Photoautotrophic Cells Research Articles

An inexpensive chamber for selecting and maintaining phototrophic plant cells

The high cost of lighted CO2 environmental chambers has prevented many laboratories from initiating and maintaining phototrophic cell cultures. The construction of an inexpensive chamber for the selection and subculture of photomixotrophic and photoautotrophic higher plant cell tissue cultures is described.

Mass spectrometric determination of the inorganic carbon species assimilated by photoautotrophic cells of Euphorbia characias L.

The chemical forms of inorganic carbon, CO2 or HCO3-, incorporated during photosynthesis in photoautotrophic Euphorbia characias cell suspension cultures were determined in experiments using 13CO2 and a mass spectrometry technique. From the equations of the CO2 hydration reaction, a kinetic model was first developed, and the effect of photosynthesis on the external CO2 concentration was simulated. It was predicted from this model that CO2 and HCO3- uptakes could be differentiated by recording only the CO2 variation rate in the external medium, successively in absence then in presence of an exogenous carbonic anhydrase activity. The results obtained with either CO2-grown or air-grown photoautotrophic cells were in good agreement with the model and demonstrated that CO2 was the sole species taken up during photosynthesis. In addition no accumulation of inorganic carbon within the cells was observed in the light. Similarly, in dark, CO2 was the only species released by respiration in the external medium.

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 μg 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 μmol 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.

Comparison of photosynthetic characteristics of two photoautotrophic cell suspension cultures of soybean

A new photoautotrophic soybean suspension culture (SB1-P) was initiated within a 14-month period, and was characterized for the purpose of comparing the physiology with that of a established soybean photoautotrophic strain, SB-P. SB1-P was found to have comparable Chl levels (1500–1700 μg g −1 fresh weight), cell size (near 30μm in diameter) cell aggregate size (150–190 μm diameter), chromosomes number ( 2n = 40), rates of growth (cell division time 4.5–5.0 days), net photosynthetic CO 2 fixation (19 and 36 μmol mg −1 Chl h −1 at atmospheric levels of CO 2 and 21 and 2% O 2, respectively) and oxygen evolution (74–76 μmol mg −1 Chl h −1), light-dependent and dark 14CO 2 fixation (49–54 and 3.5–3.7 μmol mg −1 Chl h −, respectively) and oxygen inhibition of photosynthesis (47% inhibition at 21% O 2 compared to photosynthesis at 2% O 2 with atmospheric levels of CO 2). SB1-P was similar to SB-P with regard to the activation level of RuBPcase (40% activated in cells grown at 5% CO 2), low in vitro RuBPcase activity (25–30 μmol CO 2 fixed mg −1 Chl h −1), and a low ratio of RuBPcase(initial)/PEP case (0.28–0.85). Traits shared by SB1-P and SB-P are a low photosynthetic capacity, an inability to grow at air levels of CO 2, high dark respiration, an elevated CO 2 compensation concentration in culture, and RuBPcase deactivation at air CO 2 levels. From these observations it was concluded that many of the characteristics of photoautotrophic soybean cultures are caused by the conditions employed in culture, and are not due to the specific genotype of soybean cultured or to chance occurence during initiation. The cultures which are actively growing exhibit several traits similar to those of developing leaf cells (active cell division, low photosynthetic capacity, elevated respiration, a low ratio of RuBPcase(initial)/PEPcase. This indicates the photoautotrophic cell physiology is more comparable to that of developing leaf cells, rather than to that of mature leaf cells.

Photoautotrophic cells as a screening system for herbicides inhibiting PET.

Photoautotrophic cells as a screening system for herbicides inhibiting PET.

Photosynthesis and respiration in air-grown and CO 2-grown photoautotrophic cell suspension cultures of carnation

Photoautotrophic carnation ( Dianthus caryophyllus L.) cell-suspension cultures were grown either in the presence of air enriched with CO 2 (1%, v/v) or in atmospheric air. Photosynthesis, respiration and carbohydrate content of the two types of cells were compared. Chlorophyll content and maximal rates of net photosynthesis were very similar in both cultures. High rates of photosynthesis were correlated with the exponential phase of growth and low cellular sucrose and glucose contents. The apparent affinity for CO 2 for photosynthesis was low compared to leaves of C 3 plants and only slightly higher in air-grown cells than in CO 2-grown cells. Air-grown cells had a lower growth rate, a lower carbohydrate content, and a lower respiration rate than CO 2-grown cells. Measurements of fumarase activity (a mitochondrial marker enzyme) suggest that the number of mitochondria per cell might be lower in air-grown cells than in CO 2-grown cells. Compared with intact leaves of carnation. isolated cells had about 4-times less chlorophyll and 5-times less net photosynthetic activity per mg of dry wt. However, measurements of gross and net photosynthesis with 18O 2 and a mass spectrometry technique indicate that 65–70% of the energy produced by the photosynthetic apparatus of isolated cells was used for CO 2 fixation at saturating levels of CO 2, a result roughly comparable to the 80–85% recorded with intact carnation leaves.

A comparison of effects of several herbicides on photoautotrophic, photomixotrophic and heterotrophic cultured tobacco cells and seedlings

The effects of herbicides with different primary modes of action were examined on the growth of photoautotrophic, photomixotrophic, and heterotrophic cultures of tobacco cells. These responses were compared with those of tobacco seedlings to the same herbicides. Herbicides, which primarily inhibit or disturb photosynthetic processes, suppressed the growth of photoautotrophic cells most strongly, as compared to photomixotrophic and heterotrophic cells (atrazine, diuron, paraquat). Herbicides having a primary mode of action other than the inhibition of photosynthetic processes, suppressed the growth of all types of cultured cells at similar concentrations (2,4-D, diphenamid, glyphosate, dinoseb, sodium chlorate, bialaphos, DTP), but the photoautotrophic cells were still the most sensitive to all kinds of herbicides except sodium chlorate. Furthermore, photoautotrophic cells responded to most of the herbicides as did the seedlings, with the exception of glyphosate and diphenamid. The possibility of photoautotrophically cultured cells as a model system to study the effects of herbicides are discussed.

Blue-light control of mRNA level and transcription during chloroplast differentiation in photomixotrophic and photoautotrophic cell cultures (Chenopodium rubrum L.)

In cell suspension cultures of Chenopodium rubrum maintained under photomixotrophic or photoautotrophic growth conditions the differentiation of chloroplasts is strictly blue-light-dependent. During this process of greening the steady-state concentration of mRNAs coding for plastid proteins increases rapidly in response to blue-light exposure as was determined by a dothybridization technique employing cloned DNA sequences complementary to these nuclear and plastid transcripts (light-harvesting chlorophyll a/b protein, rbcs, rbcl, psbA, atpB, atpE). Red light suppresses this response when applied at an advanced stage of chloroplast development. Indications are that blue-light dependency of chloroplast differentiation is a common feature of cultured plant cells irrespective of their metabolism. A DNA-protein complex with an active RNA polymerase ("transcriptionally active chromosome"; TAC) which specifically transcribes the plastid genes of its endogenous DNA has been isolated and purified from chloroplasts of light-grown cells. Quantitative analyses of these in-vitro transcripts show that the activity of TAC from cells grown in blue light prior to isolation is significantly higher than that of TAC from cells raised in red light under the same conditions. The results indicate that blue light enhances the transcription of plastid genes encoding prominent proteins. This response could account for the observed rise in transcript level in vivo.

Photosynthetic Characteristics of a Photoautotrophic Cell Suspension Culture of Soybean

A soybean suspension culture (SB-P) which can grow photoautotrophically in 5% CO(2) will not grow in ambient CO(2) levels. This elevated CO(2) requirement seems to be due to the additive effects of a number of factors. The in vivo activity of ribulose-1,5-bisphosphate carboxylase (RuBPcase) is much lower in the SB-P cells, compared to soybean plants. This may be due to the low light intensity used to culture the cells, which has been shown to decrease both the amount and activity in whole plants, resulting in a low rate of net photosynthesis. The RuBPcase activation level is also lowered in air CO(2) levels. The presence of the liquid medium raises the cells CO(2) compensation concentration (the CO(2) concentration reached when the rates of CO(2) fixed by photosynthesis and the CO(2) respired by the cells are equal). These factors, coupled with the high respiratory loss of CO(2) all contribute to reduced net photosynthesis in air, resulting in a photosynthetic capacity that is inadequate for cell survival. Active cell division, low photosynthetic capacity, elevated respiration, and a low ratio of RuBPcase(initial)/phosphoenolpyruvate carboxylase are traits that SB-P cells share with young leaf cells, indicating SB-P cell physiology may be comparable to that of young expanding leaves rather than to that of mature leaves.

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.

Control of nitrogen assimilation in Stichococcus bacillaris by growth conditions

Stichococcus bacillaris Naeg. (Chlorophyceae) grown on a 12 h light: 12 h dark cycle divides synchronously under photoautotrophic conditions and essentially nonsynchronously under mixotrophic conditions. Photoassimilation of carbon under photoautotrophic conditions was followed by a decline in cell carbon content during the dark period, whereas under mixotrophic conditions cell carbon increased throughout the light–dark cycle. The rates of nitrogen assimilation by cultures grown on either nitrate or ammonium declined sharply during the dark, and these declines were most pronounced under photoautotrophic conditions. Photoautotrophic cells synthesized glutamine synthetase and NADPH – glutamate dehydrogenase (GDH) exclusively in the light, whereas in mixotrophic cells about 20% of the total synthesis of these enzymes during one light–dark cycle occurred in the dark. NADH–GDH was synthesized almost continuously over the entire light–dark cycle. In the dark, both under photoautotrophic and mixotrophic conditions, the alga contained more than 50% of glutamine synthetase in an inactive form, which was reactivated in vitro in the presence of mercaptoethanol and in vivo after returning the cultures to the light. The thermal stability of glutamine synthetase activity was less in light-harvested cells than in dark-harvested cells. The inactivation of glutamine synthetase did not occur in cultures growing either heterotrophically in continuous darkness or photoautotrophically in continuous light. This enzyme appears to be under thiol control only in cells grown under alternating light–dark conditions, irrespective of whether this light regime results in synchronous cell division or not.

Photo-organotrophic growth of Euglena gracilis Z on glycolate

1. 1. Euglena gracilis Z cultured photo-organotrophically, under normal atmosphere and continuous light, in mineral medium supplemented with 25 mM glycolate as the sole carbon source was able to metabolize this substrate. 2. 2. Final productivities (weights per ml) of proteins, lipids and paramylum were twice as abundant in glycolate medium as in mineral medium. 3. 3. The generation time, which was not dependent upon glycolate (25 or 50 mM), was reduced by an increased illumination. Incorporations of NaH 14CO 3 and [1- 14C]glycolate had shown that: (i) a small amount of photorespiratory CO 2 participated in paramylum synthesis and (ii) important decarboxylations from glyoxylate and glycine took place. 4. 4. The ultrastructural characteristics of Euglena gracilis grown with glycolate resembled those of photo-autotrophic cells. 5. 5. We demonstrated that in such cells it was the RuBP carboxylase which had the preponderant role and not the PEP carboxylase which on the other hand was remarkably active in cells grown with lactate source.

Lack of effect of the photosystem II-based herbicides diuron and atrazine on growth of photoheterotrophic Chenopodium rubrum cells at concentrations inhibiting photoautotrophic growth of these cells

The effects of the photosynthetic herbicides diuron and atrazine on the growth and photosynthesis of a photoautotrophic cell suspension culture of Chenopodium rubrum have been studied with regard to the possible basis of phytotoxicity of these herbicides. The cells can also grow photoheterotrophically in the presence of sucrose while maintaining a high chlorophyll (Chl) content and photosynthetic capacity. Atrazine and diuron strongly inhibit CO2-dependent O2 evolution of both photoautotrophically and photoheterotrophically grown cells at submicromolar concentrations. Atrazine and diuron at submicromolar levels also inhibit photoautotrophic growth of the cells, but 10-fold higher concentrations of these herbicides have no effect upon photoheterotrophic growth while sucrose is still present in the culture medium. We conclude that any photooxidative damage alone caused by continued light absorption by an inhibited photosynthetic apparatus does not detectably inhibit cell growth. Only in the absence of an additional energy source are these herbicides inhibitory to growth. Such a cell culture could provide a means of screening potential photosynthetic herbicides for specificity of action. Thus, compounds inhibiting photoautotrophic growth but not photoheterotrophic growth of the same cells would be specific photosynthesis inhibitors.

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.

Alternative formation of anthraquinones and lipoquinones in heterotrophic and photoautotrophic cell suspension cultures of Morinda lucida Benth.

Photoheterotrophic and photoautotrophic cell suspension cultures were raised from a callus tissue derived from a Morinda lucida Benth. plant (Rubiaceae). The cultures were characterized with regard to fresh weight, dry weight, cell number, pH, chlorophyll and quinoid natural products. The amount of lipoquinones (phylloquinone, α-tocopherol, plastoquinone, ubiquinone) isolated from the photoautotrophic cultures matched the amount detected in an intact leaf. Anthraquinone glycosides which are found in the roots of Morinda plants were not present in the photoautotrophic culture. The photoheterotrophic culture contained only trace amounts of these pigments. Abundant anthraquinone synthesis was observed when photoautotrophic and photoheterotrophic suspension cultures were transferred into darkness, provided sucrose was present in the medium. Induction of synthesis of anthraquinone pigments coincided with a rapid disappearance of lipoquinones from the culture. Thus, in the suspension culture, photoautotrophy correlates with lipoquinone synthesis and heterotrophy correlates with anthraquinone synthesis. This reflects the situation in the intact plants where lipoquinones are chloroplast-associated whereas anthraquinones occur in the roots.

Callus production from photoautotrophic soybean cell culture protoplasts

Protoplasts were prepared from a photoautotrophic (PA) cell line of Glycine max (soybean). A yield of 75 to 90% after two to three hours digestion in a mixture of 1% Cellulase R10, 0.2% Pectolyase Y23 and 2% Driselase was obtained. Cell division and colony formation occurred from approximately 18% of the plated protoplasts. The cultured protoplasts were as sensitive to the herbicide atrazine, a photosynthetic inhibitor, as the original PA cells under the same conditions. Protoplasts and cells of a heterotrophic (HT) soybean culture were not as sensitive to atrazine. The isolated protoplasts retained the PA characteristics of the parental culture in the callus and cell suspension cultures obtained from the protoplasts. The chromosome numbers in the parental cell line and in cells derived from the isolated protoplasts (both PA and HT) were found to be largely (99%) the normal diploid number of 40.

Division and growth of mesophyll cells isolated from Psoralea bituminosa leaves

Mesophyll cells have been isolated from Psoralea bituminosa plant by gentle homogenization in a liquid nutrient medium. Between 60 and 70% of the cells can be isolated from leaves using this method, of which 50 to 60% can be recovered morphologically intact. Under light the separated cells have rates of oxygen evolution under light of 3500 μl O 2 mg −1 chlorophyll h −1 (measured with a Clark-type electrode). During growth, this rate decreases rapidly, as does cell pigments (chlorophylls and carotenoids). As a first step in obtaining a photoautotrophic cell suspension, growth factors affecting cell division in free sugar medium were investigated. The starting culture contained between 1 and 2 × 10 6 cells ml −1. The best increase in cell number was obtained on a medium composed of Joshi and Ball's elements and vitamins and containing 1 mg l −1 of naphthalene acetic acid, 0.1 mg l −1 of benzylaminopurine and the grinding juice. The optimum culture pH was between 5 and 5.3.

Kinetics and Specificity of ATP-dependent Proton Translocation Measured with Acridine Orange in Microsomal Fractions from Green Suspension Cells of Chenopodium rubrum L.

From photoautotrophic cell suspension cultures of Chenopodium rubrum L. microsomal fractions were prepared by differential and isopycnic sucrose gradient centrifugation. ATP-generated proton-accumulation was studied on a microsomal fraction (MF) of proton-translocating vesicles with a density of 1.11 to 1.12 gem(-3). This activity did not overlap with mitochondrial or chloroplast markers. MF is presumably of tonoplast origin. The assay was based upon pH-dependent acridine orange (AO) absorption shifts at 490 nm, Δ(490), due to AO-accumulation and polymerization within the vesicles, and was calibrated using pK and K(D) for AO polymers given by Zanker (Z. physikal. Chemie 199, 225, 1952). Addition of 1 mM ATP to MF yields a transmembrane ΔpH of about 2 units within 10 min (kinetics with a single time constant, τ=400s) and collapses after addition of the protonophor CCCP (τ=22s). The proton pumping activity is insensitive to vanadate and highly specific for ATP compared with GTP, UTP, CTP, and ITP, and half-saturates at 0.38 to 0.46 mM ATP, indicated by both initial rate (1 min) and steady state value of Δ(490) (10 min). Proton pumping into and proton leakage from the vesicles was cast into a feedback-scheme showing that the pump is working at a constant rate, that is, independently of the size of the created ΔpH.

Synthesis, composition and location of glycerolipids in photoautotrophic soybean cell cultures

The location, synthesis and composition of glycerolipids in photoautrophic soybean ( Glycine max L. Merr.) cell cultures were investigated to begin to elucidate the function of neutral lipids in photosynthetic tissues. The neutral lipids, diacylglycerol and triacylglycerol, were primarily located in the chloroplast which was also the primary location of diacylglycerol acyltransferase (EC 2.3.1.20), the final enzymatic step in the synthesis of triacylglycerol. The chloroplasts were also the primary location of synthesis of fatty acids from acetate and lysophosphatidic acid by glycerophosphate acyltransferase (EC 2.3.1.15). Neutral lipid made up to 20% of the total glycerolipid in dark-grown heterotrophic cells, 10% in photoautotrophic cells (both 2 weeks after subculture) and less than 1% in leaf cells. The amount of neutral lipid in photoautotrophic cells was maximal 1 week after transfer and declined to less than half that value 4 weeks after transfer. Starch content, however, increased for up to 3 weeks after transfer in parallel with dry weight accumulation. The data suggest that in these cells neutral lipids may serve for temporary storage of fatty acids and glycerol for later membrane synthesis rather than for storage of energy, a function of neutral lipids in seeds.

Photosynthesis and carbon metabolism in photoautotrophic cell suspensions of Chenopodium rubrum from different phases of batch growth

Rapidly dividing photoautotrophic cell suspensions from Chenopodium rubrum L. assimilated about 85 μmol CO2 (mg chlorophyll)−1 h−1. During the late stationary phase of culture growth, CO2 fixation rate was reduced to about 60 μmol CO2 (mg chlorophyll)−1 h−1. Actively dividing cells characteristically incorporated a smaller proportion of 14C into starch than cells from non‐dividing stationary phases. In rapidly dividing cells, [14C]‐turnover from free sugars, sugar‐phosphates, organic and amino acids was substantially higher compared to non‐dividing cells from stationary growth phase. Higher proportions of photosynthetically fixed carbon were channelled into proteins, lipids and structural components in actively dividing cells than in non‐dividing cells. In the latter. 14C was preferentially channeled into starch, and a striking increase in starch accumulation was observed. The transfer of non‐dividing, stationary growth‐phase cells into fresh culture medium resulted in an increase in the maximum extractable activities of some enzymes involved in the glycolytic and dark respiratory pathways and in the citric acid cycle. In contrast, the maximum extractable activities of the chloroplastic enzymes, ribulose‐1,5‐bisphosphate carboxylase (EC 4.1.1.38) and NADP+‐glyceraldehyde‐3‐phosphate dehydrogenase (EC 1.2.1.13) were highest after the cells had reached the stationary growth phase.