Evolution of 02 in Brown Algal Chloroplasts - NCBI

Plant Physiol. (1983) 73, 889-892 0032-0889/83/73/0889/04/$00.50/0

Evolution of 02 in Brown Algal Chloroplasts Received for publication March 29, 1983 and in revised form July 26, 1983

RADOVAN Popovic, KONRAD COLBOW, WILLIAM VIDAVER, AND DOUG BRUCE

Photobiology Group, Departments of Physics and Biological Sciences, Simon Fraser University, Burnaby, British Columbia V5A 1S6 CANADA ABSTRACI` A method is described for the isolation of photosynthetically active chloroplasts from four species of brown alpe: Fucus eaiculos, NereoLwinaiwa sa a,riaa, and Macrocystis ixtegrifoli& cystis Imetkeaa When compared to lettuce and spinach chloroplasts, the alg chloroplasts all showed lower activities for both photosystems II and L Chloroplasts from all the plants produced H20, with photosystem I functioning as the 02 reductant in the light. In contrast to the green plnts, however, brown alga chloroplasts strngly reduced 02 under conditions where both photosystems II and I remain active. Relative variable fluorescence values were lower both in intact plants and chloroplasts of the brown alge than for either spinach or lettuce. It is suggested that although light harvesting activities appear similar in all the plants, details of electron transport in brown algae may differ from those of green plants.

Isolation of chloroplasts from brown algae is complicated by the large amounts of phenolic substances present in these plants (9, 13), which tend to degrade the chloroplast membranes, inactivating the photosynthetic enzymes. In addition, abundant mucilage prevents effective filtration and centrifugation of chloroplasts, particularly in N. luetkeana, L. saccharina, and M. integrifolia. Using an efficient homogenizer (10) and a previously described method (14), resulted in a successful isolation of chloroplasts only from Fucus. To minimize the effect of mucilage on the chloroplast isolation, we have devised a procedure for the removal of mucilage and grinding of tissue of brown algae, described in detail below. Samples from fronds or blades (40 g) were cut into 2-mm squares using a chopping knife on a plastic block. These were washed by stirring in 2 L of Millipore filtered seawater at 8°C and collected from the viscous extract with a stainless steel strainer. This washing procedure was repeated 8 to 10 times to remove the greater part of the mucilage. A grinding medium, modified from Nordhom et al. (14), consisted of I M sorbitol, 1 mM MnCl2, I mM MgC92, 0.5 mM K2HPO4, 5 mm EDTA, 2 mm NaNO3, 2 mm Na-isoascorbate, 2 mM cysteine, 0.2% (w/v) BSA, 4% (w/v) soluble PVP-40 and 50 mM Mes buffer (pH 6.1). Seawater-washed tissue was rewashed three times in 150 ml of this medium at 2C. This temperature was maintained for all subsequent preparative steps. On removal from the wash, the tissue was divided into four portions and each ground separately with a mortar and pestle, gradually increasing the medium volume to 50 ml. Grinding caused the release of additional mucilage, thus the combined slurries were diluted to 400 ml of medium, passed through a 0.5 mm nylon grid, and then 8 layers of cheese cloth. Chloroplasts were then sedimented by centrifugation for 7 min at 5500g. The pellet was resuspended using a glass Teflon mixer and washed in 80 mL of a reaction medium containing I M sorbitol, 1 mM MnC12, 1 mM MgCl2, 2 mM EDTA, 0.5 mM K2HPO4, and 50 mM Hepes (pH 7.6). After a second centrifugation, chloroplasts were resuspended in 8 ml of this medium. These chloroplasts were used to measure PSII and PSI activities. Chloroplasts from fresh spinach and lettuce, obtained from the local market, were isolated by conventional procedures (22,

There are few reports of photosynthetic activity in brown algal chloroplasts (14). Isolation of chloroplasts from large marine phaeophytes is complicated by the toughness of their cell walls and the copious exudation of mucilage which are common properties of these plants (5, 11, 17). Conventional methods of chloroplast isolation (22, 24) are inapplicable to blades or fronds of these algae and we report here on a chloroplast isolation technique which has yielded photosynthetically active chloroplasts from four species of marine brown algae. Functions of the algal chloroplasts, including PSII and PSI activities and variable fluorescence emission ( 16) were compared to these functions in lettuce and spinach chloroplasts. We found both similarities and differences in certain photosynthetic parameters when the algal responses were compared to those of the green plants. Results of these comparisons suggest that although the basic mechanism oflight harvesting and energy conversion are similar in both plant groups, details of energy transfer and electron transport are probably dissimilar. Photosynthesis in brown algae has been reported to saturate at relatively low light intensity (14, 25). Low light saturation indicates that algae, such as the giant kelps, are able to utilize only a small part of the full sunlight to which they are frequently 24). exposed. Some of our observations of photosynthetic activities PSII activity was measured as 02 evolved, with K3Fe(CN)6 as of brown algal chloroplasts may reflect aspects of this energy electron acceptor, using a Clarke type 02 electrode as previously conversion limitation. described (7). Saturating red light was obtained with a 150 w tungsten projector lamp fitted with a Coming 2-62 filter and MATERIALS AND METHODS condenser lens to focus light on the sample cuvette. Neutral All algae were collected from Barkley Sound on the west coast density filters (Balzers) were used to attentuate the light beam. of Vancouver Island and kept illuminated in running seawater The absolute values of light energy shown in the figures are at 10 to 1 5C until used. Photosynthetic parameters were inves- uncorrected for absorption and focusing effects by the glass tube tigated in Fucus vesiculosus, Nereocystis luetkeana, Laminaria and water jacket surrounding the Clarke electrode and, thus, are saccharina, Macrocystis integrifolia, and in chloroplasts isolated higher than the actual intensities at the sample. from these species. Activity of PSI was assayed by 02 uptake in the apparatus 889

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Table I. 02 Evolution (PSIIJ), 02 Uptake (PSI), and Relative Fluorescence, f, in Higher Plants and Brown

Algae Mixture of I ml for 02 evolution contained reaction media 50 mM Hepes (pH 7.6), 1 mm MnCl2, I mM MgC92, 2 mM EDTA with 1.5 mM FeCN, 1 mM NH4CI and 15 to 30 Mg Chl. For 02 uptake: 3 mM sodium isoascorbate, 0.2 mM TMPD, 0.1 mM MV, 4 to 12 jtg of Chl, and 15 Mm DCMU in reaction media. Plant

PSII

PSI

Spinach Lettuce Fuictis Nereocystis Laminaria Macrocystis

270 210 103 116 57 50

2060 1281 432 520

PSI

Chloroplasts

fp in vivo

3.0 3.5 0.5 0.4 0.5 0.5

4.5 5.5 1.5 1.5

MmolO2/mg Chl - h

l

E EN

256 240

7.6 6.1 4.2 4.6 4.5 4.8

1.6 1.6

lates from all the plants after the first centrifugation were resuspended in 80 mL of reaction medium minus sorbitol. This was to break the chloroplast envelope through osmotic shock. Chloroplasts were washed, centrifuged twice, and resuspended in 1 M sorbitol, I mM MgCl2, and 50 mM Hepes (pH 7.6). This treatment effectively eliminated catalase contamination. Release of 02 by the illuminated suspension was measured by adding catalase upon darkening (15, 23). Chl fluorescence emission determination was as described previously (6). Total Chl was measured in 80% acetone using the method of Amon (2). Chl extractions were made by slowly adding chloroplast suspension to the acetone while strongly shaking the samples. This was to prevent trapping of pigment in the lipoprotein precipitate.

RESULTS In chloroplast preparations from all four algal species, both PSI and PSII were active. However, in all the algae, 02 exchange rates were lower than in either spinach or lettuce chloroplasts. At light saturation, the rates for PSII in the green plant chloroplasts were 2 to 5 times higher than for the algae; and the PSI activity was 2.5 to 8 times higher in green plant than in brown algal chloroplasts; green plant PSI/PSII activity ratios were also larger (Table I). Light saturation of 02 evolution in brown algal chloroplasts occurred at 20 to 40 w m 2, while for green plant chloroplasts saturation required around 100 w m-2. At any intensity, 0, evolution was higher for either of the green plants than any of the algal chloroplasts (Fig. 1). 100 200 300 400 500 The emission peak of variable fluorescence which occurs in W m-2 the first few seconds of illumination was determined for intact blades, fronds or leaves, and for isolated chloroplasts of all six FIG. 1. Light saturating curves of the Hill reaction in the presence of species. The data are displayed asfp = (F, - F,,)/F,,; F, is equal ferricyanide in broken chloroplasts of Fucus, Nereocystis, Laminaria, to the peak fluorescence and F,, is the stationary fluorescence Macrocvstis, spinach, and lettuce. Reaction media as in Table I. corresponding to all PSII reaction center traps open. Values for were 3 to 4 times those of the intact algae for green plant leaves f, above, with DCMU and the TMPD'-ascorbate-MV coupled and 6 to almost 9 times higher when comparing chloroplasts system previously described (1, 18). 02 uptake in light was (Table I). corrected for the slow rates of dark oxidation. Activities of PSII Addition of catalase, following illumination, to spinach or and PSI of brown algae chloroplasts were measured at 1 5C and lettuce released amounts of 02 equivalent to that in spinach and lettuce at 22C. Residual mucilage in brown algae taken upchloroplasts in the light (Fig. 2). Catalase added to nonilluminated necessitated use of a ground glass 3 mL homogenizer to mix chloroplasts yielded little 02. These results indicate that H2O0 chloroplasts with reaction media 10 s before measuring 02 evo- was mainly produced by 02 reduction during illumination in lution or uptake. In these a similar with algal chloroplasts chloroplasts. To determine MV-dependent 02 reduction, chloroplast iso- (Fig. 2), 02 was released, after experiment the addition of catalase following illumination, in amounts greater than that taken up in the light. 'Abbreviations: TMPD, N,N,N',N'-tetramethyl-p-phenylendiamine; When catalase was added to nonilluminated chloroplasts, 02 was MV, methylviologen; F,,, 0 level fluorescence; F., peak in variable released and this 02 appeared to be sufficient to account for this fluorescence during itiduction of photosynthesis.

excess.

EVOLUTION OF 0, IN BROWN ALGAL CHLOROPLASTS 0.2

Fucus

891

Nereocystis

.4

.3 Light

0.1 -UghIV

.2 Dar

co

:Z 0.2

-

c5s

.1

Catalsease

Dark

E

Macrocystis

Laminaria

Lettuce

A. 4

Light

Ught

0.1 -LUght Dark Dark

Dark

0

2eln

4A

6

e

8n 10 %

0

2n

Dark Cataa e 4A 6 80

Dark

10 r^

0

2

Cataase

4

6

8

Time (min) FIG. 2. Light dependent 02 reduction in the presence of MV and subsequent 02 release after the addition of catalase compared to 02 release in nonilluminated samples for Fucus, Nereocvstis, Laminaria, Macrocystis, spinach, and lettuce. Reaction media at 1 ml contained: 50 mM Hepes (pH 7.6), 1 mm MnCI2, I mM MgCl2, 10 to 15 Ag Chl, I mM NH4CI, 0, 2 mM MV, 6 mM isoascorbate, and 300 units of commercial catalase where indicated.

DISCUSSION The chloroplast isolation techniques reported on in "Materials and Methods" yielded photosynthetically active chloroplasts from several species of brown algae. Photosynthetic activity has been previously reported only for chloroplasts isolated from Futcuis serratuts (2). Comparison of PSII and PSI activities of these chloroplasts with those from green plants indicates that light saturation occurs at lower light intensities in all of the algal chloroplasts than in either of the green plants. Also, at any light intensity, PSII and PSI activities were always lower in the algal chloroplasts. Photosynthesis is known to saturate at relatively low light intensities in brown algae (14, 25) and these results may reflect in vivo responses. The differences in H202 generation by the green plant and algal chloroplasts as shown by the release of 02 on the addition of catalase are remarkable. In contrast to lettuce and spinach, nonilluminated algal chloroplasts produced large quantities of H202. Since the chloroplasts had been thoroughly washed, it is difficult to see how they could have produced H202 from organic substrate in the dark. It is likely that considerable 02 reduction occurred in room light during preparation of the algal chloroplasts and before the addition of MV and ascorbate. Fuctis chloroplasts prepared under a green safelight did not release any 02 upon the addition of catalase. However, these chloroplasts did generate H202 in light even when MV and ascorbate were not added (data not shown). 02 is known as an acceptor in photosynthetic electron transport (3, 4, 8, 12, 19-21) and high rates of 02 reduction in the algae could contribute to both low light saturation and the lowfp fluorescence values. The much lowerfp values attained in the light in the intact brown algae and their chloroplasts compared to the green plants are indicative of lower levels of reduced Q (the primary PSII acceptor) in the algae. These results suggest differences in some details of electron transport between the two plant groups. In contrast, the maximum fluorescence yieldfm = (Fma- F,,)/F,, in the presence of the electron transport inhibitor, DCMU, is about 4 to 5 times the stationary fluorescence (F,, = 1) in both groups. This constancy off, suggests that differences in light harvesting processes in brown algae and green plants are small (6). Similar values off", despite large variations in fp, also suggest that the low light utilization by brown algae is more related to electron transport than to light harvesting efficiency.

LITERATURE CITED

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