Objective 3-Dehydroquinic acid dehydrogenase/shikimate dehydrogenase (DHD/SDH) is the sole bifunctional enzyme in the plant shikimate pathway, catalysing both shikimic acid formation and its subsequent conversion to gallic acid. As a key regulatory node in gallic acid biosynthesis, DHD/SDH is implicated in the production of downstream derivatives, including ellagic acid. In this study, we systematically analysed the RcDHD/SDH gene family in Rubus chingii Hu ( R. chingii ), with the aim of elucidating their regulatory features and expression patterns across metabolic pathways, characterising tissue- and fruit developmental stage-specific expression profiles, and evaluating their potential association with gallic acid and ellagic acid accumulation. Methods In this study, bioinformatics approaches were employed to identify members of the DHD/SDH gene family in R. chingii . The physicochemical properties, predicted subcellular localisations, chromosomal distributions, phylogenetic relationships and promoter cis-acting element compositions of the encoded proteins were systematically analysed. Quantitative real-time PCR (qRT-PCR) was used to evaluate the expression levels of individual DHD/SDH genes across different tissues and organs, during fruit developmental stages, and in response to exogenous hormone treatments. In parallel, high-performance liquid chromatography (HPLC) was conducted to quantify gallic acid and ellagic acid contents in the corresponding tissues and fruit developmental stages. These integrated analyses enabled assessment of potential associations between DHD/SDH gene expression patterns and metabolite accumulation in R. chingii . Results A total of four DHD/SDH family members ( RcDHD/SDH1 – RcDHD/SDH4 ) were identified in Rubus chingii Hu. These genes were evenly distributed across two chromosomes and were predicted to encode proteins ranging from 440 to 1,325 amino acids in length. Phylogenetic analysis indicated that all RcDHD/SDH proteins clustered within subfamily IV. Subcellular localisation predictions suggested that RcDHD/SDH proteins are localised to both the nucleus and the cytoplasm, and that all members contain the conserved DHD/SDH functional domain. Promoter analysis revealed that RcDHD/SDH genes harbour diverse cis-acting elements, including hormone-responsive, light-responsive, and low-temperature-responsive elements. qRT-PCR analysis demonstrated that the RcDHD/SDH gene family exhibits tissue-specific expression patterns in R. chingii . RcDHD/SDH1 - RcDHD/SDH4 exhibited relatively stable expression in roots, whereas RcDHD/SDH4 displayed comparatively high expression levels in stems and leaves. During fruit development, the overall expression of RcDHD/SDH genes was highest at the early stages and subsequently declined. This expression trend closely corresponded with the rapid accumulation of gallic acid and ellagic acid from the green fruit stage to the colour transition stage, followed by stabilisation at fruit maturity. These results suggest that RcDHD/SDH genes play an important regulatory role during the early stages of fruit growth and development. Conclusion This study systematically characterised the composition, structural features, and expression patterns of the RcDHD/SDH gene family in R. chingii, providing a theoretical foundation for further investigation of their biological functions and roles in secondary metabolite regulation.

Acne is a chronic inflammatory skin disease closely associated with aberrant immune activation and inflammatory cell recruitment. In this study, acne-related expression profile data from the GSE53795 dataset were used to identify differentially expressed genes between inflammatory acne lesions and non-lesional skin tissues. Functional analysis indicated that SELE, a cell adhesion molecule involved in leukocyte adhesion, rolling, and migration, may play an important role in acne-related inflammation. Further transcription factor prediction suggested that RELA/NF-κB p65, a key regulator of inflammatory responses, may regulate SELE and related inflammatory genes. Given the anti-inflammatory, antioxidant, and immunomodulatory activities of Panax notoginseng (P. notoginseng) saponins, this study further discusses the potential application of P. notoginseng as a natural active ingredient in acne-prone skincare. With the increasing market demand for natural-derived skincare ingredients, P. notoginseng shows promising value for the development of soothing, anti-inflammatory, and functional cosmetic raw materials, providing a new strategy for its high-value utilisation.

The genus Dendrobium is a renowned traditional Chinese medicinal plant and one of the most extensively studied and widely applied species within the Dendrobium genus, possessing significant medicinal value and industrial development potential. This paper reviews the resource distribution and quality correlation, development trends, chemical constituents, biological activities, clinical applications, and current product development status of Dendrobium officinale and other medicinal plants within the Dendrobium genus. It highlights the structural characteristics and pharmacological effects of key active components such as polysaccharides, alkaloids, and dibenzyl compounds, summarizes their mechanisms of action in immune regulation, antioxidant properties, and eye health protection, and supplements the influence of different origins and cultivation models on active components. The paper also provides an outlook on the current status of product development and future trends.

Aurantio-obtusin is one of the major active components in Senna tora (S. tora) seeds. Although the ubiquitin receptor DA1 regulates plant seed size, it remains unclear if StDA1 regulates the accumulation of aurantio-obtusin in S. tora seeds. In this study, StDA1 is identified as a negative regulator of aurantio-obtusin production in S. tora seeds. Antisense overexpression of StDA1 led to larger seeds in S. tora and promoted the accumulation of aurantio-obtusin. In contrast, overexpression of StDA1 resulted in a decrease in aurantio-obtusin accumulation in seeds. Moreover, using StDA1 as a bait protein, we identified as an interacting factor, StHDR1, a key enzyme in the aurantio-obtusin biosynthesis pathway. StHDR1 is able to positively regulate aurantio-obtusin accumulation in S. tora seeds, which is the opposite effect of StDA1. Through its interaction with StHDR1, StDA1 promotes StHDR1 degradation via the 26S proteasome, thereby reducing the accumulation of aurantio-obtusin. In conclusion, StDA1 is a regulator that plays a role in the accumulation of aurantio-obtusin in S.tora seeds by modulating the stability of the StHDR1 protein. This discovery points to the StDA1-StHDR1 complex as a key regulatory node that could potentially be targeted to enhance the accumulation of aurantio-obtusin in S. tora seeds.

Ganoderic acids (GAs), the major lanostane-type triterpenoids in Ganoderma lucidum (G. lucidum), possess diverse pharmacological activities but are produced at low levels, limiting their applications. Transcriptional regulation of GA biosynthesis remains poorly understood. In this study, we functionally characterized the GlZn(2)Cys(6)-type transcription factor GlZn(2)Cys(6)_82 and elucidated its role in GA biosynthesis. Overexpression of GlZn(2)Cys(6)_82 significantly increased total triterpenoids during the mycelial stage and promoted the accumulation of multiple individual GAs at the primordia stage, whereas silencing resulted in markedly reduced GA levels. qRT-PCR analyses revealed that GlZn(2)Cys(6)_82 positively regulates key biosynthetic genes, including HMGR, SQS, and LS, with SQS showing the strongest transcriptional induction. Subcellular localization assays demonstrated that GlZn(2)Cys(6)_82 is a nuclear protein, and yeast one-hybrid analyses confirmed its direct binding to the SQS promoter, establishing SQS as a downstream target. Comparative transcriptome profiling further showed that GlZn(2)Cys(6)_82 modulates genes involved in the mevalonate pathway and secondary metabolism. Collectively, our findings identify GlZn(2)Cys(6)_82 as a positive regulator of GA biosynthesis and provide a basis for metabolic engineering to increase GA production. [GRAPHICS]

Aflatoxin contamination caused by Aspergillus flavus represents a critical threat to global food safety and agricultural sustainability, yet the mechanisms underlying its effective biological control remain poorly understood, particularly at the spatial and metabolic levels. Here, we systematically investigated the biocontrol potential of Bacillus velezensis B2 and elucidated the mechanisms of its bioactive metabolites using an integrated multi-omics strategy. Chemical profiling and purification identified surfactin-iturin complexes (SLCs) as the major antifungal components. Functional assays demonstrated that SLCs completely inhibited spore germination at 160 μg/mL (MIC = 0.125 mg/mL) and exhibited approximately 10-fold higher antifungal potency than the crude extract. SLC treatment also induced severe hyphal deformities, including swelling and membrane disruption. Spatial metabolomics using DESI-MSI revealed a dynamic metabolic shift from m/z 1020 to m/z 1034 during coculture, with surfactin-type metabolites dominating the interspecies interaction interface. Transcriptomic analysis identified 3560 differentially expressed genes (1559 upregulated and 2001 downregulated), and demonstrated that SLCs globally suppressed the aflatoxin biosynthetic pathway by downregulating key genes, including aflK, aflQ, aflP, aflO, aflM, aflE, aflH, and aflT. Consistently, no detectable AFB1 was observed in treated samples (LOD = 0.05 μg/kg). Collectively, these findings demonstrate that SLCs inhibit aflatoxin production through combined structural damage and metabolic reprogramming, and highlight a spatially resolved multi-omics framework for deciphering microbial interactions and developing effective biocontrol strategies in food safety.

OBJECTIVE:In this study, the heat shock transcription factor (HSF) gene family in Ganoderma lucidum was systematically characterized. Using genomic and transcriptomic data, we identified HSF family members and investigated their expression patterns under temperature stress and their potential regulatory roles in triterpenoid biosynthesis. METHODS:A genome-wide identification of HSF genes in G. lucidum was performed using bioinformatic approaches. A phylogenetic tree was constructed, and conserved motifs, gene structures, and protein tertiary structures were predicted. The relative expression levels of HSF genes and key mevalonate (MVA) pathway enzyme genes were examined by a quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) in mycelia subjected to temperature stress. Total triterpenoid content in fermented mycelia under temperature stress was determined using the vanillin-glacial acetic acid method. RESULTS:Eight HSF family members (GlHSF1-GlHSF8) were identified in G. lucidum. Phylogenetic analysis revealed that GlHSF proteins were closely related to PoHSF from Pleurotus ostreatus. Transcriptomic analysis showed that HSF genes exhibited relatively high expression levels during the mature stage while being barely expressed during the mycelial stage. Under heat stress (42 °C), most GlHSF genes peaked at 18 h, with GlHSF2 showing the most pronounced response (approximately 13-fold upregulation). Downstream MVA pathway genes, including IDI, PMK, and MVD, were significantly upregulated at 24 h, whereas the upstream rate-limiting enzyme gene HMGR was continuously suppressed. Despite HMGR suppression, total triterpenoid content did not decrease significantly, likely due to the activation of downstream genes. Under cold stress (14 °C), the expression of most GlHSF and MVA pathway genes decreased, accompanied by a significant reduction in total triterpenoid content. CONCLUSIONS:The HSF gene family was identified in the G. lucidum genome. Based on expression analysis, GlHSF2 showed the strongest response under heat stress, and its expression peak was correlated with the sequential activation of downstream genes in the MVA pathway. This suggests that GlHSF2 acts as a potential key regulatory node, differentially regulating upstream and downstream MVA pathway genes to influence triterpenoid biosynthesis under heat stress. These findings provide a theoretical basis for future research on the biological functions of GlHSF homeostasis.

Abstract How transcription factor condensate formation contributes to specialized metabolism in medicinal plants remains poorly understood. Here we show that SmbHLH10, a basic helix–loop–helix transcription factor from Salvia miltiorrhiza, promotes tanshinone biosynthesis. SmbHLH10 formed nuclear condensate-like structures in vivo and droplets in vitro, supporting its capacity for condensate formation under the tested conditions. IDR2, an intrinsically disordered region of SmbHLH10, was required for SmbHLH10 condensate formation and acted as a regulatory module contributing to promoter-associated transcriptional activation and tanshinone accumulation. These results support a model in which SmbHLH10 condensate formation participates in tanshinone pathway regulation and suggest that transcription factor condensates contribute to pathway-specific metabolic control in medicinal plants.

IntroductionMicroRNAs (miRNAs) are key post-transcriptional regulators of plant secondary metabolism. Their primary mechanism involves silencing target genes through mRNA cleavage or translational inhibition, which is a major focus of current research in this field. However, the specific regulatory roles of individual miRNAs in coordinating different secondary metabolic pathways in medicinal plants remain largely uncharacterized.MethodsThis study investigated the roles of Smi-miR164a in Salvia miltiorrhiza. We generated Smi-miR164a-overexpressing (OE-miR164a) transgenic lines and performed comprehensive metabolic profiling and gene expression analysis.ResultsOverexpression of Smi-miR164a resulted in significant accumulation of phenolic acids, with rosmarinic acid (RA) and salvianolic acid B (SalB) levels increased by up to 2.8-fold compared to wild-type (WT). Conversely, it markedly reduced the accumulation of tanshinones, decreasing tanshinone I (T-I) and tanshinone IIA (T-IIA) to 25-68% of WT levels. Transcriptional analysis showed that expression changes in key biosynthetic genes were tightly correlated with the metabolic alterations. Genes involved in the tanshinone pathway (e.g., HMGR1, DXS2) were downregulated, whereas those in the salvianolic acid pathway (e.g., PAL1, C4H) were upregulated, consistent with the reciprocal accumulation of their corresponding metabolites.ConclusionThese findings demonstrate that the Smi-miR164a module acts as a pivotal regulator, positively influencing phenolic acid biosynthesis while negatively regulating tanshinone production in S. miltiorrhiza. This gene presents a promising target for molecular breeding aimed at enhancing the yield of specific bioactive compounds.

The seeds of Senna tora L.are the core organs for the biosynthesis of anthraquinones,and their medicinal value is highly dependent on the content of anthraquinones.However,TCP transcription factors play a key regulatory role in the biosynthesis of plant secondary metabolites.Nevertheless,whether TCP transcription factors regulate the accumulation of anthraquinone compounds has not been reported.Therefore,in this study,qRT-PCR analysis was performed to determine the expression pattern of StTCP11 at different growth and development stages of S.tora seeds.An overexpression vector was constructed to obtain two types of S.tora lines:StTCP11-overexpressing lines and empty vector(control)lines,which were used to explore the effect of StTCP11 on the accumulation of anthraquinones in seeds.The interactions between StTCP11 and key enzymes in anthraquinone biosynthesis were analyzed by yeast one-hybrid(Y1H)and dual-luciferase(Dual-LUC)reporter gene assays.The results showed that the expression level of StTCP11 was higher at the late stage of seed development.Compared with the control,the StTCP11-overexpressing lines showed significantly increased content of aurantio-obtusin,chrysophanol,emodin,and physcion in the seeds.Y1H and Dual-LUC assays confirmed that StTCP11 could directly bind to the promoter of StDXS and activate its transcription,thereby positively regulating the accumulation of anthraquinones.This study revealed the molecular mechanism of StTCP11 in promoting anthraquinone synthesis in S.tora seeds through targeted regulation of StDXS transcription,which provided a key theoretical basis for resolving the quality regulation network of S.tora seeds and breeding new high-quality varieties.

Plant-derived natural products offer a rich source of therapeutic agents. However, sustainable and high-yield production remains a grand challenge. We engineered Salvia miltiorrhiza hairy roots to produce taxadiene, a key precursor for the anticancer drug paclitaxel, and protopanaxadiol, a precursor for ginsenosides. The heterologous expression of two key biosynthetic genes, taxadiene synthase from Taxus wallichiana and protopanaxadiol synthase from Panax notoginseng, enabled the production of taxadiene and protopanaxadiol, respectively. Our strategy combined multiple approaches to enhance terpenoid production, including genome editing to redirect metabolic flux by eliminating a competing GGPP sink (via SmCPS1 disruption), transcriptional reprogramming through SmWRKY61 overexpression to enhance terpenoid precursor pathways (MVA/MEP), and optimization of cultivation conditions. This holistic approach yielded 65.17 ± 5.25 mg/kg fresh weight (FW) taxadiene in batch cultures, and the protopanaxadiol yield reached 50.04 ± 2.94 mg/kg dry weight (DW) without optimization. These results highlight the potential of this platform for industrial-scale production. Our findings demonstrate that S. miltiorrhiza hairy roots can serve as a robust and scalable platform to produce valuable plant-derived compounds. This work paves the way for future metabolic engineering efforts to achieve cost-effective and sustainable production of high-value natural products using medicinal plant systems, addressing critical supply bottlenecks for pharmaceutical compounds.

Sucrose synthase (SUS) is a key enzyme in plant carbon metabolism, catalyzing the reversible interconversion between sucrose + uridine diphosphate (UDP) and UDP-glucose (UDP-Glc) + fructose. It plays a central role in carbon flux allocation, cell wall and starch synthesis, as well as plant development and stress responses. SUS is encoded by a multigene family whose members exhibit significant functional diversification and expression specificity across species, tissues, and subcellular compartments. This review systematically summarizes the physiological functions of SUS in source-sink regulation, seed filling, and rapidly growing tissues; describes the organ-specific expression patterns and diverse subcellular localizations of different isoenzymes in Arabidopsis and major crops; and elucidates the phylogenetic pattern of the SUS gene family into three evolutionary clades-SUS I, SUS II, and SUS III-based on a comparative analysis of selected angiosperm species. Furthermore, it integrates the multi-level regulatory mechanisms of SUS, including transcriptional and post-transcriptional regulation, as well as the dynamic control of enzyme activity, stability, and subcellular localization through post-translational modifications such as phosphorylation and ubiquitination and protein interactions. Finally, this study identifies gaps in current research regarding ubiquitination mechanisms, metabolic network integration, and crop applications. It envisions SUS-centered molecular breeding strategies, informed by integrative regulatory genomics, multi-omics, and genome editing, to redirect crop carbon fluxes and thereby enhance yield, improve quality traits, and increase stress tolerance.

Tetrastigma hemsleyanum Diels et Gilg (T. hemsleyanum) is a plant of considerable medicinal and economic value. However, the molecular mechanisms underlying its tuberous root formation remain poorly understood. To investigate the molecular basis of tuberous root formation, we analyzed hormonal metabolic levels, transcriptomic profiles, and root anatomical changes during this process. Using ultra-performance liquid chromatography-electrospray ionization tandem mass spectrometry, we quantitatively assessed the levels of eight plant hormones and their derivatives in the early stages of tuberous root formation and in adventitious roots. The results revealed significant fluctuations in hormone levels, with a marked upregulation of cytokinins (tZ, DZ, and IP) and the complete absence of gibberellin GA1 post-tuberous root formation. Jasmonic acid content decreased, while methyl jasmonate (MeJA) increased substantially. Exogenous application of MeJA further confirmed the role of the jasmonic acid pathway in tuberous root formation, underscoring the pivotal role of these hormones in root differentiation and expansion. Additionally, transcriptomic analysis identified significant alterations in biological processes associated with the cytoskeleton and cell wall during tuberous root formation. Anatomical observations indicated reduced lignification and a notable increase in vascular cambium and xylem parenchyma cells. In conclusion, this study provides valuable insights into the molecular mechanisms of tuberous root formation in T. hemsleyanum, emphasizing the critical role of plant hormones and offering new strategies for enhancing tuber growth and yield through hormonal regulation.

Metabolic dysregulation is closely linked to impaired glucose metabolism and gut microbiota imbalance, and dietary carbohydrates have emerged as important modulators of these processes. In this study, polysaccharides (DOP) from Dendrobium officinale and their enzymatically derived oligosaccharide fraction (DOO1) were systematically characterized and comparatively evaluated in vitro. Enzymatic hydrolysis markedly reduced molecular weight, increased relative mannose content, and altered glycosidic linkages, yielding DOO1 as a structurally heterogeneous glucomannan-type oligosaccharide. DOO1 mainly consisted of a mannose-rich backbone with-*4)-(I-D-Manp-(1-* and-*4,6)-(I-D-Manp-(1-* linkages, along with-*4)-(I-D-Glcp-(1-* and minor-*4)-(I-D-2-O-acetyl-Manp-(1-* units. Functionally, DOO1 displayed higher inhibition of alpha-amylase relative to native DOP and significantly enhanced glucose consumption in palmitic acid treated insulin-resistant HepG2 cells, accompanied by modulation of key metabolic pathways, including arginine biosynthesis, glycerophospholipid metabolism, and the tricarboxylic acid cycle. Resistance to upper gastrointestinal digestion and selective modulation of gut microbial communities were observed in simulated gastrointestinal processing and in vitro fecal fermentation, with a notable increase in the relative abundance of Bacillota and Actinomycetota. Collectively, these findings indicate that enzymatically derived D. officinale oligosaccharides hold considerable promise as functional ingredients for improving metabolic health and supporting gut microbiota balance.

Ganoderic acids (GAs) are high-value lanostane-type triterpenoids produced by Ganoderma lucidum (G. lucidum) and are responsible for its diverse pharmacological activities. Previous studies have elucidated key enzymatic steps of the mevalonate pathway leading to lanosterol formation. However, the transcriptional regulatory mechanisms controlling the extensive downstream oxidation and modification reactions required for GA biosynthesis remain poorly understood. In this study, two transcription factors, GlHMG_9 and GlC2H2.07, were systematically identified and functionally characterized as positive regulators of GA biosynthesis. Overexpression of either transcription factor significantly increased total triterpenoid content, whereas RNAi-mediated silencing resulted in pronounced reductions in total triterpenoids and multiple individual GAs at both mycelial and primordia stages. LC–MS analysis revealed marked decreases in representative GAs upon gene silencing. Subcellular localization confirmed nuclear localization of both proteins. Transcriptome profiling demonstrated that GlHMG_9 and GlC2H2.07 coordinately regulate genes involved in the mevalonate pathway and downstream lanosterol-derived modification steps, including cytochrome P450–mediated oxidation. Notably, GlC2H2.07 influenced a broader spectrum of biosynthesis-related genes than GlHMG_9, as evidenced by a greater number of differentially expressed genes (266 vs. 157, respectively). These findings establish GlHMG_9 and GlC2H2.07 as key transcriptional regulators controlling GA biosynthesis in G. lucidum. This study provides mechanistic insights into transcription factor–mediated metabolic regulation and identifies promising regulatory targets for transcription factor–guided strain optimization, thereby providing foundational knowledge for the future development of high-yield G. lucidum cell factories through further optimization and validation.

Peucedanum praeruptorum Dunn, known as “Qianhu”, is a perennial herb with high value in traditional Chinese medicine. However, early bolting during cultivation significantly reduces both quality and yield, posing major challenges for its agricultural and industrial application. Our study systematically examined phenotypic traits, enzyme activity, and the levels of active ingredients. We also applied exogenous hormones to assess their effects on bolting and yield. Furthermore, we integrated second- and third-generation transcriptomic sequencing to identify genes associated with bolting. Comparative phenotypic analysis showed that both aboveground and underground parts grew more rapidly in bolting plants than in unbolting individuals. We observed increased enzyme activities such as POD and SOD, and higher MDA content in bolting plants, while CAT and APX activities decreased. A total of 452,720 ROI sequences were obtained from the PacBio platform, comprising 726,045,259 bases with an average length of 1603 base pairs. Enrichment analysis of differentially expressed genes identified two critical pathways associated with bolting: the photoperiod and vernalization pathways. Weighted gene co-expression network analysis identified seven genes associated with bolting: NAC078, NFYC3, VOZ1, BRN1, CRY2, COL5, and 3-MMP. Overall, this study identifies key candidate genes and co-expression networks associated with early bolting in P. praeruptorum, providing new insights into its transcriptional regulatory mechanisms.

Ganoderic acids (GAs) are high-value lanostane-type triterpenoids derived from Ganoderma lucidum (G. lucidum) with broad applications in functional foods and nutraceuticals, yet their low natural abundance limits industrial production. In this study, an integrated life-cycle multiomics analysis combining metabolomics, transcriptomics, and proteomics was conducted across six developmental stages in four G. lucidum strains to elucidate regulatory mechanisms governing GA biosynthesis. Weighted gene coexpression network analysis identified candidate cytochrome P450 enzymes and transcription factors associated with GA accumulation. A Zn(2)Cys(6)-type transcription factor, Zn(2)Cys(6)_61, was identified as a central regulator and functionally validated through overexpression and RNA interference. Genetic manipulation of Zn(2)Cys(6)_61 expression significantly altered GA levels, with overexpression markedly enhancing GA accumulation. Further analysis demonstrated that Zn(2)Cys(6)_61 directly binds to and activates the promoter of squalene synthase, a key enzyme in triterpenoid backbone biosynthesis. Together, these findings identify Zn(2)Cys(6)_61 as an effective engineering target and provide a transcription factor-based strategy for improving GA production in medicinal mushrooms.

GA is an important phytohormone that regulates root growth and secondary metabolism. GRAS family transcription factors (TFs) are the key regulators of GA signaling. Here, we found that SmGRAS5 was co-expressed in the root periderm with tanshinones in Salvia miltiorrhiza. Overexpression (OE) of SmGRAS5 increased tanshinones accumulation and upregulated the biosynthetic genes. Antisense expression (AE) of SmGRAS5 reduced tanshinones accumulation and downregulated the biosynthetic genes. Yeast one-hybrid (Y1H), dual-luciferase (Dual-LUC), and electrophoretic mobility shift assays (EMSA) showed that SmGRAS5 promoted tanshinones biosynthesis by directly binding to the GARE motif in the promoter of SmKSL1 to induce its expression. However, overexpressing SmGRAS5 reduced GA content through downregulating the biosynthetic genes and also reduced root biomass. GA treatment further increased tanshinones accumulation and restored the root growth inhibited by overexpressing SmGRAS5. SmGRAS5 could not directly bind to the GA biosynthetic genes. Transcriptome analysis revealed the potential functions of SmGRAS5 in regulating secondary metabolism. Taken together, SmGRAS5 is involved in the regulation of GA-promoted tanshinones biosynthesis by directly activating the expression of SmKSL1, which suggests that SmGRAS5 may be a potential target for further metabolic engineering of tanshinones biosynthesis in S. miltiorrhiza.

Triacylglycerol (TAG) is a major component of plant-neutral lipids. Diacylglycerol acyltransferase 2 (DGAT2) plays an important role in plant oil accumulation by catalyzing the final step of the Kennedy pathway. In this study, ten DGAT2 sequences were originating from different oil crops into the TAG-deficient yeast strain H1246, to compare their enzyme activity of oil synthesis and filter out potential amino acid residue sites for directed evolution. Based on the synthesis efficiency of total lipids, TAGs, and the topology models of these DGAT2s, five possible amino acid sites were identified that may affect the synthesis of total lipids and TAGs. In the H1246 yeast expression system, HaDGAT2 significantly increased the total oil and TAG content; however, ClDGAT2 was weak in synthesizing both oil and TAG. Thus, building on HaDGAT2 and ClDGAT2, these amino acid substitutions were created by point-to-point mutating and substantially affected the oil or TAG synthesis ability of DGAT2s. Among the five amino acid substitutions, mutations at residue (3) successfully make HaDGAT2 less capable of synthesizing lipids and TAG, and ClDGAT2 more capable of synthesizing total lipids and TAG. Except mutations at residue (2), all residue mutations contributed to a weaker ability of fatty acid synthesis. In addition, ten mutant DGAT2s and two parental DGAT2s were overexpressed in tobacco leaves to reveal their lipid synthesis function. This approach helped us to authenticate the significance of these loci. In varying degrees, those mutations enhanced the ability of ClDGAT2 to synthesize lipids, attenuated the ability of HaDGAT2 to synthesize lipids, and altered preference for fatty acids in tobacco.