A growing global population and the increasing prevalence of diet-related health issues such as “hidden hunger”, obesity, hypertension, and diabetes necessitate a fundamental rethinking of crop design and breeding. Synthetic metabolic engineering offers a method to modify and redesign metabolic pathways to increase the nutritional value of crops. We summarize recent advances in the biofortification of key nutrients including provitamin A, vitamin C, vitamin B9, iron, zinc, anthocyanins, flavonoids, and unsaturated fatty acids. We discuss the potential of multi-gene stacking, gene editing, enzyme engineering, and artificial intelligence in synthetic metabolic engineering. We propose future research directions and potential solutions centered on leveraging AI-driven systems biology, precision gene editing, enzyme engineering, agrobacterium-mediated genotype-independent transformation, and modular metabolic engineering strategies to develop next-generation nutritionally enhanced super crops and transform global food systems.

Nutritional imbalance has led to many chronic diseases and severely affected people’s quality of life. Developing nutrient-dense crops has emerged as a strategy for improving the current state of human nutritional intake globally. We summarized recent advances in rice biotechnology breeding focusing on increasing micronutrients and active natural products, highlighting the cutting-edge metabolic engineering technologies and strategies employed. We discussed common challenges and potential solutions in metabolic engineering breeding. On this basis, the future development direction of rice nutrient metabolism industrialization was prospected.

In light of the pressing global challenges of climate change, declining crop resilience, and hidden hunger, it is imperative to overcome the limitations of conventional crop breeding to enhance both the nutritional quality and stress tolerance of crops. Synthetic metabolic engineering presents innovative strategies for the precision modification and de novo design of metabolic pathways. This approach generally encompasses three essential steps: identifying key metabolites through metabolomics, integrating multi-omics technologies to investigate the synthesis and regulation of these metabolites, and utilizing gene editing or de novo design to modify crop metabolic pathways associated with desirable agronomic traits. This review underscores the vital role of plant metabolite diversity in enhancing crop nutritional quality and stress resilience. Integrated multi-omics analyses facilitate the metabolic engineering by identifying key genes, transporters, and transcription factors that regulate metabolite biosynthesis. Precision modification strategies employ genome editing tools to reprogram endogenous metabolic networks, while de novo design reconstructs metabolic pathways through the introduction of exogenous biological elements—thereby both approaches enable the targeted enhancement of desired traits. These strategies have been effectively implemented in major food crops. However, simultaneously enhancing nutritional quality and stress resilience remains challenging due to inherent trade-offs and resource competition in distinct metabolic pathways within plants. Future research should integrate AI-driven predictive models with multi-omics datasets to decipher dynamic metabolic homeostasis and engineer climate-smart crops that maximize yield while preserving quality and environmental adaptability.

Plants produce a vast array of specialized metabolites that serve as essential defenses against herbivores and pathogens. However, the capacity to produce these compounds differs substantially among plant species and is frequently diminished during domestication. Advances in synthetic metabolic engineering enable efficient elucidation and engineering of plant specialized metabolic pathways active in crop pest and pathogen resistance. This review summarizes strategies and workflows for selecting defensive metabolic pathways, identifying candidate biosynthetic genes, and rewiring native or introducing heterologous pathways to enhance crop resistance to pests and pathogens. Strategies include weighted gene co-expression network construction, biosynthetic gene cluster scanning, and metabolite genome-wide association studies for pathway discovery, as well as transcriptional reprogramming, enzyme activity optimization, and transporter deployment for pathway engineering. We further discuss challenges in using synthetic metabolic engineering to enhance crop resistance and highlight the potential of artificial intelligence in addressing them.

Qingke, a staple crop grown on the high-altitude Tibetan Plateau, has evolved a metabolomic profile providing both environmental stress resilience and human nutrition. We review the hypothesis that the metabolites that confer cold and UV resistance on the crop also facilitate human adaptation to high-altitude stresses. Specifically, β-glucans regulate blood glucose primarily via short-chain fatty acids (SCFAs) produced through gut microbiota fermentation, which directly mediate glucose homeostasis. Phenolamides accumulate via the phenylpropanoid pathway, with chalcone isomerase (CHI) serving as a key enzyme in flavonoid biosynthesis and enhancing UV-B resistance. Under low temperatures, β-glucans improve frost tolerance by modulating osmotic balance and inhibiting ice-nucleating proteins, while lipids maintain membrane fluidity to sustain cellular function during cold stress. Importantly, we explore the hypothesis that these same metabolites, upon consumption, may facilitate human adaptation to high-altitude stresses. This hypothesis is supported by preliminary epidemiological associations between Qingke consumption and favorable health outcomes in high-altitude populations, as well as established bioactivities of the implicated metabolites in vitro and in animal models. However, direct causal evidence in humans and a comprehensive understanding of the underlying molecular mechanisms remain key knowledge gaps that warrant future investigation. Qingke as a unique resource at the interface of agricultural resilience and human nutrition. Understanding its metabolic blueprint will inform the development of functional foods and climate-resilient crops.

Biological nitrogen fixation (BNF) and photosynthetic carbon fixation underpin food production and climate mitigation, yet natural systems are constrained by oxygen sensitivity, high energy demand, and inefficient catalysts. This review synthesizes advances that recast these processes as engineering targets and proposes a conceptual roadmap that bridges synthetic symbioses with the synthetic biology of enzymes and pathways. For BNF, progress spans cross-kingdom strategies—from refactoring nif gene sets and targeting nitrogenase assembly to eukaryotic organelles, to engineering plant-associated diazotrophs, rhizosphere control circuits, and emerging nodule-like microenvironments. For carbon assimilation, new-to-nature CO₂-fixation modules and photorespiratory bypasses illustrate how pathway redesign and alternative carboxylases can circumvent key Calvin-Benson-Bassham limitations, and expanding photosynthetic light capture offers additional leverage. Across these domains, we extract common design principles: (i) nitrogenase output is increasingly governed by carbon/energy supply and electron delivery as much as by oxygen protection; (ii) robust function requires compartment-aware enzyme-chassis coordination, substrate channeling, and dynamic regulation using sensors and control circuits; and (iii) scalable implementation may benefit from distributing metabolic labor across engineered consortia rather than forcing all functions into a single host. We discuss enabling technologies—including AI-guided protein design and directed evolution, cell-free prototyping, chassis toolkits, and materials/bioelectrochemical interfaces—that can accelerate design-build-test-learn cycles and reduce barriers to deployment. Together, these insights define a path toward integrated nitrogen and carbon fixation systems for low-emission agriculture and biomanufacturing.

Plant oils are increasingly sought after as sustainable sources of bioenergy for biodiesel production and high-value biochemicals. Although oilseed crops currently serve as the primary source of plant oils, meeting the rising global demand on limited arable land, without compromising food security, remains a major challenge. Therefore, metabolic engineering of high-biomass bioenergy feedstocks has been widely explored to enhance the conversion of carbon stored in vegetative tissues into energy-dense triacylglycerol (TAG). Significant progress has been made in boosting TAG accumulation in the vegetative tissues of various plant species through bioengineering strategies. These efforts span from single-gene modifications to the coordinated expression of key lipogenic factors such as WRI1, DGAT1/2, and OLE1. The resulting fatty acid and TAG profiles, however, often vary depending on the targeted plant species and promoter(s) used. This review summarizes the roles of essential lipogenic factors in plant oil biosynthesis and highlights recent advances in metabolic engineering across diverse crop species through combinatorial expression of these factors. We also discuss future strategies for achieving high-level oil production without incurring growth penalties. By offering new perspectives on metabolic engineering, this work aims to support the development of plants as efficient biofuel feedstocks, contributing to the global effort to address energy challenges.

Acetolactate synthase (ALS)-targeting herbicides are among the most widely used weed-control chemicals globally. Mutations in the ALS gene can confer herbicide resistance in crops, thereby allowing selective elimination of weeds without harming crops. Herbicide-resistant ALS alleles were initially discovered in weeds and subsequently developed through artificial mutagenesis techniques. With the advancement of CRISPR/Cas technologies, various genome-editing tools are now available to introduce these resistant alleles, as well as novel variants, into diverse crop species. Moreover, emerging methodologies, such as directed evolution, enable the generation and screening of large populations of random ALS mutants. Consequently, ALS has become one of the most extensively targeted genes in plant gene evolution. This paper provides a comprehensive overview of both conventional and recently developed strategies for ALS evolution, with particular emphasis on CRISPR/Cas-based genome editing and directed evolution. Future perspectives on technological application are also discussed. By advancing our understanding of herbicide-resistant ALS allele development for crop improvement, these methodologies may also pave the way for their application to the evolution of other agronomically important genes.

Photorespiration consumes photosynthetically fixed carbon and reduces yields by 20%-50% in C₃ crops. In an attempt to increase photosynthetic efficiency in rice by bypassing the carbon-consuming process of photorespiration, a photorespiratory bypass consisting of Chlamydomonas reinhardtii glycolate dehydrogenase and Cucurbita maxima malate synthase (termed the GMS bypass) was introduced into the rice cultivar Zhonghua 11 and osplgg1b, a mutant of the rice chloroplast glycolate transporter, to generate GMS/ZH11 and GMS/osplgg1b transgenic plants. The GMS bypass reduced photorespiration and increased photosynthesis in the transgenic plants. The straw biomass of GMS/ZH11 and GMS/osplgg1b increased by up to 16.0% and 85.7%, respectively. The yield of GMS/ZH11 increased by 22.0%-34.7% in paddy fields. Thus, the GMS bypass can increase photosynthetic efficiency and yield in rice.

Source-sink coordination serves as the foundation for improving crop yield. Current research primarily focuses on individual factors, such as increasing the source or expanding the sink, which often leads to disrupted source-sink balance, causing trade-offs among photosynthesis, yield, and stress response. To address these limitations, we present an integrated synthetic biological framework that synergistically enhances photosynthetic efficiency (source capacity), sink optimization, and abiotic stress tolerance. We developed an editing-overexpression coupling (EOC) vector system enabling simultaneous overexpression of four photosynthesis-enhancing genes (Cyt c₆, PsbA, FBPase, OsMGT3), knockout of three yield-limiting genes (GS3, Gn1a, OsAAP5), and self-excision of selection markers, gene-editing modules, and fragment deletion cassettes. Field evaluations of CFMP-gga transgenic lines revealed significant physiological improvements, including 13%-17% increase in photosynthetic rates, improved chlorophyll fluorescence parameters, and increased stomatal conductance. These enhancements translated into remarkable agronomic gains, including 18.7%-22.3% higher grain yield, 23.1%-26.1% increased biomass, and improved panicle architecture (increased grain size and grain number per panicle). The engineered lines maintained superior thermotolerance (under 42 °C stress) and alkali tolerance (at pH 10) compared to wild-type controls. This study provides a strategy for enhancing crop yield by demonstrating that coordinated multi-gene regulation of source-sink dynamics, coupled with stress resilience engineering, achieves concurrent improvements.

Betalain, an economically valuable water-soluble natural plant pigment, is prized for its strong antioxidant activity, making it popular as a dietary supplement and a visual marker for plant transformation. However, market demand significantly outstrips current production capacity. This study reports the development of an efficient push-and-pull multigene strategy based on polycistronic expression and metabolic flux regulation to enhance betalain biosynthesis in transgenic maize (Zea mays L.) endosperm. We engineered a novel enhanced RUBY (eRUBY) system derived from the original polycistronic RUBY construct (CYP76AD1_P2ADODA1_P2ADOPA5GT unit, abbreviated CDG) by introducing arogenate dehydrogenase (ADHα) to increase the L-tyrosine substrate supply. All the genes were driven by the endosperm-specific promoter. Fusion of ADHα into a single polycistronic eRUBY construct (CDGA) produced significantly higher betanin (6.88 mg g⁻¹ dry weight) and isobetanin (1.81 mg g⁻¹ dry weight) levels than in CDG + A, which stacked the ADHα cassette independently with CDG. The high betalain accumulation in CDGA lines (which also exhibited higher transgene copy number) resulted in a 2.85-7.58-fold improvement in endosperm antioxidant capacity compared to WT (versus 2.48-2.80-fold in CDG + A). Importantly, transgenic plants maintained a normal phenotype. Transcriptome and metabolome analyses further indicated that metabolism of phenylalanine, alanine, aspartate, and glutamate contributes to betalain production. Hybridization with sweet corn successfully created a high-sugar eRUBY maize variety. Collectively, these results demonstrate the successful development of a novel maize germplasm with significantly enhanced nutritional value through high betalain accumulation.

Anthocyanin biosynthesis in plants is spatiotemporally controlled by a suite of transcription factors, with MYB proteins playing a key regulatory role. However, the evolution of the distinct roles of MYB paralogs remains poorly understood. Our previous studies have established GmMYBA2 and GmMYBA3 as the regulators of seed coat and floral anthocyanin production in soybean (Glycine max), respectively. In this study, we reveal the functional divergence of their paralog GmMYBA1 in orchestrating light-responsive anthocyanin biosynthesis in juvenile tissues and stems. In brief, hypocotyl/stem- and young leaf-predominant expression of GmMYBA1 correlates with photoprotective anthocyanin accumulation. Ectopic overexpression of GmMYBA1 induces systemic pigmentation across leaves, stems, and reproductive organs, whereas RNAi-mediated silencing of GmMYBA1 significantly reduces anthocyanin accumulation in the hypocotyl. Light-dark shift assays confirmed that GmMYBA1 is required for hypocotyl pigmentation, while dual-luciferase assays revealed the specific regulation of the GmMYBA paralogs by GmSTF1/2 (soybean TGACG-motif binding factor 1/2). GmSTF1/2 both activate GmMYBA1, with only GmSTF2 weakly inducing GmMYBA2 and neither affecting GmMYBA3. Further investigation indicated that the differential transactivation of GmMYBA promoters largely resulted from their cis-element difference, suggesting regulatory divergence as a driver of MYB paralog diversification. Our findings position GmMYBA1 as the central MYB activator integrating light signaling with anthocyanin biosynthesis, with paralog specialization reflecting evolutionary subfunctionalization post-gene duplication.

Flavonoids are crucial secondary metabolites widely distributed in plants, playing vital roles in diverse biological processes. Although the flavonoid biosynthesis pathway has been extensively characterized, the transcriptional regulatory mechanisms remain poorly understood. In this study, we identify the miR166-ATHB14-LIKE module comprising the miR166 and its target gene ATHB14-LIKE as a key regulator of flavonoid biosynthesis in soybean (Glycine max). Knockdown of miR166 or overexpression of ATHB14-LIKE upregulated multiple flavonoid biosynthesis genes, leading to increased flavonoid accumulation. Conversely, miR166 overexpression suppressed these genes and reduced flavonoid levels. We further show that ATHB14-LIKE directly activates specific flavonoid biosynthesis genes by binding to their promoters. Additionally, ATHB14-LIKE forms homodimers and heterodimers with homologous proteins to regulate downstream flavonoid biosynthesis genes. Together, our findings demonstrate that the miR166-ATHB14-LIKE module controls soybean flavonoid content by coordinating the expression of key biosynthetic genes.

Cotton production faces significant challenges from insect pests, with chemical pesticide use becoming increasingly limited by resistance and environmental concerns. This study explores the potential use of caffeine, a natural plant alkaloid, as an environmentally friendly insect resistance strategy in cotton. Exogenous caffeine application demonstrated potent insecticidal effects against cotton bollworm (Helicoverpa armigera) larvae, with concentrations ≥ 2 mg mL⁻¹ causing near-complete feeding cessation and up to 70% larval mortality. Building on this, we engineered transgenic cotton (Gossypium hirsutum cv. Jin668) for heterologous caffeine biosynthesis by introducing three key N-methyltransferase genes (CaXMT1, CaMXMT1, CaDXMT1) by multiple gene transformation. Transgenic lines expressing all three genes showed remarkable caffeine accumulation (up to 3.59 mg g⁻¹ dry weight), whereas two-gene combinations exhibited wild-type-level production. Feeding preference assays revealed that caffeine-enriched cotton strongly deterred feeding by H. armigera. Non-choice feeding trials demonstrated reduced leaf consumption and reduced larval growth in H. armigera fed on caffeine-producing cotton. The study highlights the effectiveness of synthetic biology approaches using the TGSII-UNiE multigene stacking system, despite challenges in transgene stability. This work advances plant-derived insect resistance research and provides a sustainable framework for reducing chemical pesticide reliance in cotton production, while underscoring unique potential of cotton as a synthetic biology platform for secondary metabolite engineering.

Carotenoids are lipophilic isoprenoid pigments with essential roles in plants. While the cultivated allotetraploid cottons exhibit distinct mature anther coloration — yellow in Gossypium barbadense versus predominantly white in G. hirsutum — the genetic basis of this divergence remains unclear. The purpose of this study was to identify the genetic basis of anther-color variation in cotton (Gossypium) species. We firstly identified carotenoids as the primary pigments underlying yellow-anthers coloration. Comparative transcriptomics of anthers revealed that the carotenoid biosynthesis gene GbPSY4 was expressed as a key regulator in G. barbadense. Functional validation via tissue-specific expression, subcellular localization, in vivo enzymatic assays, and virus-induced gene silencing confirmed its role in carotenoid biosynthesis and yellow pigmentation. Genome-wide association studies in a G. hirsutum population revealed GhPSY4_At, an ortholog of GbPSY4, as the causal gene of anther-color variation. We conclude that PSY4-regulated carotenoid biosynthesis governs yellow pigmentation. Furthermore, a finding that G. hirsutum accessions with yellow anthers showed greater pollen viability under high-temperature stress than those with white anthers suggests that the same pathway that governs yellow pigmentation influences heat tolerance. PSY4 is a promising target for breeding stress-tolerant cotton varieties.

Carotenoids are natural pigments that are widely distributed in the flowers, fruits, and seeds of many plant species. These compounds not only endow diverse colors but also exhibit antioxidant, immune-modulatory, anti-aging, and photoprotective properties. Although carotenoid metabolism has been studied extensively in microbial and plant science, the genetic mechanisms underlying carotenoid metabolism in cotton remain underexplored. Here, we isolated gene GbDYA that regulates a dark-yellow anther color by map-based cloning using a BC₁F₁ population derived from a cross of Gossypium barbadense acc. Hai7124 with dark-yellow anthers and G. hirsutum acc. TM-1 with light-yellow anthers backcrossed with TM-1. GbDYA encodes phytoene synthase, a key rate-limiting enzyme in the carotenoid biosynthesis pathway. A long terminal repeat retrotransposon in the first exon of GhDYA (an ortholog of GbDYA in G. hirsutum acc. TM-1) caused loss of function and led to the light-yellow anther color. GbDYA is predominantly expressed in the early stages of anther development. Transcriptome, RT-qPCR and KEGG enrichment analyses revealed that GbDYA influences the synthesis and accumulation of carotenoids in anthers by modulating expression of key genes in the carotenoid biosynthesis pathway. Integrated transcriptomic and metabolomic analyses indicated that the accumulation of lutein, violaxanthin, antherxanthin, cryptoxanthin, zeaxanthin, and β-carotene contributed to yellow coloration of anthers. Dual-luciferase and yeast one-hybrid assays confirmed that transcription factor GbMYB105 (GB_A11G3511) binds to the promoter of GbDYA and activates its expression. High-temperature stress treatment indicated that carotenoids accumulation in anthers enhances pollen antioxidant activity. This study unravels the role of GbDYA in conferring the anther coloration, and provides the potential utilization by modulating accumulation of carotenoids in anthers to enhance pollen viability in high-temperature tolerance breeding in cotton.

The evolutionarily conserved mitogen-activated protein kinase (MAPK) cascades relay extracellular signals into cells, triggering a variety of cellular responses. We previously revealed NtMPK4 as a positive regulator of nicotine biosynthesis; however, its upstream regulation remains unclear. Here, we characterized a MAPK cascade, comprising NtMEKK1b, NtMPKK2a, and NtMPK4, that promotes nicotine biosynthesis. This signaling module transduces external cues, including jasmonate and pathogen elicitors such as flg22, into post-translational modifications that enhance transcriptional activity and pathway gene expression. NtMPKK2a physically interacts with and phosphorylates NtMPK4 in vivo, confirming its role as an upstream kinase. RNAi-mediated silencing of NtMPKK2a significantly reduced the expression of nicotine pathway genes and decreased nicotine accumulation, whereas induced-overexpression of NtMPKK2a upregulated nicotine pathway genes and increased nicotine contents in tobacco hairy roots. Overexpression of NtMPKK2a in tobacco cells enhanced the transactivation activity of a NIC2-locus Ethylene Response Factor NtERF221 on Putrescine N-methyltransferase (NtPMT) promotor, further supporting its role in promoting nicotine biosynthesis. Furthermore, we identified NtMEKK1b, a tobacco MEKK that interacts with NtMAPKK2a in yeast cells. Knock-down of NtMEKK1b in transgenic tobacco plants attenuated the expression of nicotine pathway genes and reduced nicotine contents, whereas induced-overexpression of NtMEKK1b upregulated gene expression and nicotine accumulation. Our findings uncover a previously uncharacterized MAPK cascade module, NtMEKK1b-NtMPKK2a-NtMPK4, that regulates nicotine biosynthesis, highlighting the importance of posttranslational regulation in nicotine biosynthesis.

Carotenoid cleavage dioxygenase 4 (CCD4) controls the rate-limiting step of β-ionone biosynthesis, making it a valuable target for healthcare and pharmaceutical applications. Nicotiana tabacum, a carotenoid-richd crop species, is a promising source for β-ionone production. This study aimed to modify CCD4 activity to increase β-ionone yield in tobacco. We identified two isoforms of CCD4 in N. tabacum, NtCCD4a and NtCCD4b, with NtCCD4a exhibiting significantly higher expression levels than NtCCD4b. Using solid-phase microextraction gas chromatography-mass spectrometry (SPME-GC-MS), we demonstrated that NtCCD4a effectively catalyzes the cleavage of β-carotene to produce β-ionone. To improve its enzymatic activity, we applied structure-based rational design to reconstruct the active pocket of NtCCD4a, followed by high-throughput screening of mutant variants. Three single base mutants, F181G, F184L, and F337M, in NtCCD4a showed enhanced β-ionone production compared to the wild-type, with F337M yielding the highest amount. No synergistic effects were observed among the three mutants. Transgenic tobacco plants expressing the F181G, F184L, and F337M mutations had accelerated β-carotene cleavage and increased β-ionone production relative to the wild-type NtCCD4a. Our results establish a framework for the design of CCD4 in major crop species through genome editing technology.

Saikosaponins are the major pharmacologically active components in Bupleurum genus and exhibit significant application potential in multiple fields such as immune regulation and anti-tumor activity. To elucidate the biosynthetic pathway of saikosaponins, we identified two cytochrome P450 monooxygenases, CYP716A41 and CYP716Y4, in Bupleurum chinense. These enzymes catalyze the C-28 oxidation and C-16 hydroxylation of oleanane-type triterpene skeletons, respectively. The catalytic efficiency of CYP716A41 from a southern B. chinense variety was significantly higher than that from a northern variety. Molecular docking and mutagenesis experiments revealed that amino acid residues at sites 9 and 35 may contribute to this difference in catalytic efficiency. Additionally, under cold stress, the expression levels of both CYP450 genes and the saikosaponin contents in the leaves of southern varieties were significantly higher compared to those in northern varieties. The variation in the catalytic efficiency of CYP716A41 and the differential expression of the two CYP450 genes under cold stress during winter are associated with the differences in saikosaponin biosynthesis in the leaves of southern and northern B. chinense varieties. This is consistent with the distinct medicinal usage practices observed between southern and northern China.

Licochalcone A (LCA) is a characteristic compound in licorice Glycyrrhiza inflata and is widely utilized in pharmaceutical and cosmetic industries. However, the biosynthetic pathway and regulatory mechanisms of LCA remain poorly understood. In this study, we first found the accumulation of LCA is induced by methyl jasmonate (MeJA). Given that MYB transcriptional factors are well-documented as key regulators of flavonoid biosynthesis, we identified a total of 147 GiR2R3-MYB genes in G. inflata, which were classified into 28 subgroups. The chromosome distributions, sequence characteristics, gene structures, duplication events and cis-acting elements were also investigated. Through integrated analysis of GiR2R3-MYBs expression patterns across different tissues and under MeJA treatment, along with phylogenetic relationship, we identified GiMYB76—a MeJA-inducible MYB transcription factor—as a potential regulator of LCA accumulation. Functional validation showed that transgenic hairy roots overexpressing GiMYB76 exhibited a significant increase in LCA content. DAP-seq analysis of GiMYB76 revealed potential target genes involved in flavonoid biosynthesis regulation. Subsequent promoter activity assay verified that GiMYB76 can bind to the promoter and activate the expression of GiCHS4. Consistently, overexpression of GiCHS4 in G. inflata hairy roots also significantly enhanced LCA production. This study not only clarifies that GiMYB76 transcriptionally activated GiCHS4 to promote LCA biosynthesis but also provides valuable insights for basic research on licorice and the development of related industries.

Penthorum chinense Pursh has been used for centuries as an herbal medicine and food in East Asia. The main active substances in P. chinense are galloylated macrocyclic polyphenolic compounds, which have excellent medicinal properties. Galloylation and glycosylation are key steps in the formation of polyphenolic compounds, as the glycosylation of flavonoids is required for the acylation of flavonoid glycosides, and the glycosylation of gallic acid is necessary for its role as an acyl donor. Therefore, glycosylation to generate the acyl donor or acceptor is a core step in the biosynthesis of polyphenolic compounds. However, how this glycosylation occurs in P. chinense is unknown. In this study, we determined that the UDP-glucose transferase PcUGT84A82 mediates the glycosylation of gallic acid and pinocembrin to produce 1-O-Galloyl-β-D-glucose and pinocembroside, respectively. Metabolic profiling of polyphenolic compounds using UHPLC-ESI-Q-TOF/MS revealed high levels of polyphenols in flowers, leaves, and roots, and low levels in stems of P. chinense. We performed isoform-sequencing (Iso-seq) to assemble a full-length transcriptome of P. chinense, from which we identified 58 UGT family members. PcUGT84A82 is highly similar to functional UGTs in other plant species, and PcUGT84A82 transcript levels were positively correlated with the levels of various polyphenolic compounds. We validated the function of PcUGT84A82 via in vitro enzyme assays and transient expression in Nicotiana benthamiana leaves. Subcellular localization tests showed that PcUGT84A82 localizes to the nucleus and cytoplasm. In summary, PcUGT84A82 catalyzes the conversion of gallic acid to 1-O-Galloyl-β-D-glucose as the acyl donor and pinocembrin to pinocembroside as the acyl acceptor, mediating the biosynthesis of galloylated macrocyclic polyphenolic compounds in P. chinense. These findings lay the foundation for elucidating the entire biosynthetic pathway of active polyphenols in this important herbal plant species.

The yam Dioscorea alata L. is widely cultivated globally. Purple-fleshed varieties of this important crop have enhanced market value due to their high anthocyanin contents, but how anthocyanin biosynthesis in D. alata tubers is regulated remains poorly understood. In this study, we identified and functionally validated key transcription factors that regulate anthocyanin biosynthesis based on a comparative transcriptome and metabolome analysis of three D. alata cultivars with different colored tubers (dark purple, light purple, and white). The anthocyanin glycoside cyanidin-3-O-(2′′-O-glucosyl) glucoside was abundant during early tuber development, and we determined that its accumulation is regulated in opposite manners by two R2R3-MYB transcription factors: DaMYB75 and DaMYB56. Yeast two-hybrid and bimolecular fluorescence complementation assays in Nicotiana benthamiana and co-expression assays in D. alata demonstrated that DaMYB75 promotes anthocyanin biosynthesis by specifically activating the promoter of the late anthocyanin biosynthesis gene DaANS and enhancing its expression through an interaction with DabHLH72. By contrast, DaMYB56 is a negative regulator of anthocyanin biosynthesis that binds to the DaANS promoter together with DabHLH72. Furthermore, the methylation levels of the DaMYB75 promoter were significantly lower in purple tubers than in white tubers. These findings shed light on the regulation of anthocyanin biosynthesis by MYBs and provide the basis for genetically improving anthocyanin content in D. alata.

Zanthoxylum bungeanum is an economically important crop worldwide due to its high content of aroma-producing monoterpenoids, and development of varieties with enhanced flavor and overall quality is a crucial research area. However, the transcriptional regulatory mechanisms underlying monoterpenoid synthesis in Z. bungeanum remain unclear, hindering these breeding efforts. In this study, RNA sequencing, gas chromatography-mass spectrometry, and other molecular biology techniques were used to identify the underlying transcriptional regulation mechanisms. Two transcription factors, ZbbHLH2 and ZbERF6, were identified as key regulators of monoterpenoid synthesis in Z. bungeanum that upregulate various monoterpenoid synthesis-associated genes and are novel transcriptional activators of ZbIDI, which encodes the rate-limiting enzyme in plant monoterpenoid synthesis. Functional analysis revealed that the expression of three genes [1] modulates monoterpenoid accumulation in Z. bungeanum peel. These findings provide novel insights into the metabolic regulatory network of monoterpenoid synthesis in Z. bungeanum peel, offer potential strategies for the biofortification of specific monoterpenoids, and will promote the development of Z. bungeanum germplasm for targeted breeding and quality improvement.

Natural colored cotton (NCC) offers a sustainable, dye-free, and eco-friendly alternative for producing colored textiles. Carotenoids, a group of important natural liposoluble pigments, are known for their diverse color spectrum. In this study, we successfully engineered the carotenoid biosynthesis pathway specifically in cotton fibers by utilizing a fiber-specific GbEXPA2 promoter and a CaMV 35S promoter to drive the expression of two key carotenoid biosynthesis genes, CrtB and CrtI, respectively. This approach resulted in the development of a golden fiber cotton germplasm enriched with β-carotene. Notably, the pigmentation was predominantly observed during the early developmental stages of the fiber (5-20 d post-anthesis). While the presence of carotenoids had no significant effect on plant architecture and growth, it positively influenced the fiber elongation rate, albeit with a slight reduction in fiber length and strength. This study represents a pioneering strategy for the future development of NCCs through carotenoid biofortification.

Iris domestica, a perennial herb of the Iridaceae family, is widely recognized for its rich isoflavone content and broad therapeutic properties. To elucidate the biosynthetic pathway of these medicinally significant compounds, we constructed a haplotype-resolved genome assembly of this species. Transcriptomic and metabolomic analyses revealed tissue-specific accumulation of isoflavone, particularly in rhizomes and roots. Functional characterization identified two candidate isoflavone synthase genes, among which IdIFS was confirmed to promote the biosynthesis of key compounds tectorigenin and irisflorentin. The high-quality genome assembly presented here provides a foundational resource for further research into the evolution, secondary metabolite, and environmental adaptation of I. domestica.

The demand for Erigeron breviscapus, a medicinal Compositae plant with cardiovascular therapeutic properties, has been increasing by 15% annually, exceeding production capacity and necessitating improvements in yield and bioactive compound content. Genetic transformation remains essential for functional genomics, yet current Agrobacterium and biolistic methods are inefficient and expensive. In this study, we cloned the full-length sequences of the BABY BOOM, WUSCHEL and GROWTH-REGULATING FACTOR (GRF) genes of E. breviscapus and then transformed them into E. breviscapus explants. The transformation efficiency for the GRF gene reached 45%, and all the transgenic E. breviscapus plants were fertile without obvious developmental defects. Furthermore, we inserted EbGRF4 and Cas9-EbPDS-sgRNA into the same vector for Agrobacterium-mediated transformation to effectively knock out the PDS gene, resulting in albino seedlings, with a gene editing efficiency of 33.3%. These findings provide a solid foundation for functional genomic research and the genetic improvement of E. breviscapus, as well as an important reference for establishing high-efficiency genetic transformation systems for other medicinal plants.