CRISPR/Cas9-mediated mutation of Eil1 transcription factor genes affects exogenous ethylene tolerance and early flower senescence in Campanula portenschlagiana.

Improving tolerance to ethylene-induced early senescence of flowers and fruits is of major economic importance for the ornamental and food industry. Genetic modifications of genes in the ethylene-signalling pathway have frequently resulted in increased tolerance but often with unwanted side effects. Here, we used CRISPR/Cas9 to knockout the function of two CpEil1 genes expressed in flowers of the diploid ornamental plant Campanula portenschlagiana. The ethylene tolerance in flowers of the primary mutants with knockout of only one or all four alleles clearly showed increased tolerance to exogenous ethylene, although lower tolerance was obtained with one compared to four mutated alleles. The allele dosage effect was confirmed in progenies where flowers of plants with zero, one, two, three and four mutated alleles showed increasing ethylene tolerance. Mutation of the Cpeil1 alleles had no significant effect on flower longevity and endogenous flower ethylene level, indicating that CpEil1 is not involved in age-dependent senescence of flowers. The study suggests focus on EIN3/Eils expressed in the organs subjected to early senescence for obtaining tolerance towards exogenous ethylene. Furthermore, the observed allelic dosage effect constitutes a key handle for a gradual regulation of sensitivity towards exogenous ethylene, simultaneously monitoring possibly unwanted side effects.

Pollen colour morphs take different paths to fitness.

Colour phenotypes are often involved in communication and are thus under selection by species interactions. However, selection may also act on colour through correlated traits or alternative functions of biochemical pigments. Such forms of selection are instrumental in maintaining petal colour diversity in plants. Pollen colour also varies markedly, but the maintenance of this variation is little understood. In Campanula americana, pollen ranges from white to dark purple, with darker morphs garnering more pollinator visits and exhibiting elevated pollen performance under heat stress. Here, we generate an F2 population segregating for pollen colour and measure correlations with floral traits, pollen attributes and plant-level traits related to fitness. We determine the pigment biochemistry of colour variants and evaluate maternal and paternal fitness of light and dark morphs by crossing within and between morphs. Pollen colour was largely uncorrelated with floral traits (petal colour, size, nectar traits) suggesting it can evolve independently. Darker pollen grains were larger and had higher anthocyanin content (cyanidin and peonidin) which may explain why they outperform light pollen under heat stress. Overall, pollen-related fitness metrics were greater for dark pollen, and dark pollen sires generated seeds with higher germination potential. Conversely, light pollen plants produce 61% more flowers than dark, and 18% more seeds per fruit, suggesting a seed production advantage. Results indicate that light and dark morphs may achieve fitness through different means-dark morphs appear to have a pollen advantage whereas light morphs have an ovule advantage-helping to explain the maintenance of pollen colour variation.

FAD2 Gene Radiation and Positive Selection Contributed to Polyacetylene Metabolism Evolution in Campanulids.

Polyacetylenes (PAs) are bioactive, specialized plant defense compounds produced by some species in the eudicot clade campanulids. Early steps of PA biosynthesis are catalyzed by Fatty Acid Desaturase 2 (FAD2). Canonical FAD2s catalyze desaturation, but divergent forms can catalyze hydroxylation, conjugation, acetylenation, and epoxygenation. These alternate reactions give rise to valuable unusual fatty acids, including the precursors to PAs. The extreme functional diversity of FAD2 enzymes and the origin of PA biosynthesis are poorly understood from an evolutionary perspective. We focus here on the evolution of the FAD2 gene family. We uncovered a core eudicot-wide gene duplication event giving rise to two lineages: FAD2-α and FAD2-ß. Independent neofunctionalizations in both lineages have resulted in functionally diverse FAD2-LIKEs involved in unusual fatty acid biosynthesis. We found significantly accelerated rates of molecular evolution in FAD2-LIKEs and use this metric to provide a list of uncharacterized candidates for further exploration of FAD2 functional diversity. FAD2-α has expanded extensively in Asterales and Apiales, two main clades of campanulids, by ancient gene duplications. Here, we detected positive selection in both Asterales and Apiales lineages, which may have enabled the evolution of PA metabolism in campanulids. Together, these findings also imply that yet uncharacterized FAD2-α copies are involved in later steps of PA biosynthesis. This work establishes a robust phylogenetic framework in which to interpret functional data and to direct future research into the origin and evolution of PA metabolism.

A natural frameshift mutation in Campanula EIL2 correlates with ethylene insensitivity in flowers.

BACKGROUND: The phytohormone ethylene plays a central role in development and senescence of climacteric flowers. In ornamental plant production, ethylene sensitive plants are usually protected against negative effects of ethylene by application of chemical inhibitors. In Campanula, flowers are sensitive to even minute concentrations of ethylene. RESULTS: Monitoring flower longevity in three Campanula species revealed C. portenschlagiana (Cp) as ethylene sensitive, C. formanekiana (Cf) with intermediate sensitivity and C. medium (Cm) as ethylene insensitive. We identified key elements in ethylene signal transduction, specifically in Ethylene Response Sensor 2 (ERS2), Constitutive Triple Response 1 (CTR1) and Ethylene Insensitive 3- Like 1 and 2 (EIL1 and EIL2) homologous. Transcripts of ERS2, CTR1 and EIL1 were constitutively expressed in all species both throughout flower development and in response to ethylene. In contrast, EIL2 was found only in Cf and Cm. We identified a natural mutation in Cmeil2 causing a frameshift which resulted in difference in expression levels of EIL2, with more than 100-fold change between Cf and Cm in young flowers. CONCLUSIONS: This study shows that the naturally occurring 7 bp frameshift discovered in Cmeil2, a key gene in the ethylene signaling pathway, correlates with ethylene insensitivity in flowers. We suggest that transfer of the eil2 mutation to other plant species will provide a novel tool to engineer ethylene insensitive flowers.

Metabolic pathways of benzimidazole anthelmintics in harebell (Campanula rotundifolia).

Benzimidazoles anthelmintics, which enter into environment primarily through excretion in the feces or urine of treated animals, can affect various organisms and disrupt ecosystem balance. The present study was designed to test the phytotoxicity and biotransformation of the three benzimidazole anthelmintics albendazole (ABZ), fenbendazole (FBZ) and flubendazole (FLU) in the harebell (Campanula rotundifolia). This meadow plant commonly grows in pastures and comes into contact with anthelmintics through the excrements of treated animals. Suspensions of harebell cells in culture medium were used as an in vitro model system. ABZ, FLU and FBZ were not found to be toxic for harebell cells, which were able to metabolize ABZ, FLU and FBZ via the formation of a wide scale of metabolites. Ultrahigh-performance liquid chromatography coupled with high mass accuracy tandem mass spectrometry (UHPLC-MS/MS) led to the identification of 24, 18 and 29 metabolites of ABZ, FLU and FBZ, respectively. Several novel metabolites were identified for the first time. Based on the obtained results, the schemes of the metabolic pathways of these anthelmintics were proposed. Most of these metabolites can be considered deactivation products, but a substantial portion of them may readily be decomposed to biologically active substances which could negatively affect ecosystems.

A sexually dimorphic corolla appendage affects pollen removal and floral longevity in gynodioecious Cyananthus delavayi (Campanulaceae).

The floral traits of bisexual flowers may evolve in response to selection on both male and female functions, but the relative importance of selection associated with each of these two aspects is poorly resolved. Sexually dimorphic traits in plants with unisexual flowers may reflect gender-specific selection, providing opportunities for gaining an increased understanding of the evolution of specific floral traits. We examined sexually dimorphic patterns of floral traits in perfect and female flowers of the gynodioecious species Cyananthus delavayi. A special corolla appendage, the throat hair, was investigated experimentally to examine its influences on male and female function. We found that perfect flowers have larger corollas and much longer throat hairs than female flowers, while female ones have much exerted stigmas. The presence of throat hairs prolonged the duration of pollen presentation by restricting the amount of pollen removed by pollen-collecting bees during each visit. Floral longevity was negatively related to the rate of pollen removal. When pollen removal rate was limited in perfect flowers, the duration of the female phases diminished with the increased male phase duration. There was a weak negative correlation between throat hair length and seed number per fruit in female flowers, but this correlation was not significant in perfect flowers. These results suggest that throat hairs may enhance male function in terms of prolonged pollen presentation. However, throat hairs have no obvious effect on female function in terms of seed number per fruit. The marked sexual dimorphism of this corolla appendage in C. delavayi is likely to have evolved and been maintained by gender-specific selection.

Platycodin D Attenuates Airway Inflammation in a Mouse Model of Allergic Asthma by Regulation NF-κB Pathway.

In this work, the anti-asthma potential of platycodin D (PLD) was studied by investigation of its effect to suppress airway inflammation, a murine model of asthma and the possible mechanisms. A total of 50 mice were randomly assigned to five experimental groups: control, ovalbumin (OVA), OVA+dexamethasone (2 mg/kg) and OVA+PLD (40, 80 mg/kg). Airway resistance (Raw) were measured; airway histological studies were evaluated by the hematoxylin and eosin (HE) staining; interleukin-4 (IL-4), interleukin-5(IL-5), and interleukin-13 in bronchoalveolar lavage fluid (BALF) were evaluated by enzyme-linked immunosorbent assay (ELISA); NF-κBp65, p-NF-κBp65, p-IKKα, IKKα, p-IKKß, p-IкBα, and IкBα of airway were measured by Western blotting. Our study demonstrated that PLD inhibited OVA-induced increases in Raw and eosinophil count in airway; IL-4, IL-5, and IL-13 were recovered in BALF. Histological studies demonstrated that PLD substantially inhibited OVA-induced eosinophilia in airway tissue. Western blotting studies demonstrated that PLD substantially inhibited NF-κB pathway. These findings suggest that PLD may effectively ameliorate the progression of asthma and could be used as a therapy for patients with allergic asthma.

Comparative study on plant latex particles and latex coagulation in Ficus benjamina, Campanula glomerata and three Euphorbia species.

Among latex-producing plants, mainly the latex of Hevea brasiliensis has been studied in detail so far, while comprehensive comparative studies of latex coagulation mechanisms among the more than 20,000 latex-bearing plant species are lacking. In order to give new insights into the potential variety of coagulation mechanisms, the untreated natural latices of five latex-bearing plants from the families Euphorbiaceae, Moraceae and Campanulaceae were visualised using Cryo-SEM and their particle size compared using the laser diffraction method. Additionally, the laticifers of these plants species were examined in planta via Cryo-SEM. Similar latex particle sizes and shape were found in Ficus benjamina and Hevea brasiliensis. Hence, and due to other similarities, we hypothesize comparable, mainly chemical, coagulation mechanisms in these two species, whereas a physical coagulation mechanism is proposed for the latex of Euphorbia spp. The latter mechanism is based on the huge amount of densely packed particles that after evaporation of water build a large surface area, which accelerates the coagulation procedure.

Spectral effects of supplementary lighting on the secondary metabolites in roses, chrysanthemums, and campanulas.

To investigate the effect of the light spectrum on photosynthesis, growth, and secondary metabolites Rosa hybrida 'Scarlet', Chrysanthemum morifolium 'Coral Charm', and Campanula portenschlagiana 'BluOne' were grown at 24/18°C day/night temperature under purpose-built LED arrays yielding approximately 200 µmol m(-2)s(-1) at plant height for 16 h per day. The four light treatments were (1) 40% Blue/60% Red, (2) 20% Blue/80% Red, (3) 100% Red, and (4) 100% White (Control). The plant height was smallest in 40% Blue/60% Red in roses and chrysanthemums, while the biomass was smallest in the white control in roses and in 100% Red in chrysanthemums. The total biomass was unaffected by the spectrum in campanulas, while the leaf area was smallest in the 40% Blue/60% Red treatment. In 100% Red curled leaves and other morphological abnormalities were observed. Increasing the blue to red ratio increased the stomatal conductance though net photosynthesis was unaffected, indicating excess stomatal conductance in some treatments. With higher blue light ratio all phenolic acids and flavonoids increased. In view of the roles of these secondary metabolites as antioxidants, anti-pathogens, and light protectants, we hypothesize that blue light may predispose plants to better cope with stress.

Enhanced accumulation of phytosterol and triterpene in hairy root cultures of Platycodon grandiflorum by overexpression of Panax ginseng 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) catalyzes the rate-limiting step in the mevalonate pathway. To elucidate the functions of HMGR in triterpene biosynthesis, Platycodon grandiflorum was transformed with a construct expressing Panax ginseng HMGR (PgHMGR). We used PCR analysis to select transformed hairy root lines and selected six lines for further investigation. Quantitative real-time PCR showed higher expression levels of HMGR and total platycoside levels (1.5-2.5-fold increase) in transgenic lines than in controls. Phytosterols levels were also 1.1-1.6-fold higher in transgenic lines than in controls. Among these lines, line T7 produced the highest level of total platycosides (1.60 ± 0.2 mg g(-1) dry weight) and α-spinasterol (1.78 ± 0.16 mg g(-1) dry weight). These results suggest that metabolic engineering of P. grandiflorum by Agrobacterium-mediated genetic transformation may enhance production of phytosterols and triterpenoids.

Comprehensive analysis of Phyteuma orbiculare L., a wild Alpine food plant.

Plants which have been traditionally eaten by the alpine population may provide new opportunities for agricultural development in mountain regions. In this context we have investigated the chemical composition of Phyteuma orbiculare (Campanulaceae), a perennial herb whose leaves have been eaten as salad by rural populations in Valais (Switzerland). Extracts of different polarities were subjected to comprehensive metabolite profiling using a dereplication platform combining HPLC-PDA-MS, and offline NMR analysis. Twenty-three compounds, including various phenolic glycosides, a new dimeric phenylpropanoid glucoside, saponins, and fatty acids were identified online, or after targeted isolation. Selected phenolic constituents were quantitatively assessed by HPLC-PDA analysis. In addition, substances relevant for nutrition, such as ß-carotene, fatty acids, ascorbic acid and minerals were quantified in leaves and flowers. The antioxidant capacity was determined with an ORAC assay, and total phenolic compounds were quantified. Finally, the phytochemical profile was compared to that of the related species P. spicatum, P. hemisphaericum and P. ovatum.

Adaptive radiation of photosynthetic physiology in the Hawaiian lobeliads: dynamic photosynthetic responses.

Hawaiian lobeliads have radiated into habitats from open alpine bogs to densely shaded rainforest interiors, and show corresponding adaptations in steady-state photosynthetic light responses and associated leaf traits. Shaded environments are not uniformly dark, however, but punctuated by sunflecks that carry most of the photosynthetically active light that strikes plants. We asked whether lobeliads have diversified in their dynamic photosynthetic light responses and how dynamic responses influence daily leaf carbon gain. We quantified gas exchange and dynamic light regimes under field conditions for ten species representing each major Hawaiian sublineage. Species in shadier habitats experienced shorter and less numerous sunflecks: average sunfleck length varied from 1.4 +/- 1.7 min for Cyanea floribunda in shaded forest understories to 31.2 +/- 2.1 min for Trematolobelia kauaiensis on open ridges. As expected, the rate of photosynthetic induction increased significantly toward shadier sites, with assimilation after 60 s rising from ca. 30% of fully induced rates in species from open environments to 60% in those from densely shaded habitats. Uninduced light use efficiency-actual photosynthesis versus that expected under steady-state conditions-increased from 10 to 70% across the same gradient. In silico transplants-modeling daily carbon gain using one species' photosynthetic light response in its own and other species' dynamic light regimes-demonstrated the potential adaptive nature of species differences: understory Cyanea pilosa in its light regimes outperformed gap-dwelling Clermontia parviflora, while Clermontia in its light regimes outperformed Cyanea. The apparent crossover in daily photosynthesis occurred at about the same photon flux density where dominance shifts from Cyanea to Clermontia in the field. Our results further support our hypothesis that the lobeliads have diversified physiologically across light environments in Hawaiian ecosystems and that those shifts appear to maximize the carbon gain of each species in its own environment.

Selective accumulation of delphinidin derivatives in tobacco using a putative flavonoid 3',5'-hydroxylase cDNA from Campanula medium.

Blue flowers generally contain 3',5'-hydroxylated anthocyanins (delphinidin derivatives) as pigments, which are formed only in the presence of flavonoid 3',5'-hydroxylases (F3'5'H). Heterologous expression of a F3'5'H gene therefore provides an opportunity to produce novel blue flowers for a number of ornamental plants missing blue flowering varieties. However, our previous study indicated difficulties in obtaining good accumulation of delphinidin derivatives in plants expressing F3'5'H. Here we report the isolation of a putative F3'5'H cDNA (Ka1) from canterbury bells (Campanula medium) and its expression in tobacco. Surprisingly, compared with other F3'5'H cDNAs, Ka1 encoded a protein with a unique primary structure that conferred high competence in the accumulation of delphinidin derivatives (up to 99% of total anthocyanins) and produced novel purple flowers. These results suggest that, among F3'5' H cDNAs, Ka1 is the best genetic resource for the creation of fine blue flowers by genetic engineering.