A Promising Use of Trimethyl Chitosan for Removing Microcystis aeruginosa in Water Treatment Processes.

The increase in cyanobacterial blooms linked to climate change and the eutrophication of water bodies is a global concern. The harmful cyanobacterium Microcystis aeruginosa is one of the most common bloom-forming species whose removal from fresh water and, in particular, from that used for water treatment processes, remains a crucial goal. Different biodegradable and environmentally friendly coagulants/flocculants have been assayed, with chitosan showing a very good performance. However, chitosan in its original form is of limited applicability since it is only soluble in acid solution. The objective of this work was therefore to test the coagulant/flocculant capacity of trimethylchitosan (TMC), a chitosan derivative produced from residues of the fishing industry. TMC has a constitutively net positive charge enabling it to remain in solution regardless of the pH. Results show that even at alkaline pHs, common during cyanobacterial blooms, TMC is effective in removing buoyant cyanobacteria from the water column, both in test tube and Jar-Test experiments. Cell integrity was confirmed by fluorescent stain and electron microscopy. Our findings lead us to conclude that the use of TMC to remove bloom cells early in the treatment of drinking water is both feasible and promising.

TOR signaling in the green picoalga Ostreococcus tauri.

Target of rapamycin (TOR) is a master regulator that controls growth and metabolism by integrating external and internal signals. Although there was a great progress in the study of TOR in plants and in the model alga Chlamydomonas, scarce data are available in other green algae. Thus, in this work we studied TOR signaling in Ostreococcus tauri, the smallest free-living eukaryote described to date. This picoalga is particularly important because it has a key site at the base of the green lineage and is part of the marine phytoplankton, contributing to global photosynthesis. We investigated OtTOR complex in silico and experimentally, by using first- and second-generation TOR inhibitors, such as rapamycin and PP242. We analyzed the effect of TOR down-regulation on cell growth and on the accumulation of carbon reserves. The results showed that O. tauri responds to TOR inhibitors more similarly to plants than to Chlamydomonas, being PP242 a valuable tool to study this pathway. Besides, Ottor expression analysis revealed that the kinase is dynamically regulated under nutritional stress. Our data indicate that TOR signaling is conserved in O. tauri and we propose this alga as a good and simple model for studying TOR kinase and its regulation.

C-ferroptosis is an iron-dependent form of regulated cell death in cyanobacteria.

Ferroptosis is an oxidative and iron-dependent form of regulated cell death (RCD) recently described in eukaryotic organisms like animals, plants, and parasites. Here, we report that a similar process takes place in the photosynthetic prokaryote Synechocystis sp. PCC 6803 in response to heat stress. After a heat shock, Synechocystis sp. PCC 6803 cells undergo a cell death pathway that can be suppressed by the canonical ferroptosis inhibitors, CPX, vitamin E, Fer-1, liproxstatin-1, glutathione (GSH), or ascorbic acid (AsA). Moreover, as described for eukaryotic ferroptosis, this pathway is characterized by an early depletion of the antioxidants GSH and AsA, and by lipid peroxidation. These results indicate that all of the hallmarks described for eukaryotic ferroptosis are conserved in photosynthetic prokaryotes and suggest that ferroptosis might be an ancient cell death program.

Molecular characterization of a Bacillus thuringiensis strain from Argentina, toxic against Lepidoptera and Coleoptera, based on its whole-genome and Cry protein analysis.

The insecticidal proteins of Bacillus thuringiensis are used in formulations of spore-crystal complexes and their genes have been incorporated into several crops, providing a model for genetic engineering in agriculture. Despite the variability of the Cry proteins described so far, it is still necessary to look for toxins with a broad spectrum of action, since a significant number of pests are not controlled with the available Cry proteins. It is also important to provide alternatives to address the problem of insect resistance, which has already appeared with the use of formulations and with transgenic plants that express cry genes that code for insecticidal proteins. The FCC 7 strain was characterized by the ultrastructural parasporal body under optical and electronic microscopy, and for the detection of Cry8-type proteins by genomic and proteomic approaches. The identity of the strain and the presence of putative toxin encoding genes, and virulence factors analyzed by Illumina Miseq 1500 platform genomic sequencing, was confirmed. The identity of the two Cry8 proteins that make up the parasporal body was confirmed by MALDI-TOF/TOF. To expand knowledge about the insecticidal activity of this strain, we conducted preliminary tests against the cotton boll weevil, Anthonomus grandis. Here we report the characterization of a novel B. thuringiensis isolate native to Argentina (FCC 7) toxic against A. grandis. The strain shows a rounded parasporal body harboring mainly a protein of about 140 kDa and two different types of Cry8 proteins. Through whole-genome sequencing, we identified the presence of two cry8-like crystal protein genes, one vpa-like and two vpb-like genes, and multiple virulence factors, deepening the knowledge of a strain that had already been described as toxic against some lepidopterans and coleopterans, including Spodoptera frugiperda, Anticarsia gemmatalis, Tenebrio molitor and Diabrotica speciosa.

Measurement of Ascorbic Acid and Glutathione Content in Cyanobacterium Synechocystis sp. PCC 6803.

Ascorbic acid (AsA) and gluthathione (GSH) are two key components of the antioxidant machinery of eukaryotic and prokaryotic cells. The cyanobacterium Synechocystis sp. PCC 6803 presents both compounds in different concentrations (AsA, 20-100 µM and GSH, 2-5 mM). Therefore, it is important to have precise and sensitive methods to determine the redox status in the cell and to detect variations in this antioxidants. In this protocol, we describe an improved method to estimate the content of both antioxidants (in their reduced and oxidized forms) from the same sample obtained from liquid cultures of Synechocystis sp. PCC 6803.

Target of rapamycin signaling is tightly and differently regulated in the plant response under distinct abiotic stresses.

MAIN CONCLUSION: TOR signaling is finely regulated under diverse abiotic stresses and may be required for the plant response with a different time-course depending on the duration and nature of the stress. Target of rapamycin (TOR) signaling is a central regulator of growth and development in eukaryotic organisms. However, its regulation under stress conditions has not yet been elucidated. In Arabidopsis, we show that TOR transcripts and activity in planta are finely regulated within hours after the onset of salt, osmotic, cold and oxidative stress. The expression of genes encoding the partner proteins of the TOR complex, RAPTOR3G and LST8-1, is also regulated. Besides, the data indicate that TOR activity increases at some time during the adverse condition. Interestingly, in oxidative stress, the major TOR activity increment occurred transiently at the early phase of treatment, while in salt, osmotic and cold stress, it was around 1 day after the unfavorable condition was applied. Those results suggest that the TOR signaling has an important role in the plant response to an exposure to stress. Moreover, basal ROS (H2O2) levels and their modification under abiotic stresses were altered in TOR complex mutants. On the other hand, the root phenotypic analysis of the effects caused by the diverse abiotic stresses on TOR complex mutants revealed that they were differently affected, being in some cases less sensitive, than wild-type plants to long-term unfavorable conditions. Therefore, in this work, we demonstrated that TOR signaling is tightly regulated under abiotic stresses, at transcript and activity level, with different and specific time-course patterns according to the type of abiotic stress in Arabidopsis. Taking our results together, we propose that TOR signaling should be necessary during the plant stress response.

TOR signaling downregulation increases resistance to the cereal killer Fusarium graminearum.

TOR is the master regulator of growth and development that senses energy availability. Biotic stress perturbs metabolic and energy homeostasis, making TOR a good candidate to participate in the plant response. Fusarium graminearum (Fusarium) produces important losses in many crops all over the world. To date, the role of TOR in Fusarium infection has remained unexplored. Here, we show that the resistance to the pathogen increases in different Arabidopsis mutants impaired in TOR complex or in wild-type plants treated with a TOR inhibitor. We conclude that TOR signaling is involved in plant defense against Fusarium.

The riddle of mitochondrial alkaline/neutral invertases: A novel Arabidopsis isoform mainly present in reproductive tissues and involved in root ROS production.

Alkaline/neutral invertases (A/N-Inv), glucosidases that irreversibly hydrolyze sucrose into glucose and fructose, play significant roles in plant growth, development, and stress adaptation. They occur as multiple isoforms located in the cytosol or organelles. In Arabidopsis thaliana, two mitochondrial A/N-Inv genes (A/N-InvA and A/N-InvC) have already been investigated. In this study, we functionally characterized A/N-InvH, a third Arabidopsis gene coding for a mitochondrial-targeted protein. The phenotypic analysis of knockout mutant plants (invh) showed a severely reduced shoot growth, while root development was not affected. The emergence of the first floral bud and the opening of the first flower were the most affected stages, presenting a significant delay. A/N-InvH transcription is markedly active in reproductive tissues. It is also expressed in the elongation and apical meristem root zones. Our results show that A/N-InvH expression is not evident in photosynthetic tissues, despite being of relevance in developmental processes and mitochondrial functional status. NaCl and mannitol treatments increased A/N-InvH expression twofold in the columella root cap. Moreover, the absence of A/N-InvH prevented ROS formation, not only in invh roots of salt- and ABA-treated seedlings but also in invh control roots. We hypothesize that this isoform may take part in the ROS/sugar (sucrose or its hydrolysis products) signaling pathway network, involved in reproductive tissue development, cell elongation, and abiotic stress responses.

Ancient signal for nitrogen status sensing in the green lineage: Functional evidence of CDPK repertoire in Ostreococcus tauri.

Calcium-dependent protein kinases (CDPKs) regulate plant development and many stress signalling pathways through the complex cytosolic [Ca2+] signalling. The genome of Ostreococcus tauri (Ot), a model prasinophyte organism that is on the base of the green lineage, harbours three sequences homologous to those encoding plant CDPKs with the three characteristic conserved domains (protein kinase, autoregulatory/autoinhibitory, and regulatory domain). Phylogenetic and structural analyses revealed that putative OtCDPK proteins are closely related to CDPKs from other Chlorophytes. We functionally characterised the first marine picophytoeukaryote CDPK gene (OtCDPK1) and showed that the expression of the three OtCDPK genes is up-regulated by nitrogen depletion. We conclude that CDPK signalling pathway might have originated early in the green lineage and may play a key role in prasinophytes by sensing macronutrient changes in the marine environment.

Sublethal effects of acaricides and Nosema ceranae infection on immune related gene expression in honeybees.

Nosema ceranae is an obligate intracellular parasite and the etiologic agent of Nosemosis that affects honeybees. Beside the stress caused by this pathogen, honeybee colonies are exposed to pesticides under beekeeper intervention, such as acaricides to control Varroa mites. These compounds can accumulate at high concentrations in apicultural matrices. In this work, the effects of parasitosis/acaricide on genes involved in honeybee immunity and survival were evaluated. Nurse bees were infected with N. ceranae and/or were chronically treated with sublethal doses of coumaphos or tau-fluvalinate, the two most abundant pesticides recorded in productive hives. Our results demonstrate the following: (1) honeybee survival was not affected by any of the treatments; (2) parasite development was not altered by acaricide treatments; (3) coumaphos exposure decreased lysozyme expression; (4) N. ceranae reduced levels of vitellogenin transcripts independently of the presence of acaricides. However, combined effects among stressors on imagoes were not recorded. Sublethal doses of acaricides and their interaction with other ubiquitous parasites in colonies, extending the experimental time, are of particular interest in further research work.

Selection and characterization of Argentine isolates of Trichoderma harzianum for effective biocontrol of Septoria leaf blotch of wheat.

Species of the genus Trichoderma are economically important as biocontrol agents, serving as a potential alternative to chemical control. The applicability of Trichoderma isolates to different ecozones will depend on the behavior of the strains selected from each zone. The present study was undertaken to isolate biocontrol populations of Trichoderma spp. from the Argentine wheat regions and to select and characterize the best strains of Trichoderma harzianum by means of molecular techniques. A total of 84 out of the 240 strains of Trichoderma were able to reduce the disease severity of the leaf blotch of wheat. Thirty-seven strains were selected for the reduction equal to or greater than 50% of the severity, compared with the control. The percentage values of reduction of the pycnidial coverage ranged between 45 and 80%. The same last strains were confirmed as T. harzianum by polymerase chain reaction amplification of internal transcribed spacers, followed by sequencing. Inter-simple sequence repeat was used to examine the genetic variability among isolates. This resulted in a total of 132 bands. Further numerical analysis revealed 19 haplotypes, grouped in three clusters (I, II, III). Shared strains, with different geographical origins and isolated in different years, were observed within each cluster. The origin of the isolates and the genetic group were partially related. All isolates from Paraná were in cluster I, all isolates from Lobería were in cluster II, and all isolates from Pergamino and Santa Fe were in cluster III. Our results suggest that the 37 native strains of T. harzianum are important in biocontrol programs and could be advantageous for the preparation of biopesticides adapted to the agroecological conditions of wheat culture.

Sucrose in bloom-forming cyanobacteria: loss and gain of genes involved in its biosynthesis.

Bloom-forming cyanobacteria are widely distributed in freshwater ecosystems. To cope with salinity fluctuations, cyanobacteria synthesize compatible solutes, such as sucrose, to maintain the intracellular osmotic balance. The screening of cyanobacterial genomes revealed that homologues to sucrose metabolism-related genes only occur in few bloom-forming strains, mostly belonging to Nostocales and Stigonematales orders. Remarkably, among Chroococcales and Oscillatoriales strains, homologues were only found in M. aeruginosa PCC 7806 and Leptolyngbya boryana PCC 6306, suggesting a massive loss of sucrose metabolism in bloom-forming strains of these orders. After a complete functional characterization of sucrose genes in M. aeruginosa PCC 7806, we showed that sucrose metabolism depends on the expression of a gene cluster that defines a transcriptional unit, unique among all sucrose-containing cyanobacteria. It was also demonstrated that the expression of the encoding genes of sucrose-related proteins is stimulated by salt. In view of its ancestral origin in cyanobacteria, the fact that most bloom-forming strains lack sucrose metabolism indicates that the genes involved might have been lost during evolution. However, in a particular strain, like M. aeruginosa PCC 7806, sucrose synthesis genes were probably regained by horizontal gene transfer, which could be hypothesized as a response to salinity fluctuations.

Clear differences in metabolic and morphological adaptations of akinetes of two Nostocales living in different habitats.

Akinetes are resting spore-like cells formed by some heterocyst-forming filamentous cyanobacteria for surviving long periods of unfavourable conditions. We studied the development of akinetes in two model strains of cyanobacterial cell differentiation, the planktonic freshwater Anabaena variabilis ATCC 29413 and the terrestrial or symbiotic Nostoc punctiforme ATCC 29133, in response to low light and phosphate starvation. The best trigger of akinete differentiation of Anabaena variabilis was low light; that of N. punctiforme was phosphate starvation. Light and electron microscopy revealed that akinetes of both species differed from vegetative cells by their larger size, different cell morphology and large number of intracellular granules. Anabaena variabilis akinetes had a multilayer envelope; those of N. punctiforme had a simpler envelope. During akinete development of Anabaena variabilis, the amount of the storage compounds cyanophycin and glycogen increased transiently, whereas in N. punctiforme, cyanophycin and lipid droplets increased transiently. Photosynthesis and respiration decreased during akinete differentiation in both species, and remained at a low level in mature akinetes. The clear differences in the metabolic and morphological adaptations of akinetes of the two species could be related to their different lifestyles. The results pave the way for genetic and functional studies of akinete differentiation in these species.

Genetic diversity of Fusarium graminearum sensu lato isolates from wheat associated with Fusarium Head Blight in diverse geographic locations of Argentina.

Fusarium Head Blight is an important wheat disease in the Argentine Pampas region, being Fusarium graminearum the predominant pathogen. DNA polymorphism of the isolates was analyzed by IGS-RFLP and ISSR. IGS-RFLP and ISSR profiling were carried out using six endonucleases and eight primers, respectively. IGS-RFLP yielded 41 bands, 30 of which were polymorphic while ISSR produced 87 bands with 47 polymorphic bands. Both markers showed genetic variability among the analyzed isolates; however, IGS-RFLP was more efficient than ISSR, showing a higher polymorphic average (59.91%) than the latter (44.11%). The averages of polymorphic information content (PIC) were 0.211 and 0.129, respectively. Twenty haplotypes were identified by IGS-RFLP and 15 haplotypes by ISSR. Genotype clustering within dendrograms was different for both types of markers. The genetic groups obtained by IGS-RFLP showed a partial association to geographic origin. This is the first report on genetic variability of F. graminearum isolates from wheat in Argentina using IGS-RFLP and ISSR markers.

Diversidad genética de aislamientos de Fusarium graminearum sensu lato asociados con la fusariosis de la espiga en trigo de diversas localidades argentinas / Genetic diversity of Fusarium graminearum sensu lato isolates from wheat associated with Fusarium Head Blight in diverse geographic locations of Argentina

La fusariosis de la espiga de trigo es una importante enfermedad para la región pampeana Argentina; Fusarium graminearum es el principal patógeno asociado. Se estudió el polimorfismo del ADN de un conjunto de aislamientos utilizando las técnicas de IGS-RFLP e ISSR. La técnica de IGS-RFLP produjo 41 bandas, 30 de ellas fueron polimórficas. El análisis de los ISSR mostró 87 bandas con 47 bandas polimórficas. La primera de estas metodologías fue más eficiente, ya que detectó mayor promedio polimórfico (59,91%) que la segunda (44,11%). Los valores promedio del contenido de información polimórfica (PIC) fueron 0,211 y 0,129, respectivamente. Se identificaron 20 haplotipos por IGS-RFLP, mientras que el análisis de los ISSR reveló 15 haplotipos. La agrupación de genotipos obtenida en ambos dendrogramas fue diferente. Los grupos genéticos obtenidos por la técnica de IGS-RFLP mostraron una asociación parcial con el origen geográfico. Este es el primer reporte que analiza la variabilidad genética en poblaciones de F. graminearum de trigo empleando marcadores IGS-RFLP e ISSR en Argentina

Fusarium Head Blight is an important wheat disease in the Argentine Pampas region, being Fusarium graminearum the predominant pathogen. DNA polymorphism of the isolates was analyzed by IGS-RFLP and ISSR. IGS-RFLP and ISSR profiling were carried out using six endonucleases and eight primers, respectively. IGS-RFLP yielded 41 bands, 30 of which were polymorphic while ISSR produced 87 bands with 47 polymorphic bands. Both markers showed genetic variability among the analyzed isolates; however, IGS-RFLP was more efficient than ISSR, showing a higher polymorphic average (59.91%) than the latter (44.11%). The averages of polymorphic information content (PIC) were 0.211 and 0.129, respectively. Twenty haplotypes were identified by IGS-RFLP and 15 haplotypes by ISSR. Genotype clustering within dendrograms was different for both types of markers. The genetic groups obtained by IGS-RFLP showed a partial association to geographic origin. This is the first report on genetic variability of F. graminearum isolates from wheat in Argentina using IGS-RFLP and ISSR markers

Sucrose in cyanobacteria: from a salt-response molecule to play a key role in nitrogen fixation.

In the biosphere, sucrose is mainly synthesized in oxygenic photosynthetic organisms, such as cyanobacteria, green algae and land plants, as part of the carbon dioxide assimilation pathway. Even though its central position in the functional biology of plants is well documented, much less is known about the role of sucrose in cyanobacteria. In those prokaryotes, sucrose accumulation has been associated with salt acclimation, and considered as a compatible solute in low-salt tolerant strains. In the last years, functional characterizations of sucrose metabolizing enzymes, metabolic control analysis, cellular localization of gene expressions, and reverse genetic experiments have revealed that sucrose metabolism is crucial in the diazotrophic growth of heterocystic strains, and besides, that it can be connected to glycogen synthesis. This article briefly summarizes the current state of knowledge of sucrose physiological functions in modern cyanobacteria and how they might have evolved taking into account the phylogenetic analyses of sucrose enzymes.

Functional characterization of Synechococcus amylosucrase and fructokinase encoding genes discovers two novel actors on the stage of cyanobacterial sucrose metabolism.

Plants and most cyanobacteria metabolize sucrose (Suc) with a similar set of enzymes. In Synechococcus sp. PCC 7002, a marine cyanobacterium strain, genes involved in Suc synthesis (spsA and sppA) have been characterized; however, its breakdown was still unknown. Indeed, neither invertase nor sucrose synthase genes, usually found in plants and cyanobacteria, were found in that Synechococcus genome. In the present study, we functionally characterized the amsA gene that codes for an amylosucrase (AMS), a glycoside-hydrolase family 13 enzyme described in bacteria, which may catabolyze Suc in Synechococcus. Additionally, we identified and characterized the frkA gene that codes for a fructokinase (FRK), enzyme that yields fructose-6P, one of the substrates for Suc synthesis. Interestingly, we demonstrate that spsA, sppA, frkA and amsA are grouped in a transcriptional unit that were named Suc cluster, whose expression is increased in response to a salt treatment. This is the first report on the characterization of an AMS and FRK in an oxygenic photosynthetic microorganism, which could be associated with Suc metabolism.

First evidence of sucrose biosynthesis by single cyanobacterial bimodular proteins.

The net synthesis of sucrose (Suc) is catalysed by the sequential action of Suc-phosphate synthase (SPS) and Suc-phosphate phosphatase (SPP). SPS and SPP from Anabaena sp. PCC 7120 (7120-SPS and 7120-SPP) define minimal catalytic units. Bidomainal SPSs, where both units are fused, occur in plants and cyanobacteria, but they display only SPS activity. Using recombinant proteins that have fused 7120-SPS and 7120-SPP, we demonstrated that they are bifunctional chimeras and that the arrangement 7120-SPS/SPP is the most efficient to catalyse the sequential reactions to yield Suc. Moreover, we present the first evidence of a bidomainal SPS present in the cyanobacterium Synechococcus elongatus PCC 7942 with both, SPS and SPP activity.

A mitochondrial alkaline/neutral invertase isoform (A/N-InvC) functions in developmental energy-demanding processes in Arabidopsis.

Recent findings demonstrate that alkaline/neutral invertases (A/N-Invs), enzymes that catalyze the breakdown of sucrose into glucose and fructose, are essential proteins in plant life. The fact that different isoforms are present in multiple locations makes them candidates for the coordination of metabolic processes. In the present study, we functionally characterized the encoding gene of a novel A/N-Inv (named A/N-InvC) from Arabidopsis, which localizes in mitochondria. A/N-InvC is expressed in roots, in aerial parts (shoots and leaves) and flowers. A detailed phenotypic analysis of knockout mutant plants (invc) reveals an impaired growth phenotype. Shoot growth was severely reduced, but root development was not affected as reported for A/N-InvA mutant (inva) plants. Remarkably, germination and flowering, two energy demanding processes, were the most affected stages. The effect of exogenous growth regulators led us to suggest that A/N-InvC may be modulating hormone balance in relation to the radicle emergence. We also show that oxygen consumption is reduced in inva and invc in comparison with wild-type plants, indicating that both organelle isoenzymes may play a fundamental role in mitochondrion functionality. Taken together, our results emphasize the involvement of mitochondrial A/N-Invs in developmental processes and uncover the possibility of playing different roles for the two isoforms located in the organelle.

The unique nucleotide specificity of the sucrose synthase from Thermosynechococcus elongatus.

Sucrose synthase catalyzes the reversible conversion of sucrose and UDP into fructose and UDP-glucose. In filamentous cyanobacteria, the sucrose cleavage direction plays a key physiological function in carbon metabolism, nitrogen fixation, and stress tolerance. In unicellular strains, the function of sucrose synthase has not been elucidated. We report a detailed biochemical characterization of sucrose synthase from Thermosynechococcus elongatus after the gene was artificially synthesized for optimal expression in Escherichia coli. The homogeneous recombinant sucrose synthase was highly specific for ADP as substrate, constituting the first one with this unique characteristic, and strongly suggesting an interaction between sucrose and glycogen metabolism.