Primary and secondary induction requirements for flowering of Festuca rubra

Root and shoot temperatures were varied independently to determine the importance of root temperature during cold acclimation. Spinach ( Spinacia oleracea L. cvs Harbin and Bloomsdale) plants were subjected to 20/20°C. 20/5°C, 5/20°C, and 5/5°C (shoot/root) temperature treatments. Leaf freezing tolerance, water potential, stomatal resistance, osmotic potential, and water content were measured at 0.25. 1.25, 3.25, and 7.25 days of treatment. There was no change in freezing tolerance or the water relations of the 20/20°C treated plants during the course of the experiment. Freezing tolerance was increased by the 5°C shoot temperature treatments, but was not enhanced by water stress induced by the low root temperature. Leaf water potential and water content decreased and stomatal resistance increased within 6 h in the 20/5°C plants. By day 3, osmotic potential began decreasing in the 20/5°C plants. Leaf water content, osmotic potential, and water potential decreased more gradually in plants grown with 5°C shoot temperature, irrespective of root temperature. Decreased water content and osmotic potential were not correlated with increased freezing tolerance as reported for other herbaceous crop plants.     

Interaction of Temperature and Light in the Development of Freezing Tolerance in Plants

Freezing tolerance is the result of a wide range of physical and biochemical processes, such as the induction of antifreeze proteins, changes in membrane composition, the accumulation of osmoprotectants, and changes in the redox status, which allow plants to function at low temperatures. Even in frost-tolerant species, a certain period of growth at low but nonfreezing temperatures, known as frost or cold hardening, is required for the development of a high level of frost hardiness. It has long been known that frost hardening at low temperature under low light intensity is much less effective than under normal light conditions; it has also been shown that elevated light intensity at normal temperatures may partly replace the cold-hardening period. Earlier results indicated that cold acclimation reflects a response to a chloroplastic redox signal while the effects of excitation pressure extend beyond photosynthetic acclimation, influencing plant morphology and the expression of certain nuclear genes involved in cold acclimation. Recent results have shown that not only are parameters closely linked to the photosynthetic electron transport processes affected by light during hardening at low temperature, but light may also have an influence on the expression level of several other cold-related genes; several cold-acclimation processes can function efficiently only in the presence of light. The present review provides an overview of mechanisms that may explain how light improves the freezing tolerance of plants during the cold-hardening period.     

Supercooling in overwintering azalea flower buds: additional freezing parameters

Results of calorimetric, nuclear magnetic resonance, and low temperature light microscopic studies on supercooled azalea (Rhododendron kosterianum, Schneid.) floral primordia are reported. Heat release during freezing of the supercooled floral primordia is in the range predicted for supercooled pure water. Spin-lattice and spin-spin relaxation times measured by pulsed nuclear magnetic resonance spectroscopy decreased after freezing, suggesting that a redistribution of tissue water is associated with injury to the floral primordium. The calorimetric and low temperature microscopy studies showed no detectable ice formation in floral primordia until the major freezing event at low temperature. No resistance to ice growth is found to exist in the primordium tissues, indicating that a freezing barrier or thermodynamic equilibrium exists between the unfrozen primordium and other flower bud parts which contain ice at subfreezing temperatures.     

Freezing injury and root development in winter cereals

Upon exposure to 2°C, the leaves and crowns of rye (Secale cereale L. cv `Puma') and wheat (Triticum aestivum L. cv `Norstar' and `Cappelle') increased in cold hardiness, whereas little change in root cold hardiness was observed. Both root and shoot growth were severely reduced in cold-hardened Norstar wheat plants frozen to −11°C or lower and transplanted to soil. In contrast, shoot growth of plants grown in a nutrient agar medium and subjected to the same hardening and freezing conditions was not affected by freezing temperatures of −20°C while root growth was reduced at −15°C. Thus, it was apparent that lack of root development limited the ability of plants to survive freezing under natural conditions.

Generally, the temperatures at which 50% of the plants were killed as determined by the conductivity method were lower than those obtained by regrowth. A simple explanation for this difference is that the majority of cells in the crown are still alive while a small portion of the cells which are critical for regrowth are injured or killed.

Suspension cultures of Norstar wheat grown in B-5 liquid medium supplemented with 3 milligrams per liter of 2,4-dichlorophenoxyacetic acid could be cold hardened to the same levels as soil growth plants. These cultures produce roots when transferred to the same growth medium supplemented with a low rate of 2,4-dichlorophenoxyacetic acid (<1 milligram per liter). When frozen to −15°C regrowth of cultures was 50% of the control, whereas the percentage of calli with root development was reduced 50% in cultures frozen to −11°C. These results suggest that freezing affects root morphogenesis rather than just killing the cells responsible for root regeneration.

     

Does plant height determine the freezing resistance in the páramo plants?

Neotropical ecosystems between treeline and snowline, called páramos, stretch along Andean ranges from Costa Rica to northern Peru. The páramo climate is characterized by regular night frosts occurring throughout the year. Páramo plants use two strategies to deal with freezing temperatures. They either avoid ice formation in the tissues or tolerate extracellular ice formation. We tested the microclimate hypothesis, which suggests that the freezing resistance of the páramo plants is determined by plant height, that is, that taller plants experience a milder microclimate and avoid freezing, whereas smaller plants are exposed to the more extreme thermal conditions near the ground and tolerate them. We measured the temperature at which ice formed inside the plants (the ‘exotherm’), and compared it with the temperature at which 50% damage to the tissue occurred (Lt50); a significant difference between the exotherm and Lt50 would indicate freezing tolerance whereas the absence of a difference would indicate avoidance by supercooling. We analysed the freezing resistance of 38 common Ecuadorian páramo species. We found no correlation between plant height and freezing resistance mechanism or injury temperature and reject the microclimate hypothesis. Tolerant plants reach higher altitudes than avoidant plants, but their altitudinal ranges largely overlap and the Lt50 does not differ between them. These results suggest that there is no qualitative difference between the two strategies to survive the páramo frosts. Shrub leaves were injured at significantly lower temperatures than other life forms, such as herbs, which may reflect leaf anatomical differences among the plants.     

Photosynthesis in cold acclimated leaves of plants with various degrees of freezing tolerance

The objective of this study was to compare the photosynthetic changes during cold acclimation in various plant types able to acquire different degrees of freezing tolerance. Four herbaceous and six woody plants were hardened under natural or artificial conditions and – after determination of their frost resistance (LT₅₀) – the net photosynthetic rate at an ambient CO₂ of 33 Pa (P_n33), the dependencies of P_n to light and to CO₂ and the room temperature chlorophyll a fluorescence were recorded under optimal conditions. Herbaceous plants acquired freezing tolerances to temperatures between ?10 and ?15°C when hardened at temperatures around 0°C. Most leaves fully developed prior to frost hardening exhibited typical symptoms of senescence after frost hardening. In non-senescing leaves P_n33 was reduced by 15 to 50% mainly due to a reduced stomatal conductance. After hardening at temperatures around ?10°C Brassica survived down to ?24°C, but P_n33 was almost abolished as a result of disturbances in the chloroplasts. After transferring the plants to 20/15°C P_n33 recovered completely within a few days. Woody plants hardened at temperatures around 0°C tolerated – 15 to ?36°C: P_n33 was reduced by 25 to 60% and hardly recovered at 20/15°C. Hardening at ?10°C induced a tolerance of ?32 to n33 was almost totally blocked, but at 20/15°C it returned to the values of the plants hardened at 0°C within a few days. In woody plants disturbances were invariably localized in the chloroplasts. Thus, conifers, and especially Pinus cembra, can survive much lower temperatures than herbaceous plants and, at the same level of freezing tolerance, exhibit appreciably less restriction in relative P_n33.     

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.     

Freezing tolerance of winter wheat plants depends on adaptation of photosynthesis and respiration in different time intervals

This study is devoted to CO₂ gas exchange (true photosynthesis at light saturation (P), dark respiration (R), and P/R ratio) in vegetating and cold-hardened winter wheat (Triticum aestivum L.) plants (cultivar Mironovskaya 808) in relation to their freezing tolerance. Under natural cultivation conditions, freezing tolerance of plants depended on adaptive changes in the shape of P and R curves in the temperature range from 20 to ?2°C. These changes, induced by cold hardening and treatment of plants with the photosynthesis inhibitor diuron, were observed within month and week ranges. Under laboratory conditions, the P/R ratio in vegetating plants increased three times within an hour range as the temperature decreased from 22 to 0°C. The P/R ratio also decreased within a minute range as a result of partial inhibition of photosynthesis with diuron and immediately decreased when CO₂ concentration in the air was reduced from 419 to 0 μl/l. The P/R ratio decreased primarily at the expense of a decrease in P. The decrease in P/R was more pronounced at low temperatures, indicating variability of low-temperature tolerance of photosynthesis within a minute range. The possibility of plant adaptation to nonsimultaneous temperature changes under natural conditions via adaptive changes in temperature tolerance of the photosynthetic apparatus is discussed.     

Alteration of Gene Expression during the Induction of Freezing Tolerance in Brassica napus Suspension Cultures

Brassica napus suspension-cultured cells can be hardened to a lethal temperature for 50% of the sample of −20°C in eight days at room temperature with abscisic acid. During the induction of freezing tolerance, changes were observed in the electrophoretic pattern of [³⁵S]methionine labeled polypeptides. In hardening cells, a 20 kilodalton polypeptide was induced on day 2 and its level increased during hardening. The induction of freezing tolerance with nonmaximal hardening regimens also resulted in increases in the 20 kilodalton polypeptide. The 20 kilodalton polypeptide was associated with a membrane fraction enriched in endoplasmic reticulum and was resolved as a single spot by two-dimensional electrophoresis. In vitro translation of mRNA indicate alteration of gene expression during abscisic acid induction of freezing tolerance. The new mRNA encodes a 20 kilodalton polypeptide associated with increased freezing tolerance induced by either abscisic acid or high sucrose. A 20 kilodalton polypeptide was also translated by mRNA isolated from cold-hardened B. napus plants.     

Effect of cold acclimation on bulk tissue electrical impedance: I. Measurements with birdsfoot trefoil at subfreezing temperatures

The resistive and reactive components of electrical impedance were measured for birdsfoot trefoil (Lotus corniculatus L.) stems at freezing temperatures to −8°C. As temperature decreased the specific resistance at frequencies between 49 hertz and 1.11 megahertz of stems from cold acclimated plants increased more rapidly than from nonacclimated plants. This temperature dependence of specific resistance could be characterized by an Arrhenius activation energy; cold acclimated stems had a larger Arrhenius activation energy than nonacclimated stems. The low frequency resistance is believed to characterize the extracellular region of the stems and the high frequency resistance is believed to characterize the intracellular region of the stems. Cold acclimation increased the intracellular but not the extracellular resistance at nonfreezing temperatures. Cold acclimated stems were not injured by freezing to −8°C and thawing, but nonacclimated stems were injured by freezing to temperatures between −2.2 and −5.6°C and thawing. Injury to nonacclimated stems at freezing temperatures below −2.2°C was indicated by a decrease in the ratio of resistance at 49 Hz to that at 1.11 megahertz.     

Factors contributing to enhanced freezing tolerance in wheat during frost hardening in the light

The interaction between light and temperature during the development of freezing tolerance was studied in winter wheat (Triticum aestivum L. var. Mv Emese). Ten-day-old plants were cold hardened at 5 degrees C for 12 days under normal (250 micromol m(-2)s(-1)) or low light (20 micromol m(-2)s(-1)) conditions. Some of the plants were kept at 20/18 degrees C for 12 days at high light intensity (500 micromol m(-2)s(-1)), which also increased the freezing tolerance of winter wheat. The freezing survival rate, the lipid composition, the antioxidant activity, and the salicylic acid content were investigated during frost hardening. The saturation level of hexadecanoic acid decreased not only in plants hardened at low temperature, but also, to a lesser extent, in plants kept under high light irradiation at normal growth temperature. The greatest induction of the enzymes glutathione reductase (EC 1.6.4.2.) and ascorbate peroxidase (EC 1.11.1.11.) occurred when the cold treatment was carried out in normal light, but high light intensity at normal, non-hardening temperature also increased the activity of these enzymes. The catalase (EC 1.11.1.6.) activity was also higher in plants grown at high light intensity than in the controls. The greatest level of induction in the activity of the guaiacol peroxidase (EC 1.11.1.7.) enzyme occurred under cold conditions with low light. The bound ortho-hydroxy-cinnamic acid increased by up to two orders of magnitude in plants that were cold hardened in normal light. Both high light intensity and low temperature hardening caused an increase in the free and bound salicylic acid content of the leaves. This increase was most pronounced in plants that were cold treated in normal light.     

The long day leaf as a source of cold hardiness inhibitors

Short photoperiods followed by low temperatures induced cold hardiness in Acer negundo, Viburnum plicatum tomentosum, and Weigela florida. Hardiness was also obtained under long days and natural fall temperatures if the leaves were removed, either manually or by low temperature. Similarly, removal of leaves from plants exposed to long days at 5° brought about an accelerated rate of hardening. These observations suggested the presence of a hardiness inhibitor in the leaves which was counteracted by short days or removal of the leaves.     

Effects of growth stage and hardening conditions on the association between frost resistance and the expression of the cold-induced protein COR14b in barley

Sowing date, being determinant for growth stage, may play a decisive role in optimising freezing resistance of winter annual plants. In cereal species, in spite of the abundant literature analysing the factors responsible for the acquisition of frost resistance through the cold hardening process, the involvement of the growth stage per se, has been seldom considered, especially at the earlier vegetative phases. In this work the contribution of growth stage in determining resistance to freezing temperature has been analysed in field and growth chamber experiments using winter and spring barley cultivars exposed to different hardening conditions. Field damage was assessed twice during winter on plants sown at three different dates. In the growth chamber experiments several acclimation treatments at 11/7 and/or 3/1 °C (day/night) were simulated. In both field and laboratory experiments the development of cold acclimation was monitored by means of a COR14b specific antibody, since in previous studies the expression of COR14b was found genetically linked to frost resistance. The lowest resistance, found in the youngest plants and in spring cultivars, however, was not always associated with the lowest level of COR14b accumulation. COR14b accumulation correlated with frost resistance at the earlier field sampling date and in plants grown at 11/7 °C. In a following phase of the hardening process (second sampling in field and 4 weeks at 3/1 °C in growth chamber) the accumulation of COR14b was independent of plant stage and genotype, showing no association with freezing resistance. Results suggest that growth stage is crucial for the achievement of maximal resistance in barley, but not for COR14b expression.     

Effects of Hormones on Morphogenesis and Cold Resistance in Berseem Clover (Trifolium alexandrinum L.)

Plants of berseem clover (Trifolium alexandrinum L.) cv. Taborwere raised under conditions inhibiting the acquisition of coldhardiness (non-hardened) or inducing cold hardiness (hardened).All non-hardened plants developed an elongated shoot and exhibitedconsiderable frost sensitivity, as measured by the extent ofthe reduction in yield of variable chlorophyll fluorescenceafter exposure to sub-zero temperature. Hardened plants developeda shorter shoot, with fewer leaves and a greater percentageof dry matter in the root system. These parameters were associatedwith a marked increase in frost resistance. Exogenous applicationof ABA to plants effected similar morphological modificationsin both hardening and non-hardening temperature regimes; plantsdeveloped a shorter primary shoot axis and leaves exhibiteda marked increase in frost hardiness. In berseem clover ABAcan thus substitute, at least partially, for the low temperaturetreatment required to induce cold hardiness. Spraying plantsraised under hardening conditions with gibberellic acid reversedthe effects of the hardening treatment, since they developedan elongated shoot and exhibited frost sensitivity comparableto non-treated plants grown under non-hardening conditions.It is concluded that these endogenous hormones are directlyinvolved in triggering changes in morphogenesis which accompanyphysiological and metabolic events associated with the inductionof plant cold hardiness. Key words: Frost resistance, morphogenesis, abscisic acid, giberellic acid, Trifolium alexandrinum     

The Effect of Abscisic Acid on the Freezing Tolerance of Callus Cultures of Lotus corniculatus L

The effects of growth temperature (2°C and 24°C), abscisic acid (ABA) concentration, duration of exposure to ABA, and light were assessed for their ability to induce acclimation to freezing temperatures in callus cultures of Lotus corniculatus L. cv Leo, a perennial forage legume. The maximal expression of freezing tolerance was achieved on B₅ media containing 10⁻⁵ molar ABA, at 24°C for 7 or 14 days. Under these culture conditions, the freezing tolerance of the callus approximated that observed in field grown plants. In contrast, low temperatures (2°C) induced only a limited degree of freezing tolerance in these cultures. Viability was assessed by tetrazolium reduction and by regrowth of the callus. The two assays often differed in their estimates of absolute freezing tolerance. Regression analysis of the temperature profile suggested that there may be two or more distinct populations of cells differing in freezing tolerance, which may have contributed to the variability between viability assays.     

The effect of photoperiod and temperature on growth and frost resistance of winter rye root systems

Root growth, development and frost resistance were examined in winter rye ( Secale cereale L. cv. Puma) plants grown under 6 combinations of temperature and photoperiod (20/16°C or 5/3°C, day/night; 8, 16- or 24-h days). Overall root system growth is influenced by the interaction of temperature and photoperiod. Maximum shoot growth occurs at a 24-h photoperiod in 20°C plants and at a 16-h photoperiod in 5°C plants, and is correlated in both treatments with a high root:shoot ratio. Frost resistance of rye roots is affected by short photoperiods in 2 ways. First, short photoperiod and low temperature delay production of new adventitious roots so that newly developing roots are not exposed to freezing temperatures. Second, short photoperiod alone can induce several degrees of frost tolerance in existing roots during the lag phase of growth. Low temperature alone does not decrease the rate of dry weight accumulation in rye root systems, but cold temperature does retard developmental processes within the roots. Rye roots grown at 5°C develop first order lateral roots, differentiate metaxylem vessels and suberize endodermal cell walls more slowly than roots grown at 20°C.     

The importance of hardening and winter temperature for growth in mountain birch populations

Seedlings of five mountain birch populations (Betula pubescens Ehrh. ssp. czerepanovii) from Fennoscandia and Iceland were raised and grown at natural daylengths at Tromsø, Norway (69°N) and different temperatures during late summer and fall season, followed by winter temperature treatment at ambient and +4 °C above ambient temperatures at Bergen, Norway (60°N). The experiment took place during two seasons (2000/01 and 2001/02). The following summer shoot and biomass growth were reduced as a result of winter warming and subsequent premature dehardening in early flushing provenances and treatments. Biomass increased in plants grown at low hardening temperature when compared with high temperature treatment. As a conclusion, increased winter temperatures would tend to increase the risk of spring frost damage and reduce growth in birch seedlings, because the differences between the frost hardening and ambient temperatures are decreasing, and because the time from budbreak to dehardening is shortened. The results are discussed in relation to simultaneous experiments with frost hardiness in the same populations and treatments.     

Low and High Temperature Limits to PSII : A Survey Using trans-Parinaric Acid, Delayed Light Emission, and F(o) Chlorophyll Fluorescence

Many studies have shown that membrane lipids of chilling-sensitive plants begin lateral phase separation (i.e. a minor component begins freezing) at chilling temperatures and that chilling-sensitive plants are often of tropical origin. We tested the hypothesis that membranes of tropical plants begin lateral phase separation at chilling temperatures, and that plants lower the temperature of lateral phase separation as they invade cooler habitats. To do so we studied plant species in one family confined to the tropics (Piperaceae) and in three families with both tropical and temperate representatives (Fabaceae [Leguminosae], Malvaceae, and Solanaceae). We determined lateral phase separation temperatures by measuring the temperature dependence of fluorescence from trans-parinaric acid inserted into liposomes prepared from isolated membrane phospholipids. In all families we detected lateral phase separations at significantly higher temperatures, on average, in species of tropical origin. To test for associated physiological effects we measured the temperature dependence of delayed light emission (DLE) by discs cut from the same leaves used for lipid analysis. We found that the temperature of maximum DLE upon chilling was strongly correlated with lateral phase separation temperatures, but was on average approximately 4°C lower. We also tested the hypothesis that photosystem II (PSII) (the most thermolabile component of photosynthesis) of tropical plants tolerates higher temperatures than PSII of temperate plants, using DLE and F_o chlorophyll fluorescence upon heating to measure the temperature at which PSII thermally denatured. We found little difference between the two groups in PSII denaturation temperature. We also found that the temperature of maximum DLA upon heating was not significantly different from the critical temperature for F_o fluorescence. Our results indicate that plants lowered their membrane freezing temperatures as they radiated from their tropical origins. One interpretation is that the tendency for membranes to begin freezing at chilling temperatures is the primitive condition, which plants corrected as they invaded colder habitats. An alternative is that membranes which freeze at temperatures only slightly lower than the minimum growth temperature confer an advantage.     

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

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