Identification and analysis of MATE protein family in Gleditsia sinensis.

Many studies have shown that multidrug and toxic compound extrusion (MATE) is a new secondary transporter family that plays a key role in secondary metabolite transport, the transport of plant hormones and disease resistance in plants. However, detailed information on this family in Gleditsia sinensis has not yet been reported. In the present study, a total of 45 GsMATE protein members were identified and analysed in detail, including with gene classification, phylogenetic evaluation and conserved motif determination. Phylogenetic analysis showed that GsMATE proteins were divided into six subfamilies. Additionally, in order to understand these members' regulatory roles in growth and development in G. sinensis , the GsMATEs expression profiles in different tissues and different developmental stages of thorn were examined in transcriptome data. The results of this study demonstrated that the expression of all MATE genes varies in roots, stems and leaves. Notably, the expression levels of GsMATE26 , GsMATE32 and GsMATE43 differ most in the early stages of thorn development, peaking at higher levels than in later stages. Our results provide a foundation for further functional characterisation of this important class of transporter family in G. sinensis .

Transcriptome analysis reveals regulatory mechanisms of different drought-tolerant Gleditsia sinensis seedlings under drought stress.

BACKGROUND: Gleditsia sinensis is a significant tree species from both ecological and economic perspectives. However, its growth is hampered by temporary droughts during the seedling stage, thereby impeding the development of the G. sinensis industry. Drought stress and rehydration of semi-annual potted seedlings using an artificial simulated water control method. RNA sequencing (RNA-seq) analyses were conducted on leaves collected from highly resistant (HR) and highly susceptible (HS) seedling families at five different stages during the process of drought stress and rehydration to investigate their gene expression patterns. RESULTS: The differentially expressed genes (DEGs) were predominantly enriched in pathways related to "chloroplast" (GO:0009507), "photosynthesis" (GO:0015979), "plant hormone signal transduction" (map04075), "flavonoid biosynthesis" (map00941), "stress response", "response to reactive oxygen species (ROS)" (GO:0000302), "signal transduction" (GO:0007165) in G. sinensis HR and HS families exposed to mild and severe drought stress. Additionally, the pathways related to "plant hormone signal transduction" (map04075), and osmoregulation were also enriched. The difference in drought tolerance between the two families of G. sinensis may be associated with "transmembrane transporter activity" (GO:0022857), "stress response", "hormones and signal transduction" (GO:0007165), "cutin, suberine and wax biosynthesis" (map00073), "ribosome" (map03010), "photosynthesis" (map00195), "sugar metabolism", and others. An enrichment analysis of DEGs under severe drought stress suggests that the drought tolerance of both families may be related to "water-soluble vitamin metabolic process" (GO:0006767), "photosynthesis" (map00195), "plant hormone signal transduction" (map04075), "starch and sucrose metabolism" (map00500), and "galactose metabolism" (map00052). Osmoregulation-related genes such as delta-1-pyrroline-5-carboxylate synthase (P5CS), Amino acid permease (AAP), Amino acid permease 2 (AAP2) and Trehalose-phosphate synthase (TPS), as well as the antioxidant enzyme L-ascorbate peroxidase 6 (APX6), may be significant genes involved in drought tolerance in G. sinensis. Five genes were selected randomly to validate the RNA-seq results using quantitative real-time PCR (RT-qPCR) and they indicated that the transcriptome data were reliable. CONCLUSIONS: The study presents information on the molecular regulation of the drought tolerance mechanism in G. sinensis and provides a reference for further research on the molecular mechanisms involved in drought tolerance breeding of G. sinensis.

Gleditsia sinensis Lam. aqueous extract attenuates nasal inflammation in allergic rhinitis by inhibiting MUC5AC production through suppression of the STAT3/STAT6 pathway.

Allergic rhinitis (AR), a chronic respiratory inflammatory disease, is among the most common chronic diseases reported worldwide. Mucus hypersecretion is a critical feature of AR pathogenesis. Although the Gleditsia sinensis extract has several beneficial effects on human health, its effects on allergic inflammation have not yet been investigated. In this study, we examined the effects of G. sinensis aqueous extract (GSAE) on nasal inflammation in an ovalbumin (OVA)-induced AR mouse model. GSAE was administered orally for 1 week and then the clinical nasal symptoms were evaluated. The levels of histamine, OVA-specific immunoglobulin (Ig) E, and interleukin (IL)-13 were measured in the serum using an enzyme-linked immunosorbent assay (ELISA). Inflammatory cells were then counted in the nasal lavage fluid (NALF) and histopathology in the nasal epithelium was evaluated. STAT3/STAT6 phosphorylation was examined in primary human nasal epithelial cells (HNEpCs) using western blot analysis. Oral administration of GSAE to OVA-induced AR mice alleviated nasal clinical symptoms and reduced OVA-specific immunoglobulin E, interleukin (IL)-13, and histamine levels. The accumulation of eosinophils in nasal lavage fluid, nasal mucosa, mast cells, goblet cells, and mucin 5AC (MUC5AC) in the nasal epithelium was also inhibited by GSAE. Treatment with GSAE inhibited the production of MUC5AC in IL-4/IL-13-stimulated primary human nasal epithelial cells through the signal transducer and activator of transcription (STAT)3/STAT6 signaling pathway. These results indicated that GSAE reduces nasal inflammation suggesting that it is a potential treatment option for AR.

Polysaccharides from the seeds of Gleditsia sinensis Lam. attenuate DSS-induced colitis in mice via improving gut barrier homeostasis and alleviating gut microbiota dybiosis.

The seeds from Gleditsia sinensis Lam., a common ecologically and economically useful tree, have high economic and nutritional value. The protective effect of polysaccharides from Gleditsia sinensis Lam. seeds (ZJMP) against dextran sulfate sodium-induced colitis in mice was investigated in this study. ZJMP alleviated weight loss, reduced the disease activity index, prevented colon shortening, alleviated colonic tissue damage, and restored goblet cell secretion in colitic mice. Dietary ZJMP reduced proinflammatory cytokine overproduction in the colonic mucosa and serum, which was accompanied by suppression of NO levels and MPO and SOD activities. The addition of ZJMP increased the expression of Muc2 and tight junction proteins. Furthermore, dietary ZJMP partially reversed the alteration of gut microbiota in colitic mice by boosting the abundance of beneficial bacteria like Akkermansia, Lactobacillus, and Christensenella while lowering the abundance of harmful bacteria like Bacteroides, Prevotella, and Mucispirillum. Additionally, the decreased production of short-chain fatty acids in the colitic mice was recovered by ZJMP administration. The findings demonstrated the anti-inflammatory properties and mechanism of dietary ZJMP in the colon, which is essential for the sensible application of ZJMP in the prevention and amelioration of inflammation-related diseases as a nutritional supplement.

Production of manno-oligosaccharide from Gleditsia microphylla galactomannan using acetic acid and ferrous chloride.

A novel and efficient method for manno-oligosaccharides (MOS) production has been proposed by utilizing Gleditsia microphylla galactomannan as the starting material. This co-operative hydrolysis using ferrous chloride (Fe2+) and acetic acid (HAc) effectively improved the MOS yield and meanwhile decreased the amount of monosaccharide and the 5-hydroxymethyl-furfural (HMF). The highest yields under the optimum conditions were 46.7% by HAc hydrolysis (5 M HAc at 130 °C for 120 min); 37.3% by Fe2+ hydrolysis (0.1 M Fe2+ at 150 °C for 120 min); and 51.4% by co-operative hydrolysis (2 M HAc, 0.05 M Fe2+ at 160 °C for 10 min). From the changes in the value of M/G (mannose/galactose) ratios, it was deduced that Fe2+ predominantly cleaves the main chain, and HAc assists in the breakage of the side chain, thus resulting in the high-efficient co-operative hydrolysis for the production of MOS.

Valorization of Gleditsia triacanthos Invasive Plant Cellulose Microfibers and Phenolic Compounds for Obtaining Multi-Functional Wound Dressings with Antimicrobial and Antioxidant Properties.

Gleditsia triacanthos is an aggressive invasive species in Eastern Europe, producing a significant number of pods that could represent an inexhaustible resource of raw material for various applications. The aim of this study was to extract cellulose from the Gleditsia triacanthos pods, characterize it by spectrophotometric and UHPLC-DAD-ESI/MS analysis, and use it to fabricate a wound dressing that is multi-functionalized with phenolic compounds extracted from the leaves of the same species. The obtained cellulose microfibers (CM) were functionalized, lyophilized, and characterized by ATR-FTIR and SEM. The water absorption and retention capacity as well as the controlled release of phenolic compounds with antioxidant properties evaluated in temporal dynamics were also determined. The antimicrobial activity against reference and clinical multi-drug-resistant Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Acinetobacter baumannii, Enterobacter cloacae, Candida albicans, and Candida parapsilosis strains occurred immediately after the contact with the tested materials and was maintained for 24 h for all tested microbial strains. In conclusion, the multi-functionalized cellulose microfibers (MFCM) obtained from the reproductive organs of an invasive species can represent a promising alternative for the development of functional wound dressings with antioxidant and antimicrobial activity, as well as being a scalable example for designing cost-effective, circular bio-economy approaches to combat the accelerated spread of invasive species.

Comparison and characterization of galactomannan at different developmental stages of Gleditsia sinensis Lam.

Pods from a Gleditsia sinensis Lam. tree were collected and the galactomannan content and other properties were determined at their different developmental stages. In green and immature seed, galactomannan was substituted to a great extent with a mannose to galactose (M/G) ratio of 2.4 from crude polysaccharides. During late galactomannan deposition, it was substituted to a lower extent and this ratio increased rapidly, reaching a M/G ratio of 3.1. Average molecular weight (Mw) of the extracted polysaccharides first increased, reached the maximum (1.19 × 106) at 17 weeks after flowering (WAF), and then decreased. These changes might result from primary galactomannan biosynthesis and from galactose removal by α-galactosidase in the endosperm. The solubility of crude polysaccharides decreased with increased M/G ratio and maximum solubility was more than 89% that collected at 13 WAF. Rheological properties showed that apparent viscosity was largely influenced by the molecular weight and M/G ratio of galactomannans.

Identification of potential genes involved in triterpenoid saponins biosynthesis in Gleditsia sinensis by transcriptome and metabolome analyses.

Gleditsia sinensis is widely used as a medicinal plant in Asia, especially in China. Triterpenes, alkaloids, and sterols were isolated from Gleditsia species. Among them, triterpenoid saponins are very important metabolites owing to their various pharmacological activities. However, the triterpenoid saponin biosynthesis pathway has not been well characterized. In the present study, we performed de novo transcriptome assembly for 14.3 Gbps of clean reads sequenced from nine tissues of G. sinensis. The results showed that 81,511 unique transcripts (unitranscripts) (47,855 unigenes) were constructed, of which 31,717 unigenes were annotated with Gene Ontology and EC numbers by Blast2GO against the NCBI-nr protein database. We also analyzed the metabolite contents in the same nine tissues by LS-MS/MS, and saponins including gleditsioside I were found in fruit at higher levels. Many of the genes with tissue-specific expression in fruit are involved in the flavonoid biosynthesis pathway, and many of those have UDP-glucosyltransferase (UGT) activity. We constructed a saponin biosynthesis pathway and identified two key enzyme families in the triterpenoid saponin biosynthesis pathway, cytochrome P450 and UDP-glucosyltransferase, that are encoded by 37 unigenes and 77 unigenes, respectively. CYP72A, CYP716A, and CYP88D, which are known as key enzymes for saponin biosynthesis, were also identified among the P450s. Our results provide insight into the secondary metabolite biosynthesis and serve as important resources for future research and cultivation of G. sinensis.

Gleditsia sinensis thorn extract inhibits the proliferation and migration of PDGF­induced vascular smooth muscle cells.

The thorns of Gleditsia sinensis have been used to prevent or treat numerous diseases. The present study aimed to investigate the molecular mechanism of the ethanol extract of Gleditsia sinensis thorns (EEGS) on platelet-derived growth factor (PDGF)­treated vascular smooth muscle cells (VSMCs). EEGS treatment was found to inhibit DNA synthesis in PDG\F-treated VSMCs in a dose-dependent manner, without cell toxicity. These inhibitory effects were associated with G1-phase cell-cycle arrest, which was caused by the decreased expression of cyclins and cyclin-dependent kinases (CDKs) and the upregulation of p27KIP1 expression in PDGF-stimulated VSMCs. Among the pathways examined, EEGS treatment was observed to only inhibit the PDGF­induced phosphorylation of Akt. In addition, EEGS treatment suppressed the migration and invasion of VSMCs in the presence of PDGF as determined by wound-healing and Matrigel™ invasion assays. Furthermore, zymographic and western blot analyses revealed that EEGS treatment suppressed matrix metalloproteinase (MMP)-9 expression in PDGF­treated VSMCs, which was attributed to a reduction in the binding activities of nuclear factor κ-light-chain-enhancer of activated B cells (NF­κB), activator protein (AP)­1 and specificity protein (Sp)­1. These results demonstrate that EEGS induces p27KIP1­mediated G1-phase cell-cycle arrest, reduces Akt phosphorylation and prevents MMP­9 expression by decreasing the binding activities of NF­κB, AP­1 and Sp­1 in PDGF-treated VSMCs, thus resulting in growth inhibition and the suppression of migration and invasion. These results may suggest a novel perspective for the use of EEGS in the treatment and prevention of vascular proliferative diseases.

[Effects of NaCl stress on the seedling growth and K(+)- and Na(+) -allocation of four leguminous tree species].

Taking the pot-cultured seedlings of four leguminous tree species (Albizia julibrissin, Robinia pseudoacacia, Sophora japonica, and Gleditsia sinensis) as test materials, this paper studied their growth indices, critical salt concentration (C50), and K+ and Na+ allocation under different levels of NaCl stress, aimed to understand the difference of test tree species in salt tolerance. NaCl stress inhibited the seedling growth of the tree species. Under NaCl stress, the dry matter accumulation decreased, while the root/shoot ratio increased, especially for A. julibrissin and G. sinensis. Quadratic regression analysis showed that the C50 of A. julibrissin, R. pseudoacacia, S. japonica, and G. sinensis was 3.0 per thousand, 5.0 per thousand, 4.5 per thousand, and 3.9 per thousand, respectively, i.e., the salt tolerance of the four tree species was in the order of R. pseudoacacia > S. japonica > G. sinensis > A. julibrissin. In the root, stem, and leaf of the four tree species seedlings, the Na+ content increased with the increase of NaCl stress, while the K+ content (except in the root of A. julibrissin) decreased after an initial increase, resulting in a larger difference in the K+/Na+ ratio in the organs. Under the same NaCl stress, the allocation of Na+ in different organs of the four tree species seedlings decreased in the order of root>stem>leaf, while that of K+ differed with tree species and NaCl stress, and leaf was the main storage organ for K+. The K+/Na+ ratio in different organs decreased in the sequence of leaf>stem>root. R. pseudoacacia under NaCl stress accumulated more K+ and less Na+ in stem and leaf, and had higher K+/Na+ ratio in all organs and higher dry mass, being assessed to be more salt-tolerant. In contrast, A. julibrissin under high NaCl stress accumulated more Na+ in stem and leaf, and had a lower K+/Na+ ratio in all organs and lower dry mass, being evaluated to be lesser salt-tolerant. The K+ accumulation in seedling stem and leaf and the Na+ retention in seedling root could be the main reasons for the salt tolerance of leguminous tree species under NaCl stress.

Shelf life extension of ricotta cheese using coatings of galactomannans from nonconventional sources incorporating nisin against Listeria monocytogenes.

Shelf life extension of Ricotta cheese was evaluated at 4 degrees C upon the use of edible coatings made of galactomannans from Gleditsia triacanthos incorporating nisin against Listeria monocytogenes. Three different treatments were tested in cheese: samples without coating; samples with coating without nisin; and samples with coating containing 50 IU x g(-1) of nisin. To test the effectiveness of the treatments against L. monocytogenes, the surface of the cheese was inoculated with a suspension of the microorganism. Microbiological and physical-chemical analyses of the cheese samples were performed during 28 days. Results showed that the cheese coated with nisin-added galactomannan film was the treatment presenting the best results in terms of microbial growth delay (p < 0.05). The addition of nisin also affects (p < 0.05) the physical and mechanical properties of the films: O(2) permeability decreased from 1.84 to 1.35 x 10(-12) cm(3) x (Pa x s x m)(-1); CO(2) permeability increased from 1.96 to 6.31 x 10(-12) cm(3).(Pa x s x m)(-1); opacity increased from 3.68 to 4.59%; tensile strength ranged from 0.84 to 1.46 MPa; and elongation at break improved from 50.93 to 68.16%. These results demonstrate that novel galactomannan-based edible coatings, when combined with nisin, may provide consumer-friendly alternatives to reduce L. monocytogenes postcontamination on cheese products during storage.

Inhibitory effects of the ethanol extract of Gleditsia sinensis thorns on human colon cancer HCT116 cells in vitro and in vivo.

The thorns of Gleditsia sinensis have traditionally been used in the treatment of several diseases, which includes their use as anti-tumor agents, but there has been no scientific evidence of this anti-tumor effect. However, the present study has identified a novel mechanism for the anti-tumor effect of Gleditsia sinensis thorns in the treatment of colon cancer. Treatment with the ethanol extract of Gleditsia sinensis thorns (EEGS) resulted in significant growth inhibition together with G2/M-phase cell cycle arrest at a dose of 600 microg/ml (IC50) in HCT116 cells. In addition, treatment with EEGS induced p27 expression and down-regulated expression of cyclins and cyclin-dependent kinases. Moreover, EEGS treatment induced phosphorylation of extracellular signal-regulated kinases (ERK), p38 MAP kinase and JNK (c-Jun N-terminal kinases). Among the pathways examined, only PD98059 (ERK-specific inhibitor) abolished EEGS-dependent p27 expression. Similarly, suppression of ERK function reversed EEGS-mediated cell proliferation inhibition and decreased cell cycle proteins. In addition, tumor necrosis factor-alpha (TNF-alpha)-induced matrix metalloproteinase-9 (MMP-9) expression was inhibited by EEGS treatment via decreased transcriptional activity of both activator protein-1 (AP-1) and nuclear factor-kappaB. Finally, EEGS treatment significantly reduced tumor sizes in HCT116 cell-xenografted tumor tissues, which was associated with the changed levels of ERK phosphorylation, p27 and MMP-9 expression. Overall, these results have identified a novel molecular mechanism for EEGS in the treatment of colon cancer and might provide a theoretical basis for the potential therapeutic use of EEGS in the treatment of malignancies.

[Effects of NaCl stress on Hovenia dulcis and Gleditsia sinensis seedlings growth, chlorophyll fluorescence, and active oxygen metabolism].

With potted Hovenia dulcis and Gleditsia sinensis seedlings as test materials, their plant growth, chlorophyll fluorescence characteristics, and active oxygen metabolism under stress of different concentration (0, 0.15%, 0.30%, 0.45%, and 0.60%) NaCl were studied. The results showed that with increasing concentration of NaCl, the plant growth, leaf chlorophyll content, photochemical efficiency of PS II (Fv/Fm), quantum yield of PS II (phi(PS II)), and photochemical quenching (q(P)) decreased gradually, while the non-photochemical quenching of fluorescence (q(N)) was in adverse. After 10 days of 0. 15% NaCl stress, the leaf chlorophyll content, Fv/Fm, phi(PS II), and q(P) of H. dulcis seedlings decreased by 19.77%, 2.94%, 29.03%, and 8.16%, respectively, with significant differences (P<0.05) to the control, while no significant differences were observed for G. sinensis seedlings. Compared with the control, the Fv/Fm and phi(PS II), of G. sinensis seedlings in treatment 0.30% NaCl decreased significantly by 1.91% and 14.66%, and the chlorophyll content and q(P) of the seedling in treatment 0.45% NaCl decreased significantly by 29.28% and 11.36%, respectively (P<0.05). With increasing concentration of NaCl, the SOD activity of G. sinensis seedlings showed a consistent increasing trend, and that of H. dulcis seedlings increased first and decreased then. The POD and CAT activities of G. sinensis and H. dulci seedlings tended to increase first and decrease then, with the increment being higher for G. sinensis than for H. dulci, while the MDA content of the seedlings had an increasing trend, with the increment being higher for H. dulcis than for G. sinensis, suggesting that the cell membrane lipid peroxidation of H. dulcis was more serious than that of G. sinensis. It was concluded that G. sinensis had greater salt tolerance than H. dulcis, which was related toits higher anti-oxidation enzyme activities.

Gleditsia sinensis fruit extract induced growth inhibition involves basic fibroblast growth factor and nitric oxide.

Recently we have shown the antiproliferative activity of Gleditsia sinensis fruit extract (GSE) on various solid tumour and leukaemia cell lines as well as primary cultured bone marrow cells isolated from patients with acute and chronic myelogenous leukaemia. We further studied whether the growth inhibitory effect of GSE involves basic fibroblast growth factor (bFGF) in cancer cell lines including breast cancer MDA-MB231, nasopharyngeal cancer CNE-2 and prostate cancer LNCaP. We also investigated whether GSE could alter the production of nitric oxide (NO) pattern from these cancer cell lines. Growth inhibition assay was quantitated by sulforhodamine B protein staining method. Enzyme linked immunosorbent assay (ELISA) was used to quantitate the total bFGF protein. The amount of NO secreted into culture medium in terms of nitrite ion concentration was measured by the Greiss method. ELISA showed that GSE could stimulate total bFGF protein level which was dose- dependent. NO production was also stimulated from these cancer cell lines after treating with GSE. Both of the increment in total bFGF and NO levels were correlated with the degree of growth inhibition. Changes involving cell shrinkage and detachment of cancer cells could readily be observed. Taken together, our results here suggest that growth inhibition induced by GSE in these solid tumour cell lines may involve both bFGF and NO regulations.