Most studies on the Fe physiology of phytoplankton have focused on the induction of high affinity uptake pathways or the rearrangement of photosynthetic machinery to decrease cellular demand. By contrast, little attention has been given to the mechanisms of intracellular Fe storage. Proper handling and storage of Fe on timescales of generations can ensure adequate Fe nutrition in episodic environments. Furthermore short term storage of Fe is essential to "buffer" the intracellular redox-labile Fe concentration and prevent Fenton production of reactive oxygen species. Even though sufficient Fe can be stored for at least 4 cell divisions, much more than in the cases of P, N and (especially) C, our understanding of Fe storage lags far behind what is known for those elements. Since the biogeochemical cycles of Fe and C, N and P are linked via the Fe quotas of phytoplankton, it is critical that we understand the environmental and physiological controls of Fe storage. Fe can be stored in proteins such as those of the ferritin superfamily or sequestered into intracellular vacuoles. Some marine diatoms, such as Phaeodactylum tricornutum have ferritin genes. However ferritin has not been detected bioinformatically or by evolutionary PCR methods in other diatoms such as Thalassiosira pseudonana. The investigators have measured the Fe-dependent regulation of transcript and protein abundance of NRAMP, a protein likely involved in vacuolar Fe metabolism, an alternative method of Fe storage found in Arabidopsis thaliana and yeast. It is proposed that the regulation and biogeochemical significance of ferritin and vacuole-mediated Fe storage may differ for different diatom groups. The filamentous N2 fixing cyanobacterium, Trichodesmium erythraeum, possesses three ferritin/ bacterioferritin genes, suggesting specialization of these proteins. Both Fe storage and Fe buffering are likely critical functions in Trichodesmium, yet nothing is known of either aspect of Fe homeostasis. This project aims to elucidate intracellular cycling and storage of Fe in marine diatoms and N2 fixing cyanobacteria and the relationship between Fe storage and cell quota. Specific objectives are to: 1) Determine the factors that regulate ferritin transcription, apo-protein synthesis and ferritin iron content in P. tricornutum lab cultures. The underlying hypothesis is that ferritins serve as Fe storage reservoirs over long generational time scales. Because they are targeted to chloroplasts, ferritins may also buffer Fe to prevent oxidative stress during degradation and synthesis of photosynthetic components. 2) Determine the role of storage vacuoles and NRAMP in Fe storage and mobilization in lab cultures of T. pseudonana, based on the hypothesis that vacuoles store Fe and NRAMP helps mobilize Fe in T. pseudonana, T. oceanica, and possibly other centric diatoms. 3) Evaluate the relationships between Fe storage proteins and cellular quota in culture and field populations of Trichodesmium; it is proposed that one or more of these proteins serve as Fe reservoir over long generational, time scales, in which case they may indicate nutritional Fe status. It is hypothesized that one or more of these proteins are co-localized in cells specifically responsible for N2 fixation in Trichodesmium colonies as a mechanism to buffer the Fe released through the diel degradation of the Fe-rich nitrogenase proteins. The above objectives will be addressed using genetic, immunological, and synchrotron-based approaches applied to laboratory cultures of P. tricornutum, T. pseudonana,and Trichodesmium. Trichodesmium trichomes collected from the Sargasso Sea will also be analyzed to determine the biogeochemical importance of (bacterio)ferritins as a storage mechanism in this group.
Broader impacts: This project combines state-of-the-art molecular biological and micro-analytical techniques to address topics of significant oceanographic relevance. Understanding of the response of marine ecosystems to pulsed Fe inputs (including intentional fertilization experiments) requires understanding the physiological response of phytoplankton. Further, storage of Fe by diatoms and diazotrophs likely imparts a competitive advantage to these groups that may impact C export and supply of 'new' N. This project will provide training and support for two beginning investigators, a PhD student and three undergraduate students, including a member of an underrepresented group. Both PIs will be actively involved in training of undergraduates, and Kustka will continue on-going community outreach activities.
