TRANSPARENT TESTA1 interacts with R2R3-MYB factors and affects early and late steps of flavonoid biosynthesis in the endothelium of Arabidopsis thaliana seeds

Wild type seed coats of Arabidopsis thaliana are brown due to the accumulation of proanthocyanidin pigments (PAs). The pigmentation requires activation of phenylpropanoid biosynthesis genes and mutations in some of these genes cause a yellow appearance of seeds, termed transparent testa (tt) phenotype. The TT1 gene encodes a WIP‐type zinc finger protein and is expressed in the seed coat endothelium where most of the PAs accumulate in wild type plants. In this study we show that TT1 is not only required for correct expression of PA‐specific genes in the seed coat, but also affects CHS, encoding the first enzyme of flavonoid biosynthesis. Many steps of this pathway are controlled by complexes of MYB and BHLH proteins with the WD40 factor TTG1. We demonstrate that TT1 can interact with the R2R3 MYB protein TT2 and that ectopic expression of TT2 can partially restore the lack in PA production in tt1. Reduced seed coat pigmentation was obtained using a TT1 variant lacking nuclear localisation signals. Based on our results we propose that the TT2/TT8/TTG1 regulon may also comprise early genes like CHS and discuss steps to further unravel the regulatory network controlling flavonoid accumulation in endothelium cells during A. thaliana seed development.     

Identification of the Arabidopsis thaliana flavonoid 3'-hydroxylase gene and functional expression of the encoded P450 enzyme

The phenylpropanoid pathway results in the synthesis of thousands of compounds, including flavonoids like flavonols, anthocyanidins and tannins. In Arabidopsis thaliana, the lack of tannins in the seed coat (testa) causes the transparent testa (tt) phenotype. In the present study, we identified the gene responsible for the tt7 mutation. We show that TT7 encodes the enzyme flavonoid 3'-hydroxylase (F3'H), and demonstrate that this P450-dependent monooxygenase has F3'H activity. The availability of the AtF3'H gene and promoter sequence will allow us to study the coregulation of a complete set of flavonol and anthocyanidin biosynthesis genes in A. thaliana, and makes in vitro synthesis of hydroxylated flavonoids more feasible.     

Biochemical and genetic characterization of Arabidopsis flavanone 3beta-hydroxylase

Flavanone 3beta-hydroxylase (F3H; EC 1.14.11.9) is a 2-oxoglutarate dependent dioxygenase that catalyzes the synthesis of dihydrokaempferol, the common precursor for three major classes of 3-hydroxy flavonoids, the flavonols, anthocyanins, and proanthocyanidins. This enzyme also competes for flux into the 3-deoxy flavonoid branch pathway in some species. F3H genes are increasingly being used, often together with genes encoding other enzymes, to engineer flavonoid synthesis in microbes and plants. Although putative F3H genes have been cloned in a large number of plant species, only a handful have been functionally characterized. Here we describe the biochemical properties of the Arabidopsis thaliana F3H (AtF3H) enzyme and confirm the activities of gene products from four other plant species previously identified as having high homology to F3H. We have also investigated the surprising "leaky" phenotype of AtF3H mutant alleles, uncovering evidence that two related flavonoid enzymes, flavonol synthase (EC 1.14.11.23) and anthocyanidin synthase (EC 1.14.11.19), can partially compensate for F3H in vivo. These experiments further indicate that the absence of F3H in these lines enables the synthesis of uncommon 3-deoxy flavonoids in the Arabidopsis seed coat.     

Alfalfa Root Flavonoid Production Is Nitrogen Regulated

Flavonoids produced by legume roots are signal molecules acting both as chemoattractants and nod gene inducers for the symbiotic Rhizobium partner. Combined nitrogen inhibits the establishment of the symbiosis. To know whether nitrogen nutrition could act at the level of signal production, we have studied the expression of flavonoid biosynthetic genes as well as the production of flavonoids in the roots of plants grown under nitrogen-limiting or nonlimiting conditions. We show here that growth of the plant under nitrogen-limiting conditions results in the enhancement of expression of the flavonoid biosynthesis genes chalcone synthase and isoflavone reductase and in an increase of root flavonoid and isoflavonoid production as well as in the Rhizobium meliloti nod gene-inducing activity of the root extract. These results indicate that in alfalfa (Medicago sativa L.) roots, the production of flavonoids can be influenced by the nitrogen nutrition of the plant.     

Metabolic engineering and potential functions of proanthocyanidins in poplar

Poplar (Populus spp.) is a widely distributed tree genus of significant economic and ecological importance. Poplar trees accumulate proanthocyanidins (PAs) in leaves, roots, and a variety of other tissues. Damage to leaves by insects causes a rapid accumulation of PAs, both at the site of damage and distally in undamaged leaves. This rapid PA accumulation is mediated by the activation of genes encoding enzymes involved in PA synthesis. PAs have been hypothesized to deter insect feeding and reduce the nutritive value of poplar leaf tissue, but experimental evidence supporting a role for PAs as an effective inducible defense against herbivores is lacking. Our recent paper described the identification of a MYB gene that regulates the PA pathway under multiple stress conditions, and we used this gene to constitutively activate the PA pathway in poplar. Here we describe observations that suggest that poplar PAs may have roles besides insect defense, for example, responses to UV light. The PA-modified trees will be a useful tool for analyzing the biological roles of PAs in this important model tree.Key words: tannins, herbivory, flavonoid, UV light, light stress     

Evolution of pyrrolizidine alkaloids in Phalaenopsis orchids and other monocotyledons: Identification of deoxyhypusine synthase, homospermidine synthase and related pseudogenes

In order to study the evolution of pathways of plant secondary metabolism, we use the biosynthesis of pyrrolizidine alkaloids (PAs) as a model system. PAs are regarded as part of the plant’s constitutive defense against herbivores. Homospermidine synthase (HSS) is the first specific enzyme of PA biosynthesis. The gene encoding HSS has been recruited from the gene encoding deoxyhypusine synthase (DHS) from primary metabolism at least four times independently during angiosperm evolution. One of these recruitments occurred within the monocot lineage. We have used the PA-producing orchid Phalaenopsis to identify the cDNAs encoding HSS, DHS and the substrate protein for DHS, i.e., the precursor of the eukaryotic initiation factor 5A. A cDNA identified from maize was unequivocally characterized as DHS. From our study of Phalaenopsis, several pseudogenes emerged, of which one was shown to be a “processed pseudogene”, and others to be transcribed. Sequence comparison of the HSS- and DHS-encoding sequences from this investigation with those of monocot species taken from the databases suggest that HSS and probably the ability to produce PAs is an old feature within the monocot lineage. This result is discussed with respect to the recent discovery of structural related PAs within grasses.     

Recruitment of alkaloid-specific homospermidine synthase (HSS) from ubiquitous deoxyhypusine synthase: Does Crotalaria possess a functional HSS that still has DHS activity?

Quinolizidine alkaloids are the most prominent group of alkaloids occurring in legumes, except for many members of the tribe Crotalarieae that accumulate pyrrolizidine alkaloids (PAs). To study the evolution of PA biosynthesis as a typical pathway of plant secondary metabolism in this tribe, we have searched for a cDNA coding for homospermidine synthase (HSS), the enzyme catalyzing the first specific step in this biosynthesis. HSS was shown to have been recruited from deoxyhypusine synthase (DHS) by independent gene duplication in several different angiosperm lineages during evolution. Except for a cDNA sequence coding for the DHS of Crotalaria retusa, no data is available concerning the origin of PA biosynthesis within this tribe of the Fabaceae. In addition to several pseudogenes, we have identified one functional DHS in C. scassellatii and two in C. juncea. Despite C. juncea plants under study being devoid of PAs, we have found that the two sequences of C. juncea are different with respect to their genomic organization, their tissue-specific expression, and their biochemical activities. Supported by the branching pattern of a maximum likelihood analysis of these sequences, they have been classified as "class 1" and "class 2" DHS. It remains open whether the duplicated DHS belonging to class 2 is involved in the biosynthesis of PAs.     

Flower Color Modification by Engineering of the Flavonoid Biosynthetic Pathway: Practical Perspectives

The status quo of flavonoid biosynthesis as it relates to flower color is reviewed together with a success in modifying flower color by genetic engineering. Flavonoids and their colored class compounds, anthocyanins, are major contributors to flower color. Many plant species synthesize limited kinds of flavonoids, and thus exhibit a limited range of flower color. Since genes regulating flavonoid biosynthesis are available, it is possible to alter flower color by overexpressing heterologous genes and/or down regulating endogenous genes. Transgenic carnations and a transgenic rose that accumulate delphinidin as a result of expressing a flavonoid 3′,5′-hydroxylase gene and have novel blue hued flowers have been commercialized. Transgenic Nierembergia accumulating pelargonidin, with novel pink flowers, has also been developed. Although it is possible to generate white, yellow, and pink-flowered torenia plants from blue cultivars by genetic engineering, field trial observations indicate difficulty in obtaining stable phenotypes.