Volume 2, Issue 3, Pages 442-456 (May 2009) Pyridine Nucleotide Cycling and Control of Intracellular Redox State in Relation to Poly (ADP-Ribose) Polymerase Activity and Nuclear Localization of Glutathione during Exponential Growth of Arabidopsis Cells in Culture  Pellny Till K. , Locato Vittoria , Vivancos Pedro Diaz , Markovic Jelena , De Gara Laura , Pallardó Federico V. , Foyer Christine H.   Molecular Plant  Volume 2, Issue 3, Pages 442-456 (May 2009) DOI: 10.1093/mp/ssp008 Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 1 Changes in the Cellular Ascorbate and Glutathione Pools and in PARP Activity during the Growth of Dark-Grown Arabidopsis Cells in Culture. Growth parameters (optical density, OD); packed cell volume (PCV) of dark-grown Arabidopsis cell cultures (A) and accumulation of total ascorbate (B), glutathione (C), PARP Dot Blot (D) and calculated PARP activity (E). Molecular Plant 2009 2, 442-456DOI: (10.1093/mp/ssp008) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 2 The Relative Growth Rates of Dark-Grown Arabidopsis Cells in Culture through the Growth Cycle. A sigmoidal growth curve was modeled on the optical density over the growth cycle (solid line) and the relative growth rate (multiplication per day) was calculated from the parameters (broken line). The arrows (10a and 10b) indicate the points at which cells were harvested for analysis of intracellular GSH compartmentation shown in Figure 10. Molecular Plant 2009 2, 442-456DOI: (10.1093/mp/ssp008) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 3 Relationships between the Relative Growth Rates of Dark-Grown Arabidopsis Cells and the Cellular Pools of Ascorbate (A) and Glutathione (B). Molecular Plant 2009 2, 442-456DOI: (10.1093/mp/ssp008) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 4 Relationships between PARP Activity and the Cellular Pools of Ascorbate (A) and Glutathione (B). The clear symbols are data from day 1, the gray symbols are data from day 2 to 5, and the black symbols are data from day 7. Thus, in (B), the line marking the correlation involves time points 1, 2, 3 and 7. Molecular Plant 2009 2, 442-456DOI: (10.1093/mp/ssp008) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 5 Growth Cycle Profiling of Selected Transcripts Encoding Cell Cycle, Ascorbate, Glutathione, PARP, and Pyridine Nucleotide-Related Enzymes. The heat map was generated using the data from Menges et al. (2002), concerning cell cycle-regulated gene expression in Arabidopsis cells. For each array probe set, expression strength is compared across the growth cycle, with the left panel showing the expression at day 1 in a blue heat map, with lighter blue showing higher expression. Changes in expression compared to day 1 are shown in a red/green heat map on the right. Red symbolizes increases and green decreases. Numbers directly above the heat map showing relative gene expression levels indicate the harvest point in terms of the day of the growth cycle (as in Figure 1). Molecular Plant 2009 2, 442-456DOI: (10.1093/mp/ssp008) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 6 Effects of Dilution and Media Change on the Growth of Light-Grown Arabidopsis Cells in Culture. Measured via optical density (OD). Growth in control cultures (diamonds, solid line) is compared to growth in diluted cultures (circles, broken line). and cultures subjected to media change (triangle, dotted line). The actual OD values are given in (A). The calculated values in (B) were determined using the factor of dilution. Molecular Plant 2009 2, 442-456DOI: (10.1093/mp/ssp008) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 7 Effects of Dilution and Media Change on PARP1 (A, C, E) and PARP2 (B, D, F) Expression during the Growth of Light-Grown Arabidopsis Cells in Culture. Samples were harvested at the points indicated and qPCR was performed. Relative expression values are normalized to the day 1 value (symbols as in Figure 6). Molecular Plant 2009 2, 442-456DOI: (10.1093/mp/ssp008) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 8 The Influence of Dilution and Media Change on the Intracellular Pools of Oxidized and Reduced Pyridine Nucleotides in Light-Grown Arabidopsis Cells. Effects of prolongation of the logarithmic growth phase by either diluting the cultures or replenishing the media immediately after the 72-h time point on the intracellular pools of NAD (A), NADP (B), NADH (C), and NADPH (D) are shown relative to values during the normal growth cycle (symbols as in Figure 6). Molecular Plant 2009 2, 442-456DOI: (10.1093/mp/ssp008) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 9 The Influence of Dilution and Media Change on the Intracellular Pools of Oxidized, Reduced and total Glutathione in Light-Grown Arabidopsis Cells. Effects of prolongation of the logarithmic growth phase by either diluting the cultures or replenishing the media immediately after the 72-h time point on the intracellular pools on the total intracellular glutathione pool (A), reduced glutathione (GSH; (B)), and glutathione disulphide (GSSG; (C)) are shown relative to the growth cycle and with respect to the relative growth rate with regression lines shown (total intracellular glutathione pool, (D); GSH, (E); GSSG, (F)). The correlation coefficients and p-values refer to the complete Simple Linear Regression Model (symbols as in Figure 6). Molecular Plant 2009 2, 442-456DOI: (10.1093/mp/ssp008) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions

Figure 10 The Effect of Growth Cycle Stage on the Cellular Distribution of GSH in Dark-Grown Arabidopsis Cells in Culture. Cells harvested at time points (A) and (B) during the logarithmic growth phase as indicated by the arrows in Figure 2 were analyzed by confocal light microscopy. The compartmentation of GSH was detected using CMFDA (green). Nuclei were located using dihydroethidium (blue in A1 and A2) or visually in the light field in (B). Chloroplasts were detected by autofluorescence (red) in both (A) and (B). Merged images with the light field are shown in A2 and B2. Nuclei are marked in blue in A2 and chloroplasts marked in red in A2 and B2. Molecular Plant 2009 2, 442-456DOI: (10.1093/mp/ssp008) Copyright © 2009 The Authors. All rights reserved. Terms and Conditions