Components of Pink Snow Mould Resistance in Winter Wheat are Expressed Prior to Cold Hardening and in Detached Leaves

Resistance to pink snow mould, caused by Microdochium nivale, was investigated in four resistant winter wheat lines from the USDA World Cereal Collection (CI9342, CI14106, PI173440 and PI181268) and three Nordic wheat lines (Bjørke, Rida and V1004). Pink snow mould resistance was tested in non‐hardened and cold‐hardened plants incubated under artificial snow cover and in detached leaf segments mounted on water agar and incubated at either 3°C in darkness or at room temperature with light during the day. The wheat lines CI9342, CI14106 and PI181268 were more resistant than the Nordic lines, both before and after cold hardening. Thus, although cold hardening strongly increases the level of snow mould resistance in all the wheat lines, some resistance mechanisms are also present prior to cold hardening in some of the resistant lines. CI9342, CI14106 and PI181268 also had a higher level of resistance than the other lines in the detached leaf assay, indicating that these lines have some resistance mechanisms acting in the leaves. The resistance of PI173440 was expressed only in intact hardened plants and not in non‐hardened plants or in detached leaves. This indicates that this line relies on cold hardening‐related changes in the crown for its resistance. In the detached leaf assay the rate of lesion development varied greatly between leaves of different order. The highest correlation with the whole plant test was obtained when using secondary leaves and incubation at 3°C in the dark.     

Exposure to subfreezing temperature and a freeze-thaw cycle affect freezing tolerance of winter wheat in saturated soil

Winter wheat is sown in the autumn and harvested the following summer, necessitating the ability to survive subfreezing temperatures for several months. Autumn months in wheat-growing regions typically experience significant rainfall and several days or weeks of mild subfreezing temperatures at night, followed by above-freezing temperatures in the day. Hence, the wheat plants usually are first exposed to potentially damaging subfreezing temperatures when they have high moisture content, are growing in very wet soil, and have been exposed to freeze-thaw cycles for a period of time. These conditions are conducive to freezing stresses and plant responses that are different from those that occur under lower moisture conditions without freeze-thaw cycles. This study was conducted to investigate the impact of mild subfreezing temperature and a freeze-thaw cycle on the ability of 22 winter wheat cultivars to tolerate freezing in saturated soil. Seedlings that had been acclimated at +4°C for 5 weeks in saturated soil were frozen to potentially damaging temperatures under three treatment conditions: (1) without any subzero pre-freezing treatment; (2) with a 16-h period at ?3°C prior to freezing to potentially damaging temperatures; and (3) with a freeze-thaw cycle of ?3°C for 24 h followed by +4°C for 24 h, followed by a 16-h period at ?3°C prior to freezing to potentially damaging temperatures. In general, plants that had been exposed to the freeze-thaw cycle survived significantly more frequently than plants frozen under the other two treatments. Plants that had been exposed to 16 h at ?3° (without the freeze-thaw cycle) before freezing to potentially damaging temperatures survived significantly more frequently than plants that were frozen to potentially damaging temperatures without a subzero pre-freezing treatment. These results indicated that cold-acclimated wheat plants actively acclimate to freezing stress while exposed to mild subfreezing temperatures, and further acclimate when allowed to thaw at +4°C for 24 h. The cultivar Norstar had the lowest LT₅₀ (temperature predicted to be lethal to 50% of the plants) of the 22 cultivars when frozen with either of the subzero pre-freezing treatments, but several cultivars had lower LT₅₀ scores than Norstar when frozen without a subzero pre-freezing treatment. We conclude it may be possible to improve winterhardiness of wheat grown in saturated soil by combining the ability to effectively respond to mild subzero pre-freezing temperatures with a greater ability to withstand freezing to damaging temperatures without a subzero pre-freezing exposure.     

Frost tolerance and biochemical changes during hardening and dehardening in contrasting white clover populations

Successful winter survival of perennial plants, like white clover, is dependent on proper timing of both hardening and dehardening. The purpose of this study was to investigate the regulation of these processes in two cultivars (AberCrest and AberHerald) and two Norwegian ecotypes (Særheim collected at 58°46′N lat. and Bodø at 67°20′N lat.) of white clover (Trifolium repens L.). For hardening and dehardening, plants were exposed to controlled temperature conditions and frost hardiness of stolons was tested by programmed freezing at the rate of 3°C per hour. In addition, stolons were analysed for starch, soluble sugars and soluble amino acids. Cultivars AberCrest and AberHerald, selected for growth at low temperature and winter hardiness in the United Kingdom, were significantly less hardy than the Norwegian populations. After six weeks of hardening (2 weeks at 6°C and 4 weeks at 0.5°C), estimated LT₅₀ values were ?13.8, ?13.0, ?17.8 and ?20.3°C for AberCrest, AberHerald, Saerheim and Bodø, respectively. The rate of dehardening increased with increasing temperature. At low temperature (6°C), the northern ecotype from Bodø was more resistant to dehardening than AberHerald. However, at 18°C the absolute rate of dehardening (°C day^?1) was twice as high in Bodø as in AberHerald plants. Stolon elongation during dehardening was initiated at lower temperatures in AberHerald than in plants of the Bodø ecotype. The content of total soluble sugars, sucrose and the amino acids proline and arginine were significantly higher in hardy plants of Bodø than in those of AberHerald. Sucrose levels decreased during dehardening and correlations between sucrose content and LT₅₀ during this process were statistically highly significant for both Bodø and AberHerald. The least hardy populations of white clover were characterized by thick stolons, long internodes and large leaves.     

Ascorbic acid content in relation to frost hardiness injury in Citrus limon leaves

Leaves of the lemon tree have a variable content of ascorbic acid throughout the year. When they are kept under low, damaging temperatures the original content of ascorbic acid decreases sharply, so that when it loses about 70% the damage of the tissue is irreversible, as judged from the results of a test for survival in appropriate tissue culture. Losses of ascorbic acid are due to oxidation because they do not occur in the absence of oxygen. Analysis of the content of ascorbic acid can serve as a rapid and simple test for survival of the leaves to freezing temperatures. By this method critical temperatures and times are determined for two very different rates of thawing. It is also found that hardening of leaves, that takes place in fall, provides an extra resistance of about 3 °C over the leaves in spring-summer. Natural content of ascorbic acid, in contrast to that found in other plants, is lower in hardened (winter) than in dehardened leaves.     

Seasonal Changes in the Composition and Content of Dehydrins in Winter Wheat Plants

Seasonal changes in the pattern and content of dehydrins in winter wheat (Triticum aestivum) plants grown under field and laboratory conditions were studied by one-dimensional PAGE and immunochemical methods. During hardening, plants accumulated dehydrin-like polypeptides with mol wts of 209, 196, 66, 50, and 41 kD. In winter, low-molecular-weight dehydrins with mol wts of 24, 22, 17, 15, and 12 kD were synthesized and accumulated as well. Their content dropped sharply in spring when plants became unhardened. Accumulation/disappearance of these proteins corresponded to the fluctuations in wintering plant frost tolerance before winter and in spring. It is assumed that both high- and medium-molecular-weight dehydrins are involved in plant stress responses and adaptation, whereas low-molecular-weight dehydrins are evidently involved only in the process of low-temperature adaptation.     

NaHSO3对盐胁迫下小麦幼苗氮同化酶及脯氨酸含量的影响

以普通小麦(Triticum aestivumL.)为材料,研究了NaHSO3对不同盐度胁迫下小麦幼苗氮素同化酶和脯氨酸含量的调节。结果表明,盐胁迫降低了叶片中硝酸还原酸(NR)的活性,加入NaHSO3之后,NR活性表现出进一步的降低。谷氨酰胺合成酶(GS)在低浓度盐胁迫下活性增加,在高浓度盐胁迫下活性降低;NaHSO3加入时,即便在低盐浓度下GS活性也降低。依赖于NADH的谷氨酸脱氢酶(NADH-GDH)和依赖于NADP的异柠檬酸脱氢酶(NADP-ICDH)的变化趋势一致,在盐胁迫下它们的活性都明显增加;NaHSO3加入促进了它们活性的进一步增加,尤其对NADH-GDH活性的促进更为明显。游离脯氨酸在高浓度盐胁迫下大量积累,在低浓度盐胁迫下含量增加不明显;NaHSO3促进了盐胁迫下脯氨酸的积累,提示了NaHSO3促进了盐胁迫下小麦幼苗碳氮营养元素的贮存。     

Hardening, abscisic acid, proline and freezing resistance in two winter wheat varieties

Two varieties of winter wheat ( Triticum aestivum L.) differing in freezing resistance ("Holme" from Sweden, freezing resistant, and "Amandus" from Germany, less freezing resistant) were hardened for five weeks by gradually reducing the day/night temperature from 20°C/15°C during the first week to 2° C/0° C during the fifth week and the photoperiod from 15 to 9 h. This treatment increased the freezing resistance of both varieties in comparison to unhardened control plants. Hardening caused an increase in osmolarity of cell sap and in the levels of proline and abscisic acid (ABA). Increase in osmolarity preceded the increase in ABA level, and proline levels increased later than ABA levels. Holme had higher values of osmolarity as well as higher levels of ABA and proline. but the differences between the two varieties were significant only for proline. Since the pressure potential remained constant or increased slightly during the hardening period, it is suggested that the accumulation of ABA is due to the hardening process and not to simple water stress caused by cold-induced inhibition of water uptake by the root.
Spraying hardened plants with 10⁻⁴ M ABA 24 h before a freezing test increased freezing resistance in both varieties, but did not obliterate the differences in freezing resistance between the two varieties. Spraying hardened plants with an aqueous proline solution (10%, w/v) was without effect on freezing resistance. It is concluded that the hardening procedure causes an accumulation of ABA in winter wheat leaves and that ABA is involved in the chain of events leading to freezing resistance.     

Cold-induced changes of enzymes,proline, carbohydrates,and chlorophyll in wheat

Quantitative changes in total leaf soluble proteins, proline, carbohydrate content, chlorophyll fluorescence, guaiacol peroxidase (POD) and catalase (CAT) activities were determined in a less cold-hardy (LCH) spring cv. Kohdasht (LT₅₀ = −6°C), a semi cold-hardy (SCH) facultative cv. Azar 2 (LT₅₀ = −15°C), and a cold-hardy (CH) winter cv. Norstar (LT₅₀ = −26°C) of wheat (Triticum aestivum L.) exposed to 4°C for 9 weeks. Seedlings were grown in a controlled growth room for 14 days at 20°C and then transferred to 4°C (experimental day 0) for 63 days (cold treatment); otherwise they were maintained continuously at 20°C (control treatment). The samples were harvested 0, 2, 21, 28, 42, and 63 days after exposure to 4°C. The results showed significant low temperature (LT)-induced accumulation of total soluble proteins, proline, and carbohydrates and elevation in activities of CAT and POD in leaves of SCH and CH winter cultivars rather than in LCH spring cultivar. In contrast, the chlorophyll fluorescence (F _v/F _m) declined during LT treatment irrespective of cultivar. The results suggest that developmental traits such as vernalization requirement of wheat affects on cold-tolerance expression system of plants.     

Mitochondrial energy-dissipating systems (alternative oxidase,uncoupling proteins,and external NADH dehydrogenase) are involved in development of frost-resistance of winter wheat seedlings

Gene expression, protein synthesis, and activities of alternative oxidase (AOX), uncoupling proteins (UCP), adenine nucleotide translocator (ANT), and non-coupled NAD(P)H dehydrogenases (NDex, NDPex, and NDin) were studied in shoots of etiolated winter wheat (Triticum aestivum L.) seedlings after exposure to hardening low positive (2°C for 7 days) and freezing (?2°C for 2 days) temperatures. The cold hardening efficiently increased frost-resistance of the seedlings and decreased the generation of reactive oxygen species (ROS) during further cold shock. Functioning of mitochondrial energy-dissipating systems can represent a mechanism responsible for the decrease in ROS under these conditions. These systems are different in their response to the action of the hardening low positive and freezing temperatures. The functioning of the first system causes induction of AOX and UCP synthesis associated with an increase in electron transfer via AOX in the mitochondrial respiratory chain and also with an increase in the sensitivity of mitochondrial non-phosphorylating respiration to linoleic and palmitic acids. The increase in electron transfer via AOX upon exposure of seedlings to hardening freezing temperature is associated with retention of a high activity of NDex. It seems that NDex but not the NDPex and NDin can play an important role in maintaining the functional state of mitochondria in heterotrophic tissues of plants under the influence of freezing temperatures. The involvement of the mitochondrial energy-dissipating systems and their possible physiological role in the adaptation of winter crops to cold and frost are discussed.     

Accumulation of Free Proline at Low Temperatures

The accumulation of free proline in the first leaves of barley, Hordeum distichum L., and wheat, Triticum aestivum L., in response to a range of low temperatures was examined with 10-day-old plants. In barley (cv. Prior) no proline accumulated at 8°C or above, but in wheat (cv. Gabo) proline accumulated at 12°C and lower temperatures. In barley, the first leaf survived for 29 days following transfer to 5°C and continued to accumulate proline throughout this period. In contrast, the first leaves of plants maintained at 20°C survived for 13 days only and accumulated no proline. Proline accumulation at low temperature was shown to be light-dependent, both in intact plants and excised leaf sections, and the light requirement could not be replaced by supplying leaf segments with precursors of proline. Proline accumulation in response to water stress was not light-dependent at 20°C but was at 5°C. Inter-specific and intra-specific variation in the extent of accumulation in response to low temperature was also examined. Considerable variation was encountered but there was no clear relationship with geographical distribution or chilling sensitivity for the species and no correlation with accumulation in response to water stress in the cultivars of barley examined.     

Non-invasive monitoring of the light-induced cyclic photosynthetic electron flow during cold hardening in wheat leaves

The effect of irradiance during low temperature hardening was studied in a winter wheat variety. Ten-day-old winter wheat plants were cold-hardened at 5 degrees C for 11 days under light (250 micromol m(-2) S(-1)) or dark (20 micromol m(-2) s(-1)) conditions. The effectiveness of hardening was significantly lower in the dark, in spite of a slight decrease in the Fv/Fm chlorophyll fluorescence induction parameter, indicating the occurrence of photoinhibition during the hardening period in the light. Hardening in the light caused a downshift in the far-red induced AG (afterglow) thermoluminescence band. The faster dark re-reduction of P700+, monitored by 820-nm absorbance, could also be observed in these plants. These results suggest that the induction of cyclic photosynthetic electron flow may also contribute to the advantage of frost hardening under light conditions in wheat plants.     

Regulation of proline and ethylene levels in rape seedlings for freezing tolerance

This study was aimed to investigate the possibility of regulating free proline content and ethylene production in the resistant to abiotic stress cv. ‘Hornet H’ and the tolerant to stress cv. ‘Sunday’ of winter rapeseed seedlings by pretreatment with exogenous L-proline and L-glutamine in non-acclimated and cold-acclimated seedlings in relation to freezing tolerance. The ratio of proline content in acclimated (at 4°C) versus non-acclimated (18°C) ‘Hornet H’ seedlings increased 2.12-fold and in ‘Sunday’ seedlings 1.95-fold. Exogenously applied, proline and glutamine produced a positive effect on free proline content in both cold-acclimated and non-acclimated seedlings. At a temperature of -1°C the proline content significantly increased in non-acclimated and especially in cold-acclimated seedlings. At an intensified freezing temperature (?3°C, ?5°C, ?7°C), the proline content decreased in comparison with that at ?1°C, but glutamine, especially proline, in cold-acclimated seedlings takes part in free proline level increase and in seedlings’ resistance to freezing. Ethylene production increased in cold-acclimated conditions and under the effect of exogenous proline and glutamine. In freezing conditions, ethylene production decreased, but in cold-acclimated seedlings and under pretreatment of proline and glutamine the ethylene synthesis was intensive. Thus, free proline content and ethylene production increase in cold-acclimated winter rapeseed seedlings and under pretreatment with glutamine and especially with proline. Free proline is involved in the response to cold stress, and its level may be an indicator of cold-hardening and freezing tolerance, but the role of ethylene in the regulation of cold tolerance remains not quite clear.     

Accumulation of Free Proline in Citrus Leaves during Cold Hardening of Young Trees in Controlled Temperature Regimes

Free proline increased in leaves of orange (Citrus sinensis [L.] Osb. cv. Valencia) and grapefruit (Citrus paradisi Macfad. cv. Star Ruby) trees on a wide range of citrus rootstocks during cold hardening. Increases in sugars accompanied proline accumulation. During cold hardening, the rate of proline accumulation was greater in old than in young leaves. In leaves of grapefruit trees kept in the dark during cold hardening, neither proline nor sugars increased and the degree of cold hardiness was less than in trees exposed to light. Like sugar accumulations, proline accumulation does not reflect specific degrees of cold hardiness in citrus cultivars.     

Cold acclimation of wheat (Triticum aestivum): Properties of enzymes involved in proline metabolism

Two cultivars of wheat ( Triticum aestivum L.), a winter wheat, Kharkov, and a spring wheat, Glenlea, were acclimated under controlled conditions at 2 temperatures, 5°C and 25°C with a 12-h photoperiod. Water content, protein and proline concentrations were determined. Enzymatic properties (activity and apparent energy of activation) were investigated for enzymatic systems involved in 2 pathways of proline metabolism, the glutamic acid and ornithine pathways. Four enzymes were studied, proline dehydrogenase (PDH, EC 1.5.1.2), glutamate dehydrogenase (GDH, EC 1.4.1.2-4), glutamine synthetase (GS, EC 6.3.1.2) and ornithine transaminase (OT, EC 2.6.1.13). Cold acclimation led to an accumulation of proline, a decrease in water content and an increase in soluble protein, especially in winter wheat. For both cultivars, cold acclimation modulated enzyme properties of PDH and GDH. Increased activities of GS and OT were observed as a result of cold acclimation in both cultivars, with the greatest increase in Kharkov. The apparent energy of activation of these 2 enzymes decreased, particularly for Kharkov, which accumulated proline in cold conditions.     

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.     

The effect of hypothermia on the content of 310 kD stress protein in seedlings of winter rye and wheat

The effect of hypothermia on the content of 310 kD stress protein in seedlings of winter rye and wheat was studied by rocket-immunoelectrophoresis and radioactive label. The 1-h low-temperature stress was found to result in an increase in the content of this protein at both above- and below-zero temperatures. It was found that the increase in the relative content of the protein with mol. wt 310 kD, under the effect of short-term low-temperature stress, occurs due to induction of its synthesis. It has been found that during cold hardening of winter wheat the content of this protein decreases up to 64% compared to the control during the first day of hardening but starts to rise by the third day and reaches 179% by the seventh day, which is well correlated with the increase in cold resistance of winter wheat plants during cold hardening.     

Plant water uptake by hard red winter wheat (Triticum aestivum L.) genotypes at 2°C and low light intensity

Background  

Hard red winter wheat (HRWW; Triticum aestivm L.) plants from genotypes selected in the Northern Great Plains of the U.S. have less tissue water after exposure to cool autumn temperatures than plants from the Southern Great Plains. It is generally assumed that the reduced tissue water content of northern compared to southern cultivars is due to an impedance to water uptake by northern plants as a result of the low autumn temperatures. We hypothesize that if low temperature impedes water uptake then less soil water would be removed by northern than by southern-selected cultivars. This hypothesis was tested by comparing plant water uptake of a northern (FR) and a southern (FS) cultivar in relation to their foliage water content at 2°C.     

Constitutive and cold-induced resistance of rye and wheat seedlings to oxidative stress

The influence of cold hardening of rye (Secale cereale L.) and wheat (Triticum aestivum L.) seedlings on their resistance to the oxidative stress (OS) agents, namely, 50 mM hydrogen peroxide or 5 mM iron (II) sulfate was studied. Unhardened rye seedlings were more resistant to hydrogen peroxide than those of wheat, since their growth was less inhibited, and they accumulated lesser amounts of lipid peroxidation products after a treatment with H₂O₂. The interspecific differences in responses to FeSO₄ were less significant. The unhardened seedlings of rye, in comparison with those of wheat, possessed more active guaiacol peroxidase (GPO) and more levels of anthocyanins and proline. In response to the OS agents, the unhardened rye seedlings enhanced activities of superoxide dismutase and catalase, whereas the wheat seedlings enhanced GPO activity and proline content. The cold hardening (6 days at 2°C) increased activities of antioxidant (AO) enzymes, contents of proline, sugars, and anthocyanins in seedlings of both species, and made the seedlings more resistant to the OS agents. After the cold hardening, rye seedlings were more resistant to OS than wheat seedlings. The hardened seedlings of both species activated the AO enzymes in response to H₂O₂ or FeSO₄ greater than the unhardened ones. However, the hardened wheat seedlings, in contrast to the unhardened ones, did not augment the proline content in contact with the OS agents. The conclusion was drawn on different contributions of AO enzymes and low-molecular weight compounds to the basal and induced by the cold—hardening resistances of rye and wheat seedlings to OS.     

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.