- Email: [email protected]
Freezing of spinach protoplasts
ABSTRACTS,
670
13TH
thermore, inactivation in samples frozen at similar rates to temperatures between -4 and -12°C was greater than inactivation in samples frozen to -18, -29, or -40°C with low initial NaCl concentrations. Increasing the initial chloropIast concentration decreased the extent of inactivation at any given time or initial NaCl concentration up to 80 mM.
ANNUAL
MEETING
complete killing. Freely suspended plant protoplasts thus afford a method for studying the freezing and dehydrative injury and resistance in plant cells free from the complications due to the presence of a cell wall. Wl.
PLANT
HARDINESS
WORKSHOP
J. LEVITT (St. Paul, Minn.) AND J. ANDERSON(Tucson, Ariz. ). Discussants: D. KETCHIE ( Wenatchee, Wash. ), J. M. LYONS (Davis, Calif.), D. Sr~moVITCH (Ottawa, Canada), P. STEPONWS ( Ithaca, N.Y. ), R. WILLIAMS (Washington, Chairmen:
Protoplasts. STEVEN C. WIEST" AND PETER L. STEPONKUS. (Department of Floriculture and Ornamental Horticulture, Cornell University, Ithaca, New York 14853).
76. Freezing
of
Spinach
Plant protoplasts are becoming increasingly popular tools for use in a wide diversity of fields in plant physioBgy and biochemistry. In the future there will probably be an interest in freezepreservation of these modified cells, just as there presently is for calluses and suspension cultures of plant cells. In addition, freezing studies using protoplasts may enable the determination of the primary site of freezing injury in plant cells. This is believed by many to be the plasma membrane but has never been directly proven, and the exact molecular site(s) of injury has only rarely been postulated. Elimination of cell walls provides direct access of probes in solution to plasma membranes, which may be useful for such studies. A limited fraction of spinach protoplasts slowly frozen (2”C/hr) to -8°C and thawed (lO”C/hr) in the presence of 0.6 M sorbitol remained intact. Increased proportions of intact protoplasts were observed when frozen in the presence of sucrose and dimethyl sulfoxide (in addition to the 0.6 M osmoticum ). These results demonstrate that studies of freezing injury, and perhaps freeze-preservation, using plant protoplasts are feasible.
of Winter 77. Freezing and Osmotic Dehydration Rye Cell Protopkzsts. D. SIMINOVITCH, J. SINCH,* W. A. KELLER,' AND I. A. DE LA ROCHE.” (Canada Department of Agriculture, Ottawa, Canada). Free protoplasts isolated from hardened and unhardened winter rye seedlings when subjected to freezing and strong osmotic solutions exhibit the same properties as their normal whole cell counterparts except that, due to the absence of cell walls, contraction deformation of the protoplast occurs uniformly over the entire volume of the cell. The behaviour of the protoplasts under these circumstances is similar to pure lipid liposomes and suspended animal egg celIs such as sea urchin eggs subjected to extracellular freezing or osmotic dehydration. Further, as in normal cells, rapid freezing induces intracellular freezing with
D.C.), G. N. BROWN (Columbia, MO.), R. OLIEN (E. Lansing, Mich. ), B. McDANIELS (Tucson, Ariz. ), G. A. JUNG (University Park, Pa.), AND B. ZIMERER Four topics were chosen for major discussion: membrane lipids, proteins and their enzyme activities, freezing and supercooling, and the effects of pretreatments on the avoidance of freezing injury. Ketchie found there was no conversion of monogalactosyl diglycerides but there was an increase in the digalactosyl form during the hardening of alfalfa, wheat, sugar beets, and apples. There was an increase in chain hngth and a slight increase in unsaturation of lipids. Lyons described chemical treatments in an attempt to lower the phase change temperature of tomato seedlings and to produce chilling resistance. Some success was obtained with ethanolamine. Tween oleates were also tested. Although there appeared to be no change in total lipid, some shift occurred in unsaturation and some change in phospholipid compostion was noted. Siminovitch was unable to detect any relation between hardiness and either the degree of lipid unsaturation or the phase change temperature as determined by D.S.C. in black locust, wheat, or rye. The phase change occurred slightly below the freezing point, when measured by glycerol. There was an increase in unsaturation during hardening of wheat and rye and a small shift in the phase change temperature. An increase in membranous material (phospholipids) occurred, though less striking in wheat and rye than in trees. This increase could be associated with folding of the membrane in locust tree cells. Steponkus discussed in more detail some of his group’s recent work with chloroplast thylakoids. In addition, he raised the question of whether the membrane permeability is affected by the phase change that occurs during freezing. Williams explained some of his investigations of the responses of lipid films to compression and the possible changes in membrane lipid due to pressure. In addition, he discussed light melting points with reference to their relation to water.
670
13TH
thermore, inactivation in samples frozen at similar rates to temperatures between -4 and -12°C was greater than inactivation in samples frozen to -18, -29, or -40°C with low initial NaCl concentrations. Increasing the initial chloropIast concentration decreased the extent of inactivation at any given time or initial NaCl concentration up to 80 mM.
ANNUAL
MEETING
complete killing. Freely suspended plant protoplasts thus afford a method for studying the freezing and dehydrative injury and resistance in plant cells free from the complications due to the presence of a cell wall. Wl.
PLANT
HARDINESS
WORKSHOP
J. LEVITT (St. Paul, Minn.) AND J. ANDERSON(Tucson, Ariz. ). Discussants: D. KETCHIE ( Wenatchee, Wash. ), J. M. LYONS (Davis, Calif.), D. Sr~moVITCH (Ottawa, Canada), P. STEPONWS ( Ithaca, N.Y. ), R. WILLIAMS (Washington, Chairmen:
Protoplasts. STEVEN C. WIEST" AND PETER L. STEPONKUS. (Department of Floriculture and Ornamental Horticulture, Cornell University, Ithaca, New York 14853).
76. Freezing
of
Spinach
Plant protoplasts are becoming increasingly popular tools for use in a wide diversity of fields in plant physioBgy and biochemistry. In the future there will probably be an interest in freezepreservation of these modified cells, just as there presently is for calluses and suspension cultures of plant cells. In addition, freezing studies using protoplasts may enable the determination of the primary site of freezing injury in plant cells. This is believed by many to be the plasma membrane but has never been directly proven, and the exact molecular site(s) of injury has only rarely been postulated. Elimination of cell walls provides direct access of probes in solution to plasma membranes, which may be useful for such studies. A limited fraction of spinach protoplasts slowly frozen (2”C/hr) to -8°C and thawed (lO”C/hr) in the presence of 0.6 M sorbitol remained intact. Increased proportions of intact protoplasts were observed when frozen in the presence of sucrose and dimethyl sulfoxide (in addition to the 0.6 M osmoticum ). These results demonstrate that studies of freezing injury, and perhaps freeze-preservation, using plant protoplasts are feasible.
of Winter 77. Freezing and Osmotic Dehydration Rye Cell Protopkzsts. D. SIMINOVITCH, J. SINCH,* W. A. KELLER,' AND I. A. DE LA ROCHE.” (Canada Department of Agriculture, Ottawa, Canada). Free protoplasts isolated from hardened and unhardened winter rye seedlings when subjected to freezing and strong osmotic solutions exhibit the same properties as their normal whole cell counterparts except that, due to the absence of cell walls, contraction deformation of the protoplast occurs uniformly over the entire volume of the cell. The behaviour of the protoplasts under these circumstances is similar to pure lipid liposomes and suspended animal egg celIs such as sea urchin eggs subjected to extracellular freezing or osmotic dehydration. Further, as in normal cells, rapid freezing induces intracellular freezing with
D.C.), G. N. BROWN (Columbia, MO.), R. OLIEN (E. Lansing, Mich. ), B. McDANIELS (Tucson, Ariz. ), G. A. JUNG (University Park, Pa.), AND B. ZIMERER Four topics were chosen for major discussion: membrane lipids, proteins and their enzyme activities, freezing and supercooling, and the effects of pretreatments on the avoidance of freezing injury. Ketchie found there was no conversion of monogalactosyl diglycerides but there was an increase in the digalactosyl form during the hardening of alfalfa, wheat, sugar beets, and apples. There was an increase in chain hngth and a slight increase in unsaturation of lipids. Lyons described chemical treatments in an attempt to lower the phase change temperature of tomato seedlings and to produce chilling resistance. Some success was obtained with ethanolamine. Tween oleates were also tested. Although there appeared to be no change in total lipid, some shift occurred in unsaturation and some change in phospholipid compostion was noted. Siminovitch was unable to detect any relation between hardiness and either the degree of lipid unsaturation or the phase change temperature as determined by D.S.C. in black locust, wheat, or rye. The phase change occurred slightly below the freezing point, when measured by glycerol. There was an increase in unsaturation during hardening of wheat and rye and a small shift in the phase change temperature. An increase in membranous material (phospholipids) occurred, though less striking in wheat and rye than in trees. This increase could be associated with folding of the membrane in locust tree cells. Steponkus discussed in more detail some of his group’s recent work with chloroplast thylakoids. In addition, he raised the question of whether the membrane permeability is affected by the phase change that occurs during freezing. Williams explained some of his investigations of the responses of lipid films to compression and the possible changes in membrane lipid due to pressure. In addition, he discussed light melting points with reference to their relation to water.