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The gap between serious soil heavy metals pollution and inefficient soil remediation threatens human health. This study proposed a method to improve the phytoremediation efficiency using bamboo vinegar (BV) solution and the potential mechanism was discussed. The results demonstrated that the application of BV increases the content of cadmium (Cd) in vacuole and cell wall hemicellulose 2 in leaves of Perilla frutescens. Simultaneously, it enhanced enzyme activities of superoxide dismutase and catalase in leaves. Therefore, this process alleviated the damage of Cd to functional tissues of Perilla frutescens, thus improving the tolerance of plants to Cd. Moreover, the BV application reduced the Cd content bound by root cell wall pectin fractions and insoluble phosphate, subsequently improving the ability of oxalic acids to carry Cd to the aerial parts. Consequently, the aerial parts obtained a larger amount of Cd enrichment. Overall, the Transfer Factor of Cd from roots to stems and enrichment of Cd in Perilla frutescens were maximally increased by 57.70 % and 54.03 % with the application of 50-fold and 300-fold diluted BV under 2 mg·L -1 Cd stress, respectively. The results can provide a theoretical basis for the promotion of phytoremediation of Cd-contaminated soil treatment technology., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Perilla (Perilla frutescens L.) is a time-honored herbal plant with widespread applications in both medicine and culinary practices around the world. Profiling the essential organs and tissues with medicinal significance on a global scale offers valuable insights for enhancing the yield of desirable compounds in Perilla and other medicinal plants. In the present study, genome-wide RNA-sequencing (RNA-seq) and assessing the global spectrum of metabolites were carried out in the two major organs/tissues of stem (PfST) and leaf (PfLE) in Perilla. The results showed a total of 18,490 transcripts as the DEGs (differentially expressed genes) and 144 metabolites as the DAMs (differentially accumulated metabolites) through the comparative profiling of PfST vs PfLE, and all the DEGs and DAMs exhibited tissue-specific trends. An association analysis between the transcriptomics and metabolomics revealed 14 significantly enriched pathways for both DEGs and DAMs, among which the pathways of Glycine, serine and threonine metabolism (ko00260), Glyoxylate and dicarboxylate metabolism (ko00630), and Glucagon signaling pathway (ko04922) involved relatively more DEGs and DAMs. The results of qRT-PCR assays of 18 selected DEGs confirmed the distinct tissue-specific characteristics of all identified DEGs between PfST and PfLE. Notably, all eight genes associated with the flavonoid biosynthesis/metabolism pathways exhibited significantly elevated expression levels in PfLE compared to PfST. This observation suggests a heightened accumulation of metabolites related to flavonoids in Perilla leaves. The findings of this study offer a comprehensive overview of the organs and tissues in Perilla that have medicinal significance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: Endophytic fungi (EF) reside within plants without causing harm and provide benefits such as enhancing nutrients and producing bioactive compounds, which improve the medicinal properties of host plants. Selecting plants with established medicinal properties for studying EF is important, as it allows a deeper understanding of their influence. Therefore, the study aimed to investigate the impact of EF after inoculating the medicinal plant Perilla frutescens, specifically focusing on their role in enhancing medicinal properties., Results: In the current study, the impact of two EF i.e., Irpex lenis and Schizophyllum commune isolated from A. bracteosa was observed on plant Perilla frutescens leaves after inoculation. Plants were divided into four groups i.e., group A: the control group, group B: inoculated with I. lenis; group C: inoculated with S. commune and group D: inoculated with both the EF. Inoculation impact of I. lenis showed an increase in the concentration of chlorophyll a (5.32 mg/g), chlorophyll b (4.46 mg/g), total chlorophyll content (9.78 mg/g), protein (68.517 ± 0.77 mg/g), carbohydrates (137.886 ± 13.71 mg/g), and crude fiber (3.333 ± 0.37%). Furthermore, the plants inoculated with I. lenis showed the highest concentrations of P (14605 mg/kg), Mg (4964.320 mg/kg), Ca (27389.400 mg/kg), and Mn (86.883 mg/kg). The results of the phytochemical analysis also indicated an increased content of total flavonoids (2.347 mg/g), phenols (3.086 mg/g), tannins (3.902 mg/g), and alkaloids (1.037 mg/g) in the leaf extract of P. frutescens inoculated with I. lenis. Thus, overall the best results of inoculation were observed in Group B i.e. inoculated with I. lenis. GC-MS analysis of methanol leaf extract showed ten bioactive constituents, including 9-Octadecenoic acid (Z)-, methyl ester, and hexadecanoic acid, methyl ester as major constituents found in all the groups of P. frutescens leaves. The phenol (gallic acid) and flavonoids (rutin, kaempferol, and quercetin) were also observed to increase after inoculation by HPTLC analysis. The enhancement in the phytochemical content was co-related with improved anti-oxidant potential which was analyzed by DPPH (% Inhibition: 83.45 µg/ml) and FRAP (2.980 µM Fe (II) equivalent) assay as compared with the control group., Conclusion: Inoculation with I. lenis significantly enhances the uptake of nutritional constituents, phytochemicals, and antioxidant properties in P. frutescens, suggesting its potential to boost the therapeutic properties of host plants., (© 2024. The Author(s).)

Rosmarinic acid (RA) is an important medicinal metabolite and a potent food antioxidant. We discovered that exposure to high light intensifies the accumulation of RA in the leaves of perilla (Perilla frutescens (L.) Britt). However, the molecular mechanism underlying RA synthesis in response to high light stress remains poorly understood. To address this knowledge gap, we conducted a comprehensive analysis employing transcriptomic sequencing, transcriptional activation, and genetic transformation techniques. High light treatment for 1 and 48 h resulted in the upregulation of 592 and 1,060 genes, respectively. Among these genes, three structural genes and 93 transcription factors exhibited co-expression. Notably, NAC family member PfNAC2, GBF family member PfGBF3, and cinnamate-4-hydroxylase gene PfC4H demonstrated significant co-expression and upregulation under high light stress. Transcriptional activation analysis revealed that PfGBF3 binds to and activates the PfNAC2 promoter. Additionally, both PfNAC2 and PfGBF3 bind to the PfC4H promoter, thereby positively regulating PfC4H expression. Transient overexpression of PfNAC2, PfGBF3, and PfC4H, as well as stable transgenic expression of PfNAC2, led to a substantial increase in RA accumulation in perilla. Consequently, PfGBF3 acts as a photosensitive factor that positively regulates PfNAC2 and PfC4H, while PfNAC2 also regulates PfC4H to promote RA accumulation under high light stress. The elucidation of the regulatory mechanism governing RA accumulation in perilla under high light conditions provides a foundation for developing a high-yield RA system and a model to understand light-induced metabolic accumulation., Competing Interests: Conflict of interest statement. The authors declare no conflicts of interest., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The basic leucine zipper (bZIP) transcription factor (TF) family is one of the biggest TF families identified so far in the plant kingdom, functioning in diverse biological processes including plant growth and development, signal transduction, and stress responses. For Perilla frutescens , a novel oilseed crop abundant in polyunsaturated fatty acids (PUFAs) (especially α-linolenic acid, ALA), the identification and biological functions of bZIP members remain limited. In this study, 101 PfbZIPs were identified in the perilla genome and classified into eleven distinct groups (Groups A, B, C, D, E, F, G, H, I, S, and UC) based on their phylogenetic relationships and gene structures. These PfbZIP genes were distributed unevenly across 18 chromosomes, with 83 pairs of them being segmental duplication genes. Moreover, 78 and 148 pairs of orthologous bZIP genes were detected between perilla and Arabidopsis or sesame, respectively. PfbZIP members belonging to the same subgroup exhibited highly conserved gene structures and functional domains, although significant differences were detected between groups. RNA-seq and RT-qPCR analysis revealed differential expressions of 101 PfbZIP genes during perilla seed development, with several PfbZIPs exhibiting significant correlations with the key oil-related genes. Y1H and GUS activity assays evidenced that PfbZIP85 downregulated the expression of the PfLPAT1B gene by physical interaction with the promoter. PfLPAT1B encodes a lysophosphatidate acyltransferase (LPAT), one of the key enzymes for triacylglycerol (TAG) assembly. Heterogeneous expression of PfbZIP85 significantly reduced the levels of TAG and UFAs (mainly C18:1 and C18:2) but enhanced C18:3 accumulation in both seeds and non-seed tissues in the transgenic tobacco lines. Furthermore, these transgenic tobacco plants showed no significantly adverse phenotype for other agronomic traits such as plant growth, thousand seed weight, and seed germination rate. Collectively, these findings offer valuable perspectives for understanding the functions of PfbZIPs in perilla, particularly in lipid metabolism, showing PfbZIP85 as a suitable target in plant genetic improvement for high-value vegetable oil production.

The new-type tobacco varieties "Zisu" and "Luole" were obtained by distant hybridization between N. tabacum L. var. HHY and Perilla frutescens and Ocimum basilicum, with obviously different chemical composition. Smoking is the major risk factor for COPD, characterized by neutrophil-dominant inflammation. In the present study, rat COPD model was established by cigarette exposure, and the health hazard of three varieties was compared by general condition observation, pathological and morphological evaluation, total and differential cell numeration, and characterization of major inflammatory mediators and MAPK/NF-κB pathway, etc. Rats in "HHY" group developed obvious symptoms such as cough, dyspnea, mental fatigue, etc., but these symptoms were obviously mitigated in "Zisu" and "Luole" groups. H&E staining analysis, including score, MLI, MAN, wt% and WA%, showed that "Zisu" and "Luole" significantly alleviated lung injury and the degree of airway remodeling and emphysema compared to "HHY". In BALF, the number of total leukocyte and the percent neutrophils in "Zisu" and "Luole" groups were evidently lower than "HHY" group. The levels of inflammatory mediators, such as IL-8, MPO, MIP-2, LTB4, TNF-α and neutrophil elastase, in "HHY" group were obviously higher than "Zisu" and "Luole" groups. The ROS-mediated NF-κB p65 and p38MAPK pathways may play an important role. Results indicated that tobacco introduced perilla and basil genes could remarkably attenuate recruitment, infiltration and activation of neutrophils and intervene in airway inflammation, retarding disease progression, especially "Zisu". Changes in chemical composition via breeding techniques may be a novel way for tobacco harm reduction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Perilla (Perilla frutescens) is a potential specific oilseed crop with an extremely high α-linolenic acid (ALA) content in its seeds. AP2/ERF transcription factors (TFs) play important roles in multiple biological processes. However, limited information is known about the regulatory mechanism of the AP2/ERF family in perilla's oil accumulation. In this research, we identified 212 AP2/ERF family members in the genome of perilla, and their domain characteristics, collinearity, and sub-genome differentiation were comprehensively analyzed. Transcriptome sequencing revealed that genes encoding key enzymes involved in oil biosynthesis (e.g., ACCs, KASII, GPAT, PDAT and LPAAT) were up-regulated in the high-oil variety. Moreover, the endoplasmic reticulum-localized FAD2 and FAD3 were significantly up-regulated in the high-ALA variety. To investigate the roles of AP2/ERFs in lipid biosynthesis, we conducted a correlation analysis between non-redundant AP2/ERFs and key lipid metabolism genes using WGCNA. A significant correlation was found between 36 AP2/ERFs and 90 lipid metabolism genes. Among them, 12 AP2/ERFs were identified as hub genes and showed significant correlation with lipid synthase genes (e.g., FADs, GPAT and ACSL) and key regulatory TFs (e.g., LEC2, IAA, MYB, UPL3). Furthermore, gene expression analysis identified three AP2/ERFs (WRI, ABI4, and RAVI) potentially playing an important role in the regulation of oil accumulation in perilla. Our study suggests that PfAP2/ERFs are important regulatory TFs in the lipid biosynthesis pathway, providing a foundation for the molecular understanding of oil accumulation in perilla and other oilseed crops., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier B.V.)

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step in triacylglycerol (TAG) biosynthesis. However, GPAT members and their functions remain poorly understood in Perilla frutescens , a special edible-medicinal plant with its seed oil rich in polyunsaturated fatty acids (mostly α-linolenic acid, ALA). Here, 14 PfGPATs were identified from the P. frutescens genome and classified into three distinct groups according to their phylogenetic relationships. These 14 PfGPAT genes were distributed unevenly across 11 chromosomes. PfGPAT members within the same subfamily had highly conserved gene structures and four signature functional domains, despite considerable variations detected in these conserved motifs between groups. RNA-seq and RT-qPCR combined with dynamic analysis of oil and FA profiles during seed development indicated that PfGPAT9 may play a crucial role in the biosynthesis and accumulation of seed oil and PUFAs. Ex vivo enzymatic assay using the yeast expression system evidenced that PfGPAT9 had a strong GPAT enzyme activity crucial for TAG assembly and also a high substrate preference for oleic acid (OA, C18:1) and ALA (C18:3). Heterogeneous expression of PfGPAT9 significantly increased total oil and UFA (mostly C18:1 and C18:3) levels in both the seeds and leaves of the transgenic tobacco plants. Moreover, these transgenic tobacco lines exhibited no significant negative effect on other agronomic traits, including plant growth and seed germination rate, as well as other morphological and developmental properties. Collectively, our findings provide important insights into understanding PfGPAT functions, demonstrating that PfGPAT9 is the desirable target in metabolic engineering for increasing storage oil enriched with valuable FA profiles in oilseed crops.

The increasingly serious trend of soil salinization inhibits the normal growth and development of soybeans, leading to reduced yields and a serious threat to global crop production. Microsomal ω-3 fatty acid desaturase encoded by the FAD3 gene is a plant enzyme that plays a significant role in α-linolenic acid synthesis via regulating the membrane fluidity to better accommodate various abiotic stresses. In this study, PfFAD3a was isolated from perilla and overexpressed in soybeans driven by CaMV P35S, and the salt tolerance of transgenic plants was then evaluated. The results showed that overexpression of PfFAD3a increased the expression of PfFAD3a in both the leaves and seeds of transgenic soybean plants, and α-linolenic acid content also significantly increased; hence, it was shown to significantly enhance the salt tolerance of transgenic plants. Physiological and biochemical analysis showed that overexpression of PfFAD3a increased the relative chlorophyll content and PSII maximum photochemical efficiency of transgenic soybean plants under salt stress; meanwhile, a decreased accumulation of MDA, H 2 O 2 , and O2•-, increased the activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbic acid peroxidase (APX), as well as the production of proline and soluble sugar. In summary, the overexpression of PfFAD3a may enhance the salt tolerance in transgenic soybean plants through enhanced membrane fluidity and through the antioxidant capacity induced by C18:3.

Perilla as herb, ornamental, oil and edible plant is widely used in East Asia. Until now, the mechanism of regulated leaf coloration is still unclear. In this study, four different kinds of leaf colors were used to measure pigment contents and do transcriptome sequence to postulate the mechanism of leaf coloration. The measurements of chlorophyll, carotenoid, flavonoid, and anthocyanin showed that higher contents of all the aforementioned four pigments were in full purple leaf 'M357', and they may be determined front and back leaf color formation with purple. Meanwhile, the content of anthocyanin was controlled back leaf coloration. The chromatic aberration analysis and correlative analysis between different pigments and L*a*b* values analysis also suggested front and back leaf color change was correlated with the above four pigments. The genes involved in leaf coloration were identified through transcriptome sequence. The expression levels of chlorophyll synthesis and degradation related genes, carotenoid synthesis related genes and anthocyanin synthesis genes showed up-/down-regulated expression in different color leaves and were consistent of accumulation of these pigments. It was suggested that they were the candidate genes regulated perilla leaf color formation, and genes including F3'H, F3H, F3',5'H, DFR, and ANS are probably important for regulating both front and back leaf purple formation. Transcription factors involved in anthocyanin accumulation, and regulating leaf coloration were also identified. Finally, the probable scheme of regulated both full green and full purple leaf coloration and back leaf coloration was postulated., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

To improve the potential of cadmium phytoremediation, distant hybridization between tobacco (Nicotiana tabacum L. var. 78-04), a high-biomass crop, and Perilla frutescens var. frutescens, a wild Cd-hyperaccumulator, was carried out, developing a new variety N. tabacum L. var. ZSY. Seedlings at the six-leaf stage were grown in hydroponics and treated with 0 (control), 10 µM, 180 µM, and 360 µM CdCl 2 for 7 days; then, the differences in Cd tolerance and accumulation and physiological and metabolic responses were evaluated among "ZSY" and its parents. At high Cd dose, the growth of "ZSY," such as fresh weight, plant height, and root length, was evidently better than "78-04." In contrast to P. frutescens and "78-04," "ZSY" could accumulate more Cd in shoots than roots. Under the same treatment, "ZSY" accumulated greater amounts of Cd in both shoots (195-1523 mg kg -1 ) and roots (140-1281 mg kg -1 ) than "78-04" (shoots: 35-89 mg kg -1 , roots: 39-252 mg kg -1 ), followed by P. frutescens (shoots: 156-454 mg kg -1 , roots: 103-761 mg kg -1 ). BCF and TF values of "ZSY" reached 38-195 and 1.2-1.4, which were far higher than those of "78-04" (BCF: 2.2-35.3, TF: 0.35-0.9). Perilla frutescens was found with BCF and TF of 11-156 and 0.5-1.5. Cd stress obviously promoted the production of ROS and MDA in seedlings but reduced chlorophyll contents, especially in "78-04." As a response to Cd stress, "ZSY" had higher SOD and CAT activities when compared to P. frutescens and "78-04," while "78-04" produced more POD and proline than those of P. frutescens and "ZSY." Cd stress could affect the production and accumulation of alkaloids and phenolic compounds in root (endodermis and cortex) and mesophyll. At high Cd doses, P. frutescens and "ZSY" had more alkaloids in tissues than "78-04." Phenolic compounds in "78-04" were more obviously inhibited compared with P. frutescens and "ZSY." These secondary metabolites may play an important role in eliminating oxidative damage and enhancing Cd tolerance and accumulation in "ZSY" and P. frutescens. Results indicated that distant hybridization could be one of effective methods for introducing excellent genes from metal-hyperaccumulators into high biomass species, creating plants with superior phytoremediation potential., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Previous studies have indicated that the adrenergic receptor signaling pathway plays a fundamental role in chronic stress-induced cancer metastasis. In this study, we investigated whether an ethanol extract of Perilla frutescens leaves (EPF) traditionally used to treat stress-related symptoms by moving Qi could regulate the adrenergic agonist-induced metastatic ability of cancer cells. Our results show that adrenergic agonists including norepinephrine (NE), epinephrine (E), and isoproterenol (ISO) increased migration and invasion of MDA-MB-231 human breast cancer cells and Hep3B human hepatocellular carcinoma cells. However, such increases were completely abrogated by EPF treatment. E/NE induced downregulation of E-cadherin and upregulation of N-cadherin, Snail, and Slug. Such effects were clearly reversed by pretreatment with EPF, suggesting that the antimetastatic activity of EPF could be related to epithelial-mesenchymal transition (EMT) regulation. EPF suppressed E/NE-stimulated Src phosphorylation. Inhibition of Src kinase activity with dasatinib completely suppressed the E/NE-induced EMT process. Transfecting MDA-MB-231 cells with constitutively activated Src (SrcY527F) diminished the antimigration effect of EPF. Taken together, our results demonstrate that EPF can suppress the adrenergic agonist-promoted metastatic ability of cancer cells by inhibiting Src-mediated EMT. This study provides basic evidence supporting the probable use of EPF to prevent metastasis in cancer patients, especially those under chronic stress.

Perilla frutescens (L.), a traditional edible and medicinal crop, contains diverse triterpenes with multiple pharmacological properties. However, the biosynthesis of triterpenes in perilla remains rarely revelation. In this study, nine putative 2,3-oxidosqualene cyclase (OSC) genes ( PfOSC1-9 ) were screened from the P. frutescens genome and functionally characterized by heterologous expression. Camelliol C, a triterpenol with pharmacological effect, was first identified as abundant in perilla seeds, and the camelliol C synthase (PfOSC7) was first identified in P. frutescens utilizing a yeast system. In addition, PfOSC2, PfOSC4, and PfOSC9 were identified as cycloartenol, lupeol, and β-amyrin synthase, respectively. Molecular docking and site-directed mutagenesis revealed that changes in Leu253 of PfOSC4, Ala480 of PfOSC7, and Trp257 of PfOSC9 might lead to variations of catalytic specificity or efficiency. These results will provide key insights into the biosynthetic pathways of triterpenoids and have great significance for germplasm breeding in P. frutescens .

Perilla frutescens (Lamiaceae) is an important herbal plant with hundreds of bioactive chemicals, among which perillaldehyde and rosmarinic acid are the two major bioactive compounds in the plant. The leaves of red perilla are used as traditional Kampo medicine or food ingredients. However, the medicinal and nutritional uses of this plant could be improved by enhancing the production of valuable metabolites through the manipulation of key enzymes or regulatory genes using genome editing technology. Here, we generated a high-quality genome assembly of red perilla domesticated in Japan. A near-complete chromosome-level assembly of P. frutescens was generated contigs with N50 of 41.5 Mb from PacBio HiFi reads. 99.2% of the assembly was anchored into 20 pseudochromosomes, among which seven pseudochromosomes consisted of one contig, while the rest consisted of less than six contigs. Gene annotation and prediction of the sequences successfully predicted 86,258 gene models, including 76,825 protein-coding genes. Further analysis showed that potential targets of genome editing for the engineering of anthocyanin pathways in P. frutescens are located on the late-stage pathways. Overall, our genome assembly could serve as a valuable reference for selecting target genes for genome editing of P. frutescens., (© The Author(s) 2022. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.)

Excessive stress plays a critical role in the pathogenesis of mood disorders such as depression. Fermented natural products have recently attracted attention because of their health benefits. We evaluated the antidepressant-like efficacy of fermented Perilla frutescens (FPF), and its underlying mechanisms, in sleep deprivation (SD)-induced stress mice. SD-stressed mice revealed a remarkable increase in the immobility time in both forced swimming test and tail suspension test; this increase was ameliorated by treatment with FPF at doses of 100 and 150 mg/kg. FPF treatment also reduced the level of stress hormones such as corticosterone and adrenocorticotropic hormone. Additionally, FPF increased the levels of serotonin and dopamine which were significantly decreased in the brain tissues of SD-stressed mice. The increased expression of proinflammatory cytokines, such as TNF-α and IL1β, and the decreased expression of brain-derived neurotrophic factor (BDNF) in the stressed mice were significantly reversed by FPF treatment. Furthermore, FPF also increased phosphorylation of tropomyosin receptor kinase B (TrkB), extracellular regulated protein kinase (ERK), and cAMP response element binding protein (CREB). Among the six components isolated from FPF, protocatechuic acid and luteolin-7- O -glucuronide exhibited significant antidepressant-like effects, suggesting that they are major active components. These findings suggest that FPF has therapeutic potential for SD-induced stress, by correcting dysfunction of hypothalamic-pituitary-adrenal axis and modulating the BDNF/TrkB/ERK/CREB signaling pathway.

Diacylglycerol acyltransferase (DGAT) is the rate-limiting enzyme that catalyzes the final step in triacylglycerol biosynthesis, however, members of DGAT gene family of Perilla frutescens has not yet been identified and characterized. In this study, a total of 20 PfDGAT genes were identified from the genome of Perilla frutescens and were divided into four groups (PfDGAT1, PfDGAT2, PfDGAT3, PfWS/DGAT) according to their phylogenetic relationships. These were unevenly distributed across the 12 chromosomes. Sequence analysis revealed that PfDGAT members of the same subfamily have highly conserved gene structures, protein motifs and cis-acting elements in their promoters. Gene duplication analysis showed that random duplication and segmental duplication contributed to the expansion of the DGAT family in P. frutescens. RNA-seq and qRT-PCR analysis suggested that they may play a role in the growth and development of Perilla, especially in the accumulation of seed oil. Compared with the wild-type, seed length, width, and 1000-seed weight of transgenic PfDGAT2-2 and PfDGAT3-1 Arabidopsis were significantly increased, as well as the seed oil content increased by 7.36-15.83 %. Over-expression of PfDGAT2-2 could significantly increase the content of C18:3 and C20:1 in Arabidopsis, while over-expression of PfDGAT3-1 could significantly enhance the content of C18:2 and C18:3. In conclusion, in this study the characteristics and potential functions of the PfDGAT family members were elucidated. Our findings provided basic information for further functional studies and helped to increase the yield and quality of Perilla oil., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2022 Elsevier B.V. All rights reserved.)

The aim of the present study was to explore the effects of abscisic acid (ABA) on growth and selenium (Se) absorption of the medicinal plant, Perilla frutescens. A pot experiment was conducted to evaluate the effects of different ABA concentrations (0, 1, 5, 10 and 20 μmol/L) on the physiological characteristics and Se absorption capacity of P. frutescens. Application of 5, 10 and 20 μmol/L ABA increased the shoot biomass of P. frutescens, and only 5 and 10 μmol/L ABA increased the root biomass. Application of 5, 10, and 20 μmol/L ABA increased the contents of photosynthetic pigments (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid), superoxide dismutase activity, peroxidase activity, and soluble protein content of P. frutescens, and decreased the malondialdehyde content in P. frutescens. Only 5 and 10 μmol/L ABA used in the present study increased the catalase activity of P. frutescens. For the Se uptake, only 5 μmol/L ABA increased the Se content, Se extraction and Se bioconcentration factor of both roots and shoots. The findings of the present study indicate that 5 and 10 μmol/L ABA promotes the growth of P. frutescens, whereas 5 μmol/L ABA enhances the Se accumulation capacity in P. frutescens., Competing Interests: The authors have declared that no competing interests exist.

Cellular senescence is an inherent tumor suppressive process, and cancer-targeted senescence induction represents an attractive anti-tumor strategy. Here, we show that a methoxyflavanone derivative (Perilla-derived methoxyflavanone, PDMF) from the Asian medicinal herb, Perilla frutescens, induces cellular senescence in A549 human adenocarcinoma cells but not in normal human bronchial epithelial (NHBE) cells. We also provide evidence that PDMF preferentially activates the p53-p21 pathway in A549 cells, and that p53 is essential for its pro-senescent activity.

Perilla ( Perilla frutescens L.) is an important edible-medicinal oil crop, with its seed containing 46%-58% oil. Of perilla seed oil, α-linolenic acid (C18:3) accounts for more than 60%. Lysophosphatidic acid acyltransferase (LPAT) is one of the key enzymes responsible for triacylglycerol assembly in plant seeds, controlling the metabolic flow from lysophosphatidic acid to phosphatidic acid. In this study, the LPAT2 gene from the developing seeds of perilla was cloned and designated as PfLPAT2 . The expression profile of PfLPAT2 gene was examined in various tissues and different seed development stages of perilla (10, 20, 30, and 40 days after flowering, DAF) by quantitative real-time PCR (qRT-PCR). In order to detect the subcellular localization of PfLPAT2 protein, a fusion expression vector containing PfLPAT2 and GFP was constructed and transformed into Nicotiana benthamiana leaves by Agrobacterium -mediated infiltration. In order to explore the enzymatic activity and biological function of PfLPAT2 protein, an E . coli expression vector, a yeast expression vector and a constitutive plant overexpression vector were constructed and transformed into an E . coli mutant SM2-1, a wild-type Saccharomyces cerevisiae strain INVSc1, and a common tobacco ( Nicotiana tabacum , variety: Sumsun NN, SNN), respectively. The results showed that the PfLPAT2 open reading frame (ORF) sequence was 1 155 bp in length, encoding 384 amino acid residues. Functional structure domain prediction showed that PfLPAT2 protein has a typical conserved domain of lysophosphatidic acid acyltransferase. qRT-PCR analysis indicated that PfLPAT2 gene was expressed in all tissues tested, with the peak level in seed of 20 DAF of perilla. Subcellular localization prediction showed that PfLPAT2 protein is localized in cytoplasm. Functional complementation assay of PfLPAT2 in E . coli LPAAT mutant (SM2-1) showed that PfLPAT2 could restore the lipid biosynthesis of SM2-1 cell membrane and possess LPAT enzyme activity. The total oil content in the PfLPAT2 transgenic yeast was significantly increased, and the content of each fatty acid component changed compared with that of the non-transgenic control strain. Particularly, oleic acid (C18:1) in the transgenic yeast significantly increased, indicating that PfLPAT2 has a higher substrate preference for C18:1. Importantly, total fatty acid content in the transgenic tobacco leaves increased by about 0.42 times compared to that of the controls, with the C18:1 content doubled. The increased total oil content and the altered fatty acid composition in transgenic tobacco lines demonstrated that the heterologous expression of PfLPAT2 could promote host oil biosynthesis and the accumulation of health-promoting fatty acids (C18:1 and C18:3). This study will provide a theoretical basis and genetic elements for in-depth analysis of the molecular regulation mechanism of perilla oil, especially the synthesis of unsaturated fatty acids, which is beneficial to the genetic improvement of oil quality of oil crops.

We have shown that Anredera cordifolia extract improves learning and memory in a senescence-accelerated mouse model, and that α-linolenic acid (ALA)-rich Perilla frutescens seed oil (PO) improves brain function in healthy Japanese adults and elderly individuals. Herein, we present a 12-month, randomised, double-blind, parallel-armed intervention trial examining the effects of PO supplementation alone or in combination with A. cordifolia leaf powder on brain function in healthy elderly Japanese individuals. Participants were randomly divided into two groups: the PO group received 1.47 mL PO (0.88 g ALA) daily via soft gelatine capsules, and the POAC group received 1.47 mL PO and 1.12 g A. cordifolia leaf powder (1.46 mg vitexin and 1.12 mg adenosine) daily. After 12 months of intervention, the POAC group showed generally higher cognitive index scores than the PO group. The beneficial effects of combined supplementation on cognitive function were associated with increased ALA and eicosapentaenoic acid levels in red blood cell plasma membranes, increased serum biological antioxidant potential, and decreased serum triglyceride, glucose, and N -(epsilon)-carboxymethyl-lysine (CML), an advanced glycation end-product and biochemical marker of oxidative stress levels. The effects of combined supplementation on cognitive function also showed a significant negative correlation with serum CML levels after 12 months of intervention. Our findings suggest that combined long-term supplementation with PO and A. cordifolia more effectively ameliorates age-related cognitive decline than PO alone. These findings may serve as a basis for the development of new supplements for brain health. Clinical Trial Registry, UMIN000040863.

Psoriasis is reported to be a common chronic immune-mediated skin disease characterized by abnormal keratinocytes and cell proliferation. Perilla leaves are rich in essential oils, fatty acids, and flavonoids, which are recognized for their antioxidant and anti-inflammatory effects. In this study, the alleviating effect of essential oil (PO) extracted from Perilla frutescens stems and leaves on imiquimod (IMQ) -induced psoriasis-like lesions in BALB/c mice were investigated. Results showed that PO ameliorated psoriasis-like lesions in vivo, reduced the expression of lymphocyte antigen 6 complex locus G6D (Ly-6G), which is a marker of neutrophil activation, and inhibited the expression of inflammatory factors interleukin 1 (IL-1), interleukin 6 (IL-6), inducible nitric oxide synthase (iNOS), and cyclooxygenase 2 (COX2). In addition, PO significantly decreased the expression of cytokines such as IL-6, IL-1, interleukin 23 (IL-23), interleukin 17 (IL-17), and nuclear factor kappa-B (NF-κB). Furthermore, the down-regulation of mRNA levels of psoriasis-related pro-inflammatory cytokines, such as IL-17, interleukin 22 (IL-22), IL-23, interferon-α (IFN-α), and Interferon-γ (IFN-γ) was observed with the treatment of PO. All results show a concentration dependence of PO, with low concentrations showing the best results. These results suggest that PO effectively alleviated psoriasis-like skin lesions and down-regulated inflammatory responses, which indicates that PO could potentially be used for further studies on inflammation-related skin diseases such as psoriasis and for the treatment of psoriasis such as psoriasis natural plant essential oil resources.

Studies have reported that cholesterol, a molecule found mainly in animals, is also present in some plants and algae. This study aimed to determine whether cholesterol exists in three dehydrated algae species, namely, Pyropia tenera, Saccharina japonica, and Undaria pinnatifida , and in one plant species, namely, Perilla frutescens (four perilla seed oil samples were analyzed). These species were chosen for investigation because they are common ingredients in East Asian cuisine. Gas chromatography-flame ionization detection (GC-FID) analysis found that cholesterol was present in P. tenera (14.6 mg/100 g) and in all four perilla seed oil samples (0.3-0.5 mg/100 g). High-performance liquid chromatography with evaporative light-scattering detection (HPLC-ELSD) also demonstrated that cholesterol was present in P. tenera (14.2 mg/100 g) and allowed the separation of cholesterol from its isomer lathosterol. However, cholesterol could not be detected by HPLC-ELSD in the perilla seed oil samples, most likely because it is only present in trace amounts. Moreover, liquid chromatography-tandem mass spectrometry (LC-MS/MS) confirmed the presence of cholesterol in both P. tenera and perilla seed oil. MRM results further suggested that lathosterol (a precursor of cholesterol) was present in P. tenera.

Particulate matter (PM) has become a severe environmental issue, and ultrafine PM particles such as PM 2.5 or PM 1 can cause various complications and respiratory diseases to human beings. In particular, heavy metals contained in PM particles can contaminate edible plants; for example, plant leaves are exposed to PM particle-laden raindrops. The contaminated edible plants can injure the human health by ingestion, so a detailed understanding on the accumulation of PM particles inside edible plants is essential. In this study, we investigate the infiltration of PM particles in plant tissues with a hypothesis that ultrafine PM particles are absorbed through stomatal pathways. As an edible test plant, Perilla frutescens is selected. Drops of gold nanoparticle (AuNP) suspension are deposited on a leaf of P. frutescens to simulate the scenario where PM particle-laden raindrops fall on patulous stomata of the test plant. To examine AuNP adsorption on the P. frutescens foliar surface and diffusional AuNP absorption through stomatal apertures, we investigate three physical dynamics of AuNPs suspended in a sessile drop: sedimentation, evaporation-driven convective flow, and shrinkage of the drop interface. Quantitative information on the 3D spatial distribution of AuNPs in plant tissues was measured by X-ray imaging and two-photon excitation microscopy.

The leaf of Perilla frutescens (L.) Britton var. frutescens (egoma) is a rich source of polyphenolic compounds, including rosmarinic acid. However, there is still a lack of detailed information concerning the content of phenolic compounds in these leaves. Since some flavonoids were found as a conjugated form, leaves were used untreated or hydrolyzed using β-glucuronidase for analysis. Enzymatic hydrolysis method successfully identified some polyphenols, which have not been reported before. Scutellarin, a flavone glucuronide with a molecular mass similar to that of luteolin 7-O-glucuronide, was present in egoma leaves. Scutellarin was the second most abundant polyphenolic compound, after rosmarinic acid. Egoma leaves at the top of the plant contained a higher amount of rosmarinic acid and scutellarin compared to that in the leaves below. The difference in plant growth stage also influenced the rosmarinic acid and scutellarin contents, while the time of harvesting during the day did rosmarinic acid contents only. This is the first time that scutellarin, a traditional Chinese medicine, widely used for the treatment of cerebrovascular disease, was quantitatively determined in egoma leaves. The present study may help adding value to egoma leaves, developing dietary supplements, functional foods, and cosmetics.

Perillae Folium (PF), which is extensively used as a dietary vegetable and medicinal herb, contains two varietal forms corresponding to purple perilla leaf ( Perilla frutescens var. crispa ) and green perilla leaf ( Perilla frutescens var. frutescens ). However, the components and efficacy of different PF varieties remain underexplored so far. In the present work, a nontargeted rapid resolution liquid chromatography coupled with quadruple-time-of-flight mass spectrometry (RRLC-Q/TOF-MS)-based metabolomics approach was developed to investigate the difference in the chemical compositions between green PF and purple PF. A total of 71 compounds were identified or tentatively identified, among which 7 phenolic acids, 10 flavonoids, and 9 anthocyanins were characterized as differential metabolites. In addition, heatmap visualization and ultraperformance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UPLC-TQ-MS/MS)-based quantitative analysis revealed that flavonoids and anthocyanins especially had higher contents in purple PF. Furthermore, the anti-oxidative activities of two varietal PFs were evaluated in vivo zebrafish and in vitro human umbilical vein endothelial cells (HUVECs). The results showed that the purple PF had more pronounced anti-oxidative activities than did the green PF, which may be due to the presence of anthocyanins and a higher concentration of flavonoids in its phytochemical profile. The outcome of the present study is expected to provide useful insight on the comprehensive utilization of a PF resource.

In order to achieve premium quality with crop production, techniques involving the adjustment of nutrient supply and/or supplemental lighting with specific light quality have been applied. To examine the effects of low mineral supply and supplemental lighting, we performed non-targeted metabolite profiling of leaves and stems of the medicinal herb Perilla frutescens , grown under a lower (0.75×) and lowest (0.1×) supply of different minerals (N, K, or Mg) and under supplemental light-emitting diode (LED) lighting (red, blue, or red-blue combination). The lowest N supply increased flavonoids, and the lowest K or Mg slightly increased rosmarinic acid and some flavonoids in the leaves and stems. Supplemental LED lighting conditions (red, blue, or red-blue combination) significantly increased the contents of chlorophyll, most cinnamic acid derivatives, and rosmarinic acid in the leaves. LED lighting with either blue or the red-blue combination increased antioxidant activity compared with the control group without LED supplementation. The present study demonstrates that the cultivation of P. frutescens under low mineral supply and supplemental LED lighting conditions affected metabolic compositions, and we carefully suggest that an adjustment of minerals and light sources could be applied to enhance the levels of targeted metabolites in perilla.

Perilla frutescens (L.) is an important medicinal and edible plant in China with nutritional and medical uses. The extract from leaves of Perilla frutescens contains flavonoids and volatile oils, which are mainly used in traditional Chinese medicine. In this study, we analyzed the transcriptomic and metabolomic data of the leaves of two Perilla frutescens varieties: JIZI 1 and JIZI 2. A total of 9277 differentially expressed genes and 223 flavonoid metabolites were identified in these varieties. Chrysoeriol, apigenin, malvidin, cyanidin, kaempferol, and their derivatives were abundant in the leaves of Perilla frutescens, which were more than 70% of total flavonoid contents. A total of 77 unigenes encoding 15 enzymes were identified as candidate genes involved in flavonoid biosynthesis in the leaves of Perilla frutescens. High expression of the CHS gene enhances the accumulation of flavonoids in the leaves of Perilla frutescens. Our results provide valuable information on the flavonoid metabolites and candidate genes involved in the flavonoid biosynthesis pathways in the leaves of Perilla frutescens.

Screening new accumulators of heavy metal and identifying their tolerance, enrichment capacity of heavy metals are currently hot issues in phytoremediation research. A series of hydroponic experiments were conducted to analyze the effects of glutathione and phytochelatins in roots, stems, and leaves of Perilla frutescens under cadmium stress. The results showed that the non-protein thiols in roots and stems mainly existed in the form of GSH, PC 2 , PC 3 , and PC 4 under Cd stress condition, while in leaves they existed in the form of GSH, PC 2 , and PC 3 . Furthermore, the contents of GSH and PCs positively correlated with Cd, but negatively correlated with root vigor and chlorophyll content under Cd stress conditions. After 21 days of treatments, the contents of Cd in different parts of the plant were 1465.2-3092.9 mg· kg -1 in the roots, 199.6-478.4 mg·kg -1 in the stems and 61.3-96.9 mg· kg -1 in the leaves at 2, 5, 10 mg·L -1 Cd levels respectively, and the amount of Cd uptakes were up to 3547.7-5701.7 μg·plant -1 . Therefore, P. frutescens performed high capacity in Cd accumulation, and PCs played a key role in Cd tolerance. The application prospect of the plant in phytoremediation Cd polluted soil was also discussed., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Inc. All rights reserved.)

High salt accumulation, resulting from the rampant use of chemical fertilizers in greenhouse cultivation, has deleterious effects on plant growth and crop yield. Herein, we delineated the effects of magnesium (Mg) oversupply on Perilla frutescens leaves, a traditional edible and medicinal herb used in East-Asian countries. Mg oversupply resulted in significantly higher chlorophyll content coupled with lower antioxidant activities and growth, suggesting a direct effect on subtle metabolomes. The relative abundance of bioactive phytochemicals, such as triterpenoids, flavonoids, and cinnamic acids, was lower in the Mg-oversupplied plants than in the control. Correlation analysis between plant phenotypes (plant height, total fresh weight of the shoot, leaf chlorophyll content, and leaf antioxidant content) and the altered metabolomes in P. frutescens leaves suggested an acclimatization mechanism to Mg oversupply. In conclusion, P. frutescens preferentially accumulated compatible solutes, i.e., carbohydrates and amino acids, to cope with higher environmental Mg levels, instead of employing secondary and antioxidative metabolism., Competing Interests: The authors have declared that no competing interests exist.

Background: Polyunsaturated fatty acids such as linoleic acid (LA) and α-linolenic acid (ALA) are abundant in vegetable oils and are important for human health. In the body, LA and ALA are respectively converted to the omega-6 fatty acid γ-linolenic acid (GLA) and the omega-3 fatty acid stearidonic acid (SDA) by Δ6 desaturase (D6DES). Currently, dietary GLA and SDA are mainly obtained from marine organisms, but given their benefits to human health, many studies have aimed to enhance their accumulation in transgenic crops. Perilla frutescens (perilla) accumulates more ALA in its seed oil compared to other oilseed crops, making it a good candidate for the production of fatty acids via the fatty acid desaturase D6DES., Results: In this study, we cloned the D6DES gene from Phytophthora citrophthora and confirmed its function in budding yeast. We then transformed the functional D6DES gene under the control of the seed-specific vicilin promoter into the perilla cultivar Yeobsil. The resulting transgenic perilla seeds accumulated significant levels of GLA and SDA, as well as putative C18:2Δ 6,9 at minor levels. Developing seeds and leaves also accumulated GLA and SDA, although PcD6DES expression and GLA and SDA levels were much lower in leaves compared to developing seeds. GLA and SDA accumulated in both polar lipids and neutral lipids in mature perilla seeds expressing PcD6DES, especially in neutral lipids. Although the seed weight in PcD6DES perilla was 87-96% that of wild type, the total oil content per seed weight was similar between lines. The PcD6DES perilla plants contained very high content (over 45%) of both GLA and SDA in seed oil., Conclusions: Thus, PcD6DES perilla plants may represent a feasible alternative to traditional marine sources for the production of omega-3 oil capsules and to evening primrose seed oil for GLA as health food. In addition, these plants can be used to create other transgenic lines harboring additional genes to produce other desirable fish-oil like oils.

The objective of this study was to investigate the volatile compounds of the leaves of ten perilla accessions as well as to determine total polyphenols, antioxidant capacity. Essential oil (EOs) content ranged from 0.33 to 1.75 mL/100 g d.w. in PS3 and J1 respectively. In this study sixty-five compounds were identified by GC-MS and characterized with the predominance of perillaldehyde, perilla ketone, ß-dehydro-elsholtzia ketone, limonene, shisofuran, farnesene (Z, E, α), ß-caryophyllene, trans-shisool. The biogenesis and composition of EOs are probably attributed to several factors. JTD3 had a significantly higher polyphenol content as well as showed the highest antioxidant capacity, whereas a strong positive linear correlation was observed between them. PS1 and NP 606 produced the maximum biomass correspondingly, while a large glandular trichome density was recorded for J1. The results support that perilla is rich in natural compounds that could be developed as nutraceuticals and/or phytomedicine., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Background: Perilla frutescens is well known for its high α-linolenic acid (ALA) accumulation in seeds and medicinal values as well as a source of edible and general-purpose oils. However, the regulatory mechanisms of the biosynthesis of fatty acid in its seeds remain poorly understood due to the lacking of sequenced genome. For better understanding the regulation of lipid metabolism and further increase its oil content or modify oil composition, time-course transcriptome and lipid composition analyses were performed., Results: Analysis of fatty acid content and composition showed that the α-linolenic acid and oleic acid accumulated rapidly from 5 DAF to 15 DAF and then kept relatively stable. However, the amount of palmitic acid and linoleic acid decreased quickly from 5 DAF to 15DAF. No significant variation of stearic acid content was observed from 5 DAF to 25DAF. Our transcriptome data analyses revealed that 110,176 unigenes were generated from six seed libraries at 5, 10, 20 DAF. Of these, 53 (31 up, 22 down) and 653 (259 up, 394 down) genes showed temporal and differentially expression during the seed development in 5 DAF vs 10 DAF, 20 vs 10 DAF, respectively. The differentially expressed genes were annotated and found to be involved in distinct functional categories and metabolic pathways. Deep mining of transcriptome data led to the identification of key genes involved in fatty acid and triacylglycerol biosynthesis and metabolism. Thirty seven members of transcription factor family AP2, B3 and NFYB putatively involved in oil synthesis and deposition were differentially expressed during seed development. The results of qRT-PCR for selected genes showed a strong positive correlation with the expression abundance measured in RNA-seq analysis., Conclusions: The present study provides valuable genomic resources for characterizing Perilla seed gene expression at the transcriptional level and will extend our understanding of the complex molecular and cellular events of oil biosynthesis and accumulation in oilseed crops.

An open field experiment was carried out with five purple Perilla frutescens accessions (588P, GB, J3, JTD3, PS3) in 2014 and 2015. Morphological traits, production, total phenolic content (TPC), essential oil content (EOC) and composition as well as the antioxidant capacity (AOC) were investigated. Highest biomass was produced by JTD3 in both years. The antioxidant capacity and total phenolic content in the stems was lower than in the leaves in all accessions. Leaves of accession GB produced the highest AOC values (215.594 ± 1.437 in 2014 and 86.609 ± 3.602 mg AAE g -1 in 2015, respectively) while the strain 588P showed the lowest values (139.544 ± 1.934 in 2014 and 38.966 ± 4.569 mg AAE g -1 in 2015, respectively). The highest TPC values were measured by PS3 in 2014 (204.320 ± 1.822 mg GAE g -1 ) and GB in 2015 (136.450 ± mg GAE g -1 ). The 588P produced the highest essential oil content (1.432 ml 100 g -1 DM) while J3 had the lowest (0.144 ml 100 g -1 DM) in both years. Strong positive correlation was found between the density of glandular hairs and the essential oil content. Three accessions (588P, J3, JTD3) belong to the perillaldehyde chemotype while GB and PS3 to the dehydro elsholtzia ketone chemotype. All studied accessions can be cultivated in Hungary. For the biomass production the JTD3, while for the essential oil production the 588P can be recommended.

The objective of this research was to access the determination of metabolite profiles and antioxidant properties in the leaves of green perilla (Perilla frutescens), where these are considered functional and nutraceutical substances in Korea. A total of 25 compositions were confirmed as six phenolic acids, two triterpenoids, eight flavonoids, seven fatty acids, and two glucosides using an ultra high performance liquid chromatography coupled with electrospray ionization quadrupole time-of-flight mass spectrometry (UPLC-ESI-Q-TOF-MS/MS) technique from the methanol extract of this species. The individual and total compositions exhibited significant differences, especially rosmarinic acid (10), and linolenic acids (22 and 23) were detected as the predominant metabolites. Interestingly, rosmarinic acid (10) was observed to have considerable differences with various concentrations in three samples (Doryong, 6.38 μg/g; Sinseong, 317.60 μg/g; Bongmyeong, 903.53 μg/g) by UPLC analysis at 330 nm. The scavenging properties against 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS) radicals also showed potent effects with remarkable differences at a concentration of 100 μg/mL, and their abilities were as follows: Sinseong (DPPH, 86%; ABTS, 90%) > Bongmyeong (71% and 84%, respectively) > Doryong (63% and 73%, respectively). Our results suggest that the antioxidant activities of green perilla leaves are correlated with metabolite contents, especially the five major compositions 10 and 22-25. Moreover, this study may be useful in evaluating the relationship between metabolite composition and antioxidant activity., (Copyright © 2016. Published by Elsevier B.V.)

Background: Perilla (Perilla frutescens (L.) var frutescens) produces high levels of α-linolenic acid (ALA), a ω-3 fatty acid important to health and development. To uncover key genes involved in fatty acid (FA) and triacylglycerol (TAG) synthesis in perilla, we conducted deep sequencing of cDNAs from developing seeds and leaves for understanding the mechanism underlying ALA and seed TAG biosynthesis., Results: Perilla cultivar Dayudeulkkae contains 66.0 and 56.2 % ALA in seeds and leaves, respectively. Using Illumina HiSeq 2000, we have generated a total of 392 megabases of raw sequences from four mRNA samples of seeds at different developmental stages and one mature leaf sample of Dayudeulkkae. De novo assembly of these sequences revealed 54,079 unique transcripts, of which 32,237 belong to previously annotated genes. Among the annotated genes, 66.5 % (21,429 out of 32,237) showed highest sequences homology with the genes from Mimulus guttatus, a species placed under the same Lamiales order as perilla. Using Arabidopsis acyl-lipid genes as queries, we searched the transcriptome and identified 540 unique perilla genes involved in all known pathways of acyl-lipid metabolism. We characterized the expression profiles of 43 genes involved in FA and TAG synthesis using quantitative PCR. Key genes were identified through sequence and gene expression analyses., Conclusions: This work is the first report on building transcriptomes from perilla seeds. The work also provides the first comprehensive expression profiles for genes involved in seed oil biosynthesis. Bioinformatic analysis indicated that our sequence collection represented a major transcriptomic resource for perilla that added valuable genetic information in order Lamiales. Our results provide critical information not only for studies of the mechanisms involved in ALA synthesis, but also for biotechnological production of ALA in other oilseeds.

The volatile oil is the main component in the leaves of Perilla frutescens. According to the main types of monoterpenoids or aromatic compounds, it can be divided into different chemotypes and the main chemotypes of Chinese producing Perilla are PA type (mainly containing Perilla aldehyde and limonene), PK type (mainly containing perillaketone) and PP type (subdivided as PP-a type, with apiole as its main component; PP-m type, with myristicin as its main component; PP-e type, with elemicin as main component; PP-as type, with asarone as main component). Based on the biosynthetic pathways analysis, we also found that the formation of the particular chemotype is usually controlled by a single gene or a few genes, and different types have different pharmacological effects. In this paper, the classification under the species P. frutescens, main chemotypes of the volatile oil, and their biogenesis and regulation, pharmacological effect and influence factors are summarized and reviewed.

Perilla frutescens var. crispa (Labiatae) has two chemo-varietal forms, i.e. red and green forms of perilla, that differ in the production of anthocyanins. To facilitate molecular biological and biochemical studies in perilla-specialized metabolism we used Illumina RNA-sequencing technology in our comprehensive comparison of the transcriptome map of the leaves of red and green forms of perilla. Sequencing generated over 1.2 billion short reads with an average length of 101 nt. De novo transcriptome assembly yielded 47,788 and 47,840 unigenes in the red and green forms of perilla plants, respectively. Comparison of the assembled unigenes and existing perilla cDNA sequences showed highly reliable alignment. All unigenes were annotated with gene ontology (GO) and Enzyme Commission numbers and entered into the Kyoto Encyclopedia of Genes and Genomes. We identified 68 differentially expressed genes (DEGs) in red and green forms of perilla. GO enrichment analysis of the DEGs showed that genes involved in the anthocyanin metabolic process were enriched. Differential expression analysis revealed that the transcript level of anthocyanin biosynthetic unigenes encoding flavonoid 3'-hydroxylase, dihydroflavonol 4-reductase, and anthocyanidin synthase was significantly higher in red perilla, while the transcript level of unigenes encoding limonene synthase was significantly higher in green perilla. Our data serve as a basis for future research on perilla bio-engineering and provide a shortcut for the characterization of new functional genes in P. frutescens.

Perilla frutescens (L.) Britt. is a self-pollinating annual species and is widely cultivated in China, Korea and Japan as an economic crop and a source of medicine and spices. In this study, we sequenced one cultivar variety (PF98095) of P. frutescens (L.) var. frutescens Britt., which was assembled as reference and other three varieties (PF11109, weedy of var. frutescens, PF06336 and PF06353, cultivars of varieties crispa) in order to carry out comparative expression profiling within cultivar and weedy in varieties frutescens and between varieties frutescens and varieties crispa of cultivar type in P. frutescens. Assembly of PF98095, annotation mapping, DEG (differentially expressed gene) profiling, and comparative analysis were performed. We found that more than 65% of the reads were mapped to the reference of P. frutescens gene set. Moreover, we detected 22,962 DEGs in the weedy variety compared to the cultivar, and also, 22,138 and 23,845 DEGs were identified in two cultivars according to the reference, respectively. The DEGs and functional classification were developed to analyze the differences between weedy and cultivar and between varieties frutescens and varieties crispa of Perilla. Furthermore, candidate genes for the different color and seed size of Perilla were identified that could be further investigated in future study. The herein results may play a significant role, and contribute in functional transcriptome studies of Perilla., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Rosmarinic acid (α-o-caffeoyl-3,4-dihydroxyphenyllactic acid; RA) is a naturally occurring hydroxylated compound commonly found in species of the subfamily Nepetoideae of the Lamiaceae and Boraginaceae, such as Rosmarinus officinalis, Salvia officinalis, and Perilla frutescens. RA is biosynthesized from the amino acids L-phenylalanine and L-tyrosine by eight enzymes that include phenylalanine ammonia lyase and cinnamic acid 4-hydroxylase. RA can also be chemically produced by the esterification of caffeic acid and 3,4-dihydroxyphenyllactic acid. RA and its numerous derivatives containing one or two RA with other aromatic moieties are well known and include lithospermic acid, yunnaneic acid, salvianolic acid, and melitric acid. Recently, RA and its derivatives have attracted interest for their biological activities, which include anti-inflammatory, anti-oxidant, anti-angiogenic, anti-tumor, and anti-microbial functions. Clinically, RA attenuates T cell receptor-mediated signaling, attenuates allergic diseases like allergic rhinitis and asthma, and 2,4-dinitrofluorobenzene-induced atopic dermatitis-like symptoms, protects from neurotoxicity, and slows the development of Alzheimer's disease. These attributes have increased the demand for the biotechnological production and application of RA and its derivatives. The present review discusses the function and application of RA and its derivatives including the molecular mechanisms underlying clinical efficacy.

Perilla leaves are widely used in Chinese herbal medicine and in Japanese herbal agents used to treat respiratory diseases. This study aimed to investigate the anti‑inflammatory effects and the underlying mechanisms of Perilla frutescens leaf extract (PLE). Murine macrophage RAW264.7 cells were used as a model. Cell viability and morphological changes were studied by the MTT assay and microscopy. mRNA expression of pro‑inflammatory mediators was assessed by both semi‑quantitative reverse transcription‑polymerase chain reaction (RT‑PCR) and quantitative (q) RT‑PCR. Nitric oxide (NO) and prostaglandin E2 (PGE2) production were analyzed by the Griess test and sandwich enzyme‑linked immunosorbent assay (ELISA), respectively. The activation of kinase cascades was studied by immunoblotting. Our findings showed that PLE slightly affects cell viability, but alleviates LPS‑induced activation of RAW264.7 cells. Furthermore, PLE significantly reduced the LPS‑induced mRNA expression of the interleukin (IL)‑6, IL‑8, tumor necrosis factor‑α (TNF‑α), cyclooxygenase‑2 (COX‑2) and inducible nitric oxide synthase (iNOS), genes in a dose‑dependent manner. In addition, PLE reduced NO production and PGE2 secretion induced by LPS. PLE also inhibited activation of mitogen‑activated protein kinases (MAPKs), increased the cytosolic IκBα level, and reduced the level of nuclear factor (NF)‑κB. Taken together, these findings indicate that PLE significantly decreases the mRNA expression and protein production of pro‑inflammatory mediators, via the inhibition of extracellular‑signal‑regulated kinase (ERK)1/2, c‑Jun N‑terminal kinase (JNK), p38, as well as NF‑κB signaling in RAW264.7 cells stimulated with LPS.

The present investigation demonstrates a rapid biogenic approach for the synthesis of gold and silver nanoparticles using biologically active and medicinal important Perilla frutescens leaf extract as a reducing and stabilizing agent under ambient conditions. Gold and silver nanoparticles were first synthesized from Perilla frutescens leaf extract which was used as a vegetable and in traditional medicines for a long time in Korea, Japan, and China. The nanoparticles obtained were characterized by UV-vis spectroscopy, transmission electron microscopy, X-ray diffraction, and Fourier transform infrared spectroscopy. Surface plasmon resonance spectra of gold and silver nanoparticles were obtained at 540 and 430 nm and triangular and spherical shape respectively. TEM studies showed that the particle sizes of gold and silver nanoparticles ranges -50 nm and -40 nm respectively. X-ray diffraction studies confirm that the biosynthesized nanoparticles were crystalline gold and silver. Fourier transform infra-red spectroscopy revealed that biomolecules were involved in the synthesis and capping of the nanoparticles produced. XRD and EDX confirmed the formation of gold and silver nanoparticles. This is a simple, efficient and rapid method to synthesize gold and silver nanoparticles at room temperature without use of toxic chemicals. Obtained gold and silver nanoparticles can be used in various biomedical and biotechnological applications.

Unipolar depressive disorder may become one of the major leading causes of disease burden by 2030 according to the World Health Organization (WHO). Thus, the discovery of antidepressive foods is attractive and could have considerable impacts worldwide. We investigated the antidepressant-like effects of Perilla frutescens seed oil on adult male rats subjected to a forced swimming test (FST). Forty Sprague-Dawley rats were housed and fed various diets, including soybean oil-rich, eicosapentaenoic acid (EPA)-rich, and P. frutescens seed oil-rich diets for 6 weeks. After the dietary intervention, animals were tested using an FST and were sacrificed after the test. We analyzed the fatty acid profiles of red blood cells (RBCs) and the brain prefrontal cortex (PFC). Levels of brain-derived neurotrophic factor (BDNF), serotonin, and dopamine in the PFC were also determined. After the FST, the imipramine, EPA-rich, and P. frutescens seed oil-rich groups showed significant shorter immobility time and longer struggling time than the control group (p < 0.05). Levels of BDNF in the P. frutescens seed oil-rich group and levels of serotonin in the EPA-rich group were significantly (p < 0.05) higher than those of the control group. Moreover, the BDNF concentration in the PFC was significantly positively correlated with the struggling time. However, there were no significant differences in dopamine levels between the intervention groups and the control group. In conclusion, a P. frutescens seed oil-rich diet exhibited antidepressant-like properties through modulation of fatty acid profiles and BDNF expression in the brain during an FST.

This study was undertaken to optimize extraction using evolutionary operation-factorial (EVOP) design technique to elicit the antibacterial activity of Perilla frutescens var. acuta leaf against Staphylococcus aureus ATCC6538. Higher antibacterial activity was achieved at higher extraction temperature and over a longer extraction time. Antibacterial activity was not affected by differentiation of the ethanol concentration in the extraction solvent. The maximum antibacterial activity of ethanolic extract of P. frutescens leaf against S. aureus was obtained at 75 °C (R = -0.7904(**)) extraction temperature, 24 h (R = -0.7273(**)) extraction time, and 45% (R = -0.0635) ethanol concentration. The population of S. aureus was decreased from 7.535 log CFU/mL in the initial set to 4.865 log CFU/mL in the third set by EVOP factorial design technique, as well as to 2.600 log CFU/mL by extraction with ethyl acetate. Further, the ethyl acetate extract revealed the highest phenolic contents (111.3 ± 8.6 mg% of dry sample) as compared to the other extracts. Also, the scanning electronic microscopic study of the ethanolic extract of P. frutescens revealed potential detrimental effect on the morphology of S. aureus.

Objective: To find a method for improving the salt resistance of seeds and seedlings for Perilla Frutescens under NaCl stress, seed germination and physiological characteristics of P. frutescens seedlings were studied., Method: Several physiological indexes of P. frutescens seeds treated with different concentrations of Ca2+, 5-aminolevulinic acid (ALA), salicylic acid (SA) and spermidine (Spd) under NaCl stress like the germination vigor, germination rate, germination index and vigor index were measured. And other indexes like the biomass of the seedlings, the content of malondialdehyde (MDA) in leaves, the activities of superoxide (SOD), peroxidase (POD) and catalase (CAT) were also measured., Result: The germination of P. frutescens seeds under NaCl stress (100 mmol x L(-1)) was inhibited obviously. But after the treatment with Ca2+, ALA , SA and Spd, all germination indexes were increased. Ca2+ (10 mmol x L(-1)), ALA (100 mg x L(-1)), SA (50 mg x L(-1)) and Spd (0.25 mmol x L(-1)) could obviously alleviate the damage of salt stress to the seeds of P. frutescens. ALA (100 mg x L(-1)) significantly increased all indexes. The germination vigor was 65.3%, the germination rate was 89.7%, the germination index and vigor index were 15.2 and 0.1238, respectively. All treatments decreased the content of MDA in leaves. The activities of three enzymes including SOD, POD and CAT were all increased. ALA (100 mg x L(-1)) had the enzymes activity reach the maximum with 0.72, 6, 82 and 5.64 U x mg(-1), respectively., Conclusion: Ca2+ ALA , SA and Spd with appropriate concentration could significantly alleviate the damages to the seeds and seedlings of P. frutescens under NaCl stress and promote the salt resistance of the seeds and seedlings.

Background and Aims: Plants respond to the spatial and temporal heterogeneity of a resource supply. However, their responses will depend on intraspecific competition for resource acquisition. Although plants are subject to various intensities of intraspecific competition, most studies of resource heterogeneity have been carried out under a single density so that the effects of intraspecific competition on plant responses to resource heterogeneity are largely unknown., Methods: A growth experiment was performed to investigate plant responses to the temporal heterogeneity of water supply and nutrient levels under multiple plant densities. The annual plant Perilla frutescens was grown using different combinations of frequency of water supply, nutrient level and density, while providing the same total amount of water under all conditions. The effects of the treatments on biomass, allocation to roots and intensity of competition were analysed after 48 d., Key Results: Biomass and allocation to roots were larger under homogeneous than under heterogeneous water supply, and the effects of water heterogeneity were greater at high density than at low density. The effects of water heterogeneity were greater at high nutrient level than at low level for biomass, while the effects were greater at low nutrient level than high level for allocation to roots. Competition was severer under homogeneous than under heterogeneous water supply., Conclusions: Competition for water probably makes plants more sensitive to the water heterogeneity. In addition, the intensity of intraspecific competition can be affected by the temporal patterns of water supply. Because both resource heterogeneity and intraspecific competition affect resource acquisition and growth of plants, their interactive effects should be evaluated more carefully under future studies.

Objective: To study the effect of different organic amendments treated replant soil and to find the relations between the soil fungi community and the growth of Panax quinquefolium L. seedling., Methods: The soil was treated with bone meal and Perilla frutescens root meal, the rate of seedling emergence, incidence of rot root and root weight were investigated. The fungi community and the pathogen, Fusarium solani and Phytophthora cactorum population were counted by soil dilution plating technique., Results: The organic amendments especially Perilla frutescens root meal treated soil could reduce the population of Fusarium solani and Phytophthora cactorum in soil, thus lessen the root disease of continuous cropping Panax quinquefolium and increase survival of weedling., Conclusion: Adding suitable organic amendments in soil can change the soil fungi community and be beneficial to solving the replant problem of Panax quinquefolium.

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.

Myc-RP from Perilla frutescens and Delila from Antirrhinum majus, two plant basic helix-loop-helix transcription factors (bHLH TFs) involved in the flavonoid biosynthetic pathway, have been used for the improvement of transactivational properties by directed evolution. Through two rounds of DNA shuffling, Myc-RP variants with up to 70-fold increase in transcriptional activities have been identified using a yeast transactivation system. In a tobacco protoplast transient expression assay, one of the most improved variants, M2-1, also shows significant increase of transactivation. The majority of resulting mutations (approximately 53%) are localized in the acidic (activation) domains of the improved Myc-RP variants. In variant M2-1, three of the four mutations (L301P/N354D/S401F) are in the acidic domain. The fourth mutation (K157M) is localized to a helix within the N-terminal interaction domain. Combinatorial site-directed mutagenesis reveals that, while the acidic domain mutations contribute modestly to the increase in activity, the K157M substitution is responsible for 80% of the improvement observed in variant M2-1. The transactivation activity of the K157M/N354D double mutant is equal to that of M2-1. These results suggest that the interaction domain plays a critical role in transactivation of these bHLH TFs. Delila variants have also been screened for increased activities toward the Arabidopsis chalcone synthase (CHS) promoter, a pathway promoter that responds weakly to the bHLH TFs. Variants with increased activity on the CHS promoter, while maintaining wildtype-level activities on the naturally responsive dihydroflavonol reductase promoter, have been obtained. This study demonstrates that functional properties of TFs can be modified by directed evolution.

Background: Rosmarinic acid (RA) is a natural polyphenolic substance contained in many Lamiaceae herbs such as Perilla frutescens. Previous studies have shown RA has antioxidative and anti-inflammatory activity. However, little is known on the absorption, metabolism, degradation and excretion of RA., Aim of the Study: The aim of this study in healthy humans was to determine the absorption, metabolism, and urinary excretion of RA after a single intake of perilla extract (PE)., Method: Six healthy men (mean age 37.2 +/- 6.2 y and mean body mass index 22.0 +/- 1.9 kg/m(2)) were enrolled in the study that was a crossover design involving single intakes of PE containing 200 mg RA and placebo with a 10 day interval between treatments. Blood samples were collected before intake and at designated time intervals, while urine samples were collected over the periods 0-6 h, 6-24 h and 24-48 h after intake. RA and its related metabolites in plasma and urine were measured by LC-MS., Results: RA, methylated RA (methyl-RA), caffeic acid (CAA), ferulic acid (FA) and a trace of m-coumaric acid (COA) were detected in the urine after intake of PE. In plasma, RA, methyl-RA and FA were detected, with maximum levels obtained 0.5, 2 and 0.5 h after intake of PE, respectively. The majority of these components in both plasma and urine were present as conjugated forms (glucuronide and/or sulfated). The proportion of RA and its related metabolites excreted in the urine was 6.3 +/- 2.2% of the total dose, with approximately 75% of these components being excreted within 6 h after intake of PE., Conclusions: RA contained in PE was absorbed, conjugated and methylated following intake, with a small proportion of RA being degraded into various components, such as conjugated forms of CAA, FA and COA. These metabolites were then rapidly excreted in the urine.

We have investigated metabolite profiles and gene expression in two chemo-varietal forms, red and green forms, of Perilla frutescens var. crispa. Striking difference in anthocyanin content was observed between the red and green forms. Anthocyanin, mainly malonylshisonin, was highly accumulated in the leaves of the red form but not in the green form. Less obvious differences were also observed in the stems. However, there was no remarkable difference in the contents and patterns of flavones and primary metabolites such as inorganic anions, organic anions and amino acids. These results suggest that only the regulation of anthocyanin production, but not that of other metabolites, differs in red and green forms. Microscopic observation and immunohistochemical studies indicated that the epidermal cells of leaves and stems are the sites of accumulation of anthocyanins and localization of anthocyanidin synthase protein. By differential display of mRNA from the leaves of red and green forms, we could identify several genes encoding anthocyanin-biosynthetic enzymes and presumptive regulatory proteins. The possible regulatory network leading to differential anthocyanin accumulation in a form-specific manner is discussed.