Viability of cold-adapted L cells after freezing and storage in solid CO2

Mouse L cells adapted to low temperature by repeated exposures to 4 °C for 6–8 weeks were stored in solid CO₂ at −70 °C for 7 months simultaneously with unpretreated controls. The population of the cold-adapted subline LC2 contained more living cells after thawing than did the control L cells. The increased viability of the LC2 cells was expressed by a higher number of eosin-unstained cells immediately after thawing and by a higher increase in cell number after incubation at 36 °C. The difference between the two populations was more marked after longer storage.     

Cold shock induction of thermal sensitivity in Listeria monocytogenes

Cold shock at 0 to 15 degrees C for 1 to 3 h increased the thermal sensitivity of Listeria monocytogenes. In a model broth system, thermal death time at 60 degrees C was reduced by up to 45% after L. monocytogenes Scott A was cold shocked for 3 h. The duration of the cold shock affected thermal tolerance more than did the magnitude of the temperature downshift. The Z values were 8.8 degrees C for controls and 7.7 degrees C for cold-shocked cells. The D values of cold-shocked cells did not return to control levels after incubation for 3 h at 28 degrees C followed by heating at 60 degrees C. Nine L. monocytogenes strains that were cold shocked for 3 h exhibited D(60) values that were reduced by 13 to 37%. The D-value reduction was greatest in cold-shocked stationary-phase cells compared to cells from cultures in either the lag or exponential phases of growth. In addition, cold-shocked cells were more likely to be inactivated by a given heat treatment than nonshocked cells, which were more likely to experience sublethal injury. The D values of chloramphenicol-treated control cells and chloramphenicol-treated cold-shocked cells were no different from those of untreated cold-shocked cells, suggesting that cold shock suppresses synthesis of proteins responsible for heat protection. In related experiments, the D values of L. monocytogenes Scott A were decreased 25% on frankfurter skins and 15% in ultra-high temperature milk if the inoculated products were first cold shocked. Induction of increased thermal sensitivity in L. monocytogenes by thermal flux shows potential to become a practical and efficacious preventative control method.     

Effect of temperature,pH, dissolved oxygen,and hydrolysate on the formation of triple light chain antibodies in cell culture

THIOMABs are recombinant antibodies with reactive cysteine residues used for forming THIOMAB–drug conjugates (TDCs). We recently reported a new impurity associated with THIOMABs: one of the engineered cysteines forms a disulfide bond with an extra light chain (LC) to generate a triple light chain antibody (3LC). In our previous investigations, increased LC expression increased 3LC levels, whereas increased glutathione (GSH) production decreased 3LC levels. In this work, on three stably transfected CHO cell lines, we investigated the effects of temperature, pH, dissolved oxygen (DO), and hydrolysate on 3LC formation during THIOMAB fed‐batch cell culture production. Although pH between 6.8 and 7.0 had no significant impact on 3LC formation, temperature at 35°C instead of 33 or 31°C generated the lowest 3LC values for two cell lines. The decreased 3LC level correlated with increased GSH production. We implemented a 35°C temperature process for large‐scale (2,000 L) production of a THIOMAB. This process reduced 3LC levels by ～50% compared with a 33°C temperature process. By contrast, DO and hydrolysate had modest effect on 3LC levels for the model cell line studied. Overall, we did not find significant changes in LC expression under the conditions tested, whereas changes in GSH production were more evident. By investigating the impact of bioreactor process and medium conditions on 3LC levels, we identified strategies that reduced 3LC levels. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Validation of a new live cell strain system: characterization of plasma membrane stress failure.

Motivated by our interest in lung deformation injury, we report on the validation of a new live cell strain system. We showed that the system maintains a cell culture environment equivalent to that provided by conventional incubators and that its strain ouput was uniform and reproducible. With this system, we defined cell deformation dose (i.e., membrane strain amplitude)-cell injury response relationships in alveolar epithelial cultures and studied the effects of temperature on them. Deformation injury occurred in the form of reversible, nonlethal plasma membrane stress failure events and was quantified as the fraction of cells with uptake and retention of fluorescein-labeled dextran (FITC-Dx). The undeformed control population showed virtually no FITC-Dx uptake at any temperature, which was also true for cells strained by 3%. However, when the membrane strain was increased to 18%, ~5% of cells experienced deformation injury at a temperature of 37 degrees C. Moreover, at that strain, a reduction in temperature to 4 degrees C resulted in a threefold increase in the number of cells with plasma membrane breaks (from 4.8 to 15.9%; P < 0.05). Cooling of cells to 4 degrees C also lowered the strain threshold at which deformation injury was first seen. That is, at a 9% substratum strain, cooling to 4 degrees C resulted in a 10-fold increase in the number of cells with FITC-Dx staining (0.7 vs. 7.5%, P < 0.05). At that temperature, A549 cells offered a 50% higher resistance to shape change (magnetic twisting cytometry measurements) than at 37 degrees C. We conclude that the strain-injury threshold of A549 cells is reduced at low temperatures, and we consider temperature effects on plasma-membrane fluidity, cytoskeletal stiffness, and lipid trafficking as responsible mechanisms.     

Effect of reduced temperatures on protein synthesis in mouse L cells.

The rate of incorporation of leucine into protein, the rate of polypeptide elongation and termination, and the relative quantity and size of polysomes were analyzed in mouse L cells grown in suspension culture at various temperatures between 0 degrees C and 36 degrees C. Between 10 degrees C and 36 degrees C protein synthesis exhibited two different apparent activation energies (39 kcal/mole, 10-25 degrees C; 14 kcal/mole, 25-36 degrees C), whereas elongation and termination had only one (16 kcal/mole). Below 36 degrees C, the polysome level and size decreased, reaching a minimum of 30% of the control 36 degrees C values at 10 degrees C; below 10 degrees C the level increased again back to control values at 0 degrees C. The polysome decline was time dependent, requiring about 5 hr to reach the equilibrium value. This decline is completely reversible within 60 min, even in the presence of 4 mug/ml of actinomycin D, and even after 15 hr of incubation at the lower temperature. The results suggest that polypeptide initiation is rate limiting, particularly below 25 degrees C; whereas above this temperature, elongation or perhaps some other process may be limiting. These results are quite different from those obtained for E. coli and rabbit reticulocyte protein synthesis.     

The critical influence of temperature upon trifluorothymidine resistance in mouse lymphoma L5178Y/TK 3.7.2C cells

L5178Y/TK 3.7.2C cells are used for the assessment of chemical mutagenesis caused by presumptive TK gene mutations or multiple loci mutations affecting the TK locus that result in dose-related increases in resistance to the toxic thymidine analog, trifluorothymidine (TFT). This study was based upon our general observation that the incidence of TFTres in these cells could vary with the incubation temperature. As a result of these studies, we found that: (1) a substantial proportion of presumptive TK-/- variants produced by the mutagens 2-aminofluorene (2-AF), N-acetylaminofluorene (AAF), benzo[a]pyrene (B[a]P), 3-methylcholanthrene (3MCA), hycanthone methanesulfonate (Hyc), or methyl methanesulfonate (MMS) are more resistant to TFT at 37 degrees C than at 28 degrees C (or 39 degrees C than at 33 degrees C), (2) the loss of resistance to TFT was most notable in the small-colony variant population, (3) mutagen-derived variants become less resistant as the TFT concentration is increased from 4 micrograms/ml to 50 micrograms/ml, an effect that is more pronounced at 28 degrees C than at 37 degrees C, and (4) stock 3.7.2C cells develop a persistent TFTres due to sharply decreased TK activity when exposed to 40 degrees C for at least 24 h. These data demonstrate two different responses by these cells with respect to temperature stability at the TK locus and suggest that the degree of TFTres is influenced by both temperature and concentration of selective agent in this presumptive gene/chromosomal mutation assay.     

Influence of TRP53 status on FAS membrane localization, CFLAR (c-FLIP) ubiquitinylation, and sensitivity of GC-2spd (ts) cells to undergo FAS-mediated apoptosis

Previously we reported that testicular germ cells undergo FAS-mediated apoptosis after exposure of mice to the Sertoli cell toxicant mono-(2-ethylhexyl) phthalate (MEHP) and that this process is partially dependent on the TRP53 protein (p53). Recent reports have suggested that TRP53 may influence the ubiquitinylation and consequent proteosomal degradation of a negative regulator of FAS, CFLAR (L) (c-FLIP [L]), in human colon cancer cells. To further characterize the relationship between CFLAR and TRP53, we used the transformed germ cell line GC-2spd (ts), which harbors a temperature-sensitive Trp53 mutation that allows for TRP53 activation at 32 degrees C. We report here that GC-2 cells expressed a 10-fold increase in basal cell membrane FAS levels and an increased sensitivity to FAS agonistic antibody (JO2)-triggered apoptosis only when they were maintained at the permissive TRP53 temperature. After JO2 exposure, CFLAR (L) protein levels were enhanced only at the nonpermissive TRP53 temperature (37 degrees C) while real-time PCR results indicated an absence of Cflar (L) mRNA changes in GC-2 cells regardless of the temperature. Furthermore, transfection of GC-2 cells at 37 degrees C with siRNA against Cflar resulted in reduction of CFLAR (L) protein levels and increased sensitivity to JO2-mediated apoptosis. The CFLAR (L) protein was also more strongly ubiquitinylated in response to JO2 treatment at the permissive TRP53 temperature. Taken together, these data suggest that the TRP53 protein influences the sensitivity of GC-2 cells to undergo FAS-mediated apoptosis by modulating the expression of FAS on their cell membranes and subsequently influencing the degradation of the antiapoptotic protein CFLAR (L).     

Ubiquitin-activating enzyme, E1, is associated with maturation of autophagic vacuoles

The ubiquitin-activating enzyme, E1, is required for initiating a multi-step pathway for the covalent linkage of ubiquitin to target proteins. A CHO cell line containing a mutant thermolabile E1, ts20, has been shown to be defective in stress-induced degradation of proteins at restrictive temperature (Gropper et al., 1991. J. Biol. Chem. 266:3602-3610). Parental E36 cells responded to restrictive temperature by stimulating lysosome-mediated protein degradation twofold. Such a response was not observed in ts20 cells. The absence of accelerated degradation in these cells at 39.5 degrees C was accompanied by an accumulation of autolysosomes. The fractional volume of these degradative autophagic vacuoles was at least sixfold greater than that observed for either E36 cells at 30.5 degrees or 39.5 degrees C, or ts20 cells at 30.5 degrees C. These vacuoles were acidic and contained both acid phosphatase and cathepsin L, but, unlike the autolysosomes observed in E36 cells, ubiquitin-conjugated proteins were conspicuously absent. Combined, our results suggest that in ts20 cells, which are unable to generate ubiquitin-protein conjugates due to heat inactivation of E1, the formation and maturation of autophagosomes into autolysosomes is normal, but the conversion of autolysosomes into residual bodies is disrupted.     

The autophagy-related protein LC3 is processed in stallion spermatozoa during short-and long-term storage and the related stressful conditions

Use of cooled and frozen semen is becoming increasingly prevalent in the equine industry. However, these procedures cause harmful effects in the sperm cell resulting in reduced cell lifespan and fertility rates. Apoptosis and necrosis-related events are increased during semen cryopreservation. However, a third type of cell death, named autophagy, has not been studied during equine semen storage. Light chain (LC)3 protein is a key component of the autophagy pathway. Under autophagy activation, LC3-I is lipidated and converted to LC3-II. The ratio of LC3-II/LC3-I is widely used as a marker of autophagy activation. The main objective of this study was to investigate whether LC3 is processed during cooling, freezing and the stressful conditions associated with these technologies. A secondary objective was to determine if LC3 processing can be modulated and if that may improve the quality of cryopreserved semen. LC3 processing was studied by Western blot with a specific antibody that recognized both LC3-I and LC3-II. Viability was assessed by flow cytometry. Modulation of LC3-I to LC3-II was studied with known autophagy activators (STF-62247 and rapamycin) or inhibitors (chloroquine and 3-MA) used in somatic cells. The results showed that conversion of LC3-I to LC3-II increased significantly during cooling at 4°C, freezing/thawing and each of the stressful conditions tested (UV radiation, oxidative stress, osmotic stress and changes in temperature). STF-62247 and rapamycin increased the LC3-II/LC3-I ratio and decreased the viability of equine sperm, whereas chloroquine and 3-MA inhibited LC3 processing and maintained the percentage of viable cells after 2 h of incubation at 37°C. Finally, refrigeration at 4°C for 96 h and freezing at −196°C in the presence of chloroquine and 3-MA resulted in higher percentages of viable cells. In conclusion, results showed that an ‘autophagy-like’ mechanism may be involved in the regulation of sperm viability during equine semen cryopreservation. Modulation of autophagy during these reproductive technologies may result in an improvement of semen quality and therefore in higher fertility rates.     

Physiology of a temperature-sensitive mutant of Saccharomyces cerevisiae defective in phosphofructokinase activity.

In this paper, we describe a temperature-sensitive mutant of the yeast Saccharomyces cerevisiae (P5-9) which at a restrictive temperature (36 degrees C) shows a pleiotropic defect for transport of many different metabolites. The temperature sensitivity of the mutant is closely related to a reduction in phosphofructokinase activity. This conclusion is based on the following criteria. (i) Both the primary isolate, designated P5-9 (ts [rho-] Ino-), which is an inositol auxotroph and respiration deficient, and a purified derivative, SB4 (ts [rho+] Ino+ ), which is respiration competent and capable of growing in the absence of inositol, are temperature sensitive for growth and ethanol production in media containing glucose or fructose as the sole carbon source. (ii) The respiration-competent derivative SB4 is not temperature sensitive in media containing glycerol or glycerol-pyruvate; glucose inhibits its growth at 36 degrees C in these media. (iii) Assays of glycolytic enzymes in P5-9 and SB4 extracts, prepared from cells incubated for 1 to 2 h at 36 degrees C before harvesting, show selective reduction in phosphofructokinase activity. Analysis of tetrads derived from the cross of mutant and nonmutant haploids indicates that temperature sensitivity for growth is due to a single gene or to two closely linked genes. The biochemical analysis of spores from seven such tetrads revealed a uniform cosegregation of temperature sensitivity for growth and phosphofructokinase activity. Transport and ATP levels were drastically reduced in SB4 cells incubated at 36 degrees C for 1 to 2 h with glucose as the carbon source, but not when glycerol-pyruvate or lactate was the energy source. Therefore, depletion of energy as a result of phosphofructokinase inactivation appears to be the cause of the pleiotropic transport defect observed in the mutant.     

Elongation and shortening of time required for entry into S phase after release from G 1 and G 0 arrests in temperature-sensitive mutants of rat 3Y1 Cells

Temperature-sensitive (ts) mutants of rat 3Y1 fibroblasts representing four separate complementation groups (3Y1tsD123, 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203) are arrested mainly in the G1 phase when cells of randomly proliferating population at 33.8 degrees C are shifted to 39.8 degrees C (temperature arrest). We examined the time lag of the cellular entry into the S phase after release at 33.8 degrees C, both from the temperature arrest and from the arrest at 33.8 degrees C at a confluent cell density (density arrest). In the temperature-arrested cells, as the duration of temperature arrest increased, the time lag of entry into S phase after shift down to 33.8 degrees C was prolonged, in all four mutants. These observations suggest that the four different functional lesions, each causing arrest in the G1 phase, are also responsible for prolongation of the time lag of entry into the S phase in cells arrested in the G1 phase. The prolongation of the time lag in the temperature-arrested cultures was accelerated at a higher cell density, in medium supplemented with a lower concentration of serum, and at a higher restrictive temperature. In the density-arrested cells, as the duration of pre-exposure to 39.8 degrees C was increased, the time lag of entry into S phase at 33.8 degrees C after release from the arrest was drastically prolonged, in all four mutants. In 3Y1tsF121, 3Y1tsG125, and 3Y1tsH203, when the density-arrested cells were prestimulated by serum at 39.8 degrees C for various periods of time, the time lag of entry into S phase after release from the density arrest at 33.8 degrees C was initially shortened, and then, prolonged progressively as the period of prestimulation increased. These findings, taken together with other data, show that all four ts defects affect cells in states ranging from the deeper resting to mid- or late-G1 phase. It is suggested that events represented by these four mutants are required for entry into the S phase and normally operate in parallel but not in sequence in cells in states ranging from the deeper resting to the mid- or late-G1 phases, though they may affect each other.     

Viral p21 Ki-RAS protein: a potent intracellular mitogen that stimulates adenylate cyclase activity in early G1 phase of cultured rat cells

Rat kidney (NRK) cells infected with a temperature-sensitive mutant of the Kirsten sarcoma virus were arrested in the G0/G1 phase of their cell cycle by incubation in serum-deficient medium at a p21-inactivating temperature of 41 degrees C. These quiescent ts K-NRK cells were then stimulated to transit G1 and initiate DNA replication by lowering the temperature to 36 degrees C, which rapidly reactivated p21. Reactivating the viral Ki-RAS protein by temperature shift led to an increase in adenylate cyclase activity in early G1 phase. The Ki-RAS protein increased the sensitivity of adenylate cyclase to guanyl nucleotides by a mechanism that seemed to involve inactivation of the enzyme's inhibitory G1 regulatory protein.     

Sensitivity of polyamine-deficient Saccharomyces cerevisiae to elevated temperatures.

Saccharomyces cerevisiae cells that cannot synthesize spermidine or spermine because of a deletion in the gene coding for S-adenosylmethionine decarboxylase are very sensitive to elevated temperatures when incubated in a polyamine-deficient medium; i.e., growth is inhibited and the cells are killed. This sensitivity is very pronounced at 39 degrees C, but a moderate effect is noted even at 33 to 34 degrees C. These findings support findings from other studies from our laboratory on the importance of polyamines in protecting cell components against damage. The sensitivity of spermidine-deficient cells to the temperature 39 degrees C provides a useful method for screening for polyamine auxotrophs.     

An analysis of the effect of changes in growth temperature on proteolysis in vivo in the psychrophilic bacterium Vibrio sp. strain ANT-300.

In the psychrophilic bacterium Vibrio sp. strain ANT-300, the rate of protein degradation in vivo, measured at fixed temperatures, increased with elevation of the growth temperature. A shift in growth temperature induced a marked increase in this rate. Dialysed cell-free extracts hydrolysed exogenous insulin, globin and casein (in decreasing order of activity) but did not hydrolyse exogenous cytochrome c. Cells contained at least seven protease separated by DEAE-Sephacel chromatography, one of which was an ATP-dependent serine protease. The ATP-dependent proteolytic activity in extracts of cells incubated for 3 h at 16 degrees C after a shift-up from 0 degrees C increased to a level 36% and 17% higher than that of cells grown at 0 degrees C and 13 degrees C, respectively. A shift-down to 0 degrees C from 13 degrees C induced only a slight increase in the proteolytic activity. Extracts of all cells, whether exposed to temperature shifts or not, showed the same temperature dependence with respect to both ATP-dependent and ATP-independent protease activity. In all the extracts these proteases also exhibited the same heat lability. The ATP-dependent protease was inactivated by incubation at temperatures above 25 degrees C. There was an increase in ATP-independent protease activity during incubation at temperatures between 25 and 30 degrees C, but a decrease at 35 degrees C and higher. These results suggest that the marked increases in proteolysis in vivo, caused by a shift in temperature, may result not only from increases in levels of ATP-dependent serine protease(s) but also from increases in the susceptibility of proteins to degradation.     

Mitogenic action of factors secreted by avian erythroblastosis virus transformed cells

We describe here the capacity of erythroid LSCC HD3 cells, transformed with a ts mutant of avian erythroblastosis virus, to grow in a chemically defined medium without serum at 36 degrees C, but not at 41 degrees C. At this latter temperature the activity of v-erbB oncogene is suppressed. However, cell growth at 41 degrees C could take place either by addition of the medium derived from LSCC HD3 cells grown at 36 degrees C (conditioned medium), or by addition of fetal calf serum. These results show that LSCC HD3 cells, maintained under conditions in which the v-erbB oncogene is active, secrete growth factor(s) which exhibit a mitogenic effect similar to that observed with calf serum.     

Variation in the structure and bacteriophage-inactivating capacity of Salmonella anatum lipopolysaccharide as a function of growth temperature.

Growth temperature affects both the structure and the phage-inactivating capacity of Salmonella anatum A1 lipopolysaccharide. Whereas S. anatum cells normally synthesize smooth lipopolysaccharide when grown at physiological temperature (37 degrees C), a partial smooth-rough transition occurs when cells are grown at low temperature (20 to 25 degrees C). The synthesis at low growth temperature of lipopolysaccharide molecules lacking O-antigen was detected both by increased sensitivity of cells to the rough-specific bacteriophage Felix O-1 and by fractionation of oligosaccharides derived from lipopolysaccharide by mild acid hydrolysis. Growth temperature-induced changes in the structure of S. anatum A1 lipopolysaccharide also affected its ability to inactivate epsilon15, a bacteriophage that binds initially to the O-antigen portion of the molecule. Purified lipopolysaccharide prepared from cells grown at low growth temperature exhibited a higher in vitro phage-inactivating capacity than did lipopolysaccharide prepared from cells grown at physiological temperature (37 degrees C).     

Temperature-dependent enhancement of cell proliferation and mRNA expression for type I collagen and HSP70 in primary cultured goldfish cells

Goldfish (Carasius auratus) primary culture cells derived from caudal fin were incubated over a temperature range of 20-35 degrees C. The population doubling time of cells cultured at 20, 25, 30 and 35 degrees C were 34, 29, 17 and 14 h, respectively. Interestingly, cDNA-representational difference analysis revealed type I collagen alpha chain (colalpha(I)) as a candidate for a warm temperature-specific gene. mRNA levels of colalpha(I) increased with an increase of incubation temperature and days of culture. Furthermore, the cell growth rate and colalpha(I) mRNA levels were rapidly changed following temperature shifts. To examine the effects of culture temperature shift on the cellular physiological states, mRNA levels of HSP70 were additionally investigated. HSP70 mRNA levels in the cells cultured at 30 and 35 degrees C were again 2-3 times higher than those at 20 and 25 degrees C. When the culture temperature was shifted from 20 to 35 degrees C, HSP70 mRNA levels were rapidly increased within 1 h. Subsequently, mRNA levels of the 35 degrees C-treated cells decreased, but remained doubled compared with those of the 20 degrees C-treated cells, even 4 h following the temperature shift. When the culture temperature was lowered from 35 to 20 degrees C, HSP70 mRNA levels decreased to about 70% of the original levels in 4 h. These results indicate that goldfish cells cultured at different temperatures easily develop temperature-associated steady physiological states within 4 h of temperature shifts.     

Effect of oryzalin on root ultrastructure and respiration in various wheat cultivars subjected to cold hardening

The effect of anti-microtubular herbicide oryzalin (10 μM, 2–4 days) on the root ultrastructure and respiration in two cultivars of winter wheat (Triticum aestivum L.) contrasting in their frost-tolerance was studied during plant cold hardening (3°C, 3 days). The sensitivity of subcellular structures to oryzalin depended closely on cell metabolic activity and the extent of development of the cortical microtubule (MT) network. Most pronounced oryzalin-induced changes were related to enhanced cell vacuolation and the appearance of some signs of apoptosis (as judged from cytoplasm fragmentation) in some cells. In the root zone examined, cell heterogeneity increased, when, along with normally functioning cells, dramatically damaged and even completely destroyed cells appeared. Simultaneously, the activity of cyanide-resistant nonphosphorylating respiration pathway was activated, especially during cold hardening. In hardened cells, single cortical microtubules appeared in both wheat cultivars; this fact indicates that new cold-resistant subpopulations of MT were resistant to depolymerizing action of oryzalin.     

Temperature-dependent changes of cell shape during heterophyllous leaf formation in <Emphasis Type="Italic">Ludwigia </Emphasis><Emphasis Type="Italic">arcuata</Emphasis> (Onagraceae)

Although elongation of epidermal cells in submerged leaves is thought to be a common feature of heterophyllous aquatic plants, such elongation has not been observed in Ludwigia arcuata Walt. (Onagraceae). In this study we found that reduced culture temperature induced the elongation of epidermal cells of submerged leaves in L. arcuata. Since such submerged leaves also showed a reduction in the number of epidermal cells aligned across the leaf transverse axis, these data indicate that heterophyllous leaf formation in L. arcuata is partially temperature sensitive, i.e., the elongation of epidermal cells was temperature sensitive while the reduction in the number of epidermal cells did not show such temperature sensitivity. To clarify the mechanisms that cause such temperature sensitivity, we examined the effects of ethylene, which induced the formation of submerged-type leaves on aerial shoots at the relatively high culture-temperature of 28 degrees C. At 23 degrees C, ethylene induced both cell elongation and reduction in the number of epidermal cells across the leaf transverse axis, while cell elongation was not observed at 28 degrees C. Moreover, both submergence and ethylene treatment induced a change in the arrangement of cortical microtubules (MTs) in epidermal cells of developing leaves at 23 degrees C. Such changes in the arrangement of MTs was not induced at 28 degrees C. Factors involved in the temperature-sensitive response to ethylene would be critical for temperature-sensitive heterophyllous leaf formation in L. arcuata.