The synthesis and accumulation of active ingredients in medicinal plants are distributed in specific organs, tissues, and cell types, which are important for the exploitation of medicinal plants. However, the fine distribution of active ingredients is difficult to know. Here, the system of mass spectrometry imaging (MSI) integrated with single-cell RNA sequencing was established for the first time in Andrographis paniculata (A. paniculata), a medicinal plant widely utilised in China and Southeast Asia. MSI shows specific distribution of andrographolides in A. paniculata, with higher accumulation in non-veinal leaf regions and outer stem cortex (leaf > stem; outer > inner cortex), as validated by LC-QQQ-MS/MS assays. Leaf scRNA-seq demonstrates that ApCPS2 (the key terpene synthase for andrographolide biosynthesis) exhibits pronounced cell-type-specific expression in photosynthetic mesophyll subclusters, indicating mesophyll cells as the primary site for light-modulated andrographolide production. Interestingly, light may enhance the accumulation of andrographolide biosynthesis, confirming the light sensitivity of metabolism in mesophyll cells. This study explores medicinal components' multidimensional spatial distributions and biosynthetic pathways in A. paniculata via MSI combined with single-cell technology, providing a novel strategy for determining plant metabolites' fine synthesis and distribution.

Patchouli is a medicinal plant belonging to the Pogostemon genus within the Lamiaceae family. It is composed mainly of sesquiterpenoid metabolites, and has various pharmacological effects. The mechanism underlying the transcriptional regulation of the patchouli alcohol has been reported, but the mechanism underlying the regulation of transcription factors (TFs) involved in the sesquiterpenoid metabolic pathway is still unknown. In this study, a regulatory network was constructed for the sesquiterpenoid metabolic pathway in patchouli by using a gene expression database and WGCNA. It was found that 286 TFs have potential regulatory relationships with 28 genes that encode enzymes in the sesquiterpenoid metabolic pathway. The regulatory mechanism of this network was successfully verified by yeast one-hybrid (Y1H), dual luciferase reporter assay (dual-LUC), transient overexpression, and electrophoretic mobility shift assay (EMSA). In conclusion, a regulation network was constructed for sesquiterpenoid metabolic pathways in Pogostemon cablin, and all the TFs that may have regulatory functions in sesquiterpenoid metabolic pathways were identified, and the regulatiory functions of transcription factors were successfully verified through bench experiments.

Andrographis paniculata (Acanthaceae) produces andrographolide (AD), a diterpene compound with anti-inflammatory and antimicrobial activities. A. paniculata primarily occurs in tropical and subtropical regions characterized by high temperatures. However, the effects of temperature on A. paniculata development and metabolism, and the underlying regulatory mechanisms remain poorly understood. In this study, we demonstrate that warm temperature promotes plant growth and AD accumulation in A. paniculata. Metabolomic and transcriptomic analyses revealed that the transcription factors ApMYC2 and ApHSFB2b are strongly associated with AD production, and these transcription factors form a complex to regulate gene expression. Warm temperatures enhance jasmonic acid (JA) accumulation, which activates expression of the copalyl-diphosphate synthase gene ApCPS1, encoding a key enzyme in AD biosynthesis, through the ApMYC2-ApHSFB2b regulatory module. Furthermore, the jasmonate ZIM domain (JAZ) JA signaling repressors ApJAZ1 and ApJAZ9 interact with ApMYC2, thereby inhibiting activation of ApCPS1 mediated by ApMYC2 and ApHSFB2b. Our findings elucidate a temperature-dependent mechanism regulating AD biosynthesis in A. paniculata, mediated by JA signaling through the ApMYC2-ApHSFB2b module, providing critical insights into how temperature regulates terpenoid metabolism in plants.

Patchoulol, a primary component of patchouli oil which is extensively utilized in the fields of medicine, flavoring, and fragrances. Notably, previous research has indicated that light conditions significantly influence the accumulation of patchoulol in patchouli plants. In this study, we further investigated the hormone levels in patchouli under various light treatments, revealing a strong correlation between jasmonates and patchoulol biosynthesis. Both patchoulol and jasmonate biosynthesis were found to be light-dependent, exhibiting wavelength-specific responses within certain ranges showing similar trends. Additionally, gene expression analysis under light and dark conditions identified several candidate genes within the light and jasmonates signaling pathways. These findings provide a theoretical foundation for understanding the light-induced mechanisms of patchoulol biosynthesis and offer insights for advancing the patchouli industry.

Patchouli alcohol, a significant bioactive component of the herbal plant Pogostemon cablin, has considerable medicinal and commercial potential. Several genes and transcription factors involved in the biosynthesis pathway of patchouli alcohol have been identified. However, so far, regulatory factors directly interacting with patchouli synthase (PTS) have not been reported. This study was conducted to analyze the interaction between PcENO3 and PcPTS to explore the molecular regulation effect of PcENO3 on patchouli alcohol biosynthesis. PcENO3, a homologous protein of Arabidopsis ENO3 belonging to the enolase family, was identified and characterized. Subcellular localization experiments in Arabidopsis protoplast cells indicated that the PcENO3 protein was localized in both the cytoplasm and nucleus. The physical interaction between PcENO3 and PcPTS was confirmed through yeast two-hybrid (Y2H), GST pull-down, and bimolecular fluorescence complementation assays. Furthermore, the Y2H assay demonstrated that PcENO3 could also interact with JAZ proteins in the JA pathway. Enzymatic assays showed that the interaction with PcENO3 increased the catalytic activity of patchoulol synthase. Additionally, suppression of PcENO3 expression with VIGS (virus-induced gene silencing) decreased patchouli alcohol content compared to the control. These findings suggest that PcENO3 interacts with patchoulol synthase and modulates patchoulol biosynthesis by enhancing the enzymatic activity of PcPTS.

Farnesyl diphosphate synthase(FPPS) is a key enzyme at the branch point of the sesquiterpene biosynthetic pathway, but there are no reports on the transcriptional regulation of FPPS promoter in Pogostemon cabin. In the early stage of this study, we obtained the binding protein PcFBA-1 of FPPS gene promoter in P. cabin. In order to explore the possible mechanism of PcFBA-1 involved in the regulation of patchouli alcohol biosynthesis, this study performed PCR-based cloning and sequencing analysis of PcFBA-1, analyzed the expression patterns of PcFBA-1 in different tissues by fluorescence quantitative PCR and its subcellular localization using the protoplast transformation system, detected the binding of PcFBA-1 protein to the FPPS promoter in vitro with the yeast one-hybrid system, and verified its transcriptional regulatory function by dual-luciferase reporter gene assay. The findings demonstrated that the cloned PcFBA-1 had an open reading frame(ORF) of 1 131 bp, encoding a protein of 376 amino acids, containing two conserved domains named F-box-like superfamily and FBA-1 superfamily, and belonging to the F-box family. Moreover, neither signal peptide nor transmembrane domain was contained, implying that it was an unstable hydrophilic protein. In addition, as revealed by fluorescence quantitative PCR results, PcFBA-1 had the highest expression in leaves, and there was no significant difference in expression in roots or stems. PcFBA-1 protein was proved mainly located in the cytoplasm. Furthermore, yeast one-hybrid screening and dual-luciferase reporter gene assay showed that PcFBA-1 was able to bind to FPPS promoter both in vitro and in vivo to enhance the activity of FPPS promoter. In summary, this study identifies a new transcription factor PcFBA-1 in P. cabin, which directly binds to the FPPS gene promoter to enhance the promoter activity. This had laid a foundation for the biosynthesis of patchouli alcohol and other active ingre-dients and provided a basis for metabolic engineering and genetic improvement of P. cabin.

The production of patchoulol in the patchouli (Pogostemon cablin) plant determines its application value, as it is the principal active sesquiterpene of essential oil extracted from this plant. Here, the promoter of patchoulol synthase gene (PatPTSpro) was isolated and found to be methyl jasmonate (MeJA)-induced. A nucleus-localized AP2/ERF transcription factor PatDREB was identified as a transcription activator binding to PatPTSpro, regulating patchoulol biosynthesis through modulating the gene expression. PatDREB also interacts with jasmonate ZIM-domain 4 (JAZ4). Furthermore, PatDREB could physically interact with the MYB-related transcription factor PatSWC4 and synergistically facilitate patchoulol biosynthesis. However, the transcriptional activation activity of the PatDREB-PatSWC4 complex could be inhibited by PatJAZ4, and JA could reverse this interference. Overall, we demonstrated the positive roles of PatDREB and the PatDREB-PatSWC4 complex in regulating patchoulol production, which advance our understanding of the regulatory network of patchoulol biosynthesis.

目的 从广藿香Pogostemon cablin中克隆HAD(haloacid dehalogenase)超级家族蛋白水解酶基因PcHAD,验证其编码蛋白与广藿香醇合酶(patchoulol synthase,PcPTS)蛋白互作,探究其参与调控广藿香醇生物合成的作用.方法 从广藿香转录组数据中,筛选出一条具有完整编码区并且包含HAD-like结构域的HAD基因序列(PcHAD),并对该基因进行克隆和生物信息学分析.利用qRT-PCR对PcHAD的表达模式进行分析.利用酵母双杂交实验进一步验证PcHAD与广藿香PcPTS蛋白的互作情况.通过瞬时过表达PcHAD,分析其对广藿香醇甲羟戊酸合成途径(mevalonate pathway,MVA)相关基因的表达以及对广藿香醇含量的影响.结果 从广藿香中克隆得到HAD-like基因PcHAD,其开放阅读框为1149 bp,编码蛋白长度为382个氨基酸,蛋白相对分子质量为43 000,且为不稳定的亲水性蛋白,亚细胞定位结果表明该蛋白定位于叶绿体.酵母双杂交实验结果表明PcHAD完整蛋白能与PcPTS完整蛋白互作,且PcHADN与PcPTSN互作.与对照组相比,瞬时过表达PcH4D广藿香叶中的广藿香醇含量提高了 30％,MVA途径基因包含PTS、FPPS和HMGR在内表达量均显著上调.结论 从广藿香中克隆得到1个与广藿香醇合酶PcPTS互作的PcHAD基因,该基因在瞬时过表达后上调PTS和MVA途径多个基因的表达量,且能够显著提.高广藿香醇的含量,表明该基因可能在广藿香醇生物合成中发挥正向调控作用,为进一步揭示广藿香中HAD-PTS复合体的功能提供科学依据.

Farnesyl pyrophosphate synthase (FPPS) plays an important role in the synthesis of plant secondary metabolites, but its function and molecular regulation mechanism remain unclear in Pogostemon cablin. In this study, the full-length cDNA of the FPP synthase gene from P. cablin (PcFPPS) was cloned and characterized. The expressions of PcFPPS are different among different tissues (highly in P. cablin flowers). Subcellular localization analysis in protoplasts indicated that PcFPPS was located in the cytoplasm. PcFPPS functionally complemented the lethal FPPS deletion mutation in yeast CC25. Transient overexpression of PcFPPS in P. cablin leaves accelerated terpene biosynthesis, with an ~47% increase in patchouli alcohol. Heterologous overexpression of PcFPPS in tobacco plants was achieved, and it was found that the FPP enzyme activity was significantly up-regulated in transgenic tobacco by ELISA analysis. In addition, more terpenoid metabolites, including stigmasterol, phytol, and neophytadiene were detected compared with control by GC-MS analysis. Furthermore, with dual-LUC assay and yeast one-hybrid screening, we found 220 bp promoter of PcFPPS can be bound by the nuclear-localized transcription factor PcWRKY44. Overexpression of PcWRKY44 in P. cablin upregulated the expression levels of PcFPPS and patchoulol synthase gene (PcPTS), and then promote the biosynthesis of patchouli alcohol. Taken together, these results strongly suggest the PcFPPS and its binding transcription factor PcWRKY44 play an essential role in regulating the biosynthesis of patchouli alcohol.

Patchouli alcohol (patchoulol), a sesquiterpenoid specifically synthesized by herbal plant Pogostemon cablin, has a variety of pharmacological and biological activities and is widely utilized in the medical and cosmetic industries. However, there are few studies on the transcriptional modulation of patchoulol biosynthesis. Some studies have reported that basic leucine zipper (bZIP) transcription factors (TFs) participate in the biosynthesis of plant terpenoids, but there is no report that bZIP TFs are involved in the modulation of patchoulol biosynthesis. This study was conducted to explain the regulation of patchoulol biosynthesis by PcbZIP44, a bZIP TF identified in patchouli for the first time. PcbZIP44 was highly expressed in roots and stems, and its encoded protein was nuclear localized revealed by protoplast subcellular localization experiment. According to the results of the transient dual-luciferase assay and yeast one-hybrid (Y1H) assays, PcbZIP44 can bind to the PcPTS (patchoulol synthase) gene promoter to repress its activity. Overexpression of the PcbZIP44 gene significantly reduced the content of patchoulol and resulted in a significant down-regulation of the PcPTS gene at the transcriptional level. Correspondingly, virus-induced PcbZIP44 gene silencing (VIGS) significantly increased the content of patchoulol and resulted in a significant up-regulation of the PcPTS gene at the transcriptional level. Therefore, these results suggested that PcbZIP44 negatively regulates patchoulol biosynthesis by inhibiting the PcPTS gene in P. cablin.

Trihelix (TH) transcription factors (TFs) actively function in regulating growth and development of plant. Moreover, members of this gene family regulate environment-responsive secondary metabolism. Here, members of trihelix family were explored based on the full-length transcriptome of Pogostemon cablin (Blanco) Benth. (patchouli), an extensively used medicinal plant. Among them, 16 PatTHs exhibited complete opening reading frame (ORF) were successfully cloned, and were classified according to their structural properties into four subfamilies (GT-1, GT-2, SH4, and SIP1). The expression patterns of PatTHs varied in various P. cablin tissues, and most of the PatTHs were induced by methyl jasmonate (MeJA). On exposure to abiotic stresses, including salt, drought, and cold condition, each PatTH responded to at least one of the stresses. In addition, the promoter of patchouli HMGR gene was bound and repressed by PatGT-1, a homologous protein of Arabidopsis transcription factor GT-1. According to subcellular localization results, PatGT-1 is a nuclear-localized protein. Once PatGT-1 was transiently overexpressed, it significantly lowered the production of patchoulol via repressing genes in patchoulol biosynthetic pathway. In this study, PatTHs and their putative functions were identified from P. cablin, and PatGT-1 was revealed as a negative regulator in patchoulol synthesis. Our findings improve the understanding of trihelix family in patchouli and contribute to the future study on this family.

Background Patchouli alcohol is an effective component of the medicinal plant patchouli. Similar to other secondary metabolites, its synthesis is also regulated by transcription factors. Although the biosynthetic pathway of patchouli alcohol has been characterized, the regulatory mechanism of patchouli alcohol has not been fully revealed. Results This study combined the transcriptome data of patchouli leaves treated with different hormones and WGCNA to complete the coexpression network. The modules related to patchouli alcohol were identified, and PcMYB25 played a crucial role in regulating patchouli alcohol biosynthesis. The overexpression of PcMYB25 can promote the expression of PTS , thereby increasing the content of patchouli alcohol. Conclusions This is the first reporter that MYB25 regulates the secondary metabolism of patchouli. These experimental results lay the foundation for further analysis of the regulatory mechanism of patchouli alcohol synthesis.

Patchoulol, the major active ingredient in Pogostemon cablin (Blanco) Benth., has considerable clinical and industrial value. A number of genes that participate in the patchoulol biosynthesis pathway have been identified; however, little is known about the transcription factors involved in regulating patchoulol synthesis. In this study, PatSWC4, a homologous protein of Arabidopsis SWC4 belonging to the MYB (v-myb avian myeloblastosis viral oncogene homolog)-related transcription factor family, was identified and characterized. Subcellular localization showed that PatSWC4 protein localized in the nucleus. The highest expression level of PatSWC4 gene was found in old leaves of P. cablin and it was significantly induced by methyl jasmonate (MeJA). Additionally, yeast one-hybrid (Y1H) and dual-luciferase (dual-LUC) assays revealed that PatSWC4 could bind to the promoter of PatPTS gene to increase its transcriptional activity. Yeast two-hybrid (Y2H) assays also confirmed the interaction between PatSWC4 and PatJAZ4 proteins, indicating that PatSWC4 might function in the JA response network. Moreover, transient overexpression of PatSWC4 gene in P. cablin leaves markedly increased the production of patchoulol, and qRT-PCR analysis further revealed that genes in the patchoulol biosynthesis pathway were significantly upregulated at the transcriptional level. Ultimately, we proposed a work model depicting how JA signaling regulates PatPTS via the interaction between PatSWC4 and PatJAZ4, thereby regulating the biosynthesis of patchoulol. Our findings not only help to elucidate the regulatory mechanism governing JA-induced patchoulol biosynthesis, but also lay a foundation for future studies on improving patchoulol production through genetic and metabolic engineering.