AURTHO: Autoregulation of transcription factors as facilitator of cis-acting element discovery.

Transcriptional regulation is key in bacteria for providing an adequate response in time and space to changing environmental conditions. However, despite decades of research, the binding sites and therefore the target genes and the function of most transcription factors (TFs) remain unknown. Filling this gap in knowledge through conventional methods represents a colossal task which we demonstrate here can be significantly facilitated by a widespread feature in transcriptional control: the autoregulation of TFs implying that the yet unknown transcription factor binding site (TFBS) is neighboring the TF itself. In this work, we describe the "AURTHO" methodology (AUtoregulation of oRTHOlogous transcription factors), consisting of analyzing upstream regions of orthologous TFs in order to uncover their associated TFBSs. AURTHO enabled the de novo identification of novel TFBSs with an unprecedented improvement in terms of quantity and reliability. DNA-protein interaction studies on a selection of candidate cis-acting elements yielded an >90 % success rate, demonstrating the efficacy of AURTHO at highlighting true TF-TFBS couples and confirming the identification in a near future of a plethora of TFBSs across all bacterial species.

Cyclin-dependent kinase 7 contributes to myelin maintenance in the adult central nervous system and promotes myelin gene expression.

Mechanisms regulating oligodendrocyte differentiation, developmental myelination and myelin maintenance in adulthood are complex and still not completely described. Their understanding is crucial for the development of new protective or therapeutic strategies in demyelinating pathologies such as multiple sclerosis. In this perspective, we have investigated the role of Cyclin-dependent kinase 7 (Cdk7), a kinase involved in cell-cycle progression and transcription regulation, in the oligodendroglial lineage. We generated a conditional knock-out mouse model in which Cdk7 is invalidated in post-mitotic oligodendrocytes. At the end of developmental myelination, the number and diameter of myelinated axons, as well as the myelin structure, thickness and protein composition, were normal. However, in young adult and in aged mice, there was a higher number of small caliber myelinated axons associated with a decreased mean axonal diameter, myelin sheaths of large caliber axons were thinner, and the level of some major myelin-associated proteins was reduced. These defects were accompanied by the appearance of an abnormal clasping phenotype. We also used an in vitro oligodendroglial model and showed that Cdk7 pharmacological inhibition led to an altered myelination-associated morphological modification combined with a decreased expression of myelin-specific genes. Altogether, we identified novel functions for Cdk7 in CNS myelination.

LED color gradient as a new screening tool for rapid phenotyping of plant responses to light quality.

BACKGROUND: The increasing demand for local food production is fueling high interest in the development of controlled environment agriculture. In particular, LED technology brings energy-saving advantages together with the possibility of manipulating plant phenotypes through light quality control. However, optimizing light quality is required for each cultivated plant and specific purpose. FINDINGS: This article shows that the combination of LED gradient set-ups with imaging-based non-destructive plant phenotyping constitutes an interesting new screening tool with the potential to improve speed, logistics, and information output. To validate this concept, an experiment was performed to evaluate the effects of a complete range of red:blue ratios on 7 plant species: Arabidopsis thaliana, Brachypodium distachyon, Euphorbia peplus, Ocimum basilicum, Oryza sativa, Solanum lycopersicum, and Setaria viridis. Plants were exposed during 30 days to the light gradient and showed significant, but species-dependent, responses in terms of dimension, shape, and color. A time-series analysis of phenotypic descriptors highlighted growth changes but also transient responses of plant shapes to the red:blue ratio. CONCLUSION: This approach, which generated a large reusable dataset, can be adapted for addressing specific needs in crop production or fundamental questions in photobiology.

Palantir: a springboard for the analysis of secondary metabolite gene clusters in large-scale genome mining projects.

SUMMARY: To support small and large-scale genome mining projects, we present Post-processing Analysis tooLbox for ANTIsmash Reports (Palantir), a dedicated software suite for handling and refining secondary metabolite biosynthetic gene cluster (BGC) data annotated with the popular antiSMASH pipeline. Palantir provides new functionalities building on NRPS/PKS predictions from antiSMASH, such as improved BGC annotation, module delineation and easy access to sub-sequences at different levels (cluster, gene, module and domain). Moreover, it can parse user-provided antiSMASH reports and reformat them for direct use or storage in a relational database. AVAILABILITY AND IMPLEMENTATION: Palantir is released both as a Perl API available on CPAN (https://metacpan.org/release/Bio-Palantir) and as a web application (http://palantir.uliege.be). As a practical use case, the web interface also features a database built from the mining of 1616 cyanobacterial genomes, of which 1488 were predicted to encode at least one BGC. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Contribution of the ß-glucosidase BglC to the onset of the pathogenic lifestyle of Streptomyces scabies.

Common scab disease on root and tuber plants is caused by Streptomyces scabies and related species which use the cellulose synthase inhibitor thaxtomin A as the main phytotoxin. Thaxtomin production is primarily triggered by the import of cello-oligosaccharides. Once inside the cell, the fate of the cello-oligosaccharides is dichotomized: (i) the fuelling of glycolysis with glucose for the saprophytic lifestyle through the action of ß-glucosidase(s) (BGs); and (ii) elicitation of the pathogenic lifestyle by the inhibition of CebR-mediated transcriptional repression of thaxtomin biosynthetic genes. Here, we investigated the role of scab57721, encoding a putative BG (BglC), in the onset of the pathogenicity of S. scabies. Enzymatic assays showed that BglC was able to release glucose from cellobiose, cellotriose and all other cello-oligosaccharides tested. Its inactivation resulted in a phenotype opposite to that expected, as reduced production of thaxtomin was monitored when the mutant was cultivated on medium containing cello-oligosaccharides as unique carbon source. This unexpected phenotype could be attributed to the highly increased activity of alternative intracellular BGs, probably as a compensation for bglC inactivation, which then prevented cellobiose and cellotriose accumulation to reduce the activity of CebR. In contrast, when the bglC null mutant was cultivated on medium devoid of cello-oligosaccharides, it instead constitutively produced thaxtomin. This observed hypervirulent phenotype does not fit with the proposed model of the cello-oligosaccharide-mediated induction of thaxtomin production, and suggests that the role of BglC in the route to the pathogenic lifestyle of S. scabies is more complex than currently presented.

Using a Structural Root System Model to Evaluate and Improve the Accuracy of Root Image Analysis Pipelines.

Root system analysis is a complex task, often performed with fully automated image analysis pipelines. However, the outcome is rarely verified by ground-truth data, which might lead to underestimated biases. We have used a root model, ArchiSimple, to create a large and diverse library of ground-truth root system images (10,000). For each image, three levels of noise were created. This library was used to evaluate the accuracy and usefulness of several image descriptors classically used in root image analysis softwares. Our analysis highlighted that the accuracy of the different traits is strongly dependent on the quality of the images and the type, size, and complexity of the root systems analyzed. Our study also demonstrated that machine learning algorithms can be trained on a synthetic library to improve the estimation of several root system traits. Overall, our analysis is a call to caution when using automatic root image analysis tools. If a thorough calibration is not performed on the dataset of interest, unexpected errors might arise, especially for large and complex root images. To facilitate such calibration, both the image library and the different codes used in the study have been made available to the community.

Integrating roots into a whole plant network of flowering time genes in Arabidopsis thaliana.

Molecular data concerning the involvement of roots in the genetic pathways regulating floral transition are lacking. In this study, we performed global analyses of the root transcriptome in Arabidopsis in order to identify flowering time genes that are expressed in the roots and genes that are differentially expressed in the roots during the induction of flowering. Data mining of public microarray experiments uncovered that about 200 genes whose mutations are reported to alter flowering time are expressed in the roots (i.e. were detected in more than 50% of the microarrays). However, only a few flowering integrator genes passed the analysis cutoff. Comparison of root transcriptome in short days and during synchronized induction of flowering by a single 22-h long day revealed that 595 genes were differentially expressed. Enrichment analyses of differentially expressed genes in root tissues, gene ontology categories, and cis-regulatory elements converged towards sugar signaling. We concluded that roots are integrated in systemic signaling, whereby carbon supply coordinates growth at the whole plant level during the induction of flowering. This coordination could involve the root circadian clock and cytokinin biosynthesis as a feed forward loop towards the shoot.

The chlorophyll a fluorescence induction curve in the green microalga Haematococcus pluvialis: further insight into the nature of the P-S-M fluctuation and its relationship with the "low-wave" phenomenon at steady-state.

Chlorophyll fluorescence is an information-rich signal which provides an access to the management of light absorbed by PSII. A good example of this is the succession of fast fluorescence fluctuations during light-induced photosynthetic induction after dark-adaptation. During this period, the fluorescence trace exhibits several inflexion points: O-J-I-P-S-M-T. Whereas the OJIP part of this kinetics has been the subject of many studies, the processes that underly the PSMT transient are less understood. Here, we report an analysis of the PSMT phase in the green microalga Haematococcus pluvialis in terms of electron acceptors and light use by photochemistry, fluorescence and non-photochemical quenching (NPQ). We identify additional sub-phases between P and S delimited by an inflexion point, that we name Q, found in the second time scale. The P-Q phase expresses a transient photochemical quenching specifically due to alternative electron transport to oxygen. During the transition from Q to S, the NPQ increases and then relaxes during the S-M phase in about 1 min. It is suggested that this transient NPQ observed during induction is a high energy state quenching (qE) dependent on the alternative electron transport to molecular oxygen. We further show that this NPQ is of the same nature than the NPQ, known as the low-wave phenomenon, which is transiently observed after a saturating light pulse given at steady-state. In both cases, the NPQ is oxygen-dependent. This NPQ is observed at external pH 6.0, but not at pH 7.5, which seems correlated with faster saturation of the PQ pool at pH 6.0.

FLOR-ID: an interactive database of flowering-time gene networks in Arabidopsis thaliana.

Flowering is a hot topic in Plant Biology and important progress has been made in Arabidopsis thaliana toward unraveling the genetic networks involved. The increasing complexity and the explosion of literature however require development of new tools for information management and update. We therefore created an evolutive and interactive database of flowering time genes, named FLOR-ID (Flowering-Interactive Database), which is freely accessible at http://www.flor-id.org. The hand-curated database contains information on 306 genes and links to 1595 publications gathering the work of >4500 authors. Gene/protein functions and interactions within the flowering pathways were inferred from the analysis of related publications, included in the database and translated into interactive manually drawn snapshots.

"Rhizoponics": a novel hydroponic rhizotron for root system analyses on mature Arabidopsis thaliana plants.

BACKGROUND: Well-developed and functional roots are critical to support plant life and reach high crop yields. Their study however, is hampered by their underground growth and characterizing complex root system architecture (RSA) therefore remains a challenge. In the last few years, several phenotyping methods, including rhizotrons and x-ray computed tomography, have been developed for relatively thick roots. But in the model plant Arabidopsis thaliana, in vitro culture remains the easiest and preferred method to study root development, which technically limits the analyses to young seedlings. RESULTS: We present here an innovative design of hydroponic rhizotrons (rhizoponics) adapted to Arabidopsis thaliana. The setup allows to simultaneously characterize the RSA and shoot development from seedling to adult stages, i.e. from seed to seed. This system offers the advantages of hydroponics such as control of root environment and easy access to the roots for measurements or sampling. Being completely movable and low cost, it can be used in controlled cabinets. We chose the case of cadmium treatment to illustrate potential applications, from cell to organ levels. CONCLUSIONS: Rhizoponics makes possible, on the same plants of Arabidopsis, RSA measurements, root sampling and characterization of aerial development up to adult size. It therefore provides a valuable tool for addressing fundamental questions in whole plant physiology.

Extracellular peptidase hunting for improvement of protein production in plant cells and roots.

Plant-based recombinant protein production systems have gained an extensive interest over the past few years, because of their reduced cost and relative safety. Although the first products are now reaching the market, progress are still needed to improve plant hosts and strategies for biopharming. Targeting recombinant proteins toward the extracellular space offers several advantages in terms of protein folding and purification, but degradation events are observed, due to endogenous peptidases. This paper focuses on the analysis of extracellular proteolytic activities in two production systems: cell cultures and root-secretion (rhizosecretion), in Arabidopsis thaliana and Nicotiana tabacum. Proteolytic activities of extracellular proteomes (secretomes) were evaluated in vitro against two substrate proteins: bovine serum albumin (BSA) and human serum immunoglobulins G (hIgGs). Both targets were found to be degraded by the secretomes, BSA being more prone to proteolysis than hIgGs. The analysis of the proteolysis pH-dependence showed that target degradation was mainly dependent upon the production system: rhizosecretomes contained more peptidase activity than extracellular medium of cell suspensions, whereas variations due to plant species were smaller. Using class-specific peptidase inhibitors, serine, and metallopeptidases were found to be responsible for degradation of both substrates. An in-depth in silico analysis of genomic and transcriptomic data from Arabidopsis was then performed and led to the identification of a limited number of serine and metallo-peptidases that are consistently expressed in both production systems. These peptidases should be prime candidates for further improvement of plant hosts by targeted silencing.

On the necessity and biological significance of threshold-free regulon prediction outputs.

The in silico prediction of cis-acting elements in a genome is an efficient way to quickly obtain an overview of the biological processes controlled by a trans-acting factor, and connections between regulatory networks. Several regulon prediction web tools are available, designed to identify DNA motifs predicted to be bound by transcription factors using position weight matrix-based algorithms. In this paper we expose and discuss the conflicting objectives of software creators (bioinformaticians) and software users (biologists), who aim for reliable and exhaustive prediction outputs, respectively. Software makers, concerned with providing tools that minimise the number of false positive hits, often impose a stringent threshold score for a sequence to be included in the list of the putative cis-acting sites. This rigidity eventually results in the identification of strongly reliable but largely straightforward sites, i.e. those associated with genes already anticipated to be targeted by the studied transcription factor. Importantly, this biased identification of strongly bound sequences contrasts with the biological reality where, in many circumstances, a weak DNA-protein interaction is required for the appropriate gene's expression. We show here a series of transcriptionally controlled systems involving weakly bound cis-acting elements that could never have been discovered because of the policy of preventing software users from modifying the screening parameters. Proposing only trustworthy prediction outputs thus prevents biologists from fully utilising their knowledge background and deciding to analyse statistically irrelevant hits that could nonetheless be potentially involved in subtle, unexpected, though essential cis-trans relationships.

Inflorescence development in tomato: gene functions within a zigzag model.

Tomato is a major crop plant and several mutants have been selected for breeding but also for isolating important genes that regulate flowering and sympodial growth. Besides, current research in developmental biology aims at revealing mechanisms that account for diversity in inflorescence architectures. We therefore found timely to review the current knowledge of the genetic control of flowering in tomato and to integrate the emerging network into modeling attempts. We developed a kinetic model of the tomato inflorescence development where each meristem was represented by its "vegetativeness" (V), reflecting its maturation state toward flower initiation. The model followed simple rules: maturation proceeded continuously at the same rate in every meristem (dV); floral transition and floral commitment occurred at threshold levels of V; lateral meristems were initiated with a gain of V (ΔV) relative to the V level of the meristem from which they derived. This last rule created a link between successive meristems and gave to the model its zigzag shape. We next exploited the model to explore the diversity of morphotypes that could be generated by varying dV and ΔV and matched them with existing mutant phenotypes. This approach, focused on the development of the primary inflorescence, allowed us to elaborate on the genetic regulation of the kinetic model of inflorescence development. We propose that the lateral inflorescence meristem fate in tomato is more similar to an immature flower meristem than to the inflorescence meristem of Arabidopsis. In the last part of our paper, we extend our thought to spatial regulators that should be integrated in a next step for unraveling the relationships between the different meristems that participate to sympodial growth.

A root chicory MADS box sequence and the Arabidopsis flowering repressor FLC share common features that suggest conserved function in vernalization and de-vernalization responses.

Root chicory (Cichorium intybus var. sativum) is a biennial crop, but is harvested to obtain root inulin at the end of the first growing season before flowering. However, cold temperatures may vernalize seeds or plantlets, leading to incidental early flowering, and hence understanding the molecular basis of vernalization is important. A MADS box sequence was isolated by RT-PCR and named FLC-LIKE1 (CiFL1) because of its phylogenetic positioning within the same clade as the floral repressor Arabidopsis FLOWERING LOCUS C (AtFLC). Moreover, over-expression of CiFL1 in Arabidopsis caused late flowering and prevented up-regulation of the AtFLC target FLOWERING LOCUS T by photoperiod, suggesting functional conservation between root chicory and Arabidopsis. Like AtFLC in Arabidopsis, CiFL1 was repressed during vernalization of seeds or plantlets of chicory, but repression of CiFL1 was unstable when the post-vernalization temperature was favorable to flowering and when it de-vernalized the plants. This instability of CiFL1 repression may be linked to the bienniality of root chicory compared with the annual lifecycle of Arabidopsis. However, re-activation of AtFLC was also observed in Arabidopsis when a high temperature treatment was used straight after seed vernalization, eliminating the promotive effect of cold on flowering. Cold-induced down-regulation of a MADS box floral repressor and its re-activation by high temperature thus appear to be conserved features of the vernalization and de-vernalization responses in distant species.

Vernalization-induced repression of FLOWERING LOCUS C stimulates flowering in Sinapis alba and enhances plant responsiveness to photoperiod.

* Of the Brassicaceae, Sinapis alba has been intensively studied as a physiological model of induction of flowering by a single long day (LD), while molecular-genetic analyses of Arabidopsis thaliana have disclosed complex interactions between pathways controlling flowering in response to different environmental cues, such as photoperiod and vernalization. The vernalization process in S. alba was therefore analysed here. * The coding sequence of S. alba SaFLC, which is orthologous to the A. thaliana floral repressor FLOWERING LOCUS C, was isolated and the transcript levels quantified in different conditions. * Two-week-old seedlings grown in noninductive short days (SDs) were vernalized for 1-6 wk. Down-regulation of SaFLC was already marked after 1 wk of cold but 2 wk was needed for a significant acceleration of flowering. Flower buds were initiated during vernalization. When vernalization was stopped after 1 wk, repression of SaFLC was not stable but a significant increase in plant responsiveness to 16-h LDs was observed when LDs followed immediately after the cold treatment. * These results suggest that vernalization does not only work when plants experience long exposure to cold during the winter: shorter cold periods might stimulate flowering of LD plants if they occur when photoperiod is increasing, such as in spring.

Leaf carbohydrate controls over Arabidopsis growth and response to elevated CO2: an experimentally based model.

Transient starch production is thought to strongly control plant growth and response to elevated CO2. We tested this hypothesis with an experimentally based mechanistic model in Arabidopsis thaliana. Experiments were conducted on wild-type (WT) A. thaliana, starch-excess (sex1) and starchless (pgm) mutants under ambient and elevated CO2 conditions to determine parameters and validate the model. The model correctly predicted that mutant growth is approx. 20% of that in WT, and the absolute response of both mutants to elevated CO2 is an order of magnitude lower than in WT. For sex1, direct starch unavailability explained the growth responses. For pgm, we demonstrated experimentally that maintenance respiration is proportional to leaf soluble sugar concentration, which gave the necessary feedback mechanism on modelled growth. Our study suggests that the effects of sugar-starch cycling on growth can be explained by simple allocation processes, and the maximum rate of leaf growth (sink capacity) exerts a strong control over the response to elevated CO2 of herbaceous plants such as A. thaliana.

A novel high efficiency, low maintenance, hydroponic system for synchronous growth and flowering of Arabidopsis thaliana.

BACKGROUND: Arabidopsis thaliana is now the model organism for genetic and molecular plant studies, but growing conditions may still impair the significance and reproducibility of the experimental strategies developed. Besides the use of phytotronic cabinets, controlling plant nutrition may be critical and could be achieved in hydroponics. The availability of such a system would also greatly facilitate studies dealing with root development. However, because of its small size and rosette growth habit, Arabidopsis is hardly grown in standard hydroponic devices and the systems described in the last years are still difficult to transpose at a large scale. Our aim was to design and optimize an up-scalable device that would be adaptable to any experimental conditions. RESULTS: An hydroponic system was designed for Arabidopsis, which is based on two units: a seed-holder and a 1-L tank with its cover. The original agar-containing seed-holder allows the plants to grow from sowing to seed set, without transplanting step and with minimal waste. The optimum nitrate supply was determined for vegetative growth, and the flowering response to photoperiod and vernalization was characterized to show the feasibility and reproducibility of experiments extending over the whole life cycle. How this equipment allowed to overcome experimental problems is illustrated by the analysis of developmental effects of nitrate reductase deficiency in nia1nia2 mutants. CONCLUSION: The hydroponic device described in this paper allows to drive small and large scale cultures of homogeneously growing Arabidopsis plants. Its major advantages are its flexibility, easy handling, fast maintenance and low cost. It should be suitable for many experimental purposes.