Differential responses to cadmium induced oxidative stress in marine macroalga Ulva lactuca (Ulvales, Chlorophyta)

This study describes various biochemical processes involved in the mitigation of cadmium toxicity in green alga Ulva lactuca. The plants when exposed to 0.4 mM CdCl₂ for 4 days showed twofold increase in lipoperoxides and H₂O₂ content that collectively decreased the growth and photosynthetic pigments by almost 30% over the control. The activities of antioxidant enzymes such as superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR) and glutathione peroxidase (GPX) enhanced by twofold to threefold and that of catalase (CAT) diminished. Further, the isoforms of these enzymes, namely, Mn-SOD (~85 kDa), GR (~180 kDa) and GPX (~50 kDa) responded specifically to Cd²⁺ exposure. Moreover, the contents of reduced glutathione (3.01 fold) and ascorbate (1.85 fold) also increased substantially. Lipoxygenase (LOX) activity increased by two fold coupled with the induction of two new isoforms upon Cd²⁺ exposure. Among the polyunsaturated fatty acids, although n ? 3 PUFAs and n ? 6 PUFAs (18:3n ? 6 and C18:2n ? 6) showed relatively higher contents than control, the latter ones showed threefold increase indicating their prominence in controlling the cadmium stress. Both free and bound soluble putrescine increased noticeably without any change in spermidine. In contrast, spermine content reduced to half over control. Among the macronutrients analysed in exposed thalli, the decreased K content was accompanied by higher Na and Mn with no appreciable change in Ca, Mg, Fe and Zn. Induction of antioxidant enzymes and LOX isoforms together with storage of putrescine and n ? 6 PUFAs in cadmium exposed thallus in the present study reveal their potential role in Cd²⁺ induced oxidative stress in U. lactuca.     

Effects of ocean acidification on growth and physiology of Ulva lactuca (Chlorophyta) in a rockpool‐scenario

Rising atmospheric CO₂‐concentrations will have severe consequences for a variety of biological processes. We investigated the responses of the green alga Ulva lactuca (Linnaeus) to rising CO₂‐concentrations in a rockpool scenario. U. lactuca was cultured under aeration with air containing either preindustrial pCO₂ (280 μatm) or the pCO₂ predicted by the end of the 21st century (700 μatm) for 31 days. We addressed the following question: Will elevated CO₂‐concentrations affect photosynthesis (net photosynthesis, maximum relative electron transport rate (rETR(max)), maximum quantum yield (Fv/Fm), pigment composition) and growth of U. lactuca in rockpools with limited water exchange? Two phases of the experiment were distinguished: In the initial phase (day 1–4) the Seawater Carbonate System (SWCS) of the culture medium could be adjusted to the selected atmospheric pCO₂ condition by continuous aeration with target pCO₂ values. In the second phase (day 4–31) the SWCS was largely determined by the metabolism of the growing U. lactuca biomass. In the initial phase, Fv/Fm and rETR(max) were only slightly elevated at high CO₂‐concentrations, whereas growth was significantly enhanced. After 31 days the Chl a content of the thalli was significantly lower under future conditions and the photosynthesis of thalli grown under preindustrial conditions was not dependent on external carbonic anhydrase. Biomass increased significantly at high CO₂‐concentrations. At low CO₂‐concentrations most adult thalli disintegrated between day 14 and 21, whereas at high CO₂‐concentrations most thalli remained integer until day 31. Thallus disintegration at low CO₂‐concentrations was mirrored by a drastic decline in seawater dissolved inorganic carbon and HCO₃^?. Accordingly, the SWCS differed significantly between the treatments. Our results indicated a slight enhancement of photosynthetic performance and significantly elevated growth of U. lactuca at future CO₂‐concentrations. The accelerated thallus disintegration at high CO₂‐concentrations under conditions of limited water exchange indicates additional CO₂ effects on the life cycle of U. lactuca when living in rockpools.     

Acquired tolerance of tomato (Lycopersicon esculentum cv. Micro‐Tom) plants to cadmium‐induced stress

The effects of varying concentrations of cadmium (Cd) on the development of Lycopersicon esculentum cv. Micro‐Tom (MT) plants were investigated after 40 days (vegetative growth) and 95 days (fruit production), corresponding to 20 days and 75 days of exposure to CdCl₂, respectively. Inhibition of growth was clearly observed in the leaves after 20 days and was greater after 75 days of growth in 1 mM CdCl₂, whereas the fruits exhibited reduced growth following the exposure to a concentration as low as 0.1 mM CdCl₂. Cd was shown to accumulate in the roots after 75 days of growth but was mainly translocated to the upper parts of the plants accumulating to high concentrations in the fruits. Lipid peroxidation was more pronounced in the roots even at 0.05 mM CdCl₂ after 75 days, whereas in leaves, there was a major increase after 20 days of exposure to 1 mM CdCl₂, but the fruit only exhibited a slight significant increase in lipid peroxidation in plants subjected to 1 mM CdCl₂ when compared with the control. Oxidative stress was also investigated by the analysis of four key antioxidant enzymes, which exhibited changes in response to the increasing concentrations of Cd tested. Catalase (EC 1.11.1.6) activity was shown to increase after 75 days of Cd treatment, but the major increases were observed at 0.1 and 0.2 mM CdCl₂, whereas guaiacol peroxidase (EC 1.11.1.7) did not vary significantly from the control in leaves and roots apart from specific changes at 0.5 and 1 mM CdCl₂. The other two enzymes tested, glutathione reductase (EC 1.6.4.2) and superoxide dismutase (SOD, EC 1.15.1.1), did not exhibit any significant changes in activity, apart from a slight decrease in SOD activity at concentrations above 0.2 mM CdCl₂. However, the most striking results were obtained when an extra treatment was used in which a set of plants was subjected to a stepwise increase in CdCl₂ from 0.05 to 1 mM, leading to tolerance of the Cd applied even at the final highest concentration of 1 mM. This apparent adaptation to the toxic effect of Cd was confirmed by biomass values being similar to the control, indicating a tolerance to Cd acquired by the MT plants.     

Association of chlorophyll a/c(2) complexes to photosystem I and photosystem II in the cryptophyte Rhodomonas CS24

Photosynthetic supercomplexes from the cryptophyte Rhodomonas CS24 were isolated by a short detergent treatment of membranes from the cryptophyte Rhodomonas CS24 and studied by electron microscopy and low-temperature absorption and fluorescence spectroscopy. At least three different types of supercomplexes of photosystem I (PSI) monomers and peripheral Chl a/c(2) proteins were found. The most common complexes have Chl a/c(2) complexes at both sides of the PSI core monomer and have dimensions of about 17x24 nm. The peripheral antenna in these supercomplexes shows no obvious similarities in size and/or shape with that of the PSI-LHCI supercomplexes from the green plant Arabidopsis thaliana and the green alga Chlamydomonas reinhardtii, and may be comprised of about 6-8 monomers of Chl a/c(2) light-harvesting complexes. In addition, two different types of supercomplexes of photosystem II (PSII) dimers and peripheral Chl a/c(2) proteins were found. The detected complexes consist of a PSII core dimer and three or four monomeric Chl a/c(2) proteins on one side of the PSII core at positions that in the largest complex are similar to those of Lhcb5, a monomer of the S-trimer of LHCII, Lhcb4 and Lhcb6 in green plants.     

NADPH from the oxidative pentose phosphate pathway drives the operation of cyclic electron flow around photosystem I in high‐intertidal macroalgae under severe salt stress

Pyropia yezoensis (Bangiales, Rhodophyta) is a representative species of high‐intertidal macroalgae, whose blades can tolerate extreme stresses, such as salt stress and desiccation. In this study, the photosystem (PS) responses of P. yezoensis blades under salt stress were studied. Our results showed that when the effective photochemical quantum yield of PS (Y) II decreased to almost zero under high salt stress, YI still had a relatively high activity rate. PSII was therefore more sensitive to salt stress than PSI. Furthermore, in the presence of 3‐(3′, 4′‐dichlorophenyl)‐1,1‐dimethylurea (DCMU), YI rose as salinity increased. The YI values for DCMU‐treated thalli decreased in the presence of glucose‐6‐phosphate dehydrogenase (EC 1.1.1.49, G6PDH) inhibitor (glucosamine, Glucm). The YI values were ～0.09 in the presence of methyl viologen (MV) and almost zero in the presence of dibromothymoquinone (DBMIB). These results demonstrated that under severe salt stress (120‰ salinity) PSI activity was driven from a source other than PSII, and that stromal reductants probably supported the operation of PSI. Under salt stress, the starch content decreased and soluble sugar levels increased. The G6PDH and 6‐phosphogluconate dehydrogenase (EC 1.1.1.44) activities increased, but cytosolic glyceraldehyde 3‐phosphate dehydrogenase (EC 1.2.1.12) activity decreased. Furthermore, the NADPH content increased, but NADH decreased, which suggested that soluble sugar entered the oxidative pentose phosphate pathway (OPPP). All these results suggested that NADPH from OPPP increases the cyclic electron flow around PSI in high‐intertidal macroalgae under severe salt stress.     

High salt stress in the upper part of floating mats of Ulva prolifera,a species that causes green tides,enhances non‐photochemical quenching

Salt stress is a major abiotic stress factor that can induce many adverse effects on photosynthetic organisms. Plants and algae have developed several mechanisms that help them respond to adverse environments. Non‐photochemical quenching (NPQ) is one of these mechanisms. The thalli of algae in the intertidal zone that are attached to rocks can be subjected to salt stress for a short period of time due to the rise and fall of the tide. Ulva prolifera causes green tides and can form floating mats when green tides occur and the upper part of the thalli is subjected to high salt stress for a long period of time. In this study, we compared the Ulva prolifera photosynthetic activities and NPQ kinetics when it is subjected to different salinities over various periods of time. Thalli exposed to a salinity of 90 for 4 d showed enhanced NPQ, and photosynthetic activities decreased from 60 min after exposure up to 4 d. This indicated that the induction of NPQ in Ulva prolifera under salt stress was closely related to the stressing extent and stressing time. The enhanced NPQ in the treated samples exposed for 4 d may explain why the upper layer of the floating mats formed by Ulva prolifera thalli were able to survive in the harsh environment. Further inhibitor experiments demonstrated that the enhanced NPQ was xanthophyll cycle and transthylakoid proton gradient‐dependent. However, photosystem II subunit S and light‐harvesting complex stress‐related protein didn't over accumulate and may not be responsible for the enhanced NPQ.     

Drought tolerance in faster- and slower-growing black spruce (Picea mariana) progenies: I. Stomatal and gas exchange responses to osmotic stress

Sixteen years growth in height and basal stem diameter of full-sib black spruce [ Picea mariana (Mill.) B. S. P.] progenies varied with soil moisture availability. The responses to water stress of two faster-growing progenies under drought were compared with two slower-growing progenies to determine the physiological basis of drought tolerance. Six-month-old seedlings were stressed using an osmoticum, polyethylene glycol-3350 (PEG). Seedlings were passed through a series of increasing concentration: 10, 18 and 25% PEG (w/v) each for 3 days to provide solution water potentials of -0.4, -1.0, and -2.0 MPa, respectively. The stress was then relieved by returning the seedlings to nutrient solution without PEG for 24 hours. Gas exchange and water relation parameters were similar in the 4 progenies prior to the imposition of the stress but varied during the stress and after stress relief. The two progenies which grew more vigorously on the driest site maintained significantly higher stomatal conductance, leaf transpiration rate, and net photosynthesis rate during mild (10% PEG) and moderate (18% PEG), but not severe (25% PEG) osmotic stress, and also recovered faster after release of the stress than the other two slower-growing progenies. Black spruce progenies did not differ in xylem water potential or water use efficiency. Progenies capable of faster growth under drought stress were thus characterized by a greater dehydration tolerance, rather than postponement, compared with slower-growing progenies.     

Polyols and UV‐sunscreens in the Prasiola‐clade (Trebouxiophyceae,Chlorophyta) as metabolites for stress response and chemotaxonomy

In many regions of the world, aeroterrestrial green algae of the Trebouxiophyceae (Chlorophyta) represent very abundant soil microorganisms, and hence their taxonomy is crucial to investigate their physiological performance and ecological importance. Due to a lack in morphological features, taxonomic and phylogenetic studies of Trebouxiophycean algae can be a challenging task. Since chemotaxonomic markers could be a great assistance in this regard, 22 strains of aeroterrestrial Trebouxiophyceae were chemically screened for their polyol‐patterns as well as for mycosporine‐like amino acids (MAAs) in their aqueous extracts using RP‐HPLC and LC‐MS. d ‐sorbitol was exclusively detected in members of the Prasiolaceae family. The novel MAA prasiolin and a related compound (“prasiolin‐like”) were present in all investigated members of the Prasiola‐clade, but missing in all other tested Trebouxiophyceae. While prasiolin could only be detected in field material directly after extraction, the “prasiolin‐like” compound present in the other algae was fully converted into prasiolin after 24 h. These findings suggest d ‐sorbitol and prasiolin‐like compounds are suitable chemotaxonomic markers for the Prasiolaceae and Prasiola‐clade, respectively. Additional UV‐exposure experiments with selected strains show that MAA formation and accumulation can be induced, supporting their role as UV‐sunscreen.     

Auxin and its transport play a role in plant tolerance to arsenite‐induced oxidative stress in Arabidopsis thaliana

The role of auxin in plant development is well known; however, its possible function in root response to abiotic stress is poorly understood. In this study, we demonstrate a novel role of auxin transport in plant tolerance to oxidative stress caused by arsenite. Plant response to arsenite [As(III)] was evaluated by measuring root growth and markers for stress on seedlings treated with control or As(III)‐containing medium. Auxin transporter mutants aux1, pin1 and pin2 were significantly more sensitive to As(III) than the wild type (WT). Auxin transport inhibitors significantly reduced plant tolerance to As(III) in the WT, while exogenous supply of indole‐3‐acetic acid improved As(III) tolerance of aux1 and not that of WT. Uptake assays using H³‐IAA showed As(III) affected auxin transport in WT roots. As(III) increased the levels of H₂O₂ in WT but not in aux1, suggesting a positive role for auxin transport through AUX1 on plant tolerance to As(III) stress via reactive oxygen species (ROS)‐mediated signalling. Compared to the WT, the mutant aux1 was significantly more sensitive to high‐temperature stress and salinity, also suggesting auxin transport influences a common element shared by plant tolerance to arsenite, salinity and high‐temperature stress.     

Inhibition of photosystem II (PSII) electron transport as a convenient endpoint to assess stress of the herbicide linuron on freshwater plants

Effects of the herbicide linuron on photosynthesis of the freshwater macrophytes Elodea nuttallii (Planchon) St. John, Myriophyllum spicatum L., Potamogeton crispus L., Ranunculus circinatus Sibth., Ceratophyllum demersum L. and Chara globularis (Thuill.), and of the alga Scenedesmus acutus Meyen, were assessed by measuring the efficiency of photosystem II electron flow using chlorophyll fluorescence. In a series of single-species laboratory tests several plant species were exposed to linuron at concentrations ranging from 0 to 1000 μg l−1. It was found that the primary effect of linuron, inhibition of photosystem II electron flow, occurred with a half-lifetime of about 0.1 to 1.9 h after addition of linuron to the growth medium. The direct effect of the herbicide on photosynthesis appeared to be reversible. Complete recovery from the inhibition occurred with a half-lifetime of 0.5 to 1.8 h after transfer of linuron treated plants to linuron free medium. The EC50,24h of the inhibition of photosystem II electron transport by linuron was about 9–13 μg l−1 for most of the macrophytes tested. For S. acutus the EC50,72h for inhibition of photosystem II electron flow was about 17 μg l−1 for the free suspension, and 22 μg l−1 for cells encapsulated in alginate beads. In a long-term indoor microcosm experiment, the photosystem II electron flow of the macrophytes E. nuttallii, C. demersum and the alga Spirogyra sp. was determined during 4 weeks of chronic exposure to linuron. The EC50,4weeks for the long-term exposure was 8.3, 8.7 and 25.1 μg l−1 for E. nuttallii, C. demersum and Spirogyra, respectively. These results are very similar to those calculated for the acute effects. The relative biomass increase of E. nuttallii in the microcosms was determined during 3 weeks of chronic exposure and was related to the efficiency of photosystem II electron transport as assessed in the different treatments. It is concluded that effects of the photosynthesis inhibiting herbicide on aquatic macrophytes, algae and algae encapsulated in alginate beads can be conveniently evaluated by measuring photosystem II electron transport by means of chlorophyll fluorescence. This method can be used as a rapid and non-destructive technique in aquatic ecological research. This revised version was published online in August 2006 with corrections to the Cover Date.     

Co‐occurring increases of calcium and organellar reactive oxygen species determine differential activation of antioxidant and defense enzymes in Ulva compressa (Chlorophyta) exposed to copper excess

In order to analyse copper‐induced calcium release and (reactive oxygen species) ROS accumulation and their role in antioxidant and defense enzymes activation, the marine alga Ulva compressa was exposed to 10 µM copper for 7 d. The level of calcium, extracellular hydrogen peroxide (eHP), intracellular hydrogen peroxide (iHP) and superoxide anions (SA) as well as the activities of ascorbate peroxidase (AP), glutathione reductase (GR), glutathione‐S‐transferase (GST), phenylalanine ammonia lyase (PAL) and lipoxygenase (LOX) were determined. Calcium release showed a triphasic pattern with peaks at 2, 3 and 12 h. The second peak was coincident with increases in eHP and iHP and the third peak with the second increase of iHP. A delayed wave of SA occurred after day 3 and was not accompanied by calcium release. The accumulation of iHP and SA was mainly inhibited by organellar electron transport chains inhibitors (OETCI), whereas calcium release was inhibited by ryanodine. AP activation ceased almost completely after the use of OETCI. On the other hand, GR and GST activities were partially inhibited, whereas defense enzymes were not inhibited. In contrast, PAL and LOX were inhibited by ryanodine, whereas AP was not inhibited. Thus, copper stress induces calcium release and organellar ROS accumulation that determine the differential activation of antioxidant and defense enzymes.     

An NMR study elucidating the binding of Mg(II) and Mn(II) to spinach plastocyanin. Regulation of the binding of plastocyanin to subunit PsaF of photosystem I

In this work we address the question whether light-induced changes in the Mg(II) content in the chloroplast lumen can modulate the electron donation to photosystem I, in particular the electrostatic interaction between plastocyanin (Pc) and the photosystem 1 subunit PsaF. For this, we have used 2D NMR spectroscopy to study the binding of Mg(II) ions and the isolated luminal domain of PsaF to (15)N-labelled Pc. From the chemical-shift perturbations in the (1)H-(15)N HSQC spectra, dissociation constants of (4.9 ± 1.7) mM and (1.4 ± 0.2) mM were determined for the Pc-Mg(II) and Pc-PsaF complexes, respectively. In both cases, significant chemical-shift changes were observed for Pc backbone amide groups belonging to the two acidic patches, residues 42-45 and 59-61. In addition, competitive effects were observed upon the addition of Mg(II) ions to the Pc-PsaF complex, further strengthening that Mg(II) and PsaF bind to the same region on Pc. To structurally elucidate the Mg(II) binding site we have utilized Mn(II) as a paramagnetic analogue of Mg(II). The paramagnetic relaxation enhancement induced by Mn(II) results in line broadening in the Pc HSQC spectra which can be used to estimate distances between the bound ion and the affected nuclear spins. The calculations suggest a location of the bound Mn(II) ion close to Glu43 in the lower acidic patch, and most likely in the form of a hexaquo complex embedded within the hydration shell of Pc. The results presented here suggest a specific binding site for Mg(II) that may regulate the binding of Pc to photosystem 1 in vivo.     

Physiological responses of a green algae (<Emphasis Type="Italic">Ulva prolifera</Emphasis>) exposed to simulated acid rain and decreased salinity

In order to evaluate the combined effects of simulated acid rain (SAR) and salinity on the physiological responses of macroalgae, Ulva prolifera was cultured under three salinity treatments (5, 10, 25 ‰) and at different pH, i.e., at pH 4.4 (C), pH 4.4(F), where the pH of the culture increased from 4.4 to approximately 7.8 during the cultivation period, or in absence of SAR at pH 8.2(C), at 100 μmol(photon) m^–2 s^–1 and 20°C. Compared to 25‰ salinity, Relative growth rate (RGR) of U. prolifera was enhanced by 10‰ salinity, but decreased by 5‰ salinity. No significant differences in RGR were observed between the pH 8.2(C) and pH 4.4(F) treatments, but the chlorophyll a content was reduced by SAR. Negative effects of SAR on the photosynthesis were observed, especially under low salinity treatments. Based on the results, we suggested that the U. prolifera showed a tolerance to a wide range of salinity in contrast to the low pH induced by acid rain.     

Monochromatic ultraviolet light induced damage to Photosystem II efficiency and carbon fixation in the marine diatom Thalassiosira pseudonana (3H)

Low light adapted cultures of the marine diatom Thalassiosira pseudonana (3H) were cultured and incubated for 30 min under different ultraviolet (UV) wavelengths of near monochromatic light with and without background photosynthetically active radiation (PAR, 380–700 nm). Maximum damage to the quantum yield for stable charge separations was found in the UVB (280-320 nm) wavelengths without background PAR light while the damage under PAR was 30% less. UV induced damage to carbon fixation in the cells was described by a function similar to non-linear functions of inhibiting irradiance previously published with the exception that damage was slightly higher in the UVA (320–380). Various measurements of fluorescent transients were measured and the results indicate localised damage most likely on the acceptor side of the Photosystem II reaction center. However, dark adapted measurements of fluorescence transients with and without DCMU do not result in similar functions. This is also true for the relationships between fluorescence transients and carbon fixation for this species of marine diatom. The correlation between the weightings _H from measurements of carbon fixation and the quantum yield for stable charge separation as calculated from induction curves with DCMU and without DCMU is R ² 0.44 and R ² 0.78, respectively. The slopes of the two measurements are 3.8 and 1.4, respectively. The strong correlation between the weightings of the induction curves without DCMU and carbon fixation are due to a loss of electron transport from the reaction center to plastoquinone. Under these experimental conditions of constant photon flux density (PFD) this is manifested as a strong linear relationship between the decrease in the operational quantum yield of Photosystem II and carbon fixation. This revised version was published online in June 2006 with corrections to the Cover Date.     

Light‐dependent reversible phosphorylation of the minor photosystem II antenna Lhcb6 (CP24) occurs in lycophytes

Evolution of vascular plants required compromise between photosynthesis and photodamage. We analyzed representative species from two divergent lineages of vascular plants, lycophytes and euphyllophytes, with respect to the response of their photosynthesis and light‐harvesting properties to increasing light intensity. In the two analyzed lycophytes, Selaginella martensii and Lycopodium squarrosum, the medium phase of non‐photochemical quenching relaxation increased under high light compared to euphyllophytes. This was thought to be associated with the occurrence of a further thylakoid phosphoprotein in both lycophytes, in addition to D2, CP43 and Lhcb1‐2. This protein, which showed light intensity‐dependent reversible phosphorylation, was identified in S. martensii as Lhcb6, a minor LHCII antenna subunit of PSII. Lhcb6 is known to have evolved in the context of land colonization. In S. martensii, Lhcb6 was detected as a component of the free LHCII assemblies, but also associated with PSI. Most of the light‐induced changes affected the amount and phosphorylation of the LHCII assemblies, which possibly mediate PSI–PSII connectivity. We propose that Lhcb6 is involved in light energy management in lycophytes, participating in energy balance between PSI and PSII through a unique reversible phosphorylation, not yet observed in other land plants.