<p>Excess excitation energy, mechanical injury and defence against pathogens, each trigger rapid production of reactive oxygen species (ROS) in <i>Arabidopsis thaliana</i> leaves. ROS, such as hydrogen peroxide (H₂O₂), are required for the induction of systemic acquired acclimation and may lead to redox changes in photosynthetic electron transport (PET). On one hand, enhanced ROS production during stress can destroy cells, and on the other, ROS can also act as signals for the activation of stress responsive and defensive pathways.</p><p>In this work, physiological and molecular analyses of <i>Arabidopsis</i> mutants and transgenic lines were applied to investigate the signalling network controlling biotic and abiotic stress responses. A key enzyme of the antioxidant network is encoded by <i>ASCORBATE PEROXIDASE 2 (APX2</i>). Wounded leaves showed low induction of<i> APX2 </i>expression and when exposed to excess light, <i>APX2</i> expression was increased synergistically. Signalling pathways dependent upon jasmonic acid, chitosan and abscisic acid were not involved in the wound-induced expression of <i>APX2</i>, but PET was required, and APX2 induction was preceded by a depressed rate of CO₂fixation.</p><p>Analysis of<i> lsd1</i> (<i>LESION SIMULATING DISEASE 1</i>) strongly suggests that light acclimatory processes and pathogen defences are genetically and functionally linked. It is important to know that LSD1 type of mutants have mainly been studied with regard to pathogenesis. From this work, it reveals that association of LSD1 with hypersensitive response may only be supplementary.</p><p><i>GLUTATHIONE PEROXIDASES</i> <i>(GPXs)</i> are another major family of ROS scavenging enzymes. Analysis of the <i>Arabidopsis </i>genome database revealed a new open-reading frame, thus increasing the total number of <i>AtGPX </i>gene family to eight (<i>AtGPX1-AtGPX8</i>). <i>Arabidopsis thaliana</i> transgenic lines with reduced expression of both putative chloroplastic isoforms (<i>AtGPX1 </i>and <i>AtGPX7</i>) and <i>AtGPX7</i> knock-out mutant (ko-<i>GPX7</i>) were more sensitive to photo-oxidative stress but had a reduced bacterial growth rate when inoculated with virulent strains <i>Pseudomonas syringae</i> pv. <i>tomato</i> DC3000 and<i> P.s.t. maculicola</i> strain ES4326, indicating increased resistance to pathogenesis. This, to our knowledge, is the first functional and genetic analysis of chloroplastic GPXs in plants, and confirms that light and chloroplastic ROS metabolism is important for basal resistance against virulent pathogens.</p><p>The above results confirm that light sensing, light acclimatory processes and photo-produced ROS also govern pathogen defence pathways. This has a great ecological relevance for Darwinian fitness of plants growing in the natural environment, where simultaneous pathogen attack and fluctuations in light, temperature and other environmental factors make rapid acclimation a constant necessity. Molecular, biochemical and physiological analysis of pathogen responses in mutants impaired in light sensing, EEE-dissipatory mechanisms, and similar analysis of light acclimatory processes in mutants impaired in pathogen defences may prove to be seminal.</p>

<p>Metal contaminated stormwater, i.e. surface runoff in urban areas, can be treated in percolation systems, ponds, or wetlands to prevent the release of metals into receiving waters. Plants in such systems can, for example, attenuate water flow, bind sediment, and directly accumulate metals. By these actions plants affect metal mobility. This study aimed to examine the accumulation of Zn, Cu, Cd, and Pb in roots and shoots of plant species common in stormwater areas. Furthermore, submersed plants were used to examine the fate of metals: uptake, translocation, and leakage. Factors known to influence metal accumulation, such as metal ion competition, water salinity, and temperature, were also examined. The following plant species were collected in the field: terrestrial plants – <i>Impatiens parviflora</i>, <i>Filipendula ulmaria</i>, and <i>Urtica dioica</i>; emergent plants –<i> Alisma-plantago aquatica</i>, <i>Juncus effusus</i>, <i>Lythrum salicaria</i>, <i>Sagittaria sagittifolia</i>, and <i>Phalaris arundinacea</i>; free-floating plants – <i>Lemna gibba</i> and <i>Lemna minor</i>; and submersed plants – <i>Elodea canadensis</i> and <i>Potamogeton natans</i>. Furthermore, the two submersed plants, <i>E. canadensis</i> and <i>P. natans</i>, were used in climate chamber experiments to study the fate of the metals in the plant–water system.</p><p>Emergent and terrestrial plant species accumulated high concentrations of metals in their roots under natural conditions but much less so in their shoots, and the accumulation increased further with increased external concentration. The submersed and free-floating species accumulated high levels of metals in both their roots and shoots. Metals accumulated in the shoots of <i>E. canadensis</i> and <i>P. natans</i> derived mostly from direct metal uptake from the water column.</p><p>The accumulation of Zn, Cu, Cd, and Pb in submersed species was in general high, the highest concentrations being measured in the roots, followed by the leaves and stems, <i>E. canadensis</i> having higher accumulation capacity than <i>P. natans</i>. In <i>E. canadensis</i> the Cd uptake was passive, and the accumulation in dead plants exceeded the of living with time. The capacity to quickly accumulate Cd in the apoplast decreased with successive treatments. Some of the Cd accumulated was readily available for leakage. In <i>P. natans,</i> the presence of mixtures of metal ions, common in stormwater, did not alter the accumulation of the individual metals compared to when presented separately. It is therefore, proposed that the site of uptake is specific for each metal ion. In addition cell wall-bound fraction increased with increasing external concentration. Further, decreasing the temperature from 20ºC to 5ºC and increasing the salinity from 0‰ to 5‰ S reduced Zn and Cd uptake by a factor of two.</p><p>In <i>P. natans</i> the metals were not translocated within the plant, while in<i> E. canadensis </i>Cd moved between roots and shoots. Thus,<i> E. canadensis</i> as opposed to <i>P. natans</i> may increase the dispersion of metals from sediment via acropetal translocation. The low basipetal translocation implies that neither <i>E. canadensis</i> nor <i>P. natans</i> will directly mediate the immobilisation of metal to the sediment via translocation.</p><p>To conclude, emergent and terrestrial plant species seem to enhance metal stabilization in the soil/sediment. The submersed plants, when present, slightly increase the retention of metals via shoot accumulation.</p>

<p>Discoveries in recent years have disclosed the importance of marine cyano-bacteria in the context of primary production and global nitrogen cycling. It is hypothesized here that microbial mats in tropical coastal habitats harbour a rich diversity of previously uncharacterized cyanobacteria and that benthic marine nitrogen fixation in coastal zones is substantial.</p><p>A polyphasic approach was used to investigate cyanobacterial diversity in three tropical benthic marine habitats of different characters; an intertidal sand flat and a mangrove forest floor in the Indian Ocean, and a beach rock in the Pacific Ocean. In addition, nitrogenase activity was measured over diel cycles at all sites. The results revealed high cyanobacterial diversity, both morphologically and genetically. Substantial nitrogenase activity was observed, with highest rates at daytime where heterocystous species were present. However, the three habitats were dominated by non-heterocystous and unicellular genera such as <i>Microcoleus</i>, <i>Lyngbya</i>, <i>Cyanothece</i> and a large group of thin filamentous species, identified as members of the Pseudanabaenaceae family. In these consortia nocturnal nitrogenase activities were highest and <i>nifH</i> sequencing also revealed presence of non-cyanobacterial potential diazotrophs. A conclusive phylogenetic analysis of partial nifH sequences from the three sites and sequences from geographi-cally distant microbial mats revealed new clusters of benthic potentially ni-trogen-fixing cyanobacteria. Further, the non-heterocystous cyanobacterium <i>Lyngbya majuscula</i> was subjected to a physiological characterization to gain insights into regulatory aspects of its nitrogen fixation. The data demon-strated that nitrogenase activity is restricted to darkness, which called upon a re-evaluation of its diazotrophic behaviour.</p>

<p>Phylogeny and morphology were examined for the cyanobionts from the water fern <i>Azolla </i>and the cyanobacterial genus<i> Nostoc</i> originating from symbioses with different host plants (genera <i>Gunnera, Cycas,</i> <i>Dioon,</i> <i>Encephalarthos, Macrozamia, </i>and <i>Anthoceros</i>), the lichen genus<i> Pannaria</i>, and free-living <i>Nostoc</i> isolates from different habitats. <i>Nostoc</i> isolates of <i>Pannaria</i> formed a closely related group, but, in general, no monophyletic nature was attributed to the genus <i>Nostoc</i>, in contrast to the cyanobionts from <i>Azolla </i>which were contained in a unique monophyletic group. No correlation was detected between the diversity of the studied cyanobacteria and their geographical origin, while high host specificity was proved for the <i>Azolla</i> cyanobionts and the <i>Nostoc</i> isolates from the bipartite <i>Pannaria</i> lichen. Two patterns of evolution leading to symbiotically competent heterocystous cyanobacteria were distinguished, one comprising symbiotic <i>Nostoc </i>species and the other comprising cyanobacteria in association with the water fern <i>Azolla</i>.</p><p>The production of the non-protein amino acid BMAA, a potential neurotoxin, was also examined. A rapid and sensitive method involving the lysis and extraction of amino acids from cyanobacteria combined with an HPLC assay for fluorescence detection of BMAA was developed. To determine whether the plant or the cyanobacterium was the origin of the BMAA in the cyanobacterium-<i>Azolla </i>symbiosis, the cyanobacterium-free <i>Azolla pinnata</i> var <i>imbricata </i>strain 511 was examined. HPLC analysis demonstrated a significant BMAA production in the absence of the cyanobacterium. However, PCR and cloning revealed the presence of bacteria of the genus <i>Ochrobactrum </i>in the plant.</p>

<p>In the natural environment plants are confronted to a multitude of biotic and abiotic stress factors that must be perceived, transduced, integrated and signaled in order to achieve a successful acclimation that will secure survival and reproduction. Plants have to deal with excess excitation energy (EEE) when the amount of absorbed light energy is exceeding that needed for photosynthetic CO2 assimilation. EEE results in ROS formation and can be enhanced in low light intensities by changes in other environmental factors.</p><p>The lesions simulating disease resistance (lsd1) mutant of Arabidopsis spontaneously initiates spreading lesions paralleled by ROS production in long day photoperiod and after application of salicylic acid (SA) and SA-analogues that trigger systemic acquired resistance (SAR). Moreover, the mutant fails to limit the boundaries of hypersensitive cell death (HR) after avirulent pathogen infection giving rise to the runaway cell death (rcd) phenotype. This ROS-dependent phenotype pointed towards a putative involvement of the ROS produced during photosynthesis in the initiation and spreading of the lesions.</p><p>We report here that the rcd has a ROS-concentration dependent phenotype and that the light-triggered rcd is depending on the redox-state of the PQ pool in the chloroplast. Moreover, the lower stomatal conductance and catalase activity in the mutant suggested LSD1 was required for optimal gas exchange and ROS scavenging during EEE. Through this regulation, LSD1 can influence the effectiveness of photorespiration in dissipating EEE. Moreover, low and high SA levels are strictly correlated to lower and higher foliar H2O2 content, respectively. This implies an essential role of SA in regulating the redox homeostasis of the cell and suggests that SA could trigger rcd in lsd1 by inducing H2O2 production.</p><p>LSD1 has been postulated to be a negative regulator of cell death acting as a ROS rheostat. Above a certain threshold, the pro-death pathway would operate leading to PCD. Our data suggest that LSD1 may be subjected to a turnover, enhanced in an oxidizing milieu and slowed down in a reducing environment that could reflect this ROS rheostat property. Finally, the two protein disulphide isomerase boxes (CGHC) present in the protein and the down regulation of the NADPH thioredoxin reductase (NTR) in the mutant connect the rcd to a putative impairment in the reduction of the cytosolic thioredoxin system. We propose that LSD1 suppresses the cell death processes through its control of the oxidation-reduction state of the TRX pool. An integrated model considers the role of LSD1 in both light acclimatory processes and in restricting pathogen-induced cell death.</p>

<p>During photosynthesis sunlight is absorbed by photosynthetic pigments and converted into organic compounds, such as carbohydrates. Photosynthesis needs to be highly regulated, since both too much and too little light are harmful to plant. If too little light is absorbed, a plant cannot store enough energy, which will have effects on growth and fitness of the plant. With too much light absorbed, a dangerous side reaction of photosynthesis, the production of reactive oxygen species can happen. These reactive oxygen species can damage the proteins in the chloroplast and the lipids of the chloroplast.</p><p>To avoid the production of reactive oxygen species, plants have evolved many mechanisms, which act on different time-scales and different levels of organization. As a first measure, when the absorbed light is exceeding the capacity for its utilization, is to switch the light-harvesting antenna from efficient light harvesting to energy dissipation. This process is called feedback de-excitation (FDE). The protein PsbS is essential for this process as well as a functioning xanthophylls cycle with the enzyme violaxanthin de-epoxidase (VDE).</p><p>I have investigated the effects of plants with changes in their ability to dissipate excess excitation energy in the model plants species Arabidopsis thaliana. Three genotypes with either increased or decreased capacity for FDE were used during my experiments. The first genotype over-expresses the PsbS gene, having approximately two-fold increased amounts of PsbS and FDE. The second is a PsbS deletion mutant with no PsbS protein and no FDE. The third genotype cannot perform the conversion of violaxanthin to zeaxanthin, because the enzyme VDE is missing. This mutant has some FDE left. </p><p><i>Arabidopsis thaliana</i> is an annual plant, which flowers only once in its lifetime. Therefore, when counting the seeds produced an estimation of fitness can be made from the amount of seeds produced. This was done during my experiments and shown that FDE is a trait and that plants with increased FDE have a higher fitness and vice versa. </p><p>This was also the case for a collection of plants lacking a single protein from the light harvesting antenna. All of these genotypes had a fitness reduction, proving that their function is not redundant. </p><p>In an attempt to explain why the fitness is reduced in plants with altered FDE, photosynthetic measurements, as well as a determination of the transcriptome and the metabolome was performed. Plants lacking FDE had higher levels of photoinhibition, leading both to lower rates of photosynthesis and to higher repair cost. This could in part explain the reduction in fitness. These plants also had major changes in their transcriptome and their metabolome. Primary metabolism was most effected, for example carbohydrate and amino acid metabolism. But there were also changes in secondary metabolism such as an up regulation of the biosynthesis of anthocyanins.</p>

Excess excitation energy, mechanical injury and defence against pathogens, each trigger rapid production of reactive oxygen species (ROS) in Arabidopsis thaliana leaves. ROS, such as hydrogen peroxide (H2O2), are required for the induction of systemic acquired acclimation and may lead to redox changes in photosynthetic electron transport (PET). On one hand, enhanced ROS production during stress can destroy cells, and on the other, ROS can also act as signals for the activation of stress responsive and defensive pathways. In this work, physiological and molecular analyses of Arabidopsis mutants and transgenic lines were applied to investigate the signalling network controlling biotic and abiotic stress responses. A key enzyme of the antioxidant network is encoded by ASCORBATE PEROXIDASE 2 (APX2). Wounded leaves showed low induction of APX2 expression and when exposed to excess light, APX2 expression was increased synergistically. Signalling pathways dependent upon jasmonic acid, chitosan and abscisic acid were not involved in the wound-induced expression of APX2, but PET was required, and APX2 induction was preceded by a depressed rate of CO2 fixation. Analysis of lsd1 (LESION SIMULATING DISEASE 1) strongly suggests that light acclimatory processes and pathogen defences are genetically and functionally linked. It is important to know that LSD1 type of mutants have mainly been studied with regard to pathogenesis. From this work, it reveals that association of LSD1 with hypersensitive response may only be supplementary. GLUTATHIONE PEROXIDASES (GPXs) are another major family of ROS scavenging enzymes. Analysis of the Arabidopsis genome database revealed a new open-reading frame, thus increasing the total number of AtGPX gene family to eight (AtGPX1-AtGPX8). Arabidopsis thaliana transgenic lines with reduced expression of both putative chloroplastic isoforms (AtGPX1 and AtGPX7) and AtGPX7 knock-out mutant (ko-GPX7) were more sensitive to photo-oxidative stress but had a reduced bacterial growth rate when inoculated with virulent strains Pseudomonas syringae pv. tomato DC3000 and P.s.t. maculicola strain ES4326, indicating increased resistance to pathogenesis. This, to our knowledge, is the first functional and genetic analysis of chloroplastic GPXs in plants, and confirms that light and chloroplastic ROS metabolism is important for basal resistance against virulent pathogens. The above results confirm that light sensing, light acclimatory processes and photo-produced ROS also govern pathogen defence pathways. This has a great ecological relevance for Darwinian fitness of plants growing in the natural environment, where simultaneous pathogen attack and fluctuations in light, temperature and other environmental factors make rapid acclimation a constant necessity. Molecular, biochemical and physiological analysis of pathogen responses in mutants impaired in light sensing, EEE-dissipatory mechanisms, and similar analysis of light acclimatory processes in mutants impaired in pathogen defences may prove to be seminal.

Metal contaminated stormwater, i.e. surface runoff in urban areas, can be treated in percolation systems, ponds, or wetlands to prevent the release of metals into receiving waters. Plants in such systems can, for example, attenuate water flow, bind sediment, and directly accumulate metals. By these actions plants affect metal mobility. This study aimed to examine the accumulation of Zn, Cu, Cd, and Pb in roots and shoots of plant species common in stormwater areas. Furthermore, submersed plants were used to examine the fate of metals: uptake, translocation, and leakage. Factors known to influence metal accumulation, such as metal ion competition, water salinity, and temperature, were also examined. The following plant species were collected in the field: terrestrial plants – Impatiens parviflora, Filipendula ulmaria, and Urtica dioica; emergent plants – Alisma-plantago aquatica, Juncus effusus, Lythrum salicaria, Sagittaria sagittifolia, and Phalaris arundinacea; free-floating plants – Lemna gibba and Lemna minor; and submersed plants – Elodea canadensis and Potamogeton natans. Furthermore, the two submersed plants, E. canadensis and P. natans, were used in climate chamber experiments to study the fate of the metals in the plant–water system. Emergent and terrestrial plant species accumulated high concentrations of metals in their roots under natural conditions but much less so in their shoots, and the accumulation increased further with increased external concentration. The submersed and free-floating species accumulated high levels of metals in both their roots and shoots. Metals accumulated in the shoots of E. canadensis and P. natans derived mostly from direct metal uptake from the water column. The accumulation of Zn, Cu, Cd, and Pb in submersed species was in general high, the highest concentrations being measured in the roots, followed by the leaves and stems, E. canadensis having higher accumulation capacity than P. natans. In E. canadensis the Cd uptake was passive, and the accumulation in dead plants exceeded the of living with time. The capacity to quickly accumulate Cd in the apoplast decreased with successive treatments. Some of the Cd accumulated was readily available for leakage. In P. natans, the presence of mixtures of metal ions, common in stormwater, did not alter the accumulation of the individual metals compared to when presented separately. It is therefore, proposed that the site of uptake is specific for each metal ion. In addition cell wall-bound fraction increased with increasing external concentration. Further, decreasing the temperature from 20ºC to 5ºC and increasing the salinity from 0‰ to 5‰ S reduced Zn and Cd uptake by a factor of two. In P. natans the metals were not translocated within the plant, while in E. canadensis Cd moved between roots and shoots. Thus, E. canadensis as opposed to P. natans may increase the dispersion of metals from sediment via acropetal translocation. The low basipetal translocation implies that neither E. canadensis nor P. natans will directly mediate the immobilisation of metal to the sediment via translocation. To conclude, emergent and terrestrial plant species seem to enhance metal stabilization in the soil/sediment. The submersed plants, when present, slightly increase the retention of metals via shoot accumulation.

Discoveries in recent years have disclosed the importance of marine cyano-bacteria in the context of primary production and global nitrogen cycling. It is hypothesized here that microbial mats in tropical coastal habitats harbour a rich diversity of previously uncharacterized cyanobacteria and that benthic marine nitrogen fixation in coastal zones is substantial. A polyphasic approach was used to investigate cyanobacterial diversity in three tropical benthic marine habitats of different characters; an intertidal sand flat and a mangrove forest floor in the Indian Ocean, and a beach rock in the Pacific Ocean. In addition, nitrogenase activity was measured over diel cycles at all sites. The results revealed high cyanobacterial diversity, both morphologically and genetically. Substantial nitrogenase activity was observed, with highest rates at daytime where heterocystous species were present. However, the three habitats were dominated by non-heterocystous and unicellular genera such as Microcoleus, Lyngbya, Cyanothece and a large group of thin filamentous species, identified as members of the Pseudanabaenaceae family. In these consortia nocturnal nitrogenase activities were highest and nifH sequencing also revealed presence of non-cyanobacterial potential diazotrophs. A conclusive phylogenetic analysis of partial nifH sequences from the three sites and sequences from geographi-cally distant microbial mats revealed new clusters of benthic potentially ni-trogen-fixing cyanobacteria. Further, the non-heterocystous cyanobacterium Lyngbya majuscula was subjected to a physiological characterization to gain insights into regulatory aspects of its nitrogen fixation. The data demon-strated that nitrogenase activity is restricted to darkness, which called upon a re-evaluation of its diazotrophic behaviour.

Phylogeny and morphology were examined for the cyanobionts from the water fern Azolla and the cyanobacterial genus Nostoc originating from symbioses with different host plants (genera Gunnera, Cycas, Dioon, Encephalarthos, Macrozamia, and Anthoceros), the lichen genus Pannaria, and free-living Nostoc isolates from different habitats. Nostoc isolates of Pannaria formed a closely related group, but, in general, no monophyletic nature was attributed to the genus Nostoc, in contrast to the cyanobionts from Azolla which were contained in a unique monophyletic group. No correlation was detected between the diversity of the studied cyanobacteria and their geographical origin, while high host specificity was proved for the Azolla cyanobionts and the Nostoc isolates from the bipartite Pannaria lichen. Two patterns of evolution leading to symbiotically competent heterocystous cyanobacteria were distinguished, one comprising symbiotic Nostoc species and the other comprising cyanobacteria in association with the water fern Azolla. The production of the non-protein amino acid BMAA, a potential neurotoxin, was also examined. A rapid and sensitive method involving the lysis and extraction of amino acids from cyanobacteria combined with an HPLC assay for fluorescence detection of BMAA was developed. To determine whether the plant or the cyanobacterium was the origin of the BMAA in the cyanobacterium-Azolla symbiosis, the cyanobacterium-free Azolla pinnata var imbricata strain 511 was examined. HPLC analysis demonstrated a significant BMAA production in the absence of the cyanobacterium. However, PCR and cloning revealed the presence of bacteria of the genus Ochrobactrum in the plant.