Browsing by Author "Collakova, Eva"
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- Accession-Level Differentiation of Urushiol Levels, and Identification of Cardanols in Nascent Emerged Poison Ivy SeedlingsLott, Aneirin A.; Baklajian, Emily R.; Dickinson, Christopher C.; Collakova, Eva; Jelesko, John G. (MDPI, 2019-11-20)Poison ivy (Toxicodendron radicans (L.) Kuntze) shows accession-level differentiation in a variety of morphometric traits, suggesting local adaptation. To investigate whether the presumed defense compound urushiol also demonstrates accession-level accumulation differences, in vitro nascent germinated poison ivy seedlings from geographically isolated populations were germinated in vitro and then assayed for known urushiol congener accumulation levels. Significant accession-level differences in the accumulation levels of total C15- and C17-, total C15-, total C17-, specific C15 congeners, and specific C17 congeners of urushiol were identified. In addition, hereto novel C15- and C17-urushiol isomers were identified as well. Cardanols are assumed to be the penultimate metabolites giving rise to urushiols, but this assumption was not previously empirically validated. C15-cardanol congeners and isomers corresponding to expected substrates needed to produce the observed C15-urushiol congeners and isomers were identified in the same poison ivy seedling extracts. Total C15-cardanol and C15-cardanol congeners also showed significant accession-level differences. Based on the observed C15-cardanol congeners in poison ivy, the penultimate step in urushiol biosynthesis was proposed to be a cardanol-specific hydroxylase activity.
- Cell-type specificity and herbivore-induced responses of primary and terpene secondary metabolism in Arabidopsis rootsZhang, Jingyu (Virginia Tech, 2013-09-02)Plants employ diverse defense mechanisms to combat attack by harmful organisms. For instance, plants produce constitutive physical barriers or use chemical compounds such as specialized secondary metabolites to resist herbivore or pathogen invasion. Considering the cost-efficiency and energy balance between defense, growth and reproduction, defense reactions in plants have to be regulated temporally and spatially. As more cost-efficient strategies, plants may induce their defense response only in the presence of the attacker or restrict constitutive defenses to specific tissues or cells. In this study, we investigated aspects of the spatial regulation and induced changes of primary and secondary metabolism in roots of Arabidopsis thaliana. Roots represent important organs for anchoring plants in the soil and taking up water and nutrients. Hence, it is assumed that roots are as well protected as aerial tissues by different defense mechanisms. The first part of this work is focused on the cell-type-specific biosynthesis of volatile terpenes in Arabidopsis roots. Terpenes are the most abundant specialized metabolites in plants and play an important role in plant defense against pathogens or herbivores. Terpene biosynthetic enzyme activities are often coordinated in specific tissues and cellular compartments. Fine-scale transcriptome maps of Arabidopsis roots have shown that terpene biosynthesis is restricted to particular cell types. However, the reasons and significance of this cell-type specificity are not well understood. We hypothesized that the formation of terpene metabolites is not restricted to specific cells but can be supported by different cell types. We, therefore, probed the plasticity of the cell-specific formation of terpenes by swapping the expression of the terpene synthase (TPS) genes, TPS08, TPS13 and TPS25, between different root cell types in the respective mutant background. To investigate the ectopic expression of TPSs at different levels, quantitative real-time PCR (qRT-PCR), confocal microscopy, and gas chromatography-mass spectrometry (GC-MS) were performed. We found that terpene synthase TPS08, which produces the diterpene rhizathalene and is normally expressed in the root vascular tissue, is functionally active when expressed in the epidermis or cortex, although at substantially lower levels compared to the wild type. We did not find an obvious correlation between the volatile emission level and gene transcript level of TPS08, which may be attributed to a reduced activity of the expressed TPS08-yellow fluorescent protein (YFP) fusion protein. When expression of TPS13 (producing the sesquiterpene (Z)-"-bisabolene) was directed from the cortex to the epidermis or stele, TPS13 gene expression and (Z)-"-bisabolene formation was supported by these cell types although to varying levels in comparison to wild type. TPS13-YFP fluorescent signal driven by the epidermal WER and GL3 promoters was primarily detected at the root tip. Terpene production was also observed for the (E)-"-farnesene sesquiterpene synthase TPS25 when its expression was switched from the endodermis and non-hair producing epidermal cells to hair producing epidermal cells although only a weak fluorescent signal was detected from the expressed TPS25-mGFP protein. Together, the results provide preliminary evidence for a relaxed cell specificity of terpene biosynthesis in Arabidopsis roots and suggest that tissue-specific terpene metabolite patterns could change depending on different selective pressures in rhizosphere. In the second part of this study, we performed global gene transcript profiling and primary metabolite analysis of Arabidopsis roots upon feeding by the generalist root herbivore, Bradysia (fungus gnat). In a microarray analysis, we identified 451 of 22,810 genes that were up-regulated more than 2-fold. Gene ontology (GO) analysis showed that 26% of those genes with predicted or known functions play a role in primary or secondary metabolism, while 24% are involved in cell signaling or in responses to stimulating factors, such as jasmonic acid (JA), ethylene, wounding, and oxidative stress. At the metabolite level, we observed only marginal changes of amino acid, sugar and carboxylic acid relative levels over a time course of 4 days of Bradysia feeding. There was a trend for increased levels of amino acids and the relative levels of sucrose were increased significantly ("=0.05) at the fourth day of feeding. In conclusion, the study provided evidence for the induction of genes related to primary and secondary metabolism and stress responses in Arabidopsis roots, but showed only marginal changes at the primary metabolite level. In addition, the work indicated that the formation of terpene-specialized metabolites in Arabidopsis roots is not restricted to specific cells, but can be supported by different cell types.
- Changes in RNA Splicing in Developing Soybean (Glycine max) EmbryosAghamirzaie, Delasa; Nabiyouni, Mahdi; Fang, Yihui; Klumas, Curtis; Heath, Lenwood S.; Grene, Ruth; Collakova, Eva (MDPI, 2013-11-21)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.
- Characterization of the inositol monophosphatase gene family in ArabidopsisNourbakhsh, Aida; Collakova, Eva; Gillaspy, Glenda E. (Frontiers, 2015-01-09)Synthes is of myo-inositol is crucial in multicellular eukaryote 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 grow this 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.
- Characterizing glucose, illumination, and nitrogen-deprivation phenotypes of Synechocystis PCC6803 with Raman spectroscopyTanniche, Imen; Collakova, Eva; Denbow, Cynthia J.; Senger, Ryan S. (2020-03-30)Background. Synechocystis sp. PCC6803 is a model cyanobacterium that has been studied widely and is considered for metabolic engineering applications. Here, Raman spectroscopy and Raman chemometrics (Rametrix (TM)) were used to (i) study broad phenotypic changes in response to growth conditions, (ii) identify phenotypic changes associated with its circadian rhythm, and (iii) correlate individual Raman bands with biomolecules and verify these with more accepted analytical methods. Methods. Synechocystis cultures were grown under various conditions, exploring dependencies on light and/or external carbon and nitrogen sources. The Rametrix (TM) LITE Toolbox for MATLAB (R) was used to process Raman spectra and perform principal component analysis (PCA) and discriminant analysis of principal components (DAPC). The Rametrix (TM) PRO Toolbox was used to validate these models through leave-oneout routines that classified a Raman spectrum when growth conditions were withheld from the model. Performance was measured by classification accuracy, sensitivity, and specificity. Raman spectra were also subjected to statistical tests (ANOVA and pairwise comparisons) to identify statistically relevant changes in Synechocystis phenotypes. Finally, experimental methods, including widely used analytical and spectroscopic assays were used to quantify the levels of glycogen, fatty acids, amino acids, and chlorophyll a for correlations with Raman data. Results. PCA and DAPC models produced distinct clustering of Raman spectra, representing multiple Synechocystis phenotypes, based on (i) growth in the presence of 5 mM glucose, (ii) illumination (dark, light/dark [12 h/12 h], and continuous light at 20 mE), (iii) nitrogen deprivation (0-100%NaNO3 of native BG-11 medium in continuous light), and (iv) throughout a 24 h light/dark (12 h/12 h) circadian rhythm growth cycle. Rametrix (TM) PRO was successful in identifying glucose-induced phenotypes with 95.3% accuracy, 93.4% sensitivity, and 96.9% specificity. Prediction accuracy was above random chance values for all other studies. Circadian rhythm analysis showed a return to the initial phenotype after 24 hours for cultures grown in light/dark (12 h/12 h) cycles; this did not occur for cultures grown in the dark. Finally, correlation coefficients (R > 0.7) were found for glycogen, all amino acids, and chlorophyll a when comparing specific Raman bands to other experimental results.
- Characterizing metabolic stress-induced phenotypes of Synechocystis PCC6803 with Raman spectroscopyTanniche, Imen; Collakova, Eva; Denbow, Cynthia J.; Senger, Ryan S. (2020-03-30)Background. During their long evolution, Synechocystis sp. PCC6803 developed a remarkable capacity to acclimate to diverse environmental conditions. In this study, Raman spectroscopy and Raman chemometrics tools (Rametrix (TM)) were employed to investigate the phenotypic changes in response to external stressors and correlate specific Raman bands with their corresponding biomolecules determined with widely used analytical methods. Methods. Synechocystis cells were grown in the presence of (i) acetate (7.5-30 mM), (ii) NaCl (50-150 mM) and (iii) limiting levels of MgSO4 (0-62.5 mM) in BG-11 media. Principal component analysis (PCA) and discriminant analysis of PCs (DAPC) were performed with the Rametrix (TM) LITE Toolbox for MATLABR (R). Next, validation of these models was realized via Rametrix (TM) PRO Toolbox where prediction of accuracy, sensitivity, and specificity for an unknown Raman spectrum was calculated. These analyses were coupled with statistical tests (ANOVA and pairwise comparison) to determine statistically significant changes in the phenotypic responses. Finally, amino acid and fatty acid levels were measured with well-established analytical methods. The obtained data were correlated with previously established Raman bands assigned to these biomolecules. Results. Distinguishable clusters representative of phenotypic responses were observed based on the external stimuli (i.e., acetate, NaCl, MgSO4, and controls grown on BG-11 medium) or its concentration when analyzing separately. For all these cases, Rametrix (TM) PRO was able to predict efficiently the corresponding concentration in the culture media for an unknown Raman spectra with accuracy, sensitivity and specificity exceeding random chance. Finally, correlations (R > 0.7) were observed for all amino acids and fatty acids between well-established analytical methods and Raman bands.
- Comparative Metabolomics of Early Development of the Parasitic Plants Phelipanche aegyptiaca and Triphysaria versicolorClermont, Kristen; Wang, Yaxin; Liu, Siming; Yang, Zhenzhen; dePamphilis, Claude W.; Yoder, John I.; Collakova, Eva; Westwood, James H. (MDPI, 2019-06-13)Parasitic weeds of the family Orobanchaceae attach to the roots of host plants via haustoria capable of drawing nutrients from host vascular tissue. The connection of the haustorium to the host marks a shift in parasite metabolism from autotrophy to at least partial heterotrophy, depending on the level of parasite dependence. Species within the family Orobanchaceae span the spectrum of host nutrient dependency, yet the diversity of parasitic plant metabolism remains poorly understood, particularly during the key metabolic shift surrounding haustorial attachment. Comparative profiling of major metabolites in the obligate holoparasite Phelipanche aegyptiaca and the facultative hemiparasite Triphysaria versicolor before and after attachment to the hosts revealed several metabolic shifts implicating remodeling of energy and amino acid metabolism. After attachment, both parasites showed metabolite profiles that were different from their respective hosts. In P. aegyptiaca, prominent changes in metabolite profiles were also associated with transitioning between different tissue types before and after attachment, with aspartate levels increasing significantly after the attachment. Based on the results from 15N labeling experiments, asparagine and/or aspartate-rich proteins were enriched in host-derived nitrogen in T. versicolor. These results point to the importance of aspartate and/or asparagine in the early stages of attachment in these plant parasites and provide a rationale for targeting aspartate-family amino acid biosynthesis for disrupting the growth of parasitic weeds.
- CoSpliceNet: a framework for co-splicing network inference from transcriptomics dataAghamirzaie, Delasa; Collakova, Eva; Li, Song; Grene, Ruth (BMC, 2016)Background: Alternative splicing has been proposed to increase transcript diversity and protein plasticity in eukaryotic organisms, but the extent to which this is the case is currently unclear, especially with regard to the diversification of molecular function. Eukaryotic splicing involves complex interactions of splicing factors and their targets. Inference of co-splicing networks capturing these types of interactions is important for understanding this crucial, highly regulated post-transcriptional process at the systems level. Results: First, several transcript and protein attributes, including coding potential of transcripts and differences in functional domains of proteins, were compared between splice variants and protein isoforms to assess transcript and protein diversity in a biological system. Alternative splicing was shown to increase transcript and functionrelated protein diversity in developing Arabidopsis embryos. Second, CoSpliceNet, which integrates co-expression and motif discovery at splicing regulatory regions to infer co-splicing networks, was developed. CoSpliceNet was applied to temporal RNA sequencing data to identify candidate regulators of splicing events and predict RNAbinding motifs, some of which are supported by prior experimental evidence. Analysis of inferred splicing factor targets revealed an unexpected role for the unfolded protein response in embryo development. Conclusions: The methods presented here can be used in any biological system to assess transcript diversity and protein plasticity and to predict candidate regulators, their targets, and RNA-binding motifs for splicing factors. CoSpliceNet is freely available at http://delasa.github.io/co-spliceNet/.
- Detailed characterization of the UMAMIT proteins provides insight into their evolution, amino acid transport properties, and role in the plantZhao, Chengsong; Pratelli, Rejane; Yu, Shi; Shelley, Brett; Collakova, Eva; Pilot, Guillaume (Oxford University Press, 2021-09-30)Amino acid transporters play a critical role in distributing amino acids within the cell compartments and between plant organs. Despite this importance, relatively few amino acid transporter genes have been characterized and their role elucidated with certainty. Two main families of proteins encode amino acid transporters in plants: the amino acid-polyamine-organocation superfamily, containing mostly importers, and the UMAMIT (usually multiple acids move in and out transporter) family, apparently encoding exporters, totaling 63 and 44 genes in Arabidopsis, respectively. Knowledge of UMAMITs is scarce, based on six Arabidopsis genes and a handful of genes from other species. To gain insight into the role of the members of this family and provide data to be used for future characterization, we studied the evolution of the UMAMITs in plants, and determined the functional properties, the structure, and localization of the 47 Arabidopsis UMAMITs. Our analysis showed that the AtUMAMITs are essentially localized at the tonoplast or the plasma membrane, and that most of them are able to export amino acids from the cytosol, confirming a role in intra- and intercellular amino acid transport. As an example, this set of data was used to hypothesize the role of a few AtUMAMITs in the plant and the cell.
- Discovery of a Novel Microalgal Strain Scenedesmus Sp. A6 and Exploration of Its Potential as a Microbial Cell FactoryGuimaraes Braga da Silva, Pedro Ivo (Virginia Tech, 2018-08-14)Microalgae are photosynthetic organisms considered to be one of the most promising high-value chemicals and biofuel-producing organisms. However, there are several challenges for the widespread implementation of industrial processes using microalgae. The work presented in this dissertation proposes solutions to the different challenges involving the use of microalgae as microbial cell factories. To investigate the application of anaerobic digestion as a way to generate nutrients for microbial growth, salmon offal was used as substrate for anaerobic digestion, and soil from a flooded run-off pond on the Virginia Tech campus in Blacksburg, VA. A fast reduction in volatile solids and the short-chain fatty acid production profile is favorable for the growth of microalgae. A novel algae strain Scenedesmus sp. A6 was isolated from a decorative waterfountain in a hotel in Madison, IN. Mixotrophic growth trials were conducted using wastewater from the salmon offal digestion, that demostrated the A6 isolate grows six times faster in the wastewater then autotrophically. Bioassays of ethanolic cell extracts of A6 cultures demonstrated antimicrobial activity against E. coli cells at concentrations above 50 µg/ml. Genome sequencing and assembly revealed multiple copies of genes involved with acetate and ammonia metabolism, and several genes involved with secondary metabolite synthesis. An alternative to the high capital investment of photobioreactors for the cultivation of microalgae is the use of open-source and open-hardware bioreactor controller. Here, the concept of an open-hardwate bioreactor control called ``BioBrain'' is introduced. The BioBrain device is based on the Arduino Mega micro-controller board, and is capable of monitoring and controlling culture conditions during simple strain characterization studies, with an estimated construction cost of less than $800 USD. Finally, a new primer design tool for the ligation-independant cloning technique 𝜆-PCR was developed called lambdaPrimeR. The contributions of this work are the discovery and development of different tools that can overcome the challenges of the use of microalgae as microbial cell factories in industrial processes.
- Evaluating methiozolin programs for golf putting greens and investigating potential modes of actionVenner, Katelyn (Virginia Tech, 2015-10-06)Annual bluegrass is a winter annual grass that is problematic on golf putting greens due to its light green color, prolific seedhead production and intolerance to stress. On creeping bentgrass putting greens, herbicides for annual bluegrass control are limited. A new herbicide, methiozolin, developed by Moghu Research Center, LLC, in Daejeon, South Korea, safely and selectively controls annual bluegrass in creeping bentgrass and several other turfgrass species. Methiozolin typically controls annual bluegrass over several weeks, allowing desirable turfgrass time to grow into areas previously infested by annual bluegrass with little surface disruption. The mode of action of methiozolin is unknown, but has been proposed to act as either a cell wall biosynthesis inhibitor (CBI) or an inhibitor of tyrosine aminotransferase (TAT). Field studies were conducted at Virginia Tech to investigate strategies promoting surface recovery on putting greens following atypically rapid annual bluegrass loss resulting from methiozolin application, intensive core-cultivation as well as potential interactions with plant growth regulators (PGR's), like ethephon. In the rapid annual bluegrass removal study, all treatments receiving additional fertility via synthetic fertilizer with or without trinexapac-ethyl or biostimulant recovered 1 to 3 weeks more quickly than treatments that did not include additional fertility. Addition of the PGR trinexapac-ethyl inconsistently regulated speed of canopy recovery, both increasing and decreasing recovery speed. Under normal maintenance conditions, methiozolin does not negatively influence putting green recovery, however, if the putting green is exposed to droughty conditions, methiozolin can reduce recovery time by several weeks. Core-cultivation should be avoided in conjunction with methiozolin and ethephon applications because when this procedure was conducted on the same day as herbicide application it significantly damaged creeping bentgrass, reducing cover to 19% at 2000 g ai ha⁻¹, compared to the non-treated at 62%. Regarding the question of methiozoling mode of action, laboratory studies supported the claim that addition of exogenous 4-hydroxyphenylpyruvate (4-HPP) alleviates symptoms of methiozolin exposure in lesser duckweed, a model monocot species, but feeding various turfgrass species and annual bluegrass exogenous 4-HPP did not alleviate symptoms. Creeping bentgrass secondary root length and density was not affected by methiozolin, although annual bluegrass, Kentucky bluegrass and perennial ryegrass secondary root lengths were reduced. Based on these data, it does not appear that TAT inhibition is a primary mode of action of methiozolin in turfgrass. Studies were conducted to determine if methiozolin inhibited cell wall biosynthesis in desirable turfgrass species and annual bluegrass. All species exhibited decreased enrichment of ¹³C in cell-wall sugars form ¹³C-glucose in response to methiozolin and a known cell wall biosynthesis inhibitor, indaziflam. Indaziflam and methiozolin at 0.01 µM inhibited ¹³C enrichment of all sugars less than methiozolin at 1.0 µM, for xylose, arabinose and glucose, but not galactose. Addition of 4-HPP increased incorporation of ¹³C into xylose, but had no other influence on ¹³C incorporation into other cell wall sugars. Lack of species specific response indicates that cell wall biosynthesis inhibition is probably not the source of interspecific species responses observed in the field.
- Evidence for extensive heterotrophic metabolism, antioxidant action, and associated regulatory events during winter hardening in Sitka spruceCollakova, Eva; Klumas, Curtis; Suren, Haktan; Myers, Elijah; Heath, Lenwood S.; Holliday, Jason A.; Grene, Ruth (2013-04-30)Background Cold 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. Results The 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. Conclusions A 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.
- FITSelect: An Invention to Select Microbial Strains Maximizing Product Formation from a Single Culture Without High-Throughput ScreeningZhou, Rui (Virginia Tech, 2011-08-08)In metabolic engineering of prokaryotes, combinatorial approaches have developed recently that induce random genetic perturbations to achieve a desired cell phenotype. A screening strategy follows the randomized genetic manipulations to select strain(s) with the more optimal phenotype of interest. This screening strategy is often divided into two categories: (i) a growth competition assay and (ii) selection by high-throughput screening. The growth competition assay involves culturing strains together. The strain with the highest growth rate will ultimately dominate the culture. This strategy is ideal for selecting strain with cellular fitness (e.g., solvent tolerance), but it does not work for selecting a strain that can over-produce a product (e.g., an amino acid). For the case of selecting highly productive phenotypes, high-throughput screening is used. This method analyzes strains individually and is costly and time-consuming. In this research, a synthetic genetic circuit was developed to select highly productive phenotypes using a growth competition assay rather than high-throughput screening. This novel system is called Feed-back Inhibition of Transcription for Growth Selection (FITSelect), and it uses a natural feedback inhibition mechanism in the L-arginine production pathway to select strains (transformed with a random genomic library) that can over-produce L-arginine in E. coli DH10B. With FITSelect, the cell can thrive in the growth competition assay when L-arginine is over-produced (i.e., growth is tied to L-arginine production). Cell death or reduced growth results if L-arginine is not over-produced by the cell. This system was created by including an L-arginine concentration responsive argF promoter to control a ccdB cell death gene in the FITSelect system. The effects of ccdB were modulated by the antidote ccdA gene under control of an L-tryptophan responsive trp promoter. Several insights and construction strategies were required to build a system that ties the growth rate of the cell to L-arginine concentrations.
- Functional Analysis of Plant Glutamate ReceptorsPrice, Michelle B. (Virginia Tech, 2013-10-02)The plant glutamate receptors (GLRs) are homologs of mammalian ionotropic glutamate receptors (iGluRs) and are hypothesized to be potential amino acid sensors in plants. Since their first discovery in 1998, the members of plant GLRs have been implicated in diverse processes such as C/N ratio sensing, root formation, pollen germination and plant-pathogen interaction. However, the exact properties of these channels, such as the spectrum of ligands, ion specificities, and subunit compositions are still not well understood. It is well established that animal iGluRs form homo- or hetero-tetramers in order to form ligand-gated cation channels. The first aspect of this research was to determine if plant GLRs likewise require different subunits to form functional channels. A modified yeast-2-hybrid system approach was initially taken and applied to 14 of the 20 AtGLRs to identify a number of candidate interactors in yeast. Forster resonance energy transfer (FRET), which measures the transfer of energy between interacting molecules, was performed in mammalian cells to confirm interaction between a few of those candidates. Interestingly, despite an abundance of overlapping co-localization between heteromeric combinations, only homomeric interactions were identified between GLRs 1.1 and 3.4 in HEK293 cells. Further, amino acids have been implicated in signaling between plants and microbes, but the mechanisms for amino acid perception in defense responses are far from being understood. Recently it was demonstrated that calcium responses initiated by bacterial and fungal microbe-associated molecular patterns (MAMPs) were diminished in seedlings treated with known agonists and antagonists of mammalian iGluRs, suggesting potential roles of GLRs in pathogen responses. Analysis of publicly available microarray data shows altered gene expression of a sub-fraction of GLRs in response to pathogen infection and bacterial elicitors. Thus, the second goal of my PhD research was aimed at determining whether GLRs are involved in the interaction between plants and pathogens. Gene expression changes of a number of candidate GLRs as well as pathogen growth was examined in response to the plant pathogen Pseudomonas syringae pv. tomato DC3000. Interestingly, single gene and multi-gene deficient plants responded differently with regards to pathogen susceptibility, likely as a result of functional compensation between GLRs.
- Increased Expression of UMAMIT Amino Acid Transporters Results in Activation of Salicylic Acid Dependent Stress ResponseBesnard, Julien; Sonawala, Unnati; Maharjan, Bal; Collakova, Eva; Finlayson, Scott A.; Pilot, Guillaume; McDowell, John M.; Okumoto, Sakiko (2021-01-26)In addition to their role in the biosynthesis of important molecules such as proteins and specialized metabolites, amino acids are known to function as signaling molecules through various pathways to report nitrogen status and trigger appropriate metabolic and cellular responses. Moreover, changes in amino acid levels through altered amino acid transporter activities trigger plant immune responses. Specifically, loss of function of major amino acid transporter, over-expression of cationic amino acid transporter, or over-expression of the positive regulators of membrane amino acid export all lead to dwarfed phenotypes and upregulated salicylic acid (SA)-induced stress marker genes. However, whether increasing amino acid exporter protein levels lead to similar stress phenotypes has not been investigated so far. Recently, a family of transporters, namely USUALLY MULTIPLE ACIDS MOVE IN AND OUT TRANSPORTERS (UMAMITs), were identified as amino acid exporters. The goal of this study was to investigate the effects of increased amino acid export on plant development, growth, and reproduction to further examine the link between amino acid transport and stress responses. The results presented here show strong evidence that an increased expression of UMAMIT transporters induces stress phenotypes and pathogen resistance, likely due to the establishment of a constitutive stress response via a SA-dependent pathway.
- Investigating the Role of the VAL1 Transcription Factor in Arabidopsis thaliana Embryo DevelopmentSchneider, Andrew (Virginia Tech, 2015-10-05)Developing oilseeds accumulate oils and seed storage proteins synthesized by the pathways of primary metabolism. Seed development and metabolism are positively regulated at the transcriptional level through the transcription factors belonging to the LAFL regulatory network. The VAL genes encode repressors of the seed maturation program in germinating seeds, but they are also expressed during early stages of seed maturation. VAL1 was identified through a reverse genetics approach as a regulator of seed metabolism, as val1 mutant seeds accumulated elevated levels of storage proteins compared to the wild type. Two VAL1 splice variants were identified, yielding the canonical protein and a truncated protein lacking the plant-homeodomain-like domain important for epigenetic repression. Transcriptomics analysis also revealed that VAL1 is a global epigenetic and transcriptional repressor in developing embryos, though none of the transcripts encoding the LAFL network regulators, including FUSCA3, were affected in val1 embryos. However, VAL1 action is connected specifically to FUSCA3 as 38% of transcripts belonging to the FUSCA3 regulon, but not to other regulons, were largely de-repressed in the absence of VAL1. Based on our model, FUSCA3 activates expression of VAL1 to repress transcription of seed maturation genes without interfering with expression of the core LAFL regulators.
- Isoform-Specific Expression During Embryo Development in Arabidopsis and SoybeanAghamirzaie, Delasa (Virginia Tech, 2016-06-19)Almost every precursor mRNA (pre-mRNA) in a eukaryotic organism undergoes splicing, in some cases resulting in the formation of more than one splice variant, a process called alternative splicing. RNA-Seq provides a major opportunity to capture the state of the transcriptome, which includes the detection of alternative spicing events. Alternative splicing is a highly regulated process occurring in a complex machinery called the spliceosome. In this dissertation, I focus on identification of different splice variants and splicing factors that are produced during Arabidopsis and soybean embryo development. I developed several data analysis pipelines for the detection and the functional characterization of active splice variants and splicing factors that arise during embryo development. The main goal of this dissertation was to identify transcriptional changes associated with specific stages of embryo development and infer possible associations between known regulatory genes and their targets. We identified several instances of exon skipping and intron retention as products of alternative splicing. The coding potential of the splice variants were evaluated using CodeWise. I developed CodeWise, a weighted support vector machine classifier to assess the coding potential of novel transcripts with respect to RNA secondary structure free energy, conserved domains, and sequence properties. We also examined the effect of alternative splicing on the domain composition of resulting protein isoforms. The majority of splice variants pairs encode proteins with identical domains or similar domains with truncation and in less than 10% of the cases alternative splicing results in gain or loss of a conserved domain. I constructed several possible regulatory networks that occur at specific stages of embryo development. In addition, in order to gain a better understanding of splicing regulation, we developed the concept of co-splicing networks, as a group of transcripts containing common RNA-binding motifs, which are co-expressed with a specific splicing factor. For this purpose, I developed a multi-stage analysis pipeline to integrate the co-expression networks with de novo RNA binding motif discovery at inferred splice sites, resulting in the identification of specific splicing factors and the corresponding cis-regulatory sequences that cause the production of splice variants. This approach resulted in the development of several novel hypotheses about the regulation of minor and major splicing in developing Arabidopsis embryos. In summary, this dissertation provides a comprehensive view of splicing regulation in Arabidopsis and soybean embryo development using computational analysis.
- A Machine Learning Approach to Predict Gene Regulatory Networks in Seed Development in ArabidopsisGrene, Ruth; Heath, Lenwood S.; Li, Song; Collakova, Eva; Elmarakeby, Haitham A.; Ni, Ying; Aghamirzaie, Delasa (Frontiers, 2016-12-23)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.
- MALDI-MS Imaging of Urushiols in Poison Ivy StemAziz, Mina; Sturtevant, Drew; Winston, Jordan; Collakova, Eva; Jelesko, John G.; Chapman, Kent D. (MDPI, 2017-04-29)Urushiols are the allergenic components of Toxicodendron radicans (poison ivy) as well as other Toxicodendron species. They are alk-(en)-yl catechol derivatives with a 15- or 17-carbon side chain having different degrees of unsaturation. Although several methods have been developed for analysis of urushiols in plant tissues, the in situ localization of the different urushiol congeners has not been reported. Here, we report on the first analysis of urushiols in poison ivy stems by matrix-assisted laser desorption/ionization-mass spectrometry imaging (MALDI-MSI). Our results show that the urushiol congeners with 15-carbon side chains are mainly localized to the resin ducts, while those with 17-carbon side chains are widely distributed in cortex and vascular tissues. The presence of these urushiols in stem extracts of poison ivy seedlings was confirmed by GC-MS. These novel findings provide new insights into the spatial tissue distribution of urushiols that might be biosynthetically or functionally relevant.
- Metabolic and Transcriptional Reprogramming in Developing Soybean (Glycine max) EmbryosCollakova, Eva; Aghamirzaie, Delasa; Fang, Yihui; Klumas, Curtis; Tabataba, Farzaneh; Kakumanu, Akshay; Myers, Elijah; Heath, Lenwood S.; Grene, Ruth (MDPI, 2013-05-14)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.