Terpene synthases (TPSs) play pivotal roles in conferring the structural diversity of terpenoids, which are mainly emitted from flowers, whereas the genetic basis of the release of floral volatile terpenes remains largely elusive. Though quite similar in sequence, TPS allelic variants still function divergently, and how they drive floral terpene diversity in closely related species remains unknown. Here, TPSs responsible for the floral scent of wild Freesia species were characterized, and the functions of their natural allelic variants, as well as the causal amino acid residues, were investigated in depth. Besides the eight TPSs previously reported in modern cultivars, seven additional TPSs were functionally evaluated to contribute to the major volatiles emitted from wild Freesia species. Functional characterization of allelic natural variants demonstrated that allelic TPS2 and TPS10 variants changed enzymatic capacity while allelic TPS6 variants drove the diversity of floral terpene products. Further residue substitution analysis revealed the minor residues determining the enzyme catalytic activity and product specificity. The clarification of TPSs in wild Freesia species reveals that allelic TPS variants evolved differently to determine the interspecific floral volatile terpenes in the genus and might be used for modern cultivar improvement.

Floral volatile terpenes play pivotal roles in plant-pollinator interactions and ecological adaptation, yet the genetic mechanisms underlying their diversification in Aquilegia remain poorly understood. This study integrates gas chromatography-mass spectrometry (GC/MS) and functional characterization assays to investigate how terpene synthase (TPS) diversity shapes floral scent evolution across Aquilegia species and populations. We identified (+)-limonene, linalool, β-pinene, and β-sesquiphellandrene as the dominant floral terpenes, with substantial inter- and intraspecific variation driven by differential expression and sequence divergence of key TPS genes, including TPS7, TPS8, TPS9 and TPS24. Functional characterization revealed that orthologous variants of TPS7 and TPS8 influence species-specific monoterpene and sesquiterpene production, while TPS24 variants exhibit substrate plasticity in linalool synthesis despite retaining conserved catalytic motifs. Population-level analyses of A. viridiflora revealed that frame-shift mutations and expression shifts in TPSs are associated with terpene profile divergence, while residue variations in allelic TPS variants may have driven functional novelty during evolution. Our findings highlight TPS diversity as a molecular mechanism for floral scent diversification, enabling adaptive radiation through pollinator-mediated selection and specialization within distinct ecological niches. This study provides crucial insights into the evolutionary interplay between genetic variation, enzymatic plasticity, and ecological adaptation in shaping plant biodiversity.

Floral coloration in Freesia hybrida is predominantly attributed to anthocyanins, with glycosylation playing a critical role in their stability and diversity. This study investigates the molecular mechanisms underlying color variation between F. hybrida cultivars, focusing on anthocyanin 5-O-glucosyltransferases (An5GTs). HPLC analysis revealed that ‘Pink Passion’ petals accumulate 3,5-O-diglucosidic anthocyanins, absent in ‘Red River^®’ and ‘Ambiance’. RNA-seq identified seven candidate Fh5GT genes, with phylogenetic and subcellular localization analyses confirming their classification as cytosolic glycosyltransferases. Expression profiling highlighted elevated transcript levels of Fh5GT1, Fh5GT3, and Fh5GT7 in ‘Pink Passion’, correlating with its di-glucosidic anthocyanin accumulation. In vitro enzymatic assays demonstrated that Fh5GT3 and Fh5GT7 preferentially glucosylate 3-O-monoglucosidic anthocyanins to form stable 3,5-O-diglucosides, with minimal activity on anthocyanidins to generate 5-O-glucosidic anthocyanins. Heterologous expression of Fh5GT3 and Fh5GT7 in Arabidopsis complemented anthocyanin deficiency in 5gt mutants, restoring pigmentation. These findings elucidate the potential role of 5GTs in modulating floral color diversity through anthocyanin modification, providing insights for targeted breeding strategies to enhance ornamental traits in horticultural species.

Floral scent is an essential and genetically complex trait in herbaceous peonies (Paeonia lactiflora Pall.); however, specific genes related to metabolic and regulatory networks remain scantily studied. Our study integrated metabolite profiling and RNA-sequencing to screen floral scent biosynthetic genes. Hence, the major molecules identified by headspace collection combined with cultivar-specific GC-MS analysis were geraniol, β-caryophyllene, 2-phenylethanol (2-PE), citronellol, and 1,8-cineole. Genes related to terpenoids and 2-PE biosynthesis were identified after the assembly and annotation of the P. lactiflora transcriptomes. Eight angiosperm-specific terpene synthases (TPSs) from the TPS-a and TPS-b clades, as well as enzymes linked to 2-PE synthesis such as aromatic amino acid decarboxylase (AADC), phenylacetaldehyde reductase (PAR), and geranial reductase (GER) were identified. The biochemical analysis of the enzymes encoded by PlPAR1 and PlGER1 generated 2-PE from phenylacetaldehyde (PAld). The pairwise alignment of AADC1 reveals a splice variant lacking a 124 bp fragment, thus highlighting the possible role of alternative splicing in modulating floral scent composition. This study offers insights into the molecular-level biosynthesis of terpenoids and 2-PE in Peonia taxa, and provides the basis for the functional characterization, breeding, and bioengineering of prospective candidate genes for the production of floral volatiles in the Paeonia genus.

Volatile terpenes constitute a predominant class of floral scent emitted by Paeonia lactiflora. Despite their ecological and economical significance, the genetic blueprint of the underlying biosynthetic pathway remains poorly elucidated. Although a few terpene synthase (TPS) genes have been reported, the broader network of genes orchestrating terpene production in P. lactiflora is still largely unresolved. In this study, we attempted to address this gap by exploring the terpene biosynthetic pathway genes in P. lactiflora ‘Zifengyu’. β-caryophyllene, geraniol, citronellol, and 1, 8-cineole were identified as the dominant floral terpenes, and catalytic functions of key proteins—terpene synthase (PlTPS), Nudix hydrolase (PlNUDX), and prenyltransferase (PlPT) were comprehensively characterized. Briefly, biochemical analyses revealed that six of the nine identified PlTPS proteins utilized diverse prenyl diphosphates to generate both monoterpenes and sesquiterpenes, while their products specificity were determined by plastidic or cytosolic localizations in planta. In particular, PlTPS4, PlTPS5, and PlTPS9 catalyzed the production of β-caryophyllene, 1, 8-cineole, and geraniol, respectively. Besides, two amino acid residues were found to drive catalytic activity and product profiles in PlTPS4 and PlTPS5. Markedly, PlNUDX hydrolyzed GPP and NPP to yield geraniol and nerol thereby providing a plastid-independent pathway for monoterpene biosynthesis, and prenyltransferases were further functionally characterized to clarify the supply of prenyl diphosphates feeding into volatile terpenes. Collectively, these findings not only provide a mechanistic framework for understanding floral terpene biosynthesis in P. lactiflora but also reveal alternative metabolic routes that enrich its volatile profiles that could be utilized in scent improvement of ornamental plants.