Collaborative Research: Iron storage in diatoms and N2 fixing cyanobacteria: mechanisms, regulation and biogeochemical significance

大多数关于浮游植物铁生理的研究都集中在诱导高亲和力吸收途径或光合机构重排以减少细胞需求。与此相反，很少有人注意到细胞内铁储存的机制。适当的处理和储存铁的时间尺度的世代可以确保足够的铁营养在情节的环境。此外，Fe的短期储存对于“缓冲”细胞内氧化还原不稳定的Fe浓度和防止活性氧的芬顿产生是必要的。尽管足够的铁可以储存至少4次细胞分裂，远远超过P，N和（特别是）C的情况下，我们对铁储存的理解远远落后于已知的这些元素。由于铁和C，N和P的生态地球化学循环是通过浮游植物的铁配额联系在一起的，因此我们了解铁储存的环境和生理控制是至关重要的。铁可以储存在蛋白质中，如铁蛋白超家族的蛋白质，也可以被隔离在细胞内的空泡中。一些海洋硅藻，如三角褐指藻（Phaeodactylum tricornutum）具有铁蛋白基因。然而，铁蛋白还没有检测到生物信息学或进化PCR方法在其他硅藻，如海链藻。研究人员测量了NRAMP的转录和蛋白质丰度的铁依赖性调节，NRAMP是一种可能参与液泡铁代谢的蛋白质，是拟南芥和酵母中发现的铁储存的另一种方法。建议的铁蛋白和液泡介导的铁存储的调节和地球化学意义可能不同的硅藻组。丝状N2固定蓝藻，Trichodesmium pigraeum，具有三个铁蛋白/细菌铁蛋白基因，这表明这些蛋白质的专业化。铁储存和铁缓冲可能是关键功能的束毛藻，但没有什么是已知的铁稳态的任何方面。本项目旨在阐明海洋硅藻和固氮蓝藻中铁的细胞内循环和储存以及铁储存与细胞定额的关系。具体目标是：1）确定调节三角褐指藻实验室培养物中铁蛋白转录、脱辅基蛋白合成和铁蛋白铁含量的因子。基本假设是铁蛋白作为铁储存水库在很长的世代时间尺度。因为它们是针对叶绿体，铁蛋白也可以缓冲Fe，以防止氧化应激过程中的降解和光合成分的合成。2)确定贮存泡和NRAMP在T. Escherichana等人基于液泡储存铁和NRAMP有助于铁在T. Escherichia，T. oceanica和其他可能的中心硅藻。3)评价铁储存蛋白和细胞定额之间的关系，在文化和外地人口的束毛藻，它建议，这些蛋白质中的一个或多个作为铁水库在长世代，时间尺度，在这种情况下，他们可能表明营养铁状态。据推测，这些蛋白质中的一种或多种共定位在细胞中，特异性地负责束毛藻菌落中的N2固定，作为缓冲通过富Fe固氮酶蛋白质的昼夜降解释放的Fe的机制。上述目标将通过遗传学、免疫学和同步加速器为基础的方法应用于三角褐指藻、三角褐指藻和三角褐指藻的实验室培养来实现。和束毛藻属（Trichodesmium）。还将分析从马尾藻海收集的毛束藻毛状体，以确定（细菌）铁蛋白作为该组中储存机制的生物地球化学重要性。更广泛的影响：该项目结合了最先进的分子生物学和微观分析技术，以解决具有重要海洋学意义的主题。了解海洋生态系统的响应脉冲铁输入（包括有意施肥实验）需要了解浮游植物的生理反应。此外，硅藻和固氮生物的铁存储可能赋予这些群体的竞争优势，可能会影响C出口和供应的“新”N。该项目将为两名初级调查员、一名博士生和三名本科生提供培训和支持，其中包括一名代表性不足的群体成员。两个PI将积极参与本科生的培训，Kustka将继续进行社区外展活动。

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.