An Optimized in situ Quantification Method of Leaf H2O2 Unveils Interaction Dynamics of Pathogenic and Beneficial Bacteria in Wheat

Hydrogen peroxide (H2O2) functions as an important signaling molecule in plants during biotic interactions. However, the extent to which H2O2 accumulates during these interactions and its implications in the development of disease symptoms is unclear. In this work, we provide a step-by-step optimized protocol for in situ quantification of relative H2O2 concentrations in wheat leaves infected with the pathogenic bacterium Pseudomonas syringae pv. atrofaciens (Psa), either alone or in the presence of the beneficial bacterium Herbaspirillum seropedicae (RAM10). This protocol involved the use of 3-3'diaminobenzidine (DAB) staining method combined with image processing to conduct deconvolution and downstream analysis of the digitalized leaf image. The application of a linear regression model allowed to relate the intensity of the pixels resulting from DAB staining with a given concentration of H2O2. Decreasing H2O2 accumulation patterns were detected at increasing distances from the site of pathogen infection, and H2O2 concentrations were different depending on the bacterial combinations tested. Notably, Psa-challenged plants in presence of RAM10 accumulated less H2O2 in the leaf and showed reduced necrotic symptoms, pointing to a potential role of RAM10 in reducing pathogen-triggered H2O2 levels in young wheat plants.info:eu-repo/semantics/publishedVersio

Photoprotection and optimization of sucrose usage contribute to faster recovery of photosynthesis after water deficit at high temperatures in wheat

Plants are increasingly exposed to events of elevated temperature and water deficit, which threaten crop productivity. Understanding the ability to rapidly recover from abiotic stress, restoring carbon assimilation and biomass production, is important to unravel crop climate resilience. This study compared the photosynthetic performance of two Triticum aestivum L. cultivars, Sokoll and Paragon, adapted to the climate of Mexico and UK, respectively, exposed to one week water deficit and high temperatures, in isolation or combination. Measurements included photosynthetic assimilation rate, stomatal conductance, in vitro activities of Rubisco (EC 4.1.1.39) and invertase (INV, EC 3.2.1.26), antioxidant capacity and chlorophyll a fluorescence. In both genotypes, under elevated temperatures and water deficit (WD38℃), the photosynthetic limitations were mainly due to stomatal restrictions and to a decrease in the electron transport rate. Chlorophyll a fluorescence parameters clearly indicate differences between the two genotypes in the photoprotection when subjected to WD38℃ and showed faster recovery of Paragon after stress relief. The activity of the cytosolic invertase (CytINV) under these stress conditions was strongly related to the fast photosynthesis recovery of Paragon. Taken together, the results suggest that optimal sucrose export/utilization and increased photoprotection of the electron transport machinery are important components to limit yield fluctuations due to water shortage and elevated temperatures

High-throughput phenotyping of physiological traits for wheat resilience to high temperature and drought stress

Interannual and local fluctuations in wheat crop yield are majorly explained by abiotic constraints. Heatwaves and drought, which are among the top stressors, commonly co-occur, and their frequency is increasing with global climate change. High-throughput methods were optimised to phenotype wheat plants under controlled water deficit and high temperature, with the aim to identify phenotypic traits conferring adaptative stress responses. Wheat plants of 10 genotypes were grown in a fully automated plant facility under 25/18ºC day/night for 30 days, and then the temperature was increased for seven days (38/31ºC day/night) while maintaining half of the plants well irrigated and half at 30% field capacity. Thermal and multispectral images and pot weights were registered twice daily. At the end of the experiment, key metabolites and enzyme activities from the carbohydrate and antioxidant metabolisms were quantified. Regression machine learning models were successfully established to predict plant biomass using image-extracted parameters. Evapotranspiration traits expressed significant genotype-environment interactions (GxE) when acclimatization to stress was continuously monitored. Consequently, transpiration efficiency was essential to maintain the balance between water-saving strategies and biomass production in wheat under water deficit and high temperature. Stress tolerance included changes in the carbohydrate metabolism, particularly in the sucrolytic and glycolytic pathways, and in the antioxidant metabolism. The observed genetic differences in sensitivity to high temperature and water deficit can be exploited in breeding programs to improve wheat resilience to climate change. [Abstract copyright: © The Author(s) 2022. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Ecoengineering Solutions for the Impairment of Spreading and Growth of Invasive Spartina patens in Mediterranean Salt Marshes

The invasion of natural communities by non-indigenous species represents one of the most serious threats to biodiversity. Understanding the ecophysiology of invasive species can provide insights into potential physiological handicaps relative to native species. By doing so, we can leverage the development of ecoengineering solutions for the removal of non-indigenous species, preferably using non-chemical methods. Spartina patens is a known invasive species of cordgrass aggressively proliferating in Mediterranean salt marshes, producing impenetrable monospecific stands. As its occurrence is delimited by the upper high tide water level, we hypothesized that S. patens is intolerant to waterlogging. Therefore, we developed a field experiment where strands of S. patens were kept waterlogged over the entire tidal cycle for 30 days. At the end of the experimental period, plants in the trial plots exhibited severe stress symptoms at different physiological levels compared with control plots (no intervention). At the photobiological level, intervened plants exhibited lower efficiency in producing chemical energy from light, whilst at the biochemical level waterlogging impaired the antioxidant system and increased lipid peroxidation products. Furthermore, the application of chlorophyll a pulse amplitude modulated (PAM) fluorometry, a non-invasive technique, allowed us to evaluate the effectiveness of the implemented measures, being the tool that provided the best separation between the control and intervened population. Considering the physiological traits observed here, ecoengineering solutions based on increased waterlogging of S. patens stands, can be a low-cost and efficient measure to reduce the spreading and growth of this invasive species in the Mediterranean and other salt marshes worldwide with little disturbance.info:eu-repo/semantics/publishedVersio

Design of innovative clothing for pressure injury prevention: end-user evaluation in a mixed-methods study

The global relevance of pressure injury (PI) prevention technologies arise from their impact on the quality of life of people with limited mobility and the costs associated with treating these preventable injuries. The purpose of this mixed methods study is to evaluate the design of a prototype integrating Smart Health Textiles for PI prevention based on feedback from specialist nurses who care for individuals who are prone to or have PIs. This is a mixed methods study. A structured questionnaire was conducted as part of an evaluation of a prototype garment for the prevention of PIs. This questionnaire was applied during the evaluation of the prototype and afterwards focus group discussions were held with experts. Descriptive statistics techniques were used to analyze the data and thematic and integrated content analysis was conducted through concomitant triangulation. Nineteen nurses took part, aged 30 to 39 years (52.6%) and with 12.31 ± 8.96 years of experience. Participants showed that the prototype required more manipulation and physical effort, which interfered its usefulness, in addition to presenting difficulties with the openings and the material of the closure system, which interfered with the ease of use and learning. Overall satisfaction with the product was moderate, with some areas for improvement found, such as satisfaction, recommendations to colleagues, and pleasantness of use. It is concluded that areas for improvement have been found in all dimensions, including in the design of openings and the choice of materials. These findings supply significant insights for improving clothing to meet the needs of healthcare professionals and patients.The 4NoPressure project was co-financed by the Operational Program for Competitiveness and Internationalization (COMPETE 2020) under the PORTUGAL 2020 Partnership Agreement, with support from the European Regional Development Fund (ERDF), reference number POCI-01-0247- FEDER-039869

Metamitron and Shade Effects on Leaf Physiology and Thinning Efficacy of Malus × domestica Borkh

Thinning strategies, namely shade or photosynthetic inhibitors, rely on the reduction of carbon supply to the fruit below the demand, causing fruit abscission. In order to clarify the subject, seven field trials were carried out in Lleida, Girona, and Sint-Truiden (2017 + 2018), using orchards of ‘Golden’ and ‘Gala’ apple trees. At the stage of 9–14-mm fruit diameter, four treatments were implemented: (A) CTR-control, trees under natural environmental conditions; (B) SN-shaded trees, trees above which shading nets reducing 50% of irradiance were installed 24 h after metamitron application date—without application of metamitron—and removed after five days; (C) MET-trees sprayed with 247.5 ppm of metamitron; (D) MET + SN-trees submitted to the combined exposure to metamitron application and shading nets. Low radiation significantly increased metamitron absorption (36–53% in the three locations in 2018) and reduced its degradation. Net photosynthesis and stomatal conductance were strongly reduced in all treatments, with minimum values 2 days after spraying (DAS) and incomplete recovery 10 DAS in MET + SN. All treatments resulted in leaf sucrose and sorbitol decreases, leading to a negative carbon balance. SN and MET + SN promoted the highest thinning efficacy, increasing fruit weight and size, with MET + SN causing over-thinning in some trials. Leaf antioxidant enzymes showed moderate changes in activity increases under MET or MET + SN, accompanied by a rise of glutathione content and a reduction in ascorbate, however without lipid peroxidation. This work shows that environmental conditions, such as cloudy days, must be carefully considered upon metamitron application, since the low irradiance enhances metamitron efficacy and may cause over-thinninginfo:eu-repo/semantics/publishedVersio

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Measuring Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays:NADH-linked and 14C-based assays of Rubisco activity

Rubisco is central to carbon assimilation and efforts to improve the efficiency and sustainability of crop production have spurred interest in phenotyping Rubisco activity. We tested the hypothesis that microtiter plate-based methods provide comparable results to those obtained with the radiometric assay that measures the incorporation of 14CO2 into 3-phosphoglycerate (3-PGA). Three NADH-linked assays were tested that use alternative coupling enzymes: glyceraldehyde-3-phosphate-dehydrogenase and glycerolphosphate-dehydrogenase (GAPDH-GlyPDH); phosphoenolpyruvate-carboxylase and malate-dehydrogenase (PEPC-MDH); pyruvate-kinase and lactate-dehydrogenase (PK-LDH). To date there has been no thorough evaluation of their reliability by comparison with the 14C-based method. The three NADH-linked assays were used in parallel to estimate (1) the 3-PGA concentration response curve of NADH oxidation, (2) the Michaelis-Menten constant for RuBP, (3) fully active and inhibited Rubisco activities, and (4) Rubisco initial and total activities in fully illuminated and shaded leaves. All three methods correlated strongly with the 14C-based method, and the PK-LDH method showed a strong correlation and was the cheapest method. PEPC-MDH would be a suitable option for situations where ADP/ATP might interfere with the assay. GAPDH-GlyPDH proved more laborious than the other methods. Thus, we recommend the PK-LDH as a reliable, cheaper and higher throughput method to phenotype Rubisco activity for crop improvement efforts

Protocols from Sales et al. (2020) Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays

This collection of protocols includes four assays for measuring Rubisco activity that are used and discussed in the manuscript "Rubisco activity: challenges and opportunities of NADH-linked microtiter plate-based and 14C-based assays" (Sales et al. 2020). Three of the protocols are NADH-linked microtiter plate-based assays for Rubisco activity that use alternative coupling enzymes: 1. GAPDH-GlyPDH, glyceraldehyde-3-phosphate-dehydrogenase and glycerolphosphate-dehydrogenase; 2. PEPC-MDH, phosphoenolpyruvate-carboxylase and malate-dehydrogenase; 3. PK-LDH, pyruvate-kinase and lactate-dehydrogenase. The radiometric assay for Rubisco activity measures the incorporation of 14CO2 into 3-phosphoglycerate (3-PGA). The fifth protocol describes the purification of 2,3-bisphosphate-dependent phosphoglycerate mutase (dPGM), which is required for the NADH-linked microtiter plate-based assays 2 (PEPC-MDH) and 3 (PK-LDH)