Vlaams Instituut voor Biotechnologie

PlantCARE is a database of plant cis-acting regulatory elements, enhancers and repressors. Regulatory elements are represented by positional matrices, consensus sequences and individual sites on particular promoter sequences. Links to the EMBL, TRANSFAC and MEDLINE databases are provided when available. Data about the transcription sites are extracted mainly from the literature, supplemented with an increasing number of in silico predicted data. Apart from a general description for specific transcription factor sites, levels of confidence for the experimental evidence, functional information and the position on the promoter are given as well. New features have been implemented to search for plant cis-acting regulatory elements in a query sequence. Furthermore, links are now provided to a new clustering and motif search method to investigate clusters of co-expressed genes. New regulatory elements can be sent automatically and will be added to the database after curation. The PlantCARE relational database is available via the World Wide Web at http://sphinx.rug.ac.be:8080/PlantCARE/.

The lignin biosynthetic pathway has been studied for more than a century but has undergone major revisions over the past decade. Significant progress has been made in cloning new genes by genetic and combined bioinformatics and biochemistry approaches. In vitro enzymatic assays and detailed analyses of mutants and transgenic plants altered in the expression of lignin biosynthesis genes have provided a solid basis for redrawing the monolignol biosynthetic pathway, and structural analyses have shown that plant cell walls can tolerate large variations in lignin content and structure. In some cases, the potential value for agriculture of transgenic plants with modified lignin structure has been demonstrated. This review presents a current picture of monolignol biosynthesis, polymerization, and lignin structure.

FoldX is an empirical force field that was developed for the rapid evaluation of the effect of mutations on the stability, folding and dynamics of proteins and nucleic acids. The core functionality of FoldX, namely the calculation of the free energy of a macromolecule based on its high-resolution 3D structure, is now publicly available through a web server at http://foldx.embl.de/. The current release allows the calculation of the stability of a protein, calculation of the positions of the protons and the prediction of water bridges, prediction of metal binding sites and the analysis of the free energy of complex formation. Alanine scanning, the systematic truncation of side chains to alanine, is also included. In addition, some reporting functions have been added, and it is now possible to print both the atomic interaction networks that constitute the protein, print the structural and energetic details of the interactions per atom or per residue, as well as generate a general quality report of the pdb structure. This core functionality will be further extended as more FoldX applications are developed.

Sugars not only fuel cellular carbon and energy metabolism but also play pivotal roles as signaling molecules. The experimental amenability of yeast as a unicellular model system has enabled the discovery of multiple sugar sensors and signaling pathways. In plants, different sugar signals are generated by photosynthesis and carbon metabolism in source and sink tissues to modulate growth, development, and stress responses. Genetic analyses have revealed extensive interactions between sugar and plant hormone signaling, and a central role for hexokinase (HXK) as a conserved glucose sensor. Diverse sugar signals activate multiple HXK-dependent and HXK-independent pathways and use different molecular mechanisms to control transcription, translation, protein stability and enzymatic activity. Important and complex roles for Snf1-related kinases (SnRKs), extracellular sugar sensors, and trehalose metabolism in plant sugar signaling are now also emerging.

Myelodysplastic syndromes (MDS) are a heterogeneous group of chronic hematological malignancies characterized by dysplasia, ineffective hematopoiesis and a variable risk of progression to acute myeloid leukemia. Sequencing of MDS genomes has identified mutations in genes implicated in RNA splicing, DNA modification, chromatin regulation, and cell signaling. We sequenced 111 genes across 738 patients with MDS or closely related neoplasms (including chronic myelomonocytic leukemia and MDS-myeloproliferative neoplasms) to explore the role of acquired mutations in MDS biology and clinical phenotype. Seventy-eight percent of patients had 1 or more oncogenic mutations. We identify complex patterns of pairwise association between genes, indicative of epistatic interactions involving components of the spliceosome machinery and epigenetic modifiers. Coupled with inferences on subclonal mutations, these data suggest a hypothesis of genetic "predestination," in which early driver mutations, typically affecting genes involved in RNA splicing, dictate future trajectories of disease evolution with distinct clinical phenotypes. Driver mutations had equivalent prognostic significance, whether clonal or subclonal, and leukemia-free survival deteriorated steadily as numbers of driver mutations increased. Thus, analysis of oncogenic mutations in large, well-characterized cohorts of patients illustrates the interconnections between the cancer genome and disease biology, with considerable potential for clinical application.

The cytokine interleukin-10 (IL-10) has shown promise in clinical trials for treatment of inflammatory bowel disease (IBD). Using two mouse models, we show that the therapeutic dose of IL-10 can be reduced by localized delivery of a bacterium genetically engineered to secrete the cytokine. Intragastric administration of IL-10-secreting Lactococcus lactis caused a 50% reduction in colitis in mice treated with dextran sulfate sodium and prevented the onset of colitis in IL-10(-/-) mice. This approach may lead to better methods for cost-effective and long-term management of IBD in humans.

Humans are unable to synthesise L-ascorbic acid (L-AA, ascorbate, vitamin C), and are thus entirely dependent upon dietary sources to meet needs. In both plant and animal metabolism, the biological functions of L-ascorbic acid are centred around the antioxidant properties of this molecule. Considerable evidence has been accruing in the last two decades of the importance of L-AA in protecting not only the plant from oxidative stress, but also mammals from various chronic diseases that have their origins in oxidative stress. Evidence suggests that the plasma levels of L-AA in large sections of the population are sub-optimal for the health protective effects of this vitamin. Until quite recently, little focus has been given to improving the L-AA content of plant foods, either in terms of the amounts present in commercial crop varieties, or in minimising losses prior to ingestion. Further, while L-AA biosynthesis in animals was elucidated in the 1960s,1 it is only very recently that a distinct biosynthetic route for plants has been proposed.2 The characterisation of this new pathway will undoubtedly provide the necessary focus and impetus to enable fundamental questions on plant L-AA metabolism to be resolved. This review focuses on the role of L-AA in metabolism and the latest studies regarding its biosynthesis, tissue compartmentalisation, turnover and catabolism. These inter-relationships are considered in relation to the potential to improve the L-AA content of crops. Methodology for the reliable analysis of L-AA in plant foods is briefly reviewed. The concentrations found in common food sources and the effects of processing, or storage prior to consumption are discussed. Finally the factors that determine the bioavailability of L-AA and how it may be improved are considered, as well as the most important future research needs. © 2000 Society of Chemical Industry

The filamentous fungus Aspergillus niger is widely exploited by the fermentation industry for the production of enzymes and organic acids, particularly citric acid. We sequenced the 33.9-megabase genome of A. niger CBS 513.88, the ancestor of currently used enzyme production strains. A high level of synteny was observed with other aspergilli sequenced. Strong function predictions were made for 6,506 of the 14,165 open reading frames identified. A detailed description of the components of the protein secretion pathway was made and striking differences in the hydrolytic enzyme spectra of aspergilli were observed. A reconstructed metabolic network comprising 1,069 unique reactions illustrates the versatile metabolism of A. niger. Noteworthy is the large number of major facilitator superfamily transporters and fungal zinc binuclear cluster transcription factors, and the presence of putative gene clusters for fumonisin and ochratoxin A synthesis.

Reactive oxygen species (ROS) are known as toxic metabolic products in plants and other aerobic organisms. An elaborate and highly redundant plant ROS network, composed of antioxidant enzymes, antioxidants and ROS-producing enzymes, is responsible for maintaining ROS levels under tight control. This allows ROS to serve as signaling molecules that coordinate an astonishing range of diverse plant processes. The specificity of the biological response to ROS depends on the chemical identity of ROS, intensity of the signal, sites of production, plant developmental stage, previous stresses encountered and interactions with other signaling molecules such as nitric oxide, lipid messengers and plant hormones. Although many components of the ROS signaling network have recently been identified, the challenge remains to understand how ROS-derived signals are integrated to eventually regulate such biological processes as plant growth, development, stress adaptation and programmed cell death.

Measuring the dynamics of neural processing across time scales requires following the spiking of thousands of individual neurons over milliseconds and months. To address this need, we introduce the Neuropixels 2.0 probe together with newly designed analysis algorithms. The probe has more than 5000 sites and is miniaturized to facilitate chronic implants in small mammals and recording during unrestrained behavior. High-quality recordings over long time scales were reliably obtained in mice and rats in six laboratories. Improved site density and arrangement combined with newly created data processing methods enable automatic post hoc correction for brain movements, allowing recording from the same neurons for more than 2 months. These probes and algorithms enable stable recordings from thousands of sites during free behavior, even in small animals such as mice.

Receptor-interacting protein kinase 3 (RIPK3)-mediated necroptosis is thought to be the pathophysiologically predominant pathway that leads to regulated necrosis of parenchymal cells in ischemia-reperfusion injury (IRI), and loss of either Fas-associated protein with death domain (FADD) or caspase-8 is known to sensitize tissues to undergo spontaneous necroptosis. Here, we demonstrate that renal tubules do not undergo sensitization to necroptosis upon genetic ablation of either FADD or caspase-8 and that the RIPK1 inhibitor necrostatin-1 (Nec-1) does not protect freshly isolated tubules from hypoxic injury. In contrast, iron-dependent ferroptosis directly causes synchronized necrosis of renal tubules, as demonstrated by intravital microscopy in models of IRI and oxalate crystal-induced acute kidney injury. To suppress ferroptosis in vivo, we generated a novel third-generation ferrostatin (termed 16-86), which we demonstrate to be more stable, to metabolism and plasma, and more potent, compared with the first-in-class compound ferrostatin-1 (Fer-1). Even in conditions with extraordinarily severe IRI, 16-86 exerts strong protection to an extent which has not previously allowed survival in any murine setting. In addition, 16-86 further potentiates the strong protective effect on IRI mediated by combination therapy with necrostatins and compounds that inhibit mitochondrial permeability transition. Renal tubules thus represent a tissue that is not sensitized to necroptosis by loss of FADD or caspase-8. Finally, ferroptosis mediates postischemic and toxic renal necrosis, which may be therapeutically targeted by ferrostatins and by combination therapy.

An important mechanism for the evolution of phenotypic complexity, diversity and innovation, and the origin of novel gene functions is the duplication of genes and entire genomes. Recent phylogenomic studies suggest that, during the evolution of vertebrates, the entire genome was duplicated in two rounds (2R) of duplication. Later, approximately 350 mya, in the stem lineage of ray-finned (actinopterygian) fishes, but not in that of the land vertebrates, a third genome duplication occurred-the fish-specific genome duplication (FSGD or 3R), leading, at least initially, to up to eight copies of the ancestral deuterostome genome. Therefore, the sarcopterygian (lobe-finned fishes and tetrapods) genome possessed originally only half as many genes compared to the derived fishes, just like the most-basal and species-poor lineages of extant fishes that diverged from the fish stem lineage before the 3R duplication. Most duplicated genes were secondarily lost, yet some evolved new functions. The genomic complexity of the teleosts might be the reason for their evolutionary success and astounding biological diversity.

The fungus Laccaria bicolor — seen in its above-ground fruiting body presence as the 'bicoloured deceiver' mushroom — lives symbiotically on the roots of trees. Its genome has now been sequenced, and the key features of the genome characterized by transcript profiling. The study throws light on the mechanism of mycorrhizal symbiosis, the union of roots and soil fungi that is of vital important to plant productivity. And it will be of keen interest to evolutionary and plant biologists for its revelations about plant–fungus interactions shaping genomes over time. The genome of the fungus Laccaria bicolor is described; it is of keen interest to evolutionary and plant biologists for its revelations about plant–fungus interactions shaping genomes over time. Mycorrhizal symbioses—the union of roots and soil fungi—are universal in terrestrial ecosystems and may have been fundamental to land colonization by plants1,2. Boreal, temperate and montane forests all depend on ectomycorrhizae1. Identification of the primary factors that regulate symbiotic development and metabolic activity will therefore open the door to understanding the role of ectomycorrhizae in plant development and physiology, allowing the full ecological significance of this symbiosis to be explored. Here we report the genome sequence of the ectomycorrhizal basidiomycete Laccaria bicolor (Fig. 1) and highlight gene sets involved in rhizosphere colonization and symbiosis. This 65-megabase genome assembly contains ∼20,000 predicted protein-encoding genes and a very large number of transposons and repeated sequences. We detected unexpected genomic features, most notably a battery of effector-type small secreted proteins (SSPs) with unknown function, several of which are only expressed in symbiotic tissues. The most highly expressed SSP accumulates in the proliferating hyphae colonizing the host root. The ectomycorrhizae-specific SSPs probably have a decisive role in the establishment of the symbiosis. The unexpected observation that the genome of L. bicolor lacks carbohydrate-active enzymes involved in degradation of plant cell walls, but maintains the ability to degrade non-plant cell wall polysaccharides, reveals the dual saprotrophic and biotrophic lifestyle of the mycorrhizal fungus that enables it to grow within both soil and living plant roots. The predicted gene inventory of the L. bicolor genome, therefore, points to previously unknown mechanisms of symbiosis operating in biotrophic mycorrhizal fungi. The availability of this genome provides an unparalleled opportunity to develop a deeper understanding of the processes by which symbionts interact with plants within their ecosystem to perform vital functions in the carbon and nitrogen cycles that are fundamental to sustainable plant productivity.

Brassinosteroids (BRs) are a group of polyhydroxylated plant steroid hormones that are crucial for many aspects of a plant's life. BRs were originally characterized for their function in cell elongation, but it is becoming clear that they play major roles in plant growth, development, and responses to several stresses such as extreme temperatures and drought. A BR signaling pathway from cell surface receptors to central transcription factors has been well characterized. Here, we summarize recent progress toward understanding the BR pathway, including BR perception and the molecular mechanisms of BR signaling. Next, we discuss the roles of BRs in development and stress responses. Finally, we show how knowledge of the BR pathway is being applied to manipulate the growth and stress responses of crops. These studies highlight the complex regulation of BR signaling, multiple points of crosstalk between BRs and other hormones or stress responses, and the finely tuned spatiotemporal regulation of BR signaling.

The basic molecular mechanisms governing how endothelial cells, periendothelial cells and matrix molecules interact with each other and with numerous growth factors and receptors, to form blood vessels have been presented. The many insights gained from this basic knowledge are being extended to further understand pathological angiogenesis associated with disorders such as arterial stenosis, myocardial ischemia, atherosclerosis, allograft transplant stenosis. wound healing and tissue repair. As a result, novel angiogenic and anti-angiogenic molecules are rapid-ly entering the clinic, with the promise of relief from a host of medical disorders.

▪ Abstract Over 35 years ago, Susumu Ohno stated that gene duplication was the single most important factor in evolution ( 97 ). He reiterated this point a few years later in proposing that without duplicated genes the creation of metazoans, vertebrates, and mammals from unicellular organisms would have been impossible. Such big leaps in evolution, he argued, required the creation of new gene loci with previously nonexistent functions ( 98 ). Bold statements such as these, combined with his proposal that at least one whole-genome duplication event facilitated the evolution of vertebrates, have made Ohno an icon in the literature on genome evolution. However, discussion on the occurrence and consequences of gene and genome duplication events has a much longer, and often neglected, history. Here we review literature dealing with the occurence and consequences of gene duplication, begining in 1911. We document conceptual and technological advances in gene duplication research from this early research in comparative cytology up to recent research on whole genomes, “transcriptomes,” and “interactomes.” We have formerly seen that parts many times repeated are eminently liable to vary in number and structure; consequently it is quite probable that natural selection, during the long-continued course of modification, should have seized on a certain number of the primordially similar elements, many times repeated, and have adapted them to the most diverse purposes. Charles Darwin, 1859 ( 23 )

All ligands of the epidermal growth factor receptor (EGFR), which has important roles in development and disease, are released from the membrane by proteases. In several instances, ectodomain release is critical for activation of EGFR ligands, highlighting the importance of identifying EGFR ligand sheddases. Here, we uncovered the sheddases for six EGFR ligands using mouse embryonic cells lacking candidate-releasing enzymes (a disintegrin and metalloprotease [ADAM] 9, 10, 12, 15, 17, and 19). ADAM10 emerged as the main sheddase of EGF and betacellulin, and ADAM17 as the major convertase of epiregulin, transforming growth factor alpha, amphiregulin, and heparin-binding EGF-like growth factor in these cells. Analysis of adam9/12/15/17-/- knockout mice corroborated the essential role of adam17-/- in activating the EGFR in vivo. This comprehensive evaluation of EGFR ligand shedding in a defined experimental system demonstrates that ADAMs have critical roles in releasing all EGFR ligands tested here. Identification of EGFR ligand sheddases is a crucial step toward understanding the mechanism underlying ectodomain release, and has implications for designing novel inhibitors of EGFR-dependent tumors.

Significance Plant roots nurture a large diversity of soil microbes via exudation of chemical compounds into the rhizosphere. In turn, beneficial root microbiota promote plant growth and immunity. The root-specific transcription factor MYB72 has emerged as a central regulator in this process. Here, we show that MYB72 regulates the excretion of the coumarin scopoletin, an iron-mobilizing phenolic compound with selective antimicrobial activity that shapes the root-associated microbial community. Selected soil-borne fungal pathogens appeared to be highly sensitive to the antimicrobial activity of scopoletin, while two MYB72- inducing beneficial rhizobacteria were tolerant. Our results suggest that probiotic root-associated microbes that activate the iron-deficiency response during colonization stimulate MYB72-dependent excretion of scopoletin, thereby potentially improving their niche establishment and enhancing plant growth and protection.

Recent analysis of complete eukaryotic genome sequences has revealed that gene duplication has been rampant. Moreover, next to a continuous mode of gene duplication, in many eukaryotic organisms the complete genome has been duplicated in their evolutionary past. Such large-scale gene duplication events have been associated with important evolutionary transitions or major leaps in development and adaptive radiations of species. Here, we present an evolutionary model that simulates the duplication dynamics of genes, considering genome-wide duplication events and a continuous mode of gene duplication. Modeling the evolution of the different functional categories of genes assesses the importance of different duplication events for gene families involved in specific functions or processes. By applying our model to the Arabidopsis genome, for which there is compelling evidence for three whole-genome duplications, we show that gene loss is strikingly different for large-scale and small-scale duplication events and highly biased toward certain functional classes. We provide evidence that some categories of genes were almost exclusively expanded through large-scale gene duplication events. In particular, we show that the three whole-genome duplications in Arabidopsis have been directly responsible for >90% of the increase in transcription factors, signal transducers, and developmental genes in the last 350 million years. Our evolutionary model is widely applicable and can be used to evaluate different assumptions regarding small- or large-scale gene duplication events in eukaryotic genomes.

Hydrogen peroxide (H(2)O(2)) is an important signal molecule involved in plant development and environmental responses. Changes in H(2)O(2) availability can result from increased production or decreased metabolism. While plants contain several types of H(2)O(2)-metabolizing proteins, catalases are highly active enzymes that do not require cellular reductants as they primarily catalyse a dismutase reaction. This review provides an update on plant catalase genes, function, and subcellular localization, with a focus on recent information generated from studies on Arabidopsis. Original data are presented on Arabidopsis catalase single and double mutants, and the use of some of these lines as model systems to investigate the outcome of increases in intracellular H(2)O(2) are discussed. Particular attention is paid to interactions with cell thiol-disulphide status; the use of catalase-deficient plants to probe the apparent redundancy of reductive H(2)O(2)-metabolizing pathways; the importance of irradiance and growth daylength in determining the outcomes of catalase deficiency; and the induction of pathogenesis-related responses in catalase-deficient lines. Within the context of strategies aimed at understanding and engineering plant stress responses, the review also considers whether changes in catalase activities in wild-type plants are likely to be a significant part of plant responses to changes in environmental conditions or biotic challenge.