The Mode of Adaptation of the Photosynthetic Apparatus of a Pigment Mutant of Scenedesmus without Light Harvesting Complex to Different Light Intensities

J.PlantPhysiol. Vol. 135.pp. 144-149{1989}

The Mode of Adaptation of the Photosynthetic Apparatus of a Pigment Mutant of Scenedesmus without Light Harvesting Complex to Different Light Intensities NORMAN 1. BISHOp!, KLAUS HUMBECK , SUSANNE ROMER , 2

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and HORST SENGER2

Department of Botany and Plant Pathology, Oregon State University, Corvallis, Oregon, USA Fachbereich Biologie/Botanik der Philipps-Universitat, Lahnberge, D-3550 Marburg, Federal Republic of Germany

Received March 30, 1989 . Accepted May 25, 1989

Summary Cells of a mutant of Scenedesmus, lacking the light harvesting system of the photosynthetic apparatus (WT-LHC 1), were adapted to high or low intensities of white light. Phenotypically they demonstrated the same adaptational behavior, expressed by the photosynthetic light intensity curves, as sun and shade plants. But changes in the molecular structure were different. Under low light intensity the number of PS I and PS II reaction centers with their core antennae increased in a stoichiometric fashion. Efficiency measurements of PS II and PS I reactions indicate that some of the reaction centers are not coupled to the electron transport chain in low light intensity adapted cells. A loss in quantum efficiency due to a disorder in energy transfer in PS II is discussed and thought to be connected with a dihydroxyI trihydroxy-acarotene (lutein/loroxanthin) cycle. Key words: Mutant WT-LHC z 0/ Scenedesmus obliquus - adaptation to light intensity - carotenoids photosynthetic activities - photosynthetic apparatus.

Abbreviations: ChI = chlorophyll, CP = pigment-protein complex, DCMU = 3-(3',4'-dichlorophenyl)-l,l dimethyl-urea, DPC = diphenylcarbazide, DPIP = 2,5 dichlorophenol-indophenol, HPLC = high performance liquid chromatography, LDS = lithium dodecyl sulfate, LHC = light harvesting complex, MV = methylviologen, PAGE = polyacrylamide gel electrophoresis, PS = photosystem. Introduction It is a well documented fact that the photosynthetic apparatus of higher plants (Boardman, 1977; Anderson, 1986) as well as of green algae (Senger and Fleischhacker, 1978; Jeffrey, 1981) adapts to the intensities of incident light. The mechanism of adaptation to low light intensities is either a doubling of the whole pigment system (Wild, 1979; Humbeck et aI., 1988 a) or an increase in the light harvesting system (LichtenthaIer et aI., 1982; Leong and Anderson, 1983). Therefore, it is of interest to see how a mutant without a light harvesting system responds to different light intensities. In this contribution we report about the adaptation of a Scenedesmus mutant without a light harvesting system (WT-LHC 1) to different intensities of white light. © 1989 by Gustav Fischer Verlag. Stuttgart

Materials and Methods Organism, growth and illumination Mutant WT-LHC 1 of the unicellular green alga, Scenedesmus obliquus, was induced as described by Bishop (1982). The cells were grown autotrophically in a liquid culture medium (Bishop and Senger, 1971) in a light thermostat under homocontinuous conditions. These conditions were maintained by photoelectric density measurements combined with an automatic dilution device (Senger et a!., 1972). Cells were aerated by bubbling 3 % CO 2 in air through the cultures. The cultures were adapted to the different intensities of light for at least 7 days. For illumination with low (5Wm- 2) and high (20Wm- 2) intensities of white light a combination of fluorescent lamps (Osram-L40W/15-1 and Osram-L40W/25-1) was used.

Light adapt ion of a light harvesting mutant

Packed cell volume Packed cell volume (PCV) was determined by centrifugation of definite volumes of cell suspension in hematocrit tubes for 5 minutes at 1,400 g.

(444nm, 12Wm- 2) using an Aminco DW-2 spectrophotometer (Aminco, Silver Spring, Med., USA) with a filter system to screen the actinic light from the photomultiplier. For calculation of P 700 an extinction coefficient of 64 mM - 1 cm -I was employed (Hiyama and Ke, 1972).

High performance liquid chromatography (HPLC) Pigments were extracted from the algae with boiling methanol and cell debris was then removed by centrifugation for 5 minutes at 1,400g. The samples were evaporated and taken up in acetone. All operations were conducted under safe light and then extracts were stored in the dark at - 24°C under nitrogen. Chromatography of pigments was run on a Kontron (Munchen, FRG) HPLC-system which consisted of two pumps (LC 410), a programmer (Model 200), an UV /VIS-detector (Uvikon 720 LC) and a reporting integrator (Hewlett Packard 3390). The sample volume was 20 ILL The elution solvent contained CH 3 CN/CH 3 OH = 75/ 25 (v/v) superimposed by a multilinear gradient of water. The water content was diminished within 40 min from 10 % down to 0 % and then held for 10 min at 0 %. Finally, the water content was increased again to 10% within 10 min. The flow rate was 0.9 mil min. Separation was carried out using a 5p. RP-18 column (SS200/6/4 Nucleosil 5C18, Macherey & Nagel, Duren, FRG). To protect the main column, a pre-column (LiChrosorb RP-18, Merck, Darmstadt, FRG) was used. The eluted pigments were detected and recorded at 445nm

Identification and quantitative determination

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Pigments were identified by comparison of spectral characteristics with values from the literature, co-chromatography with authentic standards and analysis of peaks after chemical treatments of the samples as described in Humbeck et al. (1988 b). For quantitative determination calibration curves were obtained by injecting definite amounts of pigment standards and plotting them against the resulting area given in arbitrary units by the integrator.

Photosynthetic activities and P 700 Oxygen evolution was measured polarographically with a MicroClark-Electrode (Gilson Medical Electronics, Middleton, Wisc., USA) under various intensities of white light. Algae were suspended in 0.05 M phosphate buffer (pH = 7.0). For polarographic measurement of the p-benzoquinone Hill-reaction under red light (> 620 nm, 485 W m - 2), p-benzoquinone (final concentration 5 mM) was added to suspensions of whole cells. For the determination of the photosystem II-mediated DPIP reduction and the photosystem I-mediated methylviologen reduction, cells were broken in a Vibrogen cell mill (Buhler, Tubingen, FRG), and cell-free preparations were obtained as described by Senger and Mell (1977). DPIP reduction was assayed with an Aminco DW-2 spectrophotometer (Aminco, Silver Spring, Med., USA) at 579.5nm. Actinic light (>650nm, 370Wm- 2 ) was provided by cross illumination, and the photomultiplier was shielded by an interference filter (IL 578, Schott, Mainz, FRG). Final concentrations of DPIP and DPC (an artificial electron donor) were 4x 10- 5 M and 1 x to- 3 M, respectively. Electron transport to methylviologen was followed polarographicallyas oxygen uptake under white light (2130 W m - 2). The reaction mixture contained 0.2 mM methylviologen, 5 p.M DCMU, 0.1 mM DPIP, and 5 mM sodium ascorbate. Forthe determination of P 700 (Ogawa and Vernon, 1970) in cellfree preparations, P 700 was reduced in one aliquot by ascorbate (10 mM) and measured against an aliquot oxidized by blue light

145

Polyacrylamide gel electrophoresis Chloroplast particles to be examined by polyacrylamide gel electrophoresis (PAGE) were prepared according to the procedure outlined (Senger and Mell, 1977), washed twice with 0.05M Tricine buffer (PH = 7.3) and the final pellet stored at -20°C until used. Pigment-protein complexes were separated by gel electrophoresis slightly modified after Anderson et al. (1978). Samples were prepared by solubilization of the purified chloroplast particles at 4°C in 0.3 M Tris-HCl (pH = 8.8)/13 % (v/v) glycerol and Triton x-l00 and LDS to give a final Triton x-l00/LDS/protein weight ratio of 1.8/0.6/1. The amount of chlorophyll applied to the gel chamber was 5p.g. Analysis by PAGE was carried out using 0.041M Tris/ Boric acid (PH = 8.64) and 0.1 % (w/v) LDS for the upper buffer and 0.43 M Tris-HCl (pH = 9.35) for the lower buffer. The acrylamide to N,N'-methylen-bisacrylamide ratio was 30/0.8. The acrylamide was 4% (w/v) in the stacking gel (2.5mmx5mmx73mm), whereas the resolving gel (2.5 mm x 60 mm x 73 mm) contained a 7.5-15% (w/v) linear gradient of acrylamide. Other gel formulations were as described by Anderson et al. (1978). PAGE was performed in the dark at 4°C for 1 hour. During the first 10 minutes the current was set at 15 rnA and then changed to 30 rnA for the remaining time. The distribution of Chi among the pigment-protein complexes on the gel was determined by scanning at 670 nm in a spectrophotometer (Uvikon 820, Kontron, Munchen, FRG).

Fluorescence induction For fluorescence measurements cell suspensions were adjusted to a chl~rophyll conce~tration of 4 p.f. ml. After addition. of DCMU to a fmal concentration of 1 x 10- M, cells were kept m the dark for 2.5 minutes and then illuminated with blue light (Schott filter, 447 nm, Schott, Mainz, FRG; 1 W m - 2). Fluorescence induction was

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Fig. 1: Photosynthetic oxygen evolution of mutant WT-LHC 1 of Scenedesmus obliquus grown under low (5 W m - 2) and high (20 W m - 2) intensities of white light. Cultures were grown homocontinuously at 30°C and aerated with 3 % CO 2 in air for at least 7 d. Oxygen evolution was determined polarographically in an oxygraph under various intensities of white light.

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Fig. 2: Distribution of chlorophyll among the pigment-protein complexes and the free pigment (FP) in cells of mutant WT-LHCJ, of Scenedesmus adapted to low (5 Wm -2) and high (20Wm -2) intensities of white light. For comparison the pattern of wild-type (WT) of Scenedesmus is also shown. Separation of the pigment-protein complexes was performed by LDS-PAGE following Anderson et al. (1978) at 4°C. The profiles were obtained by scanning at 670 nm. measured at room temperature with the photomultiplier of an Aminco DW 2 spectrophotometer (Aminco, Silver Springs, Med., USA). The curves were monitored with a storage oscilloscope (Tektronix 5103N, Beaverton, Ore., USA) attached to the photomultiplier. The photomultiplier was shielded against the exciting blue light by a filter (Schott filter, 683 nm, Schott, Mainz, FRG).

Quantum requirement Quantum requirement of photosynthetic oxygen evolution was determined in monochromatic light of 682 nm following the method of Burger et al. (1988).

Table 1: Differences in chlorophyll-content, photosynthetic activities and respiration in cells of mutant WT-LHC l of Scenedesmus adapted to low (5Wm- 2) and high (20Wm- 2) intensities of light. Chlorophyll-content was determined by HPLC-analysis. For other conditions see Fig. 1. JIg ChI ChI a Photosyn- Respiration JlI PCV ChI b thesis Jlmol02 Jlmol02 5Wm- 2 6.05 20Wm- 2 4.62

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Results and Discussion Mutant WT-LHCt has only chI a and the pigment-protein complexes, CPI (PS I) and CPa (PS II), and no ChI b and light harvesting system (d. Fig. 2)_ Adapted to high (20Wm- 2) or low (5 Wm -2) intensities of white light the cells form more ChI under low intensities (Table 1) and produce typical light intensity curves of photosynthetic oxygen evolution (Fig. 1). The high intensity adapted cells have a higher respiration rate, higher photosynthetic capacity and reach the compensation point at slightly higher light intensities (Table 1). The light intensity curves for WT-LHC , compare favorably

to those previously obtained for low and high intensity adapted cells of normal WT Scenedesmus (Senger and Fleischhacker, 1978). The only major difference appears to be a decrease in slope (decrease in quantum efficiency) at low light intensity. To test the mechanism of adaptation, the differently adapted cells of WT and WT-LHC , were broken, their pigment-protein complexes solubilized and analyzed by gel electrophoresis (Fig. 2). The distribution patterns clearly demonstrate the absence of LHC-components (LHCP l ,2,3) in the mutant and that there are no clear differences for high

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Fig. 3: Absorption spectra of cells of mutant WT-LHC 1 of Scenedesmus obliquus grown under low (SWm- 2, -----) and high (20Wm- 2 , - - ) intensities of white light. The spectra were recorded with a Shimadzu spectrophotometer (MPS-SOOO, Shimadzu, Japan) at the temperature of liquid nitrogen (77K).

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and low intensity adapted cells. The difference in the amount of so-called free pigment (FP) is due to slight differences in solubilization of the pigment-protein complexes. Generally, we deduce from these results that different intensities of light cause no considerable changes in the distribution between PS II and PS I. This finding is supported by the fact that no differences are found in the low temperature absorption spectra of the cells adapted to different light intensities (Fig. 3). As an indicator for the size of the photosynthetic units we examined the fluorescence induction curves for PS II (Fig. 4) and the ChI alP 700 ratio (Table 2) for PS I. The half-rise time in fluorescence induction (t ~) is used as an indication for the size of PSII (Malkin et aI., 1981). For the cells adapted to high or low light intensities the values of t ~ are almost identical (Table 2) indicating that the sizes of the PS II units

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Table 2: Photosynthetic activities, half-rise time of fluorescence induction, ChllP 700 ratio and quantum requirement in cells of mutant WT-LHC 1 of Seenedesmus adapted to low (SWm-2) and high (20Wm -2) intensities of light. PSI was measured as MV-reduction followed by oxygen uptake, PS II either as p-benzoquinone Hill reaction or as DPIP-reduction, T u was calculated from the curves shown in Fig. 4. PSI JLmol02 SWm 2 20Wm- 2

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are virtually identical. So far the best indicator for the size of PS I is the ratio of ChI a to P 700. In the mutant utilized here this ratio might even be more valid since all LHC chlorophylls are missing. The values of the two types of cells for the ratio of ChI alP 700 presented in Table 2 show only 4 % difference. Thus we can consider the size of PS I units in mutant WT-LHCI, when adapted to high or low intensities, as identical. In contrast to the apparent equal sizes of the photosynthetic units, differences were found in their carotenoid composition (Fig. 5). Remarkable is the inverse behavior of lutein (dihydroxy-a-carotene) and loroxanthin (trihydroxya-carotene). Loroxanthin has increased in the low intensity adapted cells at the expense of lutein. These differences might indicate a trihydroxy/ dihydroxy cycle (similar to the xanthophyll cycle; Hager, 1980). In addition to the total photosynthesis (Fig. 1, Table 1), the activities of the single photo systems were determined (Table 2). PS II activities were measured as either DPIP or p-benzoquinone Hill reactions and PS I activities as oxidation of methylviologen with DPIP / ascorbate as electron-donor. Both photosystems show roughly the double activity based on the chlorophyll content in the high light intensity adapted cells when compared to the low light intensity adapted cells. Since the size of the photosynthetic units under the two light conditions remains the same, one has to assume that part of the photosystems of the low intensity adapted

148

NORMAN I. BISHOP, KLAus HUMBECK, SUSANNE Ri:>MER, and HORST SENGER documented by the higher quantum requirement. This might be due to a structural disorder in PS II. In turn, this disorder might be connected with the proportional decrease in lutein. In a separate investigation (Humbeck et al., 1988 b) we proposed lutein to be an essential prerequisite for the formation of functional PS II units. The possibility that a lutein/loroxanthin cycle might playa role in the photoregulation of the photosynthetic apparatus will be a subject for further investigations.

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cells are either not provided with sufficient electron transport systems or are partially disconnected from the existing electron transport chain. The difference in the slopes of the linear part of the light intensity curves already indicated altered quantum efficiencies of photosynthesis in the two types of adapted cells. Direct measurements of quantum requirement (Table 2) confirmed this assumption. The quantum requirement (at 682 nm) of oxygen evolution in low intensity adapted cells is ~ higher than that of the high intensity adapted cells. This result differs significantly from comparable investigations of the light-adapted wild-type cells of Scenedesmus (Senger and Fleischhacker, 1978) and of higher plants (Bjorkman et aI., 1972), whereby no change of the quantum requirements for oxygen evolution has been observed.

Conclusion The photosynthetic response of mutant WT-LHC 1 of the green alga, Scenedesmus obliquus, upon adaptation to high and low light intensities (Fig. 1) is very similar to the response of wild-type cells (Senger and Fleischhacker, 1978). The mutant cells respond in the same way as the wild-typecells to lower light intensities by an increase in chlorophyll biosynthesis in spite of the fact that the light harvesting system is missing. The strategy concerning how the adaptation is achieved on the basis of molecular structure is different for the mutant and wild-type cells. The number of PS II and PS I units is increased in a stoichiometric fashion in the mutant. But some of the photosynthetic units seem to be insufficiently linked to the electron transport chain in low intensity adapted cells. This is documented by the low efficiency of the single photosystems. Additionally, an insufficient energy transfer is

This work was supported by grants from the USDA {82-CRCR2-1079} to N. 1. Bishop and from the Deutsche Forschungsgemeinschaft to H. Senger {SFB 305}, K. Kumbeck {Habilitation grant} and to S. Romer {a scholarship of the Studienstiftung des deutschen Volkes}. We thank Dr. J. Burger for measuring the quantum requirements, Ms. K. Bolte for skillful technical assistance, Ms. H.-B. Bottner for the help during the preparation of the manuscript and Mr. H. Becker for drafting the figures.

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SENGER, H. and PH. FLEISCHHACKER: Adaptation of the photosynthetic apparatus of Scenedesmus obliquus to strong and weak light conditions. I. Differences in pigments, photosynthetic capacity, quantum yield and dark reactions. Physiol. Plant. 43, 35-42 (1978). SENGER, H. and V. MELL: Preparation of photosynthetically active particles from synchronized cultures of unicellular green algae. In: SAN PIETRO A. (ed.): Methods in Cell Biology, Vol. XV, 201-219, Academic Press, London, New York (1977). SENGER, H., J. PFAU, and K. WERTHMOLLER: Continuous automatic cultivation of homocontinuous and synchronized microalgae. In: PRESCOTT, D. M. (ed.): Methods in Cell Physiology, Vol. V, 301- 333, Academic Press, London, New York (1972). WILD, A.: Physiologie der Photosynthese Hoherer Pflanzen. Die Anpassung an die Lichtbedingungen. Ber. Dtsch. Bot. Ges. 92, 341-364 (1979).