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.     

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.

     

Contribution of Nonautotrophic Carbon Dioxide Fixation to Protein Synthesis in Suspension Cultures of Paul's Scarlet Rose

Bicarbonate-¹⁴C was provided to 5- and 11-day-old suspension cultures of Paul's Scarlet rose, and the incorporation of ¹⁴C into lipid, protein, amino acids, and organic acids was determined. The rate of bicarbonate uptake was approximately the same by 5- and 11-day-old cells, but the distribution of ¹⁴C among cell constituents was markedly different. In 5-day-old cells a larger proportion of the ¹⁴C entered protein, whereas in 11-day-old cells there was a greater tendency for ¹⁴C to accumulate in malate.     

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.     

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.     

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.     

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.     

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.     

Changes in Peroxidase Activity Associated with Cell Walls During Pine Hypocotyl Growth

Peroxidase active against 2,2'-azino-bis-[3-ethylbenzthiazoline-6-sulphonicacid] (ABTS) and guaiacol were found in the apoplastic fluid,as well as ionically and covalently associated with pine cellwalls. The highest activity was found covalently bound to cellwalls, while the lowest activity was in the apoplastic fluid.Both ABTS and guaiacol peroxidases increased with the hypocotylage in the three fractions, apoplastic, ionically and covalentlybound. Furthermore, the changes in both peroxidases along thehypocotyl were also studied. Both apoplastic ABTS- and guaiacol-peroxidasesincreased from the apical towards the basal region of the hypocotylsof 10-d-old seedlings. A relation between peroxidase activityin the apoplastic fluid and the cell wall stiffening in pinehypocotyls is proposed.Copyright 1995, 1999 Academic Press Cell wall, growth, hypocotyl, peroxidase, pine, Pinus pinaster Aiton     

怀牛膝细胞悬浮培养及多糖含量变化的研究

采用正交实验设计研究基本培养基、碳源、2,4-D、6-BA、CH及接种量对怀牛膝悬浮培养细胞生长和细胞中多糖含量的影响。结果表明,(1)6-BA是影响怀牛膝细胞生长的关键因子,其影响效应依次为6-BA>CH>接种量>基本培养基>2,4-D>碳源,细胞生长的适宜培养基为MS 30 g/L葡萄糖 2 mg/L 2,4-D 1 mg/L 6-BA 0.5 g/L CH,适宜接种量为30 g/L。(2)碳源是影响细胞中多糖含量的关键因子,其影响效应依次为:碳源>2,4-D>6-BA>CH>接种量>基本培养基,利于细胞中多糖含量提高的适宜培养基为:LS 30 g/L蔗糖 0.5 mg/L 6-BA 0.5 g/L CH,适宜接种量为30 g/L。     

Polyamines in Paul's Scarlet rose suspension cultures

Polyamine concentrations have been determined at intervals in suspension cultures of Paul's Scarlet rose cells during a culture period of 2 weeks. The mean concentrations of the putrescine, spermidine and spermine in the cells of the inocula were respectively 73, 70 and 13 nmol/g fresh weight. Putrescine at fitst increased with a peak (160 nmol/g) after 6 h, declined to a minimum (14 nmol/g) after 2–3 days, increased to a second peak (180 nmol/g) after 5–6 days, and then declined slowly to the concentration of the inoculum (taken on day 14). Spermidine rose slowly (×2.6) to a broad peak over 3–6 days (180 nmol/g), then declined slowly to the concentration in the inoculum. Spermine showed a rapid increase to a peak (130 nmol/g) after 2–3 days, and then declined rapidly, reaching the inoculum concentration by day 6. In one experiment the three amines showed a minor peak at day 11. Changes in spermine and RNA contents appeared to be correlated. DNA content reached a peak after that of the RNA (day 3) and did not appear to be correlated with the content of putrescine or the polyamines.     

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.     

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.     

Changes in protein and protease activity during the growth and senescence of Paul's Scarlet rose

A study was performed to determine the relationship between the protein content and protease activity in suspension cultures of rose (Rosa cv Paul's Scarlet) grown over a 30 day period. Protein levels and protease activity were calculated on a per culture and per cell basis. Older nongrowing 14 day-old cultures possessed the largest total protease activity, but the highest concentration of protease activity per cell was in young 4 day-old rapidly dividing cells.     

Callus and Cell Suspension Cultures of Carnation

Callus cultures of carnation, Dianthus caryophyllus L. ev. G. J. Sim, were grown on a synthetic medium of half strength Murashige and Skoog salts, 3 % sucrose, 100 mg/l of myo-inositol, 0.5 mg/l each of thiamin, HCl, pyridoxin, HCl and nicotinic acid and 10 g/l agar. Optimal concentrations of growth regulators were observed to be 3 × 10^?6M indoleacetic acid (JAA) combined with 3 × 10^?6M benzylaminopurin (BAP) or 10^?6M 2,4-dichlorophenoxy acetic acid (2,4-D) alone. IAA + BAP caused a 100 fold increase in fresh weight over 4 weeks at 25°C. Addition of casein hydrolysate increased growth further. Cell suspension cultures worked best in media containing 2,4-D in which they had a doubling time of about 2 days. Filtered suspensions were successfully plated on agar in petri dishes, but division was never observed in single cells. The cultures initiated roots at higher concentrations of IAA or NAA, but all attempts to induce formation of shoots or em-bryoids gave negative results.