An indigo-producing plant, Polygonum tinctorium, possesses a flavin-containing monooxygenase capable of oxidizing indole.

Polygonum tinctorium (P. tinctorium) is an indigo plant that is cultivated for a specific metabolite that it produces i.e., indoxyl ß-D-glucoside (indican). In this study, flavin-containing monooxygenase (PtFMO) from P. tinctorium was cloned. When recombinant PtFMO was expressed in E. coli in the presence of tryptophan, indigo production was observed. Furthermore, we measured the activity of PtFMO using the membrane fraction from E. coli and found that it could produce indigo using indole as a substrate. The co-expression of PtFMO with indoxyl ß-D-glucoside synthase (PtIGS), which catalyzes the glucosylation of indoxyl, brought about the formation of indican in E. coli. The results showed that indican was synthesized by sequential reactions of PtFMO and PtIGS. In three-week-old P. tinctorium specimens, the first leaves demonstrated higher levels of PtFMO expression than the subsequent leaves. This result coincided with that of our prior study on PtIGS expression level. Our study provides evidence that PtFMO might contribute to indican biosynthesis.

Tissue-specific and intracellular localization of indican synthase from Polygonum tinctorium.

The plant Polygonum tinctorium produces the secondary metabolite indican (indoxyl-ß-D-glucoside), a precursor of the blue dye indigo. P. tinctorium synthesizes indican through the actions of the UDP-glucosyltransferase (UGT), indican synthase. Herein, we partially purified an indican synthase from the leaves and subsequently performed peptide mass fingerprinting analysis. Consequently, we identified a fragment that was homologous to a UDP-glucosyltransferase 72B (UGT72B) family member. We named it PtIgs (P. tinctoriumindoxyl-ß-D-glucoside synthase) and obtained the full-length cDNA using rapid amplification of the cDNA ends. The primary structure of PtIGS, which PtIgs encoded, showed high identity with indican synthases (ItUGT1 and ItUGT2) from Indigofera tinctoria (Inoue et al., 2017). Moreover, in expression analyses of P. tinctorium, PtIGS mRNA was virtually found only in the leaves, was most highly expressed in the 1st leaves, and decreased with leaf age. Because PtIGS expression tended to reflect indican contents and synthesis activities, we concluded that PtIGS functions as an indican synthase in plant cells. To examine intracellular localization of PtIGS, crude leaf extracts were separated into cytosol and microsome fractions, and found PtIGS in the cytosol and in microsome fractions. Furthermore, microsomal PtIGS was soluble in the presence of detergents and urea and was strongly associated with membranes. Finally, we confirmed endoplasmic reticulum (ER) membrane localization of PtIGS using ultracentrifugation with a sucrose density gradient. These data suggest that PtIGS interacts with some kind of proteins on ER membranes to certainly carry out a delivery of substrate.

Functional Characterisation of New Sesquiterpene Synthase from the Malaysian Herbal Plant, Polygonum Minus.

Polygonum minus (syn. Persicaria minor) is a herbal plant that is well known for producing sesquiterpenes, which contribute to its flavour and fragrance. This study describes the cloning and functional characterisation of PmSTPS1 and PmSTPS2, two sesquiterpene synthase genes that were identified from P. minus transcriptome data mining. The full-length sequences of the PmSTPS1 and PmSTPS2 genes were expressed in the E. coli pQE-2 expression vector. The sizes of PmSTPS1 and PmSTPS2 were 1098 bp and 1967 bp, respectively, with open reading frames (ORF) of 1047 and 1695 bp and encoding polypeptides of 348 and 564 amino acids, respectively. The proteins consist of three conserved motifs, namely, Asp-rich substrate binding (DDxxD), metal binding residues (NSE/DTE), and cytoplasmic ER retention (RxR), as well as the terpene synthase family N-terminal domain and C-terminal metal-binding domain. From the in vitro enzyme assays, using the farnesyl pyrophosphate (FPP) substrate, the PmSTPS1 enzyme produced multiple acyclic sesquiterpenes of ß-farnesene, α-farnesene, and farnesol, while the PmSTPS2 enzyme produced an additional nerolidol as a final product. The results confirmed the roles of PmSTPS1 and PmSTPS2 in the biosynthesis pathway of P. minus, to produce aromatic sesquiterpenes.

Employing a biochemical protecting group for a sustainable indigo dyeing strategy.

Indigo is an ancient dye uniquely capable of producing the signature tones in blue denim; however, the dyeing process requires chemical steps that are environmentally damaging. We describe a sustainable dyeing strategy that not only circumvents the use of toxic reagents for indigo chemical synthesis but also removes the need for a reducing agent for dye solubilization. This strategy utilizes a glucose moiety as a biochemical protecting group to stabilize the reactive indigo precursor indoxyl to form indican, preventing spontaneous oxidation to crystalline indigo during microbial fermentation. Application of a ß-glucosidase removes the protecting group from indican, resulting in indigo crystal formation in the cotton fibers. We identified the gene coding for the glucosyltransferase PtUGT1 from the indigo plant Polygonum tinctorium and solved the structure of PtUGT1. Heterologous expression of PtUGT1 in Escherichia coli supported high indican conversion, and biosynthesized indican was used to dye cotton swatches and a garment.

Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium.

KEY MESSAGE: Two cDNAs for indole-3-glycerol phosphate lyase homolog were cloned from Polygonum tinctorium. One encoded cytosolic indole synthase possibly in indigoid synthesis, whereas the other encoded a putative tryptophan synthase α-subunit. Indigo is an old natural blue dye produced by plants such as Polygonum tinctorium. Key step in plant indigoid biosynthesis is production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit (TSA) homologs, PtIGL-short and -long, were isolated by RACE PCR from P. tinctorium. The genome of the plant contained two genes coding for IGL. The short and the long forms, respectively, encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli ΔtnaA ΔtrpA mutant on tryptophan-depleted agar plate signifying production of free indole, and thus was named indole synthase gene (PtINS). The long form, either intact or without the transit peptide sequence, did not complement the mutant and was tentatively named PtTSA. PtTSA was delivered into chloroplast as predicted by 42-residue-long targeting sequence, whereas PtINS was localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to fivefolds higher transcript level than that of PtTSA, and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA was significantly enhanced in the plant. The results indicate participation of PtINS in indigoid production.

Novel NAD+-Farnesal Dehydrogenase from Polygonum minus Leaves. Purification and Characterization of Enzyme in Juvenile Hormone III Biosynthetic Pathway in Plant.

Juvenile Hormone III is of great concern due to negative effects on major developmental and reproductive maturation in insect pests. Thus, the elucidation of enzymes involved JH III biosynthetic pathway has become increasing important in recent years. One of the enzymes in the JH III biosynthetic pathway that remains to be isolated and characterized is farnesal dehydrogenase, an enzyme responsible to catalyze the oxidation of farnesal into farnesoic acid. A novel NAD+-farnesal dehydrogenase of Polygonum minus was purified (315-fold) to apparent homogeneity in five chromatographic steps. The purification procedures included Gigacap S-Toyopearl 650M, Gigacap Q-Toyopearl 650M, and AF-Blue Toyopearl 650ML, followed by TSK Gel G3000SW chromatographies. The enzyme, with isoelectric point of 6.6 is a monomeric enzyme with a molecular mass of 70 kDa. The enzyme was relatively active at 40°C, but was rapidly inactivated above 45°C. The optimal temperature and pH of the enzyme were found to be 35°C and 9.5, respectively. The enzyme activity was inhibited by sulfhydryl agent, chelating agent, and metal ion. The enzyme was highly specific for farnesal and NAD+. Other terpene aldehydes such as trans- cinnamaldehyde, citral and α- methyl cinnamaldehyde were also oxidized but in lower activity. The Km values for farnesal, citral, trans- cinnamaldehyde, α- methyl cinnamaldehyde and NAD+ were 0.13, 0.69, 0.86, 1.28 and 0.31 mM, respectively. The putative P. minus farnesal dehydrogenase that's highly specific towards farnesal but not to aliphatic aldehydes substrates suggested that the enzyme is significantly different from other aldehyde dehydrogenases that have been reported. The MALDI-TOF/TOF-MS/MS spectrometry further identified two peptides that share similarity to those of previously reported aldehyde dehydrogenases. In conclusion, the P. minus farnesal dehydrogenase may represent a novel plant farnesal dehydrogenase that exhibits distinctive substrate specificity towards farnesal. Thus, it was suggested that this novel enzyme may be functioning specifically to oxidize farnesal in the later steps of JH III pathway. This report provides a basic understanding for recombinant production of this particular enzyme. Other strategies such as adding His-tag to the protein makes easy the purification of the protein which is completely different to the native protein. Complete sequence, structure and functional analysis of the enzyme will be important for developing insect-resistant crop plants by deployment of transgenic plant.

Purification and Characterization of a Novel NAD(P)+-Farnesol Dehydrogenase from Polygonum minus Leaves.

Juvenile hormones have attracted attention as safe and selective targets for the design and development of environmentally friendly and biorational insecticides. In the juvenile hormone III biosynthetic pathway, the enzyme farnesol dehydrogenase catalyzes the oxidation of farnesol to farnesal. In this study, farnesol dehydrogenase was extracted from Polygonum minus leaves and purified 204-fold to apparent homogeneity by ion-exchange chromatography using DEAE-Toyopearl, SP-Toyopearl, and Super-Q Toyopearl, followed by three successive purifications by gel filtration chromatography on a TSK-gel GS3000SW. The enzyme is a heterodimer comprised of subunits with molecular masses of 65 kDa and 70 kDa. The optimum temperature and pH were 35°C and pH 9.5, respectively. Activity was inhibited by sulfhydryl reagents, metal-chelating agents and heavy metal ions. The enzyme utilized both NAD+ and NADP+ as coenzymes with Km values of 0.74 mM and 40 mM, respectively. Trans, trans-farnesol was the preferred substrate for the P. minus farnesol dehydrogenase. Geometrical isomers of trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol were also oxidized by the enzyme with lower activity. The Km values for trans, trans-farnesol, cis, trans-farnesol and cis, cis-farnesol appeared to be 0.17 mM, 0.33 mM and 0.42 mM, respectively. The amino acid sequences of 4 tryptic peptides of the enzyme were analyzed by MALDI-TOF/TOF-MS spectrometry, and showed no significant similarity to those of previously reported farnesol dehydrogenases. These results suggest that the purified enzyme is a novel NAD(P)+-dependent farnesol dehydrogenase. The purification and characterization established in the current study will serve as a basis to provide new information for recombinant production of the enzyme. Therefore, recombinant farnesol dehydrogenase may provide a useful molecular tool in manipulating juvenile hormone biosynthesis to generate transgenic plants for pest control.

Functional characterization of sesquiterpene synthase from Polygonum minus.

Polygonum minus is an aromatic plant, which contains high abundance of terpenoids, especially the sesquiterpenes C15H24. Sesquiterpenes were believed to contribute to the many useful biological properties in plants. This study aimed to functionally characterize a full length sesquiterpene synthase gene from P. minus. P. minus sesquiterpene synthase (PmSTS) has a complete open reading frame (ORF) of 1689 base pairs encoding a 562 amino acid protein. Similar to other sesquiterpene synthases, PmSTS has two large domains: the N-terminal domain and the C-terminal metal-binding domain. It also consists of three conserved motifs: the DDXXD, NSE/DTE, and RXR. A three-dimensional protein model for PmSTS built clearly distinguished the two main domains, where conserved motifs were highlighted. We also constructed a phylogenetic tree, which showed that PmSTS belongs to the angiosperm sesquiterpene synthase subfamily Tps-a. To examine the function of PmSTS, we expressed this gene in Arabidopsis thaliana. Two transgenic lines, designated as OE3 and OE7, were further characterized, both molecularly and functionally. The transgenic plants demonstrated smaller basal rosette leaves, shorter and fewer flowering stems, and fewer seeds compared to wild type plants. Gas chromatography-mass spectrometry analysis of the transgenic plants showed that PmSTS was responsible for the production of ß -sesquiphellandrene.

Monoterpene alcohol metabolism: identification, purification, and characterization of two geraniol dehydrogenase isoenzymes from Polygonum minus leaves.

NADP(+)-dependent geraniol dehydrogenase (EC 1.1.1.183) is an enzyme that catalyzes the oxidation of geraniol to geranial. Stable, highly active cell-free extract was obtained from Polygonum minus leaves using polyvinylpolypyrrolidone, Amberlite XAD-4, glycerol, 2-mercaptoethanol, thiourea, and phenylmethylsulfonylfluoride in tricine-NaOH buffer (pH 7.5). The enzyme preparation was separated into two activity peaks, geraniol-DH I and II, by DEAE-Toyopearl 650M column chromatography at pH 7.5. Both isoenzymes were purified to homogeneity in three chromatographic steps. The geraniol-DH isoenzymes were similar in molecular mass, optimal temperature, and pH, but the isoelectric point, substrate specificity, and kinetic parameters were different. The K(m) values for geraniol of geraniol-DH I and II appeared to be 0.4 mM and 0.185 mM respectively. P. minus geraniol-DHs are unusual among geraniol-DHs in view of their thermal stability and optimal temperatures, and also their high specificity for allylic alcohols and NADP(+).

Biosynthesis of coumarin glycosides by transgenic hairy roots of Polygonum multiflorum.

To investigate the substrate specificity and regio-selectivity of coumarin glycosyltransferases in transgenic hairy roots of Polygonum multiflorum, esculetin (1) and eight hydroxycoumarins (2-9) were employed as substrates. Nine corresponding glycosides (10-18) involving four new compounds, 6-chloro-4-methylcoumarin 7-O-ß-D-glucopyranoside (15), 6-chloro-4-phenylcoumarin 7-O-ß-D-glucopyranoside (16), 8-hydroxy-4-methylcoumarin 7-O-ß-D-glucopyranoside (17), and 8-allyl-4-methylcoumarin 7-O-ß-D-glucopyranoside (18), were biosynthesized by the hairy roots.

Differential expression of copper-zinc superoxide dismutase gene of Polygonum sibiricum leaves, stems and underground stems, subjected to high-salt stress.

In aerobic organisms, protection against oxidative damage involves the combined action of highly specialized antioxidant enzymes, such as copper-zinc superoxide dismutase. In this work, a cDNA clone which encodes a copper-zinc superoxide dismutase gene, named PS-CuZnSOD, has been identified from P. sibiricum Laxm. by the rapid amplification of cDNA ends method (RACE). Analysis of the nucleotide sequence reveals that the PS-CuZnSOD gene cDNA clone consists of 669 bp, containing 87 bp in the 5' untranslated region; 459 bp in the open reading frame (ORF) encoding 152 amino acids; and 123 bp in 3' untranslated region. The gene accession nucleotide sequence number in GenBank is GQ472846. Sequence analysis indicates that the protein, like most plant superoxide dismutases (SOD), includes two conserved ecCuZnSOD signatures that are from the amino acids 43 to 51, and from the amino acids 137 to 148, and it has a signal peptide extension in the front of the N-terminus (1-16 aa). Expression analysis by real-time quantitative PCR reveals that the PS-CuZnSOD gene is expressed in leaves, stems and underground stems. PS-CuZnSOD gene expression can be induced by 3% NaHCO(3). The different mRNA levels' expression of PS-CuZnSOD show the gene's different expression modes in leaves, stems and underground stems under the salinity-alkalinity stress.