Identification and functional analysis of floral terpene synthase genes in Curcuma alismatifolia.

MAIN CONCLUSION: CaTPS2 and CaTPS3 were significantly expressed in flowers of Curcuma alismatifolia 'Shadow' and demonstrated bifunctional enzyme activity, CaTPS2 generated linalool and nerolidol as products, and CaTPS3 catalyzed ß-myrcene and ß-farnesene formation. This study presents the discovery and functional characterization of floral terpene synthase (TPS) genes in Curcuma alismatifolia 'Shadow', a cultivar renowned for its unique fragrance. Addressing the gap in understanding the genetic basis of floral scent in this species, we identified eight TPS genes through comprehensive transcriptome sequencing. Among these, CaTPS2 and CaTPS3 were significantly expressed in floral tissues and demonstrated bifunctional enzyme activity corresponding to the major volatile compounds detected in 'Shadow'. Functional analyses, including in vitro assays complemented with rigorous controls and alternative identification methods, elucidated the roles of these TPS genes in terpenoid biosynthesis. In vitro studies were conducted via heterologous expression in E. coli, followed by purification of the recombinant protein using affinity chromatography, enzyme assays were performed with GPP/FPP as the substrate, and volatile products were inserted into the GC-MS for analysis. Partially purified recombinant protein of CaTPS2 catalyzed GPP and FPP to produce linalool and nerolidol, respectively, while partially purified recombinant protein of CaTPS3 generated ß-myrcene and ß-farnesene with GPP and FPP as substrates, respectively. Real-time quantitative PCR further validated the expression patterns of these genes, correlating with terpenoid accumulation in different plant tissues. Our findings illuminate the molecular mechanisms underpinning floral fragrance in C. alismatifolia and provide a foundation for future genetic enhancements of floral scent in ornamental plants. This study, therefore, contributes to the broader understanding of terpenoid biosynthesis in plant fragrances, paving the way for biotechnological applications in horticulture plant breeding.

An integrated strategy to identify genes responsible for sesquiterpene biosynthesis in turmeric.

KEY MESSAGE: Metabolic module, gene expression pattern and PLS modeling were integrated to precisely identify the terpene synthase responsible for sesquiterpene formation. Functional characterization confirmed the feasibility and sensitivity of this strategy. Plant secondary metabolite biosynthetic pathway elucidation is crucial for the production of these compounds with metabolic engineering. In this study, an integrated strategy was employed to predict the gene function of sesquiterpene synthase (STS) genes using turmeric as a model. Parallel analysis of gene expression patterns and metabolite modules narrowed the candidates into an STS group in which the STSs showed a similar expression pattern. The projections to latent structures by means of partial least squares model was further employed to establish a clear relationship between the candidate STS genes and metabolites and to predict three STSs (ClTPS16, ClTPS15 and ClTPS14) involved in the biosynthesis of several sesquiterpene skeletons. Functional characterization revealed that zingiberene and ß-sesquiphellandrene were the major products of ClTPS16, and ß-eudesmol was produced by ClTPS15, both of which indicated the accuracy of the prediction. Functional characterization of a control STS, ClTPS1, produced a small amount of ß-sesquiphellandrene, as predicted, which confirmed the sensitivity of metabolite module analysis. This integrated strategy provides a methodology for gene function predictions, which represents a substantial improvement in the elucidation of biosynthetic pathways in nonmodel plants.

Isolation and Functional Characterization of New Terpene Synthase Genes from Traditional Edible Plants.

Terpene synthase (TPS) genes were isolated and functionally characterized from three traditional edible plants, Acanthopanax sciadophylloides ("Koshiabura") and Acanthopanax sieboldianus ("Himeukogi"), belonging to the family Araliaceae, and Curcuma zedoaria (zedoary, "Gajutsu"), belonging to the family Zingiberaceae. These plants emit characteristic fragrances and are used for traditional foods and folk medicines. From their fragrant tissues, i.e., sprouts of Araliaceae plants and developing rhizomes of zedoary, total RNAs were extracted and reverse transcribed. The resultant cDNAs were used for degenerate PCR followed by rapid amplification of cDNA ends. From the contig sequences obtained, full-length Tps genes were amplified by PCR with newly synthesized primer sets. The isolated full-length genes were introduced into engineered Escherichia coli cells, which can utilize acetoacetate to synthesize farnesyl diphosphate, the substrate for TPSs, through the mevalonate pathway. TPS products synthesized in the transformed E. coli cells were analysed by gas chromatography-mass spectrometry, nuclear magnetic resonance, and optical rotation. Consequently, the isolated Tps genes were found to encode ß-caryophyllene synthase, germacrene D synthase, linalool/(3S)-(+)-nerolidol synthase, ß-eudesmol synthase, and germacrene B synthase. These results lead us to expect that some of the effective ingredients in folk medicines are volatile terpenes and that intake of traditional foods including these edible plants would have some positive effects on our health.

Differential expression of CURS gene during various growth stages, climatic condition and soil nutrients in turmeric (Curcuma longa): Towards site specific cultivation for high curcumin yield.

Curcuma longa L., accumulates substantial amount of curcumin and essential oil. Little is known about the differential expression of curcumin synthase (CURS) gene and consequent curcumin content variations at different agroclimatic zones. The present study aimed to evaluate the effect of climate, soil and harvesting phase on expression of CURS gene for curcumin yield in two high yielding turmeric cultivars. Expression of CURS gene at different experimental zones as well as at different harvesting phase was studied through transcriptional analysis by qRT-PCR. Curcumin varied from 1.5 to 5% and 1.4-5% in Surama and Roma respectively. The expression of CURS also varied from 0.402 to 5.584 fold in Surama and 0.856-5.217 fold in Roma. Difference in curcumin content at a particular zone varied among different harvesting period from 3.95 to 4.31% in Surama and 3.57-3.83% in Roma. Expression of CURS gene was also effected by harvesting time of the rhizome which varied from 7.389 to 16.882 fold in Surama and 4.41-8.342 fold in Roma. The CURS gene expression was found regardless of variations in curcumin content at different experimental zones. This may be due to the effects of soil and environmental variables. Expression was positively correlated with curcumin content with different harvesting time at a particular zone. This find indicates effect of soil and environment on molecular and biochemical dynamics of curcumin biosynthesis and could be useful in genetic improvement of turmeric.

Development of intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase for discriminating Curcuma species.

Various Curcuma rhizomes have been used as medicines or spices in Asia since ancient times. It is very difficult to distinguish them morphologically, especially when they are boiled and dried, which causes misidentification leading to a loss of efficacy. We developed a method for discriminating Curcuma species by intron length polymorphism markers in genes encoding diketide-CoA synthase and curcumin synthase. This method could apply to identification of not only fresh plants but also samples of crude drugs or edible spices. By applying this method to Curcuma specimens and samples, and constructing a dendrogram based on these markers, seven Curcuma species were clearly distinguishable. Moreover, Curcuma longa specimens were geographically distinguishable. On the other hand, Curcuma kwangsiensis (gl type) specimens also showed intraspecies polymorphism, which may have occurred as a result of hybridization with other Curcuma species. The molecular method we developed is a potential tool for global classification of the genus Curcuma.

Foliar application of ß-D-glucan nanoparticles to control rhizome rot disease of turmeric.

The soilborne Oomycete Pythium aphanidermatum is the causal agent of rhizome rot disease, one of the most serious threats to turmeric crops. At present, effective fungicides are not available. Researches on nanoparticles in a number of crops have evidenced the positive changes in gene expression indicating their potential use in crop improvement. Hence, experiments were carried out to determine the effect of ß-D-glucan nanoparticles (nanobiopolymer) in protection of turmeric plants against rot disease by the way of products that reinforce plant's own defense mechanism. Foliar spray of ß-D-glucan nanoparticles (0.1%, w/v) elicited marked increase in the activity of defense enzymes such as peroxidases (E.C.1.11.1.7), polyphenol oxidases (E.C.1.14.18.1), protease inhibitors (E.C.3.4.21.1) and ß-1,3-glucanases (E.C.3.2.1.39) at various age levels. Constitutive and induced isoforms of these enzymes were investigated during this time-course study. ß-D-glucan nanoparticles (GNPs) significantly reduced the rot incidence offering 77% protection. Increased activities of defense enzymes in GNPs-applied turmeric plants may play a role in restricting the development of disease symptoms. These results demonstrated that GNPs could be used as an effective resistance activator in turmeric for control of rhizome rot disease.

Inhibition of hydroxycinnamoyl-CoA thioesterases in ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.) by lipase inhibitors.

Ginger (Zingiber officinale Rosc.) and turmeric (Curcuma longa L.), members of the Zingiberaceae, are widely used in traditional Asian cuisines and herbal medicine. Gingerols and diarylheptanoids, important compounds from these plants, appear to be produced by enzymes of the type III polyketide synthase class. Previous efforts to detect activity of such enzymes in tissues from these plants were only marginally successful in turmeric and completely unsuccessful in ginger because of very rapid hydrolysis of the hydroxycinnamoyl-CoA substrates (p-coumaroyl-CoA, feruloyl-CoA and caffeoyl-CoA) in these assays, presumably due to the presence of thioesterases in these tissues. In order to determine whether such thioesterase activities were specific and could be reduced so that the polyketide synthase activities could be better characterized, three inhibitors of the thioesterase domain of fatty acid synthase were tested in assays with leaf and rhizome crude protein extracts from these plants: orlistat, a reduced form of lipstatin, and peptide 1 and peptide 2 from hydrolysates of soybean ß-conglycinin. Results of these analyses indicated that specific thioesterases do exist in these plants and that they could indeed be inhibited, with highest inhibition occurring with a mixture of these three compounds, leading for example to a reduction of caffeoyl-CoA hydrolysis in leaves and rhizomes of ginger by 40-fold and 27-fold, respectively.

A superoxide dismutase purified from the rhizome of Curcuma aeruginosa Roxb. as inhibitor of nitric oxide production in the macrophage-like RAW 264.7 cell line.

Superoxide dismutase (SOD, EC 1.15.1.1) is a metalloenzyme or antioxidant enzyme that catalyzes the disproportionation of the harmful superoxide anionic radical to hydrogen peroxide and molecular oxygen. Due to its antioxidative effects, SOD has long been applied in medicinal treatment, cosmetic, and other chemical industries. Fifteen Zingiberaceae plants were tested for SOD activity in their rhizome extracts. The crude homogenate and ammonium sulfate cut fraction of Curcuma aeruginosa were found to contain a significant level of SOD activity. The SOD enzyme was enriched 16.7-fold by sequential ammonium sulfate precipitation, diethylaminoethyl cellulose ion exchange, and Superdex 75 gel filtration column chromatography. An overall SOD yield of 2.51 % with a specific activity of 812.20 U/mg was obtained. The enriched SOD had an apparent MW of 31.5 kDa, as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and a pH and temperature optima of 4.0 and 50 °C. With nitroblue tetrazolium and riboflavin as substrates, the K(m) values were 57.31 ± 0.012 and 1.51 ± 0.014 M, respectively, with corresponding V(max) values of 333.7 ± 0.034 and 254.1 ± 0.022 µmol min(-1) mg protein(-1). This SOD likely belongs to the Fe- or Mn-SOD category due to the fact that it was insensitive to potassium cyanide or hydrogen peroxide inhibition, but was potentially weakly stimulated by hydrogen peroxide, and stimulated by Mn(2+)and Fe(2+) ions. Moreover, this purified SOD also exhibited inhibitory effects on lipopolysaccharide-induced nitric oxide production in cultured mouse macrophage cell RAW 264.7 in a dose-dependent manner (IC(50) = 14.36 ± 0.15 µg protein/ml).

Structural and biochemical elucidation of mechanism for decarboxylative condensation of beta-keto acid by curcumin synthase.

The typical reaction catalyzed by type III polyketide synthases (PKSs) is a decarboxylative condensation between acyl-CoA (starter substrate) and malonyl-CoA (extender substrate). In contrast, curcumin synthase 1 (CURS1), which catalyzes curcumin synthesis by condensing feruloyl-CoA with a diketide-CoA, uses a ß-keto acid (which is derived from diketide-CoA) as an extender substrate. Here, we determined the crystal structure of CURS1 at 2.32 Å resolution. The overall structure of CURS1 was very similar to the reported structures of type III PKSs and exhibited the αßαßα fold. However, CURS1 had a unique hydrophobic cavity in the CoA-binding tunnel. Replacement of Gly-211 with Phe greatly reduced the enzyme activity. The crystal structure of the G211F mutant (at 2.5 Å resolution) revealed that the side chain of Phe-211 occupied the hydrophobic cavity. Biochemical studies demonstrated that CURS1 catalyzes the decarboxylative condensation of a ß-keto acid using a mechanism identical to that for normal decarboxylative condensation of malonyl-CoA by typical type III PKSs. Furthermore, the extender substrate specificity of CURS1 suggested that hydrophobic interaction between CURS1 and a ß-keto acid may be important for CURS1 to use an extender substrate lacking the CoA moiety. From these results and a modeling study on substrate binding, we concluded that the hydrophobic cavity is responsible for the hydrophobic interaction between CURS1 and a ß-keto acid, and this hydrophobic interaction enables the ß-keto acid moiety to access the catalytic center of CURS1 efficiently.

Purification and properties of cysteine protease from rhizomes of Curcuma longa (Linn.).

BACKGROUND: Turmeric rhizome (Curcuma domestica Linn.) contains proteases and has proteolytic activity. Curcumin from turmeric rhizomes has been used for healing many ailments, including cancer. The purpose of this study was to purify turmeric protease and to research their biochemical characteristics [corrected]. RESULTS: Cysteine protease from C. domestica has been purified to homogeneity using acetone precipitation followed by preparatory native polyacrylamide gel electrophoresis (PAGE). This protocol resulted in six fold purification with 28% final recovery. The purified turmeric protease showed a prominent single peak and band on high-performance liquid chromatography and sodium dodecyl sulfate-PAGE, respectively, and an estimated molecular weight of 43 KDa, and exhibited optimal activity between 37 and 60 degrees C. The protease activity of the turmeric protease was significantly inhibited by iodoacetic acid. The turmeric protease had higher alanine and glutamate content and cleaved synthetic peptides N-Cbz-Ile-Pro and N-Cbz-Phe-Leu in a time-dependent manner. Peptide mass fingerprint using matrix-assisted laser desorption/ionization-time of flight mass spectroscopy revealed peptide matches to proteasome subunit alpha type 3 of Oryza sativa ssp. japonica (Rice). The turmeric protease showed antifungal activity at 10 microg mL(-1) towards pathogens Pythium aphanidermatum, Trichoderma viride and Fusarium sp. CONCLUSION: Cysteine addition significantly activated turmeric protease. The protease inhibition test suggested that turmeric protease belonged to the cysteine type. The biochemical characteristics of turmeric protease described in this paper can provide useful information for potential end uses of turmeric protease for pharmaceutical industry applications such as therapeutics.

Curcumin alleviates ethanol-induced hepatocytes oxidative damage involving heme oxygenase-1 induction.

ETHNOPHARMACOLOGICAL RELEVANCE: Curcumin is the main bioactive constituent derived from the rhizome of turmeric (Curcuma longa Linn.), which has been used traditionally as hepatoprotective agents in ayurvedic and traditional Chinese medicine for centuries. AIM OF THE STUDY: The present study was carried out to demonstrate the potential protective effect of curcumin pretreatment against ethanol-induced hepatocytes oxidative damage, with emphasis on heme oxygenase-1 (HO-1) induction. MATERIALS AND METHODS: Rat primary hepatocytes were isolated and treated with ethanol (100mM) and diverse doses of curcumin (0-50 microM), which was pretreated at various time points (0-5h) before ethanol administration. Hepatic enzyme releases in the culture medium and redox status including HO-1 enzyme activity were detected. RESULTS: Ethanol exposure resulted in a sustained malondialdehyde (MDA) elevation, glutathione (GSH) depletion and evident release of cellular lactate dehydrogenase (LDH) and aspartate aminotransferase (AST), which was significantly ameliorated by curcumin pretreatment. In addition, dose- and time-dependent induction of HO-1 was involved in such hepatoprotective effects by curcumin. CONCLUSIONS: Curcumin exerts hepatoprotective properties against ethanol involving HO-1 induction, which provide new insights into the pharmacological targets of curcumin in the prevention of alcoholic liver disease.

Identification and characterization of multiple curcumin synthases from the herb Curcuma longa.

Curcuminoids are pharmaceutically important compounds isolated from the herb Curcuma longa. Two additional type III polyketide synthases, named CURS2 and CURS3, that are capable of curcuminoid synthesis were identified and characterized. In vitro analysis revealed that CURS2 preferred feruloyl-CoA as a starter substrate and CURS3 preferred both feruloyl-CoA and p-coumaroyl-CoA. These results suggested that CURS2 synthesizes curcumin or demethoxycurcumin and CURS3 synthesizes curcumin, bisdemethoxycurcumin and demethoxycurcumin. The availability of the substrates and the expression levels of the three different enzymes capable of curcuminoid synthesis with different substrate specificities might influence the composition of curcuminoids in the turmeric and in different cultivars.

[Study on isozymes in six species of Curcuma].

OBJECTIVE: To explore genetic relationships of the 39 materials in six species of Curcuma. METHOD: The peroxidase isozyme (POD) and esterase isozyme (EST) were studied using vertical slab polyacrylamide gel electrophoresis (PAGE) technique, and the zymograms were analyzed using the software of NTSYSpc2. 1. RESULT: The interspecific zymogramatic differences were obvious. Each species possessed its own specific zymogram distinguishing form the others. In the analysis of EST isozyme, C. phaeocaulis, C. wenyujin, C. kwangsiensis and C. chuanhuangjiang had their own specific zymogram. In the analysis of POD isozyme, just C. phaeocaulis and C. kwangsiensis had their specific zymogram. CONCLUSION: The genetic relationships are not associated with the geographical distributions and the genetic relationship between C. longa and C. sichuanensis are very close.

Identification and characterization of a super-stable Cu-Zn SOD from leaves of turmeric (Curcuma longa L.).

We report a novel super stable superoxide dismutase (SOD) extracted from the leaves of Curcuma longa L.-a post-harvest waste. The scavenging activity of this SOD remains intact both in crude and purified forms before and after heating at boiling temperatures (80-100 degrees C) up to 20 min, autoclaving (6-20 bars up to 10 min) and microwaving (frequency of 2,450 megahertz (MHz) or million cycles per second for 1-3 min). This SOD has significant shelf life at room temperature (25-35 degrees C) and is stable for at least 18 months at 4 degrees C and with the retained activity of 82% at -10 degrees C and 88% at -20 degrees C without any infection or contamination. The heat stable enzyme is present both in cytoplasm and chloroplasts. The enzyme is also stable under wide range of pH, alcohol and SDS concentrations. The heat stability of this SOD protein is not due to any associated phenolic compound as no phenolic compound was bound to the novel thermo-stable SOD. The activity staining through native PAGE and purification of the enzyme protein have shown that this form of enzyme has a native molecular weight of 30.8 kDa and has two subunits of 15 kDa as shown by SDS PAGE. The characterized novel isoform is a Cu-Zn SOD as is indicated by its sensitivity to both H2O2 and KCN. Indian, US and PCT patents have been filed and products are being developed using this hyperthermophilic enzyme.

Isolation and characterization of antioxidation enzymes from cells of zedoary (Curcuma zedoaria Roscoe) cultured in a 5-l bioreactor.

In this study, a cell suspension culture system for zedoary (Curcuma zedoaria Roscoe) was developed, using 50 g/l of fresh weight inoculum in a batch culture. The highest cell biomass obtained from a 5-l bioreactor equipped with three impellers after 14 days of culture was utilized to extract secondary metabolites (essential oil and curcumin) and determine the activities of antioxidant enzymes (peroxidase, superoxide dismutase, and catalase). For essential oil and curcumin, zedoary extracts were recovered via a variety of methods: steam distillation, volatile solvents, and Soxhlet. After 14 days of culture using volatile solvents, the optimal yield of essential oil (1.78%) was obtained when using petroleum ether at 40 degrees C in 6 h of extraction, and the best curcumin yield (9.69%) was obtained at 60 degrees C in 6 h via extraction with 90% ethanol. The activities of antioxidant enzymes from zedoary cells were also assessed. The specific activities of peroxidase, superoxide-dismutase, and catalase reached maximum values of 0.63 U/mg of protein, 16.60 U/mg of protein, and 19.59 U/mg of protein after 14 days of culture, respectively.

Biosynthesis of curcuminoids and gingerols in turmeric (Curcuma longa) and ginger (Zingiber officinale): identification of curcuminoid synthase and hydroxycinnamoyl-CoA thioesterases.

Members of the Zingiberaceae such as turmeric (Curcuma longa L.) and ginger (Zingiber officinale Rosc.) accumulate at high levels in their rhizomes important pharmacologically active metabolites that appear to be derived from the phenylpropanoid pathway. In ginger, these compounds are the gingerols; in turmeric these are the curcuminoids. Despite their importance, little is known about the biosynthesis of these compounds. This investigation describes the identification of enzymes in the biosynthetic pathway leading to the production of these bioactive natural products. Assays for enzymes in the phenylpropanoid pathway identified the corresponding enzyme activities in protein crude extracts from leaf, shoot and rhizome tissues from ginger and turmeric. These enzymes included phenylalanine ammonia lyase, polyketide synthases, p-coumaroyl shikimate transferase, p-coumaroyl quinate transferase, caffeic acid O-methyltransferase, and caffeoyl-CoA O-methyltransferase, which were evaluated because of their potential roles in controlling production of certain classes of gingerols and curcuminoids. All crude extracts possessed activity for all of these enzymes, with the exception of polyketide synthases. The results of polyketide synthase assays showed detectable curcuminoid synthase activity in the extracts from turmeric with the highest activity found in extracts from leaves. However, no gingerol synthase activity could be identified. This result was explained by the identification of thioesterase activities that cleaved phenylpropanoid pathway CoA esters, and which were found to be present at high levels in all tissues, especially in ginger tissues. These activities may shunt phenylpropanoid pathway intermediates away from the production of curcuminoids and gingerols, thereby potentially playing a regulatory role in the biosynthesis of these compounds.