Eva Collakova

Metabolic and Transcriptional Reprogramming in Developing Soybean (Glycine max) Embryos

Soybean (Glycine max) seeds are an important source of seed storage compounds, including protein, oil, and sugar used for food, feed, chemical, and biofuel production. We assessed detailed temporal transcriptional and metabolic changes in developing soybean embryos to gain a systems biology view of developmental and metabolic changes and to identify potential targets for metabolic engineering. Two major developmental and metabolic transitions were captured enabling identification of potential metabolic engineering targets specific to seed filling and to desiccation. The first transition involved a switch between different types of metabolism in dividing and elongating cells. The second transition involved the onset of maturation and desiccation tolerance during seed filling and a switch from photoheterotrophic to heterotrophic metabolism. Clustering analyses of metabolite and transcript data revealed clusters of functionally related metabolites and transcripts active in these different developmental and metabolic programs. The gene clusters provide a resource to generate predictions about the associations and interactions of unknown regulators with their targets based on “guilt-by-association” relationships. The inferred regulators also represent potential targets for future metabolic engineering of relevant pathways and steps in central carbon and nitrogen metabolism in soybean embryos and drought and desiccation tolerance in plants.

Near real-time analysis of the phenotypic responses of Escherichia coli to 1-butanol exposure using Raman spectroscopy

Raman spectroscopy was used to study the time course of phenotypic responses of Escherichia coli (DH5α) to 1-butanol exposure (1.2% [vol/vol]). Raman spectroscopy is of interest for bacterial phenotyping because it can be performed (i) in near real time, (ii) with minimal sample preparation (label-free), and (iii) with minimal spectral interference from water. Traditional off-line analytical methodologies were applied to both 1-butanol-treated and control cells to draw correlations with Raman data. Here, distinct sets of Raman bands are presented that characterize phenotypic traits of E. coli with maximized correlation to off-line measurements. In addition, the observed time course phenotypic responses of E. coli to 1.2% (vol/vol) 1-butanol exposure included the following: (i) decreased saturated fatty acids levels, (ii) retention of unsaturated fatty acids and low levels of cyclopropane fatty acids, (iii) increased membrane fluidity following the initial response of increased rigidity, and (iv) no changes in total protein content or protein-derived amino acid composition. For most phenotypic traits, correlation coefficients between Raman spectroscopy and traditional off-line analytical approaches exceeded 0.75, and major trends were captured. The results suggest that near-real-time Raman spectroscopy is suitable for approximating metabolic and physiological phenotyping of bacterial cells subjected to toxic environmental conditions.

UMAMIT14 is an amino acid exporter involved in phloem unloading in Arabidopsis roots

Highlight UMAMIT14, a member of the Usually Multiple Acids Move In and out Transporters 14 family of amino acid transporters, is involved in unloading amino acids from the phloem in roots in addition to a previously described role in seed loading.

Potential targets of VIVIPAROUS1/ABI3‐LIKE1 (VAL1) repression in developing Arabidopsis thaliana embryos

Developing Arabidopsis seeds accumulate oils and seed storage proteins synthesized by the pathways of primary metabolism. Seed development and metabolism are positively regulated by transcription factors belonging to the LAFL (LEC1, AB13, FUSCA3 and LEC2) regulatory network. The VAL gene family encodes repressors of the seed maturation program in germinating seeds, although they are also expressed during seed maturation. The possible regulatory role of VAL1 in seed development has not been studied to date. Reverse genetics revealed that val1 mutant seeds accumulated elevated levels of proteins compared with the wild type, suggesting that VAL1 functions as a repressor of seed metabolism; however, in the absence of VAL1, the levels of metabolites, ABA, auxin and jasmonate derivatives did not change significantly in developing embryos. Two VAL1 splice variants were identified through RNA sequencing analysis: a full-length form and a truncated form lacking the plant homeodomain-like domain associated with epigenetic repression. None of the transcripts encoding the core LAFL network transcription factors were affected in val1 embryos. Instead, activation of VAL1 by FUSCA3 appears to result in the repression of a subset of seed maturation genes downstream of core LAFL regulators, as 39% of transcripts in the FUSCA3 regulon were derepressed in the val1 mutant. The LEC1 and LEC2 regulons also responded, but to a lesser extent. Additional 832 transcripts that were not LAFL targets were derepressed in val1 mutant embryos. These transcripts are candidate targets of VAL1, acting through epigenetic and/or transcriptional repression.

Characterization of the inositol monophosphatase gene family in Arabidopsis

Synthesis of myo-inositol is crucial in multicellular eukaryotes for production of phosphatidylinositol and inositol phosphate signaling molecules. The myo-inositol monophosphatase (IMP) enzyme is required for the synthesis of myo-inositol, breakdown of inositol (1,4,5)-trisphosphate, a second messenger involved in Ca2+ signaling, and synthesis of L-galactose, a precursor of ascorbic acid. Two myo-inositol monophosphatase -like (IMPL) genes in Arabidopsis encode chloroplast proteins with homology to the prokaryotic IMPs and one of these, IMPL2, can complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, indicating an important role for IMPL2 in amino acid synthesis. To delineate how this small gene family functions in inositol synthesis and metabolism, we sought to compare recombinant enzyme activities, expression patterns, and impact of genetic loss-of-function mutations for each. Our data show that purified IMPL2 protein is an active histidinol-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme, which has the ability to hydrolyze D-galactose 1-phosphate, and D-myo-inositol 1-phosphate, a breakdown product of D-inositol (1,4,5) trisphosphate. Expression studies indicated that all three genes are expressed in multiple tissues, however, IMPL1 expression is restricted to above-ground tissues only. Identification and characterization of impl1 and impl2 mutants revealed no viable mutants for IMPL1, while two different impl2 mutants were identified and shown to be severely compromised in growth, which can be rescued by histidine. Analyses of metabolite levels in impl2 and complemented mutants reveals impl2 mutant growth is impacted by alterations in the histidine biosynthesis pathway, but does not impact myo-inositol synthesis. Together, these data indicate that IMPL2 functions in the histidine biosynthetic pathway, while IMP and IMPL1 catalyze the hydrolysis of inositol- and galactose-phosphates in the plant cell.

Changes in RNA Splicing in Developing Soybean (Glycine max) Embryos.

Developing soybean seeds accumulate oils, proteins, and carbohydrates that are used as oxidizable substrates providing metabolic precursors and energy during seed germination. The accumulation of these storage compounds in developing seeds is highly regulated at multiple levels, including at transcriptional and post-transcriptional regulation. RNA sequencing was used to provide comprehensive information about transcriptional and post-transcriptional events that take place in developing soybean embryos. Bioinformatics analyses lead to the identification of different classes of alternatively spliced isoforms and corresponding changes in their levels on a global scale during soybean embryo development. Alternative splicing was associated with transcripts involved in various metabolic and developmental processes, including central carbon and nitrogen metabolism, induction of maturation and dormancy, and splicing itself. Detailed examination of selected RNA isoforms revealed alterations in individual domains that could result in changes in subcellular localization of the resulting proteins, protein-protein and enzyme-substrate interactions, and regulation of protein activities. Different isoforms may play an important role in regulating developmental and metabolic processes occurring at different stages in developing oilseed embryos.

Mining and visualization of microarray and metabolomic data reveal extensive cell wall remodeling during winter hardening in Sitka spruce (Picea sitchensis)

Microarray gene expression profiling is a powerful technique to understand complex developmental processes, but making biologically meaningful inferences from such studies has always been challenging. We previously reported a microarray study of the freezing acclimation period in Sitka spruce (Picea sitchensis) in which a large number of candidate genes for climatic adaptation were identified. In the current paper, we apply additional systems biology tools to these data to further probe changes in the levels of genes and metabolites and activities of associated pathways that regulate this complex developmental transition. One aspect of this adaptive process that is not well understood is the role of the cell wall. Our data suggest coordinated metabolic and signaling responses leading to cell wall remodeling. Co-expression of genes encoding proteins associated with biosynthesis of structural and non-structural cell wall carbohydrates was observed, which may be regulated by ethylene signaling components. At the same time, numerous genes, whose products are putatively localized to the endomembrane system and involved in both the synthesis and trafficking of cell wall carbohydrates, were up-regulated. Taken together, these results suggest a link between ethylene signaling and biosynthesis, and targeting of cell wall related gene products during the period of winter hardening. Automated Layout Pipeline for Inferred NEtworks (ALPINE), an in-house plugin for the Cytoscape visualization environment that utilizes the existing GeneMANIA and Mosaic plugins, together with the use of visualization tools, provided images of proposed signaling processes that became active over the time course of winter hardening, particularly at later time points in the process. The resulting visualizations have the potential to reveal novel, hypothesis-generating, gene association patterns in the context of targeted subcellular location.

Potential targets of VIVIPAROUS1/ABI3-LIKE1 (VAL1) repression in developing Arabidopsis thaliana embryos

Methods: The toxicity of 1 μM Clioquinol (CQ) and 10 μM ZnCl2 (Zn) in human cancer cells of Hep-2 and human normal cells MRC-5 was determined by MTS assay. The radio-sensitization of CQ+Zn in Hep-2 and Hela cells was detected by colon formation measure. The effect of CQ+Zn on the NF-kB activity in Hep-2 and Hela cells is measured by the luciferase activity assay. The ATM RNA and protein expression level were determined by RT-PCR and Western blot methods.Fibrin is a protein hydrogel material responsible for stabilizing the platelet-rich blood clot over a wound in blood coagulation. Its utilitarian mechanical properties: Being stiff or deformable as needed result from the unique hierarchical organization of the polymer network. Experimental and theoretical studies have shown that single filament stretching, network rearrangements and protein unfolding are responsible for this unique mechanical behavior. In this work, we investigate the biophysics of fibrin unfolding in response to uniaxial stretch in situ using coherent Raman imaging. Fibrin hydrogels are stretched to regime where unfolding transitions of α-helical structures to β-sheet occur and spatially resolved broadband coherent anti-Stokes Raman scattering (B-CARS) spectra of fibrin gels are acquired and analyzed. The secondary structure is calculated from peak analysis of the amide I and amide III regions, which are sensitive to subtle changes in protein structure. Experiments on gels of different polymer concentration and crosslinker density show unfolding transitions that correlate with non-linear effects seen in shear rheology. Finally, imaging results show an inhomogeneous unfolding distribution near inert beads in the gel, suggesting that local stresses on platelets within biological clots is different from the bulk mechanical response. This demonstrates the use of hyperspectral B-CARS microscopy to measure local, mechanically-induced conformational changes in proteins.U (Ub)-signals virtually regulate all cellular pathways. However, two major challenges impede our ability to identify and characterize associations within ubiquitylation cascades: Ubiquitylation cascades are multiplex, i.e., few E1s, dozens E2s and hundreds of E3s ubiquitylate thousands of substrates. Moreover, many substrates possess more than one cognate E3-ligase. About a hundred deubiquitylases rapidly and efficiently reverse the ubiquitylation. To circumvent these limitations we took an integrating biology approach including structural based in silico search, bacterial genetics, biochemical, biophysicals and X-ray crystallography to establish a productive interdisciplinary research. A novel bacterial genetic selection system for ubiquitylation and its utilization in identifying and characterizing new E3s, Ub-receptors and ubiquitylation substrates will be presented. Using bacterial expression of a functional ubiquitylation apparatus we purified and crystallized and determined the structure of an ubiquitylated-Ub-receptor for the first time. We took a multidisciplinary approach and uncovered a novel UBD within this receptor. A surprising function of the Ub-receptor ubiquitylation will be presented. As the findings derived from the genetic selection system we developed and the crystal structure of ubiquitylated-ubiquitin-receptor are still under review so I will share the full result and discussion at presentation time.S development and metabolism in Arabidopsis are regulated by leafy cotyledon1 (LEC1), Abscisic acid-insensitive3 (ABI3), Fusca3 (FUS3) and LEC2. These transcription factors are part of the core LAFL (LEC1, ABI3, FUS3, and LEC2) regulatory network central to seed development. The action of the LAFL transcription factors is inhibited in germinating seedlings by VAL1 and 2 to suppress the embryonic program during the transition from the embryonic to vegetative state. However, the VALs are also expressed in maturing seed, which appears controversial, considering their role in suppressing the embryonic program. Two VAL1 splice variants were identified through RNA sequencing in developing Arabidopsis embryos: A full-length form containing four domains known to be involved in transcriptional and/or epigenetic regulation and a truncated form lacking the plant-homeodomainlike domain associated with epigenetic repression. Reverse genetics revealed limited influence of the VAL1 absence on seed and embryo metabolomes. However, 3,293 and 2,194 transcripts were up and down-regulated, respectively, in developing val1 mutant embryos relative to the wild type, suggesting that VAL1 is a global regulator of gene expression in developing embryos. Interestingly, none of the transcripts encoding the core LAFL network transcription factors were affected in val1 embryos. Instead, activation of VAL1 by FUS3 appears to result in repression of a subset of seed maturation genes downstream of core LAFL regulators. Transcripts that were not LAFL targets were also de-repressed in developing val1 embryos. Taken together, VAL1 appears to repress its targets through LAFL-dependent and independent mechanisms through epigenetic and/or transcriptional regulation.Members of the toll-like and IL-1 receptor family (TIR) are central regulators of immune and inflammatory responses. Signal activation is induced through ligand binding and controlled by system specific co-receptors. The IL-1RI coreceptor TILRR is a splice variant of FREM1. TILRR association with the signaling receptor magnifies IL-1 induced activation of the canonical and non-canonical NF-kB network by enhancing signal amplification at the level of the receptor complex and potentiates recruitment of the MyD88 adapter and PI3 kinase. TILRRcontrolled MyD88 dependent activation of the canonical pathway is regulated in a Ras-dependent manner, reflected in alterations in cytoskeletal structure and cell adhesion. The changes induced provide a process for rapid control of NF-kB, involving sequestration and release of cytoskeletal bound IkBα through a mechanism controlled by TILRR signal amplification. In silico simulations using agent based modeling of the NFkB network predict cytoskeletal control of inhibitor levels to provide a mechanism for signal calibration and to enable activation-sensitive regulation of NFkB induced inflammatory responses. Our studies have identified two functional sites within the TILRR core protein, which selectively control inflammatory and anti-apoptotic responses. The mechanisms underlying distinct network amplification and the relevance of pathway-specific regulation of canonical and non-canonical NFkB activation will be discussed.C (Manihot esculenta) is a major industrial crop which is rich in lot of therapeutic applications such as antitumor activity. Leaf, stem and petiole of cassava in vitro plantlets were cultured on solid MS-medium containing different combinations and concentrations of 2.4-D, picloram or NAA as auxins and BA as cytokinins. Stem was chosen as the best explant for callus induction, stem derived calli (25.892±0.117 g) on the best selected medium for callus production (5 mg/l 2.4-D and 0.2 mg/l BA) with subculture intervals each 40 days of culture. The phytochemical components such as fatty acids and their ester derivatives constituents were extracted from calli of cassava using different solvents, ethylacetate as aprotic polar solvent, chloroform and hexane as non-polar solvent to examine the efficiency of these extracts for inhibition the growth of the breast cancer cell. All extracts recorded positive results for inhibition cancer cell proliferation. Ethylacetate extracts have maximum efficiency for inhibition breast cancer (2.63 ug IC50), followed by hexane extracts (3.44 ug IC50) and then chloroform extracts (6 ug IC50) recorded the least value. The objective of this study was analyzed the composition of lipid profile components for ethylacetate extract, chloroform extract and hexane extract by GC-Mass and determine the efficiency of these extracts as anti-breast cancer cell growth. Ethylacetate extracts showing higher concentration of the total fatty acids (76.53%), also, included caprylic acid (14.52%) and caproic ester (3.10%), while that hexane and chloroform extracts recorded less values of total fatty acids (69.26% and 39.77%, respectively) and recorded no value of both of caprylic acid and caproic ester. Therefore it could be recommended to use ethylacetate as aprotic solvent for the extraction of calli, as including most amounts of fatty acids and their derivatives compounds due to the most effect as anti-breast cancer.N human cancers are caused by copy-number variations (CNVs) of proto-oncogenes. Yet, detecting chromosomal CNVs before they reach establishment in large cell populations is a major challenge. By screening for a potential deletion-byproduct of CNVs, the so-called extrachromosomal circular DNA (eccDNA), we reasoned that we might elucidate some of the early ongoing processes in genomic rearrangements. We developed a highly sensitive eccDNA purification method, Circle-Seq that relies on removal of all linear DNA and next-generation sequencing of circular DNA. More than a thousand eccDNAs larger than 1 kb were recorded in the eukaryotic model Saccharomyces cerevisiae (yeast) increasing the number of known eccDNAs in eukaryotes more than a hundred fold. Now we present hundreds of eccDNA profiles from a distant related yeast subspecies. A number of eccDNAs are found to be identical between the two yeast strains, advocating for conserved hotspots for DNA circularization and potential genomic reintegration. We reveal that CNVs in the form of eccDNAs are common in S. cerevisiae and we hypothesize that eccDNAs could be important players in genetic variation and evolution of eukaryotic genomes.N shape and morphology is essential to maintain the epigenetic state of the genome and is robust in differentiated cells. In contrast to nuclei in non motile tissues, nuclei in differentiated skeletal and cardiac muscles are facing iterated and altered cytoplasmic mechanical forces, produced by muscle contraction/relaxation waves. Recent findings from several labs (including ours), uncovered a muscle-specific network of nuclear associated cytoskeletal proteins, which is essential to protect muscle nuclei from the variable cytoplasmic strain induced by muscle contraction/relaxation and consequently is essential for the maintenance of myonuclear shape. We are studying Nesprin-related mechanisms essential for maintenance of robust muscle nuclear structure. Our recent studies identified intra-nuclear alterations in the distribution of chromatin elements and DNA within the muscle nuclei of Nesprin/MSP300/Klar mutants. These proteins were shown to be essential for linking the nuclear membrane with the microtubule network, as well as with muscle sarcomeres, in order to maintain robust myonuclear shape. Furthermore, we have identified a novel membrane protein, (Muscle-specific-alpha2delta), which is essential for keeping Nesprin/MSP-300 in the nuclear membrane preventing its retranslocation to the ER. Live imaging of muscles within intact Drosophila larvae with fluorescently labeled nuclei and Z-lines enabled imaging of myonuclei during muscle contraction/relaxation waves. A general recovery of myonuclear shape is detected in the course of contraction/relaxation waves of wild type larvae. In contrast, mutant nuclei became fluidic and exhibited significant deformation. We suggest that this deformation is the basis for defects in the intra-nuclear organization of chromatin, which further leads to aberrant transcriptional alterations in the mutant muscles. Such alterations might represent the cause for the numerous muscle diseases associated with mutants of the LINC complex in humans.

Evidence for extensive heterotrophic metabolism, antioxidant action, and associated regulatory events during winter hardening in Sitka spruce

BackgroundCold acclimation in woody perennials is a metabolically intensive process, but coincides with environmental conditions that are not conducive to the generation of energy through photosynthesis. While the negative effects of low temperatures on the photosynthetic apparatus during winter have been well studied, less is known about how this is reflected at the level of gene and metabolite expression, nor how the plant generates primary metabolites needed for adaptive processes during autumn.ResultsThe MapMan tool revealed enrichment of the expression of genes related to mitochondrial function, antioxidant and associated regulatory activity, while changes in metabolite levels over the time course were consistent with the gene expression patterns observed. Genes related to thylakoid function were down-regulated as expected, with the exception of plastid targeted specific antioxidant gene products such as thylakoid-bound ascorbate peroxidase, components of the reactive oxygen species scavenging cycle, and the plastid terminal oxidase. In contrast, the conventional and alternative mitochondrial electron transport chains, the tricarboxylic acid cycle, and redox-associated proteins providing reactive oxygen species scavenging generated by electron transport chains functioning at low temperatures were all active.ConclusionsA regulatory mechanism linking thylakoid-bound ascorbate peroxidase action with “chloroplast dormancy” is proposed. Most importantly, the energy and substrates required for the substantial metabolic remodeling that is a hallmark of freezing acclimation could be provided by heterotrophic metabolism.

A Machine Learning Approach to Predict Gene Regulatory Networks in Seed Development in Arabidopsis

Gene regulatory networks (GRNs) provide a representation of relationships between regulators and their target genes. Several methods for GRN inference, both unsupervised and supervised, have been developed to date. Because regulatory relationships consistently reprogram in diverse tissues or under different conditions, GRNs inferred without specific biological contexts are of limited applicability. In this report, a machine learning approach is presented to predict GRNs specific to developing Arabidopsis thaliana embryos. We developed the Beacon GRN inference tool to predict GRNs occurring during seed development in Arabidopsis based on a support vector machine (SVM) model. We developed both global and local inference models and compared their performance, demonstrating that local models are generally superior for our application. Using both the expression levels of the genes expressed in developing embryos and prior known regulatory relationships, GRNs were predicted for specific embryonic developmental stages. The targets that are strongly positively correlated with their regulators are mostly expressed at the beginning of seed development. Potential direct targets were identified based on a match between the promoter regions of these inferred targets and the cis elements recognized by specific regulators. Our analysis also provides evidence for previously unknown inhibitory effects of three positive regulators of gene expression. The Beacon GRN inference tool provides a valuable model system for context-specific GRN inference and is freely available at https://github.com/BeaconProjectAtVirginiaTech/beacon_network_inference.git.