Biopolymers as Seed-Coating Agent to Enhance Microbially Induced Tolerance of Barley to Phytopathogens.

Infections of agricultural crops caused by pathogen ic fungi are among the most widespread and harmful, as they not only reduce the quantity of the harvest but also significantly deteriorate its quality. This study aims to develop unique seed-coating formulations incorporating biopolymers (polyhydroxyalkanoate and pullulan) and beneficial microorganisms for plant protection against phytopathogens. A microbial association of biocompatible endophytic bacteria has been created, including Pseudomonas flavescens D5, Bacillus aerophilus A2, Serratia proteamaculans B5, and Pseudomonas putida D7. These strains exhibited agronomically valuable properties: synthesis of the phytohormone IAA (from 45.2 to 69.2 µg mL-1), antagonistic activity against Fusarium oxysporum and Fusarium solani (growth inhibition zones from 1.8 to 3.0 cm), halotolerance (5-15% NaCl), and PHA production (2.77-4.54 g L-1). A pullulan synthesized by Aureobasidium pullulans C7 showed a low viscosity rate (from 395 Pa·s to 598 Pa·s) depending on the concentration of polysaccharide solutions. Therefore, at 8.0%, w/v concentration, viscosity virtually remained unchanged with increasing shear rate, indicating that it exhibits Newtonian flow behavior. The effectiveness of various antifungal seed coating formulations has been demonstrated to enhance the tolerance of barley plants to phytopathogens.

Features of Photosynthesis in Arabidopsis thaliana Plants with Knocked Out Gene of Alpha Carbonic Anhydrase 2.

The knockout of the At2g28210 gene encoding α-carbonic anhydrase 2 (α-CA2) in Arabidopsis thaliana (Columbia) led to alterations in photosynthetic processes. The effective quantum yields of both photosystem II (PSII) and photosystem I (PSI) were higher in α-carbonic anhydrase 2 knockout plants (α-CA2-KO), and the reduction state of plastoquinone pool was lower than in wild type (WT). The electron transport rate in the isolated thylakoids measured with methyl viologen was higher in α-CA2-KO plants. The amounts of reaction centers of PSII and PSI were similar in WT and α-CA2-KO plants. The non-photochemical quenching of chlorophyll a fluorescence in α-CA2-KO leaves was lower at the beginning of illumination, but became slightly higher than in WT leaves when the steady state was achieved. The degree of state transitions in the leaves was lower in α-CA2-KO than in WT plants. Measurements of the electrochromic carotenoid absorbance shift (ECS) revealed that the light-dependent pH gradient (ΔpH) across the thylakoid membrane was lower in the leaves of α-CA2-KO plants than in WT plants. The starch content in α-CA2-KO leaves was lower than in WT plants. The expression levels of the genes encoding chloroplast CAs in α-CA2-KO changed noticeably, whereas the expression levels of genes of cytoplasmic CAs remained almost the same. It is proposed that α-CA2 may be situated in the chloroplasts.

Polyhydroxyalkanoates (PHAs) from Endophytic Bacterial Strains as Potential Biocontrol Agents against Postharvest Diseases of Apples.

Due to the increasing use and accumulation of petrochemical plastics in the environment and the rapid depletion of natural resources, microbial polyhydroxyalkanoates have great potential to replace them. This study provides new insights in the field of obtaining of polyhydroxyalkanoates (PHAs) from endophytic bacterial strains and applying them as potential biocontrol agents against postharvest diseases of apples. Two strains-Pseudomonas flavescens D5 and Bacillus aerophilus A2-accumulated PHAs in amounts ranging from 2.77 to 5.9 g L-1. The potential to use low-cost substrates such as beet molasses and soapstock for PHA accumulation was shown. The PHAs produced by the Ps. flavescens D5 strain had pronounced antagonistic activity against Penicillium expansum (antifungal property = 62.98-73.08%). The use of PHAs as biocontrol agents significantly reduced the severity of apple blue mold, especially in the preventive treatment option.

The Role of Carbonic Anhydrase αCA4 in Photosynthetic Reactions in Arabidopsis thaliana Studied, Using the Cas9 and T-DNA Induced Mutations in Its Gene.

An homozygous mutant line of Arabidopsis thaliana with a knocked out At4g20990 gene encoding thylakoid carbonic anhydrase αCA4 was created using a CRISPR/Cas9 genome editing system. The effects of the mutation were compared with those in two mutant lines obtained by the T-DNA insertion method. In αCA4 knockouts of all three lines, non-photochemical quenching of chlorophyll a fluorescence was lower than in the wild type (WT) plants due to a decrease in its energy-dependent component. The αCA4 knockout also affected the level of expression of the genes encoding all proteins of the PSII light harvesting antennae, the genes encoding cytoplasmic and thylakoid CAs and the genes induced by plant immune signals. The production level of starch synthesis during the light period, as well as the level of its utilization during the darkness, were significantly higher in these mutants than in WT plants. These data confirm that the previously observed differences between insertional mutants and WT plants were not the result of the negative effects of T-DNA insertion transgenesis but the results of αCA4 gene knockout. Overall, the data indicate the involvement of αCA4 in the photosynthetic reactions in the thylakoid membrane, in particular in processes associated with the protection of higher plants' photosynthetic apparatus from photoinhibition.

Effect of CO2 Content in Air on the Activity of Carbonic Anhydrases in Cytoplasm, Chloroplasts, and Mitochondria and the Expression Level of Carbonic Anhydrase Genes of the α- and ß-Families in Arabidopsis thaliana Leaves.

The carbonic anhydrase (CA) activities of the preparations of cytoplasm, mitochondria, chloroplast stroma, and chloroplast thylakoids, as well as the expression levels of genes encoding αCA1, αCA2, αCA4, ßCA1, ßCA2, ßCA3, ßCA4, ßCA5, and ßCA6, were measured in the leaves of Arabidopsis thaliana plants, acclimated to different CO2 content in the air: low (150 ppm, lCO2), normal (450 ppm, nCO2), and high (1200 ppm, hCO2). To evaluate the photosynthetic apparatus operation, the carbon assimilation and chlorophyll a fluorescence were measured under the same conditions. It was found that the CA activities of the preparations of cytoplasm, chloroplast stroma, and chloroplast thylakoids measured after two weeks of acclimation were higher, the lower CO2 concentration in the air. That was preceded by an increase in the expression levels of genes encoding the cytoplasmic form of ßCA1, and other cytoplasmic CAs, ßCA2, ßCA3, and ßCA4, as well as of the chloroplast CAs, ßCA5, and the stromal forms of ßCA1 in a short-term range 1-2 days after the beginning of the acclimation. The dependence on the CO2 content in the air was most noticeable for the CA activity of the preparations of the stroma; it was two orders higher in lCO2 plants than in hCO2 plants. The CA activity of thylakoid membranes from lCO2 plants was higher than that in nCO2 and hCO2 plants; however, in these plants, a significant increase in the expression levels of the genes encoding αCA2 and αCA4 located in thylakoid membranes was not observed. The CA activity of mitochondria and the expression level of the mitochondrial ßCA6 gene did not depend on the content of carbon dioxide. Taken together, the data implied that in the higher plants, the supply of inorganic carbon to carboxylation sites is carried out with the cooperative functioning of CAs located in the cytoplasm and CAs located in the chloroplasts.

Phosphate Mobilization by Culturable Fungi and Their Capacity to Increase Soil P Availability and Promote Barley Growth.

Large-scale screening of 848 culturable soil and endophytic filamentous fungi and yeasts for the ability to mobilize inorganic and organic P compounds was performed. Five strains of filamentous fungi having the highest level of phosphate-mobilizing ability were selected: Penicillium bilaiae Pb14, P. bilaiae C11, P. rubens EF5, Talaromyces pinophilus T14, and Aspergillus sp. D1. These strains in vitro actively solubilized Ca, Al, and Fe phosphates and Ca phytate. The amount of mobilized P negatively correlated with pH of the medium and positively correlated with fungal biomass. The proposed mechanisms for P mobilization were acidification of the medium, organic acid release, and phosphatase activity. The fungi decreased pH of the medium from 7.0 to 2.3-5.0. Ten different organic acids were produced by fungi with pyruvic acid being a major component. Acid phosphatase activity varied from 0.12 EU to 0.84 EU, and alkaline phosphatase activity varied from 0.08 EU to 0.61 EU depending on the strain. Available P concentration in soil was increased by 13-28% after introduction of the fungi. The fungi also produced phytohormones auxins, salicylic acid, and abscisic acid. All the strains, except Aspergillus sp. D1, promoted elongation and increased biomass of barley seedlings grown in soil. Shoot P concentration increased by 17-26% after inoculation with P. bilaiae Pb14, T. pinophilus T14, and Aspergillus sp. D1. It was concluded that the selected fungal strains promoted plant growth due to P mobilization and phytohormone production.

Characterization of cadmium-tolerant endophytic fungi isolated from soybean (Glycine max) and barley (Hordeum vulgare).

Cadmium stress disrupts plant-microbial interactions and reduces plant growth and development. In plants, the tolerance to stress can be increased by inoculation with endophytic microorganisms. The aim of this study was to investigate the distribution of endophytic fungi in various plant organs of barley and soybean and evaluate their Cd removal ability. Two hundred fifty-three fungal strains were isolated from various organs of barley (Hordeum vulgare cv Arna) and soybean (Glycine max cv Almaty). The colonization rate ranged from 13.6% to 57.3% and was significantly higher in the roots. Ten genera were identified: Fusarium, Penicillium, Aspergillus, Metarhizium, Beauveria, Trichoderma, Rhodotorula, Cryptococcus, Aureobasidium and Metschnikowia. Twenty-three fungal strains have a Cd tolerance index from 0.24 to 1.12. Five strains (Beauveria bassiana T7, Beauveria bassiana T15, Rhodotorula mucilaginosa MK1, Rhodotorula mucilaginosa RH2, Metschnikowia pulcherrima MP2) with the highest level of Cd tolerance have minimum inhibitory concentrations from 290 to 2400 µg/ml. These fungi were able to remove Cd up to 59%. The bioaccumulation capacity ranged from 2.3 to 11.9 mg/g. Selected fungal strains could be considered as biological agents for their potential application in the bioremediation of contaminated sites.

Advances in understanding the physiological role and locations of carbonic anhydrases in C3 plant cells.

The review describes the structures of plant carbonic anhydrases (CAs), enzymes catalyzing the interconversion of inorganic carbon forms and belonging to different families, as well as the interaction of inhibitors and activators of CA activity with the active sites of CAs in representatives of these families. We outline the data that shed light on the location of CAs in green cells of C3 plants, algae and angiosperms, with the emphasis on the recently obtained data. The proven and proposed functions of CAs in these organisms are listed. The possibility of the involvement of several chloroplast CAs in acceleration of the conversion of bicarbonate to CO2 and in supply of CO2 for fixation by Rubisco is particularly considered. Special attention is paid to CAs in various parts of thylakoids and to discussion about current knowledge of their possible physiological roles. The review states that, despite the significant progress in application of the mutants with suppressed CAs synthesis, the approach based on the use of the inhibitors of CA activity in some cases remains quite effective. Combination of these two approaches, namely determining the effect of CA activity inhibitors in plants with certain knocked-out CA genes, turns out to be very useful for understanding the functions of other CAs.

Regulation of the size of photosystem II light harvesting antenna represents a universal mechanism of higher plant acclimation to stress conditions.

We investigated acclimatory responses of Arabidopsis plants to drought and salinity conditions before the appearance of obvious signs of damage caused by these factors. We detected changes indicating an increase in the reduction level of the chloroplast plastoquinone pool (PQ pool) 5-7 days after introduction of the stress factors. After 10-14 days, a decrease in the size of PSII light harvesting antenna was observed in plants under conditions of drought and salinity. This was confirmed by a decrease in content of PSII antenna proteins and by downregulation of gene expression levels of these proteins under the stress conditions. No changes in values of performance index and maximum quantum yield of PSII were detected. Under drought and salinity, the content of hydrogen peroxide in leaves was higher than in control leaves. Thus, we propose that reduction of the size of PSII antenna represents one of the universal mechanisms of acclimation of higher plants to stress factors and the downsizing already begins to manifest under mild stress conditions. Both the PQ pool reduction state and the hydrogen peroxide content are important factors needed for the observed rearrangement.

Carbonic Anhydrases in Photosynthesizing Cells of C3 Higher Plants.

The review presents data on the location, nature, properties, number, and expression of carbonic anhydrase genes in the photosynthesizing cells of C3 plants. The available data about the presence of carbonic anhydrases in plasma membrane, cytoplasm, mitochondria, chloroplast stroma and thylakoids are scrutinized. Special attention was paid to the presence of carbonic anhydrase activities in the different parts of thylakoids, and on collation of sources of these activities with enzymes encoded by the established genes of carbonic anhydrases. The data are presented to show that the consistent incorporation of carbonic anhydrases belonging to different families of these enzymes forms a coherent system of CO2 molecules transport from air to chloroplasts in photosynthesizing cells, where they are included in organic molecules in the carboxylation reaction. It is discussed that the manifestation of the activity of a certain carbonic anhydrase depends on environmental conditions and the stage of ontogenesis.

The presence of the low molecular mass carbonic anhydrase in photosystem II of C3 higher plants.

The carrier of carbonic anhydrase (CA) activity was detected in gel among low molecular mass proteins from pea, spinach and Arabidopsis, after nondenaturing electrophoresis in PAAG of the dodecyl-ß-d-maltoside treated PSII membranes (the fragments of thylakoid membrane containing PSII complexes). The elimination of Mn-stabilizing protein PsbO by treatment of PSII membranes with salts, did not lead to a decrease in CA activity observed in the gel although it reduced the amount of this protein down to 25% compared to the original sample. The isolated protein PsbO did not demonstrated CA activity. The distinguished features of CA activity of PSII membranes were as follows: 1) resistance to heating, 2) high sensitivity to ethoxyzolamide, the specific inhibitor of CA, and 3) stimulation of this activity by acetazolamide, another specific inhibitor of CA at low concentration of the latter. CA activity was not stimulated by acetazolamide in the PSII membranes samples from Arabidopsis thaliana mutants with knocked out gene At4g20990 encoding αCA4 (according to the nomenclature by Fabre et al., 2007). Taking into account the above data and our previous findings that the energy-dependent part of nonphotochemical quenching of chlorophyll a fluorescence is highly suppressed in that mutant, we suppose that thylakoid membranes of higher plants contain in the vicinity of PSII complex a true CA belonging to the α family of CAs.

Influence of knockout of At4g20990 gene encoding α-CA4 on photosystem II light-harvesting antenna in plants grown under different light intensities and day lengths.

Effect of knockout of the At4g20990 gene encoding α-carbonic anhydrase 4 (α-CA4) in Arabidopsis thaliana in plants grown in low light (LL, 80 µmol photons m-2 s-1) or in high light (HL, 400 µmol photons m-2 s-1) under long (LD, 16 h) or short (SD, 8 h) day length was studied. In α-CA4 knockout plants, under all studied conditions, the non-photochemical quenching was lower; the decrease was more pronounced under HL. This pointed to α-CA4 implication in the processes leading to energy dissipation in PSII antenna. In this context the content of major antenna proteins Lhcb1 and Lhcb2 was lower in α-CA4 knockouts than in wild-type (WT) plants under all growth conditions. The expression level of lhcb2 gene was also lower in mutants grown under LD, LL and HL in comparison to WT. At the same time, this level was higher in mutants grown under SD, LL and it was the same under SD, HL. Overall, the data showed that the knockout of the At4g20990 gene affected both the contents of proteins of PSII light-harvesting complex and the expression level of genes encoding these proteins, with peculiarities dependent on day length. These data together with the fact of a decrease of non-photochemical quenching of leaf chlorophyll a fluorescence in α-CA4-mut as compared with that in WT plants implied that α-CA4 participates in acclimation of photosynthetic apparatus to light intensity, possibly playing important role in the photoprotection. The role of this CA can be especially important in plants growing under high illumination conditions.

Bacterial endophytes of Trans-Ili Alatau region's plants as promising components of a microbial preparation for agricultural use.

In this study, 382 isolates of bacterial endophytes from tissues of plants grown in the foothills and piedmont plains of the Trans-Ili Alatau were isolated. It was found that certain strains actively produce indole-3-acetic acid (IAA) in a medium containing l-tryptophan. Among the strains studied, 26 strains (66%) showed a positive response to production of IAA. Bacteria synthesized IAA in the range of 18.6 ± 1.1 to 82.4 ± 2.3 µg/ml. IAA was synthesized more actively by Streptosporangium sp. KK1 (44.1 µg/ml), Rhodococcus sp. KK 2 (42.5 µg/ml), Streptomyces tendae KK3 (44.9 µg/ml) strains. The most active auxin's producer is a Jeotgalicoccus halotolerans BAK1 strain, whose total level of IAA production reached 82.4 µg/ml. Such strains as Streptomyces griseorubiginosis KK4, Streptomyces sp. KK5 and Jeotgalicoccus halotolerans BAK1 were found to have a significant stimulating effect which was reflected in the increase of the length of the roots of soybean and barley. As a result, 8 promising strains with fungicidal, growth-stimulating, phosphorus dissolving and enzymatic activities were selected for the further development of highly microbiological preparations for crop research.

The effect of application of micromycetes on plant growth, as well as soybean and barley yields.

The possibility of application of micromycetes (strains Penicillium bilaiae Pb14, Aureobasidium pullulans YA05 and Rhodotorula mucilaginosa YR07) to increase yields of soybean (Glycine max cv Almaty) and barley (Hordeum vulgare cv Arna) was estimated. It was shown that the most positive effect on germination energy and seed germination after seed treatment with liquid culture, supernatant and filtrate, is achieved at 1:5 dilution. In studying the influence of cell-associated and extracellular biologically active compounds of micromycetes (liquid culture and supernatant) on biometric parameters of seedlings, the maximum stimulating effect was observed in the variants with liquid culture. These strains of micromycetes were used as a bases for various compositions of preparations - application of each strain separately and application of micromycetes mixes. In microfield experiments, the increase of soybean yield ranged from 4.5 to 9.4 quintal/ha, barley - from 2.9 to 5.9 quintal/ha. A significant increase in various parameters of structure of the yield was shown in all experimental variants when compared to the control. It was found that an increase in soybean and barley yields and yield components was higher in the variant with a mix of micromycetes when compared to the separate application of each strain. The most efficient mixture was based on the mix of fungal strains (culture filtrate of P. bilaiae Pb14 diluted 1:5 + liquid cultures of A. pullulans YA05 and Rh. mucilaginosa YR07 in a 1:5 dilution).

Plant growth-promoting and antifungal activity of yeasts from dark chestnut soil.

538 yeast strains were isolated from dark chestnut soil collected from under the plants of the legume family (Fabaceae). The greatest number of microorganisms is found at soil depth 10-20 cm. Among the 538 strains of yeast 77 (14.3%) strains demonstrated the ability to synthesize IAA. 15 strains were attributed to high IAA-producing yeasts (above 10 µg/ml). The most active strains were YA05 with 51.7 ± 2.1 µg/ml of IAA and YR07 with 45.3 ± 1.5 µg/ml. In the study of effect of incubation time on IAA production the maximum accumulation of IAA coincided with maximum rates of biomass: at 120 h for YR07 and at 144 h for strain YA05. IAA production increased when medium was supplemented with the L-tryptophan. 400 µg/ml of L-tryptophan showed maximum IAA production. 10 strains demonstrated the ability to inhibit the growth and development of phytopathogenic fungi. YA05 and YR07 strains formed the largest zones of inhibition compared to the other strains--from 21.6 ± 0.3 to 30.6 ± 0.5 mm. Maximum zone of inhibition was observed for YA05 against Phytophtora infestans and YR07 strains against Fusarium graminearum. YA05 and YR07 strains were identified as Aureobasidium pullulans YA05 (GenBank accession No JF160955) and Rhodotorula mucilaginosa YR07 (GenBank accession No JF160956).

The presence of soluble carbonic anhydrase in the thylakoid lumen of chloroplasts from Arabidopsis leaves.

Supernatant obtained after high-speed centrifugation of disrupted thylakoids that had been washed free from extrathylakoid carbonic anhydrases demonstrated carbonic anhydrase activity that was inhibited by the specific inhibitors acetazolamide and ethoxyzolamide. A distinctive feature of the effect of Triton X-100 on this activity also suggested that the source of the activity is a soluble protein. Native electrophoresis of a preparation obtained using chromatography with agarose/mafenide as an affinity sorbent revealed one protein band with carbonic anhydrase activity. The same protein was revealed in a mutant deficient in soluble stromal carbonic anhydrase ß-CA1, and this indicated that the newly revealed carbonic anhydrase is not a product of the At3g01500 gene. These data imply the presence of soluble carbonic anhydrase in the thylakoid lumen of higher plants.

Carbonic anhydrase activity in Arabidopsis thaliana thylakoid membrane and fragments enriched with PSI or PSII.

The procedure of isolating the thylakoids and the thylakoid membrane fragments enriched with either photosystem I or photosystem II (PSI- and PSII-membranes) from Arabidopsis thaliana leaves was developed. It differed from the one used with pea and spinach in durations of detergent treatment and centrifugation, and in concentrations of detergent and Mg(2+) in the media. Both the thylakoid and the fragments preserved carbonic anhydrase (CA) activities. Using nondenaturing electrophoresis followed by detection of CA activity in the gel stained with bromo thymol blue, one low molecular mass carrier of CA activity was found in the PSI-membranes, and two carriers, a low molecular mass one and a high molecular mass one, were found in the PSII-membranes. The proteins in the PSII-membranes differed in their sensitivity to acetazolamide (AA), a specific CA inhibitor. AA at 5 × 10(-7) M inhibited the CA activity of the high molecular mass protein but stimulated the activity of the low molecular mass carrier in the PSII-membranes. At the same concentration, AA moderately inhibited, by 30%, the CA activity of PSI-membranes. CA activity of the PSII-membranes was almost completely suppressed by the lipophilic CA inhibitor, ethoxyzolamide at 10(-9) M, whereas CA activity of the PSI-membranes was inhibited by this inhibitor even at 5 × 10(-7) M just the same as for AA. The observed distribution of CA activity in the thylakoid membranes from A. thaliana was close to the one found in the membranes of pea, evidencing the general pattern of CA activity in the thylakoid membranes of C3-plants.

Multiple sources of carbonic anhydrase activity in pea thylakoids: soluble and membrane-bound forms.

Carbonic anhydrase (CA) activity of pea thylakoids, thylakoid membranes enriched with photosystem I (PSI-membranes), or photosystem II (PSII-membranes) as well as both supernatant and pellet after precipitation of thylakoids treated with detergent Triton X-100 were studied. CA activity of thylakoids in the presence of varying concentrations of Triton X-100 had two maxima, at Triton/chlorophyll (triton/Chl) ratios of 0.3 and 1.0. CA activities of PSI-membranes and PSII-membranes had only one maximum each, at Triton/Chl ratio 0.3 or 1.0, respectively. Two CAs with characteristics of the membrane-bound proteins and one CA with characteristics of the soluble proteins were found in the medium after thylakoids were incubated with Triton. One of the first two CAs had mobility in PAAG after native electrophoresis the same as that of CA residing in PSI-membranes, and the other CA had mobility the same as the mobility of CA residing in PSII-membranes, but the latter was different from CA situated in PSII core-complex (Ignatova et al. 2006 Biochemistry (Moscow) 71:525-532). The properties of the "soluble" CA removed from thylakoids were different from the properties of the known soluble CAs of plant cell: apparent molecular mass was about 262 kD and it was three orders more sensitive to the specific CA inhibitor, ethoxyzolamide, than soluble stromal CA. The data are discussed as indicating the presence of, at least, four CAs in pea thylakoids.