Novel candidate genes for environmental stresses response in Synechocystis sp. PCC 6803 revealed by machine learning algorithms.

Cyanobacteria have developed acclimation strategies to adapt to harsh environments, making them a model organism. Understanding the molecular mechanisms of tolerance to abiotic stresses can help elucidate how cells change their gene expression patterns in response to stress. Recent advances in sequencing techniques and bioinformatics analysis methods have led to the discovery of many genes involved in stress response in organisms. The Synechocystis sp. PCC 6803 is a suitable microorganism for studying transcriptome response under environmental stress. Therefore, for the first time, we employed two effective feature selection techniques namely and support vector machine recursive feature elimination (SVM-RFE) and LASSO (Least Absolute Shrinkage Selector Operator) to pinpoint the crucial genes responsive to environmental stresses in Synechocystis sp. PCC 6803. We applied these algorithms of machine learning to analyze the transcriptomic data of Synechocystis sp. PCC 6803 under distinct conditions, encompassing light, salt and iron stress conditions. Seven candidate genes namely sll1862, slr0650, sll0760, slr0091, ssl3044, slr1285, and slr1687 were selected by both LASSO and SVM-RFE algorithms. RNA-seq analysis was performed to validate the efficiency of our feature selection approach in selecting the most important genes. The RNA-seq analysis revealed significantly high expression for five genes namely sll1862, slr1687, ssl3044, slr1285, and slr0650 under ion stress condition. Among these five genes, ssl3044 and slr0650 could be introduced as new potential candidate genes for further confirmatory genetic studies, to determine their roles in their response to abiotic stresses.

Co-overexpression of chitinase and ß-1,3-glucanase significantly enhanced the resistance of Iranian wheat cultivars to Fusarium.

Fusarium head blight (FHB) is a devastating fungal disease affecting different cereals, particularly wheat, and poses a serious threat to global wheat production. Chitinases and ß-glucanases are two important proteins involved in lysing fungal cell walls by targeting essential macromolecular components, including chitin and ß-glucan micro fibrils. In our experiment, a transgenic wheat (Triticum aestivum) was generated by introducing chitinase and glucanase genes using Biolistic technique and Recombinant pBI121 plasmid (pBI-ChiGlu (-)). This plasmid contained chitinase and glucanase genes as well as nptII gene as a selectable marker. The expression of chitinase and glucanase was individually controlled by CaMV35S promoter and Nos terminator. Immature embryo explants from five Iranian cultivars (Arta, Moghan, Sisun, Gascogen and A-Line) were excised from seeds and cultured on callus induction medium to generate embryonic calluses. Embryogenic calluses with light cream color and brittle texture were selected and bombarded using gold nanoparticles coated with the recombinant pBI-ChiGlu plasmid. Bombarded calluses initially were transferred to selective callus induction medium, and later, they were transfferd to selective regeneration medium. The selective agent was kanamycin at a concentration of 25 mg/l in both media. Among five studied cultivars, A-Line showed the highest transformation percentage (4.8%), followed by the Sisun, Gascogen and Arta in descending order. PCR and Southern blot analysis confirmed the integration of genes into the genome of wheat cultivars. Furthermore, in an in-vitro assay, the growth of Fusarium graminearum was significantly inhibited by using 200 µg of leaf protein extract from transgenic plants. According to our results, the transgenic plants (T1) showed the resistance against Fusarium when were compared to the non-transgenic plants. All transgenic plants showed normal fertility and no abnormal response was observed in their growth and development.

Cadmium toxicity promotes hormonal imbalance and induces the expression of genes involved in systemic resistances in barley.

Cadmium (Cd) is a widely distributed pollutant that adversely affects plants' metabolism and productivity. Phytohormones play a vital role in the acclimation of plants to metal stress. On the other hand, phytohormones trigger systemic resistances, including systemic acquired resistance (SAR) and induced systemic resistance (ISR), in plants in response to biotic interactions. The present study aimed to investigate the possible induction of SAR and ISR pathways in relation to the hormonal alteration of barley seedlings in response to Cd stress. Barley seedlings were exposed to 1.5 mg g-1 Cd in the soil for three days. The nutrient content, oxidative status, phytohormones profile, and expression of genes involved in SAR and ISR pathways of barley seedlings were examined. Cd accumulation resulted in a reduction in the nutrient content of barley seedlings. The specific activity of superoxide dismutase and the hydrogen peroxide content significantly increased in response to Cd toxicity. Abscisic acid, jasmonic acid, and ethylene content increased under Cd exposure. Cd treatment resulted in the upregulation of NPR1, PR3, and PR13 genes in SAR pathways. The transcripts of PAL1 and LOX2.2 genes in the ISR pathway were also significantly increased in response to Cd treatment. These findings suggest that hormonal-activated systemic resistances are involved in the response of barley to Cd stress.

Identification of responsive genes to multiple abiotic stresses in rice (Oryza sativa): a meta-analysis of transcriptomics data.

Abiotic stresses limit the quantity and quality of rice grain production, which is considered a strategic crop in many countries. In this study, a meta-analysis of different microarray data at seedling stage was performed to investigate the effects of multiple abiotic stresses (drought, salinity, cold situation, high temperature, alkali condition, iron, aluminum, and heavy metal toxicity, nitrogen, phosphorus, and potassium deficiency) on rice. Comparative analysis between multiple abiotic stress groups and their control groups indicated 561 differentially expressed genes (DEGs), among which 422 and 139 genes were up-regulated and down-regulated, respectively. Gene Ontology analysis showed that the process of responding to stresses and stimuli was significantly enriched. In addition, pathways such as metabolic process and biosynthesis of secondary metabolites were identified by KEGG pathway analysis. Weighted correlation network analysis (WGCNA) uncovered 17 distinct co-expression modules. Six modules were significantly associated with genes involved in response to abiotic stresses. Finally, to validate the results of the meta-analysis, five genes, including TIFY9 (JAZ5), RAB16B, ADF3, Os01g0124650, and Os05g0142900 selected for qRT-PCR analysis. Expression patterns of selected genes confirmed the results of the meta-analysis. The outcome of this study could help introduce candidate genes that may be beneficial for use in genetic engineering programs to produce more tolerant crops or as markers for selection.

Differential gene expression (DGE) analysis in persons with a history of giardiasis.

Giardiasis, which is caused by Giardia duodenalis, has clinical symptoms such as steatorrhea and can be very dangerous in children. In addition, some documents reported that this parasite is present inside the tissue of patients with cancer. In this study, we analyzed the gene expression profiles of some main genes important to apoptosis and anti-apoptosis in humans.Expression profile arrays of Genomic Spatial Event (GSE) 113666, GSE113667, and GSE113679 obtained from Gene Expression Omnibus were used for meta-analysis using R commands. Cytoscape and STRING databases used the protein-protein Interaction network. Then, the Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analysis was performed. Similar genes in Homo sapiens were identified using Basic Local Alignment Search Tool analysis. The validation was performed on eight people using real-time Polymerase chain reaction. In addition to the candidate genes, the gene expression of some other genes, including Serine/Threonine Kinase 1 (AKT1), Cyclin Dependent Kinase Inhibitor 2A (CDKN2A), Kirsten Rat Sarcoma (KRAS), and Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA) were also examined. Analysis of the expression of serum amyloid A1 (SAA1), Regenerating Islet-Derived 3 Gamma (REG3G), and REG3A genes did not show any difference between the two groups of healthy and diseased people. Examining the mean expression of the four genes AKT1, CDKN2A, KRAS, and PIK3CA showed that three genes of AKT1, CDKN2A, and KRAS had increased expression in people with a history of giardiasis compared to healthy people. We showed that the gene expression pattern differs in apoptosis and anti-apoptosis signaling in people with a history of giardiasis. Giardia duodenalis seems to induce post-non-infectious symptoms with stimulation of human gene expression.

Evaluation of antibacterial properties of nisin peptide expressed in carrots.

Nisin, derived from Lactococcus lactis, is a well-known natural food preservative. In the present study, the gene of nisin was transformed to carrot by Agrobacterium tumefaciens strain LBA4404 harboring the recombinant binary vector pBI121 containing neomycin phosphotransferase II (nptII) gene, peptide signal KDEL, and Kozak sequence. The integration of nisin and nptII transgenes into the plant genome was confirmed by polymerase chain reaction (PCR) and dot blot analysis. The gene expression was also performed by RT-PCR and Enzyme-Linked Immunosorbent Assay. The level of nisin expressed in one gram of transgenic plant ranged from 0.05 to 0.08 µg/ml. The stability of nisin varied in orange and peach juices depending on the temperature on the 70th day. The leaf protein extracted from the transgenic plant showed a significant preservative effect of nisin in peach and orange juice. A complete inhibition activity against Staphylococcus aureus and Escherichia coli in orange juice was observed within 24 h. After 24 h, log 1 and log 2 were obtained in a peach juice containing Staphylococcus aureus and Escherichia coli, respectively. Results of HPLC indicated that Chlorogenic and Chicoric acid compounds were increased in transgenic plants, but this increase was not significant. The study of determining the genetic stability of transgenic plants in comparison with non-transgenic plants showed high genetic stability between non-transgenic plants and transgenic plants. This study confirmed the significant inhibitory effect of nisin protein on gram-positive and gram-negative bacteria.

Enhancing petunia tissue culture efficiency with machine learning: A pathway to improved callogenesis.

The important feature of petunia in tissue culture is its unpredictable and genotype-dependent callogenesis, posing challenges for efficient regeneration and biotechnology applications. To address this issue, machine learning (ML) can be considered a powerful tool to analyze callogenesis data, extract key parameters, and predict optimal conditions for petunia callogenesis, facilitating more controlled and productive tissue culture processes. The study aimed to develop a predictive model for callogenesis in petunia using ML algorithms and to optimize the concentrations of phytohormones to enhance callus formation rate (CFR) and callus fresh weight (CFW). The inputs for the model were BAP, KIN, IBA, and NAA, while the outputs were CFR and CFW. Three ML algorithms, namely MLP, RBF, and GRNN, were compared, and the results revealed that GRNN (R2≥83) outperformed MLP and RBF in terms of accuracy. Furthermore, a sensitivity analysis was conducted to determine the relative importance of the four phytohormones. IBA exhibited the highest importance, followed by NAA, BAP, and KIN. Leveraging the superior performance of the GRNN model, a genetic algorithm (GA) was integrated to optimize the concentration of phytohormones for maximizing CFR and CFW. The genetic algorithm identified an optimized combination of phytohormones consisting of 1.31 mg/L BAP, 1.02 mg/L KIN, 1.44 mg/L NAA, and 1.70 mg/L IBA, resulting in 95.83% CFR. To validate the reliability of the predicted results, optimized combinations of phytohormones were tested in a laboratory experiment. The results of the validation experiment indicated no significant difference between the experimental and optimized results obtained through the GA. This study presents a novel approach combining ML, sensitivity analysis, and GA for modeling and predicting callogenesis in petunia. The findings offer valuable insights into the optimization of phytohormone concentrations, facilitating improved callus formation and potential applications in plant tissue culture and genetic engineering.

Advanced stage, high-grade primary tumor ovarian cancer: a multi-omics dissection and biomarker prediction process.

Ovarian cancer (OC) incidence and mortality rates continue to escalate globally. Early detection of OC is challenging due to extensive metastases and the ambiguity of biomarkers in advanced High-Grade Primary Tumors (HGPTs). In the present study, we conducted an in-depth in silico analysis in OC cell lines using the Gene Expression Omnibus (GEO) microarray dataset with 53 HGPT and 10 normal samples. Differentially-Expressed Genes (DEGs) were also identified by GEO2r. A variety of analyses, including gene set enrichment analysis (GSEA), ChIP enrichment analysis (ChEA), eXpression2Kinases (X2K) and Human Protein Atlas (HPA), elucidated signaling pathways, transcription factors (TFs), kinases, and proteome, respectively. Protein-Protein Interaction (PPI) networks were generated using STRING and Cytoscape, in which co-expression and hub genes were pinpointed by the cytoHubba plug-in. Validity of DEG analysis was achieved via Gene Expression Profiling Interactive Analysis (GEPIA). Of note, KIAA0101, RAD51AP1, FAM83D, CEP55, PRC1, CKS2, CDCA5, NUSAP1, ECT2, and TRIP13 were found as top 10 hub genes; SIN3A, VDR, TCF7L2, NFYA, and FOXM1 were detected as predominant TFs in HGPTs; CEP55, PRC1, CKS2, CDCA5, and NUSAP1 were identified as potential biomarkers from hub gene clustering. Further analysis indicated hsa-miR-215-5p, hsa-miR-193b-3p, and hsa-miR-192-5p as key miRNAs targeting HGPT genes. Collectively, our findings spotlighted HGPT-associated genes, TFs, miRNAs, and pathways as prospective biomarkers, offering new avenues for OC diagnostic and therapeutic approaches.

Anticancer effects of cell-free culture supernatant of Escherichia coli in bladder cancer cell line: New insight into the regulation of inflammation.

Bladder cancer (BC) is the 10th most common malignancy in worldwide, with substantial mortality and morbidity if not treated effectively. According to various research, inflammatory circumstances majorly impact the microenvironment of bladder cancer, and the chronic presence of cytokines and chemokines promotes tumor progression. In this investigation, we explored the impact of cell-free culture supernatant ofEscherichia colistrain 536 on inflammatory cytokines and chemokines in bladder cancer model microarray data (GSE162251). Then we examined in silico outcomes on human bladder cancer cell line 5637 to verify and extrapolate findings. This investigation revealed for the first time that this compound has potent suppressor effects on interleukin 1 beta (IL-1ß), C-C motif chemokine ligand 2 (CCL2), and C-X3-C motif chemokine ligand 1 (CX3CL1) gene expression as well as increased NAD(P)H quinone dehydrogenase 1 (NQO1), as an anti-oxidant agent, gene expression in 4, 8, and 24 h. Moreover, we confirmed that c-MYC, a member of the MYC proto-oncogene family, gene expression reduced in 5637 cells in 4 h and then followed up its expression in 8 and 24 h. In addition, our investigation demonstrated that the supernatant raised the BCL2-Associated X Protein/B-cell lymphoma 2 (BAX/BCL2) ratio, and subsequent flow cytometry analysis demonstrated that the supernatant induction apoptosis and necrosis. In conclusion, our findings demonstrate that this compound is a potential candidate for the suppression of bladder cancer progression.

Comprehensive RNA-Seq-based study and metabolite profiling to identify genes involved in podophyllotoxin biosynthesis in Linum album Kotschy ex Boiss. (Linaceae).

Linum album is a well-known rich source of anticancer compounds, i.e., podophyllotoxin (PTOX) and other lignans. These compounds play an important role in the plant's defensive system. The RNA-Seq data of flax (L. usitatissimum) were analyzed under various biotic and abiotic stresses to comprehend better the importance of lignans in plant defense responses. Then, the association between the lignan contents and some related gene expressions was experimented with HPLC and qRT-PCR, respectively. Transcriptomic profiling showed a specific expression pattern in different organs, and just the commonly regulated gene EP3 was detected with a significant increase under all stresses. The in silico analysis of the PTOX biosynthesis pathway identified a list of genes, including laccase (LAC11), lactoperoxidase (POD), 4-coumarate-CoA ligase (4CL), and secoisolariciresinol dehydrogenase (SDH). These genes increased significantly under individual stresses. The HPLC analysis showed that the measured lignan contents generally increased under stress. In contrast, a quantitative expression of the genes involved in this pathway using qRT-PCR showed a different pattern that seems to contribute to regulating PTOX content in response to stress. Identified modifications of critical genes related to PTOX biosynthesis in response to multiple stresses can provide a baseline for improving PTOX content in L. album.

Comparative analysis of different artificial neural networks for predicting and optimizing in vitro seed germination and sterilization of petunia.

The process of optimizing in vitro seed sterilization and germination is a complicated task since this process is influenced by interactions of many factors (e.g., genotype, disinfectants, pH of the media, temperature, light, immersion time). This study investigated the role of various types and concentrations of disinfectants (i.e., NaOCl, Ca(ClO)2, HgCl2, H2O2, NWCN-Fe, MWCNT) as well as immersion time in successful in vitro seed sterilization and germination of petunia. Also, the utility of three artificial neural networks (ANNs) (e.g., multilayer perceptron (MLP), radial basis function (RBF), and generalized regression neural network (GRNN)) as modeling tools were evaluated to analyze the effect of disinfectants and immersion time on in vitro seed sterilization and germination. Moreover, non­dominated sorting genetic algorithm­II (NSGA­II) was employed for optimizing the selected prediction model. The GRNN algorithm displayed superior predictive accuracy in comparison to MLP and RBF models. Also, the results showed that NSGA­II can be considered as a reliable multi-objective optimization algorithm for finding the optimal level of disinfectants and immersion time to simultaneously minimize contamination rate and maximize germination percentage. Generally, GRNN-NSGA-II as an up-to-date and reliable computational tool can be applied in future plant in vitro culture studies.

Transcriptome profiling of barley in response to mineral and organic fertilizers.

BACKGROUND: Nitrogen is very important for crop yield and quality. Crop producers face the challenge of reducing the use of mineral nitrogen while maintaining food security and other ecosystem services. The first step towards understanding the metabolic responses that could be used to improve nitrogen use efficiency is to identify the genes that are up- or downregulated under treatment with different forms and rates of nitrogen. We conducted a transcriptome analysis of barley (Hordeum vulgare L.) cv. Anni grown in a field experiment in 2019. The objective was to compare the effects of organic (cattle manure) and mineral nitrogen (NH4NO3; 0, 40, 80 kg N ha-1) fertilizers on gene activity at anthesis (BBCH60) and to associate the genes that were differentially expressed between treatment groups with metabolic pathways and biological functions. RESULTS: The highest number of differentially expressed genes (8071) was found for the treatment with the highest mineral nitrogen rate. This number was 2.6 times higher than that for the group treated with a low nitrogen rate. The lowest number (500) was for the manure treatment group. Upregulated pathways in the mineral fertilizer treatment groups included biosynthesis of amino acids and ribosomal pathways. Downregulated pathways included starch and sucrose metabolism when mineral nitrogen was supplied at lower rates and carotenoid biosynthesis and phosphatidylinositol signaling at higher mineral nitrogen rates. The organic treatment group had the highest number of downregulated genes, with phenylpropanoid biosynthesis being the most significantly enriched pathway for these genes. Genes involved in starch and sucrose metabolism and plant-pathogen interaction pathways were enriched in the organic treatment group compared with the control treatment group receiving no nitrogen input. CONCLUSION: These findings indicate stronger responses of genes to mineral fertilizers, probably because the slow and gradual decomposition of organic fertilizers means that less nitrogen is provided. These data contribute to our understanding of the genetic regulation of barley growth under field conditions. Identification of pathways affected by different nitrogen rates and forms under field conditions could help in the development of more sustainable cropping practices and guide breeders to create varieties with low nitrogen input requirements.

Modeling and optimizing in vitro percentage and speed callus induction of carrot via Multilayer Perceptron-Single point discrete GA and radial basis function.

BACKGROUND: Callus induction is the first step in optimizing plant regeneration. Fit embryogenesis and shooting rely on callus induction. In addition, using artificial intelligence models in combination with an algorithm can be helpful in the optimization of in vitro culture. The present study aimed to evaluate the percentage and speed of callus induction optimization in carrot with a Multilayer Perceptron-Single point discrete genetic algorithm (GA). MATERIALS AND METHODS: In this study, the outputs included callus induction percentage and speed, while inputs were different types and concentrations of plant growth regulator (0. 5, 0.2 mg/l 2,4-D, 0.3, 0.2, 0.5 mg/l BAP, 1, 0.2 mg/l Kin, and 2 mg/l NAA), different explants (shoot, root, leaf, and nodal), a different concentration compound of MS medium (1 × MS, 4× MS, and 8× MS) and time of sampling. The data were obtained in the laboratory, and multilayer perceptron (MLP) and radial basis function (RBF), two well-known ANNs, were employed to model. Then, GA was used for optimization, and sensitivity analysis was performed to indicate the inputs' importance. RESULTS: The results showed that MLP had better prediction efficiency than RBF. Based on the results, R2 in training and testing data was 95 and 95% for the percentage of callus induction, while it was 94 and 95% for the speed of callus induction, respectively. In addition, a concentration compound of MS had high sensitivity, while times of sampling had low sensitivity. Based on the MLP-Single point discrete GA, the best results were obtained for shoot explants, 1× MS media, and 0.5 mg/l 2, 4-D + 0.5 mg/l BAP. Further, a non-significant difference was observed between the test result and predicted MLP. CONCLUSIONS: Generally, MLP-Single point discrete GA is considered a potent tool for predicting treatment and fit model results used in plant tissue culture and selecting the best medium for callus induction.

Metabolomics analysis of milk thistle lipids to identify drought-tolerant genes.

Milk thistle is an oil and medicinal crop known as an alternative oil crop with a high level of unsaturated fatty acids, which makes it a favorable edible oil for use in food production. To evaluate the importance of Milk thistle lipids in drought tolerance, an experiment was performed in field conditions under three different water deficit levels (Field capacity (FC), 70% FC and 40% FC). After harvesting seeds of the plant, their oily and methanolic extracts were isolated, and subsequently, types and amounts of lipids were measured using GC-MS. Genes and enzymes engaged in biosynthesizing of these lipids were identified and their expression in Arabidopsis was investigated under similar conditions. The results showed that content of almost all measured lipids of milk thistle decreased under severe drought stress, but genes (belonged to Arabidopsis), which were involved in their biosynthetic pathway showed different expression patterns. Genes biosynthesizing lipids, which had significant amounts were selected and their gene and metabolic network were established. Two networks were correlated, and for each pathway, their lipids and respective biosynthesizing genes were grouped together. Four up-regulated genes including PXG3, LOX2, CYP710A1, PAL and 4 down-regulated genes including FATA2, CYP86A1, LACS3, PLA2-ALPHA were selected. The expression of these eight genes in milk thistle was similar to Arabidopsis under drought stress. Thus, PXG3, PAL, LOX2 and CYP86A1 genes that increased expression were selected for protein analysis. Due to the lack of protein structure of these genes in the milk thistle, modeling homology was performed for them. The results of molecular docking showed that the four proteins CYP86A1, LOX2, PAL and PXG3 bind to ligands HEM, 11O, ACT and LIG, respectively. HEM ligand was involved in production of secondary metabolites and dehydration tolerance, and HEM binding site remained conserved in various plants. CA ligands were involved in synthesis of cuticles and waxes. Overall, this study confirmed the importance of lipids in drought stress tolerance in milk thistle.

RNA-Seq Analysis of Magnaporthe grisea Transcriptome Reveals the High Potential of ZnO Nanoparticles as a Nanofungicide.

Magnaporthe grisea is one of the most destructive pathogen that encounters a challenge to rice production around the worldwide. The unique properties of ZnO nanoparticles (NPs), have high attractiveness as nanofungicide. In the present study, the response of fungi to ZnO NPs was evaluated using RNA sequencing (RNA-seq). Two different aligners (STAR and Hisat2) were used for aligning the clean reads, and the DEseq2 package was used to identify the differentially expressed genes (DEGs). In total, 1,438 and 761 fungal genes were significantly up- and down-regulated in response to ZnO NPs, respectively. The DEGs were subjected to functional enrichment analysis to identify significantly enriched biological pathways. Functional enrichment analysis revealed that "cell membrane components," "ion (calcium) transmembrane transporter activity," "steroid biosynthesis pathway" and "catalytic activity" were the contributed terms to fungal response mechanisms. The genes involved in aflatoxin efflux pumps and ribosome maturation were among the genes showing significant up- and down-regulation after ZnO NPs application. To confirm the obtained RNA-seq results, the expression of six randomly selected genes were evaluated using q-RT-PCR. Overall, the RNA-seq results suggest that ZnO NPs primarily act on the fungal cell membrane, but accumulation of ROS inside the cell induces oxidative stress, the fungal catalytic system is disrupted, resulting into the inhibition of ROS scavenging and eventually, to the death of fungal cells. Our findings provide novel insights into the effect of ZnO NPs as a promising nanofungicide for effective control of rice blast disease.

Transgenic tobacco co-expressing flavodoxin and betaine aldehyde dehydrogenase confers cadmium tolerance through boosting antioxidant capacity.

Excessive heavy metal (HM) levels in soil have become a source of concern due to their adverse effects on human health and the agriculture industry. Soil contamination by HMs leads to an accumulation of reactive oxygen species (ROSs) within the plant cell and disruption of photosynthesis-related proteins. The response of tobacco lines overexpressing flavodoxin (Fld) and betaine aldehyde dehydrogenase (BADH) to cadmium (Cd) toxicity was investigated in this study. PCR results demonstrated the expected amplicon length of each gene in the transgenic lines. Absolute qRT-PCR demonstrates a single copy of T-DNA integration into each transgenic line. Relative qRT-PCR confirmed overexpression of Fld and BADH in transgenic lines. The maximum quantum yield of photosystem II (Fv/Fm) was measured under Cd toxicity stress and revealed that transgenic lines had a higher Fv/Fm than wild-type (WT) plants. Accumulation of proline, glycine betaine (GB), and higher activity of antioxidant enzymes alongside lower levels of malondialdehyde (MDA) and hydrogen peroxide (H2O2) was indicative of a robust antioxidant system in transgenic plants. Therefore, performing a loop in reducing the ROS produced in the photosynthesis electron transport chain and stimulating the ROS scavenger enzyme activity improved the plant tolerance to Cd stress.

Molecular and morphological evaluation of transgenic Persian walnut plants harboring Fld gene under osmotic stress condition.

BACKGROUND: Soil drought stress is a limiting factor of productivity in walnut (Juglans regia L). Ferredoxin (Fd) level decreases under adverse environmental stress. Functional replacement of decreased Fd by Fld (Flavodoxin) had been shown to have protective effect under abiotic stress condition. This study aimed to evaluate four transgenic lines (L3, L4, L13 and L17) along with non-transgenic line under three osmotic stresses levels (0, 10 and 12% PEG). METHODS AND RESULTS: This experiment carried out based on a completely randomized design with four replications. To confirm that the Fld gene is successfully integrated into the walnut genome, PCR and dot blot analysis were carried out. The transgenic lines of walnut expressing Fld displayed increased tolerance to osmotic stress at 10 and 12% PEG condition. Lines expressing Fld exhibited increasing tolerance to drought stress and maintained health of plants under osmotic conditions. Results of real time PCR showed that expression level of Fld gene in L4 was higher than the others. Among transgenic lines, L4 was more tolerant than other lines under osmotic stress. CONCLUSIONS: These findings indicate that expression of Fld gene can increase tolerance to osmotic stress in Persian walnut and is useful tool for walnut production in arid and semi-arid regions.

Co-Expression Network Analysis of Soybean Transcriptome Identify Hub Genes Under Saline-Alkaline and Water Deficit Stress.

Background: Soybean is an important oilseed crop that its development and production are affected by environmental stresses (such as saline-alkaline and water deficit). Objectives: This experiment was performed with the aim of identifying candidate genes in saline-alkaline stress and water-deficit stress conditions using transcriptome analysis and to investigate the expression of these genes under water deficit stress conditions using RTqPCR. Materials and Methods: In this experiment, soybean transcriptome data under saline-alkaline and water-deficit stress were downloaded from the NCBI website, and then the co-expression modules were determined for them and the gene network was plotted for each module, and finally, the hub genes were identified. To compare the expression of genes in saline-alkaline and water deficit conditions, soybean plants were subjected to water deficit stress and their gene expression was determined using RTqPCR. Results: The filtered (Log FC above +2 and below -2) genes of soybean were grouped under saline-alkaline stress in 15 modules and under water-deficit stress in 2 different modules. Within each module, the interaction of genes was identified using the gene network, then three genes of ann11, cyp450 and zfp selected as hub genes. These hub genes are highly co-expression with other network genes, which not only display differential expression but also differential co-expression. The results of RT-PCR indicated that cyp450 gene expression was not significantly different from the control, while ann11 gene expression significantly increased under water deficit stress, but zfp gene expression decreased significantly under water deficit stress. Conclusions: We identified three genes, ann11, cyp450 and zfp, as hub genes. According to our results, ann11 gene had a significant increase in expression under water deficit stress, which can indicate the importance of this gene under drought conditions. Therefore, according to the results of this experiment as well as other researchers, we introduce this gene as a key gene in water deficit tolerance and recommend its use in genetic engineering to increase the tolerance of other plants.

Identification of the key functional genes in salt-stress tolerance of Cyanobacterium Phormidium tenue using in silico analysis.

The development of artificial biocrust using cyanobacterium Phormidium tenue has been suggested as an effective strategy to prevent soil degradation. Here, a combination of in silico approaches with growth rate, photosynthetic pigment, morphology, and transcript analysis was used to identify specific genes and their protein products in response to 500 mM NaCl in P. tenue. The results show that 500 mM NaCl induces the expression of genes encoding glycerol-3-phosphate dehydrogenase (glpD) as a Flavoprotein, ribosomal protein S12 methylthiotransferase (rimO), and a hypothetical protein (sll0939). The constructed co-expression network revealed a group of abiotic stress-responsive genes. Using the Basic Local Alignment Search Tool (BLAST), the homologous proteins of rimO, glpD, and sll0939 were identified in the P. tenue genome. Encoded proteins of glpD, rimO, and DUF1622 genes, respectively, contain (DAO and DAO C), (UPF0004, Radical SAM and TRAM 2), and (DUF1622) domains. The predicted ligand included 22B and MG for DUF1622, FS5 for rimO, and FAD for glpD protein. There was no direct disruption in ligand-binding sites of these proteins by Na+, Cl-, or NaCl. The growth rate, photosynthetic pigment, and morphology of P. tenue were investigated, and the result showed an acceptable tolerance rate of this microorganism under salt stress. The quantitative real-time polymerase chain reaction (qRT-PCR) results revealed the up-regulation of glpD, rimO, and DUF1622 genes under salt stress. This is the first report on computational and experimental analyses of the glpD, rimO, and DUF1622 genes in P. tenue under salt stress to the best of our knowledge. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-03050-w.

Plant regeneration and transformation of Trachyspermum ammi using Agrobacterium tumefaciens and zygotic embryos.

BACKGROUND: Trachyspermum ammi is one of the key medicinal plant species with many beneficial properties. Thymol is the most important substance in the essential oil of this plant. Thymol is a natural monoterpene phenol with high anti-microbial, anti-bacterial, and anti-oxidant properties. Thymol in the latest research has a significant impact on slowing the progression of cancer cells in human. In this research, embryos were employed as convenient explants for the fast and effectual regeneration and transformation of T. ammi. To regenerate this plant, Murashige and Skoog (MS) and Gamborg's B5 (B5) media were supplemented with diverse concentrations of plant growth regulators, such as 6-benzyladenine (BA), 1-naphthaleneacetic acid (NAA), 2,4-dichlorophenoxyacetic acid (2,4-D), and kinetin (kin). Transgenic Trachyspermum ammi plants were also obtained using Agrobacterium-mediated transformation and zygotic embryos explants. Moreover, two Agrobacterium tumefaciens strains (EHA101 and LBA4404) harboring pBI121-TPS2 were utilized for genetic transformation to Trachyspermum ammi. RESULTS: According to the obtained results, the highest plant-regeneration frequency was obtained with B5 medium supplemented with 0.5 mg/l BA and 1 mg/l NAA. The integrated gene was also approved using the PCR reaction and the Southern blot method. Results also showed that the EHA101 strain outperformed another strain in inoculation time (30 s) and co-cultivation period (1 day) (transformation efficiency 19.29%). Furthermore, HPLC method demonstrated that the transformed plants contained a higher thymol level than non-transformed plants. CONCLUSIONS: In this research, a fast protocol was introduced for the regeneration and transformation of Trachyspermum ammi, using zygotic embryo explants in 25-35 days. Our findings confirmed the increase in the thymol in the aerial part of Trachyspermum ammi. We further presented an efficacious technique for enhancing thymol content in Trachyspermum ammi using Agrobacterium-mediated plant transformation system that can be beneficial in genetic transformation and other plant biotechnology techniques.