Spatiotemporal Regulation of <scp> N ₂ </scp> Fixation and Photosynthesis in <i>Trichodesmium</i> : New Insights From <scp>mRNA CARD</scp> ‐ <scp>FISH</scp> and <scp>nanoSIMS</scp>

<jats:title>ABSTRACT</jats:title> <jats:p> To protect the O <jats:sub>2</jats:sub> ‐sensitive nitrogenase enzyme, diazotrophic cyanobacteria separate N <jats:sub>2</jats:sub> fixation from photosynthesis in space or time. However, in the filamentous <jats:italic>Trichodesmium</jats:italic> , technical limitations and conflicting results have so far prevented a consensus on the separation mechanism. Here, we specifically adapted single‐cell methods for simultaneously capturing both photosynthesis and N <jats:sub>2</jats:sub> fixation at the levels of gene expression, fixation and transport of carbon and nitrogen. Testing expression of <jats:italic>nifH</jats:italic> (nitrogenase) and <jats:italic>psbA</jats:italic> (photosystem II), we found that about half of the cells exclusively expressed either one of the genes at a given time, with <jats:italic>nifH</jats:italic> expression mostly in stretches of several consecutive cells within a filament. Short (30 min) stable isotope labelling enabled us to capture 25% of cells in a stage where they fixed nitrogen but not carbon. Lugol staining suggests that low O <jats:sub>2</jats:sub> levels in these cells were maintained by glycogen respiration. Half of the cells received freshly fixed nitrogen within 30 min, most likely by rapid intercellular transport. Taken together, our findings suggest that single‐cell regulation of gene expression in combination with rapid intercellular transport of freshly fixed nitrogen as well as glycogen respiration facilitate the separation of N <jats:sub>2</jats:sub> fixation and photosynthesis in <jats:italic>Trichodesmium</jats:italic> . </jats:p>