Life Sciences Commons^™

Generating Crispr Mutations In The Asymmetric Leaves1 Gene Of Arabidopsis, Gabriella J. Rueschhoff, Tara Phelps-Durr

SACAD: John Heinrichs Scholarly and Creative Activity Days

The goal of this study is to create new mutations in the ASYMMETRIC LEAVES 1 (AS1) gene of Arabidopsis using CRISPR technology. AS1 is a myb domain transcription factor that represses the KNOX genes, a group of genes involved in maintaining an undifferentiated cell state. AS1 binds to the chromatin remodeling protein Histone Repressor A (HIRA). In animals, HIRA is involved in the permanent suppression of proliferation genes required for early development. The interaction between AS1 and HIRA provides clues to how AS1 maintains suppression at the KNOX genes; however, we must be careful applying what …

A High-Efficiency Gene Silencing In Plants Using Two-Hit Asymmetrical Artificial Micrornas, Sachin Teotia, Xiaoran Wang, Na Zhou, Mengmeng Wang, Haiping Liu, Jun Qin, Dianwei Han, Chingwen Li, Christine E. Li, Shangjin Pan, Haifeng Tang, Wenjun Kang, Zhanhui Zhang, Xiaoqing Tang, Ting Peng, Guiliang Tang

Michigan Tech Publications

MicroRNAs (miRNAs) are small non-coding RNA molecules that play a crucial role in gene regulation. They are produced through an enzyme-guided process called dicing and have an asymmetrical structure with two nucleotide overhangs at the 3' ends. Artificial microRNAs (amiRNAs or amiRs) are designed to mimic the structure of miRNAs and can be used to silence specific genes of interest. Traditionally, amiRNAs are designed based on an endogenous miRNA precursor with certain mismatches at specific positions to increase their efficiency. In this study, the authors modified the highly expressed miR168a in Arabidopsis thaliana by replacing the single miR168 stem-loop / …

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 …

Understanding The Development Of Rafflesia Using A Transcriptomic Study Of Seed And Flower Bud In Comparison To Arabidopsis Thaliana, Brian Joseph Tomek

Selected Full-Text Master Theses 2021-

The holoparasitic plant genus Rafflesia is of interest for producing the largest single flowers in the world, though efforts to conserve it have been challenging with not much success in ex situ propagation due to the little that we know of its biology. Further study and understanding of the growth of Rafflesia species is necessary in order to preserve these endangered plants. Due to its potentially long growth cycle, there is a definite difficulty in studying the growth cycle through observation alone. Transcriptomic study is one tool which can be used to understand how Rafflesia species change throughout different stages …

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 …

Functional Role Of Ppal And Potential For Moss In Industrial Applications., Susana Perez Martinez

Electronic Theses and Dissertations

This dissertation is an examination and characterization of the functional roles of PPAL. PROTEIN PRENYLTRANSFERASE ALPHA SUBUNIT-LIKE (PPAL) is a recently discovered gene. PPAL homologs are present in all plants and many animals, where its function is largely unknown. It is possible that PPAL could participate in prenylation processes since it shares similarity to the α subunits of known prenylation enzymes. Prenylation is a post-translational modification of proteins that involves the addition of a lipid moiety to proteins to facilitate membrane targeting and association and promote protein-protein interactions. Prenylation has important roles in plant growth and development, including …

Pickle Associates With Histone Deacetylase 9 To Mediate Vegetative Phase Change In Arabidopsis, Tieqiang Hu, Darren Manuela, Valerie Hinsch, Mingli Xu

Faculty Publications

• The juvenile-to-adult vegetative phase change in flowering plants is mediated by a decrease in miR156 levels. Downregulation of MIR156A/MIR156C, the two major sources of miR156, is accompanied by a decrease in acetylation of histone 3 lysine 27 (H3K27ac) and an increase in trimethylation of H3K27 (H3K27me3) at MIR156A/MIR156C in Arabidopsis.

• Here, we show that histone deacetylase 9 (HDA9) is recruited to MIR156A/MIR156C during the juvenile phase and associates with the CHD3 chromatin remodeler PICKLE (PKL) to erase H3K27ac at MIR156A/MIR156C.

• H2Aub and H3K27me3 become enriched at MIR156A/MIR156C, and the recruitment of Polycomb Repressive Complex …

Characterization Of Phenotypic Traits Related To Loss-Of-Function And Ectopic Expression Of Bhlh093 And Bhlh061 In Arabidopsis Thaliana, Leila Belhadjali

WWU Graduate School Collection

bHLH093 and bHLH061 are members of sub-group IIIb of basic helix-loop-helix (bHLH) Arabidopsis thaliana transcription factors. Although bHLH proteins are the second largest transcription factor family in Arabidopsis, only a small proportion of them have been functionally characterized. Here, we investigated the phenotypic impact of bHLH061 and bHLH093 ectopic overexpression and loss-of-function to confirm previously published results and provide new insight into their role in development. bHLH093 and bHLH061 are homologs of two stomatal development genes, SCREAM1/ICE1 and SCREAM2 and have been shown to dimerize with two master regulators of stomata development, FAMA and MUTE. While no evidence was found …

The Role Of Elmo5 In Arabidopsis Thaliana Cell Adhesion, Isabel Kristina Ball

Honors Projects

Plant cell growth and development relies on proper cellular adhesion. As the extracellular matrix serves as the area of connection between two cells, its synthesis and maintenance are essential for cellular adhesion. The middle lamella region, the layer of the extracellular matrix between two adjacent cell walls, is diffuse with the polysaccharide pectin due to its delivery by Golgi vesicles early during cell division. A Ruthenium Red screen for cellular adhesion mutants identified the family of 5 ELMO proteins that are critical for proper cellular adhesion. To further our understanding of plant cellular adhesion and pathways of pectin synthesis and …

Low Light Intensity Delays Vegetative Phase Change, Mingli Xu, Tieqiang Hu, R Scott Poethig

Faculty Publications

Plants that develop under low light (LL) intensity often display a phenotype known as the “shade tolerance syndrome (STS)”. This syndrome is similar to the phenotype of plants in the juvenile phase of shoot development, but the basis for this similarity is unknown. We tested the hypothesis that the STS is regulated by the same mechanism that regulates the juvenile vegetative phase by examining the effect of LL on rosette development in Arabidopsis (Arabidopsis thaliana). We found that LL prolonged the juvenile vegetative phase and that this was associated with an increase in the expression of the master …

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 …

Genetic And Epigenetic Regulations Of Eds1-Mediated Plant Immunity And Identification Of Bacterial Type Iii Effectors Targeting De Novo Dna Methyltransferase Drm2 In Arabidopsis, Ming Zhao

Theses and Dissertations

In Arabidopsis thaliana, Enhanced Disease Susceptibility1 (EDS1) acts as an indispensable hub in plant immunity and is mainly responsible for plant basal defense, systemic acquired resistance (SAR), increase of endogenous content of salicylic acid (SA) and effector-triggered immunity (ETI). Epigenetic regulators play crucial roles in multiple important cellular processes. As one catalytic core of Polycomb Repressive Complex (PRC2), CURLY LEAF (CLF/SDG1) protein promotes H3K27me3 deposition at specific loci for genes repression. In this work, I found that EDS1 interacted with epigenetic regulators SDG1, SDG9, SDG15, SDG22 and SDG35 in yeast two-hybrid assays. The growth of the bacterial pathogen Pseudomonas …

The Role Of The Golgi Elmo Proteins In Cell Adhesion In Arabidopsis Thaliana, Wesley James Hudson

Honors Projects

Proper growth and development of plant cells is dependent upon successful cell adhesion between cells, and this is mostly mediated by pectin in the plant cell wall. Previously, the Kohorn Laboratory identified a non-enzymatic Golgi protein named ELMO1 as it is required for cell adhesion, likely acting as a scaffold for cell wall polymer synthesis. Plants with mutant ELMO1 demonstrate a weak defective cellular adhesion phenotype as well as reduced mannose content in the cell wall. ELMO1 has homologous proteins in at least 29 different vascular plants. These homologues have 2 possible deletions in their amino acid sequence, but protein …

Multiple Roles Of Nup1 In Arabidopsis Growth And Development, Raj K. Thapa

Electronic Thesis and Dissertation Repository

The nuclear pore complex (NPC) is the gateway between the nucleus and cytoplasm, which provides the passage for transport of RNA, protein, and other molecules into and out of the nucleus. NPC is conserved across all eukaryotes and plays a vital role in various cellular processes. However, compared to other organisms, the study of NPC in plants is limited. Although more than 30 different types of nucleoporin proteins in the model plant Arabidopsis thaliana have been identified, none of those proteins has been studied in detail. In this thesis, I focused on one such protein named NUCLEOPORIN1 (NUP1) and investigated …

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 …

The Effects Of Ppal-1 In Arabidopsis Gamete Development., Amanda White

College of Arts & Sciences Senior Honors Theses

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 …

Regulation Of Arabidopsis Floral Organ Development By The Transcription Factor Aintegumenta-Like6, Jorman Heflin

Theses and Dissertations

Flowers, and the fruits and seeds they produce, are of great agricultural and economic importance. Understanding the molecular mechanisms that mediate the identity, size and arrangement of floral organs within the flower may allow us to tune these elements for increased crop productivity. I examine several potential regulatory targets of the Arabidopsis AINTEGUMENTA-LIKE6 (AIL6) transcription factor that is involved in these processes and I identify protein-protein interactions that may mediate its activity in flowers. As described by the ABCE model, unique floral organ identities are specified in each whorl of the flower by different combinations of homeotic gene activities. Two …

1. Microarray Analysis Identifies Potential Genes Associated With Botrytis Cinerea And Hormonal Stresses., Kenna Marie Whitley

Biology Theses

Biotic and abiotic stresses critically influence plant survival and growth; survival depends on the ability to correctly sense and react to their environment. Certain environmental stresses must be overcome through careful manipulation of internal levels, and of the resulting signaling cascades. Hormones signaling networks and the crosstalk among each respective hormone signaling pathway are principle for response mediation via transcriptional reprogramming or altered signaling pathways. The long-term objective is to determine how plants sense biotic stresses, and how hormone signaling networks control and direct plant responses to the fungal pathogen Botrytis cinerea. This research aims to identify commonly regulated genes …

Genetic And Physical Interactions Between The Organellar Mechanosensitive Ion Channel Homologs Msl1, Msl2, And Msl3 Reveal A Role For Inter-Organellar Communication In Plant Development, Josephine S. Lee, Margaret E. Wilson, Ryan A. Richardson, Elizabeth S. Haswell

Biology Faculty Publications & Presentations

Plant development requires communication on many levels, including between cells and between organelles within a cell. For example, mitochondria and plastids have been proposed to be sensors of environmental stress and to coordinate their re- sponses. Here we present evidence for communication between mitochondria and chloroplasts during leaf and root development, based on genetic and physical inter- actions between three Mechanosensitive channel of Small conductance-Like (MSL) proteins from Arabidopsis thaliana. MSL proteins are Arabidopsis homologs of the bac- terial Mechanosensitive channel of Small conductance (MscS), which relieves cellular osmotic pressure to protect against lysis during hypoosmotic shock. MSL1 localizes to …

Transcriptional Profiling Identifies Potential Genes Associated With Multiple Biotic Stresses In Arabidopsis, Hibatullah Mustafa Al Ashram

Biology Theses

Plants are exposed to many environmental stresses that affect their growth and development. These stresses include biotic stresses (organisms) and abiotic stresses (drought and salinity). Plants respond to these stresses by transcriptional reprogramming and different signaling pathways. Arabidopsis thaliana had shown great sensitivity to the biotic stress: Botrytis cinerea, Alternaria brassicicola, Pseudomonas syringae and the herbivore insect Pieris rapae. The overall goal is to identify common regulated genes that respond to all these biotic stresses to ultimately improve plant stress tolerance in Arabidopsis. The specific aims are to: (1) determine the regulated genes in response to an …

Adaptation Mechanisms Of Two-Spotted Spider Mite, Tetranychus Urticae, To Arabidopsis Indole Glucosinolates, Golnaz Salehipourshirazi

Electronic Thesis and Dissertation Repository

The two-spotted spider mite Tetranychus urticae Koch is a key agricultural pest that causes significant yield losses in a wide range of economically important crops. Rapid development of resistance to several classes of pesticides in T. urticae necessitates introduction of alternative management strategies to control this pest. Indole glucosinolates (IGs) are secondary metabolites found in Brassicaceae plants (including Arabidopsis thaliana) that have been shown to be effective against T. urticae and could be potential candidates to control spider mites. However, a laboratory population selected on IG-containing Arabidopsis was able to evolve adaptation to this plant. The overall objective of …

The Influence Of Dose Of A Plant-Derived Volatile Cue On Arabidopsis Thaliana Resistance Against Insect Herbivores, Sarah Bissmeyer, Grace Freundlich, Christopher Frost

Kentucky Journal of Undergraduate Scholarship

Green leaf volatiles (GLVs) are plant-derived volatile organic compounds (VOCs) known to affect plant-plant communication. Specifically, GLVs can facilitate “priming”, whereby plants initiate a faster and stronger defensive response to a subsequent stress. The stress-induced GLV cis-3-hexenyl acetate (z3HAC) is a known priming cue, yet whether the concentration of the z3HAC affects plant defenses is unknown. Here, we tested the hypothesis that z3HAC concentration of would influence Arabidopsis thaliana resistance against a specialist (Trichoplusia ni) and generalist (Spodoptera exigua) herbivore. Our z3HAC treatments ranged from 0-100 ng/hr, which spanned the …

Evaluating Effects Of Gene Mutations And Light Intensity On Arabidopsis Thaliana Development, Carolanne Bekus

Undergraduate Theses and Capstone Projects

Arabidopsis thaliana is a model organism often compared to commercial crops. The completion of sequencing A. thaliana’s genome has led to the next crucial challenge of determining gene function in these plants. The discovery of gene function within these plants will provide insights on how gene function can affect commercial crop production. This work compared wild-type Columbia (Col-O) A. thaliana to single gene mutants VPI/ABI3-like 1 (VAL1) and basic region/leucine zipper motif (bZIP). These single gene mutations may affect several traits that, in turn, can result in morphological changes and/or time of development in seedlings. Wild-type and …

The Natural Variance Of The Arabidopsis Floral Secondary Metabolome, Takayuki Tohge, Monica Borghi, Alisdair R. Fernie

Biology Faculty Publications

Application of mass spectrometry-based metabolomics enables the detection of genotype-related natural variance in metabolism. Differences in secondary metabolite composition of flowers of 64 Arabidopsis thaliana (Arabidopsis) natural accessions, representing a considerable portion of the natural variation in this species are presented. The raw metabolomic data of the accessions and reference extracts derived from flavonoid knockout mutants have been deposited in the MetaboLights database. Additionally, summary tables of floral secondary metabolite data are presented in this article to enable efficient re-use of the dataset either in metabolomics cross-study comparisons or correlation-based integrative analysis of other metabolomic and phenotypic features such as …

A Rapid And Efficient Method For Enriching Mitochondrial Dna From Plants, Mackenzie M. Strehle, Emma Purfeerst, Alan C. Christensen

School of Biological Sciences: Faculty Publications

Current mitochondrial purification techniques are tedious and protracted due to their emphasis on recovering physiologically active mitochondria. However, for studies that are exclusively interested in isolating mitochondrial DNA (mtDNA) for applications such as PCR and sequencing, respiring mitochondria - and the complex procedures that stem from the need to retain their function - are unnecessary. Still, global DNA extraction methods have proven insufficient for mitochondrial DNA isolation because nuclear mitochondrial DNA segments (NUMTs) pose unique challenges to accurate mtDNA quantification and characterization. We present a rapid and simple extraction technique that maximizes recovery of mitochondrial DNA from plant cells, while …

Epigenetic Regulation Of Salicylic Acid-Mediated Plant Defense, Phillip Key

Senior Theses

Plant pathogens remain a significant threat to the stability of modern agricultural systems, and the investigation of mechanisms to improve the security of food resources has led to the partial characterization of plant immune response. With this progress, there is a newfound ability to analyze the relative pathogen resistance capability of specifically modified organisms, and in doing so, it is possible to identify individual alterations that might play a role in creating a more robust immune response. In this study, ten Arabidopsis thaliana mutants were infiltrated with Pseudomonas syringae pv. morsprunorum alongside positive and negative control Col-0 Wild-Type and npr1-2. …

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 …

Purification Of 26s Proteasomes And Their Subcomplexes From Plants, Richard S. Marshall, David C. Gemperline, Richard D. Vierstra

Biology Faculty Publications & Presentations

The 26S proteasome is a highly dynamic, multisubunit, ATP-dependent protease that plays a central role in cellular housekeeping and many aspects of plant growth and development by degrading aberrant polypeptides and key cellular regulators that are first modified by ubiquitin. Although the 26S proteasome was originally enriched from plants over 30 years ago, only recently have significant advances been made in our ability to isolate and study the plant particle. Here, we describe two robust methods for purifying the 26S proteasome and its subcomplexes from Arabidopsis thaliana; one that involves conventional chromatography techniques to isolate the complex from wild-type …

In Silico Mechanistic Study Of Abscisic Acid (Aba) Mediated Drought Tolerance In Crops, Bushra Shaheen, Tayyaba Shaheen, Shahid Ur Rehman, Mahmood Ur Rahman, Muhammad Nawaz, Muhammad Tahir Ul Qamar

Journal of Bioresource Management

Drought is one of the major environmental stresses that produces adverse effects on plants. Vast areas of agricultural land is susceptible to drought. Drought induced yield loss of crops has negative effects on the economy of a country. Among phytohormones, Abscisic Acid (ABA) induces abiotic stress tolerance in plants, and directs a complex regulatory network involving multiple transporters, up-regulation of ABA biosynthesis genes and various signalling pathways that enable plants to withstand low water availability. The current study was designed to understand ABA synthesis, its transport across the plant during stress and its mechanism to induce stomatal closure by using …