The Effect of Storage Temperature on Shoot Growth, Flowering, and Carbohydrate Metabolism in Tulip Bulbs

Dry bulbs of the cvs. ‘Apeldoorn’ and ‘Paul Richter’ at stage G of flower development were stored at 5° or 21°C for 2, 4, 6, 8, 10, 12, and 14 weeks, respectively before being planted and forced at 18°C. Samples from each treatment were taken for carbohydrate analysis. The low temperature treatment (5°C) was necessary to obtain satisfactory shoot growth and flowering after planting. The rate of shoot growth and the percentage of flowering bulbs increased with increasing duration of the 5°C treatment. Time of flowering was also precipitated. 12–14 weeks of low temperature treatment seemed optimal. High temperature (21°C), or a short period at 5°C (2–6 weeks), resulted in many non-flowering bulbs, and a very slow shoot elongation when flowering occurred. In the latter case the tips or large areas of the perianths became white, the red pigmentation being prevented. Paper chromatographic analysis of oligosaccharides revealed a substantially increased content of sucrose and fructosyl sucrose (DP ≤ 5) during the first 2–4 weeks of cooling. At the end of 12 weeks at 5°C, the content of oligosaccharides decreased. The increase in the oligosaccharide content was accompanied by a corresponding starch decrease. High temperature storage (21°) led to comparatively slight changes in the sucrose and fructosyl sucrose content of the bulbs. The significance of carbohydrate metabolism in relation to shoot elongation and flowering is discussed.     

The effect of low temperature and GA3 treatments on dormancy breaking and activity of antioxidant enzymes in Fritillaria meleagris bulblets cultured in vitro

We investigated the effect of low temperature and gibberellic acid (GA₃) treatment on dormancy in Fritillaria meleagris L. bulbs. Also, we studied the effect of dormancy breaking on the antioxidant enzymes activity. To overcome dormancy, bulbs require a period (4–8 weeks) of exposure to low temperature. Bulbs regenerated in vitro were grown in the dark on medium without growth regulators at the standard (24 °C) or at low temperatures (4 and 15 °C) for 4, 6, 8 and 10 weeks. Bulbs were collected after 3, 4 and 5 weeks of cooling at 4 °C. To investigate the influence of GA₃ on dormancy, bulbs were treated for 24 h with GA₃ solutions with 1, 2 and 3 mg l^?1 concentrations. During the period of growth of bulbs at 4 °C, regeneration of bulbs was very weak, while at 15 °C the number of regenerated bulbs increased significantly. Improved bulb sprouting was achieved by a short treatment with gibberellin. Low temperature also represents a kind of oxidative stress for the plant. The activity of superoxide dismutase, catalase (CAT) and peroxidase (POX) in bulbs of F. meleagris L. grown in vitro and ex vitro increased with decreasing temperature in contrast to glutathione reductase. POX showed generally lower activity than CAT which indicates that major role in the breaking dormancy and preparing bulbs for sprouting have catalases.     

Exogenous GA<Subscript>3</Subscript> overcomes bud deterioration in tulip (<Emphasis Type="Italic">Tulipa gesneriana</Emphasis> L.) bulbs during dry storage by promoting endogenous IAA activity in the internodes

Endogenous free IAA was examined with an immunohistochemical method for its involvement in the reduction of bud deterioration after GA₃ was injected into the bulbs. We found that tulip bulbs stored at 20°C constantly developed severe bud deterioration, whereas the symptoms of deterioration was lighter in the bulbs with GA₃ injection and not observed in the bulbs with 4°C treatment. 73% success in overcoming bud deterioration was achieved in 20°C with GA₃ treatment after 8 weeks of bulb storage, and the success rate was 7% after 12 weeks of storage. IAA was detected in the parenchyma cells in the internodes of the shoot after the bulbs were stored at 4°C or at 20°C with GA₃ injection for 4 weeks, but little was detected in the bulbs stored at 20°C constantly. Moreover, a weak IAA signal was present in between the cells of the internodes irrespective of bulb treatment. After planting, the bulbs that had been treated differently exhibited different flowering ability. The bulbs stored at 4°C for 4, 8 and 12 weeks attained high flowering percentage, which was lower in the 20°C with GA₃ treatment and lowest in the 20°C treatment. It may be concluded that GA₃ injection decreases bud deterioration of tulip bulbs during dry storage at 20°C by promoting the endogenous IAA in the internodes.     

Control of the hexose content of potato tubers

The amounts of glucose and fructose in a range of harvested tubers of Solanum tuberosum were compared with the labelling of these hexoses by [U-¹⁴C]sucrose supplied to the tubers. Hexose content varied. Fructose was more heavily labelled than glucose. There was no correlation between the amounts of glucose and fructose in the tuber and their labelling. The maximum catalytic activities of α-glucan phosphorylase, acid invertase, alkaline invertase, sucrose synthase, α-amylase and β-amylase in tubers stored for 17 weeks at 5° and at 10° were estimated. The values showed no clear correlation with hexose content, but provided sound evidence that starch breakdown was phosphorolytic. It is suggested that the amounts of glucose and fructose in mature harvested tubers may be determined more by the partitioning of the translocated sucrose during the development of the tubers than by the metabolism of the harvested tuber.     

Hydrogen-deuterium exchange of the tryptophan residues in bovine α-lactalbumin

Effects of deuteration on the Raman spectrum of a tryptophan residue have been examined. The 1386 cm^?1 line of deuterated tryptophan residue has been found to be useful for tracing the hydrogen-deuterium exchange reaction of this residue in a protein. An examination on bovine α-lactalbumin at pH 6.4 and at 20°C indicates that two of the four tryptophan residues exchange with a rate constant much greater than 9 × 10^?4 sec^?1, while the other two exchange with a rate constant of 4 × 10^?5 sec^?1. The latter two have been assigned to Trp 28 and Trp 108 of this protein. The kinetics of hydrogen-deuterium exchange reaction of completely “free” tryptophan residue have been examined by a proton magnetic resonance study on tryptophan itself. By taking the result of this examination into account, the chance of exposure to the solvent for Trp 28 or Trp 108 has been estimated to be 3 × 10^?6 at pH 6.4 and at 20°C.     

Optimizing amniotic membrane tissue banking protocols for ophthalmic use

Amniotic membrane (AM) due to its anti-inflammatory, anti-scarring and anti-angiogenic properties is used as corneal and wound grafts. When developing AM tissue banks, cell viability, membrane morphology and genomic stability should be preserved following cryopreservation. To analyze the changes rendered to the AM during the process of cryopreservation by comparing different combinations of standard cryopreservation media; fetal bovine serum (FBS), dimethyl sulfoxide (DMSO), Dulbecco’s modified eagle’s medium (DMEM) and glycerol at ?80 °C and at ?196 °C for a period of 6 weeks and at 4 °C in 70 % alcohol for 6 weeks. Following informed consent, placentae of healthy term pregnancies delivered by elective Cesarean section were collected and AM separated into 5 × 5 cm size sections and under sterile conditions stored in 9:1 DMSO:FBS and 1:1 DMEM:Glycerol at ?196 and ?80 °C for 6 weeks. Similar sections were also stored at 4 °C in 70 % alcohol for 6 weeks. After storage periods following were assessed; AM epithelial cell viability by trypan blue vital stain, epithelial cell proliferation capacity by cell doubling time, membrane morphology by haematoxylin and eosin (H&E) stain and genomic stability by conventional G-banded karyotyping. Human amniotic epithelial cells were cultured in DMEM and 10 % FBS in humidified atmosphere of 5 % carbon dioxide at 37 °C and were characterized using RT-PCR for Octamer-binding protein 4 (Oct-4) and glucose-6-phosphate dehydrogenase (G6PD) genes. All the above parameters were also assessed in fresh AM. AM obtained from 4 term placentae. Mean cell count and mean cell doubling times in days respectively; for fresh AM 3.8 × 10⁶; 1.59, after 6 weeks in DMSO:FBS at ?196 °C 3.0 × 10⁶; 2.38 and at ?80 °C 2.1 × 10⁶; 1.60, in DMEM:Glycerol at ?196 °C 3.6 × 10⁶; 2.33 at ?80 °C 23 × 10⁶; 1.66 and at 4 °C 3.3 × 10⁶; 2.14. Histology analysis of the fresh AM showed an intact epithelial monolayer, thick basement membrane (BM) and avascular stromal matrix. Amniotic membranes stored at ?196 °C showed morphology similar to fresh AM in both preservation media and AM stored at ?80 °C showed disruption of the stromal matrix. At 4 °C the epithelial monolayer showed flattening. Fresh AM karyotype was 46XX. Analyzable spreads for karyotype were not obtained from stored AMs. Human amniotic epithelial cells were positive for both Oct-4 and G6PD genes. AM is best preserved at ?196 °C either in 1:9 DMSO:FBS or 1:1 DMEM:Glycerol. In both conditions cell viability and membrane integrity were shown to be preserved up to 6 weeks. Since analyzable chromosome spreads from cell cultures were not obtained, genomic stability could not be assessed.     

Polynucleotide Phosphorylase from Acromobacter sp. KR 170-4

Eighty-five strains of bacteria were screened for selection of microorganisms suitable for industrial production of polynucleotides. Among these bacteria, Achromobacter sp. KR 170-4 (ATCC 21942) was found to be rich in polynucleotide Phosphorylase (PNPase) in its “salt-shockate” as compared with the other strains tested. PNPase was purified about 50-fold from the “salt-shockate” of Achromobacter sp. KR 170-4, and properties of the enzyme were elucidated. Optimal pH for reaction was 10.1. Stable pH range at 37°C was between pH 6.5 and 10.5. Optimal temperatures were 46°C for polymerization of ADP or IDP, and 43°C for CDP or UDP. The enzyme was stable below 55°C at pH 9.2. The enzyme required Mn²⁺ rather than Mg²⁺ unlike the other PNPases reported. Optimal concentration of Mn²⁺ was 6 mM.     

Effect of orchard and postharvest application of daminozide on ethylene synthesis by apple fruit

The effects of daminozide (butanedioic acid-2,2-dimethylhydrazide) on ethylene synthesis by apple fruits were investigated. The objective was to determine the effects of postharvest applications as compared to the standard application of diaminozide in the orchard. Immersion in a solution containing 4.25 g L^?1 active ingredient for 5 min delayed the rise in ethylene production in individual “Cox” apples at 15°C by about 2 days, whereas orchard application of 0.85 g L^?1 caused delays of about 3 days. Both modes of application depressed the maximal rate of ethylene production attained by ripe apples by about 30%. Daminozide did not affect the stimulation of respiration by ethylene treatment of “Gloster” apples, but it delayed the increase in ethylene synthesis. Daminozide applied immediately after harvest delayed the rise in ethylene synthesis in “Golden Delicious” held at 15°C, but it was less effective when applied 48 h after harvest or when apples were held at 5°C. Exposure to 1–2 μl L^?1 ethylene for 48 h was less effective in promoting the rise in ethylene in daminozidetreated “Cox” and “Gloster” apples than in untreated fruit. High (100–1000 μl L^?1) concentrations of ethylene more or less overcame the daminozide effect. Apples absorbed about 40% of surface-applied [¹⁴C]daminozide in 48 h, but more than 90% of the radioactivity in the fruit was recovered from the peel and outer 1 cm of the cortex. Daminozide was partly converted to carbon dioxide and other metabolites.     

Pyrimidine Deoxyribonucleosides Are Phosphorylated to Triphosphates at Low Temperatures Too,But Are Not Incorporated into DNA or Phospholipids.

Abstract

Thymidine (Thd) was phosphorylated to dTTP also at 0°C, both in Ehrlich ascites tumor cells and human tonsillar lymphocytes, but was not incorporated into DNA. The uptake and phosphorylation of ¹⁴C-Thd into the pool showed regular kinetics (Km 6, 6 uM), and the main metabolite was dTTP (75–84%) both at 0°C, and 37°C. Similarly, deoxycytidine (dCyd) was also transported and phosphorylated to nucleotides (76%) at low temperature, but no incorporation into DNA and phospholipid precursor liponucleotides could be detected at 0°C. Under the same conditions, at 37°C, when lymphocytes were labeled with 5-³H-dCyd, 51% of the total pool radioactivity was found in liponucleotides. Transport and phosphorylation of deoxynucleosides seem to be tightly coordinated at both temperatures, which processes are directly coupled to membrane-phospholipid and DNA biosynthesis, but only at physiological temperature while they seem “uncoupled” at low temperature. The fact that nucleoside phosphorylation occures also at low temperature has implications for several experimental techniques used in cell biology.     

GROWTH AND PROBABLE GAMETE FORMATION IN THE MARINE DINOFLAGELLATE CERATIUM SCHRANKII1

Growth and formation of probable gametes in clonal cultures of Ceratium schrankii Kofoid isolated from the Gulf of Naples were monitored at 15, 20 and 25°C (12:12h LD period). Sustained division rates (k) of 0.15 div d ^?1 at 15°C and elevated ones at 20°C (0.27 div d^?1) and 25°C (0.25 div d^?1) were obtained. “Gamete” formation occurred at each temperature with few “gametes” observed at 15°C, and the greatest numbers at 20° and 25°C. This process in each parent cell involved two successive divisions with the first division forming tow “pregametes” which, within a few hours, divided again to form four motile unicells. These “gametes” remained motile for at least 24 h and normally survived for no longer tahn 48 h. Depending on the initial population at the onset of “gamete” formation, the impact on Ceratium populations was to cause either a momentary pause in log growth or a reversible decline in the populaiton.     

The effects of starvation and temperature acclimation on pentose phosphate pathway dehydrogenases in brook trout liver

Temperature and starvation were found to be factors which affected the PPP dehydrogenase activities in brook trout liver. Fish acclimated at 5 °C possessed greater levels of G6PD, H6PD, and 6PGD activity than those fish maintained at 10 or 15 °C. This phenomenon was probably associated with increased lipogenesis during cold acclimation.During starvation hepatic G6PD and 6PGD activities decreased, whereas H6PD activity increased slightly. Upon refeeding, the G6PD level gradually increased, but the “overshoot” in enzyme activity reported in mammalian studies was not observed.When both cold acclimation and starvation were studied simultaneously, regulation by temperature was initially the dominant control factor. After 6 wk at 5 °C, there was no difference in specific activities between starved and fed fish. However, fish maintained at 5 °C for longer than 2 mo did show the normal response to starvation and refeeding. Therefore, regulation of the PPP by temperature appears to be a transitory phenomenon and may be associated with temporary metabolic reorganization in the fish.     

Physiological and Biochemical Studies of Chilling Injury in Bananas

The physiological changes in green bananas (cv. Sin-zun), which are very sensitive to chilling injury, were studied during and after exposure to low temperatures (4±1°C, 6±0.5°C) for various periods. While the fruits injured by chilling did not fail to produce CO₂ and ethylene, the pattern of both CO_2- and ethylene production in these chilled fruits (9 and 15 days at 6°C) after transfer to 20°C was not normal. The contents of acetaldehyde and ethanol in chilled fruits, both in peels and pulps, increased with the advance of chilling, injury. There was an accumulation of α-keto acids in the peels of chilled fruits. Only half the conversion of ¹⁴C (fed as succinic acid-1, 4-¹⁴C) to citric acid and isocitric acid was observed in chilled tissues as compared with healthy ones; the activity of citrate synthase in banana peels appears therefore to be inhibited by chilling injury. A histological study of the tissues showed that the browning substances (polyphenols) present in chilled fruits accumulate around the vascular tissues.     

The onset of sucrose accumulation in cold-stored potato tubers is caused by an increased rate of sucrose synthesis and coincides with low levels of hexose-phosphates, an activation of sucrose phosphate synthase and the appearance of a new form of amylase

These experiments investigate events involved in triggering sugar accumulation in the cold in tubers of Solanum tuberosum L. cv. Desirée. Sugar content, ¹⁴C-glucose metabolism, metabolite levels and activities of sucrose phosphate synthase (SPS) and starch-degrading enzymes were followed after transfer to 4°C. (i) Net sucrose accumulation began between 2 and 4 d. By 10 d, reducing sugars were also increasing. From 20 d onwards, sugar accumulation slowed. Sucrose fell, but reducing sugars continued to increase. (ii) To measure unidirectional sucrose synthesis, U-[¹⁴C]glucose was injected into tubers after various times at 4°C. The tubers were then incubated for 6 h. After 1 d at 4°C, both the absolute and the relative (expressed as a percentage of the metabolized label) rates of sucrose synthesis decreased compared to those at 20°C. Between 2 and 4 d at 4°C, labelling of sucrose increased 3-fold, to over 60% of the metabolized label. This high rate was maintained for up to 50 d in cold storage. When tissue slices were incubated with 2.5 mol m^?3 U-[¹⁴C]glucose, the rate of labelling of sucrose in slices from 6 d cold-stored material was higher than in slices from warm-stored material, irrespective of whether the incubation occurred at 4°C or at 20°C. (iii) Hexose-phosphates increased during the first day after transfer to 4°C. Their levels fell during the next 3 d, as sucrose synthesis increased. They then rose (until 20 d) and fell, in parallel with the rise and decline of sucrose levels. UDPglucose remained unaltered during the first 4 d, and then increased and decreased in parallel with sucrose. (iv) SPS activity assayed in optimal conditions and the total amount of SPS protein did not change. However, when assayed in the presence of phosphate and limiting substrate concentrations, activity rose 3–5-fold between 2 and 4 d. (v) Amylases and phosphorylases were investigated using zymograms to separate isoforms. Phosphorylases did not change. Between 2 and 4 d at 4°C, a new amylolytic activity appeared. (vi) Estimates of the specific activity of the phosphorylated intermediates and the absolute rate of sucrose synthesis (calculated from the ¹⁴C-labelling data and metabolite analysis) showed that changed kinetic properties of SPS and decreased levels of hexose-phosphate are accompanied by a 6–8-fold stimulation of sucrose synthesis. They also show that the final level of sugar is partly determined by a cycle of sugar synthesis and degradation. (vii) It is concluded that the onset of sugar accumulation in cold-stored tubers is initiated by a change in the kinetic properties of SPS and the appearance of a new amylolytic activity. It is discussed how other factors, including hexose-phosphate levels and subcellular compartmentalization, could also influence the final levels of sugars by altering the balance of sugar synthesis and remobilization.     

Induction ofent-kaurene biosynthesis by low temperature in dwarf peas

Germinating pea (Pisum sativum L.) seeds of two dwarf cultivars, “Progress No. 9” and “Green Arrow”, and two tall cultivars, “Alaska” and “Alderman”, were treated with low temperature (3–5°C) for 14 days and then transferred to normal growing conditions (19–21°C for 16 h/14.5–16.5°C for 8 h) for an additional 10 days. Biosynthesis of [¹⁴C]ent-kaurene from [¹⁴C]2-mevalonic acid (2-MVA) was assayed in cell-free enzyme extracts prepared from shoot tips 10 days after cold treatment and was compared with activity in enzyme extracts prepared from noncold-treated, 10-day-old control plants. Shoot lengths of cold-treated plants were measured throughout a 35-day period and compared with shoot lengths of plants grown without cold treatment for 25–35 days. Low temperature induced a five-to 10-fold enhancement ofent-kaurene, hence potentially gibberellin (GA), biosynthesis in seedlings of the two dwarf cultivars but not in the tall cultivars. However, the lack of an increase in growth rate in the cold-treated dwarfs indicated that endogenous GA biosynthesis remained blocked at some point beyondent-kaurene in the biosynthetic pathway. Since the late-flowering “Alderman” cultivar did not exhibit enhanced biosynthesis ofent-kaurene, it appears that if vernalization in late-flowering cultivars of peas is correlated with enhanced GA biosynthesis, it is not the early part of the biosynthetic pathway which is affected.     

Antigen binding to lymphoid cells from unimmunized mice: IV. Shedding and reappearance of multiple antigen binding Ig receptors of T- and B-lymphocytes

Patterned antigen-binding cells (ABC) can bind two antigens and show “islands” of Ig receptor with mixed specificity. However, these cells, when unfixed, lose most of their bound fluorescent antigen within minutes upon warming above 0 °C. Residual antigen moves to one pole of the cell forming a “cap” within 5 min at room temperature. If such patterned ABC are capped with a single antigen, receptors to a second antigen can be detected on a portion of the capped cells, but only in the cap. The frequency of capped, “double” ABC approximated the frequency of patterned “double” ABC originally present.If lymphoid cells are mixed with fluorescent antigens at 0 °C and then incubated for 4 hr at 37 °, no ABC are found. When the cells are then fixed and the fluorescent antigens readded, new antigen-binding Ig receptors can be shown to have reappeared on the cell surface during the 4-hr incubation. The reappearance of antigen receptor could be inhibited by prior addition of either 10^?2M sodium azide or 50 μg/ml cycloheximide, implying that the receptors were actively synthesized by the cell. These inhibitors did not prevent shedding, but azide did inhibit the capping process. Both B-cells (bone marrow or spleen cells) and T-cells (splenic T-cells or 99.5% pure cortisone-resistant T-cells) were shown to regenerate multispecific ABC to the frequency found prior to incubation.     

Activity and metabolism of 9-substituted cytokinins during lettuce seed germination

The activities of N⁶-benzyladenine (BA) and its 9-substituted methyl, methoxymethyl, tetrahydropyranyl, cyclopentyl, and cyclohexyl analogs were determined for the promotion of lettuce seed (Lactuca sativa L. cv. Grand Rapids) germination. Cytokinin concentrations used were 10^?4, 10^?5, 10^?6 and 10^?7M. All seeds were incubated under total dark conditions at 28 ± 1°C. After 48 h the percentage of germination was recorded. A comparison of means based on Duncan's Multiple Range Test allowed for a ranking of cytokinin activities for the promotion of lettuce seed germination. The activities were: BA = 9-tetrahydropyranyl BA > 9-methyl BA > 9-methoxymethyl BA > 9-cyclopentyl BA > 9-cyclohexyl BA. The results were significant at the 95% confidence level as determined by analysis of variance. In order to study the metabolism of a cytokinin, lettuce seeds were incubated with 9-methyl-BA-methylene-¹⁴C. The labeled cytokinin was prepared by refluxing benzylamine hydrochloride (methylene-¹⁴C) with an equal molar ratio of 6-chloro-9-methylpurine. Final cytokinin concentration was 10^?5M. Incubation periods were 2, 4, 8, 12, 16 and 20 h at 28 ± 1°C under total dark conditions. At the end of the various time periods the seeds were extracted with 70 percent methanol. The resulting extracts were purified and radioactive metabolites identified by solvent fractionation, Sephadex LH-20 column chromatography, and paper chromatography. Co-chromatography with authentic standards in the appropriate solvent system revealed that the metabolites were 9-methyl BA, N⁶-benzyladenosine-5′-monophosphate, and N⁶-benzyladenosine. The results lend support to the theory that the cytokinin ribonucleotide serves as a storage form which is converted to the active ribonucleoside as needed during lettuce seed germination.     

Effects of anaerobiosis on ethylene production, respiration and flowering in iris bulbs

Ethylene production of iris bulbs (Iris hollandica cv. Ideal) was very low. When stored at 30°C, production was 12–20 pmol C₂H₄ (kg fresh weight)^?1 h^?1. Higher temperatures (35°C, 40°C) enhanced the ethylene production; a treatment with 40°C for ca 7 days caused a 3 times higher ethylene production than at 30°. During anaerobic storage (in 100% N₂) ethylene production was equal to that of control bulbs. When after a 7 day period of anaerobiosis the N₂ was replaced by air, a burstlike ethylene production was observed. Twenty-four h after the replacement, ethylene production was equal to control values again. The effects of this production of ethylene on mitochondrial respiration and flowering were investigated. When mitochondria were isolated immediately after the anaerobic treatment (before the enhanced ethylene production) alternative pathway capacity was not detectable, a situation also occurring in control bulbs. When mitochondria were isolated 24 h after the end of the anaerobiosis (after the ethylene burst) uninhibited respiration did not change significantly, but a capacity of the alternative pathway was observed. The increase in alternative pathway capacity after anaerobiosis was partly inhibited by 2,5-norbornadiene (NBD), an ethylene antagonist. Fermentation occurred during anaerobiosis: ethanol concentrations increased during the treatment and decreased when air was supplied. When bulbs were exposed to ethanol vapour the alternative pathway was induced but only when very high ethanol levels in the bulbs were reached. The amount of ethanol accumulated in the bulbs during a 7 day anaerobic treatment was far too low to explain the observed induction of alternative pathway capacity. Flowering percentages were enhanced after a 24 h treatment with ethylene and after a 7 day anaerobic treatment. NBD significantly inhibited the effect of exogenous ethylene and of anaerobiosis on flowering. Ethanol was not able to induce flowering. The burst-like production of ethylene after anaerobiosis probably is responsible for the effects on respiration and flowering.     

Size distribution and maturation of newly replicated DNA through the S and G2 phases of physarum polycephalum

The size distribution of newly made DNA and the dynamics of size maturation of progeny DNA molecules were studied in the synchronous S and G2 phases of Physarum polycephalum. Pulse labeling of DNA and analysis of the products on alkaline sucrose gradients showed that synthesis of primary replication units (which will also be referred to as “Okazaki” fragments) occurred throughout the S period. Pulse and pulse-chase experiments revealed a distinct pattern of size maturation. An apparently linear increase in molecular weight of progeny DNA molecules during the first hour of the S phase occurred at a rate of approximately 4–5 × 10⁵ daltons per min at 26°C, corresponding to the joining of 6–8 Okazaki fragments. The resulting 35–45S (1.1–2.2 × 10⁷ daltons) DNA molecules may correspond to the Physarum “replicon.” The further size increases of the newly made DNA appear to occur in steps, possibly reflecting a clustering of isochronous replicons along the chromatide. These observations are discussed with regard to mechanisms of DNA replication and size maturation.     

The induction of freezing tolerance in jack pine seedlings: The role of root plasma membrane H+- ATPase and redox activities

The acquired freezing tolerance of jack pine seedlings (Pinus banksiana Lamb.) conditioned at low nonfreezing temperatures and short photoperiods was determined by comparison of seedling survival to that of nonconditioned (control) seedlings following exposure to ?5 and ?10°C. Compared to that of controls, survival of conditioned seedlings was markedly increased following exposure to freezing temperatures. A 1-week conditioning treatment significantly increased the survival of the seedlings after exposure to ?5°C, but was less effective on seedlings exposed to ?10°C. Conditioning periods of 2 and 4 weeks resulted in higher survival of seedlings exposed to both ?5 and ?10°C. The changes of two root-plasma-membrane-associated enzyme activities, H⁺-ATPase and NADH-dependent ferricyanide reductase, were studied in enriched plasma membrane fractions during conditioning and after freezing. Post-freezing activities of both enzymes were enhanced by conditioning at low temperatures and short photoperiods. These changes may be related to the increased frost hardiness also induced by conditioning.