Eukaryotic Phytoplankton Functional Diversity: Dynamics of Phytoplankton

初级生产力的命运，无论是从透光层迅速输出，转移到营养网上，或在表层有效地回收，主要取决于吸收碳的浮游植物的种类。高的新产量与大细胞有关，大多数开花的特征是单一物种的大细胞或受保护的细胞占优势（例如，硅藻、颗石藻）。间歇性上升流事件是硅藻能够大量繁殖的典型条件，通常只有少数几个种类占主导地位。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，但硅藻在硝酸盐可用时快速生长的能力必须是允许逃逸的一个因素。本计画将以真核浮游植物硝酸盐吸收与运输相关基因的调控为研究重点，探讨硅藻策略的基础。通过评估基因表达，以应对一个典型的上升流周期在蒙特雷湾，加州的氮供应量的变化，研究人员将阐明在这种系统中的硅藻优势在很大程度上可预测的结果的遗传和生理基础。这项工作建立在丰富的海洋学文献和实地研究自然种群的氮吸收动力学，以及对浮游植物生物化学和营养动力学的研究的伟大机构。拟议研究的新方面是增加了遗传水平的调节和反应。由于最近获得了硅藻中硝酸盐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测定方法上的进展，该项目的时间是正确的。定量PCR（Q-PCR）和定量逆转录酶PCR（Q-RT-PCR）将用于定量测定几种生态型硅藻、E. huxleyi和海洋绿藻。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深入了解。此外，含有几十种不同的硝酸还原酶和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的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，研究人员已经开发出必要的初步工具，调查浮游植物对营养盐有效性的反应的遗传调控。 在应用于田间样品之前，将使用培养实验来表征测定的灵敏度、分辨率和时间尺度。在进一步开发和优化检测方法的同时，项目研究人员将进行一项上涌实验，其中基因表达和细胞活性的检测方法将与15 N示踪剂孵育和尺寸分数实验结合使用，以测量N转化。这一领域的演习将进行码头孵化使用蒙特利湾的水，在模拟上升流事件的时间过程中，还原酶和转运蛋白基因的表达特定于某些生态类型（经验定义的基础上，从现场样品的序列相似性）的主要群体（硅藻，E。Huxleyi，绿藻类）进行定量。该研究的结果将允许浮游生物的生理机制的理解，负责可靠的优势硅藻在上升流regimes.The更广泛的影响，这项研究将开发从PI，博士后和学生所做的努力由该项目支持，让本科生参与研究，并丰富他们的教学和培训机会，通过这种经验。计划概述涉及到该项目的领域和实验室方面的高级论文研究本科生。这项工作的跨学科性质有助于吸引来自各个部门（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).