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Browsing by Author "Okumoto, Sakiko"

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    Arabidopsis UMAMIT24 and 25 are amino acid exporters involved in seed loading
    Besnard, Julien; Zhao, Chengsong; Avice, Jean-Christophe; Vitha, Stanislav; Hyodo, Ayumi; Pilot, Guillaume; Okumoto, Sakiko (Oxford University Press, 2018-10-12)
    Phloem-derived amino acids are the major source of nitrogen supplied to developing seeds. Amino acid transfer from the maternal to the filial tissue requires at least one cellular export step from the maternal tissue prior to the import into the symplasmically isolated embryo. Some members of UMAMIT (usually multiple acids move in an out transporter) family (UMAMIT11, 14, 18, 28, and 29) have previously been implicated in this process. Here we show that additional members of the UMAMIT family, UMAMIT24 and UMAMIT25, also function in amino acid transfer in developing seeds. Using a recently published yeast-based assay allowing detection of amino acid secretion, we showed that UMAMIT24 and UMAMIT25 promote export of a broad range of amino acids in yeast. In plants, UMAMIT24 and UMAMIT25 are expressed in distinct tissues within developing seeds; UMAMIT24 is mainly expressed in the chalazal seed coat and localized on the tonoplast, whereas the plasma membrane-localized UMAMIT25 is expressed in endosperm cells. Seed amino acid contents of umamit24 and umamit25 knockout lines were both decreased during embryogenesis compared with the wild type, but recovered in the mature seeds without any deleterious effect on yield. The results suggest that UMAMIT24 and 25 play different roles in amino acid translocation from the maternal to filial tissue; UMAMIT24 could have a role in temporary storage of amino acids in the chalaza, while UMAMIT25 would mediate amino acid export from the endosperm, the last step before amino acids are taken up by the developing embryo.
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    Characterization of the Arabidopsis glutamine dumper1 mutant reveals connections between amino acid homeostasis and plant stress responses
    Yu, Shi (Virginia Tech, 2015-04-15)
    Amino acids constitute the major organic form of transported nitrogen in plants, elements for protein synthesis, and precursors of many plant secondary metabolites, such as lignin, hormones, and flavonoids. Furthermore, amino acid metabolism lies at the crossroad of carbon and nitrogen metabolism. The Arabidopsis glutamine dumper1 (gdu1) mutant secretes glutamine from hydathodes, a phenotype caused by the overexpression of Glutamine Dumper1 (GDU1). GDU1 is a small transmembrane protein presents only in higher plants. The gdu1-1D mutant shows a pleiotropic phenotype: perturbed amino acid metabolism, tolerance to exogenous toxic concentrations of amino acids, elevated amino acid export, and activated stress/defense responses, lesions, and smaller rosettes. The biochemical function of GDU1 remains elusive. To better elucidate the biological processes leading to the complex Gdu1D phenotype, two approaches were conducted: (1) An ethyl methanesulfonate suppressor screening of the Gdu1D phenotype, which led to the isolation of intragenic mutations in GDU1 and mutations in the ubiquitin ligase LOG2 (Loss Of Gdu1D 2). Study of the intragenic mutations in GDU1 helped to characterize its structure-function relationships. Characterization of LOG2 showed that LOG2 interacts with GDU1 and is necessary for the Gdu1D phenotype. (2) The responses of the plant to the dexamethasone-induced expression of GDU1 were studied over time. This experiment identified major signaling pathways contributing to different components of the Gdu1D phenotype and the early events triggered by the perturbation of amino acid homeostasis. Our results showed that GDU1 overexpression first increases amino acid export, which is followed by amino acid imbalance and stress responses. This study sheds light on how amino acid imbalance interacts with various plant signaling pathways and stress responses, and suggests that LOG2 is involved in this process.
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    Characterizing RNA translocation in the parasitic weed Cuscuta pentagona
    LeBlanc, Megan Leanne (Virginia Tech, 2013-06-03)
    The obligate stem parasite Cuscuta pentagona is able to take up host plant mRNA through a specialized organ known as the haustorium. Direct cell-to-cell symplastic connections between two different organisms are rare, and the translocation mechanisms and fate of these RNAs in the parasite is not understood. To characterize this phenomenon, mobile Arabidopsis and tomato mRNAs were identified from microarray and transcriptome sequencing projects and quantified in the host-parasite system. Mobile RNAs were quantified using real time (qRT)-PCR and were found to vary substantially in their rate of uptake and distribution in the parasite. Transcripts of tomato Gibberellic Acid Insensitive (SlGAI) and Cathepsin D Protease Inhibitor (SlPI) can be traced over 30-cm of parasite stem. SlPI was abundant in the C. pentagona stem, but the number of copies decreased substantially within the first eight hours post detachment. Additional studies of mobile RNAs from Arabidopsis, Translationally Controlled Tumor Protein (AtTCTP), Auxin Response Factor (AtARF) and a Salt-inducible Zinc Finger Protein (AtSZFP) supported the idea that mRNA molecules differ in their mechanisms of uptake and mobility between host and parasite. Known phloem-mobile RNAs (SlGAI and AtTCTP) have uptake patterns that differ from each other as well as from other RNAs that are not reported to be phloem mobile (SlPI and AtSZF1). The function of RNAs in plants extend beyond protein translation to include post transcriptional gene silencing or long distance signaling, and mobile RNA in C. pentagona systems offers novel insights into this aspect of plant biology. Studies of cell-to-cell trafficking of RNAs and other macromolecules would be facilitated by the ability to manipulate individual cells. To this end, work was initiated to explore alternative approaches to understanding single cell biology using laser-mediated approaches. Optoperforation, or the use of multiphoton processes to form quasi-free electron plasmas to initiate transient pore formation in plasma membranes, has been demonstrated, but not in cells of an intact plant. This work details a protocol for optoperforation of Arabidopsis epidermal cells to allow for uptake of external dye-labeled dextrans and retention for up to 72 hours, and has the potential for transformation and molecular tagging applications.
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    Editorial: Amino Acids of the Glutamate Family: Functions beyond Primary Metabolism
    Okumoto, Sakiko; Funck, Dietmar; Trovato, Maurizio; Forlani, Giuseppe (Frontiers, 2016-03-21)
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    Effects of Nitrate and Cytokinin on Nitrogen Metabolism and Heat Stress Tolerance of Creeping Bentgrass
    Wang, Kehua (Virginia Tech, 2010-07-23)
    Creeping bentgrass (Agrostis stolonifera L.) is a major low-cut cool-season turfgrass used worldwide. The objectives of this research were to: 1) to gain insight into the diurnal fluctuation of N metabolism and effects of cytokinin (CK) and nitrate; 2) to characterize the impacts of N and CK on creeping bentgrass under heat stress; 3) to investigate the simultaneous effects of CK and N on the antioxidant responses of heat stressed creeping bentgrass; and 4) to examine the expression pattern of the major heat shock proteins (HSPs) in creeping bentgrass during different heat stress periods, and then to study the influence of N on the expression pattern of HSPs. The transcript abundance of nitrate reductase (NR), nitrite reductase (NIR), plastidic glutamine synthetase (GS2), ferredoxin-dependent glutamate synthase (Fd-GOGAT), and glutamate dehydrogenase (GDH) and N metabolites in shoots were monitored during the day/night cycle (14/8 h). All the measured parameters exhibited clear diurnal changes, except GS2 expression and total protein. Both NR expression and nitrate content in shoots showed a peak after 8.5 h in dark, indicating a coordinated oscillation. Nitrate nutrition increased diurnal variation of nitrate content compared to control and CKHowever, CK shifted the diurnal in vivo NR activity pattern during this period. Grass grown at high N had better turf quality (TQ), higher Fv/Fm, normalized difference vegetation index (NDVI), and chlorophyll concentration at both 15 d and 28 d of heat stress than at low N, except for TQ at 15 d. Shoot NO3-, NH4+, and amino acids increased due to the high N treatment, but not water soluble proteins. High N also induced maximum shoot nitrate reductase activity (NRmax) at 1 d. CK increased NDVI at 15 d and Fv/Fm at 28 d. In addition, grass under 100 µM CK had greatest NRmax at both 1 d and 28 d. Under high N with 100 µM CK, root tZR and iPA were 160% and 97% higher than under low N without CK, respectively. Higher O2- production, H2O2 concentration, and higher malonydialdehyde (MDA) content in roots were observed in grass grown at high N. The activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and guaiacol peroxidase (POD) in roots were enhanced by high N at 19, 22, and 24% levels, respectively, relative to low N. Twenty-eight days of heat stress resulted in either the development of new isoforms or enhanced isoform intensities of SOD, APX, and POD in roots compared to the grass responses prior to heat stress. However, no apparent differences were observed among treatments. No CK effects on these antioxidant parameters were found in this experiment. At week seven, grass at medium N had better TQ, NDVI, and Fv/Fm accompanied by lower shoot electrolyte leakage (ShEL) and higher root viability (RV), suggesting better heat resistance. All the investigated HSPs (HSP101, HSP90, HSP70, and sHSPs) were up-regulated by heat stress. Their expression patterns indicated cooperation between different HSPs and that their roles in creeping bentgrass thermotolerance were affected by N level.
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    Functional Analysis of Plant Glutamate Receptors
    Price, Michelle B. (Virginia Tech, 2013-10-02)
    The plant glutamate receptors (GLRs) are homologs of mammalian ionotropic glutamate receptors (iGluRs) and are hypothesized to be potential amino acid sensors in plants. Since their first discovery in 1998, the members of plant GLRs have been implicated in diverse processes such as C/N ratio sensing, root formation, pollen germination and plant-pathogen interaction. However, the exact properties of these channels, such as the spectrum of ligands, ion specificities, and subunit compositions are still not well understood. It is well established that animal iGluRs form homo- or hetero-tetramers in order to form ligand-gated cation channels. The first aspect of this research was to determine if plant GLRs likewise require different subunits to form functional channels. A modified yeast-2-hybrid system approach was initially taken and applied to 14 of the 20 AtGLRs to identify a number of candidate interactors in yeast. Forster resonance energy transfer (FRET), which measures the transfer of energy between interacting molecules, was performed in mammalian cells to confirm interaction between a few of those candidates. Interestingly, despite an abundance of overlapping co-localization between heteromeric combinations, only homomeric interactions were identified between GLRs 1.1 and 3.4 in HEK293 cells. Further, amino acids have been implicated in signaling between plants and microbes, but the mechanisms for amino acid perception in defense responses are far from being understood. Recently it was demonstrated that calcium responses initiated by bacterial and fungal microbe-associated molecular patterns (MAMPs) were diminished in seedlings treated with known agonists and antagonists of mammalian iGluRs, suggesting potential roles of GLRs in pathogen responses. Analysis of publicly available microarray data shows altered gene expression of a sub-fraction of GLRs in response to pathogen infection and bacterial elicitors. Thus, the second goal of my PhD research was aimed at determining whether GLRs are involved in the interaction between plants and pathogens. Gene expression changes of a number of candidate GLRs as well as pathogen growth was examined in response to the plant pathogen Pseudomonas syringae pv. tomato DC3000. Interestingly, single gene and multi-gene deficient plants responded differently with regards to pathogen susceptibility, likely as a result of functional compensation between GLRs.
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    Glutamate receptor homologs in plants: functions and evolutionary origins
    Price, Michelle B.; Jelesko, John G.; Okumoto, Sakiko (Frontiers, 2012)
    The plant glutamate-like receptor homologs (GLRs) are homologs of mammalian ionotropic glutamate receptors (iGluRs) which were discovered more than 10 years ago, and are hypothesized to be potential amino acid sensors in plants. Although initial progress on this gene family has been hampered by gene redundancy and technical issues such as gene toxicity; genetic, pharmacological, and electrophysiological approaches are starting to uncover the functions of this protein family. In parallel, there has been tremendous progress in elucidating the structure of animal glutamate receptors (iGluRs), which in turn will help understanding of the molecular mechanisms of plant GLR functions. In this review, we will summarize recent progress on the plant GLRs. Emerging evidence implicates plant GLRs in various biological processes in and beyond N sensing, and implies that there is some overlap in the signaling mechanisms of amino acids between plants and animals. Phylogenetic analysis using iGluRs from metazoans, plants, and bacteria showed that the plant GLRs are no more closely related to metazoan iGluRs as they are to bacterial iGluRs, indicating the separation of plant, other eukaryotic, and bacterial GLRs might have happened as early on as the last universal common ancestor. Structural similarities and differences with animal iGluRs, and the implication thereof, are also discussed.
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    Increased Expression of UMAMIT Amino Acid Transporters Results in Activation of Salicylic Acid Dependent Stress Response
    Besnard, Julien; Sonawala, Unnati; Maharjan, Bal; Collakova, Eva; Finlayson, Scott A.; Pilot, Guillaume; McDowell, John M.; Okumoto, Sakiko (2021-01-26)
    In addition to their role in the biosynthesis of important molecules such as proteins and specialized metabolites, amino acids are known to function as signaling molecules through various pathways to report nitrogen status and trigger appropriate metabolic and cellular responses. Moreover, changes in amino acid levels through altered amino acid transporter activities trigger plant immune responses. Specifically, loss of function of major amino acid transporter, over-expression of cationic amino acid transporter, or over-expression of the positive regulators of membrane amino acid export all lead to dwarfed phenotypes and upregulated salicylic acid (SA)-induced stress marker genes. However, whether increasing amino acid exporter protein levels lead to similar stress phenotypes has not been investigated so far. Recently, a family of transporters, namely USUALLY MULTIPLE ACIDS MOVE IN AND OUT TRANSPORTERS (UMAMITs), were identified as amino acid exporters. The goal of this study was to investigate the effects of increased amino acid export on plant development, growth, and reproduction to further examine the link between amino acid transport and stress responses. The results presented here show strong evidence that an increased expression of UMAMIT transporters induces stress phenotypes and pathogen resistance, likely due to the establishment of a constitutive stress response via a SA-dependent pathway.
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    Three genes from Solanum chacoense coding for squalene synthase
    Wadlington, William Herring (Virginia Tech, 2011-05-04)
    Squalene synthase (EC 2.5.1.2.1; SQS) is located at a branch point in the isoprenoid pathway and catalyzes the condensation of two molecules of farnesyl diphosphate to form squalene. SQS activity contributes to the formation of triterpenes and sterols, including phytosterols, brassinosteroids, cholesterol, and in potato plants, steroidal glycoalkaloids (SGAs). These compounds have diverse functions in the plant. SGAs are defense compounds that deter feeding by potato pests. The wild potato Solanum chacoense accumulates higher amounts of SGAs than cultivated potato and some of its accessions produce leptines, a rare class of SGAs that is toxic to Colorado potato beetle. Unlike most eukaryotes, higher plants have more than one gene coding for SQS. Three sqs gene homologs were isolated from S. chacoense, sqs1Sc, sqs2Sc, and sqs4Sc, that have 74 to 83% identity at the amino acid level. Some of the amino acid differences between sqs isoforms are likely to affect enzyme activity. Each of the three genes contained an intron in the 3'UTR. This feature may have a role in the nonsense-mediated decay of incomplete sqs mRNAs. A partial SQS polypeptide retaining catalytic activity but lacking the membrane anchoring domain could adversely affect a cell with the randomly distributed accumulation of squalene. The mRNA of sqs1Sc and sqs2Sc was detected in all tissues whereas sqs4Sc transcript was limited to bud tissue. The sqs2Sc transcript was less uniformly distributed in the plant than sqs1Sc and accumulated most abundantly in floral tissue. The results demonstrate that the three sqs genes have different patterns of gene expression and encode proteins with different primary structures indicating distinct roles in plant squalene metabolism.
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    Visualization of Glutamine Transporter Activities in Living Cells Using Genetically Encoded Glutamine Sensors
    Gruenwald, Katrin; Holland, John Todd; Stromberg, Verlyn; Ahmad, Altaf; Watcharakichkorn, Daisy; Okumoto, Sakiko (PLOS, 2012-06-14)
    Glutamine plays a central role in the metabolism of critical biological molecules such as amino acids, proteins, neurotransmitters, and glutathione. Since glutamine metabolism is regulated through multiple enzymes and transporters, the cellular glutamine concentration is expected to be temporally dynamic. Moreover, differentiation in glutamine metabolism between cell types in the same tissue (e.g. neuronal and glial cells) is often crucial for the proper function of the tissue as a whole, yet assessing cell-type specific activities of transporters and enzymes in such heterogenic tissue by physical fractionation is extremely challenging. Therefore, a method of reporting glutamine dynamics at the cellular level is highly desirable. Genetically encoded sensors can be targeted to a specific cell type, hence addressing this knowledge gap. Here we report the development of Föster Resonance Energy Transfer (FRET) glutamine sensors based on improved cyan and yellow fluorescent proteins, monomeric Teal Fluorescent Protein (mTFP)1 and venus. These sensors were found to be specific to glutamine, and stable to pH-changes within a physiological range. Using cos7 cells expressing the human glutamine transporter ASCT2 as a model, we demonstrate that the properties of the glutamine transporter can easily be analyzed with these sensors. The range of glutamine concentration change in a given cell can also be estimated using sensors with different affinities. Moreover, the mTFP1-venus FRET pair can be duplexed with another FRET pair, mAmetrine and tdTomato, opening up the possibility for real-time imaging of another molecule. These novel glutamine sensors will be useful tools to analyze specificities of glutamine metabolism at the single-cell level.