The glycosyltransferase UGT76B1 modulates N-hydroxy-pipecolic acid homeostasis and plant immunity

The tradeoff between growth and defense is a critical aspect of plant immunity. Therefore, the plant immune response needs to be tightly regulated. Salicylic acid (SA) is an important plant hormone regulating defense against biotrophic pathogens. Recently, N-hydroxy-pipecolic acid (NHP) was identified as another regulator for plant innate immunity and systemic acquired resistance (SAR). Although the biosynthetic pathway leading to NHP formation is already been identified, how NHP is further metabolized is unclear. Here, we present UGT76B1 as a uridine diphosphate-dependent glycosyltransferase (UGT) that modifies NHP by catalyzing the formation of 1-O-glucosyl-pipecolic acid in Arabidopsis thaliana. Analysis of T-DNA and clustered regularly interspaced short palindromic repeats (CRISPR) knock-out mutant lines of UGT76B1 by targeted and nontargeted ultra-high performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS) underlined NHP and SA as endogenous substrates of this enzyme in response to Pseudomonas infection and UV treatment. ugt76b1 mutant plants have a dwarf phenotype and constitutive defense response which can be suppressed by loss of function of the NHP biosynthetic enzyme FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1). This suggests that elevated accumulation of NHP contributes to the enhanced disease resistance in ugt76b1. Externally applied NHP can move to distal tissue in ugt76b1 mutant plants. Although glycosylation is not required for the long-distance movement of NHP during SAR, it is crucial to balance growth and defense.     

Arabidopsis UGT76B1 glycosylates N-hydroxy-pipecolic acid and inactivates systemic acquired resistance in tomato

Systemic acquired resistance (SAR) is a mechanism that plants utilize to connect a local pathogen infection to global defense responses. N-hydroxy-pipecolic acid (NHP) and a glycosylated derivative are produced during SAR, yet their individual roles in this process are currently unclear. Here, we report that Arabidopsis thaliana UGT76B1 generated glycosylated NHP (NHP-Glc) in vitro and when transiently expressed alongside Arabidopsis NHP biosynthetic genes in two Solanaceous plants. During infection, Arabidopsis ugt76b1 mutants did not accumulate NHP-Glc and accumulated less glycosylated salicylic acid (SA-Glc) than wild-type plants. The metabolic changes in ugt76b1 plants were accompanied by enhanced defense to the bacterial pathogen Pseudomonas syringae, suggesting that glycosylation of the SAR molecules NHP and salicylic acid by UGT76B1 plays an important role in modulating defense responses. Transient expression of Arabidopsis UGT76B1 with the Arabidopsis NHP biosynthesis genes ALD1 and FMO1 in tomato (Solanum lycopersicum) increased NHP-Glc production and reduced NHP accumulation in local tissue and abolished the systemic resistance seen when expressing NHP-biosynthetic genes alone. These findings reveal that the glycosylation of NHP by UGT76B1 alters defense priming in systemic tissue and provide further evidence for the role of the NHP aglycone as the active metabolite in SAR signaling.

The Arabidopsis UDP-glycosyltransferase UGT76B1 glycosylates the systemic acquired resistance-signaling metabolite NHP and can inactivate systemic defense responses when expressed in tomato.     

Enzymatic Biosynthesis of Novel Resveratrol Glucoside and Glycoside Derivatives

A UDP glucosyltransferase from Bacillus licheniformis was overexpressed, purified, and incubated with nucleotide diphosphate (NDP) d- and l-sugars to produce glucose, galactose, 2-deoxyglucose, viosamine, rhamnose, and fucose sugar-conjugated resveratrol glycosides. Significantly higher (90%) bioconversion of resveratrol was achieved with α-d-glucose as the sugar donor to produce four different glucosides of resveratrol: resveratrol 3-O-β-d-glucoside, resveratrol 4′-O-β-d-glucoside, resveratrol 3,5-O-β-d-diglucoside, and resveratrol 3,5,4′-O-β-d-triglucoside. The conversion rates and numbers of products formed were found to vary with the other NDP sugar donors. Resveratrol 3-O-β-d-2-deoxyglucoside and resveratrol 3,5-O-β-d-di-2-deoxyglucoside were found to be produced using TDP-2-deoxyglucose as a donor; however, the monoglycosides resveratrol 4′-O-β-d-galactoside, resveratrol 4′-O-β-d-viosaminoside, resveratrol 3-O-β-l-rhamnoside, and resveratrol 3-O-β-l-fucoside were produced from the respective sugar donors. Altogether, 10 diverse glycoside derivatives of the medically important resveratrol were generated, demonstrating the capacity of YjiC to produce structurally diverse resveratrol glycosides.     

Molecular genetic analysis of tandemly located glycosyltransferase genes,UGT73B1,UGT73B2, andUGT73B3, inArabidopsis thaliana

In theArabidopsis genome, approximately 120 UDP-glycosyltransferases (UGTs) have been annotated. They generally catalyze the transfer of sugars to various acceptor molecules, including flavonoids. To better understand their physiological roles, we analyzed a tandemly located putative flavonoid UGT cluster comprisingUGT73B1, UGT73B2, andUGT73B3 on Chromosome IV. We then isolated four loss-of-function mutations —ugt73b1- 1, ugt73b2- 1, ugt73b3- 1, andugt73b3- 2. In our expression analysis, the closely related UCTs exhibited tissue-specific patterns of expression that were severely altered in their respective mutant plants. For example,UGT73B2 was up-regulated inugt73b1- 1, whereasUGT73B7 was highly expressed inugt73b2- 1, ugt73b3- t, andugt73b3- 2. Interestingly, each recessive mutant was resistant to methyl viologen (paraquat), an herbicide thought to cause oxidative stress. Our results suggest thatUGTs play an important role in the glycosylation pathways when responding to oxidative stress.     

The secondary metabolism glycosyltransferases UGT73B3 and UGT73B5 are components of redox status in resistance of Arabidopsis to Pseudomonas syringae pv. tomato

Secondary metabolism plant glycosyltransferases (UGTs) ensure conjugation of sugar moieties to secondary metabolites (SMs) and glycosylation contributes to the great diversity, reactivity and regulation of SMs. UGT73B3 and UGT73B5, two UGTs of Arabidopsis thaliana (Arabidopsis), are involved in the hypersensitive response (HR) to the avirulent bacteria Pseudomonas syringae pv. tomato (Pst‐AvrRpm1), but their function in planta is unknown. Here, we report that ugt73b3, ugt73b5 and ugt73b3 ugt73b5 T‐DNA insertion mutants exhibited an accumulation of reactive oxygen species (ROS), an enhanced cell death during the HR to Pst‐AvrRpm1, whereas glutathione levels increased in the single mutants. In silico analyses indicate that UGT73B3 and UGT73B5 belong to the early salicylic acid (SA)‐induced genes whose pathogen‐induced expression is co‐regulated with genes related to cellular redox homeostasis and general detoxification. Analyses of metabolic alterations in ugt mutants reveal modification of SA and scopoletin contents which correlate with redox perturbation, and indicate quantitative modifications in the pattern of tryptophan‐derived SM accumulation after Pst‐AvrRpm1 inoculation. Our data suggest that UGT73B3 and UGT73B5 participate in regulation of redox status and general detoxification of ROS‐reactive SMs during the HR to Pst‐AvrRpm1, and that decreased resistance to Pst‐AvrRpm1 in ugt mutants is tightly linked to redox perturbation.     

The Arabidopsis glucosyltransferase UGT76B1 conjugates isoleucic acid and modulates plant defense and senescence

Plants coordinate and tightly regulate pathogen defense by the mostly antagonistic salicylate (SA)- and jasmonate (JA)-mediated signaling pathways. Here, we show that the previously uncharacterized glucosyltransferase UGT76B1 is a novel player in this SA-JA signaling crosstalk. UGT76B1 was selected as the top stress-induced isoform among all 122 members of the Arabidopsis thaliana UGT family. Loss of UGT76B1 function leads to enhanced resistance to the biotrophic pathogen Pseudomonas syringae and accelerated senescence but increased susceptibility toward necrotrophic Alternaria brassicicola. This is accompanied by constitutively elevated SA levels and SA-related marker gene expression, whereas JA-dependent markers are repressed. Conversely, UGT76B1 overexpression has the opposite effect. Thus, UGT76B1 attenuates SA-dependent plant defense in the absence of infection, promotes the JA response, and delays senescence. The ugt76b1 phenotypes were SA dependent, whereas UGT76B1 overexpression indicated that this gene possibly also has a direct effect on the JA pathway. Nontargeted metabolomic analysis of UGT76B1 knockout and overexpression lines using ultra-high-resolution mass spectrometry and activity assays with the recombinant enzyme led to the ab initio identification of isoleucic acid (2-hydroxy-3-methyl-pentanoic acid) as a substrate of UGT76B1. Exogenously applied isoleucic acid increased resistance against P. syringae infection. These findings indicate a novel link between amino acid-related molecules and plant defense that is mediated by small-molecule glucosylation.     

A flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase responsible for terminal modification of pollen‐specific flavonols in Arabidopsis thaliana

Flavonol 3‐O‐diglucosides with a 1→2 inter‐glycosidic linkage are representative pollen‐specific flavonols that are widely distributed in plants, but their biosynthetic genes and physiological roles are not well understood. Flavonoid analysis of four Arabidopsis floral organs (pistils, stamens, petals and calyxes) and flowers of wild‐type and male sterility 1 (ms1) mutants, which are defective in normal development of pollen and tapetum, showed that kaempferol/quercetin 3‐O‐β‐d ‐glucopyranosyl‐(1→2)‐β‐d ‐glucopyranosides accumulated in Arabidopsis pollen. Microarray data using wild‐type and ms1 mutants, gene expression patterns in various organs, and phylogenetic analysis of UDP‐glycosyltransferases (UGTs) suggest that UGT79B6 (At5g54010) is a key modification enzyme for determining pollen‐specific flavonol structure. Kaempferol and quercetin 3‐O‐glucosyl‐(1→2)‐glucosides were absent from two independent ugt79b6 knockout mutants. Transgenic ugt79b6 mutant lines transformed with the genomic UGT79B6 gene had the same flavonoid profile as wild‐type plants. Recombinant UGT79B6 protein converted kaempferol 3‐O‐glucoside to kaempferol 3‐O‐glucosyl‐(1→2)‐glucoside. UGT79B6 recognized 3‐O‐glucosylated/galactosylated anthocyanins/flavonols but not 3,5‐ or 3,7‐diglycosylated flavonoids, and prefers UDP‐glucose, indicating that UGT79B6 encodes flavonoid 3‐O‐glucoside:2″‐O‐glucosyltransferase. A UGT79B6‐GUS fusion showed that UGT79B6 was localized in tapetum cells and microspores of developing anthers.     

A Versatile Family 3 Glycoside Hydrolase from Bifidobacterium adolescentis Hydrolyzes β-Glucosides of the Fusarium Mycotoxins Deoxynivalenol,Nivalenol, and HT-2 Toxin in Cereal Matrices

Glycosylation plays a central role in plant defense against xenobiotics, including mycotoxins. Glucoconjugates of Fusarium toxins, such as deoxynivalenol-3-O-β-d-glucoside (DON-3G), often cooccur with their parental toxins in cereal-based food and feed. To date, only limited information exists on the occurrence of glucosylated mycotoxins and their toxicological relevance. Due to a lack of analytical standards and the requirement of high-end analytical instrumentation for their direct determination, hydrolytic cleavage of β-glucosides followed by analysis of the released parental toxins has been proposed as an indirect determination approach. This study compares the abilities of several fungal and recombinant bacterial β-glucosidases to hydrolyze the model analyte DON-3G. Furthermore, substrate specificities of two fungal and two bacterial (Lactobacillus brevis and Bifidobacterium adolescentis) glycoside hydrolase family 3 β-glucosidases were evaluated on a broader range of substrates. The purified recombinant enzyme from B. adolescentis (BaBgl) displayed high flexibility in substrate specificity and exerted the highest hydrolytic activity toward 3-O-β-d-glucosides of the trichothecenes deoxynivalenol (DON), nivalenol, and HT-2 toxin. A K_m of 5.4 mM and a V_max of 16 μmol min⁻¹ mg⁻¹ were determined with DON-3G. Due to low product inhibition (DON and glucose) and sufficient activity in several extracts of cereal matrices, this enzyme has the potential to be used for indirect analyses of trichothecene-β-glucosides in cereal samples.     

The Arabidopsis UDP‐glycosyltransferases UGT79B2 and UGT79B3, contribute to cold,salt and drought stress tolerance via modulating anthocyanin accumulation

The plant family 1 UDP‐glycosyltransferases (UGTs) are the biggest GT family in plants, which are responsible for transferring sugar moieties onto a variety of small molecules, and control many metabolic processes; however, their physiological significance in planta is largely unknown. Here, we revealed that two Arabidopsis glycosyltransferase genes, UGT79B2 and UGT79B3, could be strongly induced by various abiotic stresses, including cold, salt and drought stresses. Overexpression of UGT79B2/B3 significantly enhanced plant tolerance to low temperatures as well as drought and salt stresses, whereas the ugt79b2/b3 double mutants generated by RNAi (RNA interference) and CRISPR‐Cas9 strategies were more susceptible to adverse conditions. Interestingly, the expression of UGT79B2 and UGT79B3 is directly controlled by CBF1 (CRT/DRE‐binding factor 1, also named DREB1B) in response to low temperatures. Furthermore, we identified the enzyme activities of UGT79B2/B3 in adding UDP‐rhamnose to cyanidin and cyanidin 3‐O‐glucoside. Ectopic expression of UGT79B2/B3 significantly increased the anthocyanin accumulation, and enhanced the antioxidant activity in coping with abiotic stresses, whereas the ugt79b2/b3 double mutants showed reduced anthocyanin levels. When overexpressing UGT79B2/B3 in tt18 (transparent testa 18), a mutant that cannot synthesize anthocyanins, both genes fail to improve plant adaptation to stress. Taken together, we demonstrate that UGT79B2 and UGT79B3, identified as anthocyanin rhamnosyltransferases, are regulated by CBF1 and confer abiotic stress tolerance via modulating anthocyanin accumulation.     

beta-Furfuryl-beta-Glucoside: An Endogenous Activator of Higher Plant UDP-Glucose:(1-3)-beta-Glucan Synthase : Biological Activity, Distribution, and in Vitro Synthesis

In a recent paper (P Ohana, DP Delmer, JC Steffens, DE Matthews, R Mayer, M Benziman [1991] J Biol Chem 266: 13472-13475), we described the purification and structural characterization of β-furfuryl-β-glucoside (FG), an endogenous activator of plant UDP-glucose:(1→3)-β-glucan (callose) synthase. In the present report, we provide evidence that FG specifically stimulates callose synthase. The effects of FG on the kinetic properties of callose synthase were studied, and we ascertained that FG, or at least a very similar compound, is present in other plant systems. Chemically synthesized α-furfuryl-β-glucoside also stimulates callose synthase, exhibiting a slightly higher K_a of 80 micromolar, compared with 50 micromolar for FG. In addition, we have identified and partially characterized an enzyme that catalyzes the synthesis of FG using β-furfuryl alcohol and UDP-glucose as substrates. A model for the regulation of callose synthesis in vivo, involving changes in intracellular compartmentation of FG and Ca²⁺, is proposed.     

Genetic Determination of the Constitutive Biosynthesis of Phospho-β-Glucosidase A in Escherichia coli K-12

Escherichia coli wild-type cells form constitutively the enzyme phospho-β-glucosidase A, which has a high affinity for phosphorylated aromatic β-glucosides and a low affinity for phosphorylated β-methyl-glucoside. Phospho-β-glucosidase B and β-glucoside permease I are formed in aromatic β-glucoside-fermenting mutants. Mutants lacking phospho-β-glucosidases A and B have been isolated. These mutants showed a reduced rate of inducibility of the β-glucoside permease I. The restoration of phospho-β-glucosidase A or B activity resulted in an increased rate of induction of the β-glucoside permease I. The presence of the phospho-β-glucosidases was not required for the constitutive biosynthesis of the β-glucoside permease. Mutants selected for growth on β-methyl-glucoside as carbon source showed an increased level of constitutive phospho-β-glucosidase A activity. Gene bglD, the structural gene for phospho-β-glucosidase A, was mapped between the pyrE locus and the cluster bgl loci, whereas bglE, the regulatory site determining the hyperproduction of phospho-β-glucosidase A, was mapped between the bgl and ilv clusters. The bglE locus appears to have a regulatory effect on the expression of the bglD gene.     

High Mobility Group Box-1 Promotes Inflammation-Induced Lymphangiogenesis via Toll-Like Receptor 4-Dependent Signalling Pathway

Lymphangiogenesis in inflammation has received considerable attention in recent years. Administration of modulating lymphangiogenesis provides more possibilities of treating inflammation-associated diseases. However, the main mediators and factors governing inflammation-induced lymphangiogenesis (ILA) are yet to be defined. Here, we explored the role of HMGB1-TLR4 signalling pathway in modulating inflammation-induced lymphangiogenesis and its underlying mechanisms using an ILA mouse model and 2 cell lines. Our results show that HMGB1 promoted VEGF-C-induced HDLECs proliferation in a dose-dependent manner and TLR4 mediates HMGB1-induced LECs proliferation and tube formation in vitro. And in vivo, rHMGB1 treatment significantly promoted ILA, and the promoting effects was inhibited notably when HMGB1-TLR4 was blocked. HMGB1-associated ILA is primarily dependent on TLR4 but not on TLR2. In mechanisms, the recruitment and activation of CD11b+ cells are important cellular mechanisms in HMGB1-TLR4 associated ILA, and multiple key pro-lymphangiogenesis molecules mediates HMGB1-TLR4 associated ILA, including VEGF-C/VEGFR3, inflammatory factors IL-1β and TNF-α, MMP-2 and MMP-9 and NF-κB p65. In conclusion, HMGB1-associated ILA is primarily dependent on TLR4, and CD11b+ cells and multiple molecular mechanisms mediate HMGB1-TLR4 associated ILA. Furthermore, the ILA can be effectively modulated by HMGB1-TLR4 signalling. Consequently, administration of modulating ILA through HMGB1-TLR4 pathway may provide us more possibilities of treating inflammation and lymphangiogenesis associated diseases.     

Purification and Substrate Specificities of Two α-l-Arabinofuranosidases from Aspergillus awamori IFO 4033

α-l-Arabinofuranosidases I and II were purified from the culture filtrate of Aspergillus awamori IFO 4033 and had molecular weights of 81,000 and 62,000 and pIs of 3.3 and 3.6, respectively. Both enzymes had an optimum pH of 4.0 and an optimum temperature of 60°C and exhibited stability at pH values from 3 to 7 and at temperatures up to 60°C. The enzymes released arabinose from p-nitrophenyl-α-l-arabinofuranoside, O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose, and arabinose-containing polysaccharides but not from O-β-d-xylopyranosyl-(1→2)-O-α-l-arabinofuranosyl-(1→3)-O-β-d-xylopyranosyl-(1→4)-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. α-l-Arabinofuranosidase I also released arabinose from O-β-d-xylopy-ranosyl-(1→4)-[O-α-l-arabinofuranosyl-(1→3)]-O-β-d-xylopyranosyl-(1→4)-d-xylopyranose. However, α-l-arabinofuranosidase II did not readily catalyze this hydrolysis reaction. α-l-Arabinofuranosidase I hydrolyzed all linkages that can occur between two α-l-arabinofuranosyl residues in the following order: (1→5) linkage > (1→3) linkage > (1→2) linkage. α-l-Arabinofuranosidase II hydrolyzed the linkages in the following order: (1→5) linkage > (1→2) linkage > (1→3) linkage. α-l-Arabinofuranosidase I preferentially hydrolyzed the (1→5) linkage of branched arabinotrisaccharide. On the other hand, α-l-arabinofuranosidase II preferentially hydrolyzed the (1→3) linkage in the same substrate. α-l-Arabinofuranosidase I released arabinose from the nonreducing terminus of arabinan, whereas α-l-arabinofuranosidase II preferentially hydrolyzed the arabinosyl side chain linkage of arabinan.Recently, it has been proven that l-arabinose selectively inhibits intestinal sucrase in a noncompetitive manner and reduces the glycemic response after sucrose ingestion in animals (33). Based on this observation, l-arabinose can be used as a physiologically functional sugar that inhibits sucrose digestion. Effective l-arabinose production is therefore important in the food industry. l-Arabinosyl residues are widely distributed in hemicelluloses, such as arabinan, arabinoxylan, gum arabic, and arabinogalactan, and the α-l-arabinofuranosidases (α-l-AFases) (EC 3.2.1.55) have proven to be essential tools for enzymatic degradation of hemicelluloses and structural studies of these compounds.α-l-AFases have been classified into two families of glycanases (families 51 and 54) on the basis of amino acid sequence similarities (11). The two families of α-l-AFases also differ in substrate specificity for arabinose-containing polysaccharides. Beldman et al. summarized the α-l-AFase classification based on substrate specificities (3). One group contains the Arafur A (family 51) enzymes, which exhibit very little or no activity with arabinose-containing polysaccharides. The other group contains the Arafur B (family 54) enzymes, which cleave arabinosyl side chains from polymers. However, this classification is too broad to define the substrate specificities of α-l-AFases. There have been many studies of the α-l-AFases (3, 12), especially the α-l-AFases of Aspergillus species (2–8, 12–15, 17, 22, 23, 28–32, 36–39, 41–43, 46). However, there have been only a few studies of the precise specificities of these α-l-AFases. In previous work, we elucidated the substrate specificities of α-l-AFases from Aspergillus niger 5-16 (17) and Bacillus subtilis 3-6 (16, 18), which should be classified in the Arafur A group and exhibit activity with arabinoxylooligosaccharides, synthetic methyl 2-O-, 3-O-, and 5-O-arabinofuranosyl-α-l-arabinofuranosides (arabinofuranobiosides) (20), and methyl 3,5-di-O-α-l-arabinofuranosyl-α-l-arabinofuranoside (arabinofuranotrioside) (19).In the present work, we purified two α-l-AFases from a culture filtrate of Aspergillus awamori IFO 4033 and determined the substrate specificities of these α-l-AFases by using arabinose-containing polysaccharides and the core oligosaccharides of arabinoxylan and arabinan.     

Structural Characterization of Lignin during Pinus taeda Wood Treatment with Ceriporiopsis subvermispora

Pinus taeda wood chips were biotreated with Ceriporiopsis subvermispora under solid-state fermentation for periods varying from 15 to 90 days. Milled wood lignins extracted from sound and biotreated wood samples were characterized by wet-chemical and spectroscopic techniques. Treatment of the lignins by derivatization followed by reductive cleavage (DFRC) made it possible to detect DFRC monomers and dimers that are diagnostic of the occurrence of arylglycerol-β-O-aryl and β-β, β-5, β-1, and 4-O-5 units in the lignin structure. Quantification of these DFRC products indicated that β-O-aryl cleavage was a significant route for lignin biodegradation but that β-β, β-5, β-1, and 4-O-5 linkages were more resistant to the biological attack. The amount of aromatic hydroxyls did not increase with the split of β-O-4 linkages, suggesting that the β-O-4 cleavage products remain as quinone-type structures as detected by UV and visible spectroscopy. Nuclear magnetic resonance techniques also indicated the formation of new substructures containing nonoxygenated, saturated aliphatic carbons (CH₂ and CH₃) in the side chains of lignins extracted from biotreated wood samples.     

The isolation of oligosaccharides from the cell-wall polysaccharide of Lactobacillus casei, serological group C

1. A number of disaccharides and oligosaccharides have been isolated from the products of mild acid hydrolysis of the specific substance from Lactobacillus casei, serological group C. 2. The major disaccharide is O-β-d-glucopyranosyl-(1→3)-N-acetyl- d-galactosamine (B4) and evidence is presented for the structure of a tetrasaccharide composed of O-β-d-glucopyranosyl-(1→6)-d-galactose (B1) joined through its reducing end group to B4. 3. Disaccharide B1 is also a component of a trisaccharide O-β-d-glucopyranosyl-(1→6)-O-β- d-galactopyranosyl-(1→6)-N-acetyl-d-glucosamine (A7). 4. A number of other oligosaccharides have been shown to be related structurally. 5. The ability of certain of the oligosaccharides to inhibit the precipitin reaction has been studied. The disaccharide B1 is more effective as an inhibitor than gentiobiose and the trisaccharide A7 is considerably more effective than B1. 6. These results have been compared with those obtained previously for the composition of the cell wall.     

N-glucosyltransferase UGT76C2 is involved in cytokinin homeostasis and cytokinin response in Arabidopsis thaliana

Cytokinins are a class of phytohormones that play a crucial role in plant growth and development. The gene UGT76C2 encoding cytokinin N-glucosyltransferase of Arabidopsis thaliana has been previously identified. To determine the in planta role of UGT76C2 in cytokinin metabolism and response, we analyzed the phenotypes of its loss-of-function mutant (ugt76c2) and its overexpressors. The accumulation level of the cytokinin N-glucosides was significantly decreased in ugt76c2, but substantially increased in UGT76C2 overexpressors compared with the wild type. When treated with exogenously applied cytokinin, ugt76c2 showed more sensitivity and UGT76C2 overexpressors showed less sensitivity to cytokinin in primary root elongation, lateral root formation, Chl retention and anthocyanin accumulation. Under normal growth conditions ugt76c2 had smaller seeds than the wild type, with accompanying lowered levels of active and N-glucosylated cytokinin forms. The expression levels of cytokinin-related genes such as AHK2, AHK3, ARR1, IPT5 and CKX3 were changed in ugt76c2, suggesting homeostatic control of cytokinin activity. Studies of spatiotemporal expression patterns showed that UGT76C2 was expressed at a relatively higher level in the seedling and developing seed. In their entirety, our data, based mainly on this comparison and opposite phenotypes of knockout and overexpressors, strongly suggest that UGT76C2 is involved in cytokinin homeostasis and cytokinin response in planta through cytokinin N-glucosylation.