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The biosynthetic pathways that produce anthocyanins, the principal pigments for flower and leaf coloration in plants, have been extensively investigated. As a result, many of the enzymes involved in these pathways have been identified. Here, we make use of an inducible Arabidopsis thaliana system and demonstrate that the final step in the formation of the major anthocyanin molecule occurs via a glucosylation step catalyzed by acyl-glucose-dependent anthocyanin glucosyltransferase (AAGT). The glucosylation occurs at the 4-coumarate moiety of the anthocyanin molecule cyanidin 3-O-[2″-O-(2′″-O-(sinapoyl) xylosyl) 6″-O-(p-coumaroyl) glucoside] 5-O-[6″″-O-(malonyl) glucoside] leading to completion of the main anthocyanin structure, a reaction that has not previously been identified in studies of Arabidopsis anthocyanins. Earlier studies on flower AAGTs showed that they conjugate a glucose directly to the basic skeleton of anthocyanin. The present study provides the first evidence that an AAGT of Arabidopsis can conjugate a glucose to an acyl moiety of an anthocyanin modified with sugars and organic acids. The results from analyses of gene expression and of anthocyanin composition in a knock-out (KO) mutant and from a complementation test indicate that AtBGLU10 might encode this AAGT.

Arabidopsis thaliana, acyl-glucose-dependent anthocyanin glucosyltransferase, anthocyanin, Glycoside hydrolase family 1

Publication DOI: 10.1016/j.jplph.2012.12.001
Journal NLM ID: 9882059
Publisher: Urban & Fischer
Correspondence: nove@cc.tuat.ac.jp
Institutions: Department of Biotechnology and Life Science, Graduate School of Engineering, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184-8588, Japan
Methods: biological assays, HPLC, genetic manipulations

To identify candidate genes involved in Arabidopsis flavonoid biosynthesis, we applied transcriptome coexpression analysis and independent component analyses with 1388 microarray data from publicly available databases. Two glycosyltransferases, UGT79B1 and UGT84A2 were found to cluster with anthocyanin biosynthetic genes. Anthocyanin was drastically reduced in ugt79b1 knockout mutants. Recombinant UGT79B1 protein converted cyanidin 3-O-glucoside to cyanidin 3-O-xylosyl(1[RIGHTWARDS ARROW]2)glucoside. UGT79B1 recognized 3-O-glucosylated anthocyanidins/flavonols and uridine diphosphate (UDP)-xylose, but not 3,5-O-diglucosylated anthocyanidins, indicating that UGT79B1 encodes anthocyanin 3-O-glucoside: 2′′-O-xylosyltransferase. UGT84A2 is known to encode sinapic acid: UDP-glucosyltransferase. In ugt84a2 knockout mutants, a major sinapoylated anthocyanin was drastically reduced. A comparison of anthocyanin profiles in ugt84a knockout mutants indicated that UGT84A2 plays a major role in sinapoylation of anthocyanin, and that other UGT84As contribute the production of 1-O-sinapoylglucose to a lesser extent. These data suggest major routes from cyanidin 3-O-glucoside to the most highly modified cyanidin in the potential intricate anthocyanin modification pathways in Arabidopsis.

glycosyltransferase, flavonoid, Arabidopsis thaliana, Xylosyltransferase, anthocyanin

Publication DOI: 10.1111/j.1365-313X.2011.04779.x
Journal NLM ID: 9207397
Publisher: Oxford: Blackwell Scientific Publishers and BIOS Scientific Publishers for the Society for Experimental Biology
Correspondence: ksaito@psc.riken.jp
Institutions: CREST, Japan Science and Technology Agency, 4-1-8 Honcho, Kawaguchi, Saitama 332-0012, Japan, RIKEN Plant Science Center, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan, Graduate School of Nanobiosciences, Yokohama City University, 1-7-29, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan, Graduate School of Pharmaceutical Sciences, Chiba University, 1-33 Yayoi-cho, Inage-ku, Chiba 263-8522, Japan, Antibiotics Laboratory, RIKEN, Wako, Japan
Methods: biological assays, enzymatic assay, HPLC/PDA/ESI-MS, genetic manipulations

Members of the BAHD family of plant acyl transferases are very versatile catalytically, and are thought to be able to evolve new substrate specificities rapidly. Acylation of anthocyanins occurs in many plant species and affects anthocyanin stability and light absorption in solution. The versatility of BAHD acyl transferases makes it difficult to identify genes encoding enzymes with defined substrate specificities on the basis of structural homology to genes of known catalytic function alone. Consequently, we have used a modification to standard functional genomics strategies, incorporating co-expression profiling with anthocyanin accumulation, to identify genes encoding three anthocyanin acyl transferases from Arabidopsis thaliana. We show that the activities of these enzymes influence the stability of anthocyanins at neutral pH, and some acylations also affect the anthocyanin absorption maxima. These properties make the BAHD acyl transferases suitable tools for engineering anthocyanins for an improved range of biotechnological applications.

anthocyanins, acyl transferases, convergent evolution, functional genomics

Publication DOI: 10.1111/j.1365-313X.2007.03079.x
Journal NLM ID: 9207397
Publisher: Oxford: Blackwell Scientific Publishers and BIOS Scientific Publishers for the Society for Experimental Biology
Correspondence: cathie.martin@bbsrc.ac.uk
Institutions: John Innes Centre, Norwich Research Park, Colney, Norwich, NR4 7UH, UK, Graduate School of Pharmaceutical Sciences, Chiba University, Yayoi-cho 1-33, Inage-ku, Chiba 263-8522, Japan, Institute of Life Science, Ehime Women’s College, 421 Ibuki-cho Baba, Uwajima-shi, Ehime, 798-0025, Japan, Technologies for Systems Biology, Institute of Food Research, Norwich Research Park, Colney, Norwich, NR4 7UA, UK, Department of Applied Biology, Faculty of Textile Science and Technology, Shinshu University, 3-15-1 Tokida, Ueda, Nagano 386-8567, Japan, Institute of Advanced Technology, Suntory Ltd., 1-1-1 Wakayamadai, Shimamoto-cho, Mishima-gun, Osaka, 618-8503, Japan, RIKEN Plant Science Center, Suehiro-cho 1-7-22, Tsurumi-ku, Yokohama, 230-0045, Japan
Methods: ESI-MS, biological assays, HPLC, UV, LC-MS, enzymatic assay, genetic manipulations