Trehalose Toxicity in Cuscuta reflexa: Cell Wall Synthesis Is Inhibited upon Trehalose Feeding

α,α-Trehalose induced a rapid blackening of the terminal 2.5-centimeter region of excised Cuscuta reflexa Roxb. vine. The incorporation of radioactivity from [¹⁴C]glucose into alkali-insoluble fraction of shoot tip was markedly inhibited by 12 hours of trehalose feeding to an excised vine. This inhibition was confined to the apical segment of the vine in which cell elongation occurred. The rate of blackening of shoot tip explants was hastened by the addition of gibberellic acid A₃, which promoted elongation growth of isolated Cuscuta shoot tips. The symptom of trehalose toxicity was duplicated by 2-deoxyglucose, which has been shown to be a potent inhibitor of cell wall synthesis in yeast. The observations suggest that trehalose interferes with the synthesis of cell wall polysaccharides, the chief component of which was presumed to be cellulose.     

Utilization of nucleoside diphosphate glucoses in developing cotton fibers

The capacity for biosynthesis of hot alkali-insoluble products using uridine diphosphate (UDP)-glucose and guanosine diphosphate (GDP)-glucose as substrate has been studied in isolated cotton fibers harvested at various stages of development following anthesis. During the period of rapid elongation and primary wall synthesis (7-14 days postanthesis), incorporation of radioactivity from GDP-¹⁴C-glucose into hot alkali-insoluble product is high. This activity gradually declines and is not demonstrated in older fibers undergoing active deposition of secondary wall. With respect to all characteristics examined, the product from GDP-glucose resembles cellulose. Incorporation of UDP-¹⁴C-glucose into hot alkali-insoluble product was low in young fibers but increased to high levels in older fibers. This product was shown to be soluble in chloroform-methanol, and when chromatographed in lipid solvents it was separated into three components. Activity for the production of two of these three presumed glucolipids increased with increasing age of fibers.     

Effects of gibberellic Acid, calcium, kinetic, and ethylene on growth and cell wall composition of pea epicotyls

The influence of gibberellic acid (GA), calcium, kinetin, and ethylene on growth and cell-wall composition of decapitated pea epicotyls (Pisum sativum L. var. Alaska) was investigated. Calcium, kinetin, and ethylene each caused an inhibition of GA-induced elongation of pea stems. Gibberellic acid did not reverse the induction of swelling by Ca²⁺, kinetin, or ethylene. Both Ca²⁺ and ethylene significantly inhibited the stimulatory effects of GA on the formation of residual wall material. Although GA promoted the development of walls relatively low in pectic substances and pectic uronic acid, Ca²⁺, kinetin, and ethylene favored the formation of walls rich in these constituents. Calcium, kinetin, and GA, alone or in combination, had no effect on the production of ethylene by pea epicotyls.     

Requirement of sodium chloride for the action of gibberellic acid in stimulating hypocotyl elongation of a halophyte, Salicornia herbacea L.

NaCl stimulated hypocotyl elongation of the halophyte Salicorniaherbacea L. grown either in light or dark. Its optimal concentrationwas around 0.1–0.2 M and its promoting effect was muchmore prominent in the dark. Gibberellic acid at 10^–5 Mstimulated hypocotyl elongation in light but not in the dark.Indole-3-acetic acid and kinetin were ineffective in promotinghypocotyl elongation. In light, gibberellic acid and NaCl synergisticallyenhanced hypocotyl elongation when both were given simultaneously.The action of NaCl could be replaced by KCl, but not by mannitol.Osmotic pressure of the epidermis of the Salicornia hypocotylincreased in response to gibberellic acid and/or NaCl treatment.Na⁺ content in the hypocotyl increased with NaCl application.Gibberellic acid and NaCl when given alone increased the extensibilityof the hypocotyl cell wall. Synergistic interaction in increasingthe extensibility was observed between gibberellic acid andNaCl. Stress-relaxation analysis of mechanical properties ofthe hypocotyl wall revealed that gibberellic acid and NaCl actedsynergistically in decreasing minimum relaxation time. Basedon these results, a possible mechanism by which gibberellicacid and NaCl regulate hypocotyl elongation of Salicornia herbaceaL., a typical halophilic plant, is discussed. ¹ Present address: Laboratory of Biology, Tezukayama College,Gakuen Minami, Nara 631, Japan. (Received June 13, 1978; )     

On the nature of the physiological responses of Avena stem segments to gibberellic Acid treatment

Gibberellic acid was found to cause elongation in Avena sativa (oat) stem segments whether it was applied continuously or as a short pulse. The shorter the pulse time became, the higher was the gibberellic acid concentration needed to cause elongation; the segmental growth apparently depends upon the amount of gibberellic acid taken up by the segments. Avena segments showed a decreased growth response to gibberellic acid if the treatments were initiated at increasingly later times after excision from the plant. This decreased responsiveness to gibberellic acid was inhibited by low temperature (0-4 C), but accelerated by anaerobiosis. On the other hand, growth stimulation by a gibberellic acid pulse at the start of incubation was not altered by cold treatment but was nullified by a nitrogen atmosphere. Both the readiness of the segments for growth stimulation by gibberellic acid and its action in promoting growth clearly involve temperature-dependent, aerobic metabolism.     

Effects of Growth Regulators on Ribonucleic Acid Metabolism of Barley Leaf Segments

Illumination or gibberellic acid treatment of etiolated barley leaf segments stimulates unrolling and results in an increased level of RNA. In contrast, segments treated with abscisic acid do not unroll and have a lower content of RNA. Gibberellic acid treatment enhanced the capacity of segments to incorporate radioactivity from ³²P-orthophosphate into all the RNA components detected by gel electrophoresis; abscisic acid greatly restricted the incorporation of precursors into all the RNA fractions. In conjunction with a changed capacity for RNA synthesis it was observed that abscisic acid-treated segments had a lowered soluble DNA-dependent RNA polymerase level in comparison to gibberellic acid-treated or illuminated segments. However, the influence of growth regulators on RNA polymerase content of the segments was associated with general effects on protein level rather than a specific effect on the synthesis of polymerase enzyme.     

A Correlation between a Ribonucleic Acid Fraction Selectively Labeled in the Presence of Gibberellic Acid and Amylase Synthesis in Barley Aleurone Layers

The effects of gibberellic acid on the incorporation of radio-active uridine and adenosine into RNA of barley aleurone layers were investigated using a double labeling method combined with acrylamide gel electrophoresis. After 16 hours of incubation, gibberellic acid stimulated the incorporation of label into all species of RNA, but the effects were very small (0-10%) for ribosomal and transfer RNA and comparatively large (up to 300%) for RNA sedimenting between 5S and 14S. This result was obtained for both isolated aleurone layers and for layers still attached to the endosperm. A similar but less marked pattern occurred in layers incubated for 8 hours, but the effect was not observed after 4 hours. The gibberellic acid-enhanced RNA labeling was not due to micro-organisms. The following evidence was obtained for an association between the gibberellic acid-enhanced RNA synthesis and α-amylase synthesis: (a) synthesis of α-amylase took place in parallel with incorporation of label into gibberellic acid-RNA; (b) actinomycin D inhibited amylase synthesis and gibberellic acid-RNA by similar percentages; (c) 5-fluorouracil halved incorporation of label into ribosomal RNA but had no effect on amylase synthesis and gibberellic acid-RNA; and (d) abscisic acid had little effect on synthesis of RNA in the absence of gibberellic acid, but when it was included with gibberellic acid the synthesis of both enzyme and gibberellic acid-RNA was eliminated. We conclude that large changes in the synthesis of the major RNA species are not necessary for α-amylase synthesis to occur but that α-amylase synthesis does not occur without the production of gibberrellic acid-RNA. Gibberellic acid-RNA is probably less than 1% of the total tissue RNA, is polydisperse on acrylamide gels, and could be messenger species for α-amylase and other hydrolytic enzymes whose synthesis is under gibberellic acid control.     

Regulation of glucose metabolism and cell wall synthesis in Avena stem segments by gibberellic Acid

Gibberellic acid (GA) stimulated both the elongation of Avena sativa stem segments and increased synthesis of cell wall material. The effects of GA on glucose metabolism, as related to cell wall synthesis, have been investigated in order to find specific events regulated by GA. GA caused a decline in the levels of glucose, glucose 6-phosphate, and fructose 6-phosphate if exogenous sugar was not supplied to the segments, whereas the hormone caused no change in the levels of glucose 6-phosphate, fructose 6-phosphate, UDP-glucose, or the adenylate energy charge if the segments were incubated in 0.1 m glucose. No GA-induced change could be demonstrated in the activities of hexokinase, phosphoglucomutase, UDP-glucose pyrophosphorylase, or polysaccharide synthetases using UDP-glucose, UDP-galactose, UDP-xylose, and UDP-arabinose as substrates. GA stimulated the activity of GDP-glucose-dependent β-glucan synthetase by 2- to 4-fold over the control. When glucan synthetase was assayed using UDP-glucose as substrate, only β-1,3-linked glucan was synthesized in vitro, whereas with GDP-glucose, only β-1,4-linked glucan was synthesized. These results suggest that one part of the mechanism by which GA stimulates cell wall synthesis concurrently with elongation in Avena stem segments may be through a stimulation of cell wall polysaccharide synthetase activity.     

The priming effect of glucose in soil sterilized by γ-radiation and reinoculated withCellulomonas sp.

Mineralization of native organic matter andU-¹⁴C-glucose was followed by measuring the formation of CO₂ and its radioactivity in chernozem soil samples presterilized by γ-radiation and inoculated with a washed suspension ofCellulomonas sp. cells. The introduced bacteria mineralized the soil organic component to a higher extent in variants enriched with glucose. This so-called priming effect of glucose was observed also in the presence of chloramphenicol, inhibiting the growth of the bacteria. The increased mineralization of the native soil organic fraction was also detected in samples that were not enriched with glucose when the bacterial suspension was first disintegrated ultrasonically and the material then used for the inoculation. Possible participation of phenomena of the type of cometabolism and activation of cell membrane transport mechanisms on the occurrence of the priming effect of glucose in the soil is discussed.     

Effect of Abscisic Acid on the Activities of Photosynthetic Enzymes and 14CO2 Fixation Products in Leaves of Pennisetum typhoides Seedlings

Abscisic acid inhibited the rate of ¹⁴CO₂ fixation in leaves of Pennisetum typhoides (Burm. f.) Stapf & Hubbard seedlings, but increased the activities of phosphoenol-pyruvate-carboxylase and malic enzyme. The leaves of the seedlings grown in the presence of abscisic acid incorporated, in comparison to the control, more radioactivity in the fraction of organic acids, but less radioactivity was recorded in the amino acid fraction. On the other hand, gibberellic acid which also inhibits photosynthetic ¹⁴CO₂ assimilation and decreases the activities of photosynthetic enzymes, favours greater incorporation in alanine, and reduces that in malate. It is deduced that bio-regulants can greatly influence the flow of ¹⁴C into individual photosynthetic products. As in growth, abscisic and gibberellic acids in combination tended to antagonize each other in their effects on enzyme activity as well as in incorporation of ¹⁴CO₂ into photosynthetic products.     

Kinetin and carbohydrate metabolism in chinese cabbage

The effects of kinetin on starch and sugar levels and on ¹⁴CO₂ and ³²P-orthophosphate labeling patterns of floated Chinese cabbage (Brassica pekinensis) leaf discs were investigated. Kinetin caused gross starch degradation. Neutral sugars were depressed by 30 to 40% in leaf tissue treated with kinetin for 24 hours. ¹⁴CO₂ labeling of leaf discs pretreated with kinetin for 24 hours showed increased radioactivity in chloroform-soluble material and most sugar phosphates, and a 35 to 40% decrease in radioactivity in the neutral sugars, glucose, sucrose, and fructose. Incorporation into ATP was increased by 40% by kinetin. ³²P-Orthophosphate uptake was inhibited 30% by kinetin. When corrected for uptake, kinetin stimulated incorporation into chloroform-soluble material but had little effect on other cell fractions. These results indicate that kinetin mobilizes starch reserves and increases the flow of sugars required for the synthesis of lipids and structural materials in floated discs.     

The Conversion of d-Glucose-6-C to Cell Wall Polysaccharide Material in Zea mays in Presence of High Endogenous Levels of Myoinositol

When corn (Zea mays) roots are supplied with high concentrations of unlabeled myoinositol, the conversion of d-glucose-6-¹⁴C to cell wall galacturonic acid is significantly reduced compared to controls, although its incorporation into cell wall glucosyl units remains unchanged. This suggests that, in order to be converted to uronic acid, radiolabel from glucose must first pass through the internal myoinositol pool of the roots.     

Gibberellic Acid-Promoted Lignification and Phenylalanine Ammonia-lyase Activity in a Dwarf Pea (Pisum sativum)

The effects of gibberellic acid on lignification in seedlings of a dwarf and a tall cultivar of pea (Pisum sativum) grown under red or white light or in the darkness, were studied. Gibberellic acid (10⁻⁶-10⁻⁴ m) promoted stem elongation in both light and dark and increased the percentage of lignin in the stems of the light-grown dwarf pea. The gibberellin had no effect on the lignin content of the tall pea although high concentrations (10⁻⁴ m) promoted growth of the tall plants. Time course studies indicated that the enhanced lignification in the gibberellin-treated dwarf plants occurred only after a lag period of several days. It was concluded that gibberellic acid-enhanced ligmification had no direct relation to gibberellic acid-promoted growth. The activity of phenylalanine ammonia-lyase (E.C. 4.3.1.5) was higher in gibberellin-treated dwarf plants grown under white or red light than in untreated dwarf plants. Gibberellic acid had no detectable effect on the activity of this enzyme when the plants were grown in darkness, just as it had no effect on lignification under dark conditions. The data suggest that in gibberellin-deficient peas the activity of phenylalanine ammonia-lyase is one of the limiting factors in lignification.     

A chemical and autoradiographic study of cellulose synthesis during the encystment of Acanthamoeba castellanii

When cells of Acanthamoeba castellanii are placed in a non-nutrient medium, they differentiate into cysts which possess cellulosic walls. In the present study, the source of the glucosyl unit for cyst wall cellulose was investigated by following the encystment of trophozoites grown in the presence of ¹⁴C-labeled fatty acids (uniformly labeled palmitate or oleate) or [3-³H]glucose. Cells were fractionated at the beginning and after 30 hr of encystment using a modified Schmidt-Tannhauser procedure. In cells grown on fatty acids, 90% of the labeled material was in the lipid fractions both before and after encystment with the total amount of label/cell changing very little. Both partial and complete acid hydrolysis of the glycogen of the acidsoluble fraction and the alkali-insoluble residue of the cyst wall indicated that the glucose of both fractions was not radioactive, although Acanthamoeba is known to have a functional glyoxylate pathway.Fractionation data of cells grown on [³H]glucose indicated a sevenfold increase in radioactivity in the wall insoluble fraction and a fivefold decrease in the acid-soluble fraction with the cpm/cell of the other fractions changing very little after 30 hr of encystment. Approximately 70% of the ³H-labeled material was recovered as glucose from the 30-hr wall insoluble fraction following complete acid hydrolysis. The specific radioactivity of glucose in the cyst wall insoluble fraction was the same as that of glycogen glucose isolated from the acid soluble fraction of trophozoites. Electron microscopic autoradiography showed that the majority of nonlipid radioactivity was due to glycogen in trophozoites. Autoradiograms failed to reveal Golgi bodies or any particular region of the cell as being the specialized site of cellulose synthesis. The results of the fractionation and autoradiographic studies are consistent with the concept that glycogen is a precursor of cyst wall cellulose, and that glucosyl units of glycogen and/or other glucose derivatives are converted to cellulose without significant dilution under the experimental conditions used.     

Gibberellin estimation and biosynthesis in germinating Hordeum distichon

Gibberellic acid (GA₃) and 13-deoxy-gibberellic acid (GA₇) were identified in extracts of germinating barley as their ¹⁴C-methyl esters. The maximal level of GA₃ was estimated by an isotopic dilution procedure to be 1·5 ng per grain. Germinating barley incorporated 2-¹⁴C-mevalonic acid into several terpenes, whose specific radioactivities were measured, but incorporation into GA₃ could not be detected. Cell-free embryo extracts from germinating barley converted 2-¹⁴C-mevalonic acid into isopentenol, dimethylallyl alcohol, farnesol and squalene, while ¹⁴C-isopentenyl pyrophosphate was incorporated into geraniol, farnesol, geranylgeraniol and squalene. There was no detectable incorporation into the gibberellin intermediate ent-kaurene.     

Incorporation de phenylalanine,de glucose et d'acide cinnamique dans les flavonols de l'oignon

In Allium cepa bulb scales, incorporation of ¹⁴C-phenylalanine, cinnamic acid and glucose were studied in relation to flavonol synthesis. The best incorporation into flavonols is obtained with either cinnamic acid or phenylalanine. ¹⁴C-glucose gives a slow incorporation into flavonol aglycone; this is because there is a big pool of free glucose in the scales in which the precursor is diluted. Under certain conditions, free cinnamic acid is quickly incorporated in a complex which may be a glycoside. After short labelling experiments with phenylalanine or cinnamic acid, some free precursor can be found in the scales a few days later but it is not available for flavonol synthesis. In these conditions, flavonol analysis shows in some cases, no turnover and in others, a turnover of 10% per day due to catabolism.     

Polysaccharidmetabolismus in den Zellwänden wachsender Keimlinge von Phaseolus aureus

Summary Quantitative determinations of the cell wall constituents (pectin, hemicellulose and -cellulose) of growing Phaseolus aureus seedlings showed marked changes during early growth. The cell walls of the 2 to 4 days old seedlings were composed of approximately 30% -cellulose, 50% hemicelluloses and 20% pectin. After four weeks the proportion of the different fractions had changed to approximately 60% -cellulose, 30% hemicelluloses and 10% pectin. Quantitative sugar determinations on these polysaccharide fractions have shown that mainly the non-cellulosic fractions (hemicelluloses and pectin) underwent considerable changes in sugar composition during growth. The hemicelluloses contained non-cellulosic polysaccharides with a high glucose content, which were not starch. These were broken down in the cell walls during growth.In a series of experiments in which ¹⁴C-glucose was injected into the hypocotyls of four days old Phaseolus aureus seedlings, the transport of radioactivity to the different plant organs and its incorporation into the cell wall polysaccharides of the bean stem were studied. The major part of the radioactivity was incorporated into the cell wall of the stem tissue. Minor amounts were transported to the roots and leaves. Of the cell wall polysaccharides of the stem, the hemicellulosic fraction showed a higher rate of incorporation of the ¹⁴C-glucose than the -cellulose in the early stages of growth. With increasing age of the plant, radioactivity was transferred from the hemicellulosic fraction to the -cellulose, suggesting turnover of polysaccharides in the growing cell wall.     

Role of an acidic compartment in synthesis of disaturated phosphatidylcholine by rat granular pneumocytes

A possible role for an acidic subcellular compartment in biosynthesis of lung surfactant phospholipids was evaluated with granular pneumocytes in primary culture. Incubation with chloroquine (100μm) was used to perturb this compartment. With control cells, incorporation of [9,10-³H]palmitic acid into total lipids and into total phosphatidylcholines increased linearly with time up to 4h. Total incorporation into phosphatidylcholine during a 1h incubation was 999+85pmol of [9,10-³H]palmitic acid, 458±18pmol of [1-¹⁴C]oleic acid and 252±15pmol of [U-¹⁴C]glucose per μg of phosphatidylcholine phosphorus. The cellular content of either disaturated phosphatidylcholine or total phosphatidylcholines did not change during a 2h incubation with chloroquine. In the presence of chloroquine, the specific radioactivity of [³H]palmitic acid in disaturated phosphatidylcholine increased by 40%, and that of disaturated-phosphatidylcholine fatty acids from [U-¹⁴C]glucose increased by 125%. Incorporation of [1-¹⁴C]oleic acid into phosphatidylcholine was decreased by chloroquine by 79% and 33% in the presence or absence of palmitic acid respectively. Chloroquine stimulated phospholipase activity in intact cells, and in sonicated cells at pH4.0, but not at pH8.5. The observations indicate that chloroquine stimulates synthesis of disaturated phosphatidylcholine in granular pneumocytes from fatty acids, both exogenous and synthesized de novo, which can be due to stimulation of acidic phospholipase. This stimulation of acidic phospholipase A activity by chloroquine appears to be coupled to the synthesis of disaturated phosphatidylcholine, thereby enhancing remodelling of phosphatidylcholine synthesized de novo. Our findings, therefore, implicate the involvement of an acidic subcellular compartment in the remodelling pathway of disaturated phosphatidylcholine synthesis by granular pneumocytes.     

Regulation of pentosan biosynthesis in barley aleurone tissue by gibberellic acid

Summary Treatment of isolated barley aleurone layers with gibberellic acid (GA₃) resulted in a progressive inhibition of cell-wall synthesis after a 4-h lag period. The incorporation of both [¹⁴C]arabinose and [¹⁴C]glucose into the cell wall was inhibited by GA₃, but analysis of the labelled sugars in the polymerized product showed that the process most affected by the hormone treatment was pentosan biosynthesis. Labelling kinetics and pulse-chase analysis indicated that the pentosans were synthesized in the cytoplasm and subsequently transferred to the cell wall; GA₃ did not significantly affect the latter step. The GA₃-inhibited labelling of the cell-wall pentosans cannot be explained on the basis of an effect on uptake of radioactive cell-wall precursor, expansion of the free pentose pool, or degradation of newly-formed pentosan. GA₃ inhibited the activity of a membrane-bound arabinosyl transferase present in the aleurone layers. This inhibition may explain the inhibition of cell-wall pentosan synthesis by GA₃.Abbreviations GA gibberellin - GA₃ gibberellic acid     

Phosphorylated and nucleotide sugar metabolism in relation to cell wall production in Avena coleoptiles treated with fluroride and peroxyacetyl nitrate

Coleoptile sections of Avena sativa L. were pretreated with sodium fluoride or peroxyacetyl nitrate at levels which inhibit auxin-induced growth but did not affect glucose uptake or CO₂ production when postincubated for 30 minutes in a ¹⁴C-glucose medium without auxin. Labeling of metabolites involved in cell wall synthesis was measured. Peroxyacetyl nitrate decreased labeling, and it was concluded that the pool size of uridine di-phosphoglucose, sucrose, and cell wall polysaccharides decreased compared to control. The changes suggest that peroxyacetyl nitrate inactivated sucrose and cell wall synthesizing enzymes including cellulose synthetase and decreased cell growth by inhibiting production of cell wall constituents. Fluoride treatment had no effect on production of cell wall polysaccharides, with or without indoleacetic acid stimulation of growth. The only change after fluoride treatment was a decrease in uridine diphosphoglucose during incubation without indoleacetic acid, a decrease that disappeared when indoleacetic acid was present. It was concluded that some other aspect of cell wall metabolism, not determined here, was involved in fluoride-induced inhibition of growth.