Combined transcriptomic and metabolic analyses reveal potential mechanism for fruit development and quality control of Chinese raspberry (Rubus chingii Hu).

KEY MESSAGE: Combined transcriptomic and metabolic analyses reveal that fruit of Rubus chingii Hu launches biosynthesis of phenolic acids and flavonols at beginning of fruit set and then coordinately accumulated or converted to their derivatives. Rubus chingii Hu (Chinese raspberry) is an important dual functional food with nutraceutical and pharmaceutical values. Comprehensively understanding the mechanisms of fruit development and bioactive components synthesis and regulation could accelerate genetic analysis and molecular breeding for the unique species. Combined transcriptomic and metabolic analyses of R. chingii fruits from different developmental stages, including big green, green-to-yellow, yellow-to-orange, and red stages, were conducted. A total of 89,188 unigenes were generated and 57,545 unigenes (64.52%) were annotated. Differential expression genes (DEGs) and differentially accumulated metabolites (DAMs) were mainly involved in the biosynthesis of secondary metabolites. The fruit launched the biosynthesis of phenolic acids and flavonols at the very beginning of fruit set and then coordinately accumulated or converted to their derivatives. This was tightly regulated by expressions of the related genes and MYB and bHLH transcription factors. The core genes products participated in the biosynthesis of ellagic acid (EA) and kaempferol-3-O-rutinoside (K-3-R), such as DAHPS, DQD/SDH, PAL, 4CL, CHS, CHI, F3H, F3'H, FLS, and UGT78D2, and their corresponding metabolites were elaborately characterized. Our research reveals the molecular and chemical mechanisms of the fruit development of R. chingii. The results provide a solid foundation for the genetic analysis, functional genes isolation, fruit quality improvement and modifiable breeding of R. chingii.

The p53 signaling pathway of the large yellow croaker (Larimichthys crocea) responds to acute cold stress: evidence via spatiotemporal expression analysis of p53, p21, MDM2, IGF-1, Gadd45, Fas, and Akt.

The p53 activation is induced by stressors, such as DNA damage, oxidative stress, and activated oncogenes, and can promote cell cycle arrest, cellular senescence, and apoptosis. The large yellow croaker (Larimichthys crocea) is an important warm temperate marine fish in the Chinese aquiculture industry. However, few studies have investigated the role of p53 in the response of L. crocea to environmental stressors. Therefore, the aim of the present study was to assess the spatiotemporal mRNA expression levels of genes involved in the p53 signaling pathway of the large yellow croaker in response to cold stress. The results showed significant changes in the expression levels of p53, p21, MDM2, IGF-1, Gadd45, Fas, and Akt in various tissues of the large yellow croaker in response to cold stress for different times. As compared to the control group, p53 mRNA expression was upregulated in most of the examined tissues at 24 h with the exception of the gill. In the liver, the expression levels of p53 and Fas were significantly decreased at 12 h, while those of p21, MDM2, IGF-1, Gadd45 were dramatically increased. Akt expression was notably changed in response to cold in several tissues. These results suggested that p53 was potentially a key gene in the large yellow croaker response to cold and possibly other environmental stressors.

Gene networks and toxicity/detoxification pathways in juvenile largemouth bass (Micropterus salmoides) liver induced by acute lead stress.

Lead (Pb) is a nonessential heavy metal that can be bioconcentrated to highly toxic levels in the environment. To understand the genes and toxicity/detoxification pathways of juvenile largemouth bass, liver transcriptomes were investigated in this fish after acute 96 h Pb exposure (Pb nitrate 0, 17.8, or 100 mg/L). Acute Pb exposure induced an immune response and apoptosis pathway activation in the liver. A number of transcripts related to complement and coagulation cascades were significantly increased. Up- and downregulated genes were significantly enriched in numerous pathways, including the natural killer cell-mediated cytotoxicity pathway, the Jak-STAT and P53 signaling pathways, cancer and apoptosis. These genes included Bid，Bcl-2, JNK, and PI3K (17.8 mg/L) and PI3K, AKT, PPARδ, RAS, MMPs, c-Jun p53, and PD-L1 (100 mg/L). Comprehensive analysis of liver transcriptomic data revealed numerous pathways associated with the immune system and carcinogenesis, especially pathways related to apoptosis and systemic lupus erythematosus.

Dynamic alterations in methylation of global DNA and growth-related genes in large yellow croaker (Larimichthys crocea) in response to starvation stress.

DNA methylation is susceptible to various environmental factors such as salinity, temperature and nutritional conditions, and can affect gene function, organ metabolism, body growth and development. In order to explore the effect of starvation on growth-related genes in large yellow croaker (Larimichthys crocea), we studied methylation of the global DNA and growth-related genes (MSTN1,MSTN2,IGF1,IGF2) and the corresponding mRNA expressions, using ELISA-based technique, bisulfite sequencing PCR (BSP) technique and quantitative Real-time PCR (qRT-PCR) respectively. The results showed that the global DNA methylation levels were significantly different (p <0.05) between the experimental group and the control group at starvation 14d, 21d in muscle and at starvation 7d, 14d, 28d, and re-feeding 7d in liver. The CpG islands of MSTN1, MSTN2, IGF1 and IGF2 were enriched in exons rather than promoters. The proximal promoter of MSTN1 and IGF1 and the exon1 of MSTN2 had almost no methylation at all treatment stages. The methylation status in MSTN1 exon 1 and IGF2 exon 2 varied from different starvation time, and started to have significant differences on starvation 7d (p <0.05) both in liver and muscle. In the liver there was a strong positive correlation between IGF2 exon 2 methylation and global DNA methylation (r = 0.7558). The mRNA expression levels of these growth-related genes were significantly different at starvation 14d (p <0.05), but did not have significant correlation with the methylation of these exons. The results implied that exon methylation of these growth-related genes might affect post-transcriptional process.

Liver Transcriptome Analysis of the Large Yellow Croaker (Larimichthys crocea) during Fasting by Using RNA-Seq.

The large yellow croaker (Larimichthys crocea) is an economically important fish species in Chinese mariculture industry. To understand the molecular basis underlying the response to fasting, Illumina HiSeqTM 2000 was used to analyze the liver transcriptome of fasting large yellow croakers. A total of 54,933,550 clean reads were obtained and assembled into 110,364 contigs. Annotation to the NCBI database identified a total of 38,728 unigenes, of which 19,654 were classified into Gene Ontology and 22,683 were found in Kyoto Encyclopedia of Genes and Genomes (KEGG). Comparative analysis of the expression profiles between fasting fish and normal-feeding fish identified a total of 7,623 differentially expressed genes (P < 0.05), including 2,500 upregulated genes and 5,123 downregulated genes. Dramatic differences were observed in the genes involved in metabolic pathways such as fat digestion and absorption, citrate cycle, and glycolysis/gluconeogenesis, and the similar results were also found in the transcriptome of skeletal muscle. Further qPCR analysis confirmed that the genes encoding the factors involved in those pathways significantly changed in terms of expression levels. The results of the present study provide insights into the molecular mechanisms underlying the metabolic response of the large yellow croaker to fasting as well as identified areas that require further investigation.

Liver transcriptome sequencing and de novo annotation of the large yellow croaker (Larimichthy crocea) under heat and cold stress.

Large yellow croaker is an economically important marine fish in China. To understand the molecular mechanisms of this fish under temperature stress, liver transcriptomes of large yellow croakers undergoing heat and cold stress were investigated. The results showed that 130,246 contigs from cold stressed fish (CS), 109,203 contigs from control fish (NS) and 98,569 contigs from heat stressed fish (HS) were obtained from the liver transcriptomes with de novo analysis, respectively. And in total, 9467 (6113 up-regulated and 3364 down-regulated), 5350 (2185 up-regulated and 3165 down-regulated), 10,622 (3146 up-regulated and 7477 down-regulated) significantly differentially expressed genes were identified in CS-NS, HS-NS, and HS-CS, respectively. Pathway enrichment analysis showed that many pathways including those of energy metabolism and metabolic pathways were affected after temperature stress. Further qPCR analysis also confirmed that the expression levels of genes coding for key enzymes in metabolic pathways were dramatically changed.

[Complex effects of simulated acid rain and Cu on the physiological characteristics of Paulownia fortunei and its detoxification mechanism].

A pot experiment was conducted to study the effects of simulated acid rain (pH 4.0, 5.0) and Cu (0-200 mg x kg(-1)) on the physiological characteristics of Paulownia fortunei and its detoxification mechanism. With no Cu addition, the leaf chlorophyll, carotenoid, O2 division by, H2O2, and MDA contents of P. fortunei had no significant differences between the two acid rain treatments. However, with the addition of 100 and 200 mg Cu x kg(-1), the chlorophyll and carotenoid contents of treatment pH 4.0 were lower, while the O2 divided by, H2O2 and MDA contents were higher thanthose of treatment pH 5.0. The chlorophyll a/b ratio of treatments Cu was higher than that of the control. The leaf Cu content decreased obviously with the increasing acidity of stimulated acid rain, but the root Cu content was in reverse. With increasing Cu addition, both the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) and the total contents of phytochelatins (PCs) and glutathione (GSH) in treatment pH 5.0 increased, while the activities of SOD, POD, CAT and APX in treatment pH 4.0 decreased after an initial increase, and the total contents of glutathione (GSH) decreased greatly in treatment 200 mg Cu x kg(-1). All of these demonstrated that the oxidative stress of high Cu concentration to P. fortunei was aggravated by stimulated acid rain.