Estimated Drainage of Carbon from the Tricarboxylic Acid Cycle for Protein Synthesis in Suspension Cultures of Paul's Scarlet Rose Cells

The amount of carbon (μmoles of carbon atoms) drained from the tricarboxylic acid cycle for protein synthesis was compared with μmoles of CO₂ released from the cycle at 2-day intervals during the growth of suspension cultures of Paul's Scarlet rose. We concluded that during the period of most rapid protein synthesis (day 0-4) one-sixth as much carbon was drained from the tricarboxylic acid cycle for protein synthesis as was released as CO₂. By day 8, one-thirtieth of the amount of carbon released as CO₂ was incorporated into protein. Net protein synthesis stopped on day 8, but the evolution of CO₂/culture continued at its maximum rate until day 10.     

Metabolism of Glutamate in Suspension Cultures of Paul's Scarlet Rose Cells

When specifically labeled glutamate-1-¹⁴C was provided to 4-day-old rose cells, 87.6% of the ¹⁴C in glutamate recovered from protein was in the number 1 carbon atom of the glutamate molecule. It was concluded that newly absorbed glutamate was incorporated directly into protein without any prior metabolism.     

Changes in Cell Walls Associated with Cell Separation in Suspension Cultures of Paul's Scarlet Rose

Studies of the formation and separation of cellular aggregatesin Paul's Scarlet rose suspension cultures have been conductedin an attempt to resolve the basis for intercellular adherence.During the typical growth cycle, cellular aggregates begin tofragment spontaneously at about day 12 in culture. This fragmentationis accompanied by a sharp decrease in the galactose contentof wall polysaccharides. At approximately this same time, thereis an increase in ß-glucosidase and ß-galactosidaseactivity associated with the cell surface. The presence of aß-glucosidase inhibitor, nojirimycin, inhibits thesurface ß-glycosidase activity, reduces the extentto which cell wall galactose is decreased, and prevents theseparation of cells. No other cell wall component examined changesin a manner consistent with cell separation. The results suggestthat a galactose-containing wall polysaccharide may be involvedin the maintenance of intercellular coherency and that partialremoval of this component may be causally related to the spontaneousseparation of cells in culture.     

Ammonium Influence on the Growth and Nitrate Reductase Activity of Paul's Scarlet Rose Suspension Cultures

Suspension cultures of Paul's Scarlet rose were grown in two defined media which differed only in their inorganic nitrogen content. Both possessed equal amounts of NO(3) (+) (24 mm), but differed in that NH(4) (+) (0.91 mm) was present in control medium; whereas, no NH(4) (+) was present in the test medium. A comparison of fresh weight increases over a 14-day growth period showed that NH(4) (+) caused a 2-fold stimulation in growth and governed the pattern of development.Ammonium also caused a 2-fold increase in nitrate reductase activity but had little influence on the activity of representative enzymes from the Embden-Meyerhof pathway or citric acid cycle. Thus NH(4) (+) enhanced the nitrate reductase activity which was correlated with increased growth.Ammonium had no influence on the in vitro activity of nitrate reductase which suggested that the stimulatory influence was due to an increased synthesis of the enzyme. The enhanced synthesis did not appear to be due to an increased availability of NO(3) (+) since the uptake of NO(3) (+) by intact cells was not influenced by the presence of NH(4) (+) during the period of most rapid increase in nitrate reductase activity.     

Isolation and Identification of the Phenols of Paul's Scarlet Rose Stems and Stem-Derived Suspension Cultures

The phenols of Paul's Scarlet rose stems and stem-derived cell cultures have been analyzed using C₁₈-reversed-phase high performance liquid chromatography.

Rose stems were found to contain gallic acid, (+)catechin, (−)epicatechin, the dimers (−)epicatechin-(+)catechin and (+)catechin-(+)catechin, a polymeric procyanidin, ferulic acid, and several gallotannins. In contrast, a cell suspension of Paul's Scarlet rose which has been maintained in culture for over 25 years contained only low levels of gallic acid and (−)epicatechin-(+)catechin. The phenol content of a second rose cell line which was started from the same initial isolate in 1957, but which was maintained in a laboratory other than our own was quantitatively and qualitatively similar to the cell line kept in our laboratory for the last 20 years. A third cell line which we started 6 months ago contained a wide variety of phenols, most of which were in common with those of rose stems.

Selective subculturing of smaller cell clumps of our oldest cell line failed to enhance either the quantities or the diversity of phenols which accumulated in these cultured cells. Possible reasons for the failure of selective subculturing to enhance phenol levels in this long-established cell line are discussed.

     

Influence of Protein Synthesis on NO(3) Reduction, NH(4) Accumulation, and Amide Synthesis in Suspension Cultures of Paul's Scarlet Rose

Changes in the concentrations of NH₄⁺ and amides during the growth of suspension cultures of rose (Rosa cv. Paul's Scarlet) cells were examined. When cells were grown in medium possessing only NO₃⁻ as a nitrogen source, the concentrations of NH₄⁺ and amides increased to 4.0 × 10⁻¹ and 5.9 micromoles per gram fresh weight, respectively. The amounts of both constituents declined during the later stages of growth. When a trace amount of NH₄⁺ was added to the NO₃⁻ base starting medium, the concentration of NH₄⁺ in the cells was increased to 7.0 × 10⁻¹ micromoles per gram fresh weight.     

Some Aspects of Growth and Metabolism of Paul's Scarlet Rose Cell Suspensions

The growth pattern of suspension cultures of Paul’s Scarletrose cells has been examined. No distinct phases of cell divisionand cell expanison could be recognized. The 2-day lag phaseobserved after inoculation of stationary-phase cells into freshmedium was followed by a rapid entry into exponential growthduring which a mean cell-generation time of 36 h was recorded.Metabolic development assessed in terms of respiratory activityand RNA, DNA, and protein accumulation has been related to thephases of the growth cycle. A high metabolic activity developsimmediately after inoculation reaching a peak by early-exponentialphase. This activity sustains exponential growth until factorsin the medium become limiting. The pattern of DNA accumulationis closely correlated with increase in cell number and freshweight whereas changes in RNA levels are accompanied by similarchanges in protein levels and respiratory activity. The accumulationof phenolics follows a pattern unlike those of the other parametersexamined.     

Methylation of ribosomal RNA in growing and resting cultures of Paul's Scarlet Rose

The role of methylation of rRNA on the differential rate of ribosome accumulation was studied in exponentially growing and resting cultures of rose cells. The cells were labelled with [³H] uridine and pre-rRNA and rRNA were prepared from the nuclei and ribosomes. The results demonstrate that in resting cultures, in which the ribosome accumultaion was reduced, the pre-rRNA and rRNA were 50% and 75% less methylated respectively, than those of the growing cells.     

The Effect of Carbohydrate and Nitrogen Concentration on Phenol Synthesis in Paul's Scarlet Rose Cells Grown in Tissue Culture

The effects of exogenous concentrations of glucose and nitrate on total ethanol extractable phenols and leucoanthocyanins were studied in Paul's Scarlet Rose cells grown in either liquid suspension or solid culture. Aliquots of liquid suspension cultured cells were harvested during logarithmic, early stationary, and late stationary periods of growth for determination of fresh weight, dry weight, total ethanol extractable phenols and leucoanthocyanins. Cells produced phenols during all phases of growth, but at stationary phase, the production was greatest. Increasing concentrations of exogenous glucose in the culture medium resulted in an increased synthesis of total phenols in logarithmic cells, and an increased synthesis of total phenols and leucoanthocyanin in stationary cells. Addition of increased concentrations of exogenous nitrate to the stationary cells grown in suspension culture markedly reduced synthesis of leucoanthocyanins although total phenol synthesis was not significantly affected. Similar observations were made in cells cultured on solid medium in respect to exogenous glucose concentration, however these cells differed from the suspension cultured cells by having increased amounts of total phenol synthesis and decreased synthesis of leucoanthocyanins in the presence of increasing concentrations of exogenous nitrate in the culture medium.     

Influence of Ammonium on the Growth and Development of Suspension Cultures of Pauľs Scarlet Rose

In this work as in previous studies from this laboratory it was demonstrated that the presence of a trace amount of NH₄⁺ (72.8 μmol) stimulated the growth of Pau?s Scarlet Rose on a defined medium containing NO₃^? (1920 μmol) as the only other source of nitrogen. A kinetic analysis of several growth parameters showed that the rate of increase of dry weight, fresh weight, cell number, and cell volume were greater during early stages of growth (days 0–8) when NH₄⁺ was provided. During later stages (days 8–14) this relationship between the two cultures did not hold. The cells provided NH₄⁺ continued to increase in fresh weight and cell volume, but the cells which were not provided NH₄⁺ had a greater rate of dry weight and cell number increase. These differences led to 14-day-old cultures which were approximately equal in dry weight and cell number but differed by a factor of 2 in fresh weight. The presence of NH₄⁺ speeded up the development and growth of the cells.     

Nitrate, Ammonium and Kinetin Effects on Growth and Enzyme Activities of Paul's Scarlet Rose Callus

Factorial experiments using the three variables nitrate, ammonium, and kinetin at six different concentrations each (nitrate 4.64 to 215 mM; ammonium 2.15 to 100 mM; and kinetin 0.1 to 4.64 mg/l) were set up to measure the effects of each of these factors, and their interactions, on the fresh weight, protein, and enzyme activities of callus of Paul's Scarlet Rose. Optimum fresh weight values were obtained with nitrate at 46.4 mM. Ammonium inhibited growth at concentrations above 2.15 mM, and kinetin had no significant effect. Significant interaction between nitrate and ammonium effects on growth was found. Kinetin did not interact significantly with either nitrate or ammonium to influence the fresh weight. The specific activity of glutamate dehydrogenase (NAD) in the aminating reaction increased with increasing ammonium concentrations to 21.5; at higher concentrations the activity remained high. Glutamine synthetase specific activity was constant over a large range of nitrate and ammonium concentrations, increasing only when nitrate went from 46.4 to 100 mM. Glutamine synthetase was sensitive to the nitrate: ammonium interaction. Specific activity decreased at progressively higher ammonium levels when nitrate concentration increased. No glutamate synthase activity was detected at optimal nitrate concentrations.     

Root Carbon Dioxide Fixation by Phosphorus-Deficient Lupinus albus (Contribution to Organic Acid Exudation by Proteoid Roots)

When white lupin (Lupinus albus L.) is subjected to P deficiency lateral root development is altered and densely clustered, tertiary lateral roots (proteoid roots) are initiated. These proteoid roots exude large amounts of citrate, which increases P solubilization. In the current study plants were grown with either 1 mM P (+P-treated) or without P (-P-treated). Shoots or roots of intact plants from both P treatments were labeled independently with 14CO2 to compare the relative contribution of C fixed in each with the C exuded from roots as citrate and other organic acids. About 25-fold more acid-stable 14C, primarily in citrate and malate, was recovered in exudates from the roots of -P-treated plants compared with +P-treated plants. The rate of in vivo C fixation in roots was about 4-fold higher in -P-treated plants than in +P-treated plants. Evidence from labeling intact shoots or roots indicates that synthesis of citrate exuded by -P-treated roots is directly related to nonphotosynthetic C fixation in roots. C fixed in roots of -P-treated plants contributed about 25 and 34% of the C exuded as citrate and malate, respectively. Nonphotosynthetic C fixation in white lupin roots is an integral component in the exudation of large amounts of citrate and malate, thus increasing the P available to the plant.     

Water Conservation in Kalanchoe blossfeldiana in Relation to Carbon Dioxide Dark Fixation

The succulent Kalanchoe blossfeldiana v. Poel. var Tom Thumb was treated on long and short photoperiods for 6 weeks during which short day plants developed thicker leaves, flowered prolifically, and exhibited extensive net dark fixation of carbon dioxide. In contrast, long day plants remained vegetative and did not develop thicker leaves or exhibit net carbon dioxide dark fixation. When examined after the photoperiodic state described, long day plants showed approximately three times more water loss over a 10-day period than short day plants. Water loss is similar during light and dark periods for short day plants but long day plants exhibited two times more water loss during the day than at night. The latter plants also lost three and one-half times more water during the light period than short day plants. The water conservation by short day plants is correlated with conditions of high carbon dioxide dark fixation and effects of its related Crassulacean acid metabolism on stomatal behavior.     

不同培养方式下dCO_2对黑曲霉发酵产糖化酶的影响

通过在进气中混入部分CO_2气体,在分批培养和恒化培养两种培养方式下研究了不同dCO_2水平对黑曲霉产糖化酶的影响。在分批培养方式下,较高的dCO_2对细胞的生长具有抑制作用,并且随着dCO_2水平的增加,抑制程度加强,但是较高浓度的dCO_2对糖化酶的合成有利。而恒化培养时,低稀释率D_1=0.05/h下,较高的dCO_2对细胞的生长并没有明显的抑制作用,但有利于糖化酶合成。高稀释率D_2=0.08/h下,较高的dCO_2对细胞的生长有明显的抑制作用,并且抑制程度随着dCO_2水平的增加而加大。以上实验结果表明,dCO_2对细胞生长和糖化酶合成的影响不仅和dCO_2水平有关,也和培养方式及细胞所处的特定代谢状态有关。这对于黑曲霉产糖化酶工业放大过程中补料分批发酵中比生长速率的设计具有很好的指导作用。     

Protein synthesis during the transition from the resting to the growing state in suspension cultures of Paul's Scarlet rose cells

Cells derived from Paul's Scarlet rose ( Rosa sp. ) were grown in the chemically defined medium of Nesius. When a stationary phase culture was diluted with fresh medium, growth was initiated after a pronounced lag period. DNA replication, as revealed by thymidine labeling and autoradiography, did not begin until 36 h, and mitotic figures were not observed until 48 h after dilution. A 10–15 fold increase in the rate of protein synthesis occurred during the lag period. This was brought about by a 3.5 fold increase in the amount of ribosomal RNA per cell, plus a doubling of both the percentage of ribosomes that are present as polyribosomes and the average number of ribosomes per polyribosome. The spectrum of polypeptides synthesized by these cells during the lag and early log periods of growth was examined. Polyribosomes were extracted from the cells at intervals preceding and accompanying the initiation of proliferative growth. The polyribosomes were translated in a wheat germ cell-free protein synthesizing system and the ³⁵S-methionine-labeled translation products were separated on polyacrylamide slab gels and by 2-dimensional gel electrophoresis. Comparatively few differences were observed between stationary phase, lag phase and log phase cells in terms of the spectrum of polypeptides synthesized in vitro. However, these various phases of the growth cycle could be characterized by a relatively high rate of synthesis of a few specific polypeptides. That is, while most proteins are synthesized throughout the growth cycle and even in non-growing cells at approximately the same relative rates, there are a few variable proteins whose synthesis marks a particular phase of the growth cycle.