Cell Biology & Molecular Genetics Theses and Dissertations
Permanent URI for this collectionhttp://hdl.handle.net/1903/2750
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Item Probing the 3D Structure and Function of a Cation/H+ Exchanger in Plant Reproduction(2015) Czerny, Daniel; Sze, Heven; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Maintaining intracellular pH and K+ homeostasis are necessary for a cell to divide, grow, and communicate with other cell types. How a cell responds to stimuli and subsequently regulates intracellular pH and K+ content are largely unknown. Ion transporters, including cation/H+ exchangers are one potential determinant of intracellular pH and K+ content. A novel family of CHX transporters, predicted to exchange a cation for a H+, is found in all land plants, though their functions in the plant and the mode of transport are mostly unknown. What is the mode of transport of Arabidopsis thaliana CHX17? Model structures of the CHX17 transmembrane domain were built from two crystallized bacterial Na+/H+ antiporters. Based on protein architecture and homology, residues were selected for mutagenesis and CHX17 activity was tested in yeast. Thr170 and Lys383 in the discontinuous α-helices of transmembrane 4 and 11, and Asp201 and Lys355 in the middle of transmembranes 5 and 10 are necessary for CHX17 activity. Results suggest these are core residues that participate in cation binding and/or catalysis. Glu111 near the cytosolic surface of CHX17 was necessary for activity, suggesting CHX17 could be regulated by cytosolic pH. Thus the protein fold and mode of transport of Arabidopsis CHX17 resemble a K+/H+ exchanger. What roles do K+/H+ exchangers play in plant reproduction? chx17/18/19 mutant plants showed a 56%-77% reduction in seed set though the biological basis was unknown. Reciprocal crosses showed reduced seed set was primarily caused by defects in the male gametophyte. Mutant chx17/18/19 pollen grains developed normally and pollen tubes grew and reached most ovules. However, half the ovules receiving a mutant pollen tube failed to develop. Wild-type pistils that received chx17/18/19 pollen showed unfertilized ovules, ovules with single fertilizations, and some embryos that developed similarly to wild-type. Thus, some triple mutant pollen showed failure to complete fertilization. When fertilization was successful, embryos from self-fertilized chx17/18/19 pods showed delays in development. Our findings suggest maintenance of pH and K+ homeostasis in endomembrane compartments by CHX17 and its homologs could regulate membrane trafficking events necessary for pollen tube growth, male gamete fusion, and embryo development.Item Molecular and Genetic Analysis of Flower Development in Arabidopsis thaliana and the Diploid Strawberry, Fragaria vesca(2012) Hollender, Courtney Allison; Liu, Zhongchi; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)In a world with a warming climate and a rapidly growing population, plant biology is becoming a field of increasing importance. Deciphering the molecular and genetic mechanisms behind the development of the flower, the fruit and seed progenitor, will enhance the agricultural productivity needed to ensure a sustainable food supply. My PhD research ties in with this need by furthering the basic knowledge of the mechanisms underlying flower development in two ways. First, using Arabidopsis thaliana, the classic model plant, I investigated the regulation of a gene, SPATULA (SPT), necessary for the proper development of the gynoecium, the female flower organ that, upon fertilization, directly gives rise to fruit. For flower and fruit to properly develop, the expression of SPT, must be tightly regulated both spatially and temporally. My research examined the mechanism of transcriptional repression of SPT in the sepals and petals by several interacting transcription factors (LEUNIG, SEUSS, APETALA2) and the molecular and genetic interaction between ETTIN and SPT in patterning gynoecium. The second focus of my research was to develop Fragaria vesca (the diploid strawberry), as a model Rosaceae for the study of flower and fruit development. Arabidopsis has much value as a small, fast growing, flowering plant with a multitude of genetic and genomic resources, however the flower of this mustard family weed is not representative of all crop flowers. The Rosaceae family, including many agriculturally important fruit trees such as apple, peach, blackberry, and strawberry, warrants its own model plant to investigate the distinct mechanisms behind their unique reproductive biology. Toward developing F. vesca as the model plant for studying Rosaceae flowers, I characterized and described developmental progression of F. vesca flowers morphologically through scanning electron microscopy and histological analysis as well as molecularly through transcriptomes and in situ hybridization. In addition, I pioneered a small-scale mutagenesis screen of F. vesca that will lead to future genetic resources. My thesis work places the groundwork for future discoveries in F. vesca and Rosaceae and benefits research, education, and agricultural applications for the Rosaceae and the plant biology communities.Item Investigating the molecular mechanism of RTE1 activation of the ethylene receptor ETR1 in Arabidopsis(2011) Chang, Jianhong; Chang, Caren; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The plant hormone ethylene plays a vital role in regulating plant growth and development as well as plant defense to biotic and abiotic stresses during the entire life of the plant. In Arabidopsis, ethylene is perceived by a family of five receptors, one of which is ETR1. The Arabidopsis REVERSION-TO-ETHYLENE SENSITIVITY1 (RTE1) gene is a positive regulator of ETR1. RTE1 encodes a novel integral membrane protein that interacts with ETR1 at the Golgi apparatus and the endoplasmic reticulum (ER). Genetic evidence indicates that RTE1 is required for the formation of a functional ETR1 receptor, whereas the other ethylene receptors in Arabidopsis do not require RTE1. But the molecular mechanism by which RTE1 specifically activates ETR1 remains unknown. I took different approaches to gain insights into the molecular function of RTE1 and the basis for the specificity for activating ETR1. In a library screen for RTE1–interacting proteins using the yeast split–ubiquitin assay, an ER–localized cytochrome b5 isoform (AtCb5–D) was identified. Cb5 is a small hemoprotein that functions in oxidation/reduction reactions. Mutants of three AtCb5 isoforms show phenotypes in ethylene responses that are similar to those of the rte1 mutant, suggesting the functional parallel between AtCb5 and RTE1 in ethylene signaling. Additional genetic analyses suggest that AtCb5 might act in the same pathway as RTE1 and that AtCb5 is specific to ETR1 like RTE1. Moreover, using a hemin–agarose affinity chromatography assay, I found that RTE1 homologs are able to bind heme in vitro, raising the possibility that RTE1 carries out redox with cytochrome b5s. I also found that the specificity for regulating ETR1 by RTE1 is largely due to a unique proline (P9) conserved only in ETR1 orthologs; introduction of P9 into the Arabidopsis ERS1 ethylene receptor was sufficient to convert ERS1 into an RTE1–dependent receptor. I propose that P9 may interfere with the proper folding of ETR1 EBD and formation of the ETR1 homodimer by affecting the conserved disulfide bond–forming cysteines (C4, C6) in the ETR1 homodimer. Taken together, our results suggest a model in which RTE1, together with cytochrome b5, promotes the active conformation of ETR1 through oxidative folding.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.