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

关于浮游植物铁的生理研究大多集中在诱导高亲和力吸收途径或重排光合作用机制以减少细胞需求上。相比之下，细胞内储铁的机制却鲜有人关注。在世代的时间尺度上适当地处理和储存铁可以确保在周期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Fenton产生活性氧物种是必不可少的。尽管至少4个细胞分裂可以储存足够的铁，比P、N和(特别是)C的情况要多得多，但我们对铁储存的理解远远落后于这些元素的已知存储。由于铁与C、N和P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，因此理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中，或者隔离在细胞内的空泡中。一些海洋硅藻，如三角褐指藻，具有铁蛋白基因。然而，在其他硅藻中，如假海链藻，还没有通过生物信息学或进化的聚合酶链式反应方法检测到铁蛋白。研究人员测量了NRAMP转录本和蛋白质丰度的铁依赖调节，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另一种铁储存方法。推测铁蛋白和液泡介导的储铁作用在不同的硅藻类群中可能具有不同的调控作用和生物地球化学意义。丝状氮气固定蓝藻，红曲霉，具有三个铁蛋白/细菌铁蛋白基因，提示这些蛋白的专化性。铁储存和铁缓冲都可能是毛霉菌的关键功能，但对铁稳态的这两个方面都一无所知。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，以及铁储存与细胞配额的关系。具体目标是：1)确定调控三角褐指藻实验室培养物中铁蛋白转录、脱辅蛋白合成和铁蛋白铁含量的因素。基本的假设是，铁质在很长一代的时间尺度上作为铁的储存库。由于它们以叶绿体为目标，铁蛋白还可以缓冲铁，以防止光合作用成分降解和合成过程中的氧化应激。2)确定储藏液泡和NRAMP在假藻实验室培养中铁的储存和活化中的作用，基于液泡储存铁的假说，以及NRAMP帮助假藻、海洋假藻和其他中心硅藻中铁的活化。3)评价毛霉菌培养群体和田间群体中储铁蛋白与细胞数量之间的关系，认为其中一个或多个蛋白在世代长的时间尺度上作为储铁蛋白，在这种情况下，它们可能指示营养铁状态。据推测，这些蛋白中的一个或多个共同定位于Trichodesum菌落中负责氮气固定的细胞中，作为一种机制来缓冲通过富含Fe的固氮酶蛋白的Diel降解释放的Fe。上述目标将使用遗传、免疫学和基于同步加速器的方法应用于实验室培养的三角毛霉、假单胞菌和毛霉菌。还将对从马尾藻海收集的毛状毛进行分析，以确定(细菌)铁质作为这一组的储存机制的生物地球化学重要性。广泛的影响：该项目结合最先进的分子生物学和微观分析技术，解决具有重大海洋学意义的主题。要了解海洋生态系统对脉冲铁输入的反应(包括有意的施肥实验)，需要了解浮游植物的生理反应。此外，硅藻和重氮菌对铁的储存可能会给这些群体带来竞争优势，这可能会影响碳的出口和“新”氮的供应。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，其中包括一名代表不足的群体的成员。这两个专业人员将积极参与本科生的培训，库斯特卡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.