New Design of an Activity Assay Suitable for High-Throughput Screening of Substrates and Inhibitors of the Mitochondrial Amidoxime Reducing Component (mARC).

The mitochondrial amidoxime-reducing component (mARC) is one of the simplest molybdenum-containing enzymes. mARC is among a few known reducing enzymes playing an important role in drug metabolism in mammals. Here, an assay based on the fluorescence of NADH is reported for the rapid detection of substrates and potential inhibitors of mARC. So far unknown inhibitors might be useful for the development of drugs assigned to nonalcoholic fatty liver disease (NAFLD) and similar diseases. Kinetics of reactions catalyzed by mARC can be recorded with high sensitivity and precision. On a microtiter plate scale, the assay presented could be applied for high-throughput screening of substance libraries and detection of novel mARC substrate candidates. For instance, molnupiravir was also identified as a new substrate by this assay. For better comparison for such substances, the inhibitor or substrate-to-BAO ratio was introduced. After normalization of enzyme activities to the standard benzamidoxime, substrates can reproducibly be classified.

Peroxide-Induced Liberation of Iron from Heme Switches Catalysis during Luminol Reaction and Causes Loss of Light and Heterodyning of Luminescence Kinetics.

The peroxidation of luminol yields bright luminescence when the reaction is catalyzed by heme proteins. However, an excess of peroxide leads to less light and altered luminescence kinetics, an effect commonly referred to as "suicide inactivation". The aim of this study is to present the molecular processes causing this effect. A comprehensive set of data reported here demonstrates that suicide inactivation is due to a peroxide-induced liberation of iron from its coordinating porphyrin. Liberated iron launches catalysis of the reaction at much lower efficiency. The light-yielding efficiencies of different organic and inorganic catalysts are precisely quantified and compared. It is shown that the catalysis by free iron involves superoxide. This is explained by the formation of a ferryl-oxo-iron complex. In this context, a complete reaction mechanism involving a modified Fenton-Haber-Weiss cycle is proposed for the first time. The switch from the highly efficient biogenically catalyzed luminescence to a less efficient inorganically catalyzed reaction is accompanied by a transition from "flash-type" to "glow-type" luminescence kinetics. Ethylenediaminetetraacetic acid-mediated chelation of iron is used to demonstrate this effect and to separate both kinetics. The explanation of kinetic heterodyning is underpinned by mathematical modeling. The results are able to explain the as yet unexplained phenomena discussed in the less recent literature and to settle disputes about them. It is concluded that peroxide concentrations exceeding the level tolerated by the catalyzing heme protein negatively impact performance and precision of luminol-based assays, where the light yield is used as a quantitative measure for analyte concentrations.

Redox Modulators Determine Luminol Luminescence Generated by Porphyrin-Coordinated Iron and May Repress "Suicide Inactivation".

Iron porphyrin catalysts of the luminol reaction (horseradish peroxidase, hemoglobin, cytochrome c, and hemin) interact with diverse reducing compounds. Here, it is demonstrated how the chemiluminescence yield is modulated by such interactions. The compounds accepted as substrates protect the catalyst against the "suicide inactivation" caused by high peroxide concentrations. The reducing agents not accepted by the catalyst inhibit light production either by generating a futile redox cycle of the luminophore or by irreversibly inactivating the catalytic center. In the case of a futile cycle, light emission resumes as soon as the reducing agents in the reaction are consumed, whereas with an irreversible inactivation, light emission does not recover. The characteristics of luminescence enhancement and quenching depending on interfering agents are also reported here. They reveal details about the relative redox potentials of the involved compounds. It is discussed how this should be considered when the luminol reaction is used for quantitative analyses and when unpurified samples with a broad compound matrix are to be assayed.

Calcium improves apoplastic-cytosolic ion homeostasis in salt-stressed Vicia faba leaves.

Salinity disturbs both apoplastic and cytosolic Ca2+ and pH ([Ca2+]apo, [Ca2+]cyt, pHapo and pHcyt) homeostasis, and decreases plant growth. Seedlings of Vicia faba L. cv. Fuego were cultivated in hydroponics for 7 days under control, salinity (S), extra Ca (Ca) or salinity with extra Ca (S+Ca) conditions. The [Ca2+]apo, and pHapo in the leaves were then recorded in parallel by a pseudoratiometric method, described here for the first time. Lower [Ca2+]apo and higher pHapo were obtained under salinity, whereas extra Ca supply increased the [Ca2+]apo and acidified the pHapo. Moreover, the ratiometric imaging recorded that [Ca2+]cyt and pHcyt were highest in S+Ca plants and lowest in control plants. After all pretreatments, direct addition of NaC6H11O7 to leaves induced a decrease in [Ca2+]apo in control and S+Ca plants, but not in S and Ca plants, and only slightly affected pHapo. Addition of NaCl increased [Ca2+]cyt in protoplasts from all plants but only transiently in protoplasts from S+Ca plants. Addition of NaCl decreased pHcyt in protoplasts from Ca-pretreated plants. We conclude that Ca supply improves both apoplastic and cytosolic ion homeostasis. In addition, NaC6H11O7 probably causes transport of Ca from the apoplast into the cytosol, thereby leading to a higher resting [Ca2+]cyt.

Bacterially produced Pt-GFP as ratiometric dual-excitation sensor for in planta mapping of leaf apoplastic pH in intact Avena sativa and Vicia faba.

BACKGROUND: Ratiometric analysis with H(+)-sensitive fluorescent sensors is a suitable approach for monitoring apoplastic pH dynamics. For the acidic range, the acidotropic dual-excitation dye Oregon Green 488 is an excellent pH sensor. Long lasting (hours) recordings of apoplastic pH in the near neutral range, however, are more problematic because suitable pH indicators that combine a good pH responsiveness at a near neutral pH with a high photostability are lacking. The fluorescent pH reporter protein from Ptilosarcus gurneyi (Pt-GFP) comprises both properties. But, as a genetically encoded indicator and expressed by the plant itself, it can be used almost exclusively in readily transformed plants. In this study we present a novel approach and use purified recombinant indicators for measuring ion concentrations in the apoplast of crop plants such as Vicia faba L. and Avena sativa L. RESULTS: Pt-GFP was purified using a bacterial expression system and subsequently loaded through stomata into the leaf apoplast of intact plants. Imaging verified the apoplastic localization of Pt-GFP and excluded its presence in the symplast. The pH-dependent emission signal stood out clearly from the background. PtGFP is highly photostable, allowing ratiometric measurements over hours. By using this approach, a chloride-induced alkalinizations of the apoplast was demonstrated for the first in oat. CONCLUSIONS: Pt-GFP appears to be an excellent sensor for the quantification of leaf apoplastic pH in the neutral range. The presented approach encourages to also use other genetically encoded biosensors for spatiotemporal mapping of apoplastic ion dynamics.

A9C sensitive Cl⁻-accumulation in A. thaliana root cells during salt stress is controlled by internal and external calcium.

The involvement of chloride in salt stress symptoms and salt tolerance mechanisms in plants has been less investigated in the past. Therefore, we studied the salt-induced chloride influx in Arabidopsis expressing the GFP-based anion indicator Clomeleon. High salt concentrations induce two phases of chloride influx. The fast kinetic phase is likely caused by membrane depolarization, and is assumed to be mediated by channels. This is followed by a slower "saturation" phase, where chloride is accumulated in the cytoplasm. Both phases of chloride uptake are dependent on the presence of external calcium. In general: with high [Ca (2+)] less chloride is accumulated in the cytoplasm. Surprisingly, also the internal calcium availability has an impact on chloride transport. A complete block of the second phase of chloride influx is achieved by the anion channel blocker A9C and trivalent cations (La (3+), Gd (3+), and Al (3+)). Other channel blockers and diuretics were found to inhibit the process partially. The results suggest that several transporter species are involved here, including electroneutral cation-chloride-cotransporters, and a part of chloride possibly enters the cells through cation channels after salt application.

Calcium promotes activity and confers heat stability on plant peroxidases.

In this paper we demonstrate how peroxidase (PO) activities and their heat stability correlate with the availability of free Ca(2+) ions. Calcium ions work as a molecular switch for PO activity and exert a protective function, rendering POs heat stable. The concentration ranges of these two activities differ markedly. POs are activated by µM Ca(2+) concentration ranges, whereas heat stabilization is observed in the nM range. This suggests the existence of different Ca(2+) binding sites. The heat stability of POs depends on the source plant species. Terrestrial plants have POs that exhibit higher temperature stability than those POs from limnic and marine plants. Different POs from a single species can differ in terms of heat stability. The abundance of different POs within a plant is dependent on age and developmental stage. The heat stability of a PO does not necessarily correlate with the maximum temperature the source species is usually exposed to in its natural habitat. This raises questions on the role of POs in the heat tolerance of plants. Consequently, detailed investigations are needed to identify and characterize individual POs, with regard to their genetic origin, subcellular expression, tissue abundance, developmental emergence and their functions in innate and acquired heat tolerance.

Apoplastic calcium executes a shut-down function on plant peroxidases: a hypothesis.

Recently it was demonstrated that PO activity is switched by calcium within the typical range of apoplastic free calcium concentrations (Plieth and Vollbehr, Plant Signal Behav 2012;7: 650-660). The heat stability of POs is also dependent on calcium. Here, a scenario is put forward which assigns calcium a switch-off function under heat: Peroxidases are switched off by heat stress-triggered apoplastic calcium depletion. It is assumed that this initiates apoplastic accumulation of reactive oxygen species (ROS) and eventually triggers a self-amplifying cascade of cellular events involving plasma membrane ion transport. Calcium depletion-initiated ROS accumulation (CaDIRA) may also trigger signal percolation and the formation of systemic responses to many different stress factors in plants. This hypothesis can explain some as yet unexplained observations.

Determination of oxidative stress in wheat leaves as influenced by boron toxicity and NaCl stress.

Boron (B) toxicity symptoms are visible in the form of necrotic spots and may worsen the oxidative stress caused by salinity. Hence, the interactive effects of combined salinity and B toxicity stress on antioxidative activities (TAC, LUPO, SOSA, CAT, and GR) were investigated by novel luminescence assays and standard photometric procedures. Wheat plants grown under hydroponic conditions were treated with 2.5 µM H3BO3 (control), 75 mM NaCl, 200 µM H3BO3, or 75 mM NaCl + 200 µM H3BO3, and analysed 6 weeks after germination. Shoot fresh weight (FW), shoot dry weight (DW), and relative water content (RWC) were significantly reduced, whereas the antioxidative activity of all enzymes was increased under salinity compared with the control. High B application led to necrotic leaf spots but did not influence growth parameters. Following NaCl + B treatment, shoot DW, RWC, SOSA, GR, and CAT activities remained the same compared with NaCl alone, whereas the TAC and LUPO activities were increased under the combined stress compared with NaCl alone. However, shoot FW was significantly reduced under NaCl + B compared with NaCl alone, as an additive effect of combined stress. Thus, we found an adjustment of antioxidative enzyme activity to the interactive effects of NaCl and high B. The stress factor "salt" mainly produced more oxidative stress than that of the factor "high B". Furthermore, addition of higher B in the presence of NaCl increases TAC and LUPO demonstrating that increased LUPO activity is an important physiological response in wheat plants against multiple stresses.

Total low-molecular-weight antioxidants as a summary parameter, quantified in biological samples by a chemiluminescence inhibition assay.

Aerobic metabolism requires a complex antioxidative system to balance reactive oxygen species (ROS). When in excess, ROS disrupt cellular activities and molecular structures. Owing to the variety of ROS, there are different antioxidative activities that require various tests for their detection. The so-called 'total antioxidative capacity' inhibition assay presented in this paper can be used to quantify the overall activity of low-molecular-weight antioxidants (AOs) in biological samples. The assay is based on enhanced horseradish peroxidase-catalyzed luminol chemiluminescence. It can be fine-tuned so that the biological samples meet the requirements of the light detector. A detailed protocol describing all relevant parameters is provided. The procedure is quick, inexpensive and reproducible. The assay can be used with diverse biological materials such as plant extracts and blood plasma. Hence, it is applicable to fields as diverse as crop breeding, medical diagnostics or food sciences. The time needed for the assay depends on the number of samples and their AO content. The protocol takes one working day to complete when five samples with five replicates are measured sequentially.

A coelenterazine-based luminescence assay to quantify high-molecular-weight superoxide anion scavenger activities.

In all living cells, levels of reactive oxygen species are kept in check by antioxidative activities. Superoxide radicals are dismutated by superoxide dismutases, by other enzymes and by nonenzymatic compounds. This protocol describes the quantification of superoxide scavenging activities (SOSA). It is based on the inhibition of chemiluminescence emitted by coelenterazine when oxidized by superoxide. SOSA is a summary parameter comprising all high-molecular-weight superoxide scavengers in a biological sample. Enzymes and nonenzymatic scavengers can also be distinguished. The SOSA assay is quick, reproducible and applicable to fields as diverse as medical diagnostics, food sciences, or agriculture. The protocol presented here requires about 2 working days to complete.

Signal percolation through plants and the shape of the calcium signature.

Plants respond to almost any kind of external stimulus with transients in their cytoplasmic free calcium concentration ([Ca(2+)](c)). A huge variety of kinetics recorded by optical techniques has been reported in the past. This variety has been credited the specificity needed to explain how information about incoming stimuli is evaluated by the organism and turned into the right physiological responses which provide advantages for survival and reproduction. A physiological response often takes place away from the site of stimulation. This requires cell-to-cell communication. Hence, responding cells are not necessarily directly stimulated but rather receive an indirect stimulus via cell-to-cell communication. It appears unlikely that the '[Ca(2+)](c) signature' in the primarily stimulated cell is conveyed over long distances via cell-to-cell communication from the 'receptor cells' to the 'effector cells'. Here, a novel aspect is highlighted to explain the variety of [Ca(2+)] kinetics seen by integrating methods of [Ca(2+)](c) recording. Plants can generally be seen as cellular automata with specific morphology and capable for cell-to-cell communication. Just a few rules are needed to demonstrate how waves of [Ca(2+)](c)-increases percolate through the organism and thereby deliver a broad variety of 'signatures'. Modelling intercellular signalling may be a possible way to find explanations for different kinds of signal transmission, signal amplification, wave formation, oscillations and stimulus-response coupling. The basic examples presented here show that care has to be taken when interpreting cellular '[Ca(2+)](c) signatures' recorded by optical techniques which integrate over a big number of cells or even whole plants.

Fingerprinting antioxidative activities in plants.

BACKGROUND: A plethora of concurrent cellular activities is mobilised in the adaptation of plants to adverse environmental conditions. This response can be quantified by physiological experiments or metabolic profiling. The intention of this work is to reduce the number of metabolic processes studied to a minimum of relevant parameters with a maximum yield of information. Therefore, we inspected 'summary parameters' characteristic for whole classes of antioxidative metabolites and key enzymes. RESULTS: Three bioluminescence assays are presented. A horseradish peroxidase-based total antioxidative capacity (TAC) assay is used to probe low molecular weight antioxidants. Peroxidases are quantified by their luminol converting activity (LUPO). Finally, we quantify high molecular weight superoxide anion scavenging activity (SOSA) using coelenterazine.Experiments with Lepidium sativum L. show how salt, drought, cold, and heat influence the antioxidative system represented here by TAC, LUPO, SOSA, catalase, and glutathione reductase (GR). LUPO and SOSA run anti-parallel under all investigated stress conditions suggesting shifts in antioxidative functions rather than formation of antioxidative power. TAC runs in parallel with GR. This indicates that a majority of low molecular weight antioxidants in plants is represented by glutathione. CONCLUSION: The set of assays presented here is capable of characterising antioxidative activities in plants. It is inexpensive, quick and reproducible and delivers quantitative data. 'Summary parameters' like TAC, LUPO, and SOSA are quantitative traits which may be promising for implementation in high-throughput screening for robustness of novel mutants, transgenics, or breeds.

Aequorin as a reporter gene.

Reporter proteins allow one to monitor cellular parameters that are involved in signal transduction, development, metabolic processes, and transport. There are targeting strategies available to direct the indicator protein exactly to the locale inside the organism from which information is desired. This circumvents experimental reductionism and allows experimentation with whole intact and undisturbed organisms. The outstanding advantages of self-reporting organisms make it worth to shoulder cost- and time-consuming molecular work. Here, the luminescent Ca2+ indicator aequorin is introduced and a rough guideline is given from early planning the molecular work and assembling an experimental setup to experimentation with luminescent Arabidopsis, data processing, and control experiments.

A novel fluorescent pH probe for expression in plants.

BACKGROUND: The pH is an important parameter controlling many metabolic and signalling pathways in living cells. Recombinant fluorescent pH indicators (pHluorins) have come into vogue for monitoring cellular pH. They are derived from the most popular Aequorea victoria GFP (Av-GFP). Here, we present a novel fluorescent pH reporter protein from the orange seapen Ptilosarcus gurneyi (Pt-GFP) and compare its properties with pHluorins for expression and use in plants. RESULTS: pHluorins have a higher pH-sensitivity. However, Pt-GFP has a broader pH-responsiveness, an excellent dynamic ratio range and a better acid stability. We demonstrate how Pt-GFP expressing Arabidopsis thaliana report cytosolic pH-clamp and changes of cytosolic pH in the response to anoxia and salt-stress. CONCLUSION: Pt-GFP appears to be the better choice when used for in vivo-recording of cellular pH in plants.

Calcium: just another regulator in the machinery of life?

UNLABELLED: * BACKGROUND: Current hypotheses imply that stimulus-response systems in plants are networks of signal transduction pathways. It is usually assumed that these pathways connect receptors with effectors via chains of molecular events. Diverse intermediate signalling components (transducers) participate in these processes. However, many cellular transducers respond to several stimuli. Hence, there are no discrete chains but rather pathways that interconnect network-modules of different command structure. In particular, the cytosolic free Ca2+ concentration ([Ca2+](cyt)) is thought to perform many different tasks in a wide range of cellular events. However, this range of putative functions is so wide that it is often questioned how Ca2+ can comply with the definition of a second messenger. *THE Ca2+ SIGNATURE HYPOTHESIS: Some authors have suggested the concept of a specific signature of the ([Ca2+](cyt)) response. This implies that characteristics of the time course of changes in ([Ca2+](cyt)) and their localized sites of appearance in cells are used by the plant to identify the type and intensity of the stimulus. This hypothesis has triggered many investigations, which have yielded contradictory results. * THE CURRENT PICTURE: Much evidence suggests that the functions of calcium can be grouped into three classes: Ca2+ as a protective agent, Ca2+ as a chemical switch and Ca2+ as a 'digital' information carrier. Examples of the first two classes are presented here. The third is more controversial; while some investigations seem to support this idea, others call the Ca2+ signature hypothesis into question. Further investigations are needed to shed more light on Ca(2+)-driven signalling cascades.

Salt stress-induced chloride flux: a study using transgenic Arabidopsis expressing a fluorescent anion probe.

Salt stress leads to massive accumulation of toxic levels of Na(+) and Cl(-) ions in plants. By using the recombinant fluorescent probe CLOMELEON, we demonstrate passive anion flux under salt stress. Chloride influx is restricted in the presence of divalent cations like Mg(2+) and Ca(2+), and completely blocked by La(3+). The amount but not the rate of the reported chloride uptake is independent from the kind of corresponding permeable cation (K(+) versus Na(+)), external pH and magnitude of osmotic stress. Cl(-) efflux however seems to involve stretch-activated transport. From the influence of Ca(2+) on reported changes of cytosolic anion concentrations, we speculate that transport mechanisms of Cl(-) and Na(+) might be thermodynamically coupled under saline conditions.

Self-reporting Arabidopsis expressing pH and [Ca2+] indicators unveil ion dynamics in the cytoplasm and in the apoplast under abiotic stress.

For noninvasive in vivo measurements of intra- and extracellular ion concentrations, we produced transgenic Arabidopsis expressing pH and calcium indicators in the cytoplasm and in the apoplast. Ratiometric pH-sensitive derivatives of the green fluorescent protein (At-pHluorins) were used as pH indicators. For measurements of calcium ([Ca(2+)]), luminescent aequorin variants were expressed in fusion with pHluorins. An Arabidopsis chitinase signal sequence was used to deliver the indicator complex to the apoplast. Responses of pH and [Ca(2+)] in the apoplast and in the cytoplasm were studied under salt and "drought" (mannitol) stress. Results are discussed in the frame of ion flux, regulation, and signaling. They suggest that osmotic stress and salt stress are differently sensed, compiled, and processed in plant cells.

Reorientation of seedlings in the earth's gravitational field induces cytosolic calcium transients.

The gravitational field controls plant growth, morphology, and development. However, the underlying transduction mechanisms are not well understood. Much indirect evidence has implicated the cytoplasmic free calcium concentration ([Ca(2+)](c)) as an important factor, but direct evidence for changes in [Ca(2+)](c) is currently lacking. We now have made measurements of [Ca(2+)](c) in groups of young seedlings of Arabidopsis expressing aequorin in the cytoplasm and reconstituted in vivo with cp-coelenterazine, a synthetic high-affinity luminophore. Distinct [Ca(2+)](c) signaling occurs in response to gravistimulation with kinetics very different from [Ca(2+)](c) transients evoked by other mechanical stimuli (e.g. movement and wind). [Ca(2+)](c) changes produced in response to gravistimulation are transient but with a duration of many minutes and dependent on stimulus strength (i.e. the angle of displacement). The auxin transport blockers 2,3,5-tri-iodo benzoic acid and N-(1-naphthyl) phthalamic acid interfere with gravi-induced [Ca(2+)](c) responses and addition of methyl indole-3-acetic acid to whole seedlings induces long-lived [Ca(2+)](c) transients, suggesting that changes in auxin transport may interact with [Ca(2+)](c). Permanent nonaxial rotation of seedlings on a two-dimensional clinostat, however, produced a sustained elevation of the [Ca(2+)](c) level. This probably reflects permanent displacement of gravity-sensing cellular components and/or disturbance of cytoskeletal tension. It is concluded that [Ca(2+)](c) is part of the gravity transduction mechanism in young Arabidopsis seedlings.