Eukaryotic Phytoplankton Functional Diversity: Dynamics of Phytoplankton

初级生产的命运，无论是从光区迅速输出，向上转移营养网，还是在表层有效地循环，主要取决于吸收碳的浮游植物的种类。高新产量与大型细胞有关，大多数水华的特点是单一种类的大型或受保护的细胞(如硅藻、球藻)占优势。时断时续的上升流事件是硅藻能够开花的典型条件，在这种情况下，少数几个物种通常占主导地位。虽然放牧逃逸(通常是暂时的)可能是水华生物量积累的最终原因，但硅藻在获得硝酸盐时快速生长的能力肯定是允许这种逃逸的一个因素。本项目将通过对真核浮游植物中参与硝酸盐吸收和运输的基因的调控来研究硅藻策略的基础。通过评估基因表达对加利福尼亚州蒙特利湾典型上升流周期中氮素有效性的变化的响应，研究人员将阐明此类系统中硅藻占优势的基本可预测结果的遗传学和生理学基础。这项工作建立在丰富的海洋学和野外研究自然种群中氮素吸收动态的文献，以及大量浮游植物生物化学和营养动力学研究的基础上。这项拟议研究的新方面是增加了基因水平的调控和反应。由于最近获得了硅藻中硝酸还原酶和硝酸盐转运蛋白基因的基因序列，以及最近在基因表达分析方法方面的进展，这个项目的时机是正确的。定量聚合酶链式反应(Q-PCR)和定量逆转录酶聚合酶链式反应(Q-RT-PCR)将被用来定量测定几种生态型硅藻、绿藻和海洋绿藻中硝酸还原酶和转运蛋白基因的拷贝数和基因水平。最初的工作已经证明了这些方法的力量，并已经导致了对真核浮游植物不同策略的洞察。此外，包含数十种不同硝酸还原酶和Rubisco基因探针的微阵列的初步结果使研究人员能够确定浮游植物离散生态型的相对丰度，并独立地评估每种类型的基因表达的相对水平。因此，研究人员开发了研究浮游植物对营养物质可获得性的遗传调控所需的初步工具。在应用于田间样品之前，将使用培养实验来表征检测的灵敏度、分辨率和时间尺度。在进一步开发和优化测试的同时，项目研究人员将进行上升流实验，其中基因表达和细胞活性的分析将与15N示踪剂孵化和粒度分数实验一起部署，以测量N的转化。这项实地演习将在使用蒙特利湾水的码头孵化中进行；在模拟的上升流事件的时间过程中，将量化主要类群(硅藻、赫克斯莱伊、绿藻)的某些生态型(根据野外样本的序列相似性而经验性定义)的还原酶和转运蛋白基因的表达。这项研究的结果将有助于从机制上了解浮游生物在上升流中占据优势地位的原因。这项研究的更广泛影响将来自PIS、博士后和受项目支持的学生努力让本科生参与研究，并通过这一经历丰富他们的教学和培训机会。计划包括让本科生参与与该项目的领域和实验室方面有关的高级论文研究。这项工作的跨学科性质吸引了来自不同系(地球科学、分子生物学、生态学和进化生物学)的学生。

The fate of primary production, whether rapidly exported from the photic zone, transferred up the trophic web, or recycled efficiently in the surface layer, depends primarily on the kind of phytoplankton that assimilate the carbon. High new production is associated with large cells, and most blooms are characterized by dominance of a single species of large or protected cells (e.g., diatoms, coccolithophorids). Episodic upwelling events typify conditions in which diatoms are able to bloom, and in which a few species usually dominate. Although escape (usually temporary) from grazing may be the ultimate reason that bloom biomass accumulates, the ability of diatoms to grow rapidly when nitrate becomes available must be a factor in allowing that escape. This project will investigate the basis of the diatom strategy by focusing on the regulation of genes involved in nitrate uptake and transport in eukaryotic phytoplankton. By evaluating gene expression in response to the changing nitrogen availability of a classic upwelling cycle in Monterey Bay, California, the investigators will elucidate the genetic and physiological basis of the largely predictable outcome of diatom dominance in such systems. This work builds on the rich literature of oceanographic and field research into nitrogen uptake dynamics in natural populations, and on the great body of research on phytoplankton biochemistry and nutrient kinetics. The novel aspect of the proposed research is the addition of the genetic level of regulation and response. The time is right for this project because of the very recent acquisition of gene sequences for nitrate reductase and nitrate transporter genes in diatoms, and recent methodological progress on assays for gene expression. Quantitative PCR (Q-PCR) and quantitative reverse transcriptase PCR (Q-RT-PCR) will be used to quantify gene copy number and mRNA levels for nitrate reductase and transporter genes in several ecotypes of diatoms, E. huxleyi and marine chlorophytes. Initial work has demonstrated the power of these approaches and already has led to insights into different strategies of eukaryotic phytoplankton. In addition, preliminary results from a microarray containing several dozen different nitrate reductase and rubisco gene probes has allowed the investigators to determine the relative abundance of discrete ecotypes of phytoplankton and, independently, to assess the relative level of gene expression for each type. Thus the investigators have developed the initial tools necessary for investigation of genetic regulation of phytoplankton response to nutrient availability. Culture experiments will be used to characterize the sensitivity, resolution and time scale of the assays before application to field samples. In parallel with further assay development and optimization, the project investigators will conduct an upwelling experiments in which the assays for gene expression and cell activity will be deployed in conjunction with 15N tracer incubation and size fraction experiments to measure N transformations. This field exercise will be conducted in a dock incubation using Monterey Bay water; over the time course of a simulated upwelling event, the expression of reductase and transporter genes specific to certain ecotypes (empirically defined on the basis of sequence similarity from field samples) of the major groups (diatoms, E. huxleyi, chlorophytes) will be quantified. The results of the study will allow a mechanistic understanding of the plankton physiologies that are responsible for the reliable dominance of diatoms in upwelling regimes.The broader impacts of this study will develop from the efforts made by the PIs, post doc and students supported by the project to involve undergraduates in the research and to enrich their teaching and training opportunities by this experience. Plans are outlined to involve undergraduates in senior thesis research related to both field and lab aspects of the project. The interdisciplinary nature of this work lends itself to attracting students from various departments (geosciences, molecular biology, ecology and evolutionary biology).