Steroidal glycoalkaloids (SGAs) are predominantly found in Solanaceous plants, including tomato (Solanum lycopersicum). In addition to their roles in resistance to herbivores, pathogens, and environmental stresses, SGAs exert antifungal, antibacterial, and anticancer effects. Over the past 15 years, the biosynthesis pathway of SGAs in tomato has been progressively investigated. A growing number of intermediate compounds and novel biosynthetic enzymes have been identified. In addition, various regulatory factors and their underlying regulatory mechanisms governing SGAs biosynthesis have been increasingly elucidated. Building upon these advances in understanding the SGAs biosynthetic pathway and its regulatory network, metabolic engineering of the SGAs pathway in tomato has been achieved using techniques such as gene editing. This mini review summarizes the current understanding of SGAs biosynthesis and regulatory mechanisms in tomato, and provides an overview of recent progress and future perspectives in metabolic engineering applications targeting this pathway.

Plant prenyltransferases (PTs) are pivotal enzymes in the biosynthetic pathways of prenylated natural products (PNPs) that catalyze the transfer of isoprenyl units (C₅) to specific receptor molecules, including aromatic compounds and terpene precursors. Recent advancements in plant genomics and synthetic biology have significantly accelerated research on PTs in natural product synthesis, positioning them as a focal point for scientific investigation. Although there are existing reviews on the functions and classification of PTs, the functional diversity of plant PTs remains in-adequately understood. Consequently, this study provides an exhaustive summary of over 160 reported plant ubiquinone biosynthesis gene A(UbiA)-type PTs and selected representative isopentenyl diphosphate synthases (IDSs). We systematically reviewed the classification, function,catalytic mechanisms, and enzyme engineering strategies of plant PTs, and their application in the heterologous production of PNPs. This study aimed to advance PT characterization and application, thereby establishing a foundation for PNP-based drug development.

Medicinal plants produce important pharmaceuticals, but these compounds are often present at low levels or only in specific tissues; in addition, many medicinal plants produce small amounts of biomass and are difficult to cultivate. Genome editing for agronomic traits and metabolic engineering holds promise for improving pharmaceutical production, and genome-editing applications in medicinal plants have expanded as genome-editing techniques have advanced. For example, genome editing has been used to regulate the production of phenolic acids and tanshinone metabolites of Salvia miltiorrhiza in medicinal plants. In this review, we synthesize the current knowledge on the development and applications of gene-editing tools in medicinal plants. Furthermore, we summarize the limitations of genome editing in these species and propose solutions for addressing these challenges to fully harness this technology for improving these important plants. We focus on novel technologies to enhance the regeneration rates of transgenic plants, artificial intelligence-assisted multiomics approaches for predicting editing efficiency, key components that optimize genome-editing efficacy, and the development of innovative gene-editing systems. Finally, we offer perspectives on advancing metabolic engineering strategies for medicinal plants.

Specialized structures in medicinal plants underpin the spatial regulation of secondary metabolism, determining the biosynthesis, accumulation, and storage of pharmacologically active compounds. Specialized structures, such as glandular trichomes, roots, rhizomes, laticifer, heartwood, and so on, have evolved distinct developmental programs and metabolic regulatory networks, enabling efficient synthesis, storage, and secretion of bioactive compounds. Understanding how these tissues originate, differentiate, and coordinate metabolism is essential not only for elucidating the molecular basis of plant chemical diversity but also for decoding the biosynthetic pathways of active ingredients and improving their yields through metabolic engineering. This study summarizes recent advances in elucidating the developmental and regulatory mechanisms underlying the formation and function of specialized structures in medicinal plants, including genetic, hormonal, and environmental controls. Moreover, it also highlights the technologies that have advanced the exploration of tissue-specific metabolism, development, and differentiation mechanisms. Together, this review summarizes recent progress in elucidating the types of specialized structures responsible for active compound biosynthesis and the underlying developmental mechanisms in medicinal plants, offering new perspectives for precision breeding and metabolic engineering of medicinal plants.

Phytosterols are a diverse class of isoprenoid-derived lipids that serve as essential structural components of plant membranes and regulators of growth and reproduction. Unlike animals and fungi, which predominantly utilize cholesterol and ergosterol, plants produce a complex array of over 250 sterol molecules. These include major forms such as β-sitosterol, stigmasterol, and campesterol as well as minor components like cholesterol and various sterol biosynthetic intermediates. This review provides a comprehensive overview of plant sterols, first addressing their structural diversity and distribution across species and tissues, and then exploring their biosynthesis, transport, and functions. A key focus is placed on their role as membrane modulators, influencing fluidity, permeability, and the formation of lipid rafts. Finally, we synthesize genetic and molecular evidence, demonstrating the critical functions of sterols and their derivatives in both reproductive and vegetative development. We conclude by highlighting persistent gaps in our knowledge and proposing future research directions to unravel the multifaceted roles of these essential molecules.

Plant metabolism is increasingly being demonstrated to be partially controlled by dynamically assembled metabolons—multienzyme complexes that enable substrate channeling, insulate reactive intermediates, and permit rapid, low-energy flux control. Rigorous criteria are defined to distinguish true metabolons from generic assemblies, and evidence is synthesized across cyanogenic glucoside, phenylpropanoid/flavonoid, alkaloid, terpenoid, polyamine, sporopollenin, and auxin pathways. A practical workflow is presented in which AP-MS (Affinity purification mass spectrometry)/Co-IP (Co-immunoprecipitation), proximity labeling, BiFC (Bimolecular fluorescence complementation)/FRET (Förster resonance energy transfer)/Split-luciferase, and isotope-dilution metabolomics are integrated to resolve composition, dynamics, and direct channeling in vivo. In enzyme-based substrate channeling engineering, design rules are distilled for membrane anchoring, modular scaffolds, compartment targeting, and inducible/optogenetic control, and limitations such as metabolic burden, stoichiometry, and leakiness are noted. An AI-assisted loop is outlined in which structure-aware generative models produce binders/interfaces that are coupled to spatial optimization of enzyme order, orientation, and distance. Together, these advances reposition metabolons as a deployable technology for programmable flux in plants, enabling safer handling of labile intermediates and higher titers of valuable natural products.

Exploring the metabolic characteristics of different plant organs and tissues at a spatial level can help us to better understand the functional mechanisms of biological tissues and cells. Mass spectrometry imaging (MSI) provides a reliable tool for this purpose. However, its application for high-resolution metabolic mapping across various plant organs remains a significant challenge due to the intrinsic biological properties of plant samples and unfavorable analysis conditions. This study aimed to develop a novel MSI platform that can expand more diverse plant samples in spatial metabolomics research and enhance the detection efficiency of plant metabolites. The platform (AMG–LDI–MSI) based on an Au nanoparticles-loaded MoS₂ and doped graphene oxide (Au@MoS₂/GO) flexible film substrate combined with laser desorption/ionization (LDI)–MSI was established to enhance the detection and visualization of metabolites in various plant tissues. It has a non-sectioning, matrix-free, dual-ion mode imaging strategy, enabling high-throughput detection of metabolites and high-resolution molecular imaging within a micrometer scale. The Au@MoS₂/GO as a new substrate can offer high sensitivity and molecular coverage for diverse plant metabolites (10 classes) under the positive and negative ion modes. Moreover, the AMG–LDI–MSI platform overcomes the limitations of plant tissues (e.g., fragile leaf, water-rich fruit, or lignified roots) for in situ imaging. We successfully applied the platform to map the metabolite spatial dynamics in different types of fresh tissues (rhizome, main root, branch root, fruit, leaf, and root nodule) from medicinal plants, obtained the high-quality mass spectral imaging data, and demonstrated the universality and applicability of the platform to multiple plant tissues. These results demonstrate the significant advantages of enhancing the detection of multiple tissue metabolites in plants and their high-resolution imaging. It has overcome the limitations of previously reported MSI methods, suggesting that it could become a widely used tool for deciphering metabolic networks in plant biology.

Purine-related metabolites are central to primary metabolic pathways in plants and serve as precursors for purine alkaloid biosynthesis in caffeinated species such as tea plants (Camellia sinensis). In this study, metabolite profiling of two tissues (young and mature leaves) was performed across 183 genetically diverse tea accessions, identifying and quantifying 10 purine alkaloid-related metabolites. Metabolite genome-wide association studies revealed 17 significant loci associated with these metabolites, including both known loci such as caffeine synthase and 16 novel loci (P < 1.05 × 10⁻⁵). Through functional annotation and in vitro enzymatic assay, we characterized 5′-methylthioadenosine/S-adenosylhomocysteine nucleosidase (CsMTAN) as the causal gene underlying natural variation in adenosine and adenine content. CsMTAN can catalyze the degradation of both 5ʹ-methylthioadenosine and S-adenosylhomocysteine to release adenine. The T → A nucleotide substitution at SNP55151898, which leads to a phenylalanine → tyrosine substitution at residue 179 (F179Y), resulted in a significant alteration of enzyme activity in vitro, as evidenced by an approximately 50% reduction in adenine abundance (P < 0.05). Transient overexpression of CsMTAN-A and CsMTAN-T in Nicotiana benthamiana both significantly increased adenine content and dramatically decreased adenosine content, providing direct evidence for the functional involvement of CsMTAN in plant purine metabolism. CsMTAN-T overexpression resulted in significantly lower adenosine level than CsMTAN-A (P < 0.05). Phylogenetic analysis across 115 species and protein structural modeling revealed a distinct evolutionary divergence between plant MTAN evolution and species phylogeny, strongly suggesting the occurrence of horizontal gene transfer events in the evolutionary history of plant MTANs. This study thus furthered our understanding of the genetics and molecular mechanisms regulating purine metabolism and purine alkaloid biosynthesis in tea plants and provided novel targets for molecular breeding and synthetic biology applications.

The strigolactone (SL) and gibberellin (GA) signals jointly regulate various aspects of plant growth and development. However, the molecular regulatory network underlying the crosstalk between the SL and GA signaling pathways remains poorly understood. In this study, we found that ELONGATED HYPOCOTYL 5 (MdHY5) serves as an integrator of SL and GA signals in apple (Malus × domestica), and the WUSCHEL-related homeobox (WOX) transcription factor MdWOX8 enhances anthocyanin biosynthesis by directly activating MdHY5 expression. Furthermore, the MdWOX8-MdHY5 module is a crucial hub linking SL and GA signals by interacting with the SL signaling repressor SUPPRESSOR OF MORE AXILLARY GROWTH 2-LIKE 8 (MdSMXL8) and the GA signaling repressor REPRESSOR-of-ga1-3-LIKE 3a (MdRGL3a). MdSMXL8 inhibits the activation of the MdHY5 promoter by MdWOX8, and MdRGL3a promotes the function of MdWOX8 by enhancing its transcriptional activation of MdHY5 and disrupting the MdSMXL8–MdWOX8 interaction. Moreover, the E3 ubiquitin ligase SEVEN IN ABSENTIA 1 (MdSINA1) negatively regulates MdWOX8 homeostasis by targeting it for ubiquitination and degradation in response to SL and GA signals. Overall, our results reveal a precise regulatory network that integrates SL and GA at the transcriptional and post-translational levels, with the MdWOX8-MdHY5 module at its core.

Although terpenoids display chemical and structural diversities as well as important biological activities, they are biosynthesized through polymerization of C5 isoprene units according to the “biogenetic isoprene rule.” However, recent observations have identified irregular terpenes, such as C11, C12, C16, and C17, in some microbial species. These irregular terpenes have garnered research interest due to the “Magic Methyl Effect.” These compounds are biosynthesized from the methyl modification of terpene skeletons at a detectable level in Escherichia coli. To explore key factors in the biosynthesis of irregular terpenes, we conducted a screening of C-methyltransferases by UniKP, synthesized methyl-isopentenyl pyrophosphate as a precursor building unit, and polymerized it with C5 isoprene units to generate methyl β-elemene via farnesyl pyrophosphate synthase and germacrene A synthase. Finally, the yield of methyl β-elemene reached 6.98 mg/L, representing a 2.46-fold increase, with a ratio of methylated to native β-elemene increasing by 0.21 through the introduction S-adenosyl-L-homocysteine hydrolase and the optimization of germacrene A synthase. These findings not only expand the chemical diversity of terpenoids but also highlight the evolutionary plasticity of terpene synthases in generating structural novelty.

Tea plant (Camellia sinensis) accumulates abundant secondary metabolites under cold stress, some of which are thought to play important roles in enhancing cold tolerance. To explore novel secondary metabolites involved in cold tolerance, we conducted an untargeted metabolomics analysis of tea plants under cold stress treatment. This revealed a novel acylated flavonoid, kaempferol-3-O-(6″-p-coumaroyl)-glucoside (KCG), in which accumulation positively correlated with stress severity. The compound was purified and structurally characterized using nuclear magnetic resonance (NMR) spectroscopy. Exogenous application of this flavonoid significantly improved cold tolerance in tea plants, indicating its role as a defensive metabolite. Transcriptome sequencing identified candidate acyltransferases, with tea hydroxycinnamoyl transferase (CsHCT) emerging as a key biosynthetic gene. In vitro assays confirmed that recombinant CsHCT catalyzes the formation of KCG from kaempferol-3-O-glucoside and p-coumaroyl-CoA. Overexpression of CsHCT in tea seedlings and Arabidopsis thaliana resulted in markedly elevated levels of this flavonoid and cold resistance of these plants, validating its in vivo role. Our findings elucidate the biosynthesis of acylated flavonoids in tea plants and highlight CsHCT as a genetic target for enhancing cold resistance. This study provides foundational insights for advancing cold-resistant tea breeding programs.

Ceramide synthases (CerSs) are central to sphingolipid biosynthesis and influence plant development and defense responses. However, how CerS activity is regulated in plants remains unclear. Here, we discovered that LAG ONE HOMOLOG 2 (LOH2), the sole long-chain CerS in Arabidopsis (Arabidopsis thaliana), is post-translationally regulated by the ubiquitous kinase casein kinase 2 (CK2). CK2 interacts with LOH2 and phosphorylates serine residues S289 and S291 within its C-terminal region. We mutated these two serines to alanines and expressed the resulting non-phosphorylatable LOH2 variant in transgenic plants and protoplasts. We found that phosphorylation enhances LOH2 enzymatic activity, partially by increasing its substrate-binding affinity, but concurrently promotes LOH2 polyubiquitination and degradation via the 26S proteasome without affecting its subcellular localization. Plants expressing a non-phosphorylatable LOH2 variant showed diminished cell death, reduced C16 ceramide biosynthesis and salicylic acid (SA) accumulation, and compromised resistance to the fungal toxin Fumonisin B1 and the bacterial pathogen Pseudomonas syringae. Pathogen infection induces LOH2 phosphorylation, promoting C16 ceramide accumulation, SA production, and resistance gene expression. Collectively, our findings demonstrate that CK2 fine-tunes LOH2 enzymatic activity and stability, and thus the production of long-chain ceramides through phosphorylation, thereby regulating plant development and defense responses.

Despite decades of research on NAC–MYB master regulators, the transcription factor (TF)-mediated network governing wood development remains fragmented, hindering a systems-level understanding of secondary xylem formation. In this study, we identified GROWTH REGULATING FACTOR 20 (PagGRF20) as a key regulator within the poplar wood formation network. Compared to wild-type (WT) poplars, the PagGRF20-overexpressing lines showed a 22.1%–23.9% increase in secondary xylem thickness, a 37.7%–43.3% increase in cell wall thickness, and higher cellulose and xylan content. These phenotypic changes coincided with upregulation of genes involved in cellulose and xylan biosynthetic pathways. Conversely, PagGRF20-RNAi lines displayed opposite phenotypic traits. Molecular analyses revealed that PagGRF20 binds to the TGT[C/T]AGA cis-regulatory elements in the promoters of wood polymer biosynthesis genes, modulating polysaccharide production by activating the glycosyltransferase IRREGULAR XYLEM 14-LIKE (PagIRX14L). Crucially, overexpression of PagIRX14L in poplar increased the xylan content and decreased lignin levels, suggesting that PagGRF20-mediated transcriptional activation of PagIRX14L significantly contributes to the observed shifts in cell wall composition. Additionally, the R2R3–MYB repressor PagMYB4 physically interacts with PagGRF20, forming a transcriptional complex that suppresses lignin biosynthesis genes, including LACCASE 11 (PagLAC11), potentially altering the balance of cell wall components and affecting cellulose and xylan accumulation. Finally, we built a three-layer, PagGRF20-centered regulatory network to uncover the mechanisms and target genes underlying wood formation. Our results suggest that the PagGRF20–PagMYB4 module coordinates secondary cell wall (SCW) biosynthetic pathways, providing novel insights into engineering wood with enhanced polysaccharide deposition and low lignin content.

O-Methyltransferases (OMTs) play crucial roles in plant defense, environmental adaptation, and quality formation by catalyzing the biosynthesis of diverse methylated metabolites. Although OMT (COMT and CCoAOMT) genes have been functionally characterized in various plant species, the evolutionary trajectory of the entire OMT gene family and the functional divergence of the CCoAOMT subfamily remain to be systematically elucidated. In this study, we performed pan-genome analysis of the OMT gene family in 61 tomato (Solanum spp.) accessions and conducted phylogenetic analysis across 20 plant species (from algae to angiosperms), identifying 2,882 OMT genes. Phylogenetic reconstruction revealed that all extant plant CCoAOMT genes evolved from a single ancestral lineage (Clade I) originating before the divergence of red and green algae. In tomato, 2,199 OMT genes were classified into 42 orthogroups: nine core, five soft-core, 22 dispensable, and six private orthogroups, with 52.4% classified as dispensable genes. OMT genes in the Solanum genus have predominantly undergone purifying selection. Among all COMT orthogroups, a single tandem duplicate cluster stands out as exclusively conserved. Members of this cluster have evolved a distinct catalytic role, as evidenced by the finding that SlCOMT2c exclusively catalyzes the formation of kaempferide via the 4′-O-methylation of kaempferol. Ion mobility spectrometry showed that SlAOMT, a member of the CCoAOMT-like subfamily, catalyzes the methylation of luteolin to produce two isomeric products identified as diosmetin and chrysoeriol while losing the canonical catalytic function of the CCoAOMT subfamily. In addition, we identified a potential gene regulatory network associated with methylated flavonoid biosynthesis. This study establishes an integrative framework for elucidating OMT evolution and provides analytical tools for identifying genes involved in isomeric methylated flavonoid biosynthesis, paving the way for studying adaptive evolution and specialized metabolic pathways in plants.

Prenylated phenolic glycosides, such as nodakenin, represent a class of natural products with diverse bioactivities. Their metabolic engineering production remains largely unexplored, primarily due to the scarcity of efficient UDP-glycosyltransferases (UGTs) capable of catalyzing prenylated phenolic substrates. In this study, we characterized several UGT93 enzymes from Angelica decursiva that catalyzed the glycosylation of nodakenetin. Enzymatic assays revealed a pronounced catalytic preference of these enzymes toward various types of prenylated phenolic substrates. Notably, this substrate preference is conserved across UGT93s from other species and a reconstructed ancestral enzyme. Structural analysis and mutation experiments revealed that the preference was caused by the substrate binding with several hydrophobic and aromatic residues. This study highlights the biocatalytic potential of the UGT93 family enzymes, offering promising biocatalysts for the glycosylation of plant-derived prenylated phenolics.

Phillyrin, a unique antiviral lignan exclusive to Forsythia suspensa, shows potent anti‐influenza activity. However, its broad pharmaceutical application is hindered by low and variable natural accumulation, underscoring the need to elucidate its biosynthetic pathway for sustainable production. In this study, we used an integrated metabolomic and transcriptomic approach to investigate tissue‐specific phillyrin biosynthesis in F. suspensa. We identified two O-methyltransferases (FsE4MT01 and FsE4MT02) that catalyze the methylation of (+)‐epipinoresinol to form phillygenin and a UDP‐glycosyltransferase (FsP4′GT) that glucosylates phillygenin to yield phillyrin. Using recombinant proteins expressed in Escherichia coli, we validated their in vitro catalytic activities and determined their kinetic parameters. Molecular docking and site‐directed mutagenesis of key residues further revealed the structural basis of their catalytic mechanisms. The in vivo functions of these enzymes were confirmed through heterologous expression in Nicotiana benthamiana and F. suspensa leaves. Importantly, by developing a transient leaf‐expression system in F. suspensa, we demonstrated that overexpression of FsE4MTs and FsP4′GT substantially increased the production of phillygenin and phillyrin, respectively, whereas virus‐induced gene silencing (VIGS) of these genes reduced the accumulation of the corresponding products. Notably, enhanced phillyrin production was also achieved by expressing FsE4MTs and FsP4′GT in the related species Forsythia × intermedia, highlighting the potential of these key enzymes for metabolic engineering. Our findings elucidate the terminal steps of phillyrin biosynthesis and provide a strategic foundation for engineering the sustainable production of this pharmaceutically valuable compound.