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摄取途径或重新安排光合机制以减少细胞需求。相比之下，很少有人关注细胞内铁储存的机制。在世代的时间尺度上，适当地处理和储存铁可以确保在偶发性环境中有足够的铁营养。此外，铁的短期储存对于“缓冲”细胞内氧化还原不稳定的铁浓度和防止活性氧的芬顿产生至关重要。尽管足够的铁可以储存至少4次细胞分裂，远远超过磷、氮和（尤其是）碳，但我们对铁储存的理解远远落后于对这些元素的了解。由于铁和碳、氮、磷的生物地球化学循环是通过浮游植物的铁配额联系在一起的，因此了解铁储存的环境和生理控制至关重要。铁可以储存在诸如铁蛋白超家族的蛋白质中，也可以被隔离在细胞内液泡中。一些海洋硅藻，如三角褐指藻有铁蛋白基因。然而，铁蛋白尚未在其他硅藻（如假海藻）中进行生物信息学或进化PCR检测。研究人员测量了NRAMP转录物和蛋白质丰度的铁依赖性调节，NRAMP可能参与空泡铁代谢，这是拟南芥和酵母中发现的铁储存的另一种方法。不同硅藻群对铁蛋白和液泡介导的铁储存的调控及其生物地球化学意义可能不同。丝状固氮蓝藻赤霉病菌（Trichodesmium erythraeum）具有三个铁蛋白/细菌铁蛋白基因，表明这些蛋白具有特化作用。铁储存和铁缓冲都可能是木霉的关键功能，但对铁稳态的任何方面都一无所知。本项目旨在阐明海洋硅藻和固氮蓝藻细胞内铁的循环和储存，以及铁储存与细胞配额的关系。具体目的是：1)确定三角藻实验室培养中调节铁蛋白转录、载脂蛋白合成和铁蛋白铁含量的因素。潜在的假设是，铁蛋白在长世代的时间尺度上充当铁的储藏库。由于铁蛋白是针对叶绿体的，因此也可以缓冲铁，以防止光合成分降解和合成过程中的氧化应激。2)基于液泡储存铁和NRAMP有助于T. pseudonana、T. oceanica和可能的其他中心硅藻中铁的动员的假设，确定存储液泡和NRAMP在实验室培养中铁的储存和动员中的作用。3)评价木霉培养和田间群体中铁储存蛋白与细胞配额的关系；有人提出，这些蛋白质中的一种或多种在长代的时间尺度上充当铁的储藏库，在这种情况下，它们可能表明营养铁的状态。据推测，这些蛋白质中的一种或多种共定位在细胞中，专门负责在毛霉菌落中固定N2，作为缓冲富铁氮酶蛋白通过die降解释放的铁的机制。上述目标将采用遗传学、免疫学和基于同步辐射的方法应用于实验室培养的三角三角霉、假单胞菌和毛霉。还将分析从马尾藻海收集的毛状菌，以确定（细菌）铁蛋白作为该组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，以解决与海洋学相关的重要主题。了解海洋生态系统对脉冲铁输入（包括有意施肥实验）的响应需要了解浮游植物的生理反应。此外，硅藻和重氮营养体对铁的储存可能会给这些群体带来竞争优势，这可能会影响C的出口和“新”n的供应。该项目将为两名初级研究人员、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。这两个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.