Browsing by Author "Bartel, Bonnie"
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Item A facile forward-genetic screen forᅠArabidopsisᅠautophagy mutants reveals twenty-one loss-of-function mutations disrupting sixᅠATGᅠgenes(Taylor & Francis, 2019) Young, Pierce G.; Passalacqua, Michael J.; Chappell, Kevin; Llinas, Roxanna J.; Bartel, BonnieMacroautophagy is a process through which eukaryotic cells degrade large substrates including organelles, protein aggregates, and invading pathogens. Over 40 autophagy-related (ATG) genes have been identified through forward-genetic screens in yeast. Although homology-based analyses have identified conserved ATG genes in plants, only a few atg mutants have emerged from forward-genetic screens in Arabidopsis thaliana. We developed a screen that consistently recovers Arabidopsis atg mutations by exploiting mutants with defective LON2/At5g47040, a protease implicated in peroxisomal quality control. Arabidopsis lon2mutants exhibit reduced responsiveness to the peroxisomally-metabolized auxin precursor indole-3-butyric acid (IBA), heightened degradation of several peroxisomal matrix proteins, and impaired processing of proteins harboring N-terminal peroxisomal targeting signals; these defects are ameliorated by preventing autophagy. We optimized a lon2 suppressor screen to expedite recovery of additional atg mutants. After screening mutagenized lon2-2 seedlings for restored IBA responsiveness, we evaluated stabilization and processing of peroxisomal proteins, levels of several ATG proteins, and levels of the selective autophagy receptor NBR1/At4g24690, which accumulates when autophagy is impaired. We recovered 21 alleles disrupting 6 ATG genes: ATG2/At3g19190, ATG3/At5g61500, ATG5/At5g17290, ATG7/At5g45900, ATG16/At5g50230, and ATG18a/At3g62770. Twenty alleles were novel, and 3 of the mutated genes lack T-DNA insertional alleles in publicly available repositories. We also demonstrate that an insertional atg11/At4g30790allele incompletely suppresses lon2 defects. Finally, we show that NBR1 is not necessary for autophagy of lon2 peroxisomes and that NBR1 overexpression is not sufficient to trigger autophagy of seedling peroxisomes, indicating that Arabidopsis can use an NBR1-independent mechanism to target peroxisomes for autophagic degradation.Item A gain-of-function mutation in IAA16 confers reduced responses to auxin and abscisic acid and impedes plant growth and fertility(Springer, 2012) Rinaldi, Mauro A.; Liu, James; Enders, Tara A.; Bartel, Bonnie; Strader, Lucia C.Auxin regulates many aspects of plant development, in part, through degradation of the Aux/IAA family of transcriptional repressors. Consequently, stabilizing mutations in several Aux/IAA proteins confer reduced auxin responsiveness. However, of the 29 apparent Aux/IAA proteins in Arabidopsis thaliana, fewer than half have roles established through mutant analysis. We identified iaa16-1, a dominant gain-of-function mutation in IAA16 (At3g04730), in a novel screen for reduced root responsiveness to abscisic acid. The iaa16-1 mutation also confers dramatically reduced auxin responses in a variety of assays, markedly restricts growth of adult plants, and abolishes fertility when homozygous. We compared iaa16-1 phenotypes with those of dominant mutants defective in the closely related IAA7/AXR2, IAA14/SLR, and IAA17/AXR3, along with the more distantly related IAA28, and found overlapping but distinct patterns of developmental defects. The identification and characterization of iaa16-1 provides a fuller understanding of the IAA7/IAA14/IAA16/IAA17 clade of Aux/IAA proteins and the diverse roles of these repressors in hormone response and plant development.Item A pex1 missense mutation improves peroxisome function in a subset of Arabidopsis pex6 mutants without restoring PEX5 recycling(National Academy of Sciences, 2018) Gonzalez, Kim L.; Ratzel, Sarah E.; Burks, Kendall H.; Danan, Charles H.; Wages, Jeanne M.; Zolman, Bethany K.; Bartel, BonniePeroxisomes are eukaryotic organelles critical for plant and human development because they house essential metabolic functions, such as fatty acid β-oxidation. The interacting ATPases PEX1 and PEX6 contribute to peroxisome function by recycling PEX5, a cytosolic receptor needed to import proteins targeted to the peroxisomal matrix. Arabidopsis pex6 mutants exhibit low PEX5 levels and defects in peroxisomal matrix protein import, oil body utilization, peroxisomal metabolism, and seedling growth. These defects are hypothesized to stem from impaired PEX5 retrotranslocation leading to PEX5 polyubiquitination and consequent degradation of PEX5 via the proteasome or of the entire organelle via autophagy. We recovered a pex1 missense mutation in a screen for second-site suppressors that restore growth to the pex6-1 mutant. Surprisingly, this pex1-1 mutation ameliorated the metabolic and physiological defects of pex6-1 without restoring PEX5 levels. Similarly, preventing autophagy by introducing an atg7-null allele partially rescued pex6-1 physiological defects without restoring PEX5 levels. atg7 synergistically improved matrix protein import in pex1-1 pex6-1, implying that pex1-1 improves peroxisome function in pex6-1 without impeding autophagy of peroxisomes (i.e., pexophagy). pex1-1 differentially improved peroxisome function in various pex6 alleles but worsened the physiological and molecular defects of a pex26 mutant, which is defective in the tether anchoring the PEX1–PEX6 hexamer to the peroxisome. Our results support the hypothesis that, beyond PEX5 recycling, PEX1 and PEX6 have additional functions in peroxisome homeostasis and perhaps in oil body utilization.Item A PEX5 missense allele preferentially disrupts PTS1 cargo import into Arabidopsis peroxisomes(American Society of Plant Biologists, Society for Experimental Biology and John Wiley & Sons Ltd., 3/20/2019) Patel, Khushali J.; Kao, Yun-Ting; Llinas, Roxanna J.; Bartel, Bonnie; BioSciencesThe sorting of eukaryotic proteins to various organellar destinations requires receptors that recognize cargo protein targeting signals and facilitate transport into the organelle. One such receptor is the peroxin PEX5, which recruits cytosolic cargo carrying a peroxisome‐targeting signal (PTS) type 1 (PTS1) for delivery into the peroxisomal lumen (matrix). In plants and mammals, PEX5 is also indirectly required for peroxisomal import of proteins carrying a PTS2 signal because PEX5 binds the PTS2 receptor, bringing the associated PTS2 cargo to the peroxisome along with PTS1 cargo. Despite PEX5 being the PTS1 cargo receptor, previously identified Arabidopsis pex5 mutants display either impairment of both PTS1 and PTS2 import or defects only in PTS2 import. Here, we report the first Arabidopsis pex5 mutant with an exclusive PTS1 import defect. In addition to markedly diminished GFP‐PTS1 import and decreased pex5‐2 protein accumulation, this pex5‐2 mutant shows typical peroxisome‐related defects, including inefficient β‐oxidation and reduced growth. Growth at reduced or elevated temperatures ameliorated or exacerbated pex5‐2 peroxisome‐related defects, respectively, without markedly changing pex5‐2 protein levels. In contrast to the diminished PTS1 import, PTS2 processing was only slightly impaired and PTS2‐GFP import appeared normal in pex5‐2. This finding suggests that even minor peroxisomal localization of the PTS1 protein DEG15, the PTS2‐processing protease, is sufficient to maintain robust PTS2 processing.Item A role for the root cap in root branching revealed by the non-auxin probe naxillin(Nature America, Inc., 2012) De Rybel, Bert; Audenaert, Dominique; Xuan, Wei; Overvoorde, Paul; Strader, Lucia C.; Kepinski, Stefan; Hoye, Rebecca; Brisbois, Ronald; Parizot, Boris; Vanneste, Steffan; Liu, Xing; Gilday, Alison; Graham, Ian A.; Nguyen, Long; Jansen, Leentje; Njo, Maria Fransiska; Inze, Dirk; Bartel, Bonnie; Beeckman, TomThe acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap-specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture.Item A viable Arabidopsis pex13 missense allele confers severe peroxisomal defects and decreases PEX5 association with peroxisomes(Springer, 2014) Woodward, Andrew W.; Fleming, Wendell A.; Burkhart, Sarah E.; Ratzel, Sarah E.; Bjornson, Marta; Bartel, BonniePeroxisomes are organelles that catabolize fatty acids and compartmentalize other oxidative metabolic processes in eukaryotes. Using a forward-genetic screen designed to recover severe peroxisome-defective mutants, we isolated a viable allele of the peroxisome biogenesis gene PEX13 with striking peroxisomal defects. The pex13-4 mutant requires an exogenous source of fixed carbon for pre-photosynthetic development and is resistant to the protoauxin indole-3-butyric acid. Delivery of peroxisome-targeted matrix proteins depends on the PEX5 receptor docking with PEX13 at the peroxisomal membrane, and we found severely reduced import of matrix proteins and less organelle-associated PEX5 in pex13-4 seedlings. Moreover, pex13-4 physiological and molecular defects were partially ameliorated when PEX5 was overexpressed, suggesting that PEX5 docking is partially compromised in this mutant and can be improved by increasing PEX5 levels. Because previously described Arabidopsis pex13 alleles either are lethal or confer only subtle defects, the pex13-4 mutant provides valuable insight into plant peroxisome receptor docking and matrix protein import.Item An Arabidopsis pre-RNA processing8a (prp8a) missense allele restores splicing of a subset of mis-spliced mRNAs(Oxford University Press, 2022) Llinas, Roxanna J.; Xiong, Jia Qi; Clark, Natalie M.; Burkhart, Sarah E.; Bartel, BonnieEukaryotic precursor mRNAs often harbor noncoding introns that must be removed prior to translation. Accurate splicing of precursor messenger RNA depends on placement and assembly of small nuclear ribonucleoprotein (snRNP) sub-complexes of the spliceosome. Yeast (Saccharomyces cerevisiae) studies established a role in splice-site selection for PRE-RNA PROCESSING8 (PRP8), a conserved spliceosome scaffolding protein of the U5 snRNP. However, analogous splice-site selection studies in multicellular eukaryotes are lacking. Such studies are crucial for a comprehensive understanding of alternative splicing, which is extensive in plants and animals but limited in yeast. In this work, we describe an Arabidopsis (Arabidopsis thaliana) prp8a mutant that modulates splice-site selection. We isolated prp8a-14 from a screen for suppressors of pex14-6, which carries a splice-site mutation in the PEROXIN14 (PEX14) peroxisome biogenesis gene. To elucidate Arabidopsis PRP8A function in spliceosome fidelity, we combined prp8a-14 with various pex14 splice-site mutations and monitored the double mutants for physiological and molecular consequences of dysfunctional and functional peroxisomes that correspond to impaired and recovered splicing, respectively. prp8a-14 restored splicing and PEX14 function to alleles with mutations in the exonic guanine of the 5′-splice site but did not restore splicing or function to alleles with mutations in the intronic guanine of 5′- or 3′-splice sites. We used RNA-seq to reveal the systemic impact of prp8a-14 and found hundreds of differentially spliced transcripts and thousands of transcripts with significantly altered levels. Among differentially spliced transcripts, prp8a-14 significantly altered 5′- and 3′-splice-site utilization to favor sites resulting in shorter introns. This study provides a genetic platform for probing splicing in plants and hints at a role for plant PRP8 in splice-site selection.Item Analysis of the function and metabolism of indole-3-acetic acid conjugates in Arabidopsis thaliana(2002) LeClere, Sherry; Bartel, BonnieThe auxin indole-3-acetic acid (IAA) is involved in virtually every aspect of plant development. Plants control auxin homeostasis through complex interactions between de novo synthesis, degradation, import, export, and conjugate synthesis and hydrolysis. A thorough knowledge of these pathways and their interactions is key to understanding auxin response and plant growth. I have used genetic and biochemical methods to better understand the function and metabolism of amide-linked conjugates of IAA. Conjugates may function as storage or transport forms of IAA, or may function independently of hydrolysis. Many IAA conjugates have auxin activity in bioassays, and Arabidopsis mutants with reduced sensitivity to exogenous IAA-L-amino acid conjugates have been identified. Some of these are defective in genes encoding conjugate hydrolases, whereas others are likely to be directly or indirectly involved in some other aspect of conjugate metabolism or transport. To better understand conjugate hydrolysis pathways, I have generated and screened an overexpression library to identify cDNAs that lead to conjugate resistance when overexpressed. From this library, I have identified one mutant that cosuppresses petH, resulting in a chlorotic phenotype, and several mutants resistant to IAA-Ala that result from mutations unlinked to T-DNA inserts. In addition, I have cloned the gene defective in iar4, an IAA-alanine-resistant mutant, and found that it encodes a protein similar to a mitochondrial pyruvate dehydrogenase E1alpha subunit. To better understand conjugate hydrolysis, I have characterized the enzyme activities of four members of the amidohydrolase family. I found that bacterially expressed GST fusions of ILR1, ILL2, and IAR3 hydrolyze certain IAA-amino acids with KM values in the muM range, suggesting they are physiologically relevant. In addition, mutant plant extracts show altered rates of conjugate hydrolysis, further supporting the hypothesis that these enzymes function in vivo to cleave IAA-amino acid conjugates. I have also examined the expression profiles of the amidohydrolase family and found their expression is overlapping but distinct. My findings suggest that the activity displayed by auxin conjugates is due to their hydrolysis by the amidohydrolases to yield free IAA, and that these processes are controlled both developmentally and spatially to regulate free IAA levels.Item Biology in Bloom: A Primer on the Arabidopsis thaliana Model System(The Genetics Society of America, 4/1/2018) Woodward, Andrew W.; Bartel, Bonnie; BioSciencesArabidopsis thaliana could have easily escaped human scrutiny. Instead, Arabidopsis has become the most widely studied plant in modern biology despite its absence from the dinner table. Pairing diminutive stature and genome with prodigious resources and tools, Arabidopsis offers a window into the molecular, cellular, and developmental mechanisms underlying life as a multicellular photoautotroph. Many basic discoveries made using this plant have spawned new research areas, even beyond the verdant fields of plant biology. With a suite of resources and tools unmatched among plants and rivaling other model systems, Arabidopsis research continues to offer novel insights and deepen our understanding of fundamental biological processes.Item Cloning and characterization of IAA28, a gene involved in suppressing lateral root development and mediating auxin responses in Arabidopsis thaliana(2002) Rogg, Luise Elizabeth; Bartel, BonnieAuxins are an important class of plant hormones implicated in most aspects of plant development, and therefore influence the overall size and shape of a plant. Whereas the signal transduction pathways that sense and respond to auxin remain mysterious, a number of genes do undergo dramatic transcriptional alterations in response to auxin. The Aux/IAA genes were originally isolated based on strong and rapid transcriptional up-regulation following auxin induction. Aux/IAA genes are primary response genes whose products are thought to regulate auxin-responsive transcription. A new member of the Aux/IAA gene family, IAA28, was cloned based on the abnormal auxin responses and unusual auxin-related adult phenotypes of a gain-of-function mutant, including decreased apical dominance and extremely reduced lateral root formation. In addition to auxin, the iaa28-1 mutant is also resistant to inhibition of root elongation by cytokinin and ethylene, but responds normally to other phytohormones. Northern analysis, promoter - reporter gene fusions and Western analysis demonstrate that IAA28 is strongly expressed in roots. IAA28 may be a primary response gene, as IAA28 mRNA levels increase in response to cycloheximide. However, IAA28 transcription decreases upon auxin treatment, a novel response that differs from other characterized members of the Aux/IAA gene family. Experiments with an auxin-inducible reporter construct suggest that IAA28 is a repressor of auxin-induced transcription. Levels of tagged IAA28 protein decrease dramatically after exogenous auxin treatment, thus IAA28 degradation may increase in response to auxin signaling. In addition, microarray analysis suggests that a number of genes have altered transcript levels in the gain-of-function iaa28-1 mutant background. From these findings, IAA28 has been proposed to encode a transcriptional repressor that functions to regulate expression of genes that promote lateral root initiation in response to auxin.Item Compensatory Mutations in Predicted Metal Transporters Modulate Auxin Conjugate Responsiveness in Arabidopsis(The Genetics Society of America, 2013-01) Rampey, Rebekah A.; Baldridge, Megan T.; Farrow, David C.; Bay, Sarah N.; Bartel, BonnieLevels of the phytohormone indole-3-acetic acid (IAA) can be altered by the formation and hydrolysis of IAA conjugates. The isolation and characterization of Arabidopsis thaliana mutants with reduced IAA-conjugate sensitivity and wild-type IAA responses is advancing the understanding of auxin homeostasis by uncovering the factors needed for conjugate metabolism. For example, the discovery that the IAA-Ala-resistant mutant iar1 is defective in a protein in the ZIP family of metal transporters uncovered a link between metal homeostasis and IAA-conjugate sensitivity. To uncover additional factors impacting auxin conjugate metabolism, we conducted a genetic modifier screen and isolated extragenic mutations that restored IAA-amino acid conjugate sensitivity to the iar1 mutant. One of these suppressor mutants is defective in a putative cation diffusion facilitator, MTP5 (At3g12100; formerly known as MTPc2). Loss of MTP5 function restored IAA conjugate sensitivity to iar1 but not to mutants defective in IAA-amino acid conjugate amidohydrolases. Our results are consistent with a model in which MTP5 and IAR1 transport metals in an antagonistic fashion to regulate metal homeostasis within the subcellular compartment in which the IAA-conjugate amidohydrolases reside, and support previous suggestions that the ion composition in this compartment influences hydrolase activity.Item Developing CRISPR tools for activating silent biosynthetic gene clusters in Streptomyces spp.(2022-09-06) Ameruoso, Andrea; Chappell, James; Bartel, BonnieThe rise of antibiotic-resistant bacteria represents a major threat to global health, creating an urgent need to discover new antibiotics. Natural products derived from the genus Streptomyces represent a rich and diverse repertoire of chemical molecules from which new antibiotics are likely to be found. However, a major challenge is that the biosynthetic gene clusters (BGCs) responsible for natural product synthesis are often poorly expressed under laboratory culturing conditions, thus preventing the isolation and screening of novel chemicals. To address this, I describe a novel approach to activate silent BGCs through rewiring of the endogenous regulation using synthetic gene regulators based upon CRISPR-Cas. First, I refine CRISPR interference (CRISPRi) and create CRISPR activation (CRISPRa) systems that allow for highly programmable and effective gene repression and activation in Streptomyces. I then harness these tools to activate a silent BGC by perturbing its endogenous regulatory network. Together, this work advances the synthetic regulatory toolbox for Streptomyces and facilitates the programmable activation of silent BGCs for novel chemical discovery.Item Disparate peroxisome‐related defects in Arabidopsis pex6 and pex26 mutants link peroxisomal retrotranslocation and oil body utilization(Wiley, 2017) Gonzalez, Kim L.; Fleming, Wendell A.; Kao, Yun-Ting; Wright, Zachary J.; Venkova, Savina V.; Ventura, Meredith J.; Bartel, BonnieCatabolism of fatty acids stored in oil bodies is essential for seed germination and seedling development in Arabidopsis. This fatty acid breakdown occurs in peroxisomes, organelles that sequester oxidative reactions. Import of peroxisomal enzymes is facilitated by peroxins including PEX5, a receptor that delivers cargo proteins from the cytosol to the peroxisomal matrix. After cargo delivery, a complex of the PEX1 and PEX6 ATPases and the PEX26 tail‐anchored membrane protein removes ubiquitinated PEX5 from the peroxisomal membrane. We identified Arabidopsis pex6 and pex26 mutants by screening for inefficient seedling β‐oxidation phenotypes. The mutants displayed distinct defects in growth, response to a peroxisomally metabolized auxin precursor, and peroxisomal protein import. The low PEX5 levels in these mutants were increased by treatment with a proteasome inhibitor or by combining pex26 with peroxisome‐associated ubiquitination machinery mutants, suggesting that ubiquitinated PEX5 is degraded by the proteasome when the function of PEX6 or PEX26 is reduced. Combining pex26 with mutations that increase PEX5 levels either worsened or improved pex26 physiological and molecular defects, depending on the introduced lesion. Moreover, elevating PEX5 levels via a 35S:PEX5 transgene exacerbated pex26 defects and ameliorated the defects of only a subset of pex6 alleles, implying that decreased PEX5 is not the sole molecular deficiency in these mutants. We found peroxisomes clustered around persisting oil bodies in pex6 and pex26 seedlings, suggesting a role for peroxisomal retrotranslocation machinery in oil body utilization. The disparate phenotypes of these pex alleles may reflect unanticipated functions of the peroxisomal ATPase complex.Item Disrupting Autophagy Restores Peroxisome Function to an Arabidopsis lon2 Mutant and Reveals a Role for the LON2 Protease in Peroxisomal Matrix Protein Degradation(American Society of Plant Biologists, 2013) Farmer, Lisa M.; Rinaldi, Mauro A.; Young, Pierce G.; Danan, Charles H.; Burkhart, Sarah E.; Bartel, BonniePeroxisomes house critical metabolic reactions that are essential for seedling development. As seedlings mature, metabolic requirements change, and peroxisomal contents are remodeled. The resident peroxisomal protease LON2 is positioned to degrade obsolete or damaged peroxisomal proteins, but data supporting such a role in plants have remained elusive. Arabidopsis thaliana lon2 mutants display defects in peroxisomal metabolism and matrix protein import but appear to degrade matrix proteins normally. To elucidate LON2 functions, we executed a forward-genetic screen for lon2 suppressors, which revealed multiple mutations in key autophagy genes. Disabling core autophagy-related gene (ATG) products prevents autophagy, a process through which cytosolic constituents, including organelles, can be targeted for vacuolar degradation. We found that atg2, atg3, and atg7 mutations suppressed lon2 defects in auxin metabolism and matrix protein processing and rescued the abnormally large size and small number of lon2 peroxisomes. Moreover, analysis of lon2 atg mutants uncovered an apparent role for LON2 in matrix protein turnover. Our data suggest that LON2 facilitates matrix protein degradation during peroxisome content remodeling, provide evidence for the existence of pexophagy in plants, and indicate that peroxisome destruction via autophagy is enhanced when LON2 is absent.Item Elevated growth temperature decreases levels of the PEX5 peroxisome-targeting signal receptor and ameliorates defects of Arabidopsis mutants with an impaired PEX4 ubiquitin-conjugating enzyme(BioMed Central, 2015) Kao, Yun-Ting; Bartel, BonnieBackground: Peroxisomes house critical metabolic reactions. For example, fatty acid β-oxidation enzymes, which are essential during early seedling development, are peroxisomal. Peroxins (PEX proteins) are needed to bring proteins into peroxisomes. Most matrix proteins are delivered to peroxisomes by PEX5, a receptor that forms transient pores to escort proteins across the peroxisomal membrane. After cargo delivery, a peroxisome-tethered ubiquitin-conjugating enzyme (PEX4) and peroxisomal ubiquitin-protein ligases mono- or polyubiquitinate PEX5 for recycling back to the cytosol or for degradation, respectively. Arabidopsis pex mutants β-oxidize fatty acids inefficiently and therefore fail to germinate or grow less vigorously. These defects can be partially alleviated by providing a fixed carbon source, such as sucrose, in the growth medium. Despite extensive characterization of peroxisome biogenesis in Arabidopsis grown in non-challenged conditions, the effects of environmental stressors on peroxisome function and pex mutant dysfunction are largely unexplored. Results: We surveyed the impact of growth temperature on a panel of pex mutants and found that elevated temperature ameliorated dependence on external sucrose and reduced PEX5 levels in the pex4-1 mutant. Conversely, growth at low temperature exacerbated pex4-1 physiological defects and increased PEX5 levels. Overexpressing PEX5 also worsened pex4-1 defects, implying that PEX5 lingering on the peroxisomal membrane when recycling is impaired impedes peroxisome function. Growth at elevated temperature did not reduce the fraction of membrane-associated PEX5 in pex4-1, suggesting that elevated temperature did not restore PEX4 enzymatic function in the mutant. Moreover, preventing autophagy in pex4-1 did not restore PEX5 levels at high temperature. In contrast, MG132 treatment increased PEX5 levels, implicating the proteasome in degrading PEX5, especially at high temperature. Conclusions: We conclude that growth at elevated temperature increases proteasomal degradation of PEX5 to reduce overall PEX5 levels and ameliorate pex4-1 physiological defects. Our results support the hypothesis that efficient retrotranslocation of PEX5 after cargo delivery is needed not only to make PEX5 available for further rounds of cargo delivery, but also to prevent the peroxisome dysfunction that results from PEX5 lingering in the peroxisomal membrane.Item Elucidating the Roles of PEX19 and Prenylation in Arabidopsis Peroxisomes(2012-09-05) Stoddard, Jerrad; Bartel, Bonnie; Shamoo, Yousif; Farach-Carson, Cindy; Braam, Janet; Rudgers, Jennifer A.Peroxisomes are organelles originating from the endoplasmic reticulum. Peroxisome biogenesis requires multiple peroxins, including PEX19, a prenylated protein that helps deliver peroxisomal membrane proteins in yeast and mammals. Arabidopsis thaliana PEX19 is encoded by two isogenes, PEX19A and PEX19B. I demonstrate that pex19A and pex19B insertional mutants lack obvious abberant physiological phenotypes. I provide evidence that pex19A pex19B double mutants are inviable, that PEX19B is more abundant than PEX19A in young seedlings, that Arabidopsis PEX19 is farnesylated in vivo, and that YFP-PEX19 predominantly associates with what appears to be a subcellular membrane regardless of its prenylation state. I show that farnesyltransferase mutants apparently contain only non-prenylated PEX19 and lack phenotypes that would indicate inefficient peroxisome activity. My analysis of PEX19 suggests that PEX19 prenylation is dispensable for peroxisome biogenesis, and has generated tools for future studies of the earliest steps in peroxisome biogenesis in plants.Item ESRE Network Activation and Regulation Provide Insight into Mitochondrial Surveillance(2020-04-22) Tjahjono, Elissa; Bartel, Bonnie; Kirienko, Natalia VAll living organisms exist in a precarious state of homeostasis that requires constant maintenance. A wide variety of stresses, including hypoxia, heat, and infection by pathogens perpetually threaten to imbalance this state. Organisms use a battery of defenses to mitigate damage and restore normal function. In a pyoverdine-dependent Caenorhabditis elegans-Pseudomonas aeruginosa assay, C. elegans’ defense response utilizes the phylogenetically conserved ESRE (Ethanol and Stress Response Element) network, which has previously been shown to mitigate damage from a variety of abiotic stresses. It is intriguing that this network is involved in innate immunity; it indicates that host innate immune responses overlap with responses to abiotic stresses. I further investigated the ESRE defense network following exposure to P. aeruginosa and showed that mitochondrial damage leads to ESRE activation both in C. elegans and in mammals. Mitochondrial dysfunction contributes to a wide variety of pathologies, including neurodegenerative diseases, cancer, metabolic diseases, and aging. Mitochondrial status is actively surveilled by several different systems to ensure the preservation of cellular viability. I characterized the ESRE pathway, a novel mitochondrial surveillance system, and showed that it is robustly activated in response to increased superoxide radical. This differentiates ESRE from other existing mitochondrial surveillance pathways, such as the mitochondrial unfolded protein response (UPRmt) that monitors disruption of mitochondrial protein import and the mitochondrial MAPK (MAPKmt) pathway that responds to mitochondrial bioenergetic disruption. I also observed multiple interactions between the ESRE, UPRmt, and MAPKmt pathways. Understanding mitochondrial surveillance and the interplay that occurs among these pathways is crucial for the advancement and understanding of cell biology. Considering that the ESRE motif and its role in response to mitochondrial damage is conserved in humans, modulation of mitochondrial pathways could confer resistance to stress and improve health span, leading to a direct benefit to human health.Item Evolutionary Dynamics Leading to Tigecycline Resistance in Enterococcus faecalis(2016-09-16) Beabout-McCarthy, Kathryn; Shamoo, Yousif; Bartel, BonnieThe rising frequency of multidrug resistant pathogens poses an increasing threat to public health. There is a dire need for novel approaches to combat these deadly pathogens and maintain the efficacy of currently available antibiotics. In particular horizontal gene transfer threatens the therapeutic success of antibiotics by facilitating the rapid dissemination of resistance alleles among bacterial species. The conjugative mobile element Tn916 provides an excellent context for examining the role of adaptive parasexuality as it carries the tetracycline-resistance allele tetM and has been identified in a wide range of pathogens. For this thesis I used quantitative experimental evolution, a pipeline developed by our lab to identify clinically relevant resistance mechanisms, to study tigecycline resistance in a hospital strain of Enterococcus faecalis. Quantitative experimental evolution uses a combination of experimental evolution and allelic frequency measurements to gain insights into the adaptive trajectories leading to resistance and to predict what mechanisms of resistance are most likely to appear in the clinical setting. Here we show that antibiotic selection led to the near fixation of adaptive alleles that simultaneously altered TetM expression and produced remarkably increased levels of Tn916 horizontal gene transfer. In the absence of drug, approximately 1 in 120,000 of the non-adapted Enterococcus faecalis S613 cells had an excised copy of Tn916, whereas nearly 1 in 50 cells had an excised copy of Tn916 upon selection for resistance resulting in a more than 1,000-fold increase in conjugation rates. We also show that tigecycline, a translation inhibitor, selected for a mutation in the ribosomal S10 protein in the E. faecalis adapting populations. Furthermore, we show that mutation of S10 is an important allele for a broad range of Gram-positive and Gram-negative pathogens to adapt to tigecycline. These results show the first example of mutations that concurrently confer resistance to an antibiotic and lead to constitutive conjugal-transfer of the resistance allele. Selection created a highly parasexual phenotype and high frequency of Tn916 jumping demonstrating how the use of antibiotics can lead directly to the proliferation of resistance in, and potentially among, pathogens.Item Genes, organelles, and molecules that influence plant development through auxin regulation(2005) Woodward, Andrew W.; Bartel, BonnieHumankind depends on plants to harvest solar energy and convert it into accessible chemical energy. With booming human population growth and diminishing availability of arable land, understanding plant development is necessary for more efficient agricultural production. Auxin is a plant hormone utilized in many aspects of plant growth and environmental responses. This work examines genes that regulate or are regulated by auxin, biogenesis and function of an organelle that is an auxin source, and molecules that behave as auxins to influence plant development. Within the plant cell, peroxisomes are organelles that house many processes including fatty acid metabolism to produce energy and also proto-auxin metabolism to produce the active hormone. Peroxisomal proteins are translated in the cytoplasm and imported into peroxisomes by a host of machinery. Peroxisomal targeting signal sequences are recognized by one of two receptors; these receptors interact with each other physically and functionally in some organisms. Here, I identify the receptor machinery present in diverse organisms to predict and compare methods of peroxisomal matrix protein import. I also characterize mutants of the model plant Arabidopsis thaliana defective in import of one class of peroxisomal matrix proteins. In addition, I examine various molecules that influence plant development in an auxin-like fashion. I identify genes with mRNA accumulation regulated by a proto-auxin in a background that inefficiently converts this compound into auxin. I describe the characterization of responses to a second auxin-related molecule that impacts plant development through auxin signaling. I also describe the isolation, characterization, and cloning of a mutant with reduced sensitivity to a specific subset of auxin-like molecules. Data obtained in this work reveal a host of factors that affect auxin regulation and thereby influence plant life. The results of these experiments in plant biology highlight the diversity, complexity, and essentiality of auxin responses.Item Genetic analyses of auxin metabolism and of the transition to flowering in the model plant Arabidopsis thaliana(2000) Lasswell, Jamie Elizabeth; Bartel, BonnieThe auxins are an important class of plant hormones involved in many aspects of plant development The most common naturally occurring auxin is indole-3-acetic acid, or IAA. In Arabidopsis, up to 95% of the IAA pool is found conjugated to small molecules such as sugars and amino acids. However, the genes and enzymes involved in IAA conjugate metabolism are not yet well understood. A mutant, iar1, that is resistant to the inhibitory effects of multiple IAA-amino acid conjugates on root elongation was identified. The IAR1 gene encodes a protein with numerous transmembrane domains and several histidine-rich regions. The IAR1 protein has homologs in other organisms, including Drosophila, C. elegans, and mammals, and is similar in molecular structure to the ZIP family of zinc transporters from Arabidopsis and yeast. Plant reproduction requires precise control of the transition to flowering in response to environmental cues. We have isolated a late-flowering Arabidopsis mutant, fkf1, that is rescued by vernalization or gibberellin treatment. The mutant also exhibits a light-dependent hypocotyl elongation defect. We used a positional approach to clone FKF1, which encodes a novel protein with an N-terminal PAS domain similar to the flavin-binding region of certain photoreceptors, an F-box motif characteristic of proteins that target ubiquitin-mediated degradation, and six kelch repeats predicted to fold into a beta-propeller. FKF1 mRNA levels oscillate with a circadian rhythm and the fkf1 deletion mutation alters the rhythmic expression of other clock-regulated genes, implicating FKF1 in regulation of the circadian clock.