Photoautotrophic Suspension Cultures Research Articles

Advancement of the cultivation and upscaling of photoautotrophic suspension cultures using Chenopodium rubrum as a case study

Photoautotrophic (PA) suspension cultures combine the advantages of cell suspensions with carbon autotrophy and have been used for various applications including studies on photosynthesis, sugar signal transduction, herbicide action, and stress responses. A major practical drawback is the requirement for elevated CO2, which is typically generated by a buffer system in two-tier flasks that need to be custom made and require time-consuming handling. In this study, we substantially simplified and improved the cultivation of a PA culture of Chenopodium rubrum as a case study in Erlenmeyer flasks shaken in a CO2 enriched photoincubator. Growth rates and photosynthetic activity were found to be comparable to those in two-tier flasks but with the advantage of constant CO2 level throughout the cultivation. In addition, it was possible to establish the cultivation in a commercial laboratory-scale photobioreactor with various options to control and continuously measure the culture conditions and regimes and assess the physiological status. Although the relative biomass yield and photosynthetic performance of the batch cultures was not fully reached, the continuous cultivation system provides a good basis for future scale-up if larger amounts of biomass are needed or to impose specific cultivation regimes in combination with comprehensive online monitoring. The different improvements should contribute to the more widespread use of higher plant PA cultures and also facilitate the cultivation of microalgae.

Stress Response Monitoring of Photoautotrophic Higher Plant Suspension Cultures by Fluorescence Imaging for High-Throughput Toxic Compound Screening

Photoautotrophic suspension cultures have been established from various model and crop plants and proved to be valuable and robust experimental system to assess coordinated responses of primary and secondary metabolism to metabolic and stress related signals. The use of suspension cultures combines the ease of handling microalgae in microtiter plates with the advantage of testing physiological responses of higher plants, notably in combination with the assessment of the response of photosynthetic activity by PAM chlorophyll fluorescence imaging as well as monitoring changes in secondary metabolite production and ROS formation by steady state fluorescence of plant fluorophores or introduced fluorescent probes. Photoautotrophic cultures provide various advantages as fast, highly sensitive, robust and high-through-put experimental system for screening and characterization of the impact of toxic compounds on higher plants. This opinion article discusses and critically evaluates the potential of photoautotrophic cultures of higher plants in combination with fluorescence imaging assays in microtiter plates as a complement to existing guidelines for testing the toxicity of chemicals in plants.

Metabolizable and non-metabolizable sugars activate different signal transduction pathways in tomato.

To gain insight into the regulatory mechanisms of sugar signaling in plants, the effect of derivatives of the transport sugar sucrose (Suc), the Suc isomers palatinose and turanose, and the Suc analog fluoro-Suc were tested. Photo-autotrophic suspension culture cells of tomato (Lycopersicon peruvianum) were used to study their effect on the regulation of marker genes of source and sink metabolism, photosynthesis, and the activation of mitogen-activated protein kinases (MAPKs). Suc and glucose (Glc) resulted in reverse regulation of source and sink metabolism. Whereas the mRNA level of extracellular invertase (Lin6) was induced, the transcript level of small subunit of ribulose bisphosphate carboxylase (RbcS) was repressed. In contrast, turanose, palatinose, and fluoro-Suc only rapidly induced Lin6 mRNA level, whereas the transcript level of RbcS was not affected. The differential effect of the metabolizable and non-metabolizable sugars on RbcS mRNA regulation was reflected by the fact that only Suc and Glc inhibited photosynthesis and chlorophyll fluorescence. The activation of different signal transduction pathways by sugars was further supported by the analysis of the activation of MAPKs. MAPK activity was found to be strongly activated by turanose, palatinose, and fluoro-Suc, but not by Suc and Glc. To analyze the role of sugars in relation to pathogen perception, an elicitor preparation of Fusarium oxysporum lycopersici was used. The strong activation of MAPKs and the fast and transient induction of Lin6 expresssion by the fungal elicitor resembles the effect of turanose, palatinose, and fluoro-Suc and indicates that non-metabolizable sugars are sensed as stress-related stimuli.

Biochemical Evidence for the Activation of Distinct Subsets of Mitogen-Activated Protein Kinases by Voltage and Defense-Related Stimuli

Activation of mitogen-activated protein (MAP) kinases is a common reaction of plant cells in defense-related signal transduction pathways. To gain insight into the mechanisms that determine specificity in response to a particular stimulus, a biochemical approach has been employed. Photoautotrophic suspension culture cells of tomato (Lycopersicon peruvianum) were used as experimental system to characterize MAP kinase activation by different stress-related stimuli. An elicitor preparation of the tomato-specific pathogen Fusarium oxysporum lycopersici was shown to result in the simultaneous induction of four kinase activities that could be separated by ion-exchange chromatography. The simultaneous activation of multiple MAP kinases was further substantiated by distinct pharmacological and immunological properties: a differential sensitivity toward various protein kinase inhibitors and a differential cross-reaction with isoform-specific MAP kinase antibodies. In contrast to the two fungal elicitors chitosan and the F. oxysporum lycopersici preparation, the plant-derived stimuli polygalacturonic acid and salicylic acid were shown to activate distinctly different subsets of MAP kinases. Application of a voltage pulse was introduced as a transient stress-related stimulus that does not persist in the culture. Voltage application activates a distinct set of MAP kinases, resembling those activated by salicylic acid treatment, and generates a refractory state for the salicylic acid response. The inhibitory effect of nifedipine indicates that current application may directly affect voltage-gated calcium channels, thus, providing a tool to study various calcium-dependent pathways.

Intracellular Protons are not Involved in Elicitor Dependent Regulation of mRNAs for Defence Related Enzymes in Chenopodium rubrum

Summary Protons have been implicated in the signal transduction of elicitor-generated defence reactions. To address the possible function of cytosolic acidification we determined both intracellular and extracellular pH values and phenylalanine-ammonia-lyase (PAL) and extracellular invertase (CIN 1) mRNA levels of photoautotrophic suspension culture Chenopodium rubrum cells in response to a number of stimuli known to elicit defence responses. The results of these experiments show that there is no principle link between elicitor-induced persistent pH-shifts and the induction of the defence related mRNAs. Treatment with the fungal elicitor chitosan led to intracellular acidification, extracellular alkalinisation, and a rise of both PAL and CIN 1 mRNA levels. However, when these pH shifts are reversed by treatment with fusicoccin, a known activator of the plasma membrane ATPase, both PAL and CIN 1 mRNA levels are also elevated. A comparison between the kinetics of intracellular acidification and the rise in PAL and CIN 1 mRNA levels in response to chitosan shows that the activation of these genes precedes intracellular acidification by 1 h, indicating that protons are not involved in the analysed signal transduction pathway leading to the induction of PAL and CIN 1 mRNAs.

β-Lactam antibiotics inhibit chloroplast division in a moss ( Physcomitrella patens) but not in tomato ( Lycopersicon esculentum)

Photoautotrophic suspension cultures of moss protonema of Physcomitrella patens (Hedw.) B.S.G. and of tomato (Lycopersicon esculentum L.), respectively, were treated with three different β-lactam antibiotics. Ampicillin, cefotaxime and penicillin in the range from 10 μmol/L to 1 mmol/L had no visible effects on tomato cells but led to moss cells with giant chloroplasts. According to electron micrographs these cells are morphologically normal but contain 2–3 macrochloroplasts with well developed inner membranes. As only chloroplasts in dividing tip cells were affected, we conclude that β-lactam antibiotics specifically inhibit the plastid division process in the moss. Major proteins were not affected by antibiotic treatments as judged by SDS-PAGE and subsequent silver stain. The inhibitory effect on chloroplast division could not be compensated for by exogenous cytokinin (5 × 10−6 mol/L i6 Ade). However, upon removal of the antibiotics, macrochloroplasts started to divide. As division of bacteria and of cyanelles is sensitive to β-lactam antibiotics, we suggest that mosses conserved an ancient component of the chloroplast division mechanism that was subsequently modified or substituted during plastid evolution in land plants.

Cultivation of photoautotrophic plant cell suspensions in the bioreactor: Influence of culture conditions

A 20-1 airlift bioreactor system was established which enabled growth of photoautotrophic cell suspension cultures of Chenopodium rubrum over long periods of time. In order to find culture conditions that improve the biomass accumulation of photosynthetically active plant cell cultures in the 20-1 scale we modified the supply of cells with light and CO 2 in several batch culture experiments. Our results clearly indicate that growth was limited by the supply of cells with light. The biomass increase was 410% in 16 days and the specific growth rate μ was only 0.109 d −1 for cells growing under low light conditions (105 μ E m −2 s −1). In contrast, the biomass increase of cells growing under high light conditions (420 μ E m −2 s −1) was 1600% and μ reached 0.223 d −1. When air for aeration was supplemented with different amounts of CO 2 Chenopodium rubrum cells expressed similar growth rates, although no increase of biomass could be observed for cells growing in ambient air. Several physiological parameters (e.g., chlorophyll a + b, net-photosynthetic O 2 evolution, dark respiration activities, in vitro activities of enzymes of primary metabolism, consumption of major constituents of the MS medium) were measured routinely to describe the influence of culture conditions on the physiology of Chenopodium rubrum cells. The presented data confirm the influence of culture conditions on growth and physiology of bioreactor cultivated photoautotrophic suspension cultures.

Regulation of Sucrose Synthase Expression in Chenopodium rubrum: Characterization of Sugar Induced Expression in Photoautotrophic Suspension Cultures and Sink Tissue Specific Expression in Plants

Summary The effect of source/sink modifications on the enzyme activity and on the steady state level of mRNA of sucrose synthase has been analyzed in Chenopodium rubrum . A sucrose synthase cDNA with high homology to sucrose synthases from both monocotyledonous and dicotyledonous plants has been cloned and used as a homologous probe for Northern blot analyses. Photoautotrophic suspension culture cells, which may be shifted to mixotrophic growth by adding sugars, have been used as a model system to investigate the transition between autotrophic and heterotrophic growth. The higher activity of sucrose synthase after preincubation in the presence of D-glucose, D-fructose or sucrose correlates with an elevated level of mRNA. The sucrose synthase gene was fully induced above a concentration of 20 mM glucose and the steady state level of mRNA was already elevated 1 h after the addition of glucose. Induction of sucrose synthase by 6-desoxyglucose suggests that the non-phosphorylated glucose is the signal for the sugar induced gene expression. Both the enzyme activity and the mRNA level showed a sink tissue specific distribution in plants. The data suggest that sucrose synthase is important both for sink metabolism and the source sink transition.

Growth characteristics of liverwort cells, Marchantia paleacea var. diptera, in a photoautotrophic suspension culture

A chlorophyllous cell line of liverwort, Marchantia paleacea var. diptera, exhibited good photoautotrophic growth in a light-irradiated culture with an air supply containing 1% CO 2 as the sole carbon source. In cultures with flat, oblong glass flasks under various conditions of light intensity, a plot of light intensity against the specific growth rates of these cells revealed that light exerted both stimulatory and inhibitory effects on growth. More specifically, the specific growth rate increased with an increase in light intensity over the range of 5.4 to 60 W/m 2, while a significant decrease in the rate occurred when light intensity was greater than 60 W/m 2. Photoautotrophic suspension cultures of M. paleacea cells were conducted using an externally illuminated reactor. The distribution of light intensity in the reactor was analyzed on the basis of the relation between light transmittance and cell concentration in medium. The behavior of cell growth in the reactor could be described by taking into consideration the dependence of cell growth on light and the attenuation of light intensity in the cell-suspended medium.

Expression of a sugar-transporter gene family in a photoautotrophic suspension culture of Chenopodium rubrum L.

Photoautotrophic suspension-culture cells of Chenopodium rubrum L. were shifted to mixotrophic growth by adding glucose to investigate whether the activities of plant sugar transporters, as well as the expression of the corresponding genes, are regulated in response to sugars. The rate of D-glucose uptake was shown not to be affected by mixotrophic growth in the presence of D-glucose. The polymerase chain reaction (PCR) technique was applied to amplify cDNA and genomic fragments from monosaccharide-carrier genes. Seven members of a monosaccharide-carrier family were identified of which three were found to be expressed in the suspension-culture cells. The expression of the monosaccharide-carrier genes was independent of the presence of D-glucose.

Light acclimation potential and xanthophyll cycle pigments in photoautotrophic suspension cells of Chenopodium rubrum

The present study investigates the light acclimation potential of photoautotrophic suspension culture cells of Chenopodium rubrum L. grown in 16 h light/8 h dark cycles. Typical features of sun/shade acclimation could be demonstrated in cultures grown at photon flux densities of 30 and 150 μmol m−2 s−1. Low light grown cells had lower chlorophyll a/b ratios, lower respiration rates and lower light compensation points than high light grown cells. Maximum photosynthetic rate per cell dry weight was highest in low light conditions, indicating that the cells did not enlarge their photosynthetic machinery upon exposure to high light. Transfer of cultures to 800 μmol m−2 s−1 caused photoinhibition as indicated by a decrease in photosynthetic efficiency and by the occurrence of a slowly reversible quenching of variable chlorophyll fluorescence. Extension of the photoinhibitory treatment over six light dark cycles did not result in further dramatic changes of these parameters, whereas the chlorophyll content per dry weight and the chlorophyll a/b ratio decreased. Measurements of photochemical quenching showed that the capability of the cells to dissipate excessive energy had increased during the acclimation process. The presence of the xanthophyll cycle pigments and the operation of the cycle could be demonstrated. In agreement with the putative photoprotective function of antheraxanthin and zeaxanthin these pigments could only be detected under photoinhibitory conditions. Prolonged photoinhibitory treatment resulted in increases in the xanthophyll pigment concentration but not of the potential to deepoxidate violaxanthin. The limited potential of the cells to accumulate zeaxanthin and antheraxanthin might indicate that the xanthophyll cycle is not the main factor determining their resistance to high light stress.

Characterization of phytochrome-regulated gene expression in a photoautotrophic cell suspension: possible role for calmodulin.

A photoautotrophic suspension culture of soybeans was found to exhibit light-dependent expression of the genes encoding the major chlorophyll a- and b-binding protein (CAB). The expression was mediated by phytochrome, since it was induced by red light and reversed by far-red light. The maximal level as well as the kinetics of the induction were comparable between the suspension culture and soybean seedlings. Using this cell culture, we addressed the question of whether a calcium- and/or calmodulin-mediated step is involved in the signal transduction process between phytochrome and CAB expression. We found that W-7, a potent calmodulin antagonist, severely attenuated the induction of CAB mRNA by light, whereas W-5, a weak calmodulin antagonist, had little effect. Control experiments demonstrated that W-7 treatment did not block the induction of hsp-75 by heat shock. The addition of ionomycin, a calcium ionophore, induced a low level of CAB mRNA accumulation in the dark which could be further enhanced by light treatment. We propose that calmodulin activation by light is necessary but not sufficient to induce maximal CAB expression.

Characterization of phytochrome-regulated gene expression in a photoautotrophic cell suspension: possible role for calmodulin.

A photoautotrophic suspension culture of soybeans was found to exhibit light-dependent expression of the genes encoding the major chlorophyll a- and b-binding protein (CAB). The expression was mediated by phytochrome, since it was induced by red light and reversed by far-red light. The maximal level as well as the kinetics of the induction were comparable between the suspension culture and soybean seedlings. Using this cell culture, we addressed the question of whether a calcium- and/or calmodulin-mediated step is involved in the signal transduction process between phytochrome and CAB expression. We found that W-7, a potent calmodulin antagonist, severely attenuated the induction of CAB mRNA by light, whereas W-5, a weak calmodulin antagonist, had little effect. Control experiments demonstrated that W-7 treatment did not block the induction of hsp-75 by heat shock. The addition of ionomycin, a calcium ionophore, induced a low level of CAB mRNA accumulation in the dark which could be further enhanced by light treatment. We propose that calmodulin activation by light is necessary but not sufficient to induce maximal CAB expression.

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.

Characteristics of Five New Photoautotrophic Suspension Cultures Including Two Amaranthus Species and a Cotton Strain Growing on Ambient CO2 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 CO(2) (about 0.06 to 0.07% in the culture room) while the other strains required elevated CO(2) levels (5%). Photoautotrophy was indicated by the requirement for CO(2) 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 C(4)Amaranthus species. The cells showed high dark respiration rates and had lower net CO(2) fixation under high O(2) conditions. Dark CO(2) 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 CO(2) conditions showed the unique properties of a high RuBPcase activation level in ambient CO(2) and a stable ability to show net CO(2) fixation in 21% O(2) conditions.

Metabolism and compartmentation of dihydrozeatin exogenously supplied to photoautotrophic suspension cultures of Chenopodium rubrum

[(3)H]Dihydrozeatin supplied to photoautotrophically growing cell suspension cultures of Chenopodium rubrum was rapidly taken up and metabolized by the cells. The predominant metabolites in extracts of the cells were [(3)H]dihydrozeatin-O-glucoside and [(3)H]dihydrozeatin riboside-O-glucoside. Both these compounds could be shown to be compartmented within the vacuole, whereas [(3)H]dihydrozeatin and [(3)H]dihydrozeatin riboside, which were both present to a minor extent in cell extracts, were both present to a minor extent in cell extracts, were localized predominantly outside the vacuole. Analysis of the culture medium at the end of the 36-h incubation period showed that there had been an efflux of [(3)H]dihydrozeatin metabolites out of the cells. Whereas [(3)H]dihydrozeatin riboside was found to be the major extracellular [(3)H]dihydrozeatin metabolite, the O-glucosides of neither this compound nor [(3)H]dihydrozeatin could be detected in the medium. The differential compartmentation of [(3)H]dihydrozeatin metabolites found with the C. rubrum suspension-culture system is discussed with respect to possible mechanisms governing the metabolism of cytokinins in plants cells.

Photoautotrophic Suspension Cultures I-Obtention of Photoautotrophic Cultures from Euphorbia characias L.

Summary The aim of this study was to obtain photoautotrophic suspension cultures from Euphorbia characias L., a Mediterranean species with high isoprenoid content. Calluses were initiated in photomixotrophy on a modified Murashige and Skoog (MS) medium supplemented with NAA and BAP. Selection and subculture led to chlorophyllous and friable calluses which were used to initiate suspension cultures. Batch cultures were performed in 250 ml closed conical flasks and analysed with respect to cell density, sugar consumption and chlorophyll content. Cells in the early stationary phase exhibited good photosynthetic potency, allowing transfer into bioreactors where cells have been grown photoautotrophically since February 1984.