The relation between the photochemical yield and variable fluorescence of photosystem II in the green alga Scenedesmus obliquus

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ELSEVIER

Journal of Photochemistryand PhotobiologyB. ~iology 32 (1996) 89-95

The relation between the photochc nical yield and variable fluorescence of photosystem II in the green alga $cenedesmus obliquus Ilona Heinze, Holger Dau *, Horst Senger b'B Biologie/Botanik, Philipps-Universitiit,Lahnberge, D-35032 Marburg, Germany Received 20 March 1995; accepted 30 June 1995

Abstract The redox state of the primary quinone acceptor (QA) of photosystera II (PS ll) and specific non-photochemical effects determine the relation between PS II photochemistry and chlorophyll fluorescence. For the unicellular green alga Scenedesmus obliquuz, the fluorescence yield (F), the fluorescence yield in the presence of reduced QA (FM') and the yield of respiration-corrected oxygen evolution ( q~o,,evolution rate divided by light intensity) were determined during exposure to actinic light of various intensities (0.1-1600/zE m-2 s- t). Irrespective of the growth conditions, the parameter tl~ = (FM '~- F) IF~' was found to be proportional to ~,, for each alga! c:~!9.:re. This finding is in full agreement with non-photochemical fluorescence quenching by increased thermal deactivation of excited antenna states, but is c' L.ult to reconcile with non-photochemical quenching due to modified PS II electron transfer reactions. In particular, no indications of cyclic PS II electron flow were found.

Keywords: Chlorophyll fluorescence; Light adaptation; Non-photochemical quenching; Oxygenevolution

1. Introduction

In oxygenic photosynthesis, light is the source of beneficial photochemistry, but also of severe damage. The primary target of photodamage or photoinactivation is photosystem II (PS II) [ 1-4]. To avoid inactivation or damage resulting from excess light, plants adapt the photosynthetic apparatus to the encountered light intensity: at low light intensities, the light utilization efficiency is maximized, whereas at high light intensities, photodamage is minimized. Some of the various mechanisms involved in the light adaptation of plants are characterized by a response time of less than 30 atin (for a recent review, see Ref. [5 ] ). For the analysis of such shortterm light adaptation, the measurement of variable chlorophyll a (Chl a) fluorescence has become a prominent toot. The variable Chl a fluorescence of plants seems to result exclusively from PS II. It is determined by the redox state of the primary quinone acceptor of PS II (Q^), a phenomenon denoted as"#7~otochemical quenching", and by other factors collectively denoted as "non-photochemical quenching". The photochemical quenching is at a maximum when the Q^ of all PS II is in its oxidized state (open reaction centre state, * Corresponding author. Tel.: 49-6421-282078; fax: 49-~21-282057; email: dauh@rnai!er.uni-marb',:rg.dc. 1011-1344/96/$15.00 © 1996 Elsevier Science S.A. All rights reserved SSDI 101 1 - ! 344 ( 95 ) 07200-4

open PS H, Fo state), and is at a minimum when the QAof all PS II is singly reduced (closed reaction centre state, closed PS II, FM state). Non-photochemical fluorescence quenching is assumed to be related to short-term light adaptation. (For recent reviews on variable PS II fluorescence, see Refs. [ 58 ].) The assessment of non-photochemical quenching is possible using the saturation pulse technique [ 9-12]. Weis and Berry [ 13] investigated fl" relation between the photosynthetic electron flow and fluorescence parameters, determined by the saturation pulse technique, in leaves of higher p|ants ( Helianthus annuus and Phaseolus vul~,aris). Their results are suggestive of a linear x'elation betw,~en the non-photochemical quenching coefficient qN and the quantum yield of CO2 fixation, which was calculated from the rate of CO2 fixation and the fluorescence parameter qp (for a definition of qp and qN, see Ref. [ 11] ). Genty et al. [ 14] examined the relation between the yield of CO2 fixation (respiration-corrected rate divided by the light intensity) and the fluorescence parameter ~,. This parameter is calculated from the fluorescence yield levels of light-adapted ptants according to O p - qpOp° - (FM' - F) IFM'

with

(l)

90

L Heinze et al./Journal of Photochemis

qp = ( FM' - F) l Fv'

(2) (3)

~ O = F v , I F M,

(4)

Fv'=FM'-Fo'

where F is the fluorescence yield in the presence of actinic light, Fo' is the fluorescence yield with oxidized QA and FM' is the fluorescence yield with reduced QA. The parameter qp is the photochemical quenching coefficient, which represents the fraction of PS 1I in the open state (QA oxidized); the fluorescence parameter Op° is assumed to be closely related to Oox°, the yield of PS II electron transport. If we assume that the rate of oxygen evolution is proportional to the rate of CO2 fixation, the results of Gehty et al. [ 14] suggest that the fluorescence parameter Op is proportional to Oox, i.e. ~-COox

(5)

¢,o.=RoJ1

(6)

where Rex is the rate of photosynthetic oxygen evolutioa, I is the intensity of actinic light and c is a normalization factor discussed below. It is important to distinguish between ~ox°, ~o,,, Op a~d Op°. The photochemical yield of an individual PS II in its open state (QA oxidized) is denoted as ~ox°. In contrast, tPox is the effective photochemical yield of an ensemble of PS II with a fraction of PS 1I in the open state and the remaining fraction in the closed state. ~o~ is necessarily smaller or equal to ~ox°; for complete PS II closure (all QA are reduced), ~o~ approaches zero. The parameters ~p and Op° are both calculated from fluorescence levels. It is the goal of this study to investigate whether Op, the fluorescence parameter, is proportional to ~o,, the polarographically determined yield of photosynthetic oxygen evolution. It should be noted that ~o,, as defined by Eq. (6), is closely related, t~ut not numerically identical, to the quantum yield of photosynthetic oxyg~a evolution. The quantum yield of oxygen evolution is defined as the number of evolved 02 molecules divided by the number of absorbed quanta, whereas ~ox equals the number of evolved 02 molecules (per milligram Chl of the sample) divided by the number of incident quanta. In Eq. (5), c is a normalization factor of constant value for the sample in question. Genty et al. [ 14,15] assumed that the rate of CO2 fixation is proportional to Ro~. Based on this assumption, they were able to confirm the validity of Eq. (5) for barley and maize leaves [ 14,15 ]. Deviations from linearity in pea leaves were explained by the influence of photo~ respiration [ 16]. Other investigators have observed deviations from Eq. (5) for low intensities of actinic light [ 17-21]. These deviations may be due to a light gradient inside the leaf: the detected fluorescence emission stems mainly from the upper cell layer (high intensity of actinic light), whereas the detected oxygen evolution is due to the photosynthetic activity of all the cells.

aridPhotobiology B: Biology 32 (1996) 89-95 q

However~ Hormann et al. [ 22] observed deviations from Eq. (5) in isolated spinach thylakoids, which they attributed to the involvement of inactive PS ~tI with inefficient QA to Qn electron transfer. Based on the r~sults of a special pump and probe flash technique, Falkowski et al. [23,24] suggested that, in the green alga Chlorella pyrenoidosa, at high actinic light intensities, the relation between the photosynthetic electron flow and fluorescence emission is affected by a cyclic electron flow within or arcand PS II. Such a cyclic electron flow should result in substantial deviations from Eq. ( 5 ) a t high light intensities. In conclusion, for higher plants, the validity of Eq. (5) is still controversial; little is known about green algae. Investigations on the relation between the fluorescence parameters and the PS II electron flow are of interest because they may result in information on the molecular mechanisms involved in short-term light adaptation. Furthermore, the relation between the fluorescence parameters and the photosynthetic e!e¢tron flow is of obvious importance for numerous investigators using the saturation pulse technique for field studies on crop or algal productivity and related subjects. We have investigated the validity of Eq. (5) for the unicellular green alga Scendesmus obliquus. In this organism, the light gradient effect is essentially avoidable and the respirationcorrected rate of oxygen evolution is directly detectable.

2. Materials and methods

Autotrophic cultures of the unicellular alga Scenedesmus obliquus (strain D3 [25]) were grown at 28 °C in an inorganic nutrition medium [26] for at least 2 days. They were illuminated with white light with an intensity of about 100 /~E m -2 s - ~ (fluorescent lamps L-40 W/15-1 and L-40 W / 25-1, Osram, Berlin, Germany) and percolated with air enriched with 3% carbon dioxide. Heterotrophic cultures were grown in an incubator shaker (New Brunswick Scientific Co., New Brunswick, NJ, USA) at 30 °C and 150 rev rain-~ in the dark. For heterotrophic growth, 0.5% (w/v) glucose and 0.25% (w/v) yeast extract were added to the medium [ 27 ]. After 3 days, the cultures were transferred into light without changing the nutrition medium (mixotrophic conditions). Before the experiments started, all cultures were pre-ilhminated with white light with an intensity between 12.5 and 2000 #E m - 2 s- ~for 15 h. White light was provided by fluorescent lamps (25 #E m - 2 s - i and 100 #E m - 2 s - s see above), a xenon lamp ( 12.5/~E m -2 s- ~ and 1500/.LE m -2 s-~, xenon lamp 0102, Schoeffel Instrumen: GmbH, Trappenkamp, Germany) or a slide projector (2000/.~E m-2 S-l; Universalprojector, Leitz, Wetzlar, Germany). Immediately before starting the experiment, the cultures were centrifuged at 3000g for 5 inin, and the cell pellet was resuspended in phosphate buffer (50 mM Na2HPO4/ NaH2PO4, pH 7) or carbonate buffer (Warburg buffer No. 9, 0.1 M Na2CO3/NaHCO3, pH 9 [28] ). ~ll experiments were carried out using cell suspensions with an optical density

L Heinze et al./Journal of Photochemistry and Photobiology B: Biology 32 (1996) 89-95

(OD) of 0.3. The OD was measured at 4.36 nm against pure buffer. The Chl content was determined according to Liehtenthaler [ 29 ] after extraction of the pigments with hot methanol [30]. A relative measure of the fluorescence yield was detected by a pulse amplitude modulation fluorometer (PAM fluorometer [9]; model 101, Walz, Effeltrich, Germany). A fibreoptic delivered the weak measuring light (650 nm; intensity, less than 0 . 1 / z E m - ~ s - ~ at 1.6 ~:~,t'~z), *~-,,,~saturation --'~-^(white light; 500 ms; intensity, 13 500 #E m - 2 s - ~) and the actinic light (0--1575 #E m - 2 s-t; Prado Universalprojector, Leitz, Wetzlar, Germany; with KG1 + BG 7 filter and several neutral density filters, Sehott, Mainz, Germany). For Fo' measurements, the blue actinic light was substituted by farred light (714 nm; 0.6/zE m -2 s- ~;DIL filter, Schott, Mainz, Germany). The following fluorescence parameters were determined by the PAM fluorometer (nomenclature of Van Kooten and Snel [ 11 ] ): ( 1) Fo, fluorescence signal of darkadapted algae (5 min of dark adam,ration) in the absence of actinic light; (2) FM, maximum fluorescence signal of darkadapted algae during a saturation pulse; (3) F, fluorescence signal after 5 min of adaptation to actinic light; (4) FM', maximum fluorescence signal during a saturation pulse after 5 min of adaptation to actinic light; (5) Fo', minimum of the fluorescence signal after substitution of the actinic light by far-red light. The variable fluorescence yield Fv', the photochemical quenching coefficient qp and the parameters ~p and ~pO [ 14] were calculated according to Eqs. (1)-(4). At the same time as the fluorescence yield measurements, the oxygen gas exchange was measured with a Clark electrode [31 ]. The algal suspension was continuously stirred during the measurement. The rate of oxygen evolution was determined during the last minute of the 5 min adaptation period (adaptation to actinic light) and compensated for the respiration rate detected during the subsequent illumination with weak far-red light. The oxygen yield q~o~,which represents the yield of photosynthetic electron transport, was calculated from the compensated rate of oxygen evolution Ro~ and the light intensity I according to Eq. (6). A data set (x~, y~; i= 1..... n) was normalized with respect to a second data set (x~,z~) by choosing a normalization factor c, such that the error sum

m A I

/A E 150

~ 100 =L 0

"~

50

~

0 0

(o)

was minimal (least-square criterion).

3. R e s u l t s

Fig. 1 shows the effect of the actinic light intensity on the oxygen evolution and several fluorescence parameters. The culture was grown for 3 days under autotrophic conditions with an illumination of 25/xE m -2 s -l. In Fig. l(a), the open symbols represe,~t the oxygen evolution Roxas measured

O

go x

!

i

|

400

800

120G

• c, v

I.

1600

FM, F0' F

o

0 i

0 (13)

~

i

!

i

400

800

1200

1600

Light Intensity, i IoE m'2s'll

v .

v

qp

1o 1 O

0

(7)

i=!

c-I.l.~p

_.1

n

Zi) 2

•

Light Intensity, I [pE m'2s "1]

~.,0

x2--n-2~ ".( c y i -

91

(C)

,

,

,

400

800

1200

1600

Light Intensity, ! I~E m'2s "1]

Fig. 1. Light intensity dependence of oxygen yield and several fluorescence parameters. The culture was grown autotrophically (25 p,E m -2 s-~ for 2 days). In (a), the rate of oxygen evolution Ro~is given in/~mo102 h- ~ (rag Chl) - t, the fluorescence parameter l~/~pis normalized with respect to Ro~as described by Eq. (7). For this sample~the value of the normalization factor c is 4.0 g E tool- ~ m -2. In (b), the parameters are normalized to FM of the dark-adapted state. In (c), q~o~~ aormalized to ~p. For further details, see text.

by the Clark electrode and the filled symbols show the fluorescence parameter ~ , (Eq. ( 1) ), which is thought to reflect the yield of PS II electron transport (see Section 1), multi-

92

1. Heinze et al. / Journal of Photochemistry and Photobiology B: Biology 32 (1996) 89-95

1.0 0.5 ~

;2

V/

0.0 1.0

o

e 0.5

t/

m

~)

omm

0.0

1.0

M

0

o 0.0 0.0

0.5

1.0

0.0

0.5

1.0

Fluorescence Parameter

I

I

0.0

0.5

(])p

[r. u.]

1.0

0.0

0.5

1.0

Fig. 2. Relation between ~ox and Op. A to L differ with respect to the growth conditions (for details, see Table 1). For each data set, the light intensity dependence of the oxygenevolution and various fluorescenceparameters was recorded as shown in Fig. 1. The parameter Ooxwas normalized to Op as shown in Fig. 1(c). The values of the normalization factors c are presented irt Table 1.

plied by the actinic light intensity L The parvmeter lOp is normalized to the oxygen evolution Ro~. The rate of oxygen evolution and IOp exhibit the typical characteristics of a light response curve: a linear increase with light intensity (up to about 200/~E m - 2 s- ~), followed by a slower increase and finally saturation. Fig. 1(b) illustrates the effect of actinic light on the fluorescence parameters FM', F and Fo'. For light intensities below 400/zE m-2 s-!, the maximum fluorescence yield FM' is independent of the intensity of actinic light, but slightly reduced in comparison with FM of the dark-adapted state. For light intensities above 400/.~E m -2 s-i, FM' shows a rapid decline with increasing light intensity which reflects the increasing non-photochemical quenching. In contrast, F (the fluorescence yield after adaptation to actinic light) increases with increasing light intensity. The minimum fluorescence yield Fo' remains essentially constant. Fig. 1 (c) shows the parameters Op, qp, Op° and Oo~ for various intensities of actinic light. The photochemical quenching coefficient qp decreases rapidly with increasing light intensity, i::dicating increasing PS II closure. The parameter Op° (see Eq. (4)), which is thought to reflect the photochemical yield of open PS II, also declines. This decline is due to the decrease in FM' (see Fig. 1(b)) which results from the increased non-photochemical quenching. The parameter Op is the product of qp and Op° (see Eq. ( 1) ), and is therefore affected by both photochemical and non-photochemical quenching. After normalization of ~o~ (see Section 2), the light intensity dependence of the oxygen yield is in good agreement with the light intensity dependence of Op.

The growth conditions determine the capability of the cells to generate non-photochemical quenching [ 32]. Fig. 2 shows the relation between Oo~ and Op for cultures grown under different conditions (see Table 1). The oxygen yield Oox was normalized to Op in the same manner as shown in Fig. 1(c). In Fig. 2, the data points are scattered around a straight line which is the angle bisector. For high values of Op (low light intensities), the scatter is larger than for low values. Presumably, this behaviour is due to the oxygen evolution measurei~ients, which are less precise for weak actinic light, because inaccuracies in the determination of the respiration rate can result in large errors in Oo~ (see Section 4). Table 1 shows the result of a linear regression analysis for each individual data set. The slope of each regression line is denoted as a~ and the intercept of the y axis is denoted as ao; thus the angle bisector corresponds to a~ ffi 1 and ao ffi0. It is clearly demonstrated that, for all data sets, the deviation between the regression line and the angle bisector is small, irrespective of the value of FM'/FM (low values of FM'/FM in Table 1 correspond to high levels of non-photochemical queh~ching). Apparently, the close relationship between these parameters is unaffected by the conditions of cell growth (light intensity, nutrition medium and cell age). Moreover, this relationship is not significantly influenced by the buffer used during the measurements. There is no obvious correlation between the variations in the value of the normalization factor (values of c in Table 1) and the growth conditions. The normalization factor is roughly constant; its mean value is 3.9 E g reel- ~m - 2 (stan_ dard deviation of 17%). Thus, to a first approximation, we

L Heinze et al. /Journal of Photochemistry and Photobiology B: Biology 32 (1996) 89-95

93

Table I Growth conditions and results of linear regression analysis for the data sets shown in Fig. 2. The slope of the regression line is denoted as a~, the intercept of they axis as ao and the regression coefficient as r 2. The normalization factor is denoted as c (see Eq. (7)) Fig. 2

~ B C D E F G H I J K L

Grcwth"

2 d ant. 2 d ant. 3 d ant. 3 d ant. 3 d aut. 3 d ant. 3 d het. + 2 3 d her. + 2 3 d her. + 2 3 d het. + 2 2 d ant. 2 d ant.

d d d d

mix. mix. mix. mix.

Pre-illumination Intensity t, (/LE m - 2 s - t )

FM'/FM

25 100 25 100 1500 2000 12.5 25 100 1500 100 100

0.75 0.82 0.65 0.65 0.91 0.80 0.70 0.80 0.72 0.85 0.66 0.60

Buffer ~

after illumination with 1575/j,E m - 2 s - t Phosphate Phosphate Phosphate Phosphate Phosphate Phosphate Phosphate Phosphate Phosphate Phosphate Carbonate Carbonate

Mean values Standard deviativ ~

c (E g mol- t m -2)

ao

at

r2

3.4 3.3 4.0 3.5 4.3 4.3 3.4 4.9 4.8 4.4 4.0 2.6

0.02 0.00 -0.04 - 0.03 0.01 0.04 - 0.06 -0.01 -0.07 0.06 -0.07 - 0.05

1.03 1.00 1.07 1.02 0.96 0.89 1.09 1.01 1.10 0.88 1.12 1.05

3.9

- 0.02

1.02

0.91

+ 0.7

:1:0.04

+ 0.08

+ 0.07

0.98 0.99 0.9'~ 0.85 0.86 0.77 0.91 0.97 0.94 0.93 0.91 0.83

' Aut., autotrophic; het., heterotrophic; mix., mixotrophic, d, days. ~' Before the experiments, the cultures were pre-iiluminated for 15 h. ©During the measurement, the cells were suspended in phosphate or carbonate (Warburg) buffer.

can calculate Rox on the basis of the fluorescence parameter Oe and the light intensity ! according to

Ro,,=c-lOpl~O.9Op1 (/zmol mg -l h - l / p E s -I m -2)

(8)

with ! in units of/~E s-R m - 2 (photosynthetically active radiation) and Ro~ in units of/zmol 02 h - s (mg Chl) - 1

4. Discussion

The cell age and intensity of pre-illumination influence the capability of the green alga Scenedesmus obliquus to establish non-photochemical quenching. A more detailed analysis of the dependence of non-photochemical quenching on the growth conditions will be presented in a subsequent article. However, for all growth conditions used (Fig. 2), the relation between the fluorescence parameter ~ and the oxygen yield ~o~ is linear. Thus, in tl~e green alga Scenedesmus obliquus, the yield of photochemical electron transport can be estimated by O~,, irrespective of the extent of non-photochemical quenching. In Fig. 2, for high values of Op, the occurrence of considerable scatter is apparent. High values of Op correspond to light intensities below the oxygen evolution/respiration compensation point. For these light intensities, the determination of Oo~ is very inaccurate for the following reasons: (1) low rates of oxygen concentration changes have to be detected; (2) the correction for dark respiration is decisive for the resulting value of Oo~; (3) inaccuracies in the value of Ro, the respiration-corrected rate of oxyj;en evolution, are amplified due to the division by small light intensities (calculation of Oox by Eq. (6)). Visual inspection of the data sets shown in Fig. 2 demonstrates that the observed deviations from the

angle bisector are due to unsystematic scatter. This impression is confirmed by the regression analysis presented in Table 1: for all data sets, the r~ values are close to unity and the mean values of the slope and intercept deviate by less than 0.02 from unity and zero, respectively. For the data sets I, K and L, a systematic deviation from the angle bisector (upward curvature) cannot be immediately ruled out by visual inspection of the plots. However, it should be noted that in L and I the last data point is located below or on the angle bisector, whereas the preceding point is above. We conclude that there are no deviations from Eq. (5) which exceed the noise level. At least to a first approximation, Oox is proportional to the fluorescence parameter Op for algae grown under diverse conditions. In higher plants, deviations from a linear relation between Oox and Oe could result from a light gradient inside the leaf [ 19,20 ]. (Only the fluorescence of the upper cell layer, which is exposed to a high light intensity, reaches the fluorescence detector, whereas all cells contribute to the oxygen evolution.) In the algal suspensions used in our experiments, there is only a slight light gradient because of the low OD. Furthermore, the suspension was continuously stirred. Thus misleading results due to irregular illumination of the cells are avoided. In our experiments, photoinhibitory conditions were not considered. Therefore we cannot exclude that the relation between Op and ~o~ breaks down on prolonged exposure to light of photoinhibitory intensities. A linear relation between Op and Oco2 (the yield of CO~ fixation) was experimentally demonstrated by Genty et al. [ 14] on the basis of gas exchange experiments and theoretically derived on the basis of the bipartite Butler model. With the assumption that non-photochemical quenching is due to

94

L Heinze et al. /Journal of Photochemistry and Photobiology B: Biology 32 (1996) 89-95

increased thermal deactivation of excited antenna states ("antenna quenching"), the validity of Eq. (5) can be derived for all common PS II models, i.e. the bi partite Butler model, the single pigment pool model and the reversible radical pair model (see Ref. [6]). Furthermore, Eq. (5) should be valid inde~ndent of the extent ofPS II connectivity under the assumption of a homogeneous PS II population

[6]. The validity of Eq. (5) is in full agreement with the assumption of non-photochemical quenching by an increased rate of non-radiative decay of excited antenna states, but is difficult to reconcile with changes in the rate constants of primary charge separation or other electron transfer reactions as discussed elsewhere [6]. Weis and Berry [ 13] concluded that non-photochemical quenching results from increased non-radiative decay in some PS II units. In contrast, Genty et al. [ 14] assumed increased thermal deactivation in all PS II units. A distinction between these alternatives is difficult due to excitation energy transfer between PS U units and the relatively small values of nonphotochemical quenching [ 33]. For Scenedesmus obliquus, we found the extent of non-photochemical fluorescence quenching to be smaller than that typically observed for higher plants (see FM'/FM values presented in Table 1). Therefore no attempts were made to prove or disprove the models of Weis and Berry [ 13] and Genty et al. [ 14]. Deviations from the linear relation between Op and ~o~ may also originate from several other events. Photorespiration or the Mehler reaction [ 34 ] may consume oxygen, while the variable fluorescence remains unaffected. A cyclic electron transport around or within PS II, as suggested by Falkowski [ 23,24], which is too slow to influence the FM' level as determined by the saturation pulse technique, should not affect the variable fluorescence, but will ~'educe the net rate of oxygen evolution. PS II of the Qa non- reducing type [35,36] will affect the relation between Op and ~o~. (However, high connectivity between the different types of PS II may restore the linear relation between Op and Oox [33] .) In $cenedesmus obliquus, none of the events mentioned above seems to play an important role because significantdeviations from Eq. (5) are not observed. In particular, cyclic PS II electron flow is not relevant for the decrease in Oo~ with increasing light intensity. We were surprised to find that not only is the relation between Op and ~o~ linear, but the normalization factor c is roughly sample independent. Therefore one might be tempted to calculate the rate of photosynthetic oxygen evolution on the basis of Op (the fluorescence parameter) and I ( the intensity of photosynthetically active radiation). However, there are several possible pitfalls. The Chl concentration during the measurement was relatively low (approximately 5/zg ml- ' ). At higher Chl concentrations in the sample volume, the light intensity gradient inside the sample volume will probably result in pronounced deviations from Eq. (8). In addition, light scattering may seriously diminish the fraction of photons absorbed by the antenna Chls. Therefore morphological

changes, which affect the light scattering properties, may affect the value of c. Furthermore, at high light intensities, imprecise values of Op may cause extreme variations in the Rox value calculated by Eq. (8). The good agreement between Op and Oox, as shown in Figs. l(a) and 2, offers the possibility to use ~uore~.eance measurements instead of oxygen gas exchange measurements for the determination of Oox. The determination of Op is accurate and simple because it requires only the determination of FM' and F. Therefore the linear relation between Op and Oox is of importance for applications of Chl a fluorescence measurements in ecophysiology and outdoor experiments. However, it should be noted that we studied the validity of Eq. (5) in the laboratory only. The i'esults of this study may not be equally valid for all environmental conditions encountered in situ. In particular, further investigations are needed to clarify how the relation between Op and Oox may be modified in organisms exposed to stress conditions.

Acknowledgements Support by the Deutsche Forschungsgemeinschaft (SFB 305, Projekt B 1) is gratefully acknowledged. I.H. is a recipient of a fellowship of the Studienstiftung des deutschen Volkes.

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