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.     

Abscisic acid and mefluidide in the regulation of cold hardiness development in Solanum tuberosum L. potato

Plants of Solanum tuberosum L. potato do not cold acclimate when exposed to low temperature such as 5°C, day/night. When ABA (45 M) was added to the culture medium, stem-cultured plantlets of S. tuberosum, cv. Red Pontiac, either grown at 20°C/15°C, day/night, or at 5°C, increased in cold hardiness from –2°C (killing temperature) to –4.5°C. The increase in cold hardiness could be inhibited in both temperature regimes if cycloheximide (70 M) was added to the culture medium at the inception of ABA treatment. Cycloheximide did not inhibit cold hardiness development, however, when it was added to the culture medium 3 days after ABA treatment.When pot-grown plants were foliar sprayed with mefluidide (50 M), ABA content increased from 10 nmol to 30 nmol g^–1 dry weight and plants increased in cold hardiness from –2°C to about –3.5°C. The increases in free ABA and cold hardiness occurred only in plants grown at 20°C/15°C; neither ABA nor cold hardiness increased in plants grown at 5°C.The results suggest that an increase in ABA and a subsequent de novo synthesis of proteins are required for the development of cold hardiness in S. tuberosum regardless of temperature regime, and that the inability to synthesize ABA at low temperature, rather than protein synthesis, appears to be the reason why S. tuberosum does not cold acclimate.     

Low night temperature and inhibition of gibberellin biosynthesis override phytochrome action and induce bud set and cold acclimation, but not dormancy in PHYA overexpressors and wild-type of hybrid aspen

Juvenile trees of temperate and boreal regions cease growth and set buds in autumn in response to short day-lengths (SD) detected by phytochrome. Growth cessation and bud set are prerequisites for the development of winter dormancy and full cold hardiness. In this study we show that the SD-requirement for bud set and cold hardening can be overcome in hybrid aspen (Populus tremula L. × tremuloides Michx.) by low night temperature and inhibition of gibberellin (GA) biosynthesis. Bud set and increased cold hardiness were observed under normally non-inductive long day-length (LD) in wild-type plants, when exposed to low night temperature and paclobutrazol. In addition, the effect of PHYA overexpression could be overcome in transgenic plants, producing bud set and cold acclimation by treatment with: SD, low night temperature and paclobutrazol. After cold acclimation, the degree of bud dormancy was lower for wild-type plants prior treated with LD and transgenic plants (overexpressing PHYA), than SD-treated, wild-type plants. Thus, low night temperature in combination with reduced GA content induced bud set and promoted cold hardiness under normally non-inductive photoperiods in hybrid aspen, but was unable to affect development of dormancy. This might suggest separate signalling pathways from phytochrome regulating the induction of cold/cold hardiness and bud dormancy in hybrid aspen or alternatively, there might be one pathway that fails to complete its action in the transgenic and paclobutrazol treated plants.     

How Do Plants Survive Ice?

Plant species have had to adapt to freezing and the presenceof ice in many climatic zones. Annual plants avoid ice by seeddispersal but, for biennials and perennials to survive theymust cope with ice in various forms. Most plants that are regularlyexposed to ice during their life cycles have acquired a dormantor quiescent winter period, when they are more tolerant to freezingtemperatures. This Botanical Briefing explores some associationsbetween plants and ice, with an emphasis on processes in plantsthat alleviate stress imposed by ice cover. Examples are takenfrom winter cereals which must reach an equilibrium both withice and with freezing temperatures for survival and economicproductivity. Acclimation; anaerobiosis; anoxia; cold; flooding; hypoxia; ice; ice encasement; winter survival     

Phenolic compounds in Norway spruce as affected by boron nutrition at the end of the growing season

The increase in concentrations of phenolic compounds in boron (B) deficiency has been demonstrated in many herbaceous plant species, but information on woody plants is scarce. It has been suggested that accumulation of phenolic compounds plays a role in the development of cold hardiness in herbaceous plants but also that B deficiency decreases winter hardiness. Here we study the effects of B nutrition on phenolic compounds in Norway spruce (Picea abies L.) in the course of cold acclimation. Phenolic compounds were analysed in Norway spruce seedlings from three different B-fertilisation treatments in two harvests: non-acclimated and cold-acclimated seedlings. Norway spruce phenolic compounds consisted mainly of condensed tannins. During B deficiency, condensed tannins and monocoumaroyl–astragalin der. 1 increased in non-acclimated seedlings. The increase in tannins was 21%, which was nearly significant. However, the effect of B on phenolic compounds was almost absent in cold-acclimated seedlings. The condensed tannin concentration increased much more with time in the simulated autumn than due to B deficiency, and we conclude that the B effect was probably not large enough to be important for the hardening of the seedlings. The total phenolic concentrations more than doubled during the course of cold hardening suggesting that phenolics have a role in the winter hardiness in Norway spruce.     

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.     

Transfer and segregation of triazine tolerant chloroplasts in Brassica napus L.

Summary Hypocotyl protoplasts of 45 different genotypes of German winter oilseed rape Brassica napus L. (double zero quality: high in yield, seeds low in erucic acid and glucosinolate content) were regenerated to plants. Triazine/triazinone (tri)-tolerant chloroplasts of the Canadian spring oilseed rape variety OAC Triton were introduced into some winter oilseed rapes by means of protoplast fusion. X-ray irradiation was used to limit the transfer of nuclear DNA of Triton protoplasts and to promote the selective transfer of tri-tolerant chloroplasts. Regenerated cybrid plants survived a treatment rate of 1000 g/ha metribuzin. The presence and segregation of the tri-tolerant chloroplasts in winter oilseed rape plants, regenerated from fusion products and their progeny, was investigated by restriction fragment length polymorphism (RFLP). Our results indicate that chloroplast segregation was not completed in plants regnerated from fusion products derived from X-irradiated OAC Triton mesophyll protoplasts and German winter oilseed rape hypocotyl protoplasts. In regenerants and their progeny both chloroplast types can still be present. Chloroplasts derived from wintertype protoplasts can outcompete tritolerant chloroplasts during plant development. In some instances, even progeny plants not kept under selective conditions (metribuzin) lost tri-tolerant chloroplasts. A homogenous population of tri-tolerant chloroplasts was necessary to obtain stable tri-tolerant winter oilseed rape plants.     

Does the lack of vernalization requirement interfere with winter survival of oilseed rape plants?

Recent studies (Rapacz 1999) have shown that cultivars of spring-type oilseed rape are able to cold-acclimate to the level comparable with winter cultivars, but only after prehardening which results both in the increase of photosynthetic activity and in growth cessation. It is commonly known that under field conditions spring-type cultivars could not survive winter. Present studies were undertaken to explain the reasons for low winter hardiness of spring type rape plants. Six cultivars of spring and two of winter rape were sown in the open-air vegetation room at the end of August. The obtained results indicate that the degree of frost damage in spring-type plants increased in the course of winter and this increase was parallel to elongation of generative shoots observed after periods of warming. Each spring cultivar was completely killed by frost just after its generative shoot reached 15–20 cm, irrespective of its frost resistance level, determined previously under laboratory conditions. In the case of winter cultivars survival rate was consistent with laboratory-estimated frost resistance. It is suggested that spring rape could not survive winter because of its limited ability to prevent shoot elongation during winter at temperatures slightly above 0 °C. It was also found that less efficient photosynthetic electron transport in autumn was observed in these spring cultivars in which the elongation of generative shoots was observed already during the first warm break in winter.     

Cold hardiness and water relations parameters in Rhododendron cv. Catawbiense Boursault subjected to drought episodes

We determined whether increase in cold hardiness of Rhododendron cv. Catawbiense Boursault induced by water stress was correlated with changes in tissue water relations. Water content of the growing medium was either maintained near field capacity for the duration of the study or plants were subjected to drought episodes at different times between 15 July and 19 February. Watering during a drought episode was delayed until soil water content decreased below 0.4 m³ m⁻³ then watering was resumed at a level to maintain soil water content between 0.3 and 0.4 m³ m⁻³. Cold hardiness was evaluated in the laboratory with freeze tolerance tests on detached leaves. Water relations parameters were determined using pressure-volume analysis. Exposure to drought episodes increased cold hardiness during the cold acclimation stage in late summer and fall but not during the winter. When water-stressed plants were re-watered to field capacity, the previous gain in cold hardiness gradually disappeared. Water relations parameters correlating with seasonal changes of cold hardiness included dry matter content (r =−0.67). apoplastic water content (r =−0.60), and water potential at the turgor loss point (r = 0.40). Changes of cold hardiness in water-stressed plants in reference to well-watered plants were correlated with changes of all water relations parameters, except for osmotic potential at full turgor (r = 0.13). It is proposed that water stress reduced the hydration of cell walls, thereby increasing their rigidity. Increased rigidity of cell walls could result in a development of greater negative turgor pressures at subfreezing temperatures and therefore increased resistance to freeze dehydration.     

Short term temperature drops do not enhance cold tolerance

To successfully transplant agricultural species in the spring, prior hardening is of great significance. Low, non-freezing temperature increases cold tolerance in many species. Also, diurnal temperature drops have been suggested to improve cold tolerance, as assessed by ultrastructural studies after short term freezing of leaf discs. Pre-treatment with lower day than night temperature prior to hardening has also been reported to enhance cold resistance in winter rape. This study investigated the effect of temperature drops on cold resistance of different species. In contrast to a period of continuous low temperature, short diurnal temperature drops did not enhance cold tolerance in Arabidopsis, swede, white cabbage or pea, compared to control plants. Exposure to low temperature of 6°C for 6 days increased cold tolerance by 2–5°C compared to plants exposed to diurnal temperature drops or control plants. Pre-treatment with diurnal temperature drops in the entire growth period prior to hardening with constant low temperature did not give any additional hardening in swede and pea. In conclusion, by freeze testing of whole plants under controlled conditions we have found no evidence supporting the hypothesis that diurnal temperature drops improve cold tolerance. However, temperature drops reduce plants size like shown earlier for a number of other species, and thus is a tool to produce compact, robust plants.     

Use of intraspecific variation in thermal responses for estimating an elevational cline in the timing of cold hardening in a sub‐boreal conifer

To avoid winter frost damage, evergreen coniferous species develop cold hardiness with suitable phenology for the local climate regime. Along the elevational gradient, a genetic cline in autumn phenology is often recognised among coniferous populations, but further quantification of evolutionary adaptation related to the local environment and its responsible signals generating the phenological variation are poorly understood. We evaluated the timing of cold hardening among populations of Abies sachalinensis, based on time series freezing tests using trees derived from four seed source populations × three planting sites. Furthermore, we constructed a model to estimate the development of hardening from field temperatures and the intraspecific variations occurring during this process. An elevational cline was detected such that high‐elevation populations developed cold hardiness earlier than low‐elevation populations, representing significant genetic control. Because development occurred earlier at high‐elevation planting sites, the genetic trend across elevation overlapped with the environmental trend. Based on the trade‐off between later hardening to lengthen the active growth period and earlier hardening to avoid frost damage, this genetic cline would be adaptive to the local climate. Our modelling approach estimated intraspecific variation in two model components: the threshold temperature, which was the criterion for determining whether the trees accumulated the thermal value, and the chilling requirement for trees to achieve adequate cold hardiness. A higher threshold temperature and a lower chilling requirement could be responsible for the earlier phenology of the high‐elevation population. These thermal responses may be one of the important factors driving the elevation‐dependent adaptation of A. sachalinensis.     

Is survival after ice encasement related with sugar distribution in organs of the Antarctic plants <Emphasis Type="Italic">Deschampsia antarctica</Emphasis> Desv. (Poaceae) and <Emphasis Type="Italic">Colobanthus quitensis</Emphasis> (Kunth) Bartl. (Caryophyllaceae)?

Deschampsia antarctica and Colobanthus quitensis are usually covered by snow from April to November. It is unknown whether the leaves survive ice encasement. This study proposes that day length influences sugar distribution in C. quitensis and that sugar accumulation favors re-growths after an ice encasement period. The objectives of this work were: (1) to study the effect of day length and low temperature on sugar distribution in organs of C. quitensis and (2) to study the survival and recovery of D. antarctica and C. quitensis after a period of ice encasement. Extremely short day length (SD) (8/16 h) and long day length (LD) (21/3 h) was used, medium (MD) (16/8 h) corresponding at control day length. Also two temperatures: 4°C (cold acclimated) and 15°C (control) were evaluated. Both factors: day length and cold acclimation significantly affected sugar distribution in C. quitensis. Both species presented a high rate of survival after ice encasement. D. antarctica conserved most of their leaves green, while C. quitensis presented dead leaves and new shoots in plants from cold acclimated under SD. Only in D. antarctica the number of green leaves after ice encasement was positively correlated with sugar content in underground organs. The high sugar content in green leaves of both species suggested fast activity recovery after snow melting.     

The pattern of frost-hardening of leaves of winter cauliflower (Brassica oleracea var. botrytis cv. Clause 30)

The suitability of an electrical conductivity assay of leaf material for determining frost hardiness in winter cauliflower was established. The assay was used to monitor the changes in hardiness of field-grown plants during two seasons at two sites. Levels of frost hardiness were related to mean air temperature over the 7-day period prior to sampling (r = 0.6008, P< 0.001). There was no clear disjunction of any temperature to indicate a threshold temperature of hardening. Dehardening occurred in spring and during mild spells in winter. In the mild regions where this crop is grown commercially it is likely that plants are not fully hardened for much of the winter. This may facilitate curd growth but may put the crop at risk from frost damage.     

The effect of CCC on the nitrogen compounds content in rape plants and their frost hardiness. Relation to the conditions of day-length and temperature

Both CCC and cold (5°C) treatment gave rise to an increased content of the water-soluble proteins in leaf tissue of the winter rape, irrespective of the day length. This effect was accompanied by a decrease of the insoluble nitrogen compounds content, mainly under theLD (the 16 hour day). The applied retardant also stimulated the consumption of the structural compounds induced by low temperature. Low temperature treatment hardened plants more distinctly than the CCC application. The frost hardening effect of CCC andSD (the 8 hour day) was manifested only at 20°C and it disappeared at 5°C. Changes in frost hardiness were not correlated with the changes in the reducing sugar content and in the reducing ability of the studied tissue. The coincident effect of CCC and cold on the reducing sugar content was observed underSD conditions.     

Factors affecting the cold hardiness of the peach-potato aphid Myzus persicae

Supercooling point studies were used to investigate the factors influencing the cold hardiness of the peach-potato aphid Myzus persicae, a freezing-susceptible insect. Overwintering adults lost cold hardiness as winter progressed, with a variable proportion showing a marked reduction in supercooling ability. Cold hardiness increased in spring so that all individuals demonstrated extensive supercooling ability typical of aphids reared in the laboratory at 20°C with a long photoperiod; these levels of cold hardiness were maintained in the field during summer and early autumn. First instar nymphs demonstrated considerable cold hardiness all year. Surface moisture caused inoculative freezing in some first instar nymphs and adults when supercooled, but the majority were unaffected. In the laboratory, adults starved for 7 days at 5°C showed distinct losses of supercooling potential equivalent to those observed in the field during mid to late winter. No loss of cold hardiness was found in first instar nymphs starved under the same conditions. The results demonstrate that the cold hardiness characteristics of M. persicae are atypical of those observed in other freezing-susceptible insects and it is suggested that continued feeding during mild winter conditions allows maintenance of cold hardiness particularly in adult aphids, and provides a possible explanation for the successful anholocyclic overwintering of M. persicae during such winters.     

Ice segregation in the crown of winter cereals: Evidence for extraorgan and extratissue freezing

Meaningful improvements in winter cereal cold hardiness requires a complete model of freezing behaviour in the critical crown organ. Magnetic resonance microimaging diffusion‐weighted experiments provided evidence that cold acclimation decreased water content and mobility in the vascular transition zone (VTZ) and the intermediate zone in rye (Secale cereale L. Hazlet) compared with wheat (Triticum aestivum L. Norstar). Differential thermal analysis, ice nucleation, and localization studies identified three distinct exothermic events. A high‐temperature exotherm (?3°C to ?5°C) corresponded with ice formation and high ice‐nucleating activity in the leaf sheath encapsulating the crown. A midtemperature exotherm (?6°C and ?8°C) corresponded with cavity ice formation in the VTZ but an absence of ice in the shoot apical meristem (SAM). A low‐temperature exotherm corresponded with SAM injury and the killing temperature in wheat (?21°C) and rye (?27°C). The SAM had lower ice‐nucleating activity and freezing survival compared with the VTZ when frozen in vitro. The intermediate zone was hypothesized to act as a barrier to ice growth into the SAM. Higher cold hardiness of rye compared with wheat was associated with higher VTZ and intermediate zone desiccation resulting in the formation of ice barriers surrounding the SAM.     

The relative hardening of roots and shoots and the influence of day-length during hardening in perennial ryegrass

The influence of long and short days during the hardening period on the cold hardiness of perennial ryegrass seedlings was studied as was the relative hardiness of roots and shoots. Hardiness was assessed by the electrolyte release method which was a measure of the amount of damage subsequent to low temperature treatment. Long days promoted hardiness in shoots of Pax 0tofte plants and in one case under short days the roots were found to be hardy. Generally roots were less hardy than shoots in Pax 0tofte and S23 plants hardened under long days for 2 wk. When hardened at the fourth leaf stage for 2 wk at + 5 ^oC under long day conditions, Pax 0tofte plants were more hardy than those of S23. The long day effect on hardiness was arrived at more rapidly, there being no difference in hardiness after 3 wk in Pax 0tofte hardened under long or short days, whereas a significant degree of hardening was observed after 1 wk of hardening under long days at – 4 ^oC. The results obtained are discussed in relation to winter kill of grasses in the West of Scotland, and it is considered that root damage is not an important factor in causing winter kill. The promotive effect of long days on hardiness when hardening commences in late autumn is considered an advantage in temperate regions as it may also allow early frosts to be withstood.     

Changes in endogenous abscisic acid and cold hardiness in Actinidia treated with triazole growth retardants

Soil drench of either paclobutrazol or uniconazole (0.4 mg/pot) was applied to plants of Actinidia arguta, Ananasnaja, to determine the effect on endogenous abscisic acid measured in November, January, and March, and the concomittant cold hardiness of the treated plants. Both paclobutrazol and uniconazole treatments significantly increased cold hardiness. Paclobutrazol was more effective in increasing ABA levels compared to uniconazole. Abscisic acid was found to be highest in January, corresponding to deep dormancy, and least in March when plants were undergoing vegetative bud break. Paclobutrazol delayed vegetative bud break by 6.3 days, while uniconazole delayed bud break by 2.9 days.