Vibrio Zinc-Metalloprotease Causes Photoinactivation of Coral Endosymbionts and Coral Tissue Lesions

Background

Coral diseases are emerging as a serious threat to coral reefs worldwide. Of nine coral infectious diseases, whose pathogens have been characterized, six are caused by agents from the family Vibrionacae, raising questions as to their origin and role in coral disease aetiology.

Methodology/Principal Findings

Here we report on a Vibrio zinc-metalloprotease causing rapid photoinactivation of susceptible Symbiodinium endosymbionts followed by lesions in coral tissue. Symbiodinium photosystem II inactivation was diagnosed by an imaging pulse amplitude modulation fluorometer in two bioassays, performed by exposing Symbiodinium cells and coral juveniles to non-inhibited and EDTA-inhibited supernatants derived from coral white syndrome pathogens.

Conclusion/Significance

These findings demonstrate a common virulence factor from four phylogenetically related coral pathogens, suggesting that zinc-metalloproteases may play an important role in Vibrio pathogenicity in scleractinian corals.     

Cinnamon polyphenols attenuate the hydrogen peroxide-induced down regulation of S100β secretion by regulating sirtuin 1 in C6 rat glioma cells

Aims

It is well established that the brain is particularly susceptible to oxidative damage due to its high consumption of oxygen. The objective of this study was to investigate the protective effects of a water soluble polyphenol-rich extract of cinnamon and the possible mechanisms, under conditions of oxidative stress-induced by hydrogen peroxide, in rat C6 glioma cells.

Main methods

After 24 h of H₂O₂ incubation, the secretion and intracellular expression of S100β were determined by immunoprecitation/immunoblotting and immunofluorescence imaging.

Key findings

Cinnamon polyphenols (CP) counteracted the oxidative effects of H₂O₂ on S100β secretion and expression. CP also enhanced the impaired protein levels of sirtuins 1, 2, and 3, which are deacetylases important in cell survival. H₂O₂ also induced the overexpression of the proinflammatory factors, TNF-α, phospho-NF-κB p65, as well as of Bcl-xl, Bax and Caspase-3, which are all the members of the Bcl-2 family. CP not only suppressed the expression of these proteins but also attenuated the phosphorylation induced by H₂O₂. CP also upregulated the decreased Bcl-2 protein levels in H₂O₂ treated C6 cells. The effects of CP on H₂O₂-induced downregulation of S100β secretion were blocked by SIRT1 siRNA demonstrating that SIRT1 plays a regulatory role in CP-mediated prevention by H₂O₂.

Significance

These data demonstrate that Cinnamon polyphenols may exert neuroprotective effects in glial cells by the regulation of Bcl-2 family members and enhancing SIRT1 expression during oxidative stress.     

The effects of nitric oxide-oxidase and putative glutathione-peroxidase activities of ceruloplasmin on the viability of cardiomyocytes exposed to hydrogen peroxide

Ceruloplasmin (CP), a ferroxidase (EC 1.16.3.1) and a scavenger of reactive oxygen species, is an important extracellular antioxidant. Bovine CP indeed protects the isolated heart under ischemia–reperfusion conditions. Human CP has been shown to also exhibit, in vitro, glutathione (GSH)-peroxidase and nitric oxide (NO)-oxidase/S-nitrosating activities. This work tested, using bovine CP, the hypothesis that both activities could provide cytoprotection during oxidative stress induced by hydrogen peroxide (H₂O₂), the former activity by consuming H₂O₂ and the latter by shielding thiols from irreversible oxidation. In acellular assays, bovine CP stimulated the generation of the nitrosating NO⁺ species from the NO donors propylaminepropylamine-NONOate (PAPA/NO), S-nitroso-N-acetylpenicillamine, and S-nitrosoglutathione. This NO-oxidase activity S-nitrosated GSH as well as CP itself and was not affected by H₂O₂. In contrast to human CP, bovine CP consumed H₂O₂ in an additive rather than synergistic manner in the presence of GSH. A nonenzymatic scavenging of H₂O₂ could have masked the GSH-peroxidase activity. Cytoprotection was evaluated using neonatal rat cardiomyocytes. CP and PAPA/NO were not protective against the H₂O₂-induced loss of viability. In contrast, GSH provided a slight protection that increased more than additively in the presence of CP. This increase was canceled by PAPA/NO. CP's putative GSH-peroxidase activity can thus provide cytoprotection but is possibly affected by the S-nitrosation of a catalytically important cysteine residue.     

Ascorbate–glutathione pathways mediated by cytokinin regulate H2O2 levels in light-controlled rose bud burst

Rosebush (Rosa “Radrazz”) plants are an excellent model to study light control of bud outgrowth since bud outgrowth only arises in the presence of light and never occurs in darkness. Recently, we demonstrated high levels of hydrogen peroxide (H₂O₂) present in the quiescent axillary buds strongly repress the outgrowth process. In light, the outgrowing process occurred after H₂O₂ scavenging through the promotion of Ascorbic acid–Glutathione (AsA–GSH)-dependent pathways and the continuous decrease in H₂O₂ production. Here we showed Respiratory Burst Oxidase Homologs expression decreased in buds during the outgrowth process in light. In continuous darkness, the same decrease was observed although H₂O₂ remained at high levels in axillary buds, as a consequence of the strong inhibition of AsA–GSH cycle and GSH synthesis preventing the outgrowth process. Cytokinin (CK) application can evoke bud outgrowth in light as well as in continuous darkness. Furthermore, CKs are the initial targets of light in the photocontrol process. We showed CK application to cultured buds in darkness decreases bud H₂O₂ to a level that is similar to that observed in light. Furthermore, this treatment restores GSH levels and engages bud burst. We treated plants with buthionine sulfoximine, an inhibitor of GSH synthesis, to solve the sequence of events involving H₂O₂/GSH metabolisms in the photocontrol process. This treatment prevented bud burst, even in the presence of CK, suggesting the sequence of actions starts with the positive CK effect on GSH that in turn stimulates H₂O₂ scavenging, resulting in initiation of bud outgrowth.

Light-induced bud outgrowth in rosebush results from cytokinin-mediated peroxide scavenging and glutathione metabolism stimulation.     

Light Respiratory Processes and Gross Photosynthesis in Two Scleractinian Corals

The light dependency of respiratory activity of two scleractinian corals was examined using O₂ microsensors and CO₂ exchange measurements. Light respiration increased strongly but asymptotically with elevated irradiance in both species. Light respiration in Pocillopora damicornis was higher than in Pavona decussata under low irradiance, indicating species-specific differences in light-dependent metabolic processes. Overall, the coral P. decussata exhibited higher CO₂ uptake rates than P. damicornis over the experimental irradiance range. P. decussata also harboured twice as many algal symbionts and higher total protein biomass compared to P. damicornis, possibly resulting in self-shading of the symbionts and/or changes in host tissue specific light distribution. Differences in light respiration and CO₂ availability could be due to host-specific characteristics that modulate the symbiont microenvironment, its photosynthesis, and hence the overall performance of the coral holobiont.     

Auxin increases the hydrogen peroxide (H2O2) concentration in tomato (Solanum lycopersicum) root tips while inhibiting root growth

Background and Aims

The hormone auxin and reactive oxygen species (ROS) regulate root elongation, but the interactions between the two pathways are not well understood. The aim of this study was to investigate how auxin interacts with ROS in regulating root elongation in tomato, Solanum lycopersicum.

Methods

Wild-type and auxin-resistant mutant, diageotropica (dgt), of tomato (S. lycopersicum ‘Ailsa Craig’) were characterized in terms of root apical meristem and elongation zone histology, expression of the cell-cycle marker gene Sl-CycB1;1, accumulation of ROS, response to auxin and hydrogen peroxide (H₂O₂), and expression of ROS-related mRNAs.

Key Results

The dgt mutant exhibited histological defects in the root apical meristem and elongation zone and displayed a constitutively increased level of hydrogen peroxide (H₂O₂) in the root tip, part of which was detected in the apoplast. Treatments of wild-type with auxin increased the H₂O₂ concentration in the root tip in a dose-dependent manner. Auxin and H₂O₂ elicited similar inhibition of cell elongation while bringing forth differential responses in terms of meristem length and number of cells in the elongation zone. Auxin treatments affected the expression of mRNAs of ROS-scavenging enzymes and less significantly mRNAs related to antioxidant level. The dgt mutation resulted in resistance to both auxin and H₂O₂ and affected profoundly the expression of mRNAs related to antioxidant level.

Conclusions

The results indicate that auxin regulates the level of H₂O₂ in the root tip, so increasing the auxin level triggers accumulation of H₂O₂ leading to inhibition of root cell elongation and root growth. The dgt mutation affects this pathway by reducing the auxin responsiveness of tissues and by disrupting the H₂O₂ homeostasis in the root tip.     

Very Bright Green Fluorescent Proteins from the Pontellid Copepod Pontella mimocerami

Background

Fluorescent proteins (FP) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria have revolutionized biomedical research due to their usefulness as genetically encoded fluorescent labels. Fluorescent proteins from copepods are particularly promising due to their high brightness and rapid fluorescence development.

Results

Here we report two novel FPs from Pontella mimocerami (Copepoda, Calanoida, Pontellidae), which were identified via fluorescence screening of a bacterial cDNA expression library prepared from the whole-body total RNA of the animal. The proteins are very similar in sequence and spectroscopic properties. They possess high molar extinction coefficients (79,000 M⁻¹ cm⁻) and quantum yields (0.92), which make them more than two-fold brighter than the most common FP marker, EGFP. Both proteins form oligomers, which we were able to counteract to some extent by mutagenesis of the N-terminal region; however, this particular modification resulted in substantial drop in brightness.

Conclusions

The spectroscopic characteristics of the two P. mimocerami proteins place them among the brightest green FPs ever described. These proteins may therefore become valuable additions to the in vivo imaging toolkit.     

Arabidopsis desaturase 2 gene is involved in the regulation of cadmium and lead resistance

Aims

It was shown previously that Arabidopsis (Arabidopsis thaliana) desaturase 2 (ADS2) cDNA was isolated and it was shown that the expression of ADS2 was organ-dependent and up-regulated by low temperature. However, little is known about the role of ADS2 gene in heavy metal resistance in plants. In this study, we showed that ADS2 gene is involved in the regulation of cadmium (Cd) and lead (Pb) resistance.

Methods

For heavy metal resistance tests, seeds were germinated and grown on 1/2 MS media supplemented with the indicated concentrations of metal ions. To quantify root length, plants were grown vertically in plates. For heavy metal treatments, two-week old wild-type seedlings grown on MS media were treated with cadmium (Cd) or lead (Pb) for 24 h, and then sampled for metal content measurement and qPCR analysis.

Results

ADS2 was strongly repressed by Cd(II), and ads2-1 mutant plants showed increased Cd(II) resistance. A lower Cd content was detected in ads2-1 plants than in wild-type plants subjected to Cd(II) treatment, which was associated with activation in expression of AtPDR8 gene, a pump excluding Cd(II) and/or Cd(II)-containing toxic compounds from the cytoplasm, suggesting that ADS2-mediated Cd(II) resistance is AtPDR8 dependent. We also found that ads2-1 plants showed increased Pb(II) sensitivity, and ADS2 was strongly repressed by hydrogen peroxide (H₂O₂) but not by Pb(II). The ads2-1 mutant showed increased sensitivity to oxidative stresses mediated by H₂O₂ and paraquat, and higher levels of H₂O₂ accumulation were observed in leaves of ads2-1 plants than those of wild-type plants when subjected to Pb(II) and H₂O₂, indicating that ADS2 mediates Pb(II) resistance indirectly by impaired ROS scavenging.

Conclusions

ADS2 gene mediates Cd(II) and Pb(II) resistance, at least in part, through two distinct mechanisms, an AtPDR8-dependent mechanism and a ROS detoxification system-mediated mechanism, respectively.     

Increased prevalence of tumor-infiltrating regulatory T cells is closely related to their lower sensitivity to H2O2-induced apoptosis in gastric and esophageal cancer

Purpose and experimental design

Although an increase in regulatory T cells (Tregs) is observed in tumor microenvironments, the underlying mechanism is not fully clarified. Since it was suggested that Tregs showed a lower sensitivity toward oxidative stress in comparison with conventional T cells, in the present study, we investigated the H₂O₂ production and apoptosis of Tregs in gastric and esophageal cancer tissues, employing flow cytometric analysis using fresh samples (n = 93) and immunohistochemical analysis (n = 203).

Results

The increased tumor-infiltrating Tregs coexisted with elevated H₂O₂ production according to disease progression. The grade of apoptosis in Tregs was less pronounced than that in conventional T cells, and there was a positive correlation between H₂O₂ production and the grade of apoptosis in conventional T cells, while there was no correlation between H₂O₂ production and the grade of apoptosis in Tregs. Moreover, Tregs were less sensitive to H₂O₂-induced apoptosis compared with conventional T cells in vitro.

Conclusions

We have demonstrated that the increased prevalence of tumor-infiltrating Tregs closely related to their lower sensitivity to H₂O₂-induced apoptosis.     

Critical Role of Endothelial Hydrogen Peroxide in Post-Ischemic Neovascularization

Background

Reactive oxygen species (ROS) play an important role in angiogenesis in endothelial cells (ECs) in vitro and neovascularization in vivo. However, little is known about the role of endogenous vascular hydrogen peroxide (H₂O₂) in postnatal neovascularization.

Methodology/Principal Findings

We used Tie2-driven endothelial specific catalase transgenic mice (Cat-Tg mice) and hindlimb ischemia model to address the role of endogenous H₂O₂ in ECs in post-ischemic neovascularization in vivo. Here we show that Cat-Tg mice exhibit significant reduction in intracellular H₂O₂ in ECs, blood flow recovery, capillary formation, collateral remodeling with larger extent of tissue damage after hindlimb ischemia, as compared to wild-type (WT) littermates. In the early stage of ischemia-induced angiogenesis, Cat-Tg mice show a morphologically disorganized microvasculature. Vascular sprouting and tube elongation are significantly impaired in isolated aorta from Cat-Tg mice. Furthermore, Cat-Tg mice show a decrease in myeloid cell recruitment after hindlimb ischemia. Mechanistically, Cat-Tg mice show significant decrease in eNOS phosphorylation at Ser1177 as well as expression of redox-sensitive vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemotactic protein-1 (MCP-1) in ischemic muscles, which is required for inflammatory cell recruitment to the ischemic tissues. We also observed impaired endothelium-dependent relaxation in resistant vessels from Cat-Tg mice.

Conclusions/Significance

Endogenous ECs-derived H₂O₂ plays a critical role in reparative neovascularization in response to ischemia by upregulating adhesion molecules and activating eNOS in ECs. Redox-regulation in ECs is a potential therapeutic strategy for angiogenesis-dependent cardiovascular diseases.     

Benthic community composition affects O2 availability and variability in a Northern Red Sea fringing reef

Many coral reef ecosystems experience shifts in benthic community composition from scleractinian corals to algae. However, consequences of such phase shifts on O₂ availability, important for many reef organisms, are unresolved. This study therefore comparatively investigated potential in situ effects of different benthic cover by reef macroalgae and scleractinian corals on water column O₂ concentrations in a Northern Red Sea fringing reef. Findings revealed that mean daily O₂ concentrations at algae-dominated sites were significantly lower compared to coral-dominated sites. Minimum O₂ concentrations were significantly negatively correlated, while diurnal variability in O₂ concentration was significantly positively correlated, with increasing benthic cover by algae. In contrast, no correlation with coral cover was found. These results indicate that shifts from corals to benthic algae may likely affect both in situ O₂ availability and variability. This may be particularly pronounced in reef systems with low water exchange (e.g. closed lagoons) or under calm weather conditions and suggests potential O₂-mediated effects on reef organisms.     

Coral-associated nitrogen fixation rates and diazotrophic diversity on a nutrient-replete equatorial reef

The role of diazotrophs in coral physiology and reef biogeochemistry remains poorly understood, in part because N₂ fixation rates and diazotrophic community composition have only been jointly analyzed in the tissue of one tropical coral species. We performed field-based ¹⁵N₂ tracer incubations during nutrient-replete conditions to measure diazotroph-derived nitrogen (DDN) assimilation into three species of scleractinian coral (Pocillopora acuta, Goniopora columna, Platygyra sinensis). Using multi-marker metabarcoding (16S rRNA, nifH, 18S rRNA), we analyzed DNA- and RNA-based communities in coral tissue and skeleton. Despite low N₂ fixation rates, DDN assimilation supplied up to 6% of the holobiont’s N demand. Active coral-associated diazotrophs were chiefly Cluster I (aerobes or facultative anaerobes), suggesting that oxygen may control coral-associated diazotrophy. Highest N₂ fixation rates were observed in the endolithic community (0.20 µg N cm⁻² per day). While the diazotrophic community was similar between the tissue and skeleton, RNA:DNA ratios indicate potential differences in relative diazotrophic activity between these compartments. In Pocillopora, DDN was found in endolithic, host, and symbiont compartments, while diazotrophic nifH sequences were only observed in the endolithic layer, suggesting a possible DDN exchange between the endolithic community and the overlying coral tissue. Our findings demonstrate that coral-associated diazotrophy is significant, even in nutrient-rich waters, and suggest that endolithic microbes are major contributors to coral nitrogen cycling on reefs.Subject terms: Microbial ecology, Biogeochemistry, Stable isotope analysis     

A New Method for Xenogeneic Bone Graft Deproteinization: Comparative Study of Radius Defects in a Rabbit Model

Background and Objectives

Deproteinization is an indispensable process for the elimination of antigenicity in xenograft bones. However, the hydrogen peroxide (H₂O₂) deproteinized xenograft, which is commonly used to repair bone defect, exhibits limited osteoinduction activity. The present study was designed to develop a new method for deproteinization and compare the osteogenic capacities of new pepsin deproteinized xenograft bones with those of conventional H₂O₂ deproteinized ones.

Methods

Bones were deproteinized in H₂O₂ or pepsin for 8 hours. The morphologies were compared by HE staining. The content of protein and collagen I were measured by the Kjeldahl method and HPLC-MS, respectively. The physical properties were evaluated by SEM and mechanical tests. For in vivo study, X-ray, micro-CT and HE staining were employed to monitor the healing processes of radius defects in rabbit models transplanted with different graft materials.

Results

Compared with H₂O₂ deproteinized bones, no distinct morphological and physical changes were observed. However, pepsin deproteinized bones showed a lower protein content, and a higher collagen content were preserved. In vivo studies showed that pepsin deproteinized bones exhibited better osteogenic performance than H₂O₂ deproteinized bones, moreover, the quantity and quality of the newly formed bones were improved as indicated by micro-CT analysis. From the results of histological examination, the newly formed bones in the pepsin group were mature bones.

Conclusions

Pepsin deproteinized xenograft bones show advantages over conventional H₂O₂ deproteinized bones with respect to osteogenic capacity; this new method may hold potential clinical value in the development of new biomaterials for bone grafting.     

Hydrogen Sulfide Protects HUVECs against Hydrogen Peroxide Induced Mitochondrial Dysfunction and Oxidative Stress

Background

Hydrogen sulfide (H₂S) has been shown to have cytoprotective effects in models of hypertension, ischemia/reperfusion and Alzheimer''s disease. However, little is known about its effects or mechanisms of action in atherosclerosis. Therefore, in the current study we evaluated the pharmacological effects of H₂S on antioxidant defenses and mitochondria protection against hydrogen peroxide (H₂O₂) induced endothelial cells damage.

Methodology and Principal Findings

H₂S, at non-cytotoxic levels, exerts a concentration dependent protective effect in human umbilical vein endothelial cells (HUVECs) exposed to H₂O₂. Analysis of ATP synthesis, mitochondrial membrane potential (ΔΨm) and cytochrome c release from mitochondria indicated that mitochondrial function was preserved by pretreatment with H₂S. In contrast, in H₂O₂ exposed endothelial cells mitochondria appeared swollen or ruptured. In additional experiments, H₂S was also found to preserve the activities and protein expressions levels of the antioxidants enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione-S-transferase in H₂O₂ exposed cells. ROS and lipid peroxidation, as assessed by measuring H₂DCFDA, dihydroethidium (DHE), diphenyl-l-pyrenylphosphine (DPPP) and malonaldehyde (MDA) levels, were also inhibited by H₂S treatment. Interestingly, in the current model, D, L-propargylglycine (PAG), a selective inhibitor of cystathionine γ-lyase (CSE), abolished the protective effects of H₂S donors.

Innovation

This study is the first to show that H₂S can inhibit H₂O₂ mediated mitochondrial dysfunction in human endothelial cells by preserving antioxidant defences.

Significance

H₂S may protect against atherosclerosis by preventing H₂O₂ induced injury to endothelial cells. These effects appear to be mediated via the preservation of mitochondrial function and by reducing the deleterious effects of oxidative stress.     

Astragalin inhibits autophagy-associated airway epithelial fibrosis

Background

Fibrotic remodeling of airway and lung parenchymal compartments is attributed to pulmonary dysfunction with an involvement of reactive oxygen species (ROS) in chronic lung diseases such as idiopathic pulmonary fibrosis and asthma.

Methods

The in vitro study elucidated inhibitory effects of astragalin, kaempferol-3-O-glucoside from leaves of persimmon and green tea seeds, on oxidative stress-induced airway fibrosis. The in vivo study explored the demoting effects of astragalin on epithelial to mesenchymal transition in BALB/c mice sensitized with ovalbumin (OVA).

Results

The exposure of 20 μM H₂O₂ for 72 h accelerated E-cadherin loss and vimentin induction in airway epithelial BEAS-2B cells, which was reversed by non-toxic astragalin at 1–20 μM. Astragalin allayed the airway tissue levels of ROS and vimentin enhanced by OVA challenge. Collagen type 1 production increased in H₂O₂–exposed epithelial cells and collagen fiber deposition was observed in OVA-challenged mouse airways. This study further investigated that the oxidative stress-triggered autophagic regulation was responsible for inducing airway fibrosis. H₂O₂ highly enhanced the expression induction of the autophagy-related beclin-1 and light chains 3A/B (LC3A/B) within 4 h and astragalin blocked such induction by H₂O₂. This compound deterred the ROS-promoted autophagosome formation in BEAS-2B cells. Consistently, in OVA-sensitized mice the expression of beclin-1 and LC3A/B was highly induced, and oral administration of astragalin suppressed the autophagosome formation with inhibiting the induction of these proteins in OVA-challenged airway subepithelium. Induction of autophagy by spermidine influenced the epithelial induction of E-cadherin and vimentin that was blocked by treating astragalin.

Conclusion

These results demonstrate that astragalin can be effective in allaying ROS-promoted bronchial fibrosis through inhibiting autophagosome formation in airways.     

Stress influenced the aerotolerance of <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis> hsryfm 1301

Objective

To investigate the aerotolerance of Lactobacillus rhamnosus hsryfm 1301 and its influencing factors.

Results

The growth rate of L. rhamnosus hsryfm 1301 weakened noticeably when the concentration of supplemented H₂O₂ reached 1 mM, and only 2% of all L. rhamnosus hsryfm 1301 cells survived in MRS broth supplemented with 2 mM H₂O₂ for 1 h. After pretreatment with 0.5 mM H₂O₂, the surviving cells of L. rhamnosus hsryfm 1301 in the presence of 5 mM H₂O₂ for 1 h increased from 3.7 to 7.8 log CFU. Acid stress, osmotic stress, and heat stress at 46 °C also enhanced its aerotolerance, while heat stress at 50 °C reduced the tolerance of L. rhamnosus hsryfm 1301 to oxidative stress. Moreover, treatment with 0.5 mM H₂O₂ increased the heat stress tolerance of L. rhamnosus hsryfm 1301 by approximately 150-fold.

Conclusions

Lactobacillus rhamnosus hsryfm 1301 possesses a stress-inducible defense system against oxidative stress, and the cross-adaptation to different stresses is a promising target to increase the stress tolerance of L. rhamnosus hsryfm 1301 during probiotic food and starter culture production.

     

Directed evolution of a monomeric, bright and photostable version of Clavularia cyan fluorescent protein: structural characterization and applications in fluorescence imaging

The arsenal of engineered variants of the GFP [green FP (fluorescent protein)] from Aequorea jellyfish provides researchers with a powerful set of tools for use in biochemical and cell biology research. The recent discovery of diverse FPs in Anthozoa coral species has provided protein engineers with an abundance of alternative progenitor FPs from which improved variants that complement or supersede existing Aequorea GFP variants could be derived. Here, we report the engineering of the first monomeric version of the tetrameric CFP (cyan FP) cFP484 from Clavularia coral. Starting from a designed synthetic gene library with mammalian codon preferences, we identified dimeric cFP484 variants with fluorescent brightness significantly greater than the wild-type protein. Following incorporation of dimer-breaking mutations and extensive directed evolution with selection for blue-shifted emission, high fluorescent brightness and photostability, we arrived at an optimized variant that we have named mTFP1 [monomeric TFP1 (teal FP 1)]. The new mTFP1 is one of the brightest and most photostable FPs reported to date. In addition, the fluorescence is insensitive to physiologically relevant pH changes and the fluorescence lifetime decay is best fitted as a single exponential. The 1.19 A crystal structure (1 A=0.1 nm) of mTFP1 confirms the monomeric structure and reveals an unusually distorted chromophore conformation. As we experimentally demonstrate, the high quantum yield of mTFP1 (0.85) makes it particularly suitable as a replacement for ECFP (enhanced CFP) or Cerulean as a FRET (fluorescence resonance energy transfer) donor to either a yellow or orange FP acceptor.     

Enhanced biomass and oxidative stress tolerance of <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> PCC 7942 overexpressing the <Emphasis Type="Italic">DHAR</Emphasis> gene from <Emphasis Type="Italic">Brassica juncea</Emphasis>

Objectives

To improve the oxidative stress tolerance, biomass yield, and ascorbate/dehydroascorbate (AsA/DHA) ratio of Synechococcus elongatus PCC 7942 in the presence of H₂O₂, by heterologous expression of the dehydroascorbate reductase (DHAR) gene from Brassica juncea (BrDHAR).

Results

Under H₂O₂ stress, overexpression of BrDHAR in the transgenic strain (BrD) of S. elongatus greatly increased the AsA/DHA ratio. As part of the AsA recycling system, the oxidative stress response induced by reactive oxygen species was enhanced, and intracellular H₂O₂ level decreased. In addition, under H₂O₂ stress conditions, the BrD strain displayed increased growth rate and biomass, as well as higher chlorophyll content and deeper pigmentation than did wild-type and control strains.

Conclusion

By maintaining the AsA pool and redox homeostasis, the heterologous expression of BrDHAR increased S. elongatus tolerance to H₂O₂ stress, improving the biomass yield under these conditions. The results suggest that the BrD strain of S. elongatus, with its ability to attenuate the deleterious effects of ROS caused by environmental stressors, could be a promising platform for the generation of biofuels and other valuable bioproducts.

     

Identification, functional characterization and developmental regulation of sesquiterpene synthases from sunflower capitate glandular trichomes

Background

Besides being essential for plant structure and metabolism, soluble carbohydrates play important roles in stress responses. Sucrose has been shown to confer to Arabidopsis seedlings a high level of tolerance to the herbicide atrazine, which causes reactive oxygen species (ROS) production and oxidative stress. The effects of atrazine and of exogenous sucrose on ROS patterns and ROS-scavenging systems were studied. Simultaneous analysis of ROS contents, expression of ROS-related genes and activities of ROS-scavenging enzymes gave an integrative view of physiological state and detoxifying potential under conditions of sensitivity or tolerance.

Results

Toxicity of atrazine could be related to inefficient activation of singlet oxygen (¹O₂) quenching pathways leading to ¹O₂ accumulation. Atrazine treatment also increased hydrogen peroxide (H₂O₂) content, while reducing gene expressions and enzymatic activities related to two major H₂O₂-detoxification pathways. Conversely, sucrose-protected plantlets in the presence of atrazine exhibited efficient ¹O₂ quenching, low ¹O₂ accumulation and active H₂O₂-detoxifying systems.

Conclusion

In conclusion, sucrose protection was in part due to activation of specific ROS scavenging systems with consequent reduction of oxidative damages. Importance of ROS combination and potential interferences of sucrose, xenobiotic and ROS signalling pathways are discussed.