Phospholipid, protein, and nucleic acid increases in protoplasm and membrane structures associated with development of extreme freezing resistance in black locust tree cells

ABSTRACTrThe lipid and protein composition of the plasma membrane isolated from mulberry (Morus bombycis Koidz.) bark cells was analyzed throughout the cold acclimation period under natural and controlled environment conditions. There was a significant increase in phospholipids and unsaturation of their fatty acids during cold acclimation. The ratio of sterols to phospholipids decreased with hardiness, primarily due to the large increase in phospholipids. The fluidity of the plasma membrane, as determined by fluorescent polarization technique, increased with hardiness. Electrophoresis of plasma membrane proteins including glycoproteins revealed change in banding pattern during the early fall to winter period. Some of the protein changes could be related to growth cessation and defoliation. However, minor changes in proteins also occurred during the most active period of hardening. Changes in glycoproteins were coincident both with changes in growth stages and with the development of cold hardiness.Following a lethal frost, there is often a loss in the semipermeability properties of the plasma membrane in plants. This had led to the belief that the plasma membrane is the primary site of freezing injury (9,12,18 (24) and an increased level of phospholipids during hardening (6,16,17,23,26, 29). This has led to the speculation that the enhanced fluidity of the membrane by lipid unsaturation is related to cold hardiness. However, no preferential increase in phospholipid unsaturation could be detected in black locust (17) and poplar (29) which are both extreme cold hardy species. These findings have led to the conclusion that membrane unsaturation or fluidity is not related to the development of cold hardiness (6,14,17).In many of the studies relating lipid unsaturation to hardiness, total lipids were investigated rather than the plasma membrane which is considered to be the primary site of injury. This has been primarily due to the lack of methods to isolate sufficient quantities of pure plasma membrane from plant tissues. Recently, we have developed a method of isolating plasma membrane from plant tissues utilizing an aqueous PEG-dextran two- polymer phase system containing NaCI (20, 30). In this report, we followed the changes in lipids, proteins, and fluidity in plasma membranes of mulberry trees during cold acclimation. MATERIALS AND METHODSPlant Materials. Living bark tissues from current twigs of mulberry trees (Morus bombycis Koidz. vc Goroji) were used in the present study. Frost hardiness was evaluated by measuring the electrolyte leakage after freeze thawing of the tissues. Living bark tissues (500 mg) were cut into pieces (1.0 x 0.5 cm) and frozen in test tubes (1.5 x 15 cm) at -3°C for 2 h with small pieces ofice. Thereafter the tissues were cooled by 5°C increments at 1-h intervals. After holding at the desired temperature for 16 h, the frozen tissue pieces were then thawed at 0°C. Upon thawing, the tissues were immersed in 5 ml of distilled H20 and incubated at 25°C for 4 h with a gentle shaking and sub...

Section: Abstractrmentioning

confidence: 99%

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confidence: 99%

Chemical and Biophysical Changes in the Plasma Membrane during Cold Acclimation of Mulberry Bark Cells (Morus bombycis Koidz. cv Goroji)

Yoshida¹

1984

“…Increased phospholipid synthesis does not seem to be a prerequisite to hardening in winter wheat. However, a high rate of phospholipid synthesis may be required to maintain frost resistance.An increase in phospholipids has generally been observed during hardening of plants (4,5,7,8,11,12, 14). It is not known whether this increase is a result of low temperature or whether it is part of the frost-hardening process.…”

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confidence: 99%

Stimulation of Phospholipid Biosynthesis during Frost Hardening of Winter Wheat

Willemot¹

1975

Lipids were labeled with 3P during frost hardening of two varieties of winter wheat (Triticumn aestivum), hardy Kharkov and much less hardy Champlein. The main labeled compounds were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylglycerol. With time of incorporation the proportion of the radioactivity incorporated into the lipids increased in phosphatidylcholine, especially in Kharkov and at 1 C. During hardening, phospholipid synthesis was greatly stimulated in Kharkov, but much less in Champlein. The proportion of the phospholipids synthesized changed only little with hardening, with a trend towards an increase in phosphatidylcholine. Increased phospholipid synthesis does not seem to be a prerequisite to hardening in winter wheat. However, a high rate of phospholipid synthesis may be required to maintain frost resistance.An increase in phospholipids has generally been observed during hardening of plants (4,5,7,8,11,12, 14). It is not known whether this increase is a result of low temperature or whether it is part of the frost-hardening process. In an attempt to dissociate the two effects, phospholipid biosynthesis was measured at two temperatures during controlled hardening of a hardy and a less hardy variety of winter wheat. MATERIALS AND METHODSGrowth and Hardening of Plants. Two varieties of winter wheat (Triticuin aestivum), hardy Kharkov, and less hardy Champlein, were grown in 10-cm pots containing a 1: 1 mixture of sand and vermiculite in a growth cabinet (Controlled Environment, Winnipeg) at a 20 C day and 15 C night temperature, a 16-hr photoperiod, a relative humidity of 60%, and a light intensity of 18,000 lux. The plants were watered with Hoagland No. 1 solution (9). After 12 days they were transferred to a hardening cabinet at a constant temperature of 1 C, an 8-hr photoperiod, and a light intensity of 8,000 lux. Before and during hardening the frost resistance of the plants was tested by controlled freezing (7,13) in the pots at several temperatures. The temperature at which 50% of the plants were killed (killing temperature) was determined after 3 weeks of recovery.'Contribution No. 50 from the Canada Department of Agriculture Research Station, Ste-Foy.Labeling with "'P. The roots of triplicate samples of two plants were covered with 2 ml of a radioactive solution containing 0.05 M citrate buffer pH 5 and 3 to 8 /,Ci of 'P (H333PO4, 50 mCi/mmole, New England Nuclear) in 10-ml beakers for varying times at either 20 C or 1 C under continuous light at an intensity of 8,000 lux. During hardening experiments, feeding times were either 24 hr at 1 C or 12 hr at 20 C. At the end of the incorporation, the roots and crowns were rinsed, boiled for 3 min, covered with 6 ml of chloro-

“…1) on seedlings placed into the mist chamber at completion of the regime. The maximum degree of hardiness achieved for any one seedling was -20 C. Black locust in a natural environment has been observed to develop freezing resistance to at least -70 C (30).…”

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confidence: 99%

Ribosomal Changes during Induction of Cold Hardiness in Black Locust Seedlings

Bixby

Brown²

1975

Protein synthesis has been implicated in the cold-hardening process. Ribosomes from cold hardy and nonhardy black locust (Robinia pseudoacacia L.) seedlings were compared to determine if cold acclimation is related to alteration of ribosomal structure. Ribosomal structure, as indicated by thermal melting profiles, appears to be altered during induction of hardiness. Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins indicates at least 17 proteins from hardy seedlings that are different from those of nonhardy seedlings. These different proteins may be partially responsible for the different thermal melting profiles observed.Plants capable of developing freezing resistance demonstrate altered metabolism during low temperature acclimation (cold hardening) (23). There have been many suggestions that protein synthesis plays a role in cold hardening (1, 4, 9, 13, 23). Among the many changes found during induction of cold hardiness are those which occur in RNA (13, 26) and protein (3, 4,9,13,26) metabolism. This study was undertaken to examine the ribosome as related to induction of cold hardiness. The objective was to determine if ribosomal structure is altered during induction of hardiness. MATERLALS AND METHODSPlant Material. Black locust seedlings (Robinia pseudoacacia L.) were grown in heat-sterilized vermiculite. Moisture was maintained relatively constant. Two-month seedlings were cold hardened as outlined in Table I. At specific points in the coldhardening regime, seedlings were placed into a programmed freezer and frozen at a rate of 6 C/hr. Samples were removed every 4 C starting at -4 and allowed to thaw overnight at 5 C. Stems were then harvested, placed into a mist chamber with a 15-hr photoperiod, allowed to recover for 7 days and visually evaluated for survival. Seedlings were considered uninjured if their bark remained green and tightly bound to the stem. The bark of injured seedlings became brown and loose. Stems were assigned a value of 1 if uninjured, 0.5 if injured with 50% or more of the stem alive and 0 if dead or more than 50% injured. Values were totaled and divided by the total number of stems to determine percent survival (31).