Genetic screening identifies cyanogenesis-deficient mutants of Lotus japonicus and reveals enzymatic specificity in hydroxynitrile glucoside metabolism

Adam Matthew Takos; Daniela Lai; Lisbeth Mikkelsen; Maher Abou Hachem; Dale Andrew Shelton; Mohammed Saddik Motawie; Carl Erik Olsen; Trevor L. Wang; Catherine Rosemary Martin; Frederik Rook

doi:10.1105/tpc.109.073502

Genetic screening identifies cyanogenesis-deficient mutants of Lotus japonicus and reveals enzymatic specificity in hydroxynitrile glucoside metabolism

Adam Matthew Takos, Daniela Lai, Lisbeth Mikkelsen, Maher Abou Hachem, Dale Andrew Shelton, Mohammed Saddik Motawie, Carl Erik Olsen, Trevor L. Wang, Catherine Rosemary Martin, Frederik Rook

46 Citations (Scopus)

Abstract

Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid-derived cyanogenic glucosides (a-hydroxynitrile glucosides) by specific b-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 b-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related b-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.

Original language	English
Journal	Plant Cell
Volume	22
Pages (from-to)	1605-1619
Number of pages	15
ISSN	1040-4651
DOIs	https://doi.org/10.1105/tpc.109.073502
Publication status	Published - May 2010

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@article{3decc200901a11df928f000ea68e967b,

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 (a-hydroxynitrile glucosides) by specific b-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 b-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related b-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.",

author = "Takos, {Adam Matthew} and Daniela Lai and Lisbeth Mikkelsen and {Abou Hachem}, Maher and Shelton, {Dale Andrew} and Motawie, {Mohammed Saddik} and Olsen, {Carl Erik} and Wang, {Trevor L.} and Martin, {Catherine Rosemary} and Frederik Rook",

year = "2010",

month = may,

doi = "10.1105/tpc.109.073502",

language = "English",

volume = "22",

pages = "1605--1619",

journal = "The Plant Cell",

issn = "1040-4651",

publisher = "American Society of Plant Biologists",

}

TY - JOUR

T1 - Genetic screening identifies cyanogenesis-deficient mutants of Lotus japonicus and reveals enzymatic specificity in hydroxynitrile glucoside metabolism

AU - Takos, Adam Matthew

AU - Lai, Daniela

AU - Mikkelsen, Lisbeth

AU - Abou Hachem, Maher

AU - Shelton, Dale Andrew

AU - Motawie, Mohammed Saddik

AU - Olsen, Carl Erik

AU - Wang, Trevor L.

AU - Martin, Catherine Rosemary

AU - Rook, Frederik

PY - 2010/5

Y1 - 2010/5

N2 - Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid-derived cyanogenic glucosides (a-hydroxynitrile glucosides) by specific b-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 b-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related b-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.

AB - Cyanogenesis, the release of hydrogen cyanide from damaged plant tissues, involves the enzymatic degradation of amino acid-derived cyanogenic glucosides (a-hydroxynitrile glucosides) by specific b-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 b-glucosidase BGD2. Despite the identification of nine independent cyd2 alleles, no mutants involving the gene encoding a closely related b-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.

U2 - 10.1105/tpc.109.073502

DO - 10.1105/tpc.109.073502

M3 - Journal article

C2 - 20453117

SN - 1040-4651

VL - 22

SP - 1605

EP - 1619

JO - The Plant Cell

JF - The Plant Cell

ER -

Genetic screening identifies cyanogenesis-deficient mutants of Lotus japonicus and reveals enzymatic specificity in hydroxynitrile glucoside metabolism

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