Isolation of biochemically active chloroplasts from chlamydomonas

Plant Science, 38 (1985) 33--39

33

Elsevier Scientific Publishers Ireland Ltd.

ISOLATION OF BIOCHEMICALLY ACTIVE CHLOROPLASTS FROM CHLAMYDOMONAS

L. MENDIOLA-MORGENTHALER, S. LEU and A. BOSCHETTI* lnstitut fi~r Biochemie, Universitat Bern, Freiestrasse 3, CH-3012 Bern (Switzerland)

(Received July 9th, 1984) (Revision received November 5th, 1984) (Accepted November 24th, 1984) Chloroplasts from the cell wall mutant cw-15-2 of Chlamydomonas reinhardii were isolated by disruption of the cells in the Yeda press and fractionation through step gradients of Percoll. The resulting chloroplast fraction contained 80--85% intact chloroplasts. Electron micrographs of thin sections of the chloroplast fraction showed some cytoplasmic impurities, although almost no cytoplasmic ribosomes were detected by analysis of the ribosomal subunits. The isolated chloroplasts are active in photosynthetic O2-evolution and CO2-fixation, with the highest rates obtained in the presence of ATP. The chloroplast fraction also showed high rates of light-dependent in organello protein synthesis, with labelling of discrete ch|oroplast proteins known to be synthesized in the chloroplasts. Key words: Chlamydomonas reinhardii, chloroplast isolation; chloroplast photosynthesis; chloroplast protein syn-

thesis Introduction T h e e x i s t e n c e and c h a r a c t e r i z a t i o n o f several e x t r a c h r o m o s o m a l m u t a n t s f r o m C. reinhardii have m a d e this organism a favorite f o r s t u d y i n g c h l o r o p l a s t d e v e l o p m e n t and a u t o n o m y . H o w e v e r , certain studies have b e e n h a m p e r e d b y t h e d i f f i c u l t y in isolationg i n t a c t chloroplasts f r o m this alga. R e c e n t l y , Klein et al. [1,2] r e p o r t e d o n the isolation o f p h o t o s y n t h e t i c a l l y active chloroplasts f r o m Chlamyd o m o n a s . T h e s e w o r k e r s used auto-lysine digestion to disrupt the cell wall. B e l k n a p [3] later applied this p r o c e d u r e to isolate c h l o r o p l a s t s f r o m t w o o t h e r strains o f this alga. We have been w o r k i n g w i t h a cell wall mut a n t o f C h l a m y d o m o n a s and have previously r e p o r t e d o n the isolation o f a f r a c t i o n w h i c h was e n r i c h e d in i n t a c t chloroplasts f r o m this organism [4]. H o w e v e r , a l t h o u g h t h e chloroplasts r e t a i n e d high activities o f t h e s t r o m a l

*To whom requests for reprints should be sent.

enzyme, ribulose-l,5-bisphosphate carboxylase, t h e y displayed r a t h e r low activities in l i g h t - d e p e n d e n t p r o t e i n synthesis and p h o t o s y n t h e t i c 0 2 evolution. In t h e p r e s e n t comm u n i c a t i o n , we r e p o r t o n a m o d i f i e d p r o c e d u r e w h i c h we have d e v e l o p e d for obtaining chloroplasts w h i c h are quite active in l i g h t - d e p e n d e n t in organello p r o t e i n synthesis [ 5 ] , and also active in p h o t o s y n t h e t i c O2-evolution as well as CO2-fixation. Materials and m e t h o d s Cultures o f C. reinhardii c w 15-2 mt÷ were o b t a i n e d f r o m t h e C h l a m y d o m o n a s Genetics Center, D u k e University. T h e cells were g r o w n s y n c h r o n o u s l y with 10-h d a r k / 1 4 - h light cycles and h a r v e s t e d in the middle o f the third light period. In general, t h e cells f r o m 2 1 o f culture (1 × 107 cells/ml) were subjected to the fract i o n a t i o n p r o c e d u r e . A f t e r harvesting by c e n t r i f u g a t i o n , the cell pellets were carefully rinsed and r e s u s p e n d e d in 30 ml isolation m e d i u m (P m e d i u m ) c o n t a i n i n g 37 5 mM sorbi-

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34 tol, 35 mM HEPES/KOH (pH 7.70, 1 mM MnC12, 5 mM MgC12, and 2 mM K--EDTA. The cells were broken by passage through the Yeda pressure cell at 5 atm (5 X l 0 s Newton/ m2). The final concentration of EDTA in the homogenate was then adjusted to 8 mM by the addition of a concentrated solution of K-EDTA. After incubation for 15 min at 0°C with gentle shaking, the homogenate was centrifuged by accelerating to 3000 X g and breaking to rest in the shortest possible time. The pellet was carefully resuspended to 25 ml with P medium and an equal volume of 80% Percoll was added to the suspension. This material was layered on 2 X 11 ml of 60% Percoll. All the Percol solutions also contained the same components as P medium. After centrifugation at 5000 X g for 20 min at 4°C, the intact chloroplasts were recovered from the interface between the two Percoll layers, diluted with P medium and sedimented by centrifugation at 3000 X g and braking to rest in the shortest possible time. The chloroplasts were carefully resuspended in P medium and again treated with 8 mM EDTA, as above, sedimented and finally washed twice with P medium. When protein synthesis was to be assayed, sterile techniques were used whenever possible, from the time of harvest of the cultures.

Assay for photosynthesis Oxygen evolution was assayed at 25°C in a Clark-type electrode (Rank Brothers, Cambridge, U.K.). Chloroplast samples were added to 3-ml assay medium, such that the final chlorophyll concentration was 25--40 pg/ml. Different assay media were tested and are described in the text. NaHCO3 was added to a final concentration of 5 mM. The samples were illuminated at light intensities of about 1100 J/m 2 X s with a projector lamp. In some experiments, O2-evolution and CO2-fixation were measured simultaneously. In these cases, the assay mixture also contained NaH14CO3 (2 pCi/ml; Radiochemical Center, Amersham) such that the final spec. act. was 0.4 pCi/gmol. At 1--2 min intervals, 50-pl

samples were withdrawn, acidified with 200 pl of glacial acetic acid and the mixtures were evacuated for 5 rain. Radioactivity was determined by liquid scintillation spectrometry.

Assay for protein synthesis The assay medium was adopted from Fish and Jagendorf [6] and contained: 375 mM sorbitol; 35 mM HEPES/KOH (pH 7.7); 2 mM K--EDTA; 1 mM MnC12:1 mM MgCI~;0.1 mM Na2HPO4; 25 pM of each amino acid except for methionine; 0.5 pM unlabelled methionine and 100 pCi/ml [3SS]methionine (800--1400 Ci/mmol; New England Nuclear). Chloroplasts were added to a concentration of about 500 pg chlorophyll/ml. The samples were incubated at 25°C for 20 min and illuminated whenever light-driven protein synthesis was measured. For determining amino acid incorporation, aliquots of 20 pl were precipitated on glass fiber filters (Whatman GFA) with 10% trichloroacetic acid. The filters were washed with 5% trichloroacetic acid, ethanol and acetone. Radioactivity was measured by liquid scintillation spectrometry. Analysis of the ribosomal subunits was as previously described [ 7 ].

Electron microscopy The samples in P medium were supplemented with 1.25% glutaraldehyde and 1.5% acrolein and kept for 2 h at room temperature. After 3 washings with 1/15 M phosphate buffer, (pH 7.0), they were fixed further with 1% OsO4 for 6 h at 0°C. Dehydration in acetone was followed by embedding in the medium of Spurr according to the procedure given by the manufacturer (Polysciences, Inc., Warrington, U.S.A.). The thin sections were post-stained with lead citrate. Results and discussion The cells of the m u t a n t cw-15-2 of C. reinhardii can be broken by subjecting them to low pressure in the Yeda press. On the average, 60--70% of the cells are broken, as judged by phase contrast, microscopy. Under phase

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contrast the intact chloroplasts are crescentshaped and appear bright and refractile, whereas the stripped chloroplasts appear rounded and dark.

Subsequent centrifugation through step gradients of Percoll results in the separation of a fraction enriched in intact chloroplasts that bands at the interface between 40 and 60%

a

i

Fig. 1. Electron photomicrographs of whole cells and subcellular fractions obtained from Percoll gradients. (a) Whole cells; (b) stripped chloroplast fraction; (c) intact chloroplast fraction; (d) intact chloroplast. Bar: 2 um in (a), (b), (c) and 1 um in (d).

36 Percoll. The material which floats in the 40% Percoll layer is usually clumped and contains stripped chloroplasts, small particles and m e m b r a n e fragments as well as some intactlooking chloroplasts (cf. Fig. 1). U n b r o k e n cells sediment through the 60% Percoll layer. The final chloroplast preparation, after washings, contains, on the average, 2.5--4 mg of chlorophyll, starting with 2 1 of culture. This represents a yield of 4--6% of the chlorophyll o f the starting material. Excessive clumping of the particles can seriously hamper their fractionation. We have ro u t i n ely used media containing higher concentrations o f MgCl2 (5 mM) than normally used for isolating chloroplasts from o t h e r systems, since we f ound that this reduced clumping. At the same time, incubation with excess EDTA at certain steps of the fractionation was carried out, since this t r e a t m e n t greatly reduced co n t a m i nat i on of the chloroplast preparations by cytoplasmic 80 S-type ribosomes (see below). T h e m e t h o d described here for the isolation of chloroplasts from Chlamydomonas is based on our previous p rocedur e [4] and has been modified to obtain chloroplasts which are active in photosynthesis and protein synthesis, in organello [5]. It differs f r om t ha t r e p o r t e d by Klein et al. [1] mainly in: (1) the m e t h o d o f cell breakage, since we use a cell wall mut a n t and no detergent; (2) the much larger amounts of material we process (2 1 or more, instead of 100 ml of culture) and (3) the m e d i u m used for the isolation of the chloro-

plasts. We have also obtained intact chloroplasts, using media of lower osmolarities; i.e. 330 or 125 mM [1] instead of 375 mM sorbitol.

Intactness and purity of chloroplasts The chloroplast fractions banding between the 40% and 60% Percoll layers contained 80--85% intact particles, as determined by their ability to utilize ferricyanide as Hill oxidant [8] (Table I). Electron microscopy of the samples also revealed the predominance of intact plastids (Fig. lc,d). In comparison, the material which floated on the 40% Percoll layer, as shown in Fig. l b , contained predominantly stripped chloroplasts. However, the electron photomicrographs of the intact chloroplast fraction also revealed that some cytoplasmic impurities, such as m i t o c h o n d r i a and membranes, adhere to the chloroplasts. T h e y seem to be firmly bound, since extensive washings and even a combination of centrifugation on step and linear gradients of Percoll did n o t significantly remove these impurities. On the ot her hand, the intact chloroplast fraction contained mostly 70 S-type ribosomes and was virtually free of 80 S-type cytoplasmic ribosomes. Typical profiles of the ribosomal subunits o f the chloroplast fraction and of whole cells are shown in Fig. 2.

Photosynthetic activity The chloroplast preparations were active in bicarbonate~lependent O,-photo-evolution as well as CO2-fixation, as shown in Table II.

Table I. Ferricyanide-dependent 02 evolution by chloroplasts before and after osmotic lysis. The assay mixture contained: (a) 375 mM sorbitol; 35 ~ HEPES/KOH (pH 7.7); 1 mM MnCl:; 5 mM MgCI:; 2 mM EDTA;0.25 mM KH2PO,, 1.5 mM K~Fe(CN)6; 1.5 mM NH4C1; catalase (1000 u/ml). In (3) the assay mixture was similar, except that the concentrations of EDTA and MgClz were 5 mM and 6 raM, respectively. Expt. No.

1 2 3

umoles O~ • mg -~ chlorophyll, h ~ Intact

Lysed

Intact chloroplasts %

10 11.7 21.4

59.7 63.3 137.2

83 81 $5

37

A260

A260 50 S 40S

30S 50S

i

i

Gradient

Gradient

Fig. 2. Analysis of subunit composition on density gradients of sucrose of ribosomes from whole cells (left) and from intact chloroplastofraction (right). Chloroplasts or whole cells corresponding to 2 5 - 50 ug chlorophyll were centrifuged on exponential gradients of sucrose, as described previously [7 ]. The activities varied, depending on the assay medium used. The standard medium contained n o M g 2÷, b u t o t h e r w i s e h a d a s i m i l a r c o m p o s ition to that used for higher plant chloroplasts, a n d b y K l e i n e t al. [ 2 ] f o r Chlamydomonas chloroplasts. In this medium we measured r a t e s o f 1 4 - - 1 7 ~ m o l 0 2 , o r 13 ~ m o l C O 2 / m g chlorophyll per h for the O2-evolution or

C O s - f i x a t i o n , r e s p e c t i v e l y . A d d i t i o n o f Mg :+ p r o g r e s s i v e l y i n h i b i t e d p h o t o s y n t h e t i c as w e l l as p r o t e i n s y n t h e t i c [ 5 ] a c t i v i t i e s ; a t 1 m M M g 2÷ t h e y w e r e i n h i b i t e d b y a b o u t 25%. T h e addition of ATP, however, stimulated both O2-evolution and COs-fixation about twice (Table II). A similar stimulation by ATP of photosynthetic C O s - f i x a t i o n in c h l o r o p l a s t s

Table II. Bicarbonate-dependent 02 photo-evolution and CO 2 fixation by Chlamydomonas chloroplasts. Standard medium: (a) 375 mM sorbitol; 35 mM HEPES/KOH (pH 7.7); 1 mM MnCI~; 2 mM EDTA; 0.25 mM KH2PO , catalase (1000 u/ml); 5 mM NaHCO~. When indicated, MgCl~ (6 mM), EDTA (5 raM) and ATP (5 mM) were added. When CO 2 fixation was measured simultaneously, the assay mixture also contained NaH~4CO3 at a final spec. act. of 0.4 uCi/~mol. Chloroplasts were added to a final concentration of 25--40 ug chlorophyll/ml assay mixture. Assay medium

O 2 evolution CO 2 fixation (u moles, m g-1 chlorophyll, h ~1)

Standard + ATP MgCl 2 + EDTA ÷ MgCl~ + EDTA + ATP MgCl: ~ EDTA ~- ATP

14.4 32.7 10.7 47.9 69.7

12.7 28.8 -44.9 --

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of Sedum and peas, but not of spinach was observed also by Piazza and Gibbs [9] in a medium comparable to our standard medium, but containing in addition 1 mM free Mg2÷ and 5 mM Mg-EDTA. Chloroplast preparations tested in standard medium supplemented with Mg 2÷ and EDTA had photosynthetic activites equal to or even lower than without Mg2÷ and EDTA. However, addition of ATP resulted in a four-fold stimulation of CO2-fixation and O2-evolution (Table II). We do not know the reason for this stimulation. The response of the chloroplasts to ATP was unstable with time, since, during storage of the chloroplasts in ice, there was a more rapid decrease in photosynthetic activity when assayed in the

presence of this nucleotide than when assayed in its absence. When both O2-photo-evolution and CO2photo-assimilation were tested, the rates agreed fairly well with one another. The rates we obtained in the presence of ATP correspond closely to the values reported for Chlamydomonas chloroplasts [2,3]. They represent 25--30% of the net photosynthetic rates of the starting whole cells, when the latter were assayed in fresh culture medium (in standard medium containing sorbitol, whole cells were inactive). Similar yields were reported for Euglena chloroplasts [10]. However, it should be noted that we measured the photosynthetic activities of our chloroplasts in media with

c prn(lO -3 )

(L68lO°-/'3%"/1

15 _

control

'l

I CAP

10

30

50 min.

Fig. 3. Kinetics and the effect of inhibitors on the incorporation of [asS']methionine by isolated chloroplasts. Unless otherwise specified, incorporation was allowed to proceed in the light, as described in Materials and methods. CHI: cycloheximide, 12.5 #g/ml assay mixture. CAP: chloramphenicol, 35 ug/ml assay mixture. Insert: Autoradiograph of the products of [asS] methionine incorporation by the chloroplasts. Arrows point to the 55kDa and the 32-kDa polypeptides.

39 375 mM sorbitol in order to maintain the osmolarity used during fractionation. In contrast, Klein et al. [2] have found that in their preparations of chloroplasts from Chlamydomonas, the photosynthetic CO2-fixation was significantly inhibited by mannitol concentrations above 150 mM; at 300 mM mannitol almost no activity could be measured.

Protein synthesis When the chloroplast preparations were incubated with [3sS] methionine, the incorporation of radioactivity into acid-stable material was strongly light-dependent (Fig. 3). The kinetics of incorporation as well as the inhibition by chloramphenicol and the insensitivity to cycloheximide are typical of protein synthesis, in organello. As compared to the previously published results of in organello protein synthesis by Chlamydomonas chloroplasts [5], the presently described conditions for isolation of the chloroplasts and incubation have resulted in significantly improved protein synthetic activity. Synchronous cultures were grown to give not more than 107 cells/ml in the third light period. Chloroplasts isolated from such cultures incorporate more label into the large subunit of ribulose-l,5-bisphosphate carboxylase and other polypeptides relative to the 32kDa polypeptide (Fig. 3). Furthermore, more discrete bands appear in the region below 25 kDa instead of the heavy smearing observed before. Increasing the specific activity of the labelled amino acid resulted in an incorporation of up to 8% of the radioactivity into acid stable material, and 25 000--30 000 c.p.m./ug chlorophyll were incorporated in 20 min. These rates could be obtained by incubations containing up to 500 gg/ml chlorophyll. With higher chlorophyll concentrations the specific labelling decreases. The possibility of isolating biochemically intact chloroplasts from Chlamydomonas, as

demonstrated here, by Klein et al. [1,2] and by Belknap [3] has made this organism more amenable to the study of certain aspects of chloroplast function and biogenesis. Our procedure is limited to cell wall mutants whose cells can easily be disrupted by mild mechanical means but does not need any detergent treatment. Although providing enough material for studying protein synthesis and photosynthesis, the method could be improved upon, in order to obtain purer preparations which would be required for other kinds of studies. In this respect, none of the methods presently available for Chlarnydomonas have yielded chloroplasts of comparable purity to that obtained with higher plant chloroplasts, e.g. spinach. Acknowledgement This project was supported by the Swiss National Science Foundation.

References I U. Klein, C. Chen, M. Gibbs and A. Platt-Aloia, Plant Physiol., 72 (1983) 481. 2 U. Klein, C. Chen and M. Gibbs, Plant Physiol., 72 (1983) 488. 3 W.B. Belknap, Plant Physiol., 72 (1983) 1130. 4 L. Mendiola-Morgenthaler and A. Boschetti, in: G. Akoyunoglou (Ed.), Photosynthesis, V. Chloroplast Development, Balban Int. Sci. Service, Philadelphia, 1981, pp. 457--463. 5 S. Leu, L. Mendiola-Morgenthaler and A. Boschetti, FEBS Lett., 166 (1984) 23. 6 L.E. Fish and A.T. Jagendorf, Plant Physiol., 70 (1982) 1107. 7 R. Bolli, L. Mendiola-Morgenthaler and A. Boschetti, Biochim. Biophys. Acta, 653 (1981) 276. 8 R. McC. Lilley, M.P. Fitzgerald, K.G. Rienits and D.A. Walker, New Phytol., 75 (1975) 1. 9 G.J. Piazza and M. Gibbs, Plant Physiol.,71 (1983) 680. 10 P. Schiirmann and W. Ortiz, Planta, 154 {1982) 70.