The development of dual-target inhibitors represents a cost-effective strategy for integrated pest management. Here, we report the first dual-target inhibitors designed against the evolutionarily conserved domain of phytoene synthase (PSY), a key enzyme in carotenoid biosynthesis. Using comparative genomics, we identified structural conservation between PSY in plants and squalene synthase (erg9) in fungi. Through virtual screening and structure-based optimization of compounds targeting PSY, we identified lead compound 1c, which exhibited potent herbicidal and fungicidal activity. Invitro binding assays confirmed that 1c binds to both PSY and erg9. In plants, 1c treatment reduced chlorophyll content, downregulated photosynthesis-associated genes, and caused substrate accumulation in the carotenoid pathway. In fungi, 1c induced a mycelial morphology identical to erg9 knockout mutants. Molecular dynamics simulations revealed the differential binding conformations of 1c to PSY and erg9, elucidating its mode of action. This work establishes PSY and its homologues as a promising target for the development of novel, broad-spectrum dual-action agrochemicals based on targetome structural similarity.

Coordinated horizontal transfer of multiple genes assembles a carotenoid biosynthesis pathway in aphids.

Phytoene synthase (PSY) catalyzes the committed and rate-limiting step in carotenoid biosynthesis by condensing two geranylgeranyl diphosphate molecules into 15-cis-phytoene. As the gateway enzyme, PSY directs metabolic flux toward diverse carotenoids, which functions as essential pigments, antioxidants, and provitamin A precursors in nutrition and agriculture. PSY enzymes, widespread across biological kingdoms, have evolved lineage-specific regulatory mechanism while retaining conserved catalytic features, including hydrophobic loops and Asp-rich motifs, indicating a universal synthesis mechanism. This review summarizes evolutionary phylogeny and structure-function relationship of PSYs from prokaryotes to eukaryotes, highlighting their roles as key biotechnological targets for metabolic engineering to improve crop nutritional quality, stress resilience, and biofortification. Future research should elucidate PSY's regulation, catalytic machinery, and rate-limiting control over carotenoid profiles.

Pathogenesis and defense gene response in Crocus sativus (saffron) against the root-lesion nematode Pratylenchus penetrans.

Seed longevity, the seed's ability to stay viable over time, is an important trait in agriculture that remains a fundamental topic in plant biology. Here, we discovered a function of carotenoid metabolites in prolonging seed lifespan. We found that phytoene synthase (PSY), a major rate-limiting enzyme in carotenoid biosynthesis, modulated seed storability in Arabidopsis (Arabidopsis thaliana) under both natural and artificial aging conditions. Seeds from PSY overexpression lines exhibited significantly enhanced lifespan with low levels of reactive oxygen species (ROS), a major factor affecting longevity. In contrast, seeds from psy mutants had decreased viability with high ROS levels. Both lutein and β-carotene were detected in seeds. However, only β-carotene and its derived apocarotenoids, β-cyclocitral and β-ionone, improved seed lifespan. Seeds from a carotenoid cleavage dioxygenase 1 and 4 (ccd1/4) double mutant and ccd1/4 PSY overexpressing lines had significantly reduced seed longevity, supporting that β-carotene cleavage is necessary for preserving seed lifespan. Comparative proteomic analysis identified TIP2;2, an aquaporin protein, with differential abundances in seeds of PSY overexpression and psy mutant lines. The tip2;2 mutant had reduced seed longevity, and its promoter was transactivated by apocarotenoids. Overexpression of PSY in tip2;2 failed to fully reverse the effects of the mutation, indicating that TIP2;2 is required for the PSY-regulated seed longevity. This study uncovers a role of apocarotenoids in protecting seed longevity and highlights the importance of seed carotenoid production in strengthening agriculture.

Carotenoids and anthocyanins are the two major classes of pigments in plants and are bioactive compounds with nutritional value in fruit crops. The color diversity in citrus (Citrus spp.) is attributed to the varying levels and composition of carotenoids and anthocyanins. In blood orange (Citrus sinensis) fruits, which accumulate both carotenoids and anthocyanins, these pigments exhibit coordinated changes during ripening; however, the mechanisms underlying this accumulation remain unclear. Here, we reveal that B-BOX DOMAIN PROTEIN 24 (CsBBX24) regulates both carotenoid and anthocyanin biosynthesis during the ripening of blood orange fruits. CsBBX24 was co-expressed with the carotenoid biosynthesis gene PHYTOENE SYNTHASE 1 (CsPSY1) and the anthocyanin regulatory gene CsRuby1, which encodes a MYB-domain protein during fruit ripening. Ectopic expression of CsBBX24 in Hongkong kumquat (C. hindsii), tomato (Solanum lycopersicum), and apple (Malus domestica) calli promoted the accumulation of carotenoids and anthocyanins. An electrophoretic mobility shift assay and transient dual-luciferase assays in Nicotiana benthamiana leaves demonstrated that CsBBX24 can bind to and activate the CsPSY1 promoter for carotenoid biosynthesis and the CsRuby1 promoter for regulation of anthocyanin biosynthesis. Our findings have elucidated the role of CsBBX24 in promoting pigment accumulation during fruit ripening in blood orange, providing a valuable target gene for molecular breeding to improve citrus coloration quality.

Rosa rugosa (R. rugosa) is a commercially important ornamental species within the genus Rosa, highly valued in the horticultural market. With the increasing availability and improved annotation of Rosa genomes, establishing an efficient genetic transformation system has become essential for validating candidate gene functions. As a common intermediate tissue in plant regeneration, callus has been successfully used to establish genetic transformation systems in numerous species. In this study, we characterized the morphological and physiological differences between embryogenic and non-embryogenic calli in R. rugosa. The embryogenic callus exhibited significantly higher catalase (CAT) activity and proline (PRO) content than the non-embryogenic callus. However, its growth rate was markedly slower. Antibiotic sensitivity assays identified the optimal selection concentrations for non-embryogenic callus as 35 mg/L for kanamycin and 13 mg/L for hygromycin. We subsequently introduced the phytoene synthase (RrPSY1) gene into non-embryogenic callus, with positive transformants identified using GFP fluorescence detection and PCR analysis. The overexpression of RrPSY1 significantly increased the yellow pigment substances in the callus, confirming the establishment of an effective genetic transformation system for non-embryogenic calli in R. rugosa. This system provides a useful technical platform for the manipulation of metabolic products and the verification of related gene functions in rose.

BackgroundTerpenoids constitute a diverse group of primary and secondary metabolites that are extensively distributed in living organisms and play key roles in growth, development, and environmental adaptation. Terpenoids are derived from two isomeric precursors that are interconverted by isopentenyl-diphosphate delta-isomerase (IDI), in both the plastids and cytoplasm of plants. The plastidial pathway supplies precursors for diterpenoids and carotenoids, whereas the cytoplasmic pathway provides precursors for sesquiterpenoids and triterpenoids. A family of terpene synthases (TPSs) produce most terpenoids such as sesquiterpenes, hemiterpenes, monoterpenes, diterpenes and sesterterpenes, which in allotetraploid peanut (Arachis hypogaea L.) have been relatively underexplored.ResultsIn this study, 77 AhTPS genes were identified in the peanut genome and phylogenetically classified into five subfamilies. These AhTPSs are organized in clusters across chromosomes and exhibit conserved gene structures and motifs within each subfamily. AhTPSs in the TPS-c and -e/f subfamilies, specifically copalyl diphosphate synthase (CPS) and kaurene synthase (KS), were localized to plastids through transient expression in Nicotiana benthamiana leaves. The expression of AhCPS3 was detected a significant increase in response to abscisic acid (ABA) and methyl jasmonate (MeJA), which was notably distinct from the expression patterns of other AhCPSs and AhKS. Furthermore, 12 terpenoids were identified during seed development. Module-trait correlation analysis disclosed that the expression levels of genes encoding AhCPSs (involved in diterpene biosynthesis) and phytoene synthases (AhPSYs, involved in carotenoid biosynthesis) were significantly correlated with the abundances of soyasaponins (triterpenoids) during seed development in peanut. Additionally, the gene encoding β-amyrin synthase (AhβAS), which produces the backbone of triterpenoids, was identified in a significant module and was also induced by ABA and MeJA. Protein-protein interaction networks indicated AhCPSs, AhPSYs, and AhβAS shared a common interacting protein, AhIDI.ConclusionsThese findings provide valuable insights into the potential cross-talk in terpenoid biosynthesis across different cellular compartments.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12864-025-12013-x.

Complete genome sequence of Tenacibaculum mesophilum strain M-16, a marine bacterium from the sea of Oman with potential for zeaxanthin biosynthesis.

Capsicum chinense (habanero pepper) exhibits substantial variation in fruit pungency, color, and flavor due to its rich secondary metabolite composition, including capsaicinoids, carotenoids, and volatile organic compounds (VOCs). To dissect the genetic and regulatory basis of these traits, we conducted an integrative analysis across 244 diverse accessions using metabolite profiling, genome-wide association studies (GWAS), and transcriptome-wide association studies (TWAS). GWAS identified 507 SNPs for capsaicinoids, 304 for carotenoids, and 1176 for VOCs, while TWAS linked gene expression to metabolite levels, highlighting biosynthetic and regulatory genes in phenylpropanoid, fatty acid, and terpenoid pathways. Segmental RNA sequencing across fruit tissues of contrasting accessions revealed 7034 differentially expressed genes, including MYB31, 3-ketoacyl-CoA synthase, phytoene synthase, and ABC transporters. Notably, AP2 transcription factors and Pentatrichopeptide repeat (PPR) emerged as central regulators, co-expressed with carotenoid and VOC biosynthetic genes. High-resolution spatial transcriptomics (Stereo-seq) identified 74 genes with tissue-specific expression that overlap with GWAS and TWAS loci, reinforcing their regulatory relevance. To validate these candidates, we employed CRISPR/Cas9 to knock out AP2 and PPR genes in tomato. Widely targeted metabolomics and carotenoid profiling revealed major metabolic shifts: AP2 mutants accumulated higher levels of β-carotene and lycopene. In contrast, PPR mutants altered xanthophyll ester and apocarotenoid levels, supporting their roles in carotenoid flux and remodeling. This study provides the first integrative GWAS–TWAS–spatial transcriptomics in C. chinense, revealing key regulators of fruit quality traits. These findings lay the groundwork for precision breeding and metabolic engineering to enhance nutritional and sensory attributes in peppers.

Carotenoids serve critical biological functions through their essential contributions to organismal survival and health. As a widely consumed fruit species, peach (Prunus persica) provides humans with valuable carotenoid sources, and these compounds also substantially enhance the aromatic properties of peach fruits. The synthesis of phytoene, catalyzed by phytoene synthase (PSY), constitutes a key rate-limiting step in carotenoid biosynthesis. In this study, we investigated PSY isoforms in peaches, uncovering their tissue-specific expression patterns and identifying functional divergence among these isoforms through transgenic approaches using peach callus systems. By integrating protein structural analyses, yeast 2-hybrid assays, and engineered bacterial systems, we demonstrated that the differential interaction strengths between PSY1/PSY2 and geranylgeranyl pyrophosphate synthase (GGPPS) are likely the primary drivers of their distinct functional activities. Site-directed mutagenesis of the PSY-GGPPS interaction interface further confirmed a significant positive correlation between the strength of this interaction and the functional activity of PSY. Our findings suggest that the functional differences among peach PSY isoforms may arise from the cumulative effects of divergent residues between PSY1 and PSY2 on the PSY-GGPPS interactions. Notably, we identified a key individual residue that substantially influences the PSY-GGPPS interaction and the functional activity of PSY. This study provides insights into the molecular mechanisms driving the functional differentiation of PSY isoforms and highlights the potential for engineering PSYs rationally to develop crops with enhanced carotenoid content.

Plant breeders aim to increase provitamin A carotenoids in cassava (Manihot esculenta) storage roots to help combat vitamin A deficiency in sub-Saharan Africa, but a negative genetic correlation between total carotenoid and dry matter contents hinders progress. While genetic linkage between a major-effect variant in the phytoene synthase 2 (PSY2) gene and nearby candidate gene(s) has been thought to drive this correlation, molecular evidence suggests there may be a metabolic relationship between total carotenoid and dry matter, implying genome-wide pleiotropic effects. Bivariate genome-wide associations were used to examine the genetic architecture of the negative covariance between traits and test for pleiotropy. A population of 378 accessions in the yellow-fleshed cassava breeding program at the International Institute of Tropical Agriculture (IITA) in Ibadan, Nigeria was genotyped with DArTseqLD and phenotyped in field trials over 10 yr across three locations in Nigeria. Mixed linear models controlling for the previously identified PSY2 causal variant were used to identify multiple new pleiotropic loci. Among 17 jointly associated loci at a relaxed significance threshold, most (11 of 17) affected total carotenoid and dry matter in opposite directions, although this pattern did not reach statistical significance in a binomial test. Even after accounting for these 17 loci as covariates, significantly negative polygenic covariance between total carotenoid and dry matter remained. These findings support the hypothesis that widespread mediated pleiotropy rather than genetic linkage drives the negative genetic correlation between total carotenoid and dry matter in cassava and demonstrate a new application of multivariate genome-wide association study for interrogating the genetic architecture of correlated traits.

Astaxanthin and canthaxanthin are high-value carotenoids with growing demand due to their antioxidant properties and applications in food, cosmetic, and pharmaceutical sectors. However, natural sources are limited and current production methods are often costly or unsustainable. In this study, we developed a plant-based platform for ketocarotenoid biosynthesis using metabolically engineered Nicotiana tabacum BY-2 cell suspension cultures. Specifically, we expressed a marine bacterial crtW gene (β-carotene ketolase) alone or in combination with overexpressed plant psy (phytoene synthase) and crtI (phytoene desaturase) genes. The resulting cell lines displayed visually distinct pigmentation and accumulated different ketocarotenoid profiles based on their genetic modifications. Single-gene transformants expressing crtW produced up to 50µg g⁻¹ DW of canthaxanthin and 127µg g⁻¹ DW of astaxanthin. Co-expression of all three genes significantly increased canthaxanthin accumulation to 788µg g⁻¹ DW. Our results establish suspended undifferentiated plant cells as a scalable and sustainable system for ketocarotenoid production, offering a biological alternative to natural producers and chemical synthesis.

Phytoene synthase (PSY) is a multimeric enzyme that serves as the first enzyme in carotenoid synthesis within plant tissues and plays a crucial role in the production of carotenoids in plants. To understand the function of the PSY gene in Panax japonicus C. A. Meyer. fruit, the gene’s transcript was obtained by analyzing the transcriptome sequencing data of Panax japonicus fruit. The CDS sequence of the gene was cloned from Panax japonicus fruit using the RT-PCR cloning technique and named PjPSY1, which was then subjected to biosynthetic analysis and functional verification. The results showed that the open reading frame of the gene was 1269 bp, encoding 423 amino acids, with a protein molecular mass of 47,654.67 KDa and an isoelectric point (pI) of 8.63; the protein encoded by these amino acids was hydrophilic and localized in chloroplasts, and its three-dimensional structure was predicted by combining the pymol software to annotate the N site of action and active centre of the protein. Phylogenetic analysis demonstrated that PjPSY1 had the closest affinity to DcPSY from Daucus carota. Overexpression of PjPSY1 led to a significant increase in the content of carotenoid-related monomers in Arabidopsis thaliana, with Violaxanthin being synthesized in transgenic Arabidopsis thaliana but not in wild-type Arabidopsis thaliana. The PjPSY1 clone obtained in this study was able to promote carotenoid synthesis in the fruits of Panax japonicus, revealing that the mode of action of PjPSY1 in the carotenoid biosynthesis pathway of Panax japonicus fruits has a positive regulatory effect.

During fruit ripening in Capsicum species, substantial amounts of carotenoids accumulate in the pericarp. While the carotenoid biosynthesis pathway in Capsicum species has been extensively investigated from various angles, the transcriptional regulation of genes encoding carotenoid biosynthetic enzymes remains less understood in this non-climacteric horticultural crop compared to tomato, a climacteric fruit. In the present study, we investigated the function of the NAM, ATAF1/2 or CUC2 81 (CaNAC81) transcription factor gene. This gene was selected through RNA-Seq co-expression analysis based on the correlation between expressed transcription factor gene profiles and those of carotenoid structural genes. To determine its role in regulating the expression of biosynthetic-related carotenogenic genes, we performed Virus-Induced Gene Silencing (VIGS) assays in the Serrano-type C. annuum ‘Tampiqueño 74’. Fruits from plants infected with a pTRV2:CaNAC81 construct (silenced fruits) exhibited altered carotenoid pigmentation accumulation, manifested as yellow-orange spots, in contrast to fruits from non-agroinfected controls (NTC) and fruits from plants infected with the empty TRV2 construct (red fruits). Quantitative real-time PCR (qPCR) assays confirmed decreased transcript levels of CaNAC81 in fruits displaying altered pigmentation, along with reduced transcription of the PSY gene, which encodes the carotenoid biosynthetic enzyme phytoene synthase (PSY). High-performance liquid chromatography (HPLC) analysis revealed a distinct carotenoid pigment accumulation pattern in fruits from plants showing silencing symptoms, characterized by low concentrations of capsanthin and zeaxanthin and trace amounts of capsorubin, compared to control plants (NTC). These findings suggest the involvement of CaNAC81 in the regulatory network of the carotenoid biosynthetic pathway in chili pepper fruits.

Terpenes and terpenoids are vital components in diverse metabolic pathways, forming the terpenome-the complete spectrum of terpene-related compounds biosynthesized by an organism. Integrating bioinformatic tools has significantly enhanced the ability to assess metabolic potential by combining these computational approaches with experimental biochemical data. Furthermore, gene annotation provides critical insights into specialized targets, facilitating the identification of shared or unique features across different strains. This study investigates the presence of terpene compounds in cyanobacterial strains isolated from tropical soda lakes using a combination of gene mining, synteny analysis, phylogenetics, and metabolomics. Key enzymes, including phytoene synthase and squalene hopene cyclase, were identified, showing significant similarities and evolutionary links to gene copies in Cyanobacteria from diverse ecological environments. Metabolomic analysis complemented genomic predictions, uncovering a rich diversity of tetraterpene compounds, particularly carotenoids. Notably, triterpene hopanoids were found exclusively in a unicellular strain. These compounds show significant potential for cellular protection, metabolic adaptation, and biotechnological uses. They might support microbial communities in extreme environments, such as the saline-alkaline lakes of the Pantanal Biome in Brazil, developing unique survival and resilience strategies in these harsh conditions. This study highlights the extensive range of insights that can be obtained by integrating genetics and biochemistry in exploring cyanobacterial diversity, especially from organisms thriving in extreme environments.

Carotenoids are a family of lipophilic metabolites with essential roles in photosynthesis and photoprotection, and secondary roles as pigments providing color to non-green tissues. In addition, their cleavage generates compounds collectively known as apocarotenoids that can function as flavors, pigments, and bioactive molecules. In plants, carotenoids are synthesized and stored in plastids. Isopentenyl diphosphate and dimethylallyl diphosphate supplied by the methylerythritol 4-phosphate (MEP) pathway are used to produce geranylgeranyl diphosphate (GGPP), a common precursor for carotenoids and other plastidial isoprenoids such as chlorophylls, tocopherols, plastoquinone, phylloquinone, diterpenes, and gibberellins. The first committed step of the carotenoid pathway is the condensation of two GGPP molecules to form phytoene, which is later converted into the rest of the carotenoid products. In stark contrast to other plants such as Arabidopsis thaliana, tomato (Solanum lycopersicum) has gene families encoding the plastidial enzymes that catalyze the main rate-determining step of the MEP pathway [deoxyxylulose 5-phosphate synthase (DXS1 and DXS2)], the production of GGPP [GGPP synthase (GGPPS1-GGPPS3)], and its diversion to the carotenoid pathway [phytoene synthase (PSY1-PSY3)]. Here we review the latest advances in our understanding of how the different tomato paralogs of these three enzymes contribute to carotenoid biosynthesis in different plant tissues, developmental stages, and environmental conditions.

Phytoene synthase gene GbDYA modulates carotenoids accumulation and confers yellow anther and pollen antioxidant activity in cotton

Passion fruit (Passiflora edulis) is a perennial, woody, tropical vine. It produces edible round to oval fruit that is highly favored for its unique aroma and taste, as well as its richness in antioxidants, vitamins, and minerals. However, functional genomics studies of passion fruit are scarce, as simple and efficient genetic tools are lacking for this species. Here, we developed virus-mediated protein overexpression (VOX) and virus-induced gene silencing (VIGS) vectors based on the telosma mosaic virus (TelMV), an emerging potyvirus that infects passion fruit plants worldwide. This vector, designated pTelMV-GW, incorporates Gateway-compatible recombination sites for rapid gene cloning. Using this vector, we achieved systemic stable expression of 2 heterologous proteins in passion fruit: green fluorescent protein (GFP) and bacterial phytoene synthase (CrtB). Additionally, pTelMV-GW containing different GFP fragments also induced systemic gene silencing in GFP-transgenic Nicotiana benthamiana plants. Furthermore, we used this vector to trigger phytoene desaturase (PDS) and magnesium chelatase subunit I (ChlI) silencing in passion fruit plants. The TelMV-based VIGS was enhanced using a mild TelMV strain encoding a mutated helper-component proteinase (HC-Pro) with impaired RNA silencing suppressor activity. This upgraded vector (pTelMV-R181K-GW), containing PDS or ChlI fragments, induced clear photobleaching or yellowing phenotypes in passion fruit plants. Overall, our work presents a set of VIGS and VOX vectors for use in passion fruit plants, a crucial step towards identifying horticulturally important genes for improving passion fruit production and quality.

The identification and characterization of promoters and regulatory elements are commonly assessed using reporter genes. However, very few of these are available for haloarchaea. In this study, we describe the construction and validation of a reporter system for the haloarchaeon Haloferax volcanii based on a modified and stable version of the carotenoid biosynthesis enzyme phytoene synthase (PSY). This tool enables the analysis of a target gene expression through direct visualization of cell pigmentation and/or the extraction and quantification of carotenoid content. The modified crtB gene encoding PSY was cloned into the pTA963 vector under two regulatable promoters previously characterized in H. volcanii: PtnaA and Pxyl, inducible with tryptophan and xylose, respectively. The construct was introduced into and expressed in a non-pigmented H. volcanii strain (ΔcrtB) under varying inducer concentrations. For both promoters, a clear increase in pigmentation was visually observed in cultures and cell pellets with increasing inducer levels. These observations were corroborated by carotenoid extraction and quantification, as well as by Western blot analysis of PSY protein levels. This work presents a robust, easy-to-use, and versatile reporter system for investigating gene expression in H. volcanii, expanding the toolkit for genetic studies in haloarchaea.