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

铁(Fe)的有效性发挥着越来越广为人知的作用，调节着上升的硝酸盐的命运，并决定着海洋中浮游植物群落的大小、结构和群落组成。迄今为止进行的所有富铁实验都报告了硅藻的生物量和光合作用能力的增加。来自田间试验、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，羽状硅藻和中心硅藻在铁的生物有效性和吸收机制、存储容量和胁迫恢复方面存在根本差异。在中尺度的铁添加实验后，羽状硅藻通常在浮游植物组合中占据主导地位，部分原因是它们能够在长期的慢性铁胁迫下保持细胞的活力。这些适应的潜在分子基础几乎是未知的。初步的铁限制三角乳杆菌代谢产物数据表明，代谢重组是必要的，以满足对铁胁迫代谢产物增加的需求，如参与防御活性氧物种(ROS)和细胞内金属螯合的代谢产物。细胞的氮(N)状态，特别是谷氨酸的积累，似乎在铁胁迫的恢复中起着主要作用。这个项目利用注释非常好的三角褐指藻基因组序列来描述基因表达的全球模式，以响应铁和氮胁迫的变化以及在Diel循环的过程中。主要目标是确定限制和定义铁胁迫适应的不同阶段和水平的分子和生理过程。最近，根据铁和氮的有效性和浮游植物的生理指标的不同组合，确定了海洋生理模式。这项研究将在分子水平上深入了解防御、驯化和调节机制和途径，这些机制和途径控制着在海洋相关压力情况下的生存策略，因此具有重大的生态和生物地球化学后果。例如，EST和部分基因组微阵列的初步数据表明，伴侣和蛋白酶在监测细胞健康和平衡防御投资或激活程序性细胞死亡(PCD)之间的差异方面发挥着重要作用。这项拟议的研究将为调节这一迷人而微妙的平衡提供洞察力。这些基本的细胞过程在控制水华动态和调节颗粒通量方面发挥着重要的生物地球化学作用。对全球基因表达的分析将与使用电感耦合等离子体质谱(ICP-MS)和气相色谱-质谱仪(GC-MS)监测细胞内金属水平和初级代谢物谱的最先进水平进行比较，以确定决定细胞存活率的因素。全球基因表达谱与细胞内金属和代谢物库的分析相结合，将首次提供海洋羽状硅藻对铁胁迫的全球细胞响应的整体图景。三角P.tricornutus转录组的图谱将被评估，以了解参与识别和同化这些化合物的基因网络，这些转录组图谱是由于暴露于铁-羟甲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个主要成分)。我们将利用一个先进的反向遗传学系统来控制三角乳杆菌的基因表达水平，以评估特定基因和途径在促进铁胁迫适应中的具体作用。广泛的影响：这项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的重要当前主题，并将为控制硅藻的分布和营养生物地球化学的因素提供洞察力。通过与Affymetrix合作，通过他们的微生物计划，硅藻微阵列资源将首次开放购买和使用。作为拟议研究的一部分，将从学生人数较少的当地学校系统之一招聘一名高中教师从事相关主题的工作。在完成带薪实习后，教师将设计一项课堂活动，供下一学年使用。作为进一步的传播点，这项活动将被纳入现有移动实验室项目的海洋和浮游植物基因组学课程分期付款，该项目名为发现基因组学！，该项目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.