Nuclear Transformation of the Marine Pennate Diatom Nitzschia sp. Strain NIES-4635 by Multi-Pulse Electroporation.

Nitzschia is one of the largest genera of diatoms found in a range of aquatic environments, from freshwater to seawater. This genus contains evolutionarily and ecologically unique species, such as those that have lost photosynthetic capacity or those that live symbiotically in dinoflagellates. Several Nitzschia species have been used as indicators of water pollution. Recently, Nitzschia species have attracted considerable attention in the field of biotechnology. In this study, a transformation method for the marine pennate diatom Nitzschia sp. strain NIES-4635, isolated from the coastal Seto Inland Sea, was established. Plasmids containing the promoter/terminator of the fucoxanthin chlorophyll a/c binding protein gene (fcp, or Lhcf) derived from Nitzschia palea were constructed and introduced into cells by multi-pulse electroporation, resulting in 500 µg/mL nourseothricin-resistant transformants with transformation frequencies of up to 365 colonies per 108 cells. In addition, when transformation was performed using a new plasmid containing a promoter derived from a diatom-infecting virus upstream of the green fluorescent protein gene (gfp), 44% of the nourseothricin-resistant clones exhibited GFP fluorescence. The integration of the genes introduced into the genomes of the transformants was confirmed by Southern blotting. The Nitzschia transformation method established in this study will enable the transformation this species, thus allowing the functional analysis of genes from the genus Nitzschia, which are important species for environmental and biotechnological development.

Characterization of Chaetoceros lorenzianus-infecting DNA virus-derived promoters of genes from open reading frames of unknown function in Phaeodactylum tricornutum.

Promoters are key elements for the regulation of gene expression. Recently, we investigated the activity of promoters derived from marine diatom-infecting viruses (DIVs) in marine diatoms. Previously, we focused on potential promoter regions of the replication-associated protein gene and the capsid protein gene of the DIVs. In addition to these genes, two genes of unknown function (VP1 and VP4 genes) have been found in the DIV genomes. In this study, the promoter regions of the VP1 gene and VP4 gene derived from a Chaetoceros lorenzianus-infecting DNA virus (named ClP3 and ClP4, respectively) were newly isolated. ClP4 was found to be a constitutive promoter and displayed the highest activity. In particular, the 3' region of ClP4 (ClP4 3' region) showed a higher promoter activity than full-length ClP4. The ClP4 3' region might involve high-level promoter activity of ClP4. In addition, the ClP4 3' region may be useful for substance production and metabolic engineering of diatoms.

Erratum: Tran et al. Early Cellular Responses Induced by Sedimentary Calcite-Processed Particles in Bright Yellow 2 Tobacco Cultured Cells. Int. J. Mol. Sci. 2020, 21, 4279.

The authors would like to remove the scientific consortium 'Camille Nous' from the author list and the Author Contributions section in the published paper [...].

Biphasic activation of survival and death pathways in Arabidopsis thaliana cultured cells by sorbitol-induced hyperosmotic stress.

Hyperosmotic stresses represent some of the most serious abiotic factors that adversely affect plants growth, development and fitness. Despite their central role, the early cellular events that lead to plant adaptive responses remain largely unknown. In this study, using Arabidopsis thaliana cultured cells we analyzed early cellular responses to sorbitol-induced hyperosmotic stress. We observed biphasic and dual responses of A. thaliana cultured cells to sorbitol-induced hyperosmotic stress. A first set of events, namely singlet oxygen (1O2) production and cell hyperpolarization due to a decrease in anion channel activity could participate to signaling and osmotic adjustment allowing cell adaptation and survival. A second set of events, namely superoxide anion (O2-) production by RBOHD-NADPH-oxidases and SLAC1 anion channel activation could participate in programmed cell death (PCD) of a part of the cell population. This set of events raises the question of how a survival pathway and a death pathway could be induced by the same hyperosmotic condition and what could be the meaning of the induction of two different behaviors in response to hyperosmotic stress.

Early Cellular Responses Induced by Sedimentary Calcite-Processed Particles in Bright Yellow 2 Tobacco Cultured Cells.

Calcite processed particles (CaPPs, Megagreen®) elaborated from sedimentary limestone rock, and finned by tribomecanic process were found to increase photosynthetic CO2 fixation grapevines and stimulate growth of various cultured plants. Due to their processing, the CaPPs present a jagged shape with some invaginations below the micrometer size. We hypothesised that CaPPs could have a nanoparticle (NP)-like effects on plants. Our data show that CaPPs spontaneously induced reactive oxygen species (ROS) in liquid medium. These ROS could in turn induce well-known cellular events such as increase in cytosolic Ca2+, biotic ROS generation and activation of anion channels indicating that these CaPPs could activate various signalling pathways in a NP-like manner.

The possibility of using marine diatom-infecting viral promoters for the engineering of marine diatoms.

Marine diatoms constitute a major group of unicellular photosynthetic eukaryotes. Diatoms are widely applicable for both basic studies and applied studies. Molecular tools and techniques have been developed for diatom research. Among these tools, several endogenous gene promoters (e.g., the fucoxanthin chlorophyll a/c-binding protein gene promoter) have become available for expressing transgenes in diatoms. Gene promoters that drive transgene expression at a high level are very important for the metabolic engineering of diatoms. Various marine diatom-infecting viruses (DIVs), including both DNA viruses and RNA viruses, have recently been isolated, and their genome sequences have been characterized. Promoters from viruses that infect plants and mammals are widely used as constitutive promoters to achieve high expression of transgenes. Thus, we recently investigated the activity of promoters derived from marine DIVs in the marine diatom, Phaeodactylum tricornutum. We discuss novel viral promoters that will be useful for the future metabolic engineering of diatoms.

Development of endogenous promoters that drive high-level expression of introduced genes in the model diatom Phaeodactylum tricornutum.

The marine diatom Phaeodactylum tricornutum is attractive for basic and applied diatom research. We isolated putative endogenous gene promoters derived from genes that are highly expressed in P. tricornutum: the fucoxanthin chlorophyll a/c-binding protein (FCP) C gene, the vacuolar ATP synthase 16-kDa proteolipid subunit (V-ATPase C) gene, the clumping factor A gene and the solute carrier family 34 member 2 gene. Five putative promoter regions were isolated, linked to an antibiotic resistance gene (Sh ble) and transformed into P. tricornutum. Using quantitative RT-PCR, the promoter activities in the transformants were analyzed and compared to that of the diatom endogenous gene promoter, the FCP A gene promoter which has been used for the transformation of P. tricornutum. Among the five isolated potential promoters, the activity of the V-ATPase C gene promoter was approximately 2.73 times higher than that of the FCP A gene promoter. The V-ATPase C gene promoter drove the expression of Sh ble mRNA transcripts under both light and dark conditions at the stationary phase. These results suggest that the V-ATPase C gene promoter is a novel tool for the genetic engineering of P. tricornutum.

Methanol induces cytosolic calcium variations, membrane depolarization and ethylene production in arabidopsis and tobacco.

Background and Aims: Methanol is a volatile organic compound released from plants through the action of pectin methylesterases (PMEs), which demethylesterify cell wall pectins. Plant PMEs play a role in developmental processes but also in responses to herbivory and infection by fungal or bacterial pathogens. However, molecular mechanisms that explain how methanol could affect plant defences remain poorly understood. Methods: Using cultured cells and seedlings from Arabidopsis thaliana and tobacco BY2 expressing the apoaequorin gene, allowing quantification of cytosolic Ca2+, a reactive oxygen species (ROS) probe (CLA, Cypridina luciferin analogue) and electrophysiological techniques, we followed early plant cell responses to exogenously supplied methanol applied as a liquid or as volatile. Key Results: Methanol induces cytosolic Ca2+ variations that involve Ca2+ influx through the plasma membrane and Ca2+ release from internal stores. Our data further suggest that these Ca2+ variations could interact with different ROS and support a signalling pathway leading to well known plant responses to pathogens such as plasma membrane depolarization through anion channel regulation and ethylene synthesis. Conclusions: Methanol is not only a by-product of PME activities, and our data suggest that [Ca2+]cyt variations could participate in signalling processes induced by methanol upstream of plant defence responses.

An exception among diatoms: unique organization of genes involved in isoprenoid biosynthesis in Rhizosolenia setigera CCMP 1694.

The marine diatom Rhizosolenia setigera is unique among this group of microalgae given that it is only one of a handful of diatom species that can produce highly branched isoprenoid (HBI) hydrocarbons. In our efforts to determine distinguishing molecular characteristics in R. setigera CCMP 1694 that could help elucidate the underlying mechanisms for its ability to biosynthesize HBIs, we discovered the occurrence of independent genes encoding for two isopentenyl diphosphate isomerases (RsIDI1 and RsIDI2) and one squalene synthase (RsSQS), enzymes that catalyze non-consecutive steps in isoprenoid biosynthesis. These genes are peculiarly fused in all other genome-sequenced diatoms to date, making their organization in R. setigera CCMP 1694 a clear distinguishing molecular feature. Phylogenetic and sequence analysis of RsIDI1, RsIDI2, and RsSQS revealed that such an arrangement of individually transcribed genes involved in isoprenoid biosynthesis could have arisen through a secondary gene fission event. We further demonstrate that inhibition of squalene synthase (SQS) shifts the flux of exogenous isoprenoid precursors towards HBI biosynthesis suggesting the competition for isoprenoid substrates in the form of farnesyl diphosphate between the sterol and HBI biosynthetic pathways in this diatom.

Nuclear transformation of the diatom Phaeodactylum tricornutum using PCR-amplified DNA fragments by microparticle bombardment.

We have developed a method for marine diatom transformation by microparticle bombardment using polymerase chain reaction (PCR)-amplified DNA fragments. We constructed a circular vector (approximately 5000 bp) containing an fcpA promoter from Phaeodactylum tricornutum, antibiotic-resistance genes and terminator from Cylindrotheca fusiformis (a "gene cassette"). Then the various lengths of linear vectors (+0-+1000 linear vectors) were then PCR-amplified from the circular plasmid. The transformants of P. tricornutum transfected with the linear vectors were obtained in the triplicate experiments. Transformation efficiencies using PCR-amplified short linear vectors containing the gene cassette and additional DNA regions of 0, 50, and 500 bp at both ends of the gene cassette (+0-+500 linear vectors) did not significantly differ from one another or from the efficiency of the +1000 linear vector. Transformation efficiencies using the linear vectors were lower than that using the circular vector, but were not significantly different. The ratios of the number of transformants containing the whole region of the gene cassette to those of transformants transfected using linear vectors of various lengths were determined. An extension (≧50 bp) of DNA fragments was effective for introducing the whole region of the gene cassette into the genomic DNA. In using various amounts of the +50 linear vector (37.5-300 fmol/shot), we observed that transformation efficiencies using 37.5 fmol (52.2 ng)/shot of the linear vector were not significantly different from those obtained using 300 fmol of the linear vector. The 300 fmol quantity was set considering the quantity of the circular plasmid (1 µg=approx. 300 fmol) and the 37.5 fmol quantity was set for quick and easy preparation of approximately 500 ng of the linear short vector needed for triplicate transformation experiments in one PCR tube containing 50 µl of PCR cocktail. Integrating the gene cassette of the short linear vectors as well as that of the full length of the linear vector (+1000 linear vector) into the chromosomal DNA was determined using Southern blot analysis. The short linear vectors tended to result in smaller numbers of insertions than those of the supercoiled plasmid. This simple and time-saving transformation method using microparticle bombardment with PCR-amplified DNA fragments permitted both functional analysis of diatom-specific genes and development of diatom strains useful for further biotechnological applications.

Characterization of marine diatom-infecting virus promoters in the model diatom Phaeodactylum tricornutum.

Viruses are considered key players in phytoplankton population control in oceans. However, mechanisms that control viral gene expression in prominent microalgae such as diatoms remain largely unknown. In this study, potential promoter regions isolated from several marine diatom-infecting viruses (DIVs) were linked to the egfp reporter gene and transformed into the Pennales diatom Phaeodactylum tricornutum. We analysed their activity in cells grown under different conditions. Compared to diatom endogenous promoters, novel DIV promoter (ClP1) mediated a significantly higher degree of reporter transcription and translation. Stable expression levels were observed in transformants grown under both light and dark conditions, and high levels of expression were reported in cells in the stationary phase compared to the exponential phase of growth. Conserved motifs in the sequence of DIV promoters were also found. These results allow the identification of novel regulatory regions that drive DIV gene expression and further examinations of the mechanisms that control virus-mediated bloom control in diatoms. Moreover, the identified ClP1 promoter can serve as a novel tool for metabolic engineering of diatoms. This is the first report describing a promoter of DIVs that may be of use in basic and applied diatom research.

Effect of an Introduced Phytoene Synthase Gene Expression on Carotenoid Biosynthesis in the Marine Diatom Phaeodactylum tricornutum.

Carotenoids exert beneficial effects on human health through their excellent antioxidant activity. To increase carotenoid productivity in the marine Pennales Phaeodactylum tricornutum, we genetically engineered the phytoene synthase gene (psy) to improve expression because RNA-sequencing analysis has suggested that the expression level of psy is lower than other enzyme-encoding genes that are involved in the carotenoid biosynthetic pathway. We isolated psy from P. tricornutum, and this gene was fused with the enhanced green fluorescent protein gene to detect psy expression. After transformation using the microparticle bombardment technique, we obtained several P. tricornutum transformants and confirmed psy expression in their plastids. We investigated the amounts of PSY mRNA and carotenoids, such as fucoxanthin and ß-carotene, at different growth phases. The introduction of psy increased the fucoxanthin content of a transformants by approximately 1.45-fold relative to the levels in the wild-type diatom. However, some transformants failed to show a significant increase in the carotenoid content relative to that of the wild-type diatom. We also found that the amount of PSY mRNA at log phase might contribute to the increase in carotenoids in the transformants at stationary phase.

Production and removal of superoxide anion radical by artificial metalloenzymes and redox-active metals.

Generation of reactive oxygen species is useful for various medical, engineering and agricultural purposes. These include clinical modulation of immunological mechanism, enhanced degradation of organic compounds released to the environments, removal of microorganisms for the hygienic purpose, and agricultural pest control; both directly acting against pathogenic microorganisms and indirectly via stimulation of plant defense mechanism represented by systemic acquired resistance and hypersensitive response. By aiming to develop a novel classes of artificial redox-active biocatalysts involved in production and/or removal of superoxide anion radicals, recent attempts for understanding and modification of natural catalytic proteins and functional DNA sequences of mammalian and plant origins are covered in this review article.

Deciphering early events involved in hyperosmotic stress-induced programmed cell death in tobacco BY-2 cells.

Hyperosmotic stresses represent one of the major constraints that adversely affect plants growth, development, and productivity. In this study, the focus was on early responses to hyperosmotic stress- (NaCl and sorbitol) induced reactive oxygen species (ROS) generation, cytosolic Ca(2+) concentration ([Ca(2+)]cyt) increase, ion fluxes, and mitochondrial potential variations, and on their links in pathways leading to programmed cell death (PCD). By using BY-2 tobacco cells, it was shown that both NaCl- and sorbitol-induced PCD seemed to be dependent on superoxide anion (O2·(-)) generation by NADPH-oxidase. In the case of NaCl, an early influx of sodium through non-selective cation channels participates in the development of PCD through mitochondrial dysfunction and NADPH-oxidase-dependent O2·(-) generation. This supports the hypothesis of different pathways in NaCl- and sorbitol-induced cell death. Surprisingly, other shared early responses, such as [Ca(2+)]cyt increase and singlet oxygen production, do not seem to be involved in PCD.

A role for oxalic acid generation in ozone-induced signallization in Arabidopis cells.

Ozone (O(3) ) is an air pollutant with an impact increasingly important in our industrialized world. It affects human health and productivity in various crops. We provide the evidences that treatment of Arabidopsis thaliana with O(3) results in ascorbate-derived oxalic acid production. Using cultured cells of A. thaliana as a model, here we further showed that oxalic acid induces activation of anion channels that trigger depolarization of the cell, increase in cytosolic Ca(2+) concentration, generation of reactive oxygen species and cell death. We confirmed that O(3) reacts with ascorbate in the culture, thus resulting in production of oxalic acid and this could be part of the O(3) -induced signalling pathways that trigger programmed cell death.

Peroxyacetyl nitrate-induced oxidative and calcium signaling events leading to cell death in ozone-sensitive tobacco cell-line.

It has long been concerned that some secondary air pollutants such as smog components, ozone (O3) and peroxyacetyl nitrate (PAN), are highly phytotoxic even at low concentrations. Compared with the biology of O3, we largely lack the information on the toxicity model for PAN at the cellular signaling levels. Here, we studied the cell-damaging impact of PAN using suspension culture of smog-sensitive tobacco variety (Bel-W3). The cells were exposed to freshly synthesized PAN and the induced cell death was assessed under microscope after staining with Evans blue. Involvement of reactive oxygen species (ROS) in PAN toxicity was suggested by PAN-dependently increased intracellular H2O2 and also by the cell-protective effects of ROS scavengers and related inhibitors. Calcium chelator also lowered the level of PAN-induced cell death, indicating that Ca2+ is also involved. Using a transgenic cell line expressing aequorin, an increase in cytosolic Ca2+ concentration responsive to the pulse of PAN, but sensitive to Ca2+ channel blockers, was recorded, indicating that Ca2+ channels are activated by PAN or PAN-derived signals. Above data show some similarity between the signaling mechanisms responsive to O3 and PAN.

Copper-binding peptides from human prion protein and newly designed peroxidative biocatalysts.

A previous work suggested that peptides from the histidine-containing copper-binding motifs in human prion protein (PrP) function as peroxidase-like biocatalysts catalyzing the generation of superoxide anion radicals in the presence of neurotransmitters (aromatic monoamines) and phenolics such as tyrosine and tyrosyl residues on proteins. In this study, using various phenolic substrates, the phenol-dependent superoxide-generating activities of PrP-derived peptide sequences were compared. Among the peptides tested, the GGGTH pentapeptide was shown to be the most active catalyst for phenol-dependent reactions. Based on these results, we designed a series of oligoglycyl-histidines as novel peroxidative biocatalysts, and their catalytic performances including kinetics, heat tolerance, and freezing tolerance were analysed.

Forced symbiosis between Synechocystis spp. PCC 6803 and apo-symbiotic Paramecium bursaria as an experimental model for evolutionary emergence of primitive photosynthetic eukaryotes.

Single-cell green paramecia (Paramecium bursaria) is a swimming vehicle that carries several hundred cells of endo-symbiotic green algae. Here, a novel model for endo-symbiosis, prepared by introducing and maintaining the cells of cyanobacterium (Synechocystis spp. PCC 6803) in the apo-symbiotic cells of P. bursaria is described.

Increased anion channel activity is an unavoidable event in ozone-induced programmed cell death.

BACKGROUND: Ozone is a major secondary air pollutant often reaching high concentrations in urban areas under strong daylight, high temperature and stagnant high-pressure systems. Ozone in the troposphere is a pollutant that is harmful to the plant. PRINCIPAL FINDINGS: By exposing cells to a strong pulse of ozonized air, an acute cell death was observed in suspension cells of Arabidopsis thaliana used as a model. We demonstrated that O(3) treatment induced the activation of a plasma membrane anion channel that is an early prerequisite of O(3)-induced cell death in A. thaliana. Our data further suggest interplay of anion channel activation with well known plant responses to O(3), Ca(2+) influx and NADPH-oxidase generated reactive oxygen species (ROS) in mediating the oxidative cell death. This interplay might be fuelled by several mechanisms in addition to the direct ROS generation by O(3); namely, H(2)O(2) generation by salicylic and abscisic acids. Anion channel activation was also shown to promote the accumulation of transcripts encoding vacuolar processing enzymes, a family of proteases previously reported to contribute to the disruption of vacuole integrity observed during programmed cell death. SIGNIFICANCE: Collectively, our data indicate that anion efflux is an early key component of morphological and biochemical events leading to O(3)-induced programmed cell death. Because ion channels and more specifically anion channels assume a crucial position in cells, an understanding about the underlying role(s) for ion channels in the signalling pathway leading to programmed cell death is a subject that warrants future investigation.

A green Paramecium strain with abnormal growth of symbiotic algae.

Some hundred cells of Chlorella-like green algae are naturally enclosed within the cytoplasm of a single cell of green paramecia (Paramecium bursaria). Therefore, P. bursaria serves as an experimental model for studying the nature of endo-symbiosis made up through chemical communication between the symbiotic partners. For studying the mechanism of symbiotic regulations, the materials showing successful symbiosis are widely used. Apart from such successful model materials, some models for symbiotic distortion would be of great interest in order to understand the nature of successful symbiosis. Here, we describe a case of unsuccessful symbiosis causing unregulated growth of algae inside the hosting ciliates. Recently, we have screened some cell lines, from the mass of P. bursaria cells survived after paraquat treatment. The resultant cell lines (designated as KMZ series) show novel and unusual morphological features with heavily darker green colour distinguishable from the original pale green-coloured paramecia. In this type of isolates, endo-symbiotic algae are restricted within one or two dense spherical structures located at the center of the host cells' cytoplasm. Interestingly, this isolate maintains the host cells' circadian mating response which is known as an alga-dependent behaviour in the host cells. In contrast, we discuss that KMZ lacks the host-dependent regulation of algal growth, thus the algal complex often over-grows obviously exceeding the original size of the normal hosting ciliates. Additionally, possible use of this isolate as a novel model for symbiotic cell-to-cell communication is discussed.