Peter L. Steponkus

Cold Acclimation of Arabidopsis thaliana (Effect on Plasma Membrane Lipid Composition and Freeze-Induced Lesions)

Maximum freezing tolerance of Arabidopsis thaliana L. Heyn (Columbia) was attained after 1 week of cold acclimation at 2[deg]C. During this time, there were significant changes in both the lipid composition of the plasma membrane and the freeze-induced lesions that were associated with injury. The proportion of phospholipids increased from 46.8 to 57.1 mol% of the total lipids with little change in the proportions of the phospholipid classes. Although the proportion of di-unsaturated species of phosphatidylcholine and phosphatidylethanolamine increased, mono-unsaturated species were still the preponderant species. The proportion of cerebrosides decreased from 7.3 to 4.3 mol% with only small changes in the proportions of the various molecular species. The proportion of free sterols decreased from 37.7 to 31.2 mol%, but there were only small changes in the proportions of sterylglucosides and acylated sterylglucosides. Freezing tolerance of protoplasts isolated from either nonacclimated or cold-acclimated leaves was similar to that of leaves from which the protoplasts were isolated (-3.5[deg]C for nonacclimated leaves; -10[deg]C for cold-acclimated leaves). In protoplasts isolated from nonacclimated leaves, the incidence of expansion-induced lysis was [less than or equal to]10% at any subzero temperature. Instead, freezing injury was associated with formation of the hexagonal II phase in the plasma membrane and subtending lamellae. In protoplasts isolated from cold-acclimated leaves, neither expansion-induced lysis nor freeze-induced formation of the hexagonal II phase occurred. Instead, injury was associated with the “fracture-jump lesion,” which is manifested as localized deviations of the plasma membrane fracture plane to subtending lamellae. The relationship between the freeze-induced lesions and alterations in the lipid composition of the plasma membrane during cold acclimation is discussed.

A contrast of the plasma membrane lipid composition of oat and rye leaves in relation to freezing tolerance

The lipid composition of the plasma membrane isolated from leaves of spring oat (Avena sativa L. cv Ogle) was vastly different from that of winter rye (Secale cereale L. cv Puma). The plasma membrane of spring oat contained large proportions of phospholipids (28.8 mol% of the total lipids), cerebrosides (27.2 mol%), and acylated sterylglucosides (27.3 mol%) with lesser proportions of free sterols (8.4 mol%) and sterylglucosides (5.6 mol%). In contrast, the plasma membrane of winter rye contained a greater proportion of phospholipids (36.6 mol%), and there was a lower proportion of cerebrosides (16.4 mol%); free sterols (38.1 mol%) were the predominant sterols, with lesser proportions of sterylglucosides (5.6 mol%) and acylated sterylglucosides (2.9 mol%). Although the relative proportions of individual phospholipids, primarily phosphatidylcholine and phosphatidylethanolamine, and the molecular species of these two phospholipids were similar in oat and rye, the relative proportions of di-unsaturated species of these two phospholipids were substantially lower in oat than in rye. The relative proportions of sterol species in oat were different from those in rye; the molecular species of cerebrosides were similar in oat and rye, with only slight differences in the proportions of the individual species. After 4 weeks of cold acclimation, the proportion of phospholipids increased significantly in both oat (from 28.8 to 36.8 mol%) and rye (from 36.6 to 43.3 mol%) as a result of increases in the proportions of phosphatidylcholine and phosphatidylethanolamine. For both oat and rye, the relative proportions of di-unsaturated species increased after cold acclimation, but the increase was greater in rye than in oat. In both oat and rye, this increase occurred largely during the first week of cold acclimation. During the 4 weeks of cold acclimation, there was a progressive decrease in the proportion of cerebrosides in the plasma membrane of rye (from 16.4 to 10.5 mol%), but there was only a small decrease in oat (from 27.2 to 24.2 mol%). In both oat and rye, there were only small changes in the proportions of free sterols and sterol derivatives during cold acclimation. Consequently, the proportions of both acylated sterylglucosides and cerebrosides remained substantially higher in oat than in rye after cold acclimation. The relationship between these differences in the plasma membrane lipid composition of oat and rye and their freezing tolerance is presented.

Freeze/thaw-induced destabilization of the plasma membrane and the effects of cold acclimation.

Disruption of the plasma membrane is a primary cause of freezing injury. In this review, the mechanisms of injury resulting from freeze-induced cell dehydration are presented, including destabilization of the plasma membrane resulting from (a) freeze/thaw-induced osmotic excursions and (b) lyotropic phase transitions in the plasma membrane lipids. Cold acclimation dramatically alters the behavior of the plasma membrane during a freeze/thaw cycle—increasing the tolerance to osmotic excursions and decreasing the propensity for dehydration-induced lamellar to hexagonal-II phase transitions. Evidence for a casual relationship between the increased cryostability of the plasma membrane and alterations in the lipid composition is reviewed.

A Comparison of Freezing Injury in Oat and Rye: Two Cereals at the Extremes of Freezing Tolerance

A detailed analysis of cold acclimation of a winter rye (Secale cereale L. cv Puma), a winter oat (Avena sativa L. cv Kanota), and a spring oat cultivar (Ogle) revealed that freezing injury of leaves of nonacclimated seedlings occurred at -2[deg]C in both the winter and spring cultivars of oat but did not occur in winter rye leaves until after freezing at -4[deg]C. The maximum freezing tolerance was attained in all cultivars after 4 weeks of cold acclimation, and the temperature at which 50% electrolyte leakage occurred decreased to -8[deg]C for spring oat, -10[deg]C for winter oat, and -21[deg]C for winter rye. In protoplasts isolated from leaves of nonacclimated spring oat, expansion-induced lysis was the predominant form of injury over the range of -2 to -4[deg]C. At temperatures lower than -4[deg]C, loss of osmotic responsiveness, which was associated with the formation of the hexagonal II phase in the plasma membrane and subtending lamellae, was the predominant form of injury. In protoplasts isolated from leaves of cold-acclimated oat, loss of osmotic responsiveness was the predominant form of injury at all injurious temperatures; however, the hexagonal II phase was not observed. Rather, injury was associated with the occurrence of localized deviations of the plasma membrane fracture plane to closely appressed lamellae, which we refer to as the “fracture-jump lesion.” Although the freeze-induced lesions in the plasma membrane of protoplasts of spring oat were identical with those reported previously for protoplasts of winter rye, they occurred at significantly higher temperatures that correspond to the lethal freezing temperature.

Destabilization of the plasma membrane of isolated plant protoplasts during a freeze-thaw cycle: the influence of cold acclimation

In conclusion, isolated protoplasts are an excellent arena in which destabilization of the plasma membrane can be directly observed during a freeze-thaw cycle by cryomicroscopy. Destabilization is manifested in various ways--intracellular ice formation, loss of osmotic responsiveness, or expansion-induced lysis. The incidence of any particular form of injury will depend on the freeze-thaw protocol and hardiness of the tissue from which the protoplasts were isolated. In all cases, however, cold acclimation directly increases the stability of the plasma membrane to the multiple stresses that arise during a freeze-thaw cycle. Such observations provide for functional differences in the plasma membrane that may now be used to consider the significance of any compositional changes in the membrane that might be determined.

Freezing Sensitivity in the sfr4 Mutant of Arabidopsis Is Due to Low Sugar Content and Is Manifested by Loss of Osmotic Responsiveness

Protoplasts were tested to determine whether the freezing sensitivity of the sfr4 (sensitive to freezing) mutant of Arabidopsis was due to the mutants deficiency in soluble sugars after cold acclimation. When grown under nonacclimated conditions,sfr4 protoplasts possessed freezing tolerance similar to that of wild type, with the temperature at which 50% of protoplasts are injured (LT50) of −4.5°C. In both wild-type andsfr4 protoplasts, expansion-induced lysis was the predominant lesion between −2°C and −4°C, but its incidence was low (approximately 10%); below −5°C, loss of osmotic responsiveness (LOR) was the predominant lesion. After cold acclimation, the LT50 was decreased to only −5.6°C forsfr4 protoplasts, compared with −9.1°C for wild-type protoplasts. Although expansion-induced lysis was precluded in both types of protoplasts, the sfr4 protoplasts remained susceptible to LOR. After incubation of seedlings in Suc solution in the dark at 2°C, freezing tolerance and the incidence of freeze-induced lesions in sfr4 protoplasts were examined. The freezing tolerance of isolated protoplasts (LT50 of −9°C) and the incidence of LOR were now similar for wild type and sfr4. These results indicate that the freezing sensitivity of cold-acclimated sfr4 is due to its continued susceptibility to LOR (associated with lyotropic formation of the hexagonal II phase) and associated with the low sugar content of its cells.

Cold Hardiness and Freezing Injury of Agronomic Crops

Publisher Summary Winter hardiness of cereal grains and perennial forage crops is of considerable concern to agronomists in cold northern temperate regions of the world. Of all the factors relating to the winter hardiness complex, cold hardiness, the ability to withstand low freezing temperatures, is of paramount importance. The ability of plants to withstand low temperatures is a latent trait, which exhibits an annual periodicity. It is only through the interaction of appropriate environmental cues and the genetic potential of a species that an increase in cold hardiness is manifested. Therefore, of prime concern are the environmental factors that affect the annual process of cold acclimation and the annual process of deacclimation. Thus, to improve the winter hardiness of agronomic crops, the problem of cold hardiness must be addressed. Fundamental to such an endeavor is the need to characterize the freezing stresses that are imposed on the plant tissue, to understand the repercussions of these stresses on the cellular environment and architecture, and to determine what constitutes freezing injury at the cellular and molecular level. Only when such information is available can the physiological and biochemical aspects of cold acclimation be fully elucidated.

Mechanical study of the deformation and rupture of the plasma membranes of protoplasts during osmotic expansions

SummaryThe stress and strain (surface tension and fractional change in area) in the plasma membrane of protoplasts isolated from rye leaves (Secale cereale L. cv Puma) were measured during osmotic expansions from isotonic into a range of more dilute solutions. The membrane surface tension increases rapidly to a maximum and then decreases slowly with some protoplasts lysing in all phases of the expansion. The maximum surface tension is greater for rapid expansions, and protoplasts lyse earlier during rapid expansion. Over the range of expansion rates investigated, the area at which lysis occurs is not strongly dependent on expansion rate. The value of the maximum tension is determined by the expansion rate and the rate at which new material is incorporated into the membrane. During osmotic expansion, protoplasts isolated from cold-acclimated plants incorporate material faster than do those from nonacclimated plants and thus incur lower membrane tensions.

Cold Acclimation in Plants:Relationship Between the Lipid Composition and the Cryostability of the Plasma Membrane

Cold acclimation of plants requires an orchestration of many different, seemingly disparate processes. However, many of these processes that occur during cold acclimation ultimately contribute to the increased stability of cellular membranes during freeze-induced dehydration-the destabilization of which is the primary cause of the freezing injury. Among all cellular membranes, the plasma membrane is of primary importance to maintain its structural integrity because of the central role it plays during a freeze/ thaw cycle. We will describe here that there is a close association between the alterations of the plasma membrane lipid composition and the difference in the incidence of freeze-induced membrane lesions during cold acclimation. The stability of the plasma membrane during freezeinduced dehydration is also affected by factors associated with the endomembranes (the chloroplast envelope lipid composition) and the cytoplasm (the accumulation of sugars and the cold-regulated gene expression). Collectively, these results indicate that the structural integrity of the plasma membrane during freeze-induced dehydration is maintained by a complex but well-coordinated manner.

Dehydration-induced lamellar-to-hexagonal-II phase transitions in DOPE/DOPC mixtures

Plasma membranes of protoplasts isolated from non-acclimated rye plants undergo a transition from the bilayer to the inverted hexagonal (HII) phase during freeze-induced dehydration at -10 degrees C. It has been suggested (Bryant, G. and Wolfe, J. (1989) Eur. Biophys. J. 16, 369-372) that the differential hydration of various membrane components may induce fluid-fluid demixing of highly hydrated (e.g., PC) from poorly hydrated (PE) components during dehydration. This could yield a PE-enriched domain more prone to form the HII phase. We have examined the lyotropic phase behavior of mixtures of DOPE and DOPC at 20 degrees C by freeze-fracture electron microscopy, differential scanning calorimetry, and X-ray diffraction. HII phase formation was favored by higher proportions of DOPE and lower water contents. Mixtures of 1:1 and 1:3 DOPE/DOPC had a hydration-dependent appearance of two L alpha phases at water contents just above those at which the HII phase occurred. The hydration-dependence of the lamellar repeat spacings suggested that the DOPE-enriched domains preferentially underwent the L alpha-to-HII phase transition. Mixtures of 3:1 DOPE/DOPC did not separate into two L alpha phases during dehydration. These data suggest that the differential hydration characteristics of various membrane components may induce their lateral fluid-fluid demixing during dehydration.