Metabolomic responses of indigenous and nonindigenous plants to deer exclosure fencing and deer herbivory in a suburban forest

Trees and shrubs in suburban forest understories can be subject to chronic herbivory from abundant white-tailed deer. An undocumented consequence of this stress may be shifts in secondary metabolite production associated with defense. We aimed to learn whether plants protected from deer exhibited different metabolomic profiles compared to those exposed to deer. We tested the indigenous species Nyssa sylvatica and Lindera benzoin and the invasive, nonindigenous species Rosa multiflora and Euonymus alatus within a suburban forest understory in New Jersey, USA, in unfenced plots and plots fenced for 5.3 years. We did untargeted metabolomics by sampling leaves from three plants of each species per 6-7 fenced and unfenced plots, conducting chloroform-methanol extractions followed by LC-MS/MS, and conducting statistical analysis on Metaboanalyst. We also scored each species for deer browse frequency over eight years, and compared their heights and percent cover between unfenced and fenced plots. The analysis identified 2,333 metabolites. The global metabolome diverged significantly between fenced and unfenced plots pooled across species, but for individual species only N. sylvatica exhibited a significant fencing effect. Nyssa sylvatica was one of the most browsed species and was the only one with both greater cover and height in fenced plots, suggesting greater susceptibility to deer browsing. The metabolites most responsible for the fenced/unfenced divergence also were affected by the species-fencing combination, with increases in certain species but decreases in others. The most significant metabolites that were upregulated in fenced plants include some involved in defense-related metabolic pathways, e.g. monoterpenoid biosynthesis. Further study of more species in multiple sites is needed to learn how common metabolomic responses to deer are among forest species, how the intensity of deer pressure influences the responses, which types of metabolites are most affected, and if there are ecological consequences at the physiological, population, and/or community levels.

Production of Volatile Moth Sex Pheromones in Transgenic Nicotiana benthamiana Plants

Plant-based bio-production of insect sex pheromones has been proposed as an innovative strategy to increase the sustainability of pest control in agriculture. Here we describe the engineering of transgenic plants producing (Z)-11-hexadecen-1-ol (Z11-16OH) and (Z)-11-hexadecenyl acetate (Z11-16OAc), two main volatile components in many Lepidoptera sex pheromone blends. We assembled multiple multigene DNA constructs encoding the pheromone biosynthetic pathway and stably transformed them in Nicotiana benthamiana plants. The constructs comprised the Amyelois transitella AtrΔ11 desaturase gene, the Helicoverpa armigera farnesyl reductase HarFAR gene, and the Euonymus alatus diacylglycerol acetyltransferase EaDAct gene in different configurations. All the pheromone-producing plants showed dwarf phenotypes, whose severity correlated with pheromone levels. All but one of the recovered lines produced high levels of Z11-16OH but very low levels of Z11-16OAc, probably as a result of recurrent truncations at the level of the EaDAct gene. Only one plant line (SxPv1.2) was recovered harbouring an intact pheromone pathway and producing moderate levels of Z11-16OAc (11.8 μg g-1 FW), next to high levels of Z11-16OH (111.4 μg g-1). Z11-16OAc production was accompanied in SxPv1.2 by a partial recovery of the dwarf phenotype. SxPv1.2 was used to estimate the rates of volatile pheromone release, which resulted in 8.48 ng g-1 FW per day for Z11-16OH and 9.44 ng g-1 FW per day for Z11-16OAc. Our results suggest that pheromone release acts as a limiting factor in pheromone bio-dispenser strategies and establish a roadmap for biotechnological improvements.

Neuroprotective Effects of Euonymus alatus Extract on Scopolamine-Induced Memory Deficits in Mice

Euonymus alatus is considered to elicit various beneficial effects against cancer, hyperglycemia, menstrual discomfort, diabetic complications, and detoxification. The young leaves of this plant are exploited as food and also utilized for traditional medicine in East Asian countries, including Korea and China. Our preliminary study demonstrated that ethanolic extract from the Euonymus alatus leaf (EAE) exhibited the strongest antioxidant enzyme-inducing activity among more than 100 kinds of edible tree leaf extracts. This study investigated whether EAE could attenuate the cognitive deficits caused by oxidative stress in mice. Oral intubation of EAE at 100 mg/kg bw or higher resulted in significant improvements to the memory and behavioral impairment induced via i.p. injection of scopolamine. Furthermore, EAE enhanced the expression levels of hippocampal neurotrophic factors such as brain-derived neurotrophic factor (BDNF) and glial cell-derived neurotrophic factor in mice, activated the Nrf2, and the downstream heme oxygenase-1 (HO-1) a quintessential antioxidant enzyme. As rutin (quercetin-3-O-rutinose) was abundantly present in EAE and free quercetin was able to induce defensive antioxidant enzymes in an Nrf2-dependent manner, our findings suggested that quercetin derived from rutin via the intestinal microflora played a significant role in the protection of the mouse hippocampus from scopolamine-induced damage through BDNF-mediated Nrf2 activation, thereby dampening cognitive decline.

Dieback of Euonymus alatus (Celastraceae) Caused by Cytospora haidianensis sp. nov. in China

Euonymus alatus (Celastraceae) is widely cultivated in China for its economic value and landscape benefits. Euonymus alatus dieback occurs due to members of Cytospora and has become one of the most severe diseases affecting its cultivation in China. In this study, we examined the causal agent of bough dieback on campuses of University Road, Beijing, China. Among the strains, three were morphologically consistent with Cytospora, showing hyaline and allantoid conidia. Based on phylogenetic analyses of the concatenated actin (ACT), internal transcribed spacer (ITS), RNA polymerase II second largest subunit (RPB2), translation elongation factor 1-alpha (TEF1-α) and beta-tubulin (TUB2) gene sequences, along with morphological and physiological features, we propose C. haidianensis as a novel species. It was confirmed as a causal agent of dieback of E. alatus by pathogenicity tests. Mycelial growth of Cytospora haidianensis occurred at pH values ranging from 3.0 to 11.0, with optimum growth at 8.3, and at temperatures from 5 to 35 °C, with optimum growth at 19.8 °C. We also tested the growth of C. haidianensis in the presence of six carbon sources. Sucrose, maltose and glucose were highly efficient and xylose was the least. The ability of C. haidianensis to grow at 19.8 °C may help to explain its occurrence causing dieback of E. alatus in Beijing during the autumn season.