Induction of Frost Hardiness in Stem Cortical Tissues of Cornus stolonifera Michx. by Water Stress: II. Biochemical Changes

A decrease of protein, RNAs, and starch, and an increase of sugar were observed in 3-day water-stressed red osier dogwood plants (Cornus stolonifera Michx.) when the frost hardiness increased from −3 to −6 C. As the frost hardiness increased to −11 C after 7 days of treatment, the starch continuously decreased, however, the proteins and RNAs increased with a continuous increase of sugar. Further water stress treatment had little effect on the changes of these chemicals. Control plants in short days showed similar gradual biochemical changes in patterns. From the results of frost hardiness increases, the pattern of biochemical changes, and the mechanism of the increased freezing resistance, it appears that the water stress and short days accomplished essentially the same physiological end(s) in inducing frost hardiness in red-osier dogwood.     

Changes in Frost Hardiness of Stem Cortical Tissues of Cornus stolonifera Michx. after Recovery from Water Stress

Moderate water stress increases frost hardiness in many woody plants but little attention has been given to changes in hardiness after recovery from water stress. Tests were carried out to examine how much water stress-induced frost hardiness remained when plants were rewatered under different day length regimes. Red osier dogwood plants (Cornus stolonifera Michx.) were water-stressed at normal growing temperatures in long day (LD) or short day (SD) conditions, exposed to 6 nights of freezing temperatures, and then returned to normal growing conditions with full water supply. Water-stressed plants gained an additional 8 to 10 C of hardiness. The amount of freeze-induced hardiness in both stressed and control plants was not significant (approximately 2 C) and was not affected by photoperiod. When plants were kept in or transferred to LD, they lost nearly all of their water stress-induced hardiness within 7 days after rewatering. Water-stressed plants in SD lost the least amount of hardiness (5 C) when rewatered. In dogwood, water stress is an effective way to increase hardiness temporarily, but the photoperiod has a large effect on the retention of the acquired hardiness.     

Interactions of low temperature, water stress, and short days in the induction of stem frost hardiness in red osier dogwood

The induction of stem frost hardiness by low temperature, water stress, short days, and their combinations in 2- and 4-month-old growing dogwoods (Cornus stolonifera) were investigated. When plants were subjected to more than one factor, the increased hardiness was the sum of the effects of the individual factors involved. No interactions among these factors on hardiness were observed during a 3-week treatment. Results indicate that low temperature, water stress, and short days initially trigger independent frost-hardening mechanisms. Plant ages significantly influenced the change in low temperature-induced frost hardiness, but not the water stress or short day-induced frost hardiness.     

Plasma Membrane Alterations in Callus Tissues of Tuber-bearing Solanum Species during Cold Acclimation

Plasma membrane alterations in two tuber-bearing potato species during a 20-day cold acclimation period were investigated. Leaf-callus tissues of the frost-resistant Solanum acaule Hawkes `Oka 3878' and the frost-susceptible, commonly grown Solanum tuberosum `Red Pontiac,' were used. The former is a species that can be hardened after subjecting to the low temperature, and the latter does not harden. Samples for the electron microscopy were prepared from callus cultures after hardening at 2 C in the dark for 0, 5, 10, 15, and 20 days. After 20 days acclimation, S. acaule increased in frost hardiness from −6 to − 9 C (killing temperature), whereas frost hardiness of S. tuberosum remained unchanged (killed at −3 C). Actually, after 15 days acclimation, a −9 C frost hardiness level in S. acaule callus cultures had been achieved.     

Molecular,Physiological and Biochemical Responses of Theobroma cacao L. Genotypes to Soil Water Deficit

Six months-old seminal plants of 36 cacao genotypes grown under greenhouse conditions were subjected to two soil water regimes (control and drought) to assess, the effects of water deficit on growth, chemical composition and oxidative stress. In the control, soil moisture was maintained near field capacity with leaf water potentials (ΨWL) ranging from −0.1 to −0.5 MPa. In the drought treatment, the soil moisture was reduced gradually by withholding additional water until ΨWL reached values of between −2.0 to −2.5 MPa. The tolerant genotypes PS-1319, MO-20 and MA-15 recorded significant increases in guaiacol peroxidase activity reflecting a more efficient antioxidant metabolism. In relation to drought tolerance, the most important variables in the distinguishing contrasting groups were: total leaf area per plant; leaf, stem and total dry biomass; relative growth rate; plant shoot biomass and leaf content of N, Ca, and Mg. From the results of these analyses, six genotypes were selected with contrasting characteristics for tolerance to soil water deficit [CC-40, C. SUL-4 and SIC-2 (non-tolerant) and MA-15, MO-20, and PA-13 (tolerant)] for further assessment of the expression of genes NCED5, PP2C, psbA and psbO to water deficit. Increased expression of NCED5, PP2C, psbA and psbO genes were found for non-tolerant genotypes, while in the majority of tolerant genotypes there was repression of these genes, with the exception of PA-13 that showed an increased expression of psbA. Mutivariate analysis showed that growth variables, leaf and total dry biomass, relative growth rate as well as Mg content of the leaves were the most important factor in the classification of the genotypes as tolerant, moderately tolerant and sensitive to water deficit. Therefore these variables are reliable plant traits in the selection of plants tolerant to drought.     

Studies of Frost Hardiness in Woody Plants. II. Effect of Temperature on Hardening

The effect of temperature on hardening was studied at temperatures ranging from 0° to −20° using twigs of willow and poplar. In October and in late April when the twigs are not very frost hardy, hardening at 0° produced a considerable increase in their frost hardiness, although the effectiveness of hardening at 0° decreased with a decrease in the environmental temperature. In twigs which could withstand continuous freezing without injury, hardening at −3° to −5° was most effective in increasing the frost hardiness of the twigs. Below −20°, only negligible increase was observed either in frost hardiness or sugar content.

The rate of starch to sugar conversion differed remarkably in different twig tissues. The starch in xylem was more slowly converted to sugar than that in the cortex. The optimum temperature for converting starch into sugar during frost hardening was also found to be −3° to −5°. In addition, the greater the effectiveness of the hardening treatment, the greater the rate of conversion from starch to sugar. The frost hardiness of a twig is closely related to the sugar content of the twig, especially in the xylem.

     

Involvement of abscisic Acid in potato cold acclimation

Upon exposure to 2°C day/night (D/N), leaves of Solanum commersonii (Sc) began acclimating on the 4th day from a −5°C (killing temperature) hardy level to −12°C by the 15th day. Leaves of S. tuberosum L. (St) cv `Red Pontiac' typically failed to acclimate and were always killed at −3°C. Leaves of control (20/15°C, D/N) and treated plants (2°C, D/N) of St showed similar levels of free abscisic acid (ABA) during a 15-day sampling period. In treated Sc plants, however, free ABA contents increased 3-fold on the 4th day and then declined to their initial level thereafter. The increase was not observed in leaves of Sc control plants.

Treated St plants showed a slightly higher content of leaf soluble protein than controls. In Sc, leaves of controls maintained relatively constant soluble proteins, but leaves of treated plants showed a distinct increase. This significant increase was initiated on the 4th day, peaked on the 5th day, and remained at a high level throughout the 15-day sampling period.

Exogenously applied ABA induced frost hardiness in leaves of Sc plants whether plants were grown under a 20°C or 2°C temperature regime. When cycloheximide was added to the medium of stem-cultured plants at the beginning of 2°C acclimation, or at the beginning of the ABA treatment in the 20°C regime, it completely inhibited the development of frost hardiness. However, when cycloheximide was added to plants on the 5th day during 2°C acclimation, the induction of frost hardiness was not inhibited. The role of ABA in triggering protein synthesis needed to induce frost hardiness is discussed.

     

Environmental control of cold hardiness in woody plants

The development of cold hardiness in 2 woody plant species (Acer negundo and Viburnum plicatum tomentosum) was shown to be independent of the induction of bud dormancy. Substantial hardiness levels were obtained under controlled conditions with long days and certain low temperatures—without dormancy development as a prerequisite.

Low temperatures given during the dark period with long days induced hardiness to a level not significantly different from that of short days. Giving plants continuous 10° temperatures under long days forced plants to harden as if they were under short days, even though they were not dormant.

Development of hardiness was shown to be a photoperiodic response. Increasing weeks of short days, followed by a low temperature hardening period in darkness, brought about a progressive increase in hardiness. The short day stimulus could be reversed by long days. Following 6 weeks of short days, the rate of hardening in darkness at 5° was over twice that of plants previously exposed to long days.

     

Survival of Ice Nucleation-Active and Genetically Engineered Non-Ice-Nucleating Pseudomonas syringae Strains after Freezing

The survival after freezing of ice nucleation-active (INA) and genetically engineered non-INA strains of Pseudomonas syringae was compared. Each strain was applied to oat seedlings and allowed to colonize for 3 days, and the plants were subjected to various freezing temperatures. Plant leaves were harvested before and after freezing on two consecutive days, and bacterial populations were determined. Populations of the INA wild-type strain increased 15-fold in the 18 h after the oat plants incurred frost damage at −5 and −12°C. Plants colonized by the non-INA strain were undamaged at −5°C and exhibited no changes in population size after two freeze trials. As freezing temperatures were lowered (−7, −9, and −12°C), oat plants colonized by the non-INA strain suffered increased frost damage concomitant with bacterial population increases following 18 h. At −12°C, both strains behaved identically. The data show a relationship between frost damage to plants and increased bacterial population size during the following 18 h, indicating a potential competitive advantage of INA strains of P. syringae over non-INA strains in mild freezing environments.     

Vegetative growth and frost hardiness of cloudberry (Rubus chamaemorus) as affected by temperature and photoperiod

The effect of photoperiod and temperature on growth and induction and development of frost hardiness in cloudberry ( Rubus chamaemorus L.) was examined in two experiments. The photoperiods were 8, 12 or 24 h and the temperatures were 18, 15, 12, 9, 4, 3, –3 or –4°C depending on the experiment. The level of hardiness was expressed as LT₆₆ or LT₅₀ (the lethal temperature for 66 or 50% of the plant material) for percentage of bud break and for the degree of coloring by triphenyltetrazolium chloride for rhizomes. The vegetative growth was clearly affected by daylength; petiole elongation, leaf growth, shoot dry weight and number of shoots per plant were all reduced under short days compared with long days. However, the photoperiod had no significant effect on hardening of buds or rhizomes. Hardening increased with successively decreasing temperatures. To get the maximum hardiness, plants had to be exposed to freezing temperatures.     

Overwintering stress of Vaccinium vitis-idaea in the absence of snow cover

A snow manipulation experiment aimed to assess risks of direct freezing injury, freeze-induced dehydration and winter desiccation in the absence of snow cover on lingonberry (Vaccinium vitis-idaea). Frames with sheet-plastic sides and removable lids were used in this experiment for two purposes: to prevent accumulation of snow in mid-winter and to provide extra heat during early spring. Leaves were analyzed for frost hardiness, tissue water content and osmotic concentrations, and photoinhibition (Fv/Fm) during the period from the 10th of February to the 7th of April. The natural snow accumulation was low indicated by a minor difference in minimum temperatures between the frame treatment and naturally snow-covered plots. The heating effect of the frames started gradually at the end of February along with increasing solar elevation angles, and was highest at the beginning of April. Frost hardiness peaked in March as a consequence of cold periods, but it was practically lost by the beginning of April. Tissue water content decreased gradually at first, becoming greatly decreased later due to the extra heat. In accordance, the tissue osmotic concentrations increased first gradually, followed by a dramatic increase. Photoinhibition increased uniformly with increasing solar radiation, but at the end showed a sharp increment within a few days, obviously also indicating the effect of heating. It was concluded that neither lethal freezing stress nor significant freeze-induced dehydration occurred during the experiment. However, plants that overwintered without snow suffered from severe winter desiccation injuries due to the combination of solar heat and frozen soil. Although the desiccation stress was possibly a lethal factor, it was preceded by long-term and continued photoinhibition. It was concluded that during overwintering, chamaephyte species may suffer from both freezing and winter desiccation in the absence of protecting snow cover. However, during mild winters provided by climatic change scenarios, the risk of winter desiccation will be more probable. In relation to the future climate, it was concluded that winter desiccation and photoinhibition may develop gradually during a snowless winter and would, even if they did not reach a lethal level by themselves, possibly reduce frost hardiness.     

Cold hardening of raspberry plants in vitro is enhanced by increasing sucrose in the culture medium

The influence of exogenously applied sucrose on cold hardening of raspberry ( Rubus idaeus L.) in vitro was examined. Raspberry plants (cv. Preussen) were cultured on Murashige-Skoog (MS) media with different levels (1, 3, 5 and 7%) of sucrose and subjected to low-temperature acclimation (3/−3°C day/night temperature, 8-h photoperiod) for 14 days. Cold hardiness (LT₅₀ in controlled freezing), shoot moisture content, osmolality and the amounts of sucrose, glucose and fructose were determined. Exogenously applied sucrose was taken up by plants, but the uptake corresponded to less than 10% of total sugar reserves in the culture. Cold hardiness was primarily affected by acclimation treatment, but sucrose increased cold hardiness of nonacclimated plants and significantly enhanced the effect of acclimation treatment, 5% sucrose in the culture medium being optimal for cold hardening. LT₅₀ values ranged between −4.1 and −7.1°C for nonacclimated, and between −14.2 and −20.7°C for cold-acclimated shoots. Shoot moisture content was inversely related to medium sucrose level and declined only slightly during cold acclimation. After cold acclimation, plant osmolality predicted hardiness better than shoot moisture content. Plant osmolality and sugar content were increased by increasing the medium sucrose level and, to a greater extent, by cold acclimation. Sucrose, glucose and fructose accumulated during hardening. Sucrose was the predominant sugar, and the rate of sucrose accumulation during cold acclimation was independent of the medium sucrose level or the initial plant sucrose content. A close correlation between cold hardiness and total sugars, sucrose, glucose and fructose was established. These results suggest that sugars have more than a purely osmotic effect in protecting acclimated raspberry plants from cold.     

Characterization and Role of an Endogenous Inhibitor in the Induction of Cold Hardiness in Acer negundo

An inhibitor extracted from short day treated Acer negundo leaves was compared to abscisic acid in 4 different solvent systems. The chromatographic properties of abscisic acid and the inhibitor were in very close agreement. Treatment of Acer negundo plants under non-hardening preconditions (long days) with either the inhibitor or abscisic acid increased hardiness after a hardening period of 3 weeks at 40°. A gibberellin-inhibitor relationship was further studied by making comparison of extracts of plants subjected to either 4 weeks of long days, long days + 5° nights, or short days. These tests indicated that gibberellin-like activity was greatest when the treatment included long days. Abscisic acid-like levels were highest when the treatments consisted of short days or long days + 5° nights. Since the latter groups are the most capable of developing hardiness, the hardening process appears to be more closely related to a build-up of abscisic acid levels than a reduction of gibberellin levels.     

The Influence of Photoperiod and Temperature on the Development of Frost Hardiness in Seedlings of Pinus silvestris and Picea abies

The influence of short days and low temperature on the development of frost hardiness in seedlings of Scots pine (Pinus silvestris L.) and Norway spruce [Picea abies (L.) Karst.], grown for 6 months in glasshouses and climate chambers, was investigated. The degree of hardiness was estimated by freezing the shoots of the seedlings to predetermined temperatures. After 8 weeks in a glasshouse the viability of the seedlings was determined by establishing bud flushing. The most effective climate for the development of frost hardiness was short days (SD) and low temperature (2°C); the next most effective was SD and room temperature (20°C). However, long days (LD) and low temperature also had a marked effect on the development of hardiness. A combination of 3 weeks’treatment with SD and 20°C, and 3 weeks with SD and 2°C gave the same results as 6 weeks with SD and 2°C. The results clearly demonstrate the importance of the photoperiod prior to low temperature for the development of frost hardiness. In conclusion both short days and low temperature induce frost hardiness development. Probably this occurs by initiation of different processes in the two cases. The degree of frost hardiness development appears to depend on the sum of these different processes and on the timing between them.     

Osmotic adjustment in leaves of sorghum in response to water deficits

The relationships among the total water potential, osmotic potential, turgor potential, and relative water content were determined for leaves of sorghum (Sorghum bicolor [L.] Moench cvs. `RS 610' and `Shallu') with three different histories of water stress. Plants were adequately watered (control), or the soil was allowed to dry slowly until the predawn leaf water potential reached either −0.4 megapascal (MPa) (treatment A) or −1.6 MPa (treatment B). Severe soil and plant water deficits developed sooner after cessation of watering in `Shallu' than in `RS 610', but no significant differences in osmotic adjustment or tissue water relations were observed between the two cultivars. In both cultivars, the stress treatments altered the relationship between leaf water potential and relative water content, resulting in the previously stressed plants maintaining higher tissue water contents than control plants at the same leaf water potential. The osmotic potential at full turgor in the control sorghum was −0.7 MPa: stress pretreatment significantly lowered the osmotic potential to −1.1 and −1.6 MPa in stress treatments A and B, respectively. As a result of this osmotic adjustment, leaf turgor potentials at a given value of leaf water potential exceeded those of the control plants by 0.15 to 0.30 MPa in treatment A and by 0.5 to 0.65 MPa in treatment B. However, zero turgor potential occurred at approximately the same value of relative water content (94%) irrespective of previous stress history. From the relationship between turgor potential and relative water content there was an approximate doubling of the volumetric elastic modulus, i.e. a halving of tissue elasticity, as a result of stress preconditioning. The influence of stress preconditioning on the moisture release curve is discussed.     

Effect of photoperiod and temperature on the development of frost hardiness in three Alnus species

The influence of short day and low temperature on cold acclimation of A. crispa (Ait.) Pursh, A. glutinosa (L.) Gaertn. and A. rubra Bong, was investigated. Two clones of each species originating from in vitro propagation were exposed to three daylength/temperature treatments. Periodically plantlets were exposed to controlled freezing temperature in order to evaluate their level of frost hardiness.
Short day (SD) and cold temperature (CT) and long day (LD) and cold temperature (CT) were the most effective treatments for the development of frost hardiness in shoots and roots of the three species tested. Short day (SD) and warm temperature (WT) induced a significant increase in hardiness in shoots of all three species. However, this treatment did not trigger root hardening. A. crispa was found to be the hardiest species followed by A. glutinosa and A. rubra . Intraspecific variation was observed between the two A. glutinosa clones. A glutinosa clone AG8, a Russian provenance, showed a greater freezing resistance than A. glutinosa clone AG2, a German provenance.     

Water Relations, Stomatal Behavior, and Root Conductivity of Red Osier Dogwood during Acclimation to Freezing Temperatures

Red osier dogwood (Cornus stolonifera Michx.) was artificially acclimated by exposing plants to 8-hour short days (SD) and low (15/5 C) temperatures for 54 to 63 days. Several factors including transpiration rate, stomatal resistance, and root conductivity were correlated so that the rate of water loss in acclimating plants was higher during the first 30 to 40 days of the acclimation sequence. Six days after transferring plants to SD conditions, the stomatal resistance (r₈) decreased significantly below the r₈ of the 16-hour long day (LD) control plants at the same temperature. Transpiration rate increased by approximately 20 to 30% in the plants transferred to SD. After the initially higher transpiration rate and greater stomatal opening, the stomates closed tightly during the last 2 weeks of acclimation and the transpiration rate of the SD plants dropped to well below the LD control plants. By the end of the acclimation sequence, root conductivity to water uptake was two to three times lower in the SD plants. Leaf xylem water potentials were similar or slightly lower in the plants kept under SD conditions during the first 5 to 7 weeks of the acclimation sequence. During the last 10 to 15 days of acclimation when the stomates closed, SD leaf water potential rose significantly above the plants in the LD conditions. During acclimation, stem water content decreased by 40 to 50%. Changes in tissue hydration can be indirectly related to plant hardiness and may be affected by alteration of stomatal resistance, transpiration rate, and root conductivity during acclimation.     

Alfalfa water status and cold hardiness as influenced by cold acclimation and water stress

Abstract Water stress at a nonacclimating temperature (18–20°C) increased the cold hardiness of Medicagosativa L. (alfalfa) plants. This increased cold hardiness was retained when the previously water-stressed plants were cold acclimated (2–9°C) in the absence of water stress. Water stress during cold acclimation also increased cold hardiness. Alfalfa was demonstrated to suffer injury, measured as decreased growth following freezing, at sub-lethal temperatures. During cold acclimation the turgor potential (ψ) of watered plants increased, whereas the solute potential and the water content per unit dry weight decreased. The large positive psgrdap of acclimated plants indicates that the decreased water content per unit dry weight is related to an increased proportion of tissue dry matter rather than to tissue dehydration.     

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.

     

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.