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.     

Exogenous application of salicylic acid enhanced the rutin accumulation and influenced the expression patterns of rutin biosynthesis related genes in Fagopyrum tartaricum Gaertn leaves

Except for buckwheat, no cereal or pseudocereal contains any detectable rutin. This study was carried out to compare the rutin content in leaves treated with various concentrations of salicylic acid (SA) (50?mg?l^?1, 100?mg?l^?1, 150?mg?l^?1) for various lengths of time (2?C120?h) to those of untreated F. tartaricum. Rutin content in leaves during the early stages of SA treatment (100?mg?l^?1, 150?mg?l^?1; 24?C48?h) were higher than at other times. The highest rutin content (40.3?mg?gFW^?1) was found in leaves treated with 150?mg?l^?1 SA for 48?h. We cloned partial cDNAs of five genes known to be related to rutin biosynthesis from the leaves of F. tataricum using RACE. These genes were chalcone synthase (FtCHS), flavonol synthase (FtFLS-like), flavone 3-hydroxylase (FtF3H), 4-coumaroyl CoA ligase (Ft4CL), and 4-coumaroyl CoA ligase 2 (Ft4CL2). The protein sequences of these genes were analyzed by similarity searching and phylogenetic trees. Apart from the FtFLS-like gene, which had poor identity with F. esculentum, the other genes showed high similarity to those in F. esculentum. During the early stages of SA treatment (24?C48?h), expression levels of four genes (FtCHS, FtFLS-like, FtF3H, and Ft4CL) were higher than controls but that of Ft4CL2 was not. Most notably, the expression level of FtFLS-like 24?h after application of 150?mg?l^?1 SA was 14.79 times of the control. These results suggest that rutin content can be enhanced to great extent by SA treatment and the gene expression patterns of rutin-biosynthesis-related genes are regulated by SA.     

Genetic diversity and biological activity of Curcuma longa ecotypes from Rapa Nui using molecular markers

Curcuma Longa (CL) has been used for hundreds of years by native people from Rapa Nui for the treatment of different illness. Despite this plant was introduced from Polynesia or India, there is still scarce information about its origin. The objective of this study was to analyze the genetic variation of three CL ecotypes based on molecular phylogenetic and genotypification using internal transcribed spacer 2 (ITS2) and simple sequence repeats (SSR). Antioxidant and anti-inflammatory properties of rhizomes and leaves extracts of three CL plants were analyzed by spectrophotometric methods and cyclooxygenase 2 (COX-2) inhibition assay. Complementarily, we predicted the potential binding mode and binding energy of curcuminoids and nonsteroidals anti-inflammatory drugs (NSAIDs) into COX-2 via molecular docking. The ITS2 sequence shows two major clusters (I and II), group I consisted of Curcuma haritha and group II consisted of different species of Curcuma and Rapa Nui samples (MR-1, MR-2 and RK-2). Results of SSR markers show that genotype MR-2 was similar to MR-1 and RK-2 with 70.8 and 42.9% similarity, whereas genotype was similar to RK-2, MR-1 and MR-2 with 63.9, 43.2 and 42.9% similarity, respectively. MR-1 have better antioxidant and autoinflammatory activity than rest of CL samples due to its high concentration of polyphenols (33.68 mg/g) and curcumin (29.69 mg/g). Furthermore, docking results show that three curcuminoids of CL and selective NAIDs, as celecoxib, etodolac and meloxicam, share the same binding pocket into COX-2. However, three curcuminoids have a lower ΔG_binding than other COX-2 selective NAIDs as etodolac and meloxicam, except for Coxib family as valdecoxib, celecoxib and rofecoxib. Our findings suggest MR-1, MR-2 and MK-2 from Germplasm Bank (Mataveri Otai of CONAF) are closely related to Curcuma amada and Curcuma montana even though they have genetic variability.     

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.     

Chalcone synthase-like genes active during corolla development are differentially expressed and encode enzymes with different catalytic properties in Gerbera hybrida (Asteraceae)

Recent studies on chalcone synthase (CHS) and the related stilbene synthase (STS) suggest that the structure of chs-like genes in plants has evolved into different forms, whose members have both different regulation and capacity to code for different but related enzymatic activities. We have studied the diversity of chs-like genes by analysing the structure, expression patterns and catalytic properties of the corresponding enzymes of three genes that are active during corolla development in Gerbera hybrida. The expression patterns demonstrate that chs-like genes are representatives of three distinct genetic programmes that are active during organ differentiation in gerbera. Gchs1 and gchs3 code for typical CHS enzymes, and their gene expression pattern temporally correlates with flavonol (gchs1, gchs3) and anthocyanin (gchs1) synthesis during corolla development. Gchs2 is different. The expression pattern does not correlate with the pigmentation pattern, the amino acid sequence deviates considerably from the consensus of typical CHSs, and the catalytic properties are different. The data indicate that it represents a new member in the large superfamily of chs and chs-related genes.     

Three 1-Aminocyclopropane-1-Carboxylate Synthase Genes Regulated by Primary and Secondary Pollination Signals in Orchid Flowers

The temporal and spatial expression patterns of three 1-aminocyclopropane-1-carboxylate (ACC) synthase genes were investigated in pollinated orchid (Phalaenopsis spp.) flowers. Pollination signals initiate a cascade of development events in multiple floral organs, including the induction of ethylene biosynthesis, which coordinates several postpollination developmental responses. The initiation and propagation of ethylene biosynthesis is regulated by the coordinated expression of three distinct ACC synthase genes in orchid flowers. One ACC synthase gene (Phal-ACS1) is regulated by ethylene and participates in amplification and interorgan transmission of the pollination signal, as we have previously described in a related orchid genus. Two additional ACC synthase genes (Phal-ACS2 and Phal-ACS3) are expressed primarily in the stigma and ovary of pollinated orchid flowers. Phal-ACS2 mRNA accumulated in the stigma within 1 h after pollination, whereas Phal-ACS1 mRNA was not detected until 6 h after pollination. Similar to the expression of Phal-ACS2, the Phal-ACS3 gene was expressed within 2 h after pollination in the ovary. Exogenous application of auxin, but not ACC, mimicked pollination by stimulating a rapid increase in ACC synthase activity in the stigma and ovary and inducing Phal-ACS2 and Phal-ACS3 mRNA accumulation in the stigma and ovary, respectively. These results provide the basis for an expanded model of interorgan regulation of three ACC synthase genes that respond to both primary (Phal-ACS2 and Phal-ACS3) and secondary (Phal-ACS1) pollination signals.     

Diversity of Benzylsuccinate Synthase-Like (bssA) Genes in Hydrocarbon-Polluted Marine Sediments Suggests Substrate-Dependent Clustering

The potential of hydrocarbon biodegradation in marine sediments was determined through the detection of a functional biomarker, the bssA gene, coding for benzylsuccinate synthase, the key enzyme of anaerobic toluene degradation. Eight bssA clone libraries (409 sequences) were constructed from polluted sediments affected by the Prestige oil spill in the Atlantic Islands National Park and from hydrocarbon-amended sediment microcosms in Mallorca. The amplified products and database-derived bssA-like sequences grouped into four major clusters, as determined by phylogenetic reconstruction, principal coordinate analysis (PCoA), and a subfamily prediction tool. In addition to the classical bssA sequences that were targeted, we were able to detect sequences homologous to the naphthylmethylsuccinate synthase gene (nmsA) and the alkylsuccinate synthase gene (assA), the bssA homologues for anaerobic 2-methylnaphthalene and alkane degradation, respectively. The detection of bssA-like variants was determined by the persistence and level of pollution in the marine samples. The observed level of gene diversity was lower in the Mallorca sediments, which were dominated by assA-like sequences. In contrast, the Atlantic Islands samples, which were highly contaminated with methylnaphthalene-rich crude oil, showed a high proportion of nmsA-like sequences. Some of the detected genes were phylogenetically related to Deltaproteobacteria communities, previously described as the predominant hydrocarbon degraders at these sites. Differences between all detected bssA-like genes described to date indicate separation between marine and terrestrial sequences and further subgrouping according to taxonomic affiliation. Global analysis suggested that bssA homologues appeared to cluster according to substrate specificity. We observed undetected divergent gene lineages of bssA homologues, which evidence the existence of new degrader groups in these environments.     

The biosynthetic pathway of curcuminoid in turmeric (Curcuma longa) as revealed by 13C-labeled precursors

In order to investigate the biosynthesis of curcuminoid in rhizomes of turmeric (Curcuma longa), we established an in vitro culture system of turmeric plants for feeding (13)C-labeled precursors. Analyses of labeled desmethoxycurcumin (DMC), an unsymmetrical curcuminoid, by (13)C-NMR, revealed that one molecule of acetic acid or malonic acid and two molecules of phenylalanine or phenylpropanoids, but not tyrosine, were incorporated into DMC. The incorporation efficiencies of the same precursors into DMC and curcumin were similar, and were in the order malonic acid > acetic acid, and cinnamic acid > p-coumaric acid > ferulic acid. These results suggest the possibility that the pathway to curcuminoids utilized two cinnamoyl CoAs and one malonyl CoA, and that hydroxy- and methoxy-functional groups on the aromatic rings were introduced after the formation of the curcuminoid skeleton.     

LEAFY COTYLEDON1 is a key regulator of fatty acid biosynthesis in Arabidopsis

In plants, fatty acids are de novo synthesized predominantly in plastids from acetyl-coenzyme A. Although fatty acid biosynthesis has been biochemically well studied, little is known about the regulatory mechanisms of the pathway. Here, we show that overexpression of the Arabidopsis (Arabidopsis thaliana) LEAFY COTYLEDON1 (LEC1) gene causes globally increased expression of fatty acid biosynthetic genes, which are involved in key reactions of condensation, chain elongation, and desaturation of fatty acid biosynthesis. In the plastidial fatty acid synthetic pathway, over 58% of known enzyme-coding genes are up-regulated in LEC1-overexpressing transgenic plants, including those encoding three subunits of acetyl-coenzyme A carboxylase, a key enzyme controlling the fatty acid biosynthesis flux. Moreover, genes involved in glycolysis and lipid accumulation are also up-regulated. Consistent with these results, levels of major fatty acid species and lipids were substantially increased in the transgenic plants. Genetic analysis indicates that the LEC1 function is partially dependent on ABSCISIC ACID INSENSITIVE3, FUSCA3, and WRINKLED1 in the regulation of fatty acid biosynthesis. Moreover, a similar phenotype was observed in transgenic Arabidopsis plants overexpressing two LEC1-like genes of Brassica napus. These results suggest that LEC1 and LEC1-like genes act as key regulators to coordinate the expression of fatty acid biosynthetic genes, thereby representing promising targets for genetic improvement of oil production plants.     

A sucrose non-fermenting-1-related protein kinase 1 gene from potato, <Emphasis Type="Italic">StSnRK1</Emphasis>, regulates carbohydrate metabolism in transgenic tobacco

Sucrose non-fermenting-1-related protein kinase 1 (SnRK1) has been shown to play an essential role in regulating saccharide metabolism and starch biosynthesis of plant. The regulatory role of StSnRK1 from potato in regulating carbohydrate metabolism and starch accumulation has not been investigated. In this work, a cDNA encoding the SnRK1 protein, named StSnRK1, was isolated from potato. The open reading frame contained 1545 nucleotides encoding 514 amino acids. Subcellular localization analysis in onion epidermal cells indicated that StSnRK1 protein was localized to the nucleus. The coding region of StSnRK1 was cloned into a binary vector under the control of 35S promoter and then transformed into tobacco to obtain transgenic plants. Transgenic tobacco plants expressing StSnRK1 were shown to have a significant increased accumulation of starch content, as well as sucrose, glucose and fructose content. Real-time quantitative PCR analysis indicated that overexpression of StSnRK1 up-regulated the expression of sucrose synthase (NtSUS), ADP-glucose pyrophosphorylase (NtAGPase) and soluble starch synthase (NtSSS III) genes involved in starch biosynthesis in the transgenic plants. In contrast, the expression of sucrose phosphate synthase (NtSPS) gene was decreased in the transgenic plants. Meanwhile, enzymatic analyses indicated that the activities of major enzymes (SUS, AGPase and SSS) involved in the starch biosynthesis were enhanced, whereas SPS activity was decreased in the transgenic plants compared to the wild-type. These results suggest that the manipulation of StSnRK1 expression might be used for improving quality of plants in the future.     

Comparative analysis of GT14/GT14-like gene family in Arabidopsis, Oryza, Populus, Sorghum and Vitis

Glycosyltransferase family14 (GT14) belongs to the glycosyltransferase (GT) superfamily that plays important roles in the biosynthesis of cell walls, the most abundant source of cellulosic biomass for bioethanol production. It has been hypothesized that DUF266 proteins are a new class of GTs related to GT14. In this study, we identified 62 GT14 and 106 DUF266 genes (named GT14-like herein) in Arabidopsis, Oryza, Populus, Sorghum and Vitis. Our phylogenetic analysis separated GT14 and GT14-like genes into two distinct clades, which were further divided into eight and five groups, respectively. Similarities in protein domain, 3D structure and gene expression were uncovered between the two phylogenetic clades, supporting the hypothesis that GT14 and GT14-like genes belong to one family. Therefore, we proposed a new family name, GT14/GT14-like family that combines both subfamilies. Variation in gene expression and protein subcellular localization within the GT14-like subfamily were greater than those within the GT14 subfamily. One-half of the Arabidopsis and Populus GT14/GT14-like genes were found to be preferentially expressed in stem/xylem, indicating that they are likely involved in cell wall biosynthesis. This study provided new insights into the evolution and functional diversification of the GT14/GT14-like family genes.     

Comparative genomics and proteomics of vertebrate diacylglycerol acyltransferase (DGAT), acyl CoA wax alcohol acyltransferase (AWAT) and monoacylglycerol acyltransferase (MGAT)

BLAT (BLAST-Like Alignment Tool) analyses of the opossum (Monodelphis domestica) and zebrafish (Danio rerio) genomes were undertaken using amino acid sequences of the acylglycerol acyltransferase (AGAT) superfamily. Evidence is reported for 8 opossum monoacylglycerol acyltransferase-like (MGAT) (E.C. 2.3.1.22) and diacylglycerol acyltransferase-like (DGAT) (E.C. 2.3.1.20) genes and proteins, including DGAT1, DGAT2, DGAT2L6 (DGAT2-like protein 6), AWAT1 (acyl CoA wax alcohol acyltransferase 1), AWAT2, MGAT1, MGAT2 and MGAT3. Three of these genes (AWAT1, AWAT2 and DGAT2L6) are closely localized on the opossum X chromosome. Evidence is also reported for six zebrafish MGAT- and DGAT-like genes, including two DGAT1-like genes, as well as DGAT2-, MGAT1-, MGAT2- and MGAT3-like genes and proteins. Predicted primary, secondary and transmembrane structures for the opossum and zebrafish MGAT-, AWAT- and DGAT-like subunits and the intron–exon boundaries for genes encoding these enzymes showed a high degree of similarity with other members of the AGAT superfamily, which play major roles in triacylglyceride (DGAT), diacylglyceride (MGAT) and wax ester (AWAT) biosynthesis. Alignments of predicted opossum, zebrafish and other vertebrate DGAT1, DGAT2, other DGAT2-like and MGAT-like amino acid sequences with known human and mouse enzymes demonstrated conservation of residues which are likely to play key roles in catalysis, lipid binding or in maintaining structure. Phylogeny studies of the human, mouse, opossum, zebrafish and pufferfish MGAT- and DGAT-like enzymes indicated that the common ancestors for these genes predated the appearance of bony fish during vertebrate evolution whereas the AWAT- and DGAT2L6-like genes may have appeared more recently prior to the appearance of marsupial and eutherian mammals.