The Influence of Nitrate and Chloride Uptake on Expressed Sap pH, Organic Acid Synthesis, and Potassium Accumulation in Higher Plants

The influence of NO₃⁻ uptake and reduction on ionic balance in barley seedlings (Hordeum vulgare, cv. Compana) was studied. KNO₃ and KCl treatment solutions were used for comparison of cation and anion uptake. The rate of Cl⁻ uptake was more rapid than the rate of NO₃⁻ uptake during the first 2 to 4 hours of treatment. There was an acceleration in rate of NO₃⁻ uptake after 4 hours resulting in a sustained rate of NO₃⁻ uptake which exceeded the rate of Cl⁻ uptake. The initial (2 to 4 hours) rate of K⁺ uptake appeared to be independent of the rate of anion uptake. After 4 hours the rate of K⁺ uptake was greater with the KNO₃ treatment than with the KCl treatment, and the solution pH, cell sap pH, and organic acid levels with KNO₃ increased, relative to those with the KCl treatment. When absorption experiments were conducted in darkness, K⁺ uptake from KNO₃ did not exceed K⁺ uptake from KCl. We suggest that the greater uptake and accumulation of K⁺ in NO₃⁻-treated plants resulted from (a) a more rapid, sustained uptake and transport of NO₃⁻ providing a mobile counteranion for K⁺ transport, and (b) the synthesis of organic acids in response to NO₃⁻ reduction increasing the capacity for K⁺ accumulation by providing a source of nondiffusible organic anions.     

Resveratrol Prevents Ammonia Toxicity in Astroglial Cells

Ammonia is implicated as a neurotoxin in brain metabolic disorders associated with hyperammonemia. Acute ammonia toxicity can be mediated by an excitotoxic mechanism, oxidative stress and nitric oxide (NO) production. Astrocytes interact with neurons, providing metabolic support and protecting against oxidative stress and excitotoxicity. Astrocytes also convert excess ammonia and glutamate into glutamine via glutamine synthetase (GS). Resveratrol, a polyphenol found in grapes and red wines, exhibits antioxidant and anti-inflammatory properties and modulates glial functions, such as glutamate metabolism. We investigated the effect of resveratrol on the production of reactive oxygen species (ROS), GS activity, S100B secretion, TNF-α, IL-1β and IL-6 levels in astroglial cells exposed to ammonia. Ammonia induced oxidative stress, decreased GS activity and increased cytokines release, probably by a mechanism dependent on protein kinase A (PKA) and extracellular signal-regulated kinase (ERK) pathways. Resveratrol prevented ammonia toxicity by modulating oxidative stress, glial and inflammatory responses. The ERK and nuclear factor-κB (NF-κB) are involved in the protective effect of resveratrol on cytokines proinflammatory release. In contrast, other antioxidants (e.g., ascorbic acid and trolox) were not effective against hyperammonemia. Thus, resveratrol could be used to protect against ammonia-induced neurotoxicity.     

Impact of High-Concentrate Feeding and Low Ruminal pH on Methanogens and Protozoa in the Rumen of Dairy Cows

Non-lactating dairy cattle were transitioned to a high-concentrate diet to investigate the effect of ruminal pH suppression, commonly found in dairy cattle, on the density, diversity, and community structure of rumen methanogens, as well as the density of rumen protozoa. Four ruminally cannulated cows were fed a hay diet and transitioned to a 65% grain and 35% hay diet. The cattle were maintained on an high-concentrate diet for 3 weeks before the transition back to an hay diet, which was fed for an additional 3 weeks. Rumen fluid and solids and fecal samples were obtained prior to feeding during weeks 0 (hay), 1, and 3 (high-concentrate), and 4 and 6 (hay). Subacute ruminal acidosis was induced during week 1. During week 3 of the experiment, there was a significant increase in the number of protozoa present in the rumen fluid (P = 0.049) and rumen solids (P = 0.004), and a significant reduction in protozoa in the rumen fluid in week 6 (P = 0.003). No significant effect of diet on density of rumen methanogens was found in any samples, as determined by real-time PCR. Clone libraries were constructed for weeks 0, 3, and 6, and the methanogen diversity of week 3 was found to differ from week 6. Week 3 was also found to have a significantly altered methanogen community structure, compared to the other weeks. Twenty-two unique 16S rRNA phylotypes were identified, three of which were found only during high-concentrate feeding, three were found during both phases of hay feeding, and seven were found in all three clone libraries. The genus Methanobrevibacter comprised 99% of the clones present. The rumen fluid at weeks 0, 3, and 6 of all the animals was found to contain a type A protozoal population. Ultimately, high-concentrate feeding did not significantly affect the density of rumen methanogens, but did alter methanogen diversity and community structure, as well as protozoal density within the rumen of nonlactating dairy cattle. Therefore, it may be necessary to monitor the rumen methanogen and protozoal communities of dairy cattle susceptible to depressed pH when methane abatement strategies are being investigated.     

H2-CO2 Recirculation and pH Control for Growth of Methanogens in Mass Culture

We modified a fermentor (10-liter liquid volume) for the growth of anaerobic, H₂-CO₂-catabolizing bacteria. Gas in the fermentor (ca. 10% CO₂, 50% H₂, 40% CH₄) was recirculated by a diaphragm pump. During growth, the gas composition was maintained by the addition of a mixture of 80% H₂ and 20% CO₂, and this addition was controlled by a pH auxostat. During gas addition, gas was discharged from the recirculating gas stream and was collected by the displacement of an acidified salt solution.     

Nitrogen Mineralization, Ammonia Accumulation, and Emission of Gaseous NH3 by Soil-feeding Termites

There are numerous reports on the accumulation of ammonia in the mounds of soil-feeding termites. Here, we provided direct evidence for an effective mineralization of nitrogenous soil organic matter in the gut of Cubitermes spp., which gives rise to enormous ammonia concentrations in the intestinal tract. In Cubitermes ugandensis, the ammonia content of the nest material [24.5 μmol (g dry wt.)⁻¹] was about 300-fold higher than that of the parent soil. Large amounts of ammonia were present throughout the intestinal tract, with lowest values in the extremely alkaline gut sections (pH >12) and highest values posterior hindgut [185 μmol (g dry wt.)⁻¹]. Results obtained with other Cubitermes species were similar. Ammonia concentrations in the posterior hindgut of these humivorous species (up to 130 mM) are among the highest values ever reported for soil macroinvertebrates and are matched only by insects feeding on an extremely protein-rich diet (e.g., the sarcophageous larvae of blowflies). Volatilization of ammonia [about 10 nmol (g fresh wt.)⁻¹ h⁻¹], either directly by emission from the termite body or indirectly from their feces, led to NH₃ concentrations in the nest atmosphere of C. ugandensis that were three orders of magnitude above the ambient background – a relative accumulation that is considerably higher than that observed with CH₄ and CO₂. Together with previous results, these observations document that through their feeding activity and due to the physicochemical and biochemical properties of their digestive system, soil-feeding termites effectively catalyze the transformation of refractory soil organic nitrogen to a plant-available form that is protected from leaching by adsorption to the nest soil. Nitrogen mineralization rates of soil-feeding termites may surpass those effected by tropical earthworms and should contribute significantly to nitrogen fluxes in tropical ecosystems.     

Halophilic Nuclease of a Moderately Halophilic Bacillus sp.: Production, Purification, and Characterization

A moderately halophilic bacterium, Bacillus sp., isolated from rotting wood on the seashore in Nauru, produced an extracellular nuclease when cultivated aerobically in media containing 1 to 2 M NaCl. The enzyme was purified from the culture filtrate to an electrophoretically homogeneous state by ethanol precipitation, DEAE-Sephadex A-50 column chromatography, and Sephadex G-200 gel filtration. The enzyme consisted of two charge isomers and showed both RNase and DNase activities. Molecular weight was estimated to be 138,000 by Sephadex G-200 gel filtration. The enzyme had marked halophilic properties, showing maximal activities in the presence of 1.4 to 3.2 M NaCl or 2.3 to 3.2 M KCl. The enzyme hydrolyzed thymidine-5′-monophosphate-p-nitrophenyl ester at a rate that increased with NaCl concentration up to 4.8 M. In the presence of both Mg²⁺ and Ca²⁺, activity was greatly enhanced. The activity was lost by dialysis against water and low-salt buffer, but it was protected when 10 mM Ca²⁺ was added to the dialysis buffer. When the inactivated enzyme was dialyzed against 3.5 M NaCl buffer as much as 68% of the initial activity could be restored. The enzyme exhibited maximal activity at pH 8.5 and at 50°C on DNA and at 60°C on RNA and attacked RNA and DNA exonucleolytically and successively, producing 5′-mononucleotides.     

Methanotrophs and Methanogens in Masonry

Methanotrophs were present in 48 of 225 stone samples which were removed from 19 historical buildings in Germany and Italy. The average cell number of methanotrophs was 20 CFU per g of stone, and their activities ranged between 11 and 42 pmol of CH₄ g of stone⁻¹ day⁻¹. Twelve strains of methane-oxidizing bacteria were isolated. They belonged to the type II methanotrophs of the genera Methylocystis, Methylosinus, and Methylobacterium. In masonry, growth substrates like methane or methanol are available in very low concentrations. To determine if methane could be produced by the stone at rates sufficient to support growth of methanotrophs, methane production by stone samples under nonoxic conditions was examined. Methane production of 0.07 to 215 nmol of CH₄ g of stone⁻¹ day⁻¹ was detected in 23 of 47 stone samples examined. This indicated the presence of the so-called “mini-methane”-producing bacteria and/or methanogenic archaea. Methanotrophs occurred in nearly all samples which showed methane production. This finding indicated that methanotrophs depend on biogenic methane production in or on stone surfaces of historical buildings.     

Multifactorial Effects on Different Types of Brain Cells Contribute to Ammonia Toxicity

Effects of ammonia on astrocytes play a major role in hepatic encephalopathy, acute liver failure and other diseases caused by increased arterial ammonia concentrations (e.g., inborn errors of metabolism, drug or mushroom poisoning). There is a direct correlation between arterial ammonia concentration, brain ammonia level and disease severity. However, the pathophysiology of hyperammonemic diseases is disputed. One long recognized factor is that increased brain ammonia triggers its own detoxification by glutamine formation from glutamate. This is an astrocytic process due to the selective expression of the glutamine synthetase in astrocytes. A possible deleterious effect of the resulting increase in glutamine concentration has repeatedly been discussed and is supported by improvement of some pathologic effects by GS inhibition. However, this procedure also inhibits a large part of astrocytic energy metabolism and may prevent astrocytes from responding to pathogenic factors. A decrease of the already low glutamate concentration in astrocytes due to increased synthesis of glutamine inhibits the malate–aspartate shuttle and energy metabolism. A more recently described pathogenic factor is the resemblance between NH₄ ⁺ and K⁺ in their effects on the Na⁺,K⁺-ATPase and the Na⁺,K⁺, 2 Cl^? and water transporter NKCC1. Stimulation of the Na⁺,K⁺-ATPase driven NKCC1 in both astrocytes and endothelial cells is essential for the development of brain edema. Na⁺,K⁺-ATPase stimulation also activates production of endogenous ouabains. This leads to oxidative and nitrosative damage and sensitizes NKCC1. Administration of ouabain antagonists may accordingly have therapeutic potential in hyperammonemic diseases.     

产甲烷菌研究进展

产甲烷菌是重要的环境微生物,在自然界的碳素循环中起重要作用。迄今已有5种产甲烷菌基因组测序完成。基因组信息使人们对产甲烷茵的细胞结构、进化、代谢及环境适应性有了更深的理解。目前已知的甲烷生物合成途径有3种,它们以乙酸、甲基化合物、氢/二氧化碳为起始,通过不同的反应途径都形成了甲基辅酶M,在甲基辅酶M还原酶的催化下最终形成甲烷。     

Influence of Ca,pH and humic acid on Cd uptake

Summary Solution culture experiments were conducted to examine the effect of naturally occurring compounents of soil solutions such as Ca-ion, H-ion and organic acids on the Cd uptake of corn and snap beans. An increase in the Ca-ion concentration of solution cultures depressed the translocation of Cd from roots to tops of snap beans and corn but had no apparent deffect on the absorption of Cd by roots. Suppression of Cd translation by Ca was less marked for the corn than for the beans. No change in Cd absorption or translocation in corn was noted for solution pH values ranging from 4 to 7. Addition of humic acid to the solution decreased the Cd activity in soolution and the subsequent absorption of Cd by corn roots, indicating that Cd-ion activity in solution directly affectes Cd uptake. The addition of humic acid had no effect on Cd translation in corn plants.     

非离子氨和亚硝酸氮对虾虎鱼仔鱼的急性毒性及安全浓度评价

目的检测非离子氨和亚硝酸氮对鱼类的生态毒性效应。方法在水温（25±1）℃、溶氧（6.07～6.77）mg/L、盐度30～31、pH8.0～8.2的条件下,采用半静水式生物毒性试验方法研究了非离子氨和亚硝酸氮对裸项栉虾虎鱼（Ctenogobius gymnauchen）仔鱼的急性毒性效应（非离子氨浓度梯度设置为0、0.413、0.735、1.309、2.329、4.147 mg/L,亚硝酸氮浓度梯度设置为0、3.0、4.14、5.713、7.884、10.88 g/L）。结果非离子氨和亚硝酸氮暴露后仔鱼出现呆滞、侧游、呼吸困难、体色变白、身体弯曲等中毒症状,且随着暴露浓度的升高与暴露时间的延长,死亡率逐渐增加,存在明显的剂量效应关系和时间效应关系;非离子氨和亚硝酸氮对裸项栉虾虎鱼仔鱼96 hLC50分别为9.1 mg/L和12.405 g/L,其安全浓度分别为0.91 mg/L和1.2405 g/L。结论裸项栉虾虎鱼对非离子氨和亚硝酸氮具有较强的耐受力,非离子氨对裸项栉虾虎鱼仔鱼的毒性显著大于亚硝酸氮。     

The Effect of pH on Copper Toxicity to Blue-Green Algae

The effect of pH on copper toxicity to two planktonic blue-green algae, Aphanizomenon gracile and Oscillatoria redekei, was investigated. Growth rates of the algae without copper treatment decrease with pH, Aphanizomenon is earlier and more affected than Oscillatoria. On the other hand, pH-lowering leads sooner to a toxicity enhancement in Oscillatoria. In the acid range, toxicity retardation occurs in Aphanizomenon. At pH 5.1, shortening of the interval between copper toxicity and copper stimulus is characteristic for both species.     

Influence of vanadate on glycolysis,intracellular sodium,and pH in perfused rat hearts

Vanadium compounds have been shown to cause a variety of biological and metabolic effects including inhibition of certain enzymes, alteration of contractile function, and as an insulin like regulator of glucose metabolism. However, the influence of vanadium on metabolic and ionic changes in hearts remains to be understood. In this study we have examined the influence of vanadate on glucose metabolism and sodium transport in isolated perfused rat hearts. Hearts were perfused with 10 mM glucose and varying vanadate concentrations (0.7100 M) while changes in high energy phosphates (ATP and phosphocreatine (PCr)), intracellular pH, and intracellular sodium were monitored using 31P and 23Na NMR spectroscopy. Tissue lactate, glycogen, and (Na+, K+)-ATPase activity were also measured using biochemical assays. Under baseline conditions, vanadate increased tissue glycogen levels two fold and reduced (Na+, K+)-ATPase activity. Significant decreases in ATP and PCr were observed in the presence of vanadate, with little change in intracellular pH. These changes under baseline conditions were less severe when the hearts were perfused with glucose, palmitate and b-hydroxybutyrate. During ischemia vanadate did not limit the rise in intracellular sodium, but slowed sodium recovery on reperfusion. The presence of vanadate during ischemia resulted in attenuation of acidosis, and reduced lactate accumulation. Reperfusion in the presence of vanadate resulted in a slower ATP recovery, while intracellular pH and PCr recovery was not affected. These results indicate that vanadate alters glucose utilization and (Na+, K+)-ATPase activity and thereby influences the response of the myocardium to an ischemic insult.     

Influence of Metronidazole, CO, CO2, and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO₂ on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H₂, acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H₂ and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO₂ in the gas phase resulted in increased H₂ and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.     

Complexation and Toxicity of Copper in Higher Plants. I. Characterization of Copper Accumulation,Speciation, and Toxicity in Crassula helmsii as a New Copper Accumulator

The amphibious water plant Crassula helmsii is an invasive copper (Cu)-tolerant neophyte in Europe. It now turned out to accumulate Cu up to more than 9,000 ppm in its shoots at 10 μm (=0.6 ppm) Cu²⁺ in the nutrient solution, indicating that it is a Cu hyperaccumulator. We investigated uptake, binding environment, and toxicity of Cu in this plant under emerged and submerged conditions. Extended x-ray absorption fine structure measurements on frozen-hydrated samples revealed that Cu was bound almost exclusively by oxygen ligands, likely organic acids, and not any sulfur ligands. Despite significant differences in photosynthesis biochemistry and biophysics between emerged and submerged plants, no differences in Cu ligands were found. While measurements of tissue pH confirmed the diurnal acid cycle typical for Crassulacean acid metabolism, Δ¹³C measurements showed values typical for regular C3 photosynthesis. Cu-induced inhibition of photosynthesis mainly affected the photosystem II (PSII) reaction center, but with some unusual features. Most obviously, the degree of light saturation of electron transport increased during Cu stress, while maximal dark-adapted PSII quantum yield did not change and light-adapted quantum yield of PSII photochemistry decreased particularly in the first 50 s after onset of actinic irradiance. This combination of changes, which were strongest in submerged cultures, shows a decreasing number of functional reaction centers relative to the antenna in a system with high antenna connectivity. Nonphotochemical quenching, in contrast, was modified by Cu mainly in emerged cultures. Pigment concentrations in stressed plants strongly decreased, but no changes in their ratios occurred, indicating that cells either survived intact or died and bleached quickly.Heavy metals such as cadmium (Cd), copper (Cu), manganese, nickel (Ni), and zinc (Zn) are well known to be essential microelements for the life of plants (for Cd, see Lane and Morel, 2000). On the other hand, elevated concentrations of these metals induce inhibition of various processes in plant metabolism (for review, see Prasad and Hagemeyer, 1999; Küpper and Kroneck, 2005). Cu can occur in very high concentrations that are detrimental or even lethal to most plants. It is widely used as a pesticide in agriculture, and field runoff may easily reach concentrations of several micromolar (Gallagher et al., 2001). Photosynthetic reactions, both photochemical and biochemical ones, belong to the most important sites of inhibition by many heavy metals and in particular Cu. In the thylakoids, PSII has frequently been identified to be the main target. The exact location of its damage, however, strongly depends on the irradiance conditions, as shown originally by Cedeno-Maldonado et al. (1972) and later by Küpper et al. (1996b, 1998, 2002). The latter authors found that in low irradiance including a dark phase, the inhibition of PSII is largely due to the impairment of the correct function of the light-harvesting antenna; this mechanism was termed “shade reaction.” It results from the substitution by heavy metals of the Mg²⁺ ion in the chlorophyll (Chl) molecules of the light-harvesting complex II. In high irradiance, direct damage to the PSII reaction center (RC) occurs instead, which most likely involves insertion of Cu²⁺ into the Pheo a of the PSII RC. This was named “sun reaction” (Küpper et al., 1996b, 1998, 2002). Also, oxidative stress has often been described as a result of Cu stress; recent data have shown that in photosynthetic organisms, it is mainly a consequence of an inhibition of the photosynthetic light reactions (Rocchetta and Küpper, 2009).Plants developed a number of strategies to resist the toxicity of heavy metals, as reviewed by Cobbett and Goldsbrough (2002) and Küpper and Kroneck (2005). Such strategies include efflux pumps, sequestration in cells and intracellular compartments where metals do least harm, and binding of heavy metals inside the cells by strong ligands like phytochelatins or free amino acids. A majority of the heavy metal-resistant plants, called “excluders,” prevent the accumulation of heavy metals inside their tissues (Baker, 1981). Other resistant plants actively take up heavy metals, translocate them into the shoot, and sequester them to certain parts of the plant, where they are stored in a harmless state. These plants, which accumulate up to several percent of heavy metals in the dry mass of their aboveground parts, are called “hyperaccumulators” (Brooks et al., 1977). In their natural habitats, metal-rich soils in many parts of the world, this type of heavy metal accumulation serves as a defense against pathogens and herbivores (Boyd and Martens, 1994; Martens and Boyd, 1994; Boyd et al., 2002; Hanson et al., 2003; Jhee et al., 2005). They can now be used for the decontamination (“phytoremediation”) of anthropogenically heavy metal-contaminated soils and in some cases also for the commercial extraction (“phytomining”) of high-value metals (mainly Ni) from metal-rich soils (Baker et al., 1994; McGrath and Zhao, 2003; Chaney et al., 2005).The mechanisms by which hyperaccumulator plants accumulate the enormous amounts of heavy metals in their shoots and prevent phytotoxicity of these metals have been the subject of many studies. Nevertheless, many of these mechanisms are still under debate (Pollard et al., 2002; Küpper and Kroneck, 2005), and a short overview is given in our companion article (Mijovilovich et al., 2009) on Cu in the Cd/Zn model hyperaccumulator plant Thlaspi caerulescens. Studies of arsenic, Cd, Ni, and Zn binding in hyperaccumulators (Krämer et al., 1996; Sagner et al., 1998; Salt et al., 1999, Wang et al., 2002; Küpper et al., 2004) indicated that in such plants most of the metals are coordinated by organic acids, which are commonly found in plant vacuoles. Nonaccumulator plants, in contrast, are well known to bind heavy metals by strong sulfur ligands such as phytochelatins (mainly for Cd) and metallothioneins (mainly for Cu), as reviewed by Cobbett and Goldsbrough (2002).While hundreds of species have been found to hyperaccumulate Ni and about two dozen to hyperaccumulate Zn, true Cu hyperaccumulation in the sense of reaching thousands of ppm in the shoot dry weight has rarely been confirmed. Most species reported to be Cu hyperaccumulators before were later found to be false positives due to Cu adsorption on the leaf surface, et cetera; actually, none of the species recently revisited had a bioaccumulation factor larger than 1, which is commonly regarded as a necessary prerequisite of true hyperaccumulation (Faucon et al., 2007). But it is important in terms of the general understanding of metal metabolism in plants to identify how plants can cope with Cu toxicity other than excluding it from their metabolism and how far the mechanisms of Cu detoxification and Cu stress differ in Cu-resistant and -accumulating plants from Cu excluders and Cu-sensitive plants. Such questions are important also for breeding better Cd/Zn hyperaccumulators, since such plants (e.g. T. caerulescens) turned out to be Cu sensitive, limiting their phytoremediation potential on soils with mixed contamination (Walker and Bernal, 2004). We now analyzed Cu accumulation and Cu stress in a so far not well-characterized species, the amphibious Crassula helmsii, an aggressively invasive plant in Europe (Küpper et al., 1996a). We chose this plant because in a previous study it had turned out to be much more Cu resistant than all other investigated species (Küpper et al., 1996b), but more in summer than in winter. Moreover, preliminary experiments indicated that under high temperatures and salinity, C. helmsii switches to circadian acid metabolism (CAM), which might cause its elevated Cu resistance in summer due to the enhanced availability of malate as a Cu ligand. CAM metabolism was first reported for C. helmsii by Newman and Raven (1995).In this study, we investigated physiological mechanisms of Cu-induced inhibition of photosynthesis, Crassulacean acid metabolism induction, and Cu accumulation and complexation in C. helmsii. The most important method for our investigations of Cu stress was the two-dimensional (imaging) and spectrally resolved microscopic in vivo measurement of the transients of Chl variable fluorescence in the fluorescence kinetic microscope (FKM; Küpper et al., 2000a, 2007a). Cu ligands were investigated via EXAFS (