Activation,synthesis and turnover of nitrate reductase controlled by nitrate and ammonium in Chlorella vulgaris

Cells of Anacystis nidulans grown at 25 or 30°C were examined both by thin-section and freeze-fracture electron microscopy. Cells grown at either temperature appeared similar when fixed at the growth temperature prior to observation. When cells were chilled to near 0°C for 30 min prior to fixation, those previously grown at 25° appeared unchanged as judged by thin sectioning while those grown at 39° showed considerable morphological alteration. Freeze fracture showed particle aggregation (more pronounced in 39°-grown cells) indicating lipid-phase separation in cells chilled prior to fixation. The phase separation was totally reversed by rewarming the chilled, 25°-grown cells to their growth temperature but was only partially reversed by rewarming chilled, 39°-grown cells. These results correlate with other effects of chilling seen in Anacystis cells grown at different temperatures.     

Spectral changes in membrane fragments and artificial liposomes of Anacystis induced by chilling.

Isolated membrane fragments from Anacystis nidulans grown at 39 °C undergo visible spectral changes on chilling, suggesting a carotenoid component is altered. No such changes are seen when cells are grown at 25 °C. The magnitude of the decreased absorbance is a function of the chilling temperature and the media in which membrane fragments are suspended. The spectral decrease following chilling develops relatively slowly and is a function of the cooling rate and final temperature. The absorbance change is reversed if the fragments are heated to near 50 °C subsequent to chilling. Liposomes prepared from a total lipid extract of Anacystis undergo a spectral change on chilling which closely resembles that occurring in whole cells or isolated membrane fragments. Liposomes prepared from an extract of cells grown at 25 °C show only about 30% as great a spectral change as those from cells grown at 39 °C. The spectral bleaching is freely reversible when the liposomes are reheated, but shows a pronounced hysteresis. It is suggested that specific phase changes occur in Anacystis membranes and artificial liposomes on cooling which alter the environment of carotenoid. These changes may relate to previous observations that cells grown at 39 °C cannot survive a cold shock while those grown at 25 °C do.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385–392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15°C after the shift down of growth temperature from 39°C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15°C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39°C or 15°C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39°C or 15°C did not change the membrane fluidity (measured at 15°C) of phosphatidylcholine from whole cells grown at 39°C. On the other hand, both cardiolipins of 39°C-grown and 15°C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15°C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15°C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39°C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39°C and 15°C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39°C-grown cells and dipalmitoleoylphosphatidylcholine for 15°C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.     

Effect of Temperature on the Distribution of Membrane Particles in Streptococcus faecalis as Seen by the Freeze-Fracture Technique

When cells of Streptococcus faecalis ATCC 9790 were incubated at temperatures above 10 C before being frozen for freeze-fracture, a random distribution of particles was observed on the outer fracture face of the freeze-cleaved cell membrane. However, when cells were incubated below 10 C before freezing, particleless patches were seen on this membrane surface. The size of the patches produced on chilling could be increased by centrifugation or by storing the chilled cells overnight at about 3 C. Patch formation appeared readily reversible, since the medium and large patches that formed on chilling could not be observed in cells warmed for 10 s at 25 C. However, during the transition from the patch to patchless state, smaller patches not seen in the chilled cells were observed. This suggested that the smaller patches might have been intermediate forms produced by the fragmentation of larger patches on warming.     

A Comparison of the Effects of Chilling on Thylakoid Electron Transfer in Pea (Pisum sativum L.) and Cucumber (Cucumis sativus L.)

Experiments comparing the photosynthetic responses of a chilling-resistant species (Pisum sativum L. cv Alaska) and a chilling-sensitive species (Cucumis sativus L. cv Ashley) have shown that cucumber photosynthesis is adversely affected by chilling temperatures in the light, while pea photosynthesis is not inhibited by chilling in the light. To further investigate the site of the differential response of these two species to chilling stress, thylakoid membranes were isolated under various conditions and rates of photosynthetic electron transfer were determined. Preliminary experiments revealed that the integrity of cucumber thylakoids from 25°C-grown plants was affected by the isolation temperature; cucumber thylakoids isolated at 5°C in 400 millimolar NaCl were uncoupled, while thylakoids isolated at room temperature in 400 millimolar NaCl were coupled, as determined by addition of gramicidin. The concentration of NaCl in the homogenization buffer was found to be a critical factor in the uncoupling of cucumber thylakoids at 5°C. In contrast, pea thylakoid membranes were not influenced by isolation temperatures or NaCl concentrations. In a second set of experiments, thylakoid membranes were isolated from pea and cucumber plants at successive intervals during a whole-plant light period chilling stress (5°C). During wholeplant chilling, thylakoids isolated from cucumber plants chilled in the light were uncoupled even when the membranes were isolated at warm temperatures. Pea thylakoids were not uncoupled by the whole-plant chilling treatment. The difference in integrity of thylakoid membrane coupling following chilling in the light demonstrates a fundamental difference in photosynthetic function between these two species that may have some bearing on why pea is a chilling-resistant plant and cucumber is a chilling-sensitive plant.     

Fatty Acid Desaturation during Chilling Acclimation Is One of the Factors Involved in Conferring Low-Temperature Tolerance to Young Tobacco Leaves

The FAD7 gene, a gene for a chloroplast [omega]-3 fatty acid desaturase, is responsible for the trienoic fatty acid (TA) formation in leaf tissues. The TA content of the leaf tissue of the 25[deg]C-grown transgenic tobacco (Nicotiana tabacum cv SR1) plants, in which the FAD7 gene from Arabidopsis thaliana was overexpressed, increased uniformly by about 10%. Fatty acid unsaturation in all major leaf polar lipid species increased in the 25[deg]C-grown FAD7 transformants but was approximately the same between the control plants and the FAD7 transformants when grown at 15[deg]C. Therefore, the overexpression of the exogenous FAD7 gene leads to the same consequence in the tobacco plants as the low-temperature-induced TA production that may be catalyzed by an endogenous, temperature-regulated chloroplast [omega]-3 fatty acid desaturase. In the 25[deg]C-grown control plants, the chilling treatment caused symptoms of leaf chlorosis and suppression of leaf growth. The 25[deg]C-grown FAD7 transgenic plants conferred alleviation of these chilling-induced symptoms. A reductions of the chilling injury similar to that of the FAD7 transformants was also observed in the 15[deg]C-preincubated control plants. These results indicate that the increased TA production during chilling acclimation is one of the prerequisites for the normal leaf development at low, nonfreezing temperatures.     

Thermal adaptation of Tetrahymena membranes with special reference to mitochondria. II. Preferential interaction of cardiolipin with specific molecular species of phospholipid

A specific effect of cardiolipin on fluidity of mitochondrial membranes was demonstrated in Tetrahymena cells acclimated to a lower temperature in the previous report (Yamauchi, T., Ohki, K., Maruyama, H. and Nozawa, Y. (1981) Biochim. Biophys. Acta 649, 385-392). This study was further confirmed by the experiment using fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH). Anisotropy of DPH for microsomal and pellicular total lipids from Tetrahymena cells showed that membrane fluidity of these lipids increased gradually as the cells were incubated at 15 degrees C after the shift down of growth temperature from 39 degrees C. However, membrane fluidity of mitochondrial total lipids was kept constant up to 10 h. This finding is compatible with the result obtained using spin probe in the previous report. Additionally, the break-point temperature of DPH anisotropy was not changed in mitochondrial lipids whereas those temperatures in pellicular and microsomal lipids lowered during the incubation at 15 degrees C. Interaction between cardiolipins and various phospholipids, which were isolated from Tetrahymena cells grown at 39 degrees C or 15 degrees C and synthesized chemically, was investigated extensively using a spin labeling technique. The addition of cardiolipins from Tetrahymena cells grown at either 39 degrees C or 15 degrees C did not change the membrane fluidity (measured at 15 degrees C) of phosphatidylcholine from whole cells grown at 39 degrees C. On the other hand, both cardiolipins of 39 degrees C-grown and 15 degrees C-grown cells decreased the membrane fluidity of phosphatidylcholine from Tetrahymena cells grown at 15 degrees C. The same results were obtained for phosphatidylcholines of mitochondria and microsomes. Membrane fluidity of phosphatidylethanolamine, isolated from cells grown at 15 degrees C, was reduced to a small extent by Tetrahymena cardiolipin whereas that of 39 degrees C-grown cells was not changed. Representative molecular species of phosphatidylcholines of cells grown at 39 degrees C and 15 degrees C were synthesized chemically; 1-palmitoyl-2-oleoylphosphatidylcholine for 39 degrees C-grown cells and dipalmitoleoylphosphatidylcholine for 15 degrees C-grown ones. By the addition of Tetrahymena cardiolipin, the membrane fluidity of 1-palmitoyl-2-oleoylphosphatidylcholine was not changed but that of dipalmitoleoylphosphatidylcholine was decreased markedly. These phenomena were caused by Tetrahymena cardiolipin. However, bovine heart cardiolipin, which has a different composition of fatty acyl chains from the Tetrahymena one, exerted only a small effect.     

Inhibition of photosynthesis by chilling in the light

The photosynthetic activity of leaf slices from Spinacia oleracea L., Cucumis sativus L. and Nerium oleander L. was measured in 25° C immediately after preincubation for 2.5 h at various photon flux densities (PFD) with chilling at 4°C, or at a moderate (450 μmol m⁻² s⁻¹) PFD with various temperatures below 25°C. Inhibition of photosynthesis was evident in C. sativus and 45°C-grown N. oleander after preincubation at 4°C at all PFD. The inhibition was most severe at fluxes in excess of the moderate PFD under which the plants were grown. Photosynthesis in S. oleracea and 20°C-grown N. oleander was not inhibited at 4°C unless the PFD was in excess of this moderate PFD. The inhibition of photosynthesis was initiated in C. sativus below 13°C, and in 45°C-grown N. oleander below 8°C. A phase transition in the polar lipids from the thylakoids of these plants was detected at about the same temperatures. For S. oleracea and 20°C-grown N. oleander preincubated under the same conditions, there was no inhibition of photosynthesis and no phase transition above 0°C. These results show that some component of photosynthesis was disrupted in the light at temperatures below that of the phase transition in the thylakoid polar lipids.     

Effects of chilling on the biochemical and functional properties of thylakoid membranes

The mechanism of chilling resistance was investigated in 4-week-old plants of the chilling-sensitive cultivated tomato, Lycopersicon esculentum Mill. cv H722, and rooted cuttings of its chilling-resistant wild relative, L. hirsutum Humb. and Bonpl., which were chilled for 3 days at 2°C with a 14-hour photoperiod and light intensity of 250 micromoles per square meter per second. This chilling stress reduced the chlorophyll fluorescence ratio, stomatal conductance, and dry matter accumulation more in the sensitive L. esculentum than in the resistant L. hirsutum. Photosynthetic CO₂ uptake at the end of the chilling treatment was reduced more in the resistant L. hirsutum than in L. esculentum, but recovered at a faster rate when the plants were returned to 25°C. The reduction of the spin trap, Tiron, by isolated thylakoids at 750 micromoles per square meter per second light intensity was taken as a relative indication of the tendency for the thylakoids to produce activated oxygen. Thylakoids isolated from the resistant L. hirsutum with or without chilling treatment were essentially similar, whereas those from chilled leaves of L. esculentum reduced more Tiron than the nonchilled controls. Whole chain photosynthetic electron transport was measured on thylakoids isolated from chilled and control leaves of the two species at a range of assay temperatures from 5 to 25°C. In both species, electron transport of the thylakoids from chilled leaves was lower than the controls when measured at 25°C, and electron transport declined as the assay temperature was reduced. However, the temperature sensitivity of thylakoids from chilled L. esculentum was altered such that at all temperatures below 20°C, the rate of electron transport exceeded the control values. In contrast, the thylakoids from chilled L. hirsutum maintained their temperature sensitivity, and the electron transport rates were proportionately reduced at all temperatures. This sublethal chilling stress caused no significant changes in thylakoid galactolipid, phospholipid, or protein levels in either species. Nonchilled thylakoid membranes from L. hirsutum had fourfold higher levels of the fatty acid 16:1, than those from L. esculentum. Chilling caused retailoring of the acyl chains in L. hirsutum but not in L. esculentum. The chilling resistance of L. hirsutum may be related to an ability to reduce the potential for free radical production by close regulation of electron transport within the chloroplast.     

Nucleoid structure in freeze fractures of Streptococcus faecalis: effects of filtration and chilling.

With the techniques used in this study, the nucleoid of Streptococcus faecalis could not be seen in freeze-etch preparations unless glutaraldehyde had been added to cultures of cells before they were frozen. With time, the nucleoid became visible as a network of fibers, apparently as a result of the aggregation of individual chromosomal elements in the presence of glutaraldehyde. When glutaraldehyde was added to undisturbed cultures, the fibers that became visible were observed in small patches that were seemingly scattered throughout the cytoplasm. However, if cells were chilled or placed on filters before glutaraldehyde was added, the fibers which then developed were seen in large central areas. The appearance of centralized nucleoids in freeze fractures of cells that had been chilled or filtered could be correlated with a decrease in the central density of the cytoplasm, as seen by light microscopy, in cells embedded in gelatin or bovine serum albumin. These observations are discussed in relation to a model for the normal structure of the nucleoid which suggests that the treatments routinely used to study the morphology-physiology of cells (chilling, filtration, and fixation) result in a reorganization of the cytoplasm, leading to an increase in the centralization of nuclear material.     

The influence of chilling on photosynthesis and activities of some enzymes of sucrose metabolism in Lycopersicon esculentum Mill

The effects of chilling stress on leaf photosynthesis and sucrose metabolism were investigated in tomato plants (Lycopersicon esculentum Mill. cultivar Marmande). Twenty-one-day-old seedlings were grown in a growth chamber at 25/23 °C (day/night) (control) and at 10/8 °C (day/night) (chilled) for 7 days. The most evident effect of chilling was the marked reduction of plant growth and of CO₂ assimilation as measured after 7 days, the latter being associated with a decrease in stomatal closure and an increase in Ci. The inhibition in photosynthetic rate was also related to an impairment of photochemistry of photosystem II (PSII), as seen from the slight, but significant change in the ratio of F_v/F_m. The capacity of chilled leaves to maintain higher q_P values with respect to the controls suggests that some protection mechanism prevented excess reduction of PSII acceptors. The results of the determination of starch and soluble sugar content could show that chilling impaired sucrose translocation. The activity of leaf invertase increased significantly in chilled plants, while that of other sucrose-metabolizing enzymes was not affected by growing temperature. Furthermore, the increase in invertase (neutral and acid) activity, which is typical of senescent tissue characterized by reduced growth, seems to confirm that tomato is a plant which is not a plant genetically adapted to low temperatures.     

Candida dubliniensis and Candida albicans display surface variations consistent with observed intergeneric coaggregation

Adherence of yeasts to other microorganisms and epithelial cell surfaces is important in their colonization. Comparative studies based on the coaggregation of Candida dubliniensis versus Candida albicans with Fusobacterium nucleatum and other oral bacteria suggested differences in the surfaces of these yeasts. Transmission electron microscopy was used to test the hypothesis that there are morphologic variations in the cell surface of these two species. C. dubliniensis type strain CD36 and C. albicans ATCC 18804 were grown on Sabouraud's dextrose agar at various growth temperatures. In some experiments suspensions of yeast cells were treated with dithiothreitol. Fixation for transmission electron microscopy was accomplished using dimethylsulfoxide and alcian blue added to 3% paraformaldehyde and 1% glutaraldahyde in cacodylate buffer. The cell wall of both species was predominantly electron lucent and was visibly differentiated into several layers. A thin electron dense outer layer was seen with clearly visible fibrillar structures, closely associated to the cytoplasmic membrane. The length of the fibrils of the C. albicans cells grown at 37 degrees C was approximately two times greater than those of the cells grown at 25 degrees C. The fibrils of the 37 degrees C-grown cells were thin, distinct and tightly packed whereas those of the 25 degrees C-grown cells appeared blunt, loosely spaced and aggregated. C. dubliniensis demonstrated short, blunt fibrils appearing similar to those of the 25 degrees C-grown C. albicans cells. C. dubliniensis showed no difference in the density, length and arrangement of fibrils between the 25 degrees C and 37 degrees C growth temperatures. The shortest and most aggregated fibrils seen were of the 45 degrees C-grown C. albicans cells. Dithiothreitoltreated 37 degrees C-grown C. albicans cells revealed a distorted and partially destroyed fibrillar layer. In this investigation C. dubliniensis, unlike C. albicans, displayed an outer fibrillar layer that did not vary with variations in growth temperature. In addition, the fibrils on the C. dubliniensis cells were similar to those of the 25 degrees C-grown C. albicans in that they were considerably shorter and less dense than those of the 37 degrees C-grown C. albicans cells. It can be postulated, that C. dubliniensis exhibits constant cell surface characteristics consistent with hydrophobicity and that this property may give this species an ecological advantage. Therefore, C. dubliniensis may compete well in oral environments via enhanced attachment to oral microbes and other surfaces, perhaps even more efficiently than C. albicans.     

Growth and photosynthesis in seedlings of Hizikia fusiformis (Harvey) Okamura (Sargassaceae, Phaeophyta) cultured at two different temperatures

This study examined temperature acclimation, growth, and photosynthetic characteristics of the zygote-derived seedlings of Hizikia fusiformis (Harvey) Okamura (Sargassaceae). The seedlings were cultured at 15°C or 25°C for 4 weeks. The average relative growth rate was significantly higher in seedlings acclimated at 25°C. The photosynthetic rate measured at 15°C was much higher in seedlings grown at 15°C than those grown at 25°C, indicating photosynthetic acclimation to a lower temperature. At 35°C, the photosynthetic rate of 15°C-grown seedlings was drastically decreased, whereas that of 25°C-grown seedlings was significantly increased. The maximum relative electron transport rate (rETR_max) measured at the respective growth temperature was significantly higher in seedlings grown at 25°C than at 15°C. At a measuring temperature of 35°C, the rETR_max in both 15°C- and 25°C-grown seedlings were considerably reduced with regard to those measured at 15°C or 25°C. Our results suggested that, compared with the seedlings grown at 25°C, those acclimated at a lower temperature could be disadvantaged under adverse conditions such as increased temperatures.     

An inhibitor of catalase induced by cold in chilling-sensitive plants

An inhibitor of catalase accumulated when leaves of chilling-sensitive species were stored in the dark at 0°C. The inhibitor could be removed from crude extracts by passing them through a column of Sephadex G-25. After this treatment, the catalase activity of extracts of chilled tissues was found to be equal to that of extracts from unchilled leaves. When chilled tissues were incubated at 20°C, the inhibitor of catalase was lost, unless the tissues had been irreversibly damaged. It specifically inhibited plant catalase, and had no effect on mammalian catalase, plant malic dehydrogenase, or plant superoxide dismutase.

Despite the presence of catalase inhibitor in extracts of chilled plants, no increase in the level of H₂O₂ in chilled tissues was found, suggesting either that the inhibitor is compartmentalized and not in contact with catalase in vivo, or that the level of H₂O₂ is controlled by means other than through catalase activity. Plant tissues normally contain H₂O₂ which is destroyed by catalase when they are damaged. After chilling, H₂O₂ leaking from already injured cells would not be so readily removed by the inhibited catalase, and could contribute to further injury by acting as a source of free radical oxidants.

     

Thermal response and reversibility of prolonged larval diapause in the chestnut weevil, Curculio sikkimensis

Curculio sikkimensis undergoes prolonged larval diapause that is terminated by chilling and warming cycles. To examine the effects of warming temperatures and their duration on diapause termination, we exposed diapause larvae that had not been reactivated after chilling at 5 °C to 20 or 25 °C and chilled them again before incubation at 20 °C. With increasing warming duration at 20 °C, diapause termination after chilling increased and shorter chilling durations became effective. In contrast, few or no larvae warmed at 25 °C terminated diapause after chilling, irrespective of the warming duration. To investigate the effect of warming temperature on diapause intensity, larvae with diapause weakened by initial incubation at 20 °C after the first chilling were subsequently incubated at 15, 20, or 25 °C, then chilled at 5 °C before incubation at 20 °C. Diapause termination increased significantly after the larvae were treated at 15 or 20 °C but decreased significantly after they were treated at 25 °C. The intensification of prolonged diapause at 25 °C was reversed when the larvae were transferred to 20 °C. Diapause intensity in C. sikkimensis therefore decreases at 20 °C, increases at 25 °C, and can be reversed by alternately exposing diapause larvae to 20 and 25 °C. In C. sikkimensis, prolonged diapause does not always proceed in one direction, and its intensity fluctuates in response to ambient temperature conditions.     

Relationships between circadian rhythm of chilling resistance and acclimation to chilling in cotton seedlings

Cotton (Gossypium hirsutum L. cv. Deltapine 50) seedlings grown under light-dark cycles of 12:12h at 35°C showed rhythmic daily changes in chilling resistance. Chilling treatment (5°C, 48h) started at the beginning or middle of the daily light period resulted in a substantial growth inhibition of the seedlings upon return to 35°C whereas when chilling was started at the beginning or middle of the dark period the subsequent growth of the seedlings was much less inhibited. This rhythm in chilling resistance persisted under continuous light for three 24-h periods, indicating that it is of an endogenous nature. Seedlings grown under continuous light from germination showed no daily changes in resistance, but a rhythm was initiated by introduction of a dark period of 6h or longer. In 24-h cycles with different light and dark periods, maximal resistance was reached just before the start of dark period. Seedlings grown at 35°C could be acclimated to chilling by exposure to low, non-damaging temperatures (25–15°C). A short-term (6h) exposure to 25°C started at the resistant phase resulted in a large increase in resistance during the following otherwise sensitive phase. The resistance induced by the low temperature matched or slightly exceeded the maximal resistance reached during the resistant phase of the daily rhythm of chilling. The low-temperature-induced resistance and the daily rhythmic increase in resistance were not additive, indicating a common mechanism for the two kinds of resistances. An adaptive advantage of a combination of a rapid temperature-induced acclimation and the daily rhythmic increase in resistance is suggested.     

Growth and development temperature influences level of tolerance to high light stress

The influence of growth and development temperature on the relative tolerance of photosynthetic tissue to high light stress at chilling temperatures was investigated. Two tuber-bearing potato species, Solanum tuberosum L. cv Red Pontiac and Solanum commersonii were grown for 4 weeks, at either 12 or 24°C with 12 hours of about 375 micromoles per second per square meter of photosynthetically active radiation. Paired leaf discs were cut from directly across the midvein of leaflets of comparable developmental stage and light environment from each species at each growth temperature treatment. One disc of each pair was exposed to 1°C and about 1000 micromoles per second per square meter photosynthetically active radiation for 4 hours, and the other disc was held at 1°C in total darkness for the same duration. Photosynthetic tissue of S. tuberosum, developed at 12°C, was much more tolerant to high light and low temperature stress than tissue developed under 24°C conditions. Following the high light treatment, 24°C-grown S. tuberosum tissue demonstrated light-limited and light-saturated rates that were approximately 50% of their paired dark controls. In contrast, the 12°C-grown tissue from S. tuberosum that was subjected to the light stress showed only a 18 and 6% reduction in light-limited and light-saturated rates of photosynthetic oxygen evolution, respectively. Tissue from 24°C-grown S. commersonii was much less sensitive to the light stress than was tissue from S. tuberosum grown under the same conditions. The results presented here demonstrate that: (a) acclimation of S. tuberosum to lower temperature growth conditions with a constant light environment, results in the increased capacity of photosynthetic tissue to tolerate high light stress at chilling temperature and (b) following growth and development at relatively high temperatures S. commersonii, a frost- and heat-tolerant wild species, has a much greater tolerance to the high light stress at chilling temperature than does S. tuberosum cv Red Pontiac, a frost-sensitive cultivated species.     

Abscisic Acid-induced chilling tolerance in maize suspension-cultured cells

The induction of chilling tolerance by abscisic acid (ABA) in maize (Zea mays L. cv Black Mexican Sweet) suspension cultured cells was examined. Cell viability during exposure to chilling was estimated by triphenyl tetrazolium chloride reduction immediately after chilling and a filter paper growth assay. Both methods yielded comparable results. Chilling tolerance was induced by transferring 5-day-old cultures (late log phase) to a fresh medium containing ABA (10 to 100 micromolar). The greatest chilling tolerance was achieved with ABA at 100 micromolar. Growth of cells was inhibited at this concentration. After a 7-day exposure to 4°C in the dark, the survival of ABA-treated cells (100 micromolar ABA, 28°C for 24 h in the dark) was sevenfold greater than untreated cells. Effective induction of chilling tolerance was first observed when cells were held at 28°C for 6 hours after adding ABA. No tolerance was induced if the culture was chilled at the inception of ABA treatment. Induction of chilling tolerance was inhibited by cycloheximide. These results indicate that ABA is capable of inducing chilling tolerance when ABA-treated cells are incubated at a warm temperature before exposure to chilling, and this induction requires de novo synthesis of proteins.     

Chloroplast ultrastructure, chlorophyll fluorescence, and pigment composition in chilling-stressed soybeans

Shoots of 16-day-old soybeans (Glycine max L. Merr. cv Ransom) were chilled to 10°C for 7 days and monitored for visible signs of damage, ultrastructural changes, perturbations in fluorescence of chlorophyll (Chl), and quantitative changes in Chl a and b and associated pigments. Precautions were taken to prevent the confounding effects of water stress. A technique for the separation of lutein and zeaxanthin was developed utilizing a step gradient with the high performance liquid chromatograph. Visible losses in Chl were detectable within the first day of chilling, and regreening did not occur until the shoots were returned to 25°C. Ultrastructurally, unstacking of chloroplast grana occurred, and the envelope membranes developed protrusions. Furthermore, the lipids were altered to the point that the membranes were poorly stabilized by a glutaraldehyde/osmium double-fixation procedure. Chl fluorescence rates were greatly reduced within 2 hours after chilling began and returned to normal only after rewarming. The rapid loss of Chl that occurred during chilling was accompanied by the appearance of zeaxanthin and a decline in violaxanthin. Apparently a zeaxanthin-violaxanthin epoxidation/de-epoxidation cycle was operating. When only the roots were chilled, no substantial changes were detected in ultrastructure, fluorescence rates, or pigment levels.     

Activity of enzymatic antioxidant defense systems in chilled and heat shocked cucumber seedling radicles

Chilling whole cucumber seedlings that had 10‐mm long radicles for 4 days at 2.5°C significantly inhibited subsequent radicle growth both by increasing the time it took the seedlings to recover from chilling and attain a linear rate of radicle growth, and by decreasing the subsequent rate of linear growth. Exposing cucumber seedlings to 45°C for up to 20 min had no effect on subsequent radicle growth, while longer exposures produced reductions in growth. A heat shock at 45°C for 10 min induced the optimal protection to 4 days of chilling at 2.5°C by reducing chilling inhibition from 60 to 42%. Two hours after being chilled, heat shocked or heat shocked and then chilled, there was no difference in protein content of the apical 1 cm of the seedling radicle among these treatments and the non‐heat shocked, non‐chilled control. Two days after treatment, the protein content was still similar in tissue that had been heat shocked or heat shocked and chilled, while it was significantly reduced in tissue that had been chilled. In general, 2 h after treatment, the activity of the 5 antioxidant enzymes examined in this study [superoxide dismutase (SOD; EC 1.15.1.1), catalase (CAT; EC 1.11.1.6), ascorbate peroxidase (APX; EC 1.11.1.11), guaiacol peroxidase (GPX; EC 1.11.1.7) and glutathione reductase (GR; EC 1.6.4.2)] were reduced by chilling and unaffected or increased by heat shock. When heat shock was followed by chilling, there was a consistent effect of the heat shock treatment on preventing the loss of enzyme activity following chilling. This protective effect of the heat shock treatment was even more pronounced after 2 days of recovery at 25°C for SOD, CAT and APX. In contrast, the activity of GR and GPX was substantially higher in chilled tissue than in tissue that had been heat shocked before being chilled. Elevated levels of GR and GPX therefore appear to be correlated with the development of chilling injury, while elevated levels of SOD, CAT and APX appear to be correlated with the development of heat shock‐induced chilling tolerance.