Photosynthesis Research 49: 151-157, 1996.

© 1996 Kluwer Academic Publishers. Printed in the Netherlands.

Regular paper

Fluorescence induction curves registered from individual microalgae cenobiums in the process of population growth.

Galina Riznichenko, Galina Lebedeva, Sergei Pogosyan, Marina Sivchenko & Andrei Rubin

Department of Biophysics, Biological Faculty, Moscow Lomonosov State University, 119899 Moscow, Russia

Key words: distribution, microfluorometry, pattern, slow phase of fluorescence kinetics

Abstract

Registration of chlorophyll fluorescence induction curves (IC) from individual microalgae cenobiums was performed during Scenedesmus quadricauda culture growth. An emphasis was placed on the analysis of patterns of the slow phase of IC, since these slow fluorescence transitions reflect complex interactions between primary and secondary photosynthetic processes. Classification of IC obtained in the experiment according to the patterns of their slow phase was performed. Four different types of such patterns were distinguished. The microalgae population structure with respect to IC patterns was investigated at different stages of culture growth. The distribution of microalgae cenobiums over the patterns of IC was found to change in accordance with the stage of population development. At the stage of the population growth enhancement nonmonotone IC dominated with high steady-state level of fluorescence. The stage of linear growth was characterized by IC with monotone decay kinetics and low steady-state level of fluorescence. At the third stage including the phases of growth inhibition, stationary state and the beginning of cells death the population structure was the most heterogeneous with all IC patterns observed.

Abbreviations: CO₂ - carbon dioxide, ETC - electron transfer chain, Fl - fluorescence, FNR - ferredoxin- NADPH reductase, IC - induction curve of chlorophyll fluorescence, PQ - plastoquinone, PS I - photosystem I, PS II - photosystem II, Q_A - primary quinone acceptor of PS II

Introduction

Heterogeneity is one of the major properties of natural and artificially cultured cellular populations. Cells forming a population differ in age, size, growth rate and, what is of great importance, in reaction to environmental factors. Functional differences between individual cells and their different reactions to external influences are of the utmost importance for comprehending the adaptation of the total population to environmental conditions. That is why the analysis of distribution of cells in the population over functional characteristics and the study of its dynamics may help to determine the population state.

To determine photosynthetic activity of the autotrophic algae population fluorometric measurements of parameters of the cells suspension contained in the sample volume are usually performed. The intensity of fluorescence measured in such experiments under different conditions of illumination allows one to draw conclusions about chlorophyll contents in the sample and average efficiency of cells photosynthesis.

In the recent years there have been developed microfluorometric methods to measure fluorescence of individual cells and thereby to obtain information about the state of the photosynthetic apparatus of the cell without damaging it. Usually for these purposes researchers use flow microfluorometers which allow for the short period of time to carry out measurements of the photosynthetic parameters of a large number of cells. The work with the flow fluorometer allows one to obtain distributions of different cell characteristics using rather large samplings. Thus, during an hour tens of thousands of measurements of this kind can be made . After that statistic analysis of the distributions under different experimental conditions is usually performed. In a number of works (Li and Wood 1988, Sosic at al. 1989, Yentsch and Phinney 1989) fulfilled according to this scheme several statistical characteristics were found to change which in its turn could be used for the diagnostics of the unfavourable state of the population. However, such an approach, as a rule, does not reveal the mechanisms responsible for the shifts of the distributions of the measured parameters. In this case one usually makes a suggestion about a new subpopulation to appear with its own "shifted" distribution of the parameters resulting in visible changes of the original statistical characteristics.

A more detailed analysis of the mechanisms by the flow fluorometers is impossible since the experiments on each cell allow one to register only instantaneous magnitude of fluorescence intensity at the moment of measurement. It is practically impossible to draw any conclusions about the mechanisms of the fluorescence change without observing kinetics of the processes. The advantage of a version of the microfluorometric method used in the present work as compared to the conventional flow fluorometer is the possibility to register the cell fluorescence kinetics for a comparatively long period of time (a few minutes) and hence, to register chlorophyll fluorescence induction curves from individual cells.

In the present work the analysis of slow phase of fluorescence induction curves (IC) obtained from individual algae cenobiums has been carried out. The algae population structure with respect to IC patterns has been investigated at different stages of population development.

Methods

To carry out the experiments on registration of the chlorophyll fluorescence induction curves on individual microalgae cells we used green alga Scenedesmus quadricauda culture from the collection of the Hydrobiology Department of the Biological Faculty of Moscow State University. The cells of this microalga form colonies consisting of 4 cells (cenobiums), which were the object of our investigation.

From agar slopes algae were sown to 10% Tamiya medium . The cultivation was carried out in the flasks of 300 ml volume at temperature 26C and illumination by luminescence day-light lamps with intensity 8 W m^-2. The experiments were started on the 4th day after sowing when the number of cenobiums in 1 ml of medium reached the value of 210⁴ . The number of cenobiums was measured in Najotte chamber by the direct count method. Registration of chlorophyll fluorescence induction curves was performed with the use of luminescence microscope LUMAM-I3 with fluorescence measuring attachment FMEL-1A (LOMO, Leningrad, USSR). Photomultiplier FEU-79 (MELZ, Moscow, USSR) sensitive in spectral range of 300-820 nm was used to detect fluorescence. The time resolution of fluorescence registration was 0.1 s. Fluorescence excitation was provided by the halogen lamp KGM 9-70 with the light filter SZS-22, transmitting only blue-green part of the spectrum. Intensity of excitation light was 10 W m^-2. Light filter KS-18, transparent to light radiation in the range of wave lengths > 670 nm, was used for fluorescence registration. Objects of study on the microscope viewfield were selected on the background of blue light illumination of the sample from below, with light intensity < 0.2 W m^-2. The diameter of the photometric area was 37.5 . For photometric investigation only cenobiums consisting of 4 cells were used, selected randomly on the viewfield of the microscope. Before the beginning of the measurements algae were adapted to the dark conditions for 15-20 min. Recording of the fluorescence induction curve for each individual cenobium was performed during 200 s. In the process of algae cultivation (52 days) 846 fluorescence kinetic curves were recorded. To analyze the shape of IC we used 10 parameters F₁, F₁₀, F₂₅, F₅₀, ... F₂₀₀ which correspond to the fluorescence intensities at time moments 1, 10, 25, 50, 75, 100, 125, 150, 175 and 200 s after the beginning of light illumination.

Results

A typical fluorescence induction curve of green plants and algae with generally accepted designations related to its different kinetic phases is presented in the insert to Fig.1. Rapid changes of fluorescence yield on the OIDP portion of IC are determined mainly by initiation of primary light processes in the photosynthesis system: charge separation in the reaction center of PS II and increase in the concentration of reduced Q_A (O-I phase of IC), redox changes of the PQ pool (I-D phase) and reduction of ferredoxin and other PS I electron acceptors (D-P phase) (Dau 1994a,b).

Further slow changes of fluorescence yield on the PSMT-portion of IC result from combined effect of photochemical and nonphotochemical processes taking place in photosynthetic membranes of a plant cell. Photochemical quenching corresponds to the decrease of fluorescence emission due to oxidation of reduced Q_A by electron transport chain (ETC) carriers. It is considered that SMT-transitions of fluorescence on the induction curve as well as the appearance of additional maxima are mainly due to stimulation of dark reactions of Calvin cycle of CO₂ fixation in chloroplast stroma (Walker 1981, Walker at al. 1983, Ireland at al. 1984, Bradbury at al. 1995, Ireland at al. 1985, Walker and Osmond 1986, Seaton and Walker 1990). Nonphotochemical fluorescence quenching results from such processes as formation of proton gradient across thylakoid membrane (Krause and Laasch 1987, Quick at al. 1989, Adams at al. 1990, Dau and Hansen 1990, Havaux 1990, Noctor and Horton 1990), phosphorilation of the light-harvesting complex (Barber 1983, Hodges and Barber 1984, Allen 1992), oxidation of plastoquinone pool (Vernotte at al. 1979) and photoinhibition (Krause 1988). Thus, transients in the fluorescence quantum yield at the PSMT portion of the induction curve are multiphase and result from a number of processes proceeding in cells in response to illumination after the dark adaptation period.

The analysis of fluorescence induction curves (IC) recorded in our experiments during population growth showed that the shape of individual IC varied considerably (here and further under the shape or pattern of IC we mean kinetic pecularities of PSMT portion of fluorescence induction curve). However, one could distinguish 4 main classes of IC patterns, each of them including subclasses A and B (Fig.1).

Fig.1. Patterns of the slow phase of fluorescence induction curves, registered from individual cenobiums of Scenedesmus quadricauda. In the insert a typical fluorescence induction curve of green plants with generally accepted designations of its phases is shown. The arrow indicates the moment of switching on the light, F - fluorescence intensity.

To ascribe an experimantal IC to a definite class the following features were taken into account:

- presence of additional maximum M and minimum S on the IC in the time range 1 - 100 s of IC recording;

- location of maximum M and minimum S with respect to the steady-state level T (lower or higher). The steady-state level of Fl was considered to be reached at 200-th s of IC recording;

- location of the final portion of IC in the time range 75 - 200 s with respect to the steady-state level T (lower or higher);

- the value of ratio F₁/F₂₀₀ ;

- the rate of approaching the steady-state level of Fl at the S-T portion of IC for classes 4 A and 4B .

Time dependence of IC amplitude for type 1 is exponential with much faster decay in class 1B as compared to class 1A. The ratio F₁/F₂₀₀ for class 1B is much less than for class 1A. We suppose that IC of 1B type correspond to dying or damaged cenobiums, since their amplitude in the peak phase P was approximately 5 times lower than the average amplitude of IC of the other types.

The second maximum M appears on class 2 curves in the process of fluorescence decay . The level S of Fl in the case of 2A is higher and in the case of 2B is lower than the steady- state level T. Class 3 has complicated kinetic form. There are two intermediate minima S₁ and S₂ (both of them lower than T) after which the Fl amplitude reaches the steady state level. Maximum M is lower than T in the case of 3A, but higher than T in the case of 3B. The curves of class 4 have only one minimum S located lower than T. The curves of 4A type reach the steady-state level of Fl faster than the curves of 4B type.

The final portion of IC within 75 - 200 s time range for the types 1 and 2 reflects monotonedecrease of fluorescence intensity as it reaches the steady-state level. Contrary to that the same final portion of IC in classes 3 (S₂ - T phase) and 4 (S-T phase) corresponds to the monotone increase of fluorescence intensity in approaching its steady-state level.

So, class 1 is characterized by simple decay kinetics and low stationary level of Fl (mean value of the ratio F₁/F₂₀₀ is 2.93), class 4 is characterized by the high stationary level of Fl (mean value of the ratio F₁/F₂₀₀ is 2.1), classes 2 and 3 have more complicated IC patterns and could be considered as intermediate.

We tried to characterize population development by the dynamics of IC pattern distribution recorded in the process of algae culture growth.

We constructed scattering diagrams in coordinates F₁₀/F₂₅ against F₅₀/F₁₀₀ (Fig.2), which represent the ratio of the fluorescence intensities at definite moments during IC recording after switching on the light (10, 25, 50 and 100 s ). The latter moments were chosen to correspond to certain peculiarities of the IC shape (maxima and minima) observed at those moments. Hence, the state of each cell under study is represented by a point on the scattering diagram. Different areas of the diagram correspond to different IC patterns (Fig. 2,).

Fig.2. Dynamics of distribution of cells over the IC patterns during Scenedesmus quadricauda population growth. , and - scattering diagrams for three stages of Scenedesmus quadricauda population development. F10/F25 and F50/F100 represent the ratios of chlorophyll fluorescence intensities at definite moments during IC recording after the beginning of illumination (10, 25, 50 and 100 s); -correspondence of different IC patterns to certain areas of the scattering diagram.

It was found that during the Scenedesmus quadricauda growth cenobiums distribution with respect to IC patterns at different stages of the population development differ reliably from each other. Three stages of microalgae culture development were distinguished in accordance with the predominance of different IC patterns in the course of population growth (Fig.3).

Fig.3. Scheme of the Scenedesmus quadricauda population growth curve with designation of its stages and . The numbers over the curve stand for the IC types dominant in the population at the corresponding stage of growth.

At the first stage of culture growth including the phase of growth enhancement (4 - 6 th day of the measurements) the majority of cenobiums is characterized by IC patterns of the types 3 and 4. The number of cenobiums with IC of other types was much lower (Fig.2, ).

At the second stage which includes exponential and linear growth phases (7 - 35 days of measurements) almost all the population cells had the same pattern (1A) of the induction curve. (Fig.2, ).

The third stage includes the following phases: growth inhibition, stationary state and the beginning of cells death (36-52 days of experiments). This stage was characterized by the largest heterogeneity of the population: there were cenobiums with IC patterns of all types (Fig. 2, ).

Discussion

Apparently the analysis of the shapes of fluorescence induction curves presented here is formal and somewhat arbitrary. Nevertheless this approach allows to study the IC shapes distributions in microalgae population at different moments of the culture growth and thereby provides additional information about the dynamics of population development. In fact, registration of the fluorescence induction curve from the cell ensemble leads to “smoothing” and averaging of kinetic peculiarities of individual IC. On Fig.4 such averaged IC shapes are presented for three stages of population development.

Fig.4. Mean curves of fluorescence induction kinetics for three stages (, and ) of Scenedesmus quadricauda population growth. The ordinate axis represents the value of the ratio F/F200 averaged for definite stage of population growth. F is fluorescence intensity at current time moment of IC recording, F200 - fluorescence intensity at 200s of IC recording.

One can see that such “mean” curves provide information only about the dominant IC pattern at certain stage of culture growth whereas at each stage the population has distinct structure with respect to different IC patterns (Fig.2).

According to our results during the culture growth individual microalgae cenobiums demonstrate four main types of IC, apparently corresponding to definite types of functional activity of the cell's photosynthetic apparatus. Conventionally we call them "active type" with low steady-state fluorescence (type 1A) and "passive" type - with high steady-state level of fluorescence and correspondingly low efficiency of light energy utilization (type 4). The idea about inverse correlation of chlorophyll fluorescence emission to the performance of photosynthesis is generally recognized and widely discussed (Lichtenthaler 1992, Govindjee, 1995). In our experiments the intermediate types of IC (2,3) were also observed. The change of the ratio of the number of cenobiums with IC of different types during the culture growth can be seen on pie diagrams at Fig.5

Fig.5. Pie charts of distribution of Scenedesmus quadricauda cenobiums over the patterns of IC for three stages (, and ) of population growth.

At the first period of growth () there is only small portion of active cells of type 1A. At the linear stage of growth () the cells of this type absolutely dominate, and at the final stage () their portion in the total number of cells gradually decreases.

The question arises what is the reason for such changes in the population structure. The simplest explanation is that initially several different subpopulations with different types of photosynthetic activity and different growth rates coexist in the culture. Cells of 1A type have the highest growth rate and reproduce themselves faster than other cells and therefore they dominate at the non-limited linear stage of the growth. Further, when the density of the cells in the culture increases, the transparency of the cell culture decreases resulting in light limitation. The 1A-type cells gradually lose their dominant position and the structure of population changes so, that the fraction of cells with “passive“(4A and 4B) and “intermediate” (2 and 3) types of photosynthesis increases.

There is also another possibility, namely that structural transitions in the whole cell population are due to the reconstruction of the photosynthetic apparatus of individual cells, leading to the changing of their photosynthetic activity. From this point of view, in the process of algae culture growth definite regulatory mechanisms may operate, changing the photosynthetic activity of cells in accordance with the population state. This is manifested in the shapes of IC recorded from individual cells.

In any case the main question is how different IC shapes are related to the functioning of photosynthetic apparatus of microalgae cells. In our experiments we used light illumination of low intensity (10 W m^-2) and therefore nonphotochemical quenching did not play significant role in determining fluorescence kinetics pattern. Under these conditions of illumination fluorescence transients at PSMT portion of IC should be mainly determined by photochemical quenching due to pH-dependent FNR activation and stimulation of Calvin cycle (Pschorn at al. 1987, Hansen at al. 1993, Dau 1994b). In particular, different shapes of the slow phase of fluorescence induction curves observed in our experiments may reflect different functional states of the system of CO₂ fixation (Calvin cycle) in individual microalgae cenobiums. In fact, the decrease in fluorescence intensity after reaching the maximum M and other fluorescence transients in the time range 25 - 75 s may result from stimulation of Calvin cycle activity either by enzyme activation or by the formation of an increased ATP/ADP ratio (Dau 1994b). Further activation of the cycle of CO₂ fixation leads to the decrease of electron backpressure in ETC and therefore to the decrease in fluorescence yield. This case corresponds to induction curves of types 1 and 2. If Calvin cycle is not activated electron backpressure in ETC increases leading to the increase in fluorescence intensity (IC of classes 3 and 4). More precise distinctions between subclasses A and B in each IC class may result from the variation of the rate constant values of definite photosynthetic reactions.

To understand actual reasons leading to the changes of the IC shape it is necessary to study the relationship between fluorescence induction curve patterns and the suggested mechanisms regulating photosynthetic activity of the cells. To solve this problem it may be useful to develop a mathematical model describing interaction of primary and secondary (Calvin cycle) photosynthetic processes and to carry out an identification of its parameters corresponding to different types of experimental induction curves. The results of such mathematical simulation will be presented in the next paper.

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