L'Auto-vélo : automobilisme, cyclisme, athlétisme, yachting, aérostation, escrime, hippisme / dir. Henri Desgranges

22 avril 19271927/04/22 (A28,N9624)

Growth curves of wild type and L-ascorbic acid producing yeasts under oxidative stress.

<p>Kinetics of growth of wild type and engineered strains GRF18U (left panels) and BY4742 (right panels) as inoculated in minimal glucose media without H₂O₂ (3A and 3B) or in presence of H₂O₂ 3.5 mM (3C and 3D). • GRF18U wild type; ▪ GRF18U[<i>ScALO AtLGDH AtME AtMIP</i>]; ▴ GRF18U[<i>ScALO AtLGDH AtME AtMIP RnFGT</i>]; ○ BY4742 wild type; □ BY4742[<i>ScALO AtLGDH AtME AtMIP</i>]; ▵ BY4742[<i>ScALO AtLGDH AtME AtMIP RnFGT</i>].</p

The effects of 35S-driven expression of <i>Nt</i>ABP1 in 35S-<i>Nt</i>ABP1 tobacco BY-2 cells, and treatment with the inhibitor of auxin efflux NPA.

<p>(A–E) Morphology of three-day-old non-induced and induced cells, control and NPA-treated (10µM for three days). (A,B,D,E) Nomarski DIC images. Scale bars, 20 µm. (C) Cell length of non-treated and NPA-treated (10 µM for three days) cells. 100%, value for non-treated BY-2 cells. Error bars, SEM, n~300. Asterisks indicate significantly different means between cells non-expressing and expressing 35S-driven Nt<i>ABP1</i> gene. Two sample t-test assuming unequal variances; *P < 0.001, df = 465; **P < 0.001, df = 320. (F) Growth curves for BY-2 and 35S-<i>Nt</i>ABP1cells. Error bars, SEM, n=4. (G) Accumulation of [³H] NAA as an indicator of the auxin efflux. Difference in [³H] NAA accumulation between cells non-treated and treated with NPA is shown for one-day-old BY-2 (control) and 35S-<i>Nt</i>ABP1 cells. Radioactivity was measured 25 min after addition of radioactively labelled auxin (2 nM) without or together with NPA (10 µM). Error bars, SEM, n=3. The asterisk denotes statistical significance of difference (P = 0.018 in paired samples t-test). (H,I) Relative expression of Nt<i>ABP1</i> gene in control BY-2 and 35S-<i>Nt</i>ABP1 cell lines. (H) qRT-PCR from cDNA 24 hours after inoculation of cells into the fresh medium. Error bars, SEM, n=6. (I) PCR of Nt<i>ABP1</i> using genomic DNA (702bp fragment of endogenous gene, 256bp fragment of transgenic cDNA).</p

ABP1 inhibits fluorescence recovery after photobleaching (FRAP) of PIN1-GFP.

<p>(A–D) FRAP in the three-day-old tobacco PIN1-GFP/GVG-<i>At</i>ABP1 and PIN1-GFP cells. (A) Transversal plasma membranes decorated by PIN1-GFP in the PIN1-GFP/GVG-<i>At</i>ABP1 cells showing the situation before, immediately after and 170s after the photobleaching. Scale bars, 10 µm. (B) FRAP measured in the PIN1-GFP control cells 170 s after photobleaching. Cells were treated with DEX (1µM) in DMSO or DMSO only. Error bars, SEM, n=15. (C) Kinetics of FRAP in non-induced and induced PIN1-GFP/GVG-<i>At</i>ABP1 cells. Error bars, SEM, n=6. (D) Comparison of FRAP after 170 s in cells pre-treated with BFA (20 µM for 30 min), tyrphostin A23 (Tyr A23, 50 µM for 30 min), NAA (5 µM for 60 min), or NPA (10 µM for 25 min). Error bars, SD, Ctrl, n=6; BFA, n=6; Tyr A23, n=6; NAA, n=10; NPA, n=7. FRAP for the PIN1-GFP/GVG-<i>At</i>ABP1 non-induced cells, 100%. Asterisks indicate significant difference between <i>At</i>ABP1 non-expressing (non-induced) and expressing (induced) cells within given treatment. *P < 0.001, **P = 0.080 using independent samples t-test. The differences for Tyr A23 (P = 0.800), NAA (P = 0.412), and NPA (P = 0.332) treatments are not statistically significant. (E) Schematic depiction of a dual action of ABP1 in regulation of PIN dynamics and activity, resulting in control of auxin levels in a cell. In brackets, experimental intervention is presented. Under low intracellular auxin level, e.g. after overexpression of PIN efflux carriers, ABP1 promotes PIN endocytosis to reduce undesirable auxin export. Under high auxin level, e.g. after external addition of auxin or after inhibition of the active auxin efflux by NPA, ABP1 counteracts the endocytosis of PINs and leaves them on the PM thus promoting the active auxin efflux.</p

Additional file 1 of Secretory expression of recombinant small laccase genes in Gram-positive bacteria

Additional file 1: Supplementary figure 1. SignalP 5.0 predictions of the studied small laccases. Supplementary figure 2. The earlygrowth of S. lividans and secretion studies of ScLac ΔSP. Supplementary figure 3. The detection of ScLac signal sequence peptides in S. lividans expression system. Supplementary figure 4. The detection of ScLac signal sequence peptides in B. subtilis RIK1285 expression system. Supplementary figure 5. The detection of SvLac signal sequence peptides in B. subtilis RIK1285 expression system. Supplementary figure 6. The viability of B. subtilis  RIK1285 expressing recombinant small laccese genes using fluorescent microscopy. Supplementary figure 7.AmDyP production in B. subtilis RIK1285. Supplementary figure 8. An overview of the plasmids used in this study. Table 1. Primers used in this study

List of yeast strains constructed and used in this study

<p>List of yeast strains constructed and used in this study</p

Flow cytometric analysis of wild type and vitamin C producing yeasts under oxidative stress.

<p>Panel 5A: schematic representation of the different subpopulations that can be observed in the following panels, where wild type (5B) and recombinant strains (5C and 5D) grown in minimal glucose medium added with H₂O₂ 3.0 mM were analyzed after DHR123 and PI staining (rodamine signal is reported in the abscissa and PI signal on the ordinate axes). Upper panels: GRF18U background. Lower panels: BY background. (B): wild type. (C): [<i>ScALO AtLGDH AtME AtMIP</i>] transformed cells. (D): [<i>ScALO AtLGDH AtME AtMIP RnFGT</i>] transformed cells</p

Conversion of D-Glucose into L-ascorbic acid (milligrams/liter/OD) by transformed <i>S. cerevisiae</i> GRF18U and BY4742 cells.

<p>All strains were grown on mineral medium (2% w/v glucose, 0.67% w/v YNB), starting with an initial OD660 of 0.05 for 18 h, when samples were taken and the concentration of L-ascorbic acid inside the cells was determined (GRF18U and BY4742 correspond to the parental strains transformed with the empty plasmids harboring in the productive strains the genes of the L-AA pathway). The control cells, as well as the cells expressing <i>ScALO1</i> and <i>AtLGDH</i> can not accumulate L-ascorbic acid starting from D-glucose, therefore measured values correspond to the endogenous erythro-ascorbic acid. The standard deviation bars correspond to the data obtained from independent clones, and from independent growth and antioxidant determinations. Please note the different scale of the ordinate axes in the two graphs.</p

List of expression plasmids constructed and used in this study*

<p>Abbreviations: Sc: <i>Saccharomyces cerevisiae</i>; At: <i>Arabidopsis thaliana</i>; Rn: <i>Rattus norvegicus</i>; Zb: <i>Zygosaccharomyces bailii</i>;</p><p>TPI: Triose Phosphate Isomerase</p><p><i>URA3, HIS3, LEU2, LYS2</i>: gene markers conferring growth to auxotrophic yeast strains in the absence of uracil, histidine, leucine and lysine, respectively.</p><p>Nat^R: cassette conferring resistance to nourseotricine.</p><p>Kan^R: cassette conferring resistance to Geneticin.</p><p>CEN and INT: centromeric and integrative plasmids, respectively.</p>*<p>a complete description of plasmids construction is given in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001092#s4" target="_blank">Materials and Methods</a></p

The effects of inducible expression of <i>At</i>ABP1 in GVG-<i>At</i>ABP1 tobacco BY-2 cells, and treatment with the inhibitor of auxin efflux NPA.

<p>(A–E) Morphology of three-day-old non-induced and induced cells, control and NPA-treated (10µM for three days). (A,B,D,E) Nomarski DIC images. Scale bars, 40 µm. (C) Cell length of non-treated (Ctrl) and NPA-treated (10 µM for three days) cells. 100%, value for non-induced control. Error bars, SEM, n~300. Asterisks indicate significantly different means between cells non-expressing and expressing the At<i>ABP1</i> gene, two sample t-test assuming unequal variances; *P < 0.005, degrees of freedom (df) = 581; **P < 0.001, df = 573). (F) Growth curves for non-induced and induced cells, non-treated and treated with NPA (10 µM for three days). Error bars, SEM, n=4. (G) Accumulation of [³H] NAA as an indicator of the auxin efflux. One-day-old GVG-<i>At</i>ABP1 cells were treated with [³H] NAA (2 nM) alone (Ctrl) or in combination with NPA (10 µM), and radioactivity was measured after 25 min. Data values are percentages of non-induced, non-treated control (100%). Error bars, SEM, n=3. The differences in [³H] NAA accumulation between non-induced and induced GVG-<i>At</i>ABP1 cells either without or after NPA application are not statistically significant (P = 0.707 and P = 0.328, respectively, paired samples t-test). (H) Relative expression of the At<i>ABP1</i> gene in the GVG-<i>At</i>ABP1 cell line. qRT-PCR at 24 hours after induction with dexamethasone (DEX, 1 µM). Error bars, SEM, n=6. (I) Accumulation of [³H] NAA in BY-2 cells transformed with an empty pTA7002 vector, measured 25 min after the addition of [³H] NAA (2 nM). Data are expressed as percentages of non-treated control (100%), and represent the mean of four technical repetitions. Error bars, SEM, n=4.</p