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

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

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

The permeability transition in plant mitochondria: The missing link

The synthesis of ATP in mitochondria is dependent on a low permeability of the inner membrane. Nevertheless, mitochondria can undergo an increased permeability to solutes, named permeability transition (PT) that is mediated by a pore (PTP). PTP opening requires matrix Ca2+ and leads to mitochondrial swelling and release of intramembrane space proteins (e.g. cytochrome c). This feature has been initially observed in mammalian mitochondria and tentatively attributed to some components present either in the outer or inner membrane. Recent works on mammalian mitochondria point to mitochondrial ATP synthase dimers as physical basis for PT, a finding that has been substantiated in yeast and Drosophila mitochondria. In plant mitochondria, swelling and release of proteins have been linked to programmed cell death, but in isolated mitochondria PT has been observed in only a few cases and in plant cell cultures only indirect evidence is available. The possibility that mitochondrial ATP synthase dimers could function as PTP also in plants is discussed here on the basis of the current evidence. Finally, a hypothetical explanation for the origin of PTP is provided in the framework of molecular exaptation

Investigation of non-structural carbohydrates and xylem anatomy in petiole of grapevine varieties during water limitation and after re-irrigation

Water shortage (WS) during growing of Vitis vinifera L. can limit shoot growth and affect yield and fruit quality, as well as allocation of carbon reserves into perennial organs for the upcoming years. Varietal anatomical differences, such as specific mean xylem vessel diameter in petiole, are expected to influence water transport in canes facing water limitation. Several authors have also evidenced that non-structural carbohydrates (NSC) of adjacent living parenchyma are involved in the repair mechanism of embolized vessels. In this work, we evaluated NSC level and xylem anatomy in petiole of Cabernet Sauvignon and Syrah varieties, subjected to WS and subsequent water refilling in the summer of 2017. The anatomical analysis highlighted that Syrah had high frequency of classes of large vessels, and that the xylem differentiation of vascular bundles was also affected by WS. Moreover, petiole NSC content was significantly influenced by WS and recovery, supporting the hypothesis that starch mobilization was associated to an elevated concentration in soluble NSC. This effect was determinant for Cabernet Sauvignon, whose stress response seemed to be based mainly on NSC metabolism. Finally, Syrah, differently to Cabernet Sauvignon, sustained the WS-induced increase in soluble NSC of petiole also 18 h after re-watering

Influence of hydroxyapatite nanoparticles on germination and plant metabolism of tomato (Solanum lycopersicum L.): Preliminary evidence

The Nutrient Use Efficiency in intensive agriculture is lower than 50% for macronutrients. This feature results in unsustainable financial and environmental costs. Nanofertilizers are a promising application of nanotechnology in agriculture. The use of nanofertilizers in an efficient and safe manner calls for knowledge about the actual effects of nanoproducts on the plant metabolism and eventually on the carrier release kinetics and nutrient accumulation. Hydroxyapatite (Ca10(PO4)6(OH)2) nanoparticles (nHA) have an interesting potential to be used as nanofertilizers. In this study, the effects of different nHA solutions stabilized with carboxymethylcellulose (CMC) were evaluated on germination, seedling growth, and metabolism of Solanum lycopersicum L., used as model species. Our observations showed that the percentage germination of S. lycopersicum is not influenced by increasing concentrations of nHa, while root elongation is strongly stimulated. Tomato plants grown in hydroponics in the presence of nHA have not suffered phytotoxic effects. We conclude that nHA had nontoxic effects on our model plant and therefore it could be used both as a P supplier and carrier of other elements and molecules