<p>Juglone is a 1,4-naphthoquinone (1,4-NQ) and the allelochemical responsible for the well-known toxic effects of black walnut (<i>Juglans nigra</i>)<i> </i>and other members of the Juglandaceae. Juglone affects a variety of weed species via a mode of action unlike any commercially available herbicides, and thus has the potential to be used as a new natural product-based herbicide. However, lack of knowledge about its metabolism precludes introducing juglone biosynthesis traits into resistant crops through biotechnology. Herein, we established that juglone is derived from the phylloquinone pathway at the level of the intermediate 1,4-dihydroxy-2-naphthoic acid (DHNA). Phylloquinone is a primary 1,4-NQ made by all plants for photosynthetic electron transport. Despite the fundamental importance of phylloquinone, there are still unanswered questions about the subcellular architecture of the phylloquinone pathway. In chapter 3, we show that <i>o</i>-succinylbenzoate CoA-ligase is localized to both chloroplasts and peroxisomes and that its activity is vital in both organelles. The required dual localization of CoA ligase activity is a theme common to other plant pathways with CoA metabolic steps occurring in peroxisomes and thus leads us to propose a revised model of the phylloquinone pathway. Lastly, given the potential of introducing juglone biosynthesis as part of novel weed management strategies, we investigated the circumstances, costs, and benefits of producing allelochemicals in crops using an evolutionary game theory model. Together, this work (i) shows that the phylloquinone pathway provides crops with the biosynthetic framework to produce juglone, (ii) sheds new light on the phylloquinone pathway architecture, and (iii) reveals the circumstances in which producing an allelochemical will be an evolutionarily stable strategy. We envision these results will assist biotechnological efforts to utilize juglone as a novel, natural product-based herbicide.</p>

<p>An increasing number of chloroplast proteins have been found to interact with plant virus proteins. This is not surprising because these viruses cause various mosaic, mottles, and chlorosis symptoms on host leaves indicating damage to chloroplasts. A chloroplast protein, AtPsbP, was identified in a yeast two-hybrid screen as interacting with <i>Alfalfa mosaic virus</i> (AMV) coat protein (CP). AMV is a ssRNA virus with a wide host range including Arabidopsis. AtPsbP is an extrinsic subunit of photosystem II and with PsbQ is vital for water oxidation. We found that an RNAi knock-down of PsbP in <i>Nicotiana tabacum</i>, allowed increased replication of AMV and the development of quite severe disease symptoms in comparison to a wild-type <i>N. tabacum</i>. This suggested that PsbP plays an important role in plant resistance to AMV. PsbP, in addition to its role in photosynthesis, has been reported to interact with a wall-associated receptor kinase, WAK1, whereby it may affect plant defense signaling. We found that AtPsbP is a link between AtWAK1 and AMV CP at the plasma membrane. The formation of the AtWAK1-AtPsbP-AMV CP complex activated WAK1 kinase activity causing phosphorylation of PsbP and significant inhibition of AMV replication. We also found that the formation of the ternary complex induced the activation of the MAPK signal pathway. Analysis of the susceptibility of an Arabidopsis WAK1 knock-down indicated that WAK1, like PsbP, is critical for inhibiting AMV replication. Overall, we found a unique virus perception strategy, whereby a chloroplast protein (PsbP) interacts with a virus protein and then a Receptor-like kinase protein (WAK1) to transduce signals through the MAPK signaling pathway to activate defense responses.</p>

<p>Plants are used as a primary food source by humans. Some plants produce edible roots or leaves, but most crops used today are grown to harvest their nutrient-rich seeds which are a product of double fertilization in flowering plants. </p> <p>Cell-cell recognition, adhesion, and fusion are widespread phenomena in many biological processes, where fertilization is an exemplary process. Many players have been identified to mediate sperm-egg fusion in both animals and plants. Interestingly several of these components were shown to be structurally and functionally conserved across kingdoms. In animals Tetraspanins act as facilitators of sperm-egg fusion. Tetraspanins are known to associate in clusters in the plasma membrane of cells, where they recruit diverse signaling proteins, forming the so called Tetraspanin-enriched microdomains (TEMs). TEMs are therefore recognized as major signaling platforms mediating specific cellular processes in the plasma membrane of cells. Two <i>Arabidopsis</i>-expressed tetraspanins, <i>TET11</i> and <i>TET12</i>, are highly expressed in the sperm cells (SCs), however their function in fertilization are unknown. Using fluorescence microscopy, we quantified the expression of TETs in SCs and found evidence for the existence of a Tetraspanin-enriched microdomain (TEM) at the SC-SC adhesion interface. Sperm cell factors which are necessary for fertilization were found to accumulate at the TEM, suggesting that plant SC TEMs may function as protective platforms for fertilization factors. Sperm-expressed TETs directly interact with members of a novel, plant-specific family of unknown proteins, <i>DMP8/9</i>. DMP8/9 function as negative regulators of SC-SC adhesion and are required for double fertilization. Structural and functional analysis suggest that these two proteins may perform unique functions as membrane remodelers in SCs. In addition, we provide evidence of a new GEX2 function as a SC-SC adhesion factor and potential partner of TET-DMP complexes at the SC-SC interface.</p>

No description available.

<p>RNA-directed DNA methylation (RdDM) is a process by which epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on <i>Mediator of Paramutation 1 (Mop1</i>), a putative RNA dependent RNA polymerase. Here I show that although RdDM is essential for the maintenance of DNA methylation of a silenced <i>MuDR</i> transposon in maize, a loss of that methylation does not result in a restoration of activity of that element. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of the element, heritable silencing is mediated via H3K9 and H3K27 dimethylation, even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by H3K27 trimethylation, a mark normally associated with somatically inherited gene silencing. I find that a brief exposure of high temperature in a <i>mop1</i> mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in <i>mop1</i> wild type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once it has been reversed. To leverage the specificity of our observations made at bench, I also performed a transcriptome analysis in <i>mop1</i> mutants under heat. I found that a substantial number of genes as well as a subset of TEs are reactivated in <i>mop1</i> mutants under heat, which is consistent with the effects I observed on <i>MuDR</i>. Interestingly, I found that <i>mop1</i>-specific reactivation of TEs is closely correlated with changes in expression of nearby genes, most of which are involved in metabolic transportation and sensing. This suggests that one function of <i>MOP1</i> is to prevent inappropriate expression of genes in this pathway when they are close to TEs. Taken together, my work will provide an opportunity to better understand the causes and consequences of TE silencing and reactivation, as well as the effects of TEs on gene regulation under stress conditions.</p>

<p> As the root microbiome’s role in plant defenses against herbivory becomes clearer, scientific focus has lingered on a single side of plant defenses: resistance. Its counterpart, tolerance, is comparatively overlooked despite its power as an evolutionarily sustainable mitigator of herbivore damage. This thesis seeks to supplement our limited understanding of the extent to which tolerance to herbivory may be influenced by rhizosphere microbial communities. First, in an agricultural field setting, I (1) quantified domesticated tomato cultivar and wild ancestor tolerance to herbivory form the specialist tobacco hornworm (<em>Manduca sexta</em>) and (2) characterized the bacterial and fungal rhizosphere communities associating with high and low tolerance plants. In a subsequent greenhouse experiment, I grew these same tomato lines in either sterilized or unsterilized soil and re-challenged plants with tobacco hornworms to tease apart the contributions from host plant and rhizosphere microbiome in expressing tolerance to herbivory. In the field, wild tomato lines excelled at tolerating hornworm herbivory, while their domesticated counterparts suffered 26% yield losses under herbivory. Rhizosphere community characteristics were most reliably shaped by timepoint of rhizosphere sampling, and more subtly by tomato line and herbivory treatments. Fungal and bacterial community traits that associated with high tolerance lines include (1) high diversity, (2) resistance to community shifts under herbivory, and (3) the abundance of ASVs belonging to <em>Strenotrophomonas</em>, <em>Sphingobacterium</em>, and <em>Sphingomonas</em>. When re-challenging these lines with hornworm herbivory in the greenhouse, expressed tolerance to tobacco hornworm damage was inverted from field trends. Though wild lines suffered yield losses when grown in +microbiome treatments, we found no consistent interactions between herbivory and microbiome treatments that might indicate that +microbiome treatments either helped or hampered plant expression of tolerance to herbivory under greenhouse conditions. These experiments shed light on what role, if any, the rhizosphere microbiome plays in plant tolerance to herbivory. Ultimately, understanding the qualities of tolerance-conferring microbiomes can (1) open avenues through which plant defenses may be amended in pest management, either through microbial inoculants or plant breeding efforts aimed at enhancing crop recruitment of beneficial microbiomes; and (2) ameliorate our understanding of the tripartite interactions between host plants, their rhizospheres, and their specialist herbivores. </p>

No description available.

No description available.

Subcellular Localization of Tobacco SABP2 under Normal and Stress Conditions Salicylic acid (SA), a phytohormone, plays an important role in plant physiology. SA mediated innate immune pathway is an important pathway for plant immunity against pathogens. Plants resisting pathogen infection synthesize higher levels of Methyl Salicylate (MeSA), which is then converted to SA by the esterase activity of Salicylic Acid Binding Protein 2 (SABP2). The high level of the converted SA leads to enhanced pathogen resistance. The study of subcellular localization of a protein is critical in explaining its potential biochemical functions. SABP2 tagged with eGFP was expressed transiently in Nicotiana benthamiana leaves. The SABP2-eGFP expressing leaves were challenged with bacterial and viral pathogens and observed under confocal microscopy. Fluorescent signals were seen throughout the cell and more concentrated towards the cell periphery. To verify the localization, mCherry fluorescent organelle markers with specific targeting sequences were used. The results indicate that the SABP2 is likely a cytoplasmic protein, and there is no change in its localization upon infection by plant pathogens.

Fairway crested wheatgrass, which is identified taxonomically as Agropyron cristatum (L . ) Gaertn. (45 ), A. cristatiforme (38) , or A. pectiniforme Roem. and Schult (22), is an economically important range grass belonging to the "crested wheatgrass complex" (24, 38). The crested wheatgrass complex includes diploid, 2n = 14, tetraploid, 2n = 28, and hexaploid, 2n = 42, forms (1, 11, 22). The variety Fairway and Fairway-like derivatives are the only known diploid members of the species complex (24, 38). Meiotic chromosome behavior of Fairway diploids appears to be typical of other diploid species; however, the number of plants examined cytologically has been relatively small. Although Fairway crested wheatgrass is a good seed producer, interplant variation in fertility is high (13, 22, 25, 42). Irregular chromosome behavior is a common source of sterility and may be contributing to the variable seed set in diploid crested wheatgrass. No information is available concerning the relation of meiotic chromosome behavior to fertility in Fairway crested wheatgrass. Polyploid crested wheatgrasses are generally considered to be of autoploid origin, i.e., they are derived by duplication of the chromosome complement of a diploid prototype. Chromosome pairing in the polyploid species (31), in interspecific hybrids (12), and in polyhaploid plants (11) substantiate the autoploid derivation of polyploid crested wheatgrass. Diploid and tetraploid forms of crested wheatgrass have been hybridized by Knowles (24), and chromosome pairing in the hybrids suggest a close relation between the diploid and tetraploid genomes. Colchicine-induced tetraploids of Fairway crested wheatgrass have been produced by Knowles, 1 and these artificial tetraploids are currently being utilized in his crested wheatgrass breeding program. If the full breeding and cytogenetic potentials of diploid crested wheatgrass are to be realized, the meiotic chromosome behavior and the cytotaxonomic status of this species must be fully understood. The present investigation was designed to provide further information concerning the cytogenetic characteristics of Fairway crested wheatgrass and its autotetraploid derivatives. This investigation was established with the following objectives: 1. To examine meiotic chromosome behavior of Fairway crested wheatgrass. 2. To determine the relation of meiotic chromosome behavior to fertility in Fairway crested wheatgrass. 3. To evaluate the effectiveness of several colchicine treatments in doubling the chromosome complement of Fairway crested wheatgrass. 4. To determine the effect of induced polyploidy on plant morphology in colchicine-induced tetraploids of Fairway crested wheatgrass. 5. To determine the meiotic chromosome behavior and fertility of induced tetraploids of Fairway crested wheatgrass.