Immunostimulatory Potential of Extracellular Vesicles Isolated from an Edible Plant, Petasites japonicus, via the Induction of Murine Dendritic Cell Maturation

Extracellular vesicles (EVs) have recently been isolated from different plants. Plant-derived EVs have been proposed as potent therapeutics and drug-delivery nanoplatforms for delivering biomolecules, including proteins, RNAs, DNAs, and lipids. Herein, Petasites japonicus-derived EVs (PJ-EVs) were isolated through a series of centrifugation steps and characterized using dynamic light scattering and transmission electron microscopy. Immunomodulatory effects of PJ-EVs were assessed using dendritic cells (DCs). PJ-EVs exhibited a spherical morphology with an average size of 122.6 nm. They induced the maturation of DCs via an increase in the expression of surface molecules (CD80, CD86, MHC-I, and MHC-II), production of Th1-polarizing cytokines (TNF-α and IL-12p70), and antigen-presenting ability; however, they reduced the antigen-uptake ability. Furthermore, maturation of DCs induced by PJ-EVs was dependent on the activation and phosphorylation of MAPK and NF-κB signal pathways. Notably, PJ-EV-treated DCs strongly induced the proliferation and differentiation of naïve T cells toward Th1-type T cells and cytotoxic CD8+ T cells along with robust secretion of IFN-γ and IL-2. In conclusion, our study indicates that PJ-EVs can be potent immunostimulatory candidates with an ability of strongly inducing the maturation of DCs.

A yeast-based screening system identified bakkenolide B contained in Petasites japonicus as an inhibitor of interleukin-2 production in a human T cell line

Ca2+ signaling is related to various diseases such as allergies, diabetes, and cancer. We explored Ca2+ signaling inhibitors in natural resources using a yeast-based screening method, and found bakkenolide B from the flower buds of edible wild plant, Petasites japonicus, using the YNS17 strain (zds1Δ erg3Δ pdr1/3Δ). Bakkenolide B exhibited growth-restoring activity against the YNS17 strain and induced Li+ sensitivity of wild-type yeast cells, suggesting that it inhibits the calcineurin pathway. Additionally, bakkenolide B inhibited interleukin-2 production at gene and protein levels in Jurkat cells, a human T cell line, but not the in vitro phosphatase activity of human recombinant calcineurin, an upstream regulator of interleukin-2 production. Furthermore, bakkenolide A showed weak activity in YNS17 and Jurkat cells compared with bakkenolide B. These findings revealed new biological effects and the structure-activity relationships of bakkenolides contained in Petasites japonicus as inhibitors of interleukin-2 production in human T cells.

Persistence and Degradation Pattern of Acequinocyl and Its Metabolite, Hydroxyl-acequinocyl and Fenpyroximate in Butterburs (Petasites Japonicus Max.)

Degradation patterns and persistence of acequinocyl and its metabolite, hydroxyl-acequinocyl (acequinocyl-OH), and fenpyroximate residues in butterburs (Petasites japonicus Max.), one of the minor crops in Republic of Korea, were investigated during cultivation. Butterburs were planted in two plots (plot A for double dose; plot B for single dose) in a greenhouse. Each pesticide was applied to the foliage of butterburs at hourly intervals. Recoveries of acequinocyl and acequinocyl-OH were 78.6%−84.7% ± relative standard deviation (RSD) 1.9%−4.8% and 83.7%−95.5% ± RSD 1.0%−3.6%, respectively. The total (Ʃ) of acequinocyl residues in butterburs disappeared by 96.0% at 14 days after the final application in plot A and by 75.9% at 7 days in plot B. The biological half-life of the total (Ʃ) acequinocyl and fenpyroximate was 3.0 days and 4.0 days respectively. These results were used for setting maximum residue levels and safe use standards for the pesticide during butterbur cultivation. The risk assessment showed that the maximum % acceptable daily intake was 4.71% for Ʃ acequinocyl and 8.81% for fenpyroximate. The theoretical maximum daily intake of Ʃ acequinocyl and fenpyroximate were 24.02% and 15.24%, respectively, indicating the concentrations of Ʃ acequinocyl and fenpyroximate in butterburs pose no health risks to Koreans.

Nutrients exported from upland stream water enlarge perennial biomass crops

Rawanbuki, a variety of Japanese butterbur (Petasites japonicus subsp. giganteus), grow naturally along the Rawan River, Hokkaido, northern Japan. Most plants reach 2–3 m in height and 10 cm in diameter in 2 months and are much larger than those grown along other rivers. We examined the hypothesis that nutrients exported from upland streams enhance the growth of the Rawanbuki. Nutrient concentrations, including nitrogen, phosphorus, and base cations, in the Rawan River were much higher than those in rivers of adjacent watersheds. High nutrient concentrations and moisture contents were found in soil along the Rawan River and a significant relationship was found between physicochemical soil conditions and aboveground biomass of butterburs. This indicates that extremely large Rawanbuki plants could be caused by these high nutrient concentrations and moisture contents in the soils. A manipulation experiment showed that fertilization simulated the growth environment along the Rawan River and enhanced the stem height and stem diameter of butterburs. This study concluded that the extremely large butterburs are caused by a large amount of nutrients exported from upland areas. These results are the first demonstration of the role of stream water nutrients in enlarging agricultural crops.

Photosynthetic and Morphological Acclimation to High and Low Light Environments in Petasites japonicus subsp. giganteus

Within each species, leaf traits such as light-saturated photosynthetic rate or dark respiration rate acclimate to local light environment. Comparing only static physiological traits, however, may not be sufficient to evaluate the effects of such acclimation in the shade because the light environment changes diurnally. We investigated leaf photosynthetic and morphological acclimation for a perennial herb, butterbur (Petasites japonicus (Siebold et Zucc.) Maxim. subsp. giganteus (G.Nicholson) Kitam.) (Asteraceae), in both a well-lit clearing and a shaded understory of a temperate forest. Diurnal changes in light intensity incident on the leaves were also measured on a sunny day and an overcast day. Leaves in the clearing were more folded and upright, whereas leaves in the understory were flatter. Leaf mass per area (LMA) was approximately twofold higher in the clearing than in the understory, while light-saturated photosynthetic rate and dark respiration rate per unit mass of leaf were similar between the sites. Consequently, both light-saturated photosynthetic rate and dark respiration rate per unit area of leaf were approximately twofold higher in the clearing than in the understory, consistent with previous studies on different species. Using this experimental dataset, we performed a simulation in which sun and shade leaves were hypothetically exchanged to investigate whether such plasticity increased carbon gain at each local environment. As expected, in the clearing, the locally acclimated sun leaves gained more carbon than the hypothetically transferred shade leaves. By contrast, in the understory, the daily net carbon gain was similar between the simulated sun and shade leaves on the sunny day due to the frequent sunflecks. Lower LMA and lower photosynthetic capacity in the understory reduced leaf construction cost per area rather than maximizing net daily carbon gain. These results indicate that information on static photosynthetic parameters may not be sufficient to evaluate shade acclimation in forest understories.

First report of Tomato spotted wilt virus in Petasites japonicus in Korea

Butterbur (Petasites japonicus [Siebold & Zucc.] Maxim.) is a perennial herb of the Asteraceae family that is cultivated for medicinal and nutritional purposes. Due to long-term vegetative propagation of virus-infected native species, the yield and quality of butterbur plants have deteriorated. Five viruses have been reported to infect this species: alfalfa mosaic virus (AMV), arabis mosaic virus (ArMV), butterbur mosaic virus (ButMV), broad bean wilt virus 2 (BBWV-2), and cucumber mosaic virus (CMV) (Ham et al. 2016; Tochihara and Tamura 1976). From 2018 to 2019, butterbur plants in four greenhouses in Nonsan, South Korea (Supplementary Figure S1a, b) were found to show virus-like symptoms such as chlorotic and necrotic ring spots, necrosis, and mild mosaic on the leaves. Disease incidence was greater than 80% in one greenhouse (~1,000 m2). To identify the causal virus, we collected 17 symptomatic butterbur leaf samples from these greenhouses and performed reverse-transcription polymerase chain reaction (RT-PCR) analysis using species-specific detection primers for the five reported viruses and tomato spotted wilt virus (TSWV) (Supplementary Table S2). RT-PCR results showed that 12 samples from three greenhouses showing necrotic ring spots and mosaic symptoms were infected with a mixture of TSWV and ButMV, whereas 5 samples from one greenhouse showing mild mosaic symptoms were infected only with ButMV. TSWV (genus Orthotospovirus, family Tospoviridae) is transmitted by thrips and causes serious damage to a wide range of economically important plants (Pappu et al. 2009). ButMV (genus Carlavirus, family Betaflexiviridae) is transmitted by aphids, as well as infected vegetative propagation material (Hashimoto et al. 2009) and is the most predominant virus in butterbur in Korea (Ham et al. 2016). To isolate TSWV from butterbur, leaf extracts from symptomatic samples were mechanically inoculated on an assay host, Chenopodium quinoa, via three single-lesion passages followed by propagation in Nicotiana tabacum cv. Samsun. Thirty different indicator plant species were used for the bioassay of the TSWV isolate (TSWV-NS-BB20) by mechanical inoculation method (Supplementary Table S3). RT-PCR analysis confirmed that TSWV-NS-BB20 induced necrotic local lesions and mosaic on Nicotiana species and ring spots and mosaic on tomatoes and peppers. Notably, TSWV-NS-BB20 reproduced necrotic local lesions and mild mosaic symptoms on butterbur plants which were infected with ButMV with no obvious symptoms. To characterize TSWV-NS-BB20 genetically, the complete genome sequences of L (8914 nt), M (4751 nt), and S (2917 nt) RNA segments were obtained by RT-PCR using specific primers for TSWV as described previously (Kwak et al., 2020). The obtained sequences were deposited in GenBank under accession nos. MT643236, MT842841, and MN854654, respectively. BLASTn analysis showed that sequences of each segment had maximum nucleotide identities of 99.0, 98.9, and 98.6% to TSWV-L, M, and S (KP008128, FM163373, and KP008129) of TSWV-LL-N.05 isolate from tomato in Spain. Since 2018, TSWV outbreaks on butterbur are observed every year and thus may act as a potential source of TSWV infection for other crops of importance to Korea, such as pepper. Owing to the butterbur vegetative propagation, the identification of TSWV infection in butterbur will be helpful for future virus management to generate virus-free materials. To our knowledge, this is the first report of TSWV infection of butterbur.