Cell Biology & Molecular Genetics Theses and Dissertations
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Item Suppressors of etr1-2: I. etr1-11 is a loss-of-function mutation of the ETR1 ethylene receptor. II. REVERSION TO ETHYLENE-SENSITIVITY3 is a regulator of seedling growth.(2009) McClellan, Christopher Alan; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The plant hormone ethylene is an important regulator of plant growth and development, including senescence, abcission, fruit ripening, and responses to biotic and abiotic stresses. To find new members of the ethylene signaling pathway, a genetic screen for suppressors of the ethylene-insensitive mutant etr1-2 was performed. One mutant identified in this screen, etr1-11, is an intragenic mutation within ETR1. etr1-11 is a unique missense mutation that appears to eliminate ETR1-2 signaling. Mutant analysis further revealed that etr1-11 is a partial loss-of-function allele. The rte3 (reversion to ethylene sensitivity3) mutant was another mutant isolated in a genetic screen for suppressors of etr1-2. After testing other ethylene responses, such as leaf senescence, and performing epistasis analysis with other ethylene signaling mutants, it was determined that RTE3 is unlikely to play a direct role in the ethylene signaling pathway. Instead, RTE3 appears to be responsible for promoting hypocotyl elongation in etiolated seedlings in the ethylene triple response assay. The RTE3 gene was identified by positional cloning, and is predicted to encode a protein with an annotated SAC3/GANP domain. SAC3/GANP domains are present in proteins that participate in large multi-peptide complexes, such as the 26S proteasome regulatory subunit and the eIF3 translation initiation complex. Similarities in protein composition between these two complexes and the COP9 signalosome (CSN) suggest that a SAC3/GANP domain-containing protein may interact with members of the CSN. Interestingly, yeast two-hybrid analysis reveals that RTE3 interacts with EER5 and EIN2, proteins that have been shown to interact with members of the CSN. In addition, rte3-1 ein2-1 seedlings show a synthetic phenotype of delayed growth. Protein localization using a GFP tag reveals that RTE3 and EER5 both localize to the nucleus. These interactions suggest that RTE3, EER5, EIN2, and the CSN form a protein complex that regulates seedling growth.Item Investigation of Ethylene Signal Transduction Mechanisms: Characterizing the Novel Gene AWE1 and Testing Hypothesis of Raf-like CTR1's Function In Vivo(2009) Kendrick, Mandy Danielle; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is a gaseous plant hormone affecting multiple plant processes. Sixteen years ago the first components of the ethylene signaling pathway, the receptor ETR1 and Raf-like kinase CTR1, were identified. Since then many additional components of the pathway have been elucidated through genetic screens. Recent discoveries suggest ethylene signaling, once thought to be a linear pathway from ethylene perception at the endoplasmic reticulum to transcriptional activation at the nucleus, is more complex with multiple auto-feedback loops and potential parallel kinase cascades downstream of the receptors. Although the genetic backbone of the pathway is well established, the signaling mechanisms of the components remain unclear. ETR1 displays histidine kinase activity in vitro and physically interacts with the next-known downstream component of the pathway, CTR1. However the histidine kinase activity of ETR1 is mostly dispensable for signaling to CTR1. How then is CTR1 activated? I proposed that additional proteins, like AWE1, play a role in ETR1 to CTR1 signaling, and that the non-catalytic, amino-terminal region of CTR1 is required both for activation through direct interaction with the ETR1 receptor complex and for auto-inhibition of CTR1 kinase activity. ASSOCIATES-WITH-ETR1 (AWE1) was isolated in a yeast-two-hybrid screen for ETR1-interacting proteins and was of specific interest because the AWE1 clone also interacted with a portion of CTR1. Protein-protein interaction studies and genetic analysis of an awe1 mutant support a role of AWE1 in repressing ethylene responses. However double mutant analysis, over-expression analysis, and protein sub-cellular localization studies suggest that AWE1's function in hypocotyl elongation and cell expansion is more general. AWE1's function may require ETR1 for proper regulation but is likely to lie outside of the direct step from ETR1 to CTR1. To investigate a role of the CTR1 amino-terminal region in CTR1 regulation, I constructed transgenes consisting of truncated ETR1 receptors fused to truncated or full length CTR1 and examined how those transgenes carrying the truncated CTR1 (kinase domain only) affected Arabidopsis seedling growth compared to those transgenes expressing full length CTR1. I concluded that the CTR1 amino-terminal region may have a role in autoregulation, but additional components are required for regulation of CTR1 signaling.Item Functions of the Tobacco mosaic virus helicase domain: regulating formation of the virus replication complex and altering the activity of a host-encoded transcription factor(2008-04-23) Wang, Xiao; Culver, James N; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tobacco mosaic virus (TMV)-encoded 126-kDa and 183-kDa replicases are multidomain and multifunctional proteins. The helicase domain shared by both replicases has been shown to perform multiple tasks during the virus life cycle. In vitro structural and functional analyses demonstrated that monomers and dimers of the TMV helicase domain were the active forms for ATP hydrolysis. However, self-interaction of the helicase polypeptides resulted in the formation of higher-order structures that likely serve as structural scaffolds for the assembly of virus replication complexes (VRCs). Mutagenesis studies of the TMV helicase motifs showed that conserved amino acid residues played important roles in protein ATPase and/or RNA binding activities. A close correlation between ATPase activity of the helicase domain and assembly of wild-type VRC-like vesicles by the 126-kDa replicase further suggests that ATPase activity of the TMV helicase domain may modulate proper VRC assembly. In addition to helicase self-interaction, a novel virus-host interaction involving ATAF2, a NAC domain transcription factor was identified. Members within the NAC domain family are involved in plant developmental processes and stress/defense responses. In this study, transgenic plants overexpressing ATAF2 showed a strong developmental phenotype. Inoculation of TMV in these transgenic plants resulted in reduced virus accumulations. Additionally, transcriptional induction of ATAF2 occurred in response to TMV infection and salicylic acid treatment. Combined, these results suggest that ATAF2 is involved in a host defense response. One interesting finding was that in susceptible hosts, virus-directed induction of ATAF2 and PR1, a well-defined pathogenesis-related (PR) marker gene for host defense system, occurred only in locally-infected but not in systemically-infected tissues. Dynamic changes in the expression of host defense genes suggest that viruses have evolved certain mechanisms to actively modulate host gene expression. Interaction between the TMV helicase domain and ATAF2 may provide one way to suppress the ATAF2-mediated host defense signaling pathway. Combined these studies investigated the importance of the TMV helicase domain in VRC formation and in manipulating the host defense system. The results confirmed the functional versatility of the TMV helicase domain in establishing a successful virus life cycle.Item MOLECULAR CHARACTERIZATION OF INTERACTIONS BETWEEN TMV REPLICASE PROTEIN AND AUXIN RESPONSIVE PROTEINS: IMPLICATIONS IN DISEASE DEVELOPMENT(2006-11-25) Padmanabhan, Meenu Sreedevi; Culver, James; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Tobacco Mosaic Virus and Arabidopsis thaliana serve as ideal model systems to study the molecular aspects of virus - host interactions. Using this system, an interaction between the helicase domain within TMV replicase protein and an auxin responsive protein, IAA26 was identified. IAA26 is a member of the Aux/IAA family of transcription factors that function as repressors in signaling pathways controlled by the phytohormone auxin. Characterization of the interaction was carried out utilizing a helicase mutant defective in its interaction with IAA26 and by creating transgenic plants silenced for IAA26 expression. These studies suggest that the interaction was not essential for either viral replication or movement but promoted the development of disease symptoms. Cellular co-localization studies revealed that in TMV infected tissue, the nuclear localization and stability of IAA26 was compromised and the protein was relocalized to distinct cytoplasmic vesicles in association with the viral replicase. In keeping with its role as a transcription factor, the alterations in IAA26 function should lead to misregulation of downstream auxin responsive genes and this is supported by the fact that ~ 30% of the Arabidopsis genes displaying transcriptional alterations to TMV could be linked to the auxin signaling pathway. Aux/IAA family members share significant sequence and functional homology, and an additional interaction screen identified two more Arabidopsis Aux/IAA proteins, IAA27 and IAA18 and a putative tomato Aux/IAA protein, LeIAA26 that could interact with TMV helicase. The nuclear localization of these three proteins was disrupted by TMV and alterations in LeIAA26 levels induced virus infection-like symptoms in tomato. Additionally, transgenic plants over-expressing a proteolysis resistant mutant of IAA26 showed abnormal developmental phenotype, the severity of which was abrogated during TMV infection which blocked nuclear accumulation of the protein. Taken together, these findings suggest that TMV induced disease symptoms can partially be explained by the ability of the virus to disrupt the functioning of interacting Aux/IAA proteins within susceptible hosts. The significance of such interactions is yet to be determined but it appears that they may be advantageous to the virus while infecting tissues that are in a developmentally static stage.Item REVERSION-TO-ETHYLENE-SENSITIVITY1: A Novel Regulator of Ethylene Receptor Function in Arabidopsis thaliana(2006-11-25) Resnick, Josephine; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene is a plant hormone that has profound effects on plant growth and development. Genetic analysis has been central in the elucidation of the ethylene-signaling pathway, made possible through the isolation of ethylene-response mutants in Arabidopsis. This thesis focuses on elucidating the function of the Arabidopsis REVERSION-TO-ETHYLENE-SENSITIVITY1 (RTE1) locus, which was identified in a genetic screen for suppressors of the ethylene-insensitive receptor mutant etr1-2. The RTE1 gene was cloned by positional cloning and found to encode a novel integral membrane protein with homologs in plants and animals, but with no known molecular function. Our studies show that RTE1 is a negative regulator of the ethylene-response pathway, specifically acting as a positive regulator of the ETR1 ethylene receptor. Loss-of-function mutations in the RTE1 gene suppress the etr1-2 ethylene-insensitive phenotype, and genetic analysis suggests that loss of RTE1 results in a largely non-functional ETR1-2 mutant receptor. Similarly, wild-type ETR1 function appears to be greatly reduced in the absence of RTE1. Overexpression of the RTE1 gene confers weak ethylene insensitivity that is largely dependent on ETR1. rte1 mutations do not appear to affect the other four ethylene receptors of Arabidopsis, indicating that RTE1 specifically regulates ETR1. Sequence analysis revealed regions of conserved cysteine and histidine residues, and one rte1 loss-of-function mutant contains a point mutation at Cys161. Since such residues are common in metal binding proteins, we explored the possibility that RTE1 may be involved in facilitating the binding of an essential copper cofactor to the ETR1 receptor. However, experimental evidence suggests that this is not the likely role of RTE1. Interestingly, rte1 was unable to suppress the ethylene insensitive mutant etr1-1, indicating that the differences between etr1-2 and etr1-1 may hold a clue as to how RTE1 regulates ETR1. A suppression analysis of eleven additional etr1 insensitive mutants suggests that RTE1 plays a role in regulating signaling by the transmitter domain of ETR1. A possible role for RTE homologs in non-plant systems is also discussed, although more work is required to elucidate a detailed biochemical model for RTE1 action.Item The Importance of Sorting Calcium in Plant Cells: Uncovering the Roles of A Sarcoplasmic/Endoplasmic Reticulum-Like Calcium ATPase(2006-11-29) Li, Xiyan; Sze, Heven; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The spatial and temporal dynamics of intracellular Ca2+ in response to environmental and hormonal cues underscore the importance of Ca2+ transport during plant growth and development. The Arabidopsis thaliana genome predicts multiple genes encoding Ca2+ transporters, though the biological roles of most are unknown. Here I determine the function of AtECA3 which represents the first plant P-type 2A ATPase resembling mammalian sarcoplasmic/endoplasmic reticulum Ca ATPase (SERCA). AtECA3 (At1g10130) expressed in a yeast mutant lacking its endogenous Ca2+ pumps functionally substitutes for the defective Ca2+-ATPases. AtECA3-dependent yeast growth is blocked by thapsigargin, a specific SERCA inhibitor. The results suggest that AtECA3 is a cation pump with specificity for Ca2+ and Mn2+, and that AtECA3 enhances yeast growth on Ca2+-depleted medium or on medium with high Mn2+ by sequestrating Ca2+ or Mn2+, respectively, into endomembrane compartments. AtECA3 is expressed in pollen grains as revealed by promoter::GUS analyses and a green fluorescence protein (GFP)-tagged to AtECA3 labels endomembranes at the pollen tip. In vitro tube growth of wild-type pollen is enhanced by 10 mM Ca2+, and inhibited by thapsigargin, suggesting that AtECA3 supports tube elongation by sorting intracellular Ca2+ to appropriate compartments. This idea is supported by genetic evidence, where three T-DNA insertional mutants show 33% reduction in pollen tube length. This defect lowers sperm transmission shown as segregation distortion and decreased seed set. AtECA3 is also expressed in vascular tissues of young roots and leaves, and the GFP-tagged protein colocalizes with two Golgi markers. Three millimolar Ca2+ stimulate root growth of wild-type but not of mutants, indicating that Ca2+ accumulation in Golgi lumen is critical for growth. Root growth of eca3-4, but not of wild-type, is hypersensitive to 50 μM Mn2+. Thus loading Mn2+ into Golgi lumen by AtECA3 supports root growth. Intriguingly, mutant roots show 80% increase in apoplastic peroxidase suggesting that secretory activities became deregulated. In conclusion, I provide molecular evidence for a Golgi Ca2+/Mn2+ pump in plants. Ca2+ and Mn2+ accumulation into Golgi/secretory compartments by AtECA3 and perhaps cation release from these stores affect secretory activities critical for root growth, pollen tube elongation and male fertility.Item New Insight into Isoprenoid Biosynthesis in the Cyanobacterium Synechocystis sp. strain PCC 6803(2006-04-19) Poliquin, Kelly Ann; Gantt, Elisabeth; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In cyanobacteria, many compounds including chlorophylls, carotenoids, and quinones are synthesized from the isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These Csub5 compounds are products of the well-studied methylerythritol phosphate (MEP) pathway found in cyanobacteria, plant plastids, and many bacteria. Previous studies suggest that isoprenoid biosynthesis via the MEP pathway in the cyanobacterium Synechocystis sp. strain PCC 6803 are more complex than those proposed for a model bacterium. Most notably, in vitro isoprenoid biosynthesis in Synechocystis is stimulated by compounds of the pentose phosphate cycle (PPC) and not by intermediates of the MEP pathway. Isoprenoid biosynthesis in Synechocystis was therefore further investigated by disrupting sll1556, a gene distantly related to type 2 IPP isomerase genes. This gene is not essential under optimal photosynthetic conditions (20 µmol photons/m²/s). IPP isomerase activity could not be shown for the purified protein. Whereas in vitro PPC substrate stimulated isoprenoid biosynthesis could not be demonstrated in delta sll1556 cell-free extracts, it was restorable upon addition of the recombinant Sll1556 protein. PPC-stimulated isoprenoid biosynthesis results in a progression of isoprenoid production (Csub5 to Csub10 to Csub20) in vitro, although PPC compounds were not found to serve as direct substrates. Isoprenoid synthesis activity was unaffected when LytB, the terminal enzyme of the MEP pathway responsible for the production of both IPP and DMAPP, was immunodepleted from the cell-free extract, suggesting LytB activity is not likely to contribute to the observed in vitro isoprenoid synthesis. The physiological importance of Sll1556 was revealed at high light (200 µmol photons/m²/s). High light stress in the delta sll1556 mutant is evident by slower cell growth, a decrease in chlorophyll and carotenoid content, and in fewer thylakoids per cell. Myxoxanthophyll, but not zeaxanthin, increased in high light cells. The exact function of Sll1556 remains to be elucidated, but the combined results are consistent with a role in isoprenoid biosynthesis that is particularly important under high light stress. The mechanism by which Sll1556 is involved in PPC-stimulated isoprenoid synthesis is discussed, as are future areas of exploration for research.Item The role of auxin on the evolution of embryo development and axis formation in land plants(2005-03-10) Poli, DorothyBelle; Cooke, Todd J.; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)ABSTRACT Title of Dissertation: THE ROLE OF AUXIN ON THE EVOLUTION OF EMBRYO DEVELOPMENT AND AXIS FORMATION IN LAND PLANTS DorothyBelle Poli, Doctor of Philosophy, 2005 Dissertation directed by: Professor Todd J. Cooke, Cell Biology and Molecular Genetics This thesis examined the role of auxin in the evolution of land plants. Several approaches were used to study how auxin regulates the development in the bryophyte sporophytes. The altered growth of isolated young sporophytes exposed to applied auxin (indole-3-acetic acid) or an auxin antagonist (p-chlorophenoxyisobutyric acid) suggested that endogenous auxin regulates the rates of axial growth in all bryophyte divisions. In the hornwort Phaeoceros personii, auxin moved at very low fluxes, was insensitive to an auxin-transport inhibitor (N-[1-naphthyl]phthalamic acid), and exhibited a polarity ratio close to 1.0, implying that auxin moves by simple diffusion. The liverwort Pellia epiphylla exhibited somewhat higher auxin fluxes, which were sensitive to transport inhibitors but lacked any measurable polarity. Thus, auxin movement in liverwort sporophytes appears to result from facilitated diffusion. In the moss Polytrichum ohioensis, auxin movement was predominantly basipetal in young sporophytes and occurred at high fluxes exceeding those measured in maize coleoptiles. In older sporophytes, acropetal auxin flux had increased beyond the level measured for basipetal flux in the specimens observed in several, but not all, seasons. The evidence from both inhibitor treatments and isolated tissues is consistent with the interpretation that the cortex carries out basipetal transport in both younger and older sporophytes, whereas the central vascular tissues carries out basipetal transport in younger sporophytes and acropetal flux in older sporophytes. Given the significant differences in fall rainfall in the collection years, the purported sensitivity of vascular tissue development may account for the seasonal variation observed in these experiments. Auxin regulators and polar transport were also used to study the regulation of the embryogenesis of the fern Marsilea vestita. Auxin biosynthesis inhibitors affected initial cell proliferation resulting in the formation of aborted embryos, p-chlorophenoxyisobutyric acid delayed growth and development in all stages of embryogenesis while -naphthaleneacetic acid mediated rapid cell proliferation that caused enlarged disorganized embryos. Polar auxin transport inhibitors caused no significant abnormalities, which suggested a limited role for polar transport in fern embryogenesis. In conclusion, this evidence suggests that auxin is ultimately involved in the establishment of the body plans in all land plant sporophytes.Item Upstream Events In Ethylene Signal Transduction In Arabidopsis(2004-11-23) Shockey, Jason Alan; Chang, Caren; Molecular and Cell Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Ethylene gas has profound effects on the growth and development of higher plants. The understanding of how plants can sense this gas, and react in the appropriate manner is important for both agricultural purposes as well as the basic understanding of plant biology. While many components of this signaling pathway have been identified using classical genetics, we have little understanding of how these components work together. My work has focused on the understanding of early events in ethylene signal transduction. The interaction between the ETR1 ethylene receptor and the CTR1 Raf-like kinase was the first clue that the ethylene signaling pathway diverged from that of the yeast HOG1 osmo-sensing pathway. In this thesis, I examined the functional relevance of this interaction in the regulation of CTR1's activity. My work suggests that although CTR1 demonstrates the novel interaction with two-component receptors, the biochemical regulation of CTR1 may be similar to that of Raf1. Recent studies have suggested that histidine kinase activity of ETR1 may not play a major role in ethylene signal transduction, despite the remarkable degree of sequence conservation with functional histidine kinases from bacteria and yeast. In order to better understand the role of this highly conserved domain, either in ethylene signaling or other possible functions, I utilized biochemical assays, protein interaction studies and transgenic plants. My work indicates that phospho-relay plays no observable role in most ethylene responses, but plays an important role in recovery from ethylene treatment. Important members of this signaling system may yet be unidentified. A gene previously identified in the Chang lab, D2, was shown to have a probable role as a scaffolding protein in ethylene signaling using multiple reverse genetic techniques. This gene is unique to plants and cyanobacteria, as is the ethylene binding fold suggesting the two may have evolved together. The emerging paradigm of the ethylene signaling system reveals the pathway to be much more complex than originally thought.Item FUNCTIONAL ANALYSES OF ARABIDOPSIS RIBONUCLEOTIDE REDUCTASE SMALL SUBUNIT GENE FAMILY(2004-11-23) Wang, Chunxin; Liu, Zhongchi; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)A fundamental question in plant development is how cell division events are coordinately regulated in the context of growth and development. To address this question, I chose to study a pleiotropic mutant, tso2, which exhibited developmental defects including callus-like floral organs and fasciated shoot meristem. I isolated the TSO2 gene and showed it encodes the small subunit of ribonucleotide reductase (RNR). RNR catalyzes a rate-limiting step in the production of deoxyribonucleotides needed for DNA synthesis. Subsequently, I showed that tso2 mutants reduce the dNTP levels. To understand why tso2 mutants, defective in this essential process, are still viable, I identified two homologs of TSO2, R2A and R2B in Arabidopsis. Mutations in R2A and R2B were isolated using a reverse genetic approach. While r2a, r2b single mutants or r2a r2b double mutants fail to display any visible phenotype, r2a and r2b mutations can enhance tso2 genetically, resulting in seedling lethality and embryonic lethality in tso2 r2a and tso2 r2b, respectively. Overexpression of either R2A or R2B can rescue the tso2 mutants, suggesting that the three R2 genes are functionally redundant. In addition to the developmental defects, tso2 mutants were more sensitive to HU (hydroxyurea) and UV-C, indicating that TSO2 plays a major role in DNA repair. In tso2 r2a double mutant seedlings, increased DNA damage accumulates, leading to massive programmed cell death. In addition, release of transcriptional gene silencing was observed in tso2 r2a double mutants, suggesting that DNA damage can lead to epigenetic instability. To further identify regulators of RNR and novel components of plant DNA damage response pathways, 18 independent tso2 suppressors were isolated in a genetic screen. These suppressors fall into at least four different complementation groups. My genetic and molecular characterization of TSO2 is the first functional study of RNR in plants. My results indicated that plants could initiate programmed cell death in response to DNA damage. The developmental defects in tso2 mutants are caused by epigenetic instability and aberrant cell division. The isolation of potential tso2 suppressors will be crucial to the understanding of plant DNA damage response pathway, an understudied area in plant biology.