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The isolation of structurally intact chloroplasts
SHORT COMMUNICATIONS SC 43014
The isolation of structurally intact chloroplasts Electron-microscopical studies in several laboratories (see refs. 1-3) have recently shown that the chloroplasts (isolated in buffered 0.35 M NaCl) generally employed in biochemical studies are greatly altered structurally from their state in the leaf cell. It is most desirable that chemical and enzymic information should be obtained from suspensions in which the chloroplasts themselves are morphologically intact and in which no other cell debris is present. This paper describes a method for the aqueous isolation of structurally intact chloroplasts from young leaves of Vicia faba L. (the broad bean) in suspensions virtually freed from other cellular material. Vicia faba L. (var. Giant Windsor) plants were grown as described by LEECH 3 and placed in the dark for 24-48 h prior to chloroplast isolation. It is essential that the plants should be completely destarched before use because any starch grains present in the chloroplasts are pulled through the lamella system and bounding membrane during centrifugation causing fragmentation of the chloroplast. 10 g green expanding leaves from 2-3-week-old plants were chilled, cut into small pieces, dropped into 50 ml cold 0.15 M Na zHP0 4 -KH zP0 4 buffer (pH 7.3) containing 0.3 M sucrose and stirred firmly with a large plastic spatula for 5 min. No sand was used and violent homogenization or crushing carefully avoided. Leaf debris was removed by filtration through a single layer of nylon net. Under the light microscope and also under phase contrast, 60-80 % of the chloroplasts in the filtrate had the opaque appearance suggested- by KAHN AND VON WETTSTEIN z to be characteristic of membrane-bound chloroplasts. The filtrate was centrifuged at 600 X g for 2 min; electron-microscopical examinations showed that cell wall and nuclear fragments were completely removed during this spin. The green supernatant which contains intact chloroplasts, chloroplast fragments and numerous mitochondria was layered on top of a sucrose-density gradient. The gradient was discontinuous and consisted of 6 ml 46 % sucrose (Po = 1.20) on top of 10 ml 50 % sucrose (Po = 1.229). All the sucrose solutions were in 0.15 M Na zHP0 4 -KH 2 P0 4 buffer (pH 7.3). The tubes containing the completed gradients were spun in a swing-out head for 20 min at 1000 X g, great care being taken to increase and decrease the centrifugal force gradually. After centrifugation a discrete green band just above the partition between the two sucrose layers was found to consist almost exclusively of intact chloroplasts: chloroplasts denuded of their external membranes, chloroplast fragments and mitochondria remain higher up in the centrifuge tube. The layer containing the intact chloroplasts was carefully removed with a Pasteur pipette, diluted with an equal volume of grinding medium and the intact chloroplasts spun down at rooo X g for 10 min to give pellet II' Average yields of intact chloroplasts are rather low: a suspension containing 5-10 mg chlorophyll will be obtained from a leaf homogenate containing 50 mg chlorophyll. The best yields are obtained from leaf material grown in more than 6 h of sunshine per day and treated in small batches of 10 g. An extensive search for an alternative isolation medium in which the yield of intact chloroplasts would be increased was unsuccessful. It proved impossible to remove cell-wall fragments from suspensions in which the tonicity of sucrose was increased above 0.3 M. The addition singly to the 0.3 M sucrose of the following substances reputed to aid membrane retention, did not increase the yield of intact chloroplasts: 10-4 and 10-2 M EDTA, ro- 3 and ro- 2 M MgCI 2 , 5 mg/l kinetin,s % polyvinyl pyrrolidone, 5 mM ATP and 2 mg/ml bovine serum albumin. Biochim_ Biophvs_ Acta, 79 (1964) 637-639
SH0RT COMMUNICATIONS Almost every chloroplast in the pellet II had the appearance of a membranebound chloroplast (see ref. 2). Electron-microscopical examination was however essential to ascertain the degree of integrity of the chloroplasts and the degree of contamination of the pellet with other cytoplasmic material. If the pellet II was prepared for the electron microscope by the conventional methods involving fixation in buffered OS04 followed hy dehydration through graded alcohols the integrity of the chloroplasts was not maintained. In sections of material which, prior to fixation, had appeared to contain many intact chloroplasts, the outer chloroplast membranes were found to be distended and dragged away from the stroma matrix and broken in several places. Centrifugation of the plastids during fixation and dehydration accentuated the damage. The damage was eliminated if the tonicity of the solutions employed was reduced gradually throughout the fixation and dehydration procedures. The following method was adopted. The compacted pellet of chloroplasts (II) in a 15-ml conical centrifuge tube was fixed for I h at 0°. The fixative solution contained 2 % OS04 and 50 % sucrose in 0.15 M Na 2 HPO c KH 2 P0 4 buffer (pH 7.0). After fixation the pellet was released gently from the bottom of the tube and dehydration carried out at 0° as follows: 50 % sucrose in water; 50 % sucrose in 30 % ethanol with one change; 30 % sucrose in 40 % ethanol; 25 % sucrose in 50 % ethanol. At this stage all but 5 ml of the supernatant was removed and 50 % ethanol added dropwise with gentle shaking of the tube. (More concentrated solutions of ethanol cannot be added at this stage because precipitation of sucrose will occur.) Dehydration was then continued by successive changes into 50 %, 60 %, 70 %, 95 % and 100 % ethanol. Small pieces of the dehydrated pellet were embedded in Epon as described by LEECH 3 except that the mixtures A and B of LUFT 4 were used in the proportions 7A:3B. Plate A is an electron-micrograph of a section from material from pellet II' It can be seen that the pellet consists almost exclusively of chloroplasts with completely intact bounding membranes. The suggestion that chloroplasts having an opaque and apparently grana-free appearance in the light microscope are morphologically intact chloroplasts is vindicated. The intact chloroplasts in Plate A have a more highly electron-dense and less diffuse stroma matrix than the chloroplasts in which the outer bounding membrane has been punctured (a). The isolated chloroplasts are considerably more rounded than in the intact cell and many have characteristically folded lamella systems. The two apparently separate lamella systems observed within a single chloroplast (b) can be explained as sections of a single lamella network which has become cup-shaped during isolation of the plastid. The main contaminant of the preparations of intact chloroplasts is broken chloroplasts. Morphologically recognisable fragments of cell wall and nuclear material are completely absent. Profiles of mitochondria are infrequently encountered in the sections but those present are structurally well preserved. In a count of 200 profiles in electronmicrographs of the pellet 82 % were of intact chloroplasts, 8.5 % of chloroplasts in which the bounding membrane was present but ruptured, 5 % chloroplast fragments and 4.5 % mitochondria. 90.5 % of the total profiles were thus of intact or virtually intact chloroplasts. When resuspended in 0.61\1 sucrose intact chloroplasts will remain apparently unchanged morphologically for up to 4 h at 2°. These chloroplasts have the stroma matrix intact as in non-aqueously isolated plastids (HEBER AND TYSZKIEWICZ5 , Biochim. Biophys. Acta, 79 (1964) 637-639
SHORT COMMUNICATIONS
b39
Plate A. Section from a pellet containing intact chloroplasts prepared from leaves of Vicia faba L. as described in the text. Fixed in 2 % OS04 in buffered (pH 7.0) 50 % sucrose and dehydrated and embedded as described in the text; No. 2314. Magnification x 6750. Chloroplast with a punctured external membrane (a) and chloroplast with a folded lamella system (b).
STOCKING AND ONGUN 6 ) but in addition the aqueously isolated chloroplasts retain the bounding membrane destroyed during the non-aqueous procedure. It is however possible that the chloroplasts have lost low-molecular-weight compounds during aqueous isolation. The chemical composition and enzymic capabilities of the intact chloroplasts described in this paper are being investigated. I am very pleased to acknowledge my gratitude to Mr. A. D. GREENWOOD for preparing the electron micrograph shown in Plate A.
Department oj Botany, Imperial College, London (Great Britain) 1 2 3
4
5 6
RACHEL M. LEECH
G. JACOBI AND E. PERNER, Flora, 150 (1961) 209. A. KAHN AND D. VON VVETTSTEIN,]. Ultrastruet. Res., 5 (1961) 557. R. M. LEECH, Biochim. Biophys. Acta, 71 (1963) 253. J. H. LUFT,j. Biochem. Biophys. Cytol., 9 (1961) 409. U. HEBER AND E. TYSZKIEWICZ, j. Exptl. Botany, 13 (1962) 185. C. R. STOCKING AND A. ONGUN, Am.]. Botany, 49 (1962) 284.
Received February 20th, 1964 Biochim. Biophys. Acta, 79 ([964) 637-639
The isolation of structurally intact chloroplasts Electron-microscopical studies in several laboratories (see refs. 1-3) have recently shown that the chloroplasts (isolated in buffered 0.35 M NaCl) generally employed in biochemical studies are greatly altered structurally from their state in the leaf cell. It is most desirable that chemical and enzymic information should be obtained from suspensions in which the chloroplasts themselves are morphologically intact and in which no other cell debris is present. This paper describes a method for the aqueous isolation of structurally intact chloroplasts from young leaves of Vicia faba L. (the broad bean) in suspensions virtually freed from other cellular material. Vicia faba L. (var. Giant Windsor) plants were grown as described by LEECH 3 and placed in the dark for 24-48 h prior to chloroplast isolation. It is essential that the plants should be completely destarched before use because any starch grains present in the chloroplasts are pulled through the lamella system and bounding membrane during centrifugation causing fragmentation of the chloroplast. 10 g green expanding leaves from 2-3-week-old plants were chilled, cut into small pieces, dropped into 50 ml cold 0.15 M Na zHP0 4 -KH zP0 4 buffer (pH 7.3) containing 0.3 M sucrose and stirred firmly with a large plastic spatula for 5 min. No sand was used and violent homogenization or crushing carefully avoided. Leaf debris was removed by filtration through a single layer of nylon net. Under the light microscope and also under phase contrast, 60-80 % of the chloroplasts in the filtrate had the opaque appearance suggested- by KAHN AND VON WETTSTEIN z to be characteristic of membrane-bound chloroplasts. The filtrate was centrifuged at 600 X g for 2 min; electron-microscopical examinations showed that cell wall and nuclear fragments were completely removed during this spin. The green supernatant which contains intact chloroplasts, chloroplast fragments and numerous mitochondria was layered on top of a sucrose-density gradient. The gradient was discontinuous and consisted of 6 ml 46 % sucrose (Po = 1.20) on top of 10 ml 50 % sucrose (Po = 1.229). All the sucrose solutions were in 0.15 M Na zHP0 4 -KH 2 P0 4 buffer (pH 7.3). The tubes containing the completed gradients were spun in a swing-out head for 20 min at 1000 X g, great care being taken to increase and decrease the centrifugal force gradually. After centrifugation a discrete green band just above the partition between the two sucrose layers was found to consist almost exclusively of intact chloroplasts: chloroplasts denuded of their external membranes, chloroplast fragments and mitochondria remain higher up in the centrifuge tube. The layer containing the intact chloroplasts was carefully removed with a Pasteur pipette, diluted with an equal volume of grinding medium and the intact chloroplasts spun down at rooo X g for 10 min to give pellet II' Average yields of intact chloroplasts are rather low: a suspension containing 5-10 mg chlorophyll will be obtained from a leaf homogenate containing 50 mg chlorophyll. The best yields are obtained from leaf material grown in more than 6 h of sunshine per day and treated in small batches of 10 g. An extensive search for an alternative isolation medium in which the yield of intact chloroplasts would be increased was unsuccessful. It proved impossible to remove cell-wall fragments from suspensions in which the tonicity of sucrose was increased above 0.3 M. The addition singly to the 0.3 M sucrose of the following substances reputed to aid membrane retention, did not increase the yield of intact chloroplasts: 10-4 and 10-2 M EDTA, ro- 3 and ro- 2 M MgCI 2 , 5 mg/l kinetin,s % polyvinyl pyrrolidone, 5 mM ATP and 2 mg/ml bovine serum albumin. Biochim_ Biophvs_ Acta, 79 (1964) 637-639
SH0RT COMMUNICATIONS Almost every chloroplast in the pellet II had the appearance of a membranebound chloroplast (see ref. 2). Electron-microscopical examination was however essential to ascertain the degree of integrity of the chloroplasts and the degree of contamination of the pellet with other cytoplasmic material. If the pellet II was prepared for the electron microscope by the conventional methods involving fixation in buffered OS04 followed hy dehydration through graded alcohols the integrity of the chloroplasts was not maintained. In sections of material which, prior to fixation, had appeared to contain many intact chloroplasts, the outer chloroplast membranes were found to be distended and dragged away from the stroma matrix and broken in several places. Centrifugation of the plastids during fixation and dehydration accentuated the damage. The damage was eliminated if the tonicity of the solutions employed was reduced gradually throughout the fixation and dehydration procedures. The following method was adopted. The compacted pellet of chloroplasts (II) in a 15-ml conical centrifuge tube was fixed for I h at 0°. The fixative solution contained 2 % OS04 and 50 % sucrose in 0.15 M Na 2 HPO c KH 2 P0 4 buffer (pH 7.0). After fixation the pellet was released gently from the bottom of the tube and dehydration carried out at 0° as follows: 50 % sucrose in water; 50 % sucrose in 30 % ethanol with one change; 30 % sucrose in 40 % ethanol; 25 % sucrose in 50 % ethanol. At this stage all but 5 ml of the supernatant was removed and 50 % ethanol added dropwise with gentle shaking of the tube. (More concentrated solutions of ethanol cannot be added at this stage because precipitation of sucrose will occur.) Dehydration was then continued by successive changes into 50 %, 60 %, 70 %, 95 % and 100 % ethanol. Small pieces of the dehydrated pellet were embedded in Epon as described by LEECH 3 except that the mixtures A and B of LUFT 4 were used in the proportions 7A:3B. Plate A is an electron-micrograph of a section from material from pellet II' It can be seen that the pellet consists almost exclusively of chloroplasts with completely intact bounding membranes. The suggestion that chloroplasts having an opaque and apparently grana-free appearance in the light microscope are morphologically intact chloroplasts is vindicated. The intact chloroplasts in Plate A have a more highly electron-dense and less diffuse stroma matrix than the chloroplasts in which the outer bounding membrane has been punctured (a). The isolated chloroplasts are considerably more rounded than in the intact cell and many have characteristically folded lamella systems. The two apparently separate lamella systems observed within a single chloroplast (b) can be explained as sections of a single lamella network which has become cup-shaped during isolation of the plastid. The main contaminant of the preparations of intact chloroplasts is broken chloroplasts. Morphologically recognisable fragments of cell wall and nuclear material are completely absent. Profiles of mitochondria are infrequently encountered in the sections but those present are structurally well preserved. In a count of 200 profiles in electronmicrographs of the pellet 82 % were of intact chloroplasts, 8.5 % of chloroplasts in which the bounding membrane was present but ruptured, 5 % chloroplast fragments and 4.5 % mitochondria. 90.5 % of the total profiles were thus of intact or virtually intact chloroplasts. When resuspended in 0.61\1 sucrose intact chloroplasts will remain apparently unchanged morphologically for up to 4 h at 2°. These chloroplasts have the stroma matrix intact as in non-aqueously isolated plastids (HEBER AND TYSZKIEWICZ5 , Biochim. Biophys. Acta, 79 (1964) 637-639
SHORT COMMUNICATIONS
b39
Plate A. Section from a pellet containing intact chloroplasts prepared from leaves of Vicia faba L. as described in the text. Fixed in 2 % OS04 in buffered (pH 7.0) 50 % sucrose and dehydrated and embedded as described in the text; No. 2314. Magnification x 6750. Chloroplast with a punctured external membrane (a) and chloroplast with a folded lamella system (b).
STOCKING AND ONGUN 6 ) but in addition the aqueously isolated chloroplasts retain the bounding membrane destroyed during the non-aqueous procedure. It is however possible that the chloroplasts have lost low-molecular-weight compounds during aqueous isolation. The chemical composition and enzymic capabilities of the intact chloroplasts described in this paper are being investigated. I am very pleased to acknowledge my gratitude to Mr. A. D. GREENWOOD for preparing the electron micrograph shown in Plate A.
Department oj Botany, Imperial College, London (Great Britain) 1 2 3
4
5 6
RACHEL M. LEECH
G. JACOBI AND E. PERNER, Flora, 150 (1961) 209. A. KAHN AND D. VON VVETTSTEIN,]. Ultrastruet. Res., 5 (1961) 557. R. M. LEECH, Biochim. Biophys. Acta, 71 (1963) 253. J. H. LUFT,j. Biochem. Biophys. Cytol., 9 (1961) 409. U. HEBER AND E. TYSZKIEWICZ, j. Exptl. Botany, 13 (1962) 185. C. R. STOCKING AND A. ONGUN, Am.]. Botany, 49 (1962) 284.
Received February 20th, 1964 Biochim. Biophys. Acta, 79 ([964) 637-639