Effect of cold acclimation on the incidence of two forms of freezing injury in protoplasts isolated from rye leaves

The freezing tolerance and incidence of two forms of freezing injury (expansion-induced lysis and loss of osmotic responsiveness) were determined for protoplasts isolated from rye leaves (Secale cereale L. cv Puma) at various times during cold acclimation. During the first 4 weeks of the cold acclimation period, the LT₅₀ (i.e. the minimum temperature at which 50% of the protoplasts survived) decreased from −5°C to −25°C. In protoplasts isolated from nonacclimated leaves (NA protoplasts), expansion-induced lysis (EIL) was the predominant form of injury at the LT₅₀. However, after only 1 week of cold acclimation, the incidence of EIL was reduced to less than 10% at any subzero temperature; and loss of osmotic responsiveness was the predominant form of injury, regardless of the freezing temperature. Fusion of either NA protoplasts or protoplasts isolated from leaves of seedlings cold acclimated for 1 week (1-week ACC protoplasts) with liposomes of dilinoleoylphosphatidylcholine also decreased the incidence of EIL to less than 10%. Fusion of protoplasts with dilinoleoylphosphatidylcholine diminished the incidence of loss of osmotic responsiveness, but only in NA protoplasts or 1-week ACC protoplasts that were frozen to temperatures over the range of -5 to -10°C. These results suggest that the cold acclimation process, which results in a quantitative increase in freezing resistance, involves several different qualitative changes in the cryobehavior of the plasma membrane.     

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.     

Modification of the intracellular sugar content alters the incidence of freeze-induced membrane lesions of protoplasts isolated from Arabidopsis thaliana leaves

Sugar content and freezing tolerance of protoplasts of Arabidopsis thaliana leaves were manipulated by incubating seedlings in a sucrose solution before protoplast isolation. Incubation in a 400 mM sucrose solution at 2 °C in the dark increased their freezing tolerance equivalent to that achieved after a conventional cold acclimation at 2 °C. The increased freezing tolerance was due to a decrease in the incidence of freeze‐induced lesions: expansion‐induced lysis (EIL) between ?2 and ?4 °C and loss of osmotic responsiveness (LOR) between ?5 and ?12 °C. The concentration of sucrose in the incubation medium required to minimize the incidence of the lesions was substantially different: 10–35 mM for EIL and 30–400 mM for LOR. Incubation in the sucrose solution at 23 °C decreased LOR only at ?5 and ?6 °C but less than that incubated at 2 °C, and there was no effect on EIL. Incubation in sorbitol solutions at 2 °C also decreased LOR at ?5 and ?6 °C but much less than in the sucrose solution. These results suggest that low concentrations of sucrose act as a metabolic substrate for the low‐temperature‐induced alterations required for the amelioration of EIL and, at higher concentrations, sucrose has a direct cryoprotective effect to minimize LOR.     

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.     

Cold responses of Arabidopsis mutants impaired in freezing tolerance

Mutants of Arabidopsis thaliana L. (Heynh), characterized asdeficient in their freezing tolerance after cold acclimation,were surveyed for some of the normal responses to cold exposure.In foliar tissue, the coldinducibility of three proteins, thelevels of sucrose and glucose, the fatty acyl composition oflipids, and the accumulation of anthocyanin was examined. Fourmutations (sfr3, sfr4, sfr6, and sfr7) reduced or eliminatedthe accumulation of anthocyanin during cold acclimation. Onemutation (sfr4) prevented the normally cold-induced elevationof sucrose and glucose levels; both sfr4 and another mutation(sfr7) affected fatty acid composition after (and only after)cold acclimation. On the other hand mutations sfr1, sfr2 andsfr5 did not differ significantly from the wild type in anyof the parameters tested, suggesting that they have other, perhapshighly specific, effects on lowtemperature responses. Key words: Arabidopsis thaliana, cold acclimation, freezing tolerance, mutation     

Behavior of the Plasma Membrane of Isolated Protoplasts during a Freeze-Thaw Cycle

Cryomicroscopy of protoplasts isolated from nonacclimated (NA) rye leaves (Secale cereale L. cv Puma) revealed that the predominant form of injury following cooling to the minimum temperature for 50% survival (LT₅₀) (−5°C) was expansion-induced lysis of the plasma membrane during warming and thawing of the suspending medium when the decreasing osmolality resulted in osmotic expansion of the protoplasts. When cooled to temperatures below the LT₅₀, the predominant form of injury was loss of osmotic responsiveness following cooling so that the protoplasts were osmotically inactive during warming. Only a low incidence (<10%) of expansion-induced lysis was observed in protoplasts isolated from acclimated (ACC) leaves, and the predominant form of injury following cooling to the LT₅₀ (−25°C) was loss of osmotic responsiveness. The tolerable surface area increment (TSAI) which resulted in lysis of 50% of a population (TSAI₅₀) of NA protoplasts osmotically expanded from isotonic solutions was 1122 ± 172 square micrometers. Similar values were obtained when the protoplasts were osmotically expanded from hypertonic solutions. The TSAI determined from cryomicroscopic measurements of individual NA protoplasts was similar to the TSAI₅₀ values obtained from osmotic manipulation. The TSAI₅₀ of ACC protoplasts expanded from isotonic solutions (2145 ± 235 square micrometers) was approximately double that of NA protoplasts and increased following osmotic contraction. Osmotic contractions were readily reversible upon return to isotonic solutions. During freeze-induced dehydration, endocytotic vesicles formed in NA protoplasts whereas exocytotic extrusions formed on the surface of ACC protoplasts. During osmotic expansion following thawing of the suspending medium, the endocytotic vesicles remained in the cytoplasm of NA protoplasts and the protoplasts lysed before their original volume and surface area were regained. In contrast, the exocytotic extrusions were drawn back into the surface of ACC protoplasts as the protoplasts regained their original volume and surface area.     

The sensitive to freezing3 mutation of Arabidopsis thaliana is a cold-sensitive allele of homomeric acetyl-CoA carboxylase that results in cold-induced cuticle deficiencies

The sfr3 mutation causes freezing sensitivity in Arabidopsis thaliana. Mapping, sequencing, and transgenic complementation showed sfr3 to be a missense mutation in ACC1, an essential gene encoding homomeric (multifunctional) acetyl-CoA carboxylase. Cuticle permeability was compromised in the sfr3 mutant when plants were grown in the cold but not in the warm. Wax deposition on the inflorescence stem of cold-grown sfr3 plants was inhibited and the long-chain components of their leaf cuticular wax were reduced compared with wild-type plants. Thus, freezing sensitivity of sfr3 appears, from these results, to be due to cuticular deficiencies that develop during cold acclimation. These observations demonstrated the essential role of the cuticle in tolerance to freezing and drought.     

Freezing tolerance, cold acclimation and oxidative stress in potato. Paraquat tolerance is related to acclimation but is a poor indicator of freezing tolerance

Potato is a species commonly cultivated in temperate areas where the growing season may be interrupted by frosts, resulting in loss of yield. Cultivated potato, Solanum tuberosum, is freezing sensitive, but it has several freezing-tolerant wild potato relatives, one of which is S. commersonii. Our study was aimed to resolve the relationship between enhanced freezing tolerance, acclimation capacity and capacity to tolerate active oxygen species. To be able to characterize freezing tolerant ideotypes, a potato population (S1), which segregates in freezing tolerance, acclimation capacity and capacity to tolerate superoxide radicals, was produced by selfing a somatic hybrid between a freezing-tolerant Solanum commersonii (LT₅₀=-4.6°C) and -sensitive S. tuberosum (LT₅₀=-3.0°C). The distribution of non-acclimated freezing tolerance (NA-freezing tolerance) of the S1 population varied between the parental lines and we were able to identify genotypes, having significantly high or low NA-freezing tolerance. When a population of 25 genotypes was tested both for NA-freezing and paraquat (PQ) tolerance, no correlation was found between these two traits (R = 0.02). However, the most NA-freezing tolerant genotypes were also among the most PQ tolerant plants. Simultaneously, one of the NA-freezing sensitive genotypes (2022) (LT₅₀=-3.0°C) was observed to be PQ tolerant. These conflicting results may reflect a significant, but not obligatory, role of superoxide scavenging mechanisms in the NA-freezing tolerance of S. commersonii. The freezing tolerance after cold acclimation (CA-freezing tolerance) and the acclimation capacity (AC) was measured after acclimation for 7 days at 4/2°C. Lack of correlation between NA-freezing tolerance and AC (R =-0.05) in the S1 population points to independent genetic control of NA-freezing tolerance and AC in Solanum sp. Increased freezing tolerance after cold acclimation was clearly related to PQ tolerance of all S1 genotypes, especially those having good acclimation capacity. The rapid loss of improved PQ tolerance under deacclimation conditions confirmed the close relationship between the process of cold acclimation and enhanced PQ tolerance. Here, we report an increased PQ tolerance in cold-acclimated plants compared to non-acclimated controls. However, we concluded that high PQ tolerance is not a good indicator of actual freezing tolerance and should not be used as a selectable marker for the identification of a freezing-tolerant genotype.     

甜杨6-磷酸葡萄糖脱氢酶在抗冻性低温诱导中的作用

对-20℃低温锻炼及脱锻炼过程中甜杨(Populus suaveolens)幼苗的G6PDH、SOD和POD活性、MDA含量和半致死温度(LT50)进行了测定和分析.结果发现,低温锻炼在一定程度上提高了幼苗6-磷酸葡萄糖脱氢酶(G6PDH)、SOD和POD活性,降低了MDA含量和幼苗半致死温度(LT50).另外,将幼苗放回常温(脱锻炼)2 d能引起幼苗的G6PDH、SOD和POD活性的显著下降,并使LT50和MDA含量的迅速回升.结果表明,低温锻炼中G6PDH活性的增加有助于SOD和POD活性的提高,进而对幼苗的LT50和MDA含量的降低有明显的促进作用,G6PDH可能参与了SOD和POD活性的调节和抗冻性的低温诱导.     

Rapid degradation of starch in chloroplasts and concomitant accumulation of soluble sugars associated with ABA-induced freezing tolerance in the moss Physcomitrella patens

Abscisic acid (ABA) has been postulated to play a role in the development of freezing tolerance during the cold acclimation process in higher plants, but its role in cold tolerance in tower land plants has not been elucidated. The moss Physcomitrella patens rapidly developed freezing tolerance when its protonemata were grown in a medium containing ABA, with dramatic changes in the LT50 value from -2 degrees C to over -10 degrees C. We examined physiological and morphological alterations in protonema cells caused by ABA treatment to elucidate early cellular events responsible for rapid enhancement of freezing tolerance. Microscopic observations revealed that ABA treatment for 1 day resulted in a dramatic alteration in the appearance of intracellular organelles. ABA-treated cells had slender chloroplasts, with a reduced amount of starch grains, in comparison with those of non-treated cells. The ABA-treated cells also had several segmented vacuoles while many of non-treated cells had one central vacuole. When frozen to -4 degrees C, freezing injury-associated ultrastructural changes such as formation of aparticulate domains and fracture-jump lesions were frequently observed in the plasma membrane of non-treated protonema cells but not in that of ABA-treated cells. The ABA treatment increased the osmotic concentration of the protonema cells, in correlation with accumulation of free soluble sugars. These results suggest that ABA-induced accumulation of soluble sugars, associated with morphological changes in organelles, mitigated freezing-induced structural damage in the plasma membrane, eventually leading to enhancement of freezing tolerance in the protonema cells.     

Arabidopsis thaliana avoids freezing by supercooling

Arabidopsis thaliana (L.) Heynh. has been described as a freezing-tolerant species based on freezing-resistance assays. Nonetheless, this type of experiment does not discriminate between freezing-tolerance and freezing-avoidance mechanisms. The purpose of this paper was to determine which of these two freezing-resistance mechanisms is responsible for freezing resistance in A. thaliana. This was achieved by comparing the thermal properties (ice-nucleation temperature and the freezing temperature) of leaves and the lethal temperature to 10, 50 and 90% of the plants (LT10, LT50, and LT90, respectively). Two wild-type genotypes were used (Columbia and Ler) and their mutants (esk-1 and frs-1, respectively), which differ in their freezing resistance. This study's results indicated that the mutant esk-1, described as a freezing-tolerant species showed freezing tolerance only after a cold-acclimation period. The mutant frs-1, described as freezing sensitive, presented freezing avoidance. Both wild genotypes presented LT50 similar to or higher than the ice-nucleation temperature. Thus, the main freezing-resistance mechanism for A. thaliana is avoidance of freezing by supercooling. No injury of the photosynthetic apparatus was shown by measuring the maximal photochemical efficiency (Fv/Fm) and pigments (chlorophyll and carotenoid) during cold acclimation in all genotypes. During cold acclimation, Columbia and esk-1 increased total soluble carbohydrates in leaves. esk-1 was the only genotype that presented freezing tolerance after cold acclimation. This feature could be related to an increase in sugar accumulation in the apoplast.     

Klebsormidium flaccidum, a charophycean green alga, exhibits cold acclimation that is closely associated with compatible solute accumulation and ultrastructural changes

To elucidate the fundamental mechanisms and subsequent evolutionary aspects of plant cold acclimation, we examined the effect of cold acclimation on freezing tolerance in Klebsormidium flaccidum, a green alga belonging to Charophyceae, a sister group of land plants. Freezing tolerance of K. flaccidum was significantly enhanced by cold treatment: survival increased from 15% at -10 degrees C when grown at 18 degrees C to 55 and 85% after exposure at 2 degrees C for 2 and 7 d, respectively. Accompanying the development of freezing tolerance, soluble sugars (glucose and sucrose), a putative glycoside and amino acids, including gamma-aminobutyric acid (GABA), accumulated to high levels in the alga, suggesting that these solutes play a crucial role in the cold acclimation of K. flaccidum. Interestingly, the application of abscisic acid (ABA) did not change the freezing tolerance of the alga. We also observed changes in cell structure, including increased numbers and sizes of starch grains in chloroplasts, chloroplast enlargement, vacuole size reduction and cytoplasmic volume increase. These results suggest that K. flaccidum responds well to cold treatment and develops freezing tolerance in a process comparable to that of land plants.     

The role of abscisic acid and low temperature in chickpea (Cicer arietinum) cold tolerance. II. Effects on plasma membrane structure and function

The frost hardiness of many plants such as chickpea can be increased by exposure to low non-freezing temperatures and/or the application of abscisic acid (ABA), a process known as frost acclimation. Experiments were conducted to study the response over a 14 d period of enriched plasma membrane fractions isolated from chickpea plants exposed to low temperature and sprayed with exogenous ABA. Measurement of the temperatures inducing 50% foliar cell death (LT50), and subsequent statistical analysis suggest that, like many plants, exposure to low temperatures (5/-2 degrees C; day/night) induces a significant level (P <0.05) of frost acclimation in chickpea when compared with control plants (20/7 degrees C; day/night). Spraying plants with exogenous ABA also increased frost tolerance (P <0.05), but was not as effective as low temperature-induced frost acclimation. Both pre-exposure to low temperatures and pre-treatment with ABA increased the levels of fatty acid desaturation in the plasma membrane (measured as the double bond index, DBI). Exposure of chickpea plants to low temperatures increased the DBI by 15% at day 4 and 19% at day 14 when compared with untreated control plants. Application of ABA alone did not increase the DBI by more than 6% at any time; the effects of both treatments applied together was more than additive, inducing a DBI increase of 27% at day 14 when compared with controls. There was a good correlation (P <0.05) between the DBI and LT50, suggesting that the presence of more unsaturated lipid in the plasma membrane may prevent cell lysis at low temperatures. Both pre-exposure to low, non-freezing temperatures and pre-treatment with ABA induced measurable changes in membrane fluidity, but these changes did not correlate with changes in LT50, suggesting that physical properties of the plasma membrane other than fluidity are involved in frost acclimation in chickpea.     

Development of freezing tolerance in different altitudinal ecotypes of <Emphasis Type="Italic">Salix paraplesia</Emphasis>

Salix paraplesia was used as an experimental model to investigate the effect of short day photoperiod (SD) and low temperature (LT) on development of freezing tolerance and on endogenous abscisic acid (ABA) contents. We characterized differences in SD and LT-induced cold acclimation in three ecotypes from different altitudes. The results demonstrated that cold acclimation could be triggered by exposing the plants to SD or LT alone, and that a combination of the different treatments had an additive effect on freezing tolerance in all ecotypes studied. However, the high altitudinal ecotype was more responsive to SD and LT than the low altitudinal ecotype. Development of freezing tolerance induced by SD and LT was accompanied by changes in ABA contents which were ecotype-dependent. Although the stem had higher initial freezing tolerance, the leaves developed freezing tolerance more quickly than the stem and thus leaves may provide an interesting experimental system for physiological and molecular studies of cold acclimation in woody plants.     

Cold resistance in Antarctic angiosperms

Deschampsia antarctica Desv. (Poaceae) and Colobanthus quitensis (Kunth) Bartl. (Cariophyllaceae) are the only two vascular plants that have colonized the Maritime Antarctic. The primary purpose of the present work was to determine cold resistance mechanisms in these two Antarctic plants. This was achieved by comparing thermal properties of leaves and the lethal freezing temperature to 50% of the tissue (LT50). The grass D. antarctica was able to tolerate freezing to a lower temperature than C. quitensis. The main freezing resistance mechanism for C. quitensis is supercooling. Thus, the grass is mainly a freezing‐tolerant species, while C. quitensis avoids freezing. D. antarctica cold acclimated; thus, reducing its LT50. C. quitensis showed little cold‐acclimation capacity. Because day length is highly variable in the Antarctic, the effect of day length on freezing tolerance, growth, various soluble carbohydrates, starch, and proline contents in leaves of D. antarctica growing in the laboratory under cold‐acclimation conditions was studied. During the cold‐acclimation treatment, the LT50 was lowered more effectively under long day (21/3 h light/dark) and medium day (16/8) light periods than under a short day period (8/16). The longer the day length treatment, the faster the growth rate for both acclimated and non‐acclimated plants. Similarly, the longer the day treatment during cold acclimation, the higher the sucrose content (up to 7‐fold with respect to non‐acclimated control values). Oligo and polyfructans accumulated significantly during cold acclimation only with the medium day length treatment. Oligofructans accounted for more than 80% of total fructans. The degrees of polymerization were mostly between 3 and 10. C. quitensis under cold acclimation accumulated a similar amount of sucrose than D. antarctica, but no fructans were detected. The suggestion that survival of Antarctic plants in the Antarctic could be at least partially explained by accumulation of these substances is discussed.     

The molecular biology of the low-temperature response in plants

Plants growing in temperate regions are able to survive freezing temperatures from -5 degrees to -30 degrees C, depending on the species, through a process known as cold acclimation. In the last decade much work has been done on the molecular mechanisms of low temperature (LT) signal transduction and cold acclimation. Mutant studies and microarray analyses have revealed C-Repeat binding factor (CBF) -dependent and -independent signaling pathways in plants. Experimental evidence suggests the existence of 'potential LT sensors' but as yet there is no direct proof. A number of signal transducers such as various kinases/phosphatases have been demonstrated but the signal transduction pathways have not been elucidated. An understanding of the molecular basis of the signaling process, however, is of potential practical application. Designing new strategies to improve cold tolerance in crop varieties could increase the plant productivity and also expand the area under cultivation.