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 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.     

Metabolic and ultrastructural changes associated with flooding at low temperature in winter wheat and barley

Cold-hardened winter wheat (Triticum aestivum L. cv. Fredrick) and winter barley (Hordeum vulgare L. cv. Dover) were exposed to total flooding at 2 C. Dover seedlings were damaged more quickly than Fredrick, and after 3 weeks of flooding the survival of Dover was reduced to 10% and Fredrick to about 50%. Tissue moisture was slightly greater in Dover than Fredrick throughout the 4-week flooding period. Carbon dioxide and ethanol accumulated throughout the 4-week flooding period in both cultivars. Lactic acid increased rapidly during the 1st week of flooding, and remained relatively constant during the remainder of the flooding period. Oxygen consumption of seedling shoot tissue after exposure to flooding declined abruptly after only 1 day of flooding, but recovered somewhat during the subsequent 2 weeks. The effect of flooding was more pronounced on the ultrastructure of Dover than Fredrick. Although proliferation of endoplasmic reticulum was observed in the early stages of flooding in both cultivars, the occurrence of distinct parallel arrays and concentric whorls of membranes was prevalent in the flooded barley. Severe ultrastructural damage to a large proportion of apical cells in both cultivars was observed after 2 to 3 weeks of flooding.     

Toxicity of Anaerobic Metabolites Accumulating in Winter Wheat Seedlings during Ice Encasement

Ice encasement damages cold-hardened winter wheat without major disruption of cellular organelles. CO₂ accumulates during total ice encasement to higher levels in Kharkov than in less hardy Fredrick wheat. Partial ice encasement and exposure to a nitrogen atmosphere at -1 C allows greater CO₂ accumulation but neither treatment is as damaging as total ice encasement. Lactic acid accumulates to low levels only during the 1st day of encasement and thereafter remains constant. Exposure of plants to a combination of 50% CO₂ and 5% ethanol reduces survival, with a cultivar difference similar to that found in ice-encased plants. Plants in CO₂ and ethanol show a proliferation of membranes and nuclear condensation similar to that in cells of ice-encased plants. Permeability increases markedly in the presence of CO₂ and ethanol, to levels similar to or greater than those of iced plants. Ethanol alone does not increase permeability but in combination with CO₂ raises permeability of the less hardy Fredrick, although not of Kharkov, but reduces survival of both cultivars. A comparison of the endogenous levels of ethanol, CO₂, and lactic acid at the 50% kill point of plants due to ice encasement or due to externally supplied metabolite indicates that only CO₂ accumulates to independently toxic levels. Permeability and ultrastructural evidence suggest that CO₂ and ethanol in combination are the agents reducing plant viability during ice encasement.     

Ultrastructural and Enzymic Studies of Cell Membranes from Ice-encased and Noniced Winter Wheat Seedlings

A marked increase in the amount of cisternal-like cytoplasmic membranes was observed after ice encasement of winter wheat (Triticum aestivum L.) seedlings. Linear sucrose gradients were employed to separate the various membrane components of the microsomal membrane fraction. NADH- and NADPH-cytochrome c reductase, two specific enzyme markers for plant endoplasmic reticulum (ER) were used to locate the ER in the linear gradients. The identity of the ER fraction was confirmed by determining the effect of EDTA and Mg²⁺ in the preparative media on the distribution of NADH- and NADPH-cytochrome c reductase activity within the gradient. In the presence of EDTA which dissociates ribosomes from ER, peaks of activity for the two enzymes were observed at a density corresponding to that for “smooth” ER. When the media also contained an appropriate concentration of Mg²⁺ to maintain the attachment of ribosomes to the ER, the peaks of activity for the enzymes shifted to a density corresponding to that for “rough” ER. NADH-cytochrome c reductase activity was similar for 24 C-grown and 2 C-grown iced seedlings, but significantly lower for 2 C noniced seedlings. No preferential increase in uptake of radioactive leucine or choline in the ER was observed during ice encasement. The accumulation of electron microscopically visible membrane arrays was not inhibited by the presence of protein synthesis inhibitors at concentrations which severely inhibited incorporation of [1-¹⁴C]leucine into membrane protein, but did not affect survival and growth of the seedlings. These observations indicate that the apparent proliferation of ER during ice encasement does not result from net membrane synthesis, but rather from reorganization of existing membrane elements within the cell.     

Membrane properties of isolated winter wheat cells in relation to icing stress

Isolated cell preparations of winter wheat (Triticum aestivum L.) were utilized to examine the effect of ice encasement at −1°C and exposure to ethanol on metabolic and biochemical properties of cells. Following icing and ethanol treatments, passive efflux of amino acids increased gradually with duration of exposure to the stress, and closely paralleled the decline in viability of cells. In contrast, uptake of ⁸⁶Rb declined much more rapidly than viability following exposure to icing or ethanol. Electron spin resonance spectroscopy studies revealed no significant change in molecular ordering within the cell membranes following icing or exposure to ethanol, whereas a small but significant increase in order was detected in the noniced controls. O₂ consumption by isolated cells declined only gradually due to icing and ethanol treatments, and remained relatively high even when cell viability was severely reduced. These results indicate that the plasma membrane is a primary site of injury during ice encasement and that damage to the ion transport system is the earliest manifestation of this injury.     

Ice-Encasement Injury to Microsomal Membranes Isolated from Winter Wheat Crowns : II. Changes in Membrane Lipids during Ice Encasement

The physical properties and chemical composition of microsomal membranes were examined during a 7 day period of ice encasement in crown tissue of winter wheat (Triticum aestivum L. cv Norstar). Membrane damage, detected as an increase in microviscosity and electrolyte leakage, began between 1 and 3 days of icing, and was associated with a reduction in the recovery of microsomal membranes from stressed tissue, an increase in the microsomal free fatty acid:total fatty acid ratio, and a decrease in the phospholipid:total fatty acid ratio. These trends were amplified between 3 and 7 days of ice encasement. Examination of the free and total fatty acid fractions showed there was a slight, but not statistically significant (P = 0.05) reduction in the degree of unsaturation of the total fatty acid fraction. The composition of the free and total fatty acid fractions were very similar during ice encasement. Furthermore, analysis of phospholipid classes revealed no significant change in the relative amounts of phosphatidylcholine, phosphatidylethanolamine, phosphatidic acid, or lysophospholipids in microsomal membranes during icing. Membrane injury during ice encasement apparently involves hydrolysis of the ester bond between glycerol and the acyl groups of the phospholipid resulting in loss of the phosphate-containing polar head group and a concomitant accumulation of free fatty acids in the bilayer.     

Impact of early and late winter icing events on sub‐arctic dwarf shrubs

Polar regions are predicted to undergo large increases in winter temperature and an increased frequency of freeze–thaw cycles, which can cause ice layers in the snow pack and ice encasement of vegetation. Early or late winter timing of ice encasement could, however, modify the extent of damage caused to plants. To determine impacts of the date of ice encasement, a novel field experiment was established in sub‐arctic Sweden, with icing events simulated in January and March 2008 and 2009. In the subsequent summers, reproduction, phenology, growth and mortality, as well as physiological indicators of leaf damage were measured in the three dominant dwarf shrubs: Vaccinium uliginosum, Vaccinium vitis‐idaea and Empetrum nigrum. It was hypothesised that January icing would be more damaging compared to March icing due to the longer duration of ice encasement. Following 2 years of icing, E. nigrum berry production was 83% lower in January‐iced plots compared to controls, and V. vitis‐idaea electrolyte leakage was increased by 69%. Conversely, electrolyte leakage of E. nigrum was 25% lower and leaf emergence of V. vitis‐idaea commenced 11 days earlier in March‐iced plots compared to control plots in 2009. There was no effect of icing on any of the other parameters measured, indicating that overall these study species have moderate to high tolerance to ice encasement. Even much longer exposure under the January icing treatment does not clearly increase damage.     

Electron Microscopic Cytochemical Studies on the Glycoproteins in Cellular Membranes of Winter Wheat Seedlings with Concanavalin A-Horseradish Peroxidase Method

The localization and distribution of the glycoproteins in cellular membranes of strong cold hardy winter wheat (Triticum aestivum L. cv. Yanda 1817) seedlings were studied employing the modified cytochemical method using Concanavalin A-Horseradish peroxidase conjugates. The results obtained showed that the reaction products of Horseradish peroxidase activity which indicated the presence of glycoproteins appeared as individual particles distributed at plasmalemma, endoplasmic reticulmn, nuclear envelope and tonoplast in the leaflet cells of the seedlings grown under optimum temperature 25℃ day/20℃ night. After cold acclimation of the seedlings at low temperature during late autumn and early winter, the quantity of glycoproteins distributed in endoplasmic reticulum and nuclear envelope was significantly increased, and glycoproteins were transported into almost all of the plasmodesmata. At some sites of mitochondria and plastides newly formed glycoproteins were observed. However, no distribution of glycoproteins was for,nd in Golgi bodies during the whole observations. The role of glycoproteins for the development and stability of cold resistance in plant through blocking up the plasmodesma passage in the overwintering period of winter wheat seedlings, and the possible differences of the synthetic site and transportation pathway of glycoproteins between animal and plant systems were discussed.     

Changes in adenine nucleotides and energy charge in isolated winter wheat cells during low temperature stress

Adenylate energy charge (AEC) and adenine nucleotide levels of isolated winter wheat (Triticum aestivum L. cv Kharkov 22 MC) cells exposed to various low temperature stresses were determined. During ice encasement at −1°C, nucleotide levels decreased gradually in approximate relation to a decline in cell viability. AEC values remained high even after 5 weeks of icing when cell viability was severely reduced. When isolated cell suspensions were exposed to various cooling and freezing regimes ranging from −10 to −30°C, cell damage was dependent on the minimum temperature imposed and the duration of exposure to the freezing stress. The levels of all three adenine nucleotides declined with increasing severity of the imposed stress, but AEC values remained high even at −30°C when nearly all of the cells were killed. The addition of 10 millimolar Ca²⁺ to cell suspensions enhanced survival during low temperature stresses, but did not influence nucleotide levels other than through its effect on cell viability. These results indicate that impairment of the ion transport system during the early stages of ice encasement prior to a detectable decline in cell viability cannot be attributed to changes in the adenylate energy charge system of the cell.     

Variable increases in cold hardiness induced in winter rape by plant growth regulators

Triazole and conventional growth regulators were tested for their ability to extend cold hardiness and improve the winter survival of winter rape (Brassica napus L.). Winter rape plants were grown in the field (Ottawa 45°23 N) and in growth cabinets. Plant growth regulators (PGRs) were applied during the early vegetative stage and the plants were allowed to cold harden. Cold-hardened plants from the field and cabinet were subjected to freezing and ice encasement tests in the laboratory. Some of the triazole PGRs reduced plant size by limiting cell expansion and increasing cell numbers. While cold hardiness and ice encasement tolerance were increased by some growth regulators, these effects were not consistent with time nor were they reflected in increased winter survival. Natural cold hardening may have eclipsed the PGR-induced hardening.PGR contribution number 1381.     

抗寒锻炼中不同抗寒性小麦细胞膜糖蛋白的细胞化学研究

本研究根据植物细胞的特点,修改了在动物和人体细胞方面立的酶标Con A的电镜细胞化学方法,成功地展现了2个不同抗寒性冬小麦品种幼苗在抗寒锻炼和脱锻炼过程中细胞膜系统上糖蛋白的分布动态,显示与Con A连接的标志酶-辣根过氧化物酶活性的反应产物呈颗粒状分散分布在质膜、内质网、核膜及液泡膜的一些部位上,揭示糖蛋白在冬小麦细胞膜系统上的分布似有其特定的位点。经抗寒锻炼后,强抗寒性品种燕大1817细胞内的糖蛋白在内质网和核膜上的分布量明显地增加;同时,几乎所有的胞间连丝通道中都有糖蛋白的分布。脱锻炼后,内质网和核膜上的糖蛋白分布量又减少,胞间连丝通道中的糖蛋白也消失,基本上回复到抗寒锻炼前的分布状态。抗寒性弱的冬小麦品种郑州39-1幼苗在同样的抗寒锻炼和脱锻炼过程中不产生这些明显的变化。这些结果说明,抗寒锻炼中内质网和核膜上糖蛋白分布量的增加,以及糖蛋白输入胞间连丝的动态变化是与植物抗寒力的提高和保持稳定密切相关的。     

A survey of aphids and aphid parasitoids in cereal fields in Denmark, and the parasitoids' role in biological control

In 1996 and 1997 a field survey of the abundance and species composition of cereal aphid primary and secondary parasitoids in spring barley, winter wheat and durum wheat was conducted in Zealand, Denmark. The purpose was to create a better understanding of the mechanisms underlying aphid–parasitoid dynamics in the field. Such an understanding can be used when developing biological control methods in cereals. In both years aphid attacks in cereals began in late June and never exceeded the economic threshold. In 1996 the first aphids were found in wheat on 26 June; in 1997 the first aphids were found on 24 June on both crops. The highest densities reached in 1996 were an average of six aphids per shoot in winter wheat and one aphid per shoot in spring barley. In 1997 the highest densities reached were 11 aphids per shoot in winter wheat and four aphids per shoot in spring barley. The aphid population collapsed by the end of July to early August in 1996, but it collapsed by mid-July in 1997. The onset and peak of parasitization were delayed in comparison to aphid infestation. Parasitism was 20–60% by the end of the cropping season in spring barley, and 30–80% in winter wheat and durum wheat in 1996. In 1997 parasitism did not exceed 3–11% in barley and was less than 2% in one winter wheat field but more than 40% in the other winter wheat field sampled. In both years most parasitism was due to Aphidiidae (Hymenoptera). The two dominant species were Aphidius ervi Haliday and Aphidius rhopalosiphi De Stefani-Perez. Hyperparasitism began after primary parasitism and increased progressively during the cropping season. The two years were similar in many respects, including for species composition of aphids and parasitoids. The late start of the aphid infestation may have contributed to the high level of parasitization found in 1996, but in 1997 the aphid infestation period was so short that a parasitoid population did not have time to build up.     

Mechanisms Coordinating Wheat Seedling Growth Response as Affected by Shoot/Root Ratio

Excision of four out of five roots in 7-day-old wheat seedlings (Triticum durum Desf.) rapidly suppressed shoot growth promoted biomass accumulation by the remained root largely due to its expanded branching. Next, the rate of shoot growth increased although was not completely recovered. After the reduction of the root system, the rate of photosynthesis in the leaves of seedlings did not decrease. As compared to the intact plants, auxins and cytokinins accumulated in the remained root, whereas in the growing part of the shoot, the level of auxins rapidly declined. Shoot growth rate was assumed to decrease after the excision of a part of the root system due to lower extensibility of growing tissue, and the promotion of lateral root formation on the remained root apparently resulted from active redistribution of phytohormones and assimilates between plant organs. The prime role of hormonal signals in the coordination of shoot and root growth is discussed.     

Effects of long-term barley monoculture on plant-affecting soil microbiota

Effects of soil microbiota on shoot and root growth of barley were tested in a greenhouse tube-growing system. Tubes were filled with a mixture of pure sand and various percentages of soils sampled from plots in three long-term field experiments measuring effects of various crop rotations on yield. Using 3% soil in the sand-soil mixture, shoot dry weight of barley test plants was reduced by about 35% and root depth by about 40% in soils from monoculture plots as compared to soils from crop-rotation plots. Typical root symptoms on poorly growing barley plants started as distinct dark-brown zones which then rapidly spread over the whole root system until the root tips ceased to grow. As tested in one experiment, the barley monoculture soil also affected wheat and oats, but to a lesser degree than it did barley. Most of the depressing effects of monoculture soil on barley were eliminated when soil samples were treated with metalaxyl or heated to 65°C for 2 hours. A Pythium sp. frequently isolated from barley roots showing typical symptoms affected barley, wheat and oats in the same way as did barley monoculture soil.     

Impacts of winter icing events on the growth,phenology and physiology of sub‐arctic dwarf shrubs

The Arctic is experiencing the greatest climate change in winter, including increases in freeze–thaw cycles that can result in ice encasement of vegetation. Ice encasement can expose plants to hypoxia and greater temperature extremes, but currently the impacts of icing on plants in the field remain little understood. With this in mind, a unique field manipulation experiment was established in heathland in northern Sweden with ice encasement simulated in early March 2008, 2009 and 2010 until natural thaw each spring. In the following summers we assessed the impacts on flowering, bud phenology, shoot growth and mortality and leaf damage (measured by chlorophyll fluorescence and electrolyte leakage) of the three dominant dwarf shrub species Empetrum nigrum, Vaccinium vitis‐idaea (both evergreen) and Vaccinium myrtillus (deciduous). Two consecutive winters of icing decreased V. vitis‐idaea flowering by 57%, while flowering of V. myrtillus and E. nigrum remained unaffected. Vaccinium myrtillus showed earlier budburst but shoot growth for all species was unchanged. Shoot mortality of V. myrtillus and V. vitis‐idaea increased after the first year (by 70 and 165%, respectively) and again for V. myrtillus following the third year (by 67%), while E. nigrum shoot mortality remained unaffected, as were chlorophyll fluorescence and electrolyte leakage in all species. Overall, the sub‐arctic heathland was relatively tolerant to icing, but the considerable shoot mortality of V. myrtillus contrasting with the general tolerance of E. nigrum suggests plant community structure in the longer term could change if winters continue to see a greater frequency of icing events.     

Effect of low temperature and calcium on survival and membrane properties of isolated winter wheat cells

Isolated cells obtained by enzymic digestion of young primary leaves of cold-hardened, dark-grown Kharkov winter wheat (Triticum aestivum L.) were exposed to various low temperature stresses. The initial uptake of ⁸⁶Rb was generally decreased by increasing concentrations of Ca²⁺, but after longer periods of incubation, the inhibiting effect of high Ca²⁺ levels diminished. Viability of isolated cells suspended in water declined rapidly when ice encased at −1°C, while in the presence of 10 millimolar Ca²⁺ viability declined only gradually over a 5-week period. Ice encasement markedly reduced ⁸⁶Rb uptake prior to a significant decline in cell viability or increased ion efflux. Cell damage increased progressively when the icing temperature was reduced from −1 to −2 and −3°C, but the presence of Ca²⁺ in the suspending medium reduced injury. Cell viability and ion uptake were reduced to a greater extent following slow cooling than after rapid cooling to subfreezing temperatures ranging from −10 to −30°C. The results from this study support the view that an early change in cellular properties due to prolonged ice encasement at −1°C involves the ion transport system, whereas cooling to lower subfreezing temperatures for only a few hours results in more general membrane damage, including loss of semipermeability of the plasma membrane.     

Time-course of pathogen induced accumulation of callose as mechanical protective barrier in wheat seedlings

Results of fluorescence microscopic study and quantitative luminescent analysis of pathogen induced callose accumulation in winter wheat seedlings of two cultivars with different resistance to causal agent of eyespot disease are presented. Higher content of constitutive callose in intact seedlings of unsusceptible cultivar at the initial stages of vegetation was determined. It correlates with resistance of this cultivar to Pseudocercosporella herpotrichoides—causal agent of eyespot disease. The increased pathogen induced accumulation of callose in seedlings of susceptible cultivar was revealed, however, it did not influence it protection against pathogen.     

Dynamics of nutrient remobilisation from seed of wheat genotypes during imbibition,germination and early seedling growth

The changes in nutrient content of grain tissues and seedling parts of two wheat genotypes (Triticum aestivum L., Excalibur and Janz) with low or high seed Zn content were followed from imbibition to early seedling development (12 days). The grains were separated into seed coat, endosperm and embryo, while the seedlings were separated into roots and shoots. The dry weight of the seed coat did not change throughout the experimental period, whereas the endosperm weight declined rapidly from day 4 onward. The weight of embryo did not show any difference between and within cultivars. About a half of seed Zn was remobilised into shoot and roots during 12 days of growth, regardless of the initial seed Zn content in both genotypes. The seed coat contained 55–77% of the total seed nutrients in the two wheat genotypes, except in the case of S (around 40%). Manganese, Fe, Ca, K, and P were remobilised effectively from the seed coat as well as from the endosperm, while remobilisation of Zn and Cu was relatively less from the seed coat than from the endosperm. After 10 days of growth, all nutrients monitored were completely remobilised from the endosperm. Remobilised K was directed primarily into shoots; an increase in K content in shoots was relatively higher than the accumulation of dry matter, with a consequent increase in K concentration in shoot tissue. The remobilisation of some nutrients (eg. Fe, Ca and Zn) from various grain tissues during inbibition, germination and early growth is different from the remobilisation in more mature plants.     

Competitive ability and tolerance of organically grown wheat cultivars to natural weed infestations

Competitive ability of a traditional winter wheat cultivar (Maris Widgeon) was compared with two modem cultivars (Hereward and Genesis) when grown organically in each of four seasons in Gloucestershire, UK. In two seasons, cultivars were compared at two sowing dates (September and October or November). Cv. Maris Widgeon was the tallest cultivar and intercepted most photosynthetically active radiation (PAR), particularly if sown early. Cv. Hereward was taller than cv. Genesis when sown early. Cv. Maris Widgeon accumulated most nitrogen and dry matter early in the season (until the end of March) when sown early, but not when sown late. Relative nitrogen and dry matter accumulation by cultivars later in the season depended on season and sowing date. In one season cv. Maris Widgeon had significantly more early season ground cover. Cultivars did not differ significantly in early tiller production. Although most of these indirect measures of competitive ability were greater for the older variety cv. Maris Widgeon, infestation of Veronica spp. was greater in plots of this cultivar than in plots of either one or both of the shorter cultivars in two successive seasons. In the 1993/94 season, the soil seedbank from plots previously cropped with cv. Maris Widgeon produced more seedlings of Sinapis awensis. In the 1994 / 95 season, cv. Maris Widgeon plots contained less weed in terms of ground cover, numbers, dry matter and nitrogen than the other cultivars, and soil sampled from plots previously cropped with cv. Hereward sown late gave rise to larger numbers of weeds than cv. Maris Widgeon. Sowing date affected weed burdens and emergence to a much greater extent than wheat cultivar. In years and sowing date treatments with relatively low weed presence the shorter cultivars tended to yield more than cv. Maris Widgeon. When weed burdens were severe cv. Maris Widgeon yields were equal to or greater than those of either cvs Hereward or Genesis. The results suggest that the traditional tall cv. Maris Widgeon could be used beneficially to tolerate weeds in organic systems when high weed infestations were anticipated, but could not be relied upon to suppress weed development, and in some circumstances could actually encourage certain species.