Planta (Berl.) 76, 182--189 (1967)
Crystalline Structures in Viciafaba Chloroplasts L. K. S E t , w A Y * , T. E. WEInl~ and C. R. STOCKING Botany Department, University of California, Davis, California Received April 26, 1967
Summary. Chloroplast stroma crystals similar to those reported by PnRN~R (1962, 1963) but different from those described by other workers were demonstrat ed in Vicia/aba chloroplasts. They were absent in chloroplasts isolated in no sucrose, 0.3 M sucrose or in situ materiM either fixed in the absence of sucrose or fixed in the presence of 0.3 M sucrose. They were present in chloroplasts isolated in 0.5 M sucrose and in situ chloroplasts fixed in the presence of 0.7 1VIsucrose. Introduction
GU~NIHG (1965) d e m o n s t r a t e d the presence of aggregated fibrils, each 85 A in diameter, in the stroma of Avena sativa chloroplasts and called t h e m stroma centers. He a t t r i b u t e d their discovery to better fixation with glutarMdehyde and apparently considered the stroma center to be a normM constituent of plastids of oats. T~oMsoH et M., (1965), working with Phaseolus vulgaris, have shown t h a t organized arrays of granules measuring 85 A in diameter m a y result when plants are exposed to peroxyacetyl nitrate, a photochemically produced air pollutant. Comparison of the published micrographs leads one to suspect t h a t the two papers are describing the same phenomenon. However, THOMSON et al., (1965) reported t h a t the granules were not present in material kept in the dark, while GUNninG (1965) showed a plastid of an etiolated plant which had a stroma center. The work on crystals in plastids reported b y P n g s E g (1962, 1963) is of special interest because their presence was controlled b y conditions of isolation. H e reported t h a t the stroma of isolated spinach plastids had a crystalline structure with spacings of less t h a n 100 A. These crystals were absent in chloroplasts of intact tissue and chloroplasts isolated in isotonic medium, lie concluded t h a t t h e y resulted from osmotic dehydration during isolation in hypertonic medium. The present paper substantiates the observations of P]~R~E~ t h a t the appearance of crystals in plastid stroma is related to the isolation m e d i u m used. I t also demonstrates t h a t chloroplasts of intact tissue will contain crystals after t r e a t m e n t with hypertonic solutions. * Present address: Department of Botany, Washington State University, Pullman, Washington.
Crystalline Structures in Vicia ]aba Chloroplasts
183
Materials and Methods The chloroplasts studied were those in mature (fully expanded) leaves of greenhouse-grown Vicia/aba L. plants. For preparation of isolated chloroplasts, the leaves were ground for 30 sec in a Waring blender running on 65 volts. Grinding was in either 0.3 M or 0.5 M sucrose, buffered at psi 7.4 with 0.1 M potassium phosphate. The brei was filtered through four layers of cheese cloth and centrifuged at 270 • g for 1 min. The s u p e r n a t a n t was centrifuged a t 480 • g for 7 rain a n d the pelleted plastids were washed once with buffered sucrose. Fixation of the plastids was carried out in 6 % glutaraldehyde in 0.1 M phosphate buffer pSI 7.1. F o r fixation of chloroplasts in situ, small pieces a b o u t 1--2 m m square of Vicia/aba leaves were fixed in 6% glutaraldehyde in 0.1 M phosphate buffer, p t I 7.1, in solutions containing no sucrose, 0.3 M sucrose or 0.7 M sucrose. Both leaf pieces and isolated plastids were washed in buffer, and postfixed with 1% buffered osmium. Dehydration was in a graded series of alcohols a n d propylene oxide, followed b y embedding in araldite. Thin sections were stained with aqueous solutions of 2 % barium permanganate and examined in a n Hitachi SIU- 11 electron microscope.
Results C h l o r o p l a s t s of m a t u r e a n d y o u n g Vicia /aba l e a v e s w h e n i s o l a t e d i n 0.5 M s u c r o s e c o n t a i n c r y s t a l l i n e b o d i e s i n t h e s t r o m ~ (Fig. 1). T h e s a m e is t r u e for i n s i t n p l a s t i d s w h e n f i x e d i n t h e p r e s e n c e of 0.7 M
sucrose (Fig. 2). However,
the crystals were absent in plastids isolated
Fig. 1. Crystal bodies in a chloroplast of Vicia /aba isolated in 0.5 M sucrose. • 27,000. Here a n d in the following micrographs, the marker line is equivalent to 0.5 ~z
184
L. K. StIUNwAY, T. E. WEIEt{ and C. 1%. STOOXING:
Fig. 2. Crystal bodies in an in situ chloroplast of Vicia /aba treated with 0.7 M sucrose. • 87,000
Crystalline Structures in Vicia ]aba Chloroplasts
185
Fig. 3. Crystal body of an isolated chloroplast of Vicia ]aba showing one directional periodicity. • 144,000
186
L. K. S•cMwAY, T. E. WEIER and C. R. STOCKING:
Fig. 4. Crystal body of an isolated chloroplast of Vicia ]aba showing two directional periodicity. • 228,000
Crystalline Structures in Vicia/aba Chloroplasts
187
?ig. 5. Crystal body of an isolated chlorplast of Viola ]aba showing gradual blending of stroma and crystal body. X 156,000
188
L. K. S~uMwAu T. E. W~IER and C. R. STocJ~I~G:
in 0.3 M sucrose and in in-situ plastids either fixed in the absence of sucrose or fixed in the presence of 0.3 M sucrose. These crystalline bodies m a y show only a one directional periodicity (Fig. 3) or they m a y have two directional periodicities which intersect with an angle of 70--800 (Fig. 4). Some crystals end rather abruptly (Figs. 1 and 2) while others blend with the stroma without a sharp boundary (Fig. 5). Plastids fixed only
Fig. 6. Crystal bodies of an isolated chloroplast of Vicia /aba fixed only in glutaraldehyde. • 73,000 in glutaraldehyde show t h a t the presence of the crystals is not dependent on the use of osmium as a fixative (Fig. 6). Temperatures of dehydration of 20 ~ 4 ~ --200 and --700 had no effect on these crystals. Center to center spacing of the electron dense lines is 105--115 A for both directions. Discussion
The presence of crystals in the stroma of chloroplasts has been ascribed to better fixation (GuNnING, 1965), to plastid damage due to air pollutants (TltoNso~ et al., 1965), or to plastid isolation procedures ( P E ~ N ~ , 1962, 1963). In some instances (MASTO~, 1966), they appear to be actual storage products. Pn~Nn~ (1962, 1963) observed t h a t hypertonie solutions m a y induce crystalline body formation in chloroplasts. His findings are substantiated and extended here. These observations point to the importance of tonicity in the fixation and isolation media. The recognition of the possibility of such induced structural changes in the ultrastructure of organelles, and the proper evaluation of these changes should enhance our knowledge of the function and molecular organization of cell organelles.
Crystalline Structures in Vicia/aba Chloroplasts
189
References Gu~I~G, B. E. S. : The fine structure of chloroplast stroma following aldehyde osmium-tetroxide fixation. J. Cell Biol. 24, 79--93 (1965). MA~TO~, I.: Some possibly significant structural relations between chloroplasts and other cell components. In: Biochemistry o5 chloroplasts (T. W. GooDwIN, ed.), vol. 1, p. 23--47. New York: Academic Press 1966. PERNE~, E. : Elektronenmikroskopisehe Befunde fiber Kristallgitterstrukturen im Stroma isolierter Spinatehloroplasten. Port. Acta biol. A 6, 359--372 (1962). Kristallisationserscheinungen im Stroma isolierter Spinatehloroplasten guter Erhaltung. Naturwissenschaften 50, 134--135 (1963). THOMSOn,W. W., W. M. DUGGER,jr., and R. L. PAL~ER: Effects of peroxyacetyl nitrate on ultrastructure of chloroplasts. Bot. Gaz. Chicago 126, 66--72 (1965). -
-
L. K. S~U~WAY Botany Department, Washington State University Pullman, Washington 99163, USA
13 Planta(Berl.), Bd. 76
Crystalline Structures in Viciafaba Chloroplasts L. K. S E t , w A Y * , T. E. WEInl~ and C. R. STOCKING Botany Department, University of California, Davis, California Received April 26, 1967
Summary. Chloroplast stroma crystals similar to those reported by PnRN~R (1962, 1963) but different from those described by other workers were demonstrat ed in Vicia/aba chloroplasts. They were absent in chloroplasts isolated in no sucrose, 0.3 M sucrose or in situ materiM either fixed in the absence of sucrose or fixed in the presence of 0.3 M sucrose. They were present in chloroplasts isolated in 0.5 M sucrose and in situ chloroplasts fixed in the presence of 0.7 1VIsucrose. Introduction
GU~NIHG (1965) d e m o n s t r a t e d the presence of aggregated fibrils, each 85 A in diameter, in the stroma of Avena sativa chloroplasts and called t h e m stroma centers. He a t t r i b u t e d their discovery to better fixation with glutarMdehyde and apparently considered the stroma center to be a normM constituent of plastids of oats. T~oMsoH et M., (1965), working with Phaseolus vulgaris, have shown t h a t organized arrays of granules measuring 85 A in diameter m a y result when plants are exposed to peroxyacetyl nitrate, a photochemically produced air pollutant. Comparison of the published micrographs leads one to suspect t h a t the two papers are describing the same phenomenon. However, THOMSON et al., (1965) reported t h a t the granules were not present in material kept in the dark, while GUNninG (1965) showed a plastid of an etiolated plant which had a stroma center. The work on crystals in plastids reported b y P n g s E g (1962, 1963) is of special interest because their presence was controlled b y conditions of isolation. H e reported t h a t the stroma of isolated spinach plastids had a crystalline structure with spacings of less t h a n 100 A. These crystals were absent in chloroplasts of intact tissue and chloroplasts isolated in isotonic medium, lie concluded t h a t t h e y resulted from osmotic dehydration during isolation in hypertonic medium. The present paper substantiates the observations of P]~R~E~ t h a t the appearance of crystals in plastid stroma is related to the isolation m e d i u m used. I t also demonstrates t h a t chloroplasts of intact tissue will contain crystals after t r e a t m e n t with hypertonic solutions. * Present address: Department of Botany, Washington State University, Pullman, Washington.
Crystalline Structures in Vicia ]aba Chloroplasts
183
Materials and Methods The chloroplasts studied were those in mature (fully expanded) leaves of greenhouse-grown Vicia/aba L. plants. For preparation of isolated chloroplasts, the leaves were ground for 30 sec in a Waring blender running on 65 volts. Grinding was in either 0.3 M or 0.5 M sucrose, buffered at psi 7.4 with 0.1 M potassium phosphate. The brei was filtered through four layers of cheese cloth and centrifuged at 270 • g for 1 min. The s u p e r n a t a n t was centrifuged a t 480 • g for 7 rain a n d the pelleted plastids were washed once with buffered sucrose. Fixation of the plastids was carried out in 6 % glutaraldehyde in 0.1 M phosphate buffer pSI 7.1. F o r fixation of chloroplasts in situ, small pieces a b o u t 1--2 m m square of Vicia/aba leaves were fixed in 6% glutaraldehyde in 0.1 M phosphate buffer, p t I 7.1, in solutions containing no sucrose, 0.3 M sucrose or 0.7 M sucrose. Both leaf pieces and isolated plastids were washed in buffer, and postfixed with 1% buffered osmium. Dehydration was in a graded series of alcohols a n d propylene oxide, followed b y embedding in araldite. Thin sections were stained with aqueous solutions of 2 % barium permanganate and examined in a n Hitachi SIU- 11 electron microscope.
Results C h l o r o p l a s t s of m a t u r e a n d y o u n g Vicia /aba l e a v e s w h e n i s o l a t e d i n 0.5 M s u c r o s e c o n t a i n c r y s t a l l i n e b o d i e s i n t h e s t r o m ~ (Fig. 1). T h e s a m e is t r u e for i n s i t n p l a s t i d s w h e n f i x e d i n t h e p r e s e n c e of 0.7 M
sucrose (Fig. 2). However,
the crystals were absent in plastids isolated
Fig. 1. Crystal bodies in a chloroplast of Vicia /aba isolated in 0.5 M sucrose. • 27,000. Here a n d in the following micrographs, the marker line is equivalent to 0.5 ~z
184
L. K. StIUNwAY, T. E. WEIEt{ and C. 1%. STOOXING:
Fig. 2. Crystal bodies in an in situ chloroplast of Vicia /aba treated with 0.7 M sucrose. • 87,000
Crystalline Structures in Vicia ]aba Chloroplasts
185
Fig. 3. Crystal body of an isolated chloroplast of Vicia ]aba showing one directional periodicity. • 144,000
186
L. K. S•cMwAY, T. E. WEIER and C. R. STOCKING:
Fig. 4. Crystal body of an isolated chloroplast of Vicia ]aba showing two directional periodicity. • 228,000
Crystalline Structures in Vicia/aba Chloroplasts
187
?ig. 5. Crystal body of an isolated chlorplast of Viola ]aba showing gradual blending of stroma and crystal body. X 156,000
188
L. K. S~uMwAu T. E. W~IER and C. R. STocJ~I~G:
in 0.3 M sucrose and in in-situ plastids either fixed in the absence of sucrose or fixed in the presence of 0.3 M sucrose. These crystalline bodies m a y show only a one directional periodicity (Fig. 3) or they m a y have two directional periodicities which intersect with an angle of 70--800 (Fig. 4). Some crystals end rather abruptly (Figs. 1 and 2) while others blend with the stroma without a sharp boundary (Fig. 5). Plastids fixed only
Fig. 6. Crystal bodies of an isolated chloroplast of Vicia /aba fixed only in glutaraldehyde. • 73,000 in glutaraldehyde show t h a t the presence of the crystals is not dependent on the use of osmium as a fixative (Fig. 6). Temperatures of dehydration of 20 ~ 4 ~ --200 and --700 had no effect on these crystals. Center to center spacing of the electron dense lines is 105--115 A for both directions. Discussion
The presence of crystals in the stroma of chloroplasts has been ascribed to better fixation (GuNnING, 1965), to plastid damage due to air pollutants (TltoNso~ et al., 1965), or to plastid isolation procedures ( P E ~ N ~ , 1962, 1963). In some instances (MASTO~, 1966), they appear to be actual storage products. Pn~Nn~ (1962, 1963) observed t h a t hypertonie solutions m a y induce crystalline body formation in chloroplasts. His findings are substantiated and extended here. These observations point to the importance of tonicity in the fixation and isolation media. The recognition of the possibility of such induced structural changes in the ultrastructure of organelles, and the proper evaluation of these changes should enhance our knowledge of the function and molecular organization of cell organelles.
Crystalline Structures in Vicia/aba Chloroplasts
189
References Gu~I~G, B. E. S. : The fine structure of chloroplast stroma following aldehyde osmium-tetroxide fixation. J. Cell Biol. 24, 79--93 (1965). MA~TO~, I.: Some possibly significant structural relations between chloroplasts and other cell components. In: Biochemistry o5 chloroplasts (T. W. GooDwIN, ed.), vol. 1, p. 23--47. New York: Academic Press 1966. PERNE~, E. : Elektronenmikroskopisehe Befunde fiber Kristallgitterstrukturen im Stroma isolierter Spinatehloroplasten. Port. Acta biol. A 6, 359--372 (1962). Kristallisationserscheinungen im Stroma isolierter Spinatehloroplasten guter Erhaltung. Naturwissenschaften 50, 134--135 (1963). THOMSOn,W. W., W. M. DUGGER,jr., and R. L. PAL~ER: Effects of peroxyacetyl nitrate on ultrastructure of chloroplasts. Bot. Gaz. Chicago 126, 66--72 (1965). -
-
L. K. S~U~WAY Botany Department, Washington State University Pullman, Washington 99163, USA
13 Planta(Berl.), Bd. 76
