Fruit quality is determined by multiple metabolites, which are mainly affected by plant hormones. In this study, two genes, ethylene-related gene SlACO1 and gibberellin-related gene SlGARP, were overexpressed (OE) and knocked down through RNAi in tomato, and the profiles of carotenoids, flavonoids, volatiles, and hormones were detected in the leaves and fruit. The color index significantly increased in SlACO1-OE fruit but significantly decreased in the SlACO1-RNAi line. Similarly, total carotenoids, volatiles, salicylic acid, and ethylene significantly increased in the fruits of SlACO1-OE and SlGARP-OE, whereas ABA decreased significantly. Some compounds, such as lycopene, 3-hexenal, and d-limonene, significantly increased in the fruit of SlACO1-OE but decreased in the SlACO1-RNAi line, indicating that SlACO1 might play an important role in the accumulation of these compounds. The functional characterization of SlACO1 and SlGARP would facilitate the improvement in tomato fruit quality.

The sesquiterpene synthase BgPgS from Burkholderia gladioli produces the main product (-)-1-epi-pacifigorgia-6,10-diene, besides a few structurally related compounds. The enzyme mechanism of BgPgS was addressed through isotopic labeling experiments, revealing several intricate mechanistic problems. Unexpectedly, the isotopic labelings for the Me groups C12 and C13 occurred in different positions for the main and the side products. Moreover, as demonstrated in this study, two hydrogen atoms must change from the bottom to the top hemisphere, which is not possible through standard terpene biosynthesis routines with suprafacial hydrogen migrations. Our rational solution involves two mechanistic explanations: First, a key rearrangement may be associated with a conformational change that rotates one hydrogen from bottom to top. Second, a "break-flip-cyclize" sequence explains the change of side by the other hydrogen. DFT calculations show that the proposed terpene cyclization cascades are energetically feasible; only one problematic activation barrier (>25 kcal/mol) remains. However, several mechanistic alternatives either failed to explain the experimental results of the isotopic labeling experiments or were associated with even higher activation barriers. Our biosynthetic proposal for pacifigorgiadiene biosynthesis can be understood as a contribution that awaits further investigation and scientific debate for its ultimate resolution.

Fruit secondary metabolites play pivotal roles in plant evolution by deterring herbivores and attracting seed dispersers. However, the mechanisms by which these compounds evolve and drive diversification in citrus remain poorly understood. In this study, we demonstrate that the emergence of the bitter compound neohesperidoside (Neo) has contributed to citrus dissemination by enhancing defense against biotic stresses. Targeted metabolomic analyses revealed that Neo accumulation emerged in early-diverging citrus lineages, whereas its non-bitter counterpart rutinoside (Rut) can be traced back to Citrus-related species. Comparative genomic analyses and enzyme functional assays further revealed that Neo biosynthesis originated from the duplication of two di-glucosyltransferase genes, CmdGlcT-1 and UGT79B203, in earlydiverging citrus, followed by neofunctionalization into Cm1,2RhaT and UGT79B202, enzymes capable of synthesizing Neo. A structurally conserved amino acid residue-corresponding to Phe195 in Cm1,2RhaT and Leu201 in UGT79B203-was identified as critical for this functional transition. Compared with Rut, Neo exhibits stronger antifungal and anti-feeding activities, suggesting a role in enhanced biotic defense that may have contributed to the broader geographical distribution of early-diverging citrus species. Together, these findings provide new insights into the evolutionary origin of citrus bitterness and highlight the adaptive role of specialized metabolites in shaping plant-environment interactions.

Terpenoids constitute the largest and most structurally diverse family of natural products, and bacterial genomes harbor vast yet largely unexplored biosynthetic potential. Here, we performed large-scale genome mining combined with heterologous expression in yeast to systematically screen 313 bacterial type I terpene synthases, leading to the identification of 16 active diterpene synthases (DTSs) and the discovery of 10 previously unknown diterpenes, including 5 unprecedented carbon skeletons. The DTS ShHS from Streptomyces hundungensis produces a series of highly rearranged diterpenes featuring the complex hundungane scaffold, whereas CbCS, which shares identical early cyclization steps with ShHS, generates the structurally simpler sphaeroane skeleton. Isotope-labeling experiments in combination with density functional theory calculations reveal an intricate carbocation cascade and identify a key branching intermediate that governs skeletal divergence. The crystal structure of CbCS allowed for identification of active-site residues responsible for functional differentiation. Structure-guided mutagenesis enabled functional interconversion between complex and simple diterpene skeletons. These findings expand the known chemical space of bacterial diterpenes and demonstrate how subtle active-site features precisely control carbocation rearrangement trajectories in terpene biosynthesis.

Aroma is a key attribute of peach fruits, yet the molecular basis of aroma differences among peach types remains poorly understood. This study comprehensively characterized the aroma profiles and identified key odor-active compounds distinguishing peaches from nectarines and across different flesh colors. Sensory evaluation using Check-All-That-Apply (CATA) revealed stronger fruity, sweet, floral, creamy, and tropical notes in peaches, whereas nectarine exhibited more green and waxy attributes. HS-SPME-GC-MS analysis of 44 peach cultivars identified 80 volatile compounds, and multivariate analysis (OPLS-DA) combined with relative odor activity values (ROAVs) showed that skin hairiness and flesh color significantly influence aroma composition. Nine and eight key volatiles were identified as major contributors to peach-nectarines differentiation and flesh color variation, respectively. Partial Least Squares Regression (PLSR) analysis suggested that lactones, terpenes, and norisoprenoids drive fruity, sweet, and floral notes in peaches, while some C6 compounds contribute to green and waxy attributes in nectarines. Furthermore, aroma addition and omission tests demonstrated the importance of key compounds, including linalool, β-ionone, γ-decalactone, hexyl acetate and nonanal for peaches, and linalool, (E)-2-hexenal and hexanol for nectarines in partially reconstructing characteristic aromas. These findings provide mechanistic insights into aroma differentiation and offer practical guidance for flavor-oriented breeding strategies.

Aroma differentiation is a key trait that distinguishes citrus and other horticultural crops from staple crops. However, the mechanistic basis and sensory features of the distinctive and varied citrus-like aromas of citrus remain poorly understood. In this study, we demonstrated that γ-terpinene determines tangerine-like aroma, affects consumer preference, and has pest-repellent properties. Both forward and reverse genetic analyses uncovered the pivotal role of CreTPS3a in γ-terpinene biosynthesis. In addition, we identified a solo long terminal repeat (solo-LTR) insertion upstream of the CreTPS3a promoter in MD1-type domesticated mandarins. We found that the transcription factor CreARF2 specifically binds to this solo-LTR and positively regulates CreTPS3a expression and γ-terpinene accumulation. Notably, this regulatory mechanism may be associated with the geographic distribution patterns of tangerine germplasms. By integrating sensory evaluation with insect behavioral assays, we identified a γ-terpinene sensory threshold of approximately 50 μg/g, which optimally balances pest-repellent properties with consumer preference. Collectively, these findings reveal the molecular mechanisms that underlie the production of tangerine-like aroma, illustrate the complex interactions among citrus plants, human beings, and insects, and offer new possibilities for the development of innovative, eco-friendly strategies that may simultaneously enhance fruit aroma and strengthen plant defense against pests.

Mei (Prunus mume), the only floral-scented species within the Prunus genus, holds exceptional ornamental, edible, and medicinal value. By synergizing headspace solid-phase microextraction (HS-SPME) and organic solvent extraction (OSE) coupled with gas chromatography-mass spectrometry (GC-MS), we systematically detected 103 emitted and 83 endogenous volatiles from 67 mei cultivars and 3 congeneric species, constructing a comprehensive volatile metabolite library to date. Crucially, 17 emitted and 23 endogenous volatiles were identified as main volatiles, dominated by aldehydes, alcohols, esters, and phenols. Six distinct aroma types were identified through sensory evaluation, with the sweet aroma type exhibiting the highest discriminability and preference. Metabolomics-sensomics integration revealed benzyl alcohol, eugenol, and benzyl acetate as characteristic aroma compounds defining mei's distinctive sweet aroma. Subsequently, 26 mei cultivars with potential application value were selected. In all, the results provides solid dataset for breeding and utilization of mei resources, bridging the gap between aroma science and horticultural innovation.

Diterpene synthase plays an important role in the biosynthesis of novel diterpenoids with diverse biological activities. In this study, two uncommon diterpene synthases, AsSS and CvSS, were discovered in bacteria, showing only a 26% sequence identity. Unexpectedly, sphaerols A (1) and B (2) with an interesting rearrangement scaffold, a pair of diastereoisomers with different hydroxyl configurations at C-7, were synthesized. Their structures were elucidated using spectroscopic data and quantum chemical computational methods. The proposed cyclization mechanism of these products was validated through isotopic labeling experiments and density functional theory calculations. Through amino acid residue swapping, it was revealed that AsSS-M81 and CvSS-C82 play crucial roles in determining the stereoselective hydroxylation at C-7. Furthermore, functional interconversion between AsSS and CvSS has been successfully achieved.

Two diterpene synthases (DTSs) for peyssonnosol and peyssonnosol B were discovered in bacteria. Their enzyme mechanisms were investigated through isotopic labelling experiments, density functional theory (DFT) calculations and site-directed mutagenesis, yielding biosynthetically related molecules that gave important insights into the terpene cyclisation cascade. In the present case, several mechanisms in line with the isotopic labelling experiments could be formulated. Only an extended experimental approach in combination with computational chemistry ultimately resulted in a refined mechanistic model.

Slow-melting flesh (SMF) in peaches offers many advantages, including easy transportation, maintaining flavour after ripening, an extended harvest window, and reduced losses caused by fruit softening. However, the underlying molecular mechanism remains elusive. A high-quality genome of the SMF cultivar Chunrui was sequenced, assembled and annotated. The assembled genome was 249.6 Mb in size and characterized by a contig N50 of 12.35 Mb and a scaffold N50 of 30.27 Mb. Analysis of a segregating population indicated that a single dominant gene or major gene controlled the SMF trait. This trait was mapped to chromosome 4, which had a total length of 1.39 Mb. Fine mapping and gene expression analyses identified the receptor protein kinase THESEUS 1 (PpTHE1) as a candidate SMF gene. A Gypsy LTR-retrotransposon inserted downstream of PpTHE1 inhibited its expression. Functional analyses in peach and tomato fruits showed PpTHE1 played a positive role in maintaining fruit firmness. Screening of a yeast library using the kinase domain of PpTHE1 as the bait identified an ERF-type transcription factor PpERF61 and pectinlyase PpPL15. Luciferase complementation imaging, bimolecular fluorescence complementation and co-immunoprecipitation assays showed that PpTHE1 could interact with PpERF61 and PpPL15 in planta. Furthermore, our experimental data revealed that PpTHE1 significantly attenuates the DNA-binding capacity of PpERF61 to its target genes. These findings reveal the regulatory mechanism underlying the SMF fruit quality trait and thus provide theoretical support for breeding programmes to develop high-quality, storage-tolerant peach genotypes.

As is well known, postharvest abscisic acid (ABA) treatment of citrus fruits can significantly enhance fruit coloration by promoting the accumulation of carotenoids, however, the underlying molecular mechanism is still unclear. In this study, we further confirmed the effect of ABA treatment in promoting citrus fruit coloration by activating the expression of 4 key carotenoid metabolism genes (CsPSY, CsLCYB, CsHYD, and CsNCED2) during postharvest storage. A novel basic helix-loop-helix (bHLH) transcription factor, CsbHLH6, was identified using the promoters of 3 key ABA-induced carotenoid metabolism genes (CsLCYB, CsHYD, and CsNCED2) by yeast one-hybrid screening. Overexpression of CsbHLH6 enhanced carotenoid accumulation in citrus fruits and calli. Interference with CsbHLH6 expression in citrus calli and fruits indicated that CsbHLH6 was essential for ABA-induced carotenoid biosynthesis and fruit coloration. As a nucleus-localized transcriptional activator, CsbHLH6 directly bound to the promoters of 6 key carotenoid metabolism genes (CsPSY, CsLCYB, CsLCYE, CsHYD, CsZEP, and CsNCED2), subsequently activating their expression in vitro and in vivo. Furthermore, ABA signaling significantly raised CsbHLH6 promoter activity, thus enhancing the regulation of carotenoid accumulation by CsbHLH6. In summary, our study reveals the molecular mechanism by which CsbHLH6 regulates ABA-induced citrus fruit coloration during postharvest storage, which is crucial for improving the postharvest appearance quality of citrus fruits.

Citrus reticulata 'Chachiensis' contributes its fruit peel to the raw material of 'Guangchenpi', is renowned for its distinctive medicinal and aromatic properties, and has been utilized for hundreds of years. However, the molecular and metabolic mechanism underlining the properties remains unknown. In this study, dimethyl anthranilate was uniquely detected in 'Chachiensis' fruit peel compared to other mandarin cultivars and was further validated as the characteristic metabolic biomarker based on orthogonal partial least squares discrimination analysis analysis. Two SAMTs genes, CreSAMT1 and CreSAMT2, were screened by combined volatile profiling and transcriptome sequencing. CreSAMT1 could catalyze the methylation of N-methyl-2-aminobenzoic acid to synthesize dimethyl anthranilate, and its constant expression contributes to the specific accumulation of dimethyl anthranilate in 'Chachiensis', which was activated by CreERF35 and CreZAT11. While CreSAMT2 is highly expressed in citrus flowers and is responsible for catalyzing anthranilate to form methyl anthranilate, the main floral volatiles. Moreover, the involvement of transcription factors such as ERF were speculated in regulating its volatiles biosynthesis. The study provides a theoretical basis to elucidate the volatile metabolism, and to improve the aromatic citrus industry.

Guangchenpi (GCP, Citrus reticulata), a well-known traditional Chinese medicine and homology food condiment, is predominantly consumed in Asia. Extending its aging process enhances its medicinal properties, flavor, and profitability. However, pinpointing the aging year of GCP remains a bottleneck of the industry. This study represents the first comprehensive characterization of 32 glycosidically bound volatiles in GCP, employing a combination of Amberlite XAD-2 resin extraction and gas chromatography-mass spectrometry (GC–MS) analysis. The identified compounds include bioactive substances such as 5-hydroxymethylfurfural and acorenone B. Fourteen glycosidically bound volatiles exhibited a significant correlation with the aging duration of GCP. Additionally, the aging process was associated with dynamic shifts in the microbial community, notably an enrichment of Lactobacillus and Oceanobacillus, which potentially influenced the volatile compound profile. Machine learning integration revealed a model based on the content and relative weight of the 14 glycosidically bound volatiles, demonstrating high accuracy in determining aging years of GCP. This model could aid in detecting adulteration and standardizing the GCP market.

Bioactive compounds play an increasingly prominent role in breeding functional and nutritive fruit crops such as citrus. However, the genomic and metabolic bases for the selection and differentiation underlying bioactive compound variations in citrus remain poorly understood. In this study, we constructed a species-level variation atlas of genomes and metabolomes using 299 citrus accessions. A total of 19 829 significant SNPs were targeted to 653 annotated metabolites, among which multiple significant signals were identified for secondary metabolites, especially flavonoids. Significant differential accumulation of bioactive compounds in the phenylpropane pathway, mainly flavonoids and coumarins, was unveiled across ancestral citrus species during differentiation, which is likely associated with the divergent haplotype distribution and/or expression profiles of relevant genes, including p-coumaroyl coenzyme A 2'-hydroxylases, flavone synthases, cytochrome P450 enzymes, prenyltransferases, and uridine diphosphate glycosyltransferases. Moreover, we systematically evaluated the beneficial bioactivities such as the antioxidant and anticancer capacities of 219 citrus varieties, and identified robust associations between distinct bioactivities and specific metabolites. Collectively, these findings provide citrus breeding options for enrichment of beneficial flavonoids and avoidance of potential risk of coumarins. Our study will accelerate the application of genomic and metabolic engineering strategies in developing modern healthy citrus cultivars.

'Zong Cheng' navel orange (ZC) is a brown mutant of Lane Late navel orange (LL) and emits a more pleasant odor than that of LL. However, the key volatile compound of this aroma and underlying mechanism remains unclear. In this study, sensory evaluations and volatile profiling were performed throughout fruit development to identify significant differences in sensory perception and metabolites between LL and ZC. It revealed that the sesquiterpene content varied significantly between ZC and LL. Based on aroma extract dilution and gas chromatography-olfactometry analyses, the volatile compound leading to the background aroma of LL and ZC is Dlimonene, the orange note in LL was mainly attributed to octanal, whilst valencene, beta-myrcene, and (E)-beta-ocimene presented balsamic, sweet, and herb notes in ZC. Furthermore, Cs5g12900 and six potential transcription factors were identified as responsible for valencene accumulation in ZC, which is important for enhancing the aroma of ZC.

Mei (Prunus mume) is a traditional tree of Rosaceae rich in flavonoids that are important for flower color, medicinal use, food, and skincare applications in East Asia. However, a systematic evaluation of flavonoid resources in mei germplasms is lacking. Here, 81 flavonoid metabolites from 56 representative mei cultivars were identified and compared with those of three closely related species for the first time. It was found that flavonoids were more varied and abundant in mei germplasms compared to its congeneric species. Additionally, the flavonoids were diverse among the nine mei cultiver groups and its three relatives. It was further suggested that four flavonols could be used as biomakers to distinguish the Apricot Mei cultivars from other cultivar groups, providing new insights for systematic classification based on metabolites. On the other side, flavonoids of mei germplasms also displayed distinct flower color characteristics, and cyanidin branch derivatives affected the formation of different flower colors. Furthermore, flavonoids also exhibited some specific distributions among mei germplasms. Cultivars such as WYY, BXZS, DBLE, ELE, and JNTG, which were rich in functional flavonoids, may serve as valuable resources for exploiting the flavonoids of mei. These fundamental data and results were supposed to laid a solid foundation for the further application of mei flavonoid resources in the fields of flower color, medicinal use, food and cosmetic.

Guangchenpi (GCP), which is the peel of Citrus reticulata 'Chachiensis', is widely used as an herbal medicine, tea and food ingredient in southeast Asia. Prolonging its aging process results in a more pleasant flavor and increases its profitability. Through the integration of sensory evaluation with flavoromic analysis approaches, we evaluated the correlation between the flavor attributes and the profiles of the volatiles and flavonoids of GCP with various aging years. Notably, d-limonene, γ-terpinene, dimethyl anthranilate and α-phellandrene were the characteristic aroma compounds of GCP. Besides, α-phellandrene and nonanal were decisive for consumers' perception of GCP aging time due to changes of their odor activity values (OAVs). The flavor attributes of GCP tea liquid enhanced with the extension of aging time, and limonene-1,2-diol was identified as an important flavor enhancer. Combined with machine learning models, key flavor-related metabolites could be developed as efficient biomarkers for aging years to prevent GCP adulteration.

The dried pericarp of Citrus reticulata 'Chachi' (CRC) is commonly utilized in Traditional Chinese Medicine (TCM) and food consumption. Geqingpi (GQP), Sihuaqingpi (SHQP) and Guangchenpi (CRP) are TCM materials derived from different developmental stages of CRC pericarp, each possessing distinct medicinal properties. However, there is currently limited study on the active substances in these three stages. Metabolome data suggested that the discrepancies in flavonoid content, particularly polymethoxyflavonoids (PMFs), predominantly contribute to the variations across the three periods. Additionally, through multivariate statistical analysis, 7 potential biomarkers were identified and nobiletin (NOB) possessed the most significant contribution. Omethyltransferases (OMTs) play a crucial role to catalyze the formation of -OCH3 in PMFs. The analysis of transcriptome and genome data revealed the presence of 93 CrOMTs in CRC and 43 were expressed on pericarp. Further investigation identified that 28 of these genes were potentially responsible for catalyzing the biosynthesis of important PMFs such as NOB, sinensetin (SIN), and 3,3 ',4 ',5,6,7,8-heptamethoxyflavone (HEP). Our results may provide a new perspective for the study of GQP, SHQP and CRP, as well as the regulatory mechanism of PMFs in CRC.

Citrus fruits have abundant flavonoid glycosides (FGs), an important class of natural functional and flavor components. However, there have been few reports about the modification of UDP-glycosyltransferases (UGTs) on flavonoids in citrus. Notably, in flavonoid biosynthesis, 7-O-glucosylation is the initial and essential step of glycosylation prior to the synthesis of flavanone disaccharides, the most abundant and iconic FGs in citrus fruits. Here, based on the accumulation of FGs observed at the very early fruit development stage of two pummelo varieties, we screened six novel flavonoid 7-O-glucosyltransferase genes (7GlcTs) via transcriptomic analysis and then characterized them in vitro. The results revealed that four Cg7GlcTs possess wide catalytic activities towards various flavonoid substrates, with CgUGT89AK1 exhibiting the highest catalytic efficiency. Transient overexpression of CgUGT90A31 and CgUGT89AK1 led to increases in FG synthesis in pummelo leaves. Interestingly, these two genes had conserved sequences and consistent functions across different germplasms. Moreover, CitUGT89AK1 was found to play a role in the response of citrus to Huanglongbing infection by promoting FG production. The findings improve our understanding of flavonoid 7-O-glucosylation by identifying the key genes, and may help improve the benefits of flavonoid biosynthesis for plants and humans in the future.