Immunolocalization of Antifreeze Proteins in Winter Rye Leaves,Crowns, and Roots by Tissue Printing

During cold acclimation, antifreeze proteins (AFPs) that are similar to pathogenesis-related proteins accumulate in the apoplast of winter rye (Secale cereale L. cv Musketeer) leaves. AFPs have the ability to modify the growth of ice. To elucidate the role of AFPs in the freezing process, they were assayed and immunolocalized in winter rye leaves, crowns, and roots. Each of the total soluble protein extracts from cold-acclimated rye leaves, crowns, and roots exhibited antifreeze activity, whereas no antifreeze activity was observed in extracts from nonacclimated rye plants. Antibodies raised against three apoplastic rye AFPs, corresponding to a glucanase-like protein (GLP, 32 kD), a chitinase-like protein (CLP, 35 kD), and a thaumatin-like protein (TLP, 25 kD), were used in tissue printing to show that the AFPs are localized in the epidermis and in cells surrounding intercellular spaces in cold-acclimated plants. Although GLPs, CLPs, and TLPs were present in nonacclimated plants, they were found in different locations and did not exhibit antifreeze activity, which suggests that different isoforms of pathogenesis-related proteins are produced at low temperature. The location of rye AFPs may prevent secondary nucleation of cells by epiphytic ice or by ice propagating through the xylem. The distributions of pathogenesis-induced and cold-accumulated GLPs, CLPs, and TLPs are similar and may reflect the common pathways by which both pathogens and ice enter and propagate through plant tissues.     

Proteins accumulate in the apoplast of winter rye leaves during cold acclimation

During cold acclimation, winter rye ( Secale cereale L.) plants develop the ability to tolerate freezing temperatures by forming ice in intercellular spaces and xylem vessels. In this study, proteins were extracted from the apoplast of rye leaves to determine their role in controlling extracellular ice formation. Several polypeptides in the 15 to 32 kDa range accumulated in the leaf apoplast during cold acclimation at 5°C and decreased during deacclimation at 20°C. A second group of polypeptides (63, 65 and 68 kDa) appeared only when the leaves were maximally frost tolerant. Ice nucleation activity, as well as the previously reported antifreeze activity, was higher in apoplastic extracts from cold-acclimated than from nonacclimated rye leaves. These results indicate that apoplastic proteins exert a direct influence on the growth of ice. In addition, freezing injury was greater in extracted cold-acclimated leaves than in unextracted cold-acclimated leaves, which suggests that the proteins present in the apoplast are an important component of the mechanism by which winter rye leaves tolerate ice formation     

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.     

Immunogold localization of glucanase-like antifreeze protein in cold acclimated winter rye

Summary Apoplastic antifreeze proteins (AFPs) accumulate in winter rye (Secale cereale L. cv. Musketeer) leaves during cold acclimation. Two of the rye AFPs with molecular masses of 32 and 35 kDa are similar in their amino acid sequences and epitopes to -1, 3-endoglucanase. Localization of these AFPs, which we refer to as glucanase-like proteins (GLPs), was carried out with antiserum raised against the 32 kDa AFP. Specimens from leaves and roots of non-acclimated (NA) plants and cold acclimated (CA) plants were prepared by freeze-substitution for high resolution immunoelectron microscopy. In CA leaves, high levels of GLPs were observed in cell walls of mesophyll cells adjacent to intercellular spaces and in secondary thickenings of xylem vessels. Taken together with the absence of GLPs in vacuoles, these results confirm the apoplastic accumulation of AFPs in CA winter rye. Within the cells of CA leaves, GLPs were localized in cisternae of the rough endoplasmic reticulum, the Golgi apparatus and the plasma membrane, which indicates that GLPs are secreted via an exocytic bulk-flow pathway. The occurrence of high levels of GLPs in CA leaves, their low presence in NA leaves and the lack of GLPs in roots all suggest that there is a correlation between increased accumulation of GLPs and increased freezing tolerance of these plant materials. Furthermore, the localization of GLPs in the immediate vicinity of pathways for free water within the tissues supports the view that these proteins have an important role in the crystallization and/or recrystallization of water when the leaves of CA winter rye are exposed to freezing temperatures.Abbreviations AFP antifreeze protein - BSA bovine serum albumin - CA cold acclimated - GAR goat antirabbit antiserum conjugated with colloidal gold - GLP glucanase-like protein - NA non-acclimated - PBS phosphate buffered saline - PR pathogenesis related     

Freeze-Induced Membrane Ultrastructural Alterations in Rye (Secale cereale) Leaves

Freezing injury in protoplasts isolated from leaves of nonaccli-mated rye (Secale cereale cv Puma) is associated with the formation of the inverted hexagonal (HII) phase. However, in protoplasts from cold-acclimated rye, injury is associated with the occurrence of localized deviations in the fracture plane, a lesion referred to as the "fracture-jump lesion." To establish that these ultrastructural consequences of freezing are not unique to protoplasts, we have examined the manifestations of freezing injury in leaves of non-acclimated and cold-acclimated rye by freeze-fracture electron microscopy. At -10[deg]C, injury in nonacclimated leaves was manifested by the appearance of aparticulate domains in the plasma membrane, aparticulate lamellae subtending the plasma membrane, and by the frequent occurrence of the HII phase. The HII phase was not observed in leaves of cold-acclimated rye frozen to -35[deg]C. Rather, injury was associated with the occurrence of the fracture-jump lesion between the plasma membrane and closely appressed cytoplasmic membranes. Studies of the time dependence of HII phase formation in nonacclimated leaves indicated that freeze-induced dehydration requires longer times in leaves than in isolated protoplasts. These results demonstrate that the freeze-induced formation of the HII phase in nonacclimated rye and the fracture-jump lesion in cold-acclimated rye are not unique to protoplasts but also occur in the leaves from which the protoplasts are isolated.     

Antifreeze proteins are secreted by winter rye cells in suspension culture

During cold-acclimation, winter rye ( Secale cereale L) leaves secrete antifreeze proteins (AFPs) into the apoplast. The AFPs bind to ice and modify its growth, which is easily observed in vitro . However, it is not yet known whether in planta AFPs interact with ice or whether they exert cryoprotective effects. These experiments are difficult to conduct with intact plants, so the aim of this work was to determine whether AFPs are produced in response to cold temperature in cell culture and to examine their function by using suspension cells. We showed that suspension cells secreted three of the six known winter rye AFPs into the culture medium during acclimation at 4°C. These AFPs were not present in washed suspension cells, thus indicating that they are not firmly bound to the cell walls. In order to examine the function of extracellular AFPs, non-acclimated (NA) winter rye suspension cells and protoplasts isolated from NA winter rye leaves were then frozen and thawed in the presence of AFPs extracted from cold-acclimated winter rye leaves. The AFPs had no effect on the survival of NA protoplasts after freezing; however, they lowered the lethal temperature at which 50% of the cells are killed by freezing (LT₅₀) of NA suspension cells by 2.5°C. We conclude that low above-zero temperatures induce winter rye suspension cells to secrete AFPs free in solution where they can protect intact suspension cells, but not protoplasts, from freezing injury, presumably by interacting with extracellular ice.     

Antifreeze proteins in winter rye

Six antifreeze proteins, which have the unique ability to adsorb onto the surface of ice and inhibit its growth, have been isolated from the apoplast of winter rye leaves where ice forms at subzero temperatures. The rye antifreeze proteins accumulate during cold acclimation and are similar to plant pathogenesis-related proteins, including two endoglucanase-like, two chitinase-like and two thaumatin-like proteins. Immunolocalization of the glucanase-like antifreeze proteins showed that they accumulate in mesophyll cell walls facing intercellular spaces, in pectinaceous regions between adjoining mestome sheath cells, in the secondary cell walls of xylem vessels and in epidermal cell walls. Because the rye antifreeze proteins are located in areas where they could be in contact with ice, they may function as a barrier to the propagation of ice or to inhibit the recrystallization of ice. Antifreeze proteins similar to pathogenesis-related proteins were also found to accumulate in closely-related plants within the Triticum group but not in freezing-tolerant dicotyledonous plants. In winter wheat, the accumulation of antifreeze proteins and the development of freezing tolerance are regulated by chromosome 5. Rye antifreeze proteins may have evolved from pathogenesis-related proteins, but they retain their catalytic activities and may play a dual role in increasing both freezing and disease resistance in overwintering plants.     

Antifreeze proteins modify the freezing process in planta

During cold acclimation, winter rye (Secale cereale L. cv Musketeer) plants accumulate antifreeze proteins (AFPs) in the apoplast of leaves and crowns. The goal of this study was to determine whether these AFPs influence survival at subzero temperatures by modifying the freezing process or by acting as cryoprotectants. In order to inhibit the growth of ice, AFPs must be mobile so that they can bind to specific sites on the ice crystal lattice. Guttate obtained from cold-acclimated winter rye leaves exhibited antifreeze activity, indicating that the AFPs are free in solution. Infrared video thermography was used to observe freezing in winter rye leaves. In the absence of an ice nucleator, AFPs had no effect on the supercooling temperature of the leaves. However, in the presence of an ice nucleator, AFPs lowered the temperature at which the leaves froze by 0.3 degrees C to 1.2 degrees C. In vitro studies showed that apoplastic proteins extracted from cold-acclimated winter rye leaves inhibited the recrystallization of ice and also slowed the rate of migration of ice through solution-saturated filter paper. When we examined the possible role of winter rye AFPs in cryoprotection, we found that lactate dehydrogenase activity was higher after freezing in the presence of AFPs compared with buffer, but the same effect was obtained by adding bovine serum albumin. AFPs had no effect on unstacked thylakoid volume after freezing, but did inhibit stacking of the thylakoids, thus indicating a loss of thylakoid function. We conclude that rye AFPs have no specific cryoprotective activity; rather, they interact directly with ice in planta and reduce freezing injury by slowing the growth and recrystallization of ice.     

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.     

Antifreeze protein produced endogenously in winter rye leaves

After cold acclimation, winter rye (Secale cereale L.) is able to withstand the formation of extracellular ice at freezing temperatures. We now show, for the first time, that cold-acclimated winter rye plants contain endogenously produced antifreeze protein. The protein was extracted from the apoplast of winter rye leaves, where ice forms during freezing. After partial purification, the protein was identified as antifreeze protein because it modified the normal growth pattern of ice crystals and depressed the freezing temperature of water noncolligatively.     

Winter rye antifreeze activity increases in response to cold and drought, but not abscisic acid

Antifreeze activity increases in winter rye ( Secale cereale L.) during cold acclimation as the plants accumulate antifreeze proteins (AFPs) that are similar to glucanases, chitinases and thaumatin-like proteins (TLPs) in the leaf apoplast. In the present work, experiments were conducted to assess the role of drought and abscisic acid (ABA) in the regulation of antifreeze activity and accumulation of AFPs. Antifreeze activity was detected as early as 24 h of drought treatment at 20°C and increased as the level of apoplastic proteins increased. Apoplastic proteins accumulated rapidly under water stress and reached a level within 8 days that was equivalent to the level of apoplastic proteins accumulated when plants were acclimated to cold temperature for 7 weeks. These drought-induced apoplastic proteins had molecular masses ranging from 11 to 35 kDa and were identified as two glucanases, two chitinases, and two TLPs, by using antisera raised against cold-induced rye glucanase, chitinase, and TLP, respectively. Apoplastic extracts obtained from plants treated with ABA lacked the ability to modify the growth of ice crystals, even though ABA induced the accumulation of apoplastic proteins within 4 days to a level similar to that obtained when plants were either drought-stressed for 8 days or cold-acclimated for 7 weeks. These ABA-induced apoplastic proteins were identified immunologically as two glucanases and two TLPs. Moreover, the ABA biosynthesis inhibitor fluridone did not prevent the accumulation of AFPs in the leaves of cold-acclimated rye plants. Our results show that cold acclimation and drought both induce antifreeze activity in winter rye plants and that the pathway regulating AFP production is independent of ABA.     

Ethylene Induces Antifreeze Activity in Winter Rye Leaves

Antifreeze activity is induced by cold temperatures in winter rye (Secale cereale) leaves. The activity arises from six antifreeze proteins that accumulate in the apoplast of winter rye leaves during cold acclimation. The individual antifreeze proteins are similar to pathogenesis-related proteins, including glucanases, chitinases, and thaumatin-like proteins. The objective of this study was to study the regulation of antifreeze activity in response to ethylene and salicyclic acid, which are known regulators of pathogenesis-related proteins induced by pathogens. Nonacclimated plants treated with salicylic acid accumulated apoplastic proteins with no antifreeze activity. In contrast, when nonacclimated plants were exposed to ethylene, both antifreeze activity and the concentration of apoplastic protein increased in rye leaves. Immunoblotting revealed that six of the seven accumulated apoplastic proteins consisted of two glucanases, two chitinases, and two thaumatin-like proteins. The ethylene-releasing agent ethephon and the ethylene precursor 1-aminocyclopropane-1-carboxylate also induced high levels of antifreeze activity at 20 degrees C, and this effect could be blocked by the ethylene inhibitor AgNO(3). When intact rye plants were exposed to 5 degrees C, endogenous ethylene production and antifreeze activity were detected within 12 and 48 h of exposure to cold, respectively. Rye plants exposed to drought produced both ethylene and antifreeze activity within 24 h. We conclude that ethylene is involved in regulating antifreeze activity in winter rye in response to cold and drought.     

Antifreeze proteins in winter rye leaves form oligomeric complexes

Antifreeze proteins (AFPs) similar to three pathogenesis-related proteins, a glucanase-like protein (GLP), a chitinase-like protein (CLP), and a thaumatin-like protein (TLP), accumulate during cold acclimation in winter rye (Secale cereale) leaves, where they are thought to modify the growth of intercellular ice during freezing. The objective of this study was to characterize the rye AFPs in their native forms, and our results show that these proteins form oligomeric complexes in vivo. Nine proteins were separated by native-polyacrylamide gel electrophoresis from apoplastic extracts of cold-acclimated winter rye leaves. Seven of these proteins exhibited multiple polypeptides when denatured and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After isolation of the individual proteins, six were shown by immunoblotting to contain various combinations of GLP, CLP, and TLP in addition to other unidentified proteins. Antisera produced against individual cold-induced winter rye GLP, CLP, and TLP all dramatically inhibited glucanase activity in apoplastic extracts from cold-acclimated winter rye leaves, and each antiserum precipitated all three proteins. These results indicate that each of the polypeptides may be exposed on the surface of the protein complexes. By forming oligomeric complexes, AFPs may form larger surfaces to interact with ice, or they may simply increase the mass of the protein bound to ice. In either case, the complexes of AFPs may inhibit ice growth and recrystallization more effectively than the individual polypeptides.     

Ultrastructural and protein changes in cell suspension cultures of peach associated with low temperature-induced cold acclimation and abscisic acid treatment

Cell suspension cultures were initiated from callus derived from xylem tissues of peach [Prunus persica (L.) Batsch]. Cold acclimation was induced (LT₅₀ of-13°C) in cell suspensions at 3°C in the dark for 10 days. Freezing tolerance returned to the level of nonacclimated cells (LT₅₀ of –4.5°C) when cold-acclimated cells were transferred to 24°C (in dark) for 3 days. Addition of 75 M abscisic acid (ABA) to the growth medium failed to induce cold acclimation after cells were cultured for 5 days at 24°C. Microvacuolation, cytoplasmic augmentation and disappearance of starch grains were observed in cells that were cold-acclimated by exposure to low temperature. Similar ultrastructural alterations were not observed in ABA-treated cells. Several qualitative and quantitative changes in proteins were noted during both cold acclimation and ABA treatment. Both the ultrastructural and protein changes observed during cold acclimation were reversed during deacclimation. The relationship of these changes to cold acclimation in peach cell-cultures is discussed.Abbreviations ABA abscisic acid - 2,4-d 2,4-dichlorophenoxyacetic acid - IBA indole-3-butyric acid - Ms Murashige & Skoog - PMSF phenylmethylsulfonyl fluoride - LT₅₀ or Freezing Tolerance temperature that resulted in 50% decrease in TTC reduction - TTC 2,3,5-triphenyltetrazolium chloride     

Effect of microtubule stabilization on the freezing tolerance of mesophyll cells of spinach

Freezing, dehydration, and supercooling cause microtubules in mesophyll cells of spinach (Spinacia oleracea L. cv Bloomsdale) to depolymerize (ME Bartolo, JV Carter, Plant Physiol [1991] 97: 175-181). The objective of this study was to determine whether the LT₅₀ (lethal temperature: the freezing temperature at which 50% of the tissue is killed) of spinach leaf tissue can be changed by diminishing the extent of microtubule depolymerization in response to freezing. Also examined was how tolerance to the components of extracellular freezing, low temperature and dehydration, is affected by microtubule stabilization. Leaf sections of nonacclimated and cold-acclimated spinach were treated with 20 micromolar taxol, a microtubule-stabilizing compound, prior to freezing, supercooling, or dehydration. Taxol stabilized microtubules against depolymerization in cells subjected to these stresses. When pretreated with taxol both nonacclimated and cold-acclimated cells exhibited increased injury during freezing and dehydration. In contrast, supercooling did not injure cells with taxol-stabilized microtubules. Electrolyte leakage, visual appearance of the cells, or a microtubule repolymerization assay were used to assess injury. As leaves were cold-acclimated beyond the normal period of 2 weeks taxol had less of an effect on cell survival during freezing. In leaves acclimated for up to 2 weeks, stabilizing microtubules with taxol resulted in death at a higher freezing temperature. At certain stages of cold acclimation, it appears that if microtubule depolymerization does not occur during a freeze-thaw cycle the plant cell will be killed at a higher temperature than if microtubule depolymerization proceeds normally. An alternative explanation of these results is that taxol may generate abnormal microtubules, and connections between microtubules and the plasma membrane, such that normal cellular responses to freeze-induced dehydration and subsequent rehydration are blocked, with resultant enhanced freezing injury.     

Photoprotective mechanisms in cold-acclimated and nonacclimated needles of <Emphasis Type="Italic">Picea glehnii</Emphasis>

The response of Picea glehnii, a cold-tolerant species in the boreal zone, to air temperature (T) was investigated for its cold-acclimated needles (i.e. the ones subjected to gradual decrease in T) and nonacclimated needles (i.e. the ones subjected to a sudden decrease in T) were compared under low temperature. Cold-acclimated needles showed a greater increase of zeaxanthin and lutein contents than nonacclimated ones, whereas the nonacclimated needles showed a greater increase of thylakoid-bound ascorbate peroxidase (tAPX) activity than cold-acclimated ones under chilling conditions (after cold acclimation). These results suggest that: (1) low T induces the increase of zeaxanthin and lutein content, and tAPX activity; (2) accumulated zeaxanthin and lutein protect needles from photooxidative stress by dissipating excess energy before the reactive oxygen species (ROS) are formed in response to a gradual decrease in T (with cold acclimation and subsequent chilling condition), and by tAPX scavenging ROS formed in the case of a sudden decrease in T (without cold acclimation and chilling condition).     

Relationship between Freezing Tolerance of Root-Tip Cells and Cold Stability of Microtubules in Rye (Secale cereale L. cv Puma)

The response of cortical microtubules to low temperature and freezing was assessed for root tips of cold-acclimated and non-acclimated winter rye (Secale cereale L. cv Puma) seedlings using indirect immunofluorescence microscopy with antitubulin antibodies. Roots cooled to 0 or −3°C were fixed for immunofluorescence microscopy at these temperatures or after an additional hour at 4°C. Typical arrays of cortical microtubules were present in root-tip cells of seedlings exposed to the cold-acclimation treatment of 4°C for 2 days. Microtubules in these cold-acclimated cells were more easily depolymerized by a 0°C treatment than microtubules in root-tip cells of nonacclimated, 22°C-grown seedlings. Microtubules were still present in some cells of both nonacclimated and cold-acclimated roots at 0 and −3°C; however, the number of microtubules in these cells was lower than in controls. Microtubules remaining during the −3°C freeze were shorter than microtubules in unfrozen control cells. Repolymerization of microtubules after both the 0 and −3°C treatments occurred within 1 h. Root tips of nonacclimated seedlings had an LT-50 of −9°C. Cold acclimation lowered this value to −14°C. Treatment of 22°C-grown seedlings for 24 h with the microtubule-stabilizing drug taxol caused a decrease in the freezing tolerance of root tips, indicated by a LT-50 of −3°C. Treatment with D-secotaxol, an analog of taxol that was less effective in stabilizing microtubules, did not alter the freezing tolerance. We interpret these data to indicate that a degree of depolymerization of microtubules is necessary for realization of maximum freezing tolerance in root-tip cells of rye.     

Effect of cold exposure on cortical microtubules of rye (Secale cereale) as observed by immunocytochemistry

The responses of cortical microtubules to sub-zero temperatures were examined in non-acclimated (NA) and cold-acclimated (CA) rye ( Secale cereale L. cv. Voima) leaf and root cells, and in protoplasts isolated enzymatically from leaves. Responses of leaf and root cells to hypertonic solutions equivalent to the dehydration response of freezing (P. L. Steponkus and D. V. Lynch 1989. J. Bioenerg. Biomembr. 21: 21–41) were also examined. At the respective growth temperatures both NA and CA leaf and root cells had typical organization and abundance of cortical microtubules as observed by indirect immunofluorescence (IIF) staining. Unchanged microtubule arrays were still present in CA leaf cells after -4°C treatment, while in leaf cells of NA plants and in the root cells of both NA and CA plants microtubules were shorter and less abundant. After -10°C treatment the cortical microtubules were almost totally depolymerized in both types of root cells and in leaf cells of NA plants, while CA leaf cells still had abundant cortical microtubule arrays. Semiquantitative analyses of cortical microtubules (MTs) of protoplasts confirmed the findings with intact leaf cells. Hypertonic treatment of NA and CA leaf cells gave similar effects as exposure of cells to sub-zero temperatures. However, after the hypertonic treatment, more microtubules remained present in the CA root cells than in the NA root cells, suggesting that also in root cells cold acclimation increases the dehydration stability of MTs. In conclusion, cold acclimation induces both greater frost stability and greater osmotic tolerance in the cortical microtubules of the leaf cells, and greater osmotic tolerance in the microtubules of the root cells in winter rye.     

Induction of freezing tolerance in spinach is associated with the synthesis of cold acclimation induced proteins

Spinach (Spinacia oleracea L. cv Bloomsdale) seedlings cultured in vitro were used to study changes in protein synthesis during cold acclimation. Seedlings grown for 3 weeks postsowing on an inorganic-nutrient-agar medium were able to increase their freezing tolerance when grown at 5°C. During cold acclimation at 5°C and deacclimation at 25°C, the kinetics of freezing tolerance induction and loss were similar to that of soil-grown plants. Freezing tolerance increased after 1 day of cold acclimation and reached a maximum within 7 days. Upon deacclimation at 25°C, freezing tolerance declined within 1 day and was largely lost by the 7th day. Leaf proteins of intact plants grown at 5 and 25°C were in vivo radiolabeled, without wounding or injury, to high specific activities with [³⁵S]methionine. Leaf proteins were radiolabeled at 0, 1, 2, 3, 4, 7, and 14 days of cold acclimation and at 1, 3, and 7 days of deacclimation. Up to 500 labeled proteins were separated by two-dimensional gel electrophoresis and visualized by fluorography. A rapid and stable change in the protein synthesis pattern was observed when seedlings were transferred to the low temperature environment. Cold-acclimated leaves contained 22 polypeptides not found in nonacclimated leaves. Exposure to 5°C induced the synthesis of three high molecular weight cold acclimation proteins (CAPs) (M_r of about 160,000, 117,000, and 85,000) and greatly increased the synthesis of a fourth high molecular weight protein (M_r 79,000). These proteins were synthesized during day 1 and throughout the 14 day exposure to 5°C. During deacclimation, the synthesis of CAPs 160, 117, and 85 was greatly reduced by the first day of exposure to 25°C. However, CAP 79 was synthesized throughout the 7 day deacclimation treatment. Thus, the induction at low temperature and termination at warm temperature of the synthesis of CAPs 160, 117, and 85 was highly correlated with the induction and loss of freezing tolerance. Cold acclimation did not result in a general posttranslational modification of leaf proteins. Most of the observed changes in the two-dimensional gel patterns could be attributed to the de novo synthesis of proteins induced by low temperature. In spinach leaf tissue, heat shock altered the pattern of protein synthesis and induced the synthesis of several heat shock proteins (HSPs). One polypeptide synthesized in cold-acclimated leaves had a molecular weight and net charge (M_r 79,000, pI 4.8) similar to that of a HSP (M_r 83,000, pI 4.8). However, heat shock did not increase the freezing tolerance, and cold acclimation did not increase heat tolerance over that of nonacclimated plants, but heat-shocked leaf tissue was more tolerant to high temperatures than nonacclimated or cold-acclimated leaf tissue. When protein extracts from heat-shocked and cold-acclimated leaves were mixed and separated in the same two-dimensional gel, the CAP and HSP were shown to be two separate polypeptides with slightly different isoelectric points and molecular weights.