Hydrogen peroxide (H 2 O 2 ) is ubiquitous in cells and at the centre of developmental programmes and environmental responses. Its chemistry in cells makes H 2 O 2 notoriously hard to detect dynamically, specifically and at high resolution. Genetically encoded sensors overcome persistent shortcomings, but pH sensitivity, silencing of expression and a limited concept of sensor behaviour in vivo have hampered any meaningful H 2 O 2 sensing in living plants.We established H 2 O 2 monitoring in the cytosol and the mitochondria of Arabidopsis with the fusion protein roGFP2-Orp1 using confocal microscopy and multiwell fluorimetry.We confirmed sensor oxidation by H 2 O 2 , show insensitivity to physiological pH changes, and demonstrated that glutathione dominates sensor reduction in vivo. We showed the responsiveness of the sensor to exogenous H 2 O 2 , pharmacologically-induced H 2 O 2 release, and genetic interference with the antioxidant machinery in living Arabidopsis tissues. Monitoring intracellular H 2 O 2 dynamics in response to elicitor exposure reveals the late and prolonged impact of the oxidative burst in the cytosol that is modified in redox mutants.We provided a well defined toolkit for H 2 O 2 monitoring in planta and showed that intracellular H 2 O 2 measurements only carry meaning in the context of the endogenous thiol redox systems. This opens new possibilities to dissect plant H 2 O 2 dynamics and redox regulation, including intracellular NADPH oxidase-mediated ROS signalling.
Potato ( Solanum tuberosum L.) is the worldwide most important nongrain crop after wheat, rice, and maize. The autotetraploidy of the modern commercial potato makes breeding of new resistant and high-yielding cultivars challenging due to complicated and time-consuming identification and selection processes of desired crop features. On the other hand, plant protection of existing cultivars using conventional synthetic pesticides is increasingly restricted due to safety issues for both consumers and the environment. Chitosan is known to display antimicrobial activity against a broad range of plant pathogens and shows the ability to trigger resistance in plants by elicitation of defense responses. As chitosan is a renewable, biodegradable and nontoxic compound, it is considered as a promising next-generation plant-protecting agent. However, the molecular and cellular modes of action of chitosan treatment are not yet understood. In this study, transcriptional changes in chitosan-treated potato leaves were investigated via RNA sequencing. Leaves treated with a well-defined chitosan polymer at low concentration were harvested 2 and 5 h after treatment and their expression profile was compared against water-treated control plants. We observed 32 differentially expressed genes (fold change ≥ 1; p-value ≤ 0.05) 2 h after treatment and 83 differentially expressed genes 5 h after treatment. Enrichment analysis mainly revealed gene modulation associated with electron transfer chains in chloroplasts and mitochondria, accompanied by the upregulation of only a very limited number of genes directly related to defense. As chitosan positively influences plant growth, yield, and resistance, we conclude that activation of electron transfer might result in the crosstalk of different organelles via redox signals to activate immune responses in preparation for pathogen attack, concomitantly resulting in a generally improved metabolic state, fostering plant growth and development. This conclusion is supported by the rapid and transient production of reactive oxygen species in a typical oxidative burst in the potato leaves upon chitosan treatment. This study furthers our knowledge on the mode of action of chitosan as a plant-protecting agent, as a prerequisite for improving its ability to replace or reduce the use of less environmentally friendly agro-chemicals.
In the present study, a well-defined set of chitosans, with different degrees of acetylation (DA) and degrees of polymerization (DP), were processed by solution electrospraying from a water-based solvent. The solution properties, in terms of surface tension, conductivity, viscosity, and pH, were characterized and related to the physico-chemical properties of the chitosans. It was observed that both DA and DP values of a given chitosan, in combination with biopolymer concentration, mainly determined solution viscosity. This was, in turn, the major driving factor that defined the electrosprayability of chitosan. In addition, the physico-chemical properties of chitosans highly influenced solution conductivity and results indicated that the chitosan solutions with low or low-to-medium values of conductivity were the most optimal for electrospraying. The results obtained here also demonstrate that a good process control can be achieved by adjusting the working conditions, i.e. applied voltage, flow-rate, and tip-to-collector distance. Finally, it was also shown that electrosprayability of chitosan with inadequate physico-chemical properties can be improved by solution mixing of very different kinds of this polysaccharide. The resultant electrosprayed submicron chitosan capsules can be applied for encapsulation of food additives and to develop bioactive coatings of interest in food packaging, where these particles alone or containing functional ingredients can be released from the package into the food to promote a health benefit.
Several recent studies revealed the significant contribution of intensive agriculture to global climate change and biodiversity decline. However, synthetic pesticides and fertilizers, which are among the main reasons for these negative effects, are required to achieve the high performance of elite crops needed to feed the growing world population. Modern agro-biologics, such as biopesticides, biostimulants, and biofertilizers are intended to replace or reduce the current agro-chemicals, but the former are often difficult to combine with the latter. Chitosans, produced from the fisheries’ byproduct chitin, are among the most promising agro-biologics, and copper fungicides are among the most widely used plant protectants in organic farming. However, the two active ingredients tend to form precipitates, hindering product development. Here, we show that partial hydrolysis of a chitosan polymer can yield a mixture of smaller polymers and oligomers that act synergistically in their antifungal activity. The low molecular weight (Mw) of this hydrolysate allows its combination with copper acetate, again leading to a synergistic effect. Combined, these synergies allow a 50% reduction in copper concentration, while maintaining the antifungal activity. This is potentially a significant step towards a more sustainable agriculture.
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