Uridine Diphosphate Glucosyltransferase Research Articles

Identification and analysis of UGT genes associated with triterpenoid saponin in soapberry (Sapindus mukorossi Gaertn.)

BackgroundSoapberry (Sapindus mukorossi) is an economically important multifunctional tree species. Triterpenoid saponins have many functions in soapberry. However, the types of uridine diphosphate (UDP) glucosyltransferases (UGTs) involved in the synthesis of triterpenoid saponins in soapberry have not been clarified.ResultsIn this study, 42 SmUGTs were identified in soapberry, which were unevenly distributed on 12 chromosomes and had sequence lengths of 450 bp to 1638 bp, with an average of 1388 bp. The number of amino acids in SmUGTs was 149 to 545, with an average of 462. Most SmUGTs were acidic and hydrophilic unstable proteins, and their secondary structures were mainly α-helices and random coils. All had conserved UDPGT and PSPG-box domains. Phylogenetic analysis divided them into four subclasses, which glycosylated different carbon atoms. Prediction of cis-acting elements suggested roles of SmUGTs in plant development and responses to environmental stresses. The expression patterns of SmUGTs differed according to the developmental stage of fruits, as determined by transcriptomics and RT-qPCR. Co-expression network analysis of SmUGTs and related genes/transcription factors in the triterpenoid saponin synthesis pathway was also performed. The results indicated potential roles for many transcription factors, such as SmERFs, SmGATAs and SmMYBs. A correlation analysis showed that 42 SmUGTs were crucial in saponin synthesis in soapberry.ConclusionsOur findings suggest optimal targets for manipulating glycosylation in soapberry triterpenoid saponin biosynthesis; they also provide a theoretical foundation for further evaluation of the functions of SmUGTs and analyses of their biosynthetic mechanisms.

Uridine diphosphate glucosyltransferase is vital for fenpropathrin resistance in Bombyx mori (Lepidoptera).

The silkworm (Bombyx mori) is an important model lepidopteran insect and can be used to identify pesticide resistance-related genes of great significance for biological control of pests. Uridine diphosphate glucosyltransferases (UGTs), found in all organisms, are the main secondary enzymes involved in the metabolism of heterologous substances. However, it remains uncertain if silkworm resistance to fenpropathrin involves UGT. This study observes significant variations in BmUGT expression among B. mori strains with variable fenpropathrin resistance post-feeding, indicating BmUGT's role in fenpropathrin detoxification. Knockdown of BmUGT with RNA interference and overexpression of BmUGT significantly decreased and increased BmN cell activity, respectively, indicating that BmUGT plays an important role in the resistance of silkworms to fenpropathrin. In addition, fenpropathrin residues were significantly reduced after incubation for 12 h with different concentrations of a recombinant BmUGT fusion protein. Finally, we verified the conservation of UGT to detoxify fenpropathrin in Spodoptera exigua: Its resistance to fenpropathrin decreased significantly after knocking down SeUGT. In a word, UGT plays an important role in silkworm resistance to fenpropathrin by directly degrading the compound, a function seen across other insects. The results of this study are of great significance for breeding silkworm varieties with high resistance and for biological control of pests.

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.

Uridine diphosphate glucosyltransferases are involved in spinosad resistance in western flower thrips Frankliniella occidentalis (Pergande)

Uridine diphosphate glucosyltransferases (UGTs) play crucial roles in the insect detoxification system and are associated with pesticide resistance. Our previous transcriptomic analysis of spinosad-susceptible (Ivf03) and resistant (NIL-R) Frankliniella occidentalis revealed numerous upregulated UGT genes in the NIL-R strain, suggesting their potential contribution to spinosad resistance. To investigate this hypothesis, here we conducted UGT activity assays and spinosad induction experiments, employing RNA interference (RNAi) techniques for gene function validation. We found significantly elevated UGT activity in the NIL-R strain compared to Ivf03, with 5-nitrouracil showing a substantial synergistic effect on the resistant strain. Eighteen UGT genes were identified in F. occidentalis, with gene expansion and duplication observed within families UGT466, 467, and 468. Ten out of the eighteen UGTs exhibited higher expression levels in NIL-R, specifically FoUGT466B1, FoUGT468A3, and FoUGT468A4 consistently being upregulated across nymphs, males, and females. RNAi-based functional validation targeting these three UGT genes led to increased susceptibility to spinosad in a life stage-, sex-, and dose-dependent manner. These results indicate that UGTs are indeed involved in spinosad resistance in F. occidentalis, and the effects are dependent on life stage, sex, and dose. Therefore, sustainable control for F. occidentalis resistance should always consider these differential responses.

Reaction mechanism and regioselectivity of uridine diphosphate glucosyltransferase RrUGT3: a combined experimental and computational study

A substrate binding induced conformational change was found to be essential for the occurrence of RrUGT3 catalyzed transglycosylation reactions.

Multiomics analyses of two Leonurus species illuminate leonurine biosynthesis and its evolution

The Lamiaceae family is renowned for its terpenoid-based medicinal components, but Leonurus, which has traditional medicinal uses, stands out for its alkaloid-rich composition. Leonurine, the principal active compound found in Leonurus, has demonstrated promising effects in reducing blood lipids and treating strokes. However, the biosynthetic pathway of leonurine remains largely unexplored. Here, we present the chromosome-level genome sequence assemblies of Leonurus japonicus, known for its high leonurine production, and Leonurus sibiricus, characterized by very limited leonurine production. By integrating genomics, RNA sequencing, metabolomics, and enzyme activity assay data, we constructed the leonurine biosynthesis pathway and identified the arginine decarboxylase (ADC), uridine diphosphate glucosyltransferase (UGT), and serine carboxypeptidase-like (SCPL) acyltransferase enzymes that catalyze key reactions in this pathway. Further analyses revealed that the UGT–SCPL gene cluster evolved by gene duplication in the ancestor of Leonurus and neofunctionalization of SCPL in L. japonicus, which contributed to the accumulation of leonurine specifically in L. japonicus. Collectively, our comprehensive study illuminates leonurine biosynthesis and its evolution in Leonurus.

Ecdysteroid UDP-Glucosyltransferase Expression in Beauveria bassiana Increases Its Pathogenicity against Early Instar Silkworm Larvae.

Beauveria bassiana (B. bassiana) is a broad-spectrum entomopathogenic fungus that can control pests in agriculture and forestry. In this study, encoding ecdysteroid uridine diphosphate glucosyltransferase gene (egt) was successfully screened in B. bassiana on the medium containing 500μg/mL G418 sulfate solution through the protoplast transformation method. This enzyme has the function of 20E (20-hydroxyecdysone) inactivation, thus increasing the mortality of the early instar larvae infected with B. bassiana. In this study, we transformed B. bassiana with the egt gene, which deactivates 20-hydroxyecdysone, a key hormone in insect development. The results showed that transgenic B. bassiana killed more silkworms of the 2nd instar larvae than the wild-type with a shorter LT50 time, which was reduced by approximately 20% (day 1 of the 2nd instar silkworm infection of B. bassiana) and 26.4% (day 2 of the 2nd instar silkworm infection of B. bassiana) compared to the wild-type, and also showed a higher mortality number before molting. The transgenic B. bassiana had a higher coverage of the body surface of silkworms compared to the wild type on the 3rd instar. In summary, improving entomopathogenic fungi using biological methods such as genetic engineering is feasible.

Identifying a Detoxifying Uridine Diphosphate Glucosyltransferase (UGT), MdUGT83K2, Which Can Glycosylate the Aryloxyphenoxypropionate Herbicide

Glycosylation is a common modification reaction in plants. The products obtained upon glycosylation have different biological functions, making glycosylation an important mechanism affecting and regulating the balance of plant growth and metabolism. In this study, we first speculated that Group I in the apple glycosyltransferase family may have a predicted function like UGT83A1, according to gene chip data published online. Subsequently, by real-time PCR (polymerase chain reaction), we analyzed whether the expression of nine glycosyltransferase genes in Group I was induced by our previously reported ACCase (Acetyl-CoA carboxylase) inhibition-based herbicide QPP ((R)-ethyl·2-(4-((6-fluoro-3-methyl-4-oxo-3,4-dihydroquinazolin-2-yl)oxy) phenoxy) propanoate). It was found that expression of the MdUGT83K2 gene in Group I was significantly increased by QPP. In order to determine whether MdUGT83K2 can glycosylate QPP, we confirmed the enzymatic reaction of MdUGT83K2 in vitro and the presence of QPP glycosides in MdUGT83K2 transgenic apple seedlings by HPLC (High Performance Liquid Chromatography), and found that MdUGT83K2 can transfer glucose to QPP in vivo, which is glycosylated. In this work, we identified a novel apple glycosyltransferase, MdUGT83K2, which functions to glycosylate the ACCase-inhibiting herbicide QPP and may be involved in plant detoxification. Key Contribution: A novel apple glycosyltransferase, MdUGT83K2, was identified, which may be involved in plant detoxification by glycosylation modification of the ACCase-inhibiting herbicide.

Overexpression of the Persimmon Abscisic Acid DkUGT3 Gene Alters Plant/Fruit Development in Transgenic Tomato

Persimmon (Diospyros kaki Thunb.) contains numerous uridine diphosphate glucosyltransferases (UGT), and their roles in fruit development and quality formation have not been well studied because of limited genetic information. This study investigated a persimmon DkUGT3 which is highly expressed in young fruits and leaves during development. DkUGT3 can catalyze ABA glycosylation to form ABA-GE, thereby reducing free ABA. Tomato with overexpressed (OE) DkUGT3 significantly induces pale green color phenotypes in both transgenic young plants and fruits. DkUGT3-OE significantly weakens the tomato ABA signaling which affects the expressions of ABA-inducible transcriptional factors (TFs), such as GLK1 and GLK2 and their downstream target genes involved in chlorophyll synthesis, chloroplast development, sugar metabolism and transport, and photosynthesis, thereby impeding leaf and fruit development and quality. Conversely, DkUGT3-RNAi-treatment recovered the OE tomato fruits from yellowing phenotype to green color. This study found that chlorophyll accumulations and ABA level were increased by DkUGT3-RNAi-treatment in young persimmon leaves. These results demonstrate that DkUGT3 plays crucial roles in ABA-mediated leaf and fruit development. This study provides new evidence for the regulation of ABA in early development.

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.

Integration of the metabolome and transcriptome reveals indigo biosynthesis in Phaius flavus flowers under freezing treatment.

BackgroundIndigo-containing plant tissues change blue after a freezing treatment, which is accompanied by changes in indigo and its related compounds. Phaius flavus is one of the few monocot plants containing indigo. The change to blue after freezing was described to explore the biosynthesis of indigo in P. flavus.MethodsIn this study, we surveyed the dynamic change of P. flavus flower metabolomics and transcriptomics.ResultsThe non-targeted metabolomics and targeted metabolomics results revealed a total of 98 different metabolites, the contents of indole, indican, indigo, and indirubin were significantly different after the change to blue from the freezing treatment. A transcriptome analysis screened ten different genes related to indigo upstream biosynthesis, including three anthranilate synthase genes, two phosphoribosyl-anthranilate isomerase genes, one indole-3-glycerolphosphate synthase gene, five tryptophan synthase genes. In addition, we further candidate 37 cytochrome P450 enzyme genes, one uridine diphosphate glucosyltransferase gene, and 24 β-D-glucosidase genes were screened that may have participated in the downstream biosynthesis of indigo. This study explained the changes of indigo-related compounds at the metabolic level and gene expression level during the process of P. flavus under freezing and provided new insights for increasing the production of indigo-related compounds in P. flavus. In addition, transcriptome sequencing provides the basis for functional verification of the indigo biosynthesis key genes in P. flavus.

Construction of the UDP-Glucose Biosynthetic Enzyme Gene Coexpression Plasmid for Prunasin Production in Escherichia coli.

Microbial production of bioactive glucosides using uridine diphosphate glucosyltransferase (UGT) is an efficient glucoside production method. Here, we describe a detailed method for the construction of a UDP-glucose biosynthetic enzyme gene coexpression plasmid, that is, pCDF-PGP and the microbial production of prunasin from racemic mandelonitrile using Escherichia coli possessing UGT85A47 obtained from Japanese apricot. Furthermore, this constructed vector can find application in the production of various other glucosides that utilize other UGTs and aglycons.

Abscisic acid receptors maintain abscisic acid homeostasis by modulating UGT71C5 glycosylation activity.

Uridine diphosphate-glucosyltransferases (UGTs) maintain abscisic acid (ABA) homeostasis in Arabidopsis thaliana by converting ABA to abscisic acid-glucose ester (ABA-GE). UGT71C5 plays an important role in the generation of ABA-GE. Abscisic acid receptors are crucial upstream components of the ABA signaling pathway, but how UGTs and ABA receptors function together to modulate ABA levels is unknown. Here, we demonstrated that the ABA receptors RCAR12/13 and UGT71C5 maintain ABA homeostasis in Arabidopsis following rehydration under drought stress. Biochemical analyses show that UGT71C5 directly interacted with RCAR8/12/13 in yeast cells, and the interactions between UGT71C5 and RCAR12/13 were enhanced by ABA treatment. Enzyme activity analysis showed that ABA-GE contents were significantly elevated in the presence of RCAR12 or RCAR13, suggesting that these ABA receptors enhance the activity of UGT71C5. Determination of the content of ABA and ABA-GE in Arabidopsis following rehydration under drought stress revealed that ABA-GE contents were significantly higher in Arabidopsis plants overexpressing RCAR12 and RCAR13 than in non-transformed plants and plants overexpressing RCAR11 following rehydration under drought stress. These observations suggest that RCAR12 and RCAR13 enhance the activity of UGT71C5 to glycosylate excess ABA into ABA-GE following rehydration under drought stress, representing a rapid mechanism for regulating plant growth and development.

Microbial synthesis of salidroside

Salidroside, as one of the main active ingredients of Rhodiala plant, has the effects of anti-hypoxia, anti-radiation, anti-fatigue, anti-tumor, hypoglycemia and improving immunity. With the increasing demand for salidroside and the decreasing of plant resources, microbial production of salidroside has attracted much attention due to its advantages of short period and easy controlling. At present, microbial production of salidroside is still at the basic research stage. In order to make it easier for researchers to understand the advances of microbial synthesis of salidroside, the biosynthesis pathways, uridine diphosphate glucosyltransferases, wild strain/natural enzymes and engineered strain/recombinant enzymes were reviewed.

An abscisic acid (ABA) homeostasis regulated by its production, catabolism and transport in peanut leaves in response to drought stress.

ABA is an important messenger that acts as a signaling mediator for regulating the adaptive response of plants to drought stress. Two production pathways, de novo biosynthesis and hydrolysis of glucose-conjugated ABA by β-glucosidase (BG), increase cellular ABA levels in plants. ABA catabolism via hydroxylation by 8’-hydroxylase (CYP707A), or conjugation by uridine diphosphate glucosyltransferase (UGT), decreases cellular ABA levels. The transport of ABA through ATP-binding cassette (ABC)-containing transporter proteins, members of ABC transporter G family (ABCG), across plasma membrane (PM) is another important pathway to regulate cellular ABA levels. In this study, based on our previously constructed transcriptome of peanut leaves in response to drought stress, fourteen candidate genes involved in ABA production (including AhZEP, AhNCED1 and AhNCED3, AhABA2, AhAAO1 and AhAAO2, AhABA3, AhBG11 and AhBG24), catabolism (including AhCYP707A3, AhUGT71K1 and AhUGT73B4) and transport (including AhABCG22-1 and AhABCG22-2), were identified homologously and phylogenetically, and further analyzed at the transcriptional level by real-time RT-PCR, simultaneously determining ABA levels in peanut leaves in response to drought. The high sequence identity and very similar subcellular localization of the proteins deduced from 14 identified genes involved in ABA production, catabolism and transport with the reported corresponding enzymes in databases suggest their similar roles in regulating cellular ABA levels. The expression analysis showed that the transcripts of AhZEP, AhNCED1, AhAAO2 and AhABA3 instead of AhABA2, AhNCED3 and AhAAO1 in peanut leaves increased significantly in response to drought stress; and that the AhBG11 and AhBG24 mRNA levels were rapidly and significantly up-regulated, with a 4.83- and 4.58-fold increase, respectively at 2-h of drought stress. The genes involved in ABA catabolism AhCYP707A3, AhUGT71K1 instead of AhUGT73B4 were significantly induced in response to drought stress. The expression of two closely related peanut ABCG genes, AhABCG22.1 and AhABCG22.2, was significantly up-regulated in response to drought stress. The ABA levels rapidly began to accumulate within 2 h (a 56.6-fold increase) from the start of drought stress, and peaked at 10 h of the stress. The highly and rapidly stress up-regulated expressions of genes involved in ABA production and transport, particularly AhNCED1, AhBG11 and AhBG24, and AhABCG22.1 and AhABCG22.2, might contribute to the rapid ABA accumulation in peanut leaves in response to drought. In response to drought stress, ABA accumulation levels in peanut leaves agree well with the up-regulated expressions of ABA-producing genes (AhZEP, AhNCED1, AhAAO2, AhABA3, AhBG11 and AhBG24) and PM-localized ABA importer genes (AhABCG22-1 and AhABCG22-2), in spite of the simultaneously induced ABA catabolic genes (AhCYP707A3 and AhUGT71K1), although the induction of catabolic genes was much lower than that of biosynthetic gene (AhNCED1). This difference in induction kinetics of gene expression may define the significant accumulation of drought-induced ABA levels. These results suggest that ABA homeostasis in peanut leaves in response to drought maintained through a balance between the production, catabolism and transport, rather than simply by the biosynthesis.

The interplay of PsABAUGT1 with other abscisic acid metabolic genes in the regulation of ABA homeostasis during the development of pea seeds and germination in the presence of H2O2

Inactivation of abscisic acid (ABA) in vitro may be catalyzed either by ABA 8′-hydroxylase (ABA8′OH) or by ABA uridine diphosphate glucosyltransferase (ABAUGT), which conjugates ABA with glucose. However, the involvement of these enzymes in the control of ABA content in vivo, especially ABAUGT, has not been fully elucidated. In pea seeds, both PsABAUGT1 and PsABA8′OH1 contribute to the reduction of ABA content during seed maturation and imbibition; however, during the first hours of imbibition, a high expression of only PsABAUGT1 was observed. Imbibition of seeds with H2O2 increased the ABA content despite the oxygen availability and altered the expression of metabolic genes. The expression of the biosynthetic gene 9-cis-epoxycarotene dioxygenase (PsNCED2) was increased, while that of PsABAUGT1 was decreased in each H2O2 experiment despite O2 availability. Under hypoxia, only seeds imbibed with H2O2 germinated, while under nonlimiting oxygen conditions, the germination rate was not altered by H2O2. Under hypoxia, the germination rate of H2O2-imbibed seeds seemed to not depend on the absolute ABA content and rather on the balance between ABA and gibberellins (GA), as H2O2 increased the expression of GA synthesis genes.Overexpression of PsABAUGT1 in Arabidopsis decreases seed ABA content, accelerates germination and reduces seed sensitivity to exogenously applied ABA, confirming the ability of PsABAUGT1 to inactivate ABA. Thus, PsABAUGT1 is a new player in the regulation of ABA content in maturating and imbibed pea seeds, both under standard conditions and in response to H2O2.

Genome sequence of the corn leaf aphid (Rhopalosiphum maidis Fitch).

BackgroundThe corn leaf aphid (Rhopalosiphum maidis Fitch) is the most economically damaging aphid pest on maize (Zea mays), one of the world's most important grain crops. In addition to causing direct damage by removing photoassimilates, R. maidis transmits several destructive maize viruses, including maize yellow dwarf virus, barley yellow dwarf virus, sugarcane mosaic virus, and cucumber mosaic virus.FindingsThe genome of a parthenogenetically reproducing R. maidis clone was assembled with a combination of Pacific Biosciences (207-fold coverage) and Illumina (83-fold coverage) sequencing. The 689 assembled contigs, which have an N50 size of 9.0 megabases (Mb) and a low level of heterozygosity, were clustered using Phase Genomics Hi-C interaction maps. Consistent with the commonly observed 2n = 8 karyotype of R. maidis, most of the contigs (473 spanning 321 Mb) were successfully oriented into 4 scaffolds. The genome assembly captured the full length of 95.8% of the core eukaryotic genes, indicating that it is highly complete. Repetitive sequences accounted for 21.2% of the assembly, and a total of 17,629 protein-coding genes were predicted with integrated evidence from ab initio and homology-based gene predictions and transcriptome sequences generated with both Pacific Biosciences and Illumina. An analysis of likely horizontally transferred genes identified 2 from bacteria, 7 from fungi, 2 from protozoa, and 9 from algae. Repeat elements, transposons, and genes encoding likely detoxification enzymes (cytochrome P450s, glutathione S-transferases, carboxylesterases, uridine diphosphate–glucosyltransferases, and ABC transporters) were identified in the genome sequence. Other than Buchnera aphidicola (642,929 base pairs, 602 genes), no endosymbiont bacteria were found in R. maidis.ConclusionsA high-quality R. maidis genome was assembled at the chromosome level. This genome sequence will enable further research related to ecological interactions, virus transmission, pesticide resistance, and other aspects of R. maidis biology. It also serves as a valuable resource for comparative investigation of other aphid species.

Mutations in the uridine diphosphate glucosyltransferase 76G1 gene result in different contents of the major steviol glycosides in Stevia rebaudiana

In the metabolic glycosylation grid of steviol glycosides, UGT76G1 was shown to catalyze at least eight different glucosylation steps, including the formation of rebaudioside B (Reb B) and rebaudioside A (Reb A) (Olsson et al., 2016). In this study, the accumulation of steviolbioside, Reb B, stevioside (ST) and Reb A in more than 140 samples of stevia leaves collected from different regions in China were analyzed by high-performance liquid chromatography (HPLC), and five genotypes, ‘N01-N05’, with significantly different levels of the abovementioned glycosides were discovered. Mutations in the UGT76G1 gene cloned from cDNAs from these five genotypes were identified, and the functions of the recombinant UGT76G1 variants were ascertained by adding steviolbioside and ST substrates. In addition, homology modeling and molecular docking were used to elucidate the functional differences between variants and UGT76G1. Comparing the sequences of the five variants ‘N01-N05’ with UGT76G1 (AY345974.1) revealed that base substitutions were not observed in ‘N01’. By contrast, ‘N02’ exhibited 9 single nucleotide polymorphisms (SNPs) and 9 associated amino acid substitutions or insertions with notable variations in the protein structure; however, an enzyme assay showed similar functionalities between the variant and UGT76G1. In ‘N03’, 49 SNPs and 29 associated amino acid substitutions or insertions were identified and shown to induce significant variations in the protein structure, especially in the binding pocket, resulting in the lack of functionality of this variant in the enzyme assay. These results were in agreement with the docking profiles. Moreover, a nonsense mutation of p.1090T > G in ‘N04’ and an insertion of a 68 base fragment in ‘N05’ were found, and both produced a premature protein without any catalytic activity. Therefore, UGT76G1, which is vital to the content of main steviol glycosides, should be a key gene marker for the molecular breeding of Stevia rebaudiana. Our investigations also revealed the location and orientation of active groups of the receptors and donors in the UGT76G1 enzyme, which play key roles in determining whether the enzyme has any enzymatic activity.

Identification, Molecular Cloning, and Functional Characterization of a Wheat UDP-Glucosyltransferase Involved in Resistance to Fusarium Head Blight and to Mycotoxin Accumulation.

Plant uridine diphosphate (UDP)-glucosyltransferases (UGT) catalyze the glucosylation of xenobiotic, endogenous substrates and phytotoxic agents produced by pathogens such as mycotoxins. The Bradi5g03300 UGT-encoding gene from the model plant Brachypodium distachyon was previously shown to confer tolerance to the mycotoxin deoxynivalenol (DON) through glucosylation into DON 3-O-glucose (D3G). This gene was shown to be involved in early establishment of quantitative resistance to Fusarium Head Blight, a major disease of small-grain cereals. In the present work, using a translational biology approach, we identified and characterized a wheat candidate gene, Traes_2BS_14CA35D5D, orthologous to Bradi5g03300 on the short arm of chromosome 2B of bread wheat (Triticum aestivum L.). We showed that this UGT-encoding gene was highly inducible upon infection by a DON-producing Fusarium graminearum strain while not induced upon infection by a strain unable to produce DON. Transformation of this wheat UGT-encoding gene into B. distachyon revealed its ability to confer FHB resistance and root tolerance to DON as well as to potentially conjugate DON into D3G in planta and its impact on total DON reduction. In conclusion, we provide a UGT-encoding candidate gene to include in selection process for FHB resistance.

Suppressing ABA uridine diphosphate glucosyltransferase (SlUGT75C1) alters fruit ripening and the stress response in tomato.

Abscisic acid (ABA) glucose conjugation mediated by uridine diphosphate glucosyltransferases (UGTs) is an important pathway in regulating ABA homeostasis. In the present study, we investigated three tomato SlUGTs that are highly expressed in fruit during ripening, and these SlUGTs were localized to the cytoplasm and cell nucleus. Among these three UGTs, SlUGT75C1 catalyzes the glucosylation of both ABA and IAA invitro; SlUGT76E1 can only catalyze the conjugation of ABA; and SlUGT73C4 cannot glycosylate either ABA or IAA. Therefore, SlUGT75C1 was selected for further investigation. SlUGT75C1 RNA interference significantly up-regulated the expression level of SlCYP707A2, which encodes an ABA 8'-hydroxylase but did not affect the expression of SlNCED1, which encodes a key enzyme in ABA biosynthesis. Suppression of SlUGT75C1 significantly accelerated fruit ripening by enhancing ABA levels and promoting the early release of ethylene. SlUGT75C1-RNAi altered the expression of fruit ripening genes (genes involved in ethylene release and cell wall catabolism). SlUGT75C1-RNAi seeds showed delayed germination and root growth compared with wild-type as well as increased sensitivity to exogenous ABA. SlUGT75C1-RNAi plants were also more resistant to drought stress. These results demonstrated that SlUGT75C1 plays a crucial role in ABA-mediated fruit ripening, seed germination, and drought responses in tomato.