Proteomic Analysis of Salivary Secretions from the Tea Green Leafhopper, Empoasca flavescens Fabrecius.

Saliva plays a crucial role in shaping the compatibility of piercing-sucking insects with their host plants. Understanding the complex composition of leafhopper saliva is important for developing effective and eco-friendly control strategies for the tea green leafhopper, Empoasca flavescens Fabrecius, a major piercing-sucking pest in Chinese tea plantations. This study explored the saliva proteins of tea green leafhopper adults using a custom collection device, consisting of two layers of Parafilm stretched over a sucrose diet. A total of 152 proteins were identified using liquid chromatography-tandem mass spectrometry (LC-MS/MS) following the filter-aided sample preparation (FASP). These proteins were categorized into six groups based on their functions, including enzymes, transport proteins, regulatory proteins, cell structure proteins, other proteins, and unknown proteins. Bioinformatics analyses predicted 16 secreted proteins, which were successfully cloned and transcriptionally analyzed across various tissues and developmental stages. Genes encoding putative salivary secretory proteins, including Efmucin1, EfOBP1, EfOBP2, EfOBP3, Efmucin2, low-density lipoprotein receptor-related protein (EfLRP), EFVg1, and EFVg2, exhibited high expressions in salivary gland (SG) tissues and feeding-associated expressions at different developmental stages. These findings shed light on the potential elicitors or effectors mediating the leafhopper feeding and defense responses in tea plants, providing insights into the coevolution of tea plants and leafhoppers. The study's conclusions open avenues for the development of innovative leafhopper control technologies that reduce the reliance on pesticides in the tea industry.

The Flavor Characteristics, Antioxidant Capability, and Storage Year Discrimination Based on Backpropagation Neural Network of Organic Green Tea (Camellia sinensis) during Long-Term Storage.

The storage period of tea is a major factor affecting tea quality. However, the effect of storage years on the non-volatile major functional components and quality of green tea remains largely unknown. In this study, a comparative analysis of organic green teas with varying storage years (1-16 years) was conducted by quantifying 47 functional components, using electronic tongue and chromatic aberration technology, alongside an evaluation of antioxidative capacity. The results indicated a significant negative correlation between the storage years and levels of tea polyphenols, total amino acids, soluble sugars, two phenolic acids, four flavonols, three tea pigments, umami amino acids, and sweet amino acids. The multivariate statistical analysis revealed that 10 functional components were identified as effective in distinguishing organic green teas with different storage years. Electronic tongue technology categorized organic green teas with different storage years into three classes. The backpropagation neural network (BPNN) analysis demonstrated that the classification predictive ability of the model based on the electronic tongue was superior to the one based on color difference values and 10 functional components. The combined analysis of antioxidative activity and functional components suggested that organic green teas with shorter storage periods exhibited stronger abilities to suppress superoxide anion radicals and hydroxyl radicals and reduce iron ions due to the higher content of eight components. Long-term-stored organic green teas, with a higher content of substances like L-serine and theabrownins, demonstrated stronger antioxidative capabilities in clearing both lipid-soluble and water-soluble free radicals. Therefore, this study provided a theoretical basis for the quality assessment of green tea and prediction of green tea storage periods.

Comprehensive analysis of the effect of etiolated tea cultivars and harvest seasons on volatile compounds and in vitro antioxidant capacity in steamed green teas.

To explore the effects of harvest seasons and etiolated cultivars on the volatile compounds of steamed green teas, this study analyzed comprehensively the volatile compounds of steamed green teas using simultaneous distillation extraction-gas chromatography-mass spectrometry (SDE-GC-MS) and chemometrics analytical techniques in combination with odor activity value (OAV). Additionally, the in vitro antioxidant capacity of the steamed green teas was evaluated. The results showed that 95 volatile compounds were identified, among which aldehydes, ketones, alcohols, and acids were the main types in steamed green teas made from etiolated tea cultivars. Furthermore, the relative content of volatile compounds in steamed green tea was significantly negatively correlated with season (P < 0.05). In steamed green teas harvested in different seasons, spring tea contained a higher abundance of volatile compounds such as (+)-δ-cadinene, farnesyl acetone, carvenone, trans-ß-ionone, and nerolidol. The differences of total volatile compounds among the three etiolated tea cultivars were not remarkable (P > 0.05). Combined with the OAV, 20 and 19 key aroma compounds in steamed green teas from different harvest seasons and cultivars were identified, respectively, which could bring unique aromas to different steamed green tea samples. By comparison, summer tea had the strongest antioxidant capacity, while there was no obvious difference in the antioxidant capacity among cultivars. This study provided a scientific basis for the aroma quality of steamed green teas made from etiolated tea cultivars in different harvest seasons.

Tea green leafhopper-induced synomone attracts the egg parasitoids, mymarids to suppress the leafhopper.

BACKGROUND: The tea green leafhopper, Empoasca flavescens is the most important pest of tea plants in China. Mymarid attractants based on herbivore-induced plant volatiles (HIPVs) from leafhopper feeding and oviposition-induced plant volatiles (OIPVs) were formulated and tested as a novel pest control agent against the leafhopper in tea plantations. RESULTS: Results showed that two mymarid species, Stethynium empoascae and Schizophragma parvula, had a reducing effect on leafhopper populations. The HIPVs and OIPVs were identified and bioassayed to screen the key synomones showing strong attraction to the mymarids. They were formulated into different blends, of which Field Attractant 1, comprising linalool, methyl salicylate, (E)-2-hexenal, perillen and α-farnesene at ratio of 1:2:3:58:146 (20 mg/lure), showed the strongest attraction to the mymarids. In field trials with the attractant, the average parasitism rate (60.46 ± 23.71%) of tea leafhoppers by the two mymarids in the attractant-baited area was significantly higher than that (42.85 ± 19.24%) in the CK area. Also, the average leafhopper density (46 ± 30 per 80 tea shoots) in the attractant-baited area was significantly lower than that (110 ± 70 per 80 tea shoots) in the CK area. CONCLUSION: This study showed that a synthetic blend of key volatiles from HIPVs and OIPVs at an optimal ratio can be formulated into an attractant with the potential to attract and retain wild mymarid populations to suppress leafhopper populations in infested tea plantations, so as to reduce or avoid the spraying of insecticides. © 2023 Society of Chemical Industry.

Spatiotemporal Dynamics of Phthalate Esters in Tea Plants Growing Different Geographical Environments and an Attempt on Their Risk Assessment.

The phthalate esters (PAEs) have become ubiquitous pollutants. In the present work, we investigated their pollution on teas. Dimethyl phthalate (DMP), diethyl phthalate (DEP), diisobutyl phthalate (DiBP), di-n-butyl phthalate (DBP), and di-(2-ethyl) hexyl phthalate (DEHP) were detected in all fresh tea leaves with DBP being the major congener of PAEs in teas followed by DiBP and DEHP. Seasonal variation, spatial distribution difference, correlationship of environmental factors, and potential health risks of PAEs were analyzed. The PAEs content in one bud and two leaves was lower than that in upper mature leaves in tea plants. The PAEs content in fresh tea leaves was the lowest in spring, while it was high in autumn and winter. The correlation analysis results showed that PAEs had significantly negative correlation with ambient air temperature, while it was positively correlated with the air quality index. PAEs analysis of spring tea in Anhui and Zhejiang provinces further indicated that the factor of provincial regions had little impact on the PAEs pollution level in tea. By contrast, the different environmental areas significantly affected PAE pollution, especially the agricultural areas. The human daily intake-based (13 g/day) risk assessment indicated that both the carcinogenic and non-carcinogenic risks (1.76 × 10-7-6.12 × 10-7) of PAEs via tea consumption were acceptable, with the estrogen equivalence (1.60-6.29 ng E2/kg) being at a medium level. This study provides significant information for pollution control and risk assessment of PAEs in Chinese tea production.

Determination of Five Phthalate Esters in Tea and Their Dynamic Characteristics during Black Tea Processing.

A highly specific and high extraction-rate method for the analysis of dimethyl phthalate (DMP), diethyl phthalate (DEP), di-n-butyl phthalate (DBP), diisobutyl phthalate (DiBP), and di-(2-ethyl) hexyl phthalate (DEHP) in tea samples was developed. Based on three-factor Box-Behnken response surface design, solid-phase extraction (SPE) of five phthalate ester (PAE) residues in tea was optimized. Optimal extraction conditions were found for extraction temperature (40 °C), extraction time (12 h), and ratio of tea to n-hexane (1:20). The dynamic distribution of PAEs at each stage of black tea processing was also analyzed, and it was found that the baking process was the main stage of PAE emission, indicating that traditional processing of black tea significantly degrades PAEs. Further, principal component analysis of the physicochemical properties and processing factors of the five PAEs identified the main processing stages affecting the release of PAEs, and it was found that the degradation of PAEs during black tea processing is also related to its own physicochemical properties, especially the octanol-water partition coefficient. These results can provide important references for the detection, determination of processing losses, and control of maximum residue limits (MRLs) of PAEs to ensure the quality and safety of black tea.

A push-pull strategy for controlling the tea green leafhopper (Empoasca flavescens F.) using semiochemicals from Tagetes erecta and Flemingia macrophylla.

BACKGROUND: The tea green leafhopper, Empoasca flavescens is the most important pest in Chinese tea plantations. For decades its control has been executed almost exclusively through pesticide applications. A semiochemical-based 'push-pull' strategy was tested on the leafhopper in the study. RESULTS: The odors released from Tagetes erecta and Flemingia macrophylla significantly repelled and attracted leafhoppers, respectively. These volatile compounds (46 from T. erecta and 53 F. macrophylla) were identified and quantified via gas chromatography-mass spectometry (GC-MS) analysis. Y-tube olfactometer assays indicated that thymol anisole, thymol and camphor had significant repellent effects on the leafhoppers, resulting in a ternary repellent blend at a 4:3:13 ratio. Cis-3-hexen-1-ol, cis-3-hexenyl acetate, nonanal and α-farnesene were significantly attractive to the leafhoppers, making an attractant blend at a 17:4:1:1 ratio. In the field, the push-pull strategy with the repellent dispensers placed within the tea bushes and the attractant-baited sticky traps hung 15 cm above the tea plants showed a significant control efficacy, reaching 69% and 55% at two and 14 days post-treatment, respectively, similar to those in the insecticide control plots. Additionally, the leafhopper density in the push-pull intercropping plot was 63.2 leafhoppers/100 tea shoots/visit, much lower than those in the pull intercropping plot and nonintercropping plot. CONCLUSION: Application of the push-pull strategy using both synthetic repellent and attractant, or intercropping T. erecta and F. macrophylla with tea plants, can effectively reduce the leafhopper population. This approach might have great potential as an environmentally safe control strategy against the leafhopper. © 2022 Society of Chemical Industry.

Short-chain fatty acids contribute to neuropathic pain via regulating microglia activation and polarization.

Microglia activation and subsequent pro-inflammatory responses play a key role in the development of neuropathic pain. The process of microglia polarization towards pro-inflammatory phenotype often occurs during neuroinflammation. Recent studies have demonstrated an active role for the gut microbiota in promoting microglial full maturation and inflammatory capabilities via the production of Short-Chain Fatty Acids (SCFAs). However, it remains unclear whether SCFAs is involved in pro-inflammatory/anti-inflammatory phenotypes microglia polarization in the neuropathic pain. In the present study, chronic constriction injury (CCI) was used to induce neuropathic pain in mice, the mechanical withdrawal threshold, thermal hyperalgesia were accomplished. The levels of microglia markers including ionized calcium-binding adaptor molecule 1 (Iba1), cluster of differentiation 11b (CD11b), pro-inflammatory phenotype markers including CD68, interleukin-1ß (IL-1ß), tumor necrosis factor-α (TNF-α), and anti-inflammatory phenotype markers including CD206, IL-4 in the hippocampus and spinal cord were determined on day 21 after CCI. The results showed that CCI produced mechanical allodynia and thermal hyperalgesia, and also increased the expressions of microglia markers (Iba1, CD11b) and pro-inflammatory phenotype markers (CD68, IL-1ß, and TNF-α), but not anti-inflammatory phenotype marker (CD206, IL-4) in the hippocampus and spinal cord, accompanied by increased SCFAs in the gut. Notably, antibiotic administration reversed these abnormalities, and its effects was also bloked by SCFAs administration. In conclusion, data from our study suggest that CCI can lead to mechanical and thermal hyperalgesia, while SCFAs play a key role in the pathogenesis of neuropathic pain by regulating microglial activation and subsequent pro-inflammatory phenotype polarization. Antibiotic administration may be a new treatment for neuropathic pain by reducing the production of SCFAs and further inhibiting the process of microglia polarization.

Histone deacetylase 3 in hippocampus contributes to memory impairment after chronic constriction injury of sciatic nerve in mice.

ABSTRACT: Chronic neuropathic pain is frequently accompanied by memory impairment, yet the underlying mechanisms remain unclear. Here, we showed that mice displayed memory impairment starting at 14 days and lasting for at least 21 days after chronic constriction injury (CCI) of unilateral sciatic nerve in mice. Systemic administration of the pan histone deacetylase (HDAC) inhibitor sodium butyrate attenuated this memory impairment. More specifically, we found that hippocampus HDAC3 was involved in this process because the levels of its mRNA and protein increased significantly in the hippocampus at 14 and 21 days after CCI, but not sham surgery. Systemic administration of the selective HDAC3 antagonist RGFP966 attenuated CCI-induced memory impairment, improved hippocampal long-term potentiation impairment, and rescued reductions of dendritic spine density and synaptic plasticity-associated protein in the hippocampus. In addition, HDAC3 overexpression in the hippocampus led to memory impairment without affecting basal nociceptive responses in naive mice. Our findings suggest that HDAC3 contributes to memory impairment after CCI by impairing synaptic plasticity in hippocampus. Histone deacetylase 3 might serve as a potential molecular target for therapeutic treatment of memory impairment under neuropathic pain conditions.

Spatial repellency, antifeedant activity and toxicity of three medium chain fatty acids and their methyl esters of coconut fatty acid against stable flies.

BACKGROUND: Stable flies are one of the most detrimental arthropod pests to livestock. With changing climates and agronomic practices, they expand their roles as pests and disease vectors as well. Their painful bites reduce livestock productivity, annoy companion animals, and interfere with human recreational activities. Current management technologies are unable to effectively control stable flies. The present study reports new results concerning the contact, spatial repellency, and toxicity of a bio-based product, coconut fatty acid and their methyl ester derivatives of free fatty acids of C8:0 , C10:0 and C12:0 to stable flies. RESULTS: Three medium chain fatty acid methyl esters (C8:0 , C10:0 and C12:0 ) showed strong antifeedant activity against stable flies and their strengths were dose-dependent. Only the C8:0 acid, C8:0 - and C10:0 methyl esters elicited significant antennal responses. Laboratory single cage olfactometer bioassays revealed that coconut fatty acid and C8:0 methyl ester displayed active spatial repellency. All three methyl esters showed strong toxicity against stable flies. CONCLUSION: Antifeedant activity is the main method through which coconut fatty acid deters stable fly blood-feeding. The C8:0 , C10:0 and C12:0 methyl esters act not only as strong antifeedants, but also possess strong toxicity against stable fly adults. Limited spatial repellency was observed from coconut fatty acid and C8:0 methyl ester. © 2019 Society of Chemical Industry.

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The visual communication between insect and plant is one of the key sensory signal channels for the survival, reproduction and defense of insects. The colored sticky boards, which are developed based on insects positive phototaxis and as one of the effective green pest control measures, have been widely applied for monitoring, forecasting and mass-trapping of many diurnal agricultural and forestry insect pests. In addition, they could be used to attract beneficial insects to the target areas to prey on or parasitize harmful insects, and to aid in pollination. Here, we discussed the insect phototaxis theory and the "attract and kill" mechanism of colored sticky boards and reviewed the effects of color, shape, size, height, density, facing direction, working duration of the sticky boards, as well as plant morphological characters and insect physiological status on trapping efficacy. We summarized various application techniques of the color sticky boards on different target pest insects in tea plantations, vegetable fields and greenhouses, etc. We analyzed various application technologies of combining colored sticky boards with synthetic sex pheromone attractants and/or botanical attractants and their efficacy; then evaluated the pros and cons of using the colored sticky boards for pest control or monitoring. Accordingly, we provided suggestions for improvements and discussed the trapping efficacy assessment and cost-benefit analysis. Finally, we proposed the RD directions of next generation colored sticky boards, especially its combination with synthetic pheromones/kairomones as an important integrated pest management (IPM) measure, and the future of the colored sticky board industry.

Complete mitochondrial genome of the tea looper caterpillar, Ectropis obliqua (Lepidoptera: Geometridae) with a phylogenetic analysis of Geometridae.

The complete mitochondrial genome (mitogenome) of the Ectropis obliqua (Lepidoptera: Geometridae) was sequenced and compared with other species of Geometridae. The mitochondrial genome of E. obliqua is 15,475bp long and contains 37 genes including 13 protein coding genes (PCGs), 22 transfer RNA genes, 2 ribosomal RNA genes and an A+T-rich region. Gene cluster trnI-trnQ-trnM rearranged to trnM-trnI-trnQ comparing with the ancestral mitogenome of insects. All tRNA genes have typical cloverleaf secondary structure except for trnS (AGN). All the sequenced mitogenomes from Geometridae have negative AT-skews and positive GC-skews except for Biston suppressaria. The synonymous and nonsynonymous substitutions ratios (Ka/Ks) indicated variable selective pressures among 13 PCGs. The phylogenetic relationship among 11 species of Geometridae was recovered based on 13 protein coding genes and 2 rRNA genes using Bayesian inference. E. obliqua is grouped with other Ennominae species and recovered to be sister-relationship with Abraxas suspecta.

The complete mitochondrial genome of Tambocerus sp. (Hemiptera: Cicadellidae).

The complete mitochondrial genome of Tambocerus sp. (Hemiptera: Cicadellidae) from Zhejiang and Anhui provinces of China was sequenced. The total length of the mitogenome is 15 955 bp (GenBank accession no. KT827824) and consists of 22 transfer RNAs, 13 protein-coding genes, 2 ribosomal RNAs and 1 control region. The base composition of the heavy strand for A, T, C, and G is 41.39, 35.02, 14.00, and 9.59%, respectively. All of the protein-coding genes (PCGs) start with ATN. Five protein-coding genes use TAA as stop codons, four use TAG as stop codons, and others use incomplete stop codons ''T--'' or ''TA-''. The control region has a length of 1581 bp which is between rrnS and trnI genes with the AT content high to 85.96%. Phylogenetic analysis indicated that Tambocerus sp. was clustered in a closely related subgroup with Homalodisca vitripennis and Empoasca vitis. This is consistent with the result of the traditional taxonomy.

Complete mitochondrial genome of Empoasca vitis (Hemiptera: Cicadellidae).

The complete mitochondrial genome of Empoasca vitis was sequenced. The length of the mitogenome is 15,154 bp with 78.35% AT content (GenBank accession No. KJ815009). The genome encode 37 typical mitochondrial genes including 22 transfer RNA genes, 13 protein-coding genes, 2 ribosomal RNA genes and an A+T-rich region. The gene arrangement is similar to that of Drosophila yakuba, the presumed ancestral insect mitochondrial gene arrangement. Except for cox2 using GTG as start codon, other protein-coding genes (PCGs) share the start codons ATN. Usual termination codon TAA and incomplete stop codon T are using by 13 protein-coding genes. The A+T-rich region has a length of 977 bp with the AT content high to 88.95%.

Epigallocatechin-3-O-Gallate Protects Against Hepatic Damage and Testicular Toxicity in Male Mice Exposed to Di-(2-Ethylhexyl) Phthalate.

The aim of this study was to examine the effects of epigallocatechin-3-O-gallate (EGCG) on hepatic damage and testicular toxicity in male mice exposed to daily oral administration of di-(2-ethylhexyl) phthalate (DEHP). A mouse model was used to assess the effects of daily intraperitoneal EGCG injection on hepatic and testicular damage. Histological and mitochondrial membrane potential results revealed that EGCG treatment significantly arrested the progression of hepatic damage. EGCG treatment resulted in significant suppression of liver injury (i.e., reduced activities of alanine aminotransferase [ALT] and aspartate aminotransferase [AST]). The development of DEHP-induced hepatic and testicular damage altered the testosterone concentration in mouse serum, which could affect the reproductive ability of male mice. Moreover, EGCG treatment markedly attenuated testes lesions, sperm deformity, and spermatogenic cell apoptosis. At the molecular level, hepatic CYP3A4 expression was substantially reduced by EGCG treatment in mice exposed to DEHP compounds, whereas testicular aromatase expression was increased significantly in testes. Thus, these results demonstrate that EGCG administration may protect against liver damage and reproductive toxicity in males exposed to DEHP.

Mitochondrial COI and 16sRNA evidence for a single species hypothesis of E. vitis, J. formosana and E. onukii in East Asia.

Tea green leafhopper is one of the most damaging tea pests in main tea production regions of East Asia. For lack of recognized morphological characters, the dominant species of tea green leafhoppers in Mainland China, Taiwan and Japan have always been named as Empoasca vitis Göthe, Jacobiasca formosana Paoli and Empoasca onukii MATSUDA, respectively. Furthermore, nothing is known about the genetic relationships among them. In this study, we collected six populations from Mainland China, four populations from Japan and one population from Taiwan, and examined the genetic distances in the COI and 16sRNA regions of mtDNA among them. The results showed that the genetic distances based on single gene or the combined sequences among eleven leafhopper populations were 0.3-1.2%, which were all less than the species boundary of 2%. Moreover, there were at least two haplotypes shared by two distinct populations from different regions. The phylogenetic analysis based on single gene or combined sets also supported that tea green leafhoppers from Mainland China, Taiwan and Japan were closely related to each other, and there were at least two specimens from different regions clustered ahead of those from the same region. Therefore, we propose that the view of recognizing the dominant species of tea green leafhoppers in three adjacent tea production regions of East Asia as different species is unreliable or questionable and suggest that they are a single species.

Probing behavior of Empoasca vitis (Homoptera: Cicadellidae) on resistant and susceptible cultivars of tea plants.

Feeding activities of the tea green leafhopper, Empoasca vitis (Gothe) (Homoptera: Cicadellidae), on resistant and susceptible cultivars of tea plants (Camellia sinensis L.) were recorded and analyzed using the direct current electrical penetration graph (EPG) system. Six distinct EPG waveforms characterizing the feeding behavior of the tea green leafhopper, categorized as waveforms A, C, E, S, F, and R, were obtained during the investigation. Duration of passive ingestion, possibly of phloem (E), was the longest among all the probing waveforms on susceptible cultivars, whereas durations of the salivation (S) waveform and stylet work waveform (F) became longer on resistant cultivars. The durations of waveforms S and F on the resistant cultivar Jiandecha were slightly longer than those on the less resistant cultivar Yunguidaye, and both were significantly longer than those on the susceptible cultivars Hangzhoudaye and Zhushan-1. Waveform E was shorter on the resistant cultivar Jiandecha than on the less resistant cultivars Yunguidaye and was significantly shorter than on the susceptible cultivars (Hangzhoudaye and Zhushan-1). It is suggested that E, S, and F are the important waveforms related to leafhopper feeding behavior and tea plant resistance. Based on the results, the resistance levels of tea cultivars against the tea leafhopper can be evaluated quickly by direct current EPG.

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.

Green tea polysaccharide-conjugates protect human umbilical vein endothelial cells against impairments triggered by high glucose.

Hot-water extracts of low-grade green tea were precipitated with ethanol, deproteinized with trichloroacetic acid, neutralized with NaOH and fractionated by DEAE-cellulose DE-52 column chromatography to yield three (3) of unexplored polysaccharide-conjugate fractions termed gTPC1, gTPC2 and gTPC3. Monosaccharide and amino acid composition, contents of total neutral sugars, proteins and moistures, HPGPC distribution and Zeta potentials of gTPC1-3 were investigated. Exposure of human umbilical vein endothelial (HUVE) cells to high glucose (33 mM) for 12h significantly decreased cell viability relative to normal glucose control (p<0.001). As compared with cell injury group, gTPC1-3 at all of three dose levels (50, 150 and 300 µg/mL) were found to possess remarkably protective effects on HUVE cells against impairments induced by high glucose in a dose-dependent manner (p<0.05, p<0.001). To contribute toward our understanding of the cell-based protection mechanism of gTPC1-3, the latter were subjected to self-oxidation of 1,2,3-phentriol assay, and their scavenging effects were observed as 55.1%, 47.6% and 47.9% at the concentration of 300 µg/mL, respectively. On the basis of the fact that high glucose-induced endothelial dysfunction involves in the overproduction of reactive oxygen species (ROS) and contributes to the vascular complications in patients with diabetes, inhibitory effects of gTPC1-3 on high glucose-mediated HUVE cell loss are, at least in part, correlated with their potential scavenging potency of ROS. Taken together, gTPC1-3 could be developed as non-cytotoxic candidates of therapeutic agent for diabetic vascular complications.

Differential expression pattern of an acidic 9/13-lipoxygenase in flower opening and senescence and in leaf response to phloem feeders in the tea plant.

BACKGROUND: Lipoxygenase (LOXs) is a large family of plant enzymes that catalyse the hydroperoxidation of free polyunsaturated fatty acids into diverse biologically active compounds, collectively named phyto-oxylipins. Although multiple isoforms of LOXs have been identified in a wide range of annual herbaceous plants, the genes encoding these enzymes in perennial woody plants have not received as much attention. In Camellia sinensis (L.) O. Kuntze, no LOX gene of any type has been isolated, and its possible role in tea plant development, senescence, and defence reaction remains unknown. The present study describes the isolation, characterization, and expression of the first tea plant LOX isoform, namely CsLOX1, and seeks to clarify the pattern of its expression in the plant's defence response as well as in flower opening and senescence. RESULTS: Based on amino acid sequence similarity to plant LOXs, a LOX was identified in tea plant and named CsLOX1, which encodes a polypeptide comprising 861 amino acids and has a molecular mass of 97.8 kDa. Heterologous expression in yeast analysis showed that CsLOX1 protein conferred a dual positional specificity since it released both C-9 and C-13 oxidized products in equal proportion and hence was named 9/13-CsLOX1. The purified recombinant CsLOX1 protein exhibited optimum catalytic activity at pH 3.6 and 25°C. Real-time quantitative PCR analysis showed that CsLOX1 transcripts were detected predominantly in flowers, up-regulated during petal senescence, and down-regulated during flower bud opening. In leaves, the gene was up-regulated following injury or when treated with methyl jasmonate (MeJA), but salicylic acid (SA) did not induce such response. The gene was also rapidly and highly induced following feeding by the tea green leafhopper Empoasca vitis, whereas feeding by the tea aphid Toxoptera aurantii resulted in a pattern of alternating induction and suppression. CONCLUSIONS: Analysis of the isolation and expression of the LOX gene in tea plant indicates that the acidic CsLOX1 together with its primary and end products plays an important role in regulating cell death related to flower senescence and the JA-related defensive reaction of the plant to phloem-feeders.