Carbohydrate levels among winter wheat cultivars varying in freezing tolerance and snow mold resistance during autumn and winter

The LT₅₀ values and soluble carbohydrate levels in wheat ( Triticum aestivum L.) crowns and leaves were monitored throughout autumn and winter in cultivars varying in freezing tolerance and snow mold resistance during 1993/1994 and 1994/1995 in the field at Sapporo, Japan. During the first stage of hardening, from sowing to mid‐November, the pattern of accumulation of mono‐ and disaccharides was similar for all cultivars. During the second stage of cold hardening, from mid‐November to mid‐December, the greatest accumulation of mono‐ and disaccharides, without a corresponding increase in fructan, was observed among the freezing‐tolerant cultivars; and levels of simple saccharides rapidly decreased under snow cover. Conversely, levels of mono‐ and disaccharides in snow mold‐resistant cultivars were less than 70% of those in freezing‐tolerant cultivars before snow cover and maintained low levels throughout winter, while polysaccharide levels in snow mold‐resistant cultivars were about 120% of those in freezing‐tolerant cultivars in December. Sugar metabolism during the winter was examined using 18 cultivars in 1994/1995. LT₅₀ values were correlated to the greatest extent with total mono‐ and disaccharide and fructan content among wheat cultivars excluding snow mold‐resistant cultivars in December. Snow mold‐resistant cultivars tended to metabolize carbohydrates more slowly until the end of the snow cover period. This result suggested that the enzymatic metabolism of the synthesis of sugars and the conversion of fructan to cryoprotective sugars in response to low temperatures, especially subzero ones, might be different between the two contrasting types in resistance to winter stress.     

Carbohydrate and Proline Contents in Leaves, Roots, and Apices of Salt-Tolerant and Salt-Sensitive Wheat Cultivars1

Intra-specific variations in nonstructural carbohydrates and free proline were determined in leaves, apices, roots, and maturing seeds of two salt-tolerant cultivars (CR and Kharchia-65) and one salt-sensitive cv. Ghods of spring wheat (Triticum aestivum L.) grown in sand culture at various levels of salinity (0, 100, 200, and 300 mM NaCl and CaCl₂ at 5 : 1 molar ratio) under controlled environmental conditions. The levels of leaf, apex, and root ethanol-soluble carbohydrates, fructans, starch, and proline increased in line with elevating level of salinity in all three cultivars under investigation. The contents of proline, soluble and insoluble carbohydrates in the apex increased to levels exceeding those in the leaves and roots. Soluble carbohydrate content of salt-sensitive cv. Ghods was higher in the leaves, apices, and roots and lower in the maturing seeds than in the other cultivars at all levels of salinity except at 300 mM. The results show considerable variation in the amount of soluble, insoluble sugars, and proline among plant tissues and wheat genotypes in response to salinity. Higher soluble carbohydrates and fructan in leaves, roots and maturing seeds of stressed plants indicate that their accumulation may help plant to tolerate salinity. Salt-sensitive cv. Ghods accumulated less soluble sugars in the maturing seeds and higher soluble sugars in the apices, which might be used as an indicator in screening wheat genotypes for salinity tolerance.     

Carbohydrate content and glycosidase activities following cold hardening in two grass species

The freezing resistance of the grass species Phleum pratense L. (timothy) and Phalaris arundinaces L. increases significantly after cold hardening. The content and composition of soluble carbohydrates were determined in the plants after short day treatment, cold hardening and dehardening. The amounts of mono-, di- and trisaccharides were reduced during the short day treatment, increased during cold hardening and decreased again during dehardening. The total amounts of soluble carbohydrates (mono-, di-, tri- and polysaccharides) were the same in hardened and dehardened plants, indicating that during hardening soluble polysaccharides (fructose polymers, fructans) were converted to mono- and oligosaccharides. Sucrose increased most after hardening conditions and, in P. arundinacea , a significant increase in 1-F-fructosylsucrose (isokestose) was also observed.
Invertase (β-fructofuranosidase. EC 3.2.1.26) activity increased following cold hardening and decreased following dehardening, while the α-galactosidase (EC 3.2.1.22) activity seemed to increase after dehardening. The glycosidases are probably involved in the mobilisation of polysaccharides during cold hardening.     

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.     

Exogenous nitric oxide effect on fructan accumulation and FBEs expression in chilling-sensitive and chilling-resistant wheat

This study was to investigate the effect of exogenous nitric oxide (NO) on fructan accumulation and fructan biosynthesic enzymes (FBEs) expression in seedlings leaves of two wheat (Triticum aestivum L.) cultivars, winter wheat (Zhoumai18, ZM) and spring wheat (Yanzhan4110, YZ), under 4 °C. The seedlings of two wheat cultivars were subjected to different concentrations of sodium nitroprussiate (SNP) for 0, 24, 48, and 96 h. Relative water content (RWC) was increased by exogenous NO in YZ, but decreased in ZM. Except for glucose, fructose and fructans of degree of polymerization (DP) 3 in YZ, other soluble carbohydrates contents in the two wheat cultivars all increased to different degrees. The activities of FS (including sucrose: sucrose 1-fructosyltransferase (1-SST, EC: 2.4.1.99) and sucrose: fructan 6-fructosyltransferase (6-SFT, EC: 2.4.1.10)) were significantly higher than fructan: fructan 1-fructosyltransferase (1-FFT, EC: 2.4.1.100) in the seedlings of two wheat cultivars. The same phenomenon occurred to FBEs expression. In addition, sucrose content decreased while fructans content increased under low temperature, which was in accordance with the improved 1-FFT activity in ZM. Moreover, fructans content increased to a high level under high concentration of NO in ZM while kept at a constant low level in YZ. The expression levels of FBEs were universally higher in ZM than in YZ, which identified with the high frost resistance of the winter cultivar. It is concluded that exogenous NO treatment on wheat may be a good option to reduce chilling injury by regulating fructan accumulation in leaves. This is the first report owing that exogenous NO alleviated the negative effects of chilling stress by accumulating fructans in wheat.     

Fructan:fructan 1-fructosyltransferase,a key enzyme for biosynthesis of graminan oligomers in hardened wheat

Fructans play important roles not only as a carbon source for survival under persistent snow cover but also as agents that protect against various stresses in overwintering plants. Complex fructans having both ß-(2,1)- and ß-(2,6)-linked fructosyl units accumulate in wheat (Triticum aestivum L.) during cold hardening. We detected fructan: fructan 1-fructosyltransferase (1-FFT; EC 2.4.1.100) activity for catalyzing the formation and extension of ß-(2,1)-linked fructans in hardened wheat tissues, cloned cDNAs (wft3 and wft4) of 1-FFT, and analyzed the enzymatic properties of a wft3 recombinant protein (Wft3m) produced by yeast. Wft3m transferred ß-(2,1)-linked fructosyl units to phlein, an extension of sucrose through ß-(2,6)-linked fructosyl units, as well as to inulin, an extension of sucrose through ß-(2,1)-linked fructosyl units, but could not efficiently synthesize long inulin oligomers. Incubation of a mixture of Wft3m and another recombinant protein of wheat, sucrose:fructan 6-fructosyltransferase (6-SFT), with sucrose and 1-kestotriose produced fructans similar to those that accumulated in hardened wheat tissues. The results demonstrate that 1-FFT produces branches of ß-(2,1)-linked fructosyl units to phlein and graminan oligomers synthesized by 6-SFT and contributes to accumulation of fructans containing ß-(2,1)- and ß-(2,6)-linked fructosyl units. In combination with sucrose:sucrose 1-fructosyltransferase (1-SST; EC 2.4.1.99) and 6-SFT, 1-FFT is necessary for fructan synthesis in hardened wheat.     

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.     

The fructan syndrome: Evolutionary aspects and common themes among plants and microbes

Fructans are multifunctional fructose‐based water soluble carbohydrates found in all biological kingdoms but not in animals. Most research has focused on plant and microbial fructans and has received a growing interest because of their practical applications. Nevertheless, the origin of fructan production, the so‐called “fructan syndrome,” is still unknown. Why fructans only occur in a limited number of plant and microbial species remains unclear. In this review, we provide an overview of plant and microbial fructan research with a focus on fructans as an adaptation to the environment and their role in (a)biotic stress tolerance. The taxonomical and biogeographical distribution of fructans in both kingdoms is discussed and linked (where possible) to environmental factors. Overall, the fructan syndrome may be related to water scarcity and differences in physicochemical properties, for instance, water retaining characteristics, at least partially explain why different fructan types with different branching levels are found in different species. Although a close correlation between environmental stresses and fructan production is quite clear in plants, this link seems to be missing in microbes. We hypothesize that this can be at least partially explained by differential evolutionary timeframes for plants and microbes, combined with potential redundancy effects.     

Cold adaptation in the phytopathogenic fungi causing snow molds

Snow molds are psychrophilic or psychrotrophic fungal pathogens of forage crops, winter cereals, and conifer seedlings. These fungi can grow and attack dormant plants at low temperatures under snow cover. In this review, we describe the biodiversity and physiological and biochemical characteristics of snow molds that belong to various taxa. Cold tolerance is one of the important factors related to their geographic distribution, because snow molds develop mycelia under snow cover and because they should produce intra- and extracellular enzymes active at low temperatures for growth and infection. Basidiomycetous snow molds produce extracellular antifreeze proteins. Their physiological significance is to keep the extracellular environment unfrozen. The psychrophilic ascomycete Sclerotia borealis shows normal mycelial growth under frozen conditions, which is faster than that on unfrozen media at optimal growth temperature. This fungus does not produce extracellular antifreeze proteins, but osmotic stress tolerance enables the fungus to grow at subzero temperatures. In conclusion, different taxa of snow molds have different strategies to adapt under snow cover.     

Constituents of soybean cultivars differing in insect resistance

Leaf samples of two insect-resistant plant introductions (PI 227687 and PI 229358) and two susceptible (Ransom and Coker Hampton 266A) cultivars of soybeans, Glycine max, were analyzed at different growth stages for their contents of total nitrogen, carbohydrates, organic acids, and sterols. The two susceptible cultivars accumulated more total nitrogen and at a faster rate than did the two resistant plant introductions. At pod-filling, the two resistant cultivars had equivalent soluble carbohydrates and 33% more than the susceptible cultivars. The quantity of organic acids was essentially the same for the two susceptible cultivars. The resistant cultivars had distinctly different quantities from each other as well as from the susceptible cultivars. The quantity of total sterol of these soybean cultivars varied during the growth of the plant. The resistant cultivars accumulated sterol faster and by pod-filling contained from 20 to 50% more sterol than did the susceptible cultivars.     

Metabolic acclimation to hypoxia in winter cereals : low temperature flooding increases adenylates and survival in ice encasement

Cold hardened seedlings of winter wheat (Triticum aestivum L. em Thell) show an hypoxic hardening response: an exposure to low temperature flooding increases the tolerance of plants to a subsequent ice encasement exposure. Seedlings of winter barley (Hordeum vulgare L.) do not show such a response in similar experimental conditions. During ice encasement, there are general declines in adenylate energy charge (AEC), total adenylates and ATP:ADP ratios in the crown tissues of two winter wheat cultivars, and a winter barley, but rates of decline are faster in the barley. When the ice period is preceded by low temperature flooding of the whole plant, levels of the adenylate components are raised significantly in the wheats, and to a lesser extent in the barley. The survival of plants in ice preceded by flooding is related to the increased initial level of adenylates at the onset of the ice encasement stress, and the maintenance of higher levels of adenylates and ATP in the early stages of ice encasement as a result of accelerated rates of glycolysis. Higher survival of both winter wheat and barley plants during ice encasement in the light is also associated with significantly higher levels of AEC and adenylates in the early stages of ice encasement.     

Proline biosynthesis in winter plants due to exposure to low temperatures

The content of bound proline sharply increased in proteins of different organs of young plants of winter rape and winter wheat exposed for 72 h to temperatures from 0 to 2 °C while it decreased only in root tips of wheat plants. Free proline which at 20 °C occurs in all plant organs only in trace amounts, accumulated considerably after 72 h exposure to low temperatures in the above-ground organs and only slightly in the roots. Free proline did not accumulate during the first 24 h at 0 to 2 °C in detached leaves of winter wheat but it was incorporated into newly synthetized proteins in which proline content increased after 6 h incubation to its maximum ( + 11.75% in comparison to control); the content of free glutamate sharply decreased during the first 6 h of incubation and the accumulation of bound glutamate was belated in comparison to that of bound proline. Sucrose infiltrated into detached leaves of winter wheat strongly stimulated proline incorporation into proteins at low temperatures, but it did not influence glutamate incorporation. The results suggest that the main reason for thede novo proline biosynthesis during the first six hours of hardening of the plants is the synthesis of proteins rich in proline; free proline accumulates later predominantly in the above-ground organs as a surplus. The above-ground organs are dehydrated in the course of the hardening process approximately to the same extent both in the light and in the dark, but proline content increases much less in the dark than in tho light.     

Oxygen deficiency affects carbohydrate reserves in overwintering forage crops

Anaerobic conditions developing under an ice cover affect winter survival and spring regrowth of economically important perennial crops. The objective was to compare, during a prolonged period of low (<2%) O2 at low temperature, the concentration of carbohydrates of four plant species contrasting in their resistance to oxygen deficiency. Four perennial forage species, lucerne (Medicago sativa L.), red clover (Trifolium pratense L.), timothy (Phleum pratense L.), and cocksfoot (Dactylis glomerata L.) were subjected to a progressively developing oxygen deficiency stress by enclosing potted plants in gas-tight bags in late autumn for overwintering in an unheated greenhouse. Timothy was previously reported to be more resistant to oxygen deficiency than the three other species. Non-structural carbohydrates increased and remained at a higher concentration in timothy than in the other three species under low O2 concentration. Concentrations of sucrose, fructose, glucose, and fructans increased in response to oxygen deficiency in timothy, whereas the concentration of soluble sugars decreased under the same conditions in lucerne, red clover, and cocksfoot. The gene expression of glyceraldehyde-3-phosphate dehydrogenase increased in response to low oxygen concentration in oxygen deficiency-sensitive lucerne while it remained unchanged in the oxygen deficiency-resistant timothy. It is concluded that timothy maintains higher carbohydrate reserves under oxygen deficiency, a specific feature that could favour its winter survival and spring regrowth.     

Development of Frost Tolerance in Winter Wheat as Modulated by Differential Root and Shoot Temperature

Abstract: Winter wheat plants ( Triticum aestivum L. cv. Urban), grown in nutrient solution, were exposed to differential shoot/root temperatures (i.e., 4/4, 4/20, 20/4 and 20/20 C) for six weeks. Leaves grown at 4C showed an increase in frost tolerance from - 4C down to - 11 C, irrespective of root temperature. In 4/20 C plants, high root temperature decreased the rate of hardening of the leaves, but did not influence the final level of frost tolerance. In roots grown at 4C frost tolerance increased from - 3 C down to - 4 C, independently of shoot temperature. An accumulation of soluble sugars in the leaves was only observed when both shoot and root were grown at 4C and was not correlated with final frost tolerance achieved. However, the rate of hardening was correlated with the soluble sugar concentration. An increase in root soluble sugar concentration was exclusively observed in roots exposed to a temperature of 4C, irrespective of shoot temperature. Proline concentration only increased in plant parts exposed to a temperature of 4C. The present results indicate that the importance of root temperature in low-temperature hardening of winter wheat is limited, even though exposure to differential root and shoot temperatures brought about pronounced changes in growth, soluble sugar concentration, insoluble sugar concentration and proline concentration in roots and leaves.     

Changes in carbohydrates and freezing tolerance during cold acclimation of red raspberry cultivars grown in vitro and in vivo

Changes in LT50 and carbohydrate levels in response to cold acclimation were monitored in vitro and in vivo in red raspberry ( Rubus idaeus L.) cultivars with different levels of cold hardiness. Entire micropropagated plantlets or shoot tips from 3 cultivars were harvested before, during and after cold acclimation. Cane samples from container-grown plants of 4 cultivars were harvested before and during cold acclimation and deacclimation. Samples were evaluated for cold hardiness (LT50) by controlled freezing, then analyzed for carbohydrates, including starch, sucrose, glucose, fructose and raffinose. Hardiness of cold-acclimated 'Muskoka' and 'Festival' was superior to that of 'Titan' or 'Willamette'. In vitro plantlets had higher levels of soluble carbohydrates on a dry weight basis and higher ratios of sucrose:(glucose+fructose) than the container-grown plants. Total soluble carbohydrates, primarily sucrose, accumulated during cold acclimation in both plantlets (33–56% relative increase) and plants (143–191% relative increase). Sucrose increased 124–165% in plantlets and 253–582% in container-grown plants during acclimation and declined rapidly to the level of control plants during deacclimation. Glucose and fructose also accumulated, but to a lesser extent than sucrose. Raffinose concentrations were very low, but increased significantly during cold acclimation. In vitro, genotype hardiness was related to the high concentrations of total soluble carbohydrates, sucrose and raffinose. In vivo, hardier genotypes had lower concentrations of starch than the less hardy genotypes. These results demonstrated the importance of soluble carbohydrates, especially sucrose, in cold hardening of red raspberry and that the in vitro conditions or controlled acclimation conditions do not necessarily reflect the phenomena observed in vivo.     

Changes in the level of MACC during cold hardening and dehardening in winter wheat

Changes in the level of 1-(malonylamino)-cyclopropane-1-carboxylic acid (MACC) were determined in 6 winter wheat cultivars during cold hardening at 4°C. The cultivars differed by one degree of frost resistance within the range of degree II to VII of the COMECON scale. The time-course of changes in MACC level showed a similar pattern in all 6 cultivars; i.e. increase till day 6, no changes for the next 10 days, and then a steady decrease till the end of the hardening period. There was little difference between the final and the initial levels. The increase of MACC level, expressed as per cent of the original level, was not directly correlated with either the degree of frost resistance of the actual percentage of survival. In some cultivars. mean errors exceeded the difference in MACC accumulation between cultivars closest on the resistance scale.
The fate of MACC during the second half of hardening and after transfer of plants to 25°C was studied in cultivars Bezostaya and San Pastore. During the second half of the hardening period the level of MACC decreased in the leaves of both cultivars, but increased significantly in the roots. Within two days of transfer of the hardened plants to 25°C, the MACC level in leaves increased again, while that in the roots decreased. This finding, together with the preliminary evidence of very low MACC metabolism, strongly suggest that MACC accumulates in roots during the hardening period and when transferred to 25°C, it moves from roots to leaves.