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Browsing by Author "Yu, Shi"

Now showing 1 - 4 of 4
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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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    Detailed characterization of the UMAMIT proteins provides insight into their evolution, amino acid transport properties, and role in the plant
    Zhao, Chengsong; Pratelli, Rejane; Yu, Shi; Shelley, Brett; Collakova, Eva; Pilot, Guillaume (Oxford University Press, 2021-09-30)
    Amino acid transporters play a critical role in distributing amino acids within the cell compartments and between plant organs. Despite this importance, relatively few amino acid transporter genes have been characterized and their role elucidated with certainty. Two main families of proteins encode amino acid transporters in plants: the amino acid-polyamine-organocation superfamily, containing mostly importers, and the UMAMIT (usually multiple acids move in and out transporter) family, apparently encoding exporters, totaling 63 and 44 genes in Arabidopsis, respectively. Knowledge of UMAMITs is scarce, based on six Arabidopsis genes and a handful of genes from other species. To gain insight into the role of the members of this family and provide data to be used for future characterization, we studied the evolution of the UMAMITs in plants, and determined the functional properties, the structure, and localization of the 47 Arabidopsis UMAMITs. Our analysis showed that the AtUMAMITs are essentially localized at the tonoplast or the plasma membrane, and that most of them are able to export amino acids from the cytosol, confirming a role in intra- and intercellular amino acid transport. As an example, this set of data was used to hypothesize the role of a few AtUMAMITs in the plant and the cell.
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    Suppressor mutations in the Glutamine Dumper1 protein dissociate disturbance in amino acid transport from other characteristics of the Gdu1D phenotype
    Yu, Shi; Pratelli, Rejane; Denbow, Cynthia J.; Pilot, Guillaume (Frontiers, 2015-08-04)
    Intracellular amino acid transport across plant membranes is critical for metabolic pathways which are often split between different organelles. In addition, transport of amino acids across the plasma membrane enables the distribution of organic nitrogen through the saps between leaves and developing organs. Amino acid importers have been studied for more than two decades, and their role in this process is well-documented. While equally important, amino acid exporters are not well-characterized. The over-expression of GDU1, encoding a small membrane protein with one transmembrane domain, leads to enhancement of amino acid export by Arabidopsis cells, glutamine secretion at the leaf margin, early senescence and size reduction of the plant, possibly caused by the stimulation of amino acid exporter(s). Previous work reported the identification of suppressor mutations of the GDU1 over-expression phenotype, which affected the GDU1 and LOG2 genes, the latter encoding a membrane-bound ubiquitin ligase interacting with GDU1. The present study focuses on the characterization of three additional suppressor mutations affecting GDU1. Size, phenotype, glutamine transport and amino acid tolerance were recorded for recapitulation plants and over-expressors of mutagenized GDU1 proteins. Unexpectedly, the over-expression of most mutated GDU1 led to plants with enhanced amino acid export, but failing to display secretion of glutamine and size reduction. The results show that the various effects triggered by GDU1 over-expression can be dissociated from one another by mutagenizing specific residues. The fact that these residues are not necessarily conserved suggests that the diverse biochemical properties of the GDU1 protein are not only born by the characterized transmembrane and VIMAG domains. These data provide a better understanding of the structure/function relationships of GDU1 and may enable modifying amino acid export in plants without detrimental effects on plant fitness.
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    Testing the efficiency of plant artificial microRNAs by transient expression in Nicotiana benthamiana reveals additional action at the translational level
    Yu, Shi; Pilot, Guillaume (Frontiers, 2014-11-19)
    Artificial microRNAs (amiRNAs) have become an important tool to assess gene functions due to their high efficiency and specificity to decrease target gene expression. Based on the observed degree of complementarity between microRNAs (miRNAs) and their targets, it was widely accepted that plant miRNAs act at the mRNA stability level, while the animal miRNAs act at the translational level. Contrary to these canonical dogmas, recent evidence suggests that both plant and animal miRNAs act at both levels. Nevertheless, it is still impossible to predict the effect of an artificial miRNA on the stability or translation of the target mRNA in plants. Consequently, identifying and discarding inefficient amiRNAs prior to stable plant transformation would help getting suppressed mutants faster and at reduced cost. We designed and tested a method using transient expression of amiRNAs and the corresponding target genes in Nicotiana benthamiana leaves to test the efficacy of amiRNAs for suppression of the target protein accumulation. The ability of the amiRNAs to suppress the target gene expression in N. benthamiana was then compared to that in stably transformed Arabidopsis. It was found that the efficacy of 16 amiRNAs, targeting a total of four genes, varied greatly. The effects of amiRNAs on target mRNA accumulation did not always correlate with target protein accumulation or the corresponding phenotypes, while a similar trend of the silencing efficacy of amiRNAs could be observed between N. benthamiana and stably transformed Arabidopsis. Our results showed that, similar to endogenous plant miRNAs, plant amiRNAs could act at the translational level, a property needed to be taken into account when testing the efficacy of individual amiRNAs. Preliminary tests in N. benthamiana can help determine which amiRNA would be the most likely to suppress target gene expression in stably transformed plants.