Metabolic and Ultrastructural Changes in Winter Wheat during Ice Encasement Under Field Conditions

The effect of ice encasement on the physiological, metabolic, and ultrastructural properties of winter wheat (Triticum aestivum L.) grown under field conditions was examined by artificially encasing winter wheat in ice during early winter. Cold hardiness and survival of ice-encased seedlings declined less rapidly in Kharkov, a cold-hardy cultivar than in Fredrick, a less hardy cultivar. Ethanol did not accumulate in non-iced seedlings, but increased rapidly upon application of an ice sheet. Lactic acid accumulated in both cultivars during late autumn, prior to ice encasement, and elevated levels of lactic acid were maintained throughout the winter in seedlings from both iced and non-iced plots. The rate of O₂ consumption of shoot tissue of seedlings from non-iced plots remained relatively constant throughout the winter, but declined rapidly in seedlings from ice encased plots. Major ultrastructural changes did not occur in shoot apex cells of non-iced winter wheat seedlings during cold hardening under field conditions. However, the imposition of an ice cover in early January resulted in a proliferation of the endoplasmic reticulum membrane system of the cells, frequently resulting in the formation of concentric whorls of membranes, often enclosing cytoplasmic organelles. Electrondense areas within the cytoplasm which appeared to be associated with the expanded endoplasmic reticulum were also frequently observed.     

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.     

Responses of sub-arctic dwarf shrubs to low oxygen and high carbon dioxide conditions

Arctic plants can experience prolonged periods of ice encasement in winter, leading to hypoxia which may cause damage to plants and reduce subsequent summer growth. Further, high CO₂ concentrations, which can occur when plants respire within ice, may cause additional injury, as has been shown to occur in crops. The tolerance of arctic plants to these within-ice atmospheric conditions has previously remained unknown, yet this knowledge is essential for understanding icing impacts on plant community structure and productivity – especially given that icing events are predicted to increase in frequency as a result of climate change. Using a unique field chamber experiment, this study quantified the responses of three widespread sub-arctic dwarf shrubs, Empetrum nigrum and Vaccinium vitis-idaea (both evergreen) and V. myrtillus (deciduous), to 14 days hypoxia and high CO₂ in winter in a factorial field experiment in northern Sweden. Growth, phenology and mortality responses were used to quantify impacts in the following spring supported by electrolyte leakage and chlorophyll fluorescence to assess leaf damage in the two evergreens. Overall, all species showed high resistance to 14 days of hypoxia with no indications of damage. Increased CO₂ did lead to 33% lower bud dormancy in E. nigrum and 70% greater shoot mortality in V. vitis-idaea indicating that these species might be negatively affected by increased occurrences of ice encasement, through elevated CO₂ levels. Despite these responses, effects of high CO₂ were rare overall. Given that the impacts of hypoxia and high CO₂ were considerably less than reported for species in temperate habitats, this suggests a moderate to high degree of tolerance to short periods of icing among these species. If these results apply to longer periods of ice encasement, increasing frequency of icing may only have some species specific impacts without being a major environmental threat to arctic dwarf shrubs.     

The relationship between cell injury and osmotic volume reduction: III. Freezing injury and frost resistance in winter wheat

In order to distinguish between several possible mechanisms of frost hardening in winter wheat (Triticum aestivum L.) cells from two hardy and two tender cultivars were plasmolyzed in CaCl₂ solution at room temperature and cell volumes estimated by microscopic examination. Analyses of Boyle-van't Hoff plots of these data reveal that all cells from cultivars progressively increase their intracellular solute concentration up to 20 days hardening. This increase, which we had predicted from published calorimetric data to be the sole mechanism of hardening explained less than half of the increase in hardening seen in the most hardy cultivar, Kharkov. Hardening also increased the osmotically inactive volume.At CaCl₂ concentrations greater than 5%, plasmolyzed protoplasts departed further from the Boyle-van't Hoff prediction, remaining larger than expected until some higher concentration of CaCl₂, where protoplast volume again sharply decreased. In all cultivars except hardened Kharkov, the concentration of CaCl₂ producing this abrupt volume decrease had a freezing point corresponding to the killing temperature. If this concentration was exceeded during plasmolysis, then the protoplasts burst during deplasmolysis at some volume less than their original volume.We interpret these data to mean that, in addition to the often described hardening mechanism of increased cell solute and water binding, winter wheat shows a third mechanism, a mechanical resistance to protoplast shrinkage which produces volumes larger than those predicted during osmotic stress. The resisting element appears to be the plasma membrane itself. Shrinkage brings the membrane under compressive stress, developing tangential pressure within it. Cell injury occurs when the cell membrane area has been reduced to the point at which irreversible loss of membrane material is inevitable. Cell death occurs during deplasmolysis when the protoplast bursts because its membrane contains insufficient material to subtend the area of the cell wall.Of the cultivars tested, hardened Kharkov was unique in avoiding injury. Hardened Kharkov was injured only after the volume inflection had been greatly exceeded. Refractile droplets of lipid appeared in the cytoplasm of hardened Kharkov protoplasts during plasmolysis but disappeared during deplasmolysis suggesting that hardy Kharkov was able reversibly to store membrane lipids in cytoplasmic vesicles and return them to the membrane on deplasmolysis.     

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.     

Stimulation of Phospholipid Biosynthesis during Frost Hardening of Winter Wheat

Lipids were labeled with ³³P during frost hardening of two varieties of winter wheat (Triticum aestivum), hardy Kharkov and much less hardy Champlein. The main labeled compounds were phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, and phosphatidylglycerol. With time of incorporation the proportion of the radioactivity incorporated into the lipids increased in phosphatidylcholine, especially in Kharkov and at 1 C. During hardening, phospholipid synthesis was greatly stimulated in Kharkov, but much less in Champlein. The proportion of the phospholipids synthesized changed only little with hardening, with a trend towards an increase in phosphatidylcholine. Increased phospholipid synthesis does not seem to be a prerequisite to hardening in winter wheat. However, a high rate of phospholipid synthesis may be required to maintain frost resistance.     

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.     

Properties of chloroplast membrane-bound ferredoxin--NADP + reductase during cold hardening of wheat. No indication of qualitative membrane changes during cold hardening

Kinetic parameters of the chloroplastbound ferredoxin-NADP⁺ reductase from two varieties of wheat (Triticum aestivum), hardy Kharkov 22 MC (winter wheat) and less hardy Rescue (spring wheat), were followed during induction of frost hardiness as a means of examining possible changes in chloroplast membranes during hardening. No changes were found in the Michaelis constants for NADPH and 2,6-dichlorophenol indophenol, inhibition constants for p-chloromercuriphenylsulfonate, and activation energy values of the enzyme in either variety. The data suggest that no qualitative changes occurred in the properties of wheat chloroplast membranes related to ferredoxin-NADP⁺ reductase during cold hardening.     

The severity of wheat diseases increases when plants and pathogens are acclimatized to elevated carbon dioxide

Wheat diseases present a constant and evolving threat to food security. We have little understanding as to how increased atmospheric carbon dioxide levels will affect wheat diseases and thus the security of grain supply. Atmospheric CO₂ exceeded the 400 ppmv benchmark in 2013 and is predicted to double or even treble by the end of the century. This study investigated the impact of both pathogen and wheat acclimation to elevated CO₂ on the development of Fusarium head blight (FHB) and Septoria tritici blotch (STB) disease of wheat. Here, plants and pathogens were cultivated under either 390 or 780 ppmv CO₂ for a period (two wheat generations, multiple pathogen subcultures) prior to standard disease trials. Acclimation of pathogens and the wheat cultivar Remus to elevated CO₂ increased the severity of both STB and FHB diseases, relative to ambient conditions. The effect of CO₂ on disease development was greater for FHB than for STB. The highest FHB disease levels and associated yield losses were recorded for elevated CO₂‐acclimated pathogen on elevated CO₂‐acclimated wheat. When similar FHB experiments were conducted using the disease‐resistant cultivar CM82036, pathogen acclimation significantly enhanced disease levels and yield loss under elevated CO₂ conditions, thereby indicating a reduction in the effectiveness of the defence pathways innate to this wheat cultivar. We conclude that acclimation to elevated CO₂ over the coming decades will have a significant influence on the outcome of plant–pathogen interactions and the durability of disease resistance.     

Virus disease in wheat predicted to increase with a changing climate

Current atmospheric CO₂ levels are about 400 μmol mol^?1 and are predicted to rise to 650 μmol mol^?1 later this century. Although the positive and negative impacts of CO₂ on plants are well documented, little is known about interactions with pests and diseases. If disease severity increases under future environmental conditions, then it becomes imperative to understand the impacts of pathogens on crop production in order to minimize crop losses and maximize food production. Barley yellow dwarf virus (BYDV) adversely affects the yield and quality of economically important crops including wheat, barley and oats. It is transmitted by numerous aphid species and causes a serious disease of cereal crops worldwide. This study examined the effects of ambient (aCO₂; 400 μmol mol^?1) and elevated CO₂ (eCO₂; 650 μmol mol^?1) on noninfected and BYDV‐infected wheat. Using a RT‐qPCR technique, we measured virus titre from aCO₂ and eCO₂ treatments. BYDV titre increased significantly by 36.8% in leaves of wheat grown under eCO₂ conditions compared to aCO₂. Plant growth parameters including height, tiller number, leaf area and biomass were generally higher in plants exposed to higher CO₂ levels but increased growth did not explain the increase in BYDV titre in these plants. High virus titre in plants has been shown to have a significant negative effect on plant yield and causes earlier and more pronounced symptom expression increasing the probability of virus spread by insects. The combination of these factors could negatively impact food production in Australia and worldwide under future climate conditions. This is the first quantitative evidence that BYDV titre increases in plants grown under elevated CO₂ levels.     

Stomatal density and aperture length in four plant species grown across a subambient CO2 gradient

Stomatal density, stomatal aperture length, area/leaf, and number of stomata/leaf were measured after the annual C₃ agronomic grasses oats (Avena sativa) and wheat (Triticum aestivum), the C, woody legume honey mesquite (Prosopis glandulosa), and the perennial C₄ grass little bluestem (Schizachyrium scoparium) were grown across a subambient carbon dioxide concentration ([CO₂]) gradient from near 200 to 350 μmol/mol in a growth chamber. The purpose was to determine if the size and density of stomata vary in response to atmospheric [CO₂] during growth, across a subambient [CO₂] range representative of the doubling that has occurred since the last ice age. Changes in stomatal density and aperture length with increasing [CO₂] were small when detected. Stomatal density decreased on adaxial flag leaf surfaces of wheat, and aperture length increased slightly with [CO₂], Leaf area and number of stomata/flag leaf increased by similar proportions with [CO₂] in two wheat cultivars. No consistent relationship between [CO₂] and stomatal density or size was detected in mesquite, oats, or little bluestem. We conclude that individual plants of these species lack the plasticity to significantly alter stomatal density and aperture length in response to increasing atmospheric [CO₂] in a single generation (annuals) or growing season (perennials).     

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.     

Photosynthesis and assimilate partitioning between carbohydrates and isoprenoid products in vegetatively active and dormant guayule: physiological and environmental constraints on rubber accumulation in a semiarid shrub

The stems and roots of the semiarid shrub guayule, Parthenium argentatum, contain a significant amount of natural rubber. Rubber accumulates in guayule when plants are vegetatively and reproductively dormant, complicating the relationship between growth/reproduction and product synthesis. To evaluate the factors regulating the partitioning of carbon to rubber, carbon assimilation and partitioning were measured in guayule plants that were grown under simulated summer‐ and winter‐like conditions and under winter‐like conditions with CO₂ enrichment. These conditions were used to induce vegetatively active and dormant states and to increase the source strength of vegetatively dormant plants, respectively. Rates of CO₂ assimilation, measured under growth temperatures and CO₂, were similar for plants grown under summer‐ and winter‐like conditions, but were higher with elevated CO₂. After 5 months, plants grown under summer‐like conditions had the greatest aboveground biomass, but the lowest levels of non‐structural carbohydrates and rubber. In contrast, the amount of resin in the stems was similar under all growth conditions. Emission of biogenic volatile compounds was more than three‐fold higher in plants grown under summer‐ compared with winter‐like conditions. Taken together, the results show that guayule plants maintain a high rate of photosynthesis and accumulate non‐structural carbohydrates and rubber in the vegetatively dormant state, but emit volatile compounds at a lower rate when compared with more vegetatively active plants. Enrichment with CO₂ in the vegetatively dormant state increased carbohydrate content but not the amount of rubber, suggesting that partitioning of assimilate to rubber is limited by sink strength in guayule.     

The effect of free air carbon dioxide enrichment (FACE) and soil nitrogen availability on the photosynthetic capacity of wheat

A simple system for free air carbon dioxide enrichment (FACE) was recently developed and it is here briefly described. Such a MiniFACE system allowed the elevation of CO₂ concentration of small field plots avoiding the occurrence of large spatial and temporal fluctuations. A CO₂ enrichment field experiment was conducted in Italy in the season 1993–1994 with wheat (cv. Super-dwarf Mercia). A randomized experimental design was used with the treatment combination CO₂ × soil N, replicated twice. Gas exchange measurements showed that photosynthetic capacity was significantly decreased in plants exposed to elevated CO₂ and grown under nitrogen deficiency. Photosynthetic acclimation was, in this case, due to the occurrence of reduced rates of rubP saturated and rubP regeneration limited photosynthesis. Gas exchange measurements did not instead reveal any significant effect of elevated CO₂ on the photosynthetic capacity of leaves of plants well fertilized with nitrogen, in spite of a transitory negative effect on rubP regeneration limited photosynthesis that was detected to occur in the central part of a day with high irradiance. It is concluded that the levels of nitrogen fertilization will play a substantial role in modulating CO₂ fertilization effects on growth and yields of wheat crops under the scenario of future climate change.     

Purine biosynthesis and catabolism in soybean root nodules: incorporation of 14C from 14CO2 into xanthine

Nodulated root systems of soybean plants were exposed to ¹⁴CO₂ in the presence and absence of allopurinol. After 5 h about one-fifth of the label in the perchloric acid-soluble fraction of the nodules was found to be in xanthine in the allopurinol-treated plants. Control plants contained much lower levels of xanthine, but with similar specific activity. Hypoxanthine was not detected in either control or allopurinol-treated plants, even though it would be expected to accumulate in the latter. Degradation of labeled xanthine from allopurinol-treated plants using xanthine oxidase and uricase resulted in the loss of most of the label. The preferential incorporation and accumulation of ¹⁴C from ¹⁴CO₂ into C₆ of xanthine in allopurinol-treated plants is consistent with the involvement of phosphoribosylaminoimidazole carboxylase in the de novo synthesis of purines. The accumulation of xanthine and absence of hypoxanthine in nodules of allopurinol-treated plants confirms earlier observations. In addition, the similar specific activities of ¹⁴C in xanthine in allopurinol-treated and control plants indicate that the xanthine which accumulates in allopurinol-treated plants is the product of de novo purine biosynthesis.     

An investigation into the interaction between nitrogen nutrition,photosynthesis and photorespiration

Photosynthetic CO₂ assimilation, photorespiration and levels of glycollate oxidase and ribulose bisphosphate (RuBP) carboxylase were measured in barley, wheat and maize plants grown on media containing nitrate or ammonium or in plants transferred from nitrate to ammonium. The CO₂ compensation point and photorespiratory CO₂ release were not altered by the nitrogen growth regime nor by transfer from nitrate to ammonium. In barley and wheat plants grown on ammonium the levels of glycollate oxidase and RuBP carboxylase per unit leaf area were higher than in nitrate grown material. These differences were not evident when the results were expressed on a protein or chlorophyll basis. The ratio of glycollate oxidase activity to RuBP carboxylase activity was not altered by the nitrogen regime.     

Nitrogen assimilation and transpiration: key processes conditioning responsiveness of wheat to elevated [CO2] and temperature

Although climate scenarios have predicted an increase in [CO₂] and temperature conditions, to date few experiments have focused on the interaction of [CO₂] and temperature effects in wheat development. Recent evidence suggests that photosynthetic acclimation is linked to the photorespiration and N assimilation inhibition of plants exposed to elevated CO₂. The main goal of this study was to analyze the effect of interacting [CO₂] and temperature on leaf photorespiration, C/N metabolism and N transport in wheat plants exposed to elevated [CO₂] and temperature conditions. For this purpose, wheat plants were exposed to elevated [CO₂] (400 vs 700 µmol mol^?1) and temperature (ambient vs ambient + 4°C) in CO₂ gradient greenhouses during the entire life cycle. Although at the agronomic level, elevated temperature had no effect on plant biomass, physiological analyses revealed that combined elevated [CO₂] and temperature negatively affected photosynthetic performance. The limited energy levels resulting from the reduced respiratory and photorespiration rates of such plants were apparently inadequate to sustain nitrate reductase activity. Inhibited N assimilation was associated with a strong reduction in amino acid content, conditioned leaf soluble protein content and constrained leaf N status. Therefore, the plant response to elevated [CO₂] and elevated temperature resulted in photosynthetic acclimation. The reduction in transpiration rates induced limitations in nutrient transport in leaves of plants exposed to elevated [CO₂] and temperature, led to mineral depletion and therefore contributed to the inhibition of photosynthetic activity.     

The relationship between cell injury and osmotic volume reduction: IV. The behavior of hardy wheat membrane lipids in monolayer

Lipid extracts from two winter wheat cultivars, Kharkov and Champlein, were studied as monomolecular layers on a Langmuir trough. An abrupt collapse of the lipid monolayers from unhardened and hardened Champlein and unhardened Kharkov was observed at pressures of 22 to 25 dynes/cm with only little return of lipid to the interface on removal of pressure. In marked contrast, the more hardy cultivar, Kharkov, in hardened state, contained lipids which progressively migrated from the interface on increasing pressure but returned with decreasing pressure, the collapse pressure being 16 to 19 dynes/cm². The same trends held true for purified phospholipids from both cultivars and treatments with the exception that the collapse pressure of hardened Kharkov phospholipid rose to the same 20 to 25 dynes/ cm range as the other purified extracts.In an attempt to duplicate conditions obtaining in a plasmolyzing cell, hardened Kharkov phospholipids were layered on a diluted aqueous cell extract, intensifying the hardening effects already observed with Kharkov total lipid extract on water and permitting a complete recovery of lipid on decompression of the monolayer. We conclude that an important element of freezing injury in winter wheat is the irreversible loss of membrane material, especially lipids, from cell membranes and that the unique reversibility of this process in hardened Kharkov greatly extends its freezing resistance.     

Effect of CO2 enrichment on non-structural carbohydrates in leaves, stems and ears of spring wheat

Field-grown spring wheat (Triticum aestivum L. cv. Dragon) was exposed to ambient and elevated CO₂ concentrations (1.5 and 2 times ambient) in open-top chambers. Contents of non-structural carbohydrates were analysed enzymatically in leaves, stems and ears six times during the growing season. The impact of elevated CO₂ on wheat carbohydrates was non-significant in most harvests. However, differences in the carbohydrate contents due to elevated CO₂ were found in all plant compartments. Before anthesis, at growth stage (GS) 30 (the stem is 1 cm to the shoot apex), the plants grown in elevated CO₂ contained significantly more water soluble carbohydrates (WSC), fructans, starch and total non-structural carbohydrates (TNC) in the leaves in comparison with the plants grown in ambient CO₂. It is hypothesised that the plants from the treatments with elevated CO₂ were sink-limited at GS30. After anthesis, the leaf WSC and TNC contents of the plants from elevated CO₂ started to decline earlier than those of the plants from ambient CO₂. This may indicate that the leaves of plants grown in the chambers with elevated CO₂ senesced earlier. Elevated CO₂ accelerated grain development: 2 weeks after anthesis, the plants grown in elevated CO₂ contained significantly more starch and significantly less fructans in the ears compared to the plants grown in ambient CO₂. Elevated CO₂ had no effect on ear starch and TNC contents at the final harvest. Increasing the CO₂ concentration from 360 to 520 μmol mol^?1 had a larger effect on wheat non-structural carbohydrates than the further increase from 520 to 680 μmol mol^?1. The results are discussed in relation to the effects of elevated CO₂ on yield and yield components.