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.     

Polyphenoloxidase from six mature grape varieties and their activities towards various phenols

Juices were prepared from three white and three red grape varieties harvested at full maturity and comparative studies on their oxygen-uptake, absorbance at 420 nm (degree of browning), polyphenoloxidase (EC 1.10.3.1; PPO) activity, and their phenol compositions were done. There was no correlation among the amounts of oxygen-uptake and oxidizable phenols in the juices and their degree of browning. However, there was similarity among the PPO from the six grape varieties in their general enzymatic properties and substrate specificity towards twenty-five phenols. A partially purified PPO fraction from Koshu juice, which did not contain free phenols, showed strong activity towards (+)-catechin, (−)-epicatechin, caffeic acid, catechol, pyrogallol, and protocatechuic acid (oxidizable phenols), but had no activity towards the other fifiteen phenols. The oxidizable substrates were not always the only limiting factor in the oxidation and browning of phenols by the PPO. Some unoxidizable phenols such as gallic acid, p-cresol, and tannic acid which were not substrates for PPO inhibited the oxidation of the oxidizable phenols except pyrogallol which was not inhibited by gallic acid. On the other hand, hydroquinone promoted the oxidation of the oxidizable phenols except protocatechuic acid. These showed that there were competitive reactions and synergism during the enzymatic oxidation of phenols.     

Relationship between Vital Staining and Subculture Growth during the Senescence of Plant Tissue Cultures

The vital staining properties of rose cultures (Rosa cv Paul's Scarlet) of increasing age were compared with their ability to be subcultured. At 4-day intervals beginning on day 14, after cell division and expansion had stopped, cells were stained separately with Evans blue, fluorescein diacetate, and phenosafranine. The degree to which parent cultures stained with each of these dyes was compared to the dry weight of their subcultures harvested after 9 and 21 days of growth.

Staining with either Evans blue or fluorescein diacetate was demonstrated to be a good means of establishing when senescing cells died. However, the staining properties of aging cultures did not correlate well with their ability to be subcultured, because an increasing proportion of the living cells appeared to lose their ability to divide as senescence progressed.

     

Gibberellin-sensitive Suspension Cultures

Suspension cultures were incubated in the presence and absence of gibberellic acid (GA₃) in an attempt to define a new experimental system for study of the molecular action of gibberellins upon growth. Unlike many suspension cultures, an auxin-independent green clone from spinach (Spinacia oleracea L.) and an auxin-dependent line of “Paul's Scarlet” rose (Rosa sp.) were promoted in expansion growth by GA₃ at 10⁻¹¹ to 10⁻⁶ molar. In Rosa the cells also elongated upon GA₃ treatment whereas in Spinacia they remained isodiametric.     

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.     

Minimal organic medium for suspension cultures of Paul's scarlet rose

Summary Maximum growth of suspension cultures of Paul's Scarlet rose has been demonstrated in a minimal organic medium possessing only 4 organic compounds: napthaleneacetic acid, kinetin, myo-inositol, and sucrose. Myo-inositol was not essential for growth, but sustained growth was reduced by 90% when it was omitted. Maximum growth required nitrate plus a supplemental amount of either NH ₄ ⁺ or glutamine.     

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.     

Acetate metabolism in cell suspension cultures

Cell suspension cultures of Paul's Scarlet rose were grown over a 14-day period, during which a 50-fold increase in fresh weight occurred. Three phases could be recognized from weight, DNA determinations, and microscopic examination. From days 0 to 7, cell division was accompanied by cell expansion; from days 7 to 10, only cell expansion occurred; and from days 10 to 14, there was no further growth.     

Phenolic Compounds and Antioxidant Activity of Different Organs of Potentilla fruticosa L. from Two Main Production Areas of China

This report compared the phenolic compounds and antioxidant activity of the leaves, flowers, and stems of Potentilla fruticosa L. collected from two main production areas of P. R. China (Taibai Mountains and the Qinghai Huzhu Northern Mountains). The results indicated that there were significant differences in the phenol contents and antioxidant activities among the different organs and between the two productions. High‐performance liquid‐chromatography analysis indicated that hyperoside, (+)‐catechin, ellagic acid, and rutin were the primary compounds in leaves and flowers; for stems, the content of six phenolic compounds, from two productions, were the lowest. The 1,1‐diphenyl‐2‐picryl hydrazyl (DPPH), 2,2‐azino‐bis(3‐ethylbenzothiazoline‐6‐sulfonic acid) di‐ammonium salt (ABTS), ferric reducing power (FRAP), lipid peroxidation assays, and microbial test system (MTS) were used to evaluate the antioxidant activity. The results demonstrated that the leaves from two productions exhibited powerful antioxidant activity than other organs, which did not significantly differ from that of the positive control (rutin), followed by the flowers and stems. The correlation between the content of phytochemicals and the antioxidant activities of different organs showed that the total phenol, tannin, hyperoside, and (+)‐catechin contents may influence the antioxidant activity, and these compounds can be used as markers for the quality control of P. fruticosa.     

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.     

广西不同产地金樱根及炮制品中没食子酸和儿茶素的含量差异分析

为了建立金樱根中没食子酸和儿茶素含量的测定方法,分析广西不同产地金樱根及炮制品中没食子酸和儿茶素含量的变化,该文以没食子酸和儿茶素的含量作为指标成分,采用HPLC法对广西产金樱根生品、炒炙品、酒炙品、盐炙品及醋炙品进行测定,并采用SPSS 23.0软件进行方差分析和聚类分析。结果表明：广西不同产地金樱根及炮制品中没食子酸和儿茶素含量均存在差异,所有样品中儿茶素的含量均比没食子酸高,南部地区(除贵港桂平外)的没食子酸和儿茶素含量整体上比北部地区高,在炮制品中醋炙后没食子酸和儿茶素含量最高。该研究表明HPLC测定方法简单可行,金樱根中没食子酸和儿茶素含量的变化差异主要表现为产地地域及炮制方法的不同,可为今后金樱根资源的合理利用、质量标准制定以及临床用药的研究提供一定科学依据。     

Densitometric Quantification and Optimization of Polyphenols in Phyllanthus maderaspatensis by HPTLC

Quantifying and optimizing the polyphenol content of Phyllanthus maderaspatensis was accomplished using a single-solvent HPTLC system. Analyzing hydroalcoholic extracts for kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid, we simultaneously quantified and optimized their concentration. In the experiment, the methanol to water ratio (%), temperature (°C), and time of extraction (min) were all optimized using a Box-Behnken statistical design. Kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid were among the dependent variables analyzed. In the HPTLC separation, silica gel 60F254 plates were used, and toluene, ethyl acetate, and formic acid (5:4:1) made up the mobile phase. For kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid, densitometric measurements were carried out using the absorbance mode at 254 nm. Hydroalcoholic extract of P. maderaspatensis contains rutin (0.344), catechin (2.62), gallic acid (0.93), ellagic acid (0.172), quercetin (0.0108) and kaempferol (0.06). Further, it may be affected by more than one factor at a time, resulting in a varying degree of reaction. A negative correlation was found between X1 (extraction time (min)) and X2 (temperature), as well as X1 and X3 (solvent ratios). Taking these characteristics into consideration, the method outlined here is a validated HPTLC method for measuring kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid.     

Procyanidins (Condensed Tannins) in Green Cell Suspension Cultures of Douglas Fir Compared with Those in Strawberry and Avocado Leaves by Means of C(18)-Reversed-phase Chromatography

The procyanidins (the most common type of proanthocyanidin or condensed tannin) from cell suspension cultures derived from cotyledons of Douglas Fir have been compared with those isolated from leaves of strawberry and avocado. Seventy per cent methanol (v/v) extracts from 100 milligrams fresh weight samples were analyzed by a combination of C₁₈-reversed-phase columns with high-performance liquid chromatography, and normal phase paper chromatography. (−)-Epicatechin and its oligomers were generally retarded longer on C₁₈ columns than the corresponding units made of (+)-catechin when eluted with solvents made up of 5% acetic acid alone or mixed with methanol up to 15% (v/v). Douglas fir preparations contained the most complex set of procyanidins and consisted of oligomers of catechin and epicatechin, whereas strawberry and avocado contained mainly (+)-catechin and (−)-epicatechin derivatives, respectively.     

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.     

Biosynthesis of Protein Amino Acids in Plant Tissue Culture II Further Isotope Competition Experiments Using Protein Amino Acids

The behavior of exogenously supplied alanine, proline, glutamine, glutamic acid and aspartic acid in isotope competition experiments using cells of Paul's Scarlet Rose grown in the presence of glucose-U-¹⁴C was examined at higher concentrations than were used in part I of this series.     

Structure-Activity Relationships in Microbial Transformation of Phenols

The second-order rate constants for the microbial transformation of a series of phenols were correlated with the physicochemical properties of the phenols. The compounds studied were phenol, p-methylphenol, p-chlorophenol, p-bromophenol, p-cyanophenol, p-nitrophenol, p-acetylphenol, and p-methoxyphenol. Phenol-grown cells of Pseudomonas putida U transformed these compounds. Microbial transformation rate constants ranged from (1.5 ± 0.99) × 10⁻¹⁴ liter · organism⁻¹ · h⁻¹ for p-cyanophenol to (7.0 ± 1.3) × 10⁻¹² liter · organism⁻¹ · h⁻¹ for phenol. Linear regression analyses of rate constants and electronic, steric, and hydrophobic parameters showed that van der Waal's radii gave the best coefficient of determination (r² = 0.956). Products identified by thin-layer chromatography and liquid chromatography indicated that the phenols were microbially oxidized to the corresponding catechols.     

Presence of a glycosylphosphatidylinositol lipid anchor on rose arabinogalactan proteins.

Arabinogalactan proteins constitute a class of plant cell surface proteoglycans with widespread occurrence and suggested functions in various aspects of plant growth and development, including cell proliferation, expansion, marking, and death. Previous investigations of subcellular fractions from suspension-cultured cells of "Paul's Scarlet" rose (Rosa sp.) have revealed extensive structural similarity between some soluble arabinogalactan proteins from the cell wall space and some plasma membrane-associated arabinogalactan proteins, thus inspiring the present investigation of the mechanism through which these inherently water-soluble molecules are held on the plasma membrane. Several lines of evidence gained through a combination of methods including reversed-phase chromatography, treatment with phosphatidylinositol-specific phospholipase C, and chemical structural analysis now show that some rose arabinogalactan proteins carry a ceramide class glycosylphosphatidylinositol lipid anchor. The predominant form of the ceramide is composed of tetracosanoic acid and 4-hydroxysphinganine. Plasma membrane vesicles readily shed arabinogalactan proteins by an inherent mechanism that appears to involve a phospholipase. This finding has significance toward understanding the biosynthesis, localization, and function of arabinogalactan proteins and toward stimulating other studies that may expand the currently very short list of higher plant proteins found to carry such membrane lipid anchors.     

Influence of cycloheximide on the synthesis and utilization of amino acids in suspension cultures

Cells from 4-days old suspension cultures of Paul's Scarlet rose were incubated with acetate-U-¹⁴C for 10 minutes. After washing, cells were incubated for 2 hours in growth medium in the presence and absence of cycloheximide. The ¹⁴C content of individual amino acids in the soluble form and in protein were determined at the end of the 10 minute pulse and at intervals thereafter in control cells and those treated with cycloheximide. During the period following the pulse there was a 3-fold increase in the ¹⁴C content of protein in control cells; no such increase occurred in the presence of cycloheximide.     

Synthesis of the phytoalexin pisatin by a methyltransferase from pea

Previous labeling studies in vivo suggest that the terminal step of (+)pisatin biosynthesis in Pisum sativum L. is methylation of the phenol (+)6a-hydroxymaackiain (HMK). We have found that extracts from pea seedlings perform this reaction, using S-adenosylmethionine as the methyl donor. The enzyme activity was induced by microbial infection or treatment with CuCl₂, which elicit pisatin synthesis, though some activity was also present in healthy tissues. It has been reported that CuCl₂-treated pea tissue provided with (−)HMK or (−)maackiain can synthesize (−)pisatin. Our extract showed no methyltransferase activity dependent on either of these substrates. Methylation of (+)maackiain was detectable, but much slower than that of (+)HMK.     

Nitrate reductase activity and growth in Paul's Scarlet rose suspension cultures in relation to nitrogen source and molybdenum

Growth and nitrate reductase activity were measured in Paul's Scarlet rose cell suspensions, cultured in media purified from molybdenum and containing nitrate or urea as sole nitrogen source with or without added Mo. Urea could replace nitrate to yield 80% of the fresh weight in nitrate medium. Nitrate reductase activities were compared by in vivo and in vitro assays. The latter varied due to inactivation during extraction. Compared with activities in cells in complete NO₃ ^- medium, activity in NO₃ ^--Mo cells was reduced to 30% and, in urea-grown cells, to trace amounts. Increases in nitrate reductase activity were found when NO₃ ^- alone was added to NO₃ ^- or urea+Mo cultures. In NO₃ ^--Mo cultures, Mo alone or with NO₃ ^- caused a similar increase in activity, whereas urea-Mo cultures required both NO₃ ^- and Mo for enzyme induction.Abbreviations FAD flavin adenine dinucleotide - Mo molybdenum - NADH reduced nicotinamide adenine dinucleotide - NO₃ ^-+Mo standard MX₁ culture medium - NO₃ ^--Mo MX₁ medium purified of Mo and used for continuous subculture with nitrate - NR nitrate reductase - PSR Paul's Scarlet rose - PVP polyvinylpyrrolidone - U urea - U+Mo MX₁ medium containing urea instead of nitrate - U-Mo MX₁ medium containing urea instead of nitrate and also purified of Mo