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

合作研究：硅藻和固氮蓝藻中的铁储存：机制、调节和生物地球化学意义

• 批准号: 0913080
• 负责人: Benjamin Twining
• 金额: $ 25.7万
• 依托单位: Bigelow Laboratory for Ocean Sciences
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-08-22 至 2011-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0913080&HistoricalAwards=false
• 关键词: Collaborative; Research; Iron; storage; diatoms

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.

大多数浮游植物铁生理学研究都集中在诱导高亲和力吸收途径或重新排列光合机制以减少细胞需求。相比之下，细胞内铁储存的机制却很少受到关注。在世代时间尺度上正确处理和储存铁可以确保偶发环境中充足的铁营养。此外，Fe 的短期储存对于“缓冲”细胞内氧化还原不稳定的 Fe 浓度并防止芬顿产生活性氧物种至关重要。尽管足够的 Fe 可以储存至少 4 次细胞分裂，比 P、N 和（尤其是）C 的情况要多得多，但我们对 Fe 储存的理解远远落后于对这些元素的了解。由于铁和碳、氮和磷的生物地球化学循环是通过浮游植物的铁配额联系起来的，因此了解铁储存的环境和生理控制至关重要。 Fe 可以储存在铁蛋白超家族等蛋白质中或隔离在细胞内液泡中。一些海洋硅藻，例如三角褐指藻，具有铁蛋白基因。然而，尚未通过生物信息学或进化 PCR 方法在其他硅藻（如假微型海链藻）中检测到铁蛋白。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 是一种可能参与液泡铁代谢的蛋白质，液泡铁代谢是拟南芥和酵母中发现的另一种铁储存方法。有人提出，对于不同的硅藻类群，铁蛋白和液泡介导的铁储存的调节和生物地球化学意义可能不同。丝状 N2 固定蓝藻，Trichodesmium erythraeum，拥有三个铁蛋白/细菌铁蛋白基因，表明这些蛋白质的特化。 Fe 储存和 Fe 缓冲可能都是 Trichodesmium 的关键功能，但对 Fe 稳态的任一方面都一无所知。该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之间的关系。具体目标是： 1) 确定三角藻实验室培养物中调节铁蛋白转录、脱辅基蛋白合成和铁蛋白铁含量的因素。基本假设是铁蛋白在较长的代际时间尺度上充当铁储存库。由于铁蛋白以叶绿体为目标，因此还可以缓冲铁，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 Fe 和 NRAMP 有助于在 T. pseudonana、T. oceanica 和可能的其他中心硅藻中动员 Fe 的假设，确定储存液泡和 NRAMP 在 T. pseudonana 实验室培养物中 Fe 储存和动员中的作用。 3) 评估Trichodesmium培养物和野外种群中Fe储存蛋白与细胞配额之间的关系；有人提出，这些蛋白质中的一种或多种在长时间的代际时间尺度上充当铁库，在这种情况下，它们可能表明营养性铁状态。据推测，这些蛋白质中的一种或多种共定位于专门负责Trichodesmium菌落中N2固定的细胞中，作为缓冲通过富铁固氮酶蛋白质的昼夜降解释放的Fe的机制。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三角褐藻、假微藻和毛藻的实验室培养来实现。还将对从马尾藻海收集的毛藻毛状体进行分析，以确定（细菌）铁蛋白作为该组存储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分析技术来解决具有重要海洋学相关性的主题。了解海洋生态系统对脉冲铁输入（包括有意施肥实验）的反应需要了解浮游植物的生理反应。此外，硅藻和固氮生物对铁的储存可能会给这些群体带来竞争优势，从而影响碳的出口和“新”氮的供应。该项目将为两名新研究人员、一名博士生和三名本科生（其中包括一名代表性不足的群体的成员）提供培训和支持。两位 PI 都将积极参与本科生的培训，Kustka 将继续开展持续的社区外展活动。