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Publication Development of a Cytosolic pH Reporter for Tobacco By2 Cells(2012-01-01) Urbanowski, Michael EThe regulation of pH is a critical homeostatic function of plant cells. In addition to acting as the primary cationic species responsible for energizing the plasma membrane, protons likely act as an important regulator and messenger. Despite this importance, few studies have thoroughly described cytosolic pH patterns as the plant cell progresses through the cell cycle. To investigate pH in plant cells, I chose Nicotiana tabacum (tobacco) Bright Yellow-2 (BY-2) cells as a model system. My research has two aims. First, I will measure and report the interphase cytosolic pH of BY-2 cells. Next, I will assay the cytosolic pH as BY-2 cells progress through mitosis and cytokinesis. I hypothesize that pH patterns are be temporally or spatially associated with structures such as the mitotic spindle or the phragmoplast. To investigate cytosolic pH in BY-2 cells, I will develop a cytosolic pH reporter based on a pH sensitive ratiometric fluorescent dye. This dye will be able to resolve both temporal and spatial changes in pH throughout the cytosol while imposing a minimal amount of stress on BY-2 cells. I found that pH-GFP, a variant of eGFP, had qualities of a robust pH reporter. To introduce the dye, explored biolistic bombardment, Agrobacterium mediated transient transformation, and polyethylene glycol mediated transformation as methods for introducing the pH-GFP gene into BY-2 cells. I observed very few transformation events using these methods and my observations did not support these approaches as suitable for introducing pH-GFP into BY-2 cells.Publication Investigation of Maize Yellow-stripe1 and Iron Signaling in Arabidopsis thaliana(2012-05) Vasques, Kenneth AIron is an essential micronutrient that plays a role in essential processes in all living organisms. Because iron deficiency anemia is the number one human nutritional deficiency worldwide, research has been focused on studying biofortification, a method of plant breeding focused on increasing the nutrient content of the grain. In order to effectively implement this strategy, research to understand the molecular mechanisms surrounding iron uptake and maintenance within plants is necessary. A major goal of this work lies in exploring iron signaling in Arabidopsis thaliana and further characterization of the yellowstripe1-like1yellowstripe1-like3 (ysl1ysl3) double mutant plant. As shown here, iron signaling in arabidopsis appears to be regulated by a long distance signal derived in the shoots. The ysl1ysl3 double mutant is a plant shown to have severe mis-regulation of iron regulated genes. Here, I show ysl1ysl3 mutant plants are unable to respond to iron deficiency properly and lose signaling ability. It has been proposed that these defects are due to iron accumulation in the shoots but that appears untrue suggesting alternative functions for YSL1 and YSL3 in iron signaling. Another goal of the work included here seeks to elucidate alternative roles for ZmYS1 in maize outside of primary acquisition of iron from the soil. Here, ZmYS1 expression is shown via promoterPublication A Molecular Phylogenetic Assessment of Pseudendoclonium(2007-09) Mullins, Richard F.Pseudendoclonium was established in 1900 by N. Wille to include a crust-forming green microalga occurring near the high water line on jetties in Drobak, Norway. Ordinal and familial affiliation of the genus have remained uncertain due to a lack of distinguishing morphological characteristics and because molecular phylogenetic data have not been generated for the type species. Ribosomal SSU rDNA sequence data for Pseudendoclonium submarinum, the type species, are presented. Phylogenetic analysis of these data place Pseudendoclonium within the Ulvales. SSU rDNA sequence data from three additional species, Pseudendoclonium basiliense, Pseudendoclonium akinetum and Pseudendoclonium fucicola are included in the analyses and clearly support the hypothesis that Pseudendoclonium is polyphyletic. Based on the sequence data, P. submarinum and P. fucicola share ulvalean lineage, but these algae are not congeneric and P. fucicola must be removed from Pseudendoclonium. Sequence data support the classification of P. basiliense and P. akinetum as distinct species of a single genus. The close affiliation of these two species with Ulothrix and other Ulotrichalean genera, however, reveals their ordinal separation from P. submarinum. P. basiliense and P. akinetum must also be removed from Pseudendoclonium and require generic reassignment within the Ulotrichales.Publication Functional Analysis of Receptor-like Kinases in Pollen-Pistil Interactions in Arabidopsis thaliana(2009-09) Aggarwal, MiniRac/Rop GTPases are molecular switches in plants that control the growth of polarized cells such as pollen tubes and root hairs, differentiation, development, actin dynamics, production of reactive oxygen species (ROS) and disease resistance. These small GTPases are activated by guanine nucleotide exchange factors (GEFs) that replace GDP for GTP and are referred to as RopGEFs in plants. To identify upstream components of the RopGEF regulated signaling pathways, GEF1 from Arabidopsis thaliana was used as a bait to screen a seedling cDNA library in a yeast two-hybrid system. This yielded members of a small family of the Catharanthus roseus Receptor-like kinase (CrRLK), referred to as the Feronia-like Receptor-like kinase (FlRLK) family as the potential GEF interactors. A synergid cell-expressed member of this family, FERONIA/SIRENE (RLK-10), regulates pollen tube reception by the female gametophyte and along with the other two plasma-membrane bound receptors of this family, THESEUS (RLK-3) and HERCULES (RLK-4) promote cell elongation in Arabidopsis. I have chosen two of these FlRLKs for my studies: RLK-5, the most abundant pollen expressed member of the family and RLK10. My studies with RLK-5 suggest that it plays a significant role in pollination or fertilization since homozygous rlk-5 was never recovered from selfed heterozygous rlk-5 mutant. In addition to this, the reciprocal crosses with RLK-5/rlk-5 and wild type resulted in severe male transmission defect indicating that the rlk-5 mutation induces male sterility. This observation was found consistent with the pollen-specific expression pattern of RLK-5 suggested by microarray data and confirmed by histochemical GUS staining analysis of the RLK5p-GUS transgenic Arabidopsis. The RLK-5/rlk-5 pollen displayed no pollen-viability defects and the pollen tube growth in-vivo appears normal. Despite the unaffected pollen viability and apparent normal in-vivo pollen tube growth, the RLK-5/rlk-5 plants formed low seed set suggesting compromised fertilization. Additional analysis will be required to determine the basis of male deficiency and reduced seed sets in RLK5/rlk5 mutants. Loss of function mutations of RLK-10 (FERONIA/SIRENE) gene show failure in pollen tube growth arrest upon penetration of the female gametophyte, supernumerary pollen tube penetration of the ovule and reduced female fertility. Our studies with RLK-10 suggest that it is important for mediating a proper oxido-reductive condition within the ovule necessary for pollen tube rupture and fertilization of the female gametophyte. The multiple pollen tube entry phenotype of the Arabidopsis knockout mutant, rlk-10 correlated with decreased ROS level and de-esterified pectin in the filiform apparatus lining the synergid cell of its female gametophyte. These results establish an important signaling link between RLK-10 and ROS and pectin in a GEF-Rac/Rop regulated pathway in pollen tube-ovule interaction.Publication Publication Analysis of Myosin Viii Function in the Moss Physcomitrella Patens(2009-01-01) Ritchie, JuliePublication Site Characteristics and Plant Invasion: Light Limitation of Invasive Establishment and Impacts of Elaeagnus Umbellata on Soil Nitrogen Availability and Co-occurring Species(2008-09) Mostoller, Erin LInvasive species have become an increasing threat to many habitats worldwide. This research addressed the questions of whether several invasive woody plants can germinate, survive and grow in light levels typical of an undisturbed forest understory, and also whether alteration of the nitrogen cycle by one invader is likely to increase the rate of invasion by others.Publication Self-Incompatibility in African Lycium (Solanaceae)(2008-01-01) Feliciano, Natalie MChapter one of my thesis has been published in the May 2008 issue of the journal Evolution under the title “A TALE OF TWO CONTINENTS: BAKER’S RULE AND THE MAINTENANCE OF SELF-INCOMPATIBILITY IN LYCIUM (SOLANACEAE).” This chapter was co-authored by Dr. Jill S. Miller and Dr. Rachel Levin.Publication The Evolution of Four Hybrid Sterility Loci in Us Weedy Red Rice(2013-05) Craig, Stephanie MThe accumulation of independent mutations in two populations of an ancestral species over time often leads to reproductive isolation. Reproductive isolation between diverging populations is re-enforced by genetic mating barriers that occur either pre- or post-zygotically. Hybrid sterility is the most common form of post-zygotic isolation in plants. Four post-zygotic sterility loci have been recently cloned in Oryza sativa, Sa, s5, DPL1 and DPL2; these loci explain, in part, the limited hybridization that occurs between the domesticated cultivated rice varieties, O. sativa spp. japonica and O. sativa spp. indica. In the United States, cultivated fields of japonica rice are often invaded by conspecific weeds that have been shown to be of indica origin. Crop-weed hybrids have been identified in crop fields, but at low frequencies. This study aimed to examine the role of these known hybrid incompatibility loci in the interaction between cultivated and weedy rice, and further assess the evolutionary processes acting at these loci. We identified a novel neutral allele at Sa, designated SaFX, that seemingly acts to restore fertility. Additionally, we found a strikingly high frequency of neutral-type alleles in weedy populations at multiple loci, suggesting that weedy individuals, particularly those of the SH and BRH groups, should be able to freely hybridize with the local japonica crop.Publication Processes, Patterns, and Predictions of Soil Moisture Variation in Upland and Peatland Cranberry Farms in Massachusetts(2020-05) Brennan, RebeccaThe American Cranberry (Vaccinium macrocarpon Ait.) represents a vital sector of the economy of southeastern Massachusetts. Due to the hydrogeological and edaphic characteristics of peatlands, variations in soil drainage and soil moisture represent major management challenges for growers in Massachusetts. An emerging trend of upland (mineral soil) cranberry farms planted with new hybrid cultivars has the potential to enhance the profitability and long-term viability of cranberry production in Massachusetts. However, sparse data exist on soil moisture characteristics of peatland and upland cranberry farms. The purpose of this research was to elucidate the differences in soil moisture between upland and peatland cranberry farms, to evaluate the soil temperature-moisture relationship and its use for inferring soil moisture, and to explore the use of unmanned aircraft systems (UAS) as a soil moisture management tool in cranberry agriculture.In this thesis, we found that volumetric soil water content (qv) in upland farms ranged from 5-15%, contrasting with values of 10-40% for peatland farms. In general, soil moisture in upland farms was two times drier and four times more uniform than peatlands farms. Our results suggest that open ditches should be dredged to at least 50 cm to obtain irrigation setpoints of -5 to -2 kPa for Massachusetts cranberry farms. We found that soil temperature and near-surface temperature were accurate predictors of soil moisture but were also strongly dependent on the magnitude of differences between air and water temperature. Soil and near-surface temperatures were also better predictors of moisture in soils with lower vegetation coverage and organic matter content. Near-surface temperature collected with a UAS was consistent with field measurements of qv, suggesting that UAS could be used to assist Massachusetts cranberry farmers by predicting large-scale variation in q v and offering management insights.Publication Characterizing the Interaction Between Non-Pathogenic Fusarium Oxysporum and Arabidopsis Thaliana to Determine Beneficial Effects Conferred to the Model Plant Host(2019-09) Vescio, Kathryn IsabelleFusarium oxysporum (Fo) is a soil-borne fungal pathogen that causes vascular wilt disease on a broad range of plants, including agricultural crops and the model plant Arabidopsis thaliana. There are non-pathogenic members of the Fo species complex that confer defense benefits against other pathogens to the host plant, however alteration to the host’s physiology through interaction with one of these strains, Fo47, have not been described. In this study, we aimed to establish the Fo47-A. thaliana interaction and determine if Fo47 reduces disease severity of a pathogenic Fo isolate, Fo5176. Additionally, we sought to use bioinformatics to mine transcriptomic data of the infection between Fo47 and A. thaliana for putative effectors from the non-pathogenic isolate using a pipeline that is validated by identifying known effectors in the interaction between Fo5176 and A. thaliana. Phenotypic characterization of A. thaliana plants inoculated with Fo47 or Fo5176 has revealed a significant increase in rosette biomass of Fo47 inoculated plants when compared to mock (sterile water) inoculated plants. As is observed in other systems, treatment of plants with Fo47 prior to challenging with pathogenic Fo significantly reduces the disease severity over time. The results of this study suggest that Fo47 is a possible biocontrol agent against Fo5176, and that inoculation with non-pathogenic Fo alters the physiology of A. thaliana such that it has a higher rosette biomass without alterations to the water status of the plant. Our pipeline for extracting putative effectors using transcriptomic data as a critical filter generated 13 candidate genes for further experimentation to determine their role in the Fo47-A. thaliana interaction. This research reports the first known observation that Fo47 increases the shoot biomass of the host plant it is interacting with, and that the model plant A. thaliana can be used as a host to examine the spectrum of interactions capable within the Fusarium oxysporum species complex.Publication Impact of Oyster Mushroom Mycelium on the Growth of Kale and Forage Radish(2018-05) Lilly, LeviSaprophytic fungi can be paired with companion crops in interplant systems to increase production efficiency. However, fungal species/strain, substrate, and inoculation rate can affect the growth of companion crops. This project investigated the viability of open-field mushroom production by interplanting three strains of Pleurotus ostreatus (Elm A, Elm B, and 8801) with kale (B. oleracea var. acephala) and forage radish (Raphanus raphanistrub sub. sativus), and measured the effect of interplanting on plant yield over two field seasons. In the field, Elm A showed an increase in plant yield at a low inoculation rate and decrease in plant yield at a high inoculation rate, compared to the untreated. Conversely, 8801 showed a reduction in plant yield at high and low inoculation rates in the field. Elm B at a high rate showed a reduction in plant yield both in the field and greenhouse. Kale was grown in hydroponics with fungal secretions added at a range of concentrations (10, 100, 1,000 and 10,000 ppm). Elm A showed an overall increase in plant yield in hydroponics, and Elm B showed an overall decrease in plant yield, compared to the untreated. Mushroom production was low in field plots and was not a commercially viable option. Pleurotus ostreatus interplanting methods with companion crops need improvement to make this a commercially viable practice.Publication Integrating Cover Crop Mixtures and No-Till for Sustainable Sweet Corn Production in the Northeast(2018-05) Fine, Julie SFall-planted forage radish (Raphanus sativus L. longipinnatus) cover crops have shown successful weed suppression and recycling of fall-captured nutrients. This research evaluated the nutrient cycling and weed suppressive benefits of forage radish cover crop mixtures to develop an integrated system for no-till sweet corn (Zea mays L. var rugosa) production that improves crop yield and soil health. Treatments included forage radish (FR), oats (Avena sativa L.) and forage radish (OFR), a mixture of peas (Pisum sativum subsp arvense L.), oats and forage radish (POFR), and no cover crop control (NCC). Subplots were assigned to nitrogen fertilizer treatments to evaluate N sufficiency and timing: 0 kg N ha-1 as the control, 28 kg N ha-1 at side-dress, and 56 kg N ha-1 with application split between planting and side-dress. Results indicated that POFR and OFR provided improved N cycling and sweet corn yield compared with FR and NCC. Early season N from decomposing cover crop residue was sufficient to eliminate the need for N fertilizer at sweet corn planting, thereby reducing input costs and risks of environmental pollution.Publication Transposable Elements in Fusarium oxysporum & Growth Inhibition of Fusarium oxysporum Using Pepper Extracts(2018-05) Aguiar, TaylorThe following contains two projects focused on the fungal pathogen, Fusarium oxysporum. The first project was purely computational in the examination of transposable elements (TEs), which are mobile sequences with the ability to multiply and move in their host genome. In F. oxysporum, TEs such as miniature impala elements are associated with the secreted in xylem gene that are related to its virulence over its host. The F. oxysporum species complex can be utilized as a model system for the examination of TE content and TE expression during the infection cycle. To find whether TEs play a role in the infection process and if their expression changes when fungi are in planta, a comparison was made using RNA-seq data from a pathogenic (Fo5176) and a non-pathogenic strain (Fo47) of F. oxysporum interacting with the model plant Arabidopsis thaliana. Complementary to this, the copy numbers of the same TEs were calculated in the two aforementioned strains and in F. oxysporum f.sp. lycopersici 4287 (Fo4287) to find if there was a correlation between expression and copy number. Using these two different datasets together showed that TE expression and copy number are lower in the non-pathogenic strain and unlinked in the infection course. The second project examined the growth inhibition of Fusarium oxysporum isolates Fo32931 (the isolate pathogenic to immunocompromised humans) and Fo4287 with the use of extracts from chilies of Capsicum chinense. Pepper plants were grown from seed and the peppers were harvested for an ethanol (100%) extraction. After preparation, the optical density of growth of the F. oxysporum isolates was measured for a 48-hour period with 96-well plate containing varying concentrations of the extracts and controls. Growth curves were analyzed and normalized to a growth control. After doing High Performance Liquid Chromatography, an estimated concentration of capsaicin (the causal agent of the burning sensation from hot chilis) was established. A correlation between the amount of growth inhibition and the concentration of capsaicin was made. Taken together, the data suggests that an increase of capsaicin concentration in extracts is correlated with reduced growth for the two tested isolates of F. oxysporum.Publication Uncovering tasselsheath3. A Genomic and Phenotypic Analysis of a Maize Floral Mutant.(2018-09) Zhang, ThompsonIn the modern era, maize has become the most successful crop grown in the United States. According to the USDA over 90 million acres of land are planted to corn and 96.2% of the U.S feed grain production is made up of the cereal. Part of the success of maize is due to its floral architecture, and its pollination technique in which the flower opens, exposing stamens containing pollen into the air. A unique organ called the lodicule functions as a release mechanism, forcing the flower to open. Lodicules from grasses and eudicot petals are homologous, yet there is little known of how lodicules are specified during development. Other examples of maize mutants with defects in the lodicule have been discovered including silky1, bearded ear, and sterile tassel silky ear1, but there has been no definitive pathway found that specifies the developmental characteristics of the lodicule. My work has focused on a maize mutant, tasselsheath3 (tsh3), which displays a floral phenotype in the lodicule whorl to better understand this organ. Analysis of tsh3 was separated into two sections: a quantitative phenotypic analysis of the tsh3 floral mutant phenotype, compared to a previously unstudied floral phenotype of tasselsheath1 (tsh1), as well as a tsh1; tsh3 double mutant. I found that lodicule morphology and lodicule number was affected in tsh1, tsh3, and the tsh1; tsh3 mutants. Section two was to identify the single gene that was disrupted in tsh3 mutants. Through both fine mapping and next generation sequencing I was able to localize tsh3 to a region between 148.1mbp and 152.8mbp on chromosome 6. This 4.7mbp region of interest contains 64 protein coding genes. As evidenced by the phenotyping data, tsh3 plays a role specifying lodicule identity during development and has been localized to this region of chromosome 6 on the maize genome.Publication Investigation of Cytochrome P450 Monooxygenases in S. homoeocarpa for Chlorothalonil Biotransformation(2017-05) Green, RobertSclerotinia homoeocarpa (F.T. Bennett) is one of the most economically important pathogens on high amenity cool-season turfgrasses where it causes dollar spot. Due to decades of over-reliance and repeated chemical treatments, S. homoeocarpa has developed resistance and insensitivity to multiple classes of fungicides. To understand the genetic mechanisms of fungicide resistance, the whole genomes of two strains with varying resistance levels to fungicides, were sequenced. In unpublished data (Sang et al.), a RNA-sequencing analysis revealed three CYP450s that were validated to play a functional role in S. homoeocarpa’s resistance against different fungicide classes. We also identified CYP450 metabolic action on the multi-site mode of action fungicide chlorothalonil. Chlorothalonil is an extensively used contact fungicide and has been known to be persistent in soils. Yet, S. homoeocarpa resistance to chlorothalonil has not been reported in the field. High Performance Liquid Chromatography (HPLC) indicated faster rates of chlorothalonil biotransformation by CYP450 overexpression strains when compared to the wild-type. We show by GC-MS that the primary transformation intermediate found in soils, 4-hydroxy-2,5,6 trichloroisophthalonitrile is produced by CYP450s’ metabolism.Publication Comparative Transcriptome Analysis of Wild Tomato Species during Fruit Development(2017-02) Zhanayeva, AltynayFleshy fruits in different species can display large variation in color. A link between fruit color and seed dispersal success is suggested by previous studies showing that high intensity of fruit color increases the visitation rate in seed-dispersing birds. Wild tomato species (Solanum spp.) are excellent model organisms for research on genetic basis of differential fruit color development during the ripening process. Despite polymorphism in fruit color, all tomato species have yellow flowers due to accumulation of carotenoid pigments, which suggests that the carotenoid pathway is intact. Thus, regulatory changes controlling enzymes activity during fruit maturation are likely to have played a role in fruit color evolution. Our transcriptome analysis of wild tomato species revealed that differences in cyc-b and crtr-b2 expression could explain the diversity of fruit color. Additionally, co-expression analysis elucidated regulators of the carotenoid pathway. Combined with Sanger sequencing of carotenoid pathway genes, we identified genes that may underlie differences in fruit color during tomato evolution.Publication Variations in the Invertebrate Communities of Wild Cape Cod Cranberry Bogs(2016-02) Wagner, BarbaraAs a species domesticated only in the last century, agricultural cranberry plants (Vaccinium macrocarpon) remain little removed from their wild relatives. Thus, it is a potential model species for studies of the earliest stages of domestication; however, there is little available quantitative information on its wild population biology and ecology. As such information is vital to studies of the ecological changes occurring during domestication, the purpose of this study was to consolidate the relevant knowledge available and conduct a preliminary search for patterns in the invertebrate communities of wild bogs. The alpha diversity was found to be greater than the overall (gamma diversity), which is likely a result of the metric used and the fact that there was minimal overlap in rare species between bogs. In addition, alpha diversity was found to be significantly negatively correlated with bog age. Two pairs of species were found to be correlated with each other, Blunt-nosed (Limotettix vaccinia (Van Duzee)) and Sharp-nosed leafhopper (Scaphytopius sp.), along with fleabeetle (Sysena frontalis (F.)) and firebeetle (Cryptocephalus incertus (Oliv.). In addition, it was found that spiders are significantly more common in western bogs, while firebeetle is significantly more common in eastern bogs. However, this may be indicative of a correlation with bog age rather than a true correlation with geographic location. More work is needed to determine the true dynamics driving these findings, so that the information could eventually be used to improve the efficacy and decrease the environmental impact of pest management on agricultural bogs.Publication Natural Products for Legume Defense: Investigating Terpene Metabolism in Medicago truncatula(2024-02) Hendrickson, Hannah NPlants use natural products called terpenes as part of their chemical response to environmental stresses. Legume species represent important agricultural and feed crops that are known to produce terpene-based compounds when under herbivore or microbial stress but whose defense mechanisms are not well understood. We worked to better elucidate the biosynthetic pathways behind terpene biosynthesis through characterization of terpene synthases (TPSs) in the model legume Medicago truncatula. Our characterization efforts focused on TPSs in terpene clades TPS-a and TPS-b, which includes monoterpene (C10) and sesquiterpene (C15) synthases with products often used in herbivore or microbial defense. We characterized several TPSs through co-expression with their predicted substrates and found several of them produced known antiherbivore or antimicrobial products. Treatments with methyl jasmonate or salicylic acid meant to mimic herbivore and microbial stress responses caused an increase in expression of several TPSs in planta, supporting potential defensive roles. Our characterization of biosynthetic enzymes and evaluation of their expression in M. truncatula allows for better understanding of legume chemical response strategies.Publication Assessing Warm-Season Annual Grasses to Increase Forage Inventory(2022-09) Marroquin, AndreaSummers are expected to continue to increase in heat/dryness in the Northeast, causing issues pertaining to forage production during the summer to worsen. Many pastures grow cool season grasses, even during the summer. These grasses enter a dormant period and slowdown in production during the months of July and August, leading to what is referred to as “summer slump”. Some farms grow corn silage during the summer, and while corn silage is a valuable crop, its cultivation often does not support soil biology. This research addresses solutions for both summer slump foraging and more sustainable silage. Summer annuals grow more efficiently during the summer and can produce better quality forage compared to winter grasses. Pearl Millet and Sudangrass were evaluated at seed percentages 0-100%. Biomass of each grass was evaluated by cutting a 2x3 ft section on a bi-weekly basis to establish how the treatments vary over time by seeding ratio and type of warm-season grass. Two separate cuts evaluated yield, quality, and regrowth. Another cut looked at ensiling success and quality of Pearl millet and Sudangrass. Results showed both forage species had similar and comparable quality to cool-season grasses. With how much more Sudangrass produces in yield and the little difference in forage quality compared to Pearl millet, Sudangrass would make a good replacement for cool-season grasses. Pearl millet and Sudangrass can be ensiled successfully and have competitive forage quality compared to corn silage.