Polystyrene nanoparticles induce concerted response of plant defense mechanisms in plant cells.

Recent advances in knowledge suggest that micro- and nanoplastics pose a threat to plant health, however, the responses of plants to this stressor are not well-known. Here we examined the response of plant cell defence mechanisms to nanoparticles of commonly used plastic, polystyrene. We used plant cell cultures of widely cultivated plants, the monocots wheat and barley (Triticum aestivum L., Hordeum vulgare L.) and the dicots carrot and tomato (Daucus carota L., Solanum lycopersicum L.). We measured the activities of enzymes involved in the scavenging of reactive oxygen species and nonenzymatic antioxidants and we estimated potential damages in plant cell structures and functioning via lipid peroxidation and DNA methylation levels. Our results demonstrate that the mode of action of polystyrene nanoparticles on plant cells involves oxidative stress. However, the changes in plant defence mechanisms are dependent on plant species, exposure time and nanoplastic concentrations. In general, both monocots showed similar responses to nanoplastics, but the carrot followed more the response of monocots than a second dicot, a tomato. Higher H2O2, lipid peroxidation and lower enzyme activities scavenging H2O2 suggest that tomato cells may be more susceptible to polystyrene-induced stress. In conclusion, polystyrene nanoplastics induce oxidative stress and the response of the plant defense mechanisms involving several chain reactions leading to oxidoreductive homeostasis.

Fast and reliable method to estimate global DNA methylation in plants and fungi with high-pressure liquid chromatography (HPLC)-ultraviolet detection and even more sensitive one with HPLC-mass spectrometry.

DNA (Deoxyribonucleic acid) methylation is one of the epigenetic modifications of DNA, acting as a bridge between genotype and phenotype. Thus, disruption of DNA methylation pattern has tremendous consequences for organism development. Current methods to determine DNA methylation suffer from methodological drawbacks like high requirement of DNA and poor reproducibility of chromatograms. Here we provide a fast and reliable method using high-pressure liquid chromatography (HPLC)-ultraviolet (UV) detector and even more sensitive one with HPLC- mass spectrometry (MS) and we test this method with various plant and fungal DNA isolates. We optimized the preparation of the DNA degradation step to decrease background noise, we improved separation conditions to provide reliable and reproducible chromatograms and conditions to measure nucleotides in HPLC-MS. We showed that global DNA methylation level can be accurately and reproducibly measured with as little as 0.2 µM for HPLC-UV and 0.02 µM for HPLC-MS of methylated cytosine.

The Effects of Combined Abiotic and Pathogen Stress in Plants: Insights From Salinity and Pseudomonas syringae pv lachrymans Interaction in Cucumber.

Plants are often challenged by abiotic and biotic stresses acting in combination and the response to combinatorial stress differs from that triggered by each factor individually. Although salinity and pathogens are major stressors limiting plant growth and productivity worldwide, their interaction is poorly understood. The reactions to pathogens overlap with those to abiotic stresses, and reactive oxygen species (ROS) and stress hormones represent central nodes in the interacting signaling pathways. Usually, abiotic stress negatively affects plant susceptibility to disease. Specific focus of this review is on cucumber plants exposed to salt stress and thereafter infected with Pseudomonas syringae pv lachrymans (Psl). We addressed this problem by discussing the changes in photochemistry, the antioxidant system, primary carbon metabolism, salicylic acid (SA) and abscisic acid (ABA) contents. Salt-treated plants were more prone to infection and this effect was determined by changes in the hormonal and redox balance as well as the carboxylate metabolism and activities of some NADPH-generating enzymes. Our detailed understanding of the interactive effects of biotic and abiotic stresses is fundamental to achieve enhanced tolerance to combination stress in agronomically important crops.

Primary carbon metabolism-related changes in cucumber exposed to single and sequential treatments with salt stress and bacterial infection.

This study examines how salt stress interacts with bacterial infection at the metabolic level. We measured chlorophyll a fluorescence as well as profiles of phosphoenolpyruvate carboxylase (PEPC), NADP-malic enzyme (NADP-ME), NADP-isocitrate dehydrogenase (NADP-ICDH) and fumarase activities, malic and citric acids contents and the expression of NADP-ICDH and NADP-ME in the organ-dependent (root vs leaves) response of cucumber plants exposed to individual or sequential action of salt stress (50 mM or 100 mM NaCl) and Pseudomonas syringae pv lachrymans (Psl). NaCl treatment, Psl infection and the combination of these stresses caused disturbances in the activity of photosystem II which were suggested to specifically transmit the stress signals. PEPC and NADP-ME were induced in cucumber plants under stress, confirming that in C3 plants they function in defence responses. The profiles of malate and citrate contents, PEPC as well as NADP-ICDH and NADP-ME activities and gene expression in response to a combination of salt and pathogen stresses differed from those provoked by individual stress with respect to the direction, intensity and timing. The results indicated that the most pronounced defence response related to the readjustment of the carbon metabolism was observed in the leaves of plants exposed to combined stress. Intense activity changes of NADPH-generating enzymes, NADP-ICDH and NADP-ME, characterized the tailored response to combined stress and could be important for the integration of defence mechanisms between organs.

Automated image analysis for quantification of reactive oxygen species in plant leaves.

The paper presents an image processing method for the quantitative assessment of ROS accumulation areas in leaves stained with DAB or NBT for H2O2 and O2- detection, respectively. Three types of images determined by the combination of staining method and background color are considered. The method is based on the principle of supervised machine learning with manually labeled image patterns used for training. The method's algorithm is developed as a JavaScript macro in the public domain Fiji (ImageJ) environment. It allows to select the stained regions of ROS-mediated histochemical reactions, subsequently fractionated according to the weak, medium and intense staining intensity and thus ROS accumulation. It also evaluates total leaf blade area. The precision of ROS accumulation area detection is validated by the Dice Similarity Coefficient in the case of manual patterns. The proposed framework reduces the computation complexity, once prepared, requires less image processing expertise than the competitive methods and represents a routine quantitative imaging assay for a general histochemical image classification.