Uridine Diphosphate Glycosyltransferase Research Articles

Identification and expression of uridine diphosphate glycosyltransferase(UGT) gene family from Dendrobium officinale

Uridine diphosphate glycosyltransferase(UGT) is a highly conserved protein in plants, which usually functions in secondary metabolic pathways. This study used the Hidden Markov Model(HMM) to screen out members of UGT gene family in the whole genome of Dendrobium officinale, and 44 UGT genes were identified. Bioinformatics was used to analyze the structure, phylogeny, and promoter region components of D. officinale genes. The results showed that UGT gene family could be divided into four subfamilies, and UGT gene structure was relatively conserved in each subfamily, with nine conserved domains. The upstream promoter region of UGT gene contained a variety of cis-acting elements related to plant hormones and environmental factors, indicating that UGT gene expression may be induced by plant hormones and external environmental factors. UGT gene expression in different tissues of D. officinale was compared, and UGT gene expression was found in all parts of D. officinale. It was speculated that UGT gene played an important role in many tissues of D. officinale. Through transcriptome analysis of D. officinale mycorrhizal symbiosis environment, low temperature stress, and phosphorus deficiency stress, this study found that only one gene was up-regulated in all three conditions. The results of this study can help understand the functions of UGT gene family in Orchidaceae plants and provide a basis for further study on the molecular regulation mechanism of polysaccharide metabolism pathway in D. officinale.

Identification of a tomato UDP-arabinosyltransferase for airborne volatile reception

Volatiles from herbivore-infested plants function as a chemical warning of future herbivory for neighboring plants. (Z)-3-Hexenol emitted from tomato plants infested by common cutworms is taken up by uninfested plants and converted to (Z)-3-hexenyl β-vicianoside (HexVic). Here we show that a wild tomato species (Solanum pennellii) shows limited HexVic accumulation compared to a domesticated tomato species (Solanum lycopersicum) after (Z)-3-hexenol exposure. Common cutworms grow better on an introgression line containing an S. pennellii chromosome 11 segment that impairs HexVic accumulation, suggesting that (Z)-3-hexenol diglycosylation is involved in the defense of tomato against herbivory. We finally reveal that HexVic accumulation is genetically associated with a uridine diphosphate-glycosyltransferase (UGT) gene cluster that harbors UGT91R1 on chromosome 11. Biochemical and transgenic analyses of UGT91R1 show that it preferentially catalyzes (Z)-3-hexenyl β-d-glucopyranoside arabinosylation to produce HexVic in planta.

Direct Yeast Surface Codisplay of Sequential Enzymes with Complementary Anchor Motifs: Enabling Enhanced Glycosylation of Natural Products.

Yeast surface display is an appealing technique for constructing multienzyme cascades. This technique is commonly achieved using a scaffold for the ordered arrangement of various enzymes. However, this method is typically complicated because scaffold use may engender extra metabolic burden on the cell host. Here, we established a direct yeast surface codisplay strategy by employing two complementary anchor motifs, Agα1 and Pir1. These motifs allow for the codisplay of sequential uridine diphosphate-glycosyltransferase (UGT) and sucrose synthase (SUS) on the surface of Pichia pastoris (syn. Komagataella phaffii) for the glycosylation of natural products. We manipulated the displayed stoichiometry, amount, and assembly order of UGT and SUS by coupling them with anchor motifs. Furthermore, their effect on enzyme activity was thoroughly investigated. The surface-codisplayed strain UGT-Pir-SUS-Agα exhibited greater thermostability than the single-displayed strains and their free counterparts. Moreover, the strain UGT-Pir-SUS-Agα was successfully applied to glycyrrhetinic acid (GA) glycosylation to produce GA-3-O-Glc, with sucrose being the sugar donor in this process. This generated 7.5- to 20- and 5.3-fold higher GA-3-O-Glc concentration compared with the free counterparts (enzyme mass loading of 20-fold in excess) and mixed single-displayed strains of UGT-Agα and SUS-Pir, respectively. This increase was due to the improved biochemical properties and substrate channeling effect of strain UGT-Pir-SUS-Agα. This controllable direct surface codisplay strategy, based on complementary anchor motifs, is readily extendable to other enzyme cascades.

Evaluation of pathways to the C-glycosyl isoflavone puerarin in roots of kudzu (Pueraria montana lobata).

Kudzu ( Pueraria montana lobata) is used as a traditional medicine in China and Southeast Asia but is a noxious weed in the Southeastern United States. It produces both O‐ and C‐glycosylated isoflavones, with puerarin (C‐glucosyl daidzein) as an important bioactive compound. Currently, the stage of the isoflavone pathway at which the C‐glycosyl unit is added remains unclear, with a recent report of direct C‐glycosylation of daidzein contradicting earlier labeling studies supporting C‐glycosylation at the level of chalcone. We have employed comparative mRNA sequencing of the roots from two Pueraria species, one of which produces puerarin (field collected P. montana lobata) and one of which does not (commercial Pueraria phaseoloides ), to identify candidate uridine diphosphate glycosyltransferase (UGT) enzymes involved in puerarin biosynthesis. Expression of recombinant UGTs in Escherichia coli and candidate C‐glycosyltransferases in Medicago truncatula were used to explore substrate specificities, and gene silencing of UGT and key isoflavone biosynthetic genes in kudzu hairy roots employed to test hypotheses concerning the substrate(s) for C‐glycosylation. Our results confirm UGT71T5 as a C‐glycosyltransferase of isoflavone biosynthesis in kudzu. Enzymatic, isotope labeling, and genetic analyses suggest that puerarin arises both from the direct action of UGT71T5 on daidzein and via a second route in which the C‐glycosidic linkage is introduced to the chalcone isoliquiritigenin.

PHARMACOGENETIC ASPECTS OF THE USE OF PERIOPERATIVE DRUGS IN PEDIATRICS. Review

Relevance. It is known that in children the frequency of perioperative critical events due to the introduction of drugs is more than 5%. For example fentanyl, which is used as an adjunct to surgical anesthesia, is a major factor in increasing the number of deaths from opioid overdoses. Among the areas of reducing the frequency of severe adverse reactions to perioperative drugs - taking into account the pharmacogenetic variations of the patient.
 Objective is to consider current data on pharmacogenetic aspects of pharmacokinetics and pharmacodynamics of perioperative drugs.
 Methods. Analysis of the data presented in PubMed by keywords "pharmacogenetics", "general anesthetics", "analgesics", "muscle relaxants", "children". Search depth – 7 years (2014-2020), with a retrospective deepening of some positions until 2002.
 Results. The pharmacogenetic aspects of the pharmacokinetics of perioperative drugs are related to the genes of the enzymes that metabolize them and their transporters. Current data on the prevalence of polymorphic alleles of CYP2C9 genes (provides metabolism of nonsteroidal anti-inflammatory and anticonvulsant drugs) and CYP2D6 (metabolizes opioids, antidepressants, antiemetics) in Europe and, in particular, in Ukraine were presented. Thus, the inactive allele CYP2C19 * 2 was found in 13%, while the allele of increased activity CYP2C19 * 17 - in 25% of the population of Ukraine; allele with lost CYP2D6 function (CYP2D6 * 4) - in 18.6% of Ukrainians. Homozygotes with CYP2C9 * 3 polymorphism metabolize nonsteroidal anti-inflammatory drugs much more slowly than wild-type carriers, which may lead to their accumulation and side effects. The analgesic effect of codeine occurs only after it is metabolized in the liver by CYP2D6 to morphine. Respiratory depression, apnea and death may occur in patients with excessive metabolic rate even after a single dose of codeine; however, was noted the ineffectiveness of ondansetron due to a decrease in its concentration in the blood in thesepatients. Concomitant use of midazolam and fentanyl may prolong the effect of fentanyl by competing metabolism of midazolam by the hepatic enzyme CYP3A4, especially in patients with low metabolism. Plasma butyrylcholinesterase deficiency reduces succinylcholine inactivation in 1 in 1,500 people. Changing the genotype of uridine diphosphate glycosyltransferase causes increased glucuronidation of morphine, which may lead to a decrease in its effectiveness. As an example of pharmacogenetic changes in pharmacodynamics can be considered malignant hyperthermia, which occurs due to exposure to volatile anesthetics and depolarizing muscle relaxants in mutations in the RYR1 gene. Carriers of the minor allele (G) of the 5HT2A rs6313 gene require less propofol and 40% less time to start induction of anesthesia.
 Conclusions. The efficacy and safety of perioperative drugs are largely related to the pharmacogenetic aspects of their pharmacokinetics, especially mutations in genes of enzymes of the metabolism. Mutations in the genes of volatile anesthetic receptors and depolarizing muscle relaxants when their used cause a risk of malignant hyperthermia. The application of pharmacogenetics is the need to titrate drugs that have significant variability of action depending on the genotype. It is advisable to avoid the use of drugs with a high risk of pharmacogenetic reactions, if other alternative drugs are available.

Identification and Characterization of UDP-Glycosyltransferase Genes in a Cerambycid Beetle, Pharsalia antennata Gahan, 1894 (Coleoptera: Cerambycidae)

The cerambycid beetle, Pharsalia antennata Gahan, 1894 (Coleoptera: Cerambycidae), is a wood-boring pest that spends most of its life cycle in the trunks or under the bark of trees. These distinctive biological characteristics make it likely that this beetle will encounter a number of plant defensive compounds, coupled with a broad range of host plants, possibly resulting in the overexpression or expansion of uridine diphosphate (UDP)-glycosyltransferase (UGT) genes. Here, we identified and characterized the UGT gene family in P. antennata through transcriptome data, sequence and phylogenetic analyses, and PCR and homology modeling approaches. In total, 59 transcripts encoding UGTs were identified, 34 of which harbored full-length sequences and shared high conservation with the UGTs of Anoplophora glabripennis. Of the 34 PantUGTs, only 31.78% amino acid identity was observed on average, but catalytic and sugar binding residues were highly conserved. Phylogenetic analyses revealed four Cerambycidae-specific clades, including 30 members from P. antennata. Combining the transcriptome and PCR data showed that PantUGTs had a wide tissue expression, and the majority of the genes were presented mainly in antennae or abdomens, suggesting their putative roles in olfaction and detoxification. This study provides, for the first time, information on the molecular and genetic basis of P. antennata, greatly enhancing our knowledge of the detoxification-related UGT gene family.

Structural basis for substrate recognition in the Phytolacca americana glycosyltransferase PaGT3.

Capsaicinoids are phenolic compounds that have health benefits. However, the pungency and poor water solubility of these compounds limit their exploitation. Glycosylation is a powerful method to improve water solubility and reduce pungency while preserving bioactivity. PaGT3, a uridine diphosphate glycosyltransferase (UGT) from Phytolacca americana, is known for its ability to glycosylate capsaicinoids and other phenolic compounds. While structural information on several UGTs is available, structures of UGTs that can glycosylate a range of phenolic compounds are rare. To fill this gap, crystal structures of PaGT3 with a sugar-donor analogue (UDP-2-fluoroglucose) and the acceptors capsaicin and kaempferol were determined. PaGT3 adopts a GT-B-fold structure that is highly conserved among UGTs. However, the acceptor-binding pocket in PaGT3 is hydrophobic and large, and is surrounded by longer loops. The larger acceptor-binding pocket in PaGT3 allows the enzyme to bind a range of compounds, while the flexibility of the longer loops possibly plays a role in accommodating the acceptors in the binding pocket according to their shape and size. This structural information provides insights into the acceptor-binding mechanism in UGTs that bind multiple substrates.

Engineering of a UDP-Glycosyltransferase for the Efficient Whole-Cell Biosynthesis of Siamenoside I in Escherichia coli.

The combination of the insufficient availability and the complex structure of siamenoside I (SI), the sweetest glucoside isolated from Siraitia grosvenorii to date, limited its use as a natural sweetener. To solve this problem, an improved biocatalyst, UGT-M2, was semi-rationally created by engineering the uridine diphosphate glycosyltransferase UGT94-289-2 from S. grosvenorii for the monoglucosylation of mogroside IIIE (MG IIIE) to SI. Subsequently, an engineered Escherichia coli cell was constructed, which combined UGT-M2 with a UDP-glucose regeneration system to circumvent the need for expensive UDP-glucose to produce SI. After optimization, high-purity SI (>96.4%) was efficiently prepared from MG IIIE at a 1 L scale with a productivity of 29.78 g/(L day) and a molar yield of 76.5% and without using exogenous UDP-glucose. This study not only developed a whole-cell approach for the preparation of SI but also provided an alternative glycosyltransferase variant for SI biosynthesis with synthetic biology in the future.

Tal2b targets and activates the expression of OsF3H03g to hijack OsUGT74H4 and synergistically interfere with rice immunity.

Transcription activator-like (TAL) effectors are major virulence factors secreted by the type III secretion systems of Xanthomonas oryzae pv. oryzicola (Xoc) and X. oryzae pv. oryzae (Xoo), causing bacterial leaf streak and bacterial blight, respectively, in rice. However, the knowledge of Xoc TAL effector function in promoting bacterial virulence remains limited. Here, we isolated the highly virulent Xoc strain HGA4 from the outbreak region of Huanggang (Hubei, China), which contains four TAL effectors not found in the Chinese model strain RS105. Among these, Tal2b was selected for introduction into RS105, which resulted in a longer lesion length than that in the control. Tal2b directly binds to the promoter region of the gene and activates the expression of OsF3H03g , which encodes 2-oxoglutarate-dependent dioxygenase in rice. OsF3H03g negatively regulates salicylic acid (SA)-related defense by directly reducing SA, and it plays a positive role in susceptibility to both Xoc and Xoo in rice. OsF3H03g interacts with a uridine diphosphate-glycosyltransferase protein (OsUGT74H4), which positively regulates bacterial leaf streak susceptibility and may inactivate SA via glycosylation modification.

A uridine diphosphate-glycosyltransferase GFUGT88A1 derived from edible mushroom Grifola frondosa extends oligosaccharide chains

Fungal glucans play a key role to provide energy, support cell structure, and present biological functions. However, the biosynthetic machineries of fungal glucan chains remain to be elucidated. Hence, we aimed to elucidate the biochemical and catalytic characteristics of a novel glucan synthesis-associated enzyme uridine diphosphate (UDP)-glycosyltransferase GFUGT88A1 derived from Grifola frondosa and predict its potential catalytic mechanism. The purified recombinant GFUGT88A1 had a molecular weight of 51.7 kDa with an optimum temperature of 37 °C and pH of 7.0. GFUGT88A1 showed preference for oligosaccharides with relatively higher polymerization degrees (≥6) as acceptors to extend its chain when using UDP-glucose as a donor. Molecular modeling and docking of GFUGT88A1 with donor UDP-glucose, and acceptors laminaribiose, laminarihexaose, and laminarinonaose indicated that the predicted amino acid residues at the active sites may bind the donor/acceptor protein primarily via hydrogen bonds with various binding energy values. The present study revealed that GFUGT88A1 extended glucan chains, which provides a reference to understand the biosynthesis pathway of mushroom glucans.

Genomic-Wide Identification and Characterization of the Uridine Diphosphate Glycosyltransferase Family in Eucommia ulmoides Oliver.

Eucommia ulmoides Oliver is a woody plant with great economic and medicinal value. Its dried bark has a long history of use as a traditional medicinal material in East Asia, which led to many glycosides, such as aucubin, geniposide, hyperoside, astragalin, and pinoresinol diglucoside, being recognized as pharmacologically active ingredients. Uridine diphosphate glycosyltransferases (UGTs) catalyze a glycosyl-transferring reaction from the donor molecule uridine-5′-diphosphate-glucose (UDPG) to the substrate, which plays an important role in many biological processes, such as plant growth and development, secondary metabolism, and environmental adaptation. In order to explore the biosynthetic pathways of glycosides in E. ulmoides, 91 putative EuUGT genes were identified throughout the complete genome of E. ulmoides through function annotation and an UDPGT domain search. Phylogenetic analysis categorized them into 14 groups. We also performed GO annotations on all the EuUGTs to gain insights into their functions in E. ulmoides. In addition, transcriptomic analysis indicated that most EuUGTs showed different expression patterns across diverse organs and various growing seasons. By protein–protein interaction predication, a biosynthetic routine of flavonoids and their glycosides was also proposed. Undoubtedly, these results will help in future research into the biosynthetic pathways of glycoside compounds in E. ulmoides.

Transient expression and enzymatic assay identified uridine-diphosphate glucosyltransferases related to flavonoid glycosylation in Vernonia amygdalina leaves

Although previous metabolomics analysis revealed that flavonoid glycosylation commonly occurred at 3−OH and 7−OH positions in Vernonia amygdalina leaves, the biosynthetic mechanisms of flavonoid glycosides are still largely unknown. Based on the previous transcriptome data for V. amygdalina leaves, a total of 62 uridine-diphosphate glycosyltransferase (UGT) genes were acquired. We conducted phylogenetic analyses and tested for expression profiles to further select the potential V. amygdalina UGT (VaUGT) related to flavonoid glycosylation. Functional analyses were conducted on the following 6 VaUGT genes: VaUGT9 and VaUGT40 from Clade I, VaUGT3 and VaUGT62 from Clade Ⅳ, and VaUGT38 and VaUGT49 from Clade Ⅵ. Transient expression analysis indicated that VaUGT38 might have substrate specificity involved in the 3−OH and 7−OH positions of kaempferol glycosylation, while VaUGT40 was related to the 7−OH position of kaempferol and apigenin glycosylation. In addition, VaUGT9 and VaUGT3 displayed glycosyltransferase activities at apigenin 7−OH and 7-O-glucoside 2″−OH positions, respectively. Recombinant expression analysis verified that the VaUGT9 and VaUGT38 proteins exhibit a substrate preference to apigenin and kaempferol, respectively. The recombinant VaUGT3, VaUGT9 and VaUGT40 proteins could use apigetrin as substrate to generate apigenin, in particular, VaUGT9 and VaUGT40 were the most efficient. Recombinant VaUGT3, VaUGT38, VaUGT40, VaUGT49, and VaUGT62 proteins exhibited glycosyltransferase activity toward kaempferol, among which VaUGT38 and VaUGT40 were the most efficient. These results improve our understanding of flavonoid glycosylation in V. amygdalina leaves.

Genomic-wide Identification and Characterization of the Uri-dine Diphosphate Glycosyltransferase Family in Eucommia ulmoides Oliver

Genomic-wide Identification and Characterization of the Uri-dine Diphosphate Glycosyltransferase Family in Eucommia ulmoides Oliver

Genome-wide identification and expression analysis of glycosyltransferase gene family 1 in Quercus robur L.

Glycosyltransferase gene family 1, also known as uridine diphosphate glycosyltransferase (UGT), is the largest glycosyltransferase family in plants, playing a vital role in their growth and development. In this study, 244 UGT genes with conserved PSPG motifs were identified in the genome of Quercus robur L. The collinearity analysis results showed that tandem repeat was the main way of UGT genes expansion in Q. robur, with 21 groups of 55 tandem repeat genes. UGT genes were divided into 15 subgroups A-P; group K was lost, and the gene structure and conserved domain of the same subgroup were basically the same. Cis-element analysis showed that upstream 2,000bp promoter sequence of UGT genes contained light response elements, plant hormone response elements, and stress-related cis-elements, which indicated that UGT genes of Q. robur might be regulated by various metabolic pathways. In particular, some UGTs in group L of Q. robur contained a conserved promoter structure. The expression pattern analysis results demonstrated that UGT genes of groups B, D, E, and I were differentially expressed under Tortrix viridana L. stress. The expression of UGTs in group E decreased under stress, the expression of group L increased, and that of genes in groups D and B were different. The functions of UGT genes in E and L groups are relatively conservative, and their functions may also conserve among species. The study results have a particular reference value for further research on the function of Q. robur UGT genes.

Detoxification gene families in Phylloxera: Endogenous functions and roles in response to the environment

Phylloxera, Daktulosphaira vitifoliae, is an agronomic pest that feeds monophagously on grapevine, Vitis spp. host plants. Phylloxera manipulates primary and secondary plant metabolism to establish either leaf or root galls. We manually annotated 198 detoxification genes potentially involved in plant host manipulation, including cytochrome P450 (66 CYPs), carboxylesterase (20 CCEs), glutathione-S-transferase (10 GSTs), uridine diphosphate-glycosyltransferase (35 UGTs) and ABC transporter (67 ABCs) families. Transcriptomic expression patterns of these detoxification genes were analyzed for root and leaf galls. In addition to these transcriptomic analyses, we reanalyzed recent data from L1 and L2–3 stages feeding on tolerant and resistant rootstock. Data from two agricultural pest aphids, the generalist Myzus persicae and the Fabaceae specialist Acyrthosiphon pisum, and from the true bug vector of Chagas disease, Rhodnius prolixus, were used to perform phylogenetic analyses for each detoxification gene family. We found expansions of several gene sub-families in the genome of D. vitifoliae. Phylogenetically close genes were found to be organized in clusters in the same genomic position and orientation suggesting recent successive duplications. These results highlight the roles of the phylloxera detoxification gene repertoire in insect physiology and in adaptation to plant secondary metabolites, and provide gene candidates for further functional analyses.

Isolation and Functional Analysis of Genes Involved in Polyacylated Anthocyanin Biosynthesis in Blue Senecio cruentus.

Polyacylated anthocyanins with multiple glycosyl and aromatic acyl groups tend to make flowers display bright and stable blue colours. However, there are few studies on the isolation and functional characterization of genes involved in the polyacylated anthocyanin biosynthesis mechanism, which limits the molecular breeding of truly blue flowers. Senecio cruentus is an important potted ornamental plant, and its blue flowers contain 3′,7-polyacylated delphinidin-type anthocyanins that are not reported in any other plants, suggesting that it harbours abundant gene resources for the molecular breeding of blue flowers. In this study, using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) analysis of blue, carmine and white colours of cineraria cultivars “Venezia” (named VeB, VeC, and VeW, respectively), we found that 3′,7-polyacylated anthocyanin, cinerarin, was the main pigment component that determined the blue colour of ray florets of cineraria. Based on the transcriptome sequencing and differential gene expression (DEG) analysis combined with RT- and qRT-PCR, we found two genes encoding uridine diphosphate glycosyltransferase, named ScUGT1 and ScUGT4; two genes encoding acyl-glucoside-dependent glucosyltransferases which belong to glycoside hydrolase family 1 (GH1), named ScAGGT11 and ScAGGT12; one gene encoding serine carboxypeptidase-like acyltransferase ScSCPL2; and two MYB transcriptional factor genes ScMYB2 and ScMYB4, that were specifically highly expressed in the ray florets of VeB, which indicated that these genes may be involved in cinerarin biosynthesis. The function of ScSCPL2 was analysed by virus-induced gene silencing (VIGS) in cineraria leaves combined with HPLC-MS/MS. ScSCPL2 mainly participated in the 3′ and 7-position acylation of cinerarin. These results will provide new insight into the molecular basis of the polyacylated anthocyanin biosynthesis mechanism in higher plants and are of great significance for blue flower molecular breeding of ornamental plants.

Melatonin alleviates NaCl-induced damage by regulating ionic homeostasis, antioxidant system, redox homeostasis, and expression of steviol glycosides-related biosynthetic genes in in vitro cultured Stevia rebaudiana Bertoni

Melatonin has been shown to play a variety of roles in regulating plant growth and response to various abiotic stresses, but its main function remains unclear in response to NaCl toxicity. The aim of the current study was to evaluate the effect of melatonin (0, 5, 10, and 20 μM) on growth, photosynthetic pigments, compatible solutes, ionic homeostasis, enzymatic and non-enzymatic antioxidants, the content of steviol glycosides, and expression of kaurenoic acid hydroxylase (KAH) and uridine diphosphate glycosyltransferase (UGTs) genes in stevia under 25 mM NaCl in in vitro culture. The results showed that in the presence of 25 mM NaCl, melatonin at 5 and 10 μM improved growth parameters, photosynthetic pigments, proline, and soluble sugar contents and reduced electrolyte leakage in stevia leaves. Interestingly, melatonin (5 and 10 μM) reduced the accumulation of malondialdehyde and the level of ROS by improving the redox state of the ascorbate-glutathione cycle and the activity of antioxidant enzymes. Additionally, melatonin decreased the accumulation of Cl and Na and improved the accumulation of K, N, Ca, and P, thus maintaining the ionic balance in leaves under NaCl toxicity. Melatonin increased the accumulation of rebaudioside A and stevioside in the leaves under salinity by upregulating the expression level of KAH and UGTs genes. However, high concentrations of melatonin (20 μM) exacerbated the negative effects of salinity and further reduced stevia growth under salinity. Therefore, the current study indicates the protective role of melatonin in the appropriate concentration in stevia against NaCl toxicity in in vitro conditions.

Identification of the Key Residues of the Uridine Diphosphate Glycosyltransferase 91D2 and its Effect on the Accumulation of Steviol Glycosides in Stevia rebaudiana

Stevia (Stevia rebaudiana Bertoni) possesses substantial value for its unique sweet compounds-steviol glycosides (SGs). In the metabolic glycosylation grid of SGs, SrUGT91D2 has been shown to catalyze formation of 1,2-β-d-glucoside linkages at the C13- and C19-positions and play a crucial role in the synthesis of SGs, including the formation of stevioside (ST), rebaudioside E (RE), and rebaudioside D (RD). However, the key residues of the SrUGT91D2 enzyme and how SrUGT91D2 affects the accumulation of SGs in S. rebaudiana remain unclear. In the present study, cloning and functional analysis of full-length SrUGT91D2 gene sequences were performed in 10 different S. rebaudiana genotypes with divergent SG compositions. After sequence analysis, it was found that most of the sequences of this gene (more than 50%) in each genotype were consistent with the UGT91D2e_No.5 allele, which has been reported to exert catalytic activity on 1,2-β-d-glucoside. Moreover, six variants (UGT91D2e_No.5, SrUGT91D2-11-14, SrUGT91D2-110, SrUGT91D2-023, SrUGT91D2-N01, and SrUGT91D2-N04) of this gene were obtained, and their activities were identified. Although there were some differences among these variants, the only type of mutation was partial base substitution at a very low level. In addition, the expression analysis of SrUGT91D2 in each genotype showed that the expression level of the gene was significantly different among genotypes, and a significant positive correlation was found between the content of RD (which was closely influenced by SrUGT91D2) and the expression level of SrUGT91D2 in each genotype (correlation coefficient = 0.91). Thus, it was indicated that SrUGT91D2 was relatively conserved in S. rebaudiana, and the differential effect of SrUGT91D2 on the accumulation of related SGs mainly derived from its expression level. Furthermore, based on homologous modeling and molecular docking analysis, T84, T144, A194, S284, E285, V286, G365, E369, R404, and G409 were predicted to be key residues in the glucosylation of SGs by SrUGT91D2. After site-mutation and enzyme assays, it was confirmed that T84, T144, R404, A194, and G409 are the key residues in the SrUGT91D2 protein, especially T144 and G409. This work provided valuable information for understanding the structure-activity relationship of the SrUGT91D2 protein and the molecular mechanism of SG accumulation in stevia.

Cadmium Chloride and Silver Nitrate Affect the Gene Expression, Stevioside, and Rebaudioside: A Production in Stevia rebaudiana (Bert.)

Stevia rebaudiana (Bert.) produces steviol glycoside sweeteners that are sweeter than sucrose. Stevioside and rebaudioside A are important steviol glycosides used in food production. This study was conducted to investigate the effect of different concentrations of cadmium chloride (0, 20, 40, 60 and 80 mg/L) and silver nitrate (0, 15, 30, 45 and 60 mg/L) and different sampling times on stevioside and rebaudioside A production and Ent-kaurenoic acid 13-hydroxylase, uridine diphosphate-glycosyltransferase 74G1 and uridine diphosphate-glycosyltransferase 76G1 genes expression in in vitro conditions. The results showed that the application of cadmium nitrate and silver nitrate decreased stevioside and rebaudioside A production. Among various concentrations of cadmium chloride, the highest concentrations of stevioside and rebaudioside A were respectively 39.76 mg/g and 2.27 mg/L dry weight 96 and 72 h after elicitation with 20 mg/L cadmium chloride. Among different concentrations of silver nitrate, the highest amounts of stevioside and rebaudioside A were respectively 33.33 mg/g dry weight was 2.19 mg/L obtained at control condition after 96 h and elicitation with 60 mg/L for 24 h. In elicitation with cadmium chloride the expression all three genes were increased but silver nitrate elicitation increased uridine diphosphate-glycosyltransferase 76G1 gene expression and decreased the Ent-kaurenoic acid 13-hydroxylase and uridine diphosphateglycosyltransferase 74G1 genes expression. The correlation analysis showed that Ent-kaurenoic acid 13-hydroxylase gene expression was not correlated with uridine diphosphate-glycosyltransferase 74G1 and uridine diphosphate-glycosyltransferase 76G1 genes expression, while uridine diphosphateglycosyltransferase 74G1 was inversely correlated with uridine diphosphate-glycosyltransferase 76G1. Also, positive correlations were observed between uridine diphosphate-glycosyltransferase 74G1 gene expression with stevioside synthesis and uridine diphosphate-glycosyltransferase 76G1 gene expression with rebaudioside A.

Chromosome-level genome of the peach fruit moth Carposina sasakii (Lepidoptera: Carposinidae) provides a resource for evolutionary studies on moths.

The peach fruit moth (PFM), Carposina sasakii Matsumura, is a major phytophagous orchard pest widely distributed across Northeast Asia. Here, we report the chromosome-level genome for the PFM, representing the first genome for the family Carposinidae, from the lepidopteran superfamily Copromorphoidea. The genome was assembled into 404.83Mb sequences using PacBio long-read and Illumina short-read sequences, including 275 contigs, with a contig N50 length of 2.62Mb. All contigs were assembled into 31 linkage groups assisted by the Hi-C technique, including 30 autosomes and a Z chromosome. BUSCO analysis showed that 98.3% of genes were complete and 0.4% of genes were fragmented, while 1.3% of genes were missing in the assembled genome. In total, 21,697 protein-coding genes were predicted, of which 84.80% were functionally annotated. Because of the importance of diapause triggered by photoperiod in PFM, five circadian genes in the PFM as well as in the other related species were annotated, and potential genes related to diapause and photoperiodic reaction were also identified from transcriptome sequencing. In addition, manual annotation of detoxification gene families was undertaken and showed a higher number of glutathione S-transferase (GST) gene in PFM than in most other lepidopterans, in contrast to a lower number of uridine diphosphate (UDP)-glycosyltransferase (UGT) gene, carboxyl/cholinesterases (CCE) gene and cytochrome P450 monooxygenase (P450) gene, suggesting different detoxication pathways in this moth. The high-quality genome provides a resource for comparative evolutionary studies of this moth and its relatives within the context of radiations across Lepidoptera.