published_or_final_version / Botany / Master / Master of Philosophy

Thesis (Ph.D.)--Michigan State University. Biochemistry and Molecular Biology, 2008. / Title from PDF t.p. (viewed on July 7, 2009) Includes bibliographical references. Also issued in print.

Thesis (M.S.)--Bowling Green State University, 2009. / Document formatted into pages; contains x, 46 p. Includes bibliographical references.

Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

M.Sc. / Lipopolysaccharides (LPS) are ubiquitous, indispensable components of the cell surface of Gram-negative bacteria that have diverse roles in bacterial pathogenesis of plants. LPS as pathogen-associated molecular pattern (PAMP) molecules can be recognized by plants to directly trigger some defense—related responses. LPS can also alter the response of plants to subsequent bacterial inoculation; these delayed effects include alterations in the expression patterns of genes coding for some pathogenesis related (PR) proteins, promotion of the synthesis of the antimicrobial conjugates, and prevention of the hypersensitive reaction caused by avirulent bacteria. Prevention of the response may allow expression of resistance in the absence of catastrophic tissue damage. LPS from Burkholderia cepacia (LPSB. cep.) have been found to trigger a strong response in plants resulting in the activation of genes coding for some pathogenesis related proteins, receptor-like kinases and resistance (R) proteins. LPS are tripartite amphipathic molecules, consisting of a Lipid A moiety that is embedded in the outer leaflet of the phospholipids/protein bilayer, a core oligosaccharide, and a polysaccharide consisting of repeating units (0-Antigen/O-side chain). Typically the Lipid A consists of a bisphosphorylated glucosamine disaccharide which is substituted by amide- and ester-bound fatty acids and / or acyloxyacyl groups. The core region, a non-repetitive oligosaccharide, is usually connected to the Lipid A part via one 3-deoxy-D-manno-oct-2-ulosonic (Kdo) residue. The core is attached in turn to the 0- Antigen that consists in most cases of a repetitive polysaccharide and that represents the major part of LPS. The bond between the Lipid A section and the Kdo residue of the core is labile under mild acid hydroysis conditions; and this allows for the fractionation of the LPS molecule into a Lipid A part and an 0-Antigen part, attached to the core. Thus far the eliciting (active) parts of LPSB. cep. have not yet been identified. In general, it is known that the Lipid A is more conserved from one organism to another as compared to the 0-Antigen. In animals, Lipid A is believed to be the active part as it was found to elicit some defense-related responses. In plants, Lipid A was also found to trigger defense responses. Several structures of the 0-Antigens from different bacteria have been characterised, but their biological activities have not yet been investigated in detail.

M.Sc. (Biochemistry) / A 14-member, closely-spaced cluster of genes coding for leucine-rich repeat receptor-like kinases (LRR-RLKs) is located on chromosome 1 of Arabidopsis thaliana. Following on from previous microarray studies that found some of the members of this cluster to be upregulated in response to biotic stressors, including the bacterial elicitor flg22, the present study sought to confirm, using a luciferase-based protoplast assay, that flg22 does in fact induce the expression of the genes, and then to investigate the promoters of the genes. The promoters of At1g51790, At1g51850 and At1g51890 responded positively in this particular assay, and bioinformatic analyses determined that W-boxes are over-represented in the cloned regions. Mutational inactivation of individual W-boxes in the promoter of At1g51790 drastically reduced the flg22 response, except for the W-box closest to the start site, which seemed to increase both basal and flg22-inducible expression. In the promoter of At1g51850, mutational inactivation of either or both of its W-box dyads resulted in virtually no flg22 inducibility. The deletion of 6 W-boxes in the promoter of At1g51890, done via truncation, drastically reduced both its basal expression and its inducible response to flg22. These results provide evidence that W-box cis-elements are responsible for the upregulation of these LRR-RLKs in response to flg22. WRKYs -7, -11, -22,and -26 were found bioinformatically to have similar expression patterns to some of the genes in the cluster, and are thus good candidates to investigate as transcriptional regulators of the cluster in future studies.

D.Phil. (Biochemistry) / This thesis concerns a study of the effect of isonitrosoacetophenone on plant metabolism. Three different systems were investigated; cultured tobacco and sorghum cells as well as Arabidopsis thaliana plants, and a metabolomic approach was followed. Unlike most scientific studies, metabolomics is a discipline which is not driven by a specific hypothesis, but rather by the obtained data to add scientific insights to the topic under investigation. As such, the current study lacks a definite overarching hypothesis, but specific objectives were outlined and answered in each experimental chapter. This thesis is therefore presented as a compilation of nine chapters in which experimental/research work is described in Chapter 3- 8. It is important to note that each chapter is presented in accordance with the guidelines for the respective journal in which the corresponding manuscript was published or submitted to.

Because plants lack a circulating adaptive immune system, they have evolved multicomponent defense mechanisms to protect themselves against pathogen attack. These defense mechanisms/responses are either constitutively active in the plant, or they are inducible by pathogens. Understanding of the plant response to pathogen attack has advanced rapidly in recent years. Bacterial and fungal pathogenicity factors have been isolated, and mechanisms that are utilized by the plant to recognize the pathogen and initiate a plethora of defense mechanisms have been identified. In contrast to the well-documented effects of LPS on mammalian cells, the effects of LPS on plant cells have been far less studied. The present study focused on the involvement of lipopolysaccharides (LPS) isolated from the outer cell wall of the Gram-negative bacteria, Burkholderia cepacia (strain ASP B 2D), and yeast elicitor (YE, a cell wall preparation from Saccharomyces cerevisiae) on the molecular mechanisms and components involved in signal transduction and defense-related responses in suspension cultured cells derived from tobacco plants (Nicotiana tabacum cv. Samsun). LPS was extracted, analyzed by denaturing electrophoresis and characterized with regard to 2-keto-3-deoxyoctonate (KDO) content, carbohydrate content, and protein content. The purified LPS and YE were found to trigger defense- and resistance-related responses in the tobacco cells. These responses included a rapid influx of Ca2+ into the cytoplasm of transgenic aequorin-transformed tobacco cells, the production of reactive oxygen species (ROS) during the oxidative burst, alkalinization of the extracellular culture medium of the cells, and changes in the protein phosphorylation patterns of the cells. Time- and concentration-dependent studies for the induction of perception and signal transduction-related responses by YE and LPS indicated that 100 µg.ml-1 of either elicitor was sufficient to induce significant responses in the cells. YE and LPS both induced a rapid transient increase in cytosolic Ca2+ levels, returning to basal levels after seconds, followed by a second, larger and long-term increase in cytosolic Ca2+. The YE-induced cytosolic Ca2+ influx was 7.5 fold higher than that of LPS. Luminol-dependent chemiluminescence measurements of hydrogen peroxide (H2O2) produced during the YE- and LPS-induced oxidative burst reactions indicated 3.5 fold higher levels of H2O2 induced by YE than that induced by LPS. Total inhibition of H2O2 production by YE- and LPS-induced cells was observed upon treatment of the cells with the H2O2-degrading enzyme, catalase. ROS production was also analyzed by the H2DCF-DA-derived fluorescence assay. The degree of ROS production by YE-treated cells was larger than that of cells treated with LPS, suggesting that YE is a more potent inducer of plant defense responses than LPS. Categorization of the origins of the oxidative bursts, induced by YE and LPS, by the addition of a ROS scavenger (NAC), inhibitors of ROS production (DPI and DDC) and a nitric oxide scavenger (PTIO) indicated that YE and LPS induced different quantities of the same ROS species. The induced ROS included O2-·, H2O2 and perhaps other ROS species as well. In addition, both YE and LPS induced a remarkable burst of nitric oxide (NO), as determined by the 97% and 95% respective inhibitions of the H2DCF-DA-derived fluorescence by the nitric oxide scavenger PTIO. Alkalinization of the extracellular culture medium of the tobacco cells was observed after treatment of the cells with YE and LPS. Both of these elicitors induced a significant increase in extracellular pH from resting pH values of 5.7 to pH 6.3 by YE, and 6.0 by LPS. Notably, the YE-induced response returned to near basal pH levels after 50 min, while the LPS-induced response showed no signs of declining and fluctuated around pH 5.9 for the duration of the experiment. YE and LPS both induced the hyperphosphorylation of two distinct proteins with approximate molecular masses of 28 kDa and 2 kDa. Changes in the pattern of the [32P]-radiolabeled proteins pp28 became visible after 20 min of YE-elicitation and 30 min of LPS-elicitation and changes in pp2 phosphorylation became visible after 20 min treatment of the cells with both elicitors. Addition of the protein kinase inhibitor, staurosporine, to the cells followed by subsequent elicitation by YE or LPS, resulted in inhibition or abolishment of the elicitor-induced responses during the oxidative burst, extracellular alkalinization, and protein phosphorylation. In contrast, the addition of the protein phosphatase inhibitor, calyculin A, was found to mimic elicitor action in several aspects, including extracellular alkalinization, the oxidative burst and protein phosphorylation, even in the absence of elicitors or any other stimulus. Thus, a fine balance between the actions of certain protein kinases and protein phosphatases is an essential component of signal transduction during YE and LPS elicitation of tobacco cells but the identification and characterization of the staurosporine-sensitive protein kinases and their substrates are necessary to gain a better understanding of the chemosensory perception and signal transduction of the YE and LPS elicitor signals in plant cells. Moreover, the question of whether these perception and transduction mechanisms are connected with a reduced activity of a protein phosphatase, or with the increased activity of a protein kinase, or even a combination of both, remains to be elucidated. / Prof. I.A. Dubery

M.Sc. (Biochemistry) / Our comprehension of pathogen perception and defense response mechanisms that play key roles in the resistance of plants against pathogen attack have progressed substantially within the recent years. Recognizing the molecular mechanisms involved in pathogen perception is the basis of understanding the signalling networks that are involved, including the transcriptional regulation of plant defense genes. This has proven to be a great challenge in plant pathology and, as such, has attracted much attention. The receptor-like kinases (RLKs) constitute one of the largest classes of plant defense genes in Arabidopsis thaliana, and contains, inter alia, the well-known leucine-rich repeats-RLKs (LRR-RLK), as well as the S-domain receptor-like kinases (SD-RLKs) that have been shown to be involved in pathogen perception and not only self-incompatibility (SI) as originally discovered. Some members of these RLKs are able to detect pattern-associated molecular patterns (PAMPs), which are conserved pathogen-derived molecules, and trigger a battery of basal defense responses. The transcriptional activation and expression levels of RLKs are dependent on the variation in promoter architecture as a result of the number, location, order and class of cis-elements found in a promoter sequence. It is hypothesized that candidate RLK genes involved in PAMP surveillance are triggered and transcriptionally regulated in response to perception of PAMPs, and that the intensity of response is relative to the promoter architecture. The primary objective was to identify SD-RLKs and LRR-RLKs which demonstrated up-regulation in response to PAMPs. The SD-RLKs (At1g11330, At1g61430 and At1g61610) and LRR-RLKs (At1g51850, At2g19190 and At5g45840) were selected on the basis of microarray data (Nürnberger - TAIR accession set 100808727) and the Genevestigator database, and characterized utilizing bioinformatics tools. Here, molecular techniques were used to show that the selected RLK genes were responsive to PAMP inductions. Furthermore, this study explored which cis-elements and their corresponding transcription factors (TFs) are found in the promoter of plant defense genes and that may be involved in transcriptional regulation thereof...

M.Sc. / Chemically-induced plant defense responses were investigated in tobacco cell cultures. The inducing conditions were as follows: chitosan (C), an elicitor (E) prepared from Phytophthora nicotianae, isonicotinic acid (INA), isonicotinamide (IND) and isonitrosoacetophenone (INAP) as well as the addition of INA, IND and INAP as conditioning agents (primary elicitors) followed by secondary elicitation with either chitosan or elicitor. The defense responses investigated included determinations of phenylalanine ammonia-lyase (PAL) activity, total soluble phenolic content, specific phenolic profiles, phytoalexin content, (3- 1,3-glucanase activity and electrophoretic analyses of pathogenesis-related proteins (PR). The compounds, 4-(3-methyl-2-butenoXy)isonitrosoacetophenone (0-INAP) and 2-isonitrosoacetophenone (INAP) were successfully synthesized from the starting materials p-hydroxyacetophenone and acetophenone respectively. The organic synthesis of 0-INAP involved the formation of a prenyl ether.of p-hydroxyacetophenone, followed by a nitrosation reaction using butyl nitrite as the source of the nitroso group, on the a-carbon atom adjacent to the carbonyl group. The synthesis of INAP only required a nitrosation reaction on the a-carbon atom adjacent to the carbonyl group. The yields of 0-INAP and INAP were 12 - 15 % and 80 %, respectively. An evaluation of the properties of 0-INAP indicated that the compound, dissolved in methanol, has a molar extinction coefficient of 16 5001.mor.cm - ' at A. 302 nm. The compound possesses antifungal activity against Cladosporium cucumerinum, Penicillium expansum and Aspergillus niger as well as the ability to scavenge superoxide radicals which was indicated by a decrease in the chemiluminescence signal produced in a reaction mbdure of hydrogen peroxide, horseradish peroxidase, the chemiluminescence probe, MCLA, and increasing concentrations of 0-INAP. The addition of INA to tobacco cells at a - final concentration of 12.5 iimol.g -1 cells or 2.5 mM did not lead to significant changes in PAL activity, but conditioning with INA, followed by chitosan as well as elicitor led to a 2.5-fold and a 4.3-fold induction respectively. INA as well as INA + C and INA + E led to significant increases in the total soluble phenolic content, and the HPLC analyses of these phenolics indicated the significant induction of a phenolic-like compound with a peak at Rt = 1.7 min. which possibly indicates isonicotinic acid, for INA + C and INA + E. A whole range of phytoalexins were detectable after the addition of INA to tobacco cells and conditioning with INA followed by chitosan induced the phytoalexin, lubimin, several hundred-fold. PR proteins were also induced by INA and a prominent band of 11- 13 kDa was induced after conditioning with INA, followed by secondary elicitation with the elicitor and especially with chitosan. (3-1,3-glucanase activity was also induced by INA; INA + E and particularly INA + C led to increases of 2.5-fold and 4.5-fold in 13-1,3-glucanase activity respectively. The addition of IND to tobacco cells at a final concentration of 12.5 pmol.g -1 cells or 2.5 mM led to a 2.6-fold induction in PAL activity after only 6 h, but conditioning with IND, followed by secondary elicitation did not lead to any significant changes. IND at the earlier time interval (24 h vs. 48 h) as well as IND + C and IND + E led to increases in the total soluble phenolic content, - and the HPLC analyses of these phenolics indicated the significant induction of a phenolic-like compound with ,a peak at Rt = 1.7 min. which possibly indicates isonicotinic acid, for IND + C and IND + E. A whole range of phytoalexins were detectable after the addition of IND to tobacco cells and conditioning with IND followed by chitosan induced the phytoalexin, solavetinone, several hundred-fold. PR proteins were also induced by IND and prominent bands of 34 kDa and 39 - 40 kDa were induced for IND + ELIC. (3-1,3-glucanase activity .was also induced by IND; however, secondary elicitation with chitosan did not lead to increases in enzyme activity, although a twofold increase was detectable for IND + ELIC, compared to IND 72. The addition of INAP to tobacco cells at a final concentration of 6.3 pmol.e cells or 1.25 mM led to a 1.7-fold induction in PAL activity after only 6 h, a response that was still detectable after 30 h; however, conditioning with INAP, followed by secondary elicitation did not lead to any noteworthy changes. INAP 24 as well as INAP 48 did not lead to significant changesin the total soluble phenolic content, but INAP + C and INAP+ E led to increases of 3.3-fold and 3.5-fold, respectively. HPLC analyses of the induced phenolics showed the significant induCtion of a phenolic compound with a peak at Rt = 14.5 min. which possibly indicate p-coumaric acid, for INAP + C and INAP + E. A whole range.of phytoalexins were detectable after the addition of INAP to tobacco cells, but the addition of a secondary elicitor led to a decrease in phytoalexin accumulation. PR proteins were also induced by INAP and conditioning with INAP, followed by especially the elicitor, led to the induction of a whole range of PR proteins with molecular masses ranging from 11 - 68 kDa. (3-1,3-glucanase activity was significantly induced (60-fold compared to control) by INAP 48; however, secondary elicitation led to a decrease in (3-1,3-glucanase)