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

主动荧光技术，如快速重复率（FRR）荧光法，正被广泛用于监测海洋中的浮游植物状况，但关于这些光合势测量与浮游植物生态相关特性（如生长速度）之间的关系仍存在许多问题。光合参数，如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之间的电子转移率似乎是特定次优条件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有多重要尚不清楚。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活性荧光参数的影响，并确定浮游植物物种和自然种群中单个细胞的活性荧光反应的变化程度。具体来说，Olson和Sosik将研究氮、铁和浮游植物生长和光限制之间的关系，并通过主动荧光技术测量光合参数。在广泛的海洋区域中，活性荧光表明浮游植物处于次优状态，而在实验中，生长速度似乎与活性荧光参数不耦合，这些都提出了有关这些关系的问题。浮游植物的间歇培养和连续培养将用于研究细胞生长的瞬态和稳态限制（包括超优光强）及其对PS2光化学能转换效率（即功能光合反应中心的比例）、PS2的功能吸收截面和PS2向ps1的电子转移速率的影响。浮游植物自然种群对不断变化的环境条件的荧光反应将通过在春季和夏季对当地水域、伍兹霍尔和马尾藻海之间的横断面的观察，以及通过瓶培养实验进行评估。研究人员将使用的方法侧重于检查有效荧光参数的物种间和物种内异质性，以及对环境条件的有效荧光响应。Olson和Sosik将通过FRR荧光法（散装法）和单个细胞“泵-探针”（PDP）技术分析上述样品。PDP流式细胞仪将用于测量微型和纳米浮游植物种群和单个较大的细胞，PDP微荧光仪将用于分析大于5克的选定物种的单个细胞。因此，将在不同生长条件下的纯培养物中评估物种内部和物种之间的活性荧光参数分布，包括瞬态和稳态情况。将整体和单个细胞技术应用于自然样品将使研究人员能够确定各种浮游植物群对整体测量的影响，而对单个细胞之间特性分布的分析可能使他们能够推断浮游植物最近的营养历史。活性荧光的整体和单个细胞测量的结合将为海洋浮游植物的调节和生长提供新的见解，包括评估营养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.