title: Genetic screening identifies cyanogenesis-deficient mutants of Lotus japonicus and reveals enzymatic specificity in hydroxynitrile glucoside metabolism.
abstract: Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid-derived cyanogenic glucosides (alpha-hydroxynitrile glucosides) by specific beta-glucosidases. Release of cyanide functions as a defense mechanism against generalist herbivores. We developed a high-throughput screening method and used it to identify cyanogenesis deficient (cyd) mutants in the model legume Lotus japonicus. Mutants in both biosynthesis and catabolism of cyanogenic glucosides were isolated and classified following metabolic profiling of cyanogenic glucoside content. L. japonicus produces two cyanogenic glucosides: linamarin (derived from Val) and lotaustralin (derived from Ile). Their biosynthesis may involve the same set of enzymes for both amino acid precursors. However, in one class of mutants, accumulation of lotaustralin and linamarin was uncoupled. Catabolic mutants could be placed in two complementation groups, one of which, cyd2, encoded the beta-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related beta-glucosidase, BGD4, were identified. This indicated that BGD4 plays no role in cyanogenesis in L. japonicus in vivo. Biochemical analysis confirmed that BGD4 cannot hydrolyze linamarin or lotaustralin and in L. japonicus is specific for breakdown of related hydroxynitrile glucosides, such as rhodiocyanoside A. By contrast, BGD2 can hydrolyze both cyanogenic glucosides and rhodiocyanosides. Our genetic analysis demonstrated specificity in the catabolic pathways for hydroxynitrile glucosides and implied specificity in their biosynthetic pathways as well. In addition, it has provided important tools for elucidating and potentially modifying cyanogenesis pathways in plants.
authors.affiliation: Department of Plant Biology and Biotechnology, University of Copenhagen, Frederiksberg, Denmark.
authors.forename: Adam
authors.initials: A
authors.lastname: Takos
authors.forename: Daniela
authors.initials: D
authors.lastname: Lai
authors.forename: Lisbeth
authors.initials: L
authors.lastname: Mikkelsen
authors.forename: Maher
authors.initials: M
authors.lastname: Abou Hachem
authors.forename: Dale
authors.initials: D
authors.lastname: Shelton
authors.forename: Mohammed Saddik
authors.initials: MS
authors.lastname: Motawia
authors.forename: Carl Erik
authors.initials: CE
authors.lastname: Olsen
authors.forename: Trevor L
authors.initials: TL
authors.lastname: Wang
authors.forename: Cathie
authors.initials: C
authors.lastname: Martin
authors.forename: Fred
authors.initials: F
authors.lastname: Rook
mesh_terms: D000483:Alleles; D001681:Biological Assay; D017343:Genes, Plant / Q000235:genetics; D005816:Genetic Complementation Test; D005820:Genetic Testing; D006027:Glycosides / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D057166:High-Throughput Screening Assays; D006856:Hydrogen Cyanide / Q000378:metabolism*; D006868:Hydrolysis; D000070116:Lotus / Q000201:enzymology* / Q000235:genetics*; D055442:Metabolome; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics*; D010641:Phenotype; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030821:Plants, Genetically Modified; D013379:Substrate Specificity; D014026:Nicotiana / Q000378:metabolism
chemical_list: D006027:Glycosides; D010940:Plant Proteins; C007173:cyanogenic glycosides; D006856:Hydrogen Cyanide
references.citation: Phytochemistry. 1998 Jan;47(2):155-62
references.pmid: 9431670
references.citation: Science. 2001 Sep 7;293(5536):1826-8
references.pmid: 11474068
references.citation: Electrophoresis. 1997 Dec;18(15):2714-23
references.pmid: 9504803
references.citation: Arch Biochem Biophys. 1987 Jan;252(1):152-62
references.pmid: 3101594
references.citation: Plant Mol Biol. 1991 Aug;17(2):209-19
references.pmid: 1907511
references.citation: J Biol Chem. 1999 Dec 10;274(50):35483-91
references.pmid: 10585420
references.citation: Plant Physiol. 2008 Jul;147(3):1072-91
references.pmid: 18467457
references.citation: Phytochemistry. 2008 Jun;69(9):1795-813
references.pmid: 18472115
references.citation: Genome Res. 2004 Jun;14(6):1188-90
references.pmid: 15173120
references.citation: Insect Biochem Mol Biol. 2007 Jan;37(1):10-8
references.pmid: 17175442
references.citation: Proteins. 1995 Nov;23(3):318-26
references.pmid: 8710825
references.citation: Structure. 1995 Sep 15;3(9):951-60
references.pmid: 8535788
references.citation: Plant J. 2007 May;50(4):678-95
references.pmid: 17425720
references.citation: Plant Physiol. 1998 Apr;116(4):1469-78
references.pmid: 9536065
references.citation: Plant Physiol. 2009 Nov;151(3):1281-91
references.pmid: 19641028
references.citation: Plant Physiol. 1989 Aug;90(4):1552-9
references.pmid: 16666964
references.citation: Plant J. 2003 Mar;33(5):949-56
references.pmid: 12609035
references.citation: Bioinformatics. 2006 Jan 15;22(2):195-201
references.pmid: 16301204
references.citation: Phytochemistry. 2008 May;69(7):1457-68
references.pmid: 18353406
references.citation: Compr Rev Food Sci Food Saf. 2009 Jul;8(3):181-194
references.pmid: 33467798
references.citation: Nucleic Acids Res. 1990 Oct 25;18(20):6097-100
references.pmid: 2172928
references.citation: Genetics. 2008 May;179(1):517-26
references.pmid: 18458107
references.citation: Phytochemistry. 2008 May;69(7):1507-16
references.pmid: 18342345
references.citation: J Biol Chem. 2000 Jan 21;275(3):1966-75
references.pmid: 10636899
references.citation: Analyst. 1966 Apr;91(81):282-4
references.pmid: 5931544
references.citation: DNA Res. 2008 Aug;15(4):227-39
references.pmid: 18511435
references.citation: Plant Physiol. 2005 Sep;139(1):363-74
references.pmid: 16126856
references.citation: Plant Physiol. 2000 Apr;122(4):1311-21
references.pmid: 10759528
references.citation: Annu Rev Plant Biol. 2006;57:303-33
references.pmid: 16669764
references.citation: Plant Physiol. 2004 May;135(1):71-84
references.pmid: 15122013
references.citation: Mol Ecol. 2007 Oct;16(19):4180-93
references.pmid: 17784921