The Characterization of Sex Differences in Hypoglycemia-Induced Activation of HPA Axis on the Transcriptomic Level.

Activation of the hypothalamic-pituitary-adrenal (HPA) axis using an insulin tolerance test (ITT) is a medical diagnostic procedure that is frequently used in humans to assess the HPA and growth-hormone (GH) axes. Whether sex differences exist in the response to ITT stress is unknown. Thus, investigations into the analysis of transcripts during activation of the HPA axis in response to hypoglycemia have revealed the underlying influences of sex in signaling pathways that stimulate the HPA axis. We assessed four time points of ITT application in Balb/c mice. After insulin injection, expression levels of 192 microRNAs and 41 mRNAs associated with the HPA, GH and hypothalamic-pituitary-gonadal (HPG) axes were determined by real-time RT-PCR in the hypothalamus, pituitary and adrenal tissues, as well as blood samples (Raw data accession: https://drive.google.com/drive/folders/10qI00NAtjxOepcNKxSJnQbJeBFa6zgHK?usp=sharing ). Although the ITT is commonly used as a gold standard for evaluating the HPA axis, we found completely different responses between males and females with respect to activation of the HPA axis. While activation of several transcripts in the hypothalamus and pituitary was observed after performing the ITT in males within 10 min, females responded via the pituitary and adrenal immediately and durably over 40 min. Additionally, we found that microRNA alterations precede mRNA responses in the HPA axis. Furthermore, robust changes in the levels of several transcripts including Avpr1b and Avpr2 observed at all time points strongly suggest that transcriptional control of these genes occurs mostly via differential signaling in pituitary and blood between males and females. Male and female HPA axis responses to ITT involve a number of sophisticated regulatory signaling pathways of miRNAs and mRNAs. Our results highlight the first robust markers in several layers of HPA, HPG and GH axis involved in ITT/hypoglycemia stress-induced dynamics.

Enhanced glutathione content improves lateral root development and grain yield in rice plants.

KEY MESSAGE: Enhanced glutathione content improves lateral root development by positively regulating the transcripts of root development genes responsive to glutathione treatment, thereby increasing the overall productivity of rice plants. Glutathione is primarily known as a cellular antioxidant molecule, but its role in lateral root development in rice plants has not been elucidated. Here, we have investigated its role in lateral root development of rice Oryza sativa L. Exogenous glutathione (GSH) promoted both the number and length of lateral roots in rice, and the GSH biosynthesis inhibitor buthionine sulfoximine (BSO) significantly reduced these parameters, compared to untreated plants. The inhibition by BSO was reversed with exogenous GSH. Transcript profiling by RNA-seq revealed that expression of the transcription factor genes DREB and ERF and the hormone-related genes AOS, LOX, JAZ, and SAUR were significantly downregulated in the BSO-treated plants and, in contrast, upregulated in plants treated with GSH and with GSH and BSO together. We generated OsGS-overexpressing transgenic plants in which the transgene is controlled by the abiotic-stress-inducible OsRab21 promoter to study the effect of endogenously increased GSH levels. In cold stress, transgenic rice plants enhanced stress tolerance and lateral root development by maintaining redox homeostasis and improving upregulating the expression of transcription factors and hormone-related genes involved in lateral root development. We observed improved root growth of OsGS-overexpressing plants in paddy fields compared to the wild-type controls. These traits may have alleviated transplanting stress during early growth in the field and accounted for the increased productivity. These results provide information and perspectives on the role of GSH in gene expression, lateral root development, and grain yield in rice.

Cucumber ovaries inhibited by dominant fruit express a dynamic developmental program, distinct from either senescence-determined or fruit-setting ovaries.

Cucurbits represent an attractive model to explore the dynamics of fruit set, whose regulation is not fully understood, despite its importance for yield determination. A fertilized ovary must integrate signals from distant plant parts and 'decide' whether to set fruit, or remain inhibited and later senesce. Here, we set out to characterize first-fruit inhibition (FFI), that is, the inhibitory effect of the first fruit on subsequent development of younger ovaries during pollination-induced and parthenocarpic fruit set. After the first fertilized ovaries set fruit, younger fertilized ovaries remained in a temporary state of inhibition. Such ovaries preserved their size and green color, and if the older fruit were removed within a 1-week reversibility window, they set fruit. The FFI effect was documented in both fertilized and parthenocarpic ovaries. We compared the gene expression profiles of pollinated ovaries (committed to set fruit) with respect to those affected by FFI, and to non-pollinated ovaries (undergoing senescence). The three fates of the ovaries were characterized by wide changes in gene expression, with several specific transcripts being up- or down-regulated in response to pollination, and to the presence of inhibitory fruit. Metabolic profiling was undertaken and integrated with the transcriptomic data in order to characterize early physiological changes that occur in post-anthesis ovaries in parthenocarpic and non-parthenocarpic genotypes. The combined results are discussed with respect to current models of fruit set and specifically with regard to FFI. Moreover, these metabolome and transcriptome data provide a valuable resource for studying ovary development and fruit set.

Intervention of pumpkin seed oil on metabolic disease revealed by metabonomics and transcript profile.

BACKGROUND: Understanding the metabolic and transcription basis of pumpkin seed oil (PSO) intervention on metabolic disease (MD) is essential to daily nutrition and health. RESULTS: This study analyzed the liver metabolic variations of Wistar rats fed normal diet (CON), high-fat diet (HFD) and high-fat plus PSO diet (PSO) to establish the relationship between the liver metabolite composition/transcript profile and the effects of PSO on MD. By using proton nuclear magnetic resonance spectroscopy together with multivariate data analysis, it was found that, compared with CON rats, HFD rats showed clear dysfunctions of choline metabolism, glucose metabolism and nucleotide and amino acid metabolism. Using quantitative real-time polymerase chain reaction (qPCR), it was found that, compared with HFD rats, PSO rats showed alleviated endoplasmic reticulum stress accompanied by lowered unfolded protein response. CONCLUSION: These findings provide useful information to understand the metabolic alterations triggered by MD and to evaluate the effects of PSO intervention. © 2016 Society of Chemical Industry.

Molecular Approaches to Genetically Improve the Accumulation of Health-Promoting Secondary Metabolites in Staple Crops-A Case Study: The Lipoxygenase-B1 Genes and Regulation of the Carotenoid Content in Pasta Products.

Secondary metabolites, also known as phytochemicals, represent a large subset of plant molecules that include compounds with health-promoting effects. Indeed, a number of epidemiological studies have shown that, when taken regularly and in adequate amounts, these molecules can have long-term beneficial effects on human health, through reduction of the incidence of degenerative diseases, such as cardiovascular diseases, obesity, diabetes, and cancer. As the dietary intake of these phytochemicals is often inadequate, various strategies are in use to improve their content in staple crops, and the end-products thereof. One of the most effective strategies is crop improvement through genetic approaches, as this is the only way to generate new cultivars in which the high accumulation of a given phytochemical is stably fixed. Efforts to genetically improve quality traits are rapidly evolving, from classical breeding to molecular-assisted approaches; these require sound understanding of the molecular bases underlying the traits, to identify the genes/alleles that control them. This can be achieved through global analysis of the metabolic pathway responsible for phytochemical accumulation, to identify the link between phytochemical content and the activities of key enzymes that regulate the metabolic pathway, and between the key enzymes and their encoding genes/alleles. Once these have been identified, they can be used as markers for selection of new improved genotypes through biotechnological approaches. This review provides an overview of the major health-promoting properties shown to be associated with the dietary intake of phytochemicals, and describes how molecular approaches provide means for improving the health quality of edible crops. Finally, a case study is illustrated, of the identification in durum wheat of the Lipoxygenase-B1 genes that control the final carotenoid content in semolina-based foods, such as pasta products.

Skin mucus proteome map of European sea bass (Dicentrarchus labrax).

Skin mucus is the first barrier of fish defence. Proteins from skin mucus of European sea bass (Dicentrarchus labrax) were identified by 2DE followed by LC-MS/MS. From all the identified proteins in the proteome map, we focus on the proteins associated with several immune pathways in fish. Furthermore, the real-time PCR transcript levels in skin are shown. Proteins found include apolipoprotein A1, calmodulin, complement C3, fucose-binding lectin, lysozyme and several caspases. To our knowledge, this is the first skin mucus proteome study and further transcriptional profiling of the identified proteins done on this bony fish species. This not only contributes knowledge on the routes involved in mucosal innate immunity, but also establishes a non-invasive technique based on locating immune markers with a potential use for prevention and/or diagnosis of fish diseases.

Type 1 diacylglycerol acyltransferases of Brassica napus preferentially incorporate oleic acid into triacylglycerol.

DGAT1 enzymes (acyl-CoA:diacylglycerol acyltransferase 1, EC 2.3.1.20) catalyse the formation of triacylglycerols (TAGs), the most abundant lipids in vegetable oils. Thorough understanding of the enzymology of oil accumulation is critical to the goal of modifying oilseeds for improved vegetable oil production. Four isoforms of BnDGAT1, the final and rate-limiting step in triacylglycerol synthesis, were characterized from Brassica napus, one of the world's most important oilseed crops. Transcriptional profiling of developing B. napus seeds indicated two genes, BnDGAT1-1 and BnDGAT1-2, with high expression and two, BnDGAT1-3 and BnDGAT1-4, with low expression. The activities of each BnDGAT1 isozyme were characterized following expression in a strain of yeast deficient in TAG synthesis. TAG from B. napus seeds contain only 10% palmitic acid (16:0) at the sn-3 position, so it was surprising that all four BnDGAT1 isozymes exhibited strong (4- to 7-fold) specificity for 16:0 over oleic acid (18:1) as the acyl-CoA substrate. However, the ratio of 18:1-CoA to 16:0-CoA in B. napus seeds during the peak period of TAG synthesis is 3:1. When substrate selectivity assays were conducted with 18:1-CoA and 16:0-CoA in a 3:1 ratio, the four isozymes incorporated 18:1 in amounts 2- to 5-fold higher than 16:0. This strong sensitivity of the BnDGAT1 isozymes to the relative concentrations of acyl-CoA substrates substantially explains the observed fatty acid composition of B. napus seed oil. Understanding these enzymes that are critical for triacylglycerol synthesis will facilitate genetic and biotechnological manipulations to improve this oilseed crop.

Transcript profiles in cortical cells of maize primary root during ethylene-induced lysigenous aerenchyma formation under aerobic conditions.

BACKGROUND AND AIMS: Internal aeration is important for plants to survive during periods of waterlogging, and the ability to form aerenchyma contributes by creating a continuous gas space between the shoots and the roots. Roots of maize (Zea mays) react to prolonged waterlogging by forming aerenchyma in root cortical cells by programmed cell death (PCD) in response to ethylene. The aim of this study was to understand the molecular mechanisms of ethylene-induced aerenchyma formation by identifying genes that are either up- or downregulated by ethylene treatment in maize root cortical cells. METHODS: Three-day-old maize seedlings were treated with ethylene for several hours under aerobic conditions. Cortical cells were isolated from the primary roots using laser microdissection (LM), and transcript profiles with and without ethylene treatment were compared by microarray. In addition, the effect on ethylene-induced aerenchyma formation of diphenyleneiodonium (DPI), an inhibitor of NADPH oxidases, was examined in order to assess the involvement of reactive oxygen species (ROS). KEY RESULTS: A total of 223 genes were identified whose transcript levels were significantly increased or decreased by ethylene treatment in root cortical cells under aerobic conditions. Subsequent tissue-specific quantitative reverse-transcription PCR analyses revealed that ethylene increased the transcript levels of genes related to ethylene signalling in all of the root tissues examined (stelar cells, cortical cells and outer cell layers), whereas it increased the transcript levels of genes related to cell wall modification and proteolysis specifically in the cortical cells. DPI treatment inhibited the ethylene-induced aerenchyma formation and suppressed expression of some cell wall modification-related genes. CONCLUSIONS: Several genes related to cell wall modification and proteolysis are specifically up- or downregulated in cortical cells during lysigenous aerenchyma formation under aerobic conditions with ethylene treatment. The results suggest that ethylene is perceived in stelar cells, cortical cells and outer cell layers in the maize primary root, and that the cortical cell-specific PCD is controlled downstream of ethylene perception through subsequent gene expression, which is partly regulated by ROS, in the cortical cells.

Unraveling the drought-responsive transcriptomes in nodules of two common bean genotypes during biological nitrogen fixation.

Common bean (Phaseolus vulgaris) can efficiently fix atmospheric nitrogen when associated with Rhizobia. However, drought stress impairs plant metabolic processes, especially the biological nitrogen fixation (BNF). Here, we assessed transcriptional responses in nodules of two common bean genotypes to drought stress under BNF reliance. The RNA-Seq analysis yielded a total of 81,489,262 and 72,497,478 high quality reads for Negro Argel and BAT 477 genotypes, respectively. The reads were mapped to the Phaseolus vulgaris reference genome and expression analysis identified 145 and 1451 differentially expressed genes (DEGs) for Negro Argel and BAT 477 genotypes, respectively. Although BAT 477 had more DEGs, both genotypes shared certain drought-responsive genes, including an up-regulated heat shock protein (HSP) and a down-regulated peroxidase, indicating shared pathways activated during drought in nodule tissue. Functional analysis using MapMan software highlighted the up-regulation of genes involved in abiotic stress responses, such as HSPs and specific transcription factors (TFs), in both genotypes. There was a significant down-regulation in metabolic pathways related to antioxidant protection, hormone signaling, metabolism, and transcriptional regulation. To validate these findings, we conducted RT-qPCR experiments for ten DEGs in nodules from both genotypes, for which the expression profile was confirmed, thus reinforcing their functional relevance in the nodule responses to drought stress during BNF. BAT 477 genotype exhibited more pronounced response to drought, characterized by a high number of DEGs. The strong down-regulation of DEGs leads to transcriptional disturbances in several pathways related to stress acclimation such as hormone and antioxidant metabolism. Additionally, we identified several genes that are known to play key roles in enhancing drought tolerance, such as HSPs and crucial TFs. Our results provide new insights into the transcriptional responses in root-nodules, an underexplored tissue of plants mainly under drought conditions. This research paves the way for potential improvements in plant-bacteria interactions, contributing to common bean adaptations in the face of challenging environmental conditions.

Comprehensive analysis of miRNAs, lncRNAs and mRNAs profiles in backfat tissue between Daweizi and Yorkshire pigs.

OBJECTIVE: Daweizi (DWZ) is a famous indigenous pig breed in China and characterized by tender meat and high fat percentage. However, the expression profiles and functions of transcripts in DWZ pigs is still in infancy. The object of this study was to depict the transcript profiles in DWZ pigs and screen the potential pathway influence adipogenesis and fat deposition. METHODS: Histological analysis of backfat tissue was firstly performed between DWZ and lean-type Yorkshire pigs, and then RNA sequencing technology was utilized to explore miRNAs, lncRNAs and mRNAs profiles in backfat tissue. 18 differentially expressed (DE) transcripts were randomly selected for quantitative real-time polymerase chain reaction (QPCR) to validate the reliability of the sequencing results. Finally, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis were conducted to investigate the potential pathways influence adipocyte differentiation, adipogenesis and lipid metabolism, and a schematic model was further proposed. RESULTS: A total of 1,625 differentially expressed transcripts were identified in DWZ pigs, including 27 upregulated and 45 downregulated miRNAs, 64 upregulated and 119 downregulated lncRNA, 814 upregulated and 556 downregulated mRNAs. QPCR analysis exhibited strong consistency with the sequencing data. GO and KEGG analysis elucidated that the differentially expressed transcripts were mainly associated with cell growth and death, signal transduction, peroxisome proliferator-activated receptors (PPAR), AMP-activated protein kinase (AMPK), PI3K-Akt, adipocytokine and foxo signaling pathways, all of which are strongly involved in cell development, lipid metabolism and adipogenesis. Further analysis indicated that the BGIR9823_87926/miR-194a-5p/AQP7 network may be effective in the process of adipocyte differentiation or adipogenesis. CONCLUSION: Our study provides comprehensive insights into the regulatory network of backfat deposition and lipid metabolism in pigs from the point of view of miRNAs, lncRNAs and mRNAs.

RNA-seq Data Analysis for Differential Expression.

Changes in the surrounding environment are mirrored by changes in the transcript profile of an organism. In the case of a plant pathogen, host colonization would be a challenge that triggers changes in transcript expression patterns. Determining the transcriptional profile could provide valuable clues on how an organism responds to defined stimuli, in this case, how a pathogen colonizes its host. Several robust data analysis methods and pipelines are available that can identify these differentially expressed transcripts. In this chapter we outline the steps and other caveats that are needed to run one such pipeline.

Stress-seventy subfamily A 4, A member of HSP70, confers yeast cadmium tolerance in the loss of mitochondria pyruvate carrier 1.

Mitochondrial pyruvate carrier (MPC), which transports pyruvate into mitochondria, is a key regulatory element in the material metabolism and energy metabolism. Since MPC was firstly identified in yeast in 2012, many groups have investigated the function of MPC. As MPC is a classic material transporter, the focus of previous studies has been placed on its role in pyruvate transport. In this study, we discovered a novel Cd resistant gene, stress-seventy subfamily A 4 (SSA4), which can recover the Cd sensitive phenotype in the yeast MPC1 mutant strain. It is suggested that, except for adjusting metabolism, MPC can regulate stress tolerance by regulating downstream genes in yeast. Previously, we discovered a Cd related gene, AGP30, which is associated with MPC1 in Arabidopsis. These results indicate that MPC can regulate Cd tolerance through downstream genes in both Arabidopsis and yeast. This study will pave the way for further exploring the bypass pathways of MPC at the molecular level, and the interaction between MPC and the downstream genes in biology.

Transcriptome of 17ß-hydroxysteroid dehydrogenase type 2 plays both hormone-dependent and hormone-independent roles in MCF-7 breast cancer cells.

Breast cancer is a major cause of cancer-related death for women in western countries. 17ß-Hydroxysteroid dehydrogenases (17ß-HSDs) play important roles in the last step of sex-hormone activation and the first step of sex-hormone inactivation. 17ß-HSD2 is responsible for oxidizing the sex hormones. We used microarray technology to analyze the effect of 17ß-HSD2 on the MCF-7 cell transcript profile after knocking down 17ß-HSD2. Five hundred forty-two genes were regulated 1.5-fold or higher after treatment with 17ß-HSD2 siRNA. Knocking down 17ß-HSD2 interrupted nucleosome assembly. Pathway-Act-Network analysis showed that the MAPK and apoptosis signaling pathways were most regulated. In the gene-gene interaction network analysis, UGT2B15, which is involved in hormone metabolism, was the most regulated core gene. FOS, GREB1, and CXCL12 were the most regulated genes, and CXCL12 was related to tumor migration. Following 17ß-HSD2 knock-down, the cell viability decreased to 75.9%. The S-phase percentage decreased by 19.4%, the Q2-phase percentage in cell apoptosis testing increased by 1.5 times, and cell migration decreased to 66.0%. These results were consistent with our gene chip analysis and indicated that 17ß-HSD2 plays both hormone-dependent and hormone-independent enzymatic roles. In-depth investigations of this enzyme on the genomic level will help clarify its related molecular mechanisms.

Differential Activity and Expression Profile of Antioxidant Enzymes and Physiological Changes in Wheat (Triticum aestivum L.) Under Drought.

Wheat crop may experience water deficit at crucial stages during its life cycle, which induces oxidative stress in the plants. The antioxidant status of the plant plays an important role in providing tolerance against the water stress. The objective of this study was to investigate the impact of water stress on physiological traits, antioxidant activity and transcript profile of antioxidant enzyme related genes in four wheat genotypes (C306, AKAW3717, HD2687, PBW343) at three crucial stages of plants under medium (75% of field capacity) and severe stress (45% of field capacity) in pots. Drought was applied by withholding water for 10 days at a particular growth stage viz. tillering, anthesis and 15 days after anthesis (15DAA). For physiological traits, a highly significant effect of water stress at a particular stage and genotypic variations for resistance to drought tolerance was observed. Under severe water stress, the malondialdehyde (MDA) content increased while the relative water content (RWC) and chlorophyll index decreased significantly in all the genotypes. The drought susceptibility index (DSI) of the genotypes varied from 0.18 to 1.9. The drought treatment at the tillering and anthesis stages was found more sensitive in terms of reduction in thousand grain weight (TGW) and grain yield. Antioxidant enzyme activities [superoxide dismutase (SOD) and peroxidase (POX)] increased with the decrease in osmotic potential in drought tolerant genotypes C306 and AKAW3717. Moreover, the transcript profile of Mn-SOD upregulated significantly and was consistent with the trend of the variation in SOD activity, which suggests that Mn-SOD might play an important role in drought tolerance.