The Influence of Dark Adaptation Temperature on the Reappearance of Variable Fluorescence following Illumination

The effect of chilling temperatures (5°C) on chlorophyll fluorescence transients was used to study chilling-induced inhibition of photosynthesis in plant species with differing chilling sensitivities. A Brancker SF-20 fluorometer was used to measure induced fluorescence transients from both attached and detached leaves of chilling-sensitive cucumber (Cucumis sativus L. cv Ashley) and chilling-resistant pea (Pisum sativum L. cv Alaska). The rate of reappearance of the variable component of fluorescence (F_v), following a period of illumination at 25°C, was dependent on the temperature at which the leaf was allowed to dark adapt in chilling-sensitive cucumber, but not in chilling-resistant pea. In cucumber, dark adaptation at 25°C following illumination resulted in a much faster return of F_v than dark adaptation at 5°C following illumination. However, F_v reappearance during the dark adaptation period in chilling-resistant pea was temperature independent. The difference in the temperature response of F_v following illumination correlated with temperature sensitivity of these two species. The process responsible for the difference in F_v may represent a site of chilling sensitivity in the photosynthetic apparatus.     

Photoinactivation of Catalase Occurs under Both High- and Low-Temperature Stress Conditions and Accompanies Photoinhibition of Photosystem II

Severe photoinactivation of catalase (EC 1.11.1.6) and a decline of variable fluorescence (F_v), indicating photoinhibition of photosynthesis, were observed as rapid and specific symptoms in leaves exposed to a high heat-shock temperature of 40°C as well as in leaves exposed to low chilling temperatures in white light of only moderately high photosynthetic photon flux density of 520 μE m⁻² s⁻¹. Other parameters, such as peroxidase (EC 1.11.1.7), glycolate oxidase (EC 1.1.3.1), glutathione reductase (EC 1.6.4.2), or the chlorophyll content, were hardly affected under these conditions. At a compatible temperature of 22°C, the applied light intensity did not induce severe photoinactivations. In darkness, exposures to high or low temperatures did not affect catalase levels. Also, decline of F_v in light was not related to temperature sensitivity in darkness. The effective low-temperature ranges inducing photoinactivation of catalase differed significantly for chilling-tolerant and chilling-sensitive plants. In leaves of rye (Secale cereale L.) and pea (Pisum sativum L.), photoinactivation occurred only below 15°C, whereas inactivation occurred at 15°C in cucumber (Cucumis sativus L.) and maize (Zea mays L.). The behavior of F_v was similar, but the difference between chilling-sensitive and chilling-tolerant plants was less striking. Whereas the catalase polypeptide, although photoinactivated, was not cleaved at 0 to 4°C, the D1 protein of photosystem II was greatly degraded during the low-temperature treatment of rye leaves in light. Rye leaves did not exhibit symptoms of any major general photodamage, even when they were totally depleted of catalase after photoinactivation at 0 to 4°C, and catalase recovered rapidly at normal temperature. In cucumber leaves, the decline of catalase after exposures to bright light at 0 to 4°C was accompanied by bleaching of chlorophyll, and the recovery observed at 25°C was slow and required several days. Similar to the D1 protein of photosystem II, catalase differs greatly from other proteins by its inactivation and high turnover in light. Inasmuch as catalase and D1 protein levels depend on continuous repair synthesis, preferential and rapid declines are generally to be expected in light whenever translation is suppressed by stress actions, such as heat or chilling, and recovery will reflect the repair capacity of the plants.     

Photoinhibition at low temperature in chilling-sensitive and -resistant plants

Photoinhibition resulting from exposure at 7°C to a moderate photon flux density (300 micromoles per square meter per second, 400-700 nanometers) for 20 hours was measured in leaves of annual crops differing widely in chilling tolerance. The incidence of photoinhibition, determined as the decrease in the ratio of induced to total chlorophyll fluorescence emission at 693 nanometers (F_v/F_max) measured at 77 Kelvin, was not confined to chilling-sensitive species. The extent of photoinhibition in leaves of all chilling-resistant plants tested (barley [Hordeum vulgare L.], broad bean [Vicia faba L.], pea [Pisum sativum L.], and wheat [Triticum aestivum L.]) was about half of that measured in chilling-sensitive plants (bean [Phaseolus vulgaris L.], cucumber [Cucumis sativus L.], lablab [Lablab purpureus L.], maize [Zea mays L.], pearl millet [Pennisetum typhoides (Burm. f.) Stapf & Hubbard], pigeon pea [Cajanus cajun (L.) Millsp.], sesame [Sesamum indicum L.], sorghum [Sorghum bicolor L. Moench], and tomato [Lycopersicon esculentum Mill.]). Rice (Oryza sativa L.) leaves of the indica type were more susceptible to photoinhibition at 7°C than leaves of the japonica type. Photoinhibition was dependent both on temperature and light, increasing nonlinearly with decreasing temperature and linearly with increasing light intensity. In contrast to photoinhibition during chilling, large differences, up to 166-fold, were found in the relative susceptibility of the different species to chilling injury in the dark. It was concluded that chilling temperatures increased the likelihood of photoinhibition in leaves of both chilling-sensitive and -resistant plants. Further, while the photoinhibition during chilling generally occurred more rapidly in chilling-sensitive plants, this was not related directly to chilling sensitivity.     

Nature of photooxidative events in leaves treated with chlorosis-inducing herbicides

Leaves of rye seedlings (Secale cereale L.) grown in the presence of four chlorosis-inducing herbicides under a low light intensity of 10 lux formed chlorophyll. When segments of such dim-light-grown leaves were exposed to 30,000 lux at either 0°C or 30°C, treatments with aminotriazole or haloxidine (group 1) showed no or only minor changes of their chlorophyll contents. In treatments with San 6706 or difunon (group 2), however, rapid photodestruction of chlorophyll occurred both at 0°C and at 30°C and was accompanied by an increase of malondialdehyde that was not seen in the presence of group 1 herbicides. Unlike the in vivo behavior, virtually equal rates of chlorophyll breakdown were observed for aminotriazole and San 6706 treatments in suspensions of isolated chloroplasts from 10 lux-grown leaves after exposure to strong light. The free radical scavengers p-benzoquinone and hydroquinone and the d-penicillamine copper complex exerting superoxide dismutating activity effectively prevented photooxidation of chlorophyll in 10 lux-grown herbicide-treated leaf segments or even restored an accumulation of chlorophyll at 30,000 lux. Ascorbate and several singlet oxygen or hydroxyl radical scavengers had no protective effects. Deuterium oxide and H₂O₂ did not enhance the degradation of chlorophyll. Superoxide dismutase activity was decreased in leaves bleached in the presence of group 2 herbicides.     

Frost injury and heterogeneous ice nucleation in leaves of tuber-bearing solanum species : ice nucleation activity of external source of nucleants

The heterogeneous ice nucleation characteristics and frost injury in supercooled leaves upon ice formation were studied in nonhardened and cold-hardened species and crosses of tuber-bearing Solanum. The ice nucleation activity of the leaves was low at temperatures just below 0°C and further decreased as a result of cold acclimation. In the absence of supercooling, the nonhardened and cold-hardened leaves tolerated extracellular freezing between −3.5° and −8.5°C. However, if ice initiation in the supercooled leaves occurred at any temperature below −2.6°C, the leaves were lethally injured.

To prevent supercooling in these leaves, various nucleants were tested for their ice nucleating ability. One% aqueous suspensions of fluorophlogopite and acetoacetanilide were found to be effective in ice nucleation of the Solanum leaves above −1°C. They had threshold temperatures of −0.7° and −0.8°C, respectively, for freezing in distilled H₂O. Although freezing could be initiated in the Solanum leaves above −1°C with both the nucleants, 1% aqueous fluorophlogopite suspension showed overall higher ice nucleation activity than acetoacetanilide and was nontoxic to the leaves. The cold-hardened leaves survived between −2.5° and −6.5° using 1% aqueous fluorophlogopite suspension as a nucleant. The killing temperatures in the cold-hardened leaves were similar to those determined using ice as a nucleant. However, in the nonhardened leaves, use of fluorophlogopite as a nucleant resulted in lethal injury at higher temperatures than those estimated using ice as a nucleant.

     

Low growth temperature effects a differential inhibition of photosynthesis in spring and winter wheat

In vivo room temperature chlorophyll a fluorescence coupled with CO₂ and O₂ exchange was measured to determine photosynthetic limitation(s) for spring and winter wheat (Triticum aestivum L.) grown at cold-hardening temperatures (5°C/5°C, day/night). Plants of comparable physiological stage, but grown at nonhardening temperatures (20°C/16°C, day/night) were used in comparison. Winter wheat cultivars grown at 5°C had light-saturated rates of CO₂ exchange and apparent photon yields for CO₂ exchange and O₂ evolution that were equal to or greater than those of winter cultivars grown at 20°C. In contrast, spring wheat cultivars grown at 5°C showed 35% lower apparent photon yields for CO₂ exchange and 25% lower light-saturated rates of CO₂ exchange compared to 20°C grown controls. The lower CO₂ exchange capacity is not associated with a lower efficiency of photosystem II activity measured as either the apparent photon yield for O₂ evolution, the ratio of variable to maximal fluorescence, or the level of reduced primary quinone electron acceptor maintained at steady-state photosynthesis, and is most likely associated with carbon metabolism. The lower CO₂ exchange capacity of the spring cultivars developed following long-term exposure to low temperature and did not occur following over-night exposure of nonhardened plants to 5°C.     

Freezing tolerance of citrus, spinach, and petunia leaf tissue : osmotic adjustment and sensitivity to freeze induced cellular dehydration

Seasonal variations in freezing tolerance, water content, water and osmotic potential, and levels of soluble sugars of leaves of field-grown Valencia orange (Citrus sinensis) trees were studied to determine the ability of citrus trees to cold acclimate under natural conditions. Controlled environmental studies of young potted citrus trees, spinach (Spinacia pleracea), and petunia (Petunia hybrids) were carried out to study the water relations during cold acclimation under less variable conditions. During the coolest weeks of the winter, leaf water content and osmotic potential of field-grown trees decreased about 20 to 25%, while soluble sugars increased by 100%. At the same time, freezing tolerance increased from lethal temperature for 50% (LT₅₀) of −2.8 to −3.8°C. In contrast, citrus leaves cold acclimated at a constant 10°C in growth chambers were freezing tolerant to about −6°C. The calculated freezing induced cellular dehydration at the LT₅₀ remained relatively constant for field-grown leaves throughout the year, but increased for leaves of plants cold acclimated at 10°C in a controlled environment. Spinach leaves cold acclimated at 5°C tolerated increased cellular dehydration compared to nonacclimated leaves. Cold acclimated petunia leaves increased in freezing tolerance by decreasing osmotic potential, but had no capacity to change cellular dehydration sensitivity. The result suggest that two cold acclimation mechanisms are involved in both citrus and spinach leaves and only one in petunia leaves. The common mechanism in all three species tested was a minor increase in tolerance (about −1°C) resulting from low temperature induced osmotic adjustment, and the second in citrus and spinach was a noncolligative mechanism that increased the cellular resistance to freeze hydration.     

Variation among Species in the Temperature Dependence of the Reappearance of Variable Fluorescence following Illumination

The relationship between the thermal dependence of the reappearance of chlorophyll variable fluorescence following illumination and temperature dependence of the apparent Michaelis constant (K_m) of NADH hydroxypyruvate reductase for NADH was investigated in cool and warm season plant species. Brancker SF-20 and SF-30 fluorometers were used to evaluate induced fluorescence transients from detached leaves of wheat (Triticum aestivum L. cv TAM-101), cotton (Gossypium hirsutum L. cv Paymaster 145), tomato (Lycopersicon esculentum cv Del Oro), bell pepper (Capsicum annuum L. cv California Wonder), and petunia (Petunia hybrida cv. Red Sail). Following an illumination period at 25°C, the reappearance of variable fluorescence during a dark incubation was determined at 5°C intervals from 15°C to 45°C. Variable fluorescence recovery was normally distributed with the maximum recovery observed at 20°C in wheat, 30°C in cotton, 20°C to 25°C in tomato, 30 to 35°C in bell pepper and 25°C in petunia. Comparison of the thermal response of fluorescence recovery with the temperature sensitivity of the apparent K_m of hydroxypyruvate reductase for NADH showed that the range of temperatures providing fluorescence recovery corresponded with those temperatures providing the minimum apparent K_m values (viz. the thermal kinetic window).     

Effects of abscisic Acid treatment on the thermostability of the photosynthetic apparatus in barley chloroplasts

Thermostability of the photosynthetic apparatus of abscisic acid (ABA)-treated seedlings of barley (Hordeum vulgare) was studied by light-scattering and by fluorescence measurements of isolated chloroplasts. ABA treatment markedly decreased heat damage of the chloroplast ultrastructure; an exogenous ABA concentration of 10⁻⁵ molar was most effective. Heat-induced increase of the 77 kilodalton fluorescence ratio F₇₄₀/F₆₈₅ was also smaller at this ABA concentration. The heat-induced increase of the initial chlorophyll fluorescence level (F_o) was virtually eliminated in ABA-treated (10⁻⁵ molar) chloroplasts up to 45°C and slightly increased at 50°C, relative to control chloroplasts where F_o increased even at 35°C and reached its maximal value at 45°C. In control chloroplasts, F_o increased with a 5-minute pretreatment temperature, an effect observed as low as 35°C. F_o was maximal at 45°C. In contrast, chloroplasts treated with 10⁻⁵ molar ABA did not exhibit a heat-induced increase in F_o until 50°C.     

Effect of growth temperature and temperature shifts on spinach leaf morphology and photosynthesis

The growth kinetics of spinach plants (Spinacia oleracea L. cv Savoy) grown at 5°C or 16°C were determined to allow us to compare leaf tissues of the same developmental stage rather than chronological age. The second leaf pairs reached full expansion at a plant age of 32 and 92 days for the 16°C and 5°C plants, respectively. Growth at 5°C resulted in an increased leaf area, dry weight, dry weight per area, and leaf thickness. Despite these changes, pigment content and composition, room temperature in vivo fluorescence, and apparent quantum yield and light-saturated rates of CO₂ exchange or O₂ evolution were not affected by the growth temperature. Furthermore, 5°C expanded leaves were found to be more resistant to photoinhibition at 5°C than were 16°C expanded leaves. Thus, it is concluded that spinach grown at low temperature is not stressed. However, shifting spinach leaves from 5°C to 16°C or from 16°C to 5°C for 12 days after full leaf expansion had occurred resulted in a 20 to 25% reduction in apparent quantum yields and 50 to 60% reduction in light saturated rates of both CO₂ exchange and O₂ evolution. This was not accompanied by a change in the pigment content or composition or in the room temperature in vivo fluorescence. It appears that leaf aging during the temperature shift period can account for the reduction in photosynthesis. Comparison of cold-hardened and non-hardened winter rye (Secale cereale L. cv Muskateer) with spinach by in vivo fluorescence indicated that rye is more sensitive to both short term and longer duration temperature shifts than is spinach. Thus, susceptibility to an abrupt temperature shift appears to be species dependent.     

Ice nucleation temperature of individual leaves in relation to population sizes of ice nucleation active bacteria and frost injury

Ice nucleation temperatures of individual leaves were determined by a tube nucleation test. With this assay, a direct quantitative relationship was obtained between the temperatures at which ice nucleation occurred on individual oat (Avena sativa L.) leaves and the population sizes of ice nucleation active (INA) bacteria present on those leaves. In the absence of INA bacteria, nucleation of supercooled growth-chamber grown oat leaves did not occur until temperatures were below approximately −5°C. Both nucleation temperature and population size of INA bacteria were determined on the same individual, field-grown oat leaves. Leaves with higher ice nucleation temperatures harbored larger populations of INA bacteria than did leaves with lower nucleation temperatures. Log₁₀ mean populations of INA bacteria per leaf were 5.14 and 3.51 for leaves with nucleation temperatures of −2.5°C and −3.0°C, respectively. Nucleation frequencies (the ratio of ice nuclei to viable cells) of INA bacteria on leaves were lognormally distributed. Strains from two very different collections of Pseudomonas syringae and one of Erwinia herbicola were cultured on nutrient glycerol agar and tested for nucleation frequency at −5°C. Nucleation frequencies of these bacterial strains were also lognormally distributed within each of the three sets. The tube nucleation test was used to determine the frequency with which individual leaves in an oat canopy harbored large populations of INA bacteria throughout the growing season. This test also predicted relative frost hazard to tomato (Lycopersicon esculentum Mill) plants.     

Cold Acclimation in Arabidopsis thaliana

The abilities of two races of Arabidopsis thaliana L. (Heyn), Landsberg erecta and Columbia, to cold harden were examined. Landsberg, grown at 22 to 24°C, increased in freezing tolerance from an initial 50% lethal temperature (LT₅₀) of about −3°C to an LT₅₀ of about −6°C after 24 hours at 4°C; LT₅₀ values of −8 to −10°C were achieved after 8 to 9 days at 4°C. Similar increases in freezing tolerance were obtained with Columbia. In vitro translation of poly(A⁺) RNA isolated from control and cold-treated Columbia showed that low temperature induced changes in the population of translatable mRNAs. An mRNA encoding a polypeptide of about 160 kilodaltons (isoelectric point about 4.5) increased markedly after 12 to 24 h at 4°C, as did mRNAs encoding four polypeptides of about 47 kilodaltons (isoelectric points ranging from 5-5.5). Incubation of Columbia callus tissue at 4°C also resulted in increased levels of the mRNAs encoding the 160 kilodalton polypeptide and at least two of the 47 kilodalton polypeptides. In vivo labeling experiments using Columbia plants and callus tissue indicated that the 160 kilodalton polypeptide was synthesized in the cold and suggested that at least two of the 47 kilodalton polypeptides were produced. Other differences in polypeptide composition were also observed in the in vivo labeling experiments, some of which may be the result of posttranslational modifications of the 160 and 47 kilodalton polypeptides.     

Developmental history affects the susceptibility of spinach leaves to in vivo low temperature photoinhibition

Room temperature chlorophyll a fluorescence was used to determine the effects of developmental history, developmental stage, and leaf age on susceptibility of spinach to in vivo low temperature (5°C) induced photoinhibition. Spinach (Spinacia oleracea cv Savoy) leaves expanded at cold hardening temperatures (5°C day/night), an irradiance of 250 micromoles per square meter per second of photosynthetic proton flux density, and a photoperiod of 16 hours were less sensitive than leaves expanded at nonhardening temperatures (16 or 25°C day/night) and the same irradiance and photoperiod. This differential sensitivity to low-temperature photoinhibition was observed at high (1200) but not lower (500 or 800 micromoles per square meter per second) irradiance treatment. In spite of a differential sensitivity to photoinhibition, both cold-hardened and nonhardened spinach exhibited similar recovery kinetics at either 20 or 5°C. Shifting plants grown at 16°C (day/night) to 5°C (day/night) for 12 days after full leaf expansion did not alter the sensitivity to photoinhibition at 5°C. Conversely, shifting plants grown at 5°C (day/night) to 16°C (day/night) for 12 days produced a sensitivity to photoinhibition at 5°C similar to control plants grown at 16°C. Thus, any resistance to low-temperature photoinhibition acquired during growth at 5°C was lost in 12 days at 16°C. We conclude that leaf developmental history, developmental stage, and leaf age contribute significantly to the in vivo photoinhibitory response of spinach. Thus, these characteristics must be defined clearly in studies of plant susceptibility to photoinhibition.     

Bacterial ice nucleation: a factor in frost injury to plants

Heterogeneous ice nuclei are necessary, and the common epiphytic ice nucleation active (INA) bacteria Pseudomonas syringae van Hall and Erwinia herbicola (Löhnis) Dye are sufficient to incite frost injury to sensitive plants at −5°C. The ice nucleation activity of the bacteria occurs at the same temperatures at which frost injury to sensitive plants occurs in nature. Bacterial ice nucleation on leaves can be detected at about −2°C, whereas the leaves themselves, i.e. without INA bacteria, contain nuclei active only at much lower temperatures. The temperature at which injury to plants occurs is predictable on the basis of the ice nucleation activity of leaf discs, which in turn depends on the number and ice nucleation activity of their resident bacteria. Bacterial isolates which are able to incite injury to corn at −5°C are always active as ice nuclei at −5°C. INA bacteria incited frost injury to all of the species of sensitive plants tested.     

Photosynthesis at High Temperature in Tuber-Bearing Solanum Species : A Comparison between Accessions of Contrasting Heat Tolerance

Differences in the photosynthetic performance between pairs of heat tolerant (HT) and heat sensitive (HS) accessions of tuber-bearing Solanum species were measured at 40 °C, after treating plants at 40/30 °C. After 1 to 9 days of heat treatment, both HT and HS accessions showed progressive inhibitory effects, primarily decreased rates of CO₂ fixation, and loss of leaf chlorophyll. These effects were most pronounced in the HS accessions. Stomatal conductivity and internal CO₂ concentrations were lower for both accessions at 40 °C especially for the HS accessions, suggesting that at ambient CO₂ concentrations, stomatal conductance was limiting CO₂ availability at the higher temperature. In the HT accessions, stomatal limitations were largely attributed to differences in vapor pressure deficit between 25° and 40 °C, while the HS accessions exhibited significant nonstomatal limitations. The young expanding leaves of the HS accession showed some HT characteristics, while the oldest leaves showed severe senescence symptoms after 9 days at 40/30 °C. The data suggest that differences in heat sensitivity between HT and HS accessions are the result of accelerated senescence, chlorophyll loss, reduced stomatal conductance, and inhibition of dark reactions at high temperature.     

Zeaxanthin Synthesis, Energy Dissipation, and Photoprotection of Photosystem II at Chilling Temperatures

When leaves of a mangrove, Rhizophora mangle, were exposed to an excess of light at chilling temperatures, synthesis of zeaxanthin through violaxanthin de-epoxidation as well as nonphotochemical fluorescence quenching were markedly reduced. The results suggest a protective role of energy dissipation against the adverse effects of high light and chilling temperatures: leaves of R. mangle that had been preilluminated in 2% O₂, 0% CO₂ at low photon flux density and showed a high level of zeaxanthin, and leaves that had been kept in the dark and contained no zeaxanthin, were both exposed to high light and chilling temperatures (5°C leaf temperature) in air and then held under control conditions in low light in air at 25°C. Measurements of chlorophyll a fluorescence at room temperature showed that the photochemical efficiency of PSII and the yield of maximum fluorescence of the preilluminated leaf recovered completely within 1 to 3 hours under the control conditions. In contrast, the fluorescence responses of the predarkened leaf in high light at 5°C did not recover at all. During a dark/light transient in 2% O₂, 0% CO₂ in low light at 5°C, nonphotochemical fluorescence quenching increased linearly with an increase in the zeaxanthin content in leaves of R. mangle. In soybean (Glycine max) leaves, which contained a background level of zeaxanthin in the dark, a similar treatment with excess light induced a level of nonphotochemical fluorescence quenching that was not paralleled by an increase in the zeaxanthin content.     

Temperature Effects on Pea Plants Probed by Simultaneous Measurements of the Kinetics of Prompt Fluorescence,Delayed Fluorescence and Modulated 820 nm Reflection

Simultaneous in vivo measurements of prompt fluorescence (PF), delayed fluorescence (DF) and 820-nm reflection (MR) were made to probe response of pea leaves to 40 s incubation at high temperatures (25–50°C). We interpret our observation to suggest that heat treatment provokes an inhibition of electron donation by the oxygen evolving complex. DF, in a time range from several microseconds to milliseconds, has been thought to reflect recombination, in the dark, between the reduced primary electron acceptor Q_A ^– and the oxidized donor (P680⁺) of photosystem II (PSII). The lower electron transport rate through PSII after 45 and 50°C incubation also changed DF induction. We observed a decrease in the amplitude of the DF curve and a change in its shape and in its decay. Acceleration of P700⁺ and PC⁺ re-reduction was induced by 45°C treatment but after 50°C its reduction was slower, indicating inhibition of photosystem I. We suggest that simultaneous PF, MR and DF might provide useful information on assessing the degree of plant tolerance to different environmental stresses.     

Effect of Root Temperature on Cytokinin Activity in Root Exudate of Vitis vinifera L

Root exudates of plants of Vitis vinifera L. cv. Thompson Seedless, grown in nutrient cultures with root temperatures maintained at either 20° or 30° and with shoots at a common air temperature, were assayed for cytokinin activity. After chromatography of freeze-dried sap on paper with n-butanol/acetic acid/water (4:1:1). activity was detected with a soybean callus assay. For both root temperatures, major activity appeared between R_F 0.6 and R_F 0.8, at about the same concentration in each case. The major difference between the 2 samples was the presence of activity at R_F 0.1 to 0.2 in the 20° sample and its absence in the 30° sample.

The higher root temperature resulted in increased shoot and root elongation, increased dry matter accumulation by both shoots and roots, and also altered the morphological appearance of the roots.

     

Induction of freezing tolerance in spinach is associated with the synthesis of cold acclimation induced proteins

Spinach (Spinacia oleracea L. cv Bloomsdale) seedlings cultured in vitro were used to study changes in protein synthesis during cold acclimation. Seedlings grown for 3 weeks postsowing on an inorganic-nutrient-agar medium were able to increase their freezing tolerance when grown at 5°C. During cold acclimation at 5°C and deacclimation at 25°C, the kinetics of freezing tolerance induction and loss were similar to that of soil-grown plants. Freezing tolerance increased after 1 day of cold acclimation and reached a maximum within 7 days. Upon deacclimation at 25°C, freezing tolerance declined within 1 day and was largely lost by the 7th day. Leaf proteins of intact plants grown at 5 and 25°C were in vivo radiolabeled, without wounding or injury, to high specific activities with [³⁵S]methionine. Leaf proteins were radiolabeled at 0, 1, 2, 3, 4, 7, and 14 days of cold acclimation and at 1, 3, and 7 days of deacclimation. Up to 500 labeled proteins were separated by two-dimensional gel electrophoresis and visualized by fluorography. A rapid and stable change in the protein synthesis pattern was observed when seedlings were transferred to the low temperature environment. Cold-acclimated leaves contained 22 polypeptides not found in nonacclimated leaves. Exposure to 5°C induced the synthesis of three high molecular weight cold acclimation proteins (CAPs) (M_r of about 160,000, 117,000, and 85,000) and greatly increased the synthesis of a fourth high molecular weight protein (M_r 79,000). These proteins were synthesized during day 1 and throughout the 14 day exposure to 5°C. During deacclimation, the synthesis of CAPs 160, 117, and 85 was greatly reduced by the first day of exposure to 25°C. However, CAP 79 was synthesized throughout the 7 day deacclimation treatment. Thus, the induction at low temperature and termination at warm temperature of the synthesis of CAPs 160, 117, and 85 was highly correlated with the induction and loss of freezing tolerance. Cold acclimation did not result in a general posttranslational modification of leaf proteins. Most of the observed changes in the two-dimensional gel patterns could be attributed to the de novo synthesis of proteins induced by low temperature. In spinach leaf tissue, heat shock altered the pattern of protein synthesis and induced the synthesis of several heat shock proteins (HSPs). One polypeptide synthesized in cold-acclimated leaves had a molecular weight and net charge (M_r 79,000, pI 4.8) similar to that of a HSP (M_r 83,000, pI 4.8). However, heat shock did not increase the freezing tolerance, and cold acclimation did not increase heat tolerance over that of nonacclimated plants, but heat-shocked leaf tissue was more tolerant to high temperatures than nonacclimated or cold-acclimated leaf tissue. When protein extracts from heat-shocked and cold-acclimated leaves were mixed and separated in the same two-dimensional gel, the CAP and HSP were shown to be two separate polypeptides with slightly different isoelectric points and molecular weights.     

Structural and dielectric properties of synthetic glycolipids in mixtures with water

Three synthetically produced glycolipids, N-(β-D-glucopyranosyl)-N-octadecyl-stearoylamide (OSGA), N-(β-D-glucopyranosyl-N-octadecyl-oleoylamide (OOGA), N-(β-D-galactopyranosyl)-N-octadecyl-lauroylamide (OLGA) have been studied in different mixtures with water by x-ray diffraction and dielectric measurements with microwaves at 9.4 GHz. The measurements were performed in the temperature range -50-70°C. X-Ray diffraction revealed a direct L_β' → H transition at 20°C, 60°C, and 45°C depending on the glycolipid species but nearly not on the water content. The hexagonal phases are saturated at a water content of ≈20 wt%. The lamellar phase absorbs even less water (< 10 wt%). The dielectric data show that in the H phase the binding of water is stronger than in the L_β' phase. In the temperature range below 0°C, OSGA and OOGA show a “subzero transition” due to the freeze-out of water in a separate ice phase. This transition can be seen in an abrupt decrease of the dielectric function because the dielectric response of ice is much smaller at microwave frequencies. OLGA does not show the subzero transition but an additional transition, hexagonal → distorted hexagonal at 60°C.