VTechWorks Repository :: Browsing by Author "Tokuhisa, James G."

Browsing by Author "Tokuhisa, James G."

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An aeroponic culture system for the study of root herbivory on Arabidopsis thaliana
Vaughan, Martha M.; Tholl, Dorothea; Tokuhisa, James G. (Biomed Central, 2011-03-10)
Background Plant defense against herbivory has been studied primarily in aerial tissues. However, complex defense mechanisms have evolved in all parts of the plant to combat herbivore attack and these mechanisms are likely to differ in the aerial and subterranean environment. Research investigating defense responses belowground has been hindered by experimental difficulties associated with the accessibility and quality of root tissue and the lack of bioassays using model plants with altered defense profiles. Results We have developed an aeroponic culture system based on a calcined clay substrate that allows insect herbivores to feed on plant roots while providing easy recovery of the root tissue. The culture method was validated by a root-herbivore system developed for Arabidopsis thaliana and the herbivore Bradysia spp. (fungus gnat). Arabidopsis root mass obtained from aeroponically grown plants was comparable to that from other culture systems, and the plants were morphologically normal. Bradysia larvae caused considerable root damage resulting in reduced root biomass and water absorption. After feeding on the aeroponically grown root tissue, the larvae pupated and emerged as adults. Root damage of mature plants cultivated in aeroponic substrate was compared to that of Arabidopsis seedlings grown in potting mix. Seedlings were notably more susceptible to Bradysia feeding than mature plants and showed decreased overall growth and survival rates. Conclusions A root-herbivore system consisting of Arabidopsis thaliana and larvae of the opportunistic herbivore Bradysia spp. has been established that mimics herbivory in the rhizosphere. Bradysia infestation of Arabidopsis grown in this culture system significantly affects plant performance. The culture method will allow simple profiling and in vivo functional analysis of root defenses such as chemical defense metabolites that are released in response to belowground insect attack.
Analysis of Plant Homeodomain Proteins and the Inhibitor of Growth Family Proteins in Arabidopsis thaliana
Safaee, Natasha Marie (Virginia Tech, 2009-08-18)
Eukaryotic organisms require the ability to respond to their environments. They do so by utilizing signal transduction pathways that allow for signals to effect final biological responses. Many times, these final responses require new gene expression events that have been stimulated or repressed within the nucleus. Thus, much of the understanding of signal transduction pathways converges on the understanding of how signaling affects gene expression alterations (Kumar et al., 2004). The regulation of gene expression involves the modification of chromatin between condensed (closed, silent) and expanded (open, active) states. Histone modifications, such as acetylation, can determine the open versus closed status of chromatin. The PHD (Plant HomeoDomain) finger is a structural domain primarily found in nuclear proteins across eukaryotes. This domain specifically recognizes the epigenetic marks H3K4me2 and H3K4me3, which are di- and tri-methylated lysine 4 residues of Histone H3 (Loewith et al., 2000; Kuzmichev et al., 2002; Vieyra et al. 2002; Shiseki et al., 2003; Pedeux et al., 2005, Doyon et al., 2006). It is estimated that there are ~150 proteins that contain the PHD finger in humans (Solimon and Riabowol, 2007). The PHD finger is conserved in yeast and plants, however an analysis of this domain has only been performed done in Arabidopsis thaliana (Lee et al., 2009). The work presented in this report aims to extend the analysis of this domain in plants by identifying the PHD fingers of the crop species Oryza sativa (rice). In addition, a phylogenetic analysis of all PHD fingers in Arabidopsis and rice was undertaken. From these analyses, it was determined that there are 78 PHD fingers in Arabidopsis and 70 in rice. In addition, these domains can be categorized into classes and groups by defining features within the conserved motif. In a separate study, I investigated the function of two of the PHD finger proteins from Arabidopsis, ING1 (INhibitor of Growth1) and ING2. In humans, these proteins can be found in complexes associated with both open and closed chromatin. They facilitate chromatin remodeling by recruiting histone acetyltransferases and histone deacetylases to chromatin (Doyon et al., 2006, Pena et al., 2006). In addition, these proteins recognize H3K4me2/3 marks and are believed to be "interpreters" of the histone code (Pena et al., 2006, Shi et al., 2006). To understand the function of ING proteins in plants, I took a reverse genetics approach and characterized ing1 and ing2 mutants. My analysis revealed that these mutants are altered in time of flowering, as well as their response to nutrient and stress conditions. Lastly, I was able to show that ING2 protein interacts in vitro with SnRK1.1, a nutrient/stress sensor (Baena-Gonzalez et al., 2007). These results indicate a novel function for PHD proteins in plant growth, development and stress response.
Biochemical, Molecular and Functional Analysis of Volatile Terpene Formation in Arabidopsis Roots
Huh, Jung-Hyun (Virginia Tech, 2011-07-20)
Plants produce secondary (or specialized) metabolites to respond to a variety of environmental changes and threats. Especially, volatile compounds released by plants facilitate short and long distance interaction with both beneficial and harmful organisms. Comparatively little is known about the organization and role of specialized metabolism in root tissues. In this study, we have investigated the root-specific formation and function of volatile terpenes in the model plant Arabidopsis. As one objective, we have characterized the two root-specific terpene synthases, TPS22 and TPS25. Both enzymes catalyze the formation of several volatile sesquiterpenes with (E)-β-farnesene as the major product. TPS22 and TPS25 are expressed in the root in distinct different cell type-specific patterns and both genes are induced by jasmonic acid. Unexpectedly, both TPS proteins are localized to mitochondria, demonstrating a subcellular localization of terpene specialized metabolism in compartments other than the cytosol and plastids. (E)-β-Farnesene is produced at low concentrations suggesting posttranslational modifications of the TPS proteins and/or limited substrate availability in mitochondria. We hypothesize that the mitochondrial localization of TPS22 and TPS25 reflects evolutionary plasticity in subcellular compartmentation of TPS proteins with emerging or declining activity. Since (E)-β-farnesene inhibits Arabidopsis root growth in vitro, mitochondrial targeting of both proteins may fine tune (E)-β-farnesene concentrations to prevent possible autotoxic or inhibitory effects of this terpene in vivo. We further investigated the role of volatile terpenes in Arabidopsis roots in interaction with the soil-borne oomycete, Pythium irregulare. Infection of roots with P. irregulare causes emission of the C11-homoterpene (or better called C4-norterpene) 4,8-dimethylnona-1,3,7-triene (DMNT), which is a common volatile induced by biotic stress in aerial parts of plants but was not previously known to be produced in plant roots. We demonstrate that DMNT is synthesized by a novel, root-specific pathway via oxidative degradation of the C30-triterpene, arabidiol. DMNT exhibits inhibitory effects on P. irregulare mycelium growth and oospore germination in vitro. Moreover, arabidiol and DMNT biosynthetic mutants were found to be more susceptible to P. irregulare infection and showed higher rates of Pythium colonization in comparison to wild type plants. Together, our studies demonstrate differences and plasticity in the metabolic organization and function of terpenes in roots in comparison to aboveground plant tissues.
Biosynthesis of Steroidal Glycoakaloids in Solanum chacoense Bitter
Mweetwa, Alice Mutiti (Virginia Tech, 2009-07-24)
Steroidal glycoalkaloids (SGAs) are secondary metabolites produced by approximately 350 species in the Solanaceae family. SGAs are reported to be important for pest resistance and flavor enhancement at low concentrations but are toxic to humans and other mammals at high concentrations. Studies on sterol / SGA biosynthesis have implicated squalene synthase as a key regulatory enzyme because it catalyzes an irreversible step from the mevalonic acid pathway. However, the regulatory mechanisms of squalene synthase are not yet known. A study was conducted to elucidate the distribution pattern of SGAs and to clone the squalene synthase gene in order to determine a relationship between SGAs and gene expression levels. Solanum chacoense, a wild potato species was used as a model plant from which tissues were harvested at specified developmental stages and analyzed for SGA content. The results from the SGA analysis suggest a qualitative and quantitative tissue- and age-dependent accumulation of SGAs. Regenerative tissues such as, axiliary shoots, flowers and floral buds had the highest levels of 88, 49 and 63 Âµmole/g DW, respectively. The roots, stems and tubers showed the lowest amounts of SGAs of 1 to 8, 5 to 15 and 7 to 15 Âµmole/g DW, respectively. Stolons and tubers accumulated higher amounts of α-chaconine (59 to 67%) than α-solanine (61 to 64%) at all developmental stages analyzed. On the other hand, in young expanding, fully expanded, and old senescing leaves where leptine and leptinines tend to dominate, α-solanine and α-chaconine together accounted for only 8 to 15%, 7 to 15%, and 8 to 45%, respectively. Plant organs that showed the highest biosynthetic activity for SGA production also had high levels of transcripts coding for genes of isoprenoid biosynthesis. The results from the cloning and characterization of squalene synthase suggest that the cloned cDNA fragment is a putative S. chacoense squalene synthase gene with an open reading frame / predicted protein precursor of 411 amino acids. The cloned cDNA has high similarity (68-100%) to known plant squalene synthase genes and contains six deduced peptide domains observed in other species. The 3â untranslated regions of floral buds, young leaves (early vegetative stage), and fully expanded leaves (anthesis) were different in length with, 249, 335, and 202 nucleotides, respectively. The Southern blot analysis suggests a single copy gene although the existence of a gene family cannot be ruled out.
Cell-type specificity and herbivore-induced responses of primary and terpene secondary metabolism in Arabidopsis roots
Zhang, 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.
Functional characterization of NAC-domain transcription factors implicated in control of vascular cell differentiation in Arabidopsis and Populus
Grant, Emily H. (Virginia Tech, 2008-12-10)
Wood has a wide variety of uses and is arguably the most important renewable raw material. The composition of xylem cell types in wood determines the utility of different types of wood for distinct commercial applications. Using expression profiling and phylogenetic analysis, we identified many xylem-associated regulatory genes that may control the differentiation of cells involved in wood formation in Arabidopsis and poplar. Prominent among these are NAC-domain transcription factors (NACs). In addition to their roles as regulators of xylem differentiation, NACs are regulators of meristem development, organ elongation and separation. We studied a subset of Populus and Arabidopsis NACs with putative involvement in xylem cell expansion and elongation (XND1/ANAC104, PopNAC118, PopNAC122, PopNAC128, PopNAC129), and secondary cell wall synthesis (ANAC073, PopNAC105, PopNAC154, PopNAC156, PopNAC157). Using quantitative Real-Time PCR, we evaluated expression of the selected Populus NACs in a developmental gradient and in response to bending stress. We prepared transgenic Arabidopsis and Populus plants with increased or decreased expression of select NAC genes. For dominant repression of target gene expression, we evaluated transgenic plants expressing translational fusions of NAC-EAR (ERF amphiphilic repressor) chimeras through chimeric repressor silencing-technology (CRES-T). XND1 overexpression in Populus and Arabidopsis resulted in severe stunting and suppression of xylem differentiation. Overexpression of PopNAC122, an XND1 ortholog, yielded an analogous phenotype in Arabidopsis. Populus XND1 overexpressors lacked phloem fibers and showed a reduction in cell size and number, vessel number and frequency of rays. Knowledge gained through characterization of these wood-associated regulatory genes can be used to optimize molecular breeding and genetic engineering strategies for improved wood quality and increased biomass.
Genetic studies of candidate genes in the glycoalkaloid biosynthetic pathway of potato
Manrique Carpintero, Norma Constanza (Virginia Tech, 2013-01-24)
Potato (Solanum tuberosum L) is an outcrossing, highly heterozygous cultivated in which the elucidation of the genetic basis of quantitative traits, is more complex than in self-pollinated crops. Both a candidate gene approach and a whole genome SNP genotyping analysis were used to assess allelic variation and to identify loci associated with biosynthesis and accumulation of steroidal glycoalkaloids (SGAs). SGAs are secondary metabolites produced in Solanum species as defense against insects and pathogens. Fragments of genomic DNA coding for regions of five SGA biosynthetic candidate genes were amplified, cloned and sequenced [3-hydroxy-3-methylglutaryl coenzyme A reductase 1 and 2 (HMG1, HMG2); 2,3-squalene epoxidase (SQE); solanidine galactosyltransferase (SGT1); and solanidine glucosyltransferase (SGT2)]. A germplasm panel of six wild potato species [Solanum chacoense (chc 80-1), S. commersonii subsp. commersonii, S. demissum, S. sparsipilum, S. spegazzinii, and S. stoloniferum] and a cultivated clone S. tuberosum Group Phureja (phu DH) was used in an allelic variation analysis. A segregating interspecific F2 population phu DH ï¿½" chc 80-1 was screened to assess association with SGAs. Sequence diversity analysis showed a tendency of purifying selection and increased frequency of rare alleles in most of the candidate genes. Genes of primary metabolism (HMG1, HMG2 and SQE) had stronger selection constraints than those in secondary metabolism (SGT1 and SGT2). Sequence polymorphism in HMG2, SQE, SGT1 and SGT2 separated either the phu DH clone which produced no SGAs, or chc 80-1, the greatest SGA accumulator, from other accessions in the panel. Segregation analysis of the F2 population revealed that allelic sequences of HMG2 and SGT2 derived from chc 80-1 were significantly associated with the greatest SGA accumulation. In the whole genome analysis, SNP genotyping and cluster analysis based on putative association with SGA accumulation in the germplasm panel, allowed identification of eight informative SNPs that can be used in future studies. In the segregating F2 population, loci located on five pseudochromosomes were associated with SGA synthesis. Loci on pseudochromosomes 1 and 6 explained segregation ratios of synthesis for Î±-solanine and Î±-chaconine, the most common SGAs in most potato species. In addition, loci on seven pseudochromosomes were associated with accumulation. New candidate genes, putatively affecting synthesis and accumulation of SGAs, were identified in adjacent genomic regions of significant SNPs. This research demonstrates how the newly available genome sequence of potato and associated biotechnological tools accelerates the identification of genetic factors underling complex traits in a species with a difficult breeding structure.
Genetic studies of phenotypic variants in the woodland strawberry, (Fragaria vesca)
Holt, Sarah Hudson (Virginia Tech, 2011-09-02)
The diploid woodland strawberry (Fragaria vesca) is a rapidly developing translational model for members of the family Rosaceae and other plants. This thesis represents some of the first forward genetics studies evaluating putative T-DNA insertional mutants in F. vesca. The observed phenotypes include alterations to floral development, anthocyanin pigmentation and leaf structure. The floral development mutant named green petal (gp) was not associated with the T-DNA insertions present. Based on similar phenotypes induced by mutation of transcription factors involved in floral development of Arabidopsis thaliana, we used a BLAST search of the F. vesca genome hybrid gene models to identify 30 candidate genes that may have caused the gp phenotype. Expression analysis of these genes revealed that it was due to a 37 bp deletion in a SEPALLATA3-like E-Class MADS box transcription factor. This mutation altered organ structure in the three inner whorls of the flower, affecting fertility and fruit development. The deletion was demonstrated to segregate with the mutant phenotype in a segregating population of 92 individuals, 22 of which had green petals. The anthocyanin biosynthesis mutant named white runner (wr) lacked red pigmentation in the stems and runners. The T-DNA insertion in this line was located in a highly repetitive LTR retrotransposon region, which complicated analysis. Segregation analysis of the wr lines revealed that the phenotype was unassociated with the T-DNA insertion as well. We used a targeted expression analysis of three critical structural genes in the flavonoid biosynthesis pathway that revealed a 20 bp deletion in the gene encoding flavanone 3-hydroxylase, an enzyme necessary for the production of flavonols, anthocyanins and proanthocyanidins. In an F2 segregating population, this deletion co-segregated with the phenotype. The third mutant line presented here displayed a curly leaf (cl) phenotype and was found to harbor a T-DNA insertion in a gene encoding a putative erythroblast macrophage attacher protein (EMP). Sequence and protein domain analysis indicated that FvEMP was related to the mammalian EMP protein that functions in cytoskeletal dynamics and red blood cell enucleation. Complementation analysis confirmed that introduction of the wild type FvEMP gene into the cl mutant plants restored wild type leaf phenotype. Further morphological analysis revealed additional pleiotropic effects of the mutation, including abnormalities in seed set and germination, pollen tube growth, adhesion of the abaxial epidermal layer to the mesophyll layer and reduced petiolule length. These phenotypes are consistent with actin binding and microtubule associated protein mutants in other plant species. Insertional mutagenesis is a critical molecular tool for model crop development. These studies highlight the precautions that must be taken when evaluating insertional mutants. These mutants are excellent tools for studying their respective disrupted gene function. The in depth molecular analysis of the mutants presented in this work was only possible because of the availability of the Fragaria vesca genome which was used extensively to identify T-DNA insertion sites and recover candidate gene sequences for expression analysis.
Managing Weeds and Soilborne Pests with Fumigant and Non-Fumigant Alternatives to Methyl Bromide
McAvoy, Theodore Porter (Virginia Tech, 2012-05-02)
Methyl bromide (MBr) was widely used as a soil fumigant to manage soilborne pests in plasticulture vegetable production; however, it has been banned by the United Nations Environment Programme. Alternatives to MBr must be implemented to sustain fresh market tomato productivity. Possible MBr alternatives include new fumigant compounds, improved plastic mulch, and grafting. Methyl iodide (MeI) and dimethyl disulfide (DMDS) were tested as fumigant alternatives to MBr for the control of yellow nutsedge and soilborne pathogens of tomato. Virtually impermeable film (VIF) and totally impermeable film (TIF) were tested for fumigant retention and yellow nutsedge control in tomato. Grafting onto resistant rootstocks was tested for bacterial wilt and nematode management in tomato. In the absence of a soil fumigant, TIF suppressed yellow nutsedge better than VIF. TIF increased fumigant retention compared to VIF at similar application rates. Reduced fumigant application rates could be used in combination with TIF while maintaining fumigant concentrations, weed control, and crop yields comparable to greater use rates with VIF. Shank applied DMDS rates could be lowered to 281 L/ha under TIF from 468 L/ha under VIF; shank applied MeI application rates could be reduced to 56 L/ha under TIF compared to 93 L/ha under VIF and drip applied DMDS could be reduced from 561 L/ha under VIF film to 374 L/ha under TIF. Grafting susceptible commercial tomato cultivars onto resistant tomato hybrid rootstocks increased yields and plant survival in bacterial wilt infested fields. "Cheong Gang", "BHN 998", and "BHN 1054" were the best performing rootstocks for bacterial wilt resistance and tomato fruit yield in severely infested fields. Grafting increased tomato yield and decreased root galling from root-knot nematodes in an infested field. Hybrid rootstock "RST 106" resulted in the lowest root-knot nematode galling. In conclusion, TIF with reduced rates of DMDS or MeI is a viable MBr alternative for fresh market tomato production to retain effective doses of fumigant, manage yellow nutsedge and maintain yields. Grafting is an effective MBr alternative to manage bacterial wilt and root-knot nematode and maintain tomato yields.
De novo formation of an aggregation pheromone precursor by an isoprenyl diphosphate synthase-related terpene synthase in the harlequin bug
Lancaster, Jason; Khrimian, Ashot; Young, Sharon; Lehner, Bryan; Luck, Katrin; Wallingford, Anna K.; Ghosh, Saikat Kumar B.; Zerbe, Philipp; Muchlinski, Andrew; Marek, Paul E.; Sparks, Michael E.; Tokuhisa, James G.; Tittiger, Claus; Köllner, Tobias G.; Weber, Donald C.; Gundersen-Rindal, Dawn E.; Kuhar, Thomas P.; Tholl, Dorothea (2018-09-11)
Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (15,6S,7R)-1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication.
Potato genomics three ways: quantification of endoreduplication in tubers, a romp through the transposon terrain, and elucidation of flower color regulation
Laimbeer, Francis Parker Effingham (Virginia Tech, 2018-08-02)
Investigations of potato (Solanum tuberosum) have been hampered by its complicated genetics and high genetic load. This dissertation applies genome reduction techniques to investigate a broad swath of genomic and physiological phenomena. It begins with the presentation and evaluation of a protocol to characterize endoreduplication within potato tubers, demonstrating substantial variation between tissue types and among wild species which may facilitate research into the genesis and growth of these starchy underground stems. Next, we transitioned to explore the distribution and consequences of a specific class of transposable element, Miniature Inverted Transposable Elements (MITEs), showing that they comprise approximately 5% of the potato genome, occur more frequently in genes with stress-related functions, and may be associated with changes, especially decreases, in gene expression. We then combined homology and sparsity based approaches to predict recent MITE activity, identifying five families as especially active. Finally, we expose the gene underlying the potato flower color locus, a homolog of AN2, while showing the effects it exerts on the flavonoid biosynthesis and fruit ripening pathways. This region was shown to be particularly dynamic, replete with MITEs and structural variants which we hypothesize to be the ultimate cause of differences in AN2 expression within the germplasm we examined. While the separate topics of this dissertation are quite disparate, each addresses an important topic in potato genetics, the in-depth study of which is only possible through the utilization of genomic reduction approaches to acquire homozygous genotypes for study and currently available genomic resources.
A Rootstock Provides Water Conservation for a Grafted Commercial Tomato (Solanum lycopersicum L.) Line in Response to Mild-Drought Conditions: A Focus on Vegetative Growth and Photosynthetic Parameters
Nilsen, Erik T.; Freeman, Joshua H.; Grene, Ruth; Tokuhisa, James G. (Public Library of Science, 2014-12-22)
The development of water stress resistant lines of commercial tomato by breeding or genetic engineering is possible, but will take considerable time before commercial varieties are available for production. However, grafting commercial tomato lines on drought resistant rootstock may produce drought tolerant commercial tomato lines much more rapidly. Due to changing climates and the need for commercial production of vegetables in low quality fields there is an urgent need for stress tolerant commercial lines of vegetables such as tomato. In previous observations we identified a scion root stock combination (‘BHN 602’ scion grafted onto ‘Jjak Kkung’ rootstock hereafter identified as 602/Jjak) that had a qualitative drought-tolerance phenotype when compared to the non-grafted line. Based on this initial observation, we studied photosynthesis and vegetative above-ground growth during mild-drought for the 602/Jjak compared with another scion-rootstock combination (‘BHN 602’ scion grafted onto ‘Cheong Gang’ rootstock hereafter identified as 602/Cheong) and a non-grafted control. Overall above ground vegetative growth was significantly lower for 602/Jjak in comparison to the other plant lines. Moreover, water potential reduction in response to mild drought was significantly less for 602/Jjak, yet stomatal conductance of all plant-lines were equally inhibited by mild-drought. Light saturated photosynthesis of 602/Jjak was less affected by low water potential than the other two lines as was the % reduction in mesophyll conductance. Therefore, the Jjak Kkung rootstock caused aboveground growth reduction, water conservation and increased photosynthetic tolerance of mild drought. These data show that different rootstocks can change the photosynthetic responses to drought of a high yielding, commercial tomato line. Also, this rapid discovery of one scion-rootstock combination that provided mild-drought tolerance suggests that screening more scion-rootstock combination for stress tolerance may rapidly yield commercially viable, stress tolerant lines of tomato.
Sequence Diversity in Coding Regions of Candidate Genes in the Glycoalkaloid Biosynthetic Pathway of Wild Potato Species
Manrique-Carpintero, Norma C.; Tokuhisa, James G.; Ginzberg, Idit; Holliday, Jason A.; Veilleux, Richard E. (Genetics Society of America, 2013-09-01)
Natural variation in five candidate genes of the steroidal glycoalkaloid (SGA) metabolic pathway and whole-genome single nucleotide polymorphism (SNP) genotyping were studied in six wild [Solanum chacoense (chc 80-1), S. commersonii, S. demissum, S. sparsipilum, S. spegazzinii, S. stoloniferum] and cultivated S. tuberosum Group Phureja (phu DH) potato species with contrasting levels of SGAs. Amplicons were sequenced for five candidate genes: 3-hydroxy-3-methylglutaryl coenzyme A reductase 1 and 2 (HMG1, HMG2) and 2.3-squalene epoxidase (SQE) of primary metabolism, and solanidine galactosyl-transferase (SGT1), and glucosyltransferase (SGT2) of secondary metabolism. SNPs (n = 337) producing 354 variations were detected within 3.7 kb of sequenced DNA. More polymorphisms were found in introns than exons and in genes of secondary compared to primary metabolism. Although no significant deviation from neutrality was found, dN/dS ratios < 1 and negative values of Tajima's D test suggested purifying selection and genetic hitchhiking in the gene fragments. In addition, patterns of dN/dS ratios across the SGA pathway suggested constraint by natural selection. Comparison of nucleotide diversity estimates and dN/dS ratios showed stronger selective constraints for genes of primary rather than secondary metabolism. SNPs (n = 24) with an exclusive genotype for either phu DH (low SGA) or chc 80-1 (high SGA) were identified for HMG2, SQE, SGT1 and SGT2. The SolCAP 8303 Illumina Potato SNP chip genotyping revealed eight informative SNPs on six pseudochromosomes, with homozygous and heterozygous genotypes that discriminated high, intermediate and low levels of SGA accumulation. These results can be used to evaluate SGA accumulation in segregating or association mapping populations.
Sesquiterpene pheromone biosynthesis in stink bugs: An isopentenyl diphosphate synthase like protein produces the cyclic sesquiterpene alcohol precursor of the aggregation pheromone murgantiol in harlequin bug (Murgantia histrionica)
Lancaster, Jason; Khrimian, Ashot; Young, Sharon; Lehner, Bryan; Luck, Katrin; Wallingford, Anna K.; Ghosh, Saikat Kumar B.; Zerbe, Philipp; Muchlinski, Andrew; Marek, Paul E.; Sparks, Michael E.; Tokuhisa, James G.; Tittiger, Claus; Köllner, Tobias G.; Weber, Donald C.; Gundersen-Rindal, Dawn E.; Kuhar, Thomas P.; Tholl, Dorothea (2018-08-23)
Insects use a diverse array of specialized terpene metabolites as pheromones in intraspecific interactions. In contrast to plants and microbes, which employ enzymes called terpene synthases (TPSs) to synthesize terpene metabolites, limited information from few species is available about the enzymatic mechanisms underlying terpene pheromone biosynthesis in insects. Several stink bugs (Hemiptera: Pentatomidae), among them severe agricultural pests, release 15-carbon sesquiterpenes with a bisabolene skeleton as sex or aggregation pheromones. The harlequin bug, Murgantia histrionica, a specialist pest of crucifers, uses two stereoisomers of 10,11-epoxy-1-bisabolen-3-ol as a male-released aggregation pheromone called murgantiol. We show that MhTPS (MhIDS-1), an enzyme unrelated to plant and microbial TPSs but with similarity to trans-isoprenyl diphosphate synthases (IDS) of the core terpene biosynthetic pathway, catalyzes the formation of (1S,6S,7R)- 1,10-bisaboladien-1-ol (sesquipiperitol) as a terpene intermediate in murgantiol biosynthesis. Sesquipiperitol, a so-far-unknown compound in animals, also occurs in plants, indicating convergent evolution in the biosynthesis of this sesquiterpene. RNAi-mediated knockdown of MhTPS mRNA confirmed the role of MhTPS in murgantiol biosynthesis. MhTPS expression is highly specific to tissues lining the cuticle of the abdominal sternites of mature males. Phylogenetic analysis suggests that MhTPS is derived from a trans-IDS progenitor and diverged from bona fide trans-IDS proteins including MhIDS-2, which functions as an (E,E)-farnesyl diphosphate (FPP) synthase. Structure-guided mutagenesis revealed several residues critical to MhTPS and MhFPPS activity. The emergence of an IDS-like protein with TPS activity in M. histrionica demonstrates that de novo terpene biosynthesis evolved in the Hemiptera in an adaptation for intraspecific communication.
Three genes from Solanum chacoense coding for squalene synthase
Wadlington, William Herring (Virginia Tech, 2011-05-04)
Squalene synthase (EC 2.5.1.2.1; SQS) is located at a branch point in the isoprenoid pathway and catalyzes the condensation of two molecules of farnesyl diphosphate to form squalene. SQS activity contributes to the formation of triterpenes and sterols, including phytosterols, brassinosteroids, cholesterol, and in potato plants, steroidal glycoalkaloids (SGAs). These compounds have diverse functions in the plant. SGAs are defense compounds that deter feeding by potato pests. The wild potato Solanum chacoense accumulates higher amounts of SGAs than cultivated potato and some of its accessions produce leptines, a rare class of SGAs that is toxic to Colorado potato beetle. Unlike most eukaryotes, higher plants have more than one gene coding for SQS. Three sqs gene homologs were isolated from S. chacoense, sqs1Sc, sqs2Sc, and sqs4Sc, that have 74 to 83% identity at the amino acid level. Some of the amino acid differences between sqs isoforms are likely to affect enzyme activity. Each of the three genes contained an intron in the 3'UTR. This feature may have a role in the nonsense-mediated decay of incomplete sqs mRNAs. A partial SQS polypeptide retaining catalytic activity but lacking the membrane anchoring domain could adversely affect a cell with the randomly distributed accumulation of squalene. The mRNA of sqs1Sc and sqs2Sc was detected in all tissues whereas sqs4Sc transcript was limited to bud tissue. The sqs2Sc transcript was less uniformly distributed in the plant than sqs1Sc and accumulated most abundantly in floral tissue. The results demonstrate that the three sqs genes have different patterns of gene expression and encode proteins with different primary structures indicating distinct roles in plant squalene metabolism.
Transposon Tagging in Strawberry and Potato and Characterization of Representative Strawberry Mutants
Lu, Nan (Virginia Tech, 2013-09-25)
Strawberry and potato are both important crop species in the world providing various nutritional values. The cultivated strawberry, Fragaria ananassa, is a fruit crop with a complex genome (2n=8x=56) whereas the diploid woodland strawberry, Fragaria vesca, has a smaller genome (2n=2x=14, 240 Mb) and lots of other qualities that make it a good model for genetic and genomic study, such as high yield of seeds and efficient transformation. Potato (Solanum tuberosum, 2n=4x=48) is an important vegetable crop in the world and is highly heterozygous. The successful sequencing of the homozygous doubled monoploid clone of potato provides good insight into the study of important genes in this species in improving the pest resistance and improving yield. One approach to characterize gene function in a model system is having large populations of T-DNA insertional or transposon tagged mutants. The idea of using AcDs construct to create transposon tagged mutant populations has also been applied in many species. Here we transformed two species, Fragaria vesca and a monoploid potato, Solanum phureja 1-3-516, which is the progenitor of the sequenced doubled monoploid clone, with the same AcDs construct, Ac-DsATag-Bar_gosGFP, to generate mutant collection, compare the marker gene performance and transposition efficiency, as well as characterizing phenotypic mutants with genes of interest. Transposants were found to reinsert to unlinked sites from the launch pad site in the strawberry genome, whereas in potato transposants tended to locate locally from the launch pad position when using the same construct. One transposon based activation tagging strawberry mutant, with its insertion in the promoter region of gene of interest in strawberry from the Ac-DsATag-Bar_gosGFP population was studied. In a segregating T2 population, expression level of the candidate gene, epidermis-specific secreted glycoprotein EP1 precursor, was 670 fold higher in petioles of homozygotes than in wild type plants, suggesting the function of this gene involved in maintaining mechanical strength of petioles. Since the often-used transposase gene was cloned from the monocot species maize, the efficiency of obtaining germinal transposants was many times lower than expected in order to saturate the genome for diploid species. In order to improve the chance of getting unique transposants, we attempted to codon optimize the transposase gene, as well as switching to microspore specific promoters that had been well characterized to control timing of expression of the transposase gene. Transposants were found in both T0 primary regenerates and anther culture derived potatoes using both the pAcDs-AtSCP and pAcDs-AmDEFH125 constructs. Sequencing of the empty donor site revealed that excision occurred in different cells during anther culture. A strawberry mutant with sugar transport deficiency due to T-DNA insertion near a sucrose transporter-2 gene showing stunted phenotype with increased level of anthocyanin was also characterized. The concentrations of sucrose, glucose, and fructose were significantly greater in source leaves of the mutant than wild type plants, suggesting these compounds might be substrates of this gene in transporting to sink leaves and roots.

Browsing by Author "Tokuhisa, James G."

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