荧光动力学法浮游植物初级生产力测量中初始荧光准确获取

Accurate Acquisition of Initial Fluorescence in Phytoplankton Primary Productivity Measurement by Fluorescence Dynamics Method

针对目前初始荧光检测技术集中在特征吸收波长领域,非特征吸收波长检测灵敏度低,难以准确反映藻类捕光色素能量吸收效率的问题,提出了荧光动力学法浮游植物初级生产力测量中的初始荧光准确获取技术。利用OJIP荧光动力学技术测量原理调控电子受体氧化还原状态,获得最佳激发条件,利用积分放大技术对多波段初始荧光进行高灵敏检测。测量结果表明:初始荧光能够准确反映藻类色素的实际吸收情况,铜绿微囊藻在569 nm出现藻红蛋白(PE)吸收峰,在620 nm出现藻蓝蛋白(PC)主要吸收峰,4种淡水绿藻均在439 nm出现Chl a吸收峰,在474 nm出现Car吸收峰;5种受试藻种的荧光产率F_(PSII)与初始荧光相似度均在0.996以上,表明初始荧光能够用于反映光系统Ⅱ(PSII)吸收,且测量准确性较高。

Objective After illumination,the electron transport chain of dark-adapted phytoplankton is inhibited to bring about the gradual closure of the reaction center.Light energy absorbed by the light-harvesting pigment is released solely by fluorescence or thermal dissipation to elevate fluorescence yield.This increase initiates the chlorophyll fluorescence induction process.In the early stage of chlorophyll fluorescence induction,the photochemical reaction has not yet commenced,and the photosynthetic reaction center remains fully accessible.This stage is termed the initial fluorescence phase,and it is measured by light sources with varying wavelengths within the visible light spectrum.These measurements provide vital photosynthetic insights,including pigment content,reaction center concentration,energy absorption,and excitation energy transfer.They precisely depict the structure and composition of light-harvesting pigments in phytoplankton,along with energy absorption efficiency.Consequently,this technique critically contributes to analyzing the photosynthetic status and primary productivity of live phytoplankton.Following the revelation of chlorophyll fluorescence induction,multiple techniques for measuring initial fluorescence have emerged.For example,Schreiber et al.introduced the technique of pulse amplitude modulation(PAM)for measuring photoinduced fluorescence kinetics,Kolber et al.suggested the fast repetition rate fluorescence(FRRF)measurement technique,and Strasser et al.developed the OJIP technique for rapidly measuring chlorophyll fluorescence-induced kinetic curves by continuous excitation luminescence.Currently,research on the technical approaches predominantly centers on characteristic absorption bands.Nevertheless,the sensitivity of non-characteristic absorption bands remains limited,hindering the accurate portrayal of the structural composition of light-harvesting pigments and energy absorption efficiency.Therefore,the development of a profoundly sensitive method for initial fluorescence measurement is pivotal in advancing the investigations of phytoplankton primary productivity.Methods We employ the photosynthetic electron transport model and the OJIP fluorescence kinetics measurement technology to regulate the redox state of electron receptors proximate to the O phase,thereby attaining optimal excitation conditions.Under weak light excitation,LHCII absorbs energy at a low level,and the excitation energy is transferred to the reaction center.The electron acceptor can receive and promptly re-oxidize electrons to establish a rapid dynamic equilibrium,which leads to a consistent initial fluorescence signal.Due to the weak nature of the initial fluorescence signal,integrating and amplifying signals across various bands within the microsecond range enable the attainment of highly sensitive detection(50-150μs)of initial fluorescence.Thus,precise acquisition technology for initial fluorescence is indispensable for investigating the primary productivity of phytoplankton by fluorescence dynamics.Validation of the initial fluorescence measurement results involves comparing the PSII absorption coefficient and initial fluorescence similarity.Results and Discussions The findings from the initial fluorescence measurements indicate strong correspondence between the measurements of photosynthetic pigment absorption in phytoplankton and actual absorption patterns.For example,Microcystis aeruginosa exhibits a PE absorption peak at 569 nm and a PC absorption peak at 620 nm,and freshwater green algae show an absorption peak of Chl a at 439 nm and a Car absorption peak at 474 nm(Fig.3).Moreover,compared to the reference sample,the verification results indicate the proficient representation of PSII absorption by the initial fluorescence,thereby confirming a substantial degree of measurement accuracy.The PSII fluorescence yield closely mirrors the initial fluorescence profile,exhibiting similarity values of 0.996 for Microcystis aeruginosa,0.999 for Scenedesmus dimorphus,0.999 for Scenedesmus obliquus,0.999 for Chlorella ellipsoidea,0.998 for Oocystis lacustris,and surpassing 0.998 for all four species of freshwater green algae(Fig.4).Conclusions We address the constraints of existing initial fluorescence measurement methodologies,which predominantly concentrate on characteristic absorption bands to reduce sensitivity for absorption bands lacking distinct characteristics.As a result,these techniques inadequately represent the energy absorption efficiency of photosynthetic organs in algae.To this end,we propose a precise technology for acquiring initial fluorescence and facilitating primary productivity measurement in phytoplankton by fluorescence dynamics.This approach integrates the photosynthetic electron transfer model with the measurement principles of OJIP fluorescence dynamics technology.The results of the initial fluorescence measurements demonstrate significant correspondence between the measurements of photosynthetic pigment absorption in phytoplankton and actual absorption patterns.For example,Microcystis aeruginosa exhibits a PE absorption peak at 569 nm and a PC absorption peak at 620 nm,and freshwater green algae show an absorption peak of Chla at 439 nm and a Car absorption peak at 474 nm.Furthermore,the comparative verification results indicate a close similarity between the shapes of the PSII fluorescence yield and the initial fluorescence,affirming the capacity of the initial fluorescence to precisely mirror PSII absorption.The similarity values are noteworthy,with 0.996 for Microcystis aeruginosa,0.999 for Scenedesmus dimorphus,0.999 for Scenedesmus obliquus,0.999 for Chlorella ellipsoidea,0.998 for Oocystis lacustris.Additionally,all the four species of freshwater green algae surpass 0.998.We introduce a remarkably sensitive measurement technology for initial phytoplankton fluorescence to facilitate precise and accurate measurements.Consequently,noteworthy technical advancements are provided for investigating primary productivity in phytoplankton.