Caspase-3-like activity and proteasome degradation in grapevine suspension cell cultures undergoing silver-induced programmed cell death

Toxic metal contamination is one of the major environmental concerns of the recent decade, due to the large application of metals in industrial, healthcare and commercial products, even in the form of nanostructures and nanomaterials. Nevertheless, the effects of silver (Ag+) on plants have not yet thoroughly elucidated. Therefore, suspension cell cultures of grapevine were used as a model for investigating silver toxicity. To do this, oxidative stress and programmed cell death (PCD), evaluated as reactive oxygen species production, caspase-3-like activity and ubiquitin-proteasome system, were investigated. As a result, the highest concentration (10 \u3bcM) of Ag+ caused a rapid (within 24 h) induction of PCD (approx. 80%), accompanied by generation of reactive oxygen species and activation of caspase-3-like activity. In the presence of specific inhibitor of this enzyme, a partial recovery of cell viability and a strong inhibition of caspase- 3-like activity was observed. In addition, silver-induced PCD was accompanied either by increase of poly-ubiquitin conjugated proteins and degradation of subunit PBA1 of the proteasome 20S core, similarly to what found for metal-induced neurotoxicity in animals. The present study shows that silver could induce PCD in grapevine suspension cell cultures, mediated by caspase-3-like activity and oxidative stress. These effects were associated to accumulation of poly-ubiquitin conjugated proteins, suggesting the impairment of ubiquitin-proteasome complex, confirmed by the decrease of the PBA1 subunit. These findings indicate that animal and plant cells could share a common pathway in response to toxic metal, which involves PCD and disassembling of proteasome complex

Can agriculture be eco-friendly? Plant extracts as grapevine defense inducers

Secondary metabolites confer a first set of defenses against pathogens and herbivore attack. The use of plant extracts (PE), enriched in these compounds, could represent an additional practice in environment friendly grapevine pest management. During biotic stress, the induction of different pathogenesis-related (PR) gene-expression, such as chitinase, often occurs. In the present work, four different plant extracts were tested for their modulation of chitinase activity in either grapevine suspension cell cultures (GSC) and plants

Germination performance of alien and native species could shape community assembly of temperate grasslands under different temperature scenarios

Rising temperatures due to climate change are expected to interplay with biological invasions, and may enhance the spread and growth of some alien species upon arrival in new areas. To successfully invade, a plant species needs to overcome multiple biological barriers. Among the crucial life stages, seed germination greatly contributes to the final species assembly of a plant community. Several studies have suggested that alien plant success is related to their high seed germination and longevity in the soil. Hence, our aim is to test if the germination potential of alien seeds present in the seed bank will be further enhanced by future warming in temperate dry grasslands, an ecosystem that is among those most prone to biological invasions. We designed a laboratory germination experiment at two temperatures (20 and 28 °C), to simulate an early or late heat wave in the growing season, using seeds from nine common grassland Asteraceae species, including native, archaeophyte and neophyte species. The test was performed on both single and mixed pools of these categories of species, using a full-factorial orthogonal design. The warmer germination temperature promoted neophyte success by increasing germination probability and germination speed, while negatively impacting these parameters in seeds of native species. The co-occurrence of native and archaeophyte seeds at the lower temperature limited the invasiveness of neophytes. These results provide important information on future management actions aimed at containing alien plant invasions, by improving our knowledge on the possible seed-bank response and interaction mechanisms of common species occurring in disturbed natural areas or restored sites

Biochemical and immunochemical similarities among mammalian bilitranslocase and a plant flavonoid translocator

Flavonoids are a large class of plant secondary metabolites, belonging to polyphenol family, which possess pharmacological and nutritional properties. Their synthesis takes place only in plants, while mammals can acquire them only with diet. It has been demonstrated that flavonoid uptake occurs in rat also by the activity of bilitranslocase, a carrier that is involved in anion transport in liver cell, vascular endothelium and gastric mucosa. A sequence of bilitranslocase interacting with flavonoid moieties is already known and characterized. Antibody raised against such protein epitope were shown to exhibit cross-reactivity against plant membrane proteins in tissues involved in flavonoid transport and accumulation, such as teguments of carnation petals and skin of grape berries. Further immunolocalization studies allowed to demonstrate the presence of cross-reacting protein not only at the level of tegumental tissues, but also associated to sieve elements and seed teguments in grape berries

Vernalization affects the germination performance of the wetland endangered species Eleocharis carniolica

Eleocharis carniolica W.D.J. Koch (Cyperaceae) is an endangered wetland spike rush mainly threatened by habitat loss and fragmentation. Understanding the germination ecology of this species is essential to perform successful conservation and restoration actions. In this study, we investigated the effect of vernalization (i.e. cold stratification), gibberellic acid (GA3) and chemical scarification on seed germination of E. carniolica from wild populations in northern Italy. The results showed that vernalization (i.e. 8-weeks at 4(degrees)C) significantly improved germination probability, speed, and uniformity compared to non-stratified seeds. Gibberellic acid treatment alone or in combination with vernalization did not show a significant improvement in germination. Chemical scarification using sodium hypochlorite increased germination probability, with 8 h of scarification showing the highest success rate. However, 24-h scarification had a negative impact on germination. Overall, vernalization was found to be the most effective method to enhance germination in E. carniolica. These findings provide valuable insights into the seed germination ecology of this endangered species, aiding in its exsitu conservation, propagation, and in-situ restoration efforts. Moreover, they have important implications on future germination dynamics of this endangered species, especially with predicted climate change scenarios

Flavonoids and darkness lower PCD in senescing Vitis vinifera suspension cell cultures

Background Senescence is a key developmental process occurring during the life cycle of plants that can be induced also by environmental conditions, such as starvation and/or darkness. During senescence, strict control of genes regulates ordered degradation and dismantling events, the most remarkable of which are genetically programmed cell death (PCD) and, in most cases, an upregulation of flavonoid biosynthesis in the presence of light. Flavonoids are secondary metabolites that play multiple essential roles in development, reproduction and defence of plants, partly due to their well-known antioxidant properties, which could affect also the same cell death machinery. To understand further the effect of endogenously-produced flavonoids and their interplay with different environment (light or dark) conditions, two portions (red and green) of a senescing grapevine callus were used to obtain suspension cell cultures. Red Suspension cell Cultures (RSC) and Green Suspension cell Cultures (GSC) were finally grown under either dark or light conditions for 6 days. Results Darkness enhanced cell death (mainly necrosis) in suspension cell culture, when compared to those grown under light condition. Furthermore, RSC with high flavonoid content showed a higher viability compared to GSC and were more protected toward PCD, in accordance to their high content in flavonoids, which might quench ROS, thus limiting the relative signalling cascade. Conversely, PCD was mainly occurring in GSC and further increased by light, as it was shown by cytochrome c release and TUNEL assays. Conclusions Endogenous flavonoids were shown to be good candidates for exploiting an efficient protection against oxidative stress and PCD induction. Light seemed to be an important environmental factor able to induce PCD, especially in GSC, which lacking of flavonoids were not capable of preventing oxidative damage and signalling leading to senescence

Chitosan nanocarriers-mediated delivery of double-stranded RNA "in planta"

Agriculture is currently facing numerous challenges: the rapid rise of the world population, the consequent growth in food demand, the global decrease in crop yield. Particularly regarding the last issue, climate change is worsening the environmental stresses that commonly affect crops, and the use of resources – such as fertilizers and pesticides – is highly inefficient and pollutant. In this context, research is looking for new approaches to improve crop productivity by more efficient and environmentally friendly practices. It has been shown that nanomaterials are suitable for the development of cutting-edge technologies with the aim of improving the delivery of bioactive substances on plants and to promote their resistance to biotic and abiotic stresses. Among organic polymers, chitosan, if used in the nanoscale form, shows both properties; it can induce biological responses concerning plant defense against diseases and pathogen attack, and it is particularly suitable as a carrier for several molecules. Another innovative method for the defense of crops is the exploitation of the spray-induced gene silencing (SIGS) based on the activation of the so-called RNA-interference (RNAi). It involves exogenous double stranded RNAs (dsRNAs) targeting an essential pathogen gene, which trigger the RNAi pathway leading to the translational repression by degradation of target homologous mRNAs. In our case, the research aimed to verify the feasibility of dsRNA distribution on plant surface by means of functionalized chitosan nanoparticles (CH-NPs), thus allowing the protection of the doping agent and its efficient delivery. Here we show the preliminary results regarding the characterization of CH-NPs, their loading with dsRNAs and their interaction with the leaf surface of Nicotiana benthamiana plants. The effects of the dose-dependent distribution were analyzed by confocal microscopy upon incorporation of a fluorescent probe

Antifungal activity of chili pepper extract with potential for the control of some major pathogens in grapevine

Background: In the recent years, biofungicides have drawn increasing interest in vineyard for a more sustainable integrated and copper-limited pest management. Among alternatives, botanicals could represent valuable tools, being rich sources of biologically active compounds. Conversely to the well-known antioxidant and biological properties in relation to health benefits, investigation on bioactivity of hot pungent Capsicum sp. products against fungal phytopathogens in vineyard is still scarce. Therefore, the present study aimed at exploring the biologically active compounds profile of a chili pepper (Capsicum chinense Jacq.) pod extract and its antimicrobial properties against some of the major fungal and Oomycetes pathogens of grapevine, including Botrytis cinerea Pers., Guignardia bidwellii (Ellis) Viala & Ravaz and Plasmopara viticola (Berk. & M.A. Curtis) Berl. & De Toni. Results: The ethyl acetate-extracted oleoresin from the most pungent varieties was rich in capsaicinoids and polyphenols (371.09 and 268.5 μg mg-1 dry weight, respectively). Capsaicin and dihydrocapsaicin, hydroxycinnamic and hydroxybenzoic acids and quercetin derivatives were the most abundant, while carotenoids represented only a minor fraction. The oleoresin was efficient to inhibit all three pathogenic fungi and ED50 values were determined, evidencing that G. bidwellii was the more sensitive (0.233 ± 0.034 mg mL-1 ). Conclusion: The results suggested a potentiality of chili pepper extract for the control of some important grapevine pathogens, their possible application being helpful for the recommended limitation in extensive use of copper in vineyard. The complex mixture of high amounts of capsaicinoids, associated to specific phenolic acids and other minor bioactive components might contribute for the observed antimicrobial action of chili pepper extract

Development of a non-chemical RNAi-based strategy for Amaranthus hybridus L. weed management

Weeds are one of the major issues in cropping systems, responsible for significant yield losses. Herbicide applications are the most effective strategy to control weeds, but stricter legislation has resulted in a significant reduction in the number of herbicides available on the market. Furthermore, the recent European legislation on the sustainable use of pesticides will require farmers to drastically reduce chemical use over the next ten years while promoting integrated weed management strategies that improve environmental sustainability and lower the risks to animal and human health. In addition, the over-reliance on chemical control has resulted in the evolution of resistant biotypes. As a result, new technologies to effectively manage weeds and weed resistance should be developed. In this regard, the development of a non-chemical weed control strategy based on RNA interference (RNAi) technology could: i) represent a potential non-chemical weed control strategy, ii) provide an emerging GMO-free strategy for managing invasive and resistant weeds, and iii) provide a valid opportunity to go inside the molecular mechanisms of weed biology. In this study, the acetolactate synthase (ALS) gene of Amaranthus hybridus L. has been used as the target to assess the effectiveness and applicability of in-vitro synthesized double-stranded RNAs (dsRNAs) direct application for endogenous gene silencing and weed control. A. hybridus is a monoecious and self-pollinated weed that has evolved multiple resistance to herbicides with different sites of action, including ALS inhibitors, which are the most used herbicides in soybean. ALS represents an ideal target for the development and future application of dsRNA-mediated gene silencing because it is an intronless, nucleotide-stable, and single-copy gene. We have produced dsRNAs of various lengths (ranging from 218 to 460bp) targeting three distinct ALS regions: the 5’- and 3’-ends, and a central region. dsRNAs molecules were transcribed in-vitro by T7 RNA polymerase and externally applied to the abaxial leaf surface of A. hybridus plants at 4-6 true leaves developmental stage by: i) mechanical inoculation, or ii) high-pressure spraying. Despite the expression of ALS gene transcripts was found to be lightly downregulated when synthetic 2 ALS-dsRNAs were applied, no phenotypic effects were observed. Our current research focuses on the determination of the effectiveness of ALS-dsRNAs silencing using agroinfiltration techniques, and on dsRNAs delivery techniques through the use of nanomaterials to maximize the effectiveness of gene silencing by exogenous dsRNAs application. This second approach was preliminary studied by RNA electrophoretic mobility of functionalized nanomaterial and by means of confocal microscopy on A. hybridus leaves. In parallel, we are examining the expression patterns of genes thought to be involved in the RNAi pathway in A. hybridus to verify if their expression is triggered by dsRNA applications