Apocarotenoids are allosteric effectors of a dimeric plant glycosyltransferase involved in defense and lignin formation.

Glycosyltransferases are nature's versatile tools to tailor the functionalities of proteins, carbohydrates, lipids, and small molecules by transferring sugars. Prominent substrates are hydroxycoumarins such as scopoletin, which serve as natural plant protection agents. Similarly, C13-apocarotenoids, which are oxidative degradation products of carotenoids/xanthophylls, protect plants by repelling pests and attracting pest predators. We show that C13-apocarotenoids interact with the plant glycosyltransferase NbUGT72AY1 and induce conformational changes in the enzyme catalytic center ultimately reducing its inherent UDP-α-d-glucose glucohydrolase activity and increasing its catalytic activity for productive hydroxycoumarin substrates. By contrast, C13-apocarotenoids show no effect on the catalytic activity toward monolignol lignin precursors, which are competitive substrates. In vivo studies in tobacco plants (Nicotiana benthamiana) confirmed increased glycosylation activity upon apocarotenoid supplementation. Thus, hydroxycoumarins and apocarotenoids represent specialized damage-associated molecular patterns, as they each provide precise information about the plant compartments damaged by pathogen attack. The molecular basis for the C13-apocarotenoid-mediated interplay of two plant protective mechanisms and their function as allosteric enhancers opens up potential applications of the natural products in agriculture and pharmaceutical industry.

Acceptors and Effectors Alter Substrate Inhibition Kinetics of a Plant Glucosyltransferase NbUGT72AY1 and Its Mutants.

One of the main obstacles in biocatalysis is the substrate inhibition (SI) of enzymes that play important roles in biosynthesis and metabolic regulation in organisms. The promiscuous glycosyltransferase UGT72AY1 from Nicotiana benthamiana is strongly substrate-inhibited by hydroxycoumarins (inhibitory constant Ki < 20 µM), but only weakly inhibited when monolignols are glucosylated (Ki > 1000 µM). Apocarotenoid effectors reduce the inherent UDP-glucose glucohydrolase activity of the enzyme and attenuate the SI by scopoletin derivatives, which could also be achieved by mutations. Here, we studied the kinetic profiles of different phenols and used the substrate analog vanillin, which has shown atypical Michaelis-Menten kinetics in previous studies, to examine the effects of different ligands and mutations on the SI of NbUGT72AY1. Coumarins had no effect on enzymatic activity, whereas apocarotenoids and fatty acids strongly affected SI kinetics by increasing the inhibition constant Ki. Only the F87I mutant and a chimeric version of the enzyme showed weak SI with the substrate vanillin, but all mutants exhibited mild SI when sinapaldehyde was used as an acceptor. In contrast, stearic acid reduced the transferase activity of the mutants to varying degrees. The results not only confirm the multi-substrate functionality of NbUGT72AY1, but also reveal that the enzymatic activity of this protein can be fine-tuned by external metabolites such as apocarotenoids and fatty acids that affect SI. Since these signals are generated during plant cell destruction, NbUGT72AY1 likely plays an important role in plant defense by participating in the production of lignin in the cell wall and providing direct protection through the formation of toxic phytoalexins.

Structure-function relationship of terpenoid glycosyltransferases from plants.

Covering: up to 2021Terpenoids are physiologically active substances that are of great importance to humans. Their physicochemical properties are modified by glycosylation, in terms of polarity, volatility, solubility and reactivity, and their bioactivities are altered accordingly. Significant scientific progress has been made in the functional study of glycosylated terpenes and numerous plant enzymes involved in regio- and enantioselective glycosylation have been characterized, a reaction that remains chemically challenging. Crucial clues to the mechanism of terpenoid glycosylation were recently provided by the first crystal structures of a diterpene glycosyltransferase UGT76G1. Here, we review biochemically characterized terpenoid glycosyltransferases, compare their functions and primary structures, discuss their acceptor and donor substrate tolerance and product specificity, and elaborate features of the 3D structures of the first terpenoid glycosyltransferases from plants.

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

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

Physiological and iTRAQ-based proteomic analyses reveal the function of exogenous γ-aminobutyric acid (GABA) in improving tea plant (Camellia sinensis L.) tolerance at cold temperature.

BACKGROUND: Internal γ-Aminobutyric Acid (GABA) interacting with stress response substances may be involved in the regulation of differentially abundant proteins (DAPs) associated with optimum temperature and cold stress in tea plants (Camellia sinensis (L.) O. Kuntze). RESULTS: Tea plants supplied with or without 5.0 mM GABA were subjected to optimum or cold temperatures in this study. The increased GABA level induced by exogenous GABA altered levels of stress response substances - such as glutamate, polyamines and anthocyanins - in association with improved cold tolerance. Isobaric tags for relative and absolute quantification (iTRAQ) - based DAPs were found for protein metabolism and nucleotide metabolism, energy, amino acid transport and metabolism other biological processes, inorganic ion transport and metabolism, lipid metabolism, carbohydrate transport and metabolism, biosynthesis of secondary metabolites, antioxidant and stress defense. CONCLUSIONS: The iTRAQ analysis could explain the GABA-induced physiological effects associated with cold tolerance in tea plants. Analysis of functional protein-protein networks further showed that alteration of endogenous GABA and stress response substances induced interactions among photosynthesis, amino acid biosynthesis, and carbon and nitrogen metabolism, and the corresponding differences could contribute to improved cold tolerance of tea plants.

Genome-wide identification of UDP-glycosyltransferases in the tea plant (Camellia sinensis) and their biochemical and physiological functions.

Tea (Camellia sinensis) has been an immensely important commercially grown crop for decades. This is due to the presence of essential nutrients and plant secondary metabolites that exhibit beneficial health effects. UDP-glycosyltransferases (UGTs) play an important role in the diversity of such secondary metabolites by catalysing the transfer of an activated sugar donor to acceptor molecules, and thereby creating a huge variety of glycoconjugates. Only in recent years, thanks to the sequencing of the tea plant genome, have there been increased efforts to characterise the UGTs in C. sinensis to gain an understanding of their physiological role and biotechnological potential. Based on the conserved plant secondary product glycosyltransferase (PSPG) motif and the catalytically active histidine in the active site, UGTs of family 1 in C. sinensis are identified here, and shown to cluster into 21 groups in a phylogenetic tree. Building on this, our current understanding of recently characterised C. sinensis UGTs (CsUGTs) is highlighted and a discussion on future perspectives made.

Subfunctionalization of a monolignol to a phytoalexin glucosyltransferase is accompanied by substrate inhibition.

Uridine diphosphate-dependent glycosyltransferases (UGTs) mediate the glycosylation of plant metabolites, thereby altering their physicochemical properties and bioactivities. Plants possess numerous UGT genes, with the encoded enzymes often glycosylating multiple substrates and some exhibiting substrate inhibition kinetics, but the biological function and molecular basis of these phenomena are not fully understood. The promiscuous monolignol/phytoalexin glycosyltransferase NbUGT72AY1 exhibits substrate inhibition (Ki) at 4 µM scopoletin, whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 µM. We therefore used hydrogen/deuterium exchange mass spectrometry and structure-based mutational analyses of both proteins and introduced NbUGT72AY1 residues into StUGT72AY2 and vice versa to study promiscuity and substrate inhibition of UGTs. A single F87I and chimeric mutant of NbUGT72AY1 showed significantly reduced scopoletin substrate inhibition, whereas its monolignol glycosylation activity was almost unaffected. Reverse mutations in StUGT72AY2 resulted in increased scopoletin glycosylation, leading to enhanced promiscuity, which was accompanied by substrate inhibition. Studies of 3D structures identified open and closed UGT conformers, allowing visualization of the dynamics of conformational changes that occur during catalysis. Previously postulated substrate access tunnels likely serve as drainage channels. The results suggest a two-site model in which the second substrate molecule binds near the catalytic site and blocks product release. Mutational studies showed that minor changes in amino acid sequence can enhance the promiscuity of the enzyme and add new capabilities such as substrate inhibition without affecting existing functions. The proposed subfunctionalization mechanism of expanded promiscuity may play a role in enzyme evolution and highlights the importance of promiscuous enzymes in providing new functions.

Effects of GABA on the polyphenol accumulation and antioxidant activities in tea plants (Camellia sinensis L.) under heat-stress conditions.

Polyphenols are important active components in tea plants, which have strong biological activity and antioxidant activity. A certain degree of stress or exogenous substances can significantly increase the content of polyphenols in plants. γ-Aminobutyric acid (GABA), a natural functional amino acid, was used to study whether exogenous GABA can increase the content of polyphenols and enhance antioxidant activity in tea plants under heat-stress conditions. The results showed that the content of GABA was positively correlated with the content of polyphenols (r = 0.649), especially with the content of total catechins (r = 0.837). Most of the related genes encoding flavonoid metabolism (PAL, C4H, 4CL, CHS, CHI, F3H, F3'H, F3'5'H, DFR, LAR, ANS, ANR and FLS) as well as enzyme activities (PAL, C4H and 4CL) were upregulated. In addition, the activities of antioxidant enzymes were induced under heat-stress conditions. However, 3-mercaptopropionic acid (3-MPA), an inhibitor of GABA synthesis, exhibited opposite results under heat-stress conditions compared with GABA treatment. These results indicated that GABA plays a key role in the accumulation of polyphenols and the upregulation of the antioxidant system in tea plants under heat-stress conditions.

GABA shunt contribution to flavonoid biosynthesis and metabolism in tea plants (Camellia sinensis).

γ-Aminobutyric acid (GABA), a signal molecule, is regarded as the intersection node of carbon and nitrogen metabolism, and its contributions to flavonoid metabolism in tea plant growth and development remain unclear. The correlation between the GABA shunt and flavonoid metabolism in tea plants is worth to explore. Secondary metabolites and their correlations with the taste of tea soup made from tea plants (Camellia sinensis) during different seasons were investigated. Related secondary metabolites and transcript profiles of genes encoding enzymes in the GABA shunt, flavonoid pathway and polyamine biosynthesis were measured throughout the tea plant growth seasons and after exogenous GABA applications. In addition, the abundance of differentially expressed proteins was quantified after treatments with or without exogenous GABA. The tea leaves showed the highest metabolite concentrations in spring season. CsGAD, CsGABAT, CsSPMS, CsODC, CsF3H and CsCHS were found to be important genes in the GABA and anthocyanin biosynthesis pathways. GABA and anthocyanin concentrations showed a positive correlation, to some extent, CsF3H and CsCHS played important roles in the GABA and anthocyanin biosynthesis.

Effects of nitric oxide on the GABA, polyamines, and proline in tea (Camellia sinensis) roots under cold stress.

Tea plant often suffers from low temperature induced damage during its growth. How to improve the cold resistance of tea plant is an urgent problem to be solved. Nitric oxide (NO), γ-aminobutyric acid (GABA) and proline have been proved that can improve the cold resistance of tea plants, and signal transfer and biosynthesis link between them may enhance their function. NO is an important gas signal material in plant growth, but our understanding of the effects of NO on the GABA shunt, proline and NO biosynthesis are limited. In this study, the tea roots were treated with a NO donor (SNAP), NO scavenger (PTIO), and NO synthase inhibitor (L-NNA). SNAP could improve activities of arginine decarboxylase, ornithine decarboxylase, glutamate decarboxylase, GABA transaminase and Δ1-pyrroline-5-carboxylate synthetase and the expression level of related genes during the treatments. The contents of putrescine and spermidine under SNAP treatment were 45.3% and 37.3% higher compared to control at 24 h, and the spermine content under PTIO treatment were 57.6% lower compare to control at 12 h. Accumulation of proline of SNAP and L-NNA treatments was 52.2% and 43.2% higher than control at 48 h, indicating other pathway of NO biosynthesis in tea roots. In addition, the NO accelerated the consumption of GABA during cold storage. These facts indicate that NO enhanced the cold tolerance of tea, which might regulate the metabolism of the GABA shunt and of proline, associated with NO biosynthesis.

Transcriptomic and metabolomic profiling of Camellia sinensis L. cv. 'Suchazao' exposed to temperature stresses reveals modification in protein synthesis and photosynthetic and anthocyanin biosynthetic pathways.

To determine the mechanisms in tea plants responding to temperature stresses (heat and cold), we examined the global transcriptomic and metabolomic profiles of the tea plant cultivar 'Suchazao' under moderately low temperature stress (ML), severely low temperature stress (SL), moderately high temperature stress (MH) and severely high temperature stress (SH) using RNA-seq and high performance liquid chromatography tandem mass spectrometry/mass spectrometry (HPLC-MS/MS), respectively. The identified differentially expressed genes indicated that the synthesis of stress-resistance protein might be redirected to cope with the temperature stresses. We found that heat shock protein genes Hsp90 and Hsp70 played more critical roles in tea plants in adapting to thermal stress than cold, while late embryogenesis abundant protein genes (LEA) played a greater role under cold than heat stress, more types of zinc finger genes were induced under cold stress as well. In addition, energy metabolisms were inhibited by SH, SL and ML. Furthermore, the mechanisms of anthocyanin synthesis were different under the cold and heat stresses. Indeed, the CsUGT75C1 gene, encoding UDP-glucose:anthocyanin 5-O-glucosyl transferase, was up-regulated in the SL-treated leaves but down-regulated in SH. Metabolomics analysis also showed that anthocyanin monomer levels increased under SL. These results indicate that the tea plants share certain foundational mechanisms to adjust to both cold and heat stresses. They also developed some specific mechanisms for surviving the cold or heat stresses. Our study provides effective information about the different mechanisms tea plants employ in surviving cold and heat stresses, as well as the different mechanisms of anthocyanin synthesis, which could speed up the genetic breeding of heat- and cold-tolerant tea varieties.

γ-Aminobutyric Acid (GABA) Accumulation in Tea (Camellia sinensis L.) through the GABA Shunt and Polyamine Degradation Pathways under Anoxia.

γ-Aminobutyric acid (GABA) is an important bioactive component of tea (Camellia sinensis) providing various health benefits. We studied GABA accumulation via the GABA shunt and polyamine degradation pathways under anoxia in tea leaves. Anoxia caused a ∼20-fold increment in GABA concentration, relative to fresh tea leaves. This increment was due to the increase of glutamate decarboxylase and diamine oxidase activities. Genes involved in GABA formation, such as CsGAD1 and CsGAD2, were significantly up-regulated by anoxia. The concentrations of putrescine and spermine, two substrates for GABA production, were also increased by anoxia. Treating tea leaves with aminoguanidine completely inhibited diamine oxidase activity during anoxia, but the concentration of GABA decreased by only ∼25%. We infer that about one-fourth of GABA formed in tea leaves under anoxia comes from the polyamine degradation pathway, opening the possibility of producing GABA tea based through the regulation of metabolism.