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

Tools for Nano-Enabled Agriculture: Fertilizers Based on Calcium Phosphate, Silicon, and Chitosan Nanostructures

The Green New Deal requires a profound transformation of the agricultural sector, which will have to become more sustainable and ensure universal access to healthy food. Thus, it will be essential to introduce radical technological innovations. Nanotechnologies have the potential to produce a significant boost to the improvement of the food system. Within this context, in the next years, a strong challenge will need to be faced regarding developing new and more efficient uses of nutrients in agriculture, being the nutrient use efficiency (NUE) paramount in sustaining high crop productivity without depleting biodiversity, and altering both the natural and agricultural systems. Nutrients leaching causes environmental pollution and water eutrophication, while nutrient excess favors pest and weed widespread. Therefore, it will be mandatory to improve plant nutrition efficiency without affecting agricultural productivity and economic sustainability. A promising alternative consists of the introduction of the so-called nanomaterial enhanced fertilizers and plant growth stimulators. Such innovation includes nanotechnological solutions that can improve nutrient delivery for a more finely tuned, accurate, and saving-resources distribution of nutrients. This review provides a critical view of the latest advances in nanofertilizer research, mainly referring to nano-hydroxyapatite, silica nanoparticles, and chitosan-derived nanostructures

Structural and functional properties of plant mitochondrial F-ATP synthase

The mitochondrial F-ATP synthase is responsible for coupling the transmembrane proton gradient, generated through the inner membrane by the electron transport chain, to the synthesis of ATP. This enzyme shares a basic architecture with the prokaryotic and chloroplast ones, since it is composed of a catalytic head (F1), located in the mitochondrial matrix, a membrane-bound part (FO), together with a central and a peripheral stalk. In this review we compare the structural and functional properties of F-ATP synthase in plant mitochondria with those of yeast and mammals. We also present the physiological impact of the alteration of F-ATP synthase in plants, with a special regard to its involvement in cytoplasmic male sterility. Furthermore, we show the involvement of this enzyme in plant stress responses. Finally, we discuss the role of F-ATP synthase in shaping the curvature of the mitochondrial inner membrane and in permeability transition pore formation

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

LIPOXYGENASE ACTIVITY AND HYDROPEROXIDE FORMATION IN COFFEE (Coffea arabica L.) CHERRIES CULTIVATED BY DIFFERENT AGRONOMIC TECHNIQUES

It is widely accepted that biotic stress induces acidic lipoxygenase (LOX) activity mainly due to the effects of methyl jasmonate [1]. The activation of LOX pathway leads to the production of a large amount of oxidation products, which may affect the coffee organoleptic profile [2]. Hitherto the presence of LOX in coffee (Coffea arabica L.) plants is still scarcely examined [3], therefore the aim of this study was to investigate the possible involvement of this enzyme in oxidative stress of cherries from plants cultivated in organic and conventional manners. Primary and secondary oxidation products were evaluated in three separate cherry fractions: pulp, parchment seed and green coffee. Hydroperoxides, measured by HPLC, were highest in parchment seed and lowest in green coffee, but no significant difference was found between the samples obtained from the two cultivations. However, the pulp fraction from organic cultivated plants had a larger amount of primary peroxidation products, when compared to that from conventionally cultivated plants. The presence of LOX was then examined in all cherry fractions by immunochemical analysis. Only the pulp fraction showed an 84 kDa protein that exhibited a cross-reactivity against a pea LOX antibody. Subsequently, the presence of LOX was confirmed in heavy (28000 g) and light (100000 g) membrane pulp fractions by an enzymatic assay, immunochemical method and hydroperoxide formation. In both organic and conventional cultivation systems, the light membrane showed the highest LOX activity, measured as conjugated diene formation, at pH 8.0. Instead, the heavy membrane fraction highlighted a different pH optimum, which was found to be 5.5 in pulps from organic cultivations and 8.0 in the conventional ones, respectively. All the activities reported were inhibited by nordihydroguaiaretic acid (NDGA) and caffeic acid, well known LOX inhibitors. The NDGA-sensitive hydroperoxide production was simultaneously confirmed by RP-HPLC analysis. Oxidative stress was also detected by evaluating the antioxidant activity in all fractions. A lipophilic extraction, performed using dioxane:water (95:5, v/v), revealed that parchment seeds possessed the lowest antioxidant capacity. On the other hand, a significant difference in the antioxidative capacity was detected in parchment seeds from organic and conventionally cultivated plants. Taken together, these results show, for the first time, the presence of LOX in coffee cherries, whose activity was associated to membrane fractions. Furthermore, the cultivation techniques seem to induce the activation of different isoenzymes. In particular, the organic ones were subjected to an oxidative stress in coffee fruits leading to the expression of an acidic LOX activity, detectable in the pulp fraction. [1] Buzi A., et al. (2004). J. Phytopathol., 152: 34-42. [2] Kohlmann M., et al. (1999) Eur. J. Biochem. 260:885-895. [3] Rojas M.L., et al. (1993). Physiol. Mol. Plant Path., 43: 209-219

CHARACTERIZATION OF LIPASE ACTIVITY IN GREEN COFFEE BEANS DURING STORAGE AND GERMINATION

Lipase, green coffee, germination, storage, lipid Coffee seeds possess an intermediate storage pattern, showing a non-quiescent behaviour characterized by various metabolic reactions occurring during storage. In particular, lipase is the main enzyme involved in the mobilization of triacylglycerols, providing energy and a source of carbon skeleton during early stages of germination. During storage, the triacylglycerols might be involved in the generation of undesirable compounds (known as \u201coff-flavours\u201d), lowering both the viability of coffee seeds and the cup quality. In this work, the soluble protein fraction from coffee seeds and plantlets was extracted by acetone and then was utilised to assay lipase activity. Green coffee beans, harvested in Colombia, were stored at room temperature for 2-3 months, in order to verify the influence of prolonged storage on lipase activity. For germination experiments, the seeds were imbibed for 7 days at 30 \ub0C and transferred in perlite at 28 \ub0C and 90% R.H. for further 3 weeks. Lipase activity was detected by a colorimetric method based on specific degradation of a chromogenic substrate, at pH 8.2. Green coffee seeds exhibited an appreciable lipase activity that was slightly increased during storage. Such an activity was inhibited by tetrahydrolipstatin (THL) in a concentration-dependent manner, while it was slightly stimulated by both EGTA and EDTA. During the germination, after 10, 14, 17 and 21 days, lipase activity showed an initial increase that was followed by a gradual decrease. The effect of the presence or absence of the parchment (seed coat), during the first stages of germination, has also been investigated

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

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