Structurally diverse stilbenes from Gnetum parvifolium and their anti-neuroinflammatory activities.

Phytochemical investigation on the aerial parts of Gnetum parvifolium led to the isolation of 15 new and eight known structurally diverse stilbenes. The isolated compounds comprised (E)- or (Z)-stilbene (1-6, 15-20), dihydrostilbene (21), phenylbenzofuran (7, 8, 22), benzylated stilbene (9-11), benzylated stilbene dimer (12), and nitrogen-containing stilbene (13a, 13b, 14) types. The structures of the new compounds (1-12, 13a, 13b, 14) were established through spectroscopic analyses and experimental and calculated ECD data. Compound 12 is the first stilbene dimer connected through a benzyl group. In the anti-neuroinflammatory activity assay, compounds 4, 5, 9-11, 13b, and 16-21 displayed significant inhibitory effects against LPS-induced NO release in BV-2 microglial cells, with IC50 values of 0.35-16.1 µM. Compound 10 had the most potent activity (IC50 = 0.35 µM), and the further research indicated that it could decrease the mRNA levels of iNOS, IL-1ß, IL-6, and TNF-α in a dose-dependent manner.

Assessment of Biological Activities, Acute and Sub-chronic Toxicity of Liang (Gnetum gnemon var. tenerum) Leaves Powder, a Natural Product.

Gnetum gnemon var. tenerum (Gnetaceae) is a shrub plant native to South-East Asia. In Thailand, Liang leaves are commonly consumed in South of Thailand as vegetable. According to literature, they have an antihyperglycemic capacity because of their rich chlorophyll, fiber, and protein. However, there is need to assess the safety since natural food products are not completely devoid of toxicity. This study aimed to assess the biological activities as well as the acute and sub-chronic oral toxicity of Liang leaves powder (LLP). The evaluation of LLP for acute oral toxicity was performed at dose level 2000 mg/kg body weight in Wistar rats while the sub-chronic oral toxicity of LLP was performed at the effective dose (1.47 g/kg) for antihyperglycemic property according to Organisation for Economic Co-operation and Development (OECD)-425. The results showed that LLP demonstrated anti-inflammatory activities. It also showed no clinical signs of toxic effects and mortality in rats throughout 90 d. Thus, LLP could be classified in GHS category 5 which are of relatively low acute toxicity and then the lethal dose, 50% (LD50) cut off at 5000 mg/kg body weight to infinity (∞). Administration of LLP to the experimental rats significantly increased (p < 0.05) the concentration of triglyceride and increased concentration of creatinine as a result of kidney malfunction was also noticed in the experimental rats. Hematological alteration was not noticed in the treated female rats, but red blood cell, hemoglobin and hematocrit concentrations significantly increased in the treated male rats. The study concludes that sub-chronic administration of 1.47 g/kg LLP is relatively safe.

Effect of Resveratrol in Melinjo Seed (Gnetum gnemon L.) Extract on Type 2 Diabetes Mellitus Patients and its Possible Mechanism: A Review.

BACKGROUND AND OBJECTIVE: Diabetes mellitus is the third leading cause of death in Indonesia (6.7 %), followed by stroke (21.1 %) and coronary heart disease (12.9 %). The prevalence of diabetes worldwide continues to increase on a yearly basis, including in Indonesia. Diabetes is a significant burden for many countries due to the high costs of treatment and reduced productivity of diabetes patients. Comprehensive strategies to prevent and treat diabetes are therefore mandatory. Oral hypoglycemic drugs are the first-line therapy for diabetes mellitus patients; however, these oral drugs still have several side effects. Therefore, it is necessary to conduct studies on medicinal plants with hypoglycemic effects to identify substances that have an anti-diabetic potential resembling physiological processes in the body. Indonesian people often use herbal medicines empirically, but the benefits have not been scientifically documented. Melinjo (Gnetum gnemon L.) is a native Indonesian gymnosperm plant, and the seeds are often processed into food. Melinjo seeds extract contains many polyphenols, including trans-resveratrol. CONCLUSION: Studies on the health benefits of resveratrol are widely available, including antidiabetes and blood sugar control in patients with type 2 diabetes.

Gnetum montanum extract induces apoptosis by inhibiting the activation of AKT in SW480 human colon cancer cells.

CONTEXT: Gnetum montanum Markgr. (Gnetaceae) is used to treat rheumatic arthralgia and bruises in the clinic. OBJECTIVE: To exam the activity and mechanism of G. montanum extract (GME) against colon cancer cells SW480. MATERIALS AND METHODS: The anti-proliferative activity of GME (0-120 µg/mL) on SW480 cells was determined using MTS assay at 24, 48, and 72 h. The in vitro activity of GME (0-120 µg/mL) on SW480 cells was investigated using flow cytometry and western blotting analysis. The in vivo activity of GME was evaluated using xenograft tumour model of zebrafish and nude mice. The chemical composition of GME was detected by using HPLC-MS/MS. RESULTS: The IC50 value SW480 cells viability by GME were 126.50, 78.25, and 50.77 µg/mL, respectively, for 24, 48, and 72 h. The experiments showed that apoptotic cells and G2/M phase cells increased from 20.81 to 61.53% (p < 0.01) and 25.76 to 34.93% with 120 µg/mL GME, respectively. GME also down-regulated the protein expression of P-AKT, P-GSK-3ß, P-PDK1, P-c-Raf, caspase-3, and Bcl-2, and up-regulated the expression cleaved caspase-3, cleaved PARP, and Bax. In vivo study found that GME can significantly inhibit the growth and migration of SW480 cells in xenograft zebrafish. GME reduced the nude mice tumour weight to approximately 32.19% at 28 mg/kg/day and to 53.17% (p < 0.01) at 56 mg/kg/day. Forty-two compounds were identified from the GME. DISCUSSION AND CONCLUSIONS: GME has a significant antitumor effect on colon cancer cells SW480, and it has the potential to be developed as an anticancer agent.

Revealing the developmental dynamics in male strobilus transcriptome of Gnetum luofuense using nanopore sequencing technology.

Gnetum is a pantropical distributed gymnosperm genus. As being dioecious, Gnetum species apply female and male strobili to attract and provide nutrition to insect pollinators. Due to its unique gross morphology, a Gnetum male strobilus receives much attention in previous taxonomic and evolutionary studies. However, underlying molecular mechanisms that control male strobilus development and pollination adaptation have not been well studied. In the present study, nine full-length transcriptomes were sequenced from three developmental stages of the G. luofuense male strobili using Oxford Nanopore Technologies. In addition, weighted gene co-expression network analysis (WGCNA), and RT-qPCR analysis were performed. Our results show that a total of 3138 transcription factors and 466 long non-coding RNAs (lncRNAs) were identified, and differentially expressed lncRNAs and TFs reveal a dynamic pattern during the male strobilus development. Our results show that MADS-box and Aux/IAA TFs were differentially expressed at the three developmental stages, suggesting their important roles in the regulation of male strobilus development of G. luofuense. Results of WGCNA analysis and annotation of differentially expressed transcripts corroborate that the male strobilus development of G. luofuense is closely linked to plant hormone changes, photosynthesis, pollination drop secretion and reproductive organ defense. Our results provide a valuable resource for understanding the molecular mechanisms that drive organ evolution and pollination biology in Gnetum.

Deciphering the evolution of the ovule genetic network through expression analyses in Gnetum gnemon.

BACKGROUND AND AIMS: The ovule is a synapomorphy of all seed plants (gymnosperms and angiosperms); however, there are some striking differences in ovules among the major seed plant lineages, such as the number of integuments or the orientation of the ovule. The genetics involved in ovule development have been well studied in the model species Arabidopsis thaliana, which has two integuments and anatropous orientation. This study is approached from what is known in arabidopsis, focusing on the expression patterns of homologues of four genes known to be key for the proper development of the integuments in arabidopsis: AINTEGUMENTA (ANT), BELL1, (BEL1), KANADIs (KANs) and UNICORN (UCN). METHODS: We used histology to describe the morphoanatomical development from ovules to seeds in Gnetum gnemon. We carried out spatiotemporal expression analyses in G. gnemon, a gymnosperm, which has a unique ovule morphology with an integument covering the nucellus, two additional envelopes where the outermost becomes fleshy as the seed matures, and an orthotropous orientation. KEY RESULTS: Our anatomical and developmental descriptions provide a framework for expression analyses in the ovule of G. gnemon. Our expression results show that although ANT, KAN and UCN homologues are expressed in the inner integument, their spatiotemporal patterns differ from those found in angiosperms. Furthermore, all homologues studied here are expressed in the nucellus, revealing major differences in seed plants. Finally, no expression of the studied homologues was detected in the outer envelopes. CONCLUSIONS: Altogether, these analyses provide significant comparative data that allows us to better understand the functional evolution of these gene lineages, providing a compelling framework for evolutionary and developmental studies of seeds. Our findings suggest that these genes were most likely recruited from the sporangium development network and became restricted to the integuments of angiosperm ovules.

A new phenylheptanoid from the leaves of Gnetum gnemon L.

Phytochemical study on the EtOAC-soluble extract of the leaves of Gnetum gnemon furnished the isolation of a new phenylheptanoid, gnetumal (1), along with five known compounds (2-6). Their isolation was carried out by using the column chromatography and their structures were elucidated based on the basis of the spectral interpretation. Bioactivity assay of these compounds indicated that gnetumal (1) and p-coumaric acid (5) possessed more potent tyrosinase inhibitory activity, with IC50 values of 31.6 and 2.3 µM, respectively, than that of a positive control kojic acid (IC50; 44.6 µM).

A full-length transcriptome and gene expression analysis reveal genes and molecular elements expressed during seed development in Gnetum luofuense.

BACKGROUND: Gnetum is an economically important tropical and subtropical gymnosperm genus with various dietary, industrial and medicinal uses. Many carbohydrates, proteins and fibers accumulate during the ripening of Gnetum seeds. However, the molecular mechanisms related to this process remain unknown. RESULTS: We therefore assembled a full-length transcriptome from immature and mature G. luofuense seeds using PacBio sequencing reads. We identified a total of 5726 novel genes, 9061 alternative splicing events, 3551 lncRNAs, 2160 transcription factors, and we found that 8512 genes possessed at least one poly(A) site. In addition, gene expression comparisons of six transcriptomes generated by Illumina sequencing showed that 14,323 genes were differentially expressed from an immature stage to a mature stage with 7891 genes upregulated and 6432 genes downregulated. The expression of 14 differentially expressed transcription factors from the MADS-box, Aux/IAA and bHLH families was validated by qRT-PCR, suggesting that they may have important roles in seed ripening of G. luofuense. CONCLUSIONS: These findings provide a valuable molecular resource for understanding seed development of gymnosperms.

Endophytes from Gnetum gnemon L. can protect seedlings against the infection of phytopathogenic bacterium Ralstonia solanacearum as well as promote plant growth in tomato.

Endophytes are beneficial plant microbes which help the plants by producing various plant growth promoting substances and also by acting as biocontrol agents against various plant pathogens. In the present study, evaluation of endophytic bacteria isolated from Gnetum gnemon, an ethnomedicinal plant was carried out for their plant growth promoting (PGP) activity and antagonistic potential against bacterial wilt pathogen Ralstonia solanacearum. Initially a total of 40 endophytic bacteria were isolated which were clustered into 13 groups based on RFLP and BOX-PCR fingerprinting. These 13 representative isolates belonged to different genera of Bacillus (9), Solibacillus (1), Staphylococcus (2) and Caballeronia (1). Among them, six isolates were positive for production of IAA, the value of which ranged from 11.16 to 27.63 µg mL-1. Phosphate solubilisation in the range of 106.4 to -212.7 µg mL-1 was shown by three isolates. Eight isolates producedammonia, the value of which ranged from 1.3 to 6.1 µmol mL-1. All tested isolates were positive for siderophore production. For extracellular enzyme production, 7 isolates were positive for protease, 8 for cellulase and 10 for amylase production. The isolates were also tested for their antagonistic activity against R. solanacearum in vitro and in planta assay using tomato seedlings. Staphylococcus warneri GL1 showed the highest biocontrol efficacy of 77.67 % followed by Bacillus velezensis GL3 i.e. 70.1 %. R. solanacearum antagonistic isolates were analysed for the presence of antimicrobial peptide biosynthesis genes bmyB, srfAA, fenD and ituC. All the antagonistic isolates showed the presence of all four genes, except the isolate Bacillus velezensis GMC2, where the gene for fengycin synthetase (fenD) was absent. Based on in vitro PGP traits, three isolates Bacillus velezensis GL3, Bacillus atrophaeus GMC1 and Bacillus megaterium GS2 were selected, these three endophytic bacteria individually and their consortia were tested for in planta PGP activities in tomato plants. Application of Bacillus velezensis GL3 alone and consortia of three isolates showed significant improvement in growth parameters such as shoot length, fresh weight and dry weight in a pot experiment. Colonization of endosphere of treated tomato seedlings by the endophytic isolate Bacillus velezensis GL3 was confirmed by visualization of colony morphology and BOX-PCR fingerprinting. Our study highlights the potential of endophytes associated with unexplored plants like G. gnemon for development of bioformulation aimed at enhancing plant growth and bacterial wilt disease control.

Melinjo seed extract increases adiponectin multimerization in physiological and pathological conditions.

Melinjo seed extract (MSE) contains large amounts of polyphenols, including dimers of trans-resveratrol (e.g. gnetin C, L, gnemonoside A, B and D), and has been shown to potentially improve obesity. However, there is no clinical evidence regarding the anti-obesity effects of MSE, and its mechanisms are also unclear. We investigated the hypothesis that MSE supplementation increases the adiponectin (APN) multimerization via the up-regulation of disulfide bond A oxidoreductase-like protein (DsbA-L) under either or both physiological and obese conditions. To investigate the effect of MSE on the physiological condition, 42 healthy young volunteers were enrolled in a randomized, double-blind placebo-controlled clinical trial for 14 days. The participants were randomly assigned to the MSE 150 mg/day, MSE 300 mg/day or placebo groups. Furthermore, in order to investigate the effect of MSE on APN levels under obese conditions, we administered MSE powder (500 or 1000 mg/kg/day) to control-diet- or high-fat-diet (HFD)-fed C57BL/6 mice for 4 weeks. All participants completed the clinical trial. The administration of MSE 300 mg/day was associated with an increase in the ratio of HMW/total APN in relation to the genes regulating APN multimerization, including DsbA-L. Furthermore, this effect of MSE was more pronounced in carriers of the DsbA-L rs191776 G/T or T/T genotype than in others. In addition, the administration of MSE to HFD mice suppressed their metabolic abnormalities (i.e. weight gain, increased blood glucose level and fat mass accumulation) and increased the levels of total and HMW APN in serum and the mRNA levels of ADIPOQ and DsbA-L in adipose tissue. The present study suggests that MSE may exert beneficial effects via APN multimerization in relation to the induction of DsbA-L under both physiological and obese conditions.

Single-Molecule Long-Read Sequencing Reveals the Diversity of Full-Length Transcripts in Leaves of Gnetum (Gnetales).

The limitations of RNA sequencing make it difficult to accurately predict alternative splicing (AS) and alternative polyadenylation (APA) events and long non-coding RNAs (lncRNAs), all of which reveal transcriptomic diversity and the complexity of gene regulation. Gnetum, a genus with ambiguous phylogenetic placement in seed plants, has a distinct stomatal structure and photosynthetic characteristics. In this study, a full-length transcriptome of Gnetum luofuense leaves at different developmental stages was sequenced with the latest PacBio Sequel platform. After correction by short reads generated by Illumina RNA-Seq, 80,496 full-length transcripts were obtained, of which 5269 reads were identified as isoforms of novel genes. Additionally, 1660 lncRNAs and 12,998 AS events were detected. In total, 5647 genes in the G. luofuense leaves had APA featured by at least one poly(A) site. Moreover, 67 and 30 genes from the bHLH gene family, which play an important role in stomatal development and photosynthesis, were identified from the G. luofuense genome and leaf transcripts, respectively. This leaf transcriptome supplements the reference genome of G. luofuense, and the AS events and lncRNAs detected provide valuable resources for future studies of investigating low photosynthetic capacity of Gnetum.

Pollination Drop Proteome and Reproductive Organ Transcriptome Comparison in Gnetum Reveals Entomophilous Adaptation.

Gnetum possesses morphologically bisexual but functionally unisexual reproductive structures that exude sugary pollination drops to attract insects. Previous studies have revealed that the arborescent species (G. gnemon L.) and the lianoid species (G. luofuense C.Y.Cheng) possess different pollination syndromes. This study compared the proteome in the pollination drops of these two species using label-free quantitative techniques. The transcriptomes of fertile reproductive units (FRUs) and sterile reproductive units (SRUs) for each species were furthermore compared using Illumina Hiseq sequencing, and integrated proteomic and transcriptomic analyses were subsequently performed. Our results show that the differentially expressed proteins between FRUs and SRUs were involved in carbohydrate metabolism, the biosynthesis of amino acids and ovule defense. In addition, the differentially expressed genes between the FRUs and SRUs (e.g., MADS-box genes) were engaged in reproductive development and the formation of pollination drops. The integrated protein-transcript analyses revealed that FRUs and their exudates were relatively conservative while the SRUs and their exudates were more diverse, probably functioning as pollinator attractants. The evolution of reproductive organs appears to be synchronized with changes in the pollination drop proteome of Gnetum, suggesting that insect-pollinated adaptations are not restricted to angiosperms but also occur in gymnosperms.

Screening and Evaluation of Xanthine Oxidase Inhibitors from Gnetum parvifolium in China.

As a traditional natural medicine for treating many kinds of diseases, Gnetum parvifolium showed apparent inhibition on xanthine oxidase (XO). In this study, ultrafiltration combined with liquid chromatography-mass spectrometry (LC-MS) is used for the screening of XO inhibitors from Gnetum parvifolium. Their antioxidation, XO inhibition, and enzymic kinetic parameters are also determined. Finally, piceatannol (1), rhaponiticin (2), resveratrol (3), and isorhapontigenin (4) are screened out and identified as XO inhibitors from the extract of Gnetum parvifolium. Four inhibitors show better inhibition than allopurinol and good radical scavenging abilities. However, the antioxidant activities are weaker than ascorbic acid. The kinetic parameters illustrate the inhibition mode of XO by piceatannol is competitive type, while the inhibition modes for rhaponiticin, resveratrol and isorhapontigenin are uncompetitive types. In order to evaluate the difference among samples obtained in China, the amounts of four inhibitors and related activities in 20 samples are assessed and analyzed by partial least squares analysis. The results indicate piceatannol contribute the highest coefficients in three kinds of activities. Based on these findings, more comprehensive research on pharmaceutical and biochemical activities of these four XO inhibitors could be conducted in future.

Oligostilbenes from the leaves of Gnetum latifolium and their biological potential to inhibit neuroinflammation.

Oligostilbenes are polyphenol oligomers derived from resveratrol and are commonly produced by members of the Gnetaceae family, and many researchers have focused on their anti-inflammatory activities. The EtOAc fraction of a Gnetum latifolium extract showed inhibitory activity against neuroinflammation induced by the transfection of Aß1-42 into microglial BV-2 cells. The bioassay-guided isolation of the 70% EtOH extract of this plant resulted in three previously undescribed resveratrol oligostilbenes and ten known stilbene derivatives. The structures of the isolated compounds were established based on extensive NMR spectroscopic analysis. The absolute configurations of the three undescribed compounds were confirmed by comparison with available compounds with known stereochemistry and by ECD calculations and molecular modelling. Latifoliols A and B are the first reported oligostilbenes with a bridged 3-oxabicyclo[3.3.0]octane moiety, and latifoliol C was formed by the condensation of gnemontanin G with oxyresveratrol. Moreover, the hypothetical biogenetic pathway of latifoliols A, B and C was proposed. The potential anti-inflammatory activities of the thirteen isolated compounds were tested by measuring their effect on the secreted NO concentrations induced by transfection with plasmids expressing the Aß1-42 gene in the BV-2 cell line. Interestingly, cis- and trans-shegansu B and latifolol, whose structures contained double bonds, strongly inhibited NO secretion in BV-2 cells, supporting the double binding effect of the stilbene derivative on inhibitory activity.

Dietary intake, forest foods, and anemia in Southwest Cameroon.

BACKGROUND: Forest cover has been associated with higher dietary diversity and better diet quality in Africa. Anemia prevalence among women of reproductive age in sub-Saharan Africa is very high and diet is one known contributor of a high prevalence rate. We investigated whether living in communities with high forest cover was associated with better diet quality and lower anemia prevalence among women of reproductive age in Southwest Cameroon. METHODOLOGY: We conducted a cross-sectional survey of 247 women of reproductive age from four forest-based villages (n = 126) and four non-forest villages (n = 121). We assessed the Hemoglobin (Hb) levels, anthropometric status, and diet (by 24-hour recall), as well as anemia-related morbidity and socio-demographic characteristics. Differences between groups were assessed with Pearson's chi-square and independent T-tests. We used a number of multivariate regression models to estimate the impacts of forest proximity on adjusted hemoglobin status of women of reproductive age, as well as to identify the most likely pathway through which forest proximity was important. RESULTS: We found that women living in forest communities had higher adjusted hemoglobin levels (mean hemoglobin concentration 11.10±1.53 g/dl vs.10.68±1.55g/dl; p = 0.03 for women forest and non-forest communities respectively). Moderate to severe anemia prevalence was significantly higher in women living in the non-forest villages compared to women in forest villages (forest 63% vs. 73%; p = 0.04). Compared with women from non-forest villages, women from forest-based villages had consumed significantly more vitamin A rich fruits and vegetables and animal source foods, and more of these came from the forest (as opposed to the farm or purchased sources). We found that the consumption of Gnetum africanum (Eru), a leafy green vegetable that grows in forests of the Congo Basis, was best able to account for the higher levels of adjusted hemoglobin in women in forest communities. CONCLUSION: This study contributes to the growing evidence that in some circumstances, forests make important contributions to diet quality and nutrition. The results of this study suggest that plant foods from the forest may make important contributions to iron intake and reduce the risk of anemia in women. Efforts to prevent forest loss and maintain ecosystem services are warranted to enhance nutrition and health of forest-based communities.

Two Independent Plastid accD Transfers to the Nuclear Genome of Gnetum and Other Insights on Acetyl-CoA Carboxylase Evolution in Gymnosperms.

Acetyl-CoA carboxylase (ACCase) is the key regulator of fatty acid biosynthesis. In most plants, ACCase exists in two locations (cytosol and plastids) and in two forms (homomeric and heteromeric). Heteromeric ACCase comprises four subunits, three of them (ACCA-C) are nuclear encoded (nr) and the fourth (ACCD) is usually plastid encoded. Homomeric ACCase is encoded by a single nr-gene (ACC). We investigated the ACCase gene evolution in gymnosperms by examining the transcriptomes of newly sequenced Gnetum ula, combined with 75 transcriptomes and 110 plastomes of other gymnosperms. AccD-coding sequences are elongated through the insertion of repetitive DNA in four out of five cupressophyte families (except Sciadopityaceae) and were functionally transferred to the nucleus of gnetophytes and Sciadopitys. We discovered that, among the three genera of gnetophytes, only Gnetum has two copies of nr-accD. Furthermore, using protoplast transient expression assays, we experimentally verified that the nr-accD precursor proteins in Gnetum and Sciadopitys can be delivered to the plastids. Of the two nr-accD copies of Gnetum, one dually targets plastids and mitochondria, whereas the other potentially targets plastoglobuli. The distinct transit peptides, gene architectures, and flanking sequences between the two Gnetum accDs suggest that they have independent origins. Our findings are the first account of two distinctly targeted nr-accDs of any green plants and the most comprehensive analyses of ACCase evolution in gymnosperms to date.

Melinjo (Gnetum gnemon L.) seed extract induces uncoupling protein 1 expression in brown fat and protects mice against diet-induced obesity, inflammation, and insulin resistance.

Dietary supplementation with melinjo (Gnetum gnemon L.) seed extract (MSE) has been proposed as an anti-obesity strategy. However, it remains unclear how MSE modulates energy balance. We tested the hypothesis that dietary MSE reduces energy intake and/or increases physical activity and metabolic thermogenesis in brown and white adipose tissue (BAT and WAT) in mice. Twenty-four C57BL/6 J mice were provided with normal diet, high-fat diet (HFD), or HFD with 1% MSE added, for 17 weeks. Food intake, spontaneous locomotor activity, hepatic triglyceride (TG) content, and blood parameters were examined. Mitochondrial thermogenesis-associated molecule and inflammatory marker expression levels in BAT and WAT were examined by quantitative PCR and western blotting. Dietary MSE did not affect energy intake or spontaneous locomotor activity, but significantly suppressed HFD-induced fat accumulation, hyperglycemia, and hyperinsulinemia. Homeostasis model assessment of insulin resistance score and hepatic TG content were both lower in the MSE-supplemented HFD-fed group than in the HFD-fed group, indicating reduced insulin resistance and a less fatty liver. Dietary MSE upregulated thermogenic uncoupling protein 1 (UCP1) and mitochondrial marker cytochrome c oxidase subunit IV protein expression in BAT; this was closely associated with sirtuin 1 mRNA induction. mRNAs of adipose inflammatory markers, such as monocyte chemotactic 1 and interleukin-1, were induced by HFD but suppressed by MSE. Considering that UCP1 protein expression is the most physiologically relevant parameter to assess the thermogenic capacities of BAT, our results indicate that dietary MSE supplementation induces BAT thermogenesis and reduces obesity-associated adipose tissue inflammation, hepatic steatosis, and insulin resistance.

Ratio of sugar concentrations in the phloem sap and the cytosol of mesophyll cells in different tree species as an indicator of the phloem loading mechanism.

MAIN CONCLUSION: Sucrose concentration in phloem sap was several times higher than in the cytosol of mesophyll cells. The results suggest that phloem loading involves active steps in the analyzed tree species. Phloem loading in source leaves is a key step for carbon partitioning and passive symplastic loading has been proposed for several tree species. However, experimental evidence to prove the potential for sucrose diffusion from mesophyll to phloem is rare. Here, we analyzed three tree species (two angiosperms, Fagus sylvatica, Magnolia kobus, and one gymnosperm, Gnetum gnemon) to investigate the proposed phloem loading mechanism. For this purpose, the minor vein structure and the sugar concentrations in phloem sap as well as in the subcellular compartments of mesophyll cells were investigated. The analyzed tree species belong to the open type minor vein subcategory. The sucrose concentration in the cytosol of mesophyll cells ranged between 75 and 165 mM and was almost equal to the vacuolar concentration. Phloem sap could be collected from F. sylvatica and M. kobus and the concentration of sucrose in phloem sap was about five- and 11-fold higher, respectively, than in the cytosol of mesophyll cells. Sugar exudation of cut leaves was decreased by p-chloromercuribenzenesulfonic acid, an inhibitor of sucrose-proton transporter. The results suggest that phloem loading of sucrose in the analyzed tree species involves active steps, and apoplastic phloem loading seems more likely.

Proteome-Level Analysis of Metabolism- and Stress-Related Proteins during Seed Dormancy and Germination in Gnetum parvifolium.

Gnetum parvifolium is a rich source of materials for traditional medicines, food, and oil, but little is known about the mechanism underlying its seed dormancy and germination. In this study, we analyzed the proteome-level changes in its seeds during germination using isobaric tags for relative and absolute quantitation. In total, 1,040 differentially expressed proteins were identified, and cluster analysis revealed the distinct time points during which signal transduction and oxidation-reduction activity changed. Gene Ontology analysis showed that "carbohydrate metabolic process" and "response to oxidative stress" were the main enriched terms. Proteins associated with starch degradation and antioxidant enzymes were important for dormancy-release, while proteins associated with energy metabolism and protein synthesis were up-regulated during germination. Moreover, protein-interaction networks were mainly associated with heat-shock proteins. Furthermore, in accord with changes in the energy metabolism- and antioxidant-related proteins, indole-3-acetic acid, Peroxidase, and soluble sugar content increased, and the starch content decreased in almost all six stages of dormancy and germination analyzed (S1-S6). The activity of superoxide dismutase, abscisic acid, and malondialdehyde content increased in the dormancy stages (S1-S3) and then decreased in the germination stages (S4-S6). Our results provide new insights into G. parvifolium seed dormancy and germination at the proteome and physiological levels, with implications for improving seed propagation.