Myosin subfragment-2 (S2) is an intrinsically unstable coiled coil. This dissertation tests if the mechanical stability of myosin S2 would influence the availability of myosin S1 heads to actin thin filaments. The elevated instability in myosin S2 coiled coil could be one of the causes for hypercontractility in Familial Hypertrophic Cardiomyopathy (FHC). As hypothesized FHC mutations, namely E924K and E930del, in myosin S2 displayed an unstable myosin S2 coiled coil compared to wild type as measured by Fluorescence Resonant Energy Transfer (FRET) and gravitational force spectroscopy (GFS). To remedy this, anti-S2 peptides; the stabilizer and the destabilizer peptides by namesake were designed in our lab to increase and decrease the stability of myosin S2 coiled coil to influence the actomyosin interaction. Firstly, the effectiveness of anti-S2 peptides were tested on muscle myosin S2 peptides across MYH11 (smooth), MYH7 (cardiac), and MYH2 (skeletal) with GFS and FRET. The results demonstrated that the mechanical stability was increased by the stabilizer and decreased by the destabilizer across the cardiac and skeletal myosin S2 isoform but not for the smooth muscle isoform. The destabilizer peptide had dissociation binding constants of 9.97 × 10-1 μM to MYH7 isoform, 1.00 μM to MYH2 isoform, and no …

Flavonoids are polyphenolics compounds that constitute a major group of plant specialized metabolites, biosynthesized via the phenylpropanoid/polymalonate pathways. The resulting specialized metabolites can be due to decoration of flavonoid compounds with sugars, usually glucose, by the action of regiospecific UDP-glycosyltransferase (UGT) enzymes. In some cases, glycosylation can involve enzymatic attachment of other sugar moieties, such as glucuronic acid, galactose, rhamnose or arabinose. These modifications facilitate or impact the bioactivity, stability, solubility, bioavailability and taste of the resulting flavonoid metabolites. The present work shows the limitations of utilizing mammalian UDP-glucuronosyltransferases (UGATs) for flavonoid glucuronidation, and then proceeds to investigate plant UG(A)T candidates from the model legume Medicago truncatula for glucuronidating brain-targeted flavonoid metabolites that have shown potential in neurological protection. We identified and characterized several UG(A)T candidates from M. truncatula which efficiently glycosylate various flavonoids compounds with different/multiple regiospecificities. Biochemical characterization identified one enzyme, UGT84F9, that efficiently glucuronidates a range of flavonoid compounds in vitro. In addition, examination of the ugt84f9 gene knock-out mutation in M. truncatula indicates that UGT84F9 is the major UG(A)T enzyme that is necessary and sufficient for attaching glucuronic acid to flavonoid aglycones, particularly flavones, in this species. Finally, the identified UG(A)T candidates were analyzed via homology …

West Nile virus (WNV) is a geographically endemic mosquito-borne flavivirus that has spread across the United States infecting birds, mosquitos, humans, horses and other mammals. The wide spread nature of this virus is due to the ability of the mosquito vector to persist in broad, ecological diverse environments across the United States. In this study, mosquito populations in North Texas region were sampled for detection of Wolbachia, blood meal source, and WNV. The ultimate goal of this study was to examine the potential of a biocontrol agent, Wolbachia sp. that colonizes the hindgut of various insects, including mosquitos, as a natural means to interrupt virus transmission from mosquitos to other hosts, including humans. In Australia, Wolbachia sp. from fruit flies (Drosophila melanogaster) have been successfully used to block transmission of a similar pathogenic virus from mosquitos responsible for transmission of Dengue fever. Here, mosquitoes were collected using CDC style Gravid Traps in Denton, Texas, from October 2012 through September 2014. Collected mosquitoes were identified, sexed, and categorized as to the amount of host blood in their alimentary system using a Zeiss Axio Zoom microscope (Carl Zeiss Microscopy, LLC, Thornwood, NY). Culex quinquefaciatus was the dominant blood engorged species collected. Smaller …

The isoflavones in kudzu roots, especially the C-glycosylated isoflavone puerarin, have been linked to many health benefits. Puerarin contains a carbon-carbon glycosidic bond that can withstand hydrolysis. The C-glycosylation reaction in the biosynthesis of puerarin has not been thoroughly investigated, with conflicting reports suggesting that it could take place on daidzein, isoliquiritigenin, or 2,7,4ʹ-trihydroxyisoflavanone. Kudzu species were identified for use in comparative transcriptomics. A non-puerarin producing kudzu was identified as Pueraria phaseoloides and a puerarin producing kudzu was identified as Pueraria montana lobata. Through the use of the plant secondary product glycosyltransferase (PSPG) motif, glycosyltransferases (UGTs) were identified from the transcriptomes. The UGTs that had higher digital expression in P. m. lobata were examined further using additional tools to home in on the UGT that could be responsible for puerarin biosynthesis. One of the UGTs identified, UGT71T5, had previously been characterized from kudzu as a C-glycosyltransferase involved in puerarin biosynthesis through in vitro enzyme activity (with daidzein) and a gain of function approach in soybean hairy roots. Previous studies have not supported the end-product of a pathway such as daidzein as the target for C-glycosylation, and no genetic analysis of UGT function had been conducted in kudzu. The activity of …

Alteration in diet and knockdown of detoxification genes impacts the response of C. elegans to oxygen deprivation stress. I hypothesized that feeding worms a vitamin D3-supplementation diet would result in differential oxygen deprivation stress response. We used a combination of wet lab and transcriptomics approach to investigate the effect of a vitamin-D3 supplemented diet on the global gene expression changes and the anoxia response phenotype of C. elegans (Chapter 2). C. elegans genome consists of 143 detoxification genes (cyp and ugt). The presence of a significant number of genes in these detoxification families was a challenge with identifying and selecting specific cyp and ugt genes for detailed analysis. Our goal was to understand the evolution, phylogenetic, and expression of the detoxification enzymes CYPs and UGTs in C. elegans (Chapter 3). We undertook a phylogenetic and bioinformatics approach to analyze the C. elegans, detoxification family. Phylogenetic analysis provided insight into the association of the human and C. elegans xenobiotic/endobiotic detoxification system. Protein coding genes in C. elegans have been predicted to be human orthologs. The results of this work demonstrate the role of C. elegans in the identification and characterization of vitamin D3 induced alterations in gene expression profile and anoxia …

Previous studies have shown that human platelets and megakaryocytes carry microRNAs suggesting their role in platelet function and megakaryocyte development, respectively. However, there is limited information on microRNAs' role in zebrafish thrombopoiesis. Zebrafish thrombocytes could be used as a model to study their role in megakaryocyte maturation and platelet function because thrombocytes have both megakaryocyte features and platelet properties. In our laboratory, I identified 15 microRNAs in thrombocytes using single-cell RNA sequencing. Knockdown of three microRNAs, mir-7148, let-7b, and mir-223, by the piggyback method in zebrafish led to an increase in the percentage of thrombocytes. Functional thrombocyte analysis using plate tilt assay showed no modulatory effect of the three microRNAs on thrombocyte aggregation/agglutination. I then verified these findings in zebrafish larvae after the knockdown of the above microRNAs followed by an arterial laser thrombosis assay. I concluded mir-7148, let-7b, and mir-223 are repressors for thrombocyte production. Furthermore, I explored let-7b downstream genes in thrombocytes detected by RNA-seq analysis and chose 14 targets based on their role in cell differentiation (rorca, tgif1, rfx1a, deaf1, zbtb18, mafba, cebpa, spi1a, spi1b, fhl3b, ikzf1, irf5, irf8, and lbx1b) that are transcriptional regulators. The qRT-PCR analysis of expression levels the above genes following let-7b knockdown …

Fusarium head blight (FHB) is an important disease of small grain cereals including wheat that affects grain quality and yield. The fungus Fusarium graminearum (Fg) is the major agent of this disease. Lack of natural resistance has limited ability to control wheat losses to this disease. Developing new approaches is critical for increasing host plant resistance to this fungus. This work has identified four processes that can be targeted for enhancing host plant resistance to FHB. The first involves targeting the pattern-triggered immunity mechanism to promote host plant resistance. Two other approaches involved reducing activity of susceptibility factors in the host to enhance plant resistance. The susceptibility factors targeted include accumulation of the phytohormone jasmonic acid and the 9-lipoxygenase pathway that oxidizes fatty acids. Besides suppressing host defenses against Fg, jasmonic acid also directly acts on the fungus to promote fungal growth. 9- lipoxygenases similarly suppress host defenses to promote fungal pathogenicity. Another approach that was developed involved having the plant express double stranded RNA to target fungal virulence genes for silencing. This host-induced gene silencing approach was employed to target two fungal virulence genes, the lipase encoding FGL1 and salicylate hydroxylase encoding FgNahG, which the fungus secretes into the …

Upland cotton (Gossypium hirsutum L.) is the world's most prominent fiber crop. Cotton transformation is labor intensive and time consuming, taking 12 to 18 months for rooted T0 plants. One rate limiting step is the necessary production of somatic embryos. In other recalcitrant species, ectopic expression of three genes were shown to promote somatic embryogenesis: WUSCHEL (WUS), SHOOT MERISTEMLESS (STM), and BABY BOOM (BBM). WUS is responsible for maintaining stem-cell fate in shoot and floral meristems. STM is needed to establish and maintain shoot meristems. STM and WUS have similar functions but work in different pathways; overexpression of both together converts somatic cells to meristematic and embryogenic fate. BBM encodes an AP2/ERF transcription factor that is expressed during embryogenesis and ectopic expression of BBM reprograms vegetative tissues to embryonic growth. In prior studies, these genes were constitutively expressed, and cultures did not progress beyond embryogenesis because the embryogenic signal was not turned off. In our study, we set out to use these genes to increase the efficiency of cotton transformation and decrease the time it takes to regenerate a plant. A disarmed cotton leaf crumple virus (dCLCrV) vector delivers WUS, STM, or BBM into cotton tissue cultures through Agrobacterium tumefaciens …

Leguminous plants are able to fix nitrogen by establishing a symbiotic relationship with soil dwelling bacteria, called rhizobia. The model plant Medicago truncatula forms a partnership with Sinorhizobium meliloti whereby the plant gains bioavailable nitrogen and in exchange the bacteria gains carbohydrates. This process occurs within nodules, which are structures produced on the roots of the plants within which nitrogen is fixed. M. truncatula incomplete root elongation (MtIRE) was localized to the infection zone, which is zone II of indeterminate nodules. It was shown to encode a signaling kinase so it was anticipated to play a role in nodulation. Mutants of MtIRE in the R108 background, mutagenized with the Tnt1 retrotransposon, were obtained from reverse screen, and were assessed to determine if a disrupted MtIRE gene was the cause of nitrogen fixation defective nodules. Mutant line NF1320, having a mutant phenotype, showed typical Mendelian segregation of 3:1 when backcrossed to R108. Experimental results show that MtIRE gene is not the cause of the mutant phenotype, but was linked to the causative locus. MtIRE co-segregated with the mutant phenotype 83%. Southern blot and the first version of the M. truncatula genome (version 3.5) reported a single MtIRE gene and this was …

Hemolytic disorders are characterized by hemolysis and are prone to thrombosis. Previously, it has been shown that the RNA released from damaged blood cells activates clotting. However, the nature of RNA released from hemolysis is still elusive. We found that after hemolysis, the red blood cells from both zebrafish and humans release 5.8S rRNA. This RNA activated coagulation in zebrafish and human plasmas. Using both natural and synthetic 5.8S rRNA and its synthetic truncated fragments, we found that the 3'-end 26 nucleotide-long RNA (3'-26 RNA) and its stem-loop secondary structure were necessary and sufficient for clotting activity. Corn trypsin inhibitor (CTI), a coagulation factor XII (FXII) inhibitor blocked 3'-26 RNA-mediated coagulation activation of both zebrafish and human plasma. CTI also inhibited zebrafish coagulation in vivo. 5.8S rRNA monoclonal antibody inhibited both 5.8S rRNA- and 3'-26 RNA-mediated zebrafish coagulation activity. Both 5.8S rRNA and 3'-26 RNA activates normal human plasma but did not activate FXII-deficient human plasma. Taken together, these results suggested that the activation of zebrafish plasma is via FXII-like protein. Since zebrafish has no FXII and hepatocyte growth factor activator (Hgfac) has sequence similarities to FXII, we knocked down the hgfac in adult zebrafish. We found that plasma from …

Microtubules (MT) are regulated by multiple categories of proteins, including proteins responsible for severing MTs that are therefore called MT-severing proteins. Studies of katanin, spastin, and fidgetin in animal systems have clarified that these proteins are MT-severing. However, studies in plants have been limited to katanin p60, and little is known about spastin or fidgetin and their function in plants. I looked at plant genomes to identify MT-severing protein homologues to clarify which severing proteins exist in plants. I obtained data from a variety of eukaryotic species to look for MT-severing proteins using homology to human proteins and analyzed these protein sequences to obtain information on the evolution of MT-severing proteins in different species. I focused this analysis on MT-severing proteins in the maize and Arabidopsis thaliana genomes. I created evolutionary phylogenetic trees for katanin-p60, katanin-p80, spastin, and fidgetin using sequences from animal, plant, and fungal genomes. I focused on Arabidopsis spastin and worked to understand its functionality by identifying protein interaction partners. The yeast two-hybrid technique was used to screen an Arabidopsis cDNA library to identify putative spastin interactors. I sought to confirm the putative protein interactions by using molecular tools for protein localization such as the YFP system. …

Stomata are specialized plant structures required for gaseous exchange with the outer environment. During stomata formation, the cytoskeleton plays an important role in controlling the division of the individual cells leading to the generation of the stomata complex. Two mutants that affect microfilament and microtubule organization in subsidiary mother cells include brk1 and dcd1. While only 20% of the subsidiary cells in the brk1 and dcd1 single mutants are abnormally shaped, it was reported that there is a synergistic effect between the brk1 and dcd1 mutations in the brk1; dcd1 double mutant since 100% of the subsidiary cells are abnormal. The focus of this research is to try to understand this synergistic effect by investigating the actin cytoskeleton and nuclear position in the single and double mutants. The reported results include the observation that the size of actin patch was largest in the wild-type subsidiary mother cells (SMCs) and smallest in dcd1 and brk1; dcd1 SMCs and that brk1 and brk1; dcd1 double mutants had fewer actin patches than wild-type and dcd1 SMCs. Additionally, we observed that some SMCs that did not have actin patches still underwent nuclear migration suggesting that nuclear migration may not be solely dependent on actin …

Plant microtubules play essential roles in cell processes such as cell division, cell elongation, and organelle organization. Microtubules are arranged in highly dynamic and ordered arrays, but unlike animal cells, plant cells lack centrosomes. Therefore, microtubule nucleation and organization are governed by microtubule-associated proteins, including a microtubule-severing protein, katanin. Mutant analysis and in vitro characterization has shown that the highly conserved katanin is needed for the organization of the microtubule arrays in Arabidopsis and rice as well as in a variety of animal models. Katanin is a protein complex that is part of the AAA+ family of ATPases. Katanin is composed of two subunits, katanin-p60, a catalytic subunit and katanin-p80, a regulatory subunit. Spastin is another MT-severing protein that was identified on the basis of its homology to katanin. In animal cells, spastin is also needed for microtubule organization, but its functionality has not yet been investigated in plants. To initiate an exploration of the function of katanin-p60 and spastin in Zea mays, my research goal was to generate tools for the expression and purification of maize katanin-p60 and spastin proteins in vitro. Plasmids that express katanin-p60 and spastin with N-terminal GST tags were designed and constructed via In-Fusion® cloning …

Previous studies have shown that fish release alarming substances into the water to alert their kin to escape from danger. In our laboratory, we found that zebrafish produce trypsin and release it from their gills into the environment when they are under stress. By placing the zebrafish larvae in the middle of a small tank and then placing trypsin at one end of the tank, we observed that the larvae moved away from the trypsin zone and almost to the opposite end of the tank. This escape response was significant and did not occur in response to the control substances, bovine serum albumin (BSA), Russell's viper venom (RVV), and collagen. Also, previously, we had shown that the trypsin could act via a protease-activated receptor-2 (PAR2) on the surface of the cells. Therefore, we hypothesized that trypsin would induce a change in neuronal activity in the brain via PAR2-mediated signaling in cells on the surface of the fish body. To investigate whether the trypsin-responsive cells were surface cells, we generated a primary cell culture of zebrafish keratinocytes, confirmed these cells' identity by specific marker expression, and then incubated these cells with the calcium indicator Fluo-4 and exposed them to trypsin. By …

Synthetic biology is a rapidly growing field that aims to treat cellular biological networks in an analogous way to electrical circuits. However, the field of plant synthetic biology has not grown at the same pace as bacterial and yeast synthetic biology, leaving a dearth of characterized tools for the community. Due to the need for tools for the synthetic plant biologist, I have endeavored to create a library of well-characterized TATA box variants in the cauliflower mosaic virus (CaMV) 35S promoter using the standardized assembly method Golden Braid 2.0. I introduced single nucleotide changes in the TATA box of the CaMV 35S promoter, a genetic part widely used in plant gene expression studies and agricultural biotechnology. Using a dual-luciferase reporter system, I quantified the transcriptional strength of the altered TATA box sequences and compared to the wild-type sequence, both in transient protoplast assays and stable transgenic Arabidopsis thaliana plants. The library of TATA-box modified CaMV 35S promoters with varying transcriptional strengths created here can provide the plant synthetic biology community with a series of modular Golden Braid-adapted genetic parts that can be used dependably and reproducibly by researchers to fine-tune gene expression levels in complex, yet predictable, synthetic genetic circuits.

With effective engineering using synthetic biology approaches, plant-based platforms could conceivably be designed to minimize the production costs and wastes of high-value products such as medicines, biofuels, and chemical feedstocks that would otherwise be uneconomical. Additionally, modern agricultural crops could be engineered to be more productive, resilient, or restorative in different or rapidly changing environments and climates. To achieve these complex goals, information-processing genetic devices and circuits containing multiple interacting parts that behave predictably must be developed. A genetic Boolean AND logic gate is a device that computes the presence or absence of two inputs (signals, stimuli) and produces an output (response) only if both inputs are present. Here, we optimized individual genetic components and used synthetic protein heterodimerizing domains to rationally assemble genetic AND logic gates that integrate two hormonal inputs in whole plants. These AND gates produce an output only in the presence of both abscisic acid and auxin, but not when either or neither hormone is present. Furthermore, we demonstrate the AND gate can also integrate two plant stresses, cold temperature and bacterial infection, to produce a specific response. The design principles used here are generalizable, and therefore multiple orthogonal AND gates could be assembled and rationally …

Fatty acid amide hydrolase (FAAH) is a widely conserved amidase in eukaryotes, best known for inactivating the signal of N-acylethanolamine (NAE) lipid mediators. In the plant Arabidopsis thaliana, FAAH-mediated hydrolysis of NAEs has been associated with numerous biological processes. Recently, the phylogenetic distribution of FAAH into two major branches (group I and II FAAHs) across angiosperms outside of Arabidopsis (and in other Brassicaceae), suggests a previously unrecognized complexity of this enzyme. Although A. thaliana has long been used to assess biological questions for plants, in this case it will fall short in understanding the significance of multiple FAAHs in other plant systems. Thus, in this study, I examined the role (s) of six FAAH isoforms in upland cotton (Gossypium hirsutum L.) and two FAAHs in the legume Medicago truncatula.

GPR17, a uracil nucleotide cysteinyl leukotriene receptor, belongs to the GPCR (G protein coupled receptor) family. It has been shown recently that inhibiting this protein in the nervous system in mice can lead to blockage of oligodendrocyte maturation, which supports myelin repair. Interestingly, our laboratory found GPR17 in thrombocytes. However, we do not know whether it has any function in thrombocyte aggregation or the nature of the ligand. In this paper, we studied the role of GPR17 in hemostasis, which is a fundamental defense mechanism in the event of injury. Using zebrafish as a model system, our laboratory has studied specifically thrombocytes, which play a significant role in hemostasis. The major reasons to use zebrafish as a model system are that their thrombocytes are functionally equivalent to human platelets, the adult fish are amenable to knockdown experiments, and they are readily available in the market. This study was performed by using a piggy back knockdown method where we used a chemical hybrid of control morpholino and an antisense oligonucleotide sequence leads to the degradation the mRNA for GPR17. After knockdown GPR17 in thrombocytes, the percent difference of the thrombocytes aggregation between the control and knockdown blood samples was measured by …

In this dissertation I am using M. truncatula as a model legume that forms indeterminate nodules with rhizobia under limited nitrogen conditions. I take advantage of an M. truncatula Tnt1 mutant population that provides a useful resource to uncover and characterize novel genes. Here, I focused on several objectives. First, I carried out forward and reverse genetic screening of M. truncatula Tnt1 mutant populations to uncover novel genes involved in symbiotic nitrogen fixation. Second, I focused on reverse genetic screening of two genes, identified as encoding blue copper proteins, and characterization of their mutants' potential phenotypes. Third, I further characterized a nodule essential gene, M. truncatula vacuolar iron transporter like 8 (MtVTL8), which encodes a nodule specific iron transporter. I characterized the expression pattern, expression localization and function of MtVTL8. Additionally, I characterized several residues predicted to be essential to function using a model based on the known crystal structure of Eucalyptus grandis vacuolar iron transporter 1 (EgVIT1), a homologous protein to MtVTL8. I identified several potential essential residues of the MtVTL8 protein, mutagenized them, and through complementation experiments in planta and in yeast assessed functionality of the resulting protein. This helped us to better understand the potential mechanism by …

Although cytoplasmic lipid droplets (LDs) are the major reserves for energy-dense neutral lipids in plants, the cellular mechanisms for packaging neutral lipids into LDs remain poorly understood. To gain insights into the cellular processes of neutral lipid accumulation and compartmentalization, a necessary step forward would be to characterize functional roles of lipogenic proteins that participate in the compartmentalization of neutral lipids in plant cells. In this study, the lipogenic proteins, Arabidopsis thaliana SEIPIN homologues and mouse (Mus Musculus) fat storage-inducing transmembrane protein 2 (FIT2), were characterized for their functional roles in the biogenesis of cytoplasmic LDs in various plant tissues. Both Arabidopsis SEIPINs and mouse FIT2 supported the accumulation of neutral lipids and cytoplasmic LDs in plants. The three Arabidopsis SEIPIN isoforms play distinct roles in compartmentalizing neutral lipids by enhancing the numbers and sizes of LDs in various plant tissues and developmental stages. Further, the potential applications of Arabidopsis SEIPINs and mouse FIT2 in engineering neutral lipids and terpenes in plant vegetative tissues were evaluated by co-expressing these and other lipogenic proteins in Nicotiana benthamiana leaves. Arabidopsis SEIPINs and mouse FIT2 represent effective tools that may complement ongoing strategies to enhance the accumulation of desired neutral lipids and terpenes …

Environmental stress conditions can drastically affect plant growth and productivity. In contrast to soil moisture or salinity that can gradually change over a period of days or weeks, changes in light intensity or temperature can occur very rapidly, sometimes over the course of minutes or seconds. So, in our study we have taken an metabolomics approach to identify the rapid response of plants to light stress. In the first part we have focused on the ultrafast (0-90 sec) metabolic response of local tissues to light stress and in the second part we analyzed the metabolic response associated with rapid systemic signaling (0-12 min). Analysis of the rapid response of Arabidopsis to light stress has revealed 111 metabolites that significantly alter in their level during the first 90 sec of light stress exposure. We further show that the levels of free and total glutathione accumulate rapidly during light stress in Arabidopsis and that the accumulation of total glutathione during light stress is dependent on an increase in nitric oxide (NO) levels. We further suggest that the increase in precursors for glutathione biosynthesis could be linked to alterations in photorespiration, and that phosphoenolpyruvate could represent a major energy and carbon source for …

To better understand the nature and function of N-acylethanolamines (NAEs) in Camelina sativa, we used mass spectrometry analysis to identify and quantify NAE types in developing seeds, desiccated seeds and seedlings. Developing seeds showed a differential increase in individual NAE species and an overall increase in NAE content with seed development and maturation. The NAE composition in mature, desiccated seeds mostly reflected the total fatty acid composition in the seed tissues, except for a noted absence of 11-eicosenoic (20C monounsaturated) fatty acid in the NAE pool. During seed stratification and seedling growth, individual NAE species were depleted at similar rates. Simulated drought treatments during seedling development resulted in a significant rise in NAE levels for the major 18C NAE types compared with untreated seedlings. Arabidopsis and Camelina mutants with reported altered fatty acid profiles were analyzed for their NAE compositions; both Arabidopsis and Camelina had relatively similar changes between compositions of total seed fatty acids and NAEs. Furthermore, seeds were analyzed from transgenic Arabidopsis and Camelina with engineered, non-native, long-chain polyunsaturated fatty acids (18C, 20C and 22C), and the results showed the production of novel N-acylphosphatidylethanolamines (presumed precursors of NAEs) and NAEs with the same long acyl chains. These results …

Symbiotic nitrogen (N) fixation (SNF) occurs in specialized organs called nodules after successful interactions between legume hosts and rhizobia. Within nodule cells, N-fixing rhizobia are surrounded by plant-derived symbiosome membranes, through which the exchange of nutrients and ammonium occurs between bacteria and the host legume. Phosphorus (P) is an essential macronutrient, and N2-fixing legumes have a higher requirement for P than legumes grown on mineral N. First, I investigated the impact of P deprivation on wild-type Medicago truncatula plants. My observations that plants had impaired SNF activity, reduced growth, and accumulated less phosphate in P-deficient tissues (leaves, roots and nodules) is consistent with those of similar previous studies. Galactolipids decreased with increase in phospholipids in all P-starved organs. Matrix-assisted laser desorption/ionization–mass spectrometry imaging (MALDI-MSI) of phosphatidylcholine (PC) species in nodules showed that under low P environments distributions of some PC species changed, indicating that membrane lipid remodeling during P stress is not uniform across the nodule. Secondly, a metabolomics study was carried out to test the alterations in the metabolic profile of the nodules in P-stress. GC-MS based untargeted metabolomics showed increased levels of amino acids and sugars and decline in amounts of organic acids in P deprived nodules. Subsequently, …

Eukaryotic platelets are small cell fragments that are released into the bloodstream from megakaryocytes, and their production is initiated in the bone marrow. They are mainly involved in blood hemostasis and thrombus formation. The newly synthesized platelets are called reticulated platelets or young platelets. Zebrafish thrombocytes are equivalent to mammalian platelets and have similar characteristics and functions. Likewise, zebrafish has both young and mature thrombocytes. Only young thrombocytes as reticulated platelets are labeled with thiazole orange. Similarly, labeling zebrafish thrombocytes with a specific concentration of DiI-C18 showed two populations of thrombocytes (DiI+ and DiI-). Again, only young thrombocytes showed DiI+ labeling. The mechanism of selective labeling of young thrombocytes by is unknown. Furthermore, there is no zebrafish line where young and mature thrombocytes are differentially labeled with fluorescence proteins. Therefore, in this study, we identified and confirmed that the RFP labeled cells of Glofish were young thrombocytes. In addition, we found that myosin light chain 2 (MLC2) promoter is expressed in young thrombocytes. We also generated a transgenic zebrafish line, GloFli fish, where the young and mature thrombocytes are labeled with red and green fluorescence proteins respectively. Furthermore, this study showed a two-fold increase in glycerol-phospholipids (GP) in mature thrombocytes …