Molecular insights into plant desiccation tolerance: transcriptomics, proteomics and targeted metabolite profiling in Craterostigma plantagineum.

The resurrection plant Craterostigma plantagineum possesses an extraordinary capacity to survive long-term desiccation. To enhance our understanding of this phenomenon, complementary transcriptome, soluble proteome and targeted metabolite profiling was carried out on leaves collected from different stages during a dehydration and rehydration cycle. A total of 7348 contigs, 611 proteins and 39 metabolites were differentially abundant across the different sampling points. Dynamic changes in transcript, protein and metabolite levels revealed a unique signature characterizing each stage. An overall low correlation between transcript and protein abundance suggests a prominent role for post-transcriptional modification in metabolic reprogramming to prepare plants for desiccation and recovery. The integrative analysis of all three data sets was performed with an emphasis on photosynthesis, photorespiration, energy metabolism and amino acid metabolism. The results revealed a set of precise changes that modulate primary metabolism to confer plasticity to metabolic pathways, thus optimizing plant performance under stress. The maintenance of cyclic electron flow and photorespiration, and the switch from C3 to crassulacean acid metabolism photosynthesis, may contribute to partially sustain photosynthesis and minimize oxidative damage during dehydration. Transcripts with a delayed translation, ATP-independent bypasses, alternative respiratory pathway and 4-aminobutyric acid shunt may all play a role in energy management, together conferring bioenergetic advantages to meet energy demands upon rehydration. This study provides a high-resolution map of the changes occurring in primary metabolism during dehydration and rehydration and enriches our understanding of the molecular mechanisms underpinning plant desiccation tolerance. The data sets provided here will ultimately inspire biotechnological strategies for drought tolerance improvement in crops.

HOMECAT: consensus homologs mapping for interspecific knowledge transfer and functional genomic data integration.

MOTIVATION: Comparative studies are encouraged by the fast increase of data availability from the latest high-throughput techniques, in particular from functional genomic studies. Yet, the size of datasets, the challenge of complete orthologs findings and not last, the variety of identification formats, make information integration challenging. With HOMECAT, we aim to facilitate cross-species relationship identification and data mapping, by combining orthology predictions from several publicly available sources, a convenient interface for high-throughput data download and automatic identifier conversion into a Cytoscape plug-in, that provides both an integration with a large set of bioinformatics tools, as well as a user-friendly interface. AVAILABILITY: HOMECAT and the Supplementary Materials are freely available at http://www.cbmc.it/homecat/.

Isoprene-Emitting Tobacco Plants Are Less Affected by Moderate Water Deficit under Future Climate Change Scenario and Show Adjustments of Stress-Related Proteins in Actual Climate.

Isoprene-emitting plants are better protected against thermal and oxidative stresses, which is a desirable trait in a climate-changing (drier and warmer) world. Here we compared the ecophysiological performances of transgenic isoprene-emitting and wild-type non-emitting tobacco plants during water stress and after re-watering in actual environmental conditions (400 ppm of CO2 and 28 °C of average daily temperature) and in a future climate scenario (600 ppm of CO2 and 32 °C of average daily temperature). Furthermore, we intended to complement the present knowledge on the mechanisms involved in isoprene-induced resistance to water deficit stress by examining the proteome of transgenic isoprene-emitting and wild-type non-emitting tobacco plants during water stress and after re-watering in actual climate. Isoprene emitters maintained higher photosynthesis and electron transport rates under moderate stress in future climate conditions. However, physiological resistance to water stress in the isoprene-emitting plants was not as marked as expected in actual climate conditions, perhaps because the stress developed rapidly. In actual climate, isoprene emission capacity affected the tobacco proteomic profile, in particular by upregulating proteins associated with stress protection. Our results strengthen the hypothesis that isoprene biosynthesis is related to metabolic changes at the gene and protein levels involved in the activation of general stress defensive mechanisms of plants.

Machine Learning to Support the Presentation of Complex Pathway Graphs.

Visualization of biological mechanisms by means of pathway graphs is necessary to better understand the often complex underlying system. Manual layout of such pathways or maps of knowledge is a difficult and time consuming process. Node duplication is a technique that makes layouts with improved readability possible by reducing edge crossings and shortening edge lengths in drawn diagrams. In this article, we propose an approach using Machine Learning (ML) to facilitate parts of this task by training a Support Vector Machine (SVM) with actions taken during manual biocuration. Our training input is a series of incremental snapshots of a diagram describing mechanisms of a disease, progressively curated by a human expert employing node duplication in the process. As a test of the trained SVM models, they are applied to a single large instance and 25 medium-sized instances of hand-curated biological pathways. Finally, in a user validation study, we compare the model predictions to the outcome of a node duplication questionnaire answered by users of biological pathways with varying experience. We successfully predicted nodes for duplication and emulated human choices, demonstrating that our approach can effectively learn human-like node duplication preferences to support curation of pathway diagrams in various contexts.

B-cell clonogenic activity of HIV-1 p17 variants is driven by PAR1-mediated EGF transactivation.

Combined antiretroviral therapy (cART) for HIV-1 dramatically slows disease progression among HIV+ individuals. Currently, lymphoma represents the main cause of death among HIV-1-infected patients. Detection of p17 variants (vp17s) endowed with B-cell clonogenic activity in HIV-1-seropositive patients with lymphoma suggests their possible role in lymphomagenesis. Here, we demonstrate that the clonogenic activity of vp17s is mediated by their binding to PAR1 and to PAR1-mediated EGFR transactivation through Gq protein. The entire vp17s-triggered clonogenic process is MMPs dependent. Moreover, phosphoproteomic and bioinformatic analysis highlighted the crucial role of EGFR/PI3K/Akt pathway in modulating several molecules promoting cancer progression, including RAC1, ABL1, p53, CDK1, NPM, Rb, PTP-1B, and STAT1. Finally, we show that a peptide (F1) corresponding to the vp17s functional epitope is sufficient to trigger the PAR1/EGFR/PI3K/Akt pathway and bind PAR1. Our findings suggest novel potential therapeutic targets to counteract vp17-driven lymphomagenesis in HIV+ patients.

Rac1 activation links tau hyperphosphorylation and Aß dysmetabolism in Alzheimer's disease.

One of the earliest pathological features characterizing Alzheimer's disease (AD) is the loss of dendritic spines. Among the many factors potentially mediating this loss of neuronal connectivity, the contribution of Rho-GTPases is of particular interest. This family of proteins has been known for years as a key regulator of actin cytoskeleton remodeling. More recent insights have indicated how its complex signaling might be triggered also in pathological conditions. Here, we showed that the Rho-GTPase family member Rac1 levels decreased in the frontal cortex of AD patients compared to non-demented controls. Also, Rac1 increased in plasma samples of AD patients with Mini-Mental State Examination < 18 compared to age-matched non demented controls. The use of different constitutively active peptides allowed us to investigate in vitro Rac1 specific signaling. Its activation increased the processing of amyloid precursor protein and induced the translocation of SET from the nucleus to the cytoplasm, resulting in tau hyperphosphorylation at residue pT181. Notably, Rac1 was abnormally activated in the hippocampus of 6-week-old 3xTg-AD mice. However, the total protein levels decreased at 7-months. A rescue strategy based on the intranasal administration of Rac1 active peptide at 6.5 months prevented dendritic spine loss. This data suggests the intriguing possibility of a dual role of Rac1 according to the different stages of the pathology. In an initial stage, Rac1 deregulation might represent a triggering co-factor due to the direct effect on Aß and tau. However, at a later stage of the pathology, it might represent a potential therapeutic target due to the beneficial effect on spine dynamics.

Transcriptomic profiling of hemp bast fibres at different developmental stages.

Bast fibres are long extraxylary cells which mechanically support the phloem and they are divided into xylan- and gelatinous-type, depending on the composition of their secondary cell walls. The former, typical of jute/kenaf bast fibres, are characterized by the presence of xylan and a high degree of lignification, while the latter, found in tension wood, as well as flax, ramie and hemp bast fibres, have a high abundance of crystalline cellulose. During their differentiation, bast fibres undergo specific developmental stages: the cells initially elongate rapidly by intrusive growth, subsequently they cease elongation and start to thicken. The goal of the present study is to provide a transcriptomic close-up of the key events accompanying bast fibre development in textile hemp (Cannabis sativa L.), a fibre crop of great importance. Bast fibres have been sampled from different stem regions. The developmental stages corresponding to active elongation and cell wall thickening have been studied using RNA-Seq. The results show that the fibres sampled at each stem region are characterized by a specific transcriptomic signature and that the major changes in cell wall-related processes take place at the internode containing the snap point. The data generated also identify several interesting candidates for future functional analysis.

A single amino acid substitution confers B-cell clonogenic activity to the HIV-1 matrix protein p17.

Recent data highlight the presence, in HIV-1-seropositive patients with lymphoma, of p17 variants (vp17s) endowed with B-cell clonogenicity, suggesting a role of vp17s in lymphomagenesis. We investigated the mechanisms responsible for the functional disparity on B cells between a wild-type p17 (refp17) and a vp17 named S75X. Here, we show that a single Arginine (R) to Glycine (G) mutation at position 76 in the refp17 backbone (p17R76G), as in the S75X variant, is per se sufficient to confer a B-cell clonogenic potential to the viral protein and modulate, through activation of the PTEN/PI3K/Akt signaling pathway, different molecules involved in apoptosis inhibition (CASP-9, CASP-7, DFF-45, NPM, YWHAZ, Src, PAX2, MAPK8), cell cycle promotion and cancer progression (CDK1, CDK2, CDK8, CHEK1, CHEK2, GSK-3 beta, NPM, PAK1, PP2C-alpha). Moreover, the only R to G mutation at position 76 was found to strongly impact on protein folding and oligomerization by altering the hydrogen bond network. This generates a conformational shift in the p17 R76G mutant which enables a functional epitope(s), masked in refp17, to elicit B-cell growth-promoting signals after its interaction with a still unknown receptor(s). Our findings offer new opportunities to understand the molecular mechanisms accounting for the B-cell growth-promoting activity of vp17s.

MINERVA-a platform for visualization and curation of molecular interaction networks.

Our growing knowledge about various molecular mechanisms is becoming increasingly more structured and accessible. Different repositories of molecular interactions and available literature enable construction of focused and high-quality molecular interaction networks. Novel tools for curation and exploration of such networks are needed, in order to foster the development of a systems biology environment. In particular, solutions for visualization, annotation and data cross-linking will facilitate usage of network-encoded knowledge in biomedical research. To this end we developed the MINERVA (Molecular Interaction NEtwoRks VisuAlization) platform, a standalone webservice supporting curation, annotation and visualization of molecular interaction networks in Systems Biology Graphical Notation (SBGN)-compliant format. MINERVA provides automated content annotation and verification for improved quality control. The end users can explore and interact with hosted networks, and provide direct feedback to content curators. MINERVA enables mapping drug targets or overlaying experimental data on the visualized networks. Extensive export functions enable downloading areas of the visualized networks as SBGN-compliant models for efficient reuse of hosted networks. The software is available under Affero GPL 3.0 as a Virtual Machine snapshot, Debian package and Docker instance at http://r3lab.uni.lu/web/minerva-website/. We believe that MINERVA is an important contribution to systems biology community, as its architecture enables set-up of locally or globally accessible SBGN-oriented repositories of molecular interaction networks. Its functionalities allow overlay of multiple information layers, facilitating exploration of content and interpretation of data. Moreover, annotation and verification workflows of MINERVA improve the efficiency of curation of networks, allowing life-science researchers to better engage in development and use of biomedical knowledge repositories.

Rac1 selective activation improves retina ganglion cell survival and regeneration.

In adult mammals, after optic nerve injury, retinal ganglion cells (RGCs) do not regenerate their axons and most of them die by apoptosis within a few days. Recently, several strategies that activate neuronal intracellular pathways were proposed to prevent such degenerative processes. The rho-related small GTPase Rac1 is part of a complex, still not fully understood, intracellular signaling network, mediating in neurons many effects, including axon growth and cell survival. However, its role in neuronal survival and regeneration in vivo has not yet been properly investigated. To address this point we intravitreally injected selective cell-penetrating Rac1 mutants after optic nerve crush and studied the effect on RGC survival and axonal regeneration. We injected two well-characterized L61 constitutively active Tat-Rac1 fusion protein mutants, in which a second F37A or Y40C mutation confers selectivity in downstream signaling pathways. Results showed that, 15 days after crush, both mutants were able to improve survival and to prevent dendrite degeneration, while the one harboring the F37A mutation also improved axonal regeneration. The treatment with F37A mutant for one month did not improve the axonal elongation respect to 15 days. Furthermore, we found an increase of Pak1 T212 phosphorylation and ERK1/2 expression in RGCs after F37A treatment, whereas ERK1/2 was more activated in glial cells after Y40C administration. Our data suggest that the selective activation of distinct Rac1-dependent pathways could represent a therapeutic strategy to counteract neuronal degenerative processes in the retina.

Light-induced dissociation of an antenna hetero-oligomer is needed for non-photochemical quenching induction.

PsbS plays a major role in activating the photoprotection mechanism known as "non-photochemical quenching," which dissipates chlorophyll excited states exceeding the capacity for photosynthetic electron transport. PsbS activity is known to be triggered by low lumenal pH. However, the molecular mechanism by which this subunit regulates light harvesting efficiency is still unknown. Here we show that PsbS controls the association/dissociation of a five-subunit membrane complex, composed of two monomeric Lhcb proteins (CP29 and CP24) and the trimeric LHCII-M. Dissociation of this supercomplex is indispensable for the onset of non-photochemical fluorescence quenching in high light, strongly suggesting that protein subunits catalyzing the reaction of heat dissipation are buried into the complex and thus not available for interaction with PsbS. Consistently, we showed that knock-out mutants on two subunits participating to the B4C complex were strongly affected in heat dissipation. Direct observation by electron microscopy and image analysis showed that B4C dissociation leads to the redistribution of PSII within grana membranes. We interpreted these results to mean that the dissociation of B4C makes quenching sites, possibly CP29 and CP24, available for the switch to an energy-quenching conformation. These changes are reversible and do not require protein synthesis/degradation, thus allowing for changes in PSII antenna size and adaptation to rapidly changing environmental conditions.

Photosynthetic antenna size in higher plants is controlled by the plastoquinone redox state at the post-transcriptional rather than transcriptional level.

We analyze the effect of the plastoquinone redox state on the regulation of the light-harvesting antenna size at transcriptional and post-transcriptional levels. This was approached by studying transcription and accumulation of light-harvesting complexes in wild type versus the barley mutant viridis zb63, which is depleted in photosystem I and where plastoquinone is constitutively reduced. We show that the mRNA level of genes encoding antenna proteins is almost unaffected in the mutant; this stability of messenger level is not a peculiarity of antenna-encoding genes, but it extends to all photosynthesis-related genes. In contrast, analysis of protein accumulation by two-dimensional PAGE shows that the mutant undergoes strong reduction of its antenna size, with individual gene products having different levels of accumulation. We conclude that the plastoquinone redox state plays an important role in the long term regulation of chloroplast protein expression. However, its modulation is active at the post-transcriptional rather than transcriptional level.