Effects of Temperature on Cold Acclimation and Deacclimation in Flower Buds of Evergreen Azaleas

The effects of various storage temperature/duration combinations(5, 10 and 17°/4, 8, 12 and 16 weeks) on cold acclimationand deacclimation of flower buds were studied in four speciesof evergreen azaleas having different natural distribution andcold hardiness. The freezing process and the exotherm temperaturedistribution of florets in excised whole buds determined bydifferential thermal analysis were used as the diagnostics todetermine the degree of bud acclimation and deacclimation. Theacclimation in buds lasted for as long as 12 to 16 weeks at5°C storage, and from 8 to 12 weeks at 10°C, and itappeared to be maintained after the chilling requirement forbreaking bud dormancy had been satisfied. Therefore, bud acclimationseems to be maintained independently from bud dormancy. Thedehardening effect on acclimated buds occurred as a result ofshort exposures to higher temperatures or long exposures tolower temperatures, and there was no relation between the rateof deacclimation and the degree of hardiness in each species.Among three storage temperatures examined, 5°C was the mosteffective for the maintenance of cold acclimation in flowerbuds and the small difference of floret water contents at 5and 10°C storage is not significant. (Received August 28, 1982; Accepted February 4, 1983)     

Cold Hardiness and Acclimation Intensity in Flower Buds for Fall Bloom and Spring Bloom Clones in Rhododendron kiusianum, a Dwarf Evergreen Azalea

The relationship between the degree of cold hardiness (supercoolingability of florets) and the acclimation intensity in flowerbuds was investigated in the fall bloom and the spring bloom(typical) clones of Rhododendron kiusianum, a hardy dwarf evergreenazalea. Supercooling ability or exotherm temperature distribution(ETD) in florets was determined by differential thermal analysis(DTA) and the intensity of bud acclimation or the rate of deacclimationwas judged by the changes in ETD profiles resulting from thedehardening temperature treatment. Although the two clone typesshowed no significant differences in ETDs and water contentsin florets, they differed in their rates of bud deacclimation.The flower buds of fall bloom clones generally tend to deacclimatemore quickly than the spring bloom ones throughout the seasons.It is concluded that the degree of cold hardiness in flowerbuds of R. kiusianum does not differ between the fall bloomand spring bloom clones but the intensity of bud acclimationdoes; acclimation intensity is higher in the spring bloom clonesand the rate of deacclimation is greater in the fall bloom ones. (Received October 14, 1985; Accepted February 5, 1986)     

Changes in Sugar Content during Cold Acclimation and Deacclimation of Cabbage Seedlings

The relationship between freezing tolerance and sugar contentin cabbage seedlings was investigated. Seedlings exposed tonon-freezing low temperature (5 °C) acquired freezing tolerancedown to -6 °C. The degree of freezing tolerance increasedwith duration of exposure to low temperature (up to 10 d). Sucrose,glucose, fructose and myo -inositol were detected as solublesugars in cabbage leaves, and all soluble sugars, except formyo -inositol, and starch increased gradually during cold acclimationsuch that their levels were positively correlated with the degreeof freezing tolerance. The induced freezing tolerance was attributednot to ontogenetic changes but to cold acclimation. However,the induced freezing tolerance was lost after only 1 d of deacclimationat control temperatures, and this change was associated witha large reduction in sugar content. These results reveal that the sugar content of cabbage leavesis positively correlated with freezing tolerance. Brassica oleracea L.; cabbage; cold acclimation; deacclimation; freezing tolerance; sugars     

Relationship of Nuclear Magnetic Resonance Relaxation Time to Water Content and Cold Hardiness in Flower Buds of Evergreen Azalea

The longitudinal relaxation time (T₁) of water protons in floretsof R. ? akebono flower buds was measured with a pulse NMR spectrometerto determine the relationship of T₁ to water content and coldhardiness (supercooling ability). Seasonal changes of T₁ inflorets were closely correlated with water content and supercoolingability of florets. T₁ of florets was short for acclimated budshaving a low water content and long for non-acclimated budshaving a high water content. Flower buds collected in Novemberand stored at 0 and 5?C for 4 weeks had shorter T₁ values thanbuds stored at 10?C even though the floret water content andsupercooling ability were similar. Thus, the short T₁ of coldacclimated buds hardened naturally or by storage at low temperaturesis due to a combination of both reduced water content and temperature. (Received August 27, 1983; Accepted May 26, 1984)     

Characteristics of Cold Acclimation and Deacclimation in Tuber-bearing Solanum Species

The effect of temperatures on cold acclimation and deacclimation in foliage tissues was studied in Solanum commersonii (Oka 4583), a tuber-bearing potato. The threshold temperature for cold acclimation was about 12 C. In a temperature range of 2 to 12 C, the increase in hardiness was dependent on the acclimating temperature; the lower the acclimating temperature, the more hardiness achieved. A day/night temperature of 2 C, regardless of photoperiod, appeared to the optimum acclimating temperature for the Solanum species studied. A subfreezing temperature hardened plants less effectively. The maximum level of hardiness could be reached after 15 days of cold acclimation. However, it took only 1 day to deacclimate the hardened plants to a preacclimation level when plants were subjected to a warm regime from cold. The degree of deacclimation was dependent on the temperature of the warm regime.     

Freezing Avoidance Mechanisms by Supercooling in Some Rhododendron flower Buds with Reference to Water Relations

Excised florets of some hardy Rhododendron species did not toleratefreezing at –5°C when ice-inoculated due to intracellularfreezing. Florets in intact December buds, however, could besupercooled to about –30°C. When flower buds of R.japonicum were slowly cooled with daily decrements of 5°Cto temperatures ranging from 0 to –20°C, the exothermtemperatures of the florets drastically decreased. This wasaccompanied by a decrease in water content of florets and peduncleand an increase in that of scales. The water in florets andthe peduncle is thought to migrate to scales and other tissuesduring the early stages of freezing; the dehydrated floret hasa lower freezing point which enhances its supercooling abilityand the dehydrated peduncle helps to maintain the supercooledstate of the florets. This hypothesis would explain the dependenceon the cooling rate of supercooling in Rhododendron flower buds.Water migration within flower buds was observed in other hardyRhododendron species with some variation in ice formation siteand the quantity of migrated water. The exotherm temperatureof excised florets was inversely proportional to their watercontent. Dehydration of flower buds by wind at 0°C alsoenhanced their supercooling ability. Mechanisms of freezingavoidance by supercooling in Rhododendron flower buds and therelationship of supercooling to freezing tolerance are discussed. ¹ Contribution No. 2254 from the Institute of Low TemperatureScience ² This is a revised form of the master's thesis of the seniorauthor (M.I.) which is cited in the present and previous papers(Sakai 1979a, b, etc.). (Received August 11, 1980; Accepted June 1, 1981)     

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.     

Cold Acclimation Ability and Photosynthesis among Species of the Tropical Coffea Genus

Abstract: Three Coffea species (C. arabica cv. Icatu, C. canephora cv. Apoatã and C. dewevrei) were tested in order to identify and study the mechanisms of tolerance to low, non‐freezing temperatures. Several photosynthesis‐related parameters were monitored during a 20‐day period of gradual temperature decrease, from 25/20 °C (day/night) down to 15/10 °C, during chilling treatments (15/4 °C), and upon rewarming (25/20 °C). Differences were found among species, both during low temperature exposure and during rewarming. In general, Coffea species showed cold‐induced photoinhibition of photosynthesis, which was attributable to biochemical (in vivo ribulose‐1,5‐bisphosphate carboxylase/oxygenase activity and carbohydrate synthesis) and biophysical (antennae functioning, photosystem II efficiency and linear electron transport) inactivation, rather than to stomatal constraints. The moderately low temperature of 15/10 °C was enough to cause a negative impact on net photosynthesis (A), mostly due to low (initial) rubisco activity in all species. However, C. arabica cv. Icatu showed a higher tolerance to chilling and recovered quickly and completely upon rewarming, as assessed from the impacts on the photosynthetic machinery (e.g. A_max, F_o, F_v/F_m, F_v′/F_m′, q_P, ?_e, rubisco activity) and on carbohydrate metabolism. Such lesser effects are likely to be related to the strong increases and higher contents of zeaxanthin, lutein and β‐carotene that presumably increased the ability to dissipate excitation energy and contributed to protect the photosynthetic apparatus. During cold exposure, a significant reduction of the α/β carotene ratio, which is considered an acclimation feature, was observed solely in C. arabica cv. Icatu. However, C. canephora cv. Apoatã and, especially, C. dewevrei showed to be highly cold‐sensitive. In these latter species, the photoinhibitory impairments to photosynthesis were stronger, probably due to the lower contents of protecting pigments during chilling conditions that lead to a higher vulnerability to excess excitation energy. Moreover, the mesophyll impairments (e.g. A_max, F_v/F_m, ?_e) became significant even at moderately low temperatures of 15/10 °C, and a lower ability to recover after chilling exposure was observed. The limitation of in vivo rubisco activity and A_max may have been due to substrate limitation, but disturbances in sugar metabolism could also play an important role in the expression of chilling sensitivity in C. canephora cv. Apoatã and C. dewevrei.     

Group 3 LEA Gene HVA1 Regulation by Cold Acclimation and Deacclimation in Two Barley Cultivars with Varying Freeze Resistance

The level of expression of the group 3 late embryogenesis abundant abscisic acid-regulated gene (HVA1) to cold treatment has been studied in winter barley (Hordeum vulgare) seedling tissue. The cDNA clone (pHVA1) encoding this late embryogenesis abundant protein was used as a hybridization probe to detect the corresponding mRNA. Expression of the HVA1 gene was determined after the tissue had been subjected to a regimen of 2°C exposure (cold acclimation), followed by a return to 25°C growth conditions (deacclimation). Accumulation of HVA1 mRNA occurred upon cold acclimation of the tissue and disappeared as early as 2 hours after exposure to deacclimation conditions. A comparison of the response to cold acclimation and deacclimation was made between seedling tissue of a freeze-resistant and less freeze-resistant cultivar. In both cultivars, the HVA1 gene was expressed and modulated by cold treatment. Within 2 hours of deacclimation HVA1 mRNA was no longer detectable in either cultivar independently of freeze resistance. The level of expression of HVA1 appeared to be greater in the less freeze-resistant cultivar (Winter Malt).     

Induction of Freezing Tolerance in Spinach during Cold Acclimation

Spinach (Spinacia oleracea L.) seedlings, grown in soil or on an agar medium in vitro, became cold acclimated when exposed to a constant 5°C. Plants subjected to cold acclimation, beginning 1 week postgermination, attained freezing tolerance levels similar to that achieved by seedlings that were cold acclimated beginning 3 weeks after sowing. Seedlings at 1 week of age had only cotyledonary leaves, while 3-week-old seedlings had developed true leaves. Plants grown in vitro were able to increase in freezing tolerance, but were slightly less hardy than soil-grown plants. These results suggest that spinach, a cool-season crop that begins growth in early spring when subzero temperatures are likely, can undergo cold acclimation at the earliest stages of development following germination. Axenic seedlings, grown in vitro, were used to develop a noninjurious radiolabeling technique. Leaf proteins were radiolabeled to specific activities of 10⁵ counts per minute per microgram at 25°C or 5 × 10⁴ counts per minute per microgram at 5°C over a 24 hour period. The ability to radiolabel leaf proteins of in vitro grown plants to high specific activities at low temperature, without injury or microbial contamination, will facilitate studies of cold acclimation.     

Naringin and Neohesperidin Levels during Development of Leaves, Flower Buds, and Fruits of Citrus aurantium

The distribution of the flavanones naringin and neohesperidin has been analyzed during the development of the leaves, flower buds, and fruits of Citrus aurantium. These flavonoids are at maximum concentration in the organs studied during the logarithmic phase of growth, gradually decreasing until the organs reach maximum development. However, this decrease in the naringin and neohesperidin concentration in leaves, flower buds, and fruits is due to a dilution of the flavonoids caused by cell growth, because total content per organ continues to increase. The levels of neohesperidin are always greater than those of naringin, although the ratio between the relative concentrations is different in the three organs studied. Leaves have the highest ratios, varying between 8.83 and 5.18, followed by flowers (3.15-1.85), and fruits (2.23-1.02). These observations suggest different relationships between the respective enzymic activities in their biosynthetic pathway.     

RNA Synthesis in Spring and Winter Wheat during Cold Acclimation

The factors responsible for the low transpiration rates of citrus were investigated. Leaf resistance to water vapor exchange by orange seedlings (Citrus sinensis L. cv. Koethen) including a substantial boundary layer resistance, was as low as 1 s cm⁻¹ in humid air. Leaf resistance of well watered plants increased to values as large as 5 s cm⁻¹ when the difference in absolute humidity between leaf and air was increased. Leaf resistance was only slightly influenced by temperature between 20 and 30°C providing the humidity difference between leaf and air was kept constant. Leaf resistance increased when leaf temperature was increased between 20 and 30°C when the absolute humidity external to the leaf was kept constant. Increased humidity differences resulted in greater increases in leaf resistance during initial experiments than when the experiments were repeated with the same leaves indicating acclimation by the plant. It was concluded that the effects of humidity differences on leaf resistance are partially responsible for the low transpiration rates of citrus.     

Solute Accumulation and Compartmentation during the Cold Acclimation of Puma Rye

During cold acclimation of Puma rye (Secale cereale L. cv Puma), the intracellular osmotic potential nearly doubles. During this period, the accumulation of glycinebetaine, proline, and soluble sugars was monitored. The amount of glycinebetaine increased from 290 to 1300 micrograms per gram fresh weight during the 4-week acclimation period. Proline content did not change during the first 3 weeks of acclimation but then increased from 27 to 580 micrograms per gram fresh weight during the next 3 weeks. The total soluble sugar content more than doubled by the second week of cold acclimation, increasing from 11 to 26 milligrams per gram fresh weight. Most of this increase can be attributed to the accumulation of sucrose and raffinose, whose levels increased from 2.4 and 0 to 11 and 5 milligrams per gram fresh weight, respectively. The content of monosaccharides, predominantly glucose, remained at a constant 10 milligrams per gram fresh weight throughout the acclimation period. A comparison of the sugar content of protoplasts versus vacuoles isolated from cold-acclimated leaves revealed that the extravacuolar volume contained monosaccharides, sucrose, and raffinose. Thus, the increased amounts of sucrose and raffinose that occur during cold acclimation are present in compartments external to the vacuole and may contribute to cryoprotection.     

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.     

Adenine Nucleotide Changes during Cold Acclimation of Winter Rape Plants

During the first stage of hardening of winter rape plants (Brassica napus L. var. oleifera L., cv. Górczański), marked increase of ATP content in leaves was observed. Lowering the temperature from 5 to 0 C (the second stage of hardening) had no further effect on ATP content. In roots, not capable of hardening, pronounced decrease of ATP content was noted after prolonged exposure to cold. It was found that increased ATP content and higher energy charge in cold-treated leaves were due to light and dark processes.     

Dynamic Phosphoproteome Profiling of Zebrafish Embryonic Fibroblasts during Cold Acclimation

Temperature affects almost all aspects of the fish life. To cope with low temperature, fish have evolved the ability of cold acclimation for survival. However, intracellular signaling events underlying cold acclimation in fish remain largely unknown. Here, the formation of cold acclimation in zebrafish embryonic fibroblasts (ZF4) is monitored and the phosphorylation events during the process are investigated through a large‐scale quantitative phosphoproteomic approach. In total, 11 474 phosphorylation sites are identified on 4066 proteins and quantified 5772 phosphosites on 2519 proteins. Serine, threonine, and tyrosine (Ser/Thr/Tyr) phosphorylation accounted for 85.5%, 13.3%, and 1.2% of total phosphosites, respectively. Among all phosphosites, 702 phosphosites on 510 proteins show differential regulation during cold acclimation of ZF4 cells. These phosphosites are divided into six clusters according to their dynamic changes during cold exposure. Kinase–substrate prediction reveals that mitogen‐activated protein kinase (MAPK) among the kinase groups is predominantly responsible for phosphorylation of these phosphosites. The differentially regulated phosphoproteins are functionally associated with various cellular processes such as regulation of actin cytoskeleton and MAPK signaling pathway. These data enrich the database of protein phosphorylation sites in zebrafish and provide key clues for the elucidation of intracellular signaling networks during cold acclimation of fish.     

Peroxide-Scavenging Systems during Cold Acclimation of Apple Callus in Culture

The relationship between peroxide-scavenging systems and coldacclimation was studied in apple callus in culture during acclimationunder artificial conditions. Unacclimated callus did not survivefreezing at –10?C, whereas callus acclimated at 0?C exhibitedgradually increased resistance to freezing and, after acclimationfor 20 days, it survived at temperatures as low as –15–C.During acclimation of callus, there was an immediate and abruptincrease in the activities of ascorbate peroxidase (EC 1.11.1.11 [EC] ),peroxidase (EC 1.11.1.7 [EC] ) and catalase (EC 1.11.1.6 [EC] ), which reachedmaximum values after acclimation for 10 days, at the same timeas the very beginning of the increase in cold hardiness wasobserved. An increase in the activity of glyceraldehyde-3-phosphatedehydrogenase (EC 1.2.1.12 [EC] ) occurred during the first 5 daysof cold treatment. The activities of glucose-6-phosphate dehydrogenase(EC 1.1.1.49 [EC] ), hexokinase (EC 2.7.1.1 [EC] ), glutathione reductase(EC 1.6.4.2 [EC] ), glutathione peroxidase (EC 1.11.1.9 [EC] ) and dehydro-ascorbatereductase (EC 1.8.5.1 [EC] ) increased gradually during the cold treatment.In contrast, the activity of glucosephosphate isomerase (EC5.3.1.9 [EC] ) decreased gradually during acclimation. Furthermore,during acclimation, the levels of glucose-6-phosphate, fructose-6-phosphateand glucose-1-phosphate increased slowly and steadily, and thelevels of GSH and ascorbate remained at consistently higherlevels. In addition, acclimation caused marked cytological changes.The most striking of these changes was the microvacuolationand thickening of the cell wall. These results indicate thatthe enhancement of peroxide-scavenging systems at the time ofcold acclimation proceeds in two stages: during the first stage,the enzymatic activities involved in the degradation of peroxides(i.e., the activities of ascorbate peroxidase, peroxidase andcatalase) increase; and, in the second stage, an alternativeenzymatic system develops for detoxification of peroxides, coupledwith the pentose phosphate cycle. (Received July 20, 1990; Accepted April 16, 1991)     

Levels and Aggregation of Ribosomes during Dormancy and Dormancy Break of Peach Flower Buds

The level of ribosomes and their aggregation state (monosomes-polysomes) have been investigated in relation to dormancy in peach flower buds (Prunus persica). The ribosomes were analysed by a linear sucrose gradient. The ribosomal content hardly changes in the bud before dormancy and until dormancy breaks; on the other hand after this period there is a remarkable increase in the ribosomal level strictly related to the increase in bud weight. The ribosomal content reaches the highest value at flowering. During dormancy break the largest portion of ribosomes is localized in flower primordia, not in scales. Polysomes, which are always present in buds, remain constant during dormancy (35%), increase at dormancy breaking, reaching the highest level at flowering.     

Synthesis of Freezing Tolerance Proteins in Leaves, Crown, and Roots during Cold Acclimation of Wheat

Protein synthesis was studied in leaves, crown, and roots during cold hardening of freezing tolerant winter wheat (Triticum aestivum L. cv Fredrick and cv Norstar) and freezing sensitive spring wheat (T. aestivum L. cv Glenlea). The steady state and newly synthesized proteins, labeled with [³⁵S]methionine, were resolved by one- and two-dimensional polyacrylamide gels. The results showed that cold hardening induced important changes in the soluble protein patterns depending upon the tissue and cultivar freezing tolerance. At least eight new proteins were induced in hardened tissues. A 200 kilodalton (kD) (isoelectric point [pl] 6.85) protein was induced concomitantly in the leaves, crown, and roots. Two proteins were specifically induced in the leaves (both 36 kD, pl 5.55 and 5.70); three in the crown with M_r 150 (pl 5.30), 45 (pl 5.75), and 44 kD (pl > 6.80); and two others in the roots with M_r 64 (pl 6.20) and 52 kD (pl 5.55). In addition, 19 other proteins were synthesized at a modified rate (increased or decreased) in the leaves, 18 in the crown and 23 in the roots. Among the proteins induced or increased in hardened tissues, some were expressed at a higher level in the freezing tolerant cultivars than in the sensitive one, indicating a correlation between the synthesis and accumulation of these proteins and the degree of freezing tolerance. These proteins, suggested to be freezing tolerance proteins, may have an important role in the cellular adaptation to freezing.