Diffany: an ontology-driven framework to infer, visualise and analyse differential molecular networks.

BACKGROUND: Differential networks have recently been introduced as a powerful way to study the dynamic rewiring capabilities of an interactome in response to changing environmental conditions or stimuli. Currently, such differential networks are generated and visualised using ad hoc methods, and are often limited to the analysis of only one condition-specific response or one interaction type at a time. RESULTS: In this work, we present a generic, ontology-driven framework to infer, visualise and analyse an arbitrary set of condition-specific responses against one reference network. To this end, we have implemented novel ontology-based algorithms that can process highly heterogeneous networks, accounting for both physical interactions and regulatory associations, symmetric and directed edges, edge weights and negation. We propose this integrative framework as a standardised methodology that allows a unified view on differential networks and promotes comparability between differential network studies. As an illustrative application, we demonstrate its usefulness on a plant abiotic stress study and we experimentally confirmed a predicted regulator. AVAILABILITY: Diffany is freely available as open-source java library and Cytoscape plugin from http://bioinformatics.psb.ugent.be/supplementary_data/solan/diffany/.

SAMBA, a plant-specific anaphase-promoting complex/cyclosome regulator is involved in early development and A-type cyclin stabilization.

The anaphase-promoting complex/cyclosome (APC/C) is a large multiprotein E3 ubiquitin ligase involved in ubiquitin-dependent proteolysis of key cell cycle regulatory proteins, including the destruction of mitotic cyclins at the metaphase-to-anaphase transition. Despite its importance, the role of the APC/C in plant cells and the regulation of its activity during cell division remain poorly understood. Here, we describe the identification of a plant-specific negative regulator of the APC/C complex, designated SAMBA. In Arabidopsis thaliana, SAMBA is expressed during embryogenesis and early plant development and plays a key role in organ size control. Samba mutants produced larger seeds, leaves, and roots, which resulted from enlarged root and shoot apical meristems, and, additionally, they had a reduced fertility attributable to a hampered male gametogenesis. Inactivation of SAMBA stabilized A2-type cyclins during early development. Our data suggest that SAMBA regulates cell proliferation during early development by targeting CYCLIN A2 for APC/C-mediated proteolysis.

Physiological and transcriptomic evidence for a close coupling between chloroplast ontogeny and cell cycle progression in the pennate diatom Seminavis robusta.

Despite the growing interest in diatom genomics, detailed time series of gene expression in relation to key cellular processes are still lacking. Here, we investigated the relationships between the cell cycle and chloroplast development in the pennate diatom Seminavis robusta. This diatom possesses two chloroplasts with a well-orchestrated developmental cycle, common to many pennate diatoms. By assessing the effects of induced cell cycle arrest with microscopy and flow cytometry, we found that division and reorganization of the chloroplasts are initiated only after S-phase progression. Next, we quantified the expression of the S. robusta FtsZ homolog to address the division status of chloroplasts during synchronized growth and monitored microscopically their dynamics in relation to nuclear division and silicon deposition. We show that chloroplasts divide and relocate during the S/G2 phase, after which a girdle band is deposited to accommodate cell growth. Synchronized cultures of two genotypes were subsequently used for a cDNA-amplified fragment length polymorphism-based genome-wide transcript profiling, in which 917 reproducibly modulated transcripts were identified. We observed that genes involved in pigment biosynthesis and coding for light-harvesting proteins were up-regulated during G2/M phase and cell separation. Light and cell cycle progression were both found to affect fucoxanthin-chlorophyll a/c-binding protein expression and accumulation of fucoxanthin cell content. Because chloroplasts elongate at the stage of cytokinesis, cell cycle-modulated photosynthetic gene expression and synthesis of pigments in concert with cell division might balance chloroplast growth, which confirms that chloroplast biogenesis in S. robusta is tightly regulated.

T-loop phosphorylation of Arabidopsis CDKA;1 is required for its function and can be partially substituted by an aspartate residue.

As in other eukaryotes, progression through the cell cycle in plants is governed by cyclin-dependent kinases. Phosphorylation of a canonical Thr residue in the T-loop of the kinases is required for high enzyme activity in animals and yeast. We show that the Arabidopsis thaliana Cdc2(+)/Cdc28 homolog CDKA;1 is also phosphorylated in the T-loop and that phosphorylation at the conserved Thr-161 residue is essential for its function. A phospho-mimicry T161D substitution restored the primary defect of cdka;1 mutants, and although the T161D substitution displayed a dramatically reduced kinase activity with a compromised ability to bind substrates, homozygous mutant plants were recovered. The rescue by the T161D substitution, however, was not complete, and the resulting plants displayed various developmental abnormalities. For instance, even though flowers were formed, these plants were completely sterile as a result of a failure of the meiotic program, indicating that different requirements for CDKA;1 function are needed during plant development.

Quantitation of artemisinin and its biosynthetic precursors in Artemisia annua L. by high performance liquid chromatography-electrospray quadrupole time-of-flight tandem mass spectrometry.

This study reports the development and validation of a rapid, sensitive and selective assay for the quantitation of artemisinin, arteannuin B, artemisitene and artemisinic acid in Artemisia annua L. by reversed phase high performance liquid chromatography (HPLC) electrospray (ESI) quadrupole time of flight (Q-TOF) tandem mass spectrometry (MS/MS). A recovery of >97% for all analytes was achieved by immersing one gram of fresh plant material in chloroform for 1 min. This result supports the hypothesis that artemisinin and some of its structural analogs present in the leaves A. annua L. are localized entirely in the subcuticular space of the glands on the surface of the leaves. We validated the use of this chloroform extract, without additional sample preparation steps, for quantitative Q-TOF MS/MS. No ion suppression (matrix effect) resulting from interference with other compounds was detected. For every concentration within the range of the standard curve (0.1 to 3.00 microg/ml), accuracy was between 85% and 115%. Within- and between-day variations for the analysis of A. annua L. samples were <20%.

Gene-to-metabolite networks for terpenoid indole alkaloid biosynthesis in Catharanthus roseus cells.

Rational engineering of complicated metabolic networks involved in the production of biologically active plant compounds has been greatly impeded by our poor understanding of the regulatory and metabolic pathways underlying the biosynthesis of these compounds. Whereas comprehensive genome-wide functional genomics approaches can be successfully applied to analyze a select number of model plants, these holistic approaches are not yet available for the study of nonmodel plants that include most, if not all, medicinal plants. We report here a comprehensive profiling analysis of the Madagascar periwinkle (Catharanthus roseus), a source of the anticancer drugs vinblastine and vincristine. Genome-wide transcript profiling by cDNA-amplified fragment-length polymorphism combined with metabolic profiling of elicited C. roseus cell cultures yielded a collection of known and previously undescribed transcript tags and metabolites associated with terpenoid indole alkaloids. Previously undescribed gene-to-gene and gene-to-metabolite networks were drawn up by searching for correlations between the expression profiles of 417 gene tags and the accumulation profiles of 178 metabolite peaks. These networks revealed that the different branches of terpenoid indole alkaloid biosynthesis and various other metabolic pathways are subject to differing hormonal regulation. These networks also served to identify a select number of genes and metabolites likely to be involved in the biosynthesis of terpenoid indole alkaloids. This study provides the basis for a better understanding of periwinkle secondary metabolism and increases the practical potential of metabolic engineering of this important medicinal plant.

A functional genomics approach toward the understanding of secondary metabolism in plant cells.

Despite the tremendous importance of secondary metabolites for humans as for the plant itself, plant secondary metabolism remains poorly characterized. Here, we present an experimental approach, based on functional genomics, to facilitate gene discovery in plant secondary metabolism. Targeted metabolite analysis was combined with cDNA-amplified fragment length polymorphism-based transcript profiling of jasmonate-elicited tobacco Bright yellow 2 cells. Transcriptome analysis suggested an extensive jasmonate-mediated genetic reprogramming of metabolism, which correlated well with the observed shifts in the biosynthesis of the metabolites investigated. This method, which in addition to transcriptome data also generates gene tags, in the future might lead to the creation of novel tools for metabolic engineering of medicinal plant systems in general.