Functional diversification of a wild potato immune receptor at its center of origin.

Plant cell surface pattern recognition receptors (PRRs) and intracellular immune receptors cooperate to provide immunity to microbial infection. Both receptor families have coevolved at an accelerated rate, but the evolution and diversification of PRRs is poorly understood. We have isolated potato surface receptor Pep-13 receptor unit (PERU) that senses Pep-13, a conserved immunogenic peptide pattern from plant pathogenic Phytophthora species. PERU, a leucine-rich repeat receptor kinase, is a bona fide PRR that binds Pep-13 and enhances immunity to Phytophthora infestans infection. Diversification in ligand binding specificities of PERU can be traced to sympatric wild tuber-bearing Solanum populations in the Central Andes. Our study reveals the evolution of cell surface immune receptor alleles in wild potato populations that recognize ligand variants not recognized by others.

Botrytis cinerea infection accelerates ripening and cell wall disassembly to promote disease in tomato fruit.

Postharvest fungal pathogens benefit from the increased host susceptibility that occurs during fruit ripening. In unripe fruit, pathogens often remain quiescent and unable to cause disease until ripening begins, emerging at this point into destructive necrotrophic lifestyles that quickly result in fruit decay. Here, we demonstrate that one such pathogen, Botrytis cinerea, actively induces ripening processes to facilitate infections and promote disease in tomato (Solanum lycopersicum). Assessments of ripening progression revealed that B. cinerea accelerated external coloration, ethylene production, and softening in unripe fruit, while mRNA sequencing of inoculated unripe fruit confirmed the corresponding upregulation of host genes involved in ripening processes, such as ethylene biosynthesis and cell wall degradation. Furthermore, an enzyme-linked immunosorbent assay (ELISA)-based glycomics technique used to assess fruit cell wall polysaccharides revealed remarkable similarities in the cell wall polysaccharide changes caused by both infections of unripe fruit and ripening of healthy fruit, particularly in the increased accessibility of pectic polysaccharides. Virulence and additional ripening assessment experiments with B. cinerea knockout mutants showed that induction of ripening depends on the ability to infect the host and break down pectin. The B. cinerea double knockout Δbc polygalacturonase1 Δbc polygalacturonase2 lacking two critical pectin degrading enzymes was incapable of emerging from quiescence even long after the fruit had ripened at its own pace, suggesting that the failure to accelerate ripening severely inhibits fungal survival on unripe fruit. These findings demonstrate that active induction of ripening in unripe tomato fruit is an important infection strategy for B. cinerea.

Photosynthetic carbon and nitrogen metabolism of Camellia oleifera Abel during acclimation to low light conditions.

Tea-oil tree (Camellia oleifera Abel) is an important woody oil crop with high economic value. However, it has low photosynthetic production considering the low light intensity of its growth environment. To understand the acclimation mechanism of tea-oil trees to low light conditions, three light intensity treatments were conducted: high light (450-500 µmol. m-2. s-1), medium light (180-200 µmol. m-2. s-1), and low light (45-50 µmol. m-2. s-1). The carbon (C) and nitrogen (N) metabolism network were constructed by investigating the leaf anatomy, photosynthetic characteristics, N partitioning, transcriptome and metabolome. Results demonstrated that a larger proportion light energy was used for photochemical reactions in an environment with lower light intensity, which resulted in an increase in photosystem II photochemical efficiency and instantaneous light use efficiency (LUE) at the leaf level. As the light intensity increased, decreased electron transfer and carboxylation efficiencies, photorespiration and dark respiration rates, LUE at plant level, and N use efficiency (PNUE) were observed. Leaves trended to harvest more light using higher expression levels of light-harvesting protein genes, higher chlorophyll content, more granum and more tightly stacked granum lamella under lower light intensity. At transcriptional and metabolic levels, the TCA cycle, and the synthesis of starch and saccharides were weakened as light intensity decreased, while the Calvin cycle did not show the regularity between different treatments. Less N was distributed in Rubisco, respiration, and cell wall proteins as light decreased. Storage N was prominently accumulated in forms of amino acids (especially L-arginine) and amino acid derivatives as under medium and low light environments, to make up for C deficiency. Therefore, tea-oil trees actively improve light-harvesting capacity and enlarges the storage N pool to adapt to a low light environment, at the cost of a decrease of photosynthetic C assimilation and PNUE.

POLLEN WALL ABORTION 1 is essential for pollen wall development in rice.

The integrity of pollen wall structures is essential for pollen development and maturity in rice (Oryza sativa L.). In this study, we isolated and characterized the rice male-sterile mutant pollen wall abortion 1 (pwa1), which exhibits a defective pollen wall (DPW) structure and has sterile pollen. Map-based cloning, genetic complementation, and gene knockout experiments revealed that PWA1 corresponds to the gene LOC_Os01g55094 encoding a coiled-coil domain-containing protein. PWA1 localized to the nucleus, and PWA1 was expressed in the tapetum and microspores. PWA1 interacted with the transcription factor TAPETUM DEGENERATION RETARDATION (TDR)-INTERACTING PROTEIN2 (TIP2, also named bHLH142) in vivo and in vitro. The tip2-1 mutant, which we obtained by clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9-mediated gene editing, showed delayed tapetum degradation, sterile pollen, and DPWs. We determined that TIP2/bHLH142 regulates PWA1 expression by binding to its promoter. Analysis of the phenotype of the tip2-1 pwa1 double mutant indicated that TIP2/bHLH142 functions upstream of PWA1. Further studies suggested that PWA1 has transcriptional activation activity and participates in pollen intine development through the ß-glucosidase Os12BGlu38. Therefore, we identified a sterility factor, PWA1, and uncovered a regulatory network underlying the formation of the pollen wall and mature pollen in rice.

Oryza sativa PECTIN DEFECTIVE TAPETUM1 affects anther development through a pectin-mediated signaling pathway in rice.

Galacturonosyltransferase (GalAT) is required for the synthesis of pectin, an important component of plant cell walls that is also involved in signal transduction. Here, we describe the rice (Oryza sativa) male-sterile mutant O. sativa pectin-defective tapetum1 (ospdt1), in which GalAT is mutated. The ospdt1 mutant exhibited premature programmed cell death (PCD) of the tapetum and disordered pollen walls, resulting in aborted pollen grains. Pectin distribution in the anther sac was comparable between the mutant and the wild-type, suggesting that the structural pectin was not dramatically affected in ospdt1. Wall-associated kinases are necessary for the signal transduction of pectin, and the intracellular distribution of O. sativa indica WALL-ASSOCIATED KINASE1 (OsiWAK1), which binds pectic polysaccharides to its extracellular domain, was affected in ospdt1. OsiWAK1 RNA interference lines exhibited earlier tapetal PCD, similar to ospdt1. Furthermore, overexpression of OsiWAK1 in ospdt1 lines partially rescued the defects observed in ospdt1, suggesting that OsiWAK1 plays pivotal roles in the function of OsPDT1. These results suggest that the mutation of OsPDT1 does not dramatically affect structural pectin but affects components of the pectin-mediated signaling pathway, such as OsiWAK1, and causes male sterility.

OsMYB103 is essential for tapetum degradation in rice.

KEY MESSAGE: OsMYB103 positively regulates tapetum degradation, and functions downstream of TDR and upstream of EAT1 and PTC1. The precise regulation of programmed cell death (PCD) of the tapetum is crucial for the development of anthers and pollen in rice. In this study, we isolated and identified a male-sterile mutant of rice, osmyb103, which exhibited delayed tapetum degradation and defective mature pollen. Map-based cloning and genetic complementation revealed that OsMYB103 corresponded to the gene LOC_Os04g39470 and encoded a R2R3 MYB transcription factor. OsMYB103 was localized in the nucleus and was expressed preferentially in the tapetal cells and microspores of the anther. OsMYB103 regulated the expression of two transcription factors, ETERNAL TAPETUM 1 (EAT1) and PERSISTENT TAPETAL CELL 1 (PTC1), both of which regulated tapetum degradation positively. Moreover, the expression of OsMYB103 was directly regulated by the additional positive regulator of tapetum degradation TAPETUM DEGENERATION RETARDATION (TDR) and was able to interact with it. Genetic evidence confirmed that OsMYB103 acted upstream of EAT1. The results show that OsMYB103 is a positive regulator of tapetum degradation in rice. These findings provide a better understanding of the regulatory network that underlies degradation of the tapetum in rice.

OsFLA1 encodes a fasciclin-like arabinogalactan protein and affects pollen exine development in rice.

KEY MESSAGE: OsFLA1 positively regulates pollen exine development, and locates in the cellular membrane. Arabinogalactan proteins are a type of hydroxyproline-rich glycoprotein that are present in all plant tissues and cells and play important roles in plant growth and development. Little information is available on the participation of fasciclin-like arabinogalactan proteins in sexual reproduction in rice. In this study, a rice male-sterile mutant, osfla1, was isolated from an ethylmethanesulfonate-induced mutant library. The osfla1 mutant produced withered, shrunken, and abortive pollen. The gene OsFLA1 encoded a FLA protein and was expressed strongly in the anthers in rice. Subcellular localization showed that OsFLA1 was located in the cellular membrane. In the osfla1 mutant, abnormal Ubisch bodies and a discontinuous nexine layer of the microspore wall were observed, which resulted in pollen abortion and ultimately in male sterility. The results show the important role that OsFLA1 plays in male reproductive development in rice.

Alkyl-benzofuran dimers from Eupatorium chinense with insulin-sensitizing and anti-inflammatory activities.

Five new racemic alkyl-benzofuran dimers, (±)-dieupachinins I-M (1-5), were isolated from the root tubers of Eupatorium chinense, a well-known traditional Chinese medicine for the treatment of diphtheria in Guangdong province. The structures of these compounds, especially the first examples of 12,10'-epoxy dimer dieupachinin I (1), 12-nor-dimer dieupachinin J (2), and 12,12'-dinor-dimer dieupachinin K (3), were elucidated by spectroscopic data analysis. Chiral resolution were further carried out on a cellulose column by HPLC, and compounds 2-5 were successfully separated into two enantiomers, respectively. The absolute configurations of (+)-(2-5) and (-)-(2-5) were established by theoretical ECD calculation. All the compounds were evaluated for insulin-stimulated glucose uptake in C2C12 myotubes and (±)-dieupachinin I (1) exhibited the best activity. Compound 1 enhanced insulin-stimulated glucose uptake via activating the insulin receptor substrate 1/protein kinase B/glycogen synthase kinase-3ß signaling pathway. Moreover, all the isolates were tested for their nitric oxygen (NO) inhibitory effects in lipopolysaccharide-treated RAW264.7 macrophages, and compounds (±)-1, (±)-2, and (±)-4 showed promising inhibitory effects with IC50 values of 6.42 ± 1.85, 6.29 ± 1.94, and 16.03 ± 2.07 µM, respectively. (±)-Dieupachinin I (1) again dose-dependently suppressed LPS-induced expression of inducible NO synthase and nuclear translocation of p65.

Benzofurans from Eupatorium chinense enhance insulin-stimulated glucose uptake in C2C12 myotubes and suppress inflammatory response in RAW264.7 macrophages.

Fourteen acetylbenzofuran derivatives, including three undescribed carbon skeletons with a newly formed hexane or benzene ring on the other side of the benzofuran ring, (±)-eupatonin A (1), (±)-eupatonin B (2), and eupatonin C (3), two new benzofurans (-)-12ß-hydroxygynunone (4) and (+)-12-hydroxyl-13-noreuparin (5), as well as 9 known ones (6-14), were isolated from 95% ethanol extract of the roots of Eupatorium chinense. Their structures were determined by spectroscopic methods and quantum chemical DFT and TDDFT calculations of the NMR chemical shifts and ECD spectra, which helped in the determination of the relative configurations of 1 and 2 and the absolute configurations of 4 and 5, respectively. 1 and 2 were further identified to be racemic mixtures by chiral HPLC analysis. All compounds were evaluated for insulin-stimulated glucose uptake in differentiated C2C12 myotubes. Compounds 1, 3, 4, 5, 11, 12, and 13 markedly enhanced insulin-mediated glucose uptake. (±)-Eupatonin A (1) activated the IRS-1/Akt/GSK-3ß signaling pathway and enhanced insulin stimulated GLUT4 membrane translocation in C2C12 myotubes. On LPS stimulated RAW264.7 macrophages, several compounds exhibited significant inhibitory effect on NO production with IC50 values ranging from 4.94 to 9.70 µΜ. (±)-Eupatonin A (1) again dose-dependently suppressed LPS-induced NO production and decreased the expression of inducible NO synthase (iNOS), through inhibiting NF-κB activity.

Spotlight on fungal pectin utilization-from phytopathogenicity to molecular recognition and industrial applications.

Pectin is a complex polysaccharide with D-galacturonic acid as its main component that predominantly accumulates in the middle lamella of the plant cell wall. Integrity and depolymerization of pectic structures have long been identified as relevant factors in fungal phytosymbiosis and phytopathogenicity in the context of tissue penetration and carbon source supply. While the pectic content of a plant cell wall can vary significantly, pectin was reported to account for up to 20-25% of the total dry weight in soft and non-woody tissues with non- or mildly lignified secondary cell walls, such as found in citrus peel, sugar beet pulp, and apple pomace. Due to their potential applications in various industrial sectors, pectic sugars from these and similar agricultural waste streams have been recognized as valuable targets for a diverse set of biotechnological fermentations.Recent advances in uncovering the molecular regulation mechanisms for pectinase expression in saprophytic fungi have led to a better understanding of fungal pectin sensing and utilization that could help to improve industrial, pectin-based fermentations. Related research in phytopathogenic fungi has furthermore added to our knowledge regarding the relevance of pectinases in plant cell wall penetration during onset of disease and is therefore highly relevant for agricultural sciences and the agricultural industry. This review therefore aims at summarizing (i) the role of pectinases in phytopathogenicity, (ii) the global regulation patterns for pectinase expression in saprophytic filamentous fungi as a highly specialized class of pectin degraders, and (iii) the current industrial applications in pectic sugar fermentations and transformations.

Identification of Confusable Herbal Medicines by Mapping of Partial Degradation Products from Herbal Medicine Polysaccharides.

Partial degradation products (PDPs) of herbal medicine (HM) polysaccharides with precolumn derivatization using 1-phenyl-3-methy-5-pyrazolone (PMP) were mapped by high performance liquid chromatography (HPLC), and three groups of confusable HMs were differentiated using the PDP fingerprints assisted with cluster analysis (CA). Three variables of HPLC mobile phase, i.e. acetonitrile proportion, buffer concentration and pH value were optimized with PDP of ß-cyclodextrin. Radix Glehniae and Radix Adenophorae; Radix Sophorae Tokinensis and Rhizoma Menispermi; Radix Achyranthis Bidentatae and Radix Cyathulae were successfully distinguished by the method, respectively. The results involving mass spectrometry analysis showed that these PDPs primarily included oligosaccharides and a few monosaccharides. The method can be used as an effective approach for the identification and quality control of HMs, and can also facilitate the in-depth study of biological activity and further development of HM polysaccharides to some extent.

[Chemical constituents from roots of Viburnum setigerum].

This experiment was to study the constituents of the roots of Viburnum setigerum through various column chromatographic techniques. Thirteen compounds were obtained and their structures were identified using chemical and spectroscopic methods as (7αH, 8'αH)-4, 4', 8α-trihydroxy-3, 3', 9-trimethoxy-7, 9'-epoxylignan (1), (7αH, 8'αH)-4, 4', 8α, 9-tetrahydroxy-3, 3'-dimethoxy-7, 9'-epoxylignan (2), alashinol G (3), alashinol F (4), (-)-secoisolariciresinol (5), (7R, 7'R, 8R, 8'S)-3, 3'-dimethoxy-7, 7'-epoxylignane -4, 4', 9, 9'-tetraol (6), (7αH, 8αH, 8'ßH)-4, 4', 7'α, 9-tetrahydroxy-3, 3'-dimethoxy-7, 9'-epoxylignan (7), loganin (8), dihydroquercetin (9), protocatechuic acid (10), 4-hydroxy-3-methoxy-benzoic acid (11), adoxoside (12), and catechin (13). Compound 1 was a new compound. Compounds 3-7 and 11 were reported from the genus Viburnum for the first time. All compounds were separated from this plant for the first time.

Terpenoids from Curcuma wenyujin increased glucose consumption on HepG2 cells.

Thirty four terpenoids, including two new cadinane-type sesquiterpenoids containing conjugated aromatic-ketone moieties, curcujinone A (1) and curcujinone B (2), were isolated from 95% ethanol extract of the root tubers of Curcuma wenyujin. Their structures were determined by spectroscopic methods, especially 2D NMR and HRMS techniques. The relative and absolute configurations of 1 and 2 were identified by quantum chemical DFT and TDDFT calculations of the 13C NMR chemical shifts, ECD spectra, and specific optical rotations. All compounds and extracts were evaluated for their anti-diabetic activities with a glucose consumption model on HepG2 Cells. The petroleum fraction CWP (10µg/mL) and compounds curcumenol (4), 7α,11α-epoxy-5ß-hydroxy-9-guaiaen-8-one (5), curdione (17), (1S, 4S, 5S 10S)-germacrone (18), zederone (20), a mixture of curcumanolide A (25) and curcumanolide B (26), gajutsulactone B (27), and wenyujinin C (30) showed promising activities with over 45% increasing of glucose consumption at 10µM.

Rapid screening of different types of antitumor compound groups from traditional chinese medicine by hollow fiber cell fishing with high performance liquid chromatography.

A novel hollow fiber cell fishing with high performance liquid chromatography (HFCF-HPLC) was extended and used to screen flavonoid and anthraquinone active compound groups simultaneously from traditional Chinese medicines (TCMs). In this study, three cells (MCF-7, SGC7901, and MADB-106) were seeded on the inner wall of the hollow fiber employed to screen bioactive components from TCM water decoction. The variables influencing HFCFHPLC, such as cell seeding time, screening stirring rate and time, and active compound concentration, were investigated and optimized. The surface property of the hollow fiber seeded with cells, the cell survival rate under different conditions, the nonspecific binding between active centers in the fiber and the target compounds, and the repeatability and recovery of HFCF-HPLC were analyzed and validated. Certain structures of the compounds fished by HFCF-HPLC were identified after comparing the retention times of the reference substances. To verify preliminarily the binding site between the bioactive components and cells, we separated the cell membrane and cell organelle from live MCF-7 cells. We then employed the cell membrane, cell organelle, and the whole cells to screen simultaneously the active compounds. The cell fishing factor of the active compound was calculated and discussed as the index of cell-drug binding ability in HFCFHPLC. Tamoxifen as a positive control and indomethacin as a negative control were screened by HFCF-HPLC to verify the method. The results indicate that HFCF-HPLC is an effective and reliable method for the screening and analysis of bioactive components. Moreover, this method can be applied to predict bioactive candidates in TCMs.

Genome-wide analysis of pectate-induced gene expression in Botrytis cinerea: identification and functional analysis of putative d-galacturonate transporters.

The fungal plant pathogen Botrytis cinerea produces a spectrum of cell wall degrading enzymes for the decomposition of host cell wall polysaccharides and the consumption of the monosaccharides that are released. Especially pectin is an abundant cell wall component, and the decomposition of pectin by B. cinerea has been extensively studied. An effective concerted action of the appropriate pectin depolymerising enzymes, monosaccharide transporters and catabolic enzymes is important for complete d-galacturonic acid utilization by B. cinerea. In this study, we performed RNA sequencing to compare genome-wide transcriptional profiles between B. cinerea cultures grown in media containing pectate or glucose as sole carbon source. Transcript levels of 32 genes that are induced by pectate were further examined in cultures grown on six different monosaccharides, by means of quantitative RT-PCR, leading to the identification of 8 genes that are exclusively induced by d-galacturonic acid. Among these, the hexose transporter encoding genes Bchxt15 and Bchxt19 were functionally characterised. The subcellular location was studied of BcHXT15-GFP and BcHXT19-GFP fusion proteins expressed under control of their native promoter, in a B. cinerea wild-type strain. Both genes are expressed during growth on d-galacturonic acid and the fusion proteins are localized in plasma membranes and intracellular vesicles. Target gene knockout analysis revealed that BcHXT15 contributes to d-galacturonic acid uptake at pH 5∼5.6. The virulence of all B. cinerea hexose transporter mutants tested was unaltered on tomato and Nicotiana benthamiana leaves.

A new ionic liquid-water-organic solvent three phase microextraction for simultaneous preconcentration flavonoids and anthraquinones from traditional Chinese prescription.

A novel technique, ionic liquid-water-organic solvent three phase microextraction (ILWOS-3p-ME) was developed and introduced for simultaneous preconcentration and determination of flavonoids and anthraquinones in Chinese herbal formula and its preparations. This technique was performed in one step by using a syringe. High performance liquid chromatography with an UV-detector (HPLC/UV) was subsequently conducted. Two solvents with different densities (organic solvent and ionic liquid with densities less than and higher than water, respectively) were separately placed in a syringe, which was used as an extraction device. A cloudy emulsion was formed by manually shaking the syringe. The mixture was allowed to stand for several minutes; afterward, the emulsion readily separated into three phases: an upper organic solvent extraction phase; a middle aqueous sample phase; and a lower ionic liquid extraction phase. Both the upper and lower layers were transferred to a small Eppendorf (EP) tube. Conducting ILWOS-3P-ME with HPLC/UV, we simultaneously determined the bioactive components of flavonoids and anthraquinones in traditional Chinese medicine. ILWOS-3P-ME is a simple, rapid, practical, and effective method to extract and preconcentrate of different types of trace bioactive components from traditional Chinese medicine simultaneously.

The D-galacturonic acid catabolic pathway in Botrytis cinerea.

D-galacturonic acid is the most abundant component of pectin, one of the major polysaccharide constituents of plant cell walls. Galacturonic acid potentially is an important carbon source for microorganisms living on (decaying) plant material. A catabolic pathway was proposed in filamentous fungi, comprising three enzymatic steps, involving D-galacturonate reductase, L-galactonate dehydratase, and 2-keto-3-deoxy-L-galactonate aldolase. We describe the functional, biochemical and genetic characterization of the entire D-galacturonate-specific catabolic pathway in the plant pathogenic fungus Botrytis cinerea. The B. cinerea genome contains two non-homologous galacturonate reductase genes (Bcgar1 and Bcgar2), a galactonate dehydratase gene (Bclgd1), and a 2-keto-3-deoxy-L-galactonate aldolase gene (Bclga1). Their expression levels were highly induced in cultures containing GalA, pectate, or pectin as the sole carbon source. The four proteins were expressed in Escherichia coli and their enzymatic activity was characterized. Targeted gene replacement of all four genes in B. cinerea, either separately or in combinations, yielded mutants that were affected in growth on D-galacturonic acid, pectate, or pectin as the sole carbon source. In Aspergillus nidulans and A. niger, the first catabolic conversion only involves the Bcgar2 ortholog, while in Hypocrea jecorina, it only involves the Bcgar1 ortholog. In B. cinerea, however, BcGAR1 and BcGAR2 jointly contribute to the first step of the catabolic pathway, albeit to different extent. The virulence of all B. cinerea mutants in the D-galacturonic acid catabolic pathway on tomato leaves, apple fruit and bell peppers was unaltered.

A chemical genetic screen for modulators of exocytic transport identifies inhibitors of a transport mechanism linked to GTR2 function.

Membrane and protein traffic to the cell surface is mediated by partially redundant pathways that are difficult to perturb in ways that yield a strong phenotype. Such robustness is expected in a fine-tuned process, regulated by environmental cues, that is required for controlled cell surface growth and cell proliferation. Synthetic genetic interaction screens are especially valuable for investigating complex processes involving partially redundant pathways or mechanisms. In a previous study, we used a triple-synthetic-lethal yeast mutant screen to identify a novel component of the late exocytic transport machinery, Avl9. In a chemical-genetic version of the successful mutant screen, we have now identified small molecules that cause a rapid (within 15 min) accumulation of secretory cargo and abnormal Golgi compartment-like membranes at low concentration (<2 muM), indicating that the compounds likely target the exocytic transport machinery at the Golgi. We screened for genes that, when overexpressed, suppress the drug effects, and found that the Ras-like small GTPase, Gtr2, but not its homolog and binding partner, Gtr1, efficiently suppresses the toxic effects of the compounds. Furthermore, assays for suppression of the secretory defect caused by the compounds suggest that Gtr proteins can regulate a pathway that is perturbed by the compounds. Because avl9Delta and gtr mutants share some phenotypes, our results indicate that the small molecules identified by our chemical-genetic strategy are promising tools for understanding Avl9 function and the mechanisms that control late exocytic transport.

[Effect of gensenoside Rg3 on apoptosis of Hep-2 and expression of HIF-1alha in human laryngeal cancer cell line under anoxic conditions].

OBJECTIVE: To study the mechanism and effect of gensenoside Rg3 on Hep-2 Cell Line during the normoxia and hypoxia. METHODS: Hep-2 Human Laryngeal Cancer Cell Line was cultured under anoxic conditions, and set the normal control group and positive control group (DDP). MTT was used to observe the growth inhibition rates of Hep-2 Human Laryngeal Cancer Cell by Rg3; The cell cycle and cell apoptosis analysis were detected by FCM. Then the expression of HIF-1alpha and VEGF protein was detected by immunohistochemistry and FCM; The expression of HIF-1alpha and VEGF mRNA were detected by transcription-polymerase chain reaction (RT-PCR). RESULTS: Rg3 could significantly inhibit the growth of Hep-2 cells and arrest the cells in G0/G1 phase during normoxia and hypoxia The mRNA and protein expression of HIF-1alpha were dolon-regulated. CONCLUSION: Rg3 can inhibit Hep-2 cells growth by delaying the progress of cell cycle and inhibit the expression of HIF-1alpha during hypoxia, this may be the mechanism of its anti-tumor effect.