Effect of Electropermeabilization by Ohmic Heating for Inactivation of Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium,and Listeria monocytogenes in Buffered Peptone Water and Apple Juice

The effect of electric field-induced ohmic heating for inactivation of Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes in buffered peptone water (BPW) (pH 7.2) and apple juice (pH 3.5; 11.8 °Brix) was investigated in this study. BPW and apple juice were treated at different temperatures (55°C, 58°C, and 60°C) and for different times (0, 10, 20, 25, and 30 s) by ohmic heating compared with conventional heating. The electric field strength was fixed at 30 V/cm and 60 V/cm for BPW and apple juice, respectively. Bacterial reduction resulting from ohmic heating was significantly different (P < 0.05) from that resulting from conventional heating at 58°C and 60°C in BPW and at 55°C, 58°C, and 60°C in apple juice for intervals of 0, 10, 20, 25, and 30 s. These results show that electric field-induced ohmic heating led to additional bacterial inactivation at sublethal temperatures. Transmission electron microscopy (TEM) observations and the propidium iodide (PI) uptake test were conducted after treatment at 60°C for 0, 10, 20, 25 and 30 s in BPW to observe the effects on cell permeability due to electroporation-caused cell damage. PI values when ohmic and conventional heating were compared were significantly different (P < 0.05), and these differences increased with increasing levels of inactivation of three food-borne pathogens. These results demonstrate that ohmic heating can more effectively reduce bacterial populations at reduced temperatures and shorter time intervals, especially in acidic fruit juices such as apple juice. Therefore, loss of quality can be minimized in a pasteurization process incorporating ohmic heating.     

Differential Effects of Temperature and Starvation on Induction of the Viable-but-Nonculturable State in the Coral Pathogens Vibrio shiloi and Vibrio tasmaniensis

We compared induction of the viable-but-nonculturable (VBNC) state in two Vibrio spp. isolated from diseased corals by starving the cells and maintaining them in artificial seawater at 4 and 20°C. In Vibrio tasmaniensis, isolated from a gorgonian octocoral growing in cool temperate water (7 to 17°C), the VBNC state was not induced by incubation at 4°C after 157 days. By contrast, Vibrio shiloi, isolated from a coral in warmer water (16 to 30°C), was induced into the VBNC state by incubation at 4°C after 126 days. This result is consistent with reports of low-temperature induction in several Vibrio spp. A large proportion of the V. tasmaniensis population became VBNC after incubation for 157 days at 20°C, and V. shiloi became VBNC after incubation for 126 days at 20°C. Resuscitation of V. shiloi cells from cultures at both temperatures was achieved by nutrient addition, suggesting that starvation plays a major role in inducing the VBNC state. Our results suggest that viable V. shiloi could successfully persist in the VBNC state in seawater for significant periods at the lower temperatures that may be experienced in winter conditions, which may have an effect on the seasonal incidence of coral bleaching. For both species, electron microscopy revealed that prolonged starvation resulted in transformation of the cells from rods to cocci, together with profuse blebbing, production of a polymer-like substance, and increased membrane roughness. V. shiloi cells developed an increased periplasmic space and membrane curling; these features were absent in V. tasmaniensis.     

Effects of Temperature on Bacterial Communities and Metabolites during Fermentation of Myeolchi-Aekjeot,a Traditional Korean Fermented Anchovy Sauce

Myeolchi-aekjeot (MA) in Korea is produced outdoors without temperature controls, which is a major obstacle to produce commercial MA products with uniform quality. To investigate the effects of temperature on MA fermentation, pH, bacterial abundance and community, and metabolites were monitored during fermentation at 15°C, 20°C, 25°C, and 30°C. Initial pH values were approximately 6.0, and pH values increased after approximately 42 days, with faster increases at higher temperatures. Bacterial abundances increased rapidly in all MA samples after quick initial decreases during early fermentation and then they again steadily decreased after reaching their maxima, which were significantly greater at higher temperatures. Bacterial community analysis revealed that Proteobacteria and Tenericutes were predominant in all initial MA samples, but they were rapidly displaced by Firmicutes as fermentation progressed. Photobacterium and Mycoplasma belonging to Proteobacteria and Tenericutes, respectively, which may include potentially pathogenic strains, were dominant in initial MA, but decreased with the growth of Chromohalobacter, which occurred faster at higher temperatures––they were dominant until 273 and 100 days at 15°C and 20°C, respectively, but not detected after 30 days at 25°C and 30°C. Chromohalobacter also decreased with the appearance of subsequent genera belonging to Firmicutes in all MA samples. Tetragenococcus, halophilic lactic acid bacteria, appeared predominantly at 20°C, 25°C, and 30°C; they were most abundant at 30°C, but not detected at 15°C. Alkalibacillus and Lentibacillus appeared as dominant genera with the decrease of Tetragenococcus at 25°C and 30°C, but only Lentibacillus was dominant at 15°C and 20°C. Metabolite analysis showed that amino acids related to tastes were major metabolites and their concentrations were relatively higher at high temperatures. This study suggests that high temperatures (approximately 30°C) may be appropriate in MA fermentation, in the light of faster disappearance of potentially pathogenic genera, higher amino acids, growth of Tetragenococcus, and faster fermentation.     

Temperature Effects on Development of Meloidogyne Enterolobii and M. Floridensis

Meloidogyne enterolobii and M. floridensis are virulent species that can overcome root-knot nematode resistance in economically important crops. Our objectives were to determine the effects of temperature on the infectivity of second-stage juveniles (J2) of these two species and determine differences in duration and thermal-time requirements (degree-days [DD]) to complete their developmental cycle. Florida isolates of M. enterolobii and M. floridensis were compared to M. incognita race 3. Tomato cv. BHN 589 seedlings following inoculation were placed in growth chambers set at constant temperatures of 25°C, and 30°C, and alternating temperatures of 30°C to 25°C (day–night). Root infection by the three nematode species was higher at 30°C than at 25°C, and intermediate at 30°C to 25°C, with 33%, 15%, and 24% infection rates, respectively. There was no difference, however, in the percentages of J2 that infected roots among species at each temperature. Developmental time from infective J2 to reproductive stage for the three species was shorter at 30°C than at 25°C, and 30°C to 25°C. The shortest time and DD to egg production for the three species were 13 days after inoculation (DAI) and 285.7 DD, respectively. During the experimental timeframe of 29 d, a single generation was completed at 30°C for all three species, whereas only M. floridensis completed a generation at 30°C to 25°C. The number of days and accumulated DD for completing the life cycle (from J2 to J2) were 23 d and 506.9 DD for M. enterolobii, and 25 d and 552.3 DD for M. floridensis and M. incognita, respectively. Exposure to lower (25°C) and intermediate temperatures (30°C to 25°C) decreased root penetration and slowed the developmental cycle of M. enterolobii and M. floridensis compared with 30°C.     

Radiation Resistance and Injury of Yersinia enterocolitica

The D values of Yersinia enterocolitica strains IP134, IP107, and WA, irradiated at 25°C in Trypticase soy broth, ranged from 9.7 to 11.8 krad. When irradiated in ground beef at 25 and −30°C, the D value of strain IP107 was 19.5 and 38.8 krad, respectively. Cells suspended in Trypticase soy broth were more sensitive to storage at −20°C than those mixed in ground beef. The percentages of inactivation and of injury (inability to form colonies in the presence of 3.0% NaCl) of cells stored in ground beef for 10 days at −20°C were 70 and 23%, respectively. Prior irradiation did not alter the cell's sensitivity to storage at −20°C, nor did storage at −20°C alter the cell's resistance to irradiation at 25°C. Added NaCl concentrations of up to 4.0% in Trypticase soy agar (TSA) (which contains 0.5% NaCl) had little effect on colony formation at 36°C of unirradiated Y. enterocolitica. With added 4.0% NaCl, 79% of the cells formed colonies at 36°C; with 5.0% NaCl added, no colonies were formed. Although 2.5% NaCl added to ground beef did not sensitize Y. enterocolitica cells to irradiation, when added to TSA it reduced the number of apparent radiation survivors. Cells uninjured by irradiation formed colonies on TSA when incubated at either 36 or 5°C. More survivors of an exposure to 60 krad were capable of recovery and forming colonies on TSA when incubated at 36°C for 1 day than at 5°C for 14 days. This difference in count was considered a manifestation of injury to certain survivors of irradiation.     

Seed dormancy,germination and storage behavior of Magnolia sinica,a plant species with extremely small populations of MagnoliaceaeOA

Magnolia sinica is one of the most endangered Magnoliaceae species in China. Seed biology information concerning its long-term ex situ conservation and utilization is insufficient. This study investigated dormancy status, germination requirements and storage behavior of M. sinica. Freshly matured seeds germinated to ca. 86.5% at 25/15°C but poorly at 30°C; GA3 and moist chilling promoted germination significantly at 20°C. Embryos grew at temperatures(alternating or constant) between 20°C and 25°...     

Respiration and Alternative Oxidase in Corn Seedling Tissues during Germination at Different Temperatures

Respiration rates of Zea mays L. seedling tissues grown at 30 and 14°C were measured at 25°C at different stages of seedling growth. Accumulation of heat units was used to define the developmental stages to compare respiration between the two temperatures. At both temperatures, respiration rates of most tissues were highest at the youngest stages, then declined with age. Respiration rates of mesocotyl tissue were the most responsive to temperature, being nearly twofold higher when grown at 14 compared to 30°C. Alternative pathway respiration increased concomitantly with respiration and was higher in mesocotyls grown in the cold. When seedlings were started at 30 then transferred to 14°C, the increase in alternative pathway respiration due to cold was not observed unless the seedlings were transferred before 2 days of growth. Seedlings transferred to 14°C after growth at 30°C for 2 days had the same alternative oxidase capacity as seedlings grown at 30°C. Seedlings grown at 14°C for 10 to 12 days, then transferred to 30°C, lost alternative pathway respiratory capacity over a period of 2 to 3 days. Western blots of mitochondrial proteins indicated that this loss of capacity was due to a loss of the alternative oxidase protein. Some in vitro characteristics of mitochondria were determined. The temperature optimum for measurement of alternative oxidase capacity was 15 to 20°C. At 41°C, very little alternative oxidase was measured, i.e., the mitochondrial oxygen uptake was almost completely sensitive to cyanide. This inactivation at 41°C was reversible. After incubation at 41°C, the alternative oxidase capacity measured at 25°C was the similar to when it was measured at that temperature directly. Isolated mitochondria lost alternative oxidase capacity at the same rate when incubated at 41°C as they did when incubated at 25°C. Increasing the supply of electrons to isolated mitochondria increased the degree of engagement of the alternative pathway, whereas lower temperature decreased the degree of engagement. Lower temperatures did not increase the degree of engagement of the pathway in intact tissues. We interpret these observations to indicate that the greater capacity of alternative oxidase in cold-grown seedlings is a consequence of development at these low temperatures which results in elevated respiration rates. Low temperature itself does not cause greater capacity or engagement of the alternative oxidase in mitochondria that have developed under warm temperatures. Our hypothesis would be that the low growth temperatures require the seedlings to have a higher respiration rate for some reason, e.g., to prevent the accumulation of a toxic metabolite, and that the alternative pathway functions in that respiration.     

Microbiological Analysis of Rock Cod (Sebastes spp.) Stored Under Elevated Carbon Dioxide Atmospheres

The numbers and types of microorganisms on fresh rock cod fillets and fillets stored in air or in a modified atmosphere (MA; 80% CO₂, 20% air) at 4°C were compared. Samples were analyzed after 0, 7, 14, and 21 days of storage. The isolation plates were incubated aerobically, anaerobically, or under MA at 4, 20, or 35°C. After 7 days of storage in air, the fillets were obviously spoiled and had a 3- to 4-log cycle increase in microbial counts. Plate counts increased more slowly on MA-stored fillets. After 21 days, the counts on the latter had increased only 2 log cycles, and the fillets did not seem spoiled. The microbial flora changed greatly during MA storage. Only Lactobacillus spp. (70%) and an Aeromonas sp.-like isolate (30%) were found on plates incubated aerobically at 4 and 20°C, and only Lactobacillus spp. was found on plates incubated aerobically and anaerobically at 35 and at 20°C under MA. Isolation plates incubated at 20°C in air gave the highest counts in the shortest incubation time and the greatest diversity of bacterial types recovered. No Vibrio parahaemolyticus, Staphylococcus aureus, or Clostridium botulinum type E were isolated from the fresh or MA-stored fillets.     

Method for Investigation of Competition between Bacteria as a Function of Three Environmental Factors Varied Simultaneously

Competition between microorganisms as affected by temperature, pH, and the sodium chloride (NaCl) concentration was investigated by selective replication from gradient plates. Salmonella typhimurium was inhibited by Pseudomonas putida at 20 and 23°C but not 30 and 35°C. P. putida no longer grew at the extremes of pH and NaCl concentration, particularly at 30 and 35°C.     

Control of Lignin Peroxidase Production by Phanerochaete chrysosporium INA-12 by Temperature Shifting

There are two temperature optima connected with lignin peroxidase synthesis by Phanerochaete chrysosporium INA-12. One, at 37°C, is for the mycelium-growing phase; the other, at 30°C, is for the lignin peroxidase-producing phase. One of six extracellular proteins with ligninase activity increased when cultures were grown at 30°C for the entire fermentation period or when cultures were grown at 37°C for the first 2 days of incubation and then shifted to 30°C, compared with the activity of control cultures grown at 37°C for the entire fermentation period. The unsaturation of fatty acid (Δ/mole) of P. chrysosporium INA-12 mycelium decreased from 1.25 to 1.03 when the growth temperature was shifted from 20 to 40°C.     

Effect of Storage Temperature and Duration on Survival and Infectivity of Steinernema innovationi (Rhabditida: Steinernematidae)

Entomopathogenic nematode species differ in their optimum storage temperature; therefore, we conducted a study on the survival and infectivity of the recently described Steinernema innovationi from South Africa at five storage temperatures (5°C, 10°C, 15°C, 20°C, and 25°C) over 84 d using 20,000 infective juveniles (IJ) in 25 ml aqueous suspension containing 0.1% formalin. Our results showed that survival was highest and most stable at 15°C, ranging from 84% to 88% after 84 d. Infectivity of IJ against Galleria mellonella larvae was >90% for all temperatures except for 5°C at which survival decreased to 10% after 84 d. In addition, we stored 2.5 million IJ on a sponge formulation in 15 ml of 0.1% formalin solution for 84 d at the optimum 15°C followed by 2 wk storage at 25°C. Storage of the IJ on a sponge formulation for 14 d at 25°C post 15°C storage for 84 d did not have a detrimental effect on IJ survival (87%) or infectivity to G. mellonella (95%).     

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).     

Evolution of Bacterial Consortia in Spontaneously Started Rye Sourdoughs during Two Months of Daily Propagation

The evolution of bacterial consortia was studied in six semi-solid rye sourdoughs during long-term backslopping at different temperatures. Each rye sourdough was started spontaneously in a laboratory (dough yield 200), propagated at either 20°C or 30°C, and renewed daily at an inoculation rate of 1∶10 for 56 days. The changes in bacterial diversity over time were followed by both DGGE coupled with partial 16S rRNA gene sequencing and pyrosequencing of bar-coded 16S rRNA gene amplicons. Four species from the genus Lactobacillus (brevis, crustorum, plantarum, and paralimentarius) were detected in different combinations in all sourdoughs after 56 propagation cycles. Facultative heterofermentative lactic acid bacteria dominated in sourdoughs fermented at 30°C, while both obligate and facultative heterofermentative LAB were found to dominate in sourdoughs fermented at 20°C. After 56 propagation cycles, Kazachstania unispora (formerly Saccharomyces unisporus) was identified as the only yeast species that dominated in sourdoughs fermented at 20°C, while different combinations of strains from four yeast species (Kazachstania unispora, Saccharomyces cerevisiae, Candida krusei and Candida glabrata) were detected in sourdoughs propagated at 30°C. The evolution of bacterial communities in sourdoughs fermented at the same temperature did not follow the same time course and changes in the composition of dominant and subdominant bacterial communities occurred even after six weeks of backslopping.     

Role of Growth Temperature in Freeze-Thaw Tolerance of Listeria spp.

The food-borne pathogen Listeria monocytogenes can grow in a wide range of temperatures, and several key virulence determinants of the organism are expressed at 37°C but are strongly repressed below 30°C. However, the impact of growth temperature on the ability of the bacteria to tolerate environmental stresses remains poorly understood. In other microorganisms, cold acclimation resulted in enhanced tolerance against freezing and thawing (cryotolerance). In this study, we investigated the impact of growth temperature (4, 25, and 37°C) on the cryotolerance of 14 strains of L. monocytogenes from outbreaks and from food processing plant environments and four strains of nonpathogenic Listeria spp. (L. welshimeri and L. innocua). After growth at different temperatures, cells were frozen at −20°C, and repeated freeze-thaw cycles were applied every 24 h. Pronounced cryotolerance was exhibited by cells grown at 37°C, with a <1-log decrease after 18 cycles of freezing and thawing. In contrast, freeze-thaw tolerance was significantly reduced (P < 0.05) when bacteria were grown at either 4 or 25°C, with log decreases after 18 freeze-thaw cycles ranging from 2 to >4, depending on the strain. These findings suggest that growth at 37°C, a temperature required for expression of virulence determinants of L. monocytogenes, is also required for protection against freeze-thaw stress. The negative impact of growth at low temperature on freeze-thaw stress was unexpected and has not been reported before with this or other psychrotrophic microorganisms.Listeria monocytogenes remains a leading cause of deaths due to food-borne illness in the United States and other industrialized nations. Neonates, pregnant women, and immunocompromised people are at high risk for infection (15, 28, 37). Outbreaks of listeriosis tend to involve a relatively small number of closely related strains (“epidemic clones”), primarily of serotype 4b. Several major outbreaks have been attributed to epidemic clone I (ECI) and epidemic clone II (ECII), both of serotype 4b (5, 21).Unlike most other human food-borne pathogens, L. monocytogenes grows over a wide temperature range (1 to 45°C), with optimal growth at approximately 37°C (33). It has been known for some time that expression of several key virulence genes, including hly, encoding the hemolysin listeriolysin O and actA, encoding a protein that mediates actin polymerization required for intracellular pathogenesis, is optimal at 37°C but severely repressed at temperatures below 30°C (22). In contrast, flagellin, motility, and chemotaxis genes are repressed at 37°C but optimally expressed at temperatures below 25°C (9, 10).For an organism such as L. monocytogenes, which is commonly found in the environment but can also colonize and infect warm-blooded animals, temperature is likely to serve as a major signal differentiating environmental from vertebrate host-associated habitats (19). However, with the exception of the thermoregulated phenotypes described above (virulence factor production, motility, and chemotaxis), the impact of growth at different temperatures on specific responses and adaptations of the pathogen remains poorly understood.L. monocytogenes may be exposed to freezing as well as thawing in the course of its existence in natural environments (e.g., soils and water in temperate or cold regions), as well as during the storage and preservation of foods. The organism has been repeatedly isolated from frozen foods (e.g., ice cream) (7). After freezing (−18°C) in laboratory media or in foods and a single thawing cycle, survival depended on strain, freezing medium, and the presence of glycerol as cryoprotectant (13, 14). The freezing and thawing of bacteria grown at 30°C, in combination with essential oil, has been explored as one means to reduce the pathogen in foods (8). The possible role of the general stress sigma factor (sigma B) in survival of bacteria grown at 30°C and exposed to repeated freezing and thawing was also investigated (43). However, there is a surprising dearth of information on the possible impact of growth temperature on tolerance of L. monocytogenes to repeated freezing and thawing (cryotolerance).Studies with another gram-positive psychrotrophic bacterium (Exiguobacterium sibiricum and other Exiguobacterium spp.) revealed that growth in the cold (4°C), or growth on solid media regardless of temperature, resulted in increased tolerance of the bacteria against repeated freezing and thawing (40). It is not known whether low temperature or surface-associated growth may exert similar impacts on the cryotolerance of L. monocytogenes. The objective of the present study was to investigate the impact of growth temperature (4, 25, and 37°C) and of planktonic versus agar growth of L. monocytogenes on protection of the bacteria against repeated freezing and thawing.     

Effects of Temperature on Methanogenesis in a Thermophilic (58°C) Anaerobic Digestor

The short-term effects of temperature on methanogenesis from acetate or CO₂ in a thermophilic (58°C) anaerobic digestor were studied by incubating digestor sludge at different temperatures with ¹⁴C-labeled methane precursors (¹⁴CH₃COO⁻ or ¹⁴CO₂). During a period when Methanosarcina sp. was numerous in the sludge, methanogenesis from acetate was optimal at 55 to 60°C and was completely inhibited at 65°C. A Methanosarcina culture isolated from the digestor grew optimally on acetate at 55 to 58°C and did not grow or produce methane at 65°C. An accidental shift of digestor temperature from 58 to 64°C during this period caused a sharp decrease in gas production and a large increase in acetate concentration within 24 h, indicating that the aceticlastic methanogens in the digestor were the population most susceptible to this temperature increase. During a later period when Methanothrix sp. was numerous in the digestor, methanogenesis from ¹⁴CH₃COO⁻ was optimal at 65°C and completely inhibited at 75°C. A partially purified Methanothrix enrichment culture derived from the digestor had a maximum growth temperature near 70°C. Methanogenesis from ¹⁴CO₂ in the sludge was optimal at 65°C and still proceeded at 75°C. A CO₂-reducing Methanobacterium sp. isolated from the digestor was capable of methanogenesis at 75°C. During the period when Methanothix sp. was apparently dominant, sludge incubated for 24 h at 65°C produced more methane than sludge incubated at 60°C, and no acetate accumulated at 65°C. Methanogenesis was severely inhibited in sludge incubated at 70°C, but since neither acetate nor H₂ accumulated, production of these methanogenic substrates by fermentative bacteria was probably the most temperature-sensitive process. Thus, there was a correlation between digestor performance at different temperatures and responses to temperature by cultures of methanogens believed to play important roles in the digestor.     

Temperature Effects on the Attachment of Pasteuria penetrans Endospores to Meloidogyne arenaria Race 1

Pasteuria penetrans is a gram positive bacterium that prevents Meloidogyne spp. from reproducing and diminishes their ability to penetrate roots. The attachment of the endospores to the cuticle of the nematodes is the first step in the life cycle of the bacterium and is essential for its reproduction. As a preliminary study to a field solarization test, the effects of temperature on the attachment of P. penetrans on Meloidogyne arenaria race 1 were investigated. Preexposing second-stage juveniles (J2) of M. arenaria to approximately 30 °C in water before exposing them to endospores increased their receptivity to endospore attachment when compared to treating J2 at 25 °C or 35 °C. In tests with soil, highest attachment occurred when J2 were incubated in soil infested with endospores and maintained at 20 °C to 30 °C for 4 days. Heating J2 in soil to sublethal temperatures (35 °C to 40 °C) decreased endospore attachment. Incubating P. penetrans endospores in soil at 30 °C to 70 °C for 5 hours a day over 10 days resulted in reductions of endospore attachment to nematodes as temperatures of incubation increased to 50 °C and higher.     

Greening under High Light or Cold Temperature Affects the Level of Xanthophyll-Cycle Pigments, Early Light-Inducible Proteins, and Light-Harvesting Polypeptides in Wild-Type Barley and the Chlorina f2 Mutant

Etiolated seedlings of wild type and the chlorina f2 mutant of barley (Hordeum vulgare) were exposed to greening at either 5°C or 20°C and continuous illumination varying from 50 to 800 μmol m⁻² s⁻¹. Exposure to either moderate temperature and high light or low temperature and moderate light inhibited chlorophyll a and b accumulation in the wild type and in the f2 mutant. Continuous illumination under these greening conditions resulted in transient accumulations of zeaxanthin, concomitant transient decreases in violaxanthin, and fluctuations in the epoxidation state of the xanthophyll pool. Photoinhibition-induced xanthophyll-cycle activity was detectable after only 3 h of greening at 20°C and 250 μmol m⁻² s⁻¹. Immunoblot analyses of the accumulation of the 14-kD early light-inducible protein but not the major (Lhcb2) or minor (Lhcb5) light-harvesting polypeptides demonstrated transient kinetics similar to those observed for zeaxanthin accumulation during greening at either 5°C or 20°C for both the wild type and the f2 mutant. Furthermore, greening of the f2 mutant at either 5°C or 20°C indicated that Lhcb2 is not essential for the regulation of the xanthophyll cycle in barley. These results are consistent with the thesis that early light-inducible proteins may bind zeaxanthin as well as other xanthophylls and dissipate excess light energy to protect the developing photosynthetic apparatus from excess excitation. We discuss the role of energy balance and photosystem II excitation pressure in the regulation of the xanthophyll cycle during chloroplast biogenesis in wild-type barley and the f2 mutant.     

Isolation and Characterization of a Composite Plasmid Rms201 Mutant Temperature Sensitive for Replication

A mutant temperature-sensitive for R-plasmid replication, Rms201ts14, was isolated from composite plasmid Rms201 after mutagenesis of P1 transducing lysate with 100 mM hydroxylamine for 40 h at 37°C. When Escherichia coli ML1410(Rms201ts14)⁺ was grown at temperatures between 40 and 42°C in L broth, antibiotic-sensitive cells were segregated. When the incubation temperature of ML1410(Rms201ts14)⁺ in L-broth was shifted to 42 from 30°C, the increase in the number of antibiotic-resistant cells ceased 90 min after the temperature shift. However, the total number of cells continuously increased, and only 3% of the cells retained the plasmid at 5 h after the temperature shift to 42°C. At 30°C the amounts of covalently closed circular deoxyribonucleic acid per chromosome of Rms201ts14 and Rms201 were 3.8 and 6.3%, respectively. Incorporation of radioactive thymidine into the covalently closed circular deoxyribonucleic acid of Rms201ts14 did not take place at 42°C, whereas radioactive thymidine was incorporated into the covalently closed circular deoxyribonucleic acid of Rms201 at a rate of 4%/chromosome even at 42°C. The synthesis of plasmid covalently closed circular deoxyribonucleic acid in a cell harboring Rms201ts14 was almost completely blocked at 42°C. These results indicated that the gene(s) responsible for plasmid deoxyribonucleic acid replication was affected in the mutant Rms201ts14. Temperature-sensitive miniplasmid pMSts214, which has a molecular weight of 5.3 × 10⁶ and encodes ampicillin resistance, was isolated from Rms201ts14. Similarly, miniplasmid pMS201, which encodes single ampicillin resistance, was isolated from its parent, Rms201, and its molecular weight was 4.7 × 10⁶. These results indicate that the gene(s) causing temperature sensitivity for replication of Rms201 resides on the miniplasmid.     

Kernel Abortion in Maize : II. Distribution of C among Kernel Carbohydrates

This study was designed to compare the uptake and distribution of ¹⁴C among fructose, glucose, sucrose, and starch in the cob, pedicel, and endosperm tissues of maize (Zea mays L.) kernels induced to abort by high temperature with those that develop normally. Kernels cultured in vitro at 30 and 35°C were transferred to [¹⁴C]sucrose media 10 days after pollination. Kernels cultured at 35°C aborted prior to the onset of linear dry matter accumulation. Significant uptake into the cob, pedicel, and endosperm of radioactivity associated with the soluble and starch fractions of the tissues was detected after 24 hours in culture on labeled media. After 8 days in culture on [¹⁴C]sucrose media, 48 and 40% of the radioactivity associated with the cob carbohydrates was found in the reducing sugars at 30 and 35°C, respectively. This indicates that some of the sucrose taken up by the cob tissue was cleaved to fructose and glucose in the cob. Of the total carbohydrates, a higher percentage of label was associated with sucrose and a lower percentage with fructose and glucose in pedicel tissue of kernels cultured at 35°C compared to kernels cultured at 30°C. These results indicate that sucrose was not cleaved to fructose and glucose as rapidly during the unloading process in the pedicel of kernels induced to abort by high temperature. Kernels cultured at 35°C had a much lower proportion of label associated with endosperm starch (29%) than did kernels cultured at 30°C (89%). Kernels cultured at 35°C had a correspondingly higher proportion of ¹⁴C in endosperm fructose, glucose, and sucrose. These results indicate that starch synthesis in the endosperm is strongly inhibited in kernels induced to abort by high temperature even though there is an adequate supply of sugar.     

Concomitant changes in high temperature tolerance and heat-shock proteins in desert succulents

Raising the day/night air temperatures from 30°C/20°C to 50°C/40°C increases the high temperature tolerated by Agave deserti, Carnegiea gigantea, and Ferocactus acanthodes by 6°C to 8°C; the increase is about half completed in 3 days and fully completed in 10 days. A 25 to 27 kilodalton protein concomitantly accumulates for all three desert succulents upon transfer to 50°C/40°C, while accumulation of other heat “heat-shock” proteins is species specific. Some of the induced proteins are more abundant at 3 days, while others (including the 25-27 kilodalton protein) remain after completion of high temperature acclimation.