Unhardened Plants Research Articles

Change of Antioxidant Enzyme Activity during Low-Temperature Hardening of Nicotiana tabacum L. and Secale cereale L.

Changes in the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), and catalase (CAT) for the plants Nicotiana tabacum L. and Secale cereale L. during low-temperature hardening have been investigated. It was determined that a transient increase in the content of MDA occurs in tobacco plants at the beginning of the hardening period with subsequent activation of SOD, APX, and CAT. Unlike tobacco, winter rye has an MDA content and activity of SOD, APX, and CAT throughout the hardening period that remains at the level of unhardened plants. In tobacco cells, the majority of SOD activity was detected in chloroplasts (90%), while other cellular structures accounted for only 10% of the total activity identified in PAGE. In winter rye cells, SOD activity was distributed evenly between chloroplasts and other cellular compartments (46% in chloroplasts and 54% outside of chloroplasts). The distribution of APX and CAT activity in tobacco and winter rye cells was identical: all APX was concentrated in chloroplasts, while all catalase activity was manifested outside of chloroplasts. It is concluded that tobacco and winter rye plants showed different antioxidant defense strategies under conditions of low hardening temperatures. Tobacco plants provided protection against oxidative damage during low-temperature hardening by increasing activity of SOD, APX, and CAT. Winter rye plants were able to avoid the development of oxidative stress during hardening due to uniform distribution of SOD activity in cells, maintaining the constitutive activity of SOD, APX, and CAT, and, probably, due to the accumulation of nonenzymatic antioxidants.

The relationship between the differences in frost resistance of Arabidopsis and Thellungiella and heat shock proteins and dehydrins

Plants of extremophile Thellungiella (Thellungiella salsuginea (Pall.) OE Schulz) withstood freezing at −15°C for 2 h without hardening, whereas plants of Arabidopsis (Arabidopsis thaliana (L.) Heinh.) were damaged at −10°C and died at −15°C under these conditions. The content of heat shock proteins (HSPs) HSP101, HSP60 and constitutive HSC70 was significantly higher in unhardened Thellungiella plants than in unhardened Arabidopsis plants. The spectrum of dehydrins (DHNs) in unhardened Thellungiella plants was more diverse and their total content was higher than in unhardened Arabidopsis plants. Frost resistance of Arabidopsis increased after hardening (4°C, 7 days), and there was an increase in the content of HSP101 and HSP60, as well as in the content of the DHN with a mol wt of 70 kD. Thellungiella plants survived after hardening at −18°C, and the increase in the content of HSP101, HSP70, and HSP60 was significantly less pronounced than in Arabidopsis. At the same time, the content of DHNs in Thellungiella increased significantly during the hardening primarily because of the appearance of two DHNs (mol wts of 42 and 45 kD). It is assumed that an increased content of HSPs and DHNs and their greater diversity can be one of the factors of Thellungiella resistance to low temperatures as compared to Arabidopsis.

Low temperature and hardening effects on photosynthetic apparatus efficiency and survival of forage grass varieties

Freezing tolerance is essential for perennial plants and ability to adapt to extreme temperature is crucial for their survival in many environments. Freezing tolerance of hardened and unhardened plants of Dactylis glomerata and Lolium perenne varieties was probed by their quantum photosynthetic efficiency using the chlorophyll fluorescence technique. Quantum yield of photosystem II (PSII) electron transport (Φ<sub>PSII</sub>), maximal (F<sub>m</sub>’) and steady-state (F<sub>s</sub>) chlorophyll fluorescence yields of light-adapted samples were measured. Φ<sub>PSII</sub> depended on developmental phase, temperature and hardening process. A clear decline in PSII activity, especially after –10°C application was observed. Plant hardening during emergence phase had a positive impact on PSII activity, especially after –5°C application. After 72 h of –5°C temperature treatment, hardened plants showed quicker recovery of their photosynthetic apparatus (0.527–0.697) as compared to unhardened ones (0.224–0.330). Stress temperature of –10°C caused irreversible changes of photosynthetic apparatus of hardened and unhardened plants independently of growth phases (0.003–0.014). Φ<sub>PSII</sub> and F<sub>m</sub>’ parameters were strongly correlated with shoots survival under stress. Our results suggest that perennial plants’ hardening allows them to survive low temperatures due inter alia enhancing their photosynthetic machinery performance.

Lectin activity and composition in the wheat cell walls under the effects of low temperature and inhibitors of calcium signaling pathway

In the roots of winter wheat (Triticum aestivum L., cv. Mironovskaya 808) seedlings, the effects of neomycin (100 μM), an inhibitor of phospholipase C, and dilthiazem (250 μM), a blocker of calcium channels on lectin activity and composition at low-temperature treatment (2–3°C) were studied. Hypothermia induced the appearance of two peaks of cell wall-bound lectin activity, e.g., in 0.5 and 6 h. Under these conditions, the inhibitors suppressed lectin activity. In 0.5 h of hypothermia, substantial changes in total profile of proteins were observed: lectins with mol wts of 85, 78, and 54 kD disappeared, and novel lectins with mol wts of 110, 105, 70, 50, and 34.5 kD appeared. In the presence of dilthiazem, the set of lectin proteins remained similar to that in unhardened plants, and the increase in the lectin content and activity was observed only after 3-h exposure to low temperature. This indicates that blocking dilthiazem-sensitive calcium channels slowed plant response to stress and did not permitt the cell to start rapidly the development of defense mechanisms. The important role of lectins with mol wts of 110 and 60 kD in the formation of freeze tolerance is supposed because these lectins did not appear in the presence of dilthiazem.

Effect of cartolin on oryzalin-induced changes in lectin activity during low-temperature plant hardening

The effect of cartolin (0.33 μM), an antistress regulator of cytokinin type, on the cytoskeleton-dependent changes in lectin activity in the roots of unhardened (23°C) and cold-hardened (3°C, 7 days) 7-day-old plants of three cultivars of winter wheat (Triticum aestivum L.) was studied. In unhardened plants, cartolin increased activity of soluble and cell wall-bound lectins in a cultivar-specific mode. This is evidently important for subsequent enhancement of adaptation processes in the cell. The inhibitor of microtubule polymerization, oryzalin, reduced the activity of soluble lectins and increased that of cell wall-bound lectins. A reduced sensitivity of lectin activity to oryzalin after cartolin treatment could result from its stabilizing action on the cytoskeletal structures and on the interaction between cell-wall lectins and microtubules. The most efficient cartolin action, the suppression of oryzalin effect on lectin activity in hardened plants, was observed in the frost-sensitive wheat cultivar. It is likely that cartolin treatment is more efficient in the activation of adaptation processes occurring with the involvement of cytoskeletal structures in the cultivars of lower tolerance.

The effect of age and cold hardening on resistance to pink snow mould (Microdochium nivale) in perennial ryegrass (Lolium perenne L)

The effect of plant age and cold hardening on resistance to pink snow mould caused by Microdochium nivale was studied in perennial ryegrass. Resistance to M. nivale was estimated as relative regrowth after inoculation and incubation under artificial snow cover at 2°C. Resistance increased with increasing plant age. Cold hardened and unhardened plants of the same age displayed identical resistance. Preliminary studies indicate that expression of genes coding for the PR proteins chitinase and PR-1a increased during incubation of inoculated perennial ryegrass, but no clear difference in expression of these genes was found between plants of different ages, or in hardened versus unhardened plants.

Cold induced expression of plant defensin and lipid transfer protein transcripts in winter wheat

In winter wheat, low temperature hardening induces a non‐specific resistance to snow moulds and other fungi. In a suppressive subtractive hybridization cDNA library between winter wheat unhardened and hardened at 2°C, 17% of 186 sequenced cDNA clones were homologous to the small plant defence related proteins, γ‐thionin, γ‐purothionin or non‐specific lipid transfer proteins (LTPs), based on their nucleotide sequences or deduced from amino acid sequences. Several partial‐ and full‐length cDNA clones homologous to two defensin proteins (γ‐thionin, subfamily B‐2, γ‐purothionin, subfamily I), and non‐specific LTPs were isolated. Regulation of defensin transcripts was clearly different from that of the LTPs under controlled environments in the field. The γ‐thionin and γ‐purothionin transcripts were not expressed in unhardened plants grown at 20°C, strongly upregulated after 1–3 days hardening at 2°C, remained upregulated for 28 days hardening, and disappeared following 1 day of exposure to dehardening conditions at 20°C. The LTPs transcripts were constitutively expressed in plants growing at 20°C, gradually increased to maximum levels following 14–28 days at hardening, and remained upregulated following 1 and 7 days dehardening. In the field, the γ‐thionin and γ‐purothionin transcripts rapidly increased to maximum levels by mid‐November and decreased gradually during the winter until mid‐March LTP transcripts were highly expressed in the early autumn, further increased during December–January, then decreased during late winter and early spring. Differences in the pattern and level of expression of these transcripts were evident among cultivars; γ‐thionin expression was associated with freezing resistance among the genotypes tested under both controlled environment and field conditions.

Comparative study of frost tolerance and antioxidant activity in cereals

The aim of this study was to find a correlation between the frost tolerance of cereals and their antioxidant capacity. Frost-tolerant and frost-sensitive cereal species, including wheat, barley, rye, durum wheat and oat varieties and wheat chromosome substitution lines, were cold-hardened at low, gradually decreasing temperature for 7 weeks. Control, unhardened plants were grown at a constant temperature of 17/16 °C. Changes in the activity of antioxidant enzymes (catalase, ascorbate peroxidase, guaiacol peroxidase (GPx), glutathione-S-transferase and glutathione reductase (GR)) in the crown and leaves were determined in the control and in hardened plants. The glutathione-S-transferase in the crown and leaf, and the ascorbate peroxidase and GPx in the leaf were significantly higher in the hardened plants than in the controls. The catalase activity in the leaves was lower in the hardened plants. The highest correlation between the enzyme activity and frost tolerance was found in the case of GPx and ascorbate peroxidase from hardened leaves. Enzyme activities in the crown and in unhardened leaves showed no significant positive correlation.

Effect of plant age on water content in crowns of fall rye and winter wheat cultivars differing in snow mold resistance

Resistance to snow molds in winter wheat increases with plant age, and older plants express higher levels of resistance than young plants. Experiments were conducted to study the effect of plant age on percent crown water content (%CWC) and dry weights in fall rye and winter wheat cultivars grown under controlled environments and in the field at Lethbridge, AB. Under controlled environments, the oldest (6 wk of pre-hardening growth at 20°C) treatments accumulated the greatest dry weights following exposure of plants to 1 to 6 wk hardening conditions at 2°C, compared with younger (1 to 4 wk pre-hardening growth) treatments. Exposure of plants to hardening temperatures had the greatest effect on %CWC values, which decreased, gradually, from 82–89% (4.95–8.67 g H2O g–1 DW) in unhardened treatments to 67–72% (2.05–2.65 g H2O g–1 DW) in plants receiving the 6 wk pre-hardening and 6 wk hardening growth. However, the oldest treatments (4 to 6 wk pre-hardening growth) always exhibited the lowest %CWC values among all hardening treatments. The %CWC in the oldest (6 wk) unhardened plants was also lower ([Formula: see text] = 80.8% or 4.24 g H2O g–1 DW) than in the youngest (1wk) unhardened plants ([Formula: see text] = 91.2% or 11.31 g H2O g–1 DW ), demonstrating that water loss occurs in older plants in the absence of low hardening temperatures. In a field study at Lethbridge during the autumn, winter, and early spring of 1997–1998 and 1998–1999, different seeding dates were employed to obtain plants differing in age and developmental state. The %CWC in early-seeded treatments was lower during the autumn, and remained lower in early spring, compared with later seeded cultivars. The %CWC in crowns was negatively associated with the snow mold resistance rating of a fall rye and five winter wheat cultivars under controlled environment conditions, and among a fall rye and 13 winter wheat cultivars in the field; the highest correlation values in the field were observed from mid-November to mid-March during 1997–1998 (r = –0.84), and 1998–1999 (r = – 0.76). These results indicate that the type of snow mold resistance that increases with plant age is related to the accumulation of crown dry matter and the ability of wheat and rye plants to lose crown water in response to both extended growth at warm temperatures and hardening at low, above freezing temperatures. Key words: Carbohydrates, fructans, low temperature basidiomycete, Coprinus psychromorbidus

Glycine betaine accumulation and induction of cold tolerance in strawberry ( Fragaria X ananassa Duch.) plants

Endogenous glycine betaine levels in the leaves of strawberry ( Fragaria X ananassa Duch.) plants were determined during cold acclimation and in response to exogenous abscisic acid (ABA) application. Glycine betaine levels in the leaves increased nearly two-fold after 4 weeks of cold acclimation treatment during which the cold tolerance of leaves increased from −5.8 to −17°C. Exogenous application of ABA (100 μM) to plants triggered glycine betaine accumulation in unhardened plants. It also increased cold tolerance of leaves in both unhardened and cold-hardened plants. Similar to ABA, exogenous glycine betaine was effective in inducing cold tolerance in unhardened and cold-hardening plants. Exogenous application of glycine betaine (2 mM) to unhardened plants increased the cold tolerance of leaves almost two-fold within 72 h of application. In addition, it improved freezing survival and regrowth in whole plants. The results suggest that glycine betaine is involved in inducing cold tolerance both in response to exogenous ABA and during natural cold acclimation of strawberry plants.

Metabolic changes associated with cold‐acclimation in contrasting cultivars of barley

Cereal plants become more resistant to freezing when first exposed to a period of cold‐acclimation. Many physiological and molecular changes have been shown to occur at low temperatures, but the role and the contribution of each to frost resistance is still poorly understood. Two cultivars of barley (Hordeum vulgare L.), the winter barley Onice and the spring barley Gitane, were acclimated under controlled conditions under an 8‐h photoperiod at 4°C (light) and 2°C (dark) for 21 days. Changes in free proline, ABA, water‐soluble carbohydrates and free fatty acids were measured to assess their involvement in cold‐acclimation and to explain the different frost‐resistant capacities of the two cultivars. Exposure of barley plants to low temperature resulted in an equal increase in proline in both cultivars. During the first days of cold acclimation, ABA levels showed a peak in the frost‐resistant cultivar, lasting about 24 h, followed by a decrease. The water soluble carbohydrates reached their highest content after 3 days of hardening, although after 14 to 21 days of acclimation the carbohydrate content was similar to that of unhardened plants. The frost‐resistant Onice had a much higher free fatty acid content than the frost‐sensitive Gitane. Furthermore in Onice 86% of free farty acids was represented by unsaturated molecular species. Inolenic acid alone being 71%. In contrast, in the frost‐sensitive cultivar only 31% of free fatty acids was unsaturated and a large amount of 9‐oxo‐nonanoic acid, a product present in the linolenic acid cascade, was also detected.The ABA content after 2 days of hardening and the free fatty acid composition were clearly different between the two cultivars and may explain, at least in part, the different frost‐resistant capacities of Onice and Gitane.

Cold acclimation in eucalypt hybrids.

We evaluated cold resistance and the capacity for cold acclimation of different Eucalyptus genotypes. Seedlings of half-sib families of E. globulus and hybrids E. gunnii x globulus, E. viminalis x globulus and E. cypellocarpa x globulus were exposed daily for 56 days to a 9-h photoperiod at 14.7 degrees C, followed by 15 h in a dark cold room maintained at 2.5 degrees C with the root system maintained at 8 degrees C to cold harden the seedlings. Unhardened seedlings were maintained at about 16 degrees C during the dark period. Cold acclimation occurred in all families with decreases in the temperature causing 50% mortality (LT(50)) of between 1.5 and 3 degrees C. Both hardened and unhardened plants of hybrid families were more cold tolerant than E. globulus. A significant correlation between LT(50) and leaf osmotic pressure was observed; the increase in osmotic pressure in hardened plants was predominantly a result of an increase in the concentration of soluble sugars. Exotherm peaks were similar in hardened and unhardened plants. These results indicate that cold hardening increased the ability of eucalypts to endure extracellular ice formation. The maintenance of photosynthetic capacity in cold-hardened plants may also play a role in their response to freezing.

Photosynthetic Response to Chilling in Peanut

Recent evidence suggests peanut (Arachis hypogaea L.) may be sensitive to relatively mild night temperature in terms of both dry matter accumulation and yield. The objective of this research was to quantify the effects of a range of night temperatures (9–20 °C) on leaf CO2 exchange rate (CER) of unhardened plants and to study the mechanisms involved in the contrasting response of peanut cultivars to differing night temperature. Four peanut cultivars (Ontario Agricultural College [OAC] Ruby, OAC Garroy, Chico, and Early Bunch) were grown in either controlled‐environment cabinets or outdoors during August and September at Guelph, Ontario. Results of the outdoor study indicated a linear decline in CER as ambient night temperature fell from 20 to 13 °C for all cultivars except OAC Garroy, with OAC Ruby less sensitive to low night temperature than Chico or Early Bunch. Limitations to CER after chilling at 10 °C in the dark were greater for Chico and Early Bunch (CER reduced by ≈35%) than for OAC Ruby and OAC Garroy (CER reduced ≈15%). All cultivars showed the ability to recover CER within 24 to 48 h of alleviation of chilling conditions. Cultivars grown indoors showed differing responses to one or two successive chilling nights (Chico was more affected than all others), but after four successive chilling nights, all cultivars showed a similar linear response to night temperature (i.e., CER = 0.693 × Train + 11.8 ; r2 = 0.90). An analysis of the relative contribution of stomatal and mesophyll constraints to CER in indoor studies (chilled at 9 °C) indicated significant limitations due to each component, with the relative importance dependent on cultivar and chilling period duration. Results suggest that night temperatures may be an important factor in determining relative cultivar performance in cool production environments.

The Response of Some Australian Temperate Rain Forest Tree Species to Freezing Temperatures and Its Biogeographical Significance

The effect of freezing temperatures on hardened and unhardened plants of some Australian temperate rain forest canopy species was investigated and the results discussed in relation to their current geographic range and ecology, and to the changes in distribution and evolution apparent from the fossil record. There was less difference among unhardened plants than among hardened plants, although Eucryphia milliganii Hook. f. which is restricted to high latitudes, commonly occurring at high altitudes, showed the greatest frost resistance, and Ceratopetalum apetalum D. Don., Doryphora sassafras Endl., and Atherosperma moschatum Labill., which are common subcanopy trees and extend to lower latitudes than the other species, showed the least frost resistance of the unhardened plants. Following a hardening regime, the general order of

Host water potentials may restrict development of snow mould fungi in low temperature‐hardened grasses

The growth of the snow mould fungi Fusarium nivale (Fr.) Ces. and Typhula ishikariensis Imai at different water potentials was measured on potato dextrose broth supplemented with KCl or polyethylene glycol 6000. The dew point hygrometer method was used for determination of water potentials in both artificial growth media and plant material. Both fungi had a considerable decrease in dry weight production when water potentials of growth media were decreased from −0.7 to −3 MPa. Crown tissue from hardened plants of Phleum pratense L. (timothy, cvs Engmo, Grindstad) and Phalaris arundinacea L. (reed canary grass, cvs Hansvoll, American common, Vantage) showed a significant decrease in water potentials compared with unhardened plants. Thus, reduced availability of water may partly explain increased resistance to fungal pathogens in grasses after hardening.

LEAF CO2 EXCHANGE RATES IN WINTER RYE GROWN AT COLD-HARDENING AND NONHARDENING TEMPERATURES

CO2 gas exchange measurements were performed on cold-hardened and unhardened Puma rye (Secale cereale L.) leaves at 10 and 20 °C in the presence of 2 and 21% O2 and as a function of irradiance and CO2 concentration. A decrease in O2 concentration from 21 to 2% appeared to result in a differential stimulation of photosynthetic rates in cold-hardened and unhardened rye leaves. Under light saturating conditions the former exhibited photosynthetic rates that tended to be 1.4- to 1.5-fold higher in the presence of 2% O2 than 21% O2 when measured at either 10 or 20 °C. In contrast, unhardened rye leaves exhibited photosynthetic rates that tended to be about 1.3-fold higher in the presence of 2% O2 than 21% O2 when measured at 10 °C but about 1.8-fold higher when measured at 20 °C. Similarly, at high CO2 concentrations, leaves of unhardened plants exhibited a greater temperature-dependent stimulation of photosynthetic rates by low O2 than leaves of hardened plants. An increased capacity for CO2 utilization in cold-hardened rye could be observed when photosynthetic rates were monitored at 2% O2. Differences in transpiration rates were insufficient to account for these results. The increased capacity for CO2 utilization observed in vivo is discussed with respect to a recent report which described an increased capacity for photosynthetic electron transport in vitro in cold-hardened rye thylakoid membranes. However, photosynthetic acclimation to the contrasting growth temperatures could only be observed when CO2 exchange was measured at 2% O2. We conclude that photosynthetic acclimation in vivo may be severely limited due to the restrictions imposed by photorespiration.Key words: Cold-hardening, winter rye, CO2 exchange, photosynthesis

Simulation of in situ freezing damage of the photosynthetic apparatus by freezing in vitro of thylakoids suspended in complex media

Chloroplast thylakoid membranes were isolated from leaves of unhardened and cold-acclimated spinach (Spinacia oleracea L.). For freezethaw treatment, the membranes were suspended in complex media composed to simulate the solute concentrations in the chloroplast stroma in the unhardened and hardened states of the leaves. In particular, high concentrations of amino acids were applied for simulating the hardened state. After frost treatment, photosynthetic activities and chlorophyll fluorescence parameters of the thylakoids were tested to determine the degree of freezing damage. The results revealed a pattern of freezing injury similar to that observed upon frost treatment of thylakoids in situ. A major manifestation of damage was the inhibition of photosynthetic electron transport. Uncoupling of photophosphorylation, which is the dominating effect of freezing of thylakoids suspended in binary solutions (e.g., containing one sugar and one inorganic salt), was also visible but less pronounced in the complex media. Thylakoids obtained from cold-acclimated leaves did not exhibit an increased frost tolerance in vitro, as compared with thylakoids from unhardened plants. The results, furthermore, indicated a strong protective effect of free amino acids at the concentrations and composition found in chloroplasts of hardened leaves. The presence of inorganic salts in the complex media slightly stabilized rather than damaged the membranes during freezing. It is concluded that inactivation of thylakoids in situ may be understood as the destabilizing action of the combined solutes surrounding the thylakoids, occurring when solute concentration is raised due to freezing of water.

An appropriate physiological control for environmental temperature studies: comparative growth kinetics of winter rye

The accurate interpretation of physiological and biochemical alterations observed in plants grown under contrasting environmental conditions requires knowledge of their relative physiological ages. For this purpose, we compared the growth kinetics of winter rye (Secale cereale L. cv. Puma) at nonhardening and cold-hardening temperatures. Growth at nonhardening temperatures was characterized by a 10-day lag phase with the attainment of maximum growth after about 28 days. Growth at cold-hardening temperatures resulted in an extension of the lag phase to about 21 days with maximum growth being attained after 56 days. The calculated growth coefficient at cold-hardening temperatures was 35–40% of that at nonhardening temperatures. This relationship was consistent with growth parameters such as leaf dry weight, fresh weight, and area, but not with plant height. Although total leaf dry weight and total number of leaves per plant did not differ between nonhardened and cold-hardened plants at maximum growth, total leaf area per plant and stretched plant height was 3- to 4-times greater in nonhardened than in cold-hardened plants. This resulted in a fourfold increase in leaf dry weight per leaf area during growth at low temperature in contrast to the maintenance of a constant ratio during growth at nonhardening conditions. The increase in this ratio during low temperature growth was, in part, accounted for by a decrease in water content and an increase in cytoplasmic content. These results were confirmed by the investigation of growth on an individual leaf basis. However, the growth response of leaves 1 and 2 differed from that of leaves 3 and 4 when the leaf dry weight: leaf area ratio was measured as a function of time at cold-hardening temperatures. This indicates that the stage of leaf development influences its growth response to an altered environment. The results of the development of leaf freezing tolerance indicated that maximum vegetative growth appeared to coincide with maximum freezing tolerance of leaves from cold-hardened plants (−22 °C) but not of leaves from unhardened plants (−11 °C).

Predisposing Effects of Low Temperature on Resistance to Winter Stress Factors in Grasses

Abstract Seed propagated and vegetatively propagated plants from Phleum pratense and Phalaris arundinacea, hardened and unhardened, were exposed to the winter stress factors freezing and/or inoculation with snow mould fungi, Fusarium nivale and Typlutla ishikariensis. Exposure to both freezing and fungal inoculation in combination resulted in more severe damage than exposure to either factor alone. Artificial hardening increased resistance to the winter stress factors, the effect was especially pronounced on plants exposed to the combination of two stress factors. Resistance to freezing was significantly correlated with resistance to T. ishikariensis in hardened plants, but not in unhardened ones. Hardened and unhardened plants of the same genotype showed correlated resistance to T. ishikariensis, but not correlated freezing resistance. In the plant material investigated the variation in resistance due to environmental factors was greater than the genetic variation.

Differential Subunit Aggregation of a Purified Protein from Cold-hardened and Unhardened Puma Rye

Summary Purified ribulose-1,5-bisphosphate carboxylase-oxygenase from cold-hardened and unhardened Puma rye plants was osmotically concentrated by dialysis against buffer containing 25% (w/v) polyethylene-glycol for 16 h at 4°C in the presence or absence of β-mercaptoethanol. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of protein samples concentrated 100 to 200 fold in the absence of reducing agent indicated that the large subunit of the enzyme from unhardened rye plants aggregated to a greater extent than the large subunit of the enzyme from cold-hardened rye plants. This aggregation was reversed by the addition of β-mercaptoethanol to the concentrated enzyme samples implicating disulfide bond formation in the mechanism of aggregation. No structural alterations were observed by SDS polyacrylamide gel electrophoresis for the small subunits of the enzyme from either cold-hardened or unhardened rye plants. The relevance of these results to Levitt's sulfhydryl hypothesis is discussed.