BvCGT1-mediated differential distribution of flavonoid C-glycosides contributes to plant's response to UV-B stress.

C-glycosides are a predominant class of flavonoids that demonstrate diverse medical properties and plant physiological functions. The chemical stability, structural diversity, and differential aboveground distribution of these compounds in plants make them ideal protectants. However, little is known about the transcriptional regulatory mechanisms that play these diverse roles in plant physiology. In this study, chard was selected from 69 families for its significantly different flavonoid C-glycosides distributions between the aboveground and underground parts to investigate the role and regulatory mechanism of flavonoid C-glycosides in plants. Our results indicate that flavonoid C-glycosides are affected by various stressors, especially UV-B. Through cloning and validation of key biosynthetic genes of flavonoid C-glycosides in chard (BvCGT1), we observed significant effects induced by UV-B radiation. This finding was further confirmed by resistance testing in BvCGT1 silenced chard lines and in Arabidopsis plants with BvCGT1 overexpression. Yeast one-hybrid and dual-luciferase assays were employed to determine the underlying regulatory mechanisms of BvCGT1 in withstanding UV-B stress. These results indicate a potential regulatory role of BvDof8 and BvDof13 in modulating flavonoid C-glycosides content, through their influence on BvCGT1. In conclusion, we have effectively demonstrated the regulation of BvCGT1 by BvDof8 and BvDof13, highlighting their crucial role in plant adaptation to UV-B radiation. Additionally, we have outlined a comprehensive transcriptional regulatory network involving BvDof8 and BvDof13 in response to UV-B radiation.

Complete reconstitution of the diverse pathways of gentamicin B biosynthesis.

Gentamicin B (GB), a valuable starting material for the preparation of the semisynthetic aminoglycoside antibiotic isepamicin, is produced in trace amounts by the wild-type Micromonospora echinospora. Though the biosynthetic pathway to GB has remained obscure for decades, we have now identified three hidden pathways to GB production via seven hitherto unknown intermediates in M. echinospora. The narrow substrate specificity of a key glycosyltransferase and the C6'-amination enzymes, in combination with the weak and unsynchronized gene expression of the 2'-deamination enzymes, limits GB production in M. echinospora. The crystal structure of the aminotransferase involved in C6'-amination explains its substrate specificity. Some of the new intermediates displayed similar premature termination codon readthrough activity but with reduced toxicity compared to the natural aminoglycoside G418. This work not only led to the discovery of unknown biosynthetic routes to GB, but also demonstrated the potential to mine new aminoglycosides from nature for drug discovery.

Metabolic engineering challenges of extending N-glycan pathways in Chinese hamster ovary cells.

In mammalian cells, N-glycans may include multiple N-acetyllactosamine (poly-LacNAc) units that can play roles in various cellular functions and properties of therapeutic recombinant proteins. Previous studies indicated that ß-1,3-N-acetylglucosaminyltransferase 2 (B3GNT2) and ß-1,4-galactotransferase 1 (B4GALT1) are two of the primary glycosyltransferases involved in generating LacNAc units. In the current study, knocking out sialyltransferase genes slightly enhanced the LacNAc content (≥4 repeats per glycan) on recombinant EPO protein. Next, the role of single and dual-overexpression of B3GNT2 and B4GALT1 was explored in recombinant EPO-expressing Chinese hamster ovary (CHO) cells. While overexpression of B4GALT1 slightly enhanced the levels of large glycans on recombinant EPO, overexpression of B3GNT2 in EPO-expressing CHO cells significantly decreased the recombinant EPO LacNAc content, resulting in N-glycans terminating primarily with GlcNAc structures, a limited number of Gals, and nearly undetectable sialylation, which was also observed in sialyltransferases knock-out-B3GNT2 overexpression cell lines. Considering the nature of the binding domain motifs present on B3GNT2, which evolved from ß1,3-galactosyltransferases, its overexpression may have competed and inhibited endogenous ß1,4-galactosyltransferases for exposed GlcNAc residues on the N-glycans, resulting in premature termination of many N-glycans at GlcNAc. Furthermore, B3GNT2 overexpression enhanced intracellular UDP-GlcNAc and CMP-Neu5Ac content while slightly lowering UDP-Gal content. The presence of a sink for UDP-GlcNAc in the form of B3GNT2 with no disposition may have also elevated the intracellular levels of this nucleotide as well as its downstream product, CMP-Neu5Ac. Furthermore, we were unable to overexpress B4GALT1 at either the transcriptional or translational levels following initial B3GNT2 expression. Expression of B3GNT2 following initial expression of B4GALT1 was also problematic in that transcriptional and translational analysis indicated the accumulation of truncated B3GNT2 missing a section of the B3GNT2 trans-Golgi lumen domain while transmembrane and cytoplasmic domains were present. Given that glycosylation is a very complex intra-network process, the addition of one or more recombinant glycosyltransferases may have an unexpected influence on the expression and activities of glycosyltransferases, which can disrupt the nucleotide sugar levels and lead to unexpected modifications of the resulting N-glycan patterns.

Personalized genome sequencing coupled with iPSC technology identifies GTDC1 as a gene involved in neurodevelopmental disorders.

The cellular and molecular mechanisms underlying neurodevelopmental conditions such as autism spectrum disorders have been studied intensively for decades. The ability to generate patient-specific induced pluripotent stem cells (iPSCs) now offers a novel strategy for modelling human diseases. Recent studies have reported the derivation of iPSCs from patients with neurological disorders. The key challenge remains the demonstration of disease-related phenotypes and the ability to model the disease. Here we report a case study with signs of neurodevelopmental disorders (NDDs) harbouring chromosomal rearrangements that were sequenced using long-insert DNA paired-end tag (DNA-PET) sequencing approach. We identified the disruption of a specific gene, GTDC1. By deriving iPSCs from this patient and differentiating them into neural progenitor cells (NPCs) and neurons we dissected the disease process at the cellular level and observed defects in both NPCs and neuronal cells. We also showed that disruption of GTDC1 expression in wild type human NPCs and neurons showed a similar phenotype as patient's iPSCs. Finally, we utilized a zebrafish model to demonstrate a role for GTDC1 in the development of the central nervous system. Our findings highlight the importance of combining sequencing technologies with the iPSC technology for NDDs modelling that could be applied for personalized medicine.

A cell-free platform for rapid synthesis and testing of active oligosaccharyltransferases.

Protein glycosylation, or the attachment of sugar moieties (glycans) to proteins, is important for protein stability, activity, and immunogenicity. However, understanding the roles and regulations of site-specific glycosylation events remains a significant challenge due to several technological limitations. These limitations include a lack of available tools for biochemical characterization of enzymes involved in glycosylation. A particular challenge is the synthesis of oligosaccharyltransferases (OSTs), which catalyze the attachment of glycans to specific amino acid residues in target proteins. The difficulty arises from the fact that canonical OSTs are large (>70 kDa) and possess multiple transmembrane helices, making them difficult to overexpress in living cells. Here, we address this challenge by establishing a bacterial cell-free protein synthesis platform that enables rapid production of a variety of OSTs in their active conformations. Specifically, by using lipid nanodiscs as cellular membrane mimics, we obtained yields of up to 420 µg/ml for the single-subunit OST enzyme, "Protein glycosylation B" (PglB) from Campylobacter jejuni, as well as for three additional PglB homologs from Campylobacter coli, Campylobacter lari, and Desulfovibrio gigas. Importantly, all of these enzymes catalyzed N-glycosylation reactions in vitro with no purification or processing needed. Furthermore, we demonstrate the ability of cell-free synthesized OSTs to glycosylate multiple target proteins with varying N-glycosylation acceptor sequons. We anticipate that this broadly applicable production method will advance glycoengineering efforts by enabling preparative expression of membrane-embedded OSTs from all kingdoms of life.

Influence of the flow field on α-cyclodextrin glycosyltransferase production by Escherichia coli BL21.

The computational fluid dynamics (CFD) software package Fluent was utilized to simulate the flow field of Escherichia coli (E. coli) BL21 fermentation in a 50 L automatic bioreactor for producing α-cyclodextrin glycosyltransferase (α-CGTase) in this study. 4-down-pumping propeller (4DPP), 6-curved-blade disc turbine (6CBDT), and Rushton turbine (RT) were assembled to form eight impeller combinations (C1-C8). Through flow field simulating, four referential impeller combinations, in which C6, C7, and C8 were three layers stirring blades and C1 as a control, were selected to carry out batch fermentation experiments (TC1, TC6, TC7, and TC8) for validation. The correlation analysis between simulation results and experimental measurements indicated that TC6 (tank equipped with C6 impeller combination) exhibited lower enzymatic activity though it had the better mixing effect, fastest oxygen uptake rate (OUR), and maximum specific growth rate (µ) in the initial stage, which was just to the contrary in TC8. It was revealed by next fed-batch fermentation experiments in TC6 and TC8 that TC6 was considered as excellent flow field properties brought about the higher µ of E. coli BL21 and fast acetic acid (HAc) accumulation, which resulting in a serious inhibition on α-CGTase expression and this negative effect could not be removed. As a result, there should be a threshold of HAc accumulation rate which brought about a terrible inhibitory effect on α-CGTase expression. Moreover, the yield of α-CGTase activity reached 231.38 U mL- 1 in TC8, which elevated 31.74% compared to that obtained in TC1.

Optimization and purification of glucansucrase produced by Leuconostoc mesenteroides DRP2-19 isolated from Chinese Sauerkraut.

Strain DRP2-19 was detected to produce high yield of glucansucrase in MRS broth, which was identified to be Leuconostoc mesenteroides. In order for industrial glucansucrase production of L. mesenteroides DRP2-19, a one-factor test was conducted, then response surface method was applied to optimize its yield and discover the best production condition. Based on Plackett-Burman (PB) experiment, sucrose, Ca2+, and initial pH were found to be the most significant factors for glucansucrase production. Afterwards, effects of the three main factors on glucansucrase activity were further investigated by central composite design and the optimum composition was sucrose 35.87 g/L, Ca2+ 0.21 mmol/L, and initial pH 5.56. Optimum results showed that glucansucrase activity was increased to 3.94 ± 0.43 U/mL in 24 hr fermentation, 2.66-fold higher than before. In addition, the crude enzyme was purified using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of glucansucrase was determined as approximately 170 kDa by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 15.77-fold and showed a final specific activity of 338.56 U/mg protein.

Genetic and biochemical characterization of the GH72 family of cell wall transglycosylases in Neurospora crassa.

The Neurospora crassa genome encodes five GH72 family transglycosylases, and four of these enzymes (GEL-1, GEL-2, GEL-3 and GEL-5) have been found to be present in the cell wall proteome. We carried out an extensive genetic analysis on the role of these four transglycosylases in cell wall biogenesis and demonstrated that the transglycosylases are required for the formation of a normal cell wall. As suggested by the proteomic analysis, we found that multiple transglycosylases were being expressed in N. crassa cells and that different combinations of the enzymes are required in different cell types. The combination of GEL-1, GEL-2 and GEL-5 is required for the growth of vegetative hyphae, while the GEL-1, GEL-2, GEL-3 combination is needed for the production of aerial hyphae and conidia. Our data demonstrates that the enzymes are redundant with partially overlapping enzymatic activities, which provides the fungus with a robust cell wall biosynthetic system. Characterization of the transglycosylase-deficient mutants demonstrated that the incorporation of cell wall proteins was severely compromised. Interestingly, we found that the transglycosylase-deficient mutant cell walls contained more ß-1,3-glucan than the wild type cell wall. Our results demonstrate that the GH72 transglycosylases are not needed for the incorporation of ß-1,3-glucan into the cell wall, but they are required for the incorporation of cell wall glycoprotein into the cell wall.

Catechol glucosides act as donor/acceptor substrates of glucansucrase enzymes of Lactobacillus reuteri.

Previously, we have shown that the glucansucrase GtfA-ΔN enzyme of Lactobacillus reuteri 121, incubated with sucrose, efficiently glucosylated catechol and we structurally characterized catechol glucosides with up to five glucosyl units attached (te Poele et al. in Bioconjug Chem 27:937-946, 2016). In the present study, we observed that upon prolonged incubation of GtfA-ΔN with 50 mM catechol and 1000 mM sucrose, all catechol had become completely glucosylated and then started to reappear. Following depletion of sucrose, this glucansucrase GtfA-ΔN used both α-D-Glcp-catechol and α-D-Glcp-(1→4)-α-D-Glcp-catechol as donor substrates and transferred a glucose unit to other catechol glycoside molecules or to sugar oligomers. In the absence of sucrose, GtfA-ΔN used α-D-Glcp-catechol both as donor and acceptor substrate to synthesize catechol glucosides with 2 to 10 glucose units attached and formed gluco-oligosaccharides up to a degree of polymerization of 4. Also two other glucansucrases tested, Gtf180-ΔN from L. reuteri 180 and GtfML1-ΔN from L. reuteri ML1, used α-D-Glcp-catechol and di-glucosyl-catechol as donor/acceptor substrate to synthesize both catechol glucosides and gluco-oligosaccharides. With sucrose as donor substrate, the three glucansucrase enzymes also efficiently glucosylated the phenolic compounds pyrogallol, resorcinol, and ethyl gallate; also these mono-glucosides were used as donor/acceptor substrates.

Metabolic engineering of Bacillus subtilis for biosynthesis of heparosan using heparosan synthase from Pasteurella multocida, PmHS1.

Heparosan, the capsular polysaccharide discovered in many pathogenic bacteria, is a promising material for heparin preparation. In this study, the Pasteurella multocida heparosan synthase 1 (PmHS1) module was used to synthesize heparosan with controlled molecular weight, while tuaD/gtaB module or gcaD module was responsible for UDP-precursors production in Bacillus subtilis 168. After metabolic pathway optimization, the yield of heparosan was as high as 237.6 mg/L in strain containing PmHS1 module and tuaD/gtaB module, which indicated that these two modules were key factors in heparosan production. The molecular weight of heparosan varied from 39 to 53 kDa, which indicated that heparosan molecular weight could be adjusted by the amount of PmHS1 and the ratio of two UDP precursors. The results showed that it would be possible to produce safe heparosan with appropriate molecular weight which is useful in heparin production.

Glucansucrases produced by fructophilic lactic acid bacteria Lactobacillus kunkeei H3 and H25 isolated from honeybees.

Twenty fructophilic isolates from the stomachs of honeybee Apis mellifera ligustica from the region of Plovdiv, Bulgaria were obtained. Fructophilic isolates H3 and H25 showed formation of mucous colonies during cultivation on medium with sucrose, suggesting exopolysaccharide synthesis. The sequencing analysis of 16S rRNA identified isolates H3 and H25 as fructophilic lactic acid bacteria Lactobacillus kunkeei. The in situ analysis and periodic acid-Schiff's staining, showed that Lb. kunkeei H3 and H25 produce extracellular glucansucrases with molecular weight of about 300 kDa. In the cell-associated fractions, additional glucansucrase is detected with molecular weight of about 180 kDa. The content of α-(1 → 6) linkages in the glucans synthesized with extracellular glucansucrases from H3 and H25 after dextranase hydrolysis was significantly lower than this one of the classical dextran - about 35 and 62%, respectively. These results suggest a more branched structure of the studied polymers.

De novo biosynthesis of Gastrodin in Escherichia coli.

Gastrodin, a phenolic glycoside, is the key ingredient of Gastrodia elata, a notable herbal plant that has been used to treat various conditions in oriental countries for centuries. Gastrodin is extensively used clinically for its sedative, hypnotic, anticonvulsive and neuroprotective properties in China. Gastrodin is usually produced by plant extraction or chemical synthesis, which has many disadvantages. Herein, we report unprecedented microbial synthesis of gastrodin via an artificial pathway. A Nocardia carboxylic acid reductase, endogenous alcohol dehydrogenases and a Rhodiola glycosyltransferase UGT73B6 transformed 4-hydroxybenzoic acid, an intermediate of ubiquinone biosynthesis, into gastrodin in Escherichia coli. Pathway genes were overexpressed to enhance metabolic flux toward precursor 4-hydroxybenzyl alcohol. Furthermore, the catalytic properties of the UGT73B6 toward phenolic alcohols were improved through directed evolution. The finally engineered strain produced 545mgl(-1) gastrodin in 48h. This work creates a new route to produce gastrodin, instead of plant extractions and chemical synthesis.

STAT5-induced lunatic fringe during Th2 development alters delta-like 4-mediated Th2 cytokine production in respiratory syncytial virus-exacerbated airway allergic disease.

Notch activation plays an important role in T cell development and mature T cell differentiation. In this study, we investigated the role of Notch activation in a mouse model of respiratory syncytial virus (RSV)-exacerbated allergic airway disease. During RSV exacerbation, in vivo neutralization of a specific Notch ligand, Delta-like ligand (Dll)-4, significantly decreased airway hyperreactivity, mucus production, and Th2 cytokines. Lunatic Fringe (Lfng), a glycosyltransferase that enhances Notch activation by Dll4, was increased during RSV exacerbation. Lfng loss of function in Th2-skewed cells inhibited Dll4-Notch activation and subsequent IL-4 production. Further knockdown of Lfng in T cells in CD4Cre(+)Lfng(fl/fl) mice showed reduced Th2 response and disease pathology during RSV exacerbation. Finally, we identified STAT5-binding cis-acting regulatory element activation as a critical driver of Lfng transcriptional activation. These data demonstrate that STAT5-dependent amplification of Notch-modifying Lfng augments Th2 response via Dll4 and is critical for amplifying viral exacerbation during allergic airway disease.

Secreted expression of Leuconostoc mesenteroides glucansucrase in Lactococcus lactis for the production of insoluble glucans.

We expressed a glucansucrase, DsrI, from Leuconostoc mesenteroides that catalyzes formation of water-insoluble glucans from sucrose using a nisin-controlled gene expression system in Lactococcus lactis. These polymers have potential for production of biodegradable gels, fibers, and films. We optimized production of DsrI using several different background vectors, signal peptides, strains, induction conditions, and bioreactor parameters to increase extracellular accumulation. Optimal production of the enzyme utilized a high-copy plasmid, pMSP3535H3, which contains a nisin immunity gene, L. lactis LM0230, and bioreactors maintained at pH 6.0 to stabilize the enzyme. We were able to significantly improve growth using the lactic acid inhibitor heme and by continuous removal of lactic acid with anion exchange resins, but enzyme production was less than the controls. The recombinant enzyme under optimized conditions accumulated in the culture medium to approximately 380 mg/L, which was over 150-fold higher compared to the native L. mesenteroides strain. Methods are also included for purification of DsrI utilizing the glucan-binding domain of the enzyme.

Glycosyltransferases from oat (Avena) implicated in the acylation of avenacins.

Plants produce a huge array of specialized metabolites that have important functions in defense against biotic and abiotic stresses. Many of these compounds are glycosylated by family 1 glycosyltransferases (GTs). Oats (Avena spp.) make root-derived antimicrobial triterpenes (avenacins) that provide protection against soil-borne diseases. The ability to synthesize avenacins has evolved since the divergence of oats from other cereals and grasses. The major avenacin, A-1, is acylated with N-methylanthranilic acid. Previously, we have cloned and characterized three genes for avenacin synthesis (for the triterpene synthase SAD1, a triterpene-modifying cytochrome P450 SAD2, and the serine carboxypeptidase-like acyl transferase SAD7), which form part of a biosynthetic gene cluster. Here, we identify a fourth member of this gene cluster encoding a GT belonging to clade L of family 1 (UGT74H5), and show that this enzyme is an N-methylanthranilic acid O-glucosyltransferase implicated in the synthesis of avenacin A-1. Two other closely related family 1 GTs (UGT74H6 and UGT74H7) are also expressed in oat roots. One of these (UGT74H6) is able to glucosylate both N-methylanthranilic acid and benzoic acid, whereas the function of the other (UGT74H7) remains unknown. Our investigations indicate that UGT74H5 is likely to be key for the generation of the activated acyl donor used by SAD7 in the synthesis of the major avenacin, A-1, whereas UGT74H6 may contribute to the synthesis of other forms of avenacin that are acylated with benzoic acid.

O-acetylation of glucuronoxylan in Arabidopsis thaliana wild type and its change in xylan biosynthesis mutants.

O-Acetylglucuronoxylans (AcGX) in Arabidopsis thaliana carry acetyl residues on the 2-O and/or 3-O positions of the xylopyranosyl (Xylp) units, but the distribution of different O-acetylated Xylp units is partly unclear. We studied a possible correlation of xylan acetylation and the activities of different glycosyltransferases involved in xylan biosynthesis by analyzing the distribution of O-acetyl substituents on AcGX from Arabidopsis wild-type and mutants irx7, irx9-1, irx10, irx14 and gux1gux2. The relative contents of the Xylp structural units were determined with quantitative two-dimensional heteronuclear single quantum coherence nuclear magnetic resonance spectroscopy. In the wild type, the degree of acetylation (DA) was 60%. Mono- and diacetylated Xylp units constituted 44 and 6% of the AcGX backbone, respectively; while (4-O-methyl)-glucopyranosyluronic acid (1 → 2)-linked Xylp units, most of which also carry 3-O-acetylation, represented 13%. The DA was decreased in irx7, irx9-1 and irx14 due to the decrease in monoacetylation (2-O and 3-O), indicating a relationship between acetylation and other AcGX biosynthetic processes. The possible interactions that could lead to such changes have been discussed. No change in DA was observed in irx10 and gux1gux2, but monoacetylation was nonetheless elevated in gux1gux2. This indicates that acetylation occurs after addition of GlcpA to the xylan backbone. Mass fragmentation analysis suggests that the prevalent acetylation pattern is the acetyl group added on every other Xylp unit.

UGT74D1 catalyzes the glucosylation of 2-oxindole-3-acetic acid in the auxin metabolic pathway in Arabidopsis.

IAA is a naturally occurring auxin that plays a crucial role in the regulation of plant growth and development. The endogenous concentration of IAA is spatiotemporally regulated by biosynthesis, transport and its inactivation in plants. Previous studies have shown that the metabolism of IAA to 2-oxindole-3-acetic acid (OxIAA) and OxIAA-glucoside (OxIAA-Glc) may play an important role in IAA homeostasis, but the genes involved in this metabolic pathway are still unknown. In this study, we show that UGT74D1 catalyzes the glucosylation of OxIAA in Arabidopsis. By screening yeasts transformed with Arabidopsis UDP-glycosyltransferase (UGT) genes, we found that OxIAA-Glc accumulates in the culture media of yeasts expressing UGT74D1 in the presence of OxIAA. Further, we showed that UGT74D1 expressed in Escherichia coli converts OxIAA to OxIAA-Glc. The endogenous concentration of OxIAA-Glc decreased by 85% while that of OxIAA increased 2.5-fold in ugt74d1-deficient mutants, indicating the major role of UGT74D1 in OxIAA metabolism. Moreover, the induction of UGT74D1 markedly increased the level of OxIAA-Glc and loss of root gravitropism. These results indicate that UGT74D1 catalyzes a committed step in the OxIAA-dependent IAA metabolic pathway in Arabidopsis.

Alterations in the chondroitin sulfate chain in human osteoarthritic cartilage of the knee.

OBJECTIVE: To determine whether the structure of chondroitin sulfate (CS) in cartilage is reflected by the degree of cartilage degeneration in patients with osteoarthritis (OA) of the knee and to determine how CS biosynthesis affects cartilage degeneration. DESIGN: Two osteoarthritic cartilage samples were obtained from medial femoral condyle (MFC) and lateral femoral condyle (LFC) of 24 knees with end-stage OA. The samples were assigned to two groups as follows: lesion and remote cartilage were adjacent to and remote from the osteoarthritic cartilage, respectively. Histological grade was determined according to the Mankin score. The CS concentration and chain length were determined using high-performance liquid chromatography (HPLC) and gel filtration chromatography, respectively. Expression of the gene encoding CS glycosyltransferase was evaluated using a real-time quantitative polymerase chain reaction (qPCR) assay. These results were compared between lesion and remote cartilage. RESULTS: The Mankin score indicated that lesion cartilage was more degraded compared with remote cartilage. Although the CS levels varied among individuals, the mean CS concentration and chain length were significantly lower and shorter in lesion cartilage than in remote cartilage, respectively (concentration: 12.04 vs 14.84 µg/mg wet weight, P = 0.021; chain length: 5.36 vs 6.19 kDa, P = 0.026). Three genes encoding CS glycosyltransferases (CHPF, CSGALNACT1, CSGALNACT2) were expressed at lower levels in lesion cartilage. CONCLUSIONS: In the osteoarthritic knee, the CS concentration and chain length were reduced closer to the more degraded cartilage with decreasing CS glycosyltransferase gene expression. Inhibition of CS glycosyltransferase gene expression may reduce CS chain length, which may contribute to OA progression.

Overexpression of a glycosyltransferase gene SrUGT74G1 from Stevia improved growth and yield of transgenic Arabidopsis by catechin accumulation.

Steviol glycoside and gibberellin biosynthetic routes are known as divergent branches of a common origin in Stevia. A UDP-glycosyltransferase encoded by SrUGT74G1 catalyses the conversion of steviolbioside into stevioside in Stevia rebaudiana leaves. In the present study, transgenic Arabidopsis thaliana overexpressing SrUGT74G1 cDNA from Stevia were developed to check the probability of stevioside biosynthesis in them. However, stevioside accumulation was not evident in transgenics. Also, the transgenic Arabidopsis showed no change in GA3 content on SrUGT74G1 overexpression. Surprisingly, significant accumulation of catechin was noticed in transgenics. The transgenics showed a considerable increase in shoot length, root length and rosette area. An increase in free radical scavenging activity of transgenics was noticed. Moreover, the seed yield of transgenics was also increased by 6-15% than control. Additionally, variation in trichome branching pattern on leaf surface of transgenics was observed. The trichome branching pattern was also validated by exogenous catechin exposure (10, 50, 100 ng ml(-1)) to control plants. Hence, present study reports the probable role of SrUGT74G1 from Stevia in catechin accumulation of transgenic Arabidopsis thaliana. Thus, detailed study in present perspective has revealed the role of Stevia SrUGT74G1 gene in trichome branching pattern, improved vegetative growth, scavenging potential and seed yield by catechin accumulation in transgenic Arabidopsis.