Fishing Innate Immune System Properties through the Transcriptomic Single-Cell Data of Teleostei.

The innate immune system is the first line of defense in multicellular organisms. Danio rerio is widely considered a promising model for IIS-related research, with the most amount of scRNAseq data available among Teleostei. We summarized the scRNAseq and spatial transcriptomics experiments related to the IIS for zebrafish and other Teleostei from the GEO NCBI and the Single-Cell Expression Atlas. We found a considerable number of scRNAseq experiments at different stages of zebrafish development in organs such as the kidney, liver, stomach, heart, and brain. These datasets could be further used to conduct large-scale meta-analyses and to compare the IIS of zebrafish with the mammalian one. However, only a small number of scRNAseq datasets are available for other fish (turbot, salmon, cavefish, and dark sleeper). Since fish biology is very diverse, it would be a major mistake to use zebrafish alone in fish immunology studies. In particular, there is a special need for new scRNAseq experiments involving nonmodel Teleostei, e.g., long-lived species, cancer-resistant fish, and various fish ecotypes.

Long-term cold, freezing and drought: overlapping and specific regulatory mechanisms and signal transduction in tea plant (Camellia sinensis (L.) Kuntze).

Introduction: Low temperatures and drought are two main environmental constraints reducing the yield and geographical distribution of horticultural crops worldwide. Understanding the genetic crosstalk between stress responses has potential importance for crop improvement. Methods: In this study, Illumina RNA-seq and Pac-Bio genome resequencing were used to annotate genes and analyze transcriptome dynamics in tea plants under long-term cold, freezing, and drought. Results: The highest number of differentially expressed genes (DEGs) was identified under long-term cold (7,896) and freezing (7,915), with 3,532 and 3,780 upregulated genes, respectively. The lowest number of DEGs was observed under 3-day drought (47) and 9-day drought (220), with five and 112 genes upregulated, respectively. The recovery after the cold had 6.5 times greater DEG numbers as compared to the drought recovery. Only 17.9% of cold-induced genes were upregulated by drought. In total, 1,492 transcription factor genes related to 57 families were identified. However, only 20 transcription factor genes were commonly upregulated by cold, freezing, and drought. Among the 232 common upregulated DEGs, most were related to signal transduction, cell wall remodeling, and lipid metabolism. Co-expression analysis and network reconstruction showed 19 genes with the highest co-expression connectivity: seven genes are related to cell wall remodeling (GATL7, UXS4, PRP-F1, 4CL, UEL-1, UDP-Arap, and TBL32), four genes are related to calcium-signaling (PXL1, Strap, CRT, and CIPK6), three genes are related to photo-perception (GIL1, CHUP1, and DnaJ11), two genes are related to hormone signaling (TTL3 and GID1C-like), two genes are involved in ROS signaling (ERO1 and CXE11), and one gene is related to the phenylpropanoid pathway (GALT6). Discussion: Based on our results, several important overlapping mechanisms of long-term stress responses include cell wall remodeling through lignin biosynthesis, o-acetylation of polysaccharides, pectin biosynthesis and branching, and xyloglucan and arabinogalactan biosynthesis. This study provides new insight into long-term stress responses in woody crops, and a set of new target candidate genes were identified for molecular breeding aimed at tolerance to abiotic stresses.

Particle-Based Imaging Tools Revealing Water Flows in Maize Nodal Vascular Plexus.

In plants, water flows are the major driving force behind growth and play a crucial role in the life cycle. To study hydrodynamics, methods based on tracking small particles inside water flows attend a special place. Thanks to these tools, it is possible to obtain information about the dynamics of the spatial distribution of the flux characteristics. In this paper, using contrast-enhanced magnetic resonance imaging (MRI), we show that gadolinium chelate, used as an MRI contrast agent, marks the structural characteristics of the xylem bundles of maize stem nodes and internodes. Supplementing MRI data, the high-precision visualization of xylem vessels by laser scanning microscopy was used to reveal the structural and dimensional characteristics of the stem vascular system. In addition, we propose the concept of using prototype "Y-type xylem vascular connection" as a model of the elementary connection of vessels within the vascular system. A Reynolds number could match the microchannel model with the real xylem vessels.

Transcriptomic Data Meta-Analysis Sheds Light on High Light Response in Arabidopsis thaliana L.

The availability and intensity of sunlight are among the major factors of growth, development and metabolism in plants. However, excessive illumination disrupts the electronic balance of photosystems and leads to the accumulation of reactive oxygen species in chloroplasts, further mediating several regulatory mechanisms at the subcellular, genetic, and molecular levels. We carried out a comprehensive bioinformatic analysis that aimed to identify genetic systems and candidate transcription factors involved in the response to high light stress in Arabidopsis thaliana L. using resources GEO NCBI, string-db, ShinyGO, STREME, and Tomtom, as well as programs metaRE, CisCross, and Cytoscape. Through the meta-analysis of five transcriptomic experiments, we selected a set of 1151 differentially expressed genes, including 453 genes that compose the gene network. Ten significantly enriched regulatory motifs for TFs families ZF-HD, HB, C2H2, NAC, BZR, and ARID were found in the promoter regions of differentially expressed genes. In addition, we predicted families of transcription factors associated with the duration of exposure (RAV, HSF), intensity of high light treatment (MYB, REM), and the direction of gene expression change (HSF, S1Fa-like). We predicted genetic components systems involved in a high light response and their expression changes, potential transcriptional regulators, and associated processes.

Isolation and transcriptome analysis of a biotechnologically promising Black Sea protist, Thraustochytrium aureum ssp. strugatskii.

Background: Marine protists are an important part of the ocean ecosystem. They may possess unique sets of biosynthetic pathways and, thus, be promising model organisms for metabolic engineering for producing substances for the pharmaceutical, cosmetic, and perfume industries. Currently, full-genome data are available just for a limited number of protists hampering their use in biotechnology. Methods: We characterized the morphology of a new cultured strain of Thraustochytriaceae isolated from the Black Sea ctenophore Beroe ovata using phase-contrast microscopy. Cell culture was performed in the FAND culture medium based on fetal bovine serum and DMEM. Phylogenetic analysis was performed using the 18S rRNA sequence. We also conducted a transcriptome assembly and compared the data with the closest species. Results: The protist belongs to the genus Thraustochytrium based on the 18S rRNA sequence analysis. We designated the isolated protist as T. aureum ssp. strugatskii. The closest species with the genome assembly is Schizochytrium aggregatum. Transcriptome analysis revealed the majority of the fatty acid synthesis enzymes. Conclusion: Our findings suggest that the T. aureum ssp. strugatskii is a promising candidate for biotechnological use. Together with the previously available, our data would allow the establishment of an accurate phylogeny of the family Thraustochytriaceae. Also, it could be a reference point for studying the evolution of the enzyme families.

A Sight on Single-Cell Transcriptomics in Plants Through the Prism of Cell-Based Computational Modeling Approaches: Benefits and Challenges for Data Analysis.

Single-cell technology is a relatively new and promising way to obtain high-resolution transcriptomic data mostly used for animals during the last decade. However, several scientific groups developed and applied the protocols for some plant tissues. Together with deeply-developed cell-resolution imaging techniques, this achievement opens up new horizons for studying the complex mechanisms of plant tissue architecture formation. While the opportunities for integrating data from transcriptomic to morphogenetic levels in a unified system still present several difficulties, plant tissues have some additional peculiarities. One of the plants' features is that cell-to-cell communication topology through plasmodesmata forms during tissue growth and morphogenesis and results in mutual regulation of expression between neighboring cells affecting internal processes and cell domain development. Undoubtedly, we must take this fact into account when analyzing single-cell transcriptomic data. Cell-based computational modeling approaches successfully used in plant morphogenesis studies promise to be an efficient way to summarize such novel multiscale data. The inverse problem's solutions for these models computed on the real tissue templates can shed light on the restoration of individual cells' spatial localization in the initial plant organ-one of the most ambiguous and challenging stages in single-cell transcriptomic data analysis. This review summarizes new opportunities for advanced plant morphogenesis models, which become possible thanks to single-cell transcriptome data. Besides, we show the prospects of microscopy and cell-resolution imaging techniques to solve several spatial problems in single-cell transcriptomic data analysis and enhance the hybrid modeling framework opportunities.

Comparative transcriptome profiling of a resistant vs susceptible bread wheat (Triticum aestivum L.) cultivar in response to water deficit and cold stress.

Bread wheat (Triticum aestivum L.) is one of the most important agricultural plants wearing abiotic stresses, such as water deficit and cold, that cause its productivity reduction. Since resistance to abiotic factors is a multigenic trait, therefore modern genome-wide approaches can help to involve various genetic material in breeding. One technique is full transcriptome analysis that reveals groups of stress response genes serving marker-assisted selection markers. Comparing transcriptome profiles of the same genetic material under several stresses is essential and makes the whole picture. Here, we addressed this by studying the transcriptomic response to water deficit and cold stress for two evolutionarily distant bread wheat varieties: stress-resistant cv. Saratovskaya 29 (S29) and stress-sensitive cv. Yanetzkis Probat (YP). For the first time, transcriptomes for these cultivars grown under abiotic stress conditions were obtained using Illumina based MACE technology. We identified groups of genes involved in response to cold and water deficiency stresses, including responses to each stress factor and both factors simultaneously that may be candidates for resistance genes. We discovered a core group of genes that have a similar pattern of stress-induced expression changes. The particular expression pattern was revealed not only for the studied varieties but also for the published transcriptomic data on cv. Jing 411 and cv. Fielder. Comparative transcriptome profiling of cv. S29 and cv. YP in response to water deficit and cold stress confirmed the hypothesis that stress-induced expression change is unequal within a homeologous gene group. As a rule, at least one changed significantly while the others had a relatively lower expression. Also, we found several SNPs distributed throughout the genomes of cv. S29 and cv. YP and distinguished the studied varieties from each other and the reference cv. Chinese Spring. Our results provide new data for genomics-assisted breeding of stress-tolerant wheat cultivars.

Subcellular compartmentalization of the plant antioxidant system: an integrated overview.

The antioxidant system (AOS) maintains the optimal concentration of reactive oxygen species (ROS) in a cell and protects it against oxidative stress. In plants, the AOS consists of seven main classes of antioxidant enzymes, low-molecular antioxidants (e.g., ascorbate, glutathione, and their oxidized forms) and thioredoxin/glutaredoxin systems which can serve as reducing agents for antioxidant enzymes. The number of genes encoding AOS enzymes varies between classes, and same class enzymes encoded by different gene copies may have different subcellular localizations, functional loads and modes of evolution. These facts hereafter reinforce the complex nature of AOS regulation and functioning. Further studies can describe new trends in the behavior and functioning of systems components, and provide new fundamental knowledge about systems regulation. The system is revealed to have a lot of interactions and interplay pathways between its components at the subcellular level (antioxidants, enzymes, ROS level, and hormonal and transcriptional regulation). These facts should be taken into account in further studies during the AOS modeling by describing the main pathways of generating and utilizing ROS, as well as the associated signaling processes and regulation of the system on cellular and organelle levels, which is a complicated and ambitious task. Another objective for studying the phenomenon of the AOS is related to the influence of cell dynamics and circadian rhythms on it. Therefore, the AOS requires an integrated and multi-level approach to study. We focused this review on the existing scientific background and experimental data used for the systems biology research of the plant AOS.

Comparative Expression Analysis of Stress-Inducible Candidate Genes in Response to Cold and Drought in Tea Plant [Camellia sinensis (L.) Kuntze].

Cold and drought are two of the most severe threats affecting the growth and productivity of the tea plant, limiting its global spread. Both stresses cause osmotic changes in the cells of the tea plant by decreasing their water potential. To develop cultivars that are tolerant to both stresses, it is essential to understand the genetic responses of tea plant to these two stresses, particularly in terms of the genes involved. In this study, we combined literature data with interspecific transcriptomic analyses (using Arabidopsis thaliana and Solanum lycopersicum) to choose genes related to cold tolerance. We identified 45 stress-inducible candidate genes associated with cold and drought responses in tea plants based on a comprehensive homologous detection method. Of these, nine were newly characterized by us, and 36 had previously been reported. The gene network analysis revealed upregulated expression in ICE1-related cluster of bHLH factors, HSP70/BAM5 connected genes (hexokinases, galactinol synthases, SnRK complex, etc.) indicating their possible co-expression. Using qRT-PCR we revealed that 10 genes were significantly upregulated in response to both cold and drought in tea plant: HSP70, GST, SUS1, DHN1, BMY5, bHLH102, GR-RBP3, ICE1, GOLS1, and GOLS3. SnRK1.2, HXK1/2, bHLH7/43/79/93 were specifically upregulated in cold, while RHL41, CAU1, Hydrolase22 were specifically upregulated in drought. Interestingly, the expression of CIP was higher in the recovery stage of both stresses, indicating its potentially important role in plant recovery after stress. In addition, some genes, such as DHN3, bHLH79, PEI54, SnRK1.2, SnRK1.3, and Hydrolase22, were significantly positively correlated between the cold and drought responses. CBF1, GOLS1, HXK2, and HXK3, by contrast, showed significantly negative correlations between the cold and drought responses. Our results provide valuable information and robust candidate genes for future functional analyses intended to improve the stress tolerance of the tea plant and other species.

Stress-induced changes in the expression of antioxidant system genes for rice (Oryza sativa L.) and bread wheat (Triticum aestivum L.).

BACKGROUND: Plant cell metabolism inevitably forms reactive oxygen species (ROS), which can damage cells or lead to their death. The antioxidant system (AOS) evolved to eliminate a high concentration of ROS. For plants, this system consists of the seven classes of antioxidant enzymes and antioxidant compounds. Each enzymatic class contains a various number of genes which may vary from species to species. In such a multi-copy genetic system, the integration of evolutionary characteristics and expression data makes it possible to effectively predict promising breeding targets for the design of highly-yielding cultivars. In the plant cells, ROS production can increase as a result of abiotic stresses. Accordingly, AOS responds to stress by altering the expression of the genes of its components. Expression profiles of AOS enzymes, including their changes under stress, remains incomplete. A comprehensive study of the system behavior in response to stress for different species gives the key to identify the general mechanisms of AOS regulation. In this article, we studied stress-induced changes in the expression of AOS genes in photosynthetic tissues for rice and bread wheat. METHODS: A meta-analysis of genome-wide transcriptome data on stress-induced changes in expression profiles of antioxidant genes using microarray and next generation sequencing (NGS) experiments from the GEO NCBI database for rice and bread wheat was carried out. Experimental study of expression changes in short (6 h) and prolonged (24 h) cold stress responses for selected AOS genes of bread wheat cultivars Saratovskaya29 and Yanetzkis Probat was conducted using qPCR. RESULTS: The large-scale meta-transcriptome and complementary experimental analysis revealed a summary of fold changes in the AOS gene expression in response to cold and water deficiency for rice and bread wheat.

The evolution of gene regulatory networks controlling Arabidopsis thaliana L. trichome development.

BACKGROUND: The variation in structure and function of gene regulatory networks (GRNs) participating in organisms development is a key for understanding species-specific evolutionary strategies. Even the tiniest modification of developmental GRN might result in a substantial change of a complex morphogenetic pattern. Great variety of trichomes and their accessibility makes them a useful model for studying the molecular processes of cell fate determination, cell cycle control and cellular morphogenesis. Nowadays, a large number of genes regulating the morphogenesis of A. thaliana trichomes are described. Here we aimed at a study the evolution of the GRN defining the trichome formation, and evaluation its importance in other developmental processes. RESULTS: In study of the evolution of trichomes formation GRN we combined classical phylogenetic analysis with information on the GRN topology and composition in major plants taxa. This approach allowed us to estimate both times of evolutionary emergence of the GRN components which are mainly proteins, and the relative rate of their molecular evolution. Various simplifications of protein structure (based on the position of amino acid residues in protein globula, secondary structure type, and structural disorder) allowed us to demonstrate the evolutionary associations between changes in protein globules and speciations/duplications events. We discussed their potential involvement in protein-protein interactions and GRN function. CONCLUSIONS: We hypothesize that the divergence and/or the specialization of the trichome-forming GRN is linked to the emergence of plant taxa. Information about the structural targets of the protein evolution in the GRN may predict switching points in gene networks functioning in course of evolution. We also propose a list of candidate genes responsible for the development of trichomes in a wide range of plant species.

LSM-W2: laser scanning microscopy worker for wheat leaf surface morphology.

BACKGROUND: Microscopic images are widely used in plant biology as an essential source of information on morphometric characteristics of the cells and the topological characteristics of cellular tissue pattern due to modern computer vision algorithms. High-resolution 3D confocal images allow extracting quantitative characteristics describing the cell structure of leaf epidermis. For some issues in the study of cereal leaves development, it is required to apply the staining techniques with fluorescent dyes and to scan rather large fragments consisting of several frames. We aimed to develop a tool for processing multi-frame multi-channel 3D images obtained from confocal laser scanning microscopy and taking into account the peculiarities of the cereal leaves staining. RESULTS: We elaborated an ImageJ-plugin LSM-W2 that allows extracting data on Leaf Surface Morphology from Laser Scanning Microscopy images. The plugin is a crucial link in a workflow for obtaining data on structural properties of leaf epidermis and morphological properties of epidermal cells. It allows converting large lsm-files (laser scanning microscopy) into segmented 2D/3D images or tables with data on cells and/or nuclei sizes. In the article, we also represent some case studies showing the plugin application for solving biological tasks. Namely the plugin is applied in the following cases: defining parameters of jigsaw-puzzle pattern for maize leaf epidermal cells, analysis of the pavement cells morphological parameters for the mature wheat leaf grown under control and water deficit conditions, initiation of cell longitudinal rows, and detection of guard mother cells emergence at the initial stages of the stomatal morphogenesis in the growth zone of a wheat leaf. CONCLUSION: The proposed plugin is efficient for high-throughput analysis of cellular architecture for cereal leaf epidermis. The workflow implies using inexpensive and rapid sample preparation and does not require the applying of transgenesis and reporter genetic structures expanding the range of species and varieties to study. Obtained characteristics of the cell structure and patterns further could act as a basis for the development and verification for spatial models of plant tissues formation mechanisms accounting for structural features of cereal leaves. AVAILABILITY: The implementation of this workflow is available as an ImageJ plugin distributed as a part of the Fiji project (FijiisjustImageJ: https://fiji.sc/ ). The plugin is freely available at https://imagej.net/LSM_Worker , https://github.com/JmanJ/LSM_Worker and http://pixie.bionet.nsc.ru/LSM_WORKER/ .

Quantitative characteristics of pubescence in wheat (Triticum aestivum L.) are associated with photosynthetic parameters under conditions of normal and limited water supply.

MAIN CONCLUSION: Density and length of leaf pubescence are important factors of diversity in the response to water deficiency among wheat genotypes. Many studies evidence an important protective value of leaf hairiness in plants, especially under the conditions of drought, thermal loads and increased solar radiation. However, the physiological and adaptive roles of such traits in cereals, including cultivated plants, have not been sufficiently studied to date. The aim of this work was to study the association of morphological characteristics of leaves with parameters of gas exchange and chlorophyll fluorescence in wheat lines carrying a genetically different leaf hairiness. Isogenic and inter-varietal substitution wheat lines were used, carrying various combinations of dominant and recessive alleles of the known genes. A quantitative assessment of the pubescence was carried out in contrasting watering conditions to establish the physiological role of this trait in adaptation to drought. With the help of a portable system for studying the gas exchange and chlorophyll fluorescence, ten parameters of photosynthesis were studied, as well as morphological features of leaves and shoot biomass. It was found that gas exchange parameters are inversely proportional to the density and length of trichomes. In drought conditions, the trichome density increased and the length of trichomes decreased under the observed decrease in the level of gas exchange. A similar dependence was observed for the level of non-photochemical quenching of chlorophyll fluorescence. Under optimal conditions, the poorly haired cultivars exhibited a higher biomass than the densely haired. However, under water deficiency they significantly reduced the biomass and showed a low value of the tolerance index.

Identification of nuclear genes controlling chlorophyll synthesis in barley by RNA-seq.

BACKGROUND: Albinism in plants is characterized by lack of chlorophyll and results in photosynthesis impairment, abnormal plant development and premature death. These abnormalities are frequently encountered in interspecific crosses and tissue culture experiments. Analysis of albino mutant phenotypes with full or partial chlorophyll deficiency can shed light on genetic determinants and molecular mechanisms of albinism. Here we report analysis of RNA-seq transcription profiling of barley (Hordeum vulgare L.) near-isogenic lines, one of which is a carrier of mutant allele of the Alm gene for albino lemma and pericarp phenotype (line i:BwAlm). RESULTS: 1221 genome fragments have statistically significant changes in expression levels between lines i:BwAlm and Bowman, with 148 fragments having increased expression levels in line i:BwAlm, and 1073 genome fragments, including 42 plastid operons, having decreased levels of expression in line i:BwAlm. We detected functional dissimilarity between genes with higher and lower levels of expression in i:BwAlm line. Genes with lower level of expression in the i:BwAlm line are mostly associated with photosynthesis and chlorophyll synthesis, while genes with higher expression level are functionally associated with vesicle transport. Differentially expressed genes are shown to be involved in several metabolic pathways; the largest fraction of such genes was observed for the Calvin-Benson-Bassham cycle. Finally, de novo assembly of transcriptome contains several transcripts, not annotated in current H. vulgare genome version. CONCLUSIONS: Our results provide the new information about genes which could be involved in formation of albino lemma and pericarp phenotype. They demonstrate the interplay between nuclear and chloroplast genomes in this physiological process.

Regions of the bread wheat D genome associated with variation in key photosynthesis traits and shoot biomass under both well watered and water deficient conditions.

A quantitative trait locus (QTL) approach was taken to reveal the genetic basis in wheat of traits associated with photosynthesis during a period of exposure to water deficit stress. The performance, with respect to shoot biomass, gas exchange and chlorophyll fluorescence, leaf pigment content and the activity of various ascorbate-glutathione cycle enzymes and catalase, of a set of 80 wheat lines, each containing a single chromosomal segment introgressed from the bread wheat D genome progenitor Aegilops tauschii, was monitored in plants exposed to various water regimes. Four of the seven D genome chromosomes (1D, 2D, 5D, and 7D) carried clusters of both major (LOD >3.0) and minor (LOD between 2.0 and 3.0) QTL. A major QTL underlying the activity of glutathione reductase was located on chromosome 2D, and another, controlling the activity of ascorbate peroxidase, on chromosome 7D. A region of chromosome 2D defined by the microsatellite locus Xgwm539 and a second on chromosome 7D flanked by the marker loci Xgwm1242 and Xgwm44 harbored a number of QTL associated with the water deficit stress response.

Extraction of quantitative characteristics describing wheat leaf pubescence with a novel image-processing technique.

Leaf pubescence (hairiness) in wheat plays an important biological role in adaptation to the environment. However, this trait has always been methodologically difficult to phenotype. An important step forward has been taken with the use of computer technologies. Computer analysis of a photomicrograph of a transverse fold line of a leaf is proposed for quantitative evaluation of wheat leaf pubescence. The image-processing algorithm is implemented in the LHDetect2 software program accessible as a Web service at http://wheatdb.org/lhdetect2 . The results demonstrate that the proposed method is rapid, adequately assesses leaf pubescence density and the length distribution of trichomes and the data obtained using this method are significantly correlated with the density of trichomes on the leaf surface. Thus, the proposed method is efficient for high-throughput analysis of leaf pubescence morphology in cereal genetic collections and mapping populations.