PfbZIP85 Transcription Factor Mediates ω-3 Fatty Acid-Enriched Oil Biosynthesis by Down-Regulating PfLPAT1B Gene Expression in Plant Tissues.

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.

Tobacco introduced Perilla frutescens and Ocimum basilicum genes attenuates neutrophilic inflammation in lung tissues of COPD rats.

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.

Genome-Wide Analysis of Glycerol-3-Phosphate Acyltransferase (GPAT) Family in Perilla frutescens and Functional Characterization of PfGPAT9 Crucial for Biosynthesis of Storage Oils Rich in High-Value Lipids.

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.

Genome-wide comprehensive characterization and transcriptomic analysis of AP2/ERF gene family revealed its role in seed oil and ALA formation in perilla (Perilla frutescens).

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.

Overexpression of the Purple Perilla (Perilla frutescens (L.)) FAD3a Gene Enhances Salt Tolerance in Soybean.

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, H2O2, 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.

The revealing of a novel double bond reductase related to perilla ketone biosynthesis in Perilla frutescens.

BACKGROUND: Perilla frutescens is widely used as both a medicine and a food worldwide. Its volatile oils are its active ingredients, and, based on the different volatile constituents, P. frutescens can be divided into several chemotypes, with perilla ketone (PK) being the most common. However, the key genes involved in PK biosynthesis have not yet been identified. RESULTS: In this study, metabolite constituents and transcriptomic data were compared in leaves of different levels. The variation in PK levels was the opposite of that of isoegoma ketone and egoma ketone in leaves at different levels. Based on transcriptome data, eight candidate genes were identified and successfully expressed in a prokaryotic system. Sequence analysis revealed them to be double bond reductases (PfDBRs), which are members of the NADPH-dependent, medium-chain dehydrogenase/reductase (MDR) superfamily. They catalyze the conversion of isoegoma ketone and egoma ketone into PK in in vitro enzymatic assays. PfDBRs also showed activity on pulegone, 3-nonen-2-one, and 4-hydroxybenzalacetone. In addition, several genes and transcription factors were predicted to be associated with monoterpenoid biosynthesis, and their expression profiles were positively correlated with variations in PK abundance, suggesting their potential functions in PK biosynthesis. CONCLUSIONS: The eight candidate genes encoding a novel double bond reductase related to perilla ketone biosynthesis were identified in P. frutescens, which carries similar sequences and molecular features as the MpPR and NtPR from Nepeta tenuifolia and Mentha piperita, respectively. These findings not only reveal the pivotal roles of PfDBR in exploring and interpreting PK biological pathway but also contribute to facilitating future studies on this DBR protein family.

Comparative transcriptome sequencing analysis to postulate the scheme of regulated leaf coloration in Perilla frutescens.

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.

Sphaerisporangium perillae sp. nov., isolated from the root of Perilla frutescens (Linn.) Britt.

A novel protease-producing actinomycete, designated strain NEAU-ZS1T, was isolated from the root of Perilla frutescens (Linn.) Britt collected from Jiamusi, Heilongjiang, PR China. Comparative 16S rRNA gene sequencing showed that strain NEAU-ZS1T belonged to the genus Sphaerisporangium and was most closely related to 'Sphaerisporangium corydalis' NEAU-YHS15T (99.2%) and Sphaerisporangium cinnabarinum JCM 3291T (99.0%). Phylogenetic tree analysis revealed that strain NEAU-ZS1T formed a monophyletic clade with 'S. corydalis' NEAU-YHS15T. The genome size was 9.3 Mbp with a DNA G+C content of 70.3 mol%. Digital DNA-DNA hybridization, average nucleotide identity and average amino acid identity values between the genome sequence of strain NEAU-ZS1T and those of 'S. corydalis' NEAU-YHS15T (28.6, 83.9 and 79.1 %) and S. cinnabarinum JCM 3291T (18.5, 70.6 and 50.2 %) were below the recommended thresholds for species delineation. The strain formed spherical spore vesicles produced on the aerial hyphae. The cell wall contained meso-diaminopimelic acid and the whole-cell sugars were glucose and madurose. The polar lipids consisted of diphosphatidylglycerol, phosphatidylmethylethanolamine, phosphatidylethanolamine, phosphatidylinositol, an unidentified phospholipid and an unidentified glycolipid. The menaquinones were MK-9(H4), MK-9(H6) and MK-9(H2). The major fatty acids were iso-C16 : 0, C16 : 1 ω5c, 10-methy C17 : 0 and C17 : 1 ω7c. On the basis of the results of a polyphasic taxonomic study, it is concluded that strain NEAU-ZS1T represents a novel species of the genus Sphaerisporangium, for which the name Sphaerisporangium perillae sp. nov. is proposed. The type strain is NEAU-ZS1T (=CCTCC AA 2021019T= JCM 35655T).

Leaf Extract of Perilla frutescens (L.) Britt Promotes Adipocyte Browning via the p38 MAPK Pathway and PI3K-AKT Pathway.

The leaf of Perilla frutescens (L.) Britt (PF) has been reported to negatively affect adipocyte formation, inhibit body-fat formation, and lower body weight. However, its effect on adipocyte browning remains unknown. Thus, the mechanism of PF in promoting adipocyte browning was investigated. The ingredients of PF were acquired from the online database and filtered with oral bioavailability and drug-likeness criteria. The browning-related target genes were obtained from the Gene Card database. A Venn diagram was employed to obtain the overlapped genes that may play a part in PF promoting adipocyte browning, and an enrichment was analysis conducted based on these overlapped genes. A total of 17 active ingredients of PF were filtered, which may regulate intracellular receptor-signaling pathways, the activation of protein kinase activity, and other pathways through 56 targets. In vitro validation showed that PF promotes mitochondrial biogenesis and upregulates brite adipocyte-related gene expression. The browning effect of PF can be mediated by the p38 MAPK pathway as well as PI3K-AKT pathway. The study revealed that PF could promote adipocyte browning through multitargets and multipathways. An in vitro study validated that the browning effect of PF can be mediated by both the P38 MAPK pathway and the PI3K-AKT pathway.

A highly contiguous genome assembly of red perilla (Perilla frutescens) domesticated in Japan.

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.

[Cloning and functional characterization of a lysophosphatidic acid acyltransferase gene from Perilla frutescens].

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.

Genome-wide analysis of DGAT gene family in Perilla frutescens and functional characterization of PfDGAT2-2 and PfDGAT3-1 in Arabidopsis.

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.

Genetic variation of seed oil characteristics in native Korean germplasm of Perilla crop (Perilla frutescens L.) using SSR markers.

BACKGROUND: In order to maximize the use of valuable native Perilla germplasm in South Korea, knowledge of the Perilla seed oil content and genetic variation among native Perilla germplasm resources is very important for the conservation and development of new Perilla seed oil varieties using the native Perilla germplasm accessions preserved from the Rural Development Administration Genebank (RDA-Genebank) collection from South Korea. OBJECTIVES: In this study, we studied population structure and association mapping to identify Perilla SSR markers (PSMs) associated with the five fatty acid contents and two seed characteristics of the native Korean Perilla germplasm accessions of cultivated var. frutescens of the RDA-Genebank collected in South Korea. METHODS: For an association mapping analysis to find PSMs associated with the five fatty acid contents and two seed characteristics of the Perilla germplasm accessions of cultivated var. frutescens, we evaluated the content of five fatty acids of 280 native Korean Perilla germplasm accessions and used 29 Perilla SSR primer sets to measure the genetic diversity and relationships, population structure, and association mapping of the native Korean Perilla germplasm accessions of the RDA-Genebank collected in South Korea. RESULTS: Five fatty acids of 280 native Korean Perilla accessions were identified as follows: palmitic acid (PA) (5.30-8.66%), stearic acid (SA) (1.60-4.19%), oleic acid (OA) (9.60-22.5%), linoleic acid (LA) (8.38-25.4%), and linolenic acid (LNA) (52.7-76.4%). In a correlation analysis among the five fatty acids and two seed characteristics of the 280 Perilla accessions, the combinations of PA and SA (0.794**) and SA and OA (0.724**) showed a particularly high positive correlation coefficients compare to other combinations. By using an association analysis of the 29 PSMs and the five fatty acids in the 280 Perilla accessions, we found 17 PSMs (KNUPF1, KNUPF2, KNUPF4, KNUPF10, KNUPF16, KNUPF25, KNUPF26, KNUPF28, KNUPF37, KNUPF55, KNUPF62, KNUPF71, KNUPF74, KNUPF77, KNUPF85, KNUPF89, and KNUPF118) associated with the content of the five fatty acid components and two seed characteristics. CONCLUSIONS: These PSMs are considered to be useful molecular markers related to five fatty acid components and two seed characteristics for selecting accessions from the germplasm accessions of the Perilla crop and their related weedy types through association mapping analysis and marker-assisted selection (MAS) breeding programs.

Analysis of genetic diversity and relationships of Perilla frutescens using novel EST-SSR markers derived from transcriptome between wild-type and mutant Perilla.

BACKGROUND: Perilla frutescens (Lamiaceae) is distributed in East Asia and is classified into var. frutescens and crispa. P. frutescens is multipurpose crop for human health because of a variety of secondary metabolites such as phenolic compound and essential oil. However, a lack of genetic information has hindered the development and utilization of Perilla genotypes. METHODS AND RESULTS: This study was performed to develop expressed sequence tag-simple sequence repeat (EST-SSR) markers from P. frutescens var. crispa (wild type) and Antisperill (a mutant cultivar) and used them to assess the genetic diversity of, and relationships among, 94 P. frutescens genotypes. We obtained 65 Gb of sequence data comprising 632,970 transcripts by de novo RNA-sequencing. Of the 14,780 common SSRs, 102 polymorphic EST-SSRs were selected using in silico polymerase chain reaction (PCR). Overall, successful amplification from 58 EST-SSRs markers revealed remarkable genetic diversity and relationships among 94 P. frutescens genotypes. In total, 268 alleles were identified, with an average of 4.62 alleles per locus (range 2-11 alleles/locus). The average polymorphism information content (PIC) value was 0.50 (range 0.04-0.86). In phylogenetic and population structure analyses, the genotypes formed two major groups: Group I (var. crispa) and Group II (var. frutescens). CONCLUSION: This results suggest that 58 novel EST-SSR markers derived from wild-type cultivar (var. crispa) and its mutant cultivar (Antisperill) have potential uses for population genetics and recombinant inbred line mapping analyses, which will provide comprehensive insights into the genetic diversity and relationship of P. frutescens.

Multi-omics analysis of the bioactive constituents biosynthesis of glandular trichome in Perilla frutescens.

BACKGROUND: Perilla frutescens (L.) Britt is a medicinal and edible plant widely cultivated in Asia. Terpenoids, flavonoids and phenolic acids are the primary source of medicinal ingredients. Glandular trichomes with multicellular structures are known as biochemical cell factories which synthesized specialized metabolites. However, there is currently limited information regarding the site and mechanism of biosynthesis of these constituents in P. frutescens. Herein, we studied morphological features of glandular trichomes, metabolic profiling and transcriptomes through different tissues. RESULTS: Observation of light microscopy and scanning electron microscopy indicated the presence of three distinct glandular trichome types based on their morphological features: peltate, capitate, and digitiform glandular trichomes. The oil of peltate glandular trichomes, collected by custom-made micropipettes and analyzed by LC-MS and GC-MS, contained perillaketone, isoegomaketone, and egomaketone as the major constituents which are consistent with the components of leaves. Metabolomics and transcriptomics were applied to explore the bioactive constituent biosynthesis in the leaves, stem, and root of P. frutescens. Transcriptome sequencing profiles revealed differential regulation of genes related to terpenoids, flavonoids, and phenylpropanoid biosynthesis, respectively with most genes expressed highly in leaves. The genes affecting the development of trichomes were preliminarily predicted and discussed. CONCLUSIONS: The current study established the morphological and chemical characteristics of glandular trichome types of P. frutescens implying the bioactive constituents were mainly synthesized in peltate glandular trichomes. The genes related to bioactive constituents biosynthesis were explored via transcriptomes, which provided the basis for unraveling the biosynthesis of bioactive constituents in this popular medicinal plant.

Bulk segregant analysis identifies SSR markers associated with leaf- and seed-related traits in Perilla crop (Perilla frutescens L.).

BACKGROUND: Bulk segregant analysis (BSA) is another method of identifying significant molecular markers linked to the target gene or region for specific traits. BSA is easier and less expensive than other methods; it does not require genetic map construction and needs fewer markers than the number needed to construct a genetic map for QTL mapping. OBJECTIVES: The purpose of our study was to identify simple sequence repeat (SSR) markers linked with leaf- and seed-related traits in Perilla crop, and to allow the selection of better accessions in Perilla breeding programs with marker-assisted selection (MAS). METHODS: The genotypes of the 25 SSR markers and phenotypic data for the eight qualitative traits were used to confirm significant marker-trait associations (SMTAs) using TASSEL software. To detect SSR markers associated with leaf color, the 16 individuals of the F3 population were divided into three bulk groups based on the colors of the surface and reverse sides of the leaf, respectively: six in the green/green group, five in the green/purple group and five in the purple/purple group. RESULTS: This study detected 18 significant marker-trait associations (SMTAs) involving 12 SSR markers associated with six agronomic traits. The SSR markers KNUPF15, KNUPF21, KNUPF29, and KNUPF60 were associated with leaf surface color, and KNUPF11, KNUPF15, KNUPF21, and KNUPF60 were associated with leaf reverse side color. In addition, five SSR markers were associated with seed-related traits. KNUPF11 and KNUPF29 were associated with seed coat color, while KNUPF29 was associated with seed size. KNUPF12, KNUPF16, and KNUPF42 were associated with seed hardness. To verify the selected significant SSR markers associated with leaf color and seed-related traits, a UPGMA dendrogram for 11 individuals in the F3 population, which formed two bulk groups consisting of 6 green/green and 5 purple/purple individuals, was constructed using six SSR marker-related LC and RLC traits. CONCLUSION: These results are very important for understanding the characteristics of Perilla leaves and seeds; they may also support opportunities to effectively preserve and utilize existing accessions and to allow Perilla breeders to improve crop quality by mean of MAS.

Integrated transcriptomic and metabolomic data reveal the flavonoid biosynthesis metabolic pathway in Perilla frutescens (L.) leaves.

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.

Assessment of genetic diversity and population structure among a collection of Korean Perilla germplasms based on SSR markers.

BACKGROUND: Information on the genetic variation of genetic resource collections is very important for both the conservation and utilization of crop germplasms in genebanks. Var. frutescens of Perilla crop is extensively cultivated in South Korea as both an oil crop and a vegetable crop. OBJECTIVES: We used SSR markers to evaluate the genetic diversity, genetic relationships, and population structure of 155 accessions of var. frutescens that have been selected as genetic resources for the development of leaf vegetable cultivars and preserved in the RDA-Genebank collection from South Korea. METHODS: A total of 155 accessions of var. frutescens of Perilla crop collected in South Korea were obtained from the RDA-Genebank of the Republic of Korea. We selected 20 SSR markers representing the polymorphism of and adequately amplifying all the Perilla accessions. RESULTS: The average GD and PIC values were 0.642 and 0.592, respectively, with ranges of 0.244-0.935 and 0.232- 0.931. The genetic variability in the southern region of South Korea was higher than that in the central region. The clustering patterns were not clearly distinguished between the accessions of var. frutescens from the central and southern regions of South Korea. CONCLUSION: These results regarding the genetic diversity and population structure of the 155 accessions of var. frutescens of South Korea provide useful information for understanding the genetic variability of this crop and selecting and managing core germplasm sets in the RDA-Genebank of the Republic of Korea.

Anti-Arthritic Activities of Supercritical Carbon Dioxide Extract Derived from Radiation Mutant Perilla Frutescens Var. Crispa in Collagen Antibody-Induced Arthritis.

We investigated the anti-arthritic effects of the radiation mutant Perilla frutescens var. crispa leaf extract (SFE-M) and wild type leaf extract (SFE-W), both prepared by supercritical carbon dioxide (SC-CO2) extraction, on collagen antibody-induced arthritis (CAIA) in Balb/c mice. Animals were randomly divided into four groups: control, CAIA, CAIA + SFE-M (100 mg/kg/day), and CAIA + SFE-W (100 mg/kg/day). The mice were subjected to the respective treatments via oral gavage once daily for 4 days. Mice treated with SFE-M developed less severe arthritis than the CAIA mice. They showed significantly improved arthritic score, paw volume, and paw thickness compared to the CAIA mice from days 3 through 7. Furthermore, histopathological analysis of ankle for inflammation showed that SFE-M treatment reduced inflammatory cell infiltration and edema formation. Similarly, the neutrophil-to-lymphocyte ratio (NLR) in the whole blood was 37% lower in mice treated with SFE-M compared with the CAIA mice. However, treatment with SFE-W did not result in any significant difference compared with the CAIA group. In conclusion, SFE-M treatment delays the onset of arthritis and alleviates its clinical manifestations in CAIA mice.

Enzymatic Synthesis of Bioactive O-Glucuronides Using Plant Glucuronosyltransferases.

Many O-glucuronides exhibiting various pharmacological activities have been found in nature and in drug metabolism. The glucuronidation of bioactive natural products or drugs to generate glucuronides with better activity and druggability is important in drug discovery and research. In this study, by using two uridine diphosphate (UDP)-dependent glucuronosyltransferases (GATs, UGT88D4 and UGT88D7) from plants, we developed two glucuronidation approaches, pure enzyme catalysis in vitro and recombinant whole-cell catalysis in vivo, to efficiently synthesize bioactive O-glucuronides by the glucuronidation of natural products. In total, 14 O-glucuronides with different structures, including flavonoids, anthraquinones, coumarins, and lignans, were obtained, 7 of which were new compounds. Furthermore, one of the biosynthesized O-glucuronides, kaempferol-7- O-ß-d-glucuronide (3a), potently inhibited protein tyrosine phosphatase (PTP) 1B with an IC50 value of 8.02 × 10-6 M. Some of the biosynthesized O-glucuronides also exhibited significant antioxidant activities.