Induction of aluminum tolerance in wheat seedlings by low doses of aluminum in the nutrient solution

Preincubation of wheat (Triticum aestivum L. Thell.) seedlings in a nutrient solution containing low doses of aluminum (0.5 microgram per milliliter for tolerant cultivar Atlas 66 and 0.1 microgram per milliliter for the sensitive cultivar Grana) enabled substantial root regrowth of varieties grown in a lethal aluminum concentration, despite an increased accumulation of aluminum in root tissue of the pretreated seedlings. The distribution of aluminum in the subcellular fractions remained unchanged. The increase in tolerance was completely abolished by the addition of cycloheximide. Aluminum ions at sublethal concentrations significantly increased the incorporation of [¹⁴C]valine and [³H]thymidine in roots. The possible role of the synthesis of the inducible aluminum binding protein in the mechanism of aluminum tolerance is discussed.     

Sucrose and Glucose Uptake into Beta vulgaris Leaf Tissues : A Case for General (Apoplastic) Retrieval Systems

Concentration curves for sugar and amino acid uptake by Beta vulgaris L. leaf tissues contained both a saturable and a linear component. Similarly shaped curves were obtained for influx of sucrose, glucose, and 3-O-methyl glucose by leaf discs, whole petiole slices, petiole segments containing pith tissue only, and petiole segments containing vascular bundles, although the tissues took up the various sugars via different proportions of saturable versus linear uptake. Two millimolar p-chloromercuribenzenesulfonic acid selectively inhibited the saturable component of sucrose uptake, but had almost no effect on the linear component. Uptake of glucose and 3-O-methyl glucose remained unaffected by p-chloromercuribenzenesulfonic acid treatment. Anoxia was found to inhibit the linear component of both sucrose and 3-O-methyl glucose influx, while the saturable component remained unaffected. The linear component of sucrose uptake was also competitively inhibited by maltose, as well as being selectively promoted by certain exposures to 5 millimolar N-ethylmaleimide, 2 micrograms per milliliter cycloheximide, and high levels of mannitol acting as osmoticum. These results support the proposal that the linear component is due to a process more complex than simple, or exchange, diffusion. It would also appear that the linear transport component utilizes a separate energy source than does the saturable component of sucrose influx.

Evidence for phloem loading from the apoplast was re-examined with respect to the present findings. Saturable sucrose uptake by minor vein tissues may represent retrieval of solute from the free space, which could explain the `apoplastic loading' phenomenon.

     

Wound-induced resistance to cellulase in oat leaves

Peeling the epidermis induces the development of resistance to cellulolytic digestion in the mesophyll cell wals of the first leaf of 1- to 3-week-old oat seedlings (Avena sativa var. Victory). Development of resistance occurs between 3 and 11 hours after the abaxial epidermis is peeled from the blade, and is inhibited by actinomycin D (20 micrograms per milliliter) or cycloheximide (1 microgram per milliliter). Other methods of wounding (cutting with a razor blade, stabbing with a dissection needle or brushing with diatomaceous silica) also induce resistance in cells near the wounds. Peeling similarly induces resistance to the digestion of mesophyll cell walls by Cellulysin (Calbiochem) in pea, corn, wheat, and barley.     

Cycloheximide and heat shock induce new polypeptide synthesis in Neurospora crassa.

Treatment of Neurospora crassa with 0.1 microgram of cycloheximide per ml, a concentration which inhibited protein synthesis by about 70%, resulted in the greatly enhanced synthesis of at least three polypeptide bands with estimated molecular weights of 88,000, 30,000, and 28,000. A temperature shift from 25 to 37 degrees C resulted in the appearance of a single new polypeptide band of 70,000 daltons, the same size as the major heat shock-induced proteins observed in species of Drosophila and Dictyostelium. Synthesis of the cycloheximide-stimulated polypeptide bands was on cytoplasmic ribosomes rather than on mitochondrial ribosomes, as incorporation of isotope into the polypeptide bands was inhibited by 1.0 microgram of cycloheximide per ml but not by 1 mg of chloramphenicol per ml. In a mutant with cycloheximide-resistant ribosomes, 0.1 microgram of cycloheximide per ml failed to alter the pattern of protein synthesis from that of the controls. It is suggested that the new synthesis of the polypeptide bands reflects specific mechanisms of adaptation to different kinds of environmental stress, including inhibition of protein synthesis and temperature increases.     

Anomalous effects of cycloheximide on phenylalanine ammonia-lyase: role of synthesis and inactivation in leaf disks of helianthus annuus*

The activity of phenylalanine ammonia-lyase (PAL) increases dramatically in leaf disks of sunflower (Helianthus annuus) cultured on 0.1 M sucrose in the dark. If disks are subsequently transferred to water, PAL activity decays rapidly. After inactivation the level of PAL can be increased again by transferring the tissue back to sucrose. The initial increase in PAL activity appears to involve an increase in the rate of PAL formation and the appearance is inhibited by cycloheximide. Inactivation of the enzyme is also inhibited by cycloheximide. A comparison of cycloheximide inhibition at different concentrations showed that inactivation was much more sensitive to the inhibitor than PAL formation. The rate of PAL inactivation was very low in fresh disks placed directly on water (t 1/2 = > 1 day) but increased greatly after culture on sucrose (t_1/2 = 2 to 4 hr). Therefore, culture appears to increase PAL inactivation as well as PAL formation. Reappearance of PAL activity after inactivation is stimulated rather than inhibited by cycloheximide. The change in effect of cycloheximide from inhibition to apparent stimulation can best be explained by the observation that (1) the turnover of PAL, both formation and inactivation, increases greatly as a result of culture on sucrose and (2) inactivation is more sensitive to cycloheximide than formation. Thus, even where an anomalous cycloheximide insensitive appearance of PAL activity occurs, a mechanism other than reactivation of the enzyme may be involved.     

Molecular control of rabbit follicular testosterone production. Role of protein and RNA after stimulation with luteinizing hormone.

The time and dose dependence of the relationship between uptake of labelled precursors into protein and RNA and production of testosterone by rabbit follicles was examined. Although testosterone production was stimulated by luteinizing hormone at concentrations between 0.1 and 10 microgram/ml, the uptake of [3H]leucine into protein was significant only when the concentration of luteinizing hormone was greater than 2.5 microgram/ml. Increased production of testosterone was observed within 15 min of stimulation with luteinizing hormone whereas uptake of [3H]leucine was only significant at 90 min. Puromycin (40 microgram/ml) and cycloheximide (10 microgram/ml) in the presence of luteinizing hormone inhibited the synthesis of both testosterone and protein. However, lower concentrations of puromycin (0.1, 1 and 10 microgram/ml) and cycloheximide (1 microgram/ml) had no effect on luteinizing hormone-induced testosterone production but significantly inhibited protein synthesis by 58, 37, 31 and 71%, respectively. Actinomycin D (20, 80 and 160 microgram/ml) alone and in combination with 5 microgram luteinizing hormone/ml severely inhibited uptake of [3H]uridine into RNA without affecting testosterone production. However, with 1 microgram actinomycin/ml, testosterone production was significantly (P less than 0.01) greater than in the presence of luteinizing hormone alone. These results cast doubt on the obligatory role of RNA and protein synthesis in rabbit ovarian follicular steroidogenesis.     

Incorporation of C-Leucine into Apple Leaf Protein and Its Inhibition by Protein Synthesis Inhibitors during Growth and Senescence

Of the total ¹⁴C-leucine taken up by intact apple (Pyrus malus L., Golden Delicious) leaf discs, 44 to 62% is incorporated into protein from June to early October. Of this amount, an average of 35% is released by mild, room temperature acid hydrolysis. Prior to mid-August when leaf protein begins to decline, 15 to 20% of the ¹⁴C-leucine incorporated into protein occurs in water-(buffer) soluble protein, of which only 3% is released by mild acid hydrolysis. After mid-August, 40% of the label in protein occurs in soluble protein. The specific radio-activity of the soluble protein increases by 4- to 5-fold after mid-August, while that of total protein increases by less than 2-fold. In presenescent leaves (before the decline of protein in August) 20 micrograms per milliliter cycloheximide inhibits the incorporation of ¹⁴C-leucine into protein by 71%, and 20 micrograms per milliliter chloramphenicol inhibits it by 30%. In senescing leaves, cycloheximide inhibits ¹⁴C-leucine by 85% or more, while chloramphenicol inhibits it by less than 15%. Coincident to the initial decline of leaf protein, chloramphenicol greatly loses its ability to inhibit the incorporation of ¹⁴C-leucine into apple leaf protein. At all leaf ages, chloramphenicol increases the loss of chlorophyll from apple leaf discs. The effect of cycloheximide on leaf disc senescence changes with leaf age: in early season samples, it increases the loss of chlorophyll; in mid-season samples, it has no effect; and in late season samples, it retards the loss of chlorophyll.     

Inhibition of hepatocytic protein degradation by methylaminopurines and inhibitors of protein synthesis

Nutritional control of protein degradation in isolated rat hepatocytes can take place in the absence of protein synthesis. Suppression of degradation by amino acids (step-up) is unaffected and the enhanced degradation seen upon amino acid deprivation (step-down) is only partially inhibited by cycloheximide at a concentration (10^?3 M) which inhibits protein synthesis virtually completely. Protein degradation per se is, however, inhibited by cycloheximide as well as by puromycin, apparently at least in part by mechanisms additional or unrelated to their effect on protein synthesis. Several puromycin analogues (methylaminopurines) are stronger inhibitors of protein degradation than of protein synthesis, most notably puromycin aminonucleoside and 6-dimethylaminopurine riboside (N⁶, N⁶-dimethyladenosine). The latter compounds appear to specifically inhibit cellular autophagy, since neither the degradation of endocytosed protein (asialofetuin) nor the extralysosoma (amino acid-, propylamine- and leupeptin-resistant) degradation are affected.     

Control of the yeast cell cycle by protein synthesis

The increased synthesis of ribosomal RNA (rRNA) is correlated with enhanced cell proliferation, and it has been suggested that rRNA metabolism may have a regulatory role in the progression of the cell cycle. Alternatively, it might be the ensuing more active protein synthesis that drives the cell cycle progression. We have found that treatment with low doses of cycloheximide dissociates rRNA and protein synthesis. In fact, after the addition of cycloheximide the protein synthesis rate is strongly inhibited, whereas the rate of rRNA synthesis is unaffected for some time. The progression of the cell cycle, monitored as analysis of DNA distribution by flow cytometry and as bud emergence, is quickly and largely inhibited, thus indicating that a sustained rRNA metabolism is not sufficient to allow continuous cycle progression. The effects of cycloheximide on the daughter and mother duplication times, on the mean cell volume, and on the volume at budding were also analyzed. The results suggest that protein synthesis, rather than rRNA synthesis, may have a key role in the control of cell cycle progression in Saccharomyces cerevisiae.