Expression of endogenous UDP-glucosyltransferase in endophyte Phomopsis liquidambaris reduces deoxynivalenol contamination in wheat.

Fusarium head blight is a devastating disease that causes severe yield loses and mycotoxin contamination in wheat grain. Additionally, balancing the trade-off between wheat production and disease resistance has proved challenging. This study aimed to expand the genetic tools of the endophyte Phomopsis liquidambaris against Fusarium graminearum. Specifically, we engineered a UDP-glucosyltransferase-expressing P. liquidambaris strain (PL-UGT) using ADE1 as a selection marker and obtained a deletion mutant using an inducible promoter that drives Cas9 expression. Our PL-UGT strain converted deoxynivalenol (DON) into DON-3-G in vitro at a rate of 71.4 % after 36 h. DON inactivation can be used to confer tolerance in planta. Wheat seedlings inoculated with endophytic strain PL-UGT showed improved growth compared with those inoculated with wildtype P. liquidambaris. Strain PL-UGT inhibited the growth of Fusarium graminearum and reduced infection rate to 15.7 %. Consistent with this finding, DON levels in wheat grains decreased from 14.25 to 0.56 µg/g when the flowers were pre-inoculated with PL-UGT and then infected with F. graminearum. The expression of UGT in P. liquidambaris was nontoxic and did not inhibit plant growth. Endophytes do not enter the seeds nor induce plant disease, thereby representing a novel approach to fungal disease control.

Exploration and utilization of novel aldoxime, nitrile, and nitro compounds metabolizing enzymes from plants and arthropods.

Aldoxime (R1R2C=NOH) and nitrile (R-C≡N) are nitrogen-containing compounds that are found in species representing all kingdoms of life. The enzymes discovered from the microbial "aldoxime-nitrile" pathway (aldoxime dehydratase, nitrile hydratase, amidase, and nitrilase) have been thoroughly studied because of their industrial importance. Although plants utilize cytochrome P450 monooxygenases to produce aldoxime and nitrile, many biosynthetic pathways are yet to be studied. Cyanogenic millipedes accumulate various nitrile compounds, such as mandelonitrile. However, no such aldoxime- and nitrile-metabolizing enzymes have been identified in millipedes. Here, I review the exploration of novel enzymes from plants and millipedes with characteristics distinct from those of microbial enzymes, the catalysis of industrially useful reactions, and applications of these enzymes for nitrile compound production.

Highly efficient biosynthesis of salidroside by a UDP-glucosyltransferase-catalyzed cascade reaction.

OBJECTIVE: Salidroside is an important plant-derived aromatic compound with diverse biological properties. The main objective of this study was to synthesize salidroside from tyrosol using UDP-glucosyltransferase (UGT) with in situ regeneration of UDP-glucose (UDPG). RESULTS: The UDP-glucosyltransferase 85A1 (UGT85A1) from Arabidopsis thaliana, which showed high activity and regioselectivity towards tyrosol, was selected for the production of salidroside. Then, an in vitro cascade reaction for in situ regeneration of UDPG was constructed by coupling UGT85A1 to sucrose synthase from Glycine max (GmSuSy). The optimal UGT85A1-GmSuSy activity ratio of 1:2 was determined to balance the efficiency of salidroside production and UDP-glucose regeneration. Different cascade reaction conditions for salidroside production were also determined. Under the optimized condition, salidroside was produced at a titer of 6.0 g/L with a corresponding molar conversion of 99.6% and a specific productivity of 199.1 mg/L/h in a continuous feeding reactor. CONCLUSION: This is the highest salidroside titer ever reported so far using biocatalytic approach.

Identification of a UDP-glucosyltransferase conferring deoxynivalenol resistance in Aegilops tauschii and wheat.

Aegilops tauschii is the diploid progenitor of the wheat D subgenome and a valuable resource for wheat breeding, yet, genetic analysis of resistance against Fusarium head blight (FHB) and the major Fusarium mycotoxin deoxynivalenol (DON) is lacking. We treated a panel of 147 Ae. tauschii accessions with either Fusarium graminearum spores or DON solution and recorded the associated disease spread or toxin-induced bleaching. A k-mer-based association mapping pipeline dissected the genetic basis of resistance and identified candidate genes. After DON infiltration nine accessions revealed severe bleaching symptoms concomitant with lower conversion rates of DON into the non-toxic DON-3-O-glucoside. We identified the gene AET5Gv20385300 on chromosome 5D encoding a uridine diphosphate (UDP)-glucosyltransferase (UGT) as the causal variant and the mutant allele resulting in a truncated protein was only found in the nine susceptible accessions. This UGT is also polymorphic in hexaploid wheat and when expressed in Saccharomyces cerevisiae only the full-length gene conferred resistance against DON. Analysing the D subgenome helped to elucidate the genetic control of FHB resistance and identified a UGT involved in DON detoxification in Ae. tauschii and hexaploid wheat. This resistance mechanism is highly conserved since the UGT is orthologous to the barley UGT HvUGT13248 indicating descent from a common ancestor of wheat and barley.

Natural alleles of a uridine 5'-diphospho-glucosyltransferase gene responsible for differential endosperm development between upland rice and paddy rice.

Traditional upland rice generally exhibits insufficient grains resulting from abnormal endosperm development compared to paddy rice. However, the underlying molecular mechanism of this trait is poorly understood. Here, we cloned the uridine 5'-diphospho (UDP)-glucosyltransferase gene EDR1 (Endosperm Development in Rice) responsible for differential endosperm development between upland rice and paddy rice by performing quantitative trait loci analysis and map-based cloning. EDR1 was highly expressed in developing seeds during grain filling. Natural variations in EDR1 significantly reduced the UDP-glucosyltransferase activity of EDR1YZN compared to EDR1YD1 , resulting in abnormal endosperm development in the near-isogenic line, accompanied by insufficient grains and changes in grain quality. By analyzing the distribution of the two alleles EDR1YD1 and EDR1YZN among diverse paddy rice and upland rice varieties, we discovered that EDR1 was conserved in upland rice, but segregated in paddy rice. Further analyses of grain chalkiness in the alleles of EDR1YD1 and EDR1YZN varieties indicated that rice varieties harboring EDR1YZN and EDR1YD1 preferentially showed high chalkiness, and low chalkiness, respectively. Taken together, these results suggest that the UDP-glucosyltransferase gene EDR1 is an important determinant controlling differential endosperm development between upland rice and paddy rice.

Bombyx mori nucleopolyhedrovirus ptp and egt genes are dispensable for triggering enhanced locomotory activity and climbing behavior in Bombyx mandarina larvae.

Baculoviruses are classic pathogens that alter host behavior to enhance their dispersal and transmission. While viral protein tyrosine phosphatase (ptp) has been considered as a critical factor for inducing enhanced locomotory activity, preceding investigations have reported that viral ecdysteroid UDP-glucosyltransferase (egt) contributes to triggering climbing behavior in some virus and host species. Here we found that both egt and ptp were dispensable for these abnormal behaviors in Bombyx mandarina larvae induced by Bombyx mori nucleopolyhedrovirus, thus implying that there is an unknown core mechanism of baculovirus-induced alteration of host behaviors.

Glucosylation of (±)-Menthol by Uridine-Diphosphate-Sugar Dependent Glucosyltransferases from Plants.

Menthol is a cyclic monoterpene alcohol of the essential oils of plants of the genus Mentha, which is in demand by various industries due to its diverse sensorial and physiological properties. However, its poor water solubility and its toxic effect limit possible applications. Glycosylation offers a solution as the binding of a sugar residue to small molecules increases their water solubility and stability, renders aroma components odorless and modifies bioactivity. In order to identify plant enzymes that catalyze this reaction, a glycosyltransferase library containing 57 uridine diphosphate sugar-dependent enzymes (UGTs) was screened with (±)-menthol. The identity of the products was confirmed by mass spectrometry and nuclear magnetic resonance spectroscopy. Five enzymes were able to form (±)-menthyl-ß-d-glucopyranoside in whole-cell biotransformations: UGT93Y1, UGT93Y2, UGT85K11, UGT72B27 and UGT73B24. In vitro enzyme activity assays revealed highest catalytic activity for UGT93Y1 (7.6 nkat/mg) from Camellia sinensis towards menthol and its isomeric forms. Although UGT93Y2 shares 70% sequence identity with UGT93Y1, it was less efficient. Of the five enzymes, UGT93Y1 stood out because of its high in vivo and in vitro biotransformation rate. The identification of novel menthol glycosyltransferases from the tea plant opens new perspectives for the biotechnological production of menthyl glucoside.

UDP-glucosyltransferase UGT84B1 regulates the levels of indole-3-acetic acid and phenylacetic acid in Arabidopsis.

Auxin is a key plant growth regulator for diverse developmental processes in plants. Indole-3-acetic acid (IAA) is a primary plant auxin that regulates the formation of various organs. Plants also produce phenylacetic acid (PAA), another natural auxin, which occurs more abundantly than IAA in various plant species. Although it has been demonstrated that the two auxins have distinct transport characteristics, the metabolic pathways and physiological roles of PAA in plants remain unsolved. In this study, we investigated the role of Arabidopsis UDP-glucosyltransferase UGT84B1 in IAA and PAA metabolism. We demonstrated that UGT84B1, which converts IAA to IAA-glucoside (IAA-Glc), can also catalyze the conversion of PAA to PAA-glucoside (PAA-Glc), with a higher catalytic activity in vitro. Furthermore, we showed a significant increase in both the IAA and PAA levels in the ugt84b1 null mutants. However, no obvious developmental phenotypes were observed in the ugt84b1 mutants under laboratory growth conditions. Moreover, the overexpression of UGT84B1 resulted in auxin-deficient root phenotypes and changes in the IAA and PAA levels. Our results indicate that UGT84B1 plays an important role in IAA and PAA homeostasis in Arabidopsis.

Sesquiterpene glucosylation mediated by glucosyltransferase UGT91Q2 is involved in the modulation of cold stress tolerance in tea plants.

Plants produce and emit terpenes, including sesquiterpenes, during growth and development, which serve different functions in plants. The sesquiterpene nerolidol has health-promoting properties and adds a floral scent to plants. However, the glycosylation mechanism of nerolidol and its biological roles in plants remained unknown. Sesquiterpene UDP-glucosyltransferases were selected by using metabolites-genes correlation analysis, and its roles in response to cold stress were studied. We discovered the first plant UGT (UGT91Q2) in tea plant, whose expression is strongly induced by cold stress and which specifically catalyzes the glucosylation of nerolidol. The accumulation of nerolidol glucoside was consistent with the expression level of UGT91Q2 in response to cold stress, as well as in different tea cultivars. The reactive oxygen species (ROS) scavenging capacity of nerolidol glucoside was significantly higher than that of free nerolidol. Down-regulation of UGT91Q2 resulted in reduced accumulation of nerolidol glucoside, ROS scavenging capacity and tea plant cold tolerance. Tea plants absorbed airborne nerolidol and converted it to its glucoside, subsequently enhancing tea plant cold stress tolerance. Nerolidol plays a role in response to cold stress as well as in triggering plant-plant communication in response to cold stress. Our findings reveal previously unidentified roles of volatiles in response to abiotic stress in plants.

Functional characterization and reclassification of an enzyme previously proposed to be a limonoid UDP-glucosyltransferase.

BACKGROUND: A major problem in the orange industry is 'delayed' bitterness, which is caused by limonin, a bitter compound developing from its non-bitter precursor limonoate A-ring lactone (LARL) during and after extraction of orange juice. The glucosidation of LARL by limonoid UDP-glucosyltransferase (LGT) to form non-bitter glycosyl-limonin during orange maturation has been demonstrated as a natural way to debitter by preventing the formation of limonin. RESULT: Here, the debittering potential of heterogeneously expressed glucosyltransferase, maltose-binding protein (MBP) fused to cuGT from Citrus unishiu Marc (MBP-cuGT), which was previously regarded as LGT, was evaluated. A liquid chromatography - mass spectrometry (LC-MS) method was established to determine the concentration of limonin and its derivatives. The protocols to obtain its potential substrates, LARL and limonoate (limonin with both A and D ring open), were also developed. Surprisingly, MBP-cuGT did not exhibit any detectable effect on limonin degradation when Navel orange juice was used as the substrate; MBP-cuGT was unable to biotransform either LARL or limonoate as purified substrates. However, it was found that MBP-cuGT displayed a broad activity spectrum towards flavonoids, confirming that the enzyme produced was active under the conditions evaluated in vitro. CONCLUSION: Our results based on LC-MS demonstrated that cuGT functionality was incorrectly identified. Its active substrates, including various flavonoids but not limonoids, highlight the need for further efforts to identify the enzyme responsible for LGT activity to develop biotechnology-based approaches for producing orange juice from varietals that traditionally have a delayed bitterness. © 2020 Society of Chemical Industry.

Synthesis of bolaform biosurfactants by an engineered Starmerella bombicola yeast.

Bola-amphiphilic surfactants are molecules with fascinating properties. Their unique configuration consisting of a long hydrophobic spacer connecting two hydrophilic entities renders the molecule more water soluble than the average surfactant, but still allows formation of supramolecular structures. These properties make them extremely suitable for applications in in nanotechnology, electronics, and gene and drug delivery. In general, these compounds are obtained by chemical synthesis. We report here an efficient microbial production process for the fully green synthesis of bolaform surfactants. A sophorolipid-producing Starmerella bombicola yeast strain was disabled in its sophorolipid acetyltransferase and lactone esterase, which should logically result in synthesis of non-acetylated acidic sophorolipids; molecules with the classic amphiphilic structure. Surprisingly, also bolaform glycolipids were obtained, with an additional sophorose linked to the free carboxyl end of the acidic sophorolipids as confirmed by MS and NMR analysis. The obtained titers of 27.7 g/L total product are comparable to wild type values, and the novel molecules account for at least 74% of this. Bola-amphiphile biosynthesis proved to be attributed to the promiscuous activity of both UDP-glucosyltransferases UGTA1 and UGTB1 from the core sophorolipid pathway, displaying activity toward non-acetylated intermediates. The absence of acetyl groups seems to trigger formation of bolaform compounds starting from acidic sophorolipids. Hence, wild type S. bombicola produces these compounds only at marginal amounts in general not reaching detection limits. We created a strain knocked-out in its sophorolipid acetyltransferase and lactone esterase able to produce these novel compounds in economical relevant amounts, opening doors for the application of biological-derived bolaform structures. Biotechnol. Bioeng. 2016;113: 2644-2651. © 2016 Wiley Periodicals, Inc.

Efficient enzymatic production of rebaudioside A from stevioside.

Stevioside and rebaudioside A are the chief diterpene glycosides present in the leaves of Stevia rebaudiana. Rebaudioside A imparts a desirable sweet taste, while stevioside produces a residual bitter aftertaste. Enzymatic synthesis of rebaudioside A from stevioside can increase the ratio of rebaudioside A to stevioside in steviol glycoside products, providing a conceivable strategy to improve the organoleptic properties of steviol glycoside products. Here, we demonstrate the efficient conversion of stevioside to rebaudioside A by coupling the activities of recombinant UDP-glucosyltransferase UGT76G1 from S. rebaudiana and sucrose synthase AtSUS1 from Arabidopsis thaliana. The conversion occurred via regeneration of UDP-glucose by AtSUS1. UDP was applicable as the initial material instead of UDP-glucose for UDP-glucose recycling. The amount of UDP could be greatly reduced in the reaction mixture. Rebaudioside A yield in 30 h with 2.4 mM stevioside, 7.2 mM sucrose, and 0.006 mM UDP was 78%.

2,4-Dihydroxybenzoic Acid, a Novel SA Derivative, Controls Plant Immunity via UGT95B17-Mediated Glucosylation: A Case Study in Camellia Sinensis.

The plant hormone salicylic acid (SA) plays critical roles in plant innate immunity. Several SA derivatives and associated modification are identified, whereas the range and modes of action of SA-related metabolites remain elusive. Here, the study discovered 2,4-dihydroxybenzoic acid (2,4-DHBA) and its glycosylated form as native SA derivatives in plants whose accumulation is largely induced by SA application and Ps. camelliae-sinensis (Pcs) infection. CsSH1, a 4/5-hydroxylase, catalyzes the hydroxylation of SA to 2,4-DHBA, and UDP-glucosyltransferase UGT95B17 catalyzes the formation of 2,4-DHBA glucoside. Down-regulation reduced the accumulation of 2,4-DHBA glucosides and enhanced the sensitivity of tea plants to Pcs. Conversely, overexpression of UGT95B17 increased plant disease resistance. The exogenous application of 2,4-DHBA and 2,5-DHBA, as well as the accumulation of DHBA and plant resistance comparison, indicate that 2,4-DHBA functions as a potentially bioactive molecule and is stored mainly as a glucose conjugate in tea plants, differs from the mechanism described in Arabidopsis. When 2,4-DHBA is applied exogenously, UGT95B17-silenced tea plants accumulated more 2,4-DHBA than SA and showed induced resistance to Pcs infection. These results indicate that 2,4-DHBA glucosylation positively regulates disease resistance and highlight the role of 2,4-DHBA as potentially bioactive molecule in the establishment of basal resistance in tea plants.

UDP-glucosyltransferase 71C4 controls the flux of phenylpropanoid metabolism to shape cotton seed development.

Seeds play a crucial role in plant reproduction, making it essential to identify genes that affect seed development. In this study, we focused on UDP-glucosyltransferase 71C4 (UGT71C4) in cotton, a member of the glycosyltransferase family that shapes seed width and length, thereby influencing seed index and seed cotton yield. Overexpression of UGT71C4 results in seed enlargement owing to its glycosyltransferase activity on flavonoids, which redirects metabolic flux from lignin to flavonoid metabolism. This shift promotes cell proliferation in the ovule via accumulation of flavonoid glycosides, significantly enhancing seed cotton yield and increasing the seed index from 10.66 g to 11.91 g. By contrast, knockout of UGT71C4 leads to smaller seeds through activation of the lignin metabolism pathway and redirection of metabolic flux back to lignin synthesis. This redirection leads to increased ectopic lignin deposition in the ovule, inhibiting ovule growth and development, and alters yield components, increasing the lint percentage from 41.42% to 43.40% and reducing the seed index from 10.66 g to 8.60 g. Our research sheds new light on seed size development and reveals potential pathways for enhancing seed yield.

One-Step Purification and Immobilization of Glycosyltransferase with Zn-Ni MOF for the Synthesis of Rare Ginsenoside Rh2.

Uridine diphosphate (UDP)-glucosyltransferases (UGTs) have received increasing attention in the field of ginsenoside Rh2 conversion. By harnessing the metal chelation between transition metal ions and imidazole groups present on His-tagged enzymes, a specific immobilization of the enzyme within metal-organic frameworks (MOFs) is achieved. This innovative approach not only enhances the stability and reusability of the enzyme but also enables one-step purification and immobilization. Consequently, the need for purifying crude enzyme solutions is effectively circumvented, resulting in significant cost savings during experimentation. The use of immobilized enzymes in catalytic reactions has shown great potential for achieving higher conversion rates of ginsenoside Rh2. In this study, highly stable mesoporous Zn-Ni MOF materials were synthesized at 150 °C by a solvothermal method. The UGT immobilized on the Zn-Ni MOF (referred to as UGT@Zn-Ni MOF) exhibited superior pH adaptability and thermal stability, retaining approximately 76% of its initial activity even after undergoing 7 cycles. Furthermore, the relative activity of the immobilized enzyme remained at an impressive 80.22% even after 45 days of storage. The strong specific adsorption property of Zn-Ni MOF on His-tagged UGT was confirmed through analysis using polyacrylamide gel electrophoresis. UGT@Zn-Ni MOF was used to catalyze the conversion reaction, and the concentration of rare ginsenoside Rh2 was generated at 3.15 µg/mL. The results showed that Zn-Ni MOF is a material that can efficiently purify and immobilize His-tagged enzyme in one step and has great potential for industrial applications in enzyme purification and ginsenoside synthesis.

Expression of a viral ecdysteroid UDP-glucosyltransferase enhanced the insecticidal activity of the insect pathogenic fungus Beauveria bassiana.

BACKGROUND: Entomopathogenic fungi, such as Beauveria bassiana, hold promise as biological control agents against insect pests. However, the efficacy of these fungi can be hindered by insect immune responses. One strategy to enhance fungal virulence is to manipulate host immune by targeting key regulatory molecules like 20-hydroxyecdysone (20E). RESULTS: In this study, we engineered B. bassiana strains to constitutively express the enzyme ecdysteroid UDP-glucosyltransferase (EGT), which inactivates 20E, a crucial insect molting hormone. The engineered strain Bb::EGT-1 exhibited robust expression of EGT, leading to a significant reduction in insect 20E levels upon infection. Moreover, infection with Bb::EGT-1 resulted in accelerated larval mortality. Immune responses analysis revealed repression of insect immune response genes and decreased phenoloxidase (PO) activity in larvae infected with Bb::EGT-1. Microbiome analysis indicated alterations in bacterial composition within infected insects, with increased abundance observed during infection with Bb::EGT-1. Additionally, the presence of bacteria hindered hyphal emergence from insect cadavers, suggesting a role for microbial competition in fungal dissemination. CONCLUSIONS: Constitutive expression of EGT in B. bassiana enhances fungal virulence by reducing insect 20E levels, suppressing immune responses, and altering the insect microbiome. These findings highlighted the potential of engineered fungi as effective biocontrol agents against insect pests and provide insights into the complex interactions between entomopathogenic fungi, their hosts, and associated microbes. © 2024 Society of Chemical Industry.

Constructing an Intensified UDP Recycling System for the Glycosylation of Natural Products by Phosphorylation of Byproduct Fructose.

UDP-glucosyltransferase can be coupled with sucrose synthase to construct a two-enzyme UDP (UDP-2E) recycling system for glucosylation of natural products with inexpensive sucrose as the consumed substrate. However, sucrose hydrolysis leads to the accumulation of fructose as a byproduct, which decreases the atom economy of sucrose and suppresses in situ UDP recycling. In this study, a polyphosphate-dependent glucokinase was demonstrated to convert fructose to fructose-6-phosphate independent of expensive ATP for the first time. Then the glucokinase was introduced into the UDP-2E recycling system to construct a modified three-enzyme UDP (UDP-3E) recycling system, which showed enhanced glucosylation efficiency of triterpenoids by fructose phosphorylation to accelerate sucrose hydrolysis and UDP recycling. Finally, by further introducing a phosphofructokinase into the UDP-3E recycling system, we transformed fructose-6-phosphate into fructose-1,6-diphosphate, demonstrating that the UDP-3E recycling system can be coupled with extra enzymes to obtain final products with high added-value without compromising the glycosylation efficiency.

Identification and Functional Characterisation of Two Oat UDP-Glucosyltransferases Involved in Deoxynivalenol Detoxification.

Oat is susceptible to several Fusarium species that cause contamination with different trichothecene mycotoxins. The molecular mechanisms behind Fusarium resistance in oat have yet to be elucidated. In the present work, we identified and characterised two oat UDP-glucosyltransferases orthologous to barley HvUGT13248. Overexpression of the latter in wheat had been shown previously to increase resistance to deoxynivalenol (DON) and nivalenol (NIV) and to decrease disease the severity of both Fusarium head blight and Fusarium crown rot. Both oat genes are highly inducible by the application of DON and during infection with Fusarium graminearum. Heterologous expression of these genes in a toxin-sensitive strain of Saccharomyces cerevisiae conferred high levels of resistance to DON, NIV and HT-2 toxins, but not C4-acetylated trichothecenes (T-2, diacetoxyscirpenol). Recombinant enzymes AsUGT1 and AsUGT2 expressed in Escherichia coli rapidly lost activity upon purification, but the treatment of whole cells with the toxin clearly demonstrated the ability to convert DON into DON-3-O-glucoside. The two UGTs could therefore play an important role in counteracting the Fusarium virulence factor DON in oat.

Genome-wide analysis of UDP-glycosyltransferase gene family and identification of members involved in flavonoid glucosylation in Chinese bayberry (Morella rubra).

Glycosylation was catalyzed by UDP-glycosyltransferase (UGT) and was important for enriching diversity of flavonoids. Chinese bayberry (Morella rubra) has significant nutritional and medical values because of diverse natural flavonoid glycosides. However, information of UGT gene family was quite limited in M. rubra. In the present study, a total of 152 MrUGT genes clustered into 13 groups were identified in M. rubra genome. Among them, 139 MrUGT genes were marked on eight chromosomes and 13 members located on unmapped scaffolds. Gene duplication analysis indicated that expansion of MrUGT gene family was mainly forced by tandem and proximal duplication events. Gene expression patterns in different tissues and under UV-B treatment were analyzed by transcriptome. Cyanidin 3-O-glucoside (C3Glc) and quercetin 3-O-glucoside (Q3Glc) were two main flavonoid glucosides accumulated in M. rubra. UV-B treatment significantly induced C3Glc and Q3Glc accumulation in fruit. Based on comprehensively analysis of transcriptomic data and phylogenetic homology together with flavonoid accumulation patterns, MrUFGT (MrUGT78A26) and MrUGT72B67 were identified as UDP-glucosyltransferases. MrUFGT was mainly involved in C3Glc and Q3Glc accumulation in fruit, while MrUGT72B67 was mainly involved in Q3Glc accumulation in leaves and flowers. Gln375 and Gln391 were identified as important amino acids for glucosyl transfer activity of MrUFGT and MrUGT72B67 by site-directed mutagenesis, respectively. Transient expression in Nicotiana benthamiana tested the function of MrUFGT and MrUGT72B67 as glucosyltransferases. The present study provided valuable source for identification of functional UGTs involved in secondary metabolites biosynthesis in M. rubra.

Integrated Metabolomics and Transcriptomics Analyses Reveal the Metabolic Differences and Molecular Basis of Nutritional Quality in Landraces and Cultivated Rice.

Rice (Oryza sativa L.) is one of the most globally important crops, nutritionally and economically. Therefore, analyzing the genetic basis of its nutritional quality is a paramount prerequisite for cultivating new varieties with increased nutritional health. To systematically compare the nutritional quality differences between landraces and cultivated rice, and to mine key genes that determine the specific nutritional traits of landraces, a seed metabolome database of 985 nutritional metabolites covering amino acids, flavonoids, anthocyanins, and vitamins by a widely targeted metabolomic approach with 114 rice varieties (35 landraces and 79 cultivars) was established. To further reveal the molecular mechanism of the metabolic differences in landrace and cultivated rice seeds, four cultivars and six landrace seeds were selected for transcriptome and metabolome analysis during germination, respectively. The integrated analysis compared the metabolic profiles and transcriptomes of different types of rice, identifying 358 differentially accumulated metabolites (DAMs) and 1982 differentially expressed genes (DEGs), establishing a metabolite-gene correlation network. A PCA revealed anthocyanins, flavonoids, and lipids as the central differential nutritional metabolites between landraces and cultivated rice. The metabolite-gene correlation network was used to screen out 20 candidate genes postulated to be involved in the structural modification of anthocyanins. Five glycosyltransferases were verified to catalyze the glycosylation of anthocyanins by in vitro enzyme activity experiments. At the same time, the different mechanisms of the anthocyanin synthesis pathway and structural diversity in landrace and cultivated rice were systematically analyzed, providing new insights for the improvement and utilization of the nutritional quality of rice landrace varieties.