De novo transcriptome assembly of the wild relative of tea tree (Camellia taliensis) and comparative analysis with tea transcriptome identified putative genes associated with tea quality and stress response.

BACKGROUND: Camellia taliensis is one of the most important wild relatives of cultivated tea tree, C. sinensis. The species extensively occupies mountainous habitats representing a wide-range abiotic tolerance and biotic resistance and thus harbors valuable gene resources that may greatly benefit genetic improvement of cultivated tea tree. However, owning to a large genome size of ~3 Gb and structurally complex genome, there are fairly limited genetic information and particularly few genomic resources publicly available for this species. To better understand the key pathways determining tea flavor and enhance tea tree breeding programs, we performed a high-throughput transcriptome sequencing for C. taliensis. RESULTS: In this study, approximate 241.5 million high-quality paired-end reads, accounting for ~24 Gb of sequence data, were generated from tender shoots, young leaves, flower buds and flowers using Illumina HiSeq 2000 platform. De novo assembly with further processing and filtering yielded a set of 67,923 transcripts with an average length of 685 bp and an N50 of 995 bp. Based on sequence similarity searches against public databases, a total of 39,475 transcripts were annotated with gene descriptions, conserved protein domains or gene ontology (GO) terms. Candidate genes for major metabolic pathways involved in tea quality were identified and experimentally validated using RT-qPCR. Further gene expression profiles showed that they are differentially regulated at different developmental stages. To gain insights into the evolution of these genes, we aligned them to the previously cloned orthologous genes in C. sinensis, and found that considerable nucleotide variation within several genes involved in important secondary metabolic biosynthesis pathways, of which flavone synthase II gene (FNSII) is the most variable between these two species. Moreover, comparative analyses revealed that C. taliensis shows a remarkable expansion of LEA genes, compared to C. sinensis, which might contribute to the observed stronger stress resistance of C. taliensis. CONCLUSION: We reported the first large-coverage transcriptome datasets for C. taliensis using the next-generation sequencing technology. Such comprehensive EST datasets provide an unprecedented opportunity for identifying genes involved in several major metabolic pathways and will accelerate functional genomic studies and genetic improvement efforts of tea trees in the future.

Alpha-lipoic acid activates AMPK to protect against oxidative stress and apoptosis in rats with diabetic peripheral neuropathy.

PURPOSE: To investigate the AMPK pathway-mediated effect of alpha-lipoic acid (ALA) on the dorsal root ganglia (DRGs) of rats with diabetic peripheral neuropathy (DPN) and to attempt to elucidate the underlying mechanism. METHODS: Sprague-Dawley rats (n = 15) were randomly divided into three groups. The control group was fed a standard diet, and the other groups were fed a high-carbohydrate/high-fat diet. Diabetes was established by a single streptozotocin (STZ) (30 mg/kg) injection, and control rats were injected with an equal volume of citrate buffer. ALA (60 mg/kg/day) was administered for 12 weeks. The nerve conduction velocity (NCV) of the sciatic nerve was measured. Glutathione (GSH) and malondialdehyde (MDA) concentrations in serum were measured with the thiobarbituric acid method and biochemistry. Pathological changes in the rat DRGs were observed. AMPK, phospho-AMPK (p-AMPK), nuclear factor erythroid-2-related factor 2 (Nrf2), phospho-nuclear factor erythroid-2-related factor 2 (p-Nrf2), heme oxygenase 1 (HO-1), quinone oxidoreductase 1 (NQO1), Forkhead box O3 (FoxO3a), phospho-Forkhead box O3 (p-FoxO3a), and Bcl-2 interacting mediator of cell death (Bim) expression levels were assessed by immunohistochemistry and western blotting. RESULTS: ALA improved the motor NCV (MNCV) and sensory NCV (SNCV) of rats with DPN and reduced their mechanical pain threshold. ALA increased serum GSH concentrations and decreased serum MDA concentrations. Additionally, AMPK was activated by ALA. Nrf2, p-Nrf2, HO-1, and NQO1 expression was upregulated, while FoxO3a, p-FoxO3a, and Bim expression was downregulated. ALA reduced oxidative stress and apoptosis in DRG. CONCLUSION: ALA alleviates DPN and improves peripheral nerve function. ALA reduces oxidative stress by activating Nrf2 through AMPK and inhibits FoxO3a and Bim thereby reducing neuronal apoptosis.

Electroantennogram responses of the tea slug moth, Iragoides fasciata to some plant volatiles associated with tea, Camellia sinensis.

Electroantennogram responses to a wide range of plant volatile compounds that have been identified in tea plants Camellia sinensis L. (Ericales: Theaceae) were recorded from males and females of the tea slug moth, Iragoides fasciata Moore (Lepidoptera: Limacodidae). The responses to 26 compounds, belonging to several chemical classes, and two mixtures were evaluated. The results showed significantly different electroantennogram responses to the different chemicals, as well as significantly different responses according to gender. The green leaf volatile components elicited significantly greater responses in males. In general, the antennae of males were more sensitive, and responded more strongly, to most of the compounds. Responses to sesquiterpenoids were lower in both males and females. Dose-dependent response studies indicated differences in response between genders and concentrations, suggesting the existence of sexual dimorphism. Compounds belonging to the green leaf volatiles class appeared to be important clues in host-plant selection by this oligophagous species.

Eplerenone Prevents Cardiac Fibrosis by Inhibiting Angiogenesis in Unilateral Urinary Obstruction Rats.

Introduction: Cardiovascular disease constitutes the leading cause of mortality in patients with chronic kidney disease (CKD), which is termed cardiorenal syndrome type 4 (CRS-4). Here, we report the development of pathological cardiac remodeling and fibrosis in unilateral urinary obstruction (UUO) rats. Methods: Hematoxylin and eosin (H&E) staining was performed to observe the pathology of myocardial tissue. The degree of myocardial tissue fibrosis was observed by Masson and Sirius red staining. Immunohistochemical staining was applied to detect the expression of CD34 and CD105 in myocardial tissue, and immunofluorescent staining was performed to examine the expression of CD34, collagen I/collagen III, and alpha smooth muscle actin (α-SMA). The expression of the signal pathway-related proteins vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2), nuclear factor κB (NF-κB), and interleukin (IL)-1ß was tested by western blotting. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the mRNA levels of serum and glucocorticoid-inducible kinase (SGK)-1, NF-κB, and interleukin-1ß (IL-1ß). Results: The results showed the development of pathological cardiac remodeling and cardiac dysfunction in UUO rats. Moreover, there was more angiogenesis and endothelial-mesenchymal transition (End-MT) in the UUO group, and these effects were inhibited by eplerenone. Conclusions: The results indicated that this cardiac fibrosis was associated with angiogenesis and that End-MT was related to aldosterone and mineralocorticoid receptor (MR) activation. Moreover, in association with the MR/IL-1ß/VEGFA signaling pathway, early treatment with the MR antagonist eplerenone in rats with UUO-induced CKD may significantly attenuate MR activation and cardiac fibrosis.

Astragaloside IV Inhibits Mitochondrial-Dependent Apoptosis of the Dorsal Root Ganglion in Diabetic Peripheral Neuropathy Rats Through Modulation of the SIRT1/p53 Signaling Pathway.

PURPOSE: To investigate the effect of astragaloside IV (AS-IV) on mitochondrial-dependent apoptosis in the dorsal root ganglion of diabetic peripheral neuropathy (DPN) rats through the SIRT1/p53 pathway. METHODS: Diabetic rat model was induced by high-carbohydrate/high-fat diet and intraperitoneal injection of STZ. Diabetic rats were divided into three groups (n =16 per group): DPN group, AS-IV group (60mg/kg/d) and α-lipoic acid (ALA) group (60mg/kg/d). Weight and blood glucose levels were monitored every 4 weeks for 12 weeks. DPN was evaluated using the Von Frey Filaments Test and nerve conduction velocity. The dorsal root ganglia of rats were isolated and the pathological changes of mitochondria were observed by electron microscopy. The activity of mitochondrial electron transport chain complex, mitochondrial membrane potential, malonaldehyde (MDA) and glutathione (GSH) levels were measured. Neural apoptosis was detected using the Terminal Deoxynucleotidyl Nick-End Labeling (TUNEL) assay kit. The cleaved caspase-3, major proteins in the SIRT1/p53 pathway, including SIRT1, acetyl p53, Drp1, BAX, and BCL-2, were detected using immunohistochemistry and Western blot. Gene expression of major proteins in the SIRT1/p53 pathway was also detected. RESULTS: After 12 weeks of treatment, AS-IV and ALA did not significantly affect body weight or fasting glucose levels, but reduced mechanical abnormal pain in DPN and improved nerve conduction velocity. AS-IV and ALA increased the level of GSH and decreased the level of MDA. Both AS-IV and ALA can reduce mitochondrial damage, improve mitochondrial electron transport chain complex activity and mitochondrial membrane potential, and reduce the percentages of positive cells with DNA fragmentation and the expression of cleaved caspase-3 protein. AS-IV and ALA up-regulated the expression of SIRT1 and down-regulated the expression of acetyl-p53, Drp1 and the ratio of BAX to BCL-2. Changes in gene expression were similar. CONCLUSION: AS-IV can reduce the occurrence of mitochondrial-dependent apoptosis by regulating the SIRT1/p53 pathway. It has a similar therapeutic effect as ALA and is therefore a promising drug for the potential treatment of DPN.

Genomic Variance and Transcriptional Comparisons Reveal the Mechanisms of Leaf Color Affecting Palatability and Stressed Defense in Tea Plant.

Leaves are one of the most important organs of plants, and yet, the association between leaf color and consumable traits remains largely unclear. Tea leaves are an ideal study system with which to investigate the mechanism of how leaf coloration affects palatability, since tea is made from the leaves of the crop Camellia sinensis. Our genomic resequencing analysis of a tea cultivar ZiJuan (ZJ) with purple leaves and altered flavor revealed genetic variants when compared with the green-leaf, wild type cultivar YunKang(YK). RNA-Seq based transcriptomic comparisons of the bud and two youngest leaves in ZJ and YK identified 93%, 9% and 5% expressed genes that were shared in YK- and ZJ-specific cultivars, respectively. A comparison of both transcript abundance and particular metabolites revealed that the high expression of gene UFGT for anthocyanin biosynthesis is responsible for purple coloration, which competes with the intermediates for catechin-like flavanol biosynthesis. Genes with differential expression are enriched in response to stress, heat and defense, and are casually correlated with the environmental stress of ZJ plant origin in the Himalayas. In addition, the highly expressed C4H and LDOX genes for synthesizing flavanol precursors, ZJ-specific CLH1 for degrading chlorophyll, alternatively spliced C4H and FDR and low photosynthesis also contributed to the altered color and flavor of ZJ. Thus, our study provides a better molecular understanding of the effect of purple coloration on leaf flavor, and helps to guide future engineering improvement of palatability.

Domestication Origin and Breeding History of the Tea Plant (Camellia sinensis) in China and India Based on Nuclear Microsatellites and cpDNA Sequence Data.

Although China and India are the two largest tea-producing countries, the domestication origin and breeding history of the tea plant in these two countries remain unclear. Our previous study suggested that the tea plant includes three distinct lineages (China type tea, Chinese Assam type tea and Indian Assam type tea), which were independently domesticated in China and India, respectively. To determine the origin and historical timeline of tea domestication in these two countries we used a combination of 23 nSSRs (402 samples) and three cpDNA regions (101 samples) to genotype domesticated tea plants and its wild relative. Based on a combination of demographic modeling, NewHybrids and Neighbour joining tree analyses, three independent domestication centers were found. In addition, two origins of Chinese Assam type tea were detected: Southern and Western Yunnan of China. Results from demographic modeling suggested that China type tea and Assam type tea first diverged 22,000 year ago during the last glacial maximum and subsequently split into the Chinese Assam type tea and Indian Assam type tea lineages 2770 year ago, corresponding well with the early record of tea usage in Yunnan, China. Furthermore, we found that the three tea types underwent different breeding histories where hybridization appears to have been the most important approach for tea cultivar breeding and improvements: a high proportion of the hybrid lineages were found to be F2 and BCs. Collectively, our results underscore the necessity for the conservation of Chinese Assam type tea germplasm and landraces as a valuable resource for future tea breeding.

The Tea Tree Genome Provides Insights into Tea Flavor and Independent Evolution of Caffeine Biosynthesis.

Tea is the world's oldest and most popular caffeine-containing beverage with immense economic, medicinal, and cultural importance. Here, we present the first high-quality nucleotide sequence of the repeat-rich (80.9%), 3.02-Gb genome of the cultivated tea tree Camellia sinensis. We show that an extraordinarily large genome size of tea tree is resulted from the slow, steady, and long-term amplification of a few LTR retrotransposon families. In addition to a recent whole-genome duplication event, lineage-specific expansions of genes associated with flavonoid metabolic biosynthesis were discovered, which enhance catechin production, terpene enzyme activation, and stress tolerance, important features for tea flavor and adaptation. We demonstrate an independent and rapid evolution of the tea caffeine synthesis pathway relative to cacao and coffee. A comparative study among 25 Camellia species revealed that higher expression levels of most flavonoid- and caffeine- but not theanine-related genes contribute to the increased production of catechins and caffeine and thus enhance tea-processing suitability and tea quality. These novel findings pave the way for further metabolomic and functional genomic refinement of characteristic biosynthesis pathways and will help develop a more diversified set of tea flavors that would eventually satisfy and attract more tea drinkers worldwide.

An Integrated Metagenomics/Metaproteomics Investigation of the Microbial Communities and Enzymes in Solid-state Fermentation of Pu-erh tea.

Microbial enzymes during solid-state fermentation (SSF), which play important roles in the food, chemical, pharmaceutical and environmental fields, remain relatively unknown. In this work, the microbial communities and enzymes in SSF of Pu-erh tea, a well-known traditional Chinese tea, were investigated by integrated metagenomics/metaproteomics approach. The dominant bacteria and fungi were identified as Proteobacteria (48.42%) and Aspergillus (94.98%), through pyrosequencing-based analyses of the bacterial 16S and fungal 18S rRNA genes, respectively. In total, 335 proteins with at least two unique peptides were identified and classified into 28 Biological Processes and 35 Molecular Function categories using a metaproteomics analysis. The integration of metagenomics and metaproteomics data demonstrated that Aspergillus was dominant fungus and major host of identified proteins (50.45%). Enzymes involved in the degradation of the plant cell wall were identified and associated with the soft-rotting of tea leaves. Peroxiredoxins, catalase and peroxidases were associated with the oxidation of catechins. In conclusion, this work greatly advances our understanding of the SSF of Pu-erh tea and provides a powerful tool for studying SSF mechanisms, especially in relation to the microbial communities present.

[Effect of reduced N application on soil N residue and N loss in maize-soybean relay strip intercropping system].

A field experiment was conducted in 2012, including three planting pattern (maize-soybean relay strip intercropping, mono-cultured maize and soybean) and three nitrogen application level [0 kg N x hm(-2), 180 kg N x hm(-2) (reduced N) and 240 kg N x hm(-2) (normal N)]. Fields were assigned to different treatments in a randomized block design with three replicates. The objective of this work was to analyze the effects of planting patterns and nitrogen application rates on plant N uptake, soil N residue and N loss. After fertilization applications, NH4(+)-N and NO3(-)-N levels increased in the soil of intercropped maize but decreased in the soil of intercropped soybean. Compared with mono-crops, the soil N residue and loss of intercropped soybean were reduced, while those of intercropped maize were increased and decreased, respectively. With the reduced rate of N application, N residue rate, N loss rate and ammonia volatilization loss rate of the maize-soybean intercropping relay strip system were decreased by 17.7%, 21.5% and 0.4% compared to mono-cultured maize, but increased by 2.0%, 19.8% and 0.1% compared to mono-cultured soybean, respectively. Likewise, the reduced N application resulted in reductions in N residue, N loss, and the N loss via ammonia volatilization in the maize-soybean relay strip intercropping system compared with the conventional rate of N application adopted by local farmers, and the N residue rate, N loss rate and ammonia volatilization loss rate reduced by 12.0%, 15.4% and 1.2%, respectively.

[Effects of reduced N application rate on yield and nutrient uptake and utilization in maize-soybean relay strip intercropping system].

A field experiment with three N application rates (0, 180, 240 N kg x hm(-2), representing zero, reduced and conventional N application, respectively) and three planting patterns (maize monoculture, soybean monoculture and maize-soybean relay strip intercropping) was conducted to reveal the effects of cropping patterns and N application rates on yield, nutrient uptake and nitrogen use efficiency of maize and soybean. The results showed that the grain yield, N, P and K uptake and harvest index of the intercropped maize reduced slightly compared with the monoculture maize, however these indices of the intercropped soybean increased significantly compared with the monoculture. With the increase in nitrogen fertilizer application, the excellence of relay strip intercropping was weakened in the maize-soybean intercropping system. The grain yield, economic coefficient, N, P and K uptake, harvest index, N agronomy efficiency and N uptake efficiency of maize and soybean increased significantly at the reduced nitrogen rate (180 N kg x hm(-2)), but the rate of soil N contribution declined, compared with the conventional rate of N application by local farmers (240 N kg x hm(-2)). In the reduced nitrogen rate treatment, total soil N and P contents of the maize strip reduced, whereas the total soil N, P and K contents of soybean strip and the total K content of maize strip increased compared with the zero N application treatment. With the reduced N application, the annual total grain yield, N, P and K uptake of above-ground biomass in the maize-soybean relay strip intercropping system were higher than in the monoculture, and the land equivalent ratio (LER) was 2.28. N uptake efficiency of maize in the relay strip intercropping system was 20.2% higher than in the maize monoculture, and the index of soybean was 30.5% lower than in the monoculture. The rate of soil N contribution in the relay strip intercropping system was 20.0% and 8.8% lower than in the maize and soybean monoculture, respectively. The reduced N application in the maize-soybean relay strip intercropping system was helpful to promote annual grain yield and improve N utilization efficiency.