Based on whole-genome identification of the TPS gene family in Perilla frutescens and screening, cloning, bioinformatics, and expression analysis of the synthetic enzyme for the insect-resistant component germacrene D, this study lays the foundation for understanding the biological function of the TPS gene family and the insect resistance mechanism in P. frutescens. This study used bioinformatics tools to identify the TPS gene family of P. frutescens based on its whole genome and predicted the physicochemical properties, systematic classification, and promoter cis-elements of the proteins. The relative content of germacrene D was detected in both normal and insect-infested leaves of P. frutescens, and the germacrene D synthase was screened and isolated. Gene cloning, bioinformatics analysis, and expression profiling were then performed. The results showed that a total of 99 TPS genes were identified in the genome, which were classified into the TPS-a, TPS-b, TPS-c, TPS-e/f, and TPS-g subfamilies. Conserved motif analysis showed that the TPS in P. frutescens has conserved structural characteristics within the same subfamily. Promoter cis-element analysis predicted the presence of light-responsive elements, multiple hormone-responsive elements, and stress-responsive elements in the TPS family of P. frutescens. Transcriptome data revealed that most of the TPS genes in P. frutescens were highly expressed in the leaves. GC-MS analysis showed that the relative content of germacrene D significantly increased in insect-damaged leaves, suggesting that it may act as an insect-resistant component. The germacrene D synthase gene was screened through homologous protein binding gene expression and was found to belong to the TPS-a subfamily, encoding a 64.89 kDa protein. This protein was hydrophilic, lacked a transmembrane structure and signal peptide, and was predominantly expressed in leaves, with significantly higher expression in insect-damaged leaves compared to normal leaves. In vitro expression results showed that germacrene D synthase tended to form inclusion bodies. Molecular docking showed that farnesyl pyrophosphate(FPP) fell into the active pocket of the protein and interacted strongly with six active sites. This study provides a foundation for further research on the biological functions of the TPS gene family in P. frutescens and the molecular mechanisms underlying its insect resistance.
Perilla frutescens/chemistry* ; Plant Proteins/chemistry* ; Multigene Family ; Sesquiterpenes, Germacrane/metabolism* ; Alkyl and Aryl Transferases/chemistry* ; Phylogeny ; Gene Expression Regulation, Plant

Perilla frutescens (L.) Britt. is a characteristic oil crop rich in polyunsaturated fatty acids, particularly α-linolenic acid, which has important development and utilization value. The Dof transcription factor is one of the plant-specific transcription factor families, which is widely involved in important biological processes such as plant growth, development, and metabolic regulation. In order to explore the key Dof transcription factors involved in the oil biosynthesis and systematically analyze their regulatory mechanisms of P. frutescens seeds, a total of 56 PfDof gene family members were identified from the genome and transcriptome data of P. frutescens and classified into four subfamilies according to sequence characteristics. All PfDofs contained highly conserved C₂-C₂ zinc finger domains, with gene duplication being the primary mechanism driving their evolution and expansion. Genes within the same subgroup exhibited similar gene structures and conserved motifs. The 56 PfDofs were predicted as unstable hydrophilic proteins, with α-helixes and random coils as their predominant structural components. The RNA-seq results revealed that 11 PfDofs exhibited differential expression during different developmental stages of P. frutescens seeds. RT-qPCR was performed to further validate the expression patterns of these 11 members across various tissue samples (root, stem, leaf, and flower) of P. frutescens and at different developmental stages of its seeds. The results showed that PfDof29 exhibited the highest expression level in seeds, which was consistent with the transcriptome data. Subcellular localization studies demonstrated that PfDof29 was localized to the nucleus and had a transcriptional activation activity. Overexpression of PfDof29 in Nicotiana tabacum resulted in a significant increase in total oil content of tobacco leaves, accompanied by reductions in starch and soluble sugar content, while the protein content remained unchanged. Additionally, the metabolic balance between saturated and unsaturated fatty acids in the transgenic tobacco leaves was altered, with a significant increase in α-linolenic acid content. The expression levels of the fatty acid desaturase genes NtFAD2, NtFAD3, and NtFAD8 were significantly upregulated. A yeast one-hybrid assay revealed that PfDof29 could directly bind to the promoter region of PfFAD8, thereby regulating its expression. This study provides an initial understanding of the regulatory mechanisms of PfDof transcription factors in the synthesis and accumulation of oil in P. frutescens. These findings offer new insights into the enhancement of oil content and quality of P. frutescens seeds.
Transcription Factors/physiology* ; Perilla frutescens/metabolism* ; Plant Proteins/metabolism* ; Gene Expression Regulation, Plant ; alpha-Linolenic Acid/biosynthesis* ; Lipids/biosynthesis* ; Seeds/genetics*

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.
Acyltransferases ; Cloning, Molecular ; Escherichia coli/metabolism* ; Fatty Acids ; Perilla frutescens/metabolism* ; Plant Oils ; Plant Proteins/metabolism* ; Saccharomyces cerevisiae/metabolism* ; Seeds/chemistry* ; Tobacco/genetics*

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.
Animals ; Humans ; Oils, Volatile ; analysis ; pharmacology ; toxicity ; Perilla frutescens ; chemistry ; classification ; metabolism ; Plant Leaves ; chemistry

OBJECTIVETo 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.

METHODSeveral 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.

RESULTThe 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.

CONCLUSIONCa2+ 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.

Aminolevulinic Acid ; pharmacology ; Calcium ; pharmacology ; Catalase ; metabolism ; Dose-Response Relationship, Drug ; Germination ; drug effects ; Malondialdehyde ; metabolism ; Perilla frutescens ; drug effects ; enzymology ; metabolism ; physiology ; Peroxidase ; metabolism ; Salicylic Acid ; pharmacology ; Seedlings ; drug effects ; enzymology ; metabolism ; physiology ; Sodium Chloride ; pharmacology ; Spermidine ; pharmacology ; Stress, Physiological ; drug effects ; Superoxide Dismutase ; metabolism

OBJECTIVETo explore the method for improving the aging resistance of seeds and seedlings of Perilla frutescens through study on seed germination and physiological characteristics of P. frutescens seedlings.

METHODSeveral physiological indexes of P. frutescens seeds treated by different concentrations of ZnSO4 and PEG were measured. And other indexes like the activities of malondialdehyde (MDA), superoxide (SOD), peroxidase (POD) and catalase (CAT) were also determined.

RESULTThe germination indexes of P. frutescens aging seeds treated by different concentrations of ZnSO4 and PEG were all increased. And the seeds that treated by ZnSO4 (600 mg x L(-1)) and PEG (20%) showed the most significantly increase in every index. The germination vigor were 64.7% and 66.8%, the germination rate were 78.7% and 79.4%, the germination index were 11.8 and 12.2, the vigor index were 0.091 1 and 0.0939 respectively. The content of MDA was decreased under different treatment. The activities of three enzymes include SOD, POD and CAT were increased by different treatment of ZnSO4 (0.28, 4.71, 3.82 U x mg(-1) respectively) and PEG (0.29, 4.93, 4.18 U x mg(-1) respectively).

CONCLUSIONZnSO4 with concentration of 600 mg x L(-1) and PEG with concentration of 20% could significantly alleviate the damages to the seeds and seedlings of P. frutescens by aging and promote the aging resistance of the seeds and seedlings.

Catalase ; metabolism ; Germination ; drug effects ; Malondialdehyde ; metabolism ; Perilla frutescens ; drug effects ; enzymology ; physiology ; Peroxidase ; metabolism ; Peroxidases ; metabolism ; Plant Proteins ; metabolism ; Polyethylene Glycols ; pharmacology ; Seedlings ; enzymology ; Seeds ; enzymology ; physiology ; Zinc Sulfate ; pharmacology