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Differences in salicylic acid glucose conjugations by UGT74F1 and UGT74F2 from Arabidopsis thaliana.

ABSTRACT: Salicylic acid (SA) is a signaling molecule utilized by plants in response to various stresses. Through conjugation with small organic molecules such as glucose, an inactive form of SA is generated which can be transported into and stored in plant vacuoles. In the model organism Arabidopsis thaliana, SA glucose conjugates are formed by two homologous enzymes (UGT74F1 and UGT74F2) that transfer glucose from UDP-glucose to SA. Despite being 77% identical and with conserved active site residues, these enzymes catalyze the formation of different products: UGT74F1 forms salicylic acid glucoside (SAG), while UGT74F2 forms primarily salicylic acid glucose ester (SGE). The position of the glucose on the aglycone determines how SA is stored, further metabolized, and contributes to a defense response. We determined the crystal structures of the UGT74F2 wild-type and T15S mutant enzymes, in different substrate/product complexes. On the basis of the crystal structures and the effect on enzyme activity of mutations in the SA binding site, we propose the catalytic mechanism of SGE and SAG formation and that SA binds to the active site in two conformations, with each enzyme selecting a certain binding mode of SA. Additionally, we show that two threonines are key determinants of product specificity.

SUBMITTER: George Thompson AM

PROVIDER: S-EPMC5397973 | biostudies-literature | 2017 Apr

REPOSITORIES: biostudies-literature

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Differences in salicylic acid glucose conjugations by UGT74F1 and UGT74F2 from Arabidopsis thaliana.

George Thompson Alayna M AM Iancu Cristina V CV Neet Kenneth E KE Dean John V JV Choe Jun-Yong JY

Scientific reports 20170420

Salicylic acid (SA) is a signaling molecule utilized by plants in response to various stresses. Through conjugation with small organic molecules such as glucose, an inactive form of SA is generated which can be transported into and stored in plant vacuoles. In the model organism Arabidopsis thaliana, SA glucose conjugates are formed by two homologous enzymes (UGT74F1 and UGT74F2) that transfer glucose from UDP-glucose to SA. Despite being 77% identical and with conserved active site residues, th ...[more]

PMID: 28425481

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Project description:The nucleosome positioning regulates the gene expression and many other DNA-related processes in eukaryotes. Genome-wide mapping of nucleosome positions and correlation of genome-wide nucleosomal remodeling with the changes in the gene expression can help us understanding gene regulation on genome level.In the present study, we correlate the gene expression and the genomic nucleosomal remodeling in response to salicylic acid (SA) treatment in A. thaliana. We have mapped genome-wide nucleosomes by performing tiling microarray using 146 bp mononucleosomal template DNA. The average nucleosomal coverage is approximately 346 bp per nucleosome both under the control and the SA-treated conditions. The nucleosomal coverage is more in the coding region than in the 5' regulatory regions. We observe approximately 50% nucleosomal remodeling on SA treatment where significant nucleosomal depletion and nucleosomal enrichment around the transcription start site (TSS) occur in SA induced genes and SA repressed genes respectively in response to SA treatment. Especially in the case of the SA-induced group, the nucleosomal remodeling over the minimal promoter in response to SA is especially significant in the Non-expresser of PR1 (NPR1)-dependent genes. A detailed investigation of npr1-1 mutant confirms a distinct role of NPR1 in the nucleosome remodeling over the core promoter. We have also identified several motifs for various hormonal responses; including ABRE elements in the remodeled nucleosomal regions around the promoter region in the SA regulated genes. We have further identified that the W-box and TGACG/C motif, reported to play an important role in SA-mediated induction, are enriched in nucleosome free regions (NFRs) of the promoter region of the SA induced genes.This is the first study reporting genome-wide effects of SA treatment on the chromatin architecture of A. thaliana. It also reports significant role of NPR1 in genome-wide nucleosomal remodeling in response to SA.

Project description:Salicylic acid (SA) has an essential role in the responses of plants to pathogens. SA initiates defence signalling via binding to proteins. NPR1 is a transcriptional co-activator and a key target of SA binding. Many other proteins have recently been shown to bind SA. Amongst these proteins are important enzymes of primary metabolism. This fact could stand behind SA's ability to control energy fluxes in stressed plants. Nevertheless, only sparse information exists on the role and mechanisms of such binding. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was previously demonstrated to bind SA both in human and plants. Here, we detail that the A1 isomer of chloroplastic glyceraldehyde 3-phosphate dehydrogenase (GAPA1) from Arabidopsis thaliana binds SA with a KD of 16.7 nM, as shown in surface plasmon resonance experiments. Besides, we show that SA inhibits its GAPDH activity in vitro. To gain some insight into the underlying molecular interactions and binding mechanism, we combined in silico molecular docking experiments and molecular dynamics simulations on the free protein and protein-ligand complex. The molecular docking analysis yielded to the identification of two putative binding pockets for SA. A simulation in water of the complex between SA and the protein allowed us to determine that only one pocket-a surface cavity around Asn35-would efficiently bind SA in the presence of solvent. In silico mutagenesis and simulations of the ligand/protein complexes pointed to the importance of Asn35 and Arg81 in the binding of SA to GAPA1. The importance of this is further supported through experimental biochemical assays. Indeed, mutating GAPA1 Asn35 into Gly or Arg81 into Leu strongly diminished the ability of the enzyme to bind SA. The very same cavity is responsible for the NADP+ binding to GAPA1. More precisely, modelling suggests that SA binds to the very site where the pyrimidine group of the cofactor fits. NADH inhibited in a dose-response manner the binding of SA to GAPA1, validating our data.

Project description:BACKGROUND: Verticillium longisporum is a soil-borne vascular pathogen infecting cruciferous hosts such as oilseed rape. Quantitative disease resistance (QDR) is the major control means, but its molecular basis is poorly understood so far. Quantitative trait locus (QTL) mapping was performed using a new (Bur×Ler) recombinant inbred line (RIL) population of Arabidopsis thaliana. Phytohormone measurements and analyses in defined mutants and near-isogenic lines (NILs) were used to identify genes and signalling pathways that underlie different resistance QTL. RESULTS: QTL for resistance to V. longisporum-induced stunting, systemic colonization by the fungus and for V. longisporum-induced chlorosis were identified. Stunting resistance QTL were contributed by both parents. The strongest stunting resistance QTL was shown to be identical with Erecta. A functional Erecta pathway, which was present in Bur, conferred partial resistance to V. longisporum-induced stunting. Bur showed severe stunting susceptibility in winter. Three stunting resistance QTL of Ler origin, two co-localising with wall-associated kinase-like (Wakl)-genes, were detected in winter. Furthermore, Bur showed a much stronger induction of salicylic acid (SA) by V. longisporum than Ler. Systemic colonization was controlled independently of stunting. The vec1 QTL on chromosome 2 had the strongest effect on systemic colonization. The same chromosomal region controlled the level of abscisic acid (ABA) and jasmonic acid (JA) in response to V. longisporum: The level of ABA was higher in colonization-susceptible Ler than in colonization-resistant Bur after V. longisporum infection. JA was down-regulated in Bur after infection, but not in Ler. These differences were also demonstrated in NILs, varying only in the region containing vec1. All phytohormone responses were shown to be independent of Erecta. CONCLUSIONS: Signalling systems with a hitherto unknown role in the QDR of A. thaliana against V. longisporum were identified: Erecta mediated resistance against V. longisporum-induced stunting. Independent of Erecta, stunting was caused in a light-dependent manner with possible participation of SA and Wakl genes. ABA and JA showed a genotype-specific response that corresponded with systemic colonization by the fungus. Understanding the biological basis of phenotypic variation in A. thaliana with respect to V. longisporum resistance will provide new approaches for implementing durable resistance in cruciferous crops.

Dataset Information

Differences in salicylic acid glucose conjugations by UGT74F1 and UGT74F2 from Arabidopsis thaliana.

Publications

Differences in salicylic acid glucose conjugations by UGT74F1 and UGT74F2 from Arabidopsis thaliana.

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