Weedy rice is a persistent weed of cultivated rice (Oryza sativa ) fields worldwide, which competes with the crop and drastically reduces yields. Within the US, two main populations of genetically differentiated weedy rice exist, the straw-hulled (SH) group and the black-hulled awned (BHA) group. Current research suggests that both groups are derived from Asian cultivated rice. However, the weeds differ from the cultivated groups in various morphological traits. My research focus is on the genetic basis of two such traits: seed shattering ability and differences in flowering time. The persistence of weedy rice has been partly attributed to its ability to shatter (disperse) seed prior to crop harvesting. I have investigated the shattering phenotype in a collection of US weedy rice accessions and find that all US weedy rice groups shatter seeds easily. Additionally, I characterized the morphology of the abscission layer at the site where seed release occurs and find that weeds begin to degrade their abscission layers at least five days prior to wild plants. I also assessed allelic identity and diversity at the major shattering locus, sh4, in weedy rice and find that all cultivated and weedy rice share similar haplotypes at sh4 . These haplotypes contain a single derived mutation associated with decreased seed shattering during domestication. The combination of a shared cultivar sh4 allele and a highly shattering phenotype suggests that US weedy rice have re-acquired the shattering trait after divergence from their crop progenitors through alternative genetic mechanisms. Additionally, my investigation into flowering time in weedy rice shows that weed populations differ in their flowering times. I also assessed allelic identity and diversity at two genes involved in the transition to flowering, Hd1 and Hd3a, and again found haplotype sharing between weeds and cultivars with Hd1 only accounting for some of the flowering time differences between weeds. In order to locate genomic regions containing additional candidate genes I conducted a QTL mapping study on two F2 populations derived from crosses of weedy rice with cultivated rice. My results show sharing of QTL for flowering time between populations, yet lack of sharing of QTL for shattering.

While pollinators and leaf herbivores have been a focus of research for decades, floral antagonists have been studied significantly less. Since floral antagonists can be as common as leaf herbivores and have strong impacts on plant reproduction, it is important to understand the role of floral antagonists in the ecology and evolution of flowers. I conducted four experiments to better understand the relationship between plants, floral traits, floral antagonists, and other plant-insect interactions. First, I manipulated resources (light and soil nutrients) that are known to have impacts on plants and floral traits to test how they affect floral antagonists and other plant-insect interactions. Plentiful resources increased the proportion of floral antagonists to visit flowers, but also increase tolerance of floral antagonists. Second, I manipulated flower bud gallers, a species-specific floral herbivore that destroys flowers, to test how it affected other plant-insect interactions, floral traits, and plant reproduction. Plants with flower bud gallers tended to have more pollinator visits, but this effect is due to a shared preference by gallers and pollinators for similar plants. Third, I manipulated florivory to examine how it affects subsequent plant-arthropod interactions, floral traits, and plant reproduction. Florivory had systemic effects on other plant-insect interactions, including leaf herbivores, and shifted the plant mating system towards more selfing. Additionally, I tested how several floral antagonists respond to floral attractive and defense traits to understand which floral traits are important in mediating antagonisms. Finally, I manipulated florivory, pollination, and nectar robbing to test for effects of multiple floral interactions on subsequent plant-insect interactions, floral traits, and plant reproduction. There were significant many-way interactions between the three treatments on subsequent plant-insect interactions and reproduction, indicating that the effect of one interaction depends on what other interactions are present. Understanding the role that floral antagonists play in plant ecology can help scientists determine which interactions are most important, and may help determine why some floral traits exist in their current state. Together, this work represents some of the most comprehensive research on the community consequences of floral antagonists, as well as the interplay between floral traits and floral interactions.

No description available.

Using reverse genetics, complementation analyses, and cell biological approaches with the moss Physcomitrella patens, I assessed the in vivo function of two actin turnover proteins: actin depolymerizing factor (ADF) and its binding partner actin interacting protein 1 (AIP1). My studies identify a single ADF and AIP1 in moss. Loss-of-function analyses reveal that ADF is essential for viability, and AIP1 is required to promote normal tip cell expansion. AIP1 and ADF are diffusely cytosolic proteins that function in a common genetic pathway to promote tip growth. Specifically, ADF can partially compensate for loss of AIP1, and AIP1 requires ADF for function. Consistent with a role in actin turnover, AIP1 knockout lines and plants silencing ADF accumulate F-actin bundles along the cortex. Quantitative analysis of time-lapse F-actin movies demonstrates that AIP1 promotes and ADF is essential for cortical F-actin dynamics. The development of a complementation assay permitted dissection of the physiological relevance of regulatory mechanisms that control ADF activity. Mutant complementation analyses reveal that phosphoregulation of ADF at a conserved, N-terminal serine is important for in vivo function. Phosphomimetic ADF mutants have severe tip growth defects, but remain viable, demonstrating that ADF is critical for tip growth. A gain-of-function ADF mutant with enhanced affinity for phosphatidylinositol 4,5-bisphosphate has minor defects in tip growth, suggesting that this phospholipid regulates ADF activity in vivo. Complementation analyses with ADF/cofilin proteins from other organisms reveal that moss ADF is functionally conserved with some, but not all ADF/cofilins. Interestingly, rescue is inversely proportional to pH-sensitivity, suggesting that pH-insensitive ADF activity is important for tip growth in moss. The complementation analysis has also facilitated the identification of two temperature-sensitive mutants in moss ADF. These temperature sensitive mutants, together with the AIP1 knockout lines, will be instrumental for identifying cellular processes in plants that require actin dynamics – an open question in plant biology.

<div><br></div><div>Plants are constantly exposed to biotic and abiotic factors throughout their developmental stages which threaten their growth and productivity. Environmental stresses limit crop productivity and are likely to increase in severity due to the drastic and rapid changes in global climate. In this project, we studied the genetic factors that contribute to plant adaption to pathogens and other environmental factors in tomato. The results of these are presented in chapters 2-4 of this thesis. Chapter 1 covers background information and the review of the current literature in plant responses to biotic and abiotic stress. Chapter 2 deals with functional analysis of tomato histone methyltransferases SDG33 and SDG34 and their role in plant defense and stress tolerance. Chapter 3 focuses on the role of SDG33 and SDG34 on plant responses to Nitrogen. Finally, Chapter 4 summarizes the results from a reverse genetic screen using CRISPR cas9 genome editing to identify Receptor Like Cytoplasmic Kinases (RLCKs) required for plant resistance to fungal pathogens. </div><div>Plant responses to environmental cues are underpinned by rapid and extensive transcriptional reprogramming. Post translational modification of histones orchestrate these reprogramming and cellular responses by altering chromatin structure and establishing permissive or repressive states. Histone lysine methylation (HLM) is a principal modification of chromatin that affects various cellular processes. HLM is mediated by histone methyltransferases (HMTs) that deposit methyl groups to specific lysine residues on n-terminal histones tails. Although it is known that chromatin modifications occur in response to environmental cues, the mechanisms by which this is achieved, and the biological functions of HMTs are poorly understood. The function of tomato histone methyltransferases Set Domain Group (SDG)33 and SDG34 in biotic and abiotic stress responses were studied using tomato mutants generated through CRISPR/cas9 genome editing. </div><div>SDG33 and SDG34 genes were induced by pathogens, drought stress, the plant hormones methyl jasmonate, salicylate and abscisic acid. The sdg33 and sdg34 mutants display altered global HLMs. SDG34 is required for global H3K36 and H3K4 mono, di- and tri-methylation while SDG33 is primarily responsible for di- and tri- H3K36 and H3K4 methylation. Tomato SDG33 and SDG34 are orthologues of the Arabidopsis SDG8, an H3K4 and H3K36 methyl transferase previously implicated in plant immunity and plant growth through epigenetic control of Carotenoid Isomerase (CCR2) and other target genes. However, the tomato sdg33 or sdg34 single mutants showed no altered responses to fungal and bacterial pathogens likely due to functional redundancy of the tomato SDG33 and SDG34 genes consistent with their overlapping biochemical activities. Interestingly, tomato SDG33 or SDG34 genes rescued the disease susceptibility and early flowering phenotypes of Arabidopsis sdg8 mutant. Expression of CCR2 gene is completely inhibited in Arabidopsis sdg8 mutant attributed to loss of H3K36 di- and tri methylation at CCR2 chromatin. CCR2 gene expression was partially restored by transgenic expression of tomato SDG33 or SDG34 genes in Arabidopsis sdg8. In tomato, the single CCR2 gene is expressed independent of SDG33 or SDG33 genes suggesting that the genomic targets of the tomato HMTs are different. Unexpectedly, sdg33 and sdg34 plants were more tolerant to osmotic stress, maintain a higher water status during drought which translated to better survival after drought. Tolerance of sdg33 and sdg34 to drought stress is accompanied by higher expression of drought responsive genes. Collectively, our data demonstrate the critical role of tomato HLM in pathogen and stress tolerance likely through the regulation of gene expression.</div><div>In parallel, we characterized the role of SDGs in mediating nitrogen responses in tomato. The results are described in Chapter 2. Few studies have focused on the role of histone lysine methylation in regulating changes to nutrient availability. Transcriptome analysis in the shoot and roots showed that SDG33 and SDG34 have both overlapping and distinct regulated targets in tomato. In response to nitrogen, 509 and 245 genes are regulated by both SDG33 and SDG34 in response to nitrogen states in the roots and shoot respectively. In the roots these genes were enriched with GO terms such as ‘regulation of gene expression’, regulation of N metabolism’ and ‘regulation of hormone stimuli’. ‘Response to stimulus’, ‘photosynthesis’ and ‘N assimilation’ were the biological processes significantly enriched in the shoots. Overall, we show that SDG33 and SDG34 are involved in regulating nitrogen responsive gene expression and hence physiological nitrogen responses in the roots and shoots. </div><div>We also studied the Set Domain Group 20 (SlSDG20) an orthologue of Arabidopsis SDG25 in tomato. The details of our observations are presented in Chapter 3. SlSDG20 belongs to class III HMTs, it has the SET, Post-SET domain and GYF domain important for proline-rich sequence recognition. SlSDG20 is highly induced by B. cinerea, Methyl Jasmonate and Ethylene. To further understand the functions of SlSDG20 in tomato physiological development and plant immunity we generated slsdg20 knockout mutants through CRSIPR/Cas9. We identified one homozygous slsdg20 mutant with 151bp deletion in an exon immediately before the SET domain. Global methylation assay on the slsdg20 mutant confirmed that SlSDG20 is an H3K4 methyltransferase. The slsdg20 mutant is shorter than the wild type, produce more adventitious shoots causing prolific branching, and produce narrow leaves. Further, the mutant produces abnormal fruit and few seeds that hardly germinate. The slsdg20 mutant is highly susceptible to B. cinerea compared to the wild type. In response to Pst DC3000, slsdg20 mutant plants are comparable of the wild type. Resistance to hrcC strain of Pst DC3000 was impaired in the slsdg20 mutant, suggesting a possible role of SlSDG20 in PTI. In sum, tomato SDG20 is regulates plant immunity and plant growth including fertility.</div><div>The final chapter focuses on tomato Receptor like cytoplasmic kinases (RLCKs). Plants perceive the presence of pathogens through Pattern Recognition Receptors (PRR) which are predominantly RLKs, and subsequently recruit RLCKs to signal to downstream regulators of defense responses. Many RLCKs were characterized from Arabidopsis for their role in signalling of responses to bacterial infection. An example of RLCKs is Arabidopsis BIK1 which is implicated in signal transmission of pathogen recognition event at the cell surface. The tomato genome encodes 647 RLK/RLCKs comprising about 2% of its predicted genes. The functions of most of these predicted tomato RLCKs and RLKs have not been determined. Previously, our lab characterized the Arabidopsis BIK1 and tomato TPK1b RLCKs for fungal resistance. Here, we conducted a reverse genetic screen focused on BIK1 and TPK1b related tomato RLCKs to identify a subset with defense functions. Virus induced gene silencing and pathogen assays conducted on 15 RLCKs identified four RLCK genes with potential role in plant immunity. Then, tomato knock out mutants were generated for four RLCK genes through CRISPR/cas9 genome editing to validate the VIGS data. Subsequently, we demonstrated that TPK07, TPK09, TPK011 and TRK04 are required for resistance to B. cinerea. The data are supported by the pathogen induced expression of these genes. Furthermore, trk04 seedlings are impaired in seedling growth responses to Jasmonic acid. Our study establishes that tomato TPK07, TPK09, TPK011 and TRK04 contribute to defense against B. cinerea but their mechanism of function needs to be elucidated in future studies</div><div><br></div>

Relative growth rates (RGRs) and meristematic potential of big sagebrush (Artemisia tridentata, in some experiments ssp. tridentata Nutt. and in other experiments ssp. vaseyana [Rydb] Beetls), bitterbrush (Purshia tridentata [Pursh] DC), serviceberry (Amelanchier alnifolia Nutt.), birchleaf mountain mahogany (Cercocarpus montanus Raf.), curlleaf mountain mahogany (C. ledifolius Nutt.) and big tooth maple (Acer grandidentatum Nutt.) were quantified under both glasshouse and field conditions. The relationships between the RGRs, meristematic potential and regrowth capacity of the shrub species were then determined. Sagebrush exhibited a significantly higher RGR than the other species at both high and low nutrient levels under glasshouse conditions. Four of the other shrub species had similar RGRs. The shrub species also exhibited different patterns of biomass and nutrient allocation. Sagebrush allocated most of its resources to leaf production, and in contrast, the other species tended to allocate similar amounts of resources to both leaf and root production. In the glasshouse experiment, neither RGRs nor patterns of biomass and nutrient allocation were related to growth form. Under field conditions, curlleaf and birchleaf mountain mahogany exhibited similar RGRs while both serviceberry and maple had similar but significantly lower growth rates. Except for sagebrush, which died following simulated browsing, four other shrub species exactly compensated for lost tissues. The mean RGRs of those shrub species were correlated significantly with compensatory growth capacity. Loss of 90% of buds on previous year's growth did not cause a meristematic constraint to regrowth production. Instead, it stimulated regrowth production. The shrub species we evaluated also exhibited differences in meristematic potential. Curlleaf and birchleaf mountain mahogany had a greater number of buds and long shoots per plant but a lower percentage of long shoots at the basal position on twigs. In contrast, serviceberry and maple had fewer buds and long shoots per plant but a higher percentage of long shoots at the basal position on twigs. The meristematic potential of the shrub species correlated with regrowth capacity.

No description available.

Eutrema salsugineum is an extremophile plant that is naturally tolerant to abiotic stresses such as high salinity, drought and cold (Inan et al., 2004, Griffith et al., 2007; MacLeod et al., 2015). It is emerging as a stress tolerant model plant, due to its short life cycle and high genetic similarity to the model plant Arabidopsis (Inan et al., 2004). There are two well-studied ecotypes of Eutrema, the Shandong ecotype from the Shandong Province of China, and the Yukon ecotype that grows in the Yukon, Canada. Principal component analysis (PCA) comparing the transcriptomes of three Yukon cabinet plants, three Shandong cabinet plants, and three Yukon field plants revealed that Yukon cabinet plants load negatively along PC3, while the Shandong cabinet and Yukon field plants load positively. Of the top 50 genes that contribute to this negative loading, 12 are related to sulfur deficiency, leading to the hypothesis that the cabinet plants are deficient for sulfur. To test this hypothesis cabinet potting soil was supplemented with calcium sulfate dihydrate to raise the sulfur level to approximately that of Yukon field soil. Sulfur-treated plants were compared to those grown on unsupplemented soil and untreated plants had significantly reduced biomass and leaf area. Additionally, RT-qPCR showed that relative to sulfur-supplemented plants, untreated plants had 4 to 177-fold higher transcript levels of two sulfur deficiency marker genes, Sulfur deficiency induced 1 and γ-glutamylcyclotransferase 2;1. While these findings are consistent with untreated plants experiencing a sulfur deficiency, there was no difference in chlorophyll content, and rosette sulfur levels were only 1.1-fold higher in the sulfur-treated plants. The third most negatively loading gene on PC3 was identified as XLOC_003912, a long non-coding RNA of unknown function that is only expressed in Yukon cabinet plants. Because it shows a similar loading pattern to the 12 sulfur nutrition-related genes, it was hypothesized to be involved in sulfur homeostasis. RT-qPCR showed that XLOC_003912 expression was lower in sulfur-treated compared to untreated plants. A method was developed to suppress XLOC_003912 expression in Yukon Eutrema plants by Virus Induced Gene Silencing (VIGS). In addition to providing insight into the function of XLOC_003912, this technique can be used in future studies for determining the role of novel genes in Eutrema salsugineum. / Thesis / Master of Science (MSc) / Eutrema salsugineum is a model for studies of plant abiotic stress tolerance. I determined that the Yukon ecotype of Eutrema displays elevated expression of sulfur deficiency genes when grown in cabinets. Increasing soil sulfur level roughly 10-fold in the potting mix led to significantly increased biomass, leaf area and leaf number and reduced transcript abundance of sulfur deficiency genes. One sulfur-responsive gene encodes a novel long non-coding RNA. I adapted a method for transiently repressing its expression, an approach that can be used to explore the function of this and other novel genes in Eutrema.

No description available.

No description available.