Mitochondrial alternative NADH dehydrogenases NDA1 and NDA2 promote survival of reoxygenation stress in Arabidopsis by safeguarding photosynthesis and limiting ROS generation.

Plant submergence stress is a growing problem for global agriculture. During desubmergence, rising O2 concentrations meet a highly reduced mitochondrial electron transport chain (mETC) in the cells. This combination favors the generation of reactive oxygen species (ROS) by the mitochondria, which at excess can cause damage. The cellular mechanisms underpinning the management of reoxygenation stress are not fully understood. We investigated the role of alternative NADH dehydrogenases (NDs), as components of the alternative mETC in Arabidopsis, in anoxia-reoxygenation stress management. Simultaneous loss of the matrix-facing NDs, NDA1 and NDA2, decreased seedling survival after reoxygenation, while overexpression increased survival. The absence of NDAs led to reduced maximum potential quantum efficiency of photosystem II linking the alternative mETC to photosynthetic function in the chloroplast. NDA1 and NDA2 were induced upon reoxygenation, and transcriptional activation of NDA1 was controlled by the transcription factors ANAC016 and ANAC017 that bind to the mitochondrial dysfunction motif (MDM) in the NDA1 promoter. The absence of NDA1 and NDA2 did not alter recovery of cytosolic ATP levels and NADH : NAD+ ratio at reoxygenation. Rather, the absence of NDAs led to elevated ROS production, while their overexpression limited ROS. Our observations indicate that the control of ROS formation by the alternative mETC is important for photosynthetic recovery and for seedling survival of anoxia-reoxygenation stress.

A stress recovery signaling network for enhanced flooding tolerance in Arabidopsis thaliana.

Abiotic stresses in plants are often transient, and the recovery phase following stress removal is critical. Flooding, a major abiotic stress that negatively impacts plant biodiversity and agriculture, is a sequential stress where tolerance is strongly dependent on viability underwater and during the postflooding period. Here we show that in Arabidopsis thaliana accessions (Bay-0 and Lp2-6), different rates of submergence recovery correlate with submergence tolerance and fecundity. A genome-wide assessment of ribosome-associated transcripts in Bay-0 and Lp2-6 revealed a signaling network regulating recovery processes. Differential recovery between the accessions was related to the activity of three genes: RESPIRATORY BURST OXIDASE HOMOLOG D, SENESCENCE-ASSOCIATED GENE113, and ORESARA1, which function in a regulatory network involving a reactive oxygen species (ROS) burst upon desubmergence and the hormones abscisic acid and ethylene. This regulatory module controls ROS homeostasis, stomatal aperture, and chlorophyll degradation during submergence recovery. This work uncovers a signaling network that regulates recovery processes following flooding to hasten the return to prestress homeostasis.

Restricted suitability of BODIPY for caging in biological applications based on singlet oxygen generation.

Recent studies report the boron-dipyrromethene (BODIPY) moiety to be interesting for caging applications in photopharmacology based on its response to irradiation with wavelengths in the biooptical window. Thus, in a model study, we investigated the meso-methyl-BODIPY caged CDK2 inhibitor AZD5438 and aimed to assess the usability of BODIPY as a photoremovable protecting group in photoresponsive kinase inhibitor applications. Photochemical analysis and biological characterisation in vitro revealed significant limitations of the BODIPY-caged inhibitor concept regarding solubility and uncaging in aqueous solution. Notably, we provide evidence for BODIPY-caged compounds generating singlet oxygen/radicals upon irradiation, followed by photodegradation of the caged compound system. Consequently, instead of caging, a non-specific induction of necrosis in cells suggests the potential usage of BODIPY derivatives for photodynamic approaches.

In-vivo quantification of electron flow through photosystem I - Cyclic electron transport makes up about 35% in a cyanobacterium.

Photosynthetic electron flow, driven by photosystem I and II, provides chemical energy for carbon fixation. In addition to a linear mode a second cyclic route exists, which only involves photosystem I. The exact contributions of linear and cyclic transport are still a matter of debate. Here, we describe the development of a method that allows quantification of electron flow in absolute terms through photosystem I in a photosynthetic organism for the first time. Specific in-vivo protocols allowed to discern the redox states of plastocyanin, P700 and the FeS-clusters including ferredoxin at the acceptor site of PSI in the cyanobacterium Synechocystis sp. PCC 6803 with the near-infrared spectrometer Dual-KLAS/NIR. P700 absorbance changes determined with the Dual-KLAS/NIR correlated linearly with direct determinations of PSI concentrations using EPR. Dark-interval relaxation kinetics measurements (DIRKPSI) were applied to determine electron flow through PSI. Counting electrons from hydrogen oxidation as electron donor to photosystem I in parallel to DIRKPSI measurements confirmed the validity of the method. Electron flow determination by classical PSI yield measurements overestimates electron flow at low light intensities and saturates earlier compared to DIRKPSI. Combination of DIRKPSI with oxygen evolution measurements yielded a proportion of 35% of surplus electrons passing PSI compared to PSII. We attribute these electrons to cyclic electron transport, which is twice as high as assumed for plants. Counting electrons flowing through the photosystems allowed determination of the number of quanta required for photosynthesis to 11 per oxygen produced, which is close to published values.

Analysis of radical formation by EPR in complex starch-protein-lipid model systems and corn extrudates.

The formation of short-lived and stable radicals was investigated using electron paramagnetic resonance (EPR) spectroscopy and compared with hydroperoxides and hexanal in complex starch-protein-lipid model systems, as well as in corn extrudates. Stable radicals were detected directly in ground samples. Short-lived lipid radicals were measured ex situ in ethyl acetate extracts of model systems and extrudates by the use of the spin trap PBN. Significant adduct formation was found after 30 min at 50 °C. During storage, lipid radicals (PBN adducts) increased in model systems. Simulation of EPR spectra from bulk oil demonstrated that mainly alkoxyl radical adducts were detected, to which rapidly decomposing peroxyl radical adducts also contributed. Stable radicals in extrudates were attributed to protein radicals based on g-value of 2.00467 compared with 2.00474 found in model system prepared with zein. The signal intensity of the stable radical remained constant during storage, but increased during extrusion.

Composition of Quillaja saponin extract affects lipid oxidation in oil-in-water emulsions.

Quillaja saponin extract comprises both, surfactants and phenolic compounds, which makes it interesting, in particular, for the formulation of sensitive functional food ingredients and its protection against oxidation. The aim of this study was to investigate the antioxidant effect of Quillaja saponin extract in oil/water emulsions. Emulsions stabilised by Quillaja saponin showed decreased oxidation stability due to naturally occurring metals but stability increased to a great extent when a chelating agent was added. Antioxidant efficiency of the saponin extract was determined photometrically by 2,2'-diphenyl-1-picrylhydrazyl (DPPH) assay and by the use of electron paramagnetic resonance spectroscopy (EPR). EPR spectroscopy applying stable hydrophilic and hydrophobic radicals is advantageous, especially for characterisation of antioxidant efficiency at the interface. The extract showed antioxidant activity towards radicals in both environments, aqueous and hydrophobic, indicating the importance of phenolic compounds for the antioxidant properties of Quillaja saponin extract and their presence at the interface facilitated by saponin molecules.

Whey protein coating increases bilayer rigidity and stability of liposomes in food-like matrices.

Liposomes are suitable for encapsulating lipophilic bioactive compounds, enhancing compound solubility, stability and bioavailability. To enhance physical stability of liposomes in food-like matrices they were coated with positively charged whey protein isolate (WPI). WPI concentration, for a successful coating, was optimised by dynamic light scattering (DLS) and zeta potential measurements. Membrane properties of coated and uncoated vesicles were investigated by electron paramagnetic resonance (EPR) with site-directed and non-site-directed spin probes. Coexistence of two or three simulated spin probe populations indicated a less fluid membrane and higher concentration of water molecules in the phosphate/glycerol moiety with WPI coating. This relies on the insertion of WPI into the membrane, which is favoured by the molten globule state under investigated acidic conditions. Physical stability of liposomes benefits from WPI coating, as indicated by prolonged shelf-life, cancellation of osmotic effects in the presence of salts or sugars and a lower sensitivity towards low pH values during in vitro gastric digestion.

Partitioning of nitroxides in dispersed systems investigated by ultrafiltration, EPR and NMR spectroscopy.

HYPOTHESIS: The partitioning behavior of paramagnetic nitroxides in dispersed systems can be determined by deconvolution of electron paramagnetic resonance (EPR) spectra giving equivalent results with the validated methods of ultrafiltration techniques (UF) and pulsed-field gradient nuclear magnetic resonance spectroscopy (PFG-NMR). EXPERIMENTS: The partitioning behavior of nitroxides with increasing lipophilicity was investigated in anionic, cationic and nonionic micellar systems and 10 wt% o/w emulsions. Apart from EPR spectra deconvolution, the PFG-NMR was used in micellar solutions as a non-destructive approach, while UF based on separation of very small volume of the aqueous phase. FINDINGS: As a function of their substituent and lipophilicity, the proportions of nitroxides that were solubilized in the micellar or emulsion interface increased with increasing nitroxide lipophilicity for all emulsifier used. Comparing the different approaches, EPR deconvolution and UF revealed comparable nitroxide proportions that were solubilized in the interfaces. Those proportions were higher than found with PFG-NMR. For PFG-NMR self-diffusion experiments the reduced nitroxides were used revealing a high dynamic of hydroxylamines and emulsifiers. Deconvolution of EPR spectra turned out to be the preferred method for measuring the partitioning behavior of paramagnetic molecules as it enables distinguishing between several populations at their individual solubilization sites.

Reactive oxygen species mediate growth and death in submerged plants.

Aquatic and semi-aquatic plants are well adapted to survive partial or complete submergence which is commonly accompanied by oxygen deprivation. The gaseous hormone ethylene controls a number of adaptive responses to submergence including adventitious root growth and aerenchyma formation. Reactive oxygen species (ROS) act as signaling intermediates in ethylene-controlled submergence adaptation and possibly also independent of ethylene. ROS levels are controlled by synthesis, enzymatic metabolism, and non-enzymatic scavenging. While the actors are by and large known, we still have to learn about altered ROS at the subcellular level and how they are brought about, and the signaling cascades that trigger a specific response. This review briefly summarizes our knowledge on the contribution of ROS to submergence adaptation and describes spectrophotometrical, histochemical, and live cell imaging detection methods that have been used to study changes in ROS abundance. Electron paramagnetic resonance (EPR) spectroscopy is introduced as a method that allows identification and quantification of specific ROS in cell compartments. The use of advanced technologies such as EPR spectroscopy will be necessary to untangle the intricate and partially interwoven signaling networks of ethylene and ROS.