Photoautotrophic Growth and Photosynthesis in Cell Suspension Cultures of Chenopodium rubrum

A method is described for growing cell suspension cultures of Chenopodium rubrum photoautotrophically for prolonged periods of time. By using a two-tier culture vessel the growth medium with the cells was separated from the CO₂ reservoir. Definite CO₂ concentrations were established by a K₂CO₃/KHCO₃ buffer. Photoautotrophic growth in C. rubrum cell suspension cultures was correlated with the CO₂ level. At 0.5% CO₂ the cell cultures contained 68 μg chlorophyll/g fresh weight and showed an increase in fresh weight of about 80% in 18 days. At 1% CO₂ an increase in fresh weight of 165% in 18 days was observed. The chlorophyll content rose up to 84 μg/g fresh weight. The photoautotrophic growth was also greatly influenced by the 2,4-D content of the medium. Cell growth was enhanced by lowering the auxin concentration. Best growth was attained (210% increase in fresh weight) at 10^?8M 2,4-D. The photosynthetic activity of the cells was measured by the light dependent ¹⁴CO₂ incorporation. At 0.5% CO₂ the cell suspensions assimilated about 100 μmol CO₂/mg chlorophyll × h. In the presence of 1% CO₂ the light driven assimilation was raised up to 185 μmol CO₂/mg chlorophyll × h. In both cases, the dark incorporation of CO₂ was merely 1.8% of the values obtained in light.     

Photoautotrophic Cell Suspension Cultures from Mesembryanthemum crystallinum and their Response to Salt Stress

For the first time, photoautotrophic cell suspension cultures of Mesembryanthemum crystallinum have been established. The cells are growing in a sugar-free culture medium in the presence of 2 % (v/v) CO₂ as the sole carbon source. A 16 h light photoperiod is applied. Increase in fresh and dry weight during a 21 days growth cycle was more than 3-fold. Treatment of the cells with 200 mM NaCl from day 10 to day 21 of subculture stimulated cell culture growth, enhanced CO₂ fixation and elicited an increase in the extractable activities of enzymes related to CO₂ fixation (RubisCO; PEP carboxylase) and malic acid metabolism (NAD / NADP dependent malic enzyme and malic acid dehydrogenase). The cells performed osmotic adjustment to high salinity by uptake of K⁺, Na⁺, Cl^? and formation of proline as well as by a reduction in cell size. Although sugar and starch content of the cells changed during light/dark transition, a CAM-related diurnal fluctuation of malic acid was not observed.     

Effect of Different Sugars on Photosynthesis and Chlorophyll Fluorescence in Photoautotrophic Tomato Suspension Cell Cultures

The effects of metabolisable sugars sucrose and glucose along with non-metabolisable isomers of sucrose palatinose and turanose were tested. Rate of oxygen evolution (P), electron transport rate (ETR), and photochemical quenching (q_p) showed substantial decrease after 24 and 48 h by glucose and sucrose treatments, whereas there was no effect on all these parameters by the treatment with palatinose and turanose. Also the F_v/F_m ratio remained constant through the time of studies revealing that the maximal photochemical capacity of the cells was unchanged. Non-photochemical quenching (q_N) showed a decrease compared to the control values by all the treatments. Hence P and Chl fluorescence parameter were affected only by those sugars which are used in the metabolic pathways and not by sugar analogues.     

Characteristics of Five New Photoautotrophic Suspension Cultures Including Two Amaranthus Species and a Cotton Strain Growing on Ambient CO(2) Levels

Suspension cultures of cotton (Gossypium hirsutum), Amaranthus cruentus, A. powellii, Datura innoxia, and a Nicotiana tabacum-N. glutinosa fusion hybrid were adapted to grow photoautotrophically under continuous light. The cotton strain grew with an atmosphere of ambient CO₂ (about 0.06 to 0.07% in the culture room) while the other strains required elevated CO₂ levels (5%). Photoautotrophy was indicated by the requirement for CO₂ and for light for growth. The strains grew with doubling times near 14 days and had from 50 to 600 micrograms of chlorophyll per gram of fresh weight. The cells grew in small to moderate sized clumps with cell sizes from 40 to 70 micrometers (diameter). Like most photoautotrophic cultures described so far the ribulose 1,5-bisphosphate carboxylase (RuBPcase) activity levels were well below those of mature leaves. The phosphoenolpyruvate carboxylase levels were not elevated in the C₄Amaranthus species. The cells showed high dark respiration rates and had lower net CO₂ fixation under high O₂ conditions. Dark CO₂ fixation rates ranged from near 10 to 30% of that in light. Fluorescence emission spectra measurements show that the cell antenna pigments systems of the four strains examined are similar to that of chloroplasts of green plants. The cotton strain which was capable of growth under ambient CO₂ conditions showed the unique properties of a high RuBPcase activation level in ambient CO₂ and a stable ability to show net CO₂ fixation in 21% O₂ conditions.     

Photosynthetic Capacities and Growth Characteristics of Nicotiana tabacum (cv. Xanthi) Cell Suspension Cultures

Photoheterotrophic growth of cell suspensions of Nicotiana tabacum L. (cv. Xanthi) in organic culture medium enriched in sucrose (30 g per liter) showed a classical sigmoid growth curve. The cells developed functional chloroplast structures during the exponential growth phase, when their chlorophyll content increased steadily. A limited drop (30%) in the chlorophyll amount and structural changes of the plastids (starch accumulation) were observed during the lag phase. The measurements of photosynthetic capacities (O₂ evolution and CO₂ fixation) during the growth cycle revealed changes in the photosynthetic ratio (O₂/CO₂), which was near 1 during the lag and stationary phases and near 2 during exponential growth. During exponential growth there was also a rapid NO₃^? uptake. Analysis of label distribution among the products of ¹⁴CO₂ fixation showed that both CO₂ assimilation pathways, linked to the ribulose-biphosphate carboxylase (the autotrophic pathway) and to phosphoenolpyruvate carboxylase (the non-autotrophic pathway) were operative with an important increase of the capacity of the latter during the exponential growth phase. Maximum rate of oxygen evolution, either endogenous or with p-benzoquinone as Hill reagent, as well as the increased CO₂ Fixation capacity via the non-autotrophic pathway during the exponential phase were concomitant with a high cyanide inhibited O₂ uptake.     

Rheology and Shear Stress of Centaurea calcitrapa Cell Suspension Cultures Grown in Bioreactor

Centaurea calcitrapa suspension cultures were grown either in Erlenmeyer flasks or in a mechanically stirred bioreactor. Its rheological behaviour, when fitted to the Oswald–de Waele model (power law), showed pseudoplastic characteristics in both cases. The flow behaviour index (n) decreased over the course of a growth cycle and the consistency index (K) increased, reached a value of 1.81 N sⁿ m⁻² run on 2 l bioreactor. Bioreactor cultivation of C. calcitrapa cells at different agitation rates (30, 60, 100 and 250 rpm), highlighted the influence of shear forces on cell viability loss (90–34%) and phenol accumulation (74–140 μg l⁻¹), due to increased stirring speeds. Analysis of these results suggests that this cell line is shear-sensitive. An empirical exponential correlation was defined between apparent viscosity and biomass concentration, under the studied conditions, giving the possibility to estimate the prevailing broth regime and to optimize bioreactor design. Revisions requested 10 October 2005; Revisions received 19 December 2005     

Induction of HCO(3) Transporting Capability and High Photosynthetic Affinity to Inorganic Carbon by Low Concentration of CO(2) in Anabaena variabilis

The apparent affinity of photosynthesis for inorganic carbon in Anabaena variabilis strain M-3 increased during the course of adaptation from high to low CO₂ concentration (5% and 0.03% v/v CO₂ in air, respectively). This was attributed to an increased ability of the cells to accumulate inorganic carbon during the course of adaptation to low CO₂ conditions. The release of phycobiliproteins was used to evaluate the sensitivity of the cells to lysozyme treatment followed by osmotic shock. High CO₂-grown cells were more sensitive to this treatment than were low CO₂ ones. The efflux of inorganic carbon from cells preloaded with radioactive bicarbonate is faster in high than it is in low CO₂-adapted cells. It is postulated that the cell wall or membrane components undergo changes during the course of adaptation to low CO₂ conditions. This is supported by electron micrographs showing differences in the cell wall appearance between high and low CO₂-grown cells. The increasing ability to accumulate HCO₃⁻ and the lessened sensitivity to lysozyme during adaptation to low CO₂ conditions depends on protein synthesis. The increase in affinity for inorganic carbon during the adaptation to low CO₂ conditions is severely inhibited by the presence of spectinomycin. Incubation in the light significantly lessens the time required for the adaptation to low CO₂ conditions.     

Metabolism of 2-Chloroethylphosphonic Acid (Ethephon) in Suspension Cultures of Hevea brasiliensis

Suspension cultures of Hevea brasiliensis cells can metabolizethe growth regulator 2-chloroethylphosphonic acid to producea number of compounds, one of which appears to be an acid-labileconjugate. The general metabolic pattern closely resembles thatfound previously using Hevea leaves. Evidence that the compoundsformed are not chromatographic artefacts of a type describedby other authors is presented.     

Photosynthetic Carbon Metabolism in Leaves and Isolated Chloroplasts from Spinach Plants Grown under Short and Intermediate Photosynthetic Periods

Responses of foliar and isolated intact chloroplast photosynthetic carbon metabolism observed in spinach (Spinacia oleracea cv Wisconsin Bloomsdale) plants exposed to a shortened photosynthetic period (7-hour light/17-hour dark cycle), were used as probes to examine in vivo metabolic factors that exerted rate determination on photosynthesis (PS) and on starch synthesis. Compared with control plants propagated continuously on a 12-hour light/12-hour dark cycle, 14 to 15 days were required, subsequent to a shift from 12 to 7 hours daylength, for 7-hour plants to begin to grow at rates comparable to those of 12-hour daylength plants. Because of shorter daily durations of PS, daily demand for photosynthate by growth processes appeared to be greater in the 7-hour than in the 12-hour plants. The result was that 7-hour plants established a 1.5- to 2.0-fold higher total PS rate than 12-hour plants.

Intact chloroplasts isolated from the leaves of 7-hour plants (7-h PLD) displayed 1.5- to 2.0-fold higher PS rates than plastids isolated from 12-hour plants (12-h PLD). Plastid lamellae prepared from 7- and 12-h PLD isolates displayed equivalent rates of ferredoxin-dependent ATP and NADPH photoformation indicating that electron transport processes were not factors in the establishment of higher 7-h PLD PS rates. Analyses, both in leaves as well as intact PLD isolates, of dark to light transitional increases in Calvin cycle intermediates, e.g., ribulose-1,5-bisphosphate (RuBP) and 3-phosphoglycerate (3-PGA), as well as estimations of activities of RuBP carboxylase and fructose-1,6-bisphosphate phosphatase, indicated that 7-hour plant leaves displayed higher PS rates (than 12-hour plants), because there was a higher magnitude of activity of the Calvin cycle.

Although both the foliar level of starch and sucrose, as well as starch synthesis rate, often was higher in 7-hour compared with 12-hour plant foliage, the higher 7-hour plant total PS rates indicated that maximal sucrose and starch levels did not mediate any `feedback' inhibition of PS. The higher 7-hour plant foliar and PLD PS rates resulted in higher glucose-1-P levels as well as a higher ratio of 3-PGA:Pi, both factors of which would enhance the activity of chloroplast ADP-glucose pyrophosphorylase, and which were attributed to be causal to the higher starch synthesis rates observed in 7-hour plant foliage and PLD isolates.

     

Effect of Oxygen on Photosynthetic 14CO2 Fixation in Chroomonas sp. (Cryptophyta) III: Effect of Oxygen on Photosynthetic Carbon Metabolism

Photosynthetic carbon metabolism was studied with Chroomonassp. cells in which the rate of photosynthesis was inhibitedunder both an anaerobic condition and high concentrations ofoxygen. The time course of ¹⁴C-incorporation into photosyntheticproducts showed that 3-phosphoglycerate was the initial productof photosynthetic CO₂ fixation in Chroomonas sp. cells. During5-min photosynthesis, a considerable amount of ¹⁴C was incorporatedinto the insoluble fraction (mostly cryptomonad starch), andoxygen predominantly affected ¹⁴C-incorporation into this fraction.Although ¹⁴C-incorporation into intermediates of the photorespiratorypathway increased with increasing O₂ concentration, the amountswere much less than expected from the degree of oxygen inhibition.It is noteworthy that ¹⁴C-dihydroxyacetone phosphate accumulatedduring photosynthesis only under the anaerobic condition, whereasthe levels of the other phosphate esters were scarcely affectedby the oxygen concentration. Ribulose-1,5-bisphosphate carboxylase from Chroomonas sp. wascompetitively inhibited by oxygen, and its K_m(CO₂) value wassimilar to those of terrestrial C₃ plant enzymes. (Received November 19, 1984; Accepted May 20, 1985)     

Effect of Elevated CO2 on Photosynthesis and Chlorophyll Fluorescence of Rose Plants Grown at High Temperature and High Photosynthetic Photon Flux Density

Gas exchange and chlorophyll (Chl) fluorescence were measured on young mature leaves of rose plants (Rosa hybrida cvs. First Red and Twingo) grown in two near-to-tight greenhouses, one under control ambient CO₂ concentration, AC (355 µmol mol^–1) and one under CO₂ enrichment, EC (700 µmol mol^–1), during four flushes from late June to early November. Supply of water and mineral elements was non-limiting while temperature was allowed to rise freely during daytime. Leaf diffusive conductance was not significantly reduced at EC but net photosynthetic rate increased by more than 100 %. Although the concentration of total non-structural saccharides was substantially higher in the leaves from the greenhouse with EC, _PS2 (quantum efficiency of radiation use) around noon was not significantly reduced at EC indicating that there was no down-regulation of electron transport. Moreover, CO₂ enrichment did not cause any increase in the risk of photo-damage, as estimated by the 1 – q_P parameter. Non-photochemical quenching was even higher in the greenhouse with EC during the two summer flushes, when temperature and photosynthetic photon flux density (PPFD) were the highest. Hence rose photosynthesis benefits strongly from high concentrations of atmospheric CO₂ at both high and moderate temperatures and PPFD.     

Photosynthetic CO(2) Fixation at Air Levels of CO(2) by Isolated Spinach Chloroplasts

A system has been developed for the study of photosynthetic CO₂ fixation by isolated spinach chloroplasts at air levels of CO₂. Rates of CO₂ fixation were typically 20 to 60 micromoles/milligrams chlorophyll per hour. The rate of fixation was linear for 10 minutes but then declined to less than 10% of the initial value by 40 minutes. Ribulose 1,5-bisphosphate (RuBP) levels remained unchanged during this period, indicating that they were not the cause for the decline. The initial activity of the RuBP carboxylase in the chloroplast was high for 8 to 10 minutes and then declined similar to the rate of CO₂ fixation, suggesting that the decline in CO₂ fixation may have been caused by deactivation of the enzyme.     

Ultrastructure of Dunaliella tertiolecta Cells Grown under Low and High CO2 Concentrations1

The cells of Dunaliella tertiolecta grown under ordinary air(low-CO₂ cells) had a well developed pyrenoid with many morestarch granules than those grown under air enriched with CO₂(high-CO₂ cells). The chloroplast was located close to the plasmamembranein low-CO₂ cells, while that in high-CO₂ cells was located inthe inner area of the cells. Chloroplast envelope was electronicallydenser in low-CO₂ cells than in high-CO₂ cells, while the oppositeeffect of CO₂ was observed for the plasmamembrane. ²On leave from Institute of Biology, University of Novi Sad,Novi Sad, Yugoslavia. (Received November 7, 1985; Accepted March 5, 1986)     

Glycolate Metabolism in Low and High CO(2)-Grown Chlorella pyrenoidosa and Pavlova lutheri as Determined by O-Labeling

Photorespiration in Chlorella pyrenoidosa Chick. was assayed by measuring ¹⁸O-labeled intermediates of the glycolate pathway. Glycolate, glycine, serine, and excreted glycolate were isolated and analyzed on a gas chromatograph/mass spectrometer to determine isotopic enrichment. Rates of glycolate synthesis were determined from ¹⁸O-labeling kinetics of the intermediates, pool sizes, derived rate equations, and nonlinear regression techniques. Glycolate synthesis was higher in high CO₂-grown cells than in air-grown cells when both were assayed under the same O₂ and CO₂ concentrations. Synthesis of glycolate, for both types of cells, was stimulated by high O₂ levels and inhibited by high CO₂ levels. Glycolate synthesis in 1.5% CO₂-grown Chlorella, when exposed to a 0.035% CO₂ atmosphere, increased from about 41 to 86 nanomoles per milligram chlorophyll per minute when the O₂ concentration was increased from 21% to 40%. Glycolate synthesis in air-grown cells increased from 2 to 6 nanomoles per milligram chlorophyll per minute under the same gas levels. Synthesis was undetectable when either the O₂ concentration was lowered to 2% or the CO₂ concentration was raised to 1.5%. Glycolate excretion was also sensitive to O₂ and CO₂ concentrations in 1.5% CO₂-grown cells and the glycolate that was excreted was ¹⁸O-labeled. Air-grown cells did not excrete glycolate under any experimental condition. Indirect evidence indicated that glycolate may be excreted as a lactone in Chlorella. Photorespiratory ¹⁸O-labeling kinetics were determined for Pavlova lutheri, which unlike Chlorella and higher plants did not directly synthesize glycine and serine from glycolate. This alga did excrete a significant proportion of newly synthesized glycolate into the media.     

Light Stress and Oxidative Cell Damage in Photoautotrophic Cell Suspension of Euphorbia characias L

A photoautotrophic cell-suspension culture of Euphorbia characias L. grown at 70 [mu]mol photons m-2 s-1 was very sensitive to light stress: the gross photosynthesis measured by using a mass spectrometric 16O2/18O2 isotope technique showed a fast decrease at a rather low light intensity of 100 [mu]mol photons m-2 s-1, far below the photosynthetic saturation level. The contribution of activated oxygen species on photosystem II photoinhibition was examined for a given light intensity. A protective effect on gross photosynthesis was observed with 1% oxygen. When light stress was applied to a methyl viologen-adapted cell suspension, photoinhibition was reduced. When 50 [mu]mol L-1 methyl viologen was added, photoinhibition was slightly enhanced. These responses suggested an involvement of superoxide radicals in the photoinhibition process of E. characias photoautotrophic cells. The long-term (16 h) effects of photoinhibition were then studied. Aldehyde (malondialdehyde and 4-hydroxyalcenals) production resulting from lipid peroxidation was stimulated in long-term stressed cells. When 50 [mu]mol L-1 methyl viologen were added, increased aldehyde production was measured. Under 1% oxygen, the aldehyde production was comparable to that of nonstressed cells. The relationship among lipid peroxidation, light intensity, and net photosynthesis suggests that aldehyde production may result from cell death provoked by a prolonged energy deficit due to the inhibition of photosynthesis.     

Activity of Key Enzymes Involved in Carbohydrate Metabolism in Phaseolus vulgaris Cell Suspension Cultures

Activities of some key enzymes of glycolysis and sucrose metabolismwere investigated in relation to the physiological growth stagein bean cell suspension cultures. Activities of sucrose synthase,pyrophosphate:fructose-6-phosphate phosphotransferase, ATP:fructose-6-phosphatephosphotransferase, UDP glucose pyrophosphorylase, acid andalkaline invertase were detected. Both pyrophosphate:fructose-6-phosphatephosphotransferase and sucrose synthase activities increasedduring the active phase of cell division. Thereafter activitiesbegan to decline when sugar in the medium was depleted. Theincrease in enzyme activities coincided with a sharp decreasein the endogenous sucrose, glucose and fructose levels. Thelargest change occurred in the activity of sucrose synthase,which was more than seven fold higher in logarithmic phase cellsthan in lag hase cells. Transfer of mid-logarithmic phase cellsto fresh medium, containing 93 mmol dm^–3 sucrose, or additionof sucrose to existing medium, resulted in a further increasein PPjifructose- 6-phosphate phosphotransferase and sucrosesynthase activities. ²Present address: Plant Biotechnology Research Centre, PrivateBag X293, Pretoria 0001, Republic of South Africa.     

Turnover of O-Glucosides of Dihydrozeatin and Dihydrozeatin-9-riboside During the Cell Growth Cycle of Photoautotrophic Cell Suspension Cultures of Chenopodium rubrum

The cellular levels of O-glucosides of ³H-(diH)Z and ³H-(diH)[9R]Z, the major short-term metabolites of ³H-(diH)Z having been exogenously supplied to photoautotrophically growing suspension cell cultures of Chenopodium rubrum, decreased significantly during further culture, irrespective of whether the cells were maintained in the stationary phase or were transferred to conditions restoring cell divison. Metabolism of both compounds was more pronounced during the active growth phase than during the stationary phase. The O-glucosides were converted preferentially to polar compounds of as yet unknown nature, which were partly excreted into the medium. The cellular pools of both glycosides remained compartmented within the vacuole. In contrast to the O-glycosides, the small cellular pools of the aglycones ³H-(diH)Z and ³H-(diH)[9R]Z maintained their level during the experimental period of 30 days. Small amounts of the glucosides, as well as of the aglycones, were recovered from the medium and could have resulted from the lysis of a few cells. The results demonstrate, for the first time, that O-glucosides of cytokinins are not irreversibly deposited within the vacuole of plant cells but may serve to maintain a small, but more or less constant pool of extra-vacuolar, presumably cytosolic, aglycones. (DiH)Z and its derivatives could be demonstrated to be endogenous cytokinins of Chenopodium rubrum suspension cultured cells occurring along with those of the isopentenyladenine and zeatin types.     

Two Polypeptides Inducible by Low Levels of CO2 in Soluble Protein Fractions from Chlorella regularis Grown at Low or High pH

Previous studies suggested that certain protein(s) other thancarbonic anhydrase might play an important role in the facilitatedtransport of dissolved inorganic carbon (DIC) from the mediumto the site of CO₂ fixation by ribulose-1,5-bisphosphate carboxylase/oxygenasein the unicellular green alga Chlorella regularis adapted tolow-CO₂ (ordinary air) conditions [Shiraiwa et al. (1991) Jpn.J. Phycol. 39: 355; Satoh and Shiraiwa (1992) Research in Photosynthesis,Vol. III, p. 779]. The proteins that might be involved in thisfacilitated transport of DIC were investigated by pulse-labelingof induced proteins with ³⁵S-sulfate during adaptation of cellsgrown under high-CO₂ conditions to low CO₂. Analysis by SDS-PAGErevealed that synthesis of two polypeptides, with molecularmasses of 98 and 24 kDa, respectively, was induced under low-CO₂conditions. The 24-kDa polypeptide was induced at pH 5.5 butnot at pH 8.0, whereas the 98-kDa polypeptide was induced atboth pH 5.5 and pH 8.0. The possible role of these polypeptidesin the facilitated transport of DIC in Chlorella regularis isdiscussed. (Received October 30, 1995; Accepted February 26, 1996)     

Aspects of Growth and Metabolism in a Suspension Culture of Acer pseudoplatanus (L. ) Grown on a Glycerol Carbon Source

A suspension culture of Acer pseudoplatanus cells was transferredfrom medium containing 2% (w/v) glucose to an identical onecontaining glycerol at 2% (w/v) as the sole carbon source. Thepatterns of cell number increase, dry weight increase, and changesin packed cell volume showed marked differences as a resultof this transfer. The glucose-grown cultures contained a small proportion of cellsof exceptionally large diameter, and transfer to the glycerolcarbon source appeared to bring about a considerable increasein their number. These larger cells, in both glucose- and glycerol-containingcultures, exhibited considerable differences in cell wall architecturewhen compared with their smaller counterparts. They appearedmore irregular, and had much looser cellulose microfibril arrangementin their outer layers     

不同氮营养水平下草莓叶片光合作用对高CO2浓度的适应

研究了不同氮素水平（12mmol／L,4mmol／L,0、4mmol／L）下生长的‘丰香’草莓在富C02（700μL/L）和大气CO（390μL/L）下的光合作用。结果表明,高氮（12mmol／L）下,在富CO2环境中生长的‘丰香’草莓叶片未出现光合作用下调,富CO2下草莓叶片的净光合速率、最大羧化速率（Vc.max）、最大电子传递速率（Jmax）、碳同化的电子传递速率（Jc）和光化学猝灭系数（qp）等均显著提高;而在中氮（4mmol／L）、低氮（0．4mmol／L）下,富CO2下生长的草莓叶片的上述参数均出现不同程度的下降。富CO2下,无论氮素水平如何,草莓叶片的光呼吸电子传递速率（Jo）均降低高氮草莓叶片的非光化学猝灭系数（qN或NPQ）降低,光抑制降低,而低氮则相反。上述结果说明,氮素供应不足时草莓叶片在富CO2下光合作用出现下调,因此生产上进行CO2施肥时应适度增加氮素的供应。