Stimulation of Phospholipid Biosynthesis during Frost Hardening of Winter Wheat

Lipids were labeled with ³³P during frost hardening of two varieties of winter wheat (Triticum 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.     

Freezing of water in red-osier dogwood stems in relation to cold hardiness

Studies of stem water in red-osier dogwood (Cornus stolonifera Michx.) using nuclear magnetic resonance spectroscopy indicated that most freezing occurs at temperatures above −30 C in cold-hardy and tender stems. Hardy and tender stems had about the same amount of unfrozen water at −40 C (0.28 gram of water per gram dry weight). When hardy stems were slowly cooled below −20 C, the temperature below which little additional freezing occurs, they survived direct immersion in liquid N₂ (−196 C). Fully hardy samples not slowly precooled to at least −15 C did not survive direct immersion in liquid N₂. The results support the hypothesis that cooling rate is an unimportant factor in tissue survival at and below temperatures where there is little freezable water.     

Expression of freezing tolerance in the interspecific F1 and somatic hybrids of potatoes

 The expression of freezing tolerance was examined in interspecific F₁ and somatic hybrids of potatoes using 20 species and 34 different combinations between hardy and sensitive species. In the field, the frost tolerance of hybrids resembled either that of the hardy parent, the sensitive parent, or the parental mean, depending on the species combination and the genomic ratio (ratio of the number of sets of chromosomes contributed from each parent). Similar phenomena were observed when the non-acclimated freezing tolerance (NA) and the acclimation capacity (ACC) (two independent genetic components of freezing tolerance) were evaluated separately under controlled environments. In general, the expression level of freezing tolerance was higher in hybrids with more genomes contributed from the hardy parent than from the sensitive parent. In addition, the effectiveness or combining ability of genes conferring freezing tolerance from the hardy species also showed some influence on the expression of freezing tolerance. All three parameters, namely NA, ACC and acclimated freezing tolerance (AA) (NA plus ACC), were significantly correlated to the frost tolerance exhibited in the field. This indicates that the controlled freezing test used in this study could provide a good estimate of field performance. The implications of these results in breeding for freezing tolerance in potatoes are discussed. Received: 21 July 1998 / Accepted: 29 September 1998     

Evergreen alpine shrubs have high freezing resistance in spring,irrespective of snowmelt timing and exposure to frost: an investigation from the Snowy Mountains,Australia

Over winter, alpine plants are protected from low-temperature extremes by a blanket of snow. Climate change predictions indicate an overall reduction in snowpack and an earlier thaw; a situation which could expose the tips of shrubs which extend above the snowpack to freezing events in early spring, and cause foliar frost damage during the onset of physiological activity. We assessed the photosynthetic responses of freezing-damaged shrub leaves from an assay of freezing temperatures in the Snowy Mountains in south-eastern Australia, using chlorophyll fluorometery ex situ. We sampled leaves that were exposed early during the spring thaw and leaves that were buried in snow for up to two extra weeks, from four evergreen shrub species at monthly intervals following the period of snowmelt. Freezing resistance (estimated from LT₅₀) was poorest at the earliest spring sampling time, in both exposed above-snow and protected below-snow foliage in all species. Protected foliage in early spring had lower freezing resistance than exposed foliage, but not significantly so. By the third sampling time, freezing resistance was significantly better in the lower protected foliage (LT₅₀ of ? 14) compared with the upper exposed foliage (LT₅₀ of ? 10) in one species. Over the course of spring, freezing resistance improved significantly in all species, with LT₅₀ values of between ? 10 and ? 15 °C by the third sampling time, which is lower than the minimum air temperatures recorded at that time (> ? 5 °C). The results indicate that the dominant evergreen shrub species in this area may only be susceptible to freezing events very early in spring, before a period of frost-hardening occurs after snowmelt. Later in spring, these alpine shrubs appear frost hardy, thus further perpetuating the positive feedbacks surrounding shrub expansion in alpine areas.     

Divergent mechanisms of frost-hardiness in two populations of the gall fly, Eurosta solidaginsis

Two populations of the gall fly Eurosta solidaginsis utilize different strategies to endure seasonal exposure to temperatures below freezing. Both populations are freezing tolerant. In north temperate populations, supercooling points rise from ?10.2°C to ?6.2°C following exposures to temperatures below freezing. This level is maintained throughout winter and ensures frequent and prolonged periods of tissue freezing. South temperate populations depress the supercooling point to ?14.2°C during autumn and early winter, and this depression precludes extracellular ice formation during periods of supra-optimal temperature fluctuations. During mid-winter, supercooling points rise to the same level as in northern groups.Both populations accumulate three principal cryoprotective agents following first frost exposures (glycerol, sorbitol and trehalose). Cryoprotectants levels do not peak in northern populations until 4–6 weeks after first frost. In southern populations the accumulation profile is characterized by a high initial rate of synthesis, a protective overshoot and pronounced seasonal fluctuations. The relative survival advantages of each strategy are discussed.     

Properties of chloroplast membrane-bound ferredoxin--NADP + reductase during cold hardening of wheat. No indication of qualitative membrane changes during cold hardening

Kinetic parameters of the chloroplastbound ferredoxin-NADP⁺ reductase from two varieties of wheat (Triticum aestivum), hardy Kharkov 22 MC (winter wheat) and less hardy Rescue (spring wheat), were followed during induction of frost hardiness as a means of examining possible changes in chloroplast membranes during hardening. No changes were found in the Michaelis constants for NADPH and 2,6-dichlorophenol indophenol, inhibition constants for p-chloromercuriphenylsulfonate, and activation energy values of the enzyme in either variety. The data suggest that no qualitative changes occurred in the properties of wheat chloroplast membranes related to ferredoxin-NADP⁺ reductase during cold hardening.     

Synthesis of Freezing Tolerance Proteins in Leaves, Crown, and Roots during Cold Acclimation of Wheat

Protein synthesis was studied in leaves, crown, and roots during cold hardening of freezing tolerant winter wheat (Triticum aestivum L. cv Fredrick and cv Norstar) and freezing sensitive spring wheat (T. aestivum L. cv Glenlea). The steady state and newly synthesized proteins, labeled with [³⁵S]methionine, were resolved by one- and two-dimensional polyacrylamide gels. The results showed that cold hardening induced important changes in the soluble protein patterns depending upon the tissue and cultivar freezing tolerance. At least eight new proteins were induced in hardened tissues. A 200 kilodalton (kD) (isoelectric point [pl] 6.85) protein was induced concomitantly in the leaves, crown, and roots. Two proteins were specifically induced in the leaves (both 36 kD, pl 5.55 and 5.70); three in the crown with M_r 150 (pl 5.30), 45 (pl 5.75), and 44 kD (pl > 6.80); and two others in the roots with M_r 64 (pl 6.20) and 52 kD (pl 5.55). In addition, 19 other proteins were synthesized at a modified rate (increased or decreased) in the leaves, 18 in the crown and 23 in the roots. Among the proteins induced or increased in hardened tissues, some were expressed at a higher level in the freezing tolerant cultivars than in the sensitive one, indicating a correlation between the synthesis and accumulation of these proteins and the degree of freezing tolerance. These proteins, suggested to be freezing tolerance proteins, may have an important role in the cellular adaptation to freezing.     

Phospholipid degradation in frozen plant cells associated with freezing injury

A striking degradation of phosphatidylcholine into phosphatidic acid was observed in the cortical tissues of less hardy poplar (Poplus euramericani cv. gelrica), when the tissues were frozen below a lethal temperature. No change in phospholipids was detected during freezing or even after thawing in the cortical tissues of hardy poplar which survived slow freezing to −30 C or even immersion in liquid N₂ after prefreezing to −50 C. The degradation of phosphatidylcholine during freezing appears to be intimately associated with freezing injury.     

Effects of freezing and hardening on the sulfhydryl groups of protein fractions from cabbage leaves

Disc electrophoresis was used to separate water soluble proteins from hardy, non-hardy, and frost killed cabbage (Brassica oleracea var. capitata) leaves. Amidoschwarz staining failed to reveal any new bands as a result of hardening although the relative amounts of proteins in individual bands changed. Sulfhydryl groups in the protein bands were stained with 2,2-dihydroxy-6,6-dinaphthyl disulfide and labeled with ¹⁴C p-chloromercuribenzoate. Significant decreases in the sulfhydryl content of the total water soluble protein were found during hardening and as a result of frost death. The decrease during hardening was paralleled by a significant increase in the water soluble protein. There was a significant increase in the sulfhydryl content per unit high molecular weight protein but a decrease in the sulfhydryl content per total protein as a result of frost death. This was interpreted as evidence for intermolecular disulfide bond formation during freezing.     

The relationship between cell injury and osmotic volume reduction: III. Freezing injury and frost resistance in winter wheat

In order to distinguish between several possible mechanisms of frost hardening in winter wheat (Triticum aestivum L.) cells from two hardy and two tender cultivars were plasmolyzed in CaCl₂ solution at room temperature and cell volumes estimated by microscopic examination. Analyses of Boyle-van't Hoff plots of these data reveal that all cells from cultivars progressively increase their intracellular solute concentration up to 20 days hardening. This increase, which we had predicted from published calorimetric data to be the sole mechanism of hardening explained less than half of the increase in hardening seen in the most hardy cultivar, Kharkov. Hardening also increased the osmotically inactive volume.At CaCl₂ concentrations greater than 5%, plasmolyzed protoplasts departed further from the Boyle-van't Hoff prediction, remaining larger than expected until some higher concentration of CaCl₂, where protoplast volume again sharply decreased. In all cultivars except hardened Kharkov, the concentration of CaCl₂ producing this abrupt volume decrease had a freezing point corresponding to the killing temperature. If this concentration was exceeded during plasmolysis, then the protoplasts burst during deplasmolysis at some volume less than their original volume.We interpret these data to mean that, in addition to the often described hardening mechanism of increased cell solute and water binding, winter wheat shows a third mechanism, a mechanical resistance to protoplast shrinkage which produces volumes larger than those predicted during osmotic stress. The resisting element appears to be the plasma membrane itself. Shrinkage brings the membrane under compressive stress, developing tangential pressure within it. Cell injury occurs when the cell membrane area has been reduced to the point at which irreversible loss of membrane material is inevitable. Cell death occurs during deplasmolysis when the protoplast bursts because its membrane contains insufficient material to subtend the area of the cell wall.Of the cultivars tested, hardened Kharkov was unique in avoiding injury. Hardened Kharkov was injured only after the volume inflection had been greatly exceeded. Refractile droplets of lipid appeared in the cytoplasm of hardened Kharkov protoplasts during plasmolysis but disappeared during deplasmolysis suggesting that hardy Kharkov was able reversibly to store membrane lipids in cytoplasmic vesicles and return them to the membrane on deplasmolysis.     

Assessment of the frost sensitivity of Trifolium species by chlorophyll fluorescence analysis

The potential of the chlorophyll fluorescence technique in screening for frost sensitivity in a range of Trifolium species from different geographical origins was assessed by measuring the decrease in variable chlorophyll fluorescence (F_var) of leaves after freezing at - 5°C for 60 min. The method was rapid and the results obtained agreed well with a visual assessment of freezing injury carried out after leaves were returned to optimal growth conditions for 72 h. Trifolium alexandrinum (Berseem clover) cv. Tabor originating from Israel was shown to be the most frost sensitive species studied and Trifolium subterraneum (subterranean clover) cv. Mt. Barker, from temperate regions of Australia, the most frost resistant. On extended periods of freezing, frost damage increased and this was associated with a further reduction in variable chlorophyll fluorescence and in quenching capacity of the thylakoid membranes. These results thus indicate that substantial thylakoid membrane dysfunction is induced at freezing temperatures. Furthermore, it was found that frost hardening of the frost sensitive species T. alexandrinum for 21 days at 5°C reduced the extent of damage sustained by the thylakoid membranes as shown by higher fluorescence quenching capacity, smaller reduction in variable fluorescence (F_var) and higher initial fluorescence (F_o) when leaves of hardened plants were frozen at -5°C and -7°C.     

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.     

Enhancement of frost tolerance in olive shoots in vitro by cold acclimation and sucrose increase in the culture medium

The evaluation of frost tolerance in olive shoots in vitro has been successfully accomplished. The behavior of in vitro shoots at freezing temperatures was comparable to that of intact plants. Cold acclimation was found to increase frost tolerance in cv. Moraiolo and the LT₅₀ was about 4 °C lower compared to nonacclimated shoots. Damage in acclimated shoots occurred at –15 °C, whereas control shoots were damaged at –10 °C. Olive shoots were unable to withstand freezing temperatures of –20 °C, even when acclimated. The effects of sucrose were also determined. 6% (w/v) sucrose in the medium conferred the highest frost tolerance in both acclimated and nonacclimated plants.     

Deep supercooling enabled by surface impregnation with lipophilic substances explains the survival of overwintering buds at extreme freezing

The frost survival mechanism of vegetative buds of angiosperms was suggested to be extracellular freezing causing dehydration, elevated osmotic potential to prevent freezing. However, extreme dehydration would be needed to avoid freezing at the temperatures down to ?45°C encountered by many trees. Buds of Alnus alnobetula, in common with other frost hardy angiosperms, excrete a lipophilic substance, whose functional role remains unclear. Freezing of buds was studied by infrared thermography, psychrometry, and cryomicroscopy. Buds of A. alnobetula did not survive by extracellular ice tolerance but by deep supercooling, down to ?45°C. An internal ice barrier prevented ice penetration from the frozen stem into the bud. Cryomicroscopy revealed a new freezing mechanism. Until now, supercooled buds lost water towards ice masses that form in the subtending stem and/or bud scales. In A. alnobetula, ice forms harmlessly inside the bud between the supercooled leaves. This would immediately trigger intracellular freezing and kill the supercooled bud in other species. In A. alnobetula, lipophilic substances (triterpenoids and flavonoid aglycones) impregnate the surface of bud leaves. These prevent extrinsic ice nucleation so allowing supercooling. This suggests a means to protect forestry and agricultural crops from extrinsic ice nucleation allowing transient supercooling during night frosts.     

Quantitative and qualitative changes in carbohydrates associated with spring deacclimation in contrasting Hydrangea species

Cold deacclimation and associated changes in soluble carbohydrates and water status of two Hydrangea species differing in susceptibility to frost injuries was followed under natural conditions. In fully cold hardy plants of H. macrophylla stem freezing tolerance fluctuated in parallel with changes in air temperature, while in a seasonal perspective increased temperatures caused a sigmoid deacclimation pattern in both H. macrophylla and H. paniculata. Timing of deacclimation was approximately synchronized in the two species, but H. paniculata, the hardier species based on mid-winter hardiness, deacclimated faster than H. macrophylla, indicating that deacclimation kinetics were not correlated with mid-winter hardiness. In both species concentrations of soluble sugars decreased during deacclimation and were highly correlated with stem cold hardiness and air temperatures. This suggests that sugar hydrolysis may be an important temperature-driven mechanism of deacclimation in Hydrangea. Accumulation patterns of specific carbohydrates differed between the two species, suggesting that they utilize different strategies to overcome cold. In H. paniculata, deacclimation was associated with an increase in stem water content, which occurred shortly before bud burst and hence may be a prerequisite for leafing out.     

Application of Impedance Spectroscopy for Selecting Frost Hardy Varieties of English Ryegrass

The frost hardening and frost damage of 12 varieties of Englishryegrass (Lolium perenne) was studied by electrical impedancespectroscopy. For the measurement of the impedance spectrum(80 Hz to 1 MHz) a 10 mm length sample was cut from the stemabove the growing point, but the growing point was included.The impedance spectra were analysed by an asymmetric distributedcircuit model. The impedance spectra were measured at two phasesof hardiness and after freezing, i.e. (a) before hardening,(b) after hardening in controlled conditions, and (c) aftercontrolled frost exposure at -16 °C after hardening. Twomodel parameters, i.e. intra- and extracellular resistance,increased with hardening. The intracellular resistance and theskewness factor before hardening, and the ratio between thosetwo parameters before and after hardening, strongly correlatedwith hardening of different varieties of English ryegrass. Theextracellular resistance and the relaxation time decreased asa result of the frost exposure at -16 °C. Cold acclimation; electrical impedance; English ryegrass; frost hardiness; impedance spectroscopy; Lolium perenne     

Determination of unfrozen water in winter cereals at subfreezing temperatures

The freezing of water in acclimated and nonacclimated cereals was studied using pulsed nuclear magnetic resonance spectroscopy. The quantity of unfreezable water per unit dry matter was not strongly dependent on the degree of cold acclimation. In contrast, the fraction of water frozen which was tolerated by nonacclimated winter cereals and by an acclimated spring wheat (Triticum aestivum L.) was less than in acclimated hardy cereals. The freezing curves had the following form:L_T = L₀ΔTm/T + KL_T and L₀ are liquid water per unit dry matter at T and 0 C, respectively. ΔTm is the melting point depression and K is the liquid water which does not freeze.     

N uptake and growth responses to sub-lethal freezing in the grass Poa pratensis L.

Background and aims

Climate warming has the potential to increase both the exposure and vulnerability of grass roots to frost in temperate regions by reducing snow cover and altering the timing of cold acclimation. Despite a strong research focus on the direct effects of freezing on grass mortality, the direct sub-lethal effects of freezing on grass performance have not been well-characterized. We examined sub-lethal responses of the grass Poa pratensis to variation in the timing, severity, rate and length of freezing.

Methods

We assessed short term root functional responses (¹⁵N uptake) and longer term plant growth responses to freezing administered both under controlled conditions in a refrigerated incubator, and in the field by manipulating snow and litter cover.

Results

In fall and spring, ¹⁵N uptake declined in response to 1?day of freezing down to ?10?°C or to 3?days of freezing at ?5?°C, whereas in winter, ¹⁵N uptake was insensitive to freezing. Long term growth responses were similar, with reduced growth only occurring for grasses frozen for 3?days at ?5?°C in spring, but not for grasses frozen in fall or winter. Snow and litter removal intensified soil freezing over winter, but did not significantly affect plant growth.

Conclusions

Our results demonstrate that while P. pratensis is relatively tolerant to frost damage over winter, it may be vulnerable to sub-lethal frost effects in fall, and particularly in spring. These sub-lethal effects occur at temperatures approximately 15–20?°C warmer than the published LT₅₀ values for this species.     

Survival of Ice Nucleation-Active and Genetically Engineered Non-Ice-Nucleating Pseudomonas syringae Strains after Freezing

The survival after freezing of ice nucleation-active (INA) and genetically engineered non-INA strains of Pseudomonas syringae was compared. Each strain was applied to oat seedlings and allowed to colonize for 3 days, and the plants were subjected to various freezing temperatures. Plant leaves were harvested before and after freezing on two consecutive days, and bacterial populations were determined. Populations of the INA wild-type strain increased 15-fold in the 18 h after the oat plants incurred frost damage at −5 and −12°C. Plants colonized by the non-INA strain were undamaged at −5°C and exhibited no changes in population size after two freeze trials. As freezing temperatures were lowered (−7, −9, and −12°C), oat plants colonized by the non-INA strain suffered increased frost damage concomitant with bacterial population increases following 18 h. At −12°C, both strains behaved identically. The data show a relationship between frost damage to plants and increased bacterial population size during the following 18 h, indicating a potential competitive advantage of INA strains of P. syringae over non-INA strains in mild freezing environments.