Sensitivity of antioxidant status of plant cells to furostanol glycosides

The present paper reports that in vitro plant objects (test tube plants and cell cultures), when subjected to furostanol glycosides (FG), underwent nonspecific reactions related to antioxidant status—decrease in peroxidation of lipids (POL) and increase in guaiacol-dependent peroxidase activity. The level of superoxide increased as early as after 5 min from contact with yam (Dioscorea deltoidea Wall) cells with FG. In this case, changes in POL processes and in activities of peroxidase and aldehyde-disposing emzymes were also observed. Upon a short-term cell exposure to FG, the levels of the primary POL products (conjugated dienes) increased, and that of the secondary POL products decreased compared to the control. These events were preceded by a rise in SOD activity and in an antioxidant activity of peroxidase along with a concurrent decrease in its oxidase (prooxidant) activity. The elevated activities of aldehyde-disposing enzymes aldehyde dehydrogenase and aldehyde reductase favored the reduction in the content of the secondary products of POL. Upon a long contact of FG with cells, the effect of FG was seen only at the initial and final phases of the culture growth cycle. Namely, FG diminished the POL level at the exponential growth phase and at the end of the cell degradation phase but had no effect at the stationary phase and the onset of the degradation phase. Therefore, the treatment with FG retarded the cell culture degradation and made the fall in cell viability not so dramatic by the end of the growth cycle. Actually, by the end of the degradation phase, the viability diminished down to 40% in the control but remained at 70% in the FG-treated counterpart.     

Enhanced Resistance of Pea Plants to Oxidative Stress Caused by Paraquat during Colonization by Aerobic Methylobacteria

The influence of colonization of the pea (Pisum sativum L.) by aerobic methylobacteria of five different species (Methylophilus flavus Ship, Methylobacterium extorquens G10, Methylobacillus arboreus Iva, Methylopila musalis MUSA, Methylopila turkiensis Side1) on plant resistance to paraquat-induced stresses has been studied. The normal conditions of pea colonization by methylobacteria were characterized by a decrease in the activity of antioxidant enzymes (superoxide dismutase, catalase, and peroxidases) and in the concentrations of endogenous H₂O₂, proline, and malonic dialdehyde, which is a product of lipid peroxidation and indicator of damage to plant cell membranes, and an increase in the activity of the photosynthetic apparatus (the content of chlorophylls а, b and carotenoids). In the presence of paraquat, the colonized plants had higher activities of antioxidant enzymes, stable photosynthetic indices, and a less intensive accumulation of the products of lipid peroxidation as compared to noncolonized plants. Thus, colonization by methylobacteria considerably increased the adaptive protection of pea plants to the paraquat-induced oxidative stress.     

<Emphasis Type="Italic">Xenopus laevis</Emphasis> peroxiredoxins: Gene expression during development and characterization of the enzymes

Reactive oxygen species (ROS) are produced via catabolic and anabolic processes during normal embryonic development, and ROS content in the cell is maintained at a certain level. Peroxiredoxins are a family of selenium-independent peroxidases and play a key role in maintaining redox homeostasis of the cell. In addition to regulating the ROS level, peroxiredoxins are involved in intracellular and intercellular signaling, cell differentiation, and tissue development. The time course of peroxiredoxin gene (prx1–6) expression was studied in Xenopus laevis during early ontogeny (Nieuwkoop and Faber stages 10–63). The highest expression level was observed for prx1 at these developmental stages. The prx1, prx3, and prx4 expression level changed most dramatically in response to oxidative stress artificially induced in X. laevis embryos. In X. laevis adults, prx1–6 were all intensely expressed in all organs examined, the prx1 expression level being the highest. The X. laevis prx1–6 genes were cloned and expressed in Escherichia coli, and physico-chemical characteristics were compared for the recombinant enzymes. The highest peroxidase activity and thermal stability were observed for Prx1 and Prx2. It was assumed that Prx1 plays a leading role in X. laevis early development.     

A <Emphasis Type="Italic">pqr2</Emphasis> mutant encodes a defective polyamine transporter and is negatively affected by ABA for paraquat resistance in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the paraquat-resistant mutants that have been reported in plants, this study identified a novel A. thaliana mutant (pqr2) from an XVE inducible activation library based on its resistance to 2 μM paraquat. The pqr2 mutant exhibited a termination mutation in the exon of AT1G31830/PAR1/PQR2, encoded a polyamine uptake transporter AtPUT2/PAR1/PQR2. The PQR2 mutation could largely reduce superoxide accumulation and cell death in the pqr2 plants under paraquat treatment. Moreover, compared with wild type, the pqr2 mutant exhibited much reduced tolerance to putrescine, a classic polyamine compound, which confirmed that PQR2 encoded a defective polyamine transporter. Notably, co-treated with ABA and paraquat, both pqr2 mutant and wild type exhibited a lethal phenotype from seed germination, but the wild type like pqr2 mutant, could remain paraquat-resistance while co-treated with high dosage of Na₂WO₄, an ABA synthesis inhibitor. Gene expression analysis suggested that ABA signaling should widely regulate paraquat-responsive genes distinctively in wild type and pqr2 mutant. Hence, this study has for the first time reported about ABA negative effect on paraquat-resistance in A. thaliana, providing insight into the ABA signaling involved in the oxidative stress responses induced by paraquat in plants.     

Protective effect of fucoidans from tropical seaweeds against oxidative stress in HepG2 cells

Fucoidans are sulfated polysaccharides with proven pharmacological effects localized in the cell wall of marine brown algae. The majority of studies have been performed with temperate brown algal species, but in recent years, the evaluation of species from tropical areas has been growing. The aim of this study was to determine the protective effect of fucoidans extracted from the tropical brown seaweeds Dictyota ciliolata, Padina sanctae-crucis, and Sargassum fluitans, against oxidative stress (OS). The D. ciliolata fucoidan (FDc) exhibited the highest reactive oxygen species (ROS) scavenging activity (26%), followed by P. sanctae-crucis fucoidan (FPs) (22%) and S. fluitans fucoidan (FSf) (14%). No cytotoxic effect was detected for any of the extracted fucoidans at a concentration of 2 mg mL^?1. Not only did the fucoidans tested show protective effect against OS by reducing ROS generation, but they also increased the glutathione (GSH) level and restored catalase (CAT) activity. Fucoidans obtained from tropical seaweeds could be used as a potential natural ingredient for functional foods.     

Biotic Interactions between Two Species of Microalgae in Mixed Culture

Biotic interactions in a mixed culture of two microalgae species—Scenedesmus quadricauda (Turp.) Breb. and Monoraphidium arcuatum (Korsch.) Hind.—used in bioassay in monocultures as test objects were studied. The toxic effect of cell-free filtrates from different “age” monoculture (2, 7, 10, 15, 21, and 28 days) of S. quadricauda on the growth of the “young” test culture of M. arcuatum and, conversely, the toxic effect of cell-free filtrates from the different “age” (2, 7, 10, 15, 21, and 28 days) monoculture of M. arcuatum on the growth of the “young” test culture of S. quadricauda was evaluated. Simultaneously, the toxicity of their own filtrates of different “ages” was monitored by a test culture of each species. The interactions of the species in the mixed culture can be regarded as negative, as an antagonistic one, when both populations inhibit the growth of each other through metabolites and food resource competition, while the effect of S. quadricauda on M. arcuatum is much stronger. The main factor constraining the growth of monoculture S. quadricauda is the rapid depletion of the food resource from the medium and not the inhibition of growth by its own metabolites. The depletion of the food resources from the medium in monoculture of M. arcuatum occurs much later than in monoculture of S. quadricauda. Metabolites of S. quadricauda cause a strong inhibitory effect on the growth of M. arcuatum, and the metabolites of M. arcuatum cause a weak inhibitory effect on the growth of S. quadricauda. The filtrates of the “old” culture of S. quadricauda (21–28 days) cause the greatest inhibitory effect on cell division of M. arcuatum. The filtrates of the “old” culture of S. quadricauda (21–28 days) cause the greatest inhibitory effect on cell division of M. arcuatum. Comparative analysis of the cell number dynamics of two species, S. quadricauda and M. arcuatum, in mono- and two-species algal cultures, as well as experiments with filtrates of these monocultures, showed that the interaction of species can be explained by the food resource competition and allelopathic interaction (exometabolite effect).     

Antigenotoxic activity of biologically active substances from <Emphasis Type="Italic">Inula britannica</Emphasis> and <Emphasis Type="Italic">Limonium gmelini</Emphasis>

The antigenotoxic and antioxidant activities of biologically active substances of extracts from Inula britannica L. and Limonium gmelinii (Willd.) Kuntze in E. coli strains MG1655 (pColD-lux), MG1655 (pSoxS-lux), and MG1655 (pKatG-lux) were studied by the bioluminescent test. Plant extracts from I. britannica and L. gmelinii in all used concentrations (0.5, 5.0, 50.0, and 500.0 μg/mL) had no genotoxic or oxidant activity. The extracts statistically significantly reduced the bioluminescence intensity of the pColD-lux, pKatG-lux, and pSoxS-lux sensors (p < 0.05) induced by 4-NQO and dioxidine, hydrogen peroxide, and paraquat, respectively. The activity of the extracts depended on their concentration; the greatest antigenotoxic and antioxidant effects were detected at a concentration of 500.0 μg/mL.     

Control of postharvest soft rot caused by <Emphasis Type="Italic">Erwinia carotovora</Emphasis> of vegetables by a strain of <Emphasis Type="Italic">Bacillus amyloliquefaciens</Emphasis> and its potential modes of action

Erwinia carotovora subsp. carotovora (Ecc), the causal agent of bacterial soft rot, is one of the destructive pathogens of postharvest vegetables. In this study, a bacterial isolate (BGP20) from the vegetable farm soil showed strong antagonistic activity against Ecc in vitro, and its twofold cell-free culture filtrate showed excellent biocontrol effect in controlling the postharvest bacterial soft rot of potatoes at 25 °C. The anti-Ecc metabolites produced by the isolate BGP20 had a high resistance to high temperature, UV-light and protease K. Based on the colonial morphology, cellular morphology, sporulation, and partial nucleotide sequences of 16S rRNA and gyrB gene, the isolate BGP20 was identified as Bacillus amyloliquefaciens subsp. plantarum. Further in vivo assays showed that the BGP20 cell culture was more effective in controlling the postharvest bacterial soft rot of green peppers and Chinese cabbages than its twofold cell-free culture filtrate. In contrast, the biocontrol effect and safety of the BGP20 cell culture were very poor on potatoes. In the wounds of potatoes treated with both the antagonist BGP20 and the pathogen Ecc, the viable count of Ecc was 31,746 times that of BGP20 at 48 h of incubation at 25 °C. But in the wounds of green peppers, the viable count of BGP20 increased 182.3 times within 48 h, and that of Ecc increased only 51.3 %. In addition, the treatment with both BGP20 and Ecc induced higher activity of phenylalanine ammonia-lyase (PAL) than others in potatoes. But the same treatment did not induce an increase of PAL activity in green peppers. In conclusion, the present study demonstrated that the isolate BGP20 is a promising candidate in biological control of postharvest bacterial soft rot of vegetables, but its main mode of action is different among various vegetables.     

The initial analysis of a serine proteinase gene (<Emphasis Type="Italic">AccSp10</Emphasis>) from <Emphasis Type="Italic">Apis cerana cerana</Emphasis>: possible involvement in pupal development,innate immunity and abiotic stress responses

Serine proteinases play important roles in innate immunity and insect development. We isolated a serine proteinase gene, designated AccSp10, from the Chinese honeybees (Apis cerana cerana). RT-qPCR and a Western blot analysis at different pupal development stages indicated that AccSp10 might be involved in melanin formation in pupae and promote pupal development. In adult workers, the expression of AccSp10 was upregulated by treatments mimicking harmful environments such as the presence of Bacillus bombysepticus, different temperatures (4, 24 and 42 °C), HgCl₂, H₂O₂ and paraquat; the exception was treatment with VC (vitamin C), which did not upregulate AccSp10 expression. Western blot confirmed the results. A disc diffusion assay indicated that recombinant AccSp10 accelerated E. coli cell death during stimulation with harmful substances (HgCl₂, paraquat and cumene hydroperoxide). These findings suggest that AccSp10 may be involved in the pupal development of Chinese honeybees and protection against microorganisms and abiotic harms.     

The aromatic cytokinin <Emphasis Type="Italic">meta-</Emphasis>topolin promotes in vitro propagation,shoot quality and micrografting in <Emphasis Type="Italic">Corylus colurna</Emphasis> L.

The role of 4.1 or 8.2 μM meta-topolin (mT) on shoot multiplication, rooting and ex vitro acclimatization of micropropagated Corylus colurna L., a promising non-suckering rootstock for hazelnut (Corylus avellana L.), was examined in comparison to N⁶-benzyladenine (BA), the most used cytokinin in tissue culture of Corylus spp. The influence of 8.2 μM mT and BA on photosynthetic pigments content and antioxidant enzymes activity, catalase (CAT) and guaiacol peroxidase (POD), in regenerated shoots, and on the preparation of the rootstock for micrografting was also evaluated. The highest shoot multiplication was recorded on medium containing 8.2 μM mT and an overall positive effect of mT on growth and quality of micropropagated shoots was found. The highest chlorophyll a content (1.236 mg g^?1 fresh weight, FW) and chlorophyll a/b ratio (2.48), and the lowest total carotenoids content (0.292 mg g^?1 FW) and CAT activity (25.8 μmol min^?1 mg^?1 protein) were detected after 8.2 μM mT application, while no significant differences were found in chlorophyll b content and POD activity between the two cytokinins. The best rhizogenesis response (98% for 4.1 μM and 100% for 8.2 μM mT) and ex vitro acclimatization competence (higher than 78%) were exhibited from shoots multiplied on mT. Furthermore, the multiplication of rootstock on mT allowed obtaining the highest (70%) response of successful micrografting. The present findings provide the first evidence of the successful applicability of mT in C. colurna tissue culture and development of micrografted plantlets.     

Property of Fish Gelatin gel as Affected by the Incorporation of Gellan and Calcium Chloride

Properties of fish gelatin (FG) gel as affected by gellan (GL) at different levels (2.5–7.5% FG substitution) in combination with calcium chloride (CaCl₂) at various concentrations (3–9 mM) were studied. Gel strength and hardness of FG/GL mixed gel increased as the levels of GL increased (P < 0.05). Increasing CaCl₂ concentration also resulted in the increases in both gel strength and hardness of mixed gel when GL at the same level was incorporated (P < 0.05). Conversely, the increasing GL and CaCl₂ levels caused a decrease in springiness but an increase in syneresis of mixed gels (P < 0.05). Gelling and melting temperatures were increased in the mixed gel as levels of GL and CaCl₂ increased. L*- and b*-values of mixed gels decreased, whereas ?E*-value increased with increasing GL and CaCl₂ levels (P < 0.05). Microstructure studies revealed that denser structure with smaller voids in gel network was observed in the mixed gel in the presence of CaCl₂ at higher levels. However, mixed gels incorporated with GL above 5%, regardless of CaCl₂ levels, yielded the lower likeness score than FG gel (control) (P < 0.05). The addition of GL at low level (2.5%) with CaCl₂ (up to 6 mM) had no adverse effect on sensory property of mixed gels but could improve gelling property of FG via increasing gel strength and gelling point.     

<Emphasis Type="Italic">Heterorhabditis amazonensis</Emphasis> for biological control of fungus gnats <Emphasis Type="Italic">Bradysia difformis</Emphasis> in Daisy gerberas

Fungus gnats (FG) have been reported in Venezuelan greenhouses recently. Bradysia difformis Frey (Diptera, Mycetophilidae) was found attacking Daisy gerbera plants [Gerbera sp. L. (Asterales: Asteraceae)] and many other crops in the country causing serious damages. Heterorhabditis amazonensis Andaló et al. (Rhabditida: Heterorhabditidae), was used to assess the mortality of larvae and adults of FG, number of invader nematodes per larvae and to compare the mortality of FG using nematodes and/or ciromazine under laboratory conditions. In a commercial flower farm, different doses of these nematodes were applied for eight weeks to control FG. The results showed 95% of mortality of the B. difformis larvae under laboratory conditions and performed better in the field when 50,000 nematodes were applied in every pot as a base dose to initiate a control programme. According to our results, H. amazonensis has the potential to become a regular organism for controlling fungus gnat larvae in tropical greenhouses.     

Regulation of isoprene synthase promoter by environmental and internal factors

Isoprene synthase (ISPS) catalyzes the formation of isoprene, an important volatile terpenoid with strong effects on global atmospheric chemistry and protective physiological functions in plant leaves. Many terpene synthase genes including isoprene synthase, a member of the TPS-b cluster of this numerous gene family, were already functionally analysed but much less is known about regulation of their promoters. To study regulation of the PcISPS gene in detail we developed transgenic Grey poplar (Populus × canescens) and Arabidopsis thaliana plants in which the PcISPS promoter is fused to enhanced green fluorescent protein (E-GFP) and β-glucuronidase (GUS) reporter genes. We analysed these reporters during plant development, for organ specificity and in plants subjected to different light and temperature regimes. We observed low promoter activity in non-isoprene emitting tissue like roots where ISPS gene is transcribed but no active enzyme is detectable. In leaves we demonstrate that light and temperature directly modulate ISPS promoter activity. Moreover, with confocal laser scanning microscopy we show a cell specific gradient of ISPS promoter activity within the leaf parenchyma depending on light direction. Our results indicate that ISPS promoter activity, which correlates with basal isoprene emission capacity, is not uniformly distributed within leaf tissue and that it can adapt rapidly towards internal as well as external environmental stimuli.     

Autocrine Survival Factors of a Cytotoxic CTLL-2 Cell Line

Cell survival in multicellular organisms is controlled by numerous cytokines, growth factors, and autocrine survival factors. Autocrine survival factors remain the least studied. The aim of this work was to study the autocrine factors which control survival of a CTLL-2 cytotoxic cell line: isolation and characterization of biologic activity along with physicochemical features of the active molecules have been performed. The conditioned medium of CTLL-2 cells containing autocrine factors was separated by gel filtration into four fractions: A, B, C, and D (according to the order of their efflux from the column). The biological activity of the fractions was tested by the MTT assay with the low density 5 days culture as a cell survival model. The testing of the ability of the fractions to support the cell survival in culture has shown that fractions A and B were active, whereas fractions C and D were not. The presence of a peptide of the molecular mass of 1157 Da in active fractions A and B has been detected by MALDI-TOF-mass-spectrometry. Considerable amount of lactate in fractions A and B, which flowed out from the column along with the peptide, has been detected with an enzymatic lactic acid assay. The lactate concentration in fraction A was 3.72 ± 0.11 mM and it was 0.83 ± 0.06 mM in fraction B. The obtained data suggest that fractions A and B contain supramolecular complexes of the peptide (M 1157 Da) with different lactate content. The peptide in a free form has not been found in the CTLL-2 cell conditioned medium.     

Elicitor enhanced production of protoberberine alkaloids from in vitro cell suspension cultures of <Emphasis Type="Italic">Tinospora cordifolia</Emphasis> (Willd.) Miers ex Hook. F. &amp; Thoms

The present research investigates the effect of Piriformospora indica, an endophytic fungus, on production of protoberberine alkaloids in in vitro cell suspension cultures of Tinospora cordifolia. Although T. cordifolia produces a number of protoberberine alkaloids, the simultaneous production of jatrorrhizine and palmatine in cell suspension cultures of T. cordifolia was observed for the first time with the use of P. indica as biotic elicitor. The cells in suspension cultures were elicitated with P. indica on 14th day of culture initiation and the production of the alkaloids on 16th day was monitored. The autoclaved as well as filter sterilized cultures of P. indica were used in addition to the use of fungal cell extract. The elicitor effect of P. indica was analyzed and compared with other abiotic elicitor (methyl jasmonate) and biotic elicitors (chitin and chitosan). The culture filtrate of P. indica in the filter sterilized (5.0% v/v) form gave better response with enhanced 4.2-fold production of jatrorrhizine (10.72 mg/g DW) and 4.0-fold production of palmatine (4.39 mg/g DW). The production of these compounds was at par with that achieved in methyl jasmonate (at 250 µM) treated cell suspension cultures.     

Rootstock × scion interactions on <Emphasis Type="Italic">Theobroma cacao</Emphasis> resistance to witches’ broom: photosynthetic,nutritional and antioxidant metabolism responses

Cacao (Theobroma cacao L.) is one of the most important perennial crops in the world. In Brazil, the decrease in production in the last 25 years was caused by the entry and dissemination of witches’ broom (WB) (Moniliophthora perniciosa Aime & Phillips-Mora) in the cacao region of Bahia, the main producing region of the country. This disease increases costs and reduces crop yields. The main objective of this study was to evaluate interactions between scion and rootstocks for WB resistance through gas exchange and chlorophyll fluorescence measurements, determinations of the activity of enzymes involved in the antioxidant metabolism and of macro and micronutrient concentration at the leaf level. The experiment was conducted under greenhouse conditions using two cacao clones as rootstock, a tolerant (SCA-6) and a susceptible (SIC-876) to WB and CCN-51 as scion, a reference material due to tolerance to the disease, good productivity and high beans butter concentration. No grafted plants of SCA-6, SIC-876 and CCN-51 were used as controls. Were observed either in the grafted and non-grafted genotypes that M. perniciosa infection provoked alterations in the photochemical and biochemical phases of photosynthesis, in activity of enzymes involved in the antioxidant metabolism and in macro and micronutrients concentrations. It was concluded that interactions between scion and rootstock in T. cacao made the scion more tolerant to M. perniciosa infection. SIC-876 rootstock, considered susceptible to WB, had a positive effect on the performance of CCN-51 graft with respect to tolerance to the disease. The higher tolerance of CCN-51 graft to WB, provided by the SIC-876 rootstock, was helped by the increased activity of ascorbate and guaiacol peroxidases, enzymes involved in the antioxidant metabolism.     

Morphological changes and increase of resistance to oxidative stress by overexpression of the <Emphasis Type="Italic">LebZIP2</Emphasis> gene in <Emphasis Type="Italic">Nicotiana benthamiana</Emphasis>

The tomato bZIP2-encoding gene was inserted into the Nicotiana benthamiana genome using Agrobacterium-mediated transformation to characterize resistance to oxidative stress and two herbicides, glyphosate and paraquat. We produced transgenic tobacco plants using the LebZIP2 gene, which were then utilized to examine salt stress and herbicide resistance through oxidative mechanisms. Transgenic LebZIP2-overexpressing plants were examined using specific primers for selection marker genes (PCR using genomic DNA) and target genes (RT-PCR). Based on microscopic examination, we observed an increase in leaf thickness and cell number in transgenic plants. The electrolyte leakage of leaves suggested that LebZIP2-overexpressing lines were weak tolerant to NaCl stress and resistant to methyl viologen. During our analysis, transgenic lines were exposed to different herbicides. Transgenic plants showed an increased tolerance based on visual injury, as well as an increased biomass. Based on these results, the LebZIP2 gene may be involved in oxidative stress tolerance and cell development in plants.