Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Purified Anabaena Flavodoxin Mediates NADP⁺ Photoreduction by Tobacco Thylakoids. Water-driven NADP⁺ photoreduction by thylakoids isolated from 6-week old wild-type plants was measured as indicated in Methods, using either 20 μM ferredoxin (Fd) or 20 μM flavodoxin (Fld). Results depicted are the average ± s. e. of 3 independent determinations.
• Supplemental Table 4 - Effect of Flavodoxin Expression on the Activation State of Thioredoxin-dependent Chloroplast Enzymes in Plants Subjected to Stress Conditions.
• Supplemental Figure 2 - Accumulation of Flavodoxin in Transgenic Tobacco Plants. (A) Flavodoxin (Fld) levels in leaves from nontransformed plants (WT) and sampled primary transformants expressing the flavoprotein in plastids (pfld) or cytosol (cfld). Leaves were ground at the temperature of liquid nitrogen and cleared lysates corresponding to 16 mm² of tissue were resolved by SDS-PAGE on 15% acrylamide gels, and analyzed by immunoblotting with Fld antisera. (B) Segregation of the progeny obtained by self-pollination of primary transformant pfld5 (A), showing typical homozygous (pfld5-8, pfld5-22), heterozygous (pfld5-6) and segregant (pfld5-7) plants. Conditions for sample preparation and immunoblotting were those of panel (A). U, unprocessed Fld precursor; M, mature-sized Fld. (C) Fld contents are maintained through generations in pfld5-8 and cfld1-4 transgenic lines. Conditions for sample preparation and immunoblotting were those of panel (A). (D) Titration of Fld levels in various independent transgenic lines of the T₆ generation. Different amounts of purified Fld and cleared leaf extracts corresponding to 2 (cfld1-4), 4 (pfld5-8, pfld4-2) and 7 (pfld12-4) mg of leaf fresh weight were applied to each lane. Only the signals corresponding to the mature-sized flavoprotein were integrated for estimations of Fld contents. Extracts were prepared from young leaves (node 8) of 8-week old plants cultured in soil under growth chamber conditions.
• Supplemental Figure 3 - Methyl viologen Toxicity in Leaf Discs from Wild-type and Flavodoxin-expressing Plants. Transformants belonging to the T₃ generation and their wild-type parentals were grown in soil for 2 months. Discs were punched from node-8 leaves and exposed to methyl viologen (MV) as described in Methods. Bleaching reflects degradation of chlorophyll, whose levels were determined spectrophotometrically in acetone extracts. Quantitative estimations of chlorophyll losses at the different MV concentrations are provided in Figure 4 of the article.
• Supplemental Figure 4 - Increased Tolerance of Transgenic Tobacco Plants Expressing Bacterial Flavodoxin in Chloroplasts to Different Sources of Environmental Stress. (A) Two-weeks old seedlings were illuminated for 18 h at 500 μmol quanta m^-2 s^-1 and 40°C in MS agar. (B) Seven-weeks old plants were cultured for 20 days at 500 μmol quanta m^-2 s^-1 and 9°C in MS agar. (C) Leaves from 2-months old plants (node 8) grown in soil were exposed for 18 h to a focused light beam of 2,000 μmol quanta m^-2 s^-1. (D) Seven-weeks old plants cultured in hydroponia were exposed to UV-AB radiation for 24 h. (E) Two-months old specimens grown in soil were subjected to a 3-day water deprivation regime. (F) Two-months old plants grown in soil were exposed for 20 min to UV-C radiation and photographed after 3 days. Homozygous transformants of the T₃ generation (flavodoxin expressed in plastids, pfld, or in the cytosol, cfld) and their wild-type (WT) siblings were exposed to the stress treatments as described in Methods. The panels illustrate characteristic phenotypes, with the arrows indicating sites of tissue damage (C and D).
• Supplemental Figure 5 - Effect of Flavodoxin Accumulation on the Patterns of Leaf Antioxidant Enzyme Isoforms. Leaf extracts from soil-cultured 2-month old control (WT) and transgenic plants of the pfld5-8 line (T₅ generation) that had been exposed or not to 30 μM methyl viologen (MV) for 18 h at 500 μmol quanta m^-2 s^-1 were resolved by nondenaturing PAGE. The presence of superoxide dismutase (SOD, A) and ascorbate peroxidase (APX, B) isoenzymes was visualized after activity staining of the gels as described in Methods. The various isoenzymes were named as in Beauchamp and Fridovich (1971), and Mittler and Zilinskas (1993), with cAPX and pAPX indicating cytosolic and plastidic APX, respectively.
• Supplemental Figure 6 - The Levels of Components of the Thioredoxin/Ferredoxin-thioredoxin Reductase Pathway are not Affected by Flavodoxin Expression or Stress. Leaf extracts were prepared from the youngest fully expanded leaves (node 8) by the freeze-clamp procedure. Cleared lysates corresponding to 10 μg of protein were resolved on 15% polyacrylamide gels, transferred to nitrocellulose membranes and decorated with polyclonal antibodies raised against spinach ferredoxin-thioredoxin reductase (FTR), thioredoxin (Trx) m or Trx f.
• Supplemental Table 1 - Phenotypic Analysis of Wild-type and Transgenic Tobacco Plants Expressing Flavodoxin in Chloroplasts.
• Supplemental Table 2 - Effect of Flavodoxin Expression on the Levels of Antioxidant Enzymes and Metabolites in Plants Exposed to MV.
• Supplemental Table 3 - Flavodoxin Expression Prevents Overoxidation of 2-Cys Peroxiredoxin in Stressed Tobacco Plants.