Genome-wide identification of SAMS gene family in Cucurbitaceae and the role of ClSAMS1 in watermelon tolerance to abiotic stress.

S-Adenosyl-L-methionine (SAM) is widely involved in plant growth, development, and abiotic stress response. SAM synthetase (SAMS) is the key enzyme that catalyzes the synthesis of SAM from methionine and ATP. However, the SAMS gene family has not been identified and their functions have not been characterized in most Cucurbitaceae plants. Here, a total of 30 SAMS genes were identified in nine Cucurbitaceae species and they were categorized into 3 subfamilies. Physicochemical properties and gene structure analysis showed that the SAMS protein members are tightly conserved. Further analysis of the cis-regulatory elements (CREs) of SAMS genes' promoter implied their potential roles in stress tolerance. To further understand the molecular functions of SAMS genes, watermelon SAMSs (ClSAMSs) were chosen to analyze the expression patterns in different tissues and under various abiotic stress and hormone responses. Among the investigated genes, ClSAMS1 expression was observed in all tissues and found to be up-regulated by abiotic stresses including salt, cold and drought treatments as well as exogenous hormone treatments including ETH, SA, MeJA and ABA. Furthermore, knockdown of ClSAMS1 via virus-induced gene silencing (VIGS) decreased SAM contents in watermelon seedings. The pTRSV2-ClSAMS1 plants showed reduced susceptibility to drought, cold and NaCl stress, indicating a positive role of ClSAMS1 in abiotic stresses tolerance. Those results provided candidate SAMS genes to regulate plant resistance against abiotic stresses in Cucurbitaceae plants.

An ncRNA transcriptomics-based approach to design siRNA molecules against SARS-CoV-2 double membrane vesicle formation and accessory genes.

BACKGROUND: The corona virus SARS-CoV-2 is the causative agent of recent most global pandemic. Its genome encodes various proteins categorized as non-structural, accessory, and structural proteins. The non-structural proteins, NSP1-16, are located within the ORF1ab. The NSP3, 4, and 6 together are involved in formation of double membrane vesicle (DMV) in host Golgi apparatus. These vesicles provide anchorage to viral replicative complexes, thus assist replication inside the host cell. While the accessory genes coded by ORFs 3a, 3b, 6, 7a, 7b, 8a, 8b, 9b, 9c, and 10 contribute in cell entry, immunoevasion, and pathological progression. METHODS: This in silico study is focused on designing sequence specific siRNA molecules as a tool for silencing the non-structural and accessory genes of the virus. The gene sequences of NSP3, 4, and 6 along with ORF3a, 6, 7a, 8, and 10 were retrieved for conservation, phylogenetic, and sequence logo analyses. siRNA candidates were predicted using siDirect 2.0 targeting these genes. The GC content, melting temperatures, and various validation scores were calculated. Secondary structures of the guide strands and siRNA-target duplexes were predicted. Finally, tertiary structures were predicted and subjected to structural validations. RESULTS: This study revealed that NSP3, 4, and 6 and accessory genes ORF3a, 6, 7a, 8, and 10 have high levels of conservation across globally circulating SARS-CoV-2 strains. A total of 71 siRNA molecules were predicted against the selected genes. Following rigorous screening including binary validations and minimum free energies, final siRNAs with high therapeutic potential were identified, including 7, 2, and 1 against NSP3, NSP4, and NSP6, as well as 3, 1, 2, and 1 targeting ORF3a, ORF7a, ORF8, and ORF10, respectively. CONCLUSION: Our novel in silico pipeline integrates effective methods from previous studies to predict and validate siRNA molecules, having the potential to inhibit viral replication pathway in vitro. In total, this study identified 17 highly specific siRNA molecules targeting NSP3, 4, and 6 and accessory genes ORF3a, 7a, 8, and 10 of SARS-CoV-2, which might be used as an additional antiviral treatment option especially in the cases of life-threatening urgencies.

Transcriptome analysis reveals pathogenesis-related gene 1 pathway against salicylic acid treatment in grapevine (Vitis vinifera L).

Salicylic acid (SA) is a well-studied phenolic plant hormone that plays an important role in plant defense against the hemi-biothrophic and biothrophic pathogens and depends on the living cells of host for the successful infection. In this study, a pathogenesis test was performed between Vitis davidii and V. vinifera cultivars against grape white rot disease (Coniella diplodiella). V. davidii was found to be resistant against this disease. SA contents were found to be higher in the resistant grape cultivar after different time points. RNA-seq analysis was conducted on susceptible grapevine cultivars after 12, 24, and 48 h of SA application with the hypothesis that SA may induce defense genes in susceptible cultivars. A total of 511 differentially expressed genes (DEGs) were identified from the RNA-seq data, including some important genes, VvWRKY1/2, VvNPR1, VvTGA2, and VvPR1, for the SA defense pathway. DEGs related to phytohormone signal transduction and flavonoid biosynthetic pathways were also upregulated. The quantitative real-time PCR (qRT-PCR) results of the significantly expressed transcripts were found to be consistent with the transcriptome data, with a high correlation between the two analyses. The pathogenesis-related gene 1 (VvPR1), which is an important marker gene for plant defense, was selected for further promoter analysis. The promoter sequence showed that it contains some important cis-elements (W-box, LS7, as-1, and TCA-element) to recruit the transcription factors VvWRKY, VvNPR1, and VvTGA2 to express the VvPR1 gene in response to SA treatment. Furthermore, the VvPR1 promoter was serially deleted into different fragments (-1,837, -1,443, -1,119, -864, -558, -436, and -192 ) bp and constructed vectors with the GUS reporter gene. Deletion analysis revealed that the VvPR1 promoter between -1837 bp to -558 bp induced significant GUS expression with respect to the control. On the basis of these results, the -558 bp region was assumed to be an important part of the VvPR1 promoter, and this region contained the important cis-elements related to SA, such as TCA-element (-1,472 bp), LS7 (-1,428 bp), and as-1 (-520 bp), that recruit the TFs and induce the expression of the VvPR1 gene. This study expanded the available information regarding SA-induced defense in susceptible grapes and recognized the molecular mechanisms through which this defense might be mediated.

Integrated methylome and transcriptome analysis unravel the cold tolerance mechanism in winter rapeseed(Brassica napus L.).

BACKGROUND: Cytosine methylation, the main type of DNA methylation, regulates gene expression in plant response to environmental stress. The winter rapeseed has high economic and ecological value in China's Northwest, but the DNA methylation pattern of winter rapeseed during freezing stress remains unclear. RESULT: This study integrated the methylome and transcriptome to explore the genome-scale DNA methylation pattern and its regulated pathway of winter rapeseed, using freezing-sensitive (NF) and freezing-resistant (NS) cultivars.The average methylation level decreased under freezing stress, and the decline in NF was stronger than NS after freezing stress. The CG methylation level was the highest among the three contexts of CG, CHG, and CHH. At the same time, the CHH proportion was high, and the methylation levels were highest 2 kb up/downstream, followed by the intron region. The C sub-genomes methylation level was higher than the A sub-genomes. The methylation levels of chloroplast and mitochondrial DNA were much lower than the B. napus nuclear DNA, the SINE methylation level was highest among four types of transposable elements (TEs), and the preferred sequence of DNA methylation did not change after freezing stress. A total of 1732 differentially expressed genes associated with differentially methylated genes (DMEGs) were identified in two cultivars under 12 h and 24 h in three contexts by combining whole-genome bisulfite sequencing( and RNA-Seq data. Function enrichment analysis showed that most DMEGs participated in linoleic acid metabolism, alpha-linolenic acid metabolism, carbon fixation in photosynthetic organisms, flavonoid biosynthesis, and plant hormone signal transduction pathways. Meanwhile, some DMEGs encode core transcription factors in plant response to stress. CONCLUSION: Based on the findings of DNA methylation, the freezing tolerance of winter rapeseed is achieved by enhanced signal transduction, lower lipid peroxidation, stronger cell stability, increased osmolytes, and greater reactive oxygen species (ROS) scavenging. These results provide novel insights into better knowledge of the methylation regulation of tolerance mechanism in winter rapeseed under freezing stress.

Effect of Salinity Stress on Germination, Seedling Growth, Mineral Uptake and Chlorophyll Contents of Three Cucurbitaceae Species

Abstract This study was performed to screen out the various species of 'Cucurbitaceae' family, musk melon (Kalash and Durga), bottle gourd (Crystal Long and Nuefield) and squash (Green Round, and Squash Malika) against the salt stress. All genotypes were treated with five different levels of NaCl (T0 = control, T1 = 1.5 dS m-1, T2 = 3.0 dS m-1, T3 = 4.5 dS m-1 and T4 = 6.0 dS m-1) and half strength of Hoagland's nutrients solution as the base nutrient solution. Results showed that the bottle gourd varieties "Nuefield" and "Crystal Long" performed best by maintaining the highest germination (93.2% and 85.6%), number of leaves per plant (4.5 and 5.7), shoot length (16.84 cm and 16.14 cm), root length (13.48 cm and 13.00 cm), plant fresh weight (942.2 g and 918.6 g), plant dry weight (118.4 g and 107.5 g), leaf area (171.2 cm2 and 169.1 cm2), chlorophyll content (3.5 μg/cm-2 and 3.4 μg/cm-2) with low chloride (1.57 ppm and 1.59 ppm) and sodium content (0.47 ppm and 0.51 ppm) under salt stress followed by varieties of Squash (Green Round, and Squash Malika) and musk melon (Kalash and Durga). It was also found that a higher level of salinity (4.5 dS m-1 and 6.0 dS m-1) has more adverse effects on the performance of all selected genotypes. Conclusively, it can be recommended that as compared to all tested species, bottle gourd varieties "Nuefield" and "Crystal Long" have the ability to withstand against salinity stress and should be planted under salt stress conditions.

High-Throughput LC-ESI-MS/MS Metabolomics Approach Reveals Regulation of Metabolites Related to Diverse Functions in Mature Fruit of Grafted Watermelon.

Grafting has been reported as a factor regulating the metabolome of a plant. Therefore, a comprehensive metabolic profile and comparative analysis of metabolites were conducted from fully mature fruit of pumpkin-grafted watermelon (PGW) and a self-rooted watermelon (SRW). Widely targeted LC-ESI-MS/MS metabolomics approach facilitated the simultaneous identification and quantification of 339 metabolites across PGW and SRW. Regardless of grafting, delta-aminolevulinic acid hydrochloride, sucrose, mannose-6-phosphate (carbohydrates), homocystine, 2-phenylglycine, s-adenosyl-L-homocysteine (amino acids and derivatives), malic, azelaic, H-butanoic acid ethyl ester-hexoside isomer 1, (organic acids), MAG (18:3) isomer1, LysoPC 16:0, LysoPC 18:2 2n isomer (lipids) p-coumaric acid, piperidine, and salicylic acid-o-glycoside (secondary metabolites) were among the dominant metabolite. Dulcitol, mono-, and disaccharide sugars were higher in PGW, while polysaccharides showed complex behavior. In PGW, most aromatic and nitrogen-rich amino acids accumulated greater than 1.5- and 1-fold, respectively. Intermediates of the tricarboxylic acid cycle (TCA), stress-related metabolites, vitamin B5, and several flavonoids were significantly more abundant in PGW. Most lipids were also significantly higher in grafted watermelon. This is the first report providing a comprehensive picture of watermelon metabolic profile and changes induced by grafting. Hence, the untargeted high-throughput LC-ESI-MS/MS metabolomics approach could be suitable to provide significant differences in metabolite contents between grafted and ungrafted plants.

Identification of candidate genes influencing anthocyanin biosynthesis during the development and ripening of red and white strawberry fruits via comparative transcriptome analysis.

Strawberries are one of the most economically important berry fruits worldwide and exhibit colours ranging from white to dark red, providing a rich genetic resource for strawberry quality improvement. In the present study, we conducted transcriptome analyses of three strawberry cultivars, namely, 'Benihoppe', 'Xiaobai', and 'Snow White', and compared their gene expression profiles. Among the high-quality sequences, 5,049 and 53,200 differentially expressed genes (DEGs) were obtained when comparing the diploid and octoploid strawberry genomes and analysed to identify anthocyanin-related candidate genes. Sixty-five DEGs in the diploid genome (transcriptome data compared to the diploid strawberry genome) and 317 DEGs in the octoploid genome (transcriptome data compared to the octoploid strawberry genome) were identified among the three cultivars. Among these DEGs, 19 and 70 anthocyanin pathway genes, six and 42 sugar pathway genes, 23 and 101 hormone pathway genes, and 17 and 104 transcription factors in the diploid and octoploid genomes, respectively, correlated positively or negatively with the anthocyanin accumulation observed among the three cultivars. Real-time qPCR analysis of nine candidate genes showed a good correlation with the transcriptome data. For example, the expression of PAL was higher in 'Benihoppe' and 'Xiaobai' than in 'Snow White', consistent with the RNA-seq data. Thus, the RNA-seq data and candidate DEGs identified in the present study provide a sound basis for further studies of strawberry fruit colour formation.

Non-targeted metabolomics reveal the impact of phenanthrene stress on root exudates of ten urban greening tree species.

Different root exudations can modify the bioavailability of persistent organic pollutants (POPs). Among these exudations, the low molecular weight organic acids play an imperative role in this process. The study was conducted to analyze the effect of phenanthrene (PHE) stress on root exudation variations and changes in its chemical composition in ten urban greening tree species, namely Loropetalum chinense, Gardenia ellis, Photinia fraseri, Ligustrum japonicum, Rhododendron simsii, Osmanthus fragrans, Gardenia jasminoides, Buxus sinica, Camellia sasanqua, and Euonymus japonicas. The experiment was carried out in three PHE concentration treatments (0 mg kg-1 (CK), 200 mg kg-1 (PHEL), 2000 mg kg-1 (PHEH)). The root exudates were collected and analyzed by GC-MS method. In total, 673 compounds were identified either with high or low abundance among all species and treatments. Compounds identified in CK, PHEL, and PHEH were 240, 180, and 256, respectively. The results illustrated that carbohydrates, phenols, and esters were the dominant compounds, accounted for more than 92%. Principal component analysis depicted that tree species grown in PHEH showed obvious alteration in compounds of root exudation, whereas little difference was noticed between PHEL and CK. Phenols (80%) were the most abundant, while nitriles contributed a small portion. Moreover, among all species, R. simsii released the maximum number of compounds, and L. japonicum released the least number of compounds accounting for 89 and 46, respectively. The results achieved here to illustrate that plant type, and PHE stress can significantly change the concentrations and species of root exudates. This study provides the scientific reference for understanding the phenanthrene responsive changes in root exudates and phytoremediation of polycyclic aromatic hydrocarbons (PAHs), as well as a screening of urban greening tree species.

Comparative analysis of primary metabolites and transcriptome changes between ungrafted and pumpkin-grafted watermelon during fruit development.

Grafting has been reported as a factor that influences fruit quality. However, a comprehensive study of the metabolic profile related to fruit quality and the underlying molecular mechanism in grafted watermelon has not been carried out. Metabolomics and transcriptome analysis were performed on both pumpkin-grafted watermelon and ungrafted watermelon at different developmental stages. In total, 56 primary metabolites were identified with either high or low abundance between ungrafted and pumpkin-grafted watermelon. The results indicated that ornithine, arginine, lysine (amino acids), glucose, sucrose, glucosamine (sugars), malic acid, fumaric acid and succinic acid (organic acids) were among the dominant metabolites influencing fruit quality. Additionally, comparative RNA sequence analysis on grafted and ungrafted watermelon yielded 729, 174, 128 and 356 differentially expressed genes at 10, 18, 26 and 34 days after pollination (DAP), respectively. Functional annotations of these genes indicated that grafting significantly altered the biological and metabolic processes related to fruit quality. Our comparative metabolomics and transcriptome analysis revealed that FBA2, FK, SuSy, SPS, IAI, AI and sugar transporter gene (SWT3b) might play a central role in the accumulation of glucose and sucrose, whereas higher malic acid content was attributed to high down regulation of ALMT13 and ALMT8 in pumpkin-grafted watermelon. Changes in the ornithine, glutamine, alanine, tyrosine, valine, asparagine, phenylalanine, arginine and tryptophan contents were consistent with the transcript level of their metabolic genes such as NAOD, GS, AGT, TaT, aDH1, OGDH, aDC, 4CL 1, PaL, CaT and two nitrate transporter genes (NRT1) in pumpkin-grafted watermelon. This study provides the basis for understanding the graft-responsive changes in the metabolic profile and regulatory mechanism related to fruit quality.

BNCT induced immunomodulatory effects contribute to mammary tumor inhibition.

In the United States, breast cancer is one of the most common and the second leading cause of cancer-related death in women. Treatment modalities for mammary tumor are surgical removal of the tumor tissue followed by either chemotherapy or radiotherapy or both. Radiation therapy is a whole body irradiation regimen that suppresses the immune system leaving hosts susceptible to infection or secondary tumors. Boron neutron capture therapy (BNCT) in that regard is more selective, the cells that are mostly affected are those that are loaded with 109 or more 10B atoms. Previously, we have described that liposomal encapsulation of boron-rich compounds such as TAC and MAC deliver a high payload to the tumor tissue when injected intravenously. Here we report that liposome-mediated boron delivery to the tumor is inversely proportional to the size of the murine mammary (EMT-6) tumors. The plausible reason for the inverse ratio of boron and EMT-6 tumor size is the necrosis in these tumors, which is more prominent in the large tumors. The large tumors also have receding blood vessels contributing further to poor boron delivery to these tumors. We next report that the presence of boron in blood is essential for the effects of BNCT on EMT-6 tumor inhibition as direct injection of boron-rich liposomes did not provide any added advantage in inhibition of EMT-6 tumor in BALB/c mice following irradiation despite having a significantly higher amount of boron in the tumor tissue. BNCT reaction in PBMCs resulted in the modification of these cells to anti-tumor phenotype. In this study, we report the immunomodulatory effects of BNCT when boron-rich compounds are delivered systemically.

Construction of A High-Density Genetic Map and Mapping of Fruit Traits in Watermelon (Citrullus Lanatus L.) Based on Whole-Genome Resequencing.

Watermelon (Citrullus lanatus L.) is an important horticultural crop that is grown worldwide and has a high economic value. To dissect the loci associated with important horticultural traits and to analyze the genetic and genomic information of this species, a high-density genetic map was constructed based on whole-genome resequencing (WGR), a powerful high-resolution method for single-nucleotide polymorphism (SNP) marker development, genetic map construction, and gene mapping. Resequencing of both parental lines and 126 recombinant inbred lines (RIL) resulted in the detection of 178,762 single-nucleotide polymorphism (SNP) markers in the parental lines at a sequencing depth greater than four-fold. Additionally, 2132 recombination bin markers comprising 103,029 SNP markers were mapped onto 11 linkage groups (LGs). Substantially more SNP markers were mapped to the genetic map compared with other recent studies. The total length of the linkage map was 1508.94 cM, with an average distance of 0.74 cM between adjacent bin markers. Based on this genetic map, one locus for fruit bitterness, one locus for rind color, and one locus for seed coat color with high LOD scores (58.361, 18.353, 26.852) were identified on chromosome 1, chromosome 8, and chromosome 3, respectively. These prominent loci were identified in a region of 6.16 Mb, 2.07 Mb, and 0.37 Mb, respectively. On the basis of current research, the high-density map and mapping results will provide a valuable tool for identifying candidate genes, map-based gene cloning, comparative mapping, and marker-assisted selection (MAS) in watermelon breeding.

Vocal coselection in rat pup ultrasonic vocalizations.

Selective breeding and natural selection that select for one trait often bring along other correlated traits via coselection. Selective breeding for an infantile trait, high or low call rates of isolation-induced ultrasonic vocalization of rat pups, also alters functions of some brain systems and emotional behaviors throughout life. We examined the effect of breeding for call rate on acoustic parameters that are of communicative significance. Selecting for higher call rate produced calls of significantly increased amplitude and bandwidth relative to a randomly bred line. Selecting for lower rate produced calls of decreased duration. These nonmorphological, functional trait changes demonstrate enhanced communicatory potential and energy expenditure for the High line and the opposite for the Low line. This demonstration of coselection in a communicatory system suggests an underlying heritable suite of linked acoustic vocalization characteristics that in noisy environments could enhance dam-pup communication and lead to selection of emotionality traits with beneficial responses to stress.

Leonurus sibiricus L. (honeyweed): A review of its phytochemistry and pharmacology

Leonurus sibiricus is a herbaceous plant found in many countries in Asia and America. This plant is widely practiced as a remedy for the treatment of diabetes, menstrual irregularities, and bronchitis. The approval of therapeutic implications of any drugs depends on the well characterized mode of actions of the compounds. The bioactive compounds like diterpenes, triterpenes, flavonoids and phenolic acids in Leonurus sibiricus show analgesic, anti-inflammatory, anti-oxidant, anti-atherogenic and anti-hemorrhagic, anti-diabetic, anti-bacterial and allelopathic potency. Interestingly, the expression level of some genes is altered by the crude extract treatments, which are effective against cancers, diabetes and cardiovascular diseases where the molecular mechanisms are yet to be explored. Intriguingly, the extracts significantly induce nitric oxide production by endothelial nitric oxide synthase, a signaling molecule of vasodilation in combination with interferon-γ indicating positive effect on atherosclerosis. Further investigations are required to unlock the effects of bioactive compounds found in extracts at clinical settings.

IL-2 regulates SEB induced toxic shock syndrome in BALB/c mice.

BACKGROUND: Toxic Shock Syndrome (TSS) is characterized by fever, rash, hypotension, constitutional symptoms, and multi-organ involvement and is caused by Staphylococcus aureus enterotoxins such as Staphylococcal Enterotoxin B (SEB). SEB binds to the MHC-IIalpha chain and is recognized by the TCRbeta chain of the Vbeta8 TCR(+) T cells. The binding of SEB to Vbeta chain results in rapid activation of T cells and production of inflammatory cytokines, such as Interleukin-2 (IL-2), Interferon-gamma and Tumor Necrosis Factor-alpha which mediate TSS. Although IL2 was originally identified as the T cell growth factor and was proposed to contribute to T cell differentiation, its role in TSS remains unexplored. METHODOLOGY/PRINCIPAL FINDINGS: Mice were injected with D-Gal (25 mg/mouse). One hour after D-Galactosamine (D-Gal) injection each mouse was injected with SEB (20 microg/mouse. Mice were then observed for 72 hrs and death was recorded at different times. We tested Interleukin-12, IFNgamma, and IL-2 deficient mice (IL-2(-/-)), but only the IL-2 deficient mice were resistant to SEB induced toxic shock syndrome. More importantly reconstitution of IL-2 in IL-2 deficient mice restored the shock. Interestingly, SEB induced IL-2 production from T cells was dependent on p38MAPK activation in macrophages as inhibition of it in macrophages significantly inhibited IL-2 production from T cells. CONCLUSION: This study shows the importance of IL -2 in TSS which has not been previously explored and it also shows that regulating macrophages function can regulate T cells and TSS.

An assay for macrophage-mediated regulation of endothelial cell proliferation.

We have developed an assay that quantifies the potential of macrophages to regulate proliferation of endothelial cells. We show that young mice macrophages can be distinguished from old mice macrophages by their ability to inhibit vascular endothelial cell proliferation. While young mice macrophages robustly inhibit proliferation, old mice macrophages fail to do so and actually promote the proliferation of endothelial cells. In this report, we outline a technique that directly assesses the effect of macrophages on modulation of endothelial cell proliferation. This assay will help us in understanding the mechanisms of macrophage function in several disease states characterized by abnormal angiogenesis including cancers, angiogenic eye disease and atherosclerotic heart disease.

SEB-induced signaling in macrophages leads to biphasic TNF-alpha.

APCs express MHC-II molecules. Binding of enterotoxins to MHC-II generates a signal resulting in the production of TNF-alpha that mediates toxic shock syndrome. However, the signaling events that lead to TNF-alpha production in macrophages are not well understood. We, for the first time, demonstrate that binding of staphylococcal enterotoxin B to MHC-II results in activation of TNF-alpha-converting enzyme, epidermal growth factor receptor, p38MAPK, and NF-kappaB inducing biphasic TNF-alpha production. Paraformaldehyde-fixed, peptide-specific T cells also activate MHC-II signaling and TNF-alpha induction in peptide-pulsed macrophages. Our results reveal a novel MHC-II signaling and bidirectional macrophage-T cell interaction regulating macrophage functions. This knowledge may help to develop novel, macrophage-directed, therapeutic strategies.