Evolutionary trajectory of transcription factors and selection of targets for metabolic engineering.

Transcription factors (TFs) provide potentially powerful tools for plant metabolic engineering as they often control multiple genes in a metabolic pathway. However, selecting the best TF for a particular pathway has been challenging, and the selection often relies significantly on phylogenetic relationships. Here, we offer examples where evolutionary relationships have facilitated the selection of the suitable TFs, alongside situations where such relationships are misleading from the perspective of metabolic engineering. We argue that the evolutionary trajectory of a particular TF might be a better indicator than protein sequence homology alone in helping decide the best targets for plant metabolic engineering efforts. This article is part of the theme issue 'The evolution of plant metabolism'.

Herbicidal Sorbicillinoid Analogs Cause Lignin Accumulation in Aspergillus aculeatus TE-65L.

Five new sorbicillinoid derivatives, including (±)-aspersorbicillin A [(±)-1], a pair of enantiomers at C-9, and aspersorbicillins B-D (2-4), together with two known analogs (5 and 6) were isolated from the endophytic fungus Aspergillus aculeatus TE-65L. Their structures including absolute configurations were determined by detailed spectroscopic analyses and electronic circular dichroism calculations. The herbicidal activity of sorbicillinoids on the germ and radicle elongation of various weed types was reported for the first time. Compound 1 displayed significant herbicidal activity against Eleusine indica germ elongation (IC50 = 28.8 µg/mL), while compound 6 inhibited radicle elongation (IC50 = 25.6 µg/mL). Both were stronger than those of glyphosate (66.2 and 30.9 µg/mL, respectively). Further transcriptomic and LC-MS/MS metabolomic analysis indicated that 6 induced the transcriptional expressions of genes related to the lignin biosynthetic pathway, resulting in lignin accumulation. Transmission electron microscopy confirmed the cell wall thickening of seeds treated with 6, suggesting weed growth inhibition. This study reveals new lead compounds for fabricating natural herbicides and expands the agricultural use of sorbicillinoid analogs.

In vivo assembly in tobacco cells to elucidate and engineer the biosynthesis of 4-hydroxydihydrocinnamaldehyde from Gloriosa superba.

KEY MESSAGE: This study described the biosynthesis of 4-hydroxydihydrocinnamaldehyde sharing with monolignol pathway and supplemented the biosynthesis of colchicine in G. superba, 4-hydroxydihydrocinnamaldehyde produced in tobacco BY2 cells provided an important stepstone. The precursor, 4-hydroxydihydrocinnamaldehyde (4-HDCA), participates in the biosynthesis of the carbon skeleton of colchicine, which is derived from L-phenylalanine. However, one hypothesis proposed that 4-HDCA is synthesized by sharing the early part of the monolignol pathway in G. superba. In this study, we validated this prediction and identified the enzymatic functions involved in this pathway. GsDBR1 is a crucial enzyme to illustrate 4-HDCA diverging from monolignol pathway, we first confirmed its reductase activity on 4-coumaraldehyde, an important intermediate compound in monolignol biosynthesis. Then, the biochemical function of recombinant enzymes belonging to the other four families were verified to elucidate the entire process of 4-HDCA biosynthesis from L-phenylalanine. After reconstruction, the 4-HDCA was 78.4 ng/g with fresh weight (FW) of transgenic tobacco cells, and the yield increased to 168.22 ng/g·FW after improved treatment with methyl jasmonate (MeJA). The elucidation of 4-HDCA biosynthesis sharing the monolignol pathway supplemented the biosynthesis of colchicine in G. superba, and the production of 4-HDCA in tobacco cells provides an important step in the development of plant cell cultures as heterologous bio-factories for secondary metabolite production.

MicroRNA-146b-5p/FDFT1 mediates cisplatin sensitivity in bladder cancer by redirecting cholesterol biosynthesis to the non-sterol branch.

Chemotherapy against muscle-invasive bladder cancer is increasingly challenged by the prevalence of chemoresistance. The cholesterol biosynthesis pathway has garnered attention in studies of chemoresistance, but conflicting clinical and molecular findings necessitate a clearer understanding of its underlying mechanisms. Recently, we identified farnesyl-diphosphate farnesyltransferase 1 (FDFT1)-the first specific gene in this pathway-as a tumor suppressor and chemoresistance modulator. Raman spectroscopy revealed higher levels of FDFT1-related metabolites in chemotherapy-sensitive bladder cancer tissue compared to resistant tissue; however, this observation lacks mechanistic insight. FDFT1 expression was reduced in our cisplatin-resistant bladder cancer cells (T24R) compared to parental cisplatin-sensitive cells (T24). Using functional knockdown and ectopic overexpression in T24/T24R cells, we mechanistically demonstrate the pathway through which FDFT1 mediates cisplatin sensitivity in bladder cancer cells. Bioinformatics analysis and rescue experiments showed that microRNA-146b-5p directly targets and downregulates FDFT1, reducing the cisplatin sensitivity of T24 cells, which can be restored by forced FDFT1 expression. Further investigation into the downstream cholesterol pathway revealed that FDFT1 suppression redirects its substrate toward the non-sterol branch of the pathway, as evidenced by the upregulation of non-sterol branch-associated genes and a reduced total cholesterol level in the sterol branch. Since the non-sterol pathway leads to the prenylation of isoprenoids and activation of Ras and Rho family proteins involved in cancer progression and chemoresistance, our findings suggest that redirection of the cholesterol biosynthesis pathway is a key mechanism underlying FDFT1-mediated cisplatin resistance in bladder cancer. The miR-146b-5p/FDFT1 axis represents a promising target for overcoming chemoresistance in bladder cancer.

Integrated metabolome and transcriptome analysis provide insight into the biosynthesis of flavonoids in Panax japonicus.

Panax japonicus is an important medicinal plant, and flavonoids are one of its main secondary metabolites. In this study, the main roots, fibrous roots, stems, leaves and flowers of P. japonicus were analyzed using transcriptomics and widely targeted metabolomics. Through correlation analysis of transcription and metabolism, the flavonoid biosynthesis pathway in P. japonicus was analyzed, and the accumulation of flavonoid metabolites and the expression of related genes were investigated. Metabolomics revealed a total of 209 flavonoid metabolites in P. japonicus, among which flavonoids, flavonols, flavanones and flavanonols significantly accumulated in the flowers and leaves. Transcriptome sequencing revealed that key genes in the flavonoid pathway exhibited increased expression in the flowers and leaves. The expression patterns of key genes involved in flavonoid biosynthesis, including PjC4H, Pj4CL, PjCHS, PjCHI, PjF3H, PjF3'H, PjCYP, and PjPAL, are consistent with their upstream and downstream metabolites, demonstrating a significant positive correlation among them. In addition, the PjUGT gene is highly expressed in five tissues of P. japonicus, indicating that PjUGT is one of the key factors for the diversity of flavonoid glycosides. The WGCNA results showed that WRKY transcription factors exist widely in the candidate modules, and it was possible that PjWRKY transcription factors are involved in regulating the expression of key genes involved in flavonoid biosynthesis and the biosynthesis of flavonoid metabolites. This study reveals spatial differences in the accumulation patterns of flavonoid metabolites in different tissues and provides important clues for further understanding the regulatory mechanisms of flavonoid metabolism in P. japonicus, thus contributing to the optimization of germplasm resources of P. japonicus and the promotion of genetic diversity analysis.

Functional activities of three Rehmannia glutinosa enzymes: Elucidation of the Rehmannia glutinosa salidroside biosynthesis pathway in Saccharomyces cerevisiae.

Rehmannia glutinosa produces many phenylethanoid glycoside (PhG) compounds, including salidroside, which not only possesses various biological activities but also is a core precursor of some medicinal PhGs, so it is very important to elucidate the species' salidroside biosynthesis pathway to enhance the production of salidroside and its derivations. Although some plant copper-containing amine oxidases (CuAOs), phenylacetaldehyde reductases (PARs) and UDP-glucose glucosyltransferases (UGTs) are thought to be vital catalytic enzymes involved in the downstream salidroside biosynthesis pathways, to date, none of these proteins or the associated genes in R. glutinosa have been characterized. To verify a postulated R. glutinosa salidroside biosynthetic pathway starting from tyrosine, this study identified and characterized a set of R. glutinosa genes encoding RgCuAO, RgPAR and RgUGT enzymes for salidroside biosynthesis. The functional activities of these proteins were tested in vitro by heterologous expression of these genes in Escherichia coli, confirming these catalytic abilities in these corresponding reaction steps of the biosynthetic pathway. Importantly, four enzyme-encoding genes (including the previously reported RgTyDC2 encoding tyrosine decarboxylase and the RgCuAO1, RgPAR1 and RgUGT2 genes) were cointegrated into Saccharomyces cerevisiae to reconstitute the R. glutinosa salidroside biosynthetic pathway, achieving an engineered strain that produced salidroside and validating these enzymes' catalytic functions. This study elucidates the complete R. glutinosa salidroside biosynthesis pathway from tyrosine metabolism in S. cerevisiae, establishing a basic platform for the efficient production of salidroside and its derivatives.

Towards a thorough understanding of mammalian glycosylphosphatidylinositol-anchored protein biosynthesis.

Glycosylphosphatidylinositols (GPIs) are glycolipids found ubiquitously in eukaryotes. They consist of a glycan and an inositol phospholipid, and act as membrane anchors of many cell-surface proteins by covalently linking to their C-termini. GPIs also exist as unlinked, free glycolipids on the cell surface. In human cells, at least 160 proteins with various functions are GPI-anchored proteins (GPI-APs). Because the attachment of GPI is required for the cell-surface expression of GPI-APs, a thorough knowledge of the molecular basis of mammalian GPI-AP biosynthesis is important for understanding the basic biochemistry and biology of GPI-APs and their medical significance. In this paper, I review our previous knowledge of the biosynthesis of mammalian GPI-APs and then examine new findings made since 2020.

Exploring stachydrine: from natural occurrence to biological activities and metabolic pathways.

Stachydrine, also known as proline betaine, is a prominent constituent of traditional Chinese herb Leonurus japonicus, renowned for its significant pharmacological effects. Widely distributed in plants like Leonurus and Citrus aurantium, as well as various bacteria, stachydrine serves pivotal physiological functions across animal, plant, and bacterial kingdoms. This review aims to summarizes diverse roles and mechanisms of stachydrine in addressing cardiovascular and cerebrovascular diseases, neuroprotection, anticancer activity, uterine regulation, anti-inflammatory response, obesity management, and respiratory ailments. Notably, stachydrine exhibits cardioprotective effects via multiple pathways encompassing anti-inflammatory, antioxidant, anti-apoptotic, and modulation of calcium handling functions. Furthermore, its anti-cancer properties inhibit proliferation and migration of numerous cancer cell types. With a bi-directional regulatory effect on uterine function, stachydrine holds promise for obstetrics and gynecology-related disorders. In plants, stachydrine serves as a secondary metabolite, contributing to osmotic pressure regulation, nitrogen fixation, pest resistance, and stress response. Similarly, in bacteria, it plays a crucial osmoprotective role, facilitating adaptation to high osmotic pressure environments. This review also addresses ongoing research on the anabolic metabolism of stachydrine. While the biosynthetic pathway remains incompletely understood, the metabolic pathway is well-established. A deeper understanding of stachydrine biosynthesis holds significance for elucidating its mechanism of action, advancing the study of plant secondary metabolism, enhancing drug quality control, and fostering new drug development endeavors.

[Metabolic engineering of Escherichia coli for thymidine production].

Thymidine, as a crucial precursor of anti-AIDS drugs (e.g., zidovudine and stavudine), has wide application potential in the pharmaceutical industry. In this study, we introduced the thymidine biosynthesis pathway into the wild-type Escherichia coli MG1655 by systems metabolic engineering to improve the thymidine production in E. coli. Firstly, deoA, tdk, udp, rihA, rihB, and rihC were successively deleted to block the thymidine degradation pathway and salvage pathway in the wild-type E. coli MG1655. Then, the pyrimidine nucleoside operons from Bacillus subtilis F126 were introduced to enlarge the metabolic flux of the uridylic acid synthesis pathway. Finally, the expression of uridylate kinase, ribonucleoside diphosphate reductase, thymidine synthase, and 5'-nucleotidase in the thymidine biosynthesis pathway was optimized to enhance the metabolic flux from uridylic acid to thymidine. The engineered THY6-2 strain produced 11.10 g/L thymidine in a 5 L bioreactor with a yield of 0.04 g/g glucose and productivity of 0.23 g/(L·h). In this study, we constructed a strain that used glucose as the only carbon source for efficient production of thymidine and did not harbor plasmids, which provided a reference for the research on other pyrimidine nucleosides.

Progresses in biosynthesis pathway, regulation mechanism and potential application of 2-acetyl-1-pyrroline in fragrant rice.

The formation of rice aroma is a complex process that is influenced by genetic and environmental factors. More than 500 fragrance compounds have been documented in fragrant rice, among which 2-AP dominates the aroma of rice. This paper introduced the identification of OsBadh2 in the biosynthesis of 2-AP in rice. Then, non-enzymatic and enzymatic pathways of the 2-AP biosynthesis have been comprehensively investigated. In detail, 2-AP biosynthesis-associated enzyme, such as OsBADH2, OsP5CS, OsGAD, OsGAPDH, OsProDH, OsOAT, OsODC and OsDAO, have been summarized, while MG and fatty acids are also implicated in modulating the biosynthesis of 2-AP by providing the acetyl groups. Moreover, extensive collections of traditional fragrant rice varieties have been collated, together with the OsBadh2 haplotypes of 312 fragrant rice germplasm in China. And finally, genetic engineering of OsBadh2 and other genes in the 2-AP biosynthesis to develop fragrant rice are discussed.

Structure and Inhibition of Insect UDP-N-acetylglucosamine Pyrophosphorylase: A Key Enzyme in the Hexosamine Biosynthesis Pathway.

UDP-N-acetylglucosamine pyrophosphorylase (UAP) catalyzes the last step in the hexosamine biosynthesis pathway to directly produce UDP-N-acetylglucosamine (UDP-GlcNAc). Because UAPs play important physiological and pathological roles in organisms, they are considered potential targets for drug and pesticide development. However, the lack of efficient and selective inhibitors is a bottleneck that must be overcome. This study reports the first crystal structure of the insect UAP from Spodoptera frugiperda (SfUAP) in complex with UDP-GlcNAc. SfUAP has two insect-specific structural characteristics in the active pocket, namely, a free Cys (Cys334) and a Mg2+ binding site, which differentiate it from human UAP (HsAGX1) and fungal UAP (AfUAP) in terms of substrate and inhibitor binding. N-(4-Nitrophenyl)maleimide (pNPMI) and myricetin are discovered as potent covalent and noncovalent inhibitors of SfUAP, respectively. Moreover, myricetin can significantly reduce the level of cellular O-GlcNAcylation by inhibiting both UAP and O-GlcNAc transferase. These findings provide novel insights into the development of UAP-based drugs and pesticides.

FOXM1 Upregulates O-GlcNAcylation Level Via The Hexosamine Biosynthesis Pathway to Promote Angiogenesis in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) presents significant challenges in treatment and prognosis because of its aggressive nature and high metastatic potential. This study aims to investigate the role of the hexosamine biosynthesis pathway (HBP) and its association with HCC progression and prognosis. We identified SPP1 and FOXM1 as hub genes within the HBP pathway, showing their correlation with poor prognosis and late-stage progression. In addition, the analysis uncovered the complex participation of the HBP pathway in nutrients and oxygen reactions, PI3K-AKT signaling, AMPK activation, and angiogenesis regulation. The disruption of these pathways is pivotal in influencing the growth and progression of HCC. Targeting the HBP presents a promising therapeutic approach to modulate the tumor microenvironment, thereby enhancing the efficacy of immunotherapy. In addition, FOXM1 was identified as the HBP pathway regulator, influencing cellular O-GlcNAcylation level and VEGF secretion, thereby promoting angiogenesis in HCC. Inhibition of O-GlcNAcylation significantly hindered angiogenesis, which is suggested as a potential avenue for therapeutic intervention. Our research demonstrates the practicality of using the HBP-related gene as a prognostic marker in liver cancer patients and suggests targeting FOXM1 as a novel avenue for personalized therapy.

Muscle-specific lack of Gfpt1 triggers ER stress to alleviate misfolded protein accumulation.

Pathogenic variants in GFPT1, encoding a key enzyme to synthesize UDP-N-acetylglucosamine (UDP-GlcNAc), cause congenital myasthenic syndrome (CMS). We made a knock-in (KI) mouse model carrying a frameshift variant in Gfpt1 exon 9, simulating that found in a patient with CMS. As Gfpt1 exon 9 is exclusively expressed in striated muscles, Gfpt1-KI mice were deficient for Gfpt1 only in skeletal muscles. In Gfpt1-KI mice, (1) UDP-HexNAc, CMP-NeuAc and protein O-GlcNAcylation were reduced in skeletal muscles; (2) aged Gfpt1-KI mice showed poor exercise performance and abnormal neuromuscular junction structures; and (3) markers of the unfolded protein response (UPR) were elevated in skeletal muscles. Denervation-mediated enhancement of endoplasmic reticulum (ER) stress in Gfpt1-KI mice facilitated protein folding, ubiquitin-proteasome degradation and apoptosis, whereas autophagy was not induced and protein aggregates were markedly increased. Lack of autophagy was accounted for by enhanced degradation of FoxO1 by increased Xbp1-s/u proteins. Similarly, in Gfpt1-silenced C2C12 myotubes, ER stress exacerbated protein aggregates and activated apoptosis, but autophagy was attenuated. In both skeletal muscles in Gfpt1-KI mice and Gfpt1-silenced C2C12 myotubes, maladaptive UPR failed to eliminate protein aggregates and provoked apoptosis.

GFPT2 expression is induced by gemcitabine administration and enhances invasion by activating the hexosamine biosynthetic pathway in pancreatic cancer.

Our previous studies revealed a novel link between gemcitabine (GEM) chemotherapy and elevated glutamine-fructose-6-phosphate transaminase 2 (GFPT2) expression in pancreatic cancer (PaCa) cells. GFPT2 is a rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP). HBP can enhance metastatic potential by regulating epithelial-mesenchymal transition (EMT). The aim of this study was to further evaluate the effect of chemotherapy-induced GFPT2 expression on metastatic potential. GFPT2 expression was evaluated in a mouse xenograft model following GEM exposure and in clinical specimens of patients after chemotherapy using immunohistochemical analysis. The roles of GFPT2 in HBP activation, downstream pathways, and cellular functions in PaCa cells with regulated GFPT2 expression were investigated. GEM exposure increased GFPT2 expression in tumors resected from a mouse xenograft model and in patients treated with neoadjuvant chemotherapy (NAC). GFPT2 expression was correlated with post-operative liver metastasis after NAC. Its expression activated the HBP, promoting migration and invasion. Treatment with HBP inhibitors reversed these effects. Additionally, GFPT2 upregulated ZEB1 and vimentin expression and downregulated E-cadherin expression. GEM induction upregulated GFPT2 expression. Elevated GFPT2 levels promoted invasion by activating the HBP, suggesting the potential role of this mechanism in promoting chemotherapy-induced metastasis.

HY5 and PIF antagonistically regulate HMGR expression and sterol biosynthesis in Arabidopsis thaliana.

Secondary metabolites play multiple crucial roles in plants by modulating various regulatory networks. The biosynthesis of these compounds is unique to each species and is intricately controlled by a range of developmental and environmental factors. While light's role in certain secondary metabolites is evident, its impact on sterol biosynthesis remains unclear. Previous studies indicate that ELONGATED HYPOCOTYL5 (HY5), a bZIP transcription factor, is pivotal in skotomorphogenesis to photomorphogenesis transition. Additionally, PHYTOCHROME INTERACTING FACTORs (PIFs), bHLH transcription factors, act as negative regulators. To unveil the light-dependent regulation of the mevalonic acid (MVA) pathway, a precursor for sterol biosynthesis, mutants of light signaling components, specifically hy5-215 and the pifq quadruple mutant (pif 1,3,4, and 5), were analyzed in Arabidopsis thaliana. Gene expression analysis in wild-type and mutants implicates HY5 and PIFs in regulating sterol biosynthesis genes. DNA-protein interaction analysis confirms their interaction with key genes like AtHMGR2 in the rate-limiting pathway. Results strongly suggest HY5 and PIFs' pivotal role in light-dependent MVA pathway regulation, including the sterol biosynthetic branch, in Arabidopsis, highlighting a diverse array of light signaling components finely tuning crucial growth pathways.

Application of Silicon Influencing Grain Yield and Some Grain Quality Features in Thai Fragrant Rice.

Silicon (Si) is a beneficial nutrient that has been shown to increase rice productivity and grain quality. Fragrant rice occupies the high end of the rice market with prices at twice to more than three times those of non-fragrant rice. Thus, this study evaluated the effects of increasing Si on the yield and quality of fragrant rice. Also measured were the content of proline and the expression of the genes associated with 2AP synthesis and Si transport. The fragrant rice varieties were found to differ markedly in the effect of Si on their quality, as measured by the grain 2AP concentration, while there were only slight differences in their yield response to Si. The varieties with low 2AP when the Si supply is limited are represented by either PTT1 or BNM4 with only slight increases in 2AP when Si was increased. Si affects the gene expression levels of the genes associated with 2AP synthesis, and the accumulation of 2AP in fragrant rice mainly occurred through the upregulation of Badh2, DAO, OAT, ProDH, and P5CS genes. The findings suggest that Si is a potential micronutrient that can be utilized for improving 2AP and grain yield in further aromatic rice breeding programs.

Enhancing Phycocyanobilin Production Efficiency in Engineered Corynebacterium glutamicum: Strategies and Potential Application.

Phycocyanobilin, an algae-originated light-harvesting pigment known for its antioxidant properties, has gained attention as it plays important roles in the food and medication industries and has surged in demand owing to its low-yield extraction from natural resources. In this study, engineered Corynebacterium glutamicum was developed to achieve high PCB production, and three strategies were proposed: reinforcement of the heme biosynthesis pathway with the introduction of two PCB-related enzymes, strengthening of the pentose phosphate pathway to generate an efficient cycle of NADPH, and fed-batch fermentation to maximize PCB production. Each approach increased PCB synthesis, and the final engineered strain successfully produced 78.19 mg/L in a flask and 259.63 mg/L in a 5 L bioreactor, representing the highest bacterial production of PCB reported to date, to our knowledge. The strategies applied in this study will be useful for the synthesis of PCB derivatives and can be applied in the food and pharmaceutical industries.

Large Flux Supply of NAD(H) under Aerobic Conditions by Candida glycerinogenes.

NAD is a redox coenzyme and is the center of energy metabolism. In metabolic engineering modifications, an insufficient NAD(H) supply often limits the accumulation of target products. In this study, Candida glycerinogenes was found to be able to supply NAD(H) in large fluxes, up to 7.6 times more than Saccharomyces cerevisiae in aerobic fermentation. Aerobic fermentation in a medium without amino nitrogen sources demonstrated that C. glycerinogenes NAD synthesis was not dependent on NAD precursors in the medium. Inhibition by antisense RNA and the detection of transcript levels indicated that the main NAD supply pathway is the de novo biosynthesis pathway. It was further demonstrated that NAD(H) supply was unaffected by changes in metabolic flow through C. glycerinogenes ΔGPD aerobic fermentation (80 g/L ethanol). In conclusion, the ability of C. glycerinogenes to supply NAD(H) in large fluxes provides a new approach to solving the NAD(H) supply problem in synthetic biology.

Advances in hyaluronic acid production: Biosynthesis and genetic engineering strategies based on Streptococcus - A review.

Hyaluronic acid (HA), which is a highly versatile glycosaminoglycan, is widely applied across the fields of food, cosmetics, and pharmaceuticals. It is primary produced through Streptococcus fermentation, but the product presents inherent challenges concerning consistency and potential pathogenicity. However, recent strides in molecular biology have paved the way for genetic engineering, which facilitates the creation of high-yield, nonpathogenic strains adept at synthesizing HA with specific molecular weights. This comprehensive review extensively explores the molecular biology underpinning pivotal HA synthase genes, which elucidates the intricate mechanisms governing HA synthesis. Moreover, it delineates various strategies employed in engineering HA-producing strains.

Complex petal spot formation in the Beetle Daisy (Gorteria diffusa) relies on spot-specific accumulation of malonylated anthocyanin regulated by paralogous GdMYBSG6 transcription factors.

Gorteria diffusa has elaborate petal spots that attract pollinators through sexual deception, but how G. diffusa controls spot development is largely unknown. Here, we investigate how pigmentation is regulated during spot formation. We determined the anthocyanin composition of G. diffusa petals and combined gene expression analysis with protein interaction assays to characterise R2R3-MYBs that likely regulate pigment production in G. diffusa petal spots. We found that cyanidin 3-glucoside pigments G. diffusa ray floret petals. Unlike other petal regions, spots contain a high proportion of malonylated anthocyanin. We identified three subgroup 6 R2R3-MYB transcription factors (GdMYBSG6-1,2,3) that likely activate the production of spot pigmentation. These genes are upregulated in developing spots and induce ectopic anthocyanin production upon heterologous expression in tobacco. Interaction assays suggest that these transcription factors regulate genes encoding three anthocyanin synthesis enzymes. We demonstrate that the elaboration of complex spots in G. diffusa begins with the accumulation of malonylated pigments at the base of ray floret petals, positively regulated by three paralogous R2R3-MYB transcription factors. Our results indicate that the functional diversification of these GdMYBSG6s involved changes in the spatial control of their transcription, and modification of the duration of GdMYBSG6 gene expression contributes towards floral variation within the species.