ATP-binding cassette transporters are involved in the active transport of a wide variety of metabolites in prokaryotes and eukaryotes. One subfamily, the Pleiotropic Drug Resistance (PDR) transporters, or full-size ABCG transporters, are found only in fungi and plants. NtPDR1 was originally identified in Nicotiana tabacum suspension cells (BY2), in which its expression was induced by microbial elicitors. To obtain information on its expression in plants, we generated NtPDR1-specific antibodies and, using Western blotting, found that this transporter is localized in roots, leaves, and flowers and this was confirmed in transgenic plants expressing the ß-glucuronidase reporter gene fused to the NtPDR1 promoter region. Expression was seen in the lateral roots and in the long glandular trichomes of the leaves, stem, and flowers. Western blot analysis and in situ immunolocalization showed NtPDR1 to be localized in the plasma membrane. Induction of NtPDR1 expression by various compounds was tested in N. tabacum BY2 cells. Induction of expression was observed with the hormones methyl jasmonate and naphthalene acetic acid and diterpenes. Constitutive ectopic expression of NtPDR1 in N. tabacum BY2 cells resulted in increased resistance to several diterpenes. Transport tests directly demonstrated the ability of NtPDR1 to transport diterpenes. These data suggest that NtPDR1 is involved in plant defense through diterpene transport.

The plasma membrane separates the cell from the external environment and plays an important role in the stress response of the cell. In this study, we compared plasma membrane proteome modifications of yeast cells exposed to mild (0.4 M NaCl) or high (1 M NaCl) salt stress for 10, 30, or 90 min. Plasma membrane-enriched fractions were isolated, purified, and subjected to iTRAQ labeling for quantitative analysis. In total, 88 -109 plasma membrane proteins were identified and quantified. The quantitative analysis revealed significant changes in the abundance of several plasma membrane proteins. Mild salt stress caused an increase in abundance of 12 plasma membrane proteins, including known salt-responsive proteins, as well as new targets. Interestingly, 20 plasma membrane proteins, including the P-type H ؉ -ATPase Pma1, ABC transporters, glucose and amino acid transporters, t-SNAREs, and proteins involved in cell wall biogenesis showed a significant and rapid decrease in abundance in response to both 0.4 M and 1 M NaCl. We propose that rapid protein internalization occurs as a response to hyper-osmotic and/or ionic shock, which might affect plasma membrane morphology and ionic homeostasis. This rapid response might help the cell to survive until the transcriptional response takes place. Molecular & Cellular Proteomics 10: 10.1074/mcp.M111.009589, 1-18, 2011.Exposure of yeast cells to saline stress implies exposure to both specific cation toxicity and osmotic stress (1). Certain ions, such as Na ϩ or Li ϩ , are toxic for cells because of their ability to inhibit specific metabolic pathways, probably through inhibition of specific targets, as has been shown to be the case for the yeast Hal2 protein and certain RNA-processing enzymes (2, 3). In addition, high salinity results in an imbalance in the membrane potential and thus affects the activity of membrane transporters (4) and disrupts ion homeostasis within cells. The yeast cell response to high salinity has been extensively studied and serves as a model for changes in gene expression in response to external stimuli (5, 6). The response is mediated by several stress-responsive signaling pathways, with the high osmolarity glycerol mitogen-activated protein kinase pathway playing a major role. This pathway is involved in sensing an increase in turgor pressure and transducing the appropriate signals to the gene expression program (1). Moreover, Saccharomyces cerevisiae cells respond to high extracellular NaCl concentrations by increasing both potassium uptake and sodium efflux in order to maintain an appropriate Na ϩ /K ϩ ratio (5, 7-11).Transcript expression has been examined as a response of yeast cells to saline stress. The expression of about 7% of the genes in the yeast genome is increased by more than fivefold after a mild and brief saline shock (0.4 M NaCl, 10 min) and most responsive genes show a transient expression pattern, as mRNA levels rapidly decline after 20 min of stress. A similar set of genes shows increased expression in cells subjected to higher s...

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The ability to control the glycosylation pattern of recombinant viral glycoproteins represents a major prerequisite before their use as vaccines. The aim of this study consisted of expressing the large soluble ectodomain of glycoprotein B (gB) from Human Cytomegalovirus (HMCV) in Nicotiana tabacum Bright Yellow-2 (BY-2) suspension cells and of comparing its glycosylation profile with that of gB produced in Chinese hamster ovary (CHO) cells. gB was secreted in the BY-2 culture medium at a concentration of 20 mg/L and directly purified by ammonium sulfate precipitation and size exclusion chromatography. We then measured the relative abundance of N-glycans present on 15 (BY-2) and 17 (CHO) out of the 18 N-sites by multienzymatic proteolysis and mass spectrometry. The glycosylation profile differed at each N-site, some sites being occupied exclusively by oligomannosidic type N-glycans and others by complex N-glycans processed in some cases with additional Lewis A structures (BY-2) or with beta-1,4-galactose and sialic acid (CHO). The profiles were strikingly comparable between BY-2- and CHO-produced gB. These results suggest a similar gB conformation when glycoproteins are expressed in plant cells as site accessibility influences the glycosylation profile at each site. These data thus strengthen the BY-2 suspension cultures as an alternative expression system.

Transcription promoters of heat shock protein (HSP) genes have been used to control the expression of heterologous proteins in plants and plant cells. To obtain a strong HSP promoter that is functionally active in Nicotiana tabacum BY-2 cells, we set out to identify a promoter of an endogenous gene showing high activation of expression by heat. An N. tabacum BY-2 cell culture was treated for 8 h at 37°C and the cell protein extract analyzed by two-dimensional electrophoresis. A major spot was identified by mass spectrometry as belonging to the small HSP family. The promoter regions and the 5' and 3' untranslated regions of two genes, NtHSP3A and NtHSP3B, with sequences fitting the protein identified were cloned and fused to a hybrid reporter gene coding for β-glucuronidase (GUS) and a yellow fluorescent protein. These constructs were introduced into N. tabacum BY2 cells by Agrobacterium tumefaciens-mediated transformation. Both promoters conferred similar heat-induced GUS expression. In the best heat shock condition, GUS activity was increased 200 fold and reached 285 pmol min(-1) μg protein(-1). Up-scaling in a 4-l bioreactor resulted in similar heat-induced expression. The NtHSP3A promoter was then used to drive the expression of NtPDR1, a plasma membrane transporter belonging to the pleiotropic drug resistance family. No expression was observed at 25°C, while, at 37°C, expression was similar to that obtained using a strong constitutive promoter. These data show that the HSP promoters isolated are useful for high heat-induced expression in N. tabacum BY-2 cells.