Lotus japonicus flowers are defended by a cyanogenic ß-glucosidase with highly restricted expression to essential reproductive organs.

Flowers and leaves of Lotus japonicus contain α-, ß-, and γ-hydroxynitrile glucoside (HNG) defense compounds, which are bioactivated by ß-glucosidase enzymes (BGDs). The α-HNGs are referred to as cyanogenic glucosides because their hydrolysis upon tissue disruption leads to release of toxic hydrogen cyanide gas, which can deter herbivore feeding. BGD2 and BGD4 are HNG metabolizing BGD enzymes expressed in leaves. Only BGD2 is able to hydrolyse the α-HNGs. Loss of function mutants of BGD2 are acyanogenic in leaves but fully retain cyanogenesis in flowers pointing to the existence of an alternative cyanogenic BGD in flowers. This enzyme, named BGD3, is identified and characterized in this study. Whereas all floral tissues contain α-HNGs, only those tissues in which BGD3 is expressed, the keel and the enclosed reproductive organs, are cyanogenic. Biochemical analysis, active site architecture molecular modelling, and the observation that L. japonicus accessions lacking cyanogenic flowers contain a non-functional BGD3 gene, all support the key role of BGD3 in floral cyanogenesis. The nectar of L. japonicus flowers was also found to contain HNGs and additionally their diglycosides. The observed specialisation in HNG based defence in L. japonicus flowers is discussed in the context of balancing the attraction of pollinators with the protection of reproductive structures against herbivores.

The evolutionary appearance of non-cyanogenic hydroxynitrile glucosides in the Lotus genus is accompanied by the substrate specialization of paralogous ß-glucosidases resulting from a crucial amino acid substitution.

Lotus japonicus, like several other legumes, biosynthesizes the cyanogenic α-hydroxynitrile glucosides lotaustralin and linamarin. Upon tissue disruption these compounds are hydrolysed by a specific ß-glucosidase, resulting in the release of hydrogen cyanide. Lotus japonicus also produces the non-cyanogenic γ- and ß-hydroxynitrile glucosides rhodiocyanoside A and D using a biosynthetic pathway that branches off from lotaustralin biosynthesis. We previously established that BGD2 is the only ß-glucosidase responsible for cyanogenesis in leaves. Here we show that the paralogous BGD4 has the dominant physiological role in rhodiocyanoside degradation. Structural modelling, site-directed mutagenesis and activity assays establish that a glycine residue (G211) in the aglycone binding site of BGD2 is essential for its ability to hydrolyse the endogenous cyanogenic glucosides. The corresponding valine (V211) in BGD4 narrows the active site pocket, resulting in the exclusion of non-flat substrates such as lotaustralin and linamarin, but not of the more planar rhodiocyanosides. Rhodiocyanosides and the BGD4 gene only occur in L. japonicus and a few closely related species associated with the Lotus corniculatus clade within the Lotus genus. This suggests the evolutionary scenario that substrate specialization for rhodiocyanosides evolved from a promiscuous activity of a progenitor cyanogenic ß-glucosidase, resembling BGD2, and required no more than a single amino acid substitution.

Towards a molecular understanding of the biosynthesis of amaryllidaceae alkaloids in support of their expanding medical use.

The alkaloids characteristically produced by the subfamily Amaryllidoideae of the Amaryllidaceae, bulbous plant species that include well know genera such as Narcissus (daffodils) and Galanthus (snowdrops), are a source of new pharmaceutical compounds. Presently, only the Amaryllidaceae alkaloid galanthamine, an acetylcholinesterase inhibitor used to treat symptoms of Alzheimer's disease, is produced commercially as a drug from cultivated plants. However, several Amaryllidaceae alkaloids have shown great promise as anti-cancer drugs, but their further clinical development is restricted by their limited commercial availability. Amaryllidaceae species have a long history of cultivation and breeding as ornamental bulbs, and phytochemical research has focussed on the diversity in alkaloid content and composition. In contrast to the available pharmacological and phytochemical data, ecological, physiological and molecular aspects of the Amaryllidaceae and their alkaloids are much less explored and the identity of the alkaloid biosynthetic genes is presently unknown. An improved molecular understanding of Amaryllidaceae alkaloid biosynthesis would greatly benefit the rational design of breeding programs to produce cultivars optimised for the production of pharmaceutical compounds and enable biotechnology based approaches.

Identification of key amino acids for the evolution of promoter target specificity of anthocyanin and proanthocyanidin regulating MYB factors.

A complex of R2R3-MYB and bHLH transcription factors, stabilized by WD40 repeat proteins, regulates gene transcription for plant cell pigmentation and epidermal cell morphology. It is the MYB component of this complex which specifies promoter target activation. The Arabidopsis MYB TT2 regulates proanthocyanidin (PA) biosynthesis by activating the expression of ANR (anthocyanidin reductase), the gene product of which catalyzes the first committed step of this pathway. Conversely the closely related MYB PAP4 (AtMYB114) regulates the anthocyanin pathway and specifically activates UFGT (UDP-glucose:flavonoid-3-O-glucosyltransferase), encoding the first enzyme of the anthocyanin pathway. Both at the level of structural and regulatory genes, evolution of PA biosynthesis proceeded anthocyanin biosynthesis and we have identified key residues in these MYB transcription factors for the evolution of target promoter specificity. Using chimeric and point mutated variants of TT2 and PAP4 we found that exchange of a single amino acid, Gly/Arg(39) in the R2 domain combined with an exchange of a four amino acid motif in the R3 domain, could swap the pathway selection of TT2 and PAP4, thereby converting in planta specificity of the PA towards the anthocyanin pathway and vice versa. The general importance of these amino acids for target specificity was also shown for the grapevine transcription factors VvMYBPA2 and VvMYBA2 which regulate PAs and anthocyanins, respectively. These results provide an insight into the evolution of the different flavonoid regulators from a common ancestral gene.

Why biosynthetic genes for chemical defense compounds cluster.

In plants, the genomic clustering of non-homologous genes for the biosynthesis of chemical defense compounds is an emerging theme. Gene clustering is also observed for polymorphic sexual traits under balancing selection, and examples in plants are self-incompatibility and floral dimorphy. The chemical defense pathways organized as gene clusters are self-contained biosynthetic modules under opposing selection pressures and adaptive polymorphisms, often the presence or absence of a functional pathway, are observed in nature. We propose that these antagonistic selection pressures favor closer physical linkage between beneficially interacting alleles as the resulting reduction in recombination maintains a larger fraction of the fitter genotypes. Gene clusters promote the stable inheritance of functional chemical defense pathways in the dynamic ecological context of natural populations.

Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway.

Cyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific ß-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.

The grapevine transcription factor VvMYBPA1 regulates proanthocyanidin synthesis during fruit development.

Proanthocyanidins (PAs; or condensed tannins) can protect plants against herbivores, contribute to the taste of many fruits, and act as dietary antioxidants beneficial for human health. We have previously shown that in grapevine (Vitis vinifera) PA synthesis involves both leucoanthocyanidin reductase (LAR) and anthocyanidin reductase (ANR). Here we report the characterization of a grapevine MYB transcription factor VvMYBPA1, which controls expression of PA pathway genes including both LAR and ANR. Expression of VvMYBPA1 in grape berries correlated with PA accumulation during early berry development and in seeds. In a transient assay, VvMYBPA1 activated the promoters of LAR and ANR, as well as the promoters of several of the general flavonoid pathway genes. VvMYBPA1 did not activate the promoter of VvUFGT, which encodes the anthocyanin-specific enzyme UDP-glucose:flavonoid-3-O-glucosyltransferase, suggesting VvMYBPA1 is specific to regulation of PA biosynthesis in grapes. The Arabidopsis (Arabidopsis thaliana) MYB transcription factor TRANSPARENT TESTA2 (TT2) regulates PA synthesis in the seed coat of Arabidopsis. By complementing the PA-deficient seed phenotype of the Arabidopsis tt2 mutant with VvMYBPA1, we confirmed the function of VvMYBPA1 as a transcriptional regulator of PA synthesis. In contrast to ectopic expression of TT2 in Arabidopsis, constitutive expression of VvMYBPA1 resulted in accumulation of PAs in cotyledons, vegetative meristems, leaf hairs, and roots in some of the transgenic seedlings. To our knowledge, this is the first report of a MYB factor that controls genes of the PA pathway in fruit, including both LAR and ANR, and this single MYB factor can induce ectopic PA accumulation in Arabidopsis.

Light-induced expression of a MYB gene regulates anthocyanin biosynthesis in red apples.

Anthocyanins are secondary metabolites found in higher plants that contribute to the colors of flowers and fruits. In apples (Malus domestica Borkh.), several steps of the anthocyanin pathway are coordinately regulated, suggesting control by common transcription factors. A gene encoding an R2R3 MYB transcription factor was isolated from apple (cv Cripps' Pink) and designated MdMYB1. Analysis of the deduced amino acid sequence suggests that this gene encodes an ortholog of anthocyanin regulators in other plants. The expression of MdMYB1 in both Arabidopsis (Arabidopsis thaliana) plants and cultured grape cells induced the ectopic synthesis of anthocyanin. In the grape (Vitis vinifera) cells MdMYB1 stimulated transcription from the promoters of two apple genes encoding anthocyanin biosynthetic enzymes. In ripening apple fruit the transcription of MdMYB1 was correlated with anthocyanin synthesis in red skin sectors of fruit. When dark-grown fruit were exposed to sunlight, MdMYB1 transcript levels increased over several days, correlating with anthocyanin synthesis in the skin. MdMYB1 gene transcripts were more abundant in red skin apple cultivars compared to non-red skin cultivars. Several polymorphisms were identified in the promoter of MdMYB1. A derived cleaved amplified polymorphic sequence marker designed to one of these polymorphisms segregated with the inheritance of skin color in progeny from a cross of an unnamed red skin selection (a sibling of Cripps' Pink) and the non-red skin cultivar Golden Delicious. We conclude that MdMYB1 coordinately regulates genes in the anthocyanin pathway and the expression level of this regulator is the genetic basis for apple skin color.