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.     

Induction of Frost Hardiness in Stem Cortical Tissues of Cornus stolonifera Michx. by Water Stress: I. Unfrozen Water in Cortical Tissues and Water Status in Plants and Soil

Water supply and day length were varied in cold hardiness studies of red osier dogwood plants (Cornus stolonifera Michx.). The frost killing temperature, the content and freezing of stem cortical tissue water along with soil moisture content and tension were evaluated. Seven days of water stress in long and short day photoperiod regimes caused a rapid decrease in soil moisture content and plant water potential. During the same period, the frost hardiness increased from −3 to −11 C. Further water stress treatment had little effect. Control plants in short days showed only a gradual decrease in plant water potential and only gradually increased in frost hardiness while control plants in long days were unchanged. Freezing studies using nuclear magnetic resonance showed that increased hardiness in water-stressed plants resulted from both an increased tolerance of freezing and an increased avoidance of freezing, the latter resulting from higher solute concentration in the tissue solutions. The short day controls also showed similar changes; however, the changes were smaller over the 21 days of the study.     

Induction of Cold Acclimation in Cornus stolonifera Michx

A warm (20 to 15 Celsius day or night) preconditioning treatment enhanced cold acclimation of Cornus stolonifera bark under short-day conditions when plants were preconditioned for at least 4 weeks. Warm preconditioning inhibited the acclimation of plants subjected to long photoperiods. Removing leaves from plants exposed to low temperatures and short days inhibited acclimation. Removal of buds did not affect acclimation. Plants did not acclimate unless they were exposed to at least 4 weeks of short photoperiods prior to defoliation. Plants began to acclimate to cold at the time of growth cessation but not before. When half of the leaves were removed from plants, the defoliated and foliated branches both acclimated as well as branches on completely foliated plants. Girdling the phloem between foliated and defoliated branches prevented acclimation of the latter regardless of the position of the girdle in relation to the root system and the defoliated branch. When all of the leaves of plants were covered with aluminum foil to exclude light after 0 or 4 weeks of exposure to short days, the results resembled a defoliation study, i.e., plants with leaves covered at the start of the experiment failed to acclimate, and those covered after 4 weeks acclimated to some extent but less than uncovered control plants. Under longday conditions plants with all leaves covered failed to acclimate, and plants with none or half of their leaves covered acclimated equally and to a limited extent. Under short-day conditions, however, the covered branches of partially covered plants acclimated more than their uncovered counterparts or branches of totally uncovered plants.     

Water Permeability and Cold Hardiness of Cortex Cells in Cornus stolonifera Michx.-A Preliminary Report

The relationship of freezing resistance to water permeability of cortex cells was studied in stems of red osier dogwood (Cornus stolonifera Michx.). Permeability was estimated by determining the diffusion flux of tritiated water from cortex slices previously equilibrated in tritiated water. Energy of activation and diffusion time comparisons of tritiated water flux from living cortex slices and slices killed by immersion in liquid N₂ verified that intact membranes of uninjured cortex cells limited water flux.     

Ultrastructural Changes Related to Frost Hardiness in the Cortical Parenchyma Cells from Mulberry Twigs

Ultrastructural changes in the cortical parenchyma cells ofmulberry (Morus bombyciz Koidz. cv. ‘Gorogi’) twigswere related closely to the seasonal cycle of frost hardiness.Changes in hardiness from summer to winter and vice versa tookplace particularly in the plasmamembrane. During stages that result in a rapid increase in hardiness,the sequence of cytological changes for the replacement of theplasmamembrane was marked invaginations of the plasmamembrane;pinching off into vesicles in the peripheral cytoplasm; engulfmentof these vesicles into vacuoles; generation of numerous microvesiclesby the Golgi apparatus; the appearance of these microvesicleswith the plasmamembrane. Similar cytological changes that areassociated with increased hardiness occur during artificialhardening at 0°C for 5 to 20 days in both the autumn andspring. In the midwinter cells, however, the plasmamembranehas a fairly smooth structure. Thus, the highly folded structureof the plasmamembrane is not always characteristic of extremelyhardy cells. Ultrastructural changes associated with hardinessare discussed in terms of membrane renewal. ¹Contribution No. 2267 from the Institute of Low TemperatureScience. (Received October 11, 1980; Accepted December 5, 1980)     

Biochemical Changes in Tuber-bearing Solanum Species in Relation to Frost Hardiness during Cold Acclimation

Biochemical changes in potato leaves during cold acclimation have been examined and compared between a frost-tolerant S. acaule and a frost-susceptible S. tuberosum species. Changes were also examined in S. tuberosum, S. acaule, and S. commersonii species when they were hardened at different temperatures to varying hardiness levels.     

Environmental and Seasonal Factors Affecting the Frost-induced Stage of Cold Acclimation in Cornus stolonifera Michx

Stem tissues of red-osier dogwood (Cornus stolonifera Michx.) acclimated from −3 C to −40 or −50 C in 8 to 10 weeks under a short photoperiod (9 hours) and controlled temperature conditions. During the summer months plants did not acclimate as well as at other times. The sequence of day/night temperature regimes which induced maximum acclimation was 20/15 C for 5 to 6 weeks; 15/5 C for 2 to 3 weeks; 15/5 C plus 1 hour of frost per day for 1 week. The duration of exposure to each temperature regime influenced the rate and intensity of frost-induced acclimation. Less than 5 weeks of warm temperature preconditioning at 20/15 C reduced subsequent frost-induced acclimation. The inductive influence of frost on cold acclimation was additive over 5 days of repeated exposure, but its effects after the first exposure(s) were not immediate—requiring 1 to 4 days of 15/5 C following the frost treatments for the expression of the frost-induced acclimation to be manifest. There was a 75% increase in rRNA following 3 days of frost exposure and plants in an O₂-free atmosphere during frost exposure failed to acclimate. The results suggest that seasonal acclimation behavior was due to endogenous rhythms rather than developmental stage, and that the frost-induced phase of acclimation involves aerobic metabolic processes.     

Relationship of Electrical Conductance at Two Frequencies to Cold Injury and Acclimation in Cornus stolonifera Michx

The ratio of electrical conductance measured at two frequencies can be used to predict the cold hardiness of stem sections of Cornus stolonifera Michx. during the first stage of cold acclimation. Electrical conductance at 50 hertz divided by electrical conductance at 100 kilohertz gave a better estimate of hardiness than measurements at either frequency alone. The observed increase in the electrical conductance ratio as hardiness increased is consistent with an increase in membrane permeability. After plants were exposed to nonlethal frost, hardiness increased rapidly, and the relation between the conductance ratio and hardiness changed. This change indicates that ice crystallization induces a significant physiological alteration in the plants. Contrary to expectations, stem sections exposed to lethal temperatures could not consistently be separated from sections exposed to nonlethal temperatures by electrical conductance ratio measurements made immediately after thawing.     

Effects of Red and Far Red Light on the Initiation of Cold Acclimation in Cornus stolonifera Michx

Red and far red light distinctly influence the initial phytochrome-mediated phase of cold acclimation in red-osier dogwood (Cornus stolonifera). Under controlled conditions, short days and end-of-day far red light exposure after long days promote growth cessation, cold acclimation, and subsequent cold hardening of dogwood stems in response to low temperature. Nuclear magnetic resonance absorption spectra of the water in internode stem sections imply that the short day-induced phase of cold acclimation involves a change in tissue hydration, at least in part, due to a substantial reduction in bulk phase water as a result of senescence and loss of water from the pith. Seasonal responses to light and an attempt to induce early acclimation under natural conditions with end-of-day far red light are discussed.     

Effects of Mild Water Stress and Diurnal Changes in Temperature and Humidity on the Stable Oxygen and Hydrogen Isotopic Composition of Leaf Water in Cornus stolonifera L

In this paper we make comparisons between the observed stable isotopic composition of leaf water and the predictions of the Craig-Gordon model of isotopic enrichment when plants (Cornus stolonifera L.) were exposed to natural, diurnal changes in temperature and humidity in a glasshouse. In addition, we determined the effects of mild water stress on the isotopic composition of leaf water. The model predicted different patterns of diurnal change for the oxygen and hydrogen isotopic composition of leaf water. The observed leaf water isotopic composition followed qualitatively similar patterns of diurnal change to those predicted by the model. At midday, however, the model always predicted a higher degree of heavy isotope enrichment than was actually observed in leaves. There was no effect of mild water stress on the hydrogen isotopic composition of leaf water. For the oxygen isotopic composition of leaf water, there was either no significant difference between control and water-stressed plants or the stressed plants had lower δ¹⁸O values, despite the enriched stem water isotopic composition observed for the stressed plants.     

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.

     

Induction of Non-Targeted Stress Responses in Mammary Tissues by Heavy Ions

Purpose

Side effects related to radiation exposures are based primarily on the assumption that the detrimental effects of radiation occur in directly irradiated cells. However, several studies have reported over the years of radiation-induced non-targeted/ abscopal effects in vivo that challenge this paradigm. There is evidence that Cyclooxygenase-2 (COX2) plays an important role in modulating non-targeted effects, including DNA damages in vitro and mutagenesis in vivo. While most reports on radiation-induced non-targeted response utilize x-rays, there is little information available for heavy ions.

Methods and Materials

Adult female transgenic gpt delta mice were exposed to an equitoxic dose of either carbon or argon particles using the Heavy Ion Medical Accelerator in Chiba (HIMAC) at the National Institute of Radiological Sciences (NIRS) in Japan. The mice were stratified into 4 groups of 5 animals each: Control; animals irradiated under full shielding (Sham-irradiated); animals receiving whole body irradiation (WBIR); and animals receiving partial body irradiation (PBIR) to the lower abdomen with a 1 x 1 cm² field. The doses used in the carbon ion group (4.5 Gy) and in argon particle group (1.5 Gy) have a relative biological effectiveness equivalent to a 5 Gy dose of x-rays. 24 hours after irradiation, breast tissues in and out of the irradiated field were harvested for analysis. Induction of COX2, 8-hydroxydeoxyguanosine (8-OHdG), phosphorylated histone H2AX (γ-H2AX), and apoptosis-related cysteine protease-3 (Caspase-3) antibodies were examined in the four categories of breast tissues using immunohistochemical techniques. Analysis was performed by measuring the intensity of more than 20 individual microscopic fields and comparing the relative fold difference.

Results

In the carbon ion group, the relative fold increase in COX2 expression was 1.01 in sham-irradiated group (p > 0.05), 3.07 in PBIR (p < 0.05) and 2.50 in WBIR (p < 0.05), respectively, when compared with controls. The relative fold increase in 8-OHdG expression was 1.29 in sham-irradiated (p > 0.05), 11.31 in PBIR (p < 0.05) and 11.79 in WBIR (p < 0.05), respectively, when compared with controls. A similar increase in γ-H2AX expression was found in that, compared to controls, the increase was 1.41 fold in sham-irradiated (p > 0.05), 8.41 in PBIR (p < 0.05) and 10.59 in WBIR (p < 0.05). Results for the argon particle therapy group showed a similar magnitude of changes in the various biological endpoints examined. There was no statistical significance observed in Caspase-3 expression among the 4 groups.

Conclusions

Our data show that both carbon and argon ions induced non-targeted, out of field induction of COX2 and DNA damages in breast tissues. These effects may pose new challenges to evaluate the risks associated with radiation exposure and understanding radiation-induced side effects.     

Ribosomal Changes during Induction of Cold Hardiness in Black Locust Seedlings

Protein synthesis has been implicated in the cold-hardening process. Ribosomes from cold hardy and nonhardy black locust (Robinia pseudoacacia L.) seedlings were compared to determine if cold acclimation is related to alteration of ribosomal structure. Ribosomal structure, as indicated by thermal melting profiles, appears to be altered during induction of hardiness. Two-dimensional polyacrylamide gel electrophoresis of ribosomal proteins indicates at least 17 proteins from hardy seedlings that are different from those of nonhardy seedlings. These different proteins may be partially responsible for the different thermal melting profiles observed.     

Mechanism of Cold-induced Peach Stem Infection by Pseudomonas s. pv. persicae: Water Congestion in Tissues

We have previously shown that water congestion in tissues, caused by thawing after a period of slightly negative temperatures, results in the absorption and diffusion of bacteria in the bark tissues of peach stems (Prunus persica). In the present study we demonstrate that in peach branches acclimated to cold and inoculated with the bacterium Pseudomonas syringae pv. persicae, this freezing-thawing process is sufficient to induce, with no further treatment than incubation at + 10 °C, the development of cankers very similar to those observed in orchards in the winter. The first step of the process induces a particularly effective form of contamination and thus corresponds to an epidemiological phenomenon. The diffused bacterium, then, initiates pathogenic activity in locations favorable to its development. Moreover, an additional freezing-thawing sequence can result in the extension of ongoing infection by causing diffusion of bacteria from infected tissue to healthy areas.     

Biochemical Changes in Excised Leaves of Oryza sativa Subjected to Water Stress

Excised rice (Oryza sativa L. cv. Ratna) leaves were used to compare the changes in the levels of various biochemical intermediates and enzyme activities during senescence in turgid and water-stressed conditions. Chlorophyll, total protein and soluble protein content decreased but α-amino nitrogen content increased during the senescence of turgid leaves. In the leaves subjected to water stress, these changes were accelerated, the acceleration being greater with higher degree of water stress. Starch, soluble sugars, total carbohydrates and non-reducing sugar content decreased during senescence of turgid leaves. Water stress accelerated the changes in the levels of starch and non-reducing sugar, but the changes in the levels of soluble sugars and total carbohydrates were retarded. Reducing sugar content increased at first and then decreased in the turgid leaves, and water stress accelerated the change. The decline in the catalase activity and the increase in the peroxidase activity with time was faster in the water-stressed leaves than in the turgid leaves. Acid inorganic pyrophosphatase activity increased, but alkaline inorganic pyrophosphatase activity decreased during the senescence of turgid leaves, and such changes were accelerated by water stress. The results suggest that water stress does not accelerate all the processes connected with leaf senescence.     

Seasonal variations in freezing curves of stem sections of Cornus stolonifera MICHX.I

Comparisons of freezing curves have been used to determine theviability of plant parts exposed to stress. To gain understandingof the natural seasonal variations in freezing curves, uniformtwig sections of red-osier dogwood (Cornus stolonifera MICHX.)were collected throughout the year from a single clone and subjectedto controlled freezing while the tissue temperature was recorded.The supercooling of samples ranges from –2 to –7,but the variation was random and unpredictable. There was noapparent relationship between supercooling and the season ofthe year or the hardiness of the tissue. The freezing pointdepression, as estimated by the temperature of the first freezingplateau, was always between –0.25 and –1.0 andbore no relationship to hardiness or season. The freezing curveswere basically of three types: Summer and winter curves withtwo distinct freezing points; Early autumn curves with 3distinctfreezing points and spring curves with one prominent first freezingpoint which tended to mask the second freezing point. ¹Scientific Journal Series paper No. 6628, Minnesota AgriculturalExperiment Station. This research was supported in part by agrant from the Louis W. and MAUD HILL Family Foundation. ²Present Address: Horticulture Department, University of Wisconsin,Madison, Wisconsin, U.S.A.     

Metabolic inhibition of root water flow in red-osier dogwood (Cornus stolonifera) seedlings.

The short-term effects of sodium azide (NaN(3)) on water flow in red-osier dogwood (Cornus stolonifera Michx.) seedlings were examined in excised roots at a constant pressure of 0.3 MPa. NaN(3) significantly decreased root water flow rates (Q(v)). It also induced a significant reduction in root respiration and reduced stomatal conductance to a greater extent in intact seedlings than in excised shoots. Apoplastic flow of water increased with the NaN(3)-induced decreases in Q(v). Mercuric chloride (HgCl(2)) was also used to characterize the water flow responses and respiration of dogwood roots. Similarly to NaN(3), 0.1 and 0.3 mM HgCl(2) decreased root respiration rates and Q(v). The lower, 0.05 mM HgCl(2) treatment, reduced Q(v), but had no significant effect on root oxygen uptake. The reduction of Q(v) in HgCl(2)-treated plants was only partly reversed by 50 mM mercaptoethanol. The mercurial inhibition of Q(v) suggested the presence of Hg-sensitive water channels in dogwood roots. The results indicate that root-absorbed NaN(3) metabolically inhibited water channel activities in roots and in shoots and resulted in stomatal closure. It is suggested that the inhibition of respiration that occurs in plants stressed with environmental factors such as flooding, cold soils, and drought may be responsible for the closure of water channels in root cells and inhibition of root water flow.     

Parathyroid Glands of Amphibians. II. Structural and Biochemical Changes in Amphibian Tissues Elicited by Parathyroid Hormone Under Varying Conditions of Season and Temperature

The parathyroid glands have received relatively little attentionwith regard to their impact on the internal milieu in amphibians.Present information, however, clearly indicates that distributionof mineral in frog tissues and body fluids is under hormonalregulation; further modifications are imposed by season andtemperature. Under conditions of cold acclimation or duringwinter normal amphibian bone shows minimal rates of carbohydrateutilization: simultaneously, phosphate is conserved by reducedexcretion and increased incorporation in tissue. Exogenous parathyroidhormone, injected under these conditions when endogenous titersmay be low due to glandular degeneration, reverses these effects,and phosphate is lost in a hyperphosphaturic urine. During summeror periods of warm acclimation, hormone promotes incorporationof phosphate in tissues, and increases rates of carbohydratemetabolism in bone; citric acid, which may act in solubilizingbone mineral, is elevated in the bone. Concurrently, pre-existingsulfated mucopolysaccharides of "old" bone may be degraded,while epiphyseal zones enlarge and may exhibit accelerated orabnormal elaboration of sulfated components of cartilage and/orosteoid matrix.