Metabolite profiling and quantitative genetics of natural variation for flavonoids in Arabidopsis

Little is known about the range and the genetic bases of naturally occurring variation for flavonoids. Using Arabidopsis thaliana seed as a model, the flavonoid content of 41 accessions and two recombinant inbred line (RIL) sets derived from divergent accessions (Cvi-0×Col-0 and Bay-0×Shahdara) were analysed. These accessions and RILs showed mainly quantitative rather than qualitative changes. To dissect the genetic architecture underlying these differences, a quantitative trait locus (QTL) analysis was performed on the two segregating populations. Twenty-two flavonoid QTLs were detected that accounted for 11–64% of the observed trait variations, only one QTL being common to both RIL sets. Sixteen of these QTLs were confirmed and coarsely mapped using heterogeneous inbred families (HIFs). Three genes, namely TRANSPARENT TESTA (TT)7, TT15, and MYB12, were proposed to underlie their variations since the corresponding mutants and QTLs displayed similar specific flavonoid changes. Interestingly, most loci did not co-localize with any gene known to be involved in flavonoid metabolism. This latter result shows that novel functions have yet to be characterized and paves the way for their isolation

Complexity and robustness of the flavonoid transcriptional regulatory network revealed by comprehensive analyses of MYB-bHLH-WDR complexes and their targets in Arabidopsis seed.

In Arabidopsis thaliana, proanthocyanidins (PAs) accumulate in the innermost cell layer of the seed coat (i.e. endothelium, chalaza and micropyle). The expression of the biosynthetic genes involved relies on the transcriptional activity of R2R3-MYB and basic helix-loop-helix (bHLH) proteins which form ternary complexes (\u27MBW\u27) with TRANSPARENT TESTA GLABRA1 (TTG1) (WD repeat protein). The identification of the direct targets and the determination of the nature and spatio-temporal activity of these MBW complexes are essential steps towards a comprehensive understanding of the transcriptional mechanisms that control flavonoid biosynthesis. In this study, various molecular, genetic and biochemical approaches were used. Here, we have demonstrated that, of the 12 studied genes of the pathway, only dihydroflavonol-4-reductase (DFR), leucoanthocyanidin dioxygenase (LDOX), BANYULS (BAN), TRANSPARENT TESTA 19 (TT19), TT12 and H(+) -ATPase isoform 10 (AHA10) are direct targets of the MBW complexes. Interestingly, although the TT2-TT8-TTG1 complex plays the major role in developing seeds, three additional MBW complexes (i.e. MYB5-TT8-TTG1, TT2-EGL3-TTG1 and TT2-GL3-TTG1) were also shown to be involved, in a tissue-specific manner. Finally, a minimal promoter was identified for each of the target genes of the MBW complexes. Altogether, by answering fundamental questions and by demonstrating or invalidating previously made hypotheses, this study provides a new and comprehensive view of the transcriptional regulatory mechanisms controlling PA and anthocyanin biosynthesis in Arabidopsis

A new system for fast and quantitative analysis of heterologous gene expression in plants

Large-scale analysis of transcription factor–cis-acting element interactions in plants, or the dissection of complex transcriptional regulatory mechanisms, requires rapid, robust and reliable systems for the quantification of gene expression.Here, we describe a new system for transient expression analysis of transcription factors, which takes advantage of the fast and easy production and transfection of Physcomitrella patens protoplasts, coupled to flow cytometry quantification of a fluorescent protein (green fluorescent protein). Two small-sized and high-copy Gateway® vectors were specifically designed, although standard binary vectors can also be employed. As a proof of concept, the regulation of BANYULS (BAN), a key structural gene involved in proanthocyanidin biosynthesis in Arabidopsis thaliana seeds, was used. In P. patens, BAN expression is activated by a complex composed of three proteins (TT2/AtMYB123, TT8/bHLH042 and TTG1), and is inhibited by MYBL2, a transcriptional repressor, as in Arabidopsis. Using this approach, two new regulatory sequences that are necessary and sufficient for specific BAN expression in proanthocyanidin-accumulating cells were identified. This one hybrid-like plant system was successfully employed to quantitatively assess the transcriptional activity of four regulatory proteins, and to identify their target recognition sites on the BAN promoter

Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein

BACKGROUND: Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1. RESULTS: A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named Ank(GAG)1D4 (16.5 kDa) was isolated. Ank(GAG)1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of K(d) ~ 1 muM, and the Ank(GAG)1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing Ank(GAG)1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. Ank(GAG)1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The Ank(GAG)1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of Ank(GAG)1D4-CA with the Gag assembly and budding pathway. CONCLUSIONS: The resistance of Ank(GAG)1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin Ank(GAG)1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules

Directed evolution of artificial repeat proteins as habit modifiers for the morphosynthesis of (111)-terminated gold nanocrystals

Natural biocomposites are shaped by proteins that have evolved to interact with inorganic materials. Protein directed evolution methods which mimic Darwinian evolution have proven highly successful to generate improved enzymes or therapeutic antibodies but have rarely been used to evolve protein–material interactions. Indeed, most reported studies have focused on short peptides and a wide range of oligopeptides with chemical binding affinity for inorganic materials have been uncovered by phage display methods. However, their small size and flexible unfolded structure prevent them from dictating the shape and crystallinity of the growing material. In the present work, a specific set of artificial repeat proteins (αRep), which exhibit highly stable 3D folding with a well-defined hypervariable interacting surface, is selected by directed evolution of a very efficient home-built protein library for their high and selective affinity for the Au(111) surface. The proteins are built from the extendable concatenation of self-compatible repeated motifs idealized from natural HEAT proteins. The high-yield synthesis of Au(111)-faceted nanostructures mediated by these αRep proteins demonstrates their chemical affinity and structural selectivity that endow them with high crystal habit modification performances. Importantly, we further exploit the protein shell spontaneously assembled on the nanocrystal facets to drive protein-mediated colloidal self-assembly and on-surface enzymatic catalysis. Our method constitutes a generic tool for producing nanocrystals with determined faceting, superior biocompatibility and versatile bio-functionalization towards plasmon-based devices and (bio)molecular sensors

Functionalized artificial bidomain proteins based on an α-solenoid protein repeat scaffold : a new class of artificial diels-alderases

This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.αRep is a family of entirely artificial repeat proteins. Within the previously described αRep library, some variants are homodimers displaying interdomain cavities. Taking advantage of these properties, one of these homodimers called αRep A3 was converted into entirely artificial single chain bidomain metalloenzymes. A nonmutated A3 domain was covalently linked with an A3' domain bearing a unique cysteine on a chosen mutated position (F119C or Y26C). This single mutation ensured the covalent coupling of a 1:1 copper(II)/phenanthroline or copper(II)/terpyridine complex as a catalytic center within the interdomain cavity which was maintained large enough to accommodate two substrates of the Diels-Alder (D-A) reaction. This allowed us to obtain four new artificial Diels-Alderases that were fully characterized by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, UV-vis spectroscopy, and size exclusion chromatography analyses and were then further used for the catalysis of the D-A reaction. They were found to be able to catalyze the enantioselective D-A reaction of azachalcone with cyclopentadiene with up to 38% yield and 52% enantiomeric excess, which validates the proposed strategy. Moreover, the data were rationalized with a computational strategy suggesting the key factors of the selectivity. These results suggest that artificial metalloenzymes based on bidomain A3-A3 proteins modified with nitrogen donor ligands may be suitable for further catalyst optimization and may constitute valuable tools toward more efficient and selective artificial biocatalysts

Yellow Fever Outbreak, Southern Sudan, 2003

In May 2003, an outbreak of fatal hemorrhagic fever, caused by yellow fever virus, occurred in southern Sudan. Phylogenetic analysis showed that the virus belonged to the East African genotype, which supports the contention that yellow fever is endemic in East Africa with the potential to cause large outbreaks in humans