Geraniol and linalool synthases from wild species of perilla

Geraniol and linalool synthases were isolated from three pure strains of Perilla hirtella and Perilla setoyensis, which are wild species of perilla. Their amino acid sequences were very similar to those of Perilla citriodora and Perilla frutescens that were reported previously. However, comparison of the sequences of the same functional synthases derived from different species of Perilla demonstrated that the similarities were high among P. citriodora, P. hirtella and P. frutescens, but low between P. setoyensis and any of the others. This result corresponds well with our previous results showing that P. setoyensis is remotely related to the other perilla species. Both geraniol and linalool synthases utilize geranyl diphosphate (GDP) as their catalytic substrate and they were expressed simultaneously in perilla. The linalool synthase is considered to be the enzyme whose metabolite seems not to be oxidized nor reduced in the plant body and the geraniol and limonene synthases are the initial-step-catalyzing enzymes for a variety of oil compounds. The regulation of the substrate flow between them would be interesting for further study.     

Analysis of expressed sequence tags from a normalized cDNA library of perilla (Perilla frutescens)

Perilla (Perilla frutescens), an herbal plant belonging to the Lamiaceae family, has long been cultivated in Asia. Perilla is notable as an aroma-rich leaf vegetable and as the oilseed crop richest in omega-3 fatty acids. However, molecular analysis of this herbal plant is lacking due to insufficient genetic resources. Here, we constructed a normalized cDNA library from whole young perilla plants and analyzed expressed sequence tags (ESTs). A total of 4,582 uniESTs were generated from analysis of 5,435 ESTs. Among these, 307 uniESTs (6.7%) were identified as unique in perilla and 3,625 uniESTs were assigned at least one GO term through similarity searches in public databases. The most frequent GO terms were related to abiotic and biotic stress responses. In addition, we identified 141 uniESTs involved in lipid metabolism, of which four genes encoded fatty acid desaturases. We found one new candidate omega-3 fatty acid desaturase gene, in addition to the four that were previously reported. This analysis of uniESTs from perilla provides a valuable genetic resource to elucidate the molecular underpinnings of lipid metabolism and for molecular breeding of perilla species.     

Molecular Cloning and Expression Analysis of 3-Ketoacyl-ACP Synthases in the Immature Seeds of Perilla frutescens

We report the isolation and expression analysis of two cDNAs encoding 3-ketoacyl-acyl carrier protein synthases (KAS) that are involved in the de novo synthesis of fatty acids in plastids of perilla (Perilla frutescens L.). The cDNAs, designated PfFAB1 and PfFAB24, encoded polypeptides with high sequence identities to those of KAS I and KAS II/IV, respectively, of various plants. Genomic Southern blots revealed that there was a single PfFAB1 gene but two PfFAB24 genes in the perilla genome. Of interest is that the expression of both genes was developmentally regulated in seeds. Their mRNA expression patterns in seeds were also discussed in comparison with the profile of fatty acid accumulation.     

Essential oil variation of cultivated and wild Perilla analyzed by GC/MS

Twenty components extracted from the essential oil in the leaves of 172 samples of Perilla frutescens var. crispa (vegetable crop form), P. frutescens var. frutescens (oil crop form), the wild/weedy form of P. frutescens, and three wild Perilla species, Perilla citriodora, Perilla hirtella and Perilla setoyensis were analyzed using GC/MS. A wide range of essential oil components were found among the wild/weedy form of P. frutescens, whereas distinctive components were detected in each wild Perilla species. Egomaketone, asaron, methyleugenol and 4,6-dimethoxy- or 4,7-dimethoxy-5-(2-propenyl)-1,3-dioxaindan were detected from Perilla for the first time. Limonene derivatives, piperitone and piperitenone, were detected from P. citriodora. Discovery of the limonene derivatives in this Perilla species provides evidence of this wild species being a genome donor of P. frutescens, while limonene synthase has been considered to be a specific enzyme in cultivated P. frutescens. These results will be useful for the evaluation and utilization of Perilla genetic resources.     

Molecular cloning and overexpression of the tyrosine aminotransferase (TAT) gene leads to increased rosmarinic acid yield in Perilla frutescens

Perilla frutescens is a medicinal plant that produces various bioactive compounds, including unsaturated fatty acids and phenolic compounds such as rosmarinic acid (α-O-caffeoyl-3, 4 -dihydroxyphenylacetic acid, RA). Tyrosine aminotransferase (TAT) catalyzes the first step in the tyrosine-derived branch of RA biosynthesis, and TAT is presumed to have a role in RA accumulation. Here, we report the isolation of full-length TAT cDNA (designated PfTAT) from P. frutescens. Sequence analysis revealed that PfTAT contained 1,535 bp long and an open reading frame of 1,233 bp encoding 411 amino acid residues. Analysis of PfTAT genomic DNA revealed 7 exons and 5 introns. The 5′ flanking sequence of PfTAT was also cloned, and a group of putative cis-acting elements such TATA box, CAAT box, TC-rich repeats and G box were identified. Quantitative real-time PCR analysis indicated that constitutive expression of PfTAT in leaves was much higher than in roots and stems. A vector was constructed containing the PfTAT gene derived by the cauliflower mosaic virus 35S promoter. Transgenic P. frutescens overexpressing PfTAT was obtained with an Agrobacterium tumefaciens-mediated transformation system and overexpression was confirmed by PCR and Southern blot. PfTAT mRNA expression in transgenic plant lines measured by real-time quantitative PCR was 2–3 times greater than PfTAT expression in the untransformed plant line. Also, enhanced gene expression corresponded to significantly increased RA in PfTAT-transgenic lines, as quantified by HPLC. Our data emphasize the importance of PfTAT in the production of RA in P. frutescens.     

Phenotypic and Genetic Diversity of Local <Emphasis Type="Italic">Perilla</Emphasis> (<Emphasis Type="Italic">Perilla frutescens</Emphasis> (L.) Britt.) from Northern Thailand

Phenotypic and Genetic Diversity of Local Perilla ( Perilla frutescens (L.) Britt.) from Northern Thailand. Perilla frutescens (L.) Britt., an important oil and culinary crop in Asia, is a valuable genetic resource. Despite its nutritional value and historic and cultural importance, research on Perilla has been scarce, particularly as far as its genetic diversity is concerned. The aims of the present study were to assess variability within and between 29 seed samples of P. frutescens collected from farmers in northern Thailand, and evaluation conducted of their genetic, morphological, and agronomic characteristics, and the seed composition, including polyunsaturated fatty acids omega-3, omega-6, and omega-9, and the vitamin E γ-tocopherols. Perilla frutescens (L.) Britt. of northern Thailand is genetically variable, and structured according to origin of collection which was the consequence of local adaptation. The discovery of high levels of polyunsaturated fatty acids, namely α-linolenic acid and γ-tocopherols, in some Perilla samples indicates the potential for utilizing Perilla for its high omega-3 content including as a vitamin E supplement for humans, a prospect that should be taken into account when planning conservation strategies or when Perilla variability is used in breeding programs.     

Seed specific expression of perilla γ-tocopherol methyltransferase gene increases α-tocopherol content in transgenic perilla (<Emphasis Type="Italic">Perilla frutescens</Emphasis>)

Increasing vitamin E activity in economically important oil crops such as perilla will enhance the nutritional value of these crops. Perilla (Perilla frutescens Britt) represents an important oil crop in Asian countries, including Korea. Using Agrobacterium-mediated transformation, we have engineered perilla with the γ-tocopherol methyltransferase (γ-TMT) gene under the control of seed-specific vicillin promoter. Molecular characterization including PCR, Southern and Northern blots confirmed that the γ-TMT transgene was successfully inherited to and expressed in the progeny plants. The γ -TMT transgene was specifically expressed in immature seeds of transgenic plants, leading to efficient conversion of γ-tocopherol to α-tocopherol and dramatic increase in seed α-tocopherol content, as detected by high performance liquid chromatography analysis. We also showed that such a high α-tocopherol content phenotype was transmitted to the progeny plants. In addition, there was no significant change in fatty acid composition in transgenic seeds as compared with untransformed control Yeupsil cultivar, suggesting the lack of interplay between the fatty acid and tocopherol biosynthesis pathways. This was the first report on over expression of the γ-TMT gene in transgenic perilla displaying desirable high α-tocopherol content phenotype. Since α-tocopherol has the highest vitamin E activity, the transgenic perilla with high α-tocopherol content in seeds developed in this study will benefit both human and animal health.     

Competition between two similar plant varieties,green shrunk perilla and red shrunk perilla,in mixed cultures

Influences of plant density and time after seeding on the growth of two horticultural forms of perilla (Perilla frutescens var.crispa), green shrunk perilla (f.viridi-crispa) and red shrunk perilla (f.crispa), were examined in a mixed culture experiment. Relationships between mean individual plant weight and plant density in mixed populations were approximated by Ogawa's non-interaction type (NI-type) reciprocal equation. The density conversion factors in the equation for green and red perillas were always, respectively, smaller and larger than unity, suggesting that effects of a green perilla on the other individuals were always stronger than those of a red one in a mixed population. All coefficients in the NI-type reciprocal equation were expressed as functions of time after seeding. As a result, time trends of mean individual plant weights for both species in mixed populations could be reasonably estimated for different plant densities and mixed proportions. The results were also applied to Lotka-Volterra's equation. Time trends of Lotka-Volterra's competition coefficients for both plants could be calculated and were compared with those of density conversion factors.     

Comparative effects of perilla and fish oils on the activity and gene expression of fatty acid oxidation enzymes in rat liver

The activity and mRNA level of hepatic enzymes in fatty acid oxidation and synthesis were compared in rats fed diets containing either 15% saturated fat (palm oil), safflower oil rich in linoleic acid, perilla oil rich in α-linolenic acid or fish oil rich in eicosapentaenoic (EPA) and docosahexaenoic acids (DHA) for 15 days. The mitochondrial fatty acid oxidation rate was 50% higher in rats fed perilla and fish oils than in the other groups. Perilla and fish oils compared to palm and safflower oils approximately doubled and more than tripled, respectively, peroxisomal fatty acid oxidation rate. Compared to palm and safflower oil, both perilla and fish oils caused a 50% increase in carnitine palmitoyltransferase I activity. Dietary fats rich in n-3 fatty acids also increased the activity of other fatty acid oxidation enzymes except for 3-hydroxyacyl-CoA dehydrogenase. The extent of the increase was greater with fish oil than with perilla oil. Interestingly, both perilla and fish oils decreased the activity of 3-hydroxyacyl-CoA dehydrogenase measured using short- and medium-chain substrates. Compared to palm and safflower oils, perilla and fish oils increased the mRNA level of many mitochondrial and peroxisomal enzymes. Increases were generally greater with fish oil than with perilla oil. Fatty acid synthase, glucose-6-phosphate dehydrogenase, and pyruvate kinase activity and mRNA level were higher in rats fed palm oil than in the other groups. Among rats fed polyunsaturated fats, activities and mRNA levels of these enzymes were lower in rats fed fish oil than in the animals fed perilla and safflower oils. The values were comparable between the latter two groups. Safflower and fish oils but not perilla oil, compared to palm oil, also decreased malic enzyme activity and mRNA level. Examination of the fatty acid composition of hepatic phospholipid indicated that dietary α-linolenic acid is effectively desaturated and elongated to form EPA and DHA. Dietary perilla oil and fish oil therefore exert similar physiological activity in modulating hepatic fatty acid oxidation, but these dietary fats considerably differ in affecting fatty acid synthesis.     

Subinhibitory concentrations of perilla oil affect the expression of secreted virulence factor genes in Staphylococcus aureus

Background

The pathogenicity of staphylococcus aureus is dependent largely upon its ability to secrete a number of virulence factors, therefore, anti-virulence strategy to combat S. aureus-mediated infections is now gaining great interest. It is widely recognized that some plant essential oils could affect the production of staphylococcal exotoxins when used at subinhibitory concentrations. Perilla [Perilla frutescens (L.) Britton], a natural medicine found in eastern Asia, is primarily used as both a medicinal and culinary herb. Its essential oil (perilla oil) has been previously demonstrated to be active against S. aureus. However, there are no data on the influence of perilla oil on the production of S. aureus exotoxins.

Methodology/Principal Findings

A broth microdilution method was used to determine the minimum inhibitory concentrations (MICs) of perilla oil against S. aureus strains. Hemolysis, tumour necrosis factor (TNF) release, Western blot, and real-time RT-PCR assays were performed to evaluate the effects of subinhibitory concentrations of perilla oil on exotoxins production in S. aureus. The data presented here show that perilla oil dose-dependently decreased the production of α-toxin, enterotoxins A and B (the major staphylococcal enterotoxins), and toxic shock syndrome toxin 1 (TSST-1) in both methicillin-sensitive S. aureus (MSSA) and methicillin-resistant S. aureus (MRSA).

Conclusions/Significance

The production of α-toxin, SEA, SEB, and TSST-1 in S. aureus was decreased by perilla oil. These data suggest that perilla oil may be useful for the treatment of S. aureus infections when used in combination with β-lactam antibiotics, which can increase exotoxins production by S. aureus at subinhibitory concentrations. Furthermore, perilla oil could be rationally applied in food systems as a novel food preservative both to inhibit the growth of S. aureus and to repress the production of exotoxins, particularly staphylococcal enterotoxins.     

Comparison of photoperiodic sensitivity of green-leafed and red-leafed perilla

The flowering responses of Perilla frutescens (L.) Britt. (green-leafed Perilla) and P. crispa (Thunb.) Tanaka (red-leafed Perilla) to different 24-hour photoperiodic regimes were compared quantitatively. Although both taxa are short-day plants, P. frutescens was less strict in its short-day requirement, forming macroscopically visible inflorescence primordia and open flowers even in continuous light after 77 to 85 and 132 days, respectively, compared to 12 and 22 days in 8-hour days. P. crispa took only 4 days longer to reach the same stages in 8-hour short days, but exhibited a sharply defined critical day-length between 14 and 16 hours. Two-hour light-breaks in the middle of a 16-hour night were more effective in inhibiting floral development in P. crispa than in P. frutescens, for which they had a small effect compared to true long-days, particularly on the formation of inflorescence primordia. The number of short days needed to obtain the flowering in P. frutescens plants raised in continuous light decreased in a linear manner with the age of the plants upon transfer to short days.     

Antimicrobial activity of perilla seed polyphenols against oral pathogenic bacteria

A perilla seed (Perilla frutescens Britton var. japonica Hara) extract was examined for its antimicrobial activity against oral cariogenic streptococci and periodontopathic Porphyromonas gingivalis. Luteolin, one of the components of perilla seed, showed the strongest antimicrobial effect among the phenolic compounds. According to our results, perilla seed may be the source of an antimicrobial agent that could prevent dental caries and periodontal diseases.     

Identification of genetic variations of cultivated and weedy types of Perilla species in Korea and Japan using morphological and SSR markers

To better understand the genetic diversity and relationships of the two cultivated types of Perilla crop and their weedy types in Korea and Japan, we evaluated the genetic variations of 56 accessions by assessing five morphological characteristics and 18 SSR markers. The two cultivated types of var. frutescens and var. crispa were clearly distinguished by seed size, whereas most accessions of cultivated and weedy types of var. crispa cannot be distinguished strictly by seed characteristics. A total of 165 alleles with the SSR analysis were detected with an average number of 9.2 alleles per locus among the 56 Perilla accessions. The number of alleles per locus ranged from two for KWPE-56 and KWPE-39 to 21 for GBPFM-204. Additionally, the genetic diversity of each locus ranged from 0.497 at KWPE-56 and KWPE-39 to 0.959 at GBPFM-204, with an average of 0.692. The average genetic diversity values were 0.549, 0.685, 0.451 and 0.557 for cultivated and weedy types of var. frutescens and for cultivated and weedy types of var. crispa, respectively. The weedy type accessions of var. frutescens and var. crispa evidenced greater variation than the corresponding cultivated type accessions. The accessions of the cultivated and weedy types of var. frutescens and var. crispa from Korea exhibited greater SSR diversity than those of Japan. An UPGMA phylogenetic tree revealed three major groups, which was congruent with their morphological characteristics except for a few odd accessions. SSR markers clarified the genetic relationships between var. frutescens and var. crispa and helped improve our understanding of the genetic diversity of the two cultivated types of P. frutescens and their weedy types in Korea and Japan.     

A new flavanone and other flavonoids from green perilla leaf extract inhibit nitric oxide production in interleukin 1β-treated hepatocytes

A new flavanone, shisoflavanone A (1), and several flavonoids were purified from the ethyl acetate-soluble fraction of green perilla leaves (Perilla frutescens Britton var. crispa form viridis), and their structures were identified. Shisoflavanone A was elucidated as 8-hydroxy-6,7-dimethoxyflavanone based on its spectral data. Other constituents of the ethyl acetate-soluble fraction, i.e. 5,8-dihydroxy-7-methoxyflavanone (2), negletein (5,6-dihydroxy-7-methoxyflavone) (3), luteolin (4), apigenin (5), esculetin (6), and protocatechuic acid (7), were identified. This is the first time that constituents 2, 3, and 6 have been found in green perilla. Shisoflavanone A and the other constituents (except 7) significantly inhibited nitric oxide production in interleukin 1β-stimulated rat hepatocytes, which have been used to monitor the anti-inflammatory effects of herbal constituents. The present findings suggest that these constituents, including shisoflavanone A, may be involved in the anti-inflammatory effects of green perilla leaves.     

Yield,size, nutritional value,and antioxidant activity of oyster mushrooms grown on perilla stalks

Perilla is an edible medical plant with rapidly increasing acreage in China. In this study, we investigated the potential of perilla stalks (PSs) as an alternative substrate for the cultivation of oyster mushrooms (Pleurotus ostreatus). P. ostreatus was cultivated on cottonseed hulls (CSH) alone or mixed with PSs in different ratios. The production parameters, physical characteristics, nutritional values, and antioxidant activity of mushrooms cultivated on different substrate mixtures were determined. The addition of PSs to CSH significantly improved the growth rate, yield, biological efficiency, and proximate composition and shortened the cultivation cycle. Cultivation on PSs alone increased the amino acid content in P. ostreatus fruiting bodies and the antioxidant activity of mushroom extracts. The PS75 (25% CSH + 75% PS) substrate was deduced to be the most effective substrate on the basis of yield and biological efficiency obtained in a large area where perilla had been planted. The results demonstrate that mixtures of PS with CSHs could be used as novel, practical, and easily accessible alternative substrates for P. ostreatus cultivation.     

Increased uncoupling protein2 mRNA in white adipose tissue, and decrease in leptin, visceral fat, blood glucose, and cholesterol in KK-Ay mice fed with eicosapentaenoic and docosahexaenoic acids in addition to linolenic acid.

The effects of n-3 polyunsaturated fatty acids (n-3PUFA) on obesity and diabetes were examined using KK-Ay mice fed with perilla oil (P), soybean oil (S), or lard (L), and those containing 30% fish oil (PF, SF, or LF), containing eicosapentaenoic acid (EPA = 9.9%) and docosahexaenoic acid (DHA = 18.0%). Perilla oil contained the largest proportion of linolenic acid (LNA = 61.9%). Computerized tomography (CT) scans showed narrower areas of visceral fat in the abdominal cross sections of groups given fish oil (PF, SF, and LF) and lower leptin levels (p < 0.05-p < 0.001) compared with controls (P, S, and L), without significant changes in energy intake and body weight. The highest plasma n-3PUFA content (21.31 +/- 0.35%) was attained with PF. This group contained 2.6-fold more plasma DHA (p < 0.001), and expressed 2.7-fold more UCP2 mRNA in white adipose tissue (p < 0.01) than in the P group. The epididymal fat pad (p < 0.05) weighed less, and levels of blood glucose (p < 0.05) and total cholesterol (p < 0.01) were reduced in PF compared with P.     

温度和紫外照射对紫苏FAD7基因表达的影响

植物ω-3脂肪酸脱氢酶(ω-3FAD)基因是催化亚油酸转化为α-亚麻酸(ALA)的关键酶基因,通过调节该基因的表达,可以提高植物ALA的含量。为了研究温度和紫外照射对紫苏PfFAD7的影响,通过RTPCR方法分析了紫苏地上组织的特异性表达和温度、紫外胁迫下紫苏叶片和茎中PfFAD7基因的积累情况。分析表明,PfFAD7基因在紫苏全植株中均有表达,但在叶片中表达量最高,温度和紫外照射均影响PfFAD7基因的表达,低温可诱导PfFAD7基因的表达,而高温则抑制PfFAD7基因的表达;UV-B照射下,PfFAD7基因在叶片和茎中表达量均表现为先升高再降低。本试验对于紫苏ω-3脂肪酸脱氢酶的研究有利于高水平ALA的积累,更有利于紫苏资源的开发和利用,同时对于进一步了解不饱和脂肪酸的积累和代谢过程以及关键基因PfFAD7在此过程中的功能提供了依据。