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Browsing by Author "Ramonell, Katrina M."

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    Arabidopsis Toxicos en Levadura 12 (ATL12): A Gene Involved in Chitin-Induced, Hormone-Related and NADPH Oxidase-Mediated Defense Responses
    (MDPI, 2021) Kong, Feng; Guo, Tingwei; Ramonell, Katrina M.; University of Alabama Tuscaloosa; University of Southern California
    Plants, as sessile organisms, have evolved complex systems to respond to changes in environmental conditions. Chitin is a Pathogen-Associated-Molecular Pattern (PAMP) that exists in the fungal cell walls, and can be recognized by plants and induce plant pattern-triggered immunity (PTI). Our previous studies showed that Arabidopsis Toxicos en Levadura 12 (ATL12) is highly induced in response to fungal infection and chitin treatment. We used the model organism Arabidopsis thaliana to characterize ATL12 and explore its role in fungal defense. Histochemical staining showed that pATL12-GUS was continually expressed in roots, leaves, stems, and flowers. Subcellular co-localization of the ATL12-GFP fusion protein with the plasma membrane-mcherry marker showed that ATL12 localizes to the plasma membrane. Mutants of atl12 are more susceptible to Golovinomyces cichoracearum infection, while overexpression of ATL12 increased plant resistance to the fungus. ATL12 is highly induced by chitin after two hours of treatment and ATL12 may act downstream of MAPK cascades. Additionally, 3,3 '-diaminobenzidine (DAB) staining indicated that atl12 mutants generate less reactive oxygen species compared to wild-type Col-0 plants and RT-PCR indicated that ATL12-regulated ROS production may be linked to the expression of respiratory burst oxidase homolog protein D/F (AtRBOHD/F). Furthermore, we present evidence that ATL12 expression is upregulated after treatment with both salicylic acid and jasmonic acid. Taken together, these results suggest a role for ATL12 in crosstalk between hormonal, chitin-induced, and NADPH oxidase-mediated defense responses in Arabidopsis.
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    Arabidopsis Toxicos en Levadura 12 Modulates Salt Stress and ABA Responses in Arabidopsis thaliana
    (MDPI, 2022) Kong, Feng; Ramonell, Katrina M.; University of Alabama Tuscaloosa; University of Georgia
    Salt is one of the most common abiotic stresses, causing ionic and osmotic pressure changes that affect plant growth and development. In this work, we present molecular and genetic evidence that Arabidopsis Toxicos en Levadura 12 (ATL12) is involved in both salt stress and in the abscisic acid response to this stress. We demonstrate that ATL12 is highly induced in response to salt stress and that atl12 mutants have a lower germination rate, decreased root length, and lower survival rate compared to the Col-0 wild-type in response to salt stress. Overexpression of ATL12 increases expression of the salt stress-associated genes SOS1/2, and ABA-responsive gene RD29B. Additionally, higher levels of reactive oxygen species are detected when ATL12 is overexpressed, and qRT-PCR showed that ATL12 is involved in the AtRBOHD/F-mediated signaling. ATL12 expression is also highly induced by ABA treatment. Mutants of atl12 are hypersensitive to ABA and have a shorter root length. A decrease in water loss and reduced stomatal aperture were also observed in atl12 mutants in response to ABA. ABA-responsive genes RD29B and RAB18 were downregulated in atl12 mutants but were upregulated in the overexpression line of ATL12 in response to ABA. Taken together our results suggest that ATL12 modulates the response to salt stress and is involved in the ABA signaling pathway in Arabidopsis thaliana.
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    Developmental and Reproductive Effects of Iron Oxide Nanoparticles in Arabidopsis thaliana
    (MDPI, 2015) Bombin, Sergey; LeFebvre, Mitchell; Sherwood, Jennifer; Xu, Yaolin; Bao, Yuping; Ramonell, Katrina M.; University of Alabama Tuscaloosa
    Increasing use of iron oxide nanoparticles in medicine and environmental remediation has led to concerns regarding exposure of these nanoparticles to the public. However, limited studies are available to evaluate their effects on the environment, in particular on plants and food crops. Here, we investigated the effects of positive (PC) and negative (NC) charged iron oxide (Fe2O3) nanoparticles (IONPs) on the physiology and reproductive capacity of Arabidopsis thaliana at concentrations of 3 and 25 mg/L. The 3 mg/L treated plants did not show evident effects on seeding and root length. However, the 25 mg/L treatment resulted in reduced seedling (positive-20% and negative-3.6%) and root (positive-48% and negative-negligible) length. Interestingly, treatment with polyethylenimine (PEI; IONP-PC coating) also resulted in reduced root length (39%) but no change was observed with polyacrylic acid (PAA; IONP-NC coating) treatment alone. However, treatment with IONPs at 3 mg/L did lead to an almost 5% increase in aborted pollen, a 2%-6% reduction in pollen viability and up to an 11% reduction in seed yield depending on the number of treatments. Interestingly, the treated plants did not show any observable phenotypic changes in overall size or general plant structure, indicating that environmental nanoparticle contamination could go dangerously unnoticed.
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    The E3 Ubiquitin Ligase ATL9 Affects Expression of Defense Related Genes, Cell Death and Callose Deposition in Response to Fungal Infection
    (MDPI, 2022) Guo, Tingwei; Kong, Feng; Burton, Carter; Scaglione, Steven; Beagles, Blake; Ray, Justin; Ramonell, Katrina M.; University of Alabama Tuscaloosa; University of Southern California
    Plants use diverse strategies to defend themselves from biotic stresses in nature, which include the activation of defense gene expression and a variety of signal transduction pathways. Previous studies have shown that protein ubiquitination plays a critical role in plant defense responses, however the details of its function remain unclear. Our previous work has shown that increasing expression levels of ATL9, an E3 ubiquitin ligase in Arabidopsis thaliana, increased resistance to infection by the fungal pathogen, Golovinomyces cichoracearum. In this study, we demonstrate that the defense-related proteins PDF1.2, PCC1 and FBS1 directly interact with ATL9 and are targeted for degradation to the proteasome by ATL9. The expression levels of PDF1.2, PCC1 and FBS1 are decreased in T-DNA insertional mutants of atl9 and T-DNA insertional mutants of pdf1.2, pcc1 and fbs1 are more susceptible to fungal infection. In addition, callose is more heavily deposited at infection sites in the mutants of atl9, fbs1, pcc1 and pdf1.2. Overexpression of ATL9 and of mutants in fbs1, pcc1 and pdf1.2 showed increased levels of cell death during infection. Together these results indicate that ubiquitination, cell death and callose deposition may work together to enhance defense responses to fungal pathogens.
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    ESI-MS and EPR spin trapping study of xanthophylls: structural and environmental impacts on reactivity
    (University of Alabama Libraries, 2015) Magyar, Adam S.; Kispert, Lowell D.; Bowman, Michael K.; University of Alabama Tuscaloosa
    Carotenoids are lipophilic pigments that provide many of the colors found in nature, including the colors found in plants, flowers, and animals. The main interest in carotenoids concerns their participation in light harvesting in biological systems and prevention of light-induced oxidative damage. Another reason for the interest in the redox properties of carotenoids is related to their use as antioxidants in medicinal formulations as a result of radical-mediated processes that occur frequently in living systems. A specific group of carotenoids, known as xanthophylls, are active in photoprotection and radical scavenging. What makes xanthophylls differ from other carotenoids is that they contain oxygen. This dissertation focuses on the interaction of three xanthophylls, Zeaxanthin, Lutein, and Astaxanthin, with damaging energetic species. Chapter 3 is focused on the deprotonation of naturally-occurring zeaxanthin (Zea) radical cations (Zea*+) to form neutral radicals (#Zea*) and their involvement in the qE portion of non-photochemical quenching (NPQ) in the model organism Arabidopsis thaliana. An H/D exchange method was developed to test for the presence of #Zea* and was detected via liquid chromatography/mass spectrometry (LC/MS) methods and analyzed using analysis of variance (ANOVA) techniques. Chapter 4 examined the characteristics of xanthophyll carotenoids which self-assemble in aqueous solution to form J- and H-type aggregates. This aggregation significantly changes the photo-physical and optical properties of these xanthophylls, and has an impact on solar energy conversion and light induced oxidative damage. This study applied electron paramagnetic resonance (EPR) and optical absorption spectroscopy to investigate how complexation can affect the aggregation ability of the three xanthophylls mentioned above, their photostability, and antioxidant activity. Chapter 5 demonstrates the different chemistries of geometrical isomers of zeaxanthin isolated from various sources in mass spectrometry using electrospray ionization sources. Zeaxanthin exhibits antioxidant activity and also plays a role in photo-protection in the retina. These properties have led to it being marketed as a dietary supplement as it is not formed by the body and must be consumed as part of the diet. Understanding the different chemical properties of zeaxanthin isomers is important with regards to the nutraceutical and pharmaceutical industries.
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    Expression and regulation of ATL9, an E3 ubiquitin ligase involved in plant defense
    (PLOS, 2017) Deng, Fengyan; Guo, Tingwei; Lefebvre, Mitchell; Scaglione, Steven; Antico, Christopher J.; Jing, Tao; Yang, Xin; Shan, Weixing; Ramonell, Katrina M.; University of Alabama Tuscaloosa; Northwest A&F University - China
    Plants are continually exposed to a variety of pathogenic organisms, including bacteria, fungi and viruses. In response to these assaults, plants have developed various defense pathways to protect themselves from pathogen invasion. An understanding of the expression and regulation of genes involved in defense signaling is essential to controlling plant disease. ATL9, an Arabidopsis RING zinc finger protein, is an E3 ubiquitin ligase that is induced by chitin and involved in basal resistance to the biotrophic fungal pathogen, Golovi-nomyces cichoracearum (G. cichoracearum). To better understand the expression and regulation of ATL9, we studied its expression pattern and the functions of its different protein domains. Using p(ATL9):GUS transgenic Arabidopsis lines we found that ATL9 is expressed in numerous tissues at various developmental stages and that GUS activity was induced rapidly upon wounding. Using a GFP control protein, we showed that ATL9 is a short-lived protein within plant cells and it is degraded via the ubiquitin-proteasome pathway. ATL9 contains two transmembrane domains (TM),a RING zinc-finger domain, and a PEST domain. Using a series of deletion mutants, we found that the PEST domain and the RING domain have effects on ATL9 degradation. Further infection assays with G. cichoracearum showed that both the RING domain and the TM domains are important for ATL9's resistance phenotype. Interestingly, the PEST domain was also shown to be significant for resistance to fungal pathogens. This study demonstrates that the PEST domain is directly coupled to plant defense regulation and the importance of protein degradation in plant immunity.
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    Mass Spectrometry Studies on the Dissociation of Metal-Adducted Oligosaccharides, Glycopeptides, and Fatty Acids
    (University of Alabama Libraries, 2024) Akor, Chioma Jane-Frances; Cassady, Carolyn J.; Szulczewski, Gregory J.
    Analyzing oligosaccharides, glycopeptides, and fatty acids poses a significant analytical challenge due to their structural complexity and diversity. Structural alterations in these biomolecules have direct implications for health and diseases, necessitating the development of analytical methods capable of providing comprehensive structural information. Mass spectrometry (MS) stands out as a leading technique for this purpose, although its efficacy is hindered by poor ionization and dissociation of these biomolecules. Consequently, derivatization is commonly employed to enhance ionization and dissociation efficiency.This dissertation focuses on exploring the use of in-source decay (ISD) and high-energy collision-induced dissociation (HE-CID) performed on a matrix-assisted laser desorption ionization (MALDI)/tandem time-of-flight (TOF-TOF) mass spectrometer to obtain complete structural information on underivatized oligosaccharides, glycopeptides, and fatty acids. Extensive experimentation with various MALDI matrices and metal cations led to the identification of optimal conditions for ionization and dissociation.Dissociation of the metal-adducted underivatized biomolecules by HE-CID generated extensive fragmentation and structural information not attainable through dissociation of the protonated biomolecule, or collision-induced dissociation (CID) performed on quadrupole-based mass spectrometers. The glycosidic bond cleavage, cross-ring cleavage, and internal cleavage ions generated by HE-CID of metal-adducted oligosaccharides and glycopeptides provided valuable information on sequence, linkage type, and monosaccharide ring content. Also, HE-CID of the structural isomeric tetrasaccharides studied produced distinct mass spectra and product ions necessary to distinguish between the isomers. In addition, HE-CID of metal-adducted unsaturated fatty acids facilitated the determination of double bond positions, distinguished double bond positional isomers, and cis/trans isomers. While ISD proved less useful for fatty acids and glycopeptides, ISD complemented HE-CID in providing structural information for metal-adducted oligosaccharides.Remarkably, derivatization of oligosaccharides through permethylation was unnecessary for obtaining good ionization and structural information. Metal ions, particularly lithium (Li+), significantly enhanced the ionization and structural characterization of oligosaccharides, glycopeptides, and fatty acids. The choice of metal ions influenced the type and intensity of product ions, with lithium (Li+) consistently producing the most structural information. Furthermore, trihydroxyacetophenone (THAP) emerged as the optimal MALDI matrix for ionization and HE-CID, 2,5-dihydroxybenzoic acid (DHB) for ISD and HE-CID of long-chain oligosaccharides, and 4-dimethylaminobenzaldehyde (DMABA) for ISD of tetrasaccharides and pentasaccharides.