was weak for early developmental stages and strong for later developmental stages, with no evidence of outbreeding depression. CONCLUSIONS: The rare hexaploid E. teretifolium is largely self incompatible and suffers from late-acting inbreeding depression. Reproductive success in natural populations was accomplished through high pollinator visitation rates consistent with a lack of pollen limitation. Future reproductive health for this species will require large population sizes with sufficient mates and a robust pollinator community.

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.

A comparison of leaf and petal senescence in wallflower reveals common and distinct patterns of gene expression and physiology.

Petals and leaves share common evolutionary origins but perform very different functions. However, few studies have compared leaf and petal senescence within the same species. Wallflower (Erysimum linifolium), an ornamental species closely related to Arabidopsis (Arabidopsis thaliana), provide a good species in which to study these processes. Physiological parameters were used to define stages of development and senescence in leaves and petals and to align these stages in the two organs. Treatment with silver thiosulfate confirmed that petal senescence in wallflower is ethylene dependent, and treatment with exogenous cytokinin and 6-methyl purine, an inhibitor of cytokinin oxidase, suggests a role for cytokinins in this process. Subtractive libraries were created, enriched for wallflower genes whose expression is up-regulated during leaf or petal senescence, and used to create a microarray, together with 91 senescence-related Arabidopsis probes. Several microarray hybridization classes were observed demonstrating similarities and differences in gene expression profiles of these two organs. Putative functions were ascribed to 170 sequenced DNA fragments from the libraries. Notable similarities between leaf and petal senescence include a large proportion of remobilization-related genes, such as the cysteine protease gene SENESCENCE-ASSOCIATED GENE12 that was up-regulated in both tissues with age. Interesting differences included the up-regulation of chitinase and glutathione S-transferase genes in senescing petals while their expression remained constant or fell with age in leaves. Semiquantitative reverse transcription-polymerase chain reaction of selected genes from the suppression subtractive hybridization libraries revealed more complex patterns of expression compared with the array data.