Insights into the Transcriptional Reprogramming of Peach Leaves Inoculated with Taphrina deformans.

The dimorphic fungus Taphrina deformans is the causal agent of peach leaf curl disease, which affects leaves, flowers, and fruits. An RNA-seq approach was employed to gain insights into the transcriptional reprogramming of a peach cultivar during leaf inoculation with the yeast phase of the fungus across a compatible interaction. The results uncovered modulations of specific peach differentially expressed genes (DEGs) in peaches and pathways related to either the induction of host defense responses or pathogen colonization and disease spread. Expression profiles of DEGs were shown to be highly time-dependent and related to the presence of the two forms of the fungal growth, the inoculated yeast form and the later biotrophic phase during mycelial development. In parallel, this differential reprogramming was consistent with a diphasic detection of fungal load in the challenged leaves over the 120 h after inoculation (HAI) period. Leaf defense responses either occurred during the early yeast phase inoculation at 24 HAI, mediated primarily by cell wall modification processes, or more pronouncedly during the biotrophic phase at 72 HAI, as revealed by the activation of DEGs related to pathogen perception, signaling transduction, and secondary metabolism towards restraining further hypha proliferation. On the contrary, the expression patterns of specific DEGs at 120 HAI might further contribute to host susceptibility. These findings will further allow us to elucidate the molecular responses beyond the peach-T. deformans interaction.

Exploiting the beneficial effects of Salvia officinalis L. extracts in human health and assessing their activity as potent functional regulators of food microbiota.

Salvia officinalis L. has attracted scientific and industrial interest due to its pharmacological properties. However, its detailed phytochemical profile and its correlation with beneficial effects in the human microbiome and oxidative stress remained elusive. To unveil this, S. officinalis was collected from the region of Epirus and its molecular identity was verified with DNA barcoding. Phytochemical profile for both aqueous and ethanol-based extracts was determined by high-pressure liquid chromatography-tandem mass spectrometry and 103 phytochemicals were determined. The effect of S. officinalis extracts as functional regulators of food microbiota by stimulating the growth of Lacticaseibacillus rhamnosus strains and by suppressing evolution of pathogenic bacteria was verified. Furthermore, we recorded that both extracts exhibited a significant cellular protection against H2O2-induced DNA damage. Finally, both extracts exhibited strong inhibitory effect towards LDL oxidation. This study provides a comprehensive characterization of S. officinalis on its phytochemical components as also its potential impact in human microbiome and oxidative stress.

Molecular Characterization of Local Walnut (Juglans regia) Genotypes in the North-East Parnon Mountain Region of Greece.

Walnut is one of the most important nuts regarding their production and consumption. The available but uncharacterized genetic resources of walnut are important for the development and breeding of local varieties. Greece holds an important number of genetically uncharacterized walnut landraces, especially within the area of Parnon, which is considered to play a significant role as an in situ gene bank, due to its unique location traits. However, the genetic characterization and further use of these resources has been insufficient, due to the absence of genetic studies. In this study, we implemented SSR molecular markers, both to genetically characterize the walnut tree genetic diversity of the Parnon area and to identify its unique genetic structure, which will form the starting material for subsequent breeding programs. Overall, high levels of genetic variation were found among the individual walnut accessions that were collected in the Parnon mountain region.

Microalgae as a Sustainable Source of Antioxidants in Animal Nutrition, Health and Livestock Development.

Microalgae are a renewable and sustainable source of bioactive compounds, such as essential amino acids, polyunsaturated fatty acids, and antioxidant compounds, that have been documented to have beneficial effects on nutrition and health. Among these natural products, the demand for natural antioxidants, as an alternative to synthetic antioxidants, has increased. The antioxidant activity of microalgae significantly varies between species and depends on growth conditions. In the last decade, microalgae have been explored in livestock animals as feed additives with the aim of improving both animals' health and performance as well as product quality and the environmental impact of livestock. These findings are highly dependent on the composition of microalgae strain and their amount in the diet. The use of carbohydrate-active enzymes can increase nutrient bioavailability as a consequence of recalcitrant microalgae cell wall degradation, making it a promising strategy for monogastric nutrition for improving livestock productivity. The use of microalgae as an alternative to conventional feedstuffs is becoming increasingly important due to food-feed competition, land degradation, water deprivation, and climate change. However, the cost-effective production and use of microalgae is a major challenge in the near future, and their cultivation technology should be improved by reducing production costs, thus increasing profitability.

Stalactites Core Prospect as Environmental "Microbial Ark": The Actinomycetota Diversity Paradigm, First Reported from a Greek Cave.

Speleothems found in caves worldwide are considered the natural libraries of paleontology. Bacteria found in these ecosystems are generally limited to Proteobacteria and Actinomycetota, but rare microbiome and "Dark Matter" is generally under-investigated and often neglected. This research article discusses, for the first time to our knowledge, the diachronic diversity of Actinomycetota entrapped inside a cave stalactite. The planet's environmental microbial community profile of different eras can be stored in these refugia (speleothems). These speleothems could be an environmental "Microbial Ark" storing rare microbiome and "Dark Matter" bacterial communities evermore.

Genetic and Metabolite Variability among Commercial Varieties and Advanced Lines of Vicia faba L.

Vicia faba L. (faba bean) is one of the most promising pulse crops due to its nutritional value and high nitrogen fixation capacity. The aim of the present study was to compare the genetic diversity and the seed metabolite profiles of five genetic materials of faba bean. Specifically, three newly developed advanced lines (KK18, KK14 and KK10) and two commercial cultivars (POLIKARPI and TANAGRA), were evaluated for this purpose. Genetic diversity among populations was assessed by SCoT molecular markers. Through UPGMA dendrogram, genetic distances between populations were estimated. Untargeted metabolomics analysis of the seeds was performed employing GC/EI/MS. The cultivar POLYKARPI exhibited the highest polymorphism. All varieties showed a higher within-cultivars and advanced lines variability than between. POLYKARPI and KK14 had the lowest genetic distances, while KK18 and TANAGRA presented the highest ones. The advanced line KK18 displayed the best nutritional profile, the highest concentration of desirable metabolites (lactic acid and trehalose), the lowest concentration of anti-nutritional factors (oxalic acid) and the lowest concentration of saturated fatty acids (palmitic and stearic acid). According to the results of the present study, KK18 line is a very promising material for further exploration and utilization in breeding programs.

Seasonal Shifts in Soil Microbiome Structure Are Associated with the Cultivation of the Local Runner Bean Variety around the Lake Mikri Prespa.

Leguminous crops play a key role in food production and agroecosystem sustainability. However, climate change and agricultural intensification have a significant impact on the available arable land, soil microbiome functions, and ultimately, crop productivity. The "Prespa bean" (Phaseolous coccineous L.) is an important leguminous crop for the agricultural economy of the rural areas surrounding the lake, Mikri Prespa, which is of significant ecological importance. The seasonal effects on soil microbiome structure, diversity and functions associated with the runner bean cultivation were investigated using 16S rRNA amplicon sequencing. The results indicated that the presence of the runner bean differentially shaped the soil microbial community structure. The runner bean was implicated in the recruitment of specific bacteria, by favouring or excluding specific classes or even phyla. Soil functions involved in nutrient availability and carbon metabolism, among other pathways, were associated with microbiome-plant interactions. The temporal relative abundance shifts could be explained by the impact of soil organic matter, the fertilization regime, and the equilibrium in carbon metabolic processes. This research has shown the effect of runner bean cultivation on the soil microbiome which, in future, may potentially contribute to research into sustainable agricultural productivity and the protection of soil ecosystem services.

Diagnosis of Induced Resistance State in Tomato Using Artificial Neural Network Models Based on Supervised Self-Organizing Maps and Fluorescence Kinetics.

The aim of this study was to develop three supervised self-organizing map (SOM) models for the automatic recognition of a systemic resistance state in plants after application of a resistance inducer. The pathosystem Fusarium oxysporum f. sp. radicis-lycopersici (FORL) + tomato was used. The inorganic, defense inducer, Acibenzolar-S-methyl (benzo-[1,2,3]-thiadiazole-7-carbothioic acid-S-methyl ester, ASM), reported to induce expression of defense genes in tomato, was applied to activate the defense mechanisms in the plant. A handheld fluorometer, FluorPen FP 100-MAX-LM by SCI, was used to assess the fluorescence kinetics response of the induced resistance in tomato plants. To achieve recognition of resistance induction, three models of supervised SOMs, namely SKN, XY-F, and CPANN, were used to classify fluorescence kinetics data, in order to determine the induced resistance condition in tomato plants. To achieve this, a parameterization of fluorescence kinetics curves was developed corresponding to fluorometer variables of the Kautsky Curves. SKN was the best supervised SOM, achieving 97.22% to 100% accuracy. Gene expression data were used to confirm the accuracy of the supervised SOMs.

Barcoding High Resolution Melting (Bar-HRM) enables the discrimination between toxic plants and edible vegetables prior to consumption and after digestion.

The consumption of poisonous plants can lead to serious health problems or even casualties due to various factors, including easy access to poisonous plants due to their common distribution, co-occurrence and resemblance with edible plants, and the lack of regulation in the food product supply chain. Clinical diagnosis of intoxications usually relies on the availability of the plant consumed by the patient and on the morphology of the plant parts found in the patient's stomach. Therefore, given the fragmented nature of ingested plant material, species identification may face serious difficulties, can be inaccurate, and time-consuming. This highlights the need for rapid and reliable tools to identify toxic species. In the present study, we developed an ITS2-high-resolution melting (HRM) assay for: (1) the discrimination of common toxic plants and their edible lookalikes, and (2) the detection of toxic plants in digested samples. More specifically, we designed species-specific ITS2 primers for the authentication of poisonous species in simulated mixtures and verified them with Bar-HRM. Moreover, the developed HRM-based molecular tool was capable of quantifying the toxic species Datura stramonium in simulated mixtures with the edible Amaranthus retroflexus down to at least 0.5% v/v. This study shows that species-specific ITS2 primers can amplify the DNA from fragmented and/or artificially digested samples and that Bar-HRM is capable of detecting poisonous plant species in digested samples even after 4 h. The developed Bar-HRM protocol has important implications for application in medicine, forensics, and the agricultural industry, either to accurately detect the cause of plant intoxications or as a tool for quality control in the supply chain. PRACTICAL APPLICATION: In this work, we established a high-resolution melting DNA-based protocol capable of discriminating between phenotypically similar common toxic and edible plant species in mixtures, even at very low quantities. This technology also proved efficient in detecting the toxic species in mixtures digested in artificial gastric acid, as it would be the case after accidental ingestion. This work is expected to have important implications for application in medicine, forensics, and the agricultural industry, either for identifying the cause of plant intoxications or as a tool for quality control in different steps of the supply chain.

Environmental DNA detection of giant snakehead in Thailand's major rivers for wild stock assessment.

Capture-based aquaculture is now gaining much attention in Southeast Asia. This system was used to produce several fish species with social and economic implications, including the giant snakehead (Channa micropeltes). As wild harvesting of organisms for seed stock is one of main practices in capture-based aquaculture, abundance and distribution of the wild stock are essential for both environmental impact evaluation and stock management. Mark and recapture, visual observation and physical capture of target species are costly, ineffective, and labour intensive for fish surveys in several cases. Detection of target organisms using eDNA (environmental DNA) could be a good alternative as it has proved to be a non-invasive, rapid, and sensitive method for aquatic species monitoring and surveying. Here, we developed a TaqMan assay that targets the 16S region of giant snakehead DNA to amplify eDNA captured in water samples. 300 µl of water samples were collected from 15 sites located in the Chao Phraya River Basin (Ping, Wang, Yom, Nan, and Chao Phraya River) and filtered with 0.7 µm glass fibre membrane filter. Giant snakehead eDNA was detected in most tributaries (60%) with concentrations ranging from 74.0 copies/ml in Wang River sites to 7.4 copies/ml in Nan River sites. As intensification of capture-based aquaculture could lead to depleting of wild fish stocks, urgent management is needed. However, the existing conventional approaches for assessment of fish overexploitation, survey and monitoring have several limitations.

DNA-Based Identification of Eurasian Vicia Species Using Chloroplast and Nuclear DNA Barcodes.

Many legume species of the Vicia L. genus (Fabaceae Lindl.) are key components of the Mediterranean diet and have an integral role in sustainable agriculture. Given the importance of the Vicia species for Eurasian culture, it is necessary to implement methodologies, such as DNA barcoding, that can enable the effective authentication and identification of species in the genus. In this study, we analysed the chloroplast trnL and rpoC1, as well as the nuclear ITS2 DNA barcoding regions, to identify 71 Vicia specimens of Eurasian descent. Both the trnL and ITS2 regions were highly effective in discriminating the analysed taxa, while the more conserved rpoC1 region could not identify all of the selected species due to high sequence conservation or non-annotated or absent rpoC1 species sequences in GenBank. A dendrographic representation of the generated trnL data showed sufficient clustering for most of the analysed taxa, although some topological discrepancies were observed. ITS2 and rpoC1 reconstructions were also used for resolving the topological discrepancies observed in the trnL tree. Our analysis suggests that a combination of DNA barcoding regions is essential for accurate species discrimination within the Vicia genus, while single-locus analyses do not provide the necessary resolution.

The perennial fruit tree proteogenomics atlas: a spatial map of the sweet cherry proteome and transcriptome.

Genome-wide transcriptome analysis provides systems-level insights into plant biology. Due to the limited depth of quantitative proteomics our understanding of gene-protein-complex stoichiometry is largely unknown in plants. Recently, the complexity of the proteome and its cell-/tissue-specific distribution have boosted the research community to the integration of transcriptomics and proteomics landscapes in a proteogenomic approach. Herein, we generated a quantitative proteome and transcriptome abundance atlas of 15 major sweet cherry (Prunus avium L., cv 'Tragana Edessis') tissues represented by 29 247 genes and 7584 proteins. Additionally, 199 984 alternative splicing events, particularly exon skipping and alternative 3' splicing, were identified in 23 383 transcribed regions of the analyzed tissues. Common signatures as well as differences between mRNA and protein quantities, including genes encoding transcription factors and allergens, within and across the different tissues are reported. Using our integrated dataset, we identified key putative regulators of fruit development, notably genes involved in the biosynthesis of anthocyanins and flavonoids. We also provide proteogenomic-based evidence for the involvement of ethylene signaling and pectin degradation in cherry fruit ripening. Moreover, clusters of genes and proteins with similar and different expression and suppression trends across diverse tissues and developmental stages revealed a relatively low RNA abundance-to-protein correlation. The present proteogenomic analysis allows us to identify 17 novel sweet cherry proteins without prior protein-level annotation evidenced in the currently available databases. To facilitate use by the community, we also developed the Sweet Cherry Atlas Database (https://grcherrydb.com/) for viewing and data mining these resources. This work provides new insights into the proteogenomics workflow in plants and a rich knowledge resource for future investigation of gene and protein functions in Prunus species.

ITS Metabarcoding Reveals the Effects of Oregano Essential Oil on Fusarium oxysporum and Other Fungal Species in Soil Samples.

The destructive effects of Fusarium wilts are known to affect the production of many crops. The control of Fusarium oxysporum and other soilborne pathogens was mainly based on soil fumigation (methyl bromide), which has long been prohibited and, nowadays, is based on a limited number of available fungicides due to legislation restrictions on residue tolerances and environmental impacts. Alternatively, natural and environmentally safe compounds, such as essential oils, are being investigated for their efficacy in the control of soilborne diseases. The great fungicidal ability of the oregano essential oil components (carvacrol and thymol) has been reported to inhibit the germination and the mycelial development of several fungal species, including F. oxysporum. The aim of our study was to demonstrate how the metabarcoding approach can provide valuable information about the positive or negative impacts of two different doses of oregano essential oil on Fusarium oxysporum and other fungal species which were present in the studied soil samples through the amplification of the ITS1 and ITS2 regions, which were analyzed on a MiSeq platform. A higher dose of oregano essential oil decreased the abundance of F. oxysporum, along with other fungal species, but also had negative effects on Trichoderma evansii and Mortierella chlamydospora, species with possible fungicidal properties. Soil properties, essential oil properties, the fungal composition, and interactions between fungal species should be considered as factors influencing the effectiveness of essential oils as biological control agents for soilborne pathogens.

Biosolid-Amended Soil Enhances Defense Responses in Tomato Based on Metagenomic Profile and Expression of Pathogenesis-Related Genes.

Biosolid application is an effective strategy, alternative to synthetic chemicals, for enhancing plant growth and performance and improving soil properties. In previous research, biosolid application has shown promising results with respect to tomato resistance against Fusarium oxysporum f. sp. radicis-lycopersici (Forl). Herein, we aimed at elucidating the effect of biosolid application on the plant-microbiome response mechanisms for tomato resistance against Forl at a molecular level. More specifically, plant-microbiome interactions in the presence of biosolid application and the biocontrol mechanism against Forl in tomato were investigated. We examined whether biosolids application in vitro could act as an inhibitor of growth and sporulation of Forl. The effect of biosolid application on the biocontrol of Forl was investigated based on the enhanced plant resistance, measured as expression of pathogen-response genes, and pathogen suppression in the context of soil microbiome diversity, abundance, and predicted functions. The expression of the pathogen-response genes was variably induced in tomato plants in different time points between 12 and 72 h post inoculation in the biosolid-enriched treatments, in the presence or absence of pathogens, indicating activation of defense responses in the plant. This further suggests that biosolid application resulted in a successful priming of tomato plants inducing resistance mechanisms against Forl. Our results have also demonstrated that biosolid application alters microbial diversity and the predicted soil functioning, along with the relative abundance of specific phyla and classes, as a proxy for disease suppression. Overall, the use of biosolid as a sustainable soil amendment had positive effects not only on plant health and protection, but also on growth of non-pathogenic antagonistic microorganisms against Forl in the tomato rhizosphere and thus, on plant-soil microbiome interactions, toward biocontrol of Forl.

Metataxonomic Analysis of Bacteria Entrapped in a Stalactite's Core and Their Possible Environmental Origins.

Much is known about microbes originally identified in caves, but little is known about the entrapment of microbes (bacteria) in stalactites and their possible environmental origins. This study presents data regarding the significant environmental distribution of prokaryotic bacterial taxa of a Greek stalactite core. We investigated the involvement of those bacteria communities in stalactites using a metataxonomic analysis approach of partial 16S rRNA genes. The metataxonomic analysis of stalactite core material revealed an exceptionally broad ecological spectrum of bacteria classified as members of Proteobacteria, Actinobacteria, Firmicutes, Verrucomicrobia, and other unclassified bacteria. We concluded that (i) the bacterial transport process is possible through water movement from the upper ground cave environment, forming cave speleothems such as stalactites, (ii) bacterial genera such as Polaromonas, Thioprofundum, and phylum Verrucomicrobia trapped inside the stalactite support the paleoecology, paleomicrobiology, and paleoclimate variations, (iii) the entrapment of certain bacteria taxa associated with water, soil, animals, and plants such as Micrococcales, Propionibacteriales, Acidimicrobiales, Pseudonocardiales, and α-, ß-, and γ-Proteobacteria.

Exploring plant diversity through soil DNA in Thai national parks for influencing land reform and agriculture planning.

BACKGROUND: The severe deforestation, as indicated in national forest data, is a recurring problem in many areas of Northern Thailand, including Doi Suthep-Pui National Park. Agricultural expansion in these areas, is one of the major drivers of deforestation, having adverse consequences on local plant biodiversity. Conserving biodiversity is mainly dependent on the biological monitoring of species distribution and population sizes. However, the existing conventional approaches for monitoring biodiversity are rather limited. METHODS: Here, we explored soil DNA at four forest types in Doi Suthep-Pui National Park in Northern Thailand. Three soil samples, composed of different soil cores mixed together, per sampling location were collected. Soil biodiversity was investigated through eDNA metabarcoding analysis using primers targeting the P6 loop of the plastid DNA trnL (UAA) intron. RESULTS: The distribution of taxa for each sample was found to be similar between replicates. A strong congruence between the conventional morphology- and eDNA-based data of plant diversity in the studied areas was observed. All species recorded by conventional survey with DNA data deposited in the GenBank were detected through the eDNA analysis. Moreover, traces of crops, such as lettuce, maize, wheat and soybean, which were not expected and were not visually detected in the forest area, were identified. It is noteworthy that neighboring land and areas in the studied National Park were once used for crop cultivation, and even to date there is still agricultural land within a 5-10 km radius from the forest sites where the soil samples were collected. The presence of cultivated area near the forest may suggest that we are now facing agricultural intensification leading to deforestation. Land reform for agriculture usage necessitates coordinated planning in order to preserve the forest area. In that context, the eDNA-based data would be useful for influencing policies and management towards this goal.

Systems biology reveals key tissue-specific metabolic and transcriptional signatures involved in the response of Medicago truncatula plant genotypes to salt stress.

Salt stress is an important factor limiting plant productivity by affecting plant physiology and metabolism. To explore salt tolerance adaptive mechanisms in the model legume Medicago truncatula, we used three genotypes with differential salt-sensitivity: TN6.18 (highly sensitive), Jemalong A17 (moderately sensitive), and TN1.11 (tolerant). Cellular damage was monitored in roots and leaves 48 h after 200 mM NaCl treatment by measuring lipid peroxidation, nitric oxide, and hydrogen peroxide contents, further supported by leaf stomatal conductance and chlorophyll readings. The salt-tolerant genotype TN1.11 displayed the lowest level of oxidative damage, in contrast to the salt sensitive TN6.18, which showed the highest responses. Metabolite profiling was employed to explore the differential genotype-related responses to stress at the molecular level. The metabolic data in the salt tolerant TN1.11 roots revealed an accumulation of metabolites related to the raffinose pathway. To further investigate the sensitivity to salinity, global transcriptomic profiling using microarray analysis was carried out on the salt-stressed sensitive genotypes. In TN6.18, the transcriptomic analysis identified a lower expression of many genes related to stress signalling, not previously linked to salinity, and corresponding to the TIR-NBS-LRR gene class. Overall, this global approach contributes to gaining significant new insights into the complexity of stress adaptive mechanisms and to the identification of potential targets for crop improvement.

Marker-Free Transplastomic Plants by Excision of Plastid Marker Genes Using Directly Repeated DNA Sequences.

Excision of marker genes using DNA direct repeats makes use of the efficient native homologous recombination pathway present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and green algae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes (plastomes). Release of selection allows the accumulation of marker-free plastomes generated by marker excision, which is a spontaneous and unidirectional process. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastomes during growth, development and flowering of T0 plants allows for the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The procedure enables precise plastome engineering involving insertion of transgenes, point mutations and deletion of genes without the inclusion of any extraneous DNA. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.

Characterization of the Genetic Diversity Present in a Diverse Sesame Landrace Collection Based on Phenotypic Traits and EST-SSR Markers Coupled With an HRM Analysis.

A selection of sesame (Sesamum indicum L.) landraces of different eco-geographical origin and breeding history have been characterized using 28 qualitative morpho-physiological descriptors and seven expressed sequence tag-simple sequence repeat (EST-SSR) markers coupled with a high-resolution melting (HRM) analysis. The most variable qualitative traits that could efficiently discriminate landraces, as revealed by the correlation analyses, were the plant growth type and position of the branches, leaf blade width, stem pubescence, flowering initiation, capsule traits and seed coat texture. The agglomerative hierarchical clustering analysis based on a dissimilarity matrix highlighted three main groups among the sesame landraces. An EST-SSR marker analysis revealed an average polymorphism information content (PIC) value of 0.82, which indicated that the selected markers were highly polymorphic. A principal coordinate analysis and dendrogram reconstruction based on the molecular data classified the sesame genotypes into four major clades. Both the morpho-physiological and molecular analyses showed that landraces from the same geographical origin were not always grouped in the same cluster, forming heterotic groups; however, clustering patterns were observed for the Greek landraces. The selective breeding of such traits could be employed to unlock the bottleneck of local phenotypic diversity and create new cultivars with desirable traits.

Overexpression of A Biotic Stress-Inducible Pvgstu Gene Activates Early Protective Responses in Tobacco under Combined Heat and Drought.

Drought and heat stresses are major factors limiting crop growth and productivity, and their effect is more devastating when occurring concurrently. Plant glutathione transferases (GSTs) are differentially expressed in response to different stimuli, conferring tolerance to a wide range of abiotic stresses. GSTs from drought-tolerant Phaseolus vulgaris var. "Plake Megalosperma Prespon" is expected to play an important role in the response mechanisms to combined and single heat and drought stresses. Herein, we examined wild-type N. tabacum plants (cv. Basmas Xanthi) and T1 transgenic lines overexpressing the stress-induced Pvgstu3-3 and Pvgstu2-2 genes. The overexpression of Pvgstu3-3 contributed to potential thermotolerance and greater plant performance under combined stress. Significant alterations in the primary metabolism were observed in the transgenic plants between combined stress and stress-free conditions. Stress-responsive differentially expressed genes (DEGs) and transcription factors (TFs) related to photosynthesis, signal transduction, starch and sucrose metabolism, osmotic adjustment and thermotolerance, were identified under combined stress. In contrast, induction of certain DEGs and TF families under stress-free conditions indicated that transgenic plants were in a primed state. The overexpression of the Pvgstu3-3 is playing a leading role in the production of signaling molecules, induction of specific metabolites and activation of the protective mechanisms for enhanced protection against combined abiotic stresses in tobacco.