{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,19]],"date-time":"2026-05-19T19:46:46Z","timestamp":1779220006865,"version":"3.51.4"},"reference-count":46,"publisher":"Oxford University Press (OUP)","issue":"5","license":[{"start":{"date-parts":[[2021,1,19]],"date-time":"2021-01-19T00:00:00Z","timestamp":1611014400000},"content-version":"vor","delay-in-days":3916,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"published-print":{"date-parts":[[2010,6,28]]},"abstract":"<jats:title>Abstract<\/jats:title>\n               <jats:p>Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid\u2013derived cyanogenic glucosides (\u03b1-hydroxynitrile glucosides) by specific \u03b2-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 \u03b2-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related \u03b2-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.<\/jats:p>","DOI":"10.1105\/tpc.109.073502","type":"journal-article","created":{"date-parts":[[2010,5,8]],"date-time":"2010-05-08T02:15:03Z","timestamp":1273284903000},"page":"1605-1619","source":"Crossref","is-referenced-by-count":59,"title":["Genetic Screening Identifies Cyanogenesis-Deficient Mutants of <i>Lotus japonicus<\/i> and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism\n\u00a0"],"prefix":"10.1093","volume":"22","author":[{"given":"Adam","family":"Takos","sequence":"first","affiliation":[{"name":"Department of Plant Biology and Biotechnology, University of Copenhagen, 1871 Frederiksberg, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Daniela","family":"Lai","sequence":"additional","affiliation":[{"name":"Department of Plant Biology and Biotechnology, University of Copenhagen, 1871 Frederiksberg, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Lisbeth","family":"Mikkelsen","sequence":"additional","affiliation":[{"name":"Department of Plant Biology and Biotechnology, University of Copenhagen, 1871 Frederiksberg, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Maher","family":"Abou Hachem","sequence":"additional","affiliation":[{"name":"Department of Systems Biology, Technical University of Denmark, 2800 Kongens Lyngby, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dale","family":"Shelton","sequence":"additional","affiliation":[{"name":"Department of Plant Biology and Biotechnology, University of Copenhagen, 1871 Frederiksberg, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mohammed Saddik","family":"Motawia","sequence":"additional","affiliation":[{"name":"Department of Plant Biology and Biotechnology, University of Copenhagen, 1871 Frederiksberg, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Carl Erik","family":"Olsen","sequence":"additional","affiliation":[{"name":"Department of Basic Sciences and Environment, University of Copenhagen, 1871 Frederiksberg, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Trevor L.","family":"Wang","sequence":"additional","affiliation":[{"name":"Department of Metabolic Biology, John Innes Centre, NR4 7UH Norwich, United Kingdom"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cathie","family":"Martin","sequence":"additional","affiliation":[{"name":"Department of Plant Biology and Biotechnology, University of Copenhagen, 1871 Frederiksberg, Denmark"},{"name":"Department of Metabolic Biology, John Innes Centre, NR4 7UH Norwich, United Kingdom"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Fred","family":"Rook","sequence":"additional","affiliation":[{"name":"Department of Plant Biology and Biotechnology, University of Copenhagen, 1871 Frederiksberg, Denmark"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"286","published-online":{"date-parts":[[2010,5,7]]},"reference":[{"key":"2021040621400483600_b1","doi-asserted-by":"crossref","first-page":"1966","DOI":"10.1074\/jbc.275.3.1966","article-title":"Cytochrome P-450 from cassava (Manihot esculenta Crantz) catalyzing the first steps in the biosynthesis of the cyanogenic glucosides linamarin and lotaustralin","volume":"275","author":"Andersen","year":"2000","journal-title":"J. Biol. Chem."},{"key":"2021040621400483600_b2","doi-asserted-by":"crossref","first-page":"262","DOI":"10.1098\/rspb.1913.0021","article-title":"Herbage Studies. II.- Variation in Lotus corniculatus and Trifolium repens (cyanophoric plants)","volume":"86","author":"Armstrong","year":"1913","journal-title":"Proc. R. Soc. Lond. B. Biol. Sci."},{"key":"2021040621400483600_b3","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1093\/bioinformatics\/bti770","article-title":"The SWISS-MODEL workspace: A web-based environment for protein structure homology modelling","volume":"22","author":"Arnold","year":"2006","journal-title":"Bioinformatics"},{"key":"2021040621400483600_b4","doi-asserted-by":"crossref","first-page":"309","DOI":"10.1007\/s11101-006-9033-1","article-title":"Cyanogenic glycosides: A case study for evolution and application of cytochromes P450","volume":"5","author":"Bak","year":"2006","journal-title":"Phytochem. 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Saf."},{"key":"2021040621400483600_b29","doi-asserted-by":"crossref","first-page":"1072","DOI":"10.1104\/pp.107.109512","article-title":"The \u03b2-glucosidases responsible for bioactivation of hydroxynitrile glucosides in Lotus japonicus","volume":"147","author":"Morant","year":"2008","journal-title":"Plant Physiol."},{"key":"2021040621400483600_b30","doi-asserted-by":"crossref","first-page":"1795","DOI":"10.1016\/j.phytochem.2008.03.006","article-title":"\u03b2-Glucosidases as detonators of plant chemical defense","volume":"69","author":"Morant","year":"2008","journal-title":"Phytochemistry"},{"key":"2021040621400483600_b31","doi-asserted-by":"crossref","first-page":"2043","DOI":"10.1002\/jsfa.3337","article-title":"Rising African cassava production, diseases due to high cyanide intake and control measures","volume":"88","author":"Nhassico","year":"2008","journal-title":"J. Sci. 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Biol."},{"key":"2021040621400483600_b37","doi-asserted-by":"crossref","first-page":"1281","DOI":"10.1104\/pp.109.142190","article-title":"TILLING in Lotus japonicus identified large allelic series for symbiosis genes and revealed a bias in functionally defective EMS alleles towards glycine replacements","volume":"151","author":"Perry","year":"2009","journal-title":"Plant Physiol."},{"key":"2021040621400483600_b38","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1002\/prot.340230306","article-title":"Evaluation of comparative protein modelling by MODELLER","volume":"23","author":"Sali","year":"1995","journal-title":"Proteins"},{"key":"2021040621400483600_b39","doi-asserted-by":"crossref","first-page":"227","DOI":"10.1093\/dnares\/dsn008","article-title":"Genome structure of the legume, Lotus japonicus","volume":"15","author":"Sato","year":"2008","journal-title":"DNA Res."},{"key":"2021040621400483600_b40","doi-asserted-by":"crossref","first-page":"6097","DOI":"10.1093\/nar\/18.20.6097","article-title":"Sequence logos: A new way to display consensus sequences","volume":"18","author":"Schneider","year":"1990","journal-title":"Nucleic Acids Res."},{"key":"2021040621400483600_b41","doi-asserted-by":"crossref","first-page":"487","DOI":"10.1016\/j.plantsci.2005.10.001","article-title":"Condensed tannin biosynthesis genes are regulated separately from other flavonoid biosynthesis genes in apple fruit skin","volume":"170","author":"Takos","year":"2006","journal-title":"Plant Sci."},{"key":"2021040621400483600_b42","doi-asserted-by":"crossref","first-page":"1826","DOI":"10.1126\/science.1062249","article-title":"Resistance to an herbivore through engineered cyanogenic glucoside synthesis","volume":"293","author":"Tattersall","year":"2001","journal-title":"Science"},{"key":"2021040621400483600_b43","doi-asserted-by":"crossref","first-page":"949","DOI":"10.1046\/j.1365-313X.2003.01676.x","article-title":"An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus","volume":"33","author":"Voinnet","year":"2003","journal-title":"Plant J."},{"key":"2021040621400483600_b44","first-page":"193","article-title":"Consequences for animal production of cyanogenesis in sorghum forage and hay \u2013 A review","volume":"23","author":"Wheeler","year":"1989","journal-title":"Trop. 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