Biology Commons^™

Investigation Of The Function Of Protein Acyl Transferases Using Crispr-Cas9 To Create Null Mutants, Claire E. Christopher

Honors Theses and Capstones

24 Protein Acyl Transferases (PATs) have been identified in the model organism Arabidopsis thaliana. Despite knowing the enzymatic function of these genes (palmitoylation), the specific subcellular pathways, protein targets, and cellular activities of these proteins remain unknown. To investigate the pathways that PAT genes are involved in, deletion mutations were created in several genes and the phenotype of the organisms carrying homozygous mutations was observed. To introduce mutations, the CRISPR/Cas9 system was inserted into the plant’s genome using Agrobacterium tumefaciens to cause deletions in PAT genes of interest. To date, no CRISPR-induced pat mutants have a noticeable phenotype. These …

Genetic Characterization Of Centromere-Mediated Uniparental Genome Elimination In Arabidopsis, Diana Spencer

Electronic Theses and Dissertations

The production of haploids through crossing was first discovered from instances of interspecies crosses in Nicotiana spp. in 1924. Since then, haploid induction crosses has been used to improve plant breeding programs and commercially utilized in a number of crop industries. The generation of doubled haploid instantaneously creates a pure homozygous line, therefore eliminating the need for several generations of inbreeding. There are several pathways to induce haploids in plants: of these methods, centromere-mediated genome elimination pathway engenders the highest haploid induction rate (HIR) with up to 45% in Arabidopsis compared to 15% through phospholipase-mediated haploid induction in maize. Centromere-mediated …

The Effects Of Ppal-1 In Arabidopsis Gamete Development, Amanda J White, Susana Perez-Martinez, Mark P. Running

The Cardinal Edge

Prenylation is a type of post-translational modification in which a 15- or 20-carbon lipid is added to the carboxyl (C) terminus of the protein. Arabidopsis thaliana contains the PROTEIN PRENYLTRANSFERASE ALPHA SUBUNIT-LIKE (PPAL) gene, which encodes a protein with homology to the α-subunits of the three known prenylation enzymes, PFT, PGGT, and Rab-GGT. We previously identified two mutations in PPAL, one of which is ppal-1, which contains a T-DNA insertion in the fourth intron. We have previously observed that self-fertilizing heterozygous ppal-1 plants produce progeny in which homozygous ppal-1 is underrepresented. This project attempts to ascertain …

Mutations In Several Auxin Biosynthesis Genes And Their Effects On Plant Phenotypes In Arabidopsis, Gabriela Hernandez, Lauren Huebner, Bethany Karlin Zolman

Undergraduate Research Symposium

Auxins are important hormones in plants that regulate growth and development. Disruptions in the auxin biosynthesis pathway result in morphological changes in phenotypes in the model plant Arabidopsis thaliana, including differences in root and leaf formation. Mutations in the Tryptophan Aminotransferase of Arabidopsis (TAA1) and YUCCA (YUC4) genes interfere with the plant's ability to synthesize Indole-3-acetic acid (IAA), the primary auxin involved in plant development. IBR1 and IBR3 act in the multistep conversion of indole-3-butyric acid (IBA) to IAA. ILL2, IAR3, and ILR1 hydrolyze IAA-amino acid conjugates into free IAA. The goal of …

Characterizing The Role Of Β-Amylase3 In Cold Stress Response And Recovery In Arabidopsis Thaliana, Isabelle G. Houston

Senior Honors Projects, 2020-current

Starch is a polymer of glucose that is used as an energy store in plants. Mobilization of starch has implications in abiotic stress survival and recovery. While the importance of carbon and energy allocation in plant survival has been explored, the specific roles of starch degrading enzymes in plant responses to stress are still unclear. β-Amylase3, or BAM3, is the principle starch degrading enzyme at night and is transcriptionally upregulated in response to cold stress in the plant Arabidopsis thaliana. Using single and quadruple knockout mutant plants, I aimed to clarify the role of BAM3 in the response to …

Msl1 Is A Mechanosensitive Ion Channel That Dissipates Mitochondrial Membrane Potential And Maintains Redox Homeostasis In Mitochondria During Abiotic Stress, Chun Pong Lee, Grigory Maksaev, Gregory S. Jensen, Monika W. Murcha, Margaret E. Wilson, Mark Fricker, Ruediger Hell, Elizabeth S. Haswell, A Harvey Millar, Lee J. Sweetlove

Biology Faculty Publications & Presentations

Mitochondria must maintain tight control over the electrochemical gradient across their inner membrane to allow ATP synthesis while maintaining a redox-balanced electron transport chain and avoiding excessive reactive oxygen species production. However, there is a scarcity of knowledge about the ion transporters in the inner mitochondrial membrane that contribute to control of membrane potential. We show that loss of MSL1, a member of a family of mechanosensitive ion channels related to the bacterial channel MscS, leads to increased membrane potential of Arabidopsis mitochondria under specific bioenergetic states. We demonstrate that MSL1 localises to the inner mitochondrial membrane. When expressed in …

Identification Of N-Acylethanolamine Hydrolyzing Enzyme In Solanum Lycopersicum, Derek A. Stuffle

Undergraduate Honors Theses

N-acylethanolamines (NAEs) are fatty acid derivatives that occur naturally in plant and animal systems. In mammals, they regulate physiological functions, including neurotransmission, immune responses, vasodilation, embryo development and implantation, feeding behavior, and cell proliferation. NAEs are metabolized by fatty acid amide hydrolase (FAAH), which belongs to the amidase signature family. It is hypothesized that putative FAAH functions as the catalyst in the metabolism of N-acylethanolamine in tomato plants. To test the hypothesis, FAAH protein homologs were identified in tomato via in silico analysis. Among the six homologs identified, FAAH1 and FAAH2 were selected for further validation. This study …

Insights Into Triterpene Metabolism In Model Monocotyledonous And Oilseed Plants Genetically Engineered With Genes From Botryococcus Braunii, Chase F. Kempinski

Theses and Dissertations--Plant and Soil Sciences

Isoprenoids are one of the most diverse classes of natural products and are all derived from universal five carbon, prenyl precursors. Squalene and botryococcene are linear, hydrocarbon triterpenes (thirty carbon compounds with six prenyl units) that have industrial and medicinal values. Squalene is produced by all eukaryotes as it is the first committed precursor to sterols, while botryococcene is uniquely produced by the green algae, Botryococcus braunii (race B). Natural sources for these compounds exist, but there is a desire for more renewable production platforms. The model plant Arabidopsis thaliana was engineered to accumulate botryococcene and squalene in its oil …

Detecting Genomic Regions Responsible For Resistance In Arabidopsis, Valeria Cancino, Anjali Iyer-Pascuzzi, Rucha Karve

The Summer Undergraduate Research Fellowship (SURF) Symposium

Ralstonia solanacearum is a soil-borne plant root colonizing pathogen and the casual agent of bacterial wilt (BW) disease. BW leads to severe yield loss in a wide variety of agricultural commodity crops, such as tomato, banana, and pepper. In this study, we look at the plant-pathogen interaction between Ralstonia solanacearum and various ecotypes of Arabidopsis thaliana with the goal of finding resistant ecotypes. To identify resistant ecotypes, seeds are first sterilized and left to soak in the dark. Then the seeds are plated on agar media, transferred to a growth chamber, and allowed to grow for 5 days. On day …

Descriptive Analyses Of Pollen Surface Morphologies In The Model Systems Brassica Rapa And Arabidopsis Thaliana And Three Arabidopsis Pollen Wall Mutants By Scanning Electron Microscopy, Andrew B. Kirkpatrick

Theses and Dissertations

The mechanisms behind the construction of the pollen wall are equally elaborate and mysterious. Previous studies primarily used sectioned tissue to elucidate the events involved in proper pollen development. This study proposed and evaluated a protocol for exposing developing microspores to be examined by Scanning Electron Microscopy (SEM). Utilizing this protocol, comparative analyses of the superficial features present at the early, middle, and late tetrad as well as at released microspore stages of the model plants Brassica rapa and Arabidopsis thaliana were conducted. The utility of the technique was then evaluated through the examination of three Arabidopsis pollen wall mutants …

Heterologous Expression Of Arabidopsis Thaliana Purple Acid Phosphatase Gene (Atpap15) In Crops For Phytoremediation Of Sites Contaminated With Excess Phosphorus, Jane Jeruto Bartonjo

Masters Theses & Specialist Projects

AtPAP15 is one of the purple acid phosphatases expressed by Arabidopsis thaliana that has been extensively studied. Purified AtPAP15 has been shown to exhibit both phytase and phosphomonoesterase activities in acidic pH with maximal activity at pH 4.5. AtPAP15 is a phosphorus starvation inducible (PSI) gene that is expressed highly during phosphorus deficient conditions. In the current study, AtPAP15 was overexpressed in Nicotiana tabaccum under cauliflower mosaic virus (CaMV35S) constitutive promoter. After PCR confirmation of the gene, plants were transferred to the greenhouse and allowed to grow in pots. The pots contained Sta-Green potting mix (Lowe’s Inc., Mooresville, North Carolina,U.S.). …

Dirigent Domain-Containing Protein Is Part Of The Machinery Required For Formation Of The Lignin-Based Casparian Strip In The Root, Prashant S. Hosmani, Takehiro Kamiya, John Danku, Sadaf Naseer, Niko Geldner, Mary Lou Guerinot, David Salt

Dartmouth Scholarship

The endodermis acts as a "second skin" in plant roots by providing the cellular control necessary for the selective entry of water and solutes into the vascular system. To enable such control, Casparian strips span the cell wall of adjacent endodermal cells to form a tight junction that blocks extracellular diffusion across the endodermis. This junction is composed of lignin that is polymerized by oxidative coupling of monolignols through the action of a NADPH oxidase and peroxidases. Casparian strip domain proteins (CASPs) correctly position this biosynthetic machinery by forming a protein scaffold in the plasma membrane at the site where …

Characterization Of A Putative Activation Domain In The Hulk Gene Family, Christopher Doan

Electronic Thesis and Dissertation Repository

The HULK gene family participates in regulation of both flowering time and development in the plant Arabidopsis thaliana. The proteins encoded by these genes share conserved domain structures including a proline-rich region (PRR) in the carboxyl-terminus. Based on sequence analysis and the presence of a proline-rich domain, it has been suggested that the HULKs are putative transcription factors in which HUA2 is known to regulate several late-flowering genes: FLC, FLM and MAF2.

To investigate the putative transcriptional activation domain in the carboxyl-terminus of the HULKs, full-length HULKs and deletion constructs were 3-AT titrated in yeast-one hybrids. It …

Functions Of The Arabidopsis Kinesin Superfamily Of Microtubule-Based Motor Proteins, Chuanmei Zhu, Ram Dixit

Biology Faculty Publications & Presentations

Plants possess a large number of microtubule-based kinesin motor proteins. While the kinesin-2, 3, 9, and 11 families are absent from land plants, the kinesin-7 and 14 families are greatly expanded. In addition, some kinesins are specifically present only in land plants. The distinctive inventory of plant kinesins suggests that kinesins have evolved to perform specialized functions in plants. Plants assemble unique microtubule arrays during their cell cycle, including the interphase cortical microtubule array, preprophase band, anastral spindle and phragmoplast. In this review, we explore the functions of plant kinesins from a microtubule array viewpoint, focusing mainly on Arabidopsis kinesins. …

Arabidopsis Bhlh100 And Bhlh101 Control Iron Homeostasis Via A Fit-Independent Pathway, Alicia B. Sivitz, Victor Hermand, Catherine Curie, Grégory Vert

Dartmouth Scholarship

Iron deficiency induces a complex set of responses in plants, including developmental and physiological changes, to increase iron uptake from soil. In Arabidopsis, many transporters involved in the absorption and distribution of iron have been identified over the past decade. However, little is known about the signaling pathways and networks driving the various responses to low iron. Only the basic helix–loop–helix (bHLH) transcription factor FIT has been shown to control the expression of the root iron uptake machinery genes FRO2 and IRT1. Here, we characterize the biological role of two other iron-regulated transcription factors, bHLH100 and bHLH101, in iron homeostasis. …

Computer Simulation And Mathematical Models Of The Noncentrosomal Plant Cortical Microtubule Cytoskeleton, Ezgi Can Eren, Natarajan Gautam, Ram Dixit

Biology Faculty Publications & Presentations

There is rising interest in modeling the noncentrosomal cortical microtubule cytoskeleton of plant cells, particularly its organization into ordered arrays and the mechanisms that facilitate this organization. In this review, we discuss quantitative models of this highly complex and dynamic structure both at a cellular and molecular level. We report differences in methodologies and assumptions of different models as well as their controversial results. Our review provides insights for future studies to resolve these controversies, in addition to underlining the common results between various models. We also highlight the need to compare the results from simulation and mathematical models with …

Single-Molecule Analysis Of The Microtubule Cross-Linking Protein Map65-1 Reveals A Molecular Mechanism For Contact-Angle-Dependent Microtubule Bundling, Amanda Tulin, Sheri Mcclerklin, Yue Huang, Ram Dixit

Biology Faculty Publications & Presentations

Bundling of microtubules (MTs) is critical for the formation of complex MT arrays. In land plants, the interphase cortical MTs form bundles specifically following shallow-angle encounters between them. To investigate how cells select particular MT contact angles for bundling, we used an in vitro reconstitution approach consisting of dynamic MTs and the MT-cross-linking protein MAP65-1. We found that MAP65-1 binds to MTs as monomers and inherently targets antiparallel MTs for bundling. Dwell-time analysis showed that the affinity of MAP65-1 for antiparallel overlapping MTs is about three times higher than its affinity for single MTs and parallel overlapping MTs. We also …

Transcriptomic Characterization Of A Synergistic Genetic Interaction During Carpel Margin Meristem Development In Arabidopsis Thaliana, April N. Wynn, Elizabeth E. Rueschhoff, Robert G. Franks

Biological Sciences Research

In flowering plants the gynoecium is the female reproductive structure. In Arabidopsis thalianaovules initiate within the developing gynoecium from meristematic tissue located along the margins of the floral carpels. When fertilized the ovules will develop into seeds. SEUSS (SEU) and AINTEGUMENTA (ANT) encode transcriptional regulators that are critical for the proper formation of ovules from the carpel margin meristem (CMM). The synergistic loss of ovule initiation observed in the seu ant double mutant suggests that SEU and ANT share overlapping functions during CMM development. However the molecular mechanism underlying this synergistic interaction is unknown. Using …

Single Molecule Analysis Of The Arabidopsis Fra1 Kinesin Shows That It Is A Functional Motor Protein With Unusually High Processivity, Chuanmei Zhu, Ram Dixit

Biology Faculty Publications & Presentations

The Arabidopsis FRA1 kinesin contributes to the organization of cellulose microfibrils through an unknown mechanism. The cortical localization of this kinesin during interphase raises the possibility that it transports cell wall-related cargoes along cortical microtubules that either directly or indirectly influence cellulose microfibril patterning. To determine whether FRA1 is an authentic motor protein, we combined bulk biochemical assays and single molecule fluorescence imaging to analyze the motor properties of recombinant, GFP-tagged FRA1 containing the motor and coiled-coil domains (designated as FRA1(707)–GFP). We found that FRA1(707)–GFP binds to microtubules in an ATP-dependent manner and that its ATPase activity is dramatically stimulated …