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Phenylpropanoid volatile components are found in various plants and are useful in medicines, health foods, and fragrances. While the pharmacological actions and toxicities of these compounds have been investigated, there are few reports of the cloning of genes that encode biosynthetic enzymes involved in substituent formation at diverse positions and numbers. Previously, using the expressed sequence tag (EST) libraries of pure perilla strains that have been maintained for over 30 years for their oil type, we characterized the P450 enzyme that produces an intermediate for dillapiole by adding a hydroxy group to myristicin. In this study, we selected a P450 enzyme involved in nothoapiole biosynthesis from the EST library. Heterologous expression of this enzyme in yeast showed that it is a hydroxylase that synthesizes an intermediate to produce nothoapiole from apiole and dillapiole. The enzyme has high amino acid sequence similarity with a previously cloned enzyme and is categorized into the CYP71D subfamily. Furthermore, we investigated the presence or absence of essential oil components and intermediates believed to be involved in nothoapiole biosynthesis by component analysis of perilla essential oil using GC-MS to help elucidate the biosynthetic pathway of nothoapiole. Only a small number of plant species contain nothoapiole as their principal component and thus few studies have reported the biosynthetic genes involved or the drug efficacy and toxicity of nothoapiole. The present study will aid in understanding the biosynthesis of phenylpropanoid volatile compounds, thereby contributing to further research on potentially useful compounds such as nothoapiole.

Many O-glucuronides exhibiting various pharmacological activities have been found in nature and in drug metabolism. The glucuronidation of bioactive natural products or drugs to generate glucuronides with better activity and druggability is important in drug discovery and research. In this study, by using two uridine diphosphate (UDP)-dependent glucuronosyltransferases (GATs, UGT88D4 and UGT88D7) from plants, we developed two glucuronidation approaches, pure enzyme catalysis in vitro and recombinant whole-cell catalysis in vivo, to efficiently synthesize bioactive O-glucuronides by the glucuronidation of natural products. In total, 14 O-glucuronides with different structures, including flavonoids, anthraquinones, coumarins, and lignans, were obtained, 7 of which were new compounds. Furthermore, one of the biosynthesized O-glucuronides, kaempferol-7- O-β-d-glucuronide (3a), potently inhibited protein tyrosine phosphatase (PTP) 1B with an IC 50 value of 8.02 × 10 -6 M. Some of the biosynthesized O-glucuronides also exhibited significant antioxidant activities.

Omega-3 fatty acid desaturase (ω-3 FAD, D15D) is a key enzyme for α-linolenic acid (ALA) biosynthesis. Both chia (Salvia hispanica) and perilla (Perilla frutescens) contain high levels of ALA in seeds. In this study, the ω-3 FAD gene family was systematically and comparatively cloned from chia and perilla. Perilla FAD3, FAD7, FAD8 and chia FAD7 are encoded by single-copy (but heterozygous) genes, while chia FAD3 is encoded by 2 distinct genes. Only 1 chia FAD8 sequence was isolated. In these genes, there are 1 to 6 transcription start sites, 1 to 8 poly(A) tailing sites, and 7 introns. The 5'UTRs of PfFAD8a/b contain 1 to 2 purine-stretches and 2 pyrimidine-stretches. An alternative splice variant of ShFAD7a/b comprises a 5'UTR intron. Their encoded proteins harbor an FA_desaturase conserved domain together with 4 trans-membrane helices and 3 histidine boxes. Phylogenetic analysis validated their identity of dicot microsomal or plastidial ω-3 FAD proteins, and revealed some important evolutionary features of plant ω-3 FAD genes such as convergent evolution across different phylums, single-copy status in algae, and duplication events in certain taxa. The qRT-PCR assay showed that the ω-3 FAD genes of two species were expressed at different levels in various organs, and they also responded to multiple stress treatments. The functionality of the ShFAD3 and PfFAD3 enzymes was confirmed by yeast expression. The systemic molecular and functional features of the ω-3 FAD gene family from chia and perilla revealed in this study will facilitate their use in future studies on genetic improvement of ALA traits in oilseed crops.

This study evaluated the effects of enhanced concentrations of α-tocopherol and phenolic compounds on the resistance and stability of Perilla oil in transgenic Perilla frutescens plants against various tested pathogenic bacteria by over-expressing the γ-tmt gene. The concentration of phenolic compounds in the non-transgenic samples was 9313.198 ± 18.887 μg g -1 dry weight (DW), whereas the total concentration of the transgenic samples ranged from 9118.015 ± 18.822 to 10527.612 ± 20.411 μg g -1 DW. The largest increases in phenolic compounds in the transgenic plants in comparison with the control plants were observed in gallic acid, pyrogallol, 5-sulfosalicylic acid, catechin, chlorogenic acid, vanillin, syringic acid, naringenin, salicylic acid, quercetin, o-coumaric acid, kaempferol, and hesperetin. o-coumaric and benzoic acid acid were the most abundant phenolic acids found in the transgenic plants. Gram-negative bacteria (Salmonella typhimurium) were the most susceptible microorganism against transgenic ethyl acetate extracts with lower measurement of minimum inhibitory concentration (MICs) (0.25 ± 0.03 mg/ml) at an extract concentration of 2 mg/ml in dried plant material. The same extracts were more effective against gram-positive bacteria (Bacillus subtilis) when compared to control plants with MICs values of 0.52 ± 0.02 mg/ml. The suplementation of 20 μg of α-tocopherol (1000 ppm) in combination with ethyl acetate extracts enhanced the antimicrobial activity against S. typhimurium and B. subtilis, compared to the non-transgenic plants. The acid value of transgenic Perilla oil improved by 91.2% and 35.54% relative to the non-transgenic control oil and commercial Perilla oil, respectively. The low acid value suggests that the oil will be less susceptible to lipase action, and more economically viable and thus, may also improve the oil quality for industrial purposes. In addition, extracts obtained from transgenic plants could be a potential source of antimicrobial agents for the treatment of bacterial infections., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Perilla produces the cyclohexanoid monoterpene perillaldehyde as a major constituent of an essential oil that is accumulated in its glandular trichomes. Perillaldehyde is a marker compound for quality control of soyo and has biological activities such as antibacterial, sedative, or vasodilatory effects. The predicted perillaldehyde formation involves the cyclization of geranyl diphosphate, hydroxylation, and oxidation, and cytochrome P450 plays a crucial role in perillaldehyde biosynthesis. In this study, a cytochrome P450-type enzyme with perillyl alcohol and perillaldehyde synthase activities was isolated by analyzing an expressed sequence tag library from several oil types of pure lines of perilla. A recombinant protein with a sequence that was highly specific for the type of perillaldehyde was expressed in Saccharomyces cerevisiae and evaluated by an in vitro enzymatic reaction. The recombinant protein catalyzed the hydroxylation and oxidation of limonene to perillyl alcohol and perillaldehyde. Cytochrome P450 limonene-7-hydroxylase cDNA from Perilla frutescens has been previously isolated. The cytochrome P450 isolated in this study shares 37% amino-acid identity with the previously isolated enzyme; however, it may have different characteristics., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Key Message: We described identification, expression, subcellular localization, and functions of genes that encode fatty acid desaturase enzymes in Perilla frutescens var. frutescens. Perilla (Perilla frutescens var. frutescens) seeds contain approximately 40 % of oil, of which α-linolenic acid (18:3) comprise more than 60 % in seed oil and 56 % of total fatty acids (FAs) in leaf, respectively. In perilla, endoplasmic reticulum (ER)-localized and chloroplast-localized ω-3 FA desaturase genes (PfrFAD3 and PfrFAD7, respectively) have already been reported, however, microsomal oleate 12-desaturase gene (PfrFAD2) has not yet. Here, four perilla FA desaturase genes, PfrFAD2-1, PfrFAD2-2, PfrFAD3-2 and PfrFAD7-2, were newly identified and characterized using random amplification of complementary DNA ends and sequence data from RNAseq analysis, respectively. According to the data of transcriptome and gene cloning, perilla expresses two PfrFAD2 and PfrFAD3 genes, respectively, coding for proteins that possess three histidine boxes, transmembrane domains, and an ER retrieval motif at its C-terminal, and two chloroplast-localized ω-3 FA desaturase genes, PfrFAD7-1 and PfrFAD7-2. Arabidopsis protoplasts transformed with perilla genes fused to green fluorescence protein gene demonstrated that PfrFAD2-1 and PfrFAD3-2 were localized in the ER, and PfrFAD7-1 and PfrFAD7-2 were localized in the chloroplasts. PfrFAD2 and perilla ω-3 FA desaturases were functional in budding yeast (Saccharomyces cerevisiae) indicated by the presence of 18:2 and 16:2 in yeast harboring the PfrFAD2 gene. 18:2 supplementation of yeast harboring ω-3 FA desaturase gene led to the production of 18:3. Therefore, perilla expresses two functional FAD2 and FAD3 genes, and two chloroplast-localized ω-3 FA desaturase genes, which support an evidence that P. frutescens cultivar is allotetraploid plant.

The chemotypes found in various plant species are the good subjects for the studies to understand the regulatory mechanism in secondary metabolism. The biochemistry and molecular biology of flavonoid biosynthesis was studied using chemotypes of Perilla frutescens var. crispa differing anthocyanin accumulation. The expression of the most of structural genes for anthocyanin biosynthesis was coordinately regulated in chemotype-specific manner and by light. However, the genes for shared enzymes between anthocyanin and flavone pathway were expressed both chemotypes. Biochemical characteristics of enzymes involved in anthocyanin biosynthesis were investigated in this plant. Furthermore, the candidates of regulatory factors, members of MYB-bHLH-WD complex, of anthocyanin production were characterized in this plant.

Cytochrome P450 mono-oxygenases from peppermint, spearmint and perilla (all members of the family Lamiaceae) mediate the regiospecific hydroxylation of the parent olefin (-)-limonene to produce essential oil components oxygenated at C3, C6 and C7, respectively. Cloning, expression and mutagenesis of cDNAs encoding the peppermint limonene-3-hydroxylase and the spearmint limonene-6-hydroxylase have allowed the identification of a single amino acid residue which determines the regiospecificity of oxygenation by these two enzymes. A hybridization strategy provided a cytochrome P450 limonene hydroxylase cDNA from perilla with which to further evaluate the structural determinants of regiospecificity for oxygenation of the common substrate (-)-limonene. The perilla cDNA was a partial clone of 1550bp (lacking the N-terminal membrane insertion domain), and shared 66% identity with the peppermint 3-hydroxylase and spearmint 6-hydroxylase at the amino acid level. The perilla cytochrome P450 was expressed in Escherichia coli as a chimeric protein fused with the N-terminal membrane insertion domain of the limonene-3-hydroxylase. The kinetically competent recombinant protein was characterized and shown to produce a mixture of C3-, C6- and C7-hydroxylated limonene derivatives with a distribution of 33%, 14% and 53%, respectively., (Copyright 2010 Elsevier Ltd. All rights reserved.)

Metabolic engineering aimed at monoterpene production has become an intensive research topic in recent years, although most studies have been limited to herbal plants including model plants such as Arabidopsis. The genus Eucalyptus includes commercially important woody plants in terms of essential oil production and the pulp industry. This study attempted to modify the production of monoterpenes, which are major components of Eucalyptus essential oil, by introducing two expression constructs containing Perilla frutescens limonene synthase (PFLS) cDNA, whose gene products were designed to be localized in either the plastid or cytosol, into Eucalyptus camaldulensis. The expression of the plastid-type and cytosol-type PFLS cDNA in transgenic E. camaldulensis was confirmed by real-time polymerase chain reaction (PCR). Gas chromatography with a flame ionization detector analyses of leaf extracts revealed that the plastidic and cytosolic expression of PFLS yielded 2.6- and 4.5-times more limonene than that accumulated in wild-type E. camaldulensis, respectively, while the ectopic expression of PFLS had only a small effect on the emission of limonene from the leaves of E. camaldulensis. Surprisingly, the high level of PFLS in Eucalyptus was accompanied by a synergistic increase in the production of 1,8-cineole and alpha-pinene, two major components of Eucalyptus monoterpenes. This genetic engineering of monoterpenes demonstrated a new potential for molecular breeding in woody plants.

Differential screening by PCR-select subtraction was carried out for cDNAs from leaves of red and green perilla, two chemovarietal forms of Perilla frutescens regarding anthocyanin accumulation. One hundred and twenty cDNA fragments were selected as the clones preferentially expressed in anthocyanin-accumulating red perilla over the nonaccumulating green perilla. About half of them were the cDNAs encoding the proteins related presumably to phenylpropanoid-derived metabolism. The cDNAs encoding glutathione S-transferase (GST), PfGST1, and chalcone isomerase (CHI), PfCHI1, were further characterized. The expression of PfGST1 in an Arabidopsis thaliana tt19 mutant lacking the GST-like gene involved in vacuole transport of anthocyanin rescued the lesion of anthocyanin accumulation in tt19, indicating a function of PfGST1 in vacuole sequestration of anthocyanin in perilla. The recombinant PfCHI1 could stereospecifically convert naringenin chalcone to (2S)-naringenin. PfGST1 and PfCHI1 were preferentially expressed in the leaves of red perilla, agreeing with the accumulation of anthocyanin and expression of other previously identified genes for anthocyanin biosynthesis. These results suggest that the genes of the whole anthocyanin biosynthetic pathway are regulated in a coordinated manner in perilla.

Rubisco large subunit methyltransferase (PsLSMT) is a SET domain protein responsible for the trimethylation of Lys-14 in the large subunit of Rubisco. The polypeptide substrate specificity determinants for pea Rubisco large subunit methyltransferase were investigated using a fusion protein construct between the first 23 amino acids from the large subunit of Rubisco and human carbonic anhydrase II. A total of 40 conservative and non-conservative amino acid substitutions flanking the target Lys-14 methylation site (positions P(-3) to P(+3)) were engineered in the fusion protein. The catalytic efficiency (k(cat)/K(m)) of PsLSMT was determined using each of the substitutions and a polypeptide consensus recognition sequence deduced from the results. The consensus sequence, represented by X-(Gly/Ser)-(Phe/Tyr)-Lys-(Ala/Lys/Arg)-(Gly/Ser)-pi, where X is any residue, Lys is the methylation site, and pi is any aromatic or hydrophobic residue, was used to predict potential alternative substrates for PsLSMT. Four chloroplast-localized proteins were identified including gamma-tocopherol methyltransferase (gamma-TMT). In vitro methylation assays using PsLSMT and a bacterially expressed form of gamma-TMT from Perilla frutescens confirmed recognition and methylation of gamma-TMT by PsLSMT in vitro. RNA interference-mediated knockdown of the PsLSMT homologue (NtLSMT) in transgenic tobacco plants resulted in a 2-fold decrease of alpha-tocopherol, the product of gamma-TMT. The results demonstrate the efficacy of consensus sequence-driven identification of alternative substrates for PsLSMT as well as identification of functional attributes of protein methylation catalyzed by LSMT.

Geraniol synthases were isolated from five pure strains of Perilla citriodora and Perilla frutescens which vary in essential oil type, the main compounds of which were citral, elsholtziaketone, perillaketone, and perillene, respectively. This result supports the putative biosynthetic pathways of these three furylalkenes which are all produced by way of citral. Nucleotide sequences of geraniol synthases from three oil types of P. citriodora were identical, and almost the same as the sequence from P. frutescens, a species with twice the chromosome number of P. citriodora. This identity in sequence between P. citriodora and P. frutescens, together with other previous results, indicates that P. frutescens was formed as an amphidiploid of P. citriodora and an unknown wild species.

Tocopherols, with antioxidant properties, are synthesized by photosynthetic organisms and play important roles in human and animal nutrition. In soybean, gamma-tocopherol, the biosynthetic precursor to alpha-tocopherol, is the predominant form found in the seed, whereas alpha-tocopherol is the most bioactive component. This suggests that the final step of the alpha-tocopherol biosynthetic pathway catalyzed by gamma-tocopherol methyltransferase (gamma-TMT) is limiting in soybean seed. Soybean oil is the major edible vegetable oil consumed, so manipulating the tocopherol biosynthetic pathway in soybean seed to convert tocopherols into more active alpha-tocopherol form could have significant health benefits. In order to increase the soybean seed alpha-tocopherol content, the gamma-TMT gene isolated from Perilla frutescens was overexpressed in soybean using a seed-specific promoter. One transgenic plant was recovered and the progeny was analyzed for two generations. Our results demonstrated that the seed-specific expression of the P. frutescens gamma-TMT gene resulted in a 10.4-fold increase in the alpha-tocopherol content and a 14.9-fold increase in the beta-tocopherol content in T2 seed. Given the relative contributions of different tocopherols to vitamin E activity, the activity in T2 seed was calculated to be 4.8-fold higher than in wild-type seed. In addition, the data obtained on lipid peroxidation indicates that alpha-tocopherol may have a role in preventing oxidative damage to lipid components during seed storage and seed germination. The increase in the alpha-tocopherol content in the soybean seed could have a potential to significantly increase the dietary intake of vitamin E.

The glucosylation of 5-position of anthocyanin molecules is one of most important modification step, which is responsible for the stability and color hue of anthocyanin molecules. However, anthocyanin 5-O-glucosyltransferase (5-GT) is unstable and it had been difficult to purify this enzyme by biochemical methods. The isolation of cDNA encoding 5-GT was succeeded first by mRNA differential display (DD) of red and green chemotypes of Perilla frutescens var. crispa. The full-length cDNA was isolated by the screening of perilla cDNA library with cDNA fragments, which was displayed specifically in red perilla. The biological characters of this enzyme were investigated by using the recombinant protein expressed in yeast. We have shown an example of the application of DD technique to the molecular biology in plant secondary metabolisms.

cDNA cloning of a monoterpene synthase from Perilla frutescens whose steam-distilled oil contains 92.9% perillaketone, was performed by the PCR method using primers designed based on limonene synthase. The full-length nucleotide sequence of this cDNA consisted of 1978 bp including a 1827-bp translational region encoding a deduced protein of 608 amino acids, which was similar to that of limonene synthase from P. frutescens (85% identity). Functional expression of this clone in Escherichia coli yielded an active monoterpene synthase enzyme, which converted geranyl diphosphate into 53.8% myrcene, 20.9% sabinene, 19.8% linalool and 5.5% limonene. As for the extraction of reaction products, we performed SPME (solid phase micro extraction) as well as conventional solvent extraction, and compared these two extraction methods.