Collaborative Research: Expression profiling and functional genomics of a pennate diatom: Mechanisms of iron acquisition, stress acclimation, and recovery

铁（Fe）有效性在调节上涌硝酸盐的命运和决定海洋浮游植物组合的大小结构和群落组成方面发挥着越来越重要的作用。迄今为止进行的所有铁富集实验都报告了硅藻生物量和光合能力的增加。来自野外实验、详细的生理调查和基因组序列数据的越来越多的证据表明，pennate和centric硅藻在铁的生物利用度和吸收机制、储存能力和胁迫恢复方面存在根本差异。在中尺度铁添加实验后，Pennate硅藻通常在浮游植物组合中占主导地位，部分原因是它们能够在长时间的慢性铁胁迫下维持细胞活力。这些适应的潜在分子基础实际上是未知的。铁限制性三角藻的初步代谢物数据表明，代谢重组是满足铁胁迫代谢物需求增加的必要条件，如参与防御活性氧（ROS）和细胞内金属螯合的代谢物。细胞氮(N)状态，特别是谷氨酸的积累，似乎可能在铁胁迫下的恢复中起主要作用。该项目利用极好注释的三角褐指藻基因组序列来表征基因表达的全球模式，以响应进入和退出铁和氮胁迫以及在饮食周期的过程中。主要目的是确定约束和定义铁胁迫驯化不同阶段和水平的分子和生理过程。近年来，人们根据浮游植物的铁氮有效性和生理指标的不同组合来定义海洋生理机制。这项研究将提供分子水平上的见解，以了解在海洋相关压力情况下控制生存策略的防御、驯化和调节机制和途径，从而产生重大的生态和生物地球化学后果。例如，初步EST和部分基因组微阵列数据表明，伴侣蛋白和蛋白酶在监测细胞健康和平衡防御投资或程序性细胞死亡（PCD）激活之间的差异方面发挥着重要作用。拟议的研究将为这种迷人而微妙的平衡的调节提供见解。这些基本的细胞过程在控制水华动力学和调节颗粒通量方面起着重要的生物地球化学作用。全球基因表达分析将与使用ICP-MS和气相色谱-质谱（GC-MS）对细胞内金属水平和初级代谢物谱的最新监测进行比较，以确定决定细胞生存能力的因素。结合全球基因表达谱和细胞内金属和代谢物池的分析，将首次提供海洋pennate硅藻对铁胁迫的全球细胞反应的整体图景。将评估暴露于铁-羟酸铁载体和血红素结合铁（两类铁结合配体，据信构成海水中铁的两种主要成分）的三角螺转录组谱，以了解参与识别和同化这些化合物的基因网络。一个先进的反向遗传系统操纵水平基因表达的三角三角木将被用来评估特定的基因和途径在促进铁胁迫适应中的具体作用。更广泛的影响：本研究整合了生物地球化学、微生物生态学、海洋科学和基因组生物学等重要的当前主题，并将深入了解控制硅藻分布和营养生物地球化学的因素。通过与Affymetrix合作，通过他们的微生物学项目，硅藻微阵列资源将首次可供公开购买和使用。作为拟议研究的一部分，将从一个拥有大量未被充分代表的学生群体的当地学校系统中招募一名高中教师，从事相关主题的工作。在完成带薪实习后，老师将设计一个课堂活动，以供下一学年使用。作为进一步的传播点，该活动将被纳入一个现有的名为DISCOVER genomics的移动实验室项目的海洋和浮游植物基因组学课程。，它与华盛顿特区大都市区的中学生互动。

Iron (Fe) availability plays an increasingly well known role regulating the fate of upwelled nitrate and determining the size structure and community composition of phytoplankton assemblages in the ocean. All Fe enrichment experiments conducted to date have reported increases in the biomass and photosynthetic capacity of diatoms. Mounting evidence from field experiments, detailed physiological investigation, and genomic sequence data suggest fundamental differences in Fe bioavailability and uptake mechanisms, storage capacity, and stress recovery between pennate and centric diatoms. Pennate diatoms often dominate the phytoplankton assemblage after mesoscale Fe addition experiments because, in part, they are able to maintain cell viability during long periods of chronic Fe stress. The underlying molecular bases for these adaptations are virtually unknown. Preliminary primary metabolite data of Fe-limited P. tricornutum suggest that metabolic reconfigurations are necessary to meet increased demand for Fe-stress metabolites such as those involved in defense from reactive oxygen species (ROS) and intracellular metal chelation. Cellular nitrogen (N) status, and the accumulation of glutamate in particular, appears likely to play a primary role in recovery from Fe stress. This project capitalizes on the extremely well annotated Phaeodactylum tricornutum genome sequence to characterize global patterns of gene expression in response to shifts into and out of Fe and N stress and over the course of the diel cycle. The primary goal is to determine the molecular and physiological processes that constrain and define different phases and levels of Fe-stress acclimation. Oceanic physiological regimes have recently been defined according to different combinations of Fe and N availability and physiological indicators of the resident phytoplankton. This research will provide molecular-level insights into defense, acclimation, and regulatory mechanisms and pathways that govern survival strategies in situations of oceanographically-relevant stress and thus are of major ecological and biogeochemical consequence. Preliminary EST and partial genome microarray data, for example, indicate that chaperones and proteases play a significant role in monitoring cellular health and balancing the difference between investment in defense or activation of programmed cell death (PCD). The proposed research will provide insights into the regulation of this fascinating and delicate balance. Such basic cellular processes play an important biogeochemical role in controlling bloom dynamics and regulating particle flux. Analysis of global gene expression will be compared with state of the art monitoring of intracellular metal levels and primary metabolite profiles using ICP-MS and gas chromatograph-mass spectroscopy (GC-MS) to determine the factors that determine cell survivability. The combination of global gene expression profiling and analysis of intracellular metal and metabolite pools will supply, for the first time, a holistic picture of the global cellular response of a marine pennate diatom to Fe-stress. P. tricornutum transcriptome profiles resulting from exposure to Fe - hydroxamate siderophores and heme-bound Fe (two classes of Fe binding ligands that are believed to comprise two major components of Fe in seawater) will be evaluated to understand the network of genes involved in recognizing and assimilating these compounds. An advanced reverse-genetics system for manipulating levels gene expression in P. tricornutum will be used to evaluate the specific role of particular genes and pathways in facilitating Fe stress acclimation.Broader Impacts: This research integrates important current themes in biogeochemistry, microbial ecology, marine sciences, and genome biology and will provide insight into factors that control the distribution and nutrient biogeochemistry of diatoms. By partnering with Affymetrix, through their Microbiology Program, a diatom microarray resource will be made available for the first time for open purchase and use. As part of the proposed research, a high school teacher from one of the local school systems with large underrepresented student populations will be recruited to work on a related topic. Upon completion of his/her paid internship, the teacher will design a classroom activity for use the following school year. As a further point of dissemination, the activity will be incorporated into a curriculum installment focused on marine and phytoplankton genomics for an existing mobile laboratory program called DISCOVER GENOMICS!, which interacts with middle school students in the Washington, D.C. Metropolitan area.