Biosynthesis of cell-wall polysaccharides in cultured carrot cells

Biosynthesis of the cell wall in carrot cells (Daucus carota L.) cultured in a synthetic liquid medium was studied by measuring the incorporation of radioactive glucose and myo-inositol (MI). When the cells were fed with [¹⁴C]glucose in the presence of 0.01% MI, the label soon appeared in the neutral sugars in the cell wall but little radioactivity was found in the uronic-acid residues even after a prolonged incubation. On the other hand, radioactivity derived from [³H]MI was found to be distributed among uronic acids and pentoses but not in the hexose residues in the wall. The data indicate that MI is an important intermediate for the synthesis of acidic sugars in the wall of cultured carrot cells.Abbreviation MI myo-inositol     

Pectic polysaccharides in carrot cells growing in suspension culture

Pectic polysaccharides in the cell wall of suspension-cultured carrot cells (Daucus carota L.) were fractionated into high- and low-molecular-weight components by molecular-sieve chromatography with a Sepharose 4B column. During the phase of cell-wall expansion, the relative content of low-molecular-weight polymers rapidly increased. Electrophoretic analyses of these fractions showed that the high-molecular-weight components were largely composed of neutral and weakly acidic polymers while the low-molecular-weight fraction contained, in addition to neutral polymers, strongly acidic polyuronides in which the content of neutral sugars was very small. The accumulation of a large amount of the strongly acidic polyuronides occurred in a late stage of cell-wall growth, concomitant with a marked decrease in the high-molecular-weight components.Abbreviation MW molecular weight     

Biosynthesis of myo-inositol and its role as a precursor of cell-wall polysaccharides in suspension cultures of wild-carrot cells

The biosynthesis of myo-inositol (MI) and its role as a precursor of cell-wall polysaccharides was studied in supension cultures of wild carrot (Daucus carota L.) cells. Suspension cultures, grown in the presence or absence of 2,4-dichlorophenoxyacetic acid for 7 and 14d were incubated with [U-¹⁴C]glucose and [2-³H]MI in the presence of different concentrations of unlabeled MI. Synthesis of [¹⁴C]MI from [U-¹⁴C]glucose occurred under all conditions. The amount of MI synthesized from glucose was sharply reduced when 10 mM MI was provided in the medium. Substantial quantities of ³H were incorporated in arabinose, xylose and galacturonic acid isolated and purified from the cell-wall polysaccharides of the cell cultures in various stages of growth or embryogenesis. No ³H was present in the glucose or galactose units of cell-wall polysaccharides. At the four stages of growth and states of development of the carrot cultures used, the MI oxidation pathway contributed to the synthesis of pentosyl and galacturonosyl units of the cell wall. However, the data indicate that the contribution of the MI oxidation pathway to pentosyl and galacturonosyl units is small.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MI myo-inositol     

Behaviour of phenylalanine ammonia-lyase in carrot cells in suspension cultures

The high PAL activity in carrot cells in suspension culture was found at the linear and early stationary phases, with concomitant increases in phenylal     

Nutrient utilization during biomass and anthocyanin accumulation in suspension cultures of wild carrot cells

The medium used for the growth of anthocyanin-accumulating wild carrot (D. carota) suspension cultures contained ammonia as a sole nitrogen source and was buffered with succinate. Ammonia was the first nutrient to be completely utilized.The uptake of carbohydrate, phosphate and succinate continued after ammonia depletion. Biomass accumulation was faster and greater when sucrose was initially present in the medium than when glucose was present. When sucrose was provided in the medium it was rapidly hydrolysed to glucose and fructose and the fructose was used preferentially to glucose. Anthocyanin accumulation was rapid after ammonia fell below 3 mM and until the pH of the medium rose from 4.5 to 5.1 or 5.2.Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Degradation of pectic polysaccharides extracted from suspension cultures of carrot by purified exo-polygalacturonase

Highly purified exo-polygalacturonase was obtained from suspension cultures of carrot ( Daucus carota L. cv. Kintoki) by dialysis at pH 5.2, chromatography on DEAE-Sephadex A-50 and on Sephadex G-150, and preparative polyacrylamide disc gel electrophoresis. The enzyme did not attack the isolated carrot cell walls directly, but it had some effect on pectic polysaccharides extracted from the walls. The extracted polysaccharides were fractionated by DEAE-Sephadex A-50 column chromatography yielding four carbohydrate fractions. The major fraction (P-3) was then reacted with the exo-polygalacturonase. The enzyme treatment resulted in hydrolysis of approximately 18% of the glycosyl linkages of fraction P-3 with the release of galacturonic acids. The molecular size estimated by Bio-Gel A-5m gel filtration was not markedly affected by the enzyme action, but the percentage of galacturonosyl residues was clearly reduced. The specific activity of exo-polygalacturonase changed during the growth cycle, in relation to the cell growth.     

Changes in cell-wall polysaccharides from the mesocarp of grape berries during veraison

Softening of grape berries ( Vitis vinifera L. × V. labruscana cv. Kyoho) was evaluated by studying changes in composition and degradation of cell-wall polysaccharides. The grape berry softens at the beginning of the second growth cycle many weeks before harvest. The softening stage is called 'veraison' by viticulturists. On day 50 after full bloom, green hard berries (before veraison [BV]), softening berries (veraison [V]) and partly peel colored berries (C) were selected from the same clusters. In addition, mature berries (M) were collected on day 78 after full bloom. Mesocarp tissues at each stage were fractionated into hot water-soluble (WS), hot EDTA-soluble (pectin), alkali-soluble (hemicellulose) and residual (cellulose) fractions. Neutral and acidic sugar contents of WS and pectin fractions decreased only after the V stage, while the neutral sugar content of the hemicellulose fraction decreased from the BV to V stages. Cellulose content constantly decreased as the berry ripened, but the large decrease was found from the BV to V stages. Molecular masses of pectic and hemicellulosic polysaccharides decreased from the BV to V stages. Hemicellulosic xyloglucan was markedly depolymerized from the BV to V stages. The neutral and acidic sugar composition of each fraction changed little during the berry ripening. These data indicated that softening of berry during veraison involved the depolymerization of pectin and xyloglucan molecules and decrease in the amounts of hemicellulose and cellulose.     

Changes in the cell-wall polysaccharides of outer pericarp tissues of kiwifruit during development.

Changes in pectin, hemicelluloses and cellulose in the cell walls of outer pericarp tissues of kiwifruit (Actinidia deliciosa cv. Hayward) were determined during development. An extensive amylase digestion was employed to remove possible contaminating starch before and after fractionation of wall polysaccharides. An initial treatment of crude cell walls with alpha-amylase and iso-amylase or DMSO, was found to be insufficient removing the contaminating starch from wall polysaccharides. After EDTA and alkaline extraction, the pectic and hemicellulose fractions were again treated with the combination of alpha-amylase and iso-amylase. The amounts of predominant pectic sugars Gal, Rha and Ara, unaffected by the first and second amylase digestion, decreased markedly during the early fruit enlargement (8-12 weeks after anthesis, WAA), then increased during 16-20 WAA, and finally declined during fruit maturity (20-25 WAA). The molecular-mass of pectic polysaccharides decreased during fruit enlargement (8-16 WAA), and then changed little during fruit maturity. The higher molecular-mass components of hemicelluloses in HC-I and HC-II fractions detected at the early stage of fruit enlargement (8-12 WAA) were degraded at the late stage of fruit enlargement (16 WAA), but then remained stable at the much lower molecular-mass till fruit maturity. The amount of Xyl in the HC-II fraction decreased during the early fruit enlargement and fruit maturity, an observation that was consistent with xyloglucan (XG) content. The gel permeation profiles of XG showed a slight increase in higher molecular-mass components during 8-12 WAA, but thereafter there was no significant down-shift of molecular-mass until harvest time. The cellulose fraction increased steadily during fruit enlargement through maturity, but the XG contents in HC-I and HC-II fractions remained at a low level during these stages. Methylation analysis of HC-I and HC-II fractions confirmed the low level of XG in the hemicellulosic fractions. It was suggested that pectin in the outer pericarp of kiwifruit was degraded at the early stage of fruit enlargement, but XG remains constant during fruit enlargement and maturation.     

The effect of growth retardants on anthocyanin production in carrot cell suspension cultures

The effect of growth retardants on anthocyanin production was studied in wild carrot (Daucus carota) cell suspension cultures. Paclobutrazol [(2RS,3RS) — 1 — (4-chlorophenyl) — 4,4 —dimethyl-2-(1,2,4-triazol-1-yl) pentan-3-ol], uniconazole [(E)-1-(4-chlorophenyl-4,4 —) dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol], tetcyclacis [5-(4-chloro-phenyl) -3,4,5,9,10-pentaaza-tetracyclo-5, 4, 10^2,6, O^8,11 — dodeca-3, 9-diene], ancymidol [-cyclopropyl — 4 — methoxy-(pyrimidine-5-yl)benzyl alcohol] and CCC (2-chloro-ethyltrimethylammonium chloride) increased anthocyanin accumulation. AMO-1618 [(2-isopropyl-5-methyl-4-trimethyl-ammonium-chloride)-phenyl-1-piperidinium carboxylate] did not increase anthocyanin accumulation in the first passage but did increase it during the second passage on medium for improved anthocyanin accumulation. Prohexadione (3,5-dioxo-4-propionylcyclohexane carboxylic acid) decreased anthocyanin accumulation by 10%–12.5%.The inhibitory effect of gibberellin on anthocyanin accumulation was reversed by paclobutrazol. Paclobutrazol together with 10^–6M GA₃ increased anthocyanin level from 33% of control in GA₃ treated cell suspension to 76%. These results are consistent growth retardants increasing anthocyanin accumulation in carrot cell suspension cultures by inhibiting gibberellin biosynthesis.     

Changes in cell wall polysaccharides during elongation in a 2, 4-D free medium in a carrot suspension culture

Cell elongation occurred when carrot (Daucus carota L. ev. Kurodagosun) cells subcultured through sieving (Y. Ozeki and A. Komamine, Physiol. Plant. 53: 570-577. 1981) were transferred to a medium lacking auxin, while the cells showed no elongation in a medium containing 2, 4-D. Changes in polysaccharides of the cell walls and in their sugar composition during elongation were investigated. All wall components, EDTA-soluble pectic substance, 5 and 24%, KOH-soluble hemicelluloses and cellulose increased markedly during elongation. The increase of hemicelluloses correlated especially with elongation. In the 5% KOH-soluble hemicellulose, galactose and arabinose contents in the walls increased significantly both in amounts (per fresh weight) and relative contents (% in total neutral sugars) during elongation, while the relative contents of glucose and xylose decreased rapidly in the 5 and 24% KOH-soluble hemicelluloses. The methylation analysis tentatively indicated that larger amounts of galactan and/or arabinogalactan and lower amount of xyloglucan were found as components of the two hemicelluloses of elongating cells than those of non-elongating cells. The amounts of total carbohydrate and of uronic acid of extracellular polysaccharides secreted into the medium increased to a larger extent in the elongation culture than in the non-elongation culture. The contents of galactose and arabinose in extracellular polysaccharides increased rapidly in the elongation culture. The biochemical aspects of cell elongation in the absence of auxin were discussed from the viewpoint of the results obtained here.     

Isolation of organelles from carrot cell suspension cultures

Summary Organelles isolated from carrot cell suspension cultures by density gradient centrifugation and identified by their specific marker enzymes were found at the following mean densities on the sucrose gradient: microbodies 1.25 g/cm³ (catalase), mitochondria 1.18 (fumarase), endoplasmic reticulum 1.09 g/cm³ (NADH-cytochrome c reductase). Further enzyme assays were done for characterization of microbodies from carrot cultures.This work was supported by the Deutsche Forschungsgemeinschaft.     

Ethanol metabolism in suspension cultured carrot cells

Ethanol production in plant tissues deprived of oxygen is a well known process. Nevertheless, little information is available on the toxic effects of ethanol on plant cells and tissues, or on the possible role of acetaldehyde, the first oxidative product of ethanol, in inducing toxic effects in plants. Data on the metabolism of ethanol in suspension cultured cells of carrot ( Daucus carola L. cv. S. Valery, cell line T22), a system highly sensitive to the presence of ethanol in the culture medium, indicate that carrot cells oxidize only small amounts of ethanol to CO₂. Instead, they convert ethanol mainly to acetaldehyde, which accumulates in the culture medium. This suggests a possible role of acetaldehyde in causing ethanol-induced injury to carrot cells.     

Quantification of organelle changes in plant suspension cultures during growth

Stereological techniques were used to quantify ultrastructural changes which occurred during maturation of cultured Paul's Scarlet rose cells. The volume and ultrastructural composition of young, dividing, unsynchronized 5-day-old cells were compared to that of mature, nondividing 14-day-old cells. The volume of the 14-day-old cells was 4-fold greater than that of the 5-day-old cells, primarily due to vacuole expansion. Numerous quantitative changes occurred in the organelle composition during cell maturation, but distinctive differences were observed in the magnitude and direction of change among the different types of organelles. There was an overall decline in the plastid population as measured by both percent of cell volume and numbers of plastids per cell. The percent of cell volume and numbers of lipid bodies increased, whereas the percent volume of the mitochondria remained relatively constant while the number per cell declined.     

Changes in vacuolar pH of carrot cells in suspension culture grown under saline conditions

The effect of salinity on vacuolar pH was studied in carrot (Daucus carota L.) cells grown in liquid suspension culture either in the absence or presence of 150 mM NaCl. Both vacuolar and cytoplasmic pH were determined by several independent techniques. These techniques were NMR spectrometry, distribution of radioactive probes and spectrophotometric measurement of the absorbance changes of a naturally occurring vacuolar pH indicator. There was no difference in the cytoplasmic pH between cells grown in the presence or the absence of NaCl, but the vacuolar pH of cells grown in the presence of NaCl was higher by 0.38 to 1.05 pH units (depending on the technique that was used) than the vacuolar pH of cells grown in the absence of NaCl.     

Calcium increases the yield of somatic embryos in carrot embryogenic suspension cultures

An upward shift in the concentration of calcium present in the medium during somatic embryogenesis increased the number of embryos produced approximately two-fold. This was observed when embryogenic suspension cells grown in 2,4-D medium with the normal calcium concentration of 10^–3 M were transferred to hormone-free medium containing 10^–2 M calcium and when embryogenic suspension cells grown in 2,4-D medium containing 10^–4 M calcium were transferred to hormone-free medium with 10^–3 M calcium. At calcium concentrations between 6·10^–3 and 10^–2 M globular stage somatic embryos were found in cultures supplemented with 2·10^–6 M of 2,4-D indicating that elevated calcium counteracts the inhibitory effect of 2,4-D on somatic embryogenesis. No qualitative changes were found in the pattern of extracellular polypeptides as a result of growth and embryogenesis in media with different calcium concentrations.     

Influence of carbohydrates on quantitative aspects of growth and embryo formation in wild carrot suspension cultures

The rate of kaurene biosynthesis from mevalonate in a cell-free enzyme preparation from the endosperm of immature seeds of Marah macrocarpus is regulated by adenylate energy charge. The response curve is typical of a biosynthetic energy-utilizing sequence in which the rate of biosynthesis increases sharply as the energy charge is increased above 0.80. ADP proved to be an effective inhibitor of this process. AMP gave no inhibition at concentrations up to 2 mm and orthophosphate gave no inhibition up to 15 mm. Measurement of the pool sizes of intermediates in the sequence showed that the presence of ADP caused an increase in the levels of 5-phosphomevalonate and 5-pyrophosphomevalonate and a decrease in the levels of isopentenyl pyrophosphate and kaurene. These results indicate that pyrophosphomevalonate decarboxylase is the enzyme most subject to regulation by adenylate energy charge. The rate of conversion of isopentenyl pyrophosphate to kaurene and the rate of utilization of mevalonate by mevalonate kinase were not influenced by variations in the adenylate energy charge.     

Regulation of lysine and threonine synthesis in carrot cell suspension cultures and whole carrot roots

Aspartokinase (EC 2.7.2.4), homoserine-dehydrogenase (EC 1.1.1.3) and dihydrodipicolinic-acid-synthase (EC 4.2.1.52) activities were examined in extracts from 1-year-old and 11-year-old cell suspension cultures and whole roots of garden carrot (Daucus carota L.). Aspartokinase activity from suspension cultures was inhibited 85% by 10 mM L-lysine and 15% by 10mM L-threonine. In contrast, aspartokinase activity from whole roots was inhibited 45% by 10 mM lysine and 55% by 10 mM threonine. This difference may be based upon alterations in the ratios of the two forms (lysine-and threonine-sensitive) of aspartokinase, since the activity is consistently inhibited 100% by lysine+threonine. Only one form each of homoserine dehydrogenase and of dihydrodipicolinic acid synthase was found in extracts from cell suspension cultures and whole roots. The regulatory properties of either enzyme were identical from the two sources. In both the direction of homoserine formation and aspartic--semialdehyde formation, homoserine dehydrogenase activities were inhibited by 10mM threonine and 10 mM L-cysteine in the presence of NADH or NADPH. KCl increased homoserine dehydrogenase activity to 185% of control values and increased the inhibitory effect of threonine. Dihydrodipicolinic acid synthase activities from both sources were inhibited over 80% by 0.5 mM lysine. Aspartokinase was less sensitive to inhibition by low concentrations of lysine and threonine than were dihydrodipicolinic acid synthase and homoserine dehydrogenase to inhibition by the respective inhibitors.     

NAA对胡萝卜（Daucus carota）悬浮细胞茄红素合成的影响

通过对胡萝卜新透心红 DaucuscarotaCV.XinTouxinhong 细胞悬浮系单位鲜重细胞的茄红素含量、细胞总产量以及茄红素总产量的分析比较,研究了培养基中不同NAA浓度对胡萝卜细胞悬浮系茄红素代谢的影响.结果表明,NAA具有增强细胞茄红素代谢水平、提高单位鲜重细胞中茄红素的含量的作用,但对细胞的生长具有抑制作用.从茄红素单产和总产量综合评价,培养基中加入3.0mg·L-1NAA效果最佳.