Segregation of Nitrogen Fixation and Oxygenic Photosynthesis in the Marine Cyanobacterium Trichodesmium Ilana Berman-Frank, Pernilla Lundgren, Yi-Bu Chen, Hendrik Küpper, Zbigniew Kolber, Birgitta Bergman, and Paul Falkowski

Supplementary Material

To further test the importance of PSI in preventing oxidative stress and damage to nitrogenase we applied ascorbate and 1,4-dithiothreitol (DTT) to cultures under both aerobic and anaerobic conditions (Web fig 1). Ascorbate is an important reducing substrate for H₂O₂ detoxification via the ascorbate-glutathione pathway (1) . (Web fig. 2). DTT is a thiol reductant that reduces thioredoxin on the acceptor side of PSI (2, 3).

Addition of DTT (100M) to Trichodesmium IMS101 cultures during the photoperiod, slightly stimulated or did not affect nitrogenase activity compared to control samples for both aerobic and anaerobic incubations (Web fig. 1). Under aerobic conditions, ascorbate (100 M) always inhibited nitrogenase activity (from 10 to 70% of controls), while under anaerobic conditions; ascorbate did not cause any significant inhibition of activity. Addition of DTT to samples incubated with ascorbate aerobically reversed inhibition by ~ 25% (Web fig. 1). Under aerobic conditions, ascorbate addition stimulates the ascorbate-glutathione cycle (Web fig. 2) and increases demand for reductant (NADPH). This may be at the expense of N₂-fixation and may explain the declining nitrogenase activity (Web fig. 2). Under anaerobic conditions, ascorbate action as a scavenger is not necessary and NADPH can be diverted to nitrogen fixation. The slight stimulation of nitrogen fixation by DTT under aerobic conditions indicates that reduced thiol bonds in thioredoxin may alleviate some of the oxygen stress by detoxification of H₂O₂ via the activity of thioredoxin peroxidases, which are coupled to the electron transport chain (4, 5).

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Supplemental Figure 1. Influence of DCMU (10 M), DBMIB (2.5 M), ascorbic acid (100 M), and DTT (100 M) on nitrogenase activity. The effects of the inhibitors were tested for cultures incubated under aerobic (empty bars) and anaerobic conditions (shaded bars). Changes in nitrogenase activity were measured by acetylene reduction. Anaerobic conditions were obtained by bubbling the serum bottles with N₂ for 1 min. and immediately sealing the bottles. Inhibitors were added 3 h after beginning of the photoperiod and nitrogenase activity was measured after 2 h of incubation with the inhibitors.

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Supplemental Figure 2. Schematic model showing the interactions between nitrogen fixation, photosynthesis, respiration and oxygen consuming reactions (Mehler and the ascorbate-glutathione pathway) in Trichodesmium spp. Photosynthetic electrons are shuttled via PSI to several pathways. Linear photosynthesis supports carbohydrate synthesis and substrates for respiration and is required for operation of the Mehler reaction which reduces oxygen produced at PSII to H₂O₂ by superoxide dismutase (SOD) and subsequently to water via ascorbate peroxidase (APX) and the ascorbate-glutathione pathway (GSH - glutathione, GSSG - glutathione disulphide, GR - glutathione reductase, DHA - dehydroascorbate, DHAR - dehydroascorbate reductase, MDHA - monodehydroascorbate, MDHAR - monodehydroascorbate reductase). The Mehler reaction may also supply ATP for nitrogen fixation. For nitrogen fixation to occur, six electrons are shuttled through pyruvate (product of respiratory glycolosis), coenzyme-A, and ferredoxin or flavodoxin -(that form part of PSI ). These six electrons are required to sequentially reduce the enzyme dinitrogenase reductase . The highly reduced form of the enzyme combines with 16 ATP, 2H⁺ and 2 more electrons to fix 1 molecule of N₂ and produce 1 molecule of H₂ and 2NH₄⁺ (6).

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Supplemental References:

1. G. Noctor and C. H. Foyer, Ann. Rev. Plant Physiol. Plant Mol. Biol. 49, 249-279 (1998).
2. K. Sippola, E.-M. Aro, Plant Mol.Bio. 41, 425-433 (1999).
3. Thioredoxin may be reduced or oxidized by the relative flow of photosynthetic electrons shuttling via PSI to ferrodoxin and the enzyme ferrodoxin-thioredoxin reductase [K. Sippola, E.-M. Aro, Photochemistry and Photbiology 71, 706-714 (2000)].
4. M. Tichy, W. Vermaas, J. Bacteriol. 181, 1875-1882 (1999).
5. H. Yamamoto et al., FEBS Lett. 447, 269-273 (1999).
6. Postgate, J.R. and R.R. Eady, The evolution of biological nitrogen fixation, in Nitrogen Fixation: One Hundred Years After, H. Bothe, DeBruijn, F. J., Newton, W.E, Editor. 1988, Gustav Fischer: Stuttgart. p. 31-40.]".