The Conservation and Study of Macromycetes in the Komarov Botanical Institute Basidiomycetes Culture Collection-Their Taxonomical Diversity and Biotechnological Prospects.

Culture collections (CCs) play an important role in the ex situ conservation of biological material and maintaining species and strains, which can be used for scientific and practical purposes. The Komarov Botanical Institute Basidiomycetes Culture Collection (LE-BIN) preserves a large number of original dikaryon strains of various taxonomical and ecological groups of fungi from different geographical regions. Started in the late 1950s for the investigation of Basidiomycetes' biological activity, today, in Russia, it has become a unique specialized macromycetes collection, preserving 3680 strains from 776 species of fungi. The Collection's development is aimed at ex situ conservation of fungal diversity, with an emphasis on preserving rare and endangered species, ectomycorrhizal fungi, and strains useful for biotechnology and medicine. The main methods applied in the collection for maintaining and working with cultures are described, and the results are presented. Some problems for the isolation and cultivation of species are discussed. The taxonomical structure and variety of the strains in the collection fund are analyzed, and they show that the taxonomical diversity of fungi in the LE-BIN is commensurable with the largest CCs in the world. The achievements from the ex situ conservation of the diversity of macromycetes and the main results from the screening and investigation of the collection's strains demonstrate that a number of strains can be prospective producers of enzymes (oxidoreductases and proteases), lipids, and biologically active compounds (terpenoids, phthalides, etc.) for biotechnology and medicine.

Heterogeneous Distribution of Phospholipid Molecular Species in the Surface Culture of Flammulina velutipes: New Facts about Lipids Containing α-Linolenic Fatty Acid.

Mycelial fungi grow as colonies consisting of polar growing hyphae, developing radially from spore or inoculum. Over time, the colony develops, hyphae are subject to various exogenous or endogenous stimuli, and mycelium becomes heterogeneous in growth, gene expression, biosynthesis, and secretion of proteins and metabolites. Although the biochemical and molecular mechanisms of mycelium heterogeneity have been the subject of many studies, the role of lipids in colony development and zonality is still not understood. This work was undertaken to extend our knowledge of mycelium heterogeneity and to answer the question of how different lipid molecular species are distributed in the surface colony of the basidial fungus Flammulina velutipes and how this distribution correlates with its morphology. The heterogeneity in the lipid metabolism and lipid composition of the fungal mycelium was demonstrated. According to the real-time PCR and LC-MS/MS results, the expression of genes of PC metabolism, accumulation of phospholipid classes, and degree of unsaturation of PC and PE increased in the direction from the center to the periphery of the colony. The peripheral zone of the colony was characterized by a higher value of the PC/PE ratio and a higher level of phospholipids esterified by linolenic acid. Considering that the synthesis of phospholipids in fungi occurs in different ways, we also conducted experiments with deuterium-labeled phospholipid precursors and found out that the Kennedy pathway is the predominant route for PC biosynthesis in F. velutipes. The zonal differences in gene expression and lipid composition can be explained by the participation of membrane lipids in polar growth maintenance and regulation.

Uptake and Metabolic Conversion of Exogenous Phosphatidylcholines Depending on Their Acyl Chain Structure in Arabidopsis thaliana.

Fungi and plants are not only capable of synthesizing the entire spectrum of lipids de novo but also possess a well-developed system that allows them to assimilate exogenous lipids. However, the role of structure in the ability of lipids to be absorbed and metabolized has not yet been characterized in detail. In the present work, targeted lipidomics of phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs), in parallel with morphological phenotyping, allowed for the identification of differences in the effects of PC molecular species introduced into the growth medium, in particular, typical bacterial saturated (14:0/14:0, 16:0/16:0), monounsaturated (16:0/18:1), and typical for fungi and plants polyunsaturated (16:0/18:2, 18:2/18:2) species, on Arabidopsis thaliana. For comparison, the influence of an artificially synthesized (1,2-di-(3-(3-hexylcyclopentyl)-propanoate)-sn-glycero-3-phosphatidylcholine, which is close in structure to archaeal lipids, was studied. The phenotype deviations stimulated by exogenous lipids included changes in the length and morphology of both the roots and leaves of seedlings. According to lipidomics data, the main trends in response to exogenous lipid exposure were an increase in the proportion of endogenic 18:1/18:1 PC and 18:1_18:2 PC molecular species and a decrease in the relative content of species with C18:3, such as 18:3/18:3 PC and/or 16:0_18:3 PC, 16:1_18:3 PE. The obtained data indicate that exogenous lipid molecules affect plant morphology not only due to their physical properties, which are manifested during incorporation into the membrane, but also due to the participation of exogenous lipid molecules in the metabolism of plant cells. The results obtained open the way to the use of PCs of different structures as cellular regulators.

Balance of Δ5-and Δ7-sterols and stanols in halophytes in connection with salinity tolerance.

Sterols (STs) have a key role in regulating the fluidity and permeability of membranes in plants (phytosterols) that have wide structural diversity. We studied the effect of structural STs diversity on salt tolerance in halophytes. Specifically, we used gas chromatography-mass spectrometry (GC-MS), including two-dimensional gas chromatography-mass spectrometry (GCxGC-MS), to assess the STs composition in leaves of 21 species of wild-growing halophytes from four families (Asteraceae, Chenopodiaceae, Plumbaginaceae, Tamaricaceae) and three ecological groups (Euhalophytes (Eu), recretophytes (Re), salt excluders (Ex)). Fifteen molecular species of STs from three main groups, Δ5-, Δ7-and Δ0- STs (stanols), were detected. Plants of the genus Artemisia were characterized by a high content of stigmasterol (30-49% of the total STs), while ß-sitosterol was the major compound in two Limonium spp., where it comprised 84-92% of the total STs. Species of Chenopodiaceae were able to accumulate both Δ5-and Δ7-STs and stanols. The content of the predominant Δ5-STs decreased in the order Ex → Re → Eu. Molecular species with a saturated steroid nucleus were identified in Eu and Re, suggesting their special salt-accumulating and salt-releasing functions. The structural analogues of stigmasterol, having a double bond C-22, were stigmasta-7,22-dien-3ß-ol (spinasterol) and stigmast-22-en-3ß-ol (Δ7--sitosterol). The ratio of Δ5-stigmasterol/Δ5-ß-sitosterol increased in Ex plants, and spinasterol/Δ7--sitosterol and 22-stigmastenol/sitostanol increased in Eu plants. These data support the well-known role of stigmasterol and its isomers in plant responses to abiotic and biotic factors. The variability in STs types and their ratios suggested some involvement of the sterol membrane components in plant adaptation to growth conditions. The balance of Δ5-, Δ7-and stanols, as well as the accumulation of molecular analogues of stigmasterol, was suggested to be associated with salt tolerance of the plant species in this investigation.

Diversity of ESI-MS Based Phosphatidylcholine Profiles in Basidiomycetes.

Phosphatidylcholines (PC) are the main membrane lipid constituents comprising more than 50% of total glycerophospholipids. They coordinate a number of cell functions, particularly cell growth, homeostasis, secretion, recognition and communication. In basidial fungi PC are synthesized via the Kennedy pathway as well as through methylation of phosphatidylethanolamines (PE) and then undergo remodeling in Lands cycle that replaces fatty acids in PC molecules. The molecular profile of PC is determined by the genetic features that are characteristic for every species and depend on the environment. Here we present the results of ESI-MS based analyses of PC profiles of 38 species of basidiomycetes belonging to Agaricales (12), Polyporales (17), Russulales (5), Gleophyllales (2), Cantharellales (1), Auriculariales (1), Phallales (1). Although the variety of PC molecular species of basidiomycetes is rather diverse (20-38 molecular species in every profile), only 1-3 main molecular species represent 70-90% of total PC content. The most abundant of them are C36:4 and C36:3, followed by C34:1, C34:2, C36:5, C36:2. In the majority of basidiomycetes, C36:4 reaches up to 50-70% of total PC molecular species. Based on the results of hierarchical cluster analysis four main types of PC profiles which characterized the studied fungi independently from their taxonomic position, ecology, trophic status, and hyphal differentiation have been revealed. Comparative analyses of studied fungi using PCA method have shown that species of Polyporales differ from those of Agaricales by higher variability of PC profiles.

Role of Lipids of the Evergreen Shrub Ephedra monosperma in Adaptation to Low Temperature in the Cryolithozone.

Lipids are the fundamental components of cell membranes and they play a significant role in their integrity and fluidity. The alteration in lipid composition of membranes has been reported to be a major response to abiotic environmental stresses. Seasonal dynamics of membrane lipids in the shoots of Ephedra monosperma J.G. Gmel. ex C.A. Mey. growing in natural conditions of permafrost ecosystems was studied using HPTLC, GC-MS and ESI-MS. An important role of lipid metabolism was established during the autumn-winter period when the shoots of the evergreen shrub were exposed to low positive (3.6 °C), negative (-8.3 °C) and extremely low temperatures (-38.4 °C). Maximum accumulation of phosphatidic acid (PA), the amount of which is times times greater than the sum of phosphatidylcholine and phosphatidylethanolamine (PC + PE) was noted in shoots of E. monosperma in the summer-autumn period. The autumn hardening period (3.6 °C) is accompanied by active biosynthesis and accumulation of membrane lipids, a decrease of saturated 34:1 PCs, 34:1 PEs and 34:1 PAs, and an increase in unsaturated long-chain 38:5 PEs, 38:6 PEs, indicating that the adaptation of E. monosperma occurs not at the level of lipid classes but at the level of molecular species. At a further decrease of average daily air temperature in October (-8.3 °C) a sharp decline of PA level was registered. At an extreme reduction of environmental temperature (-38.4 °C) the content of non-bilayer PE and PA increases, the level of unsaturated fatty acids (FA) rises due to the increase of C18:2(Δ9,12) and C18:3(Δ9,12,15) acids and the decrease of C16:0 acids. It is concluded that changes in lipid metabolism reflect structural and functional reorganization of cell membranes and are an integral component of the complex process of plant hardening to low temperatures, which contributes to the survival of E. monosperma monocotyledonous plants in the extreme conditions of the Yakutia cryolithozone.

Influence of Extremely Low Temperatures of the Pole of Cold on the Lipid and Fatty-Acid Composition of Aerial Parts of the Horsetail Family (Equisetaceae).

The lipid composition of two species of vascular plants, Equisetum variegatum Schleich. ex. Web. and E. scirpoides Michx., growing in the permafrost zone (Northeastern Yakutia, the Pole of Cold of the Northern Hemisphere), with average daily air temperatures in summer of +17.8 °C, in autumn of +0.6 °C, and in winter of -46.7 °C, was comparatively studied. The most significant seasonal trend of lipid composition was an accumulation of PA in both horsetail species in the autumn-winter period. Cold acclimation in autumn was accompanied by a decrease in the proportion of bilayer-forming lipids (phosphatidylcholine in the non-photosynthetic membranes and MGDG in photosynthetic membranes), an increase in the desaturation degree due to the accumulation of triene fatty acids (E. scirpoides), and an accumulation of betaine lipids O-(1,2-diacylglycero)-N,N,N-trimethylhomoserine (DGTS). The inverse changes in some parameters were registered in the winter period, including an increase in the proportion of "bilayer" lipids and decrease in the unsaturation degree. According to the data obtained, it can be concluded that high levels of accumulation of membrane lipids and polyunsaturated FAs (PUFAs), as well as the presence of Δ5 FAs in lipids, are apparently features of cold hardening of perennial herbaceous plants in the cryolithozone.

Lipid and Metabolite Profiling of Serpula lacrymans Under Freezing Stress.

Basidiomycete fungus Serpula lacrymans is one of the most dangerous indoor fungus causing dry rot of timber. The physiology of this fungus deserves more attention as a basis for development of methods of dry rot treatment. We observed an increase in the freezing resistance of S. lacrymans after pre-cultivation of mycelia at elevated temperatures. To examine the biochemical mechanisms underlying this phenomenon the lipid composition and metabolite profiling of mycelia subjected to freezing and thawing were investigated. An analysis is made of the growth rate and metabolism of "daughter" cultures derived from a frozen mycelia. According to the results, sphingolipids and water-soluble metabolites such as mannitol, glycerol, sugar alcohols, some amino- and organic acids are able to function as protective compounds providing a cross-resistance between heat shock and freeze-thaw stress in S. lacrymans.

Role of lipids in the thermal plasticity of basidial fungus Favolaschia manipularis.

In this study, we examined the lipid composition of two strains of the tropical basidiomycete Favolaschia manipularis (Berk.) Teng, which differ in their adaptive potential to high (35 °C) and low (5 °C) temperatures. The results suggest that adaptation to extreme temperatures involves a change in the molecular composition of sterols, in addition to other well-known mechanisms of regulating membrane thickness and fluidity, such as changes in the lipid unsaturation and in the proportion of bilayer- and non-bilayer-forming lipids. It was demonstrated for the first time that adaptation to high temperature stress in fungi is accompanied by the accumulation of 9(11)-dehydroergosterol and ergosterol peroxide. Furthermore, increased thermal plasticity correlates with high storage lipid (triglycerides) content, accumulation of phosphatidic acid in the membrane, and an equal proportion of bilayer and non-bilayer lipids in the membrane.

Corynebacterium godavarianum sp. nov., isolated from the Godavari river, India.

A Gram-stain-positive, rod-shaped, non-motile bacterium, strain PRD07T, was isolated from Godavari river, India during the world's largest spiritual and religious mass bathing event 'Kumbh Mela'. Molecular analysis using 16S rRNA gene sequencing and phylogenetic analysis reveals the distinct phylogenetic positioning of strain PRD07T within the genus Corynebacterium. The strain demonstrated highest sequence similarity to Corynebacterium imitans DSM 44264T (97.9 %), Corynebacterium appendicis DSM 44531T (97.1 %) and <96.7 % with all other members of the genus Corynebacterium. The G+C content of PRD07T was 68.5 mol% (Tm) and the DNA-DNA hybridization depicts 61.09 % genomic relatedness with C. imitans DSM 44264T. Chemotaxonomic assessment of strain PRD07T suggested presence of C16 : 0 (31.6 %), C18 : 0 (3.5 %) and C18 : 1ω9c (58.6 %) as the major cellular fatty acids. The major polar lipids of strain PRD07T were phosphatidylglycerol, diphosphatidylglycerol and glycophospholipid. Differentiating molecular, phylogenetic and chemotaxonomic characteristics of strain PRD07T with its closest relatives necessitated the description of strain PRD07T as a novel species of genus Corynebacterium for which the name Corynebacteriumgodavarianum sp. nov., has been proposed. The type strain is PRD07T (=MCC 3388T=KCTC 39803T=LMG 29598T).

Key Roles of Size and Crystallinity of Nanosized Iron Hydr(oxides) Stabilized by Humic Substances in Iron Bioavailability to Plants.

Availability of Fe in soil to plants is closely related to the presence of humic substances (HS). Still, the systematic data on applicability of iron-based nanomaterials stabilized with HS as a source for plant nutrition are missing. The goal of our study was to establish a connection between properties of iron-based materials stabilized by HS and their bioavailability to plants. We have prepared two samples of leonardite HS-stabilized iron-based materials with substantially different properties using the reported protocols and studied their physical chemical state in relation to iron uptake and other biological effects. We used Mössbauer spectroscopy, XRD, SAXS, and TEM to conclude on iron speciation, size, and crystallinity. One material (Fe-HA) consisted of polynuclear iron(III) (hydr)oxide complexes, so-called ferric polymers, distributed in HS matrix. These complexes are composed of predominantly amorphous small-size components (<5 nm) with inclusions of larger crystalline particles (the mean size of (11 ± 4) nm). The other material was composed of well-crystalline feroxyhyte (δ'-FeOOH) NPs with mean transverse sizes of (35 ± 20) nm stabilized by small amounts of HS. Bioavailability studies were conducted on wheat plants under conditions of iron deficiency. The uptake studies have shown that small and amorphous ferric polymers were readily translocated into the leaves on the level of Fe-EDTA, whereas relatively large and crystalline feroxyhyte NPs were mostly sorbed on the roots. The obtained data are consistent with the size exclusion limits of cell wall pores (5-20 nm). Both samples demonstrated distinct beneficial effects with respect to photosynthetic activity and lipid biosynthesis. The obtained results might be of use for production of iron-based nanomaterials stabilized by HS with the tailored iron availability to plants. They can be applied as the only source for iron nutrition as well as in combination with the other elements, for example, for industrial production of "nanofortified" macrofertilizers (NPK).

Label Distribution in Tissues of Wheat Seedlings Cultivated with Tritium-Labeled Leonardite Humic Acid.

Humic substances (HS) play important roles in the biotic-abiotic interactions of the root plant and soil contributing to plant adaptation to external environments. However, their mode of action on plants remains largely unknown. In this study the HS distribution in tissues of wheat seedlings was examined using tritium-labeled humic acid (HA) derived from leonardite (a variety of lignites) and microautoradiography (MAR). Preferential accumulation of labeled products from tritiated HA was found in the roots as compared to the shoots, and endodermis was shown to be the major control point for radial transport of label into vascular system of plant. Tritium was also found in the stele and xylem tissues indicating that labeled products from tritiated HA could be transported to shoot tissues via the transpiration stream. Treatment with HA lead to an increase in the content of polar lipids of photosynthetic membranes. The observed accumulation of labeled HA products in root endodermis and positive impact on lipid synthesis are consistent with prior reported observations on physiological effects of HS on plants such as enhanced growth and development of lateral roots and improvement/repairs of the photosynthetic status of plants under stress conditions.

Diacylglyceryltrimethylhomoserine content and gene expression changes triggered by phosphate deprivation in the mycelium of the basidiomycete Flammulina velutipes.

Diacylglyceryltrimethylhomoserines (DGTS) are betaine-type lipids that are phosphate-free analogs of phosphatidylcholines (PC). DGTS are abundant in some bacteria, algae, primitive vascular plants and fungi. In this study, we report inorganic phosphate (Pi) deficiency-induced DGTS synthesis in the basidial fungus Flammulina velutipes (Curt.: Fr.) Sing. We present results of an expression analysis of the BTA1 gene that codes for betaine lipid synthase and two genes of PC biosynthesis (CHO2 and CPT1) during phosphate starvation of F. velutipes culture. We demonstrate that FvBTA1 gene has increased transcript abundance under phosphate starvation. Despite depletion in PC, both CHO2 and CPT1 were determined to have increased expression. We also describe the deduced amino acid sequence and genomic structure of the BTA1 gene in F. velutipes. Phylogenetic relationships between putative orthologs of BTA1 proteins of basidiomycete fungi are discussed.