Effect of cold hardening on sensitivity of winter and spring wheat leaves to short-term photoinhibition and recovery of photosynthesis

Photoinhibition of photosynthesis and its recovery were studied in wheat (Triticum aestivum L.) leaves grown at nonhardening (20°C) and cold-hardening (5°C) temperatures. Cold-hardened wheat leaves were less susceptible to photoinhibition at 5°C than nonhardened leaves, and the winter cultivars, Kharkov and Monopol, were less susceptible than the spring cultivar, Glenlea. The presence of chloramphenicol, a chloroplastic protein synthesis inhibitor, increased the susceptibility to photoinhibition, but cold-hardened leaves still remained less susceptible to photoinhibition than nonhardened leaves. Recovery at 50 μmol m⁻² s⁻¹ photosynthetic photon flux density and 20°C was at least biphasic, with a fast and a slow phase in all cultivars. Cold-hardened leaves recovered maximum fluorescence and maximum variable fluorescence in the dark-adapted state during the fast phase at a rate of 42% h⁻¹ compared with 22% h⁻¹ for nonhardened leaves. The slow phase occurred at similar rates (2% h⁻¹) in cold-hardened and nonhardened leaves. Full recovery required up to 30 h. Fast-recovery phase was not reduced by either lowering the recovery temperature to 5°C or by the presence of chloramphenicol. Slow-recovery phase was inhibited by both treatments. Hence, the fast phase of recovery does not require de novo chloroplast protein synthesis. In addition, only approximately 60% of the photochemical efficiency lost through photoinhibition at 5°C was associated with lost [¹⁴C]atrazine binding and, hence, with damage to the secondary quinone electron acceptor for photosystem II-binding site. We conclude that the decrease in susceptibility to photoinhibition exhibited following cold hardening of winter and spring cultivars is not due to an increased capacity for repair of photoinhibitory damage at 5°C but reflects intrinsic properties of the cold-hardened photosynthetic apparatus. A model to account for the fast component of recovery is discussed.     

Mitochondrial Activity and Ethanol Accumulation in Ice-encased Winter Cereal Seedlings

Cold-hardened dark-grown seedlings of winter wheat (Triticum aestivum L.) and winter rye (Secale cereale L.) are killed during total encasement in ice at −1 C at a rate related to the initial cold hardiness of the cultivars. Few plants remain alive after 7 days of encasement. Nonhardened seedlings are rapidly killed in ice. The respiratory properties of mitochondria isolated from plants after increasing periods of ice encasement decline slowly, and activity is little impaired when intact plants are about 50% killed. Electron microscopy indicates that mitochondrial structure is not disrupted until 3 weeks of ice encasement. Ethanol accumulates in hardened and nonhardened plants in ice, but at levels which are not toxic to the plants.     

Metabolic changes associated with vernalization of wheat. I. Carbohydrate and nitrogen patterns

A spring wheat (Triticum aestivum) and an obligate winter wheat (Triticum compactum) variety were each grown for 5 weeks in controlled environments at 2° and 25°. The threshold for flower induction in the winter wheat was 4 to 5 weeks at 2°, whereas the spring wheat had no low temperature requirement for flowering. Changes in the levels of carbohydrate and nitrogen fractions in the wheat leaves were determined during their growth in the cold and warm environments. There was an enhanced accumulation of the 5 carbohydrate fractions in both wheat varieties grown at 2° compared to 25°. Highly significant differences in the levels of sucrose, oligosaccharides, and starch were found between the spring and winter varieties grown at 2°. The winter wheat seedlings grown at 2° accumulated much more of these carbohydrates than the corresponding spring wheat. The carbohydrate patterns in both varieties grown at 25° were nearly identical except for the final 2 weeks of growth.

The level of nitrogenous substances in the tissues grown at 2° was much higher than in the corresponding tissues grown at 25°. The only significant difference between the spring and winter varieties was in the soluble protein fraction. This fraction rose nearly 3-fold in the winter variety grown at 2°, whereas it remained nearly constant in the similarly grown spring wheat. Most of the changing chemical patterns observed in relation to the vernalization treatment appear to be metabolic alterations associated with low temperature rather than alterations directly related with the vernalization response.

     

Low growth temperature effects a differential inhibition of photosynthesis in spring and winter wheat

In vivo room temperature chlorophyll a fluorescence coupled with CO₂ and O₂ exchange was measured to determine photosynthetic limitation(s) for spring and winter wheat (Triticum aestivum L.) grown at cold-hardening temperatures (5°C/5°C, day/night). Plants of comparable physiological stage, but grown at nonhardening temperatures (20°C/16°C, day/night) were used in comparison. Winter wheat cultivars grown at 5°C had light-saturated rates of CO₂ exchange and apparent photon yields for CO₂ exchange and O₂ evolution that were equal to or greater than those of winter cultivars grown at 20°C. In contrast, spring wheat cultivars grown at 5°C showed 35% lower apparent photon yields for CO₂ exchange and 25% lower light-saturated rates of CO₂ exchange compared to 20°C grown controls. The lower CO₂ exchange capacity is not associated with a lower efficiency of photosystem II activity measured as either the apparent photon yield for O₂ evolution, the ratio of variable to maximal fluorescence, or the level of reduced primary quinone electron acceptor maintained at steady-state photosynthesis, and is most likely associated with carbon metabolism. The lower CO₂ exchange capacity of the spring cultivars developed following long-term exposure to low temperature and did not occur following over-night exposure of nonhardened plants to 5°C.     

Sucrose Synthase Expression during Cold Acclimation in Wheat

When wheat (Triticum aestivum) seedlings are exposed to a cold temperature (2-4°C) above 0°C, sucrose accumulates and sucrose synthase activity increases. The effect of a cold period on the level of sucrose synthase (SS) was investigated. Using antibodies against wheat germ SS, Western blots studies showed that the amount of the SS peptide increased during 14 days in the cold, when plants were moved from 23°C to 4°C. The level of SS diminished when plants were moved back to 23°C. Northern blots of poly(A)⁺ RNA, confirmed a five- to sixfold induction of SS in wheat leaves during cold acclimation. These results indicate that SS is involved in the plant response to a chilling stress.     

Frost injury and heterogeneous ice nucleation in leaves of tuber-bearing solanum species : ice nucleation activity of external source of nucleants

The heterogeneous ice nucleation characteristics and frost injury in supercooled leaves upon ice formation were studied in nonhardened and cold-hardened species and crosses of tuber-bearing Solanum. The ice nucleation activity of the leaves was low at temperatures just below 0°C and further decreased as a result of cold acclimation. In the absence of supercooling, the nonhardened and cold-hardened leaves tolerated extracellular freezing between −3.5° and −8.5°C. However, if ice initiation in the supercooled leaves occurred at any temperature below −2.6°C, the leaves were lethally injured.

To prevent supercooling in these leaves, various nucleants were tested for their ice nucleating ability. One% aqueous suspensions of fluorophlogopite and acetoacetanilide were found to be effective in ice nucleation of the Solanum leaves above −1°C. They had threshold temperatures of −0.7° and −0.8°C, respectively, for freezing in distilled H₂O. Although freezing could be initiated in the Solanum leaves above −1°C with both the nucleants, 1% aqueous fluorophlogopite suspension showed overall higher ice nucleation activity than acetoacetanilide and was nontoxic to the leaves. The cold-hardened leaves survived between −2.5° and −6.5° using 1% aqueous fluorophlogopite suspension as a nucleant. The killing temperatures in the cold-hardened leaves were similar to those determined using ice as a nucleant. However, in the nonhardened leaves, use of fluorophlogopite as a nucleant resulted in lethal injury at higher temperatures than those estimated using ice as a nucleant.

     

Fructan metabolism in wheat in alternating warm and cold temperatures

The objective of this research was to develop a system in which the direction of fructan metabolism could be controlled. Three-week-old wheat seedlings (Triticum aestivum L. cv Caldwell) grown at 25°C were transferred to cold temperature (10°C) to induce fructan synthesis and then were transferred to continuous darkness at 25°C after defoliation and fructan degradation monitored. The total fructan content increased significantly 1 day after transferring from 25°C to 10°C in both leaf blades and the remainder of the shoot tissue, 90% of which was leaf sheath tissue. Leaf sheaths contained higher concentrations of fructan and greater portions of high molecular weight fructan than did leaf blades. Fructan content in leaf sheaths declined rapidly and was gone completely within 48 hours following transfer to 25°C in darkness. In leaf blades the invertase activity fluctuated during cold treatment. The activity of sucrose:sucrose fructosyl transferase increased markedly during cold treatment, while fructan hydrolase activity decreased slightly. In leaf sheaths, however, the activity of invertase decreased rapidly upon transfer to cold temperature and remained low. Trends in sucrose:sucrose fructosyl transferase and hydrolase activity in sheaths were the same as those of leaf blades. Sheath invertase and hydrolase activity increased when plants were transferred back to darkness at 25°C, while sucrose:sucrose fructosyl transferase activity decreased. These results indicate that changing leaf sheath temperature can be utilized to control the direction of fructan metabolism and thus provide a system in which the synthesis or degradation of fructan can be examined.     

Apoplastic Sugars, Fructans, Fructan Exohydrolase, and Invertase in Winter Oat: Responses to Second-Phase Cold Hardening

Changes in apoplastic carbohydrate concentrations and activities of carbohydrate-degrading enzymes were determined in crown tissues of oat (Avena sativa L., cv Wintok) during cold hardening. During second-phase hardening (−3°C for 3 d) levels of fructan, sucrose, glucose, and fructose in the apoplast increased significantly above that in nonhardened and first-phase-hardened plants. The extent of the increase in apoplastic fructan during second-phase hardening varied with the degree of fructan polymerization (DP) (e.g. DP3 and DP4 increased to a greater extent than DP7 and DP > 7). Activities of invertase and fructan exohydrolase in the crown apoplast increased approximately 4-fold over nonhardened and first-phase-hardened plants. Apoplastic fluid extracted from nonhardened, first-phase-hardened, and second-phase-hardened crown tissues had low levels, of symplastic contamination, as determined by malate dehydrogenase activity. The significance of these results in relation to increases in freezing tolerance from second-phase hardening is discussed.     

Temperature-Induced Change in the Water Relations of Abies amabilis (Dougl.) Forbes

Water conductance through Abies amabilis seedlings was measured while the roots were exposed to temperatures from 15 to 0.25°C. Before conductance was measured, the seedlings were preconditioned for 3 months at either a high temperature (23°C) or a low temperature (3°C). For both groups of seedlings, conductance decreased as root temperature decreased. Conductance was lowest at 0.25°C. In addition, preconditioning at 3°C for 3 months significantly lowered conductance to water at all root temperatures. Under the same environmental conditions, seedlings preconditioned at 3°C had less than 25% of the transpirational water loss of seedlings preconditioned at high temperature. A decrease in leaf osmotic potential also resulted from low temperature preconditioning. In trees growing in the subalpine forest, which is the natural habitat of Abies amabilis, both decreased leaf conductance to water vapor and lower osmotic potentials were evident in winter. Since in winter the temperature of the soil in the subalpine zone remains less than 1°C for many months, lowered leaf conductance and decreased osmotic potentials appear to be mechanisms which aid in preventing desiccation damage.     

Interactions among Flooding, Freezing, and Ice Encasement in Winter Wheat

Exposure of winter wheat (Triticum aestivum L.) to various combinations of flooding and freezing stresses induces much greater damage than the individual stresses. Cold-hardened plants flooded for 1 week or exposed to −6°C for 1 week show 100% survival, while survival of plants exposed to both stresses simultaneously is reduced by 20 to 30%, and cold hardiness decreases by several degrees. The level of nonstructural carbohydrates increases in crown tissue during cold acclimation, but decreases when the plants are exposed to flooding or to −6°C for 1 week. The respiratory capacity of crown tissue segments declines when the plants are stressed. Uptake of ⁸⁶Rb by the roots of intact seedlings declines after exposure to either freezing or flooding, whereas passive efflux of amino acids is observed after freezing but not following flooding. This study has shown that detectable stress-induced metabolic changes occur in winter wheat before the applied stress is severe enough to reduce survival.     

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Respiration rates of Zea mays L. seedling tissues grown at 30 and 14°C were measured at 25°C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 30°C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 14°C, the increase in alternative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 14°C after growth at 30°C for 2 days had the same alternative oxidase capacity as seedlings grown at 30°C. Seedlings grown at 14°C for 10 to 12 days, then transferred to 30°C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondria were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 20°C. At 41°C, very little alternative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 41°C was reversible. After incubation at 41°C, the alternative oxidase capacity measured at 25°C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 41°C as they did when incubated at 25°C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.     

Role of Abscisic Acid in the Induction of Freezing Tolerance in Brassica napus Suspension-Cultured Cells

Brassica napus suspension-cultured cells could be hardened in 6 days at 25°C by the addition of mefluidide or ABA to the culture medium. Cells treated with mefluidide (10 milligrams per liter) or ABA (50 micromolar) attained an LT₅₀ of −17.5°C or −18°C, respectively, while the LT₅₀ for the comparable nonhardened control (sucrose) was −10°C. The increased freezing tolerance of mefluidide-treated cells was paralleled by a 4- to 23-fold increase in ABA, as measured by gas-liquid chromatography using electron capture detection. Application of 1 milligram per liter of fluridone, an inhibitor of abscisic acid biosynthesis, prevented the mefluidide-induced increase in freezing tolerance and the accumulation of ABA. Both these inhibitory effects of fluridone were overridden by 50 micromolar ABA in the culture medium. On the basis of these results, we concluded that increased ABA levels are important for the induction of freezing tolerance in suspension-cultured cells.     

Temperature characteristics and adaptive potential of wheat ribosomes

The translational efficiency of wheat ribosomes was studied as a function of an in vivo temperature pretreatment of wheat seedlings (Triticum aestivum L.). Ribosomes were isolated from heat-pretreated (36°C) and reference (4°C, 20°C) wheat seedlings. The efficiency of the ribosomes in translating polyuridylic acid was assayed. Ribosomes from heat-pretreated seedlings exhibit a threefold enhanced incorporation rate of phenylalanine as compared to ribosomes from wheat seedlings adapted to 20 or 4°C. This difference develops within 24 hours after onset of the heat treatment of seedlings following a 3 hour lag phase. The temperature induced changes can be traced back to the cytoplasmic ribosomes, since cycloheximide inhibits translation almost completely. Thermal inactivation of ribosomes occurs at 45°C, irrespective of the temperature pretreatment of the wheat seedlings. Specific differences in the yield of ribosomes, in the polyribosomal profiles, and in the apparent Arrhenius' activation energy of protein synthesis were observed depending on the age and the temperature pretreatments. The results presented here are considered an important molecular correlation to phenotypical temperature adaptation of in vivo protein synthesis in wheat (M Weidner, C Mathée, FK Schmitz 1982 Plant Physiol 69: 1281-1288).     

Spatial and Temporal Association of Outbreaks of H5N1 Influenza Virus Infection in Wild Birds with the 0°C Isotherm

Wild bird movements and aggregations following spells of cold weather may have resulted in the spread of highly pathogenic avian influenza virus (HPAIV) H5N1 in Europe during the winter of 2005–2006. Waterbirds are constrained in winter to areas where bodies of water remain unfrozen in order to feed. On the one hand, waterbirds may choose to winter as close as possible to their breeding grounds in order to conserve energy for subsequent reproduction, and may be displaced by cold fronts. On the other hand, waterbirds may choose to winter in regions where adverse weather conditions are rare, and may be slowed by cold fronts upon their journey back to the breeding grounds, which typically starts before the end of winter. Waterbirds will thus tend to aggregate along cold fronts close to the 0°C isotherm during winter, creating conditions that favour HPAIV H5N1 transmission and spread. We determined that the occurrence of outbreaks of HPAIV H5N1 infection in waterbirds in Europe during the winter of 2005–2006 was associated with temperatures close to 0°C. The analysis suggests a significant spatial and temporal association of outbreaks caused by HPAIV H5N1 in wild birds with maximum surface air temperatures of 0°C–2°C on the day of the outbreaks and the two preceding days. At locations where waterbird census data have been collected since 1990, maximum mallard counts occurred when average and maximum surface air temperatures were 0°C and 3°C, respectively. Overall, the abundance of mallards (Anas platyrhynchos) and common pochards (Aythya ferina) was highest when surface air temperatures were lower than the mean temperatures of the region investigated. The analysis implies that waterbird movements associated with cold weather, and congregation of waterbirds along the 0°C isotherm likely contributed to the spread and geographical distribution of outbreaks of HPAIV H5N1 infection in wild birds in Europe during the winter of 2005–2006.     

Freezing injury and root development in winter cereals

Upon exposure to 2°C, the leaves and crowns of rye (Secale cereale L. cv `Puma') and wheat (Triticum aestivum L. cv `Norstar' and `Cappelle') increased in cold hardiness, whereas little change in root cold hardiness was observed. Both root and shoot growth were severely reduced in cold-hardened Norstar wheat plants frozen to −11°C or lower and transplanted to soil. In contrast, shoot growth of plants grown in a nutrient agar medium and subjected to the same hardening and freezing conditions was not affected by freezing temperatures of −20°C while root growth was reduced at −15°C. Thus, it was apparent that lack of root development limited the ability of plants to survive freezing under natural conditions.

Generally, the temperatures at which 50% of the plants were killed as determined by the conductivity method were lower than those obtained by regrowth. A simple explanation for this difference is that the majority of cells in the crown are still alive while a small portion of the cells which are critical for regrowth are injured or killed.

Suspension cultures of Norstar wheat grown in B-5 liquid medium supplemented with 3 milligrams per liter of 2,4-dichlorophenoxyacetic acid could be cold hardened to the same levels as soil growth plants. These cultures produce roots when transferred to the same growth medium supplemented with a low rate of 2,4-dichlorophenoxyacetic acid (<1 milligram per liter). When frozen to −15°C regrowth of cultures was 50% of the control, whereas the percentage of calli with root development was reduced 50% in cultures frozen to −11°C. These results suggest that freezing affects root morphogenesis rather than just killing the cells responsible for root regeneration.

     

Resistance to low temperature photoinhibition is not associated with isolated thylakoid membranes of winter rye

In vivo measurements of chlorophyll a fluorescence indicate that cold-hardened winter rye (Secale cereale L. cv Musketeer) develops a resistance to low temperature-induced photoinhibition compared with nonhardened rye. After 7.2 hours at 5°C and 1550 micromoles per square meter per second, the ratio of variable fluorescence/maximum fluorescence was depressed by only 23% in cold-hardened rye compared with 46% in nonhardened rye. We have tested the hypothesis that the principal site of this resistance to photoinhibition resides at the level of rye thylakoid membranes. Thylakoids were isolated from cold-hardened and nonhardened rye and exposed to high irradiance (1000-2600 micromoles per square meter per second) at either 5 or 20°C. The photoinhibitory response measured by room temperature fluorescence induction, photosystem II electron transport, photoacoustic spectroscopy, or [¹⁴C]atrazine binding indicates that the differential resistance to low temperature-induced photoinhibition in vivo is not observed in isolated thylakoids. Similar results were obtained whether isolated rye thylakoids were photoinhibited or thylakoids were isolated from rye leaves preexposed to a photoinhibitory treatment. Thus, we conclude that increased resistance to low temperature-induced photoinhibition is not a property of thylakoid membranes but is associated with a higher level of cellular organization.     

Leucine transport in cells isolated from cold-hardened and nonhardened winter rye

The properties of the leucine transport systems of cells isolated from dark-grown cold-hardened and nonhardened winter rye (Secale cereale L. cv. Puma) epicotyls were remarkably similar. After 1 hour of incubation, leucine was accumulated in the cells 80- to 100-fold above that of the external medium, but the transported leucine was not metabolized. Approximately one-third of the accumulated leucine was present in the vacuole after 40 minutes of incubation. At 25°C, efflux of leucine from the vacuole was 6 to 10 times slower than it was from the cytoplasm, while at 5°C efflux from the cells was inhibited.     

Developmental history affects the susceptibility of spinach leaves to in vivo low temperature photoinhibition

Room temperature chlorophyll a fluorescence was used to determine the effects of developmental history, developmental stage, and leaf age on susceptibility of spinach to in vivo low temperature (5°C) induced photoinhibition. Spinach (Spinacia oleracea cv Savoy) leaves expanded at cold hardening temperatures (5°C day/night), an irradiance of 250 micromoles per square meter per second of photosynthetic proton flux density, and a photoperiod of 16 hours were less sensitive than leaves expanded at nonhardening temperatures (16 or 25°C day/night) and the same irradiance and photoperiod. This differential sensitivity to low-temperature photoinhibition was observed at high (1200) but not lower (500 or 800 micromoles per square meter per second) irradiance treatment. In spite of a differential sensitivity to photoinhibition, both cold-hardened and nonhardened spinach exhibited similar recovery kinetics at either 20 or 5°C. Shifting plants grown at 16°C (day/night) to 5°C (day/night) for 12 days after full leaf expansion did not alter the sensitivity to photoinhibition at 5°C. Conversely, shifting plants grown at 5°C (day/night) to 16°C (day/night) for 12 days produced a sensitivity to photoinhibition at 5°C similar to control plants grown at 16°C. Thus, any resistance to low-temperature photoinhibition acquired during growth at 5°C was lost in 12 days at 16°C. We conclude that leaf developmental history, developmental stage, and leaf age contribute significantly to the in vivo photoinhibitory response of spinach. Thus, these characteristics must be defined clearly in studies of plant susceptibility to photoinhibition.     

Evaluation of Polyamine and Proline Levels during Low Temperature Acclimation of Citrus

The polyamines (PA) putrescine (Put), spermidine (Spd), and spermine (Spm) were measured during 3 weeks exposure to cold hardening (15.6°C day and 4.4°C night) and nonhardening (32.2°C day and 21.1°C night) temperature regimes in three citrus cultivars: sour orange (SO) (Citrus aurantium L.), `valencia' (VAL) (Citrus sinensis L. Osbeck), and rough lemon (RL) (Citrus jambhiri Lush). The changes in PA were compared to the amount of free proline, percent wood kill and percent leaf kill. A 2- to 3-fold increase in Spd concentrations were observed in hardened RL, SO, and VAL leaves compared to nonhardened leaves. Spermidine reached its highest level of approximately 200 nanomoles per gram fresh weight after 1 week of acclimation in both SO and VAL leaves, while RL spermidine content continued to increase up to the third week of acclimation. Spm levels in acclimated VAL and RL leaves increased 1- to 4-fold. However, SO leaves Spm content decreased with acclimation. Putrescine levels in SO and VAL increased 20 to 60% during the first 2 weeks of acclimation then declined after 3 weeks. RL putrescine content was not affected by cold acclimation. The data presented here provided direct relationship between increased Spd concentration and citrus cold hardiness. Free proline was 3- to 6-fold higher in acclimated than in nonacclimated trees. Results also demonstrate that in acclimated versus nonacclimated citrus trees the absolute amount rather than the ratio of increase in free proline is more important in predicting their ability to survive freezing stress.     

Characterization of Plasma membrane of winter wheat (TritiCum aestivum L.) Seedlings and Changes in ATP hydrolysing activity under low temparature stress

Plasma membranes were isolated from young seedlings of cold hardened and non-hardened winter wheat using aqueous polymer two phase system consisting of dextran-polyethyleneglycol. Electron microscopic analysis and activities of various marker enzymes revealed that the upper phase was enriched in plasma membrane and free from contamination by other membranes. The fraction thus obtained possessed Mg ATP hydrolysing activity with an optimum pH 6.5 and the activity was stimulated by K⁺. The activity was highly sensitive to orthovanadate, diethylstilbesterol, dicyclohexylcarbodimide, insensitive to azide and nitrate. It was suggested that the ATP hydrolysis was catalysed by H⁺ ATPase. Mg ATP hydrolysing activity in cold hardened seedlings was 38% higher than that from non-hardened seedlings.