Nucleotide and derived amino acid sequence of the cyanogenic ?-glucosidase (linamarase) from white clover (Trifolium repens L.)

Oxtoby, Elli; Dunn, Michael; Pancoro, Adi; Hughes, Monica A.

doi:10.1007/bf00039495

Cited by 65 publications

(39 citation statements)

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“…Although Li and its product have been well characterized at the nucleotide and protein levels (Oxtoby et al 1991;Barrett et al 1995;Olsen et al 2007), the molecular identity of Ac has remained undetermined. Below we discuss evidence that Ac is very likely to be the cytochrome P450 gene CYP79D15.…”

Section: Discussionmentioning

confidence: 99%

Evidence on the Molecular Basis of the Ac/ac Adaptive Cyanogenesis Polymorphism in White Clover (Trifolium repens L.)

2008

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White clover is polymorphic for cyanogenesis, with both cyanogenic and acyanogenic plants occurring in nature. This chemical defense polymorphism is one of the longest-studied and best-documented examples of an adaptive polymorphism in plants. It is controlled by two independently segregating genes: Ac/ac controls the presence/absence of cyanogenic glucosides; and Li/li controls the presence/absence of their hydrolyzing enzyme, linamarase. Whereas Li is well characterized at the molecular level, Ac has remained unidentified. Here we report evidence that Ac corresponds to a gene encoding a cytochrome P450 of the CYP79D protein subfamily (CYP79D15), and we describe the apparent molecular basis of the Ac/ac polymorphism. CYP79D orthologs catalyze the first step in cyanogenic glucoside biosynthesis in other cyanogenic plant species. In white clover, Southern hybridizations indicate that CYP79D15 occurs as a single-copy gene in cyanogenic plants but is absent from the genomes of ac plants. Gene-expression analyses by RT-PCR corroborate this finding. This apparent molecular basis of the Ac/ac polymorphism parallels our previous findings for the Li/li polymorphism, which also arises through the presence/ absence of a single-copy gene. The nature of these polymorphisms may reflect white clover's evolutionary origin as an allotetraploid derived from cyanogenic and acyanogenic diploid progenitors. C YANOGENESIS (cyanide release following tissue damage) occurs in .2650 plant species, including ferns, gymnosperms, monocots, and dicots (Seigler and Brinker 1993). In its most common form, cyanogenesis involves the interaction of two compounds, cyanogenic glycosides and their hydrolyzing enzymes; these are separated in intact tissue and brought into contact with tissue disruption. White clover (Trifolium repens L., Fabaceae) is polymorphic for cyanogenesis, with both cyanogenic and acyanogenic plants occurring in natural populations (Armstrong et al. 1913;Ware 1925). This polymorphism arises through the presence/absence of both of the underlying cyanogenic components. Inheritance of cyanogenesis in white clover follows a simple Mendelian two-locus, two-allele model (Coop 1940;Melville and Doak 1940;Corkill 1942): the gene Ac/ac controls the presence/absence of cyanogenic glucosides; and the independently segregating gene Li/li controls the presence/absence of linamarase, a cyanogenic b-glucosidase (reviewed by Hughes 1991). Plants that carry at least one dominant (functional) allele at both genes (Ac and Li) are cyanogenic, while the occurrence of two nonfunctional alleles at either gene confers the acyanogenic phenotype.White clover is a common legume of fields, lawns, and pastures in mesic temperate climates. It is a native of Eurasia but has been introduced worldwide as a forage crop and lawn plant. It is a perennial and an insectpollinated, obligate outcrosser; plants also spread vegetatively by stolons. Chromosome number and genetic map data indicate that white clover is an allotetraploid (e.g., Barrett et al. 200...

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Section: Discussionmentioning

confidence: 99%

Evidence on the Molecular Basis of the Ac/ac Adaptive Cyanogenesis Polymorphism in White Clover (Trifolium repens L.)

2008

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“…Both enzymes show high specificity for dhurrin but differ in their expression patterns and reactivity toward synthetic substrates (Hö sel et al, 1987;Cicek and Esen, 1998). A single cyanogenic b-glucosidase called linamarase is present in white clover (Trifolium repens), and segregation of alleles of the gene encoding linamarase (Li) underpins some of the natural variation in cyanogenesis in this species (Dunn and Hughes, 1983;Oxtoby et al, 1991;Olsen et al, 2007). Cyanogenic glucosides are a-hydroxynitrile glucosides and release HCN on hydrolysis.…”

Section: Introductionmentioning

confidence: 99%

Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism

et al. 2010

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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.

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“…The PSORT program did not identify any organellar targeting signals in SLW3. SLW3 had one N-glycosylation site (N-X-S/T) and shared 56% identity with the Trifolium repens cyanogenic ␤-glucosidase (Oxtoby et al, 1991). Similar extents of identity were found with plant ␤-glucosidases that hydrolyze other substrates and belong to the glycosyl hydrolase family 1 (Rojas and Romeu, 1996).…”

Section: Slw1 Is An M20b Metallopeptidase-like Proteinmentioning

confidence: 76%

Local and Systemic Changes in Squash Gene Expression in Response to Silverleaf Whitefly Feeding

Ven¹,

LeVesque²,

Perring³

et al. 2000

The Plant Cell

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Squash genes ( SLW1 and SLW3 ) induced systemically after silverleaf whitefly feeding were identified. Differences in the local and systemic expression of SLW1 and SLW3 after feeding by the closely related silverleaf and sweetpotato whiteflies were observed. Temporal and spatial studies showed that SLW1 and SLW3 were induced when second, third, and fourth nymphal instars were feeding. Although only barely detected after wounding and bacterial infection, SLW1 and SLW3 RNAs were abundant during water-deficit stress. Treatments with wound/defense signal molecules showed that SLW1 RNAs accumulated in response to methyl jasmonate and ethylene, whereas SLW3 was not regulated by known wound/defense signals, suggesting utilization of a novel mechanism for defense signal transduction. SLW1 RNAs accumulated during floral and fruit development, whereas SLW3 RNAs were not detected during vegetative or reproductive development. The potential roles of SLW1, an M20b peptidase-like protein, and SLW3, a ␤ -glucosidase-like protein, in defense and the leaf-silvering disorder are discussed. INTRODUCTIONAlthough the signal transduction pathways that are activated by mechanical wounding and pathogen invasion have been studied intensively (Ryals et al., 1996;Ryan and Pearce, 1998; Dempsey et al., 1999;Pieterse and van Loon, 1999), there is limited understanding of changes in plant gene expression in response to insect feeding. Given the similarities of mechanical wounding and the damage incurred by insects that masticate foliage, it is not surprising that wound-response transcripts and proteins (i.e., proteinase inhibitors, polyphenol oxidases, and leucine aminopeptidase) accumulate locally and systemically in response to caterpillar feeding (Green and Ryan, 1982;Pautot et al., 1993;Stout et al., 1996; Karban and Baldwin, 1997). However, insect feeding and wounding are not equivalent. Feeding by Manduca sexta larvae, for example, enhances expression of wound-response genes relative to wounding but also induces expression of a novel set of plant genes that are not induced by wounding alone (Korth and Dixon, 1997). Although these herbivory-induced genes have not been identified, some of their gene products may be involved in biosynthesis or release of volatiles that are important in mediating plant-herbivore-predator interactions (Páre and Tumlinson, 1999).Far less is known about the changes in gene expression in response to insects that use other modes of feeding (Stout et al., 1994). Only a few recent studies have investigated plant responses to phloem-feeding whiteflies. Unlike insects that consume foliage, whiteflies do not induce the woundresponse genes that are modulated by the octadecanoid pathway. Whitefly feeding on tomatoes primarily induces the salicylic acid (SA)-independent, jasmonic acid (JA)/ethylene-dependent cascade of defense signal transduction (Chao et al., 1999;Pieterse and van Loon, 1999; D.P. Puthoff and L.L. Walling, manuscript submitted; D.P. Puthoff, C.S. LeVesque, T.M. Perring, and L.L. Walling, manuscript ...

show abstract

Nucleotide and derived amino acid sequence of the cyanogenic ?-glucosidase (linamarase) from white clover (Trifolium repens L.)

Cited by 65 publications

References 29 publications

Evidence on the Molecular Basis of the Ac/ac Adaptive Cyanogenesis Polymorphism in White Clover (Trifolium repens L.)

Evidence on the Molecular Basis of the Ac/ac Adaptive Cyanogenesis Polymorphism in White Clover (Trifolium repens L.)

Genetic Screening Identifies Cyanogenesis-Deficient Mutants of Lotus japonicus and Reveals Enzymatic Specificity in Hydroxynitrile Glucoside Metabolism

Local and Systemic Changes in Squash Gene Expression in Response to Silverleaf Whitefly Feeding

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