The effect of cadaverine on redox homeostasis of wheat seedling roots and their resistance to damage heating

Polyamines are plant metabolites involved in many processes under physiologically normal and stressful conditions. Cadaverine is one of the least studied plant polyamines. The relationship between its physiological effects and the formation of signaling mediators, in particular, reactive oxygen species (ROS), has hardly been specially studied. The aim of this work was to study the possible protective effect of cadaverine on wheat (Triticum aestivum L.) seedlings under heat stress and its relationship with the formation and detoxification of ROS by antioxidant enzymes. Etiolated seedlings of soft winter wheat variety Doskonala were used in the work. We treated three-day-old seedlings with cadaverine at concentrations ranging from 0.05 to 2.5 mM by adding it to the root incubation medium. In some variants of the experiment, we treated seedlings with a hydrogen peroxide scavenger dimethylthiourea (DMTU - 150 μM), a diamine oxidase inhibitor aminogunidine (1 mM) or an inhibitor NADPH oxidase imidazole (10 μM), as well as the indicated inhibitors in combination with cadaverine. The hydrogen peroxide content and the activity of antioxidant enzymes were determined in the roots of seedlings a certain time after treatment with the studied compounds. One day after the treatment of seedlings with cadaverine, ROS antagonists, and a combination of effectors, the seedlings were subjected to damaging heating in a water thermostat (10 min at 45 °C). 24 h after heating, we assessed the content of the products of lipid peroxidation (LPO) in the roots and, after 3 days, the survival of seedlings. Incubation in the presence of cadaverine increased the resistance of seedlings to damaging heat (See Fig. 1). The highest relative number of surviving seedlings was observed in the variant with 1 mM cadaverine treatment. Under the effect of cadaverine, the content of hydrogen peroxide in the roots increased (See Fig. 2). We observed a noticeable effect 1-4 h after the start of treatment, with a maximum after 2 h. Treatment of seedlings with a scavenger of hydrogen peroxide DMTU removed the manifestation of the effect of an increase in the content of H2 O2 in the roots caused by the action of cadaverine (See Fig. 3). This effect was also completely eliminated by the diamine oxidase inhibitor aminoguanidine and was almost unchanged in the presence of the NADPH oxidase inhibitor imidazole. The effect of heat stress on seedlings caused an increase in the content of the LPO products in them. Treatment with cadaverine markedly reduced this manifestation of oxidative stress. The antioxidant DMTU and the diamine oxidase inhibitor aminoguanidine largely neutralized the protective effect of cadaverine (See Fig. 4a). At the same time, the NADPH oxidase inhibitor imidazole had almost no effect on the manifestation of the effect of cadaverine on the LPO products content in roots. Under the influence of DMTU and aminoguanidine, but not imidazole, the positive effect of cadaverine on the survival of seedlings after damaging heating was also leveled out (See Fig. 4b). The treatment of seedlings with cadaverine caused a change in the activity of antioxidant enzymes in the roots (superoxide dismutase - SOD, catalase, and guaiacol peroxidase) (See Fig. 5). DMTU and aminoguanidine neutralized the effect of cadaverine-induced increase in the activity of catalase and guaiacol peroxidase, but had almost no effect on the increase in SOD activity in roots induced by this diamine (See Fig. 6). The NADPH oxidase inhibitor imidazole did not significantly affect the manifestation of the effect of increasing the activity of antioxidant enzymes when seedlings are treated with cadaverine. We can conclude that one of the signaling mediators involved in the regulation activity of catalase and guaiacol peroxidase and in the induction of heat resistance of wheat seedlings by exogenous cadaverine is hydrogen peroxide, which is formed during the oxidation of cadaverine by diamine oxidase. At the same time, the modification of SOD activity in the roots of wheat seedlings with cadaverine, apparently, can occur without the participation of ROS.

Comparative analysis of shape variation in the cone scales of Larix dahurica and L. cajanderi (Pinaceae)

Geometric morphometric analysis of shape variation in the cone scales of two closely related larch species, Larix dahurica Laws. (=Larix gmelinii (Rupr.) Rupr) and L. cajanderi Mayr, was carried out. The data on the taxonomy and distribution of L. dahurica and L. cajanderi are contradictory. The taxonomic status of L. cajanderi has been confirmed by the genetic and morphological studies performed in Russia and based on considerable evidence, but the species has not been recognized internationally, being considered as a synonym of Larix gmelinii var. gmelinii. In the systematics of larch, morphological characters of the generative organs are mainly used as diagnostic markers, among the most important being the shape variation of the cone scales. The aim of this study was to test geometric morphometrics as a tool for analyzing differentiation of L. dahurica and L. cajanderi in the shape of their cone scales. Characterization of shape variations in cone scales using geometric morphometric methods consists in digitizing points along an outline of scales followed by analysis of partial warps, describing individual differences in coordinates of the outline points. We studied the populations of L. dahurica from Evenkia and the Trans-Baikal region and six L. cajanderi populations from Yakutia and Magadan Oblast. In each population, we analyzed samples of 100-150 cones collected from 20-30 trees. Scales taken from the middle part of the cones were scanned using an Epson Perfection V500 Photo. On the scanned images, outline points were placed with a TPSDig program (Rolf, 2010), using angular algorithm (Oreshkova et al., 2015). The data were processed and analyzed using Integrated Morphometrics Programs (IMP) software (http://www.canisius.edu/~sheets/ morphsoft.html, Sheets, 2001), following the guidelines on geometric morphometrics in biology (Pavlinov, Mikeshina, 2002; Zelditch et al., 2004). Initial coordinates of the scale landmarks were aligned with the mean structure for L. dahurica and L. cajanderi cone scales using Procrustes superimposition in the CoordGen6 program. PCA based on covariances of partial warp scores was applied to reveal directions of variation in the shape of the cone scales. The relative deformations of the cone scales (PCA scores) were used as shape variables for statistical comparisons of these two larch species with canonical discriminant analysis. Morphotypes of the cone scales were distinguished in L. dahurica populations by pairwise comparison of samples from trees in the TwoGroup6h program using Bootstrap resampling-based Goodall’s F-test (Sheets, 2001). Samples from the trees in which the cone scales differed significantly (p < 0.01) were considered to belong to different morphotypes. Morphotypes distinguished in L. dahurica populations were compared with the morphotypes that we had previously determined in L. cajanderi populations. The composition and the frequency of occurrence of morphotypes were used to determine phenotypic distances between populations (Zhivotovskii, 1991). Multidimensional scaling matrix of the phenotypic distances was applied for ordination of larch populations. In this research, we revealed differentiation of L. dahurica and L. cajanderi using geometric morphometric analysis of the shape variation of cone scales. The results of PCA of partial warp scores exposed four principal components, which account for 90% of total explained variance in the shape of the cone scales in the two larch species. Graphical representations of these shape transformations in the vector form characterized directions of shape variability in scales corresponding to the maximum and minimum values of four principal components (See Fig. 2). PCA-ordination of the larch populations revealed some difference in the shape variation of the cone scales in L. dahurica and L. cajanderi (See Fig. 3). The results of canonical discriminant analysis of relative deformations of scales showed differentiation of the populations of the two larch species (See Fig. 4). Eleven morphotypes were identified in L. dahurica cones from Evenkia and nine morphotypes in the Ingoda population, three of the morphotypes being common for both populations (See Fig. 5). The shape of L. dahurica cone scales varied from spatulate to oval and their apical margins from weakly sinuate to distinctly sinuate. The Trans-Baikal population was dominated by scales with obtuse (truncate) and rounded apexes. The obtained morphotypes were compared with 25 cone scale morphotypes previously distinguished in the Yakut and the Magadan L. cajanderi populations (See Fig. 3). Four similar morphotypes of cone scales were revealed in the North-Yeniseisk population of L. dahurica and the Yakut populations of L. cajanderi. The differences between them in the populations of the two larch species were nonsignificant (p > 0.01). All morphotypes of cone scales from the Ingoda population of L. dahurica differed significantly from L. cajanderi cone scale morphotypes. The results of multidimensional scaling phenotypic distance matrix calculated based on the similarity of morphotypes of L. dahurica and L. cajanderi populations were consistent with the results of their differentiation based on relative deformations of scales obtained using canonical discriminant analysis (See Fig. 4 and Fig. 7). In spite of the differences in the shape of the cone scales between the North-Yeniseisk and the Trans-Baikal populations of L. dahurica, they both differed from L. cajanderi populations. Thus, phenotypic analysis confirmed differentiation of these two larch species. Despite the similarities between a number of morphotypes, the Yakut L. cajanderi populations were differentiated from L. dahurica populations. Significant differences were noted between intraspecific groups: between L. cajanderi populations from Okhotsk-Kolyma Upland and Yakutia and between L. dahurica populations from Evenkia and the Trans-Baikal region (See Fig. 4). The similarities between species and intraspecific differences may be attributed to the ongoing processes of hybridization and species formation in the region where the ranges of the larches overlap with the ranges of L. czekanowskii Szafer and L. dahurica×L. cajanderi hybrids. Geometric morphometrics can be used as an effective tool for analyzing differentiation of L. dahurica and L. cajanderi in the shape of their cone scales.

Spatial distribution of macrozoobenthos communities in a plain river of a semi-desert zone

Current climate changes require special attention to the implementation of environmental activities in arid regions. The study of the biotic component of water bodies of such ecosystems and the patterns of their spatial distribution is an important area of scientific research. The river network of the semi-desert zone of the Russian Plain is one of the least studied lotic systems in the Lower Volga basin. In this river network, the plain Yeruslan River is of the greatest importance because it largely determines the environmental characteristics of this arid territory. Therefore, it is important to study the structural indicators and spatial dynamics of macrozoobenthos communities in the Yeruslan River. The aim of the work was to study species composition, the structural and quantitative indicators of macrozoobenthos from the source to the mouth of the Yeruslan River and to determine the conceptual belonging of the bottom communities of the plain river of the semidesert zone to a certain type of distribution. The Yeruslan River (51°18'3''N, 47°46'19''E) flows through the semi-desert zone of the Russian Plain (Volgograd region, Russia) and it is a tributary of Volgograd reservoir. The length of the Yeruslan River is 282 km, with a catchment area of 55700 km2. We collected samples of macrozoobenthos at 9 stations of the Yeruslan River (See Fig. 1) in June 2015 and July 2016. In the ripal zone, the integrated samples for quantitative macrozoobenthos analysis were taken using an Ekman-type grab sampler (surface area 25 cm2) in replicates (8X) and a handle blade trawl (0.2 м × 0.5 м). In the medial zone, samples were taken by an Ekman-type grab sampler (surface area 250 cm2) in replicates (2X). Samples were washed in the field using a mesh screen with 300-310-μm mesh size and preserved in 4% formaldehyde. At each station of the Yeruslan River, we used field analytical instruments for measuring pH and oxygen content. Water samples were taken for hydrochemical analysis at different sections of the river (See Table 1). We used the model of isolation by distance (Malécot, 1948), Monmonier’s maximum difference algorithm (Manni et al., 2004) and the Dickey-Fuller test (Dickey and Fuller, 1979) to perform statistical analysis of changes in the species structure of macrozoobenthos. The Yeruslan River flows within the geochemical province of continental salinity, which is characterized by an evaporative type of natural water regime, leading to progressive accumulation of salts. In this research, we found out that water was brackish at several stations of the river (1250-1420 mgl-1) due to water drainage of saline soils. We revealed that the Yeruslan River is polluted with nitrite nitrogen (at station 1) and phosphorus compounds (at stations 4 and 8) but concentrations of ammonium nitrogen, nitrate nitrogen, cadmium, copper, zinc and lead did not exceed the MPC. Comparative analysis has shown that from the source to the mouth of the river there are no significant changes in the speed of water velocity flow, and the physical and chemical conditions are specific for each station. In the river, we collected 132 species: 47 - Diptera, 20 - Oligochaeta, 11 - Mollusca, 11 - Grustacea, 11 - Coleoptera, 7 - Trichoptera, 7 - Heteroptera, 6 - Hirudinea, 4 - Odonata, 4 - Ephemeroptera, 1 - Lepidoptera, Hydracarina, Polychaeta and Megaloptera. The macrozoobenthos of the river is represented by limnophilic species in the upper, middle and lower reaches. This is due to the small slope of the Yeruslan River and the presence of permanent and temporary dams. In the mouth reaches, the macrozoobenthos communities included species of the Ponto-Caspian and Ponto-Azov zoogeographic complexes. At all stations of the river, Oligochaeta and Chironomidae were of high density. Also, in the river mouth, Mollusca were of high density (See Fig. 2). Statistical analysis of sequences of hydrobiological characteristics along the longitudinal gradient of the Yeruslan River using the Dickey- Fuller test showed that the presence of a stationary distribution trend with random “wandering” is typical of the series of total density and biomass of macrozoobenthos, the number of worms of the family Tubificidae, larvae of chironomids of the subfamily Tanytarsini and mayflies of the family Baetidae. For the other series of observations, the presence of a nonlinear trend is noted (See Table 3 and Fig. 3). The selection of a sequence of borders (barrier) between river communities within the ecosystem by Montmonier’s method using a matrix of species distances by the Bray-Curtis method made it possible to identify the source (station 1) with a high level of nitritic nitrogen in the water as one of the specific areas. The second most important border separates station 3 with a low content of dissolved oxygen, and the third one allocates the mouth reaches (station 9) as an independent area, where there is a cohabitation of river and reservoir species (See Fig. 4). Based on the analysis of fauna and using statistical methods, we found out that macrozoobenthos communities do not change from the source to the mouth of the river in accordance with the “the river continuum concept”. The habitat of taxa depends on local abiotic and biotic factors at each river station, therefore, we can assume that the distribution of macrozoobenthos communities, generally, corresponds to “the patch dynamics concept”. At the same time, stations 1, 3 and 9 form fairly isolated hydrogeomorphological areas, which is postulated by the concept of “the functional process zones”. It seems that the spatial distribution of macrozoobenthos communities in the Yeruslan River can be explained by a complex combination of two concepts: “the patch dynamics concept” and “the functional process zones”. This type of distribution seems to be typical of plain rivers with very low water velocity and the presence of dams.

The structure of Gluconacetobacter hansenii GH 1/2008 population cultivated in static conditions on various sources of carbon

Bacterial cellulose (BC) is a natural polymer that has a number of unique properties that determine the need to synthesize large amounts of it and to search the ways to increase the productivity of strains and to optimize the nutritive media. It is known that the choice of the producer for BC synthesis has an impact on its final properties and on the productivity of this polymer production. Under static liquid phase cultivation conditions, all cellulose-producing bacteria form a uniform film on the medium surface that serves as a scaffold for cells immobilization, thus providing them with the access to the air/liquid interface, where the access to oxygen is not limited. Meanwhile, when cultivation goes under agitating conditions, most of Gluconacetobacter xylinus strains produce less cellulose in form of globules of various sizes, despite the better oxygen access. Several authors explain the lower cellulose outcome when cultivated under agitated conditions by the appearance of spontaneous mutants that do not produce cellulose in the population. It was revealed that when grown on agarized media, cellulosenon- producing mutants form colonies of a specific mucoid type, while non-mucoid phenotype cells form smooth colonies of non-mucoid type. To our knowledge, there is no published research on the impact of cultivation conditions and nutritive medium composition on the appearance of spontaneous phenotype mutations in the population of Gluconacetobacter hansenii representatives. The aim of the present research is to elucidate the impact of the carbon source on the productivity of G. hansenii strain and the appearance of cellulose-negative mutants under static cultivation conditions. We studied the strain G. hansenii GH 1/2008 (VKPM В-10547) as a BC source. Liquid phase static cultivation of G. hansenii GH 1/2008 was carried out using the modified Hestrin-Schramm (HS) medium, containing 4% of monosaccharides (glucose, fructose and galactose) or disaccharides (sucrose, maltose, lactose) as carbon sources. The occurrence of mutants was calculated considering phenotypes of colonies obtained by seeding the samples of cultural liquid and wash-offs of cells from films produced by the cultivation of the producer on modified agarized HS media. The polymer outcome was expressed as the film absolute dry weight (a.d.w.) per cultivation medium volume unit. We studied the morphology of the producer’s wild type and mutant cells by means of atomic force microscopy (AFM) (See Fig. 8). The structural organization of the produced films and gel was revealed by means of scanning electron microscopy (SEM) performed after freeze-drying. The composition of the fibers was checked acquiring FTIR Spectroscopy. We established that G. hansenii GH 1/2008 produces a dense film on media containing fructose, glucose and sucrose, while the polymer has gel consistence when grown on maltose, galactose and lactose (See Fig. 1). The maximal quantity (a.d.w.) of polymer was produced on fructose- and sucrose-containing media. The overall number of immobilized producer cells was considerably higher when grown on media with glucose, fructose and sucrose than on gels grown on those containing maltose, galactose and lactose (See Table 1). SEM imaging revealed considerable difference in the microscale organization of films and gels produced by G. hansenii GH 1/2008 on various carbon sources (See Fig. 2). Fructose-containing medium yields the densest structure with dense layers separated by 2μm thick areas filled with non-ordered BC fibrils. The microscale organization of sucrose- and glucose-based films were very similar and had a cell-like structure. In cases where the synthesized polymer had squeezable gel consistence, its microstructure was not layered but close to isotropic. The studies of gels by means of FTIR spectroscopy showed that the gels are also formed of BC molecules; the spectra were almost identical (See Fig. 4). The only difference, i.e. the intensity of the 1638 см-1 peak, can be explained by the presence of a higher amount of bound water in the latter. It is known that some strains of this species may produce glucuronic acid oligomers under unfavorable conditions, but peaks corresponding to carboxyl or carbonyl groups were not revealed in the spectra. This is the evidence that no detectable amounts of glucuronic acid were produced under conditions studied. The analysis of colonies of G. hansenii GH 1/2008 cultivated under static conditions on media containing various carbon sources revealed colonies with two dominating phenotypes: non-mucoid smooth convex colonies and mucoid flat ones (See Fig. 5). The number of cells forming smooth non-mucoid colonies on agarized media was maximal in the inoculations of cultural liquids after the cultivation on media containing fructose and sucrose, i.e. those carbon sources that demonstrated high productivity per 1L of cultural liquid (See Fig. 6). In the inoculations of the cultural liquid and wash-offs of cells immobilized on gels obtained by the cultivation on media containing galactose and lactose, the number of mucoid colonies was considerably higher (See Table 2). The clones forming mucoid type colonies did not produce BC films when reinoculated in liquid media, while those forming colonies of mucoid (smooth) type produce films on the 3rd day of cultivation (See Fig. 7). The analysis of cells shape and sizes by means of AFM did not reveal any statistically valid difference between the mutants and the wild type. The present research shows that the source of carbon is a selective factor in the formation of the inner composition of the population of clones of the bacterial cellulose producer Gluconacetobacter hansenii GH 1/2008. The proliferation of cellulosenegative cells arouses competition for the substrate with cellulose-positive cells of G. hansenii GH 1/2008 that reduces the number of the latter and the production of the exopolymer.

The influence of co-treatment with Bacillus thuringiensis B-5351 and salicylic acid on the resistance of potato plants to Phytophthora infestans (Mont.) de Bary

Biocontrol agents based on strains of microorganisms that participate in mutualistic relationships with host plants, including those based on strains of endophytic bacteria, can be an alternative to chemical pesticides. There are endophytic B. thuringiensis strains that produce insect-toxic proteins and induce systemic resistance of plants to pathogens. An important issue is the possibility of regulation of the relationship of endophytic bacterial strains with the host-plant and their ability to induce plant defense reactions against pathogens by signaling molecules, including salicylic acid (SA). The aim of this work was to study the effect of SA and the endophytic bacterial strain B. thuringiensis B-5351 on the activity of potato plants reactions associated with signaling and biosynthesis of SA, as well as the activity of a potato trypsin inhibitor upon infection with the late blight pathogen. We used sterile test tube potato plants (Solanum tuberosum L.) of the Early Rose cultivar. 20-days-old potato plants were inoculated with a bacterial suspension of B. thuringiensis B-5351 (1*108 cells / ml) in distilled water, or in 1 μM SA solution by applying 5 μl of bacterial suspension to 4 upper leaves (20 μl / plant). Control plants were treated with distilled water, and some plants were treated with 1 μM SA solution in the same volume. On the 7th day after inoculation with B. thuringiensis B-5351, the plants were infected with oomycete P. infestans spores (5 μl of 1*105 spore/ ml suspension per leaf). To assay the damaged area, leaves were photographed; the images were analyzed using the ImageJ software. 24 h after infection with late blight pathogen spores, plants were fixed to assess the transcriptional activity of PR6, PR1 and PAL genes (See Table 1) (using CFX Connect Real-Time PCR Detection System Bio-Rad (USA)), and after 6, 24 and 48 h - to analyze the activity of plant proteinase inhibitors and the content of hydrogen peroxide. We estimated the number of colonyforming units (CFU) of microorganisms in plant tissues after their surface sterilization on the 7th day after inoculation of plants with B. thuringiensis B-5351 bacteria. The significance of the differences between the samples was accessed using Student’s t-test (p<0.05). Tables and Figures show data as the mean of the replicates and their standard deviations (M±SD). All experiments were conducted in three-five biological and three analytical replicates. In this work, we showed that SA presence increased the content of living cells of B. thuringiensis B-5351 bacteria in the internal tissues of plants (2,3±1,5×106 CFU/g) as compared to individual treatment with the bacterial strain under investigation (7,3±1,6×105 CFU/g) (See Table 2). Using the method of RAPD analysis, we established the identity of bacteria isolated from internal tissues of potato plants and the initial strain of B. thuringiensis B-5351 (See Fig. 1). Apparently, such titer of bacterial population regulated by plant signaling molecules in plant tissues is an important factor in the formation of plant resistance. Thus, we did not observe a decrease in the area of late blight symptoms on the leaves of potato plants treated only with B. thuringiensis B-5351, while under the individual action of SA this parameter decreased by half, and upon combined treatment of plants with SA and bacteria B. thuringiensis B-5351 - almost 4 times as compared to water-treated plants (See Fig. 2). Treatment with bacteria B. thuringiensis B-5351 increased the activity of proteinase inhibitors in potato plants only 6 h after infection with the late blight pathogen; in plants treated with bacteria cells of B. thuringiensis B-5351 together with SA, there was an almost a twofold increase in the activity of proteinase inhibitors after 6 and 24 h after infection with oomycete P. infestans. Under the influence of SA, a 15-20% increase in the content of H2O2 in intact plants was observed in all variants. In infected plants treated with SA, the H2O2 content exceeded the control values by more than 40%. In plants treated with a suspension of bacteria B. thuringiensis B-5351 and SA plants, the presence of the late blight pathogen caused a threefold increase in the H2O2 content 6 h after infection (See Fig. 3). In the variant with combined treatment with SA and bacteria B. thuringiensis B-5351 of noninfected plants, the content of PR6 gene transcripts exceeded the control level by 25%, as in infected plants treated with SA. Treatment of plants with SA together with bacteria B. thuringiensis B-5351 increased the number of PR6 gene transcripts by more than 50% relative to water-treated non-infected plants (See Fig. 4). In this work, we found that the strain of endophytic bacteria under investigation promoted the accumulation of transcripts of genes encoding PR1 and PAL proteins to the same extent as SA, both individually and when used together. In the presence of the late blight pathogen, more than a twofold (relative to this level in water-treated intact plants) increase in the content of PAL gene mRNA in potato plants was observed in cases of both individual and combined application of SA and B. thuringiensis B-5351 cells. Thus, treatment with B. thuringiensis B-5351 bacteria together with SA induces salicylate-dependent defense reactions (PR1, PAL), as well as an increase in the transcriptional activity of the PR6 gene, which, according to the literature, is a jasmonate-dependent gene. Our results showed the effectiveness of the composition based on live bacteria B. thuringiensis B-5351 and SA in protecting potato plants from late blight by inducing systemic defense responses in plants. Taking into account the insecticidal activity of the strain under investigation against Russian wheat aphids and Colorado potato beetle, which we previously described, the possibility of increasing plant resistance to late blight by biocontrol agent combining B. thuringiensis B-5351 with SA can open one of the approaches to the development of multifunctional protection of plants from biotic influences.