Pathogen derived resistance (PDR) is a recent breakthrough where plant hosts can be made to be resistant to viral infections through transformation with conserved viral genes. Given the severity of Tospovirus diseases worldwide (particularly in tomato), PDR has the potential to garner large yield returns where pathogen populations have overcome the established resistance. Tomato breeding lines FLA7804, FLA8044, and the research line MP1 were used in transformation experiments with potions of the Tomato spotted wilt virus (TSWV) N-gene, and two other chimerical viral nucleocapsid gene constructs from, Impatiens necrotic spot virus (INSV), and Groundnut ringspot virus (GRSV). We conducted 19 independent transformations consisting of 300 to 700 14-day old whole cotyledons each for a total number of approximately 9,000 potentially transformed explants. Of those, approximately 6,300 explants failed to produce regenerants, 2,419 explants underwent abnormal development on elongation media, 187 failed to root, and 215 plants to be characterized genetically. Of the 215 plants, 9 were from FLA 7804, 96 from FLA 8044, and 110 from MP1. Both PCR and Southern blot hybridization analysis later confirmed that none of the 215 plants were transgenic. Opposite to tomato, we were able to transform Arabidopsis thaliana ecotype wassilewskija (Ws) via floral dip with the above listed constructs demonstrating that constructs were not deleterious within a plant once fully introgressed. Sixteen independent transformants in the T0 generation resulted from 19,000 germinated seed from three dipped plants resulting in a total transformation rate of 0.08%. Of the 1,000 T1 seed germinated on kanamycin media from each of the 16 putative Arabidopsis plants transformed with the construct containing elements of the N-gene from all three of the aforementioned tospoviruses, four populations exhibited simple Mendelian inheritance of the transgene. DNA walking analysis yielded amplification of the unknown region outside the nptII region of the insert for three of the four remaining transformants, which was subsequently sequenced and mapped to chromosomes 1, 3, and 4. There were 25 T1 individuals selected from each population and transferred to soil for DNA extraction and zygosity determination. Homozygous T2 seed was collected for future resistance studies.

Plants have two organelles outside the nucleus which carry their own DNA, mitochondria and chloroplasts. These organelles are descendants of bacteria that were engulfed by their host according to the endosymbiotic theory. Over time, DNA has been exchanged between these organelles and the nucleus. Two polymerases, DNA Polymerases Gamma I and II, are encoded in the nucleus and remain under nuclear control, but are transported into the mitochondria and chloroplasts. DNA polymerases gamma I and II are two organelle polymerases which have been studied through sequence analysis and shown to localize to both mitochondria and chloroplasts. Little has been done to characterize the activities of these polymerases. Work in tobacco showed the homology of these polymerases to each other and to DNA Polymerase I in bacteria. They have been characterized as being part of the DNA Polymerase A family of polymerases. In my research I have studied the effect of T-DNA insertions within the DNA Polymerase Gamma I and II genes. Since these DNA Polymerases are targeted to the mitochondria and chloroplasts, I studied the effect of knocking out these genes. A plant heterozygous for an insert in DNA Polymerase Gamma I grows slightly slower than wild type plants with an approximately 20% reduction in mitochondrial and chloroplast DNA copy number. A plant homozygous for an insert in this same gene has a drastic phenotype with stunted plants that grow to around 1 inch tall, with floral stems, and have an approximately 50-55% reduction in mitochondrial and chloroplast DNA copy number. Wild type plants can grow to a height of 12-18 inches with floral stems as a comparison. A plant heterozygous for an insert in the DNA Polymerase Gamma II gene grows slightly slower than wild type plants and has an approximately 15% reduction in mitochondrial DNA copy number and a 50% reduction in chloroplast DNA copy number. These plants also produce much less seed than do other mutants and wild type plants.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22128号 / 農博第2374号 / 新制||農||1073(附属図書館) / 学位論文||R1||N5236(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 髙野 義孝, 教授 田中 千尋, 教授 寺内 良平 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Superoxide dismutases (SODs) catalyze the dismutation of superoxide radicals to oxygen and hydrogen peroxide. Mn SOD is localized in mitochondria, Cu-Zn SOD is in the cytosol and chloroplast, and Fe SOD is in chloroplasts. The effects of a chloroplast-localized oxidative stress, caused by methyl viologen or 3-(3, 4-dichlorphenyl)-1-1′ dimethylurea (DCMU) on SOD populations were investigated. A cloned Arabidopsis thaliana Fe SOD gene was expressed in Escherichia coli and was purified from transformed cells. This protein was used to raise antibodies against A. thaliana Fe SOD which in turn were used to quantify the effects of oxidative stress on Fe SOD protein. Effects of oxidative stress on enzyme activity were measured in native gels. Fe SOD responded to oxidative stress with an increase in activity, but not in antibody reactive protein. Two novel forms of Fe SOD activity, with faster migration rates in activity gels, were detected. Mn SOD, a mitochondrial enzyme, responded to the stress with an increase in activity. In contrast, the activity or amount of Cu-Zn SOD protein did not respond to this oxidative stress. In light of these results, we propose that SODs respond to oxidative stress at the enzyme and gene levels. Mitochondrial Mn SOD responded to a chloroplast-localized stress with an increase in activity, suggesting either that the site of action for methyl viologen is not exclusively in the chloroplast or that there are other signals among the compartments of the cell. Fe SOD, but not Cu-Zn SOD responded to stress, suggesting that Fe SOD may be the stress responsive enzyme in this organelle. Evolutionary relationships among different isoforms were investigated based on the known primary, secondary, and tertiary structures of these isoforms. The three dimensional structure of A. thaliana Fe SOD was modeled by using structures of crystallized E. coli and Pseudomonas ovalis Fe SODs as templates. Comparison of prokaryotic Fe SOD with eukaryotic isoforms showed that Fe and Mn SODs are structurally homologous, whereas Cu Zn SOD is not. / Ph. D.

The influence of pollinators on the evolution of flower morphology has been extensively explored. Yet, the effect of other ecological factors, such as genetic drift, environmental filtering, and allometric constraints, gained less attention. In this study, we addressed the importance of those drivers in a predominantly selfing species. 400 worldwide Arabidopsis thaliana accessions were gathered and grown in semi-controlled climatic settings to explore the association between flower organ size, genotypes, and habitats. In our dataset, petal area was the most variable trait. Petal size was phenotypically and genetically correlated with other flowering structures, but no genetic allometry constraints were found to affect petal size evolution. The negative correlation of petal size with fitness and the traces of selective constraints in petal associated genes suggest that petal size is currently under selection in this species. We found paucity of genotypes harbouring large petals at low suitability regions, which points to the presence of environmental filtering. The novelty of this project relies on the pluralistic integration of factors studied and highlights the role of the climate on flower size evolution. Our results suggest that resource allocation is an important driver of flower size evolution in self-fertilising species but that its effect is largely determined by local environmental pressures.

Glutamate is essential for proper brain function as it is our nervous systems principal excitatory neurotransmitter, a signal that stimulates nerve cells to send messages to other cells. Glutamate activates ionotropic glutamate receptors (iGluRs), which are linked to several neurological diseases in cases when they are improperly regulated. iGluRs are transmembrane channels that allow calcium, as well as other cations, into the post synaptic neuron upon binding of glutamate or other agonists.Interestingly, iGluR homologs in plants also mediate calcium signaling upon glutamate activation and were accordingly named glutamate receptor-like channels (GLRs). Cell signaling is critical for plant survival to mediate rapid response to growth, defense, and other environmental cues. GLRs are found in all plants and vital for their health, hardiness, and adaptation for growth and survival in unfavorable conditions, such as drought, nutrient poor soil, temperature extremes, pathogens, and predators. Plant research is important with vast applications. Firstly, crops are our primary source of nutrition. In addition, plants are used as sources of drugs that we employ for treating diseases. Some examples of plant-derived neuroactive compounds include caffeine in coffee beans, nicotine in tobacco, and opium from poppy plants. In short, optimizing plant growth is beneficial to maintaining our own survival and potentially achievable by understanding GLRs role in plant health and hardiness. Despite their importance for cell signaling and implication in plant defense and regeneration, the structural basis underlying the function of these channels remains ambiguous, representing a critical barrier to our understanding of GLR function. To address this problem, I dedicated my thesis work to study the structure of GLRs and gain insight into their function. There are 20 GLRs in the model plant organism, Arabidopsis thaliana, classified into 3 different clades (AtGLR1-3). To narrow down which AtGLRs to focus our structural studies on, we investigated clade 3 representatives, as many of these GLR3s have been extensively studied in different plant species, especially crops. For example, studying AtGLR3.4 could provide useful information to how the homolog in rice, OsGLR3.4, contributes to growth and production in rice. Studying AtGLR3.4’s structure may elucidate how agonistic or antagonistic targets bind and gate the channel, potentially revealing “druggable” targets to alter plant response for defense and regeneration. Without any structural information available for GLRs, I started my studies by first focusing on their mammalian homologs, iGluRs. I first designed multiple constructs for heterologous expression and purification from cell culture (for example HEK293S GnTI- cells). Then, I optimized protein extraction and purification to obtain pure protein samples. Purified proteins were then subjected to cryo-electron microscopy (cryo-EM) which eventually allowed us to solve the structure of AtGLR3.4, the first full-length GLR structure. AtGLR3.4’s structure revealed similarities to structures of its mammalian homologs, iGluRs. In comparison to iGluRs, our GLR structure also showed tetrameric subunit assembly, with a three-layer architecture that includes the ligand binding domain (LBD) in the middle, sandwiched between the extracellular amino terminal domain (ATD) at the top and the transmembrane domain (TMD) at the bottom. In contrast to the majority of iGluR structures, however, AtGLR3.4 displayed unique symmetry and domain arrangement with the non-swapped extracellular ATD and LBD domains. We also provided further evidence supporting ligand binding promiscuity that was previously revealed in isolated LBD crystal structures from other AtGLR3s. Surprisingly, we found endogenous glutathione bound to the ATDs and demonstrated its contribution to channel activity. It is important to fill the gaps in knowledge about GLR structure to understand how these channels are activated and gated. In doing so, we will learn more about iGluRs as well as better understand plant defense and growth, which has the potential to enhance crop production for food security and our overall survival.

Lipid transfer proteins (LTPs) belong to the pathogenesis-related protein family (PR-14) and are thought to participate in plant defense mechanisms. In this study, we characterize the function of an Arabidopsis thaliana mutant ltp12 (AT3G51590), a homologous lipid transfer protein to SIP470 from Nicotiana tabacum for its role in abiotic and biotic stress. SIP470, a lipid transfer protein, was found to interact with SABP2 in a yeast-two hybrid screen. SABP2 in tobacco is required for inducing a robust SAR response. The objective of this research is to understand the role of LTP12 in mediating abiotic stress as salicylic acid plays an important role in both abiotic and biotic stress in plants. For this research, stressor chemicals, NaCl (salinity), mannitol (osmotic stress), and drought (no water or PEG) will be used. Seedlings were initially germinated and grown on artificial plant growth MS media. The similar-sized young seedlings were transferred to MS media plates supplemented with or without stressor chemicals. Oxidative stress analysis of various antioxidant enzymes, such as catalase (CAT), superoxide dismutase (SOD), and peroxidase (POD) will be performed. The Na+ homeostasis for salinity stress will be studied using CoroNaTM dye and confocal microscopy. Our lab has T-DNA insertion knockout mutants of LTP12 that we will be used in the proposed studies. Here, we hypothesize that mutant ltp12 plants will be hypersensitive to abiotic stressors like NaCl, mannitol, and drought, while wildtype Col-0 will be markedly more tolerant. Reports also suggest that knockout lines of other lipid transfer proteins show a defective growth phenotype and lower expression of systemic acquired resistance (SAR). Moreover, to gain a better understanding of both lines' responses to abiotic stress, we need to carry out further studies on the soil as well. The study will also discuss the subcellular localization of ltp12 in Arabidopsis, which will provide an idea of its functional mechanism. Understanding the role of lipid transfer proteins can lead to the development of transgenic plants that are more tolerant to abiotic stresses and climate change.

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