Active Fluorescence Assays of Phytoplankton Physiological State: Population and Individual Cell Measurements

诸如快速重复频率(FRR)荧光测量等主动荧光技术正成为监测海洋浮游植物状况的常用方法，但这些光合作用潜力的测量与浮游植物生长速率等生态相关特性之间的关系仍然存在许多问题。功能反应中心的比例、光系统2(PS2)的功能吸收截面以及PS2和PSI之间的电子传递速率等光合作用参数似乎是特定次优条件下的有价值的诊断指标，但我们如何定量地解释这些指标，或者浮游植物物种和个体之间的异质性在这种解释中可能发挥的作用尚不清楚。因此，Olson和Sosik将进行一系列研究，旨在调查环境条件对浮游植物生长和主动荧光参数的影响，并确定自然种群中浮游植物物种和单个细胞主动荧光反应的可变性程度。具体地说，Olson和Sosik将调查氮、铁和光对浮游植物生长的限制与通过主动荧光技术测量的光合作用参数之间的关系。对活跃荧光表明浮游植物条件不佳的大片海洋区域的观测，以及生长速度似乎与活跃荧光参数脱钩的实验，都提出了关于这些关系的问题。浮游植物的间歇和连续培养将被用来研究细胞生长的瞬时和稳态限制(包括超理想的光强)及其对PS2的光化学能量转换效率(即功能光合作用反应中心的比例)、PS2的功能吸收截面和从PS2到PS1的电子转移速率的影响。通过观察当地水域、春季和夏季伍兹霍尔和马尾藻海之间的横断面和瓶子培养实验，评估浮游植物自然种群对环境条件变化的荧光响应。研究人员将使用的方法侧重于检查主动荧光参数的物种间和物种内的异质性，以及对环境条件的主动荧光反应。Olson和Sosik将同时使用FRR荧光法(一种整体方法)和单个细胞的“探测中泵浦”(PDP)技术来分析上述样品。PDP流式细胞仪将用于测量微米和纳米浮游植物种群以及个体较大的细胞，PDP微型荧光仪将用于分析大于5克的选定物种的个体细胞。因此，活性荧光参数在物种内和物种之间的分布将在不同生长条件下的纯培养中进行评估，包括瞬时和稳态情况。将散体和个体细胞技术应用于自然样品，将使研究人员能够确定不同浮游植物种群对个体测量的影响，分析个体细胞之间的特性分布可能使他们能够推断浮游植物最近的营养历史。结合活性荧光的个体和个体细胞测量将为海洋浮游植物的调节和生长提供新的见解，包括一种评估营养物质有效性动态的新方法。

Olson9819206Active fluorescence techniques such as Fast Repetition Rate (FRR) fluorometry are becoming commonly used to monitor phytoplankton condition in the sea, but many questions remain about the relationships between these measurements of photosynthetic potential and ecologically relevant phytoplankton properties such as growth rate. Photosynthetic parameters such as the fraction of functional reaction centers, the functional absorption cross section of photosystem 2 (PS2), and the rate of electron transfer between PS2 and PSI appear to be valuable diagnostic indicators of specific suboptimal conditions, but it is not clear how quantitatively we can interpret these indicators, or how important a role heterogeneity among phytoplankton species and individuals might play in such interpretation. Olson and Sosik therefore will conduct a series of studies designed to investigate the effects of environmental conditions on phytoplankton growth and active fluorescence parameters, and to determine the extent of variability in active fluorescence responses among phytoplankton species and individual cells in natural populations. Specifically, Olson and Sosik will investigate relationships between nitrogen-, iron-, and light-limitation on phytoplankton growth and photosynthetic parameters as measured by active fluorescence techniques. Observations of wide areas of ocean in which active fluorescence indicates suboptimal phytoplankton condition, and experiments in which growth rate appears to be uncoupled from active fluorescence parameters raise questions about these relationships. Batch and continuous cultures of phytoplankton will be used to study transient and steady-state limitation (including supraoptimal light intensities) of cell growth and the accompanying effects on photochemical energy conversion efficiency of PS2 (i.e., the fraction of functional photosynthetic reaction centers), the functional absorption cross section of PS2, and the rate of electron transfer from PS2 to PS 1. Fluorescence responses of natural populations of phytoplankton to changing environmental conditions will be assessed through observations of local waters, transects between Woods Hole and the Sargasso Sea in spring and summer, and through bottle incubation experiments. The approach the investigators will use focuses on examining inter- and intra-species heterogeneity in active fluorescence parameters, and in active fluorescence responses to environmental conditions. Olson and Sosik will analyze the samples described above by both FRR fluorometry (a bulk method) and individual cell "pump-during-probe" (PDP) techniques. A PDP flow cytometer will be used to measure pico- and nanophytoplankton populations and individual larger cells, and a PDP microfluorometer will be used to analyze individual cells of selected species larger than 5 gm. The distribution of active fluorescence parameters both within and between species will thus be assessed in pure cultures under different growth conditions, including transient and steady state situations. Application of both bulk and individual cell techniques to natural samples will allow the investigators to determine the influence of various phytoplankton groups on the bulk measurements, and analysis of the distribution of properties among individual cells may enable them to deduce the recent nutrient history of the phytoplankton.The combination of bulk and individual-cell measurements of active fluorescence will provide new insights into the regulation and growth of marine phytoplankton, including a new way to evaluate the dynamics of nutrient availability.