The effect of constant darkness and short light periods on the survival and physiological fitness of two phytoplankton species and their growth potential after re-illumination

We tested the survival potential and fitness of two different algae strains (the diatom Thalassiosira weissflogii and the cryptophyceae Rhodomonas sp.) under different growth conditions (complete darkness and short light intervals, simulating conditions in a deep mixed water column) at different temperatures, plus the effect of these conditions on the physiological fitness and growth after re-illumination was examined. Both species survived the experimental conditions without significant cell loss or physiological damage. Two different survival strategies were observed: (1) the diatom T. weissflogii immediately reduced its metabolic rate and stopped cell division. The effect on chlorophyll a (chl-a) content and photosynthetic capacity was negligible. At 10 °C, T. weissflogii used the short light windows to metabolize carbohydrates and growth. (2) The cryptophyte Rhodomonas sp. initially continued to grow after transfer into all trials. However, the cell number decreased after day 6. Carbohydrate and chl-a content went on to decrease dramatically (70 and 50%, respectively). After 3 days of re-illumination, T. weissflogii grew faster than of Rhodomonas sp.. The diatom seemed to benefit from better start conditions and would out-compete the cryptophyte during a spring bloom. Our results highlight that these algae groups have different strategies in dealing with darkness, which potentially endow diatoms with a competitive advantage in deep mixed waters and in the season of early spring.     

Intrapopulation heterogeneity of the fluorescence parameters of the marine plankton alga <Emphasis Type="Italic">Thalassiosira weissflogii</Emphasis> at various nitrogen levels

Fluorescence of the marine alga Thalassiosira weissflogii (Grunow) Fryxell et Hasle with open (F _o ) and closed (F _m ) reaction centers of photosystem 2 (PS 2) and its relative variable fluorescence (F _v/F _m ) were measured at various levels of inorganic nitrogen. A significant heterogeneity of the population in terms of these parameters was revealed. Some cells within the population were more sensitive to nitrogen deficiency, and their photosynthetic apparatus was disrupted to a greater extent. The cells within a population also differed in terms of their ability to recover after incubation at low nitrogen levels. Enhancement of nitrogen deficiency resulted in an increase in the variability of the F _o and F _v/F _m values of the cells. Fluorescence variability decreased at a less pronounced deficiency. Fluorescence variability should be taken into consideration in the studies concerning responses of algae to changes in nutrient contents.     

Fluorescence parameters of marine plankton algae at the assimilation of organic nitrogen

fluorescence parameters of marine plankton algae Pseudo-nitzschis delicatissima, Thalassiosira weissflogii, and Tetraselmis viridis were estimated after the addition of organic (urea and glycine) and inorganic (nitrate and ammonia) nitrogen to nitrogen-limited cultures acclimated to limited and saturated irradiance. The photochemical efficiency of photosystem 2, the maximum relative electron transport, and the light saturation index increased in the algae assimilating organic nitrogen. The dynamics of parameters depended species specifically on the nitrogen source and irradiance. The ecological role of organic nitrogen in the seasonal dynamics and vertical distribution of phytoplankton is discussed.     

Light absorption and scattering by cell suspensions of some cyanobacteria and microalgae

Absorption spectra of cyanobacteria (Anacystis nidulans, Anabaena variabilis, and Chlorogloeopsis fritschii), red (Cyanidium caldarum and Porphyridium cruentum), green (Dunaliella maritima and Dunaliella salina) and diatom (Thalassiosira weisflogii) alga cell suspensions are presented; the spectra were obtained by using an approach developed earlier to compensate for scattering [1, 2]. In all species, the shapes of the absorption spectra were independent of the cell concentration. For Th. weisflogii and D. maritima, the analysis of selective and nonselective scattering was carried out. The effect of mechanical cell disruption on optical properties (absorption, scattering, and “package” effect) on D. maritima was studied. The character and dynamics of optical changes in D. salina under the influence of sodium chloride were followed.     

Characterization of the effects of <Emphasis Type="Italic">n</Emphasis>-butanol on the cell envelope of <Emphasis Type="Italic">E. coli</Emphasis>

Biofuel alcohols have severe consequences on the microbial hosts used in their biosynthesis, which limits the productivity of the bioconversion. The cell envelope is one of the most strongly affected structures, in particular, as the external concentration of biofuels rises during biosynthesis. Damage to the cell envelope can have severe consequences, such as impairment of transport into and out of the cell; however, the nature of butanol-induced envelope damage has not been well characterized. In the present study, the effects of n-butanol on the cell envelope of Escherichia coli were investigated. Using enzyme and fluorescence-based assays, we observed that 1 % v/v n-butanol resulted in the release of lipopolysaccharides from the outer membrane of E. coli and caused ‘leakiness’ in both outer and inner membranes. Higher concentrations of n-butanol, within the range of 2–10 % (v/v), resulted in inner membrane protrusion through the peptidoglycan observed by characteristic blebs. The findings suggest that strategies for rational engineering of butanol-tolerant bacterial strains should take into account all components of the cell envelope.     

Paul Henry Latimer (1925–2011): discoverer of selective scattering in photosynthetic systems

The response of marine phytoplankton to the ongoing increase in atmospheric pCO₂ reflects the consequences of both increased CO₂ concentration and decreased pH in surface seawater. In the model diatom Thalassiosira weissflogii, we explored the effects of varying pCO₂ and pH, independently and in concert, on photosynthesis and respiration by incubating samples in water enriched in H₂ ¹⁸O. In long-term experiments (~6-h) at saturating light intensity, we observed no effects of pH or pCO₂ on growth rate, photosynthesis or respiration. This absence of a measurable response reflects the very small change in energy used by the carbon concentrating mechanism (CCM) compared to the energy used in carbon fixation. In short-term experiments (~3 min), we also observed no effects of pCO₂ or pH, even under limiting light intensity. We surmise that in T. weissflogii, it is the photosynthetic production of NADPH and ATP, rather than the CO₂-saturation of Rubisco that controls the rate of photosynthesis at low irradiance. In short-term experiments, we observed a slightly higher respiration rate at low pH at the onset of the dark period, possibly reflecting the energy used for exporting H⁺ and maintaining pH homeostasis. Based on what is known of the biochemistry of marine phytoplankton, our results are likely generalizable to other diatoms and a number of other eukaryotic species. The direct effects of ocean acidification on growth, photosynthesis and respiration in these organisms should be small over the range of atmospheric pCO₂ predicted for the twenty-first century.     

A physiological comparative study of acid tolerance of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> ZDY 2013 and <Emphasis Type="Italic">L. plantarum</Emphasis> ATCC 8014 at membrane and cytoplasm levels

This study aimed to disclose the acid tolerance mechanism of Lactobacillus plantarum by comparing L. plantarum ZDY 2013 with the type strain L. plantarum ATCC 8014 in terms of cell membrane, energy metabolism, and amino acid metabolism. L. plantarum ZDY 2013 had a superior growth performance under acidic condition with 100-fold higher survival rate than that of L. plantarum ATCC 8014 at pH 2.5. To determine the acid tolerance physiological mechanism, cell integrity was investigated through scanning electron microscopy. The study revealed that L. plantarum ZDY 2013 maintained cell morphology and integrity, which is much better than L. plantarum ATCC 8014 under acid stress. Analysis of energy metabolism showed that, at pH 5.0, L. plantarum ZDY 2013 enhanced the activity of Na⁺/K⁺-ATPase and decreased the ratio of NAD⁺/NADH in comparison with L. plantarum ATCC 8014. Similarly, amino acid metabolism of intracellular arginine, glutamate, and alanine was improved in L. plantarum ZDY 2013. Correspondingly, the activity of arginine deiminase and glutamate decarboxylase of L. plantarum ZDY 2013 increased by 1.2-fold and 1.3-fold compared with L. plantarum ATCC 8014 in acid stress. In summary, it is demonstrated that the special physiological behaviors (integrity of cell membrane, enhanced energy metabolism, increased amino acid and enzyme level) of L. plantarum ZDY 2013 can protect the cells from acid stress.     

Toxicity,membrane binding and uptake of the <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> agglutinin (SSA) in different insect cell lines

The fungal lectin purified from Sclerotinia sclerotiorum, further referred to as Sclerotinia sclerotiorum agglutinin or SSA, possesses insecticidal activity against important pest insects such as pea aphids (Acyrthosiphon pisum). This paper aims at a better understanding of its activity at cellular level. Therefore, different insect cell lines were treated with SSA. These cell lines were derived from different tissues and represent the three major orders of insects important in agriculture: CF-203 (midgut Choristoneura fumiferana, Lepidoptera), GUTAW1 (midgut, Helicoverpa zea, Lepidoptera), High5 cells (ovary, Trichoplusia ni, Lepidoptera), Sf9 (ovary cells from Spodoptera frugiperda, Lepidoptera), S2 (hemocyte, Drosophila melanogaster, Diptera), and TcA (whole body, Tribolium castaneum, Coleoptera). Although the sensitivity to SSA differs between the cell lines, SSA clearly showed toxicity in all six cell lines with median effect concentrations (EC₅₀) ranging between 9 and 42 μg/ml. An in-depth analysis of the mechanism of uptake in the cells revealed superior amounts of FITC-SSA at the membrane of CF-203 cells compared to Sf9 cells, while a similar small amount of SSA was internalized in both cell lines. Pre-incubation with the clathrin-mediated endocytosis inhibitor phenylarsine oxide inhibited the internalization of SSA into the CF-203 and Sf9 cells with a respective reduction of 6- and 1.7-fold. The data are discussed in relation to the importance of cellular uptake mechanism for SSA binding and cytotoxicity.     

IutB participates in the ferric-vulnibactin utilization system in <Emphasis Type="Italic">Vibrio vulnificus</Emphasis> M2799

Vibrio vulnificus, an opportunistic pathogen that causes a serious, often fatal, infection in humans, requires iron for its growth. This bacterium utilizes iron from the environment via the vulnibactin-mediated iron uptake system. The mechanisms of vulnibactin biosynthesis, vulnibactin export, and ferric-vulnibactin uptake systems have been reported, whereas the ferric-vulnibactin reduction mechanism in the cell remains unclear. The results of our previous study showed that VuuB, a member of the flavin adenine dinucleotide-containing siderophore-interacting protein family, is a ferric-vulnibactin reductase, but there are other reductases that can complement for the defective vuuB. The aim of this study was to identify these proteins that can complement the loss of function of VuuB. We constructed mutants of genes encoding putative reductases in V. vulnificus M2799, and analyzed their growth under low-iron conditions. Complementation analyses confirmed that IutB, which functions as a ferric-aerobactin reductase, participates in ferric-vulnibactin reduction in the absence of VuuB. This is the first genetic evidence that ferric-vulnibactin is reduced by a member of the ferric-siderophore reductase protein family. In the aerobactin-utilization system, IutB plays a major role in ferric-aerobactin reduction in V. vulnificus M2799, and VuuB and DesB can compensate for the defect of IutB. Furthermore, the expression of iutB and desB was found to be regulated by iron and a ferric uptake regulator.     

<Emphasis Type="Italic">PAD4</Emphasis>, <Emphasis Type="Italic">LSD1</Emphasis> and <Emphasis Type="Italic">EDS1</Emphasis> regulate drought tolerance,plant biomass production,and cell wall properties

Key message

Arabidopsis and poplar with modified PAD4, LSD1 and EDS1 genes exhibit successful growth under drought stress. The acclimatory strategies depend on cell division/cell death control and altered cell wall composition.

Abstract

The increase of plant tolerance towards environmental stresses would open much opportunity for successful plant cultivation in these areas that were previously considered as ineligible, e.g. in areas with poor irrigation. In this study, we performed functional analysis of proteins encoded by PHYTOALEXIN DEFICIENT 4 (PAD4), LESION SIMULATING DISEASE 1 (LSD1) and ENHANCED DISEASE SUSCEPTIBILITY 1 (EDS1) genes to explain their role in drought tolerance and biomass production in two different species: Arabidopsis thaliana and Populus tremula × tremuloides. Arabidopsis mutants pad4-5, lsd1-1, eds1-1 and transgenic poplar lines PAD4-RNAi, LSD1-RNAi and ESD1-RNAi were examined in terms of different morphological and physiological parameters. Our experiments proved that Arabidopsis PAD4, LSD1 and EDS1 play an important role in survival under drought stress and regulate plant vegetative and generative growth. Biomass production and acclimatory strategies in poplar were also orchestrated via a genetic system of PAD4 and LSD1 which balanced the cell division and cell death processes. Furthermore, improved rate of cell division/cell differentiation and altered physical properties of poplar wood were the outcome of PAD4- and LSD1-dependent changes in cell wall structure and composition. Our results demonstrate that PAD4, LSD1 and EDS1 constitute a molecular hub, which integrates plant responses to water stress, vegetative biomass production and generative development. The applicable goal of our research was to generate transgenic plants with regulatory mechanism that perceives stress signals to optimize plant growth and biomass production in semi-stress field conditions.

     

<Emphasis Type="Italic">In vivo</Emphasis> RNAi screen identifies candidate signaling genes required for collective cell migration in <Emphasis Type="Italic">Drosophila</Emphasis> ovary

Collective migration of loosely or closely associated cell groups is prevalent in animal development, physiological events, and cancer metastasis. However, our understanding of the mechanisms of collective cell migration is incomplete. Drosophila border cells provide a powerful in vivo genetic model to study collective migration and identify essential genes for this process. Using border cell-specific RNAi-silencing in Drosophila, we knocked down 360 conserved signaling transduction genes in adult flies to identify essential pathways and genes for border cell migration. We uncovered a plethora of signaling genes, a large proportion of which had not been reported for border cells, including Rack1 (Receptor of activated C kinase) and brk (brinker), mad (mother against dpp), and sax (saxophone), which encode three components of TGF-β signaling. The RNAi knock down phenotype was validated by clonal analysis of Rack1 mutants. Our data suggest that inhibition of Src activity by Rack1 may be important for border cell migration and cluster cohesion maintenance. Lastly, results from our screen not only would shed light on signaling pathways involved in collective migration during embryogenesis and organogenesis in general, but also could help our understanding for the functions of conserved human genes involved in cancer metastasis.     

Halophytic NHXs confer salt tolerance by altering cytosolic and vacuolar K<Superscript>+</Superscript> and Na<Superscript>+</Superscript> in <Emphasis Type="Italic">Arabidopsis</Emphasis> root cell

While the role of the vacuolar NHX Na⁺/H⁺ exchangers in plant salt tolerance has been demonstrated on numerous occasions, their control over cytosolic ionic relations has never been functionally analysed in the context of subcellular Na⁺ and K⁺ homeostasis. In this work, PutNHX1 and SeNHX1 were cloned from halophytes Puccinellia tenuiflora and Salicornia europaea and transiently expressed in Arabidopsis wild type Col-0 and the nhx1 mutant. Phylogentic analysis, topological prediction, analysis of evolutionary conservation, the topology structure and analysis of hydrophobic or polar regions of PutNHX1 and SeNHX1 indicated that they are unique tonoplast Na⁺/H⁺ antiporters with characteristics for salt tolerance. As a part of the functional assessment, cytosolic and vacuolar Na⁺ and K⁺ in different root tissues and ion fluxes from root mature zone of Col-0, nhx1 and their transgenic lines were measured. Transgenic lines sequestered large quantity of Na⁺ into root cell vacuoles and also promoted high cytosolic and vacuolar K⁺ accumulation. Expression of PutNHX1 and SeNHX1 led to significant transient root Na⁺ uptake in the four transgenic lines upon recovery from salt treatment. In contrast, the nhx1 mutant maintained a prolonged Na⁺ efflux and the nhx1:PutNHX1 and nhx1:SeNHX1 lines started to actively pump Na⁺ out of the cell. Overall, our findings suggest that PutNHX1 and SeNHX1 improve Na⁺ sequestration in the vacuole and K⁺ retention in the cytosol and vacuole of root cells of Arabidopsis, and that they interact with other regulatory mechanisms to provide a highly orchestrated regulation of ionic relations among intracellular cell compartments.     

Identification of Microalgae by Laser Desorption/Ionization Mass Spectrometry Coupled with Multiple Nanomatrices

In this study, we demonstrate a simple method to identify microalgae by surface-assisted laser desorption/ionization mass spectrometry (SALDI-MS) using three different substrates: HgSe, HgTe, and HgTeSe nanostructures. The fragmentation/ionization processes of complex molecules in algae varied according to the heat absorption and transfer efficiency of the nanostructured matrices (NMs). Therefore, the mass spectra obtained for microalgae showed different patterns of m/z values for different NMs. The spectra contained both significant and nonsignificant peaks. Constructing a Venn diagram with the significant peaks obtained for algae when using HgSe, HgTe, and HgTeSe NMs in m/z ratio range 100–1000, a unique relationship among the three sets of values was obtained. This unique relationship of sets is different for each species of microalgae. Therefore, by observing the particular relationship of sets, we successfully identified different algae such as Isochrysis galbana, Emiliania huxleyi, Thalassiosira weissflogii, Nannochloris sp., Skeletonema cf. costatum, and Tetraselmis chui. This simple and cost-effective SALDI-MS analysis method coupled with multi-nanomaterials as substrates may be extended to identify other microalgae and microorganisms in real samples.

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Graphical Abstract Identification of microalgae by surface-assisted laser desorption/ionization mass spectrometry coupled with three different mercury-based nanosubstrates

     

Roles of nitrogen and phosphorus in growth responses and toxin production (using LC-MS/MS) of tropical <Emphasis Type="Italic">Microcystis ichthyoblabe</Emphasis> and <Emphasis Type="Italic">M. flos-aquae</Emphasis>

In experiments investigating nutrient effects on tropical Microcystis, increasing nitrogen and phosphorus concentrations were found to have a significant positive effect on maximum cell yields of two strains of Microcystis ichthyoblabe (from Lower Peirce and Tengeh Reservoirs) and one strain of Microcystis flos-aquae isolated (Lower Peirce Reservoir) from Singapore. However, only increasing nitrogen concentration had a positive effect on growth rates of M. ichthyoblabe and M. flos-aquae from Lower Peirce Reservoir. MC-RR and MC-LR were produced by all three strains with MC-RR being the dominant variant. Phosphorus played an important role in MC production with increases in phosphorus from medium to high concentrations leading to decreases in MC-RR cell quotas for all three strains at the two highest nitrogen levels tested. The different growth and toxin production responses between M. ichthyoblabe strains could be due to location-specific differences.     

Enhancement of Pyruvate Productivity in <Emphasis Type="Italic">Candida glabrata</Emphasis> by Deleting the <Emphasis Type="Italic">CgADE13</Emphasis> Gene to Improve Acid Tolerance

Acid tolerance is one of the critical factors to evaluate the quality of the industrial production strains, especially organic acid producing microorganisms. To circumvent this problem, we investigated the physiological function of adenylosuccinate lyase in AMP metabolism from Candida glabrata by deleting the corresponding gene, CgADE13. At pH 4.0, CgADE13 deletion resulted in a 68.3% and 112.0% increase in biomass and cell viability compared to those of wild type strain (wt), respectively. In addition, CgADE13 deletion also protected cell morphology and counteracted ROS production. Further, the intracellular ATP level of strain Cgade13Δ was decreased by 25.0%, and its H+-ATPase activity was increased by 15.0%. Finally, pyruvate production with strain Cgade13Δ in a 30-L batch bioreactor at pH 4.0 reached 53.9 g/L, and pyruvate productivity was increased by 166.7% compared to that of wt. This is the first report regarding tolerance engineering of C. glabrata for enhancing pyruvate productivity, which provides a good starting point for metabolic engineering to achieve the industrial production of other chemicals.     

Connection between proliferation rate and temozolomide sensitivity of primary glioblastoma cell culture and expression of <Emphasis Type="Italic">YB-1</Emphasis> and <Emphasis Type="Italic">LRP/MVP</Emphasis>

Glioblastomas (GBL) are the most common and aggressive brain tumors. They are distinguished by high resistance to radiation and chemotherapy. To find novel approaches for GBL classification, we obtained 16 primary GBL cell cultures and tested them with real-time PCR for mRNA expression of several genes (YB-1, MGMT, MELK, MVP, MDR1, BCRP) involved in controlling cell proliferation and drug resistance. The primary GBL cultures differed in terms of proliferation rate, wherein a group of GBL cell cultures with low proliferation rate demonstrated higher resistance to temozolomide. We found that GBL primary cell cultures characterized by high proliferation rate and lower resistance to temozolomide expressed higher mRNA level of the YB-1 and MDR1 genes, whereas upregulated expression of MVP/LRP mRNA was a marker in the group of GBL with low proliferation rate and high resistance. A moderate correlation between expression of YB-1 and MELK as well as YB-1 and MDR1 was found. In the case of YB-1 and MGMT expression, no correlation was found. A significant negative correlation was revealed between mRNA expression of MVP/LRP and MELK, MDR1, and BCRP. No correlation in expression of YB-1 and MVP/LRP genes was observed. It seems that mRNA expression of YB-1 and MVP/LRP may serve as a marker for GBL cell cultures belonging to distinct groups, each of which is characterized by a unique pattern of gene activity.