Diverse triterpene skeletons are derived from the expansion and divergent evolution of 2,3-oxidosqualene cyclases in plants.

Triterpenoids are one of the largest groups of secondary metabolites and exhibit diverse structures, which are derived from C30 skeletons that are biosynthesized via the isoprenoid pathway by cyclization of 2,3-oxidosqualene. Triterpenoids have a wide range of biological activities, and are used in functional foods, drugs, and as industrial materials. Due to the low content levels in their native plants and limited feasibility and efficiency of chemical synthesis, heterologous biosynthesis of triterpenoids is the most promising strategy. Herein, we classified 121 triterpene alcohols/ketones according to their conformation and ring numbers, among which 51 skeletons have been experimentally characterized as the products of oxidosqualene cyclases (OSCs). Interestingly, 24 skeletons that have not been reported from nature source were generated by OSCs in heterologous expression. Comprehensive evolutionary analysis of the identified 152 OSCs from 75 species in 25 plant orders show that several pentacyclic triterpene synthases repeatedly originated in multiple plant lineages. Comparative analysis of OSC catalytic reaction revealed that stabilization of intermediate cations, steric hindrance, and conformation of active center amino acid residues are primary factors affecting triterpene formation. Optimization of OSC could be achieved by changing of side-chain orientations of key residues. Recently, methods, such as rationally design of pathways, regulation of metabolic flow, compartmentalization engineering, etc., were introduced in improving chassis for the biosynthesis of triterpenoids. We expect that extensive study of natural variation of large number of OSCs and catalytical mechanism will provide basis for production of high level of triterpenoids by application of synthetic biology strategies.

Jujube metabolome selection determined the edible properties acquired during domestication.

Plants supply both food and medicinal compounds, which are ascribed to diverse metabolites produced by plants. However, studies on domestication-driven changes in the metabolome and genetic basis of bioactive molecules in perennial fruit trees are generally lacking. Here, we conducted multidimensional analyses revealing a singular domestication event involving the genomic and metabolomic selection of jujube trees (Ziziphus jujuba Mill.). The genomic selection for domesticated genes was highly enriched in metabolic pathways, including carbohydrates and specialized metabolism. Domesticated metabolome profiling indicated that 187 metabolites exhibited significant divergence as a result of directional selection. Malic acid was directly selected during domestication, and the simultaneous selection of specialized metabolites, including triterpenes, consequently lead to edible properties. Cyclopeptide alkaloids (CPAs) were specifically targeted for the divergence between dry and fresh cultivars. We identified 1080 significantly associated loci for 986 metabolites. Among them, 15 triterpenes were directly selected at six major loci, allowing the identification of a homologous cluster containing seven 2,3-oxidosqualene cyclases (OSCs). An OSC gene was found to contribute to the reduction in the content of triterpenes during domestication. The complete pathway for synthesizing ursolic acid was dissected by integration of the metabolome and transcriptome. Additionally, an N-methyltransferase involved in the biosynthesis of CPA and responsible for inter-cultivar content variation was identified. The present study promotes our understanding of the selection process of the global metabolome subsequent to fruit tree domestication and facilitates the genetic manipulation of specialized metabolites to enhance their edible traits.

Cytochrome P450 family member CYP96B5 hydroxylates alkanes to primary alcohols and is involved in rice leaf cuticular wax synthesis.

Odd-numbered primary alcohols are components of plant cuticular wax, but their biosynthesis remains unknown. We isolated a rice wax crystal-sparse leaf 5 (WSL5) gene using a map-based cloning strategy. The function of WSL5 was illustrated by overexpression and knockout in rice, heterologous expression in Arabidopsis and transient expression in tobacco leaves. WSL5 is predicted to encode a cytochrome P450 family member CYP96B5. The wsl5 mutant lacked crystalloid platelets on the surface of cuticle membrane, and its cuticle membrane was thicker than that of the wild-type. The wsl5 mutant is more tolerant to drought stress. The load of C23 -C33 alkanes increased, whereas the C29 primary alcohol reduced significantly in wsl5 mutant and WSL5 knockout transgenic plants. Overexpression of WSL5 increased the C29 primary alcohol and decreased alkanes in rice leaves. Heterologous expression of WSL5 increased the C29 primary alcohol and decreased alkanes, secondary alcohol, and ketone in Arabidopsis stem wax. Transient expression of WSL5 in tobacco leaves also increased the production C29 primary alcohol. WSL5 catalyzes the terminal hydroxylation of alkanes, yielding odd-numbered primary alcohols, and is involved in the formation of epidermal wax crystals on rice leaf, affecting drought sensitivity.

An Efficient System for Ds Transposon Tagging in Brachypodium distachyon.

Transposon tagging is a powerful tool that has been widely applied in several species for insertional mutagenesis in plants. Several efforts have aimed to create transfer-DNA (T-DNA) insertional mutant populations in Brachypodium distachyon, a monocot plant used as a model system to study temperate cereals, but there has been a lack of research aimed at using transposon strategies. Here, we describe the application of a maize (Zea mays) Dissociation (Ds) transposon tagging system in B distachyon The 35S::AcTPase cassette and Ds element were constructed within the same T-DNA and transformed into B distachyon plants. The Ds element was readily transposed to other chromosomes or to the same chromosome under the function of Activator (Ac) transposase. Through homologous chromosome synapsis, recombination, and segregation, the Ds element separated from the Ac element. We selected stable Ds-only plants using G418 and GFP assays and analyzed 241 T0 lines, some of which were highly efficient at producing Ds-only progeny. Through thermal asymmetric interlaced PCR, we isolated 710 independent Ds flanking sequences from Ds-only plants. Furthermore, we identified a large collection of mutants with visible developmental phenotypes via this transposon tagging system. The system is relatively simple and rapid in comparison to traditional T-DNA insertion strategies, because once efficiency lines are obtained they can be reused to generate more lines from nontransposed plants without the use of time-consuming tissue culture steps.

Characterization of a 2,3-oxidosqualene cyclase in the toosendanin biosynthetic pathway of Melia toosendan.

Toosendanin, bearing a furan ring, is a limonoid belonging to the group of tetranortriterpenoids. Toosendanin is a phytochemical found in the medicinal plant Melia toosendan Sieb. et Zucc. of the Meliaceae family. Toosendanin and its derivatives demonstrate high insecticidal activity and are important pesticides derived from plants. Despite intensive investigation of limonoids over several decades, the biosynthetic pathway of these triterpenoids is less understood. To identify the key enzymes involved in the toosendanin biosynthetic pathway, we analyzed the contents of toosendanin in various plant tissues and parts and found that the highest level of toosendanin was found in the developing fruit and gradually decreased as the fruit matured. More than 346 116 transcripts were assembled based on 394 million paired-end Illumina reads and 6 million PacBio reads from the pooled RNA samples of fruits, leaves and young barks. A total of 186 263 genes were predicted. Six 2,3-oxidosqualene cyclase (OSC) genes were identified by analyzing the association between gene expression and metabolite profiles. Functional analyses using the Nicotiana benthamiana transient expression assay showed that MtOSC1 catalyzed 2,3-oxidosqualene to produce a tetracyclic triterpene skeleton, tirucalla-7,24-dien-3ß-ol, which is predicted as the precursor for toosendanin biosynthesis. We identified another OSC, MtOSC6, which is a lupeol synthase. Using synthetic biology methods, these identified enzymes could be used to model a biosynthetic pathway to produce large quantities of toosendanin.

CsMYB60 is a key regulator of flavonols and proanthocyanidans that determine the colour of fruit spines in cucumber.

Spine colour is an important fruit quality trait that influences the commercial value of cucumber (Cucumis sativus). However, little is known about the metabolites and the regulatory mechanisms of their biosynthesis in black spine varieties. In this study, we determined that the pigments of black spines are flavonoids, including flavonols and proanthocyanidins (PAs). We identified CsMYB60 as the best candidate for the previously identified B (Black spine) locus. Expression levels of CsMYB60 and the key genes involved in flavonoid biosynthesis were higher in black-spine inbred lines than that in white-spine lines at different developmental stages. The insertion of a Mutator-like element (CsMULE) in the second intron of CsMYB60 decreased its expression in a white-spine line. Transient overexpression assays indicated that CsMYB60 is a key regulatory gene and Cs4CL is a key structural gene in the pigmentation of black spines. In addition, the DNA methylation level in the CsMYB60 promoter was much lower in the black-spine line compared with white-spine line. The CsMULE insert may decrease the expression level of CsMYB60, causing hindered synthesis of flavonols and PAs in cucumber fruit spines.

Identification of key amino acid residues determining product specificity of 2,3-oxidosqualene cyclase in Oryza species.

Triterpene synthases, also known as 2,3-oxidosqualene cyclases (OSCs), synthesize diverse triterpene skeletons that form the basis of an array of functionally divergent steroids and triterpenoids. Tetracyclic and pentacyclic triterpene skeletons are synthesized via protosteryl and dammarenyl cations, respectively. The mechanism of conversion between two scaffolds is not well understood. Here, we report a promiscuous OSC from rice (Oryza sativa) (OsOS) that synthesizes a novel pentacyclic triterpene orysatinol as its main product. The OsOS gene is widely distributed in indica subspecies of cultivated rice and in wild rice accessions. Previously, we have characterized a different OSC, OsPS, a tetracyclic parkeol synthase found in japonica subspecies. Phylogenetic and protein structural analyses identified three key amino acid residues (#732, #365, #124) amongst 46 polymorphic sites that determine functional conversion between OsPS and OsOS, specifically, the chair-semi(chair)-chair and chair-boat-chair interconversions. The different orientation of a fourth amino acid residue Y257 was shown to be important for functional conversion The discovery of orysatinol unlocks a new path to triterpene diversity in nature. Our findings also reveal mechanistic insights into the cyclization of oxidosqualene into tetra- and pentacyclic skeletons, and provide a new strategy to identify key residues determining OSC specificity.

Functional Divergence of Diterpene Syntheses in the Medicinal Plant Salvia miltiorrhiza.

The medicinal plant Salvia miltiorrhiza produces various tanshinone diterpenoids that have pharmacological activities such as vasorelaxation against ischemia reperfusion injury and antiarrhythmic effects. Their biosynthesis is initiated from the general diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate by sequential reactions catalyzed by copalyl diphosphate synthase (CPS) and kaurene synthase-like cyclases. Here, we report characterization of these enzymatic families from S. miltiorrhiza, which has led to the identification of unique pathways, including roles for separate CPSs in tanshinone production in roots versus aerial tissues (SmCPS1 and SmCPS2, respectively) as well as the unique production of ent-13-epi-manoyl oxide by SmCPS4 and S. miltiorrhiza kaurene synthase-like2 in floral sepals. The conserved SmCPS5 is involved in gibberellin plant hormone biosynthesis. Down-regulation of SmCPS1 by RNA interference resulted in substantial reduction of tanshinones, and metabolomics analysis revealed 21 potential intermediates, indicating a complex network for tanshinone metabolism defined by certain key biosynthetic steps. Notably, the correlation between conservation pattern and stereochemical product outcome of the CPSs observed here suggests a degree of correlation that, especially when combined with the identity of certain key residues, may be predictive. Accordingly, this study provides molecular insights into the evolutionary diversification of functional diterpenoids in plants.

Modularity of plant metabolic gene clusters: a trio of linked genes that are collectively required for acylation of triterpenes in oat.

Operon-like gene clusters are an emerging phenomenon in the field of plant natural products. The genes encoding some of the best-characterized plant secondary metabolite biosynthetic pathways are scattered across plant genomes. However, an increasing number of gene clusters encoding the synthesis of diverse natural products have recently been reported in plant genomes. These clusters have arisen through the neo-functionalization and relocation of existing genes within the genome, and not by horizontal gene transfer from microbes. The reasons for clustering are not yet clear, although this form of gene organization is likely to facilitate co-inheritance and co-regulation. Oats (Avena spp) synthesize antimicrobial triterpenoids (avenacins) that provide protection against disease. The synthesis of these compounds is encoded by a gene cluster. Here we show that a module of three adjacent genes within the wider biosynthetic gene cluster is required for avenacin acylation. Through the characterization of these genes and their encoded proteins we present a model of the subcellular organization of triterpenoid biosynthesis.

Glycosyltransferases from oat (Avena) implicated in the acylation of avenacins.

Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid.

Discovery of rare mutations in extensively pooled DNA samples using multiple target enrichment.

Chemical mutagenesis is routinely used to create large numbers of rare mutations in plant and animal populations, which can be subsequently subjected to selection for beneficial traits and phenotypes that enable the characterization of gene functions. Several next-generation sequencing (NGS)-based target enrichment methods have been developed for the detection of mutations in target DNA regions. However, most of these methods aim to sequence a large number of target regions from a small number of individuals. Here, we demonstrate an effective and affordable strategy for the discovery of rare mutations in a large sodium azide-induced mutant rice population (F2 ). The integration of multiplex, semi-nested PCR combined with NGS library construction allowed for the amplification of multiple target DNA fragments for sequencing. The 8 × 8 × 8 tridimensional DNA sample pooling strategy enabled us to obtain DNA sequences of 512 individuals while only sequencing 24 samples. A stepwise filtering procedure was then elaborated to eliminate most of the false positives expected to arise through sequencing error, and the application of a simple Student's t-test against position-prone error allowed for the discovery of 16 mutations from 36 enriched targeted DNA fragments of 1024 mutagenized rice plants, all without any false calls.

Identification and fine mapping of quantitative trait loci for seed vigor in germination and seedling establishment in rice.

Seed vigor is an index of seed quality that is used to describe the rapid and uniform germination and the establishment of strong seedlings in any environmental conditions. Strong seed vigor in low-temperature germination conditions is particularly important in direct-sowing rice production systems. However, seed vigor has not been selected as an important breeding trait in traditional breeding programs due to its quantitative inherence. In this study, we identified and mapped eight quantitative trait loci (QTLs) for seed vigor by using a recombinant inbred population from a cross between rice (Oryza sativa L. ssp. indica) cultivars ZS97 and MH63. Conditional QTL analysis identified qSV-1, qSV-5b, qSV-6a, qSV-6b, and qSV-11 influenced seedling establishment and that qSV-5a, qSV-5c, and qSV-8 influenced only germination. Of these, qSV-1, qSV-5b, qSV-6a, qSV-6b, and qSV-8 were low-temperature-specific QTLs. Two major-effective QTLs, qSV-1, and qSV-5c were narrowed down to 1.13-Mbp and 400-kbp genomic regions, respectively. The results provide tightly linked DNA markers for the marker-assistant pyramiding of multiple positive alleles for increased seed vigor in both normal and low-temperature germination environments.

Biosynthesis and transport of pollen coat precursors in angiosperms.

The pollen coat is a hydrophobic mixture on the pollen grain surface, which plays an important role in protecting male gametes from various environmental stresses and microorganism attacks, and in pollen-stigma interactions during pollination in angiosperms. An abnormal pollen coat can result in humidity-sensitive genic male sterility (HGMS), which can be used in two-line hybrid crop breeding. Despite the crucial functions of the pollen coat and the application prospect of its mutants, few studies have focused on pollen coat formation. In this Review, the morphology, composition and function of different types of pollen coat are assessed. On the basis of the ultrastructure and development process of the anther wall and exine found in rice and Arabidopsis, the genes and proteins involved in the biosynthesis of pollen coat precursors and the possible transport and regulation process are sorted. Additionally, current challenges and future perspectives, including potential strategies utilizing HGMS genes in heterosis and plant molecular breeding, are highlighted.

Evaluation of Cooked Rice for Eating Quality and Its Components in Geng Rice.

At present, ''eating well" is increasingly desired by people instead of merely ''being full". Rice provides the majority of daily caloric needs for half of the global human population. However, eating quality is difficult to objectively evaluate in rice breeding programs. This study was carried out to objectively quantify and predict eating quality in Geng rice. First, eating quality and its components were identified by trained panels. Analysis of variance and broad-sense heritability showed that variation among varieties was significant for all traits except hardness. Among them, viscosity, taste, and appearance were significantly correlated with eating quality. We established an image acquisition and processing system to quantify cooked rice appearance and optimized the process of measuring cooked rice viscosity with a texture analyzer. The results show that yellow areas of the images were significantly correlated with appearance, and adhesiveness was significantly correlated with viscosity. Based on these results, multiple regression analysis was used to predict eating quality: eating quality = 0.37 × adhesiveness - 0.71 × yellow area + 0.89 × taste - 0.34, R2 = 0.85. The correlation coefficient between the predicted and actual values was 0.86. We anticipate that this predictive model will be useful in future breeding programs for high-eating-quality rice.

Knockout of CAFFEOYL-COA 3-O-METHYLTRANSFERASE 6/6L enhances the S/G ratio of lignin monomers and disease resistance in Nicotiana tabacum.

Background: Nicotiana tabacum is an important economic crop, which is widely planted in the world. Lignin is very important for maintaining the physiological and stress-resistant functions of tobacco. However, higher lignin content will produce lignin gas, which is not conducive to the formation of tobacco quality. To date, how to precisely fine-tune lignin content or composition remains unclear. Results: Here, we annotated and screened 14 CCoAOMTs in Nicotiana tabacum and obtained homozygous double mutants of CCoAOMT6 and CCoAOMT6L through CRSIPR/Cas9 technology. The phenotype showed that the double mutants have better growth than the wild type whereas the S/G ratio increased and the total sugar decreased. Resistance against the pathogen test and the extract inhibition test showed that the transgenic tobacco has stronger resistance to tobacco bacterial wilt and brown spot disease, which are infected by Ralstonia solanacearum and Alternaria alternata, respectively. The combined analysis of metabolome and transcriptome in the leaves and roots suggested that the changes of phenylpropane and terpene metabolism are mainly responsible for these phenotypes. Furthermore, the molecular docking indicated that the upregulated metabolites, such as soyasaponin Bb, improve the disease resistance due to highly stable binding with tyrosyl-tRNA synthetase targets in Ralstonia solanacearum and Alternaria alternata. Conclusions: CAFFEOYL-COA 3-O-METHYLTRANSFERASE 6/6L can regulate the S/G ratio of lignin monomers and may affect tobacco bacterial wilt and brown spot disease resistance by disturbing phenylpropane and terpene metabolism in leaves and roots of Nicotiana tabacum, such as soyasaponin Bb.

Divergent evolution of oxidosqualene cyclases in plants.

Triterpenes are one of the largest classes of plant metabolites and have important functions. A diverse array of triterpenoid skeletons are synthesized via the isoprenoid pathway by enzymatic cyclization of 2,3-oxidosqualene. The genomes of the lower plants Chlamydomonas reinhardtii and moss (Physcomitrella patens) contain just one oxidosqualene cyclase (OSC) gene (for sterol biosynthesis), whereas the genomes of higher plants contain nine to 16 OSC genes. Here we carry out functional analysis of rice OSCs and rigorous phylogenetic analysis of 96 OSCs from higher plants, including Arabidopsis thaliana, Oryza sativa, Sorghum bicolor and Brachypodium distachyon. The functional analysis identified an amino acid sequence for isoarborinol synthase (OsIAS) (encoded by Os11g35710/OsOSC11) in rice. Our phylogenetic analysis suggests that expansion of OSC members in higher plants has occurred mainly through tandem duplication followed by positive selection and diversifying evolution, and consolidated the previous suggestion that dicot triterpene synthases have been derived from an ancestral lanosterol synthase instead of directly from their cycloartenol synthases. The phylogenetic trees are consistent with the reaction mechanisms of the protosteryl and dammarenyl cations which parent a wide variety of triterpene skeletal types, allowing us to predict the functions of the uncharacterized OSCs.

Identification and validation of a major quantitative trait locus for slow-rusting resistance to stripe rust in wheat.

Stripe (yellow) rust, caused by Puccinia striiformis Westend. f. sp. tritici Eriks (Pst), is one of the most important wheat (Triticum aestivum L.) diseases and causes significant yield losses. A recombinant inbred (RI) population derived from a cross between Yanzhan 1 and Xichang 76-9 cultivars was evaluated for resistance to wheat stripe rust strain CYR32 at both the seedling and adult plant stages. Four resistance quantitative trait loci (QTLs) were detected in this population, in which the major one, designated as Yrq1, was mapped on chromosome 2DS. The strategy of using the Brachypodium distachyon genome, wheat expressed sequence tags and a draft DNA sequences (scaffolds) of the D-genome (Aegilops tauschii Coss.) for the development of simple sequence repeat (SSR) markers was successfully used to identify 147 SSRs in hexaploid wheat. Of the 19 polymorphic SSRs in the RI population, 17 SSRs were mapped in the homeologous group 2 chromosomes near Yrq1 region and eight SSRs were genetically mapped in the 2.7 cM region of Yrq1, providing abundant DNA markers for fine-mapping of Yrq1 and marker-assisted selection in wheat breeding program. The effectiveness of Yrq1 was validated in an independent population, indicating that this resistance QTL can be successfully transferred into a susceptible cultivar for improvement of stripe rust resistance.

The global integrative network: integration of signaling and metabolic pathways.

The crosstalk between signaling and metabolic pathways has been known to play key roles in human diseases and plant biological processes. The integration of signaling and metabolic pathways can provide an essential reference framework for crosstalk analysis. However, current databases use distinct structures to present signaling and metabolic pathways, which leads to the chaos in the integrated networks. Moreover, for the metabolic pathways, the metabolic enzymes and the reactions are disconnected by the current widely accepted layout of edges and nodes, which hinders the topological analysis of the integrated networks. Here, we propose a novel "meta-pathway" structure, which uses the uniformed structure to display the signaling and metabolic pathways, and resolves the difficulty in linking the metabolic enzymes to the reactions topologically. We compiled a comprehensive collection of global integrative networks (GINs) by merging the meta-pathways of 7077 species. We demonstrated the assembly of the signaling and metabolic pathways using the GINs of four species-human, mouse, Arabidopsis, and rice. Almost all of the nodes were assembled into one major network for each of the four species, which provided opportunities for robust crosstalk and topological analysis, and knowledge graph construction. Supplementary Information: The online version contains supplementary material available at 10.1007/s42994-022-00078-1.

Improving bread wheat yield through modulating an unselected AP2/ERF gene.

Crop breeding heavily relies on natural genetic variation. However, additional new variations are desired to meet the increasing human demand. Inflorescence architecture determines grain number per spike, a major determinant of bread wheat (Triticum aestivum L.) yield. Here, using Brachypodium distachyon as a wheat proxy, we identified DUO-B1, encoding an APETALA2/ethylene response factor (AP2/ERF) transcription factor, regulating spike inflorescence architecture in bread wheat. Mutations of DUO-B1 lead to mild supernumerary spikelets, increased grain number per spike and, importantly, increased yield under field conditions without affecting other major agronomic traits. DUO-B1 suppresses cell division and promotes the expression of BHt/WFZP, whose mutations could lead to branched 'miracle-wheat'. Pan-genome analysis indicated that DUO-B1 has not been utilized in breeding, and holds promise to increase wheat yield further.

The genome of Dioscorea zingiberensis sheds light on the biosynthesis, origin and evolution of the medicinally important diosgenin saponins.

Diosgenin saponins isolated from Dioscorea species such as D. zingiberensis exhibit a broad spectrum of pharmacological activities. Diosgenin, the aglycone of diosgenin saponins, is an important starting material for the production of steroidal drugs. However, how plants produce diosgenin saponins and the origin and evolution of the diosgenin saponin biosynthetic pathway remain a mystery. Here we report a high-quality, 629-Mb genome of D. zingiberensis anchored on 10 chromosomes with 30 322 protein-coding genes. We reveal that diosgenin is synthesized in leaves ('source'), then converted into diosgenin saponins, and finally transported to rhizomes ('sink') for storage in plants. By evaluating the distribution and evolutionary patterns of diosgenin saponins in Dioscorea species, we find that diosgenin saponin-containing may be an ancestral trait in Dioscorea and is selectively retained. The results of comparative genomic analysis indicate that tandem duplication coupled with a whole-genome duplication event provided key evolutionary resources for the diosgenin saponin biosynthetic pathway in the D. zingiberensis genome. Furthermore, comparative transcriptome and metabolite analysis among 13 Dioscorea species suggests that specific gene expression patterns of pathway genes promote the differential evolution of the diosgenin saponin biosynthetic pathway in Dioscorea species. Our study provides important insights and valuable resources for further understanding the biosynthesis, evolution, and utilization of plant specialized metabolites such as diosgenin saponins.