The glycosyltransferase UGT76E1 significantly contributes to 12-O-glucopyranosyl-jasmonic acid formation in wounded Arabidopsis thaliana leaves

Jasmonoyl-isoleucine (JA-Ile) is a phytohormone that orchestrates plant defenses in response to wounding, feeding insects, or necrotrophic pathogens. JA-Ile metabolism has been studied intensively, but its catabolism as a potentially important mechanism for the regulation of JA-Ile–mediated signaling is not well-understood. Especially the enzyme(s) responsible for specifically glycosylating 12-hydroxy-jasmonic acid (12-OH-JA) and thereby producing 12-O-glucopyranosyl-jasmonic acid (12-O-Glc-JA) is still elusive. Here, we used co-expression analyses of available Arabidopsis thaliana transcriptomic data, identifying four UDP-dependent glycosyltransferase (UGT) genes as wound-induced and 12-OH-JA–related, namely, UGT76E1, UGT76E2, UGT76E11, and UGT76E12. We heterologously expressed and purified the corresponding proteins to determine their individual substrate specificities and kinetic parameters. We then used an ex vivo metabolite-fingerprinting approach to investigate these proteins in conditions as close as possible to their natural environment, with an emphasis on greatly extending the range of potential substrates. As expected, we found that UGT76E1 and UGT76E2 are 12-OH-JA-UGTs, with UGT76E1 contributing a major in vivo UGT activity, as deduced from Arabidopsis mutants with abolished or increased UGT gene expression. In contrast, recombinant UGT76E11 acted on an unidentified compound and also glycosylated two other oxylipins, 11-hydroxy-7,9,13-hexadecatrienoic acid (11-HHT) and 13-hydroxy-9,11,15-octadecatrienoic acid (13-HOT), which were also accepted by recombinant UGT76E1, UGT76E2, and UGT76E12 enzymes. UGT76E12 glycosylated 12-OH-JA only to a low extent, but also accepted an artificial hydroxylated fatty acid and low amounts of kaempferol. In conclusion, our findings have elucidated the missing step in the wound-induced synthesis of 12-O-glucopyranosyl-jasmonic acid in A. thaliana.

[1]  Benjamin G. Davis,et al.  Functional and informatics analysis enables glycosyltransferase activity prediction , 2018, Nature Chemical Biology.

[2]  G. Howe,et al.  Resolution of growth-defense conflict: mechanistic insights from jasmonate signaling. , 2018, Current opinion in plant biology.

[3]  Sudhir Kumar,et al.  MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. , 2018, Molecular biology and evolution.

[4]  C. Wasternack,et al.  The Oxylipin Pathways: Biochemistry and Function. , 2018, Annual review of plant biology.

[5]  Abraham J. Koo.  Metabolism of the plant hormone jasmonate: a sentinel for tissue damage and master regulator of stress response , 2018, Phytochemistry Reviews.

[6]  Z. Shah,et al.  Comparative genomic and transcriptomic analyses of Family-1 UDP glycosyltransferase in three Brassica species and Arabidopsis indicates stress-responsive regulation , 2018, Scientific Reports.

[7]  Kengo Kinoshita,et al.  ATTED-II in 2018: A Plant Coexpression Database Based on Investigation of the Statistical Property of the Mutual Rank Index , 2017, Plant & cell physiology.

[8]  T. Heitz,et al.  Jasmonic Acid Oxidase 2 Hydroxylates Jasmonic Acid and Represses Basal Defense and Resistance Responses against Botrytis cinerea Infection. , 2017, Molecular plant.

[9]  K. Neet,et al.  Differences in salicylic acid glucose conjugations by UGT74F1 and UGT74F2 from Arabidopsis thaliana , 2017, Scientific Reports.

[10]  Kabin Xie,et al.  CRISPR-P 2.0: An Improved CRISPR-Cas9 Tool for Genome Editing in Plants. , 2017, Molecular plant.

[11]  G. Van den Ackerveken,et al.  Arabidopsis JASMONATE-INDUCED OXYGENASES down-regulate plant immunity by hydroxylation and inactivation of the hormone jasmonic acid , 2017, Proceedings of the National Academy of Sciences.

[12]  I. Feussner,et al.  Functional Characterization of CYP94-Genes and Identification of a Novel Jasmonate Catabolite in Flowers , 2016, PloS one.

[13]  Lingling Chen,et al.  Recent Advances in Genome Editing Using CRISPR/Cas9 , 2016, Front. Plant Sci..

[14]  T. Heitz,et al.  Dynamics of Jasmonate Metabolism upon Flowering and across Leaf Stress Responses in Arabidopsis thaliana , 2016, Plants.

[15]  W. Schwab,et al.  Glucosylation of aroma chemicals and hydroxy fatty acids. , 2015, Journal of biotechnology.

[16]  Tong Zhang,et al.  Hormone crosstalk in wound stress response: wound-inducible amidohydrolases can simultaneously regulate jasmonate and auxin homeostasis in Arabidopsis thaliana , 2015, Journal of experimental botany.

[17]  Xuecheng Wang,et al.  Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single generation , 2015, Genome Biology.

[18]  Hidemasa Bono,et al.  CRISPRdirect: software for designing CRISPR/Cas guide RNA with reduced off-target sites , 2014, Bioinform..

[19]  Xuecheng Wang,et al.  A CRISPR/Cas9 toolkit for multiplex genome editing in plants , 2014, BMC Plant Biology.

[20]  Burkhard Morgenstern,et al.  MarVis-Pathway: integrative and exploratory pathway analysis of non-targeted metabolomics data , 2014, Metabolomics.

[21]  A. J. Koo,et al.  Endoplasmic Reticulum-associated Inactivation of the Hormone Jasmonoyl-l-Isoleucine by Multiple Members of the Cytochrome P450 94 Family in Arabidopsis* , 2014, The Journal of Biological Chemistry.

[22]  Yang Lei,et al.  CRISPR-P: a web tool for synthetic single-guide RNA design of CRISPR-system in plants. , 2014, Molecular plant.

[23]  H. Kawaide,et al.  CYP94B3 activity against jasmonic acid amino acid conjugates and the elucidation of 12-O-β-glucopyranosyl-jasmonoyl-L-isoleucine as an additional metabolite. , 2014, Phytochemistry.

[24]  Burkhard Morgenstern,et al.  Meta-Analysis of Pathway Enrichment: Combining Independent and Dependent Omics Data Sets , 2014, PloS one.

[25]  T. Heitz,et al.  The Amidohydrolases IAR3 and ILL6 Contribute to Jasmonoyl-Isoleucine Hormone Turnover and Generate 12-Hydroxyjasmonic Acid Upon Wounding in Arabidopsis Leaves* , 2013, The Journal of Biological Chemistry.

[26]  C. Wasternack,et al.  Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth and development. An update to the 2007 review in Annals of Botany. , 2013, Annals of botany.

[27]  Tim Iven,et al.  Arabidopsis mutants of sphingolipid fatty acid α-hydroxylases accumulate ceramides and salicylates. , 2012, The New phytologist.

[28]  C. Olsen,et al.  UDP-Glycosyltransferases from the UGT73C Subfamily in Barbarea vulgaris Catalyze Sapogenin 3-O-Glucosylation in Saponin-Mediated Insect Resistance1[W][OA] , 2012, Plant Physiology.

[29]  V. Bulone,et al.  Radiometric and spectrophotometric in vitro assays of glycosyltransferases involved in plant cell wall carbohydrate biosynthesis , 2012, Nature Protocols.

[30]  C. Zubieta,et al.  Structural Basis for Prereceptor Modulation of Plant Hormones by GH3 Proteins , 2012, Science.

[31]  D. Werck-Reichhart,et al.  Cytochromes P450 CYP94C1 and CYP94B3 Catalyze Two Successive Oxidation Steps of Plant Hormone Jasmonoyl-isoleucine for Catabolic Turnover , 2012, The Journal of Biological Chemistry.

[32]  P. Schmitt‐Kopplin,et al.  The Arabidopsis Glucosyltransferase UGT76B1 Conjugates Isoleucic Acid and Modulates Plant Defense and Senescence[C][W][OA] , 2011, Plant Cell.

[33]  H. Kawaide,et al.  Arabidopsis CYP94B3 encodes jasmonyl-L-isoleucine 12-hydroxylase, a key enzyme in the oxidative catabolism of jasmonate. , 2011, Plant & cell physiology.

[34]  G. Howe,et al.  Cytochrome P450 CYP94B3 mediates catabolism and inactivation of the plant hormone jasmonoyl-L-isoleucine , 2011, Proceedings of the National Academy of Sciences.

[35]  N. Uozumi,et al.  12-Hydroxyjasmonic Acid Glucoside Is a COI1-JAZ-Independent Activator of Leaf-Closing Movement in Samanea saman1[OA] , 2011, Plant Physiology.

[36]  H. Matsui,et al.  Identification of a beta-glucosidase hydrolyzing tuberonic acid glucoside in rice (Oryza sativa L.). , 2010, Phytochemistry.

[37]  N. Watanabe,et al.  C14-oxylipin glucosides isolated from Lemna paucicostata. , 2010, Phytochemistry.

[38]  M. Reichelt,et al.  Disruption of Adenosine-5′-Phosphosulfate Kinase in Arabidopsis Reduces Levels of Sulfated Secondary Metabolites[W] , 2009, The Plant Cell Online.

[39]  I. Feussner,et al.  On the Substrate Binding of Linoleate 9-Lipoxygenases , 2009, Lipids.

[40]  H. Matsui,et al.  Purification and cDNA cloning of a wound inducible glucosyltransferase active toward 12-hydroxy jasmonic acid. , 2009, Phytochemistry.

[41]  Jesús Vicente-Carbajosa,et al.  DNA-free RNA isolation protocols for Arabidopsis thaliana, including seeds and siliques , 2008, BMC Research Notes.

[42]  P. Green,et al.  Impact of transcriptional, ABA-dependent, and ABA-independent pathways on wounding regulation of RNS1 expression , 2008, Molecular Genetics and Genomics.

[43]  A. Shevchenko,et al.  Lipid extraction by methyl-tert-butyl ether for high-throughput lipidomics. , 2008, Journal of lipid research.

[44]  G. Howe,et al.  Plant immunity to insect herbivores. , 2008, Annual review of plant biology.

[45]  I. Feussner,et al.  Phosphoinositide and inositolpolyphosphate signalling in defense responses of Arabidopsis thaliana challenged by mechanical wounding. , 2008, Molecular plant.

[46]  C. Wasternack,et al.  Hydroxylated jasmonates are commonly occurring metabolites of jasmonic acid and contribute to a partial switch-off in jasmonate signaling. , 2007, The New phytologist.

[47]  M. Nakajima,et al.  Identification and characterization of an Ipomoea nil glucosyltransferase which metabolizes some phytohormones. , 2007, Biochemical and biophysical research communications.

[48]  E. Bornberg-Bauer,et al.  The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses. , 2007, The Plant journal : for cell and molecular biology.

[49]  D. Bowles,et al.  Glycosyltransferases of lipophilic small molecules. , 2006, Annual review of plant biology.

[50]  C. Ford,et al.  Structure of a flavonoid glucosyltransferase reveals the basis for plant natural product modification , 2006 .

[51]  F. Studier,et al.  Protein production by auto-induction in high density shaking cultures. , 2005, Protein expression and purification.

[52]  J. Dean,et al.  The formation, vacuolar localization, and tonoplast transport of salicylic acid glucose conjugates in tobacco cell suspension cultures , 2005, Planta.

[53]  T. Douki,et al.  The upstream oxylipin profile of Arabidopsis thaliana: a tool to scan for oxidative stresses. , 2004, The Plant journal : for cell and molecular biology.

[54]  D. Bowles,et al.  Arabidopsis glycosyltransferases as biocatalysts in fermentation for regioselective synthesis of diverse quercetin glucosides , 2004, Biotechnology and bioengineering.

[55]  E. Esmans,et al.  Metabolic Fate of Jasmonates in Tobacco Bright Yellow-2 Cells1 , 2004, Plant Physiology.

[56]  Kazuo Shinozaki,et al.  A collection of 11 800 single-copy Ds transposon insertion lines in Arabidopsis. , 2004, The Plant journal : for cell and molecular biology.

[57]  J. Dean,et al.  Formation and vacuolar localization of salicylic acid glucose conjugates in soybean cell suspension cultures , 2003 .

[58]  C. Wasternack,et al.  Biochemical and Molecular Characterization of a Hydroxyjasmonate Sulfotransferase from Arabidopsis thaliana * , 2003, The Journal of Biological Chemistry.

[59]  Kazuo Shinozaki,et al.  A New Resource of Locally Transposed DissociationElements for Screening Gene-Knockout Lines in Silico on the Arabidopsis Genome1,212 , 2002, Plant Physiology.

[60]  Yi Li,et al.  The Activity of ArabidopsisGlycosyltransferases toward Salicylic Acid, 4-Hydroxybenzoic Acid, and Other Benzoates* , 2002, Journal of Biological Chemistry.

[61]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[62]  D. Bowles,et al.  Identification of Glucosyltransferase Genes Involved in Sinapate Metabolism and Lignin Synthesis in Arabidopsis * , 2001, The Journal of Biological Chemistry.

[63]  Yi Li,et al.  Higher plant glycosyltransferases , 2001, Genome Biology.

[64]  Patrik R. Jones,et al.  Glycosyltransferases in plant natural product synthesis: characterization of a supergene family. , 2000, Trends in plant science.

[65]  C. Wasternack,et al.  Metabolic profiling of oxylipins upon salicylate treatment in barley leaves — preferential induction of the reductase pathway by salicylate 1 , 1999, FEBS letters.

[66]  S. Clough,et al.  Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. , 1998, The Plant journal : for cell and molecular biology.

[67]  D W Nebert,et al.  The UDP glycosyltransferase gene superfamily: recommended nomenclature update based on evolutionary divergence. , 1997, Pharmacogenetics.

[68]  William R. Taylor,et al.  The rapid generation of mutation data matrices from protein sequences , 1992, Comput. Appl. Biosci..

[69]  M. Hamberg,et al.  Oxylipin metabolism in the red alga Gracilariopsis lemaneiformis: mechanism of formation of vicinal dihydroxy fatty acids. , 1991, Archives of biochemistry and biophysics.

[70]  M. Hamberg,et al.  An OPR3-independent pathway uses 4,5-didehydrojasmonate for jasmonate synthesis. , 2018, Nature chemical biology.

[71]  Thomas D. Schmittgen,et al.  Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2 2 DD C T Method , 2022 .