Plant Biology Graduate Program Dissertations

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Understanding the Role of a Chemical Defense Activator, Acibenzolar-S-Methyl, on Abiotic Stress Resistance in Agrostis Stolonifera
(2024-09) Lu, Jefferson
Turfgrass systems are constantly challenged by biotic and abiotic stresses that affect their growth and function. During the growing season, periods of drought and heat are important abiotic stresses that impact overall turfgrass health and susceptibility to pests such as diseases and weed infestation. Efforts to improve abiotic stress resistance have mainly relied on basic agronomic practices and use of adapted cultivars or species. In recent years, an additional tool to help enhance plant health is through the application of plant health activators. Although these compounds were first developed to primarily protect against biotic stresses (e.g. diseases), limited evidence suggests that some priming compounds may also enhance turfgrass function when exposed to heat and drought stresses. Plant health activators are used alone or mixed with other products to enhance plant health, in an attempt to reduce pesticide usage amid environmental concerns. A common Plant health activator used in turfgrass management is acibenzolar-S-methyl (ASM) which activates the systemic acquired resistance (SAR) pathway. However, little is known about the physiological costs and benefits that may be associated with the continuous activation of these resistance pathways, and how they may affect abiotic stress resistance. Creeping bentgrass was selected because it is the most widely utilized species for fine turf on golf course greens and fairways in northern climates, and the species is generally considered susceptible to summer stress. In this study the drought tolerance of creeping bentgrass was found to be improved with the application of ASM. Under controlled environment conditions, treatment with ASM resulted in plants with improved turf quality and overall health. This improvement was dependent on cultivar, specifically the stress susceptible cultivar showed a greater response to treatment with ASM in measured visual and physiological symptoms. Often, in field conditions ASM is applied with a fungicide to help control disease and improve plant health. In this study no differences were seen between the fungicide alone or in combination with ASM. Further metabolic analysis showed that treatment with ASM resulted in large change in the accumulation and depletion of a variety compounds such as amino acid derivatives, carboxylic acids, and phenylpropanoids. Creeping bentgrass grown under low nitrogen conditions showed a decline in turf quality and plant health, however the application of ASM helped plants maintain a higher turf quality in both optimal and high temperature treatments. Additionally, in both controlled environment and field conditions nitrogen status did not negatively affect the ability of creeping bentgrass to accumulate compounds which were previously found to accumulate. The results of these studies provide a better understanding of how plant health activators impact abiotic stress tolerance and help to provide a better understanding of how these products can be effectively integrated with current management practices.
Interactions between floral mutualists and antagonists, and consequences for plant reproduction
(2013-02) Soper Gorden, Nicole Leland
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.
The Evolutionary Genetics of Seed Shattering and Flowering Time, Two Weed Adaptive Traits in US Weedy Rice
(2012-09) Thurber, Carrie S.
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.
Novel Systems for the Functional Characterization of Genes Related to Paclitaxel Metabolism in Taxus Cell Cultures
(2011-05) Vongpaseuth, Khamkeo
Human society has benefited greatly from plant secondary metabolites, often utilizing a variety of compounds as dyes, food additives, and drugs. In particular, pharmaceutical development has benefited greatly from plant secondary metabolites. One example of this utility is paclitaxel, a highly substituted diterpene approved in the treatment of breast cancer, ovarian cancer, non-small cell lung cancer, and the AIDSrelated Kaposi’s sarcoma. Demand of paclitaxel is likely to increase, due to the current examination of paclitaxel in numerous clinical trials against a variety of other cancers. Taxus cell culture represents a production source of paclitaxel to meet future demand. However, paclitaxel production through Taxus cell culture is often variable and low. Targeted metabolic engineering of Taxus to produce superior paclitaxelaccumulating lines is a viable strategy to address variable and low yields. To facilitate the production of genetically engineered Taxus cell lines, stable transformation is required to examine the long-term effect of gene expression in vitro. Additionally, suitable transient transformation systems are necessary to characterize novel Taxus genes related to paclitaxel accumulation. A transient particle bombardment-mediated transformation protocol was developed to introduce transgenes into Taxus cells in vitro. Additionally, agroinfiltration in Nicotiana benthamiana was examined as a system to express genes related to paclitaxel biosynthesis and lead to the accumulation of the first dedicated taxane, taxa- 4(5), 11(12)-diene. In regard to stable transformation, an Agrobacterium-mediated transformation protocol was developed, though this method requires further optimization for reliability and increased transformation efficiency. These transformation technologies will aid in the creation of elite paclitaxel-accumulating Taxus cell lines.
Conservation While Under Invasion: Insights from a rare Hemiparasitic Plant, Swamp Lousewort (Pedicularis lanceolata Michx.)
(2010-09) Record, Sydne
with non-native invasive species is considered a major threat to many rare native species. As such, invasives removals are a common management strategy. Rare native species that interact uniquely with other organisms in their community (e.g., hemiparasitic plants) may be adversely affected by removing invasives. A management plan for a regionally rare hemiparasitic plant in Massachusetts, Pedicularis lanceolata Michx., identified invasives as a threat, but more quantitative evidence is needed to determine how P. lanceolata‟s persistence is influenced by its co-occurrence with native or invasive hosts. This research asks how P. lanceolata is affected by growth with native versus invasive hosts. Chapter I describes the species associated with P. lanceolata throughout its range, comparing areas where it is considered common and rare. Relative abundances of natives, non-native invasives, non-native non-invasives, and species with both native and non-native genotypes growing with P. lanceolata did not differ significantly at sites where the species is considered common in the Midwest compared to sites where the species is considered rare in the east. Chapter II outlines greenhouse and field removal experiments in which the types of host plants growing with P. lanceolata were manipulated. In the greenhouse, P. lanceolata growth, survival, and flowering were lower when it was growing with invasive compared to native graminoids. However, differences in P. lanceolata growth and survival when natives versus non-native were removed in the field varied from year to year due to succession of native shrubs at the site during the study. Chapter III asks how the population growth of P. lanceolata differs in uninvaded and invaded patches using an Integral Projection Model to perform population projections, sensitivity and elasticity analyses, and a life table response experiment. The population growth rate of P. lanceolata in uninvaded patches was lower than in invaded patches due to the succession of native shrubs in uninvaded patches. Chapter IV describes a metapopulation model for the invaded population of P. lanceolata in Massachusetts. The quasi-extinction probability was significantly affected by probabilities of dispersal, positive correlations in vital rates between sites, and catastrophes. These data will be used to update the management plan for P. lanceolata.
Analyses of Arabidopsis Yellow Stripe-Like (YSL) Family of Metal Transporters
(2010-02) Chu, Heng-Hsuan
Iron is one of the most important micronutrients used by living organisms. Iron is frequently a limiting nutrient for plant growth, and plants are a major source of iron for human nutrition. The most prominent symptom of iron deficiency in plants is interveinal chlorosis, or yellowing between the veins, which appears first in the youngest leaves. Iron deficiency anemia (IDA) is the number one human nutritional deficiency worldwide. In order to solve the problem of iron deficiency, it is desirable to breed plants that have increased iron in those parts that are consumed by humans. To do this, we must first understand the molecular basis of Fe uptake, transport, and storage in plants. In soil, iron is quickly oxidized to Fe(III), and Fe(III) is relatively insoluble, thus difficult for plants to obtain. Our lab has been working on metal ion homeostasis mechanisms in plants and the ultimate goal of our research is to understand the mechanisms by which plants maintain the correct levels of iron, zinc and copper in each cell and tissue. The Yellow Stripe-like (YSL) family of proteins has been identified based on sequence similarity to maize Yellow stripe 1 (YS1). YS1 transports Fe(III) that is complexed by phytosiderophores (PS), strong Fe(III) chelators of the mugineic acid family of compounds. Non-grass species of plants neither make nor use PS, yet YSL family members are found in non-grass species including Arabidopsis thaliana. YSLs in non-grasses have been hypothesized to transport metals that are complexed by nicotianamine (NA), an iron chelator that is structurally similar to PS and which is found in all higher plants. In this dissertation, Arabidopsis YSL1 and YSL3 are demonstrated to be important in iron transport and also responsible for loading Fe, Cu, and Zn from leaves into seeds. Arabidopsis YSL4 and YSL6 are demonstrated to be involved in iron transport and metal mobilization into seeds. The transport function of Arabidopsis YSL1 and YSL2 are shown be partially overlapping to the function of Arabidopsis YSL3 in vegetative structures, but distinct in reproductive organs. Arabidopsis YSL3 and YSL6 are shown to have distinct functions in planta.
The Effect of Management Practices on Bacterial, Fungal, and Nematode Communities on Cool Season Turfgrass
(2017-05) Allan-Perkins, Elisha
Golf courses comprise 50 million acres in the United States of highly managed turf susceptible to abiotic and biotic stressors. A growing area of interest is utilizing microbes to improve plant growth, increase disease and stress tolerance, and reduce pathogens. In order to develop these new practices, we must gain an understanding of turfgrass microbial communities and how they are affected by management practices. We characterized bacteria, fungi, and nematodes on three golf courses: one organic, one with reduced inputs, and one conventional. We took samples from three management areas on each course representing different management intensities (roughs, fairways, and putting greens). This is the first study to our knowledge to use metagenomics to describe bacteria and fungi on all three management areas of golf courses. The conventional and hybrid putting green were most similar to one another in nematode and microbe community composition than to the roughs and fairways of their respective courses or of the organic putting green. The organic putting green differed markedly in the high number of beneficial bacterivore nematodes and low number of herbivore nematodes compared to the conventional and hybrid putting greens. Management intensity affected fungal but not bacterial abundance, diversity, and richness. Canonical correspondence analysis and multiple stepwise regression analyses revealed pH, phosphorous, and organic matter were positively related to increased herbivore nematodes and negatively related to increased bacterivore nematodes, however there was no separation of fungal or bacterial communities based on soil properties. Lastly, we investigated the abundance of bacteria, fungi, and specifically the turf pathogen Sclerotinia homoeocarpa in the soil and thatch of the three golf courses on the three management areas and determined that fungal abundance is always greater in the thatch. S. homoeocarpa abundance did not vary among management areas on the soil or thatch, suggesting the fungal inoculum is unaffected by different management intensities. The results of our study provide baseline data on the nematode, bacterial, and fungal communities on golf courses under different management intensities. The results will help in developing future research studies to examine how cultural practices can be used to increase turf health and decrease disease severity, optimizing biocontrol organism activity, and decreasing herbivore nematode populations while increasing beneficial bacterivores.
Uncovering the Genetic Basis for Biofuel-Related Traits in Brachypodium Distachyon
(2016-09) Lee, Scott J
Biofuels derived from plant biomass present a promising avenue to address the negative aspects of fossil-fuel dependence. The sustainability of biofuel production relies in part on the efficient degradation of lignocellulosic feedstocks. In order to capitalize on the potential of lignocellulosic biofuels, the genes underlying natural genetic variation for conversion efficiency must be determined. We have developed a robust and high-throughput assay to measure feedstock quality using the anaerobic bacterium Clostridium phytofermentans. We have measured biomass accumulation phenotypes and utilized this assay to perform quantitative trait locus (QTL) mapping and a genome-wide association study (GWAS) in the model grass species Brachypodium distachyon. We detected four biomass accumulation QTLs and one bioconversion QTL, BIOFUEL1 (BFL1). We additionally found four significant total biomass accumulation marker-trait associations (MTAs) and two bioconversion MTAs within our GWAS. We developed near-isogenic lines and confirmed the effect of the BFL1 QTL and provide evidence that Bradi2g01480, a glucosyltransferase belonging to CaZY family 61 is the current best candidate for underling this QTL. Additionally, we have performed whole-genome resequencing on a total of 42 B. distachyon accessions at an average coverage of 72x to accelerate candidate gene discovery. These accessions will join the growing genetic resources for B. distachyon, enabling even more robust association studies in the future. The discovery of genomic regions significantly associated with biomass accumulation and conversion phenotypes should enable more rapid gene discovery. Only by uncovering the genes regulating these biofuel-related phenotypes can there be efficient and targeted development of improved, dedicated biofuel feedstocks.
The Role of the Formin Protein Family in Membrane Dynamics
(2016-05) van Gisbergen, Petrus Adrianus Cornelis
Using molecular genetics, and high end imaging techniques, I assessed the function of the formin protein family in the moss Physcomitrella patens. Formins are proteins that can nucleate and elongate actin filaments. P. patens has 9 formins, divided over three classes. I found that a class II formin (For2A) is essential for polarized growth and specifically binds to the phosphoinositide PI(3,5)P2. Additionally, I show that this formin polymerizes actin filaments in vivo. I demonstrated that binding PI(3,5)P2 is essential for formin function. My work also shows that one of the class I formins (For1F) is involved in exocytosis and likely is a part of the exocyst tethering complex, directly linking exocytosis to the actin cytoskeleton in plants. For1F is an essential gene, but its deletion can be rescued by overexpression of For1D, another class I formin, suggesting that class I formins are involved in exocytosis. Class I formins associate with actin filaments, but their interaction with actin differs from class II formin interaction with actin. Drug treatments show that their dynamics are dependent on both microtubules and actin filaments. This is in contrast to class II formins that do localize to endocytic sites and whose dynamics are only dependent on actin filaments. An endocytic marker can be seen traveling with the processive formin For2A when For2A is polymerizing an actin filament. Quantification of the activity of For2A along the length of tip growing cells reveals that For2A preferentially generates actin filaments towards the tip of the cell. This provides an actin array that is predominantly tip-oriented and could serve as a scaffold for myosins to transport cargo along towards the cell tip.
Chemically Mediated Interactions Between Hosts, Parasitic Plants and Insect Herbivores
(2016-05) Tjiurutue, Muvari C
Species interactions, by changing phenotypic traits, can alter the outcome of subsequent interactions. Plant-mediated responses to herbivores have been extensively studied, but little is known about plant-mediated responses involving parasitic plants within a broader community context that also includes herbivores. Because parasitic plants are important components of many ecosystems and can shape community structure, it is important to understand how host-mediated interactions influence parasite preference and success. The goal of this thesis is to examine interactions between hosts, parasitic plants and herbivores mediated by chemical traits. We first examined the effects of dodder (Cuscuta sp.) parasitism on induced defenses in cranberry, and asked how cranberry chemistry affected dodder preference and performance. We found dodder preference for some cultivars, and dodder parasitism induced many changes in cranberry chemistry, which could influence other interactions with cranberry hosts. We next examined the effects of gypsy moth herbivory on cranberry chemistry, and how plant-mediated changes affected subsequent dodder preference. Herbivory delayed and reduced the number of dodder plants that attached to cranberry hosts. Herbivory also induced changes in cranberry phenolic acids and phytohormones, which could mediate defenses against dodder parasitism. We also assessed the effects of previous herbivory (by tobacco hornworm or mechanical) and previous dodder parasitism on subsequent dodder preference on tomato hosts. Previous attachment followed by removal of dodder slowed subsequent dodder attachment on tomato hosts, but prior herbivory did not affect subsequent dodder attachment. Lastly, we asked whether damage to host induced changes in the host, and if attached parasites assimilated host defenses in response to host damage. Damage to host plants induces higher jasmonic acid in both hosts and attached parasites, and herbivores fed on leaves from parasites attached to damaged hosts ate more than herbivores fed on leaves attached to undamaged parasites. In summary, these studies demonstrate that parasites can induce changes in host responses that can potentially shape other interactions with the same hosts. Similarly, both herbivores and host responses can influence parasite preference, which could alter behavior of herbivores and pollinators, shaping community dynamics.
The Roles of Myosin XI and ROP in Moss Tip Growth
(2014) Burkart, Graham M
Because of the large number of myosin XI and ROP genes found in many angiosperms, it has been difficult to determine their precise role with respect to tip growth. In contrast, there are only two myosin XI genes in four ROP genes in the moss Physcomitrella patens. To determine their role in tip growth using a loss-of-function approach, I used RNA interference (RNAi) and found that both of these proteins are essential for tip growth. Consistent with a role in tip growth, I show that a functional, full-length fusion of mEGFP to myosin XI accumulates at a subcortical, apical region of actively growing protonemal cells. Myosin XI RNAi plants also appear to have decreased cellulose in the cell wall, suggesting a role in secretion of cellulose synthases. I found that silencing ROP increases cortical actin dynamics but does not appear to have a specific affect on the microtubule cytoskeleton. Further investigation found that ROP recruits class II formins to the cell cortex where they actively nucleate and elongate actin filaments. Loss of ROP also causes a decrease in intracellular adhesion. Unlike myosin XI RNAi plants, examination of the crystalline cellulose content of the cell wall shows that the deposition of the cell wall is not inhibited in the absence of ROP. Taken together my findings suggest that ROP defines a membrane region where myosin XI delivers secretory vesicles containing cellulose synthase and other materials needed to build new cell wall during tip growth.
Evaluation of Spring Frost Control Methods and an Assessment of Cold Hardiness in Cranberry (Vaccinium macrocarporn Ait.)
(2015-09) Ndlovu, Faith
The American cranberry (Vaccinium macrocarpon Ait.) is an important temperate woody shrub crop whose fruit has human health benefits. Cranberry acquires cold hardiness in the autumn and loses it in spring, following deacclimation. Frost protection is necessary in cranberry production as a means to reduce bud damage due to low spring temperatures. The objectives of the field studies were to evaluate two methods of sprinkler irrigation for frost protection, the conventional approach consisting of continuous irrigation throughout the night (CON) and intermittent cycling of sprinklers (INT) incorporating cycling on and off throughout the night, by (i) assessing bud damage and yield for cranberry cultivars 'Early Black', 'Howes', and 'Stevens' managed under both methods and (ii) to determine the volume of water used in each method. The objectives of the laboratory studies were to (i) evaluate and quantify carbohydrates and lipids synthesized by cranberry during the cold acclimation period under a controlled environment and to (ii) determine the cold hardiness (LT50) and lowest survival temperature (LST) of buds of economically important cranberry cultivars during acclimation. For INT to be an effective frost control method, temperature set points should be cultivar specific. Despite differences in bud damage, cranberry yield data did not show any significant differences between the two frost management methods. Since cranberry plants produce more flowers than the number of set fruit that can be supported, the remaining flowers in damaged buds may have been sufficient to produce similar yields in both methods. Substantial water savings were obtained under INT, especially on mild frost nights. Greater concentrations of total non-structural carbohydrates (TNSC) and membrane stabilizing lipids and a higher fatty acid unsaturation index were associated with low acclimation temperatures. This result suggests the importance of these compounds in increasing cold hardiness in cranberry during acclimation. In addition, a progression of freezing tolerance, determined as the LT50 and LST, was noted in the fall for all the cranberry cultivars. Knowledge of bud hardiness in the fall is important in considering the need to protect buds. Differences in hardiness should be considered when implementing frost protection in the fall.
The Evolution and Development of Awns in the Grass Subfamily Pooideae
(2024-02) Patterson, Erin L
This research focuses on a specific example of replicated evolution: the grass awn. Awns are typically extensions of the lemma, but may also appear on glumes or paleas. The lemma is a leaf-like organ on the exterior of the grass flower, the glumes are a pair of bracts subtending the basic unit of grass inflorescences, the spikelet, and the palea is the floral organ opposite the lemma. Awns are often described as "hair-" or "bristle-" like, but appear in many different shapes. Many awns are “twisted & geniculate", in which the awn has two sections, a lower twisted column, and an upper straight section (the "subule"). These sections are separated by a bend, or "knee", which gives rise to the term "geniculate". Awns may also be attached, or inserted, to the lemma at different points: either at the tip, apically, or at a point on the back of the lemma, abaxially. Awns are critical for several functions in grasses, and likely contribute to grass dispersal, seedling establishment, and reproductive success. Some awns, like those in barley and wheat, photosynthesize and increase grain weight. Awns are part of a suite of characters that have likely played a key role in the spread of grasses to thrive in almost all terrestrial biomes. I performed a series of experiments aimed at understanding the replicated evolution of awns. I hypothesized that the awned lemma shares homology with the grass leaf, and that conserved leaf genes have been recruited for awn development. To determine the evolutionary history of awns in the Pooideae (the largest grass sub-family), and to select focal species for further study, I performed several ancestral state reconstructions for awn traits in the Pooideae. I discovered that awns have been independently derived at least 30 times in the Pooideae. To test the hypothesis that lemmas and leaves are homologous, I examined the anatomy and micromorphology of several awns from Pooideae species, and found that leaf traits are shared between awns and leaves. Similarly, I examined awn development in three species with independently derived awns and found that they all shared underlying ontogeny with grass leaves. To determine whether the function of straight awns in photosynthesis and contributing to grain weight is conserved in Brachypodium distachyon, I performed an awn removal experiment, and found that awns in B. distachyon do not have a significant role in contributing to grain weight. Finally, I characterized and cloned an awnless mutant in B. distachyon, and showed that a gene with conserved functions in leaf development is responsible for the awnless trait. Further, I find that the ortholog of this gene is also necessary for awn development in Alopecurus myosuroides, a species with independently derived awns. These experiments all support the main hypothesis that lemmas are homologous to leaves, and share the same genetic pathways for development. I find that these instances of replicated evolution are likely supported by developmental constraints, and that heterochrony has a key role in shaping awn development in different species.
DEVELOPMENT, REGULATION, AND FUNCTIONAL CHARACTERIZATION OF SECONDARY CELL WALL FORTIFIED TISSUES IN THE GRASS SHOOT-BORNE ROOT SYSTEM
(2024-02) McCahill, Ian W
Plants depend on their anchorage to the soil to support their own mass and maintain a vertical orientation. Failure to maintain this establishment is called lodging and results in a number of deleterious outcomes, including wounding, reduced photosynthetic output and exposure to hazardous micorenvironments. This work synthesizes findings from the disparate fields of biophysics and plant molecular genetics to uncover a novel strategy by which plants deposit thick secondary cell walls to proactively maintain their root anchorage. In the first chapter, I present a review of plant secondary cell wall regulation, including recent findings relating to environmental modulation of cell wall development. In the second chapter, I describe the development, and the genetic and environmental regulation of secondary cell wall fortified cortex cells in the grass shoot-borne root system.
ESCAPING DOMESTICATION: HOW PARALLEL MODIFICATIONS OF SEED SHATTERING LED TO RECURRENT WEEDY RICE EVOLUTION
(2024-02) Li, Xiang
As a typical example of convergent evolution, seed shattering, or seed dispersal, has undergone repeated modifications throughout the evolutionary history of rice. This trait has been actively selected against in independently domesticated cultivated rice (Oryza sativa) populations but has been reacquired in independently evolved weedy rice (Oryza spp.) populations during de-domestication. My Ph.D. projects are dedicated to exploring the morphological and genetic bases of seed shattering in both cultivated and weedy rice from three distinct evolutionary lineages, including aus, indica and japonica. Through examining the morphology of the abscission zone (AZ), the specialized tissue responsible for seed detachment, across these cultivated and weedy rice populations, I found distinctive morphological adaptations employed by different cultivars to achieve their low shattering abilities. In contrast, high shattering weedy rice individuals exhibited a high degree of AZ convergence, despite their diverse origins. To ascertain whether the same or different loci contribute to the evolution of high shattering in independently evolved weedy rice populations, I conducted Quantitative Trait Loci (QTL) mapping in F2 progeny of a cross between a cultivar and its derived weed in the aus lineage. I identified five QTLs for seed shattering, some of which overlapped with regions showing signatures of positive selection. I found, however, that few detected QTLs overlapped with seed shattering QTLs identified in other weed x cultivar crosses, indicating a lack of genetic convergence in the evolution of shattering across weedy populations. Additionally, I performed comparative tissue-specific transcriptomic analyses to profile the gene expression in the AZ and its surrounding tissues in cultivated and weedy rice from distinct lineages across development. Different genes were found to explain the AZ morphological differences between cultivated and weedy rice in different lineages. The few overlapping genes between the lineages are likely involved in determining and maintaining AZ cell identity, potentially through modifying lignin and secondary cell wall deposition in these cells. My projects not only increase our understanding of seed shattering from both morphological and genetic perspectives, but also illustrate the extent of genetic and morphological convergence underlying the convergent evolution of seed shattering in distinct lineages.
Elucidating Mechanisms of Shoot-to-root Iron Deficiency Signaling in Arabidopsis thaliana
(2024-02) Bakirbas, Ahmet
Iron is an essential micronutrient for plant development and is required in many enzymatic processes. Because excess iron could cause oxidative damage in cells, plants strictly regulate iron uptake to ensure that only the required amount of iron is present in cells and tissues. For these reasons, plants have developed complex mechanisms to tightly regulate iron uptake, use, and storage while adapting to changes in iron concentration in their environment. The primary objective of this dissertation is to explore the mechanisms underlying iron homeostasis in Arabidopsis thaliana. While working on iron signaling, I explored potential signals in the phloem, as well as key genes that regulate metal concentrations in vascular tissues, where iron sensing is hypothesized to occur. The first chapter details the discovery of a novel long non-coding RNA (lncRNA), CAN OF SPINACH (COS), which regulates iron deficiency responses in Arabidopsis. Transcriptome analysis of whole shoots during iron deficiency led to the discovery of COS and also provided a broad overview of gene expression changes in response to iron levels in whole shoots. Using genetic analysis of COS mutants, the subtle but significant roles of this novel lncRNA were uncovered. The second chapter investigates the small RNA landscape in the phloem sap of Arabidopsis, focusing on the sRNAs altered in response to iron deficiency. I identified a group of small RNAs with altered expression in phloem sap during iron-deficient conditions. This work also established for the first time that most sRNA in Arabidopsis phloem exudates are tRNA-derived fragments, specifically 5’ tRFs and 5’ tRNA halves. The results from this chapter suggest that there is selectivity for loading, or perhaps for retaining, particular sRNAs in the phloem sap. In the last chapter, I characterized the roles and interplay of iron transporters YSL1, YSL3, and OPT3 in the vascular system of Arabidopsis. Through the use of transgenic plants, confocal microscopy, genetic crosses, and careful phenotyping, I revealed the contributions of these transporters in iron redistribution and cellular iron availability, thereby offering new insights into the complex regulation of iron in Arabidopsis. These findings collectively contribute to a more nuanced and comprehensive understanding of iron homeostasis in Arabidopsis. They hold significant implications for plant molecular genetics and agriculture, where soil nutrient management is crucial for sustainable crop production. My Ph.D. research may open avenues for targeted genetic modifications to improve iron uptake and storage in crop plants, potentially addressing issues of nutrient deficiency in agriculture.
UNCOVERING NEW ELEMENTS OF IRON HOMEOSTASIS IN MAIZE
(2023-09) Fili, Stavroula N
This research aimed to contribute to the current understanding of the molecular mechanisms of iron homeostasis in plants, specifically in grasses. Iron (Fe) is a micronutrient essential for plant growth and development, involved in processes such as photosynthesis and respiration. Iron is also crucial in the human diet, as insufficient iron intake leads to iron deficiency anemia, which affects about 25% of the world's population. Plants acquire iron from the soil through the roots and translocate it to other tissues, including the edible grains. These processes are highly regulated through multi-component networks, to ensure that potential toxic effects of excess iron are managed. Because iron is naturally insoluble in the soil, plants have developed mechanisms to increase its uptake. There are two distinct uptake mechanisms, Strategy I, performed by non-grass plant species and Strategy II, performed by grass species. Our knowledge of the mechanisms taking place in grasses is less detailed than in non-grasses. Most cereal crops are grasses, highlighting the need to uncover more aspects of their iron response. My research focused on gene discovery, a common approach for the nutritional improvement of crops. During this time, I aimed to identify new components of iron homeostasis in maize and understand their role in iron uptake. As a first step, I analyzed the maize transcriptomic response to iron deficiency and established a set of iron-responding genes in maize. To determine their possible roles in iron homeostasis, individual genes were investigated for similarity to those in other, better-characterized species. Additionally, I analyzed novel iron-deficient maize mutants that expand the list of critical iron-homeostasis genes. These were identified by screening mutant stocks for the iron-deficient (yellow-striped) phenotype. Mutants were analyzed using genomic approaches for variations that cause detrimental gene effects. One of these mutants, ivy (interveinal yellow), was determined to be iron deficient, and the underlying gene was confirmed as ZmCCD8, a key enzyme in the biosynthesis of strigolactones. Even though these hormones have been found to participate in various plant processes, they have not been previously linked to iron homeostasis. Mutant plants had altered iron-regulated gene expression. Comparison of the transcriptomes of wild-type iron-deficient plants and maize ccd8 mutants revealed suppression of several genes of the iron uptake machinery in ccd8. These genes are normally upregulated during iron deficiency and include the iron-regulated transcription factor IRO2 and genes involved in the biosynthesis of iron chelators and transporters. Additionally, external supply of synthetic strigolactone in the mutants alleviated chlorosis and returned iron-regulated gene expression to basal levels. The gene expression in the ccd8 mutants responded normally during limited iron conditions, indicating that strigolactones are not required for the iron deficiency response. I have hypothesized that strigolactones contribute to normal expression of iron-regulated genes in maize under sufficient iron conditions and may act to enhance iron uptake during iron deficiency.
INVESTIGATING RHIZOBIA AND MOLECULAR DETERMINANTS OF SUCCESSFUL SYMBIOTIC NITROGEN FIXATION IN LEGUMES
(2023-09) Chilufya, Jedaidah Yombwe
Legumes can thrive in nitrogen-limited soil by forming a symbiotic relationship with nitrogen-fixing bacteria called rhizobia. This allows them to grow sustainably beyond their native regions. Examples include sunn hemp (Crotalaria juncea L.) and barrel clover (Medicago truncatula) that have successfully expanded from India and the Mediterranean. Symbiotic nitrogen fixation occurs on specialized root structures called nodules and involves complex interactions between the symbiotic partners with initial molecular communication leading to nodule formation and subsequent nitrogen fixation in nodules. However, not all legume - rhizobia interactions result in effective nitrogen fixation due to factors including compatibility between legume and rhizobia, and transport of host molecules to the rhizobia. Therefore, to address these issues, this dissertation investigates the compatibility between tropical sunn hemp and US-native rhizobia for nodule formation and fixation. Additionally, this study develops a CRISPR/Cas9 gene-editing tool as a resource to examine the functional importance of nodule-specific protein molecules, nodulin-22 (NOD22) and nodulin-25 (NOD25) in producing nitrogen-fixing nodules in Medicago truncatula. I show that Massachusetts-native rhizobia fail to effectively fix nitrogen for sunn hemp, resulting in suboptimal growth benefits. This contrasts with non-native rhizobia found in commercial inoculants, which proved more effective at fixing nitrogen and promoting sunn hemp growth. These non-native rhizobia in commercial inoculant persisted in the soil as nitrogen fixers for sunn hemp for up to three years. Additionally, it was discovered that the Massachusetts-native rhizobia that did not benefit sunn hemp, were instead compatible with hairy vetch (Vicia villosa), another legume cover crop previously established in the region. Moreover, compatibility studies conducted in different parts of the US revealed varying results, including nodulation and nitrogen fixation incompatibilities.Next, I developed a gene-knockout tool using a vector-based CRISPR/Cas9 system to facilitate the assessment of the functional significance of two legume nodule - specific genes namely nodulin-22 (NOD22) and nodulin-25 (NOD25), in producing nitrogen fixing nodules in Medicago truncatula. Previously, these protein molecules have been studied individually, but their roles in nitrogen fixation remain unknown. Analyses via PCR-genotyping and Sanger sequencing demonstrated the successful deletion of a substantial 12 kb DNA fragment by my CRISPR/Cas9 vector, potentially inactivating NOD22 and NOD25 genes. Medicago plants exhibiting this deletion displayed stunted shoot growth and contained small, round and white nonfixing nodules. When compared to wild type pink nodules that fixed nitrogen for Medicago plants, the non-fixing nodules had very few rhizobia. The availability of the CRISPR/Cas9-NOD22/25 vector is expected to furnish mutants, thereby advancing functional studies regarding nodulin proteins. Collectively, understanding how root nodules function in nitrogen fixation in legumes is crucial for farmers to identify rhizobia that facilitates nitrogen fixation, supporting the growth of non-native legumes in the US. Additionally, researchers can gain insights into the biological significance of legume protein molecules during the process of symbiotic nitrogen fixation.
UNCOVERING TRANSCRIPTOMICS RESPONSES AND REGULATIONS DURING FUSARIUM OXYSPORUM – PLANT INTERACTIONS
(2023-09) Yu, Houlin
Fusarium oxysporum causes devastating wilt diseases in more than one hundred crops. To provide a foundation for developing technologies to enhance plant health, sustain a healthy ecosystem, and feed a continuously growing human population, my dissertation research has focused on uncovering transcriptional responses and regulations during F. oxysporum and plant interactions from fungi and plant host aspects. Through a literature review (Chapter 1), we learned that accessory chromosomes (ACs) and transcriptional regulators are essential for the host-specific virulence of F. oxysporum. Transcription factors, key regulatory elements in the sensory and response networks of these fungi, undoubtedly play a fundamental role in establishing the adaptability of this group. We compared the TFome (Chapter 2), which consists of all TFs found within a genome, to look at a species' evolutionary history of its regulatory mechanisms. The study revealed both the conservation and diversity of F. oxysporum TFome. The ACs from each F. oxysporum genome likely dictate strain-specific interactions with a particular host. This allows a comparative study that minimizes genetic differences between strains to address the underlying mechanism that results in distinct phenotypes (e.g., pathogenic vs. non-pathogenic outcomes). We performed a comparative transcriptomics study (Chapter 3) of infection by an endophytic (Fo47) and a pathogenic (Fo5176) strain of F. oxysporum in the context of the F. oxysporum-Arabidopsis pathosystem, which revealed the transcriptional plasticity of plant defense responses. F. oxysporum penetrates the root epidermis, propagating and moving toward the vasculature. These pathogens' occupation of the vascular system blocks water and nutrient transport, further causing devastating wilt disease. Extensive efforts have been taken by bulk transcriptome profiling to probe the integral plant responses to the F. oxysporum stress. However, as the pathogen journeys through multiple layers to establish the infection, different root cell types likely respond differently. We performed a single-nucleus RNA sequencing analysis in the Arabidopsis-F. oxysporum pathosystem (Chapter 4). Our research revealed a unique pathogen-induced cell cluster enriched for defense-related functions that could aid in developing new strategies to improve plant defense mechanisms.
MODULATION OF PLANT IMMUNITY DURING THE ESTABLISHMENT OF THE NITROGEN-FIXING SYMBIOSIS
(2023-02) Hernandez-Romero, Miriam
Nitrogen is essential for plant tissue growth but is often a limited resource in soils. Many legumes overcome this limitation by entering a symbiotic association with soil microbes, called rhizobia, which provide nitrogen to the plant while rhizobia receive fixed carbon. To successfully form a symbiosis, the host and symbiont exchange a series of molecular signals. One major obstacle during this interaction is the host's innate immune system, which becomes active upon rhizobial detection. It is therefore the main focus of this thesis to identify the mechanisms that modulate host immunity. In the subsequent chapters, we focus on a rhizobial mechanism of defense response evasion relevant in early stages of root infection, and a later stage, nodule-specific mechanism of host defense response suppression. Here, we demonstrated that the abundantly expressed rhizobial exopolysaccharide-I (EPS-I) of Sinorhizobium meliloti provided a survival advantage in the Medicago truncatula mutant, called defective in nitrogen fixation 2 (dnf2) which exhibits enhanced host defenses in nodules. We then determined that rhizobial EPS-I provided protection against MAMP-triggered immune defenses activated by host pattern recognition receptors of the plant family of LysM domain-containing proteins, called LYM1 and LYM2. We determined that the underlying mechanism of protection conferred by EPS-I was the suppression of the reactive oxygen species burst upon detection of rhizobia. In addition, we provided insight into the nodule-specific mechanism of host defense suppression during nodule cell invasion by rhizobia. We showed that inactive LYM1 and LYM2 proteins within nodules of the dnf2 mutant allowed for the progression of infection by rhizobia.

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