Photoautotrophic cell suspension cultures of periwinkle (Catharanthus roseus (L.) G. Don): Transition from heterotrophic to photoautotrophic growth

Chlorophyllous, heterotrophic periwinkle (Catharanthus roseus (L.) G. Don) cells were capable of sustained photoautotrophic growth in sugar-free B5 medium containing naphthaleneacetic acid and kinetin when provided with a CO₂-enriched atmosphere. An increase in cell fresh weight, first observed approximately 2 weeks after transfer from heterotrophic to photoautotrophic conditions, coincided with the development of maximum chlorophyll content and photosynthetic activity. Electron micrographs revealed that chloroplasts of cells cultured photoautotrophically in continuous light contained large starch granules and exhibited a less extensive thylakoid system than did periwinkle mesophyll chloroplasts. Photoautotrophic cells did not accumulate vindoline or dimeric alkaloids.Abbreviations Chl chlorophyll - dry wt dry weight - fr wt fresh weight - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA naphthaleneacetic acid     

Production of ethanol by adsorbed yeast cells

Summary Various ion exchange resins were tested for their ability to adsorb cells of Saccharomyces cerivisiae with the ultimate intention of developing a packed bed immobilized cell reactor for the continuous production of ethanol. The resins varied greatly in their ability to adsorb cells - the least effective resins retained less than 1 mg S. cerivisiae cells (dry weight)/g of resin (dry weight), and the most effective, 130–140 mg cells/g of resin. A column reactor packed with adsorbed yeast cells was operated continuously for over 200 hours using a 12% (w/v) glucose medium at dilution rates of 1.1 h^-1 and 1.44 h^-1 (based on void volume). High ethanol productivities of 53.1 and 62.0 g ethanol/l-h were obtained.     

Photoautotrophic growth of cell suspension cultures fromChenopodium rubrum in an airlift fermenter

Summary This communication describes the construction and operation of an airlift fermenter for the photoautotrophic growth of cell suspension cultures fromChenopodium rubrum. The basic batch culture unit provides a culture of 1.51 volume, sufficient to permit frequent aseptic sampling. It can be maintained at any desired temperature and aerated to different extents. Using an initial cell density of about 400,000 cells per ml suspension, the increase in cell number is 270% after a 14 days' growth period, although the stationary phase of growth is not yet reached. The transfer of photoautotrophic cell suspensions fromChenopodium rubrum from stationary growth into the large volume of fresh culture medium in the airlift fermenter results in an immediate protein formation, followed by an exponential phase of cell division, whereas rapid chlorophyll accumulation is delayed by 2 days.The growth capacities of photoautotrophic fermenter cultures including protein and chlorophyll formation as well asin vitro activities of the ribulosebisphosphate carboxylase and the phosphoenolpyruvate carboxylase are greatly lower as compared to photoautotrophic cells propagated in standard two-tier culture vessels using 30 ml culture medium. However the pattern of change in the activities of carboxylation enzymes is quite similar in both culture systems.Photoautotrophic cell suspensions fromChenopodium rubrum grown in an airlift fermenter assimilate about 90 mol CO₂/mg chlorophyll × hour. Dark CO₂ fixation is about 1.5% of the light values.Abbreviations PEF phosphoenolpyruvate - RuDP ribulosebisphosPhate - NS ground glass joints of standardized size made from Duran glass, Schott, Germany     

Chlorophyll synthesis in chlorella I. Occurrence of a lag phase on initiation of a dilute culture

A culture of Chlorella established by 30-fold dilution of a culture already grown to a level of 15 ml packed cell volume per liter produces little chlorophyll for approximately 12 hours. Investigation of other characteristics such as nitrogen incorporation, increase in packed cell volume and dry weight as well as RNA level show all of these to increase without any significant lag. α-Linolenate, which can be considered as a chloroplast marker, increased markedly. Photosynthetic oxygen evolution and respiration as well as the heme enzyme, catalase, increase also, indicating that the lag in chlorophyll synthesis is not due to a general inability to produce the porphyrin moiety.     

Stable endosperm cell suspension cultures from wild-type and opaque-2 maize

Stable cell suspension cultures have been established from immature endosperms of A69Y wild-type and opaque-2 maize (Zea mays L.). Cultured cells are capable of storage protein (zein) synthesis and accumulation throughout the growth period. Electrophoretic patterns of zeins show, for opaque-2 cells, the preferential inhibition of the accumulation of 22 kDa peptides typical of the mutation. Viable protoplasts, able to regenerate cell walls, as well as to divide and to express foreign DNA in transient expression experiments, can be obtained with high yields from cultures of both genotypes.Abbreviations 02 opaque-2 - wt wild-type - DAP days after pollination - PCV packed cell volume - f.w. fresh weight - SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - PEG polyethylene glycol - CAMV cauliflower mosaic virus - CAT chloramphenicol-acetyl-transferase     

Initiation and characterization of a cotton (Gossypium hirsutum L.) photoautotrophic cell suspension culture

A heterotrophic cotton (Gossypium hirsutum L. cv. Stoneville 825) cell suspension culture was adapted to grow photoautotrophically. After two years in continuous photoautotrophic culture at 5% CO₂ (balance air), the maximum growth rate of the photoautotrophic cell line was a 400% fresh weight increase in eight days. The Chl concentration was approximately 500 g per g fresh weight.Elevated CO₂ (1%–5%) was required for culture growth, while the ambient air of the culture room (600 to 700 ul CO₂ 1^–1) or darkness were lethal. The cell line had no net photosynthesis at 350 ul 1^–1 CO₂, 2% O₂, and dark respiration ranged from 29 to 44 mol CO₂ mg^–1 Chl h^–1. Photosynthesis was inhibited by O₂. The approximate 1:1 ratio of ribulose 1,5-bisphosphate carboxylase (RuBPcase) to phosphoenolpyruvate carboxylase (PEPcase) (normally about 6:1 in mature leaves of C₃ plants) was due to low RuBPcase activity relative to that of C₃ leaves, not to high PEPcase activity. The PEPcase activity per unit Chl in the cell line was identical to that of spinach leaves, while the RuBPcase activity was only 15% of the spinach leaf RuBPcase activity. RuBPcase activity in the photoautotrophic cells was not limited by a lack of activation in vivo, since the enzyme in a rapidly prepared cell extract was 73% activated. No evidence of enzyme inactivation by secondary compounds in the cells was found as can be found with cotton leaves. Low RuBPcase activity and high respiration rates are most likely important factors in the low photosynthetic efficiency of the cells at ambient CO₂.Abbreviations Chl chlorophyll - COT heterotrophic cotton cell line - COT-P photoautotrophic cotton cell line - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase - RuBP ribulose 1,5-bisphosphate - RuBPcase RuBP carboxylase - PEP phosphoenolpyruvate - PEPcase phosphoenolpyruvate carboxylase - MX Murashige and Skoog medium with 0.4 mg 1^–1 2,4-D - KT photomixotrophic medium with 1% sucrose - KT^o KT medium with no carbohydrate - KTP^o KT^o medium supplemented with 0.3 M Picloram - CER CO₂ exchange rate - PCER CO₂ exchange rate in the light     

Extracts of Marine cyanobacteria stimulated somatic embryogenesis of Daucus carota L.

Twenty five strains of marine cyanobacteria were screened for their ability to promote carrot somatic embryogenesis. Hot water extracts prepared from 21 of these strains promoted plantlet formation. Extracts from four strains increased plantlet numbers to an average of over 3.7-fold. Dialysates and nondialysates of each of these extracts also increased plantlet formation. For extracts from filamentous cyanobacteria, Nostoc sp. and Anabaena sp., dialysate was more effective (4.2-fold increase) than nondialysate (3.0-fold increase), whereas for unicellular strains Synechococcus sp. and Xenococcus sp., nondialysate was more effective (5.2-fold increase) than the dialysate (3.2-fold increase). These cyanobacterial extracts also promoted embryolike structure formation from two-year old carrot cell cultures which were unable to produce plantlets using the usual methods. Here, we demonstrate the existence in marine cyanobacterial extracts of low and high molecular weight factors which strongly promote somatic embryogenesis in carrot cell cultures.Abbreviations MS Murashige and Skoog medium - 2, 4-D 2, 4-dichlorophenoxyacetic acid - PCV packed cell volume     

Yeast cells entrapped in low-gelling temperature agarose for the continuous production of ethanol

Summary Continuous ethanol production byS. uvarum immobilized in a low-gelling temperature agarose namely SeaPlaque agarose was studied in a packed bed reactor at 30°C using sugarcane molasses containing 13.5% fermentable sugars as feed. The productivity at 95% conversion was 23 g/l.h (on reactor volume basis). The bioreactor was run continuously at a fixed dilution rate and it retained 60% of its initial activity upto 80 days.     

Production of shikonin derivatives by cell suspension cultures of Lithospermum erythrorhizon

Differences in the production of shikonin derivatives by callus and suspension cultures of Lithospermum erythrorhizon Sieb. et Zucc. were examined. When Linsmaier and Skoog medium was used in suspension cultures, cell growth was not accompanied by the production of shikonin compounds. Shikonin derivatives were produced, however, when this medium was used in callus cultures. Differences in shikonin production were examined in terms of the nutrient supply, the effect of the agar itself, and the oxygen supply. Shikonin derivatives could be produced without agar by keeping the cells exposed to air while providing an adequate supply of nutrients. In callus cultures, the production of shikonin compounds was reduced remarkedly when the oxygen concentration in the atmosphere was lowered, evidence that shikonin production during L. erythrorhizon cell growth on Linsmaier and Skoog agar medium is enhanced by an abundant supply of oxygen.     

Alkaloid formation by Catharanthus roseus cells in a packed column biofilm reactor

Summary Catharanthus roseus cells producing indole alkaloids were grown on surfaces of Ca-alginate beads within the interspacial volume of a packed column. Production media was circulated through the packed column in an upflow mode. Growth and indole alkaloid formation were quantified and compared with suspension culture of cells. Final alkaloid concentration and alkaloid yield obtained in the packed bed was superior to those obtained in suspension culture. This is thought to be due to improved cell-cell contact and interaction in the packed column.     

Cell division arrest by gamma-irradiation in photoautotrophic suspension culture of Euphorbia characias: Maintenance of photosynthetic capacity and overaccumulation of sucrose

Gamma-irradiation (250 Gy) applied to photoautotrophic cell suspensions of Euphorbia characias L. in the exponential growth phase led to the arrest of cell division and to a subsequent overaccumulation of sucrose and dry matter. From the fourth day of culture, the chlorophyll content and gross photosynthesis were not depressed by gamma-treatment nor by sugar accumulation. In both cultures, no difference was observed between oxygen uptake in the light at CO₂ saturating concentration and in the dark, suggesting that no change in energy-dissipative reactions took place after irradiation. A slight increase in oxygen uptake in both light and dark was observed in irradiated cells during the first four days. However, in the absence of limiting factors, the photosynthetic capacities of the dividing and irradiated non-dividing photoautotrophic cells were identical but higher than that of the non-dividing cells in the stationary growth phase. This suggests that gamma-irradiation arrests cell division by a mechanism different to that occuring in stationary-phase cultures. This may be of value in investigating the metabolism of secondary products.     

Cytological changes associated with induction of anthraquinone synthesis in photoautotrophic cell suspension cultures of Morinda lucida

Differences in subcellular structures between anthraquinone-producing and non-producing cells were investigated using photoautotrophic and photoheterotrophic cell suspension cultures of Morinda lucida. Irregular or distorted plastids containing starch grains were observed in the anthraquinone-producing cells, together with a highly elongated rough endoplasmic reticulum. The possible participation of plastids and rough endoplasmic reticulum in the anthraquinone biosynthesis is discussed.     

Characterization of a novel N-methyltransferase (NMT) from Catharanthus roseus plants

Young leaves from Catharanthus roseus plants contain a novel N-methyltransferase which transfers the methyl group from S-adenosyl-L-methionine specifically to position 1 of (2R, 3R)-2,3-dihydro-3-hydroxytabersonine, producing the N-methylated product. The enzyme shows a high degree of specificity toward substrates containing a reduced double bond at position 2,3 of tabersonine derivatives but the more substituted N-desmethyldeacetylvindoline did not act as a substrate. The enzyme catalyses the third last step in vindorosine and vindoline biosynthesis, and is associated with chlorophyll-containing fractions in partially purified enzyme preparations. The lack of vindoline accumulation in cell suspension cultures is correlated with the lack of expression of this enzyme activity as well as that of an acetyltransferase which catalyses the last step in vindoline biosynthesis. Neither fungal elicitor treatment of cell line #615 nor transfer to alkaloid production medium resulted in expression of these two enzyme activities, nor was either enzyme activity detected in photoautotrophic or hormone autotrophic cultures. Cell lines #200, 615–767 and 916 could not be induced to produce DAT or NMT enzyme activities.     

Establishment and characterization of photoautotrophic protoplast-derived cultures ofNicotiana plumbaginifolia

A procedure is described for the rapid establishment of photoautotrophic protoplast-derived cultures ofNicotiana plumbaginifolia. Photoautotrophic growth was induced by lowering the glucose concentration to 2.5 g.l^–1 in the protoplast culture medium and by omitting glucose from the subsequent dilution medium. Four week-old highly viable suspensions were plated on an agar-medium without glucose in unsealed Petri dishes and kept in illuminated chambers flushed with 0.05 % or 2 % CO₂. Air-grown calli had net photosynthesis rates of 1.8 and 17 moles CO₂.g^–1 fresh wt.h^–1 in air at 0.034 % CO₂ and in air enriched with 1 % CO₂, respectively. Calli grown in 2 % CO₂ exhibited lower rates of net photosynthesis at the two CO₂ concentrations tested (0 and 7.5 moles CO₂.g^–1 fresh wt.h^–1, respectively). The contribution of photosynthesis to growth was estimated to be 80 % in air-grown calli and more than 90 % in calli grown in 2 % CO₂. The suitability of this photoautotrophic culture procedure is discussed with regard to the screening of photosynthetic mutants or transformants from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - BAP 6-benzylaminopurine - IAA indoleacetic acid - Rubisco ribulose 1,5-bisphosphate carboxylase/oxygenase     

Cultivation of cell cultures of Berberis wilsonae in 20-l airlift bioreactors

Suspension cultures of Berberis wilsonae produce 4 berberine-type alkaloids: berberine, palmatine, columbamine and jatrorrhizine. In particular the formation of the phenolic alkaloids columbamine and jatrorrhizine and of berberine proves to be dependent on the concentration of dissolved oxygen. With higher aeration rates, berberine and jatrorrhizine yields can be increased considerably. Thus we reached an alkaloid yield of more than 3 g × 1^–1 with 50% dissolved oxygen tension in the medium. As far as we know this is one of the best results in fermenting of alkaloid-producing cell cultures.Abbreviations pO₂ dissolved oxygen concentration in % saturation (using air) - HPLC high-performance liquid chromatography - vvm volume air × volume medium^–1 × minute^–1 - rpm revolutions per minute - IAA indole-3-acetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid     

Hypoxia enhances prostaglandin synthesis in renal mesangial cell cultures

In view of recent findings which suggest that renal prostaglandins mediate the effect of hypoxia on erythropoietin production, we have studied whether hypoxia is a stimulus for in vitro prostaglandin synthesis. Studies were carried out in rat renal mesangial cell cultures which produce erythropoietin in an oxygen-dependent manner. Production rates of PGE₂ and in specified samples also of 6-keto-PGF_1α, as a measure of PGI₂, and PGF_2α were determined by radioimmunoassay after incubation at either 20% O₂ (normoxic) or 2% O₂ (hypoxic) in gas permeable dishes for 24 hrs. Considerable variation in PGE₂ production was noted among independent cell lines. PGE₂ production appeared to be inversely correlated to the cellular density of the cultures. In addition, PGE₂ production was enhanced in hypoxic cell cultures. The mean increase was 50 to 60%. PGF_2α and 6-keto-PGF_1α increased by about the same rate. These results indicate that hypoxia is a stimulus for in vitro prostaglandin production.     

Vital DNA staining of agarose-embedded protoplasts and cell suspensions of Nicotiana plumbaginifolia

The DNA of agarose-embedded protoplasts of Nicotiana plumbaginifolia was stained with Hoechst 33342 by immersing microscope slides, coated with immobilized protoplasts, into Erlenmeyer flasks containing consecutively dye solution, pH-correcting washing solutions and culture medium. After staining, protoplasts regenerated cell walls, started to divide and proliferated to calli. The culture system with immobilized protoplasts permits rapid change of culture media and accurate control of experimental conditions. The staining technique offers the opportunity for continuous observation of chromosomal behaviour and cell dynamics in individual plant cells.The same staining procedure was successfully applied to DNA of plant cells in suspension. Flow cytometric analysis revealed a retarding effect of the dye on the cell cycle, but within hours the cells recovered and showed their normal growth characteristics as compared to the controls.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxy acetic acid - DAPI 4'6-diamidino-2-phenylindole - FDA fluorescein diacetate - LMT low melting temperature - MES 2(N-morpholino)ethanesulfonic acid - MS Murashige and Skoog-medium - NAA -naphthaleneacetic acid - PCV packed cell volume - Tris Tris(hydroxymethyl)amino methane     

THE DETERMINATION OF CHLOROPHYLL a IN WATER CONTAINING SEDIMENT

SUMMARY

The presence of sediment at concentrations of 0,2% by mass in water samples significantly lowered the amount of chlorophyll a that could be measured. Two types of sediment differed in their ability to lower the chlorophyll a concentration. The chemically more active sediment had a marked depressive effect on the chlorophyll a concentration and the relationship between chlorophyll a and cell numbers in the samples was non-linear. It is recommended that the use of chlorophyll a as an indicator of biomass in water containing sediment should be approached with care.     

Chlorophyll Synthesis in Chlorella II. Effect of Glucose and Light Intensity on the Lag Phase

Dense cultures of Chlorella grown under our conditions contain large amounts of chlorophyll on a packed cell basis. Upon dilution the cells are, in effect, put into an environment in which light is not limiting and chlorophyll seemingly in excess (at least temporarily). Thus it is not economical for the cell to produce more chlorophyll until it is the limiting factor. It is our contention that the cell may sense this situation by the piling up of products such as glucose or starch. The observed light intensity effect which causes a stimulation of chlorophyll production at lowered intensities is analogous to that previously reported by others and is felt to be related to the rate of photosynthate production.     

Random mutagenesis in the large extrinsic loop E and transmembrane α-helix VI of the CP 47 protein of Photosystem II

The intrinsic chlorophyll-protein CP 47 is a component of Photosystem II which functions in both light-harvesting and oxygen evolution. Using the Escherichia coli mutator strain XL-1 Red, we introduced mutations at 14 sites in the large extrinsic loop E of CP 47 and its adjacent transmembrane -helix VI. Four mutant cell lines were recovered in which the histidyl residues 455H, 466H and 469H were altered. The cell lines H455T, H455Y, H469Y, and the double mutant F432L,H466R exhibited phenotypes that supported the identification of the histidyl residues 455H, 466H and 469H as chlorophyll ligands. Four additional mutant cell lines were recovered which contained mutations at positions 448R in the large extrinsic loop of CP 47. These mutants, R448K, R448Q, R448S, and R448W, exhibited variable phenotypes ranging from moderate alteration of photoautotrophic growth and oxygen evolution rates to a complete inhibition of these parameters. Those mutants exhibiting photoautotrophic growth and oxygen evolution capability under standard conditions were unable to grow photoautotrophically or evolve oxygen when grown at low chloride concentrations. Finally, a mutant cell line exhibiting a substitution at position 342G was recovered. The mutant G342D exhibited moderate alterations of photoautotrophic growth and oxygen evolution. In addition to these alterations, mutants were recovered in which deletions and insertions (leading to frame shifts) and stop codons were introduced. These mutants uniformly lacked the ability to either grow photoautotrophically or evolve oxygen.