<p>The actin cytoskeleton is required for cell expansion and is implicated in cellular responses to the plant growth hormone auxin. However, the molecular and cellular mechanisms that coordinate auxin signaling, cytoskeletal remodeling, and cell expansion are poorly understood. Previous studies have examined actin cytoskeleton responses to long-term auxin treatment, but plants respond to auxin over short timeframes, and growth changes within minutes of exposure to the hormone. To correlate actin arrays with degree of cell expansion, we used quantitative imaging tools to establish a baseline of actin organization, as well as of individual filament behaviors in root epidermal cells under control conditions and after treatment with a known inhibitor of root growth, the auxin indole-3-acetic acid (IAA). We found that cell length was highly predictive of actin array in control roots, and that short-term IAA treatment stimulated denser, more longitudinal, and more parallel arrays by inducing filament unbundling within minutes. By demonstrating that actin filaments were more “organized” after a treatment that stopped elongation, we show there is no direct relationship between actin organization and cell expansion and refute the hypothesis that “more organized” actin universally correlates with more rapidly growing root cells. The plasma membrane-bound auxin transporter AUXIN RESISTANT 1 (AUX1) has previously been shown necessary for archetypal short-term root growth inhibition in the presence of IAA. Although AUX1 was not previously suspected of being upstream of cytoskeletal responses to IAA, we used <i>aux1</i>mutants to demonstrate that AUX1 is necessary for the full complement of actin rearrangements in response to auxin, and that cytoplasmic auxin in the form of the membrane permeable auxin 1‑naphthylacetic acid (NAA) is sufficient to stimulate a partial actin response. Together, these results are the first to quantitate actin cytoskeleton response to short-term auxin treatments and demonstrate that AUX1 is necessary for short-term actin remodeling.</p>

Cell-to-cell communication is the driving force behind successful reproduction in flowering plants. Extensive extracellular communication events occur between the male and female gametophytes during pollen tube reception to facilitate successful fertilization. These signaling events culminate into a product of great importance for both animals and plants: the seed. In this study, the pathogen defense regulator PATTERN-TRIGGERED IMMUNITY COMPROMISED RECEPTOR-LIKE CYTOPLASMIC KINASE 1 (PCRK1) was identified to function in pollen tube reception from both the male and female gametophytes in the flowering plant <i>Arabidopsis thaliana</i> using a forward genetic screen. A knockout of <i>pcrk1</i> suppresses the pollen tube overgrowth phenotype leading to infertility in <i>nortia</i> mutants. In addition, <i>pcrk1</i> pollen affected the pollen tube overgrowth phenotypes of pollen tube reception mutants <i>feronia</i> and <i>turan</i>. Shared molecular components of pollen tube reception and pathogen invasion have been reported. This study reveals another link between pathogen defense and pollen tube reception. By studying the links between fertility and disease in plants, we may be able to uncover potential trade-offs with fertility when breeding for pathogen resistance.<br>

<p dir="ltr">The actin cytoskeleton is a dynamic structure that regulates various important cellular processes, such as cell protrusion, migration, transport, and cell shape changes. Cells employ different actin architectures best suited for each of these functions. We have employed an agent-based model to illuminate how the actin cytoskeleton plays such functions in animal and plant cells, via dynamic interactions between molecular players.</p><p dir="ltr">Lamellipodia found in animal cells are two-dimensional actin protrusion formed on the leading edge of cells, playing an important role in sensing surrounding mechanical environments via focal adhesions. Various molecular players, architecture, and dynamics of the lamellipodia have been investigated extensively during recent decades. Nevertheless, it still remains elusive how each component in the lamellipodia mechanically interacts with each other to attain a stable, dynamic steady state characterized by a retrograde flow emerging in the branched actin network. Using the agent-based model, we investigated how the balance between different subcellular processes is achieved for the dynamic steady state. We simulated a branched network found in the lamellipodia, consisting of actin filament (F-actin), myosin motor, Arp2/3 complex, and actin crosslinking protein. We found the importance of a balance between F-actin assembly at the leading edge of cells and F-actin disassembly at the rear end of the lamellipodia. We also found that F-actin severing is crucial to allow for the proper disassembly of an actin bundle formed via network contraction induced by motor activity. In addition, it was found that various dynamic steady states can exist.</p><p dir="ltr">The actin cytoskeleton in plant cells plays a crucial role in intracellular transport and cytoplasmic streaming, and its structure is very different from the actin cytoskeleton in animal cells. The plant actin cytoskeleton is known to show distinct dynamic behaviors with homeostasis. We used the agent-based model to simulate the plant actin cytoskeleton with the consideration of the key governing mechanisms, including F-actin polymerization/depolymerization, different types of F-actin nucleation events, severing, and capping. We succeeded in reproducing experimental observations in terms of F-actin density, length, nucleation frequency, and rates of severing, polymerization, and depolymerization. We found that the removal of nucleators results in lower F-actin density in the network, which supports recent experimental findings.</p>

Infection of barley (Hordeum vulgare) leaves with the rust fungus, Puccinia hordei, causes changes in the host protein synthesis. This thesis analyses these changes in the barley cultivar Triumph following inoculation of 7-day-old leaves with either a virulent or an avirulent race of P. hordei. The initial approach was to isolate membrane-bound polysomes from infected leaves, translate them in vitro and analyse the translation products. These products include the integral membrane proteins which were expected to be involved in the response of the host to the pathogen. A method based on differential centrifugation in the presence of a ribonuclease-inhibiting buffer was developed for separating membrane-bound polysomes from the rest of the cytoplasmic polysomes. Membrane-bound polysomes were found to comprise one fifth of the total polysomes in the leaves. Analysis of the translation products of membrane-bound polysomes by SDS-PAGE showed them to be of higher average molecular weight than those from free polysomes. Comparison of polypeptides produced by membrane-bound polysomes from healthy and inoculated plants showed some differences however the low yield of membrane-bound polysomes made it difficult to obtain conclusive results. Thus it was decided to isolate total polysomes by including 1% Triton X-100 in the extraction buffer. Polysomes were extracted from 12 to 72 h after inoculation. Infection caused a decline in yield of polysomes during this period when compared with healthy leaves of the same age. Polysomes isolated 16 h after inoculation with the virulent race were 20% less efficient at translation than polysomes from control leaves. In contrast polysome isolated from leaves inoculated with the avirulent race were 20% more efficient. Analysis of the labelled translation products by SDS-PAGE and fluorography showed relative increases in the synthesis of some proteins by 16 h after inoculation with either race when compared to products from healthy leaves. Protein synthesis in the infected plants was further analysed by in vivo labelling and one- and two-dimensional PAGE. The fluorographs revealed increased synthesis of a group of proteins from 58 to 116 kDa starting 12 h after inoculation with either race of P. hordei; confirming the results from the polysome translations. Two polypeptides with molecular weights of about 66 kDa were found to increase following infection only with the virulent race. By three days after inoculation with either fungal race the most obvious change in protein synthesis was a marked decrease in the synthesis of the two most prominent polypeptides with molecular weights of 15 and 51 kDa which were considered to be the subunits of ribulose bisphosphate carboxylase. The elicitor hypothesis, in attempting to explain cultivar-specific resistance in plants, postulates that resistance is controlled by the interaction of specific fungal elicitors and plant receptors and that this interaction which only occurs between resistant hosts and avirulent pathogens triggers specific gene expression leading to resistance. This hypothesis does not fit the situation in the barley-P. hordei interaction as protein synthesis showed similar changes following infection with either a virulent or an avirulent race.

The sensitivity, speed, and reproducibility of modern mass spectrometers enable in-depth new functional looks into the cellular proteome. Thousands of proteins can be detected in a single sample. In Co-Fractionation Mass Spectrometry (CF-MS) method, the input sample is fractionated by any biochemical method of choice. The reduced complexity of each fractionated sample leads to better proteome coverage. The separation profiles provide functional information on the proteins. This application has been used to predict organelle localization based on co-purification with marker proteins. More recently, CF-MS is being used to measure the apparent masses and determine the localization of soluble or membrane-associated protein complexes. This Ph.D. dissertation focuses on the extension of the boundary of CF-MS application to learn how protein complex evolution and protein complex composition have been accomplished. In the first part of this dissertation, the data will be presented on the degree to which variation in protein oligomerization across plant species is present, how proteomics in phylogenetic analysis (phyloproteomics/evolutionary proteomics) helps understand the evolutionary changes, and how oligomerization drives neofunctionalization during plant evolution. The latter part will describe that CF-MS coupled with multiple orthogonal chromatographic separations increases the resolving power of the profiling technique, enabling the composition of protein complexes to be predicted in the subaleurone layers of rice endosperm. Lots of novel protein complexes involved in RNA binding protein, translation, and the tissue-species metabolism will be discussed.

<p dir="ltr">H3K27me3 is a transcriptional repressive epigenetic mark that plays vital roles in many biological processes in <i>Arabidopsis thaliana</i>. A number of biochemical and functional characterizations of PKL, an ATP-dependent CHD chromatin remodeler, suggest that PKL contributes to maintain the homeostasis of H3K27me3. To identify other factors that act with PKL together to contribute to the homeostasis of H3K27me3, we undertook an EMS-mutagenesis screen for <i>pkl</i>-associated phenotypes. This genetic screen suggests that PKL may contribute to maintaining the homeostasis of H3K27me3 in an H2A.Z associated or a Mediator associated pathway.</p><p dir="ltr">Here, we took advantage of a combined genetic and bioinformatic method to characterize the contribution of PKL in these two pathways as described above. Our analysis revealed a robust genetic interaction between <i>HTA9</i>, <i>HTA11</i>, and <i>PKL</i> in maintaining proper H2A.Z distribution and enrichment of H3K27me3. In addition, the characterization also sheds light on unexpected roles of PKL in promoting the homeostasis of H3K4me3 and acting with histone demethylases to promote removal of H3K27me3 in an H2A.Z dependent manner. Furthermore, our result also raised the possibility that the tail module of the Mediator complex also plays a critical role in the homeostasis of H3K27me3. While we were examining <i>PKL</i>-dependent chromatin features, we largely optimized the protocol for preparation ChIP-seq samples and libraries and implemented a gene-centric ChIP-seq bioinformatics pipeline for providing robust analysis.</p><p dir="ltr">Ultimately, the work presented in this thesis highlights several divergent pathways that PKL contributes to maintain chromatin homeostasis. By and large, the combined observation from this thesis advances our knowledge of how PKL interacts with other chromatin-associated machineries together to maintain proper epigenetic states and promote other more emergent DNA-templated processes, including replication and transcription.</p>

Nitrogen (N), an essential plant macronutrient, is among the most limiting factors of crop yield. To sustain modern agriculture, N is often amended in soil in the form of chemical N fertilizer, a major anthropogenic contributor to nutrient pollution that affects climate, biodiversity and human health. To achieve agricultural sustainability, a comprehensive understanding of the regulation of N response in plants is required, in order to engineer crops with higher N use efficiency. Recently, epigenetic mechanisms, such as histone modifications, have gained increasing importance as a new layer of regulation of biological processes. However, our understanding of how epigenetic processes regulate N uptake and assimilation is still in its infancy. To fill this knowledge gap, we first performed a meta-analysis that combined functional genomics and network inference approaches to identify a set of N-responsive epigenetic regulators and predict their effects in regulating epigenome and transcriptome during plant N response. Our analysis suggested that histone modifications could serve as a regulatory mechanism underlying the global transcriptomic reprogramming during plant N response. To test this hypothesis, I applied chromatin immunoprecipitation-sequencing (ChIP-Seq) to monitor the genome-wide changes of four histone marks (H3K27ac, H3K4me3, H3K36me3 and H3K27me3) in response to N supply in tomato plants, followed by RNA-Seq to profile the transcriptomic changes. To investigate the organ specificity of histone modifications, I assayed shoots and roots separately. My results suggest that up to two-thirds of differentially expressed genes (DEGs) are modified in at least one of the four histone marks, supporting an integral role of histone modification in regulating N response. I observed a synergistic modification of active histone marks (H3K27ac, H3K4me3 and H3K36me3) at gene loci functionally relevant to N uptake and assimilation. Surprisingly, I uncovered a non-canonical role of H3K27me3, which is conventionally associated with repressed genes, in modulating active gene expression. Interestingly, such regulatory role of H3K27me3 is specifically associated with highly expressed genes or low expressed genes, depending on the organ context. Overall, I revealed the multi-faceted role of histone marks in mediating the plant N response, which will guide breeding and engineering of better crops with higher N use efficiency

<p>Hydrocephalus is a neurological disorder characterised by the pathological accumulation of cerebrospinal fluid (CSF) within the brain ventricles. Surgical interventions, including shunt placement, remain the gold standard treatment option for this life-threatening condition, despite these often requiring further revision surgeries. Unfortunately, there is currently no effective, pharmaceutical therapeutic agent available for the treatment of hydrocephalus. CSF is primarily produced by the choroid plexus (CP), a specialized, branched structure found in the ventricles of the brain. The CP comprises a high resistance epithelial monolayer surrounding a fenestrated capillary network, forming the blood-CSF barrier (BCSFB). The choroid plexus epithelium (CPe) critically modulates CSF production by regulating ion and water transport from the blood into the intraventricular space. This process is thought to be controlled by a host of intracellular mediators, as well as transporter proteins present on either the apical or basolateral membrane of the CPe. Though many of these proteins have been identified in the native tissue, exactly how they interact and modulate signal cascades to mediate CSF secretion remains less clear.</p> <p><br></p> <p>Transient potential receptor vanilloid 4 (TRPV4) is a non-selective cation channel that can be activated by a range of stimuli and is expressed in the CP. TRPV4 has been implicated in the regulation of CSF production through stimulating ion flux across the CPe. In a continuous CP cell line, activation of TRPV4, through the addition of a TRPV4 specific agonist GSK1016790A, stimulated a change in net transepithelial ion flux and increase in conductance. In order to develop a pharmaceutical therapeutic for the treatment of hydrocephalus, we must first understand the mechanism of CSF secretion in health and disease. Therefore, a representative <em>in vitro</em> model is critical to elucidate the signaling pathways orchestrating CSF production in the CP.</p> <p><br></p> <p>This research aims to characterize an <em>in vitro</em> culture model that can be utilized to study both the BCSFB and CSF production, to investigate and identify additional transporters, ion channels and intracellular mediators involved in TRPV4-mediated signaling in the CPe, primarily through a technique called Ussing-style electrophysiology which considers electrogenic ion flux across a monolayer. These studies implicated several potential modulators, specifically phospholipase C (PLC), phosphoinositide 3-kinase (PI3K), protein kinase C (PKC), intermediate conductance K+ channel (IK), transmembrane member 16A (TMEM16A), cystic fibrosis transmembrane conductance regulator (CFTR) and protein kinase A (PKA), in TRPV4-mediated ion flux.</p>

<p>Neutrophils are the first cells recruited to an immune stimulus stemming from infection or sterile injuries via a mixture of chemoattractant cues. In addition to eliminating pathogens, neutrophils coordinate the overall inflammation by activating and producing inflammatory signals in the tissue while modulating the activation of other immune cells which in some cases leads to adverse tissue damage. Over amplified or chronic neutrophil recruitment directly leads to autoimmune diseases including rheumatic arthritis, diabetes, neurodegenerative diseases, and cancer. Dampening neutrophil recruitment is a strategy to intervene in neutrophil-orchestrated chronic inflammation. Despite intensive research over the past several decades, clinical studies targeting neutrophil migration have been largely unsuccessful, possibly due to the prominent redundancy of adhesion receptors and chemokines. Additional challenges lie in the balance of dampening detrimental inflammation while preserving immunity. Neutrophils are terminally differentiated cells that are hard to study in cell culture. Mouse models are often used to study hematopoiesis, migration, and chemotaxis of neutrophils but is very labor intensive. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA (miRNA) overexpression screen in zebrafish and identified eight miRNAs as potent suppressors of neutrophil migration. We have generated transgenic zebrafish lines that overexpresses these candidate miRNAs where we recapitulated the mitigation in neutrophil motility and chemotaxis to tissue injury or infection. Among those we further characterized two miRNAs which have not been reported to regulate neutrophil migration, namely miR-722 and miR-199.</p> <p> </p> <p>MiR-722 downregulates the transcript level of <i>rac2</i> through binding to the <i>rac2</i> 3'UTR. Furthermore, miR-722-overexpressing larvae display improved outcomes in both sterile and bacterial systemic models, which correlates with a robust upregulation of the anti-inflammatory cytokines in the whole larvae and isolated neutrophils. miR-722 protects zebrafish from lethal lipopolysaccharide challenge. In addition, overexpression of mir-722 reduced chemotaxis of human neutrophil like cells, indicating that miR-722 is a potential agent to reduce inflammation in humans. </p> <p>MiR-199<i>,</i> decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (<i>cdk2</i>), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting Cdk2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils without inducing cell death. Human neutrophil-like cells deficient in CDK2 fail to polarize and display altered signaling downstream of the formyl peptide receptor. Chemotaxis of primary human neutrophils is also reduced upon CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. </p> <p> </p> <p>In summary, our results reveal previously unknown functions of these miRNAs, and provide potential avenues to modulate neutrophil migration as well as lead to discoveries of novel factors which can regulate this process. We have also discovered a non-classical role of CDK2 in regulating neutrophil migration which provides directions for alleviating systemic inflammation and a better understanding of neutrophil biology. </p>