SUMMARYCyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific b-glucosidases following tissue damage results in the release of hydrogen cyanide. The cyanogenesis deficient1 (cyd1) mutant of Lotus japonicus carries a partial deletion of the CYP79D3 gene, which encodes a cytochrome P450 enzyme that is responsible for the first step in cyanogenic glucoside biosynthesis. The genomic region surrounding CYP79D3 contains genes encoding the CYP736A2 protein and the UDP-glycosyltransferase UGT85K3. In combination with CYP79D3, these genes encode the enzymes that constitute the entire pathway for cyanogenic glucoside biosynthesis. The biosynthetic genes for cyanogenic glucoside biosynthesis are also co-localized in cassava (Manihot esculenta) and sorghum (Sorghum bicolor), but the three gene clusters show no other similarities. Although the individual enzymes encoded by the biosynthetic genes in these three plant species are related, they are not necessarily orthologous. The independent evolution of cyanogenic glucoside biosynthesis in several higher plant lineages by the repeated recruitment of members from similar gene families, such as the CYP79s, is a likely scenario.

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.

Isoflavonoids are a class of phenylpropanoids made by legumes, and consumption of dietary isoflavonoids confers benefits to human health. Our aim is to understand the regulation of isoflavonoid biosynthesis. Many studies have shown the importance of transcription factors in regulating the transcription of one or more genes encoding enzymes in phenylpropanoid metabolism. In this study, we coupled bioinformatics and coexpression analysis to identify candidate genes encoding transcription factors involved in regulating isoflavonoid biosynthesis in Lotus (Lotus japonicus). Genes encoding proteins belonging to 39 of the main transcription factor families were examined by microarray analysis of RNA from leaf tissue that had been elicited with glutathione. Phylogenetic analyses of each transcription factor family were used to identify subgroups of proteins that were specific to L. japonicus or closely related to known regulators of the phenylpropanoid pathway in other species. R2R3MYB subgroup 2 genes showed increased expression after treatment with glutathione. One member of this subgroup, LjMYB14, was constitutively overexpressed in L. japonicus and induced the expression of at least 12 genes that encoded enzymes in the general phenylpropanoid and isoflavonoid pathways. A distinct set of six R2R3MYB subgroup 2-like genes was identified. We suggest that these subgroup 2 sister group proteins and those belonging to the main subgroup 2 have roles in inducing isoflavonoid biosynthesis. The induction of isoflavonoid production in L. japonicus also involves the coordinated down-regulation of competing biosynthetic pathways by changing the expression of other transcription factors.

The recently reported red fluorescent protein DsRed from the reef coral Discosoma sp. represents a new marker that has been codonoptimized for high expression in mammalian cells. To facilitate expression of DsRed in ascomycete fungi, we used the clone pDsRedExpress (Clontech) for constructing a plasmid vector, pPgpd-DsRed, containing the constitutive Aspergillus nidulans glyceraldehyde 3-phosphate (gpd) promoter. This vector was used for co-transformation of Penicillium paxilli, Trichoderma harzianum and Trichoderma virens (syn. Gliocladium virens) together with either pAN7-1 or gGFP, both containing a gene for hygromycin resistance for transformant selection. In addition, gGFP contains a green fluorescent protein (GFP) gene for expression in Ascomycetes. Expression of DsRedExpress was obtained in all three fungi, indicating that DsRed can be used as a highly effective vital marker in Ascomycetes. Dual marked transformants expressed both DsRed-Express and GFP in the same mycelium and were used for non-quantitative comparison of the intensity of the fluorescence using confocal laser scanning microscopy.

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Festuca populations (Festuca arundinacea, Festuca pratensis, and Festuca rubra) from Italy, Spain, and Denmark were investigated for Neotyphodium infection, ergovaline production, and 14 microsatellite markers. Endophytes were detected in 57, 54, and 100% of the locations surveyed in Italy, Spain, and Denmark, respectively. This is the first report of F. arundinacea endophytes from seminatural grasslands in Denmark. Sixty‐seven percent of the F. rubra and 100% of the F. pratensis populations were infected. Ergovaline production varied, even within populations. A dendrogram based on microsatellite length polymorphisms separated endophytes of each Festuca species. In addition, Danish F. arundinacea endophytes were separate from the other F. arundinacea endophytes. Analysis of molecular variance (AMOVA) demonstrated a pronounced genetic variation of F. arundinacea endophytes between countries and within the Italian and Spanish locations. Sampling strategy of endophyte‐infected Festuca spp. was evaluated by occurrence and genetic diversity. Sampling a large number of plants within locations for each of the “European geographical subgroups” is the suggested strategy for obtaining a genetically diverse array of Neotyphodium endophytes.

The lack of a gene marker directly affecting starch biosynthesis in the potato tuber is documented. The absence of a 454 bp amplified fragment length polymorphism (AFLP) Solanum tuberosum fragment was identified in the wild potato species Solanum sandemanii and its absence results in a combined increased-amylose/high-sugar tuber chemotype. The trait is recessive, termed IAm (Increased Amylose) and was transferred to modern tetraploid S. tuberosum potato cultivars by marker-assisted crossing. Compared to controls, IAm plants had a larger number of stems and air exposed stolons, their tubers were smaller, elongated, and they were irregularly shaped. IAm starch had 28-59% higher amylose content than control starch, the starch granules were small and grossly misshaped, they had reduced crystallinity, swelling, and viscosity, reduced in vitro digestion rates with increased resistant starch fraction. The primary gene(s) responsible for the IAm phenotype is not known, but increased granule-associated phosphorylase (Pho1) and reduced starch synthase (SS) protein and enzyme activity in the IAm plants might explain the effects on starch structure. The data support the establishment of non-genetically modified crops with health-related slowly digestible carbohydrate.

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