Characterization of the cholesterol biosynthetic pathway in Dioscorea transversa.

Cholesterol is the precursor of bioactive plant metabolites such as steroidal saponins. An Australian plant, Dioscorea transversa, produces only two steroidal saponins: 1ß-hydroxyprotoneogracillin and protoneogracillin. Here, we used D. transversa as a model in which to elucidate the biosynthetic pathway to cholesterol, a precursor to these compounds. Preliminary transcriptomes of D. transversa rhizome and leaves were constructed, annotated, and analyzed. We identified a novel sterol side-chain reductase as a key initiator of cholesterol biosynthesis in this plant. By complementation in yeast, we determine that this sterol side-chain reductase reduces Δ24,28 double bonds required for phytosterol biogenesis as well as Δ24,25 double bonds. The latter function is believed to initiate cholesterogenesis by reducing cycloartenol to cycloartanol. Through heterologous expression, purification, and enzymatic reconstitution, we also demonstrate that the D. transversa sterol demethylase (CYP51) effectively demethylates obtusifoliol, an intermediate of phytosterol biosynthesis and 4-desmethyl-24,25-dihydrolanosterol, a postulated downstream intermediate of cholesterol biosynthesis. In summary, we investigated specific steps of the cholesterol biosynthetic pathway, providing further insight into the downstream production of bioactive steroidal saponin metabolites.

Cloning and overexpression of the ascorbate peroxidase gene from the yam (Dioscorea alata) enhances chilling and flood tolerance in transgenic Arabidopsis.

Minghuai 1 (MH1) is a yam (Dioscorea alata) cultivar with high tolerance to flooding but sensitivity to chilling. MH1 responded differently to chilling and flooding according to various physiological parameters and antioxidant enzymes. Flooding led to an increase in ascorbate peroxidase (APX) activity in both roots and leaves, while chilling did not affect APX activity. The full length DaAPX ORF sequence from MH1 (750 bp) was then cloned. Phylogenetic analysis showed that plant cytosolic APXs into four major clusters and DaAPX was closely related to Oncidium. The DaAPX gene driven by a 35S promoter was transferred into Arabidopsis. The gene expression and enzyme activity of APX in the DaAPX transgenic lines 1-3 were significantly higher than in wild type (WT) plants. Compared to WT plants, seedling growth characteristics were significantly better in all transgenic lines under chilling, flooding, and oxidative stresses, indicating that the overexpression of DaAPX in Arabidopsis enhanced tolerance to several abiotic stresses. MH1 plants supplied with H2O2 presented an increase in the activity of APX leading to enhanced tolerance to chilling. Functional characterization of the APX gene should improve our understanding of the chilling- and flood-response mechanism in the yam.

Functional characterization of squalene epoxidase and NADPH-cytochrome P450 reductase in Dioscorea zingiberensis.

Dioscorea zingiberensis is a perennial medicinal herb rich in a variety of pharmaceutical steroidal saponins. Squalene epoxidase (SE) is the key enzyme in the biosynthesis pathways of triterpenoids and sterols, and catalyzes the epoxidation of squalene in coordination with NADPH-cytochrome P450 reductase (CPR). In this study, we cloned DzSE and DzCPR gene sequences from D. zingiberensis leaves, encoding proteins with 514 and 692 amino acids, respectively. Recombinant proteins were successfully expressed in vitro, and enzymatic analysis indicated that, when SE and CPR were incubated with the substrates squalene and NADPH, 2,3-oxidosqualene was formed as the product. Subcellular localization revealed that both the DzSE and DzCPR proteins are localized to the endoplasmic reticulum. The changes in transcription of DzSE and DzCPR were similar in several tissues. DzSE expression was enhanced in a time-dependent manner after methyl jasmonate (MeJA) treatments, while DzCPR expression was not inducible.

Darkening, damage and oxidative protection are stimulated in tissues closer to the yam cut, attenuated or not by the environment.

BACKGROUND: This study investigated how the activities of the enzymes and metabolites of oxidative metabolism are affected in different regions of cut tissue, associating these changes with the evolution of browning in fresh-cut yam. Samples were collected from yam at 0-5 and 5-10 mm from the cut site. Fresh-cut yams were stored at 5 or 26 °C for days or hours respectively, simulating commercialization, with or without packaging. RESULTS: The results demonstrated that the injury to the yam was a physical inducer of changes in the levels of lipid peroxidation and phenolic compounds and in the activities of superoxide dismutase, catalase, ascorbate peroxidase, polyphenol oxidase and peroxidase. These responses were significant in the tissue closest to the wound but also observed, although less intense, in the more distant tissue. The combined effects of wounding and dehydration during storage intensified the above responses. Conversely, refrigeration attenuated the transmission of the wounding response through the adjacent tissue. CONCLUSION: The results of this work provide the first evidence in cut yam roots that the membrane degradation products, enzymes involved in oxidative protection and enzymes that modulate the oxidation of phenolic compounds are intertwined mechanisms that cause tissue darkening. © 2018 Society of Chemical Industry.

Rapidly screening of α-glucosidase inhibitors from Dioscorea opposita Thunb. peel based on rGO@Fe3O4 nanocomposites microreactor.

Present study aimed to immobilise the α-glucosidase on suitable supports to construct enzymatic microreactors and their subsequent applicability in efficient inhibitor screening from the Chinese Yam (Dioscorea opposita Thunb.) peel. A type of lamellar and porous composites (rGO@Fe3O4) were synthesised with a facile one-step solvothermal method and employed as carriers to construct enzymatic microreactors for screening α-glucosidase ligand from the Chinese Yam peel in league with the high performance liquid chromatography and mass spectrometry (HPLC-MS). The immobilisation amount of α-glucosidase on rGO@Fe3O4 under the optimised conditions was about 40 µg α-glucosidase/mg carriers. Furthermore, the binding capacities of screened inhibitors, 2,4-dimethoxy-6,7-dihydroxyphenanthrene and batatasin I, were 35.6 and 68.2%, respectively. Hence, considering their high screening efficiency and excellent magnetic separation ability, these as-prepared nanocomposite consisting of rGO and Fe3O4 may be potential supports for the enzyme (such as α-glucosidase) immobilisation for rapid α-glucosidase inhibitors screening from the diverse nature resources.

Exploration of surface plasmon resonance for yam tyrosinase characterization.

Tyrosinase is a ubiquitous enzyme in nature. It catalyzes the reaction in melanin formation. In this investigation, tyrosinase from a Yam tuber (Amorphophallus paeoniifolius) was extracted and purified by ultra-centrifugal filtration followed by ion exchange chromatography. The purified enzyme was obtained with purification fold of 12.65 and specific activity of 60.25 U/mg. The molecular weight of purified enzyme was confirmed to be 45 kDa by SDS-PAGE. The enzyme activity was optimal at pH 6 and 30 °C with Km 10 mM. Further the kinetic study of purified yam tyrosinase was carried out using Surface Plasmon Resonance method. In brief, enzyme was immobilized on chip surface by amine coupling method and different small molecules were analyzed for their binding affinities at different concentrations (mM). The affinity of each compound for yam tyrosinase was different with KD values as tannic acid (5.13 × 10-5), gallic acid (2.05 × 10-8), ascorbic acid (0.004544), caffeic acid (3.09 × 10-9), pyrogallol (2.13 × 10-4) and catechol (1.09 × 10-4). The kinetics data results were confirmed by inhibition assays and IC50 values were calculated. This data will help to study the role of tyrosinase in hyperpigmentation which will create an avenue for tyrosinase inhibitors. Tyrosinase inhibitors have wide range of applications in cosmetics, medical and food industries.

Asada-Halliwell pathway maintains redox status in Dioscorea alata tuber which helps in germination.

Reactive Oxygen Species (ROS) are important regulatory molecules governing physiological processes. In the present study a biochemical and proteome level comparison of two contrasting growth stages of Dioscorea alata tuber namely germinating and mature tuber was performed in order to understand the tuber physiology and biochemistry. Existence of all the component enzymes [APx (ascorbate peroxidase), GR (glutathione reductase), DHAR (dehydroascorbate reductase), MDHAR (mono-dehydroascorbate reductase)] and major products [ascorbate (ASC) and glutathione (GSH)] of the cycle showed an operational Asada-Halliwell cycle in the tuber. A 2.65 fold increase in ASC content & a 3.8 fold increase in GR activity fortified the redox milieu during germination. In contrast a 5 fold higher H2O2 content (due to 3.08 fold lower APx activity) and accumulation of reactive nitrogen species (RNS) such as nitric oxide (NO, 2.4-fold) and S-nitrosothiol (SNO, 2.08 fold) contributed to overall oxidative conditions in the mature tuber. The carbonic anhydrase (CA, 7.5 fold), DHAR (5.31 fold) and MDHAR (7 fold) activities were higher in the germinating tuber in comparison with the mature tuber. GSNO negatively regulated the CA (3.6 & 3.95 fold), MDHAR (7.5 & 1.5 fold) and APx (2.3 & 1.81 fold) while another NO donor, CysNO negatively regulated the DHAR (2.24 & 1.32 fold) activity in the mature and germinating stages respectively indicating again that the lesser inhibition by NO (via nitrosylation) may be because of overall reducing environment in the germinating tuber. Increased SNO leading to S-nitrosylation of dioscorin was confirmed by Biotin switch assay. This is the first report showing dioscorin nitrosylation. The present analysis showed differential redox regulation and also suggests the physiological relevance of CA, DHAR, MDHAR, APx & GR in tuber germination for the first time. These enzymes may be used as potential markers of tuber germination in future.

Prevention of enzymatic browning of Chinese yam (Dioscorea spp.) using electrolyzed oxidizing water.

In this study, the effects of electrolyzed oxidizing water (EOW) on the prevention of enzymatic browning of fresh-cut "Jiu Jinhuang" Chinese yam were investigated. The yams were immersed in the inhibitors for 25 min at 20 °C. Compared with the tap water (TW) treatment, the chromatic attributes were significantly different after 72 h of storage (P < 0.05). The activities of polyphenol oxidase (PPO, EC 1.10.3.1), peroxidase (POD, EC 1.11.1.7), and L -phenylalanine ammonia lyase (PAL, EC 4.3.1.5) were inhibited when measured at 24 h. The contents of phenolic acids, including gallic and chlorogenic acid, in the group treated with the slightly acidic electrolyzed water (SAEW) were higher than those treated with TW and neutral electrolyzed water (NEW). The group treated with NEW had the highest total phenol content (P < 0.05, at 24 h), while the group treated with SAEW had the highest flavonoid content (P < 0.05) during storage. Without being treated with inhibitors, the Km and Vmax values of yam PPO were 0.0044 mol/L and 0.02627 U/min, respectively, and the Ki of samples treated with SAEW and citric acid (CA) were 15.6607 and 2.3969 µmol/L, respectively. These results indicate that EOW is beneficial as a browning inhibitor.

Characterization of a yam class IV chitinase produced by recombinant Pichia pastoris X-33.

A yam (Dioscorea opposita Thunb) class IV chitinase, whose genomic DNA was cloned by Mitsunaga et al. (2004), was produced by the recombinant Pichia pastoris X-33 in high yields such as 66 mg/L of culture medium. The chitinase was purified by column chromatography after Endoglycosidase H treatment and then characterized. It showed properties similar to the original chitinase E purified from the yam tuber reported by Arakane et al. (2000). This Pichia-produced chitinase also showed strong lytic activity against Fusarium oxysporum and Phytophthora nicotianae, wide pH and thermal stability, optimum activity at higher temperature such as 70 °C, and high substrate affinity, indicating that one can use this Pichia-produced yam chitinase as a bio-control agent.

Molecular cloning and differential expression analysis of a squalene synthase gene from Dioscorea zingiberensis, an important pharmaceutical plant.

Diosgenin is a steroid derived from cholesterol in plants and used as a typical initial intermediate for synthesis of numerous steroidal drugs in the world. Commercially, this compound is extracted mainly from the rhizomes or tubers of some Dioscorea species. Squalene synthase (SQS: EC 2.5.1.21) catalyzes the condensation of two molecules of farnesyl diphosphate to form squalene, the first committed step for biosynthesis of plant sterols including cholesterol, and is thought to play an important role in diosgenin biosynthesis. A full-length cDNA of a putative squalene synthase gene was cloned from D. zingiberensis and designated as DzSQS (Genbank Accession Number KC960673). DzSQS was contained an open reading frame of 1,230 bp encoding a polypeptide of 409 amino acids with a predicted molecular weight of 46 kDa and an isoelectric point of 6.2. The deduced amino acid sequence of DzSQS shared over 70 % sequence identity with those of SQSs from other plants. The truncated DzSQS in which 24 amino acids were deleted from the carboxy terminus was expressed in Escherichia coli, and the resultant bacterial crude extract was incubated with farnesyl diphosphate and NADPH. GC-MS analysis showed that squalene was detected in the in vitro reaction mixture. Quantitative real-time PCR analysis revealed that DzSQS was expressed from highest to lowest order in mature leaves, newly-formed rhizomes, young leaves, young stems, and two-year-old rhizomes of D. zingiberensis.

Genetic structure and diversity in the Dioscorea cayenensis/D. rotundata complex revealed by morphological and isozyme markers.

Of the 600 known yam species, only 10 are utilized as food, and the Dioscorea cayenensis/D. rotundata species complex is among the most cultivated. In Brazil, these species are commercially cultivated in the northeast region and are cultivated in the south and southeast regions as subsistence crops by traditional agriculturists. This study aimed to evaluate the genetic diversity of 21 local varieties of D. cayenensis and 2 D. rotundata accessions using 7 isozymic loci and 24 morphological markers, and to investigate the diversity distribution in different levels of organization, such as swidden fields and communities of Vale do Ribeira. Cluster analyses for both the isozymic and morphological data separated the 2 D. rotundata accessions from the D. cayenensis accessions from Vale do Ribeira. The analysis with morphological characteristics showed the presence of 2 subgroups (Iguape and Cananéia) within group I, which included all of the local varieties from Vale do Ribeira; this result may indicate the influence of the cultural units on the morphological variation. Molecular analysis of variance indicated that most of the isozymic variability was concentrated among swiddens within communities (42.5%) and within communities (40.3%). Most of the morphological variability was also concentrated among swidden fields within communities (44.8%). The correlation between geographic and genetic distances indicated that neither morphological (r = 0.17) nor isozymic diversity (r = -0.15) is structured in space. Thus, the traditional agriculturists of Vale do Ribeira maintain and manage a great diversity of D. cayenensis varieties in their communities.

Cloning of genes and enzymatic characterizations of novel dioscorin isoforms from Dioscorea japonica.

Dioscorin, the major tuber storage protein of yam, has been shown to possess carbonic anhydrase, trypsin inhibitor, dehydroascorbate reductase, and monodehydroascorbate reductase activities. In the present study, dioscorin from Dioscorea japonica was confirmed as a glycoprotein using the enhanced concanavalin A-peroxidase staining method, and the protein was shown to have both N- and O-glycans. Following the gene cloning, four full-length isoforms of dioscorin were expressed in Escherichia coli and purified by affinity purification and anion-exchange chromatography for structural and biochemical experiments. It was clearly observed that the recombinant dioscorins had carbonic anhydrase, trypsin inhibitor, dehydroascorbate reductase, and monodehydroascorbate reductase activities. However, the dehydroascorbate reductase and monodehydroascorbate reductase activities were markedly decreased in recombinant dioscorins compared with native dioscorin. The decreased activities were closely related to the loss of the glycosylation from the protein.

Amylolytic hydrolysis of native starch granules affected by granule surface area.

Initial stage of hydrolysis of native starch granules with various amylolytic enzymes, alpha-amylase from Bacillus subtilis, glucoamylase I (GA-I) and II (GA-II) from Aspergillus niger, and beta-amylase from sweet potato showed that the reaction was apparently affected by a specific surface area of the starch granules. The ratios of the reciprocal of initial velocity of each amylolytic hydrolysis for native potato and maize starch to that for rice with the amylolytic enzymes were nearly equivalent to the ratio of surface area per mass of the 2 starch granules to that of rice, that is, 6.94 and 2.25, respectively. Thus, the reciprocal of initial velocity of each enzymatic hydrolysis as expressed in a Lineweaver-Burk plot was a linear function of the reciprocal of surface area for each starch granule. As a result, it is concluded that amylolytic hydrolysis of native starch granules is governed by the specific surface area, not by the mass concentration, of each granule.

Biochemical origin of browning during the processing of fresh Yam (Dioscorea spp.) into dried product.

This study was undertaken to follow the kinetics of polyphenoloxidase (PPO), peroxidase (POD), and phenolic compounds during yam blanching at different temperatures and after drying and to identify by high-performance liquid chromatography (HPLC) the main phenolic compounds present in yam products. PPO activity was 50% higher in nonprocessed freeze-dried Florido (Dioscorea alata) than in nonprocessed freeze-dried Deba (Dioscorea rotundata). It decreased progressively during blanching. Forty-five percent of PPO activity remained after 50 min of blanching at 60 or 65 degrees C, whereas the POD activity dropped sharply to less than 20% of the initial activity after 10 min of blanching, whatever the blanching temperature. No anthocyanidins could be detected by HPLC at 520 nm in nonprocessed freeze-dried yam. Flavanols with a maximum absorption wavelength (lambda(max)) at 280 nm and cinnamic acid compounds with 320 nm lambda(max) were detected. Catechin was identified as the major flavanol with concentrations ranging from 0.26 to 0.41 microM g(-)(1) depending on cultivar. One cinnamic compound, ferulic acid, was identified and assessed in both cultivars (0.03-0.04 microM g(-)(1)). Total phenol, flavanol, and cinnamic contents decreased during blanching independently of temperature whereas some unresolved peaks were detected by HPLC in dried product. The latter was probably due to the consumption of coloring precursors and the appearance of polymerized or complex colored products.

Molecular cloning of a genomic DNA encoding yam class IV chitinase.

Genomic DNA for a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves and sequenced. The deduced amino acid sequence shows 50 to 59% identity to class IV chitinases from other plants. The yam chitinase, however, has an additional sequence of 8 amino acids (a C-terminal extension) following the cysteine that was reported as the last amino acid for other class IV chitinases; this extension is perhaps involved in subcellular localization. A homology model based on the structure of a class II chitinase from barley was used as an aid to interpreting the available data. The analysis suggests that the class IV enzyme recognizes an even shorter segment of the substrate than class I or II enzymes. This observation might help to explain why class IV enzymes are better suited to attack against pathogen cell walls.

Intracellular localization of a class IV chitinase from yam.

Genomic DNA encoding a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves in previous research (Biosci. Biotechnol. Biochem., 68, 1508-1517 (2004)). But this chitinase had an additional sequence composed of eight amino acids (a C-terminal extension) at the C-terminal, compared with class IV chitianses from other plants. In order to clarify the role of this C-terminal extension in cellular localization, plants and suspension-cultured cells of Nicotiana tabacum were transformed with either the cloned yam class IV chitinase gene carrying the C-terminal extension or its truncated gene by the Agrobacterium-mediated method, and then their localization was investigated. The results suggest that the C-terminal extension of yam class IV chitinase plays a role as a targeting signal for plant vacuoles. This is the first report presenting the existence of vacuolar type class IV chitinase.

Tissue and subcellular localization of oligofurostanosides and their specific degrading beta-glucosidase in Dioscorea caucasica Lipsky.

The application of the histochemical technique using the Ehrlich reagent showed that the oligofurostanosides--the steroid glycosides of the furostan series--are localized in idioblasts (special cells--receptacles) of the epidermal layer of Dioscorea caucasica leaves. The activity of oligofurostanoside-specific beta-glucosidase was localized mainly in the membrane fraction of the thylakoides and was also found in the Dioscorea caucasica leaves. We have suggested, that the differential tissue, the subcellular localization of the oligofurostanosides and their degrading enzymes provide the maintenance of the furostanol structure of steroid glycosides which is necessary for their transport within the leaf and from the leaf to the rhizome.

Prediction of sensorial properties (color and taste) of amala, a paste from yam chips flour of West Africa, through flour biochemical properties.

Color and taste are permanent features of amala, a traditional thick paste obtained from yam chips flour. To assess these attributes, 23 yam chips presenting various quality attributes were processed. The sensory attributes of their derived amala were determined and some biochemical characteristics of yam flours measured. A panel defined five main taste attributes for amala: sweetness, bitterness, acidity, fermented, and roasted tastes. Amala color was measured instrumentally and sensory scores were highly correlated with flour biochemical analyses; amala sweetness was positively correlated with glucose and fructose content of the flour, whereas amala acidity and fermented taste were linked to organic acids and lactic acid contents of flour, respectively. In addition, darkness, bitterness, and roasted tastes of amala could be tightly predicted by multiple regression analysis from phenolic compound and glucose-fructose contents. Phenolic content of yam flour plays thus a key role on sensorial quality of amala. However, polyphenol oxidase and peroxidase activities were almost null in yam flours and their specific role on yam flour phenolic content needs to be clarified.

Plant chitinase as a possible biocontrol agent for use instead of chemical fungicides.

We investigated whether a plant chitinase can be used as a biocontrol agent instead of chemical fungicides by spraying chitinase E (family 19; class IV) from a yam (Dioscorea opposita Thunb) alone or together with beta-1,3-glucanase directly onto the surface of a powdery mildew infecting strawberry berries and leaves. Results were observed by eye and with a scanning electron microscope. The powdery mildew infecting the strawberries was degraded, mainly by the chitinase, and the disease did not appear again for more than 2 weeks. These results indicated that this kind of plant chitinase might be safe and biodegradable biocontrol agent for use instead of conventional fungicides.