The lipids in photoautotrophic and heterotrophic cell suspension cultures of Chenopodium rubrum

Resembling the lipids in the leaves and other green organs of intact plants, the lipids in photoautotrophic cell cultures of Chenopodium rubrum were found to contain high proportions of monogalactosyldiacylglycerols and digalactosyldiacylglycerols, as well as fair amounts of sulfoquinovosyldiacylglycerols and diacylglycerophosphoglycerols. Conversely, the heterotrophic cell cultures, from which the photoautotrophic cultures had been derived, contained only traces of these compounds. The heterotrophic cultures were rich in sterols, sterol esters, sterol glycosides, and esterified sterol glycosides. The lipids of photoautotrophic cell cultures contained higher proportions of constituent linolenic acid, but lower concentrations of linoleic acid than those of heterotrophic cultures. In the photoautotrophic cultures, as in green leaves, linolenic acid was predominantly estrified in monogalactosyldiacylglycerols and digalactosyldiacylglycerols. This investigation shows that it is possible to select strains of cell cultures, which are capable of grosing photoautotrophically, with the aim of activating the biosynthesis of specific metabolites.     

Electrical membrane potential and resistance in photoautotrophic suspension cells of Chenopodium rubrum L.

On photoautotrophically grown, suspension-cultured cells of Chenopodium rubrum L. the electrical potential difference V _mand the electrical resistance across plasmalemma and tonoplast have been measured using one or two intracellular micro-electrodes. In a mineral test-medium of 5.8 mM ionic strength V _mvalues between 100 and 250 mV, 40% thereof between 170 and 200 mV, and a mean value (±S.E.M.) of 180.6±3.4 mV have been recorded. The average membrane input resistance R _mwas 269±36 M, corresponding to an average membrane resistivity r _mof 3.0 m². V _mand r _mare sensitive to light, temperature, and addition of cyanide, suggesting the presence of an electrogenic hyperpolarizing ion pump, and are ascribed essentially to the plasmalemma. A hexose-specific saturable electrogenic membrane channel is identified through a decrease of V _mand r _mupon addition of hexoses. The hexoseconcentration-dependent depolarization V _msaturates at 92 mV and returns half-saturating concentrations (apparent k _mvalues) of 0.16 mM galactose, 0.28 mM glucose, and 0.48 mM fructose. The magnitude of V _mand r _mwell agrees with pertinent data from mesophyll cells in situ (where only V _mdata are available) and from photoautotrophic lower plant cells. However, V _mis markedly higher than reported for heterotrophically grown suspension cells of different higher plants (with which r _mdata have not been reported so far). It is concluded from the present study and a companion paper on water transport (Büchner et al., Planta, in press) that photoautotrophically grown Chenopodium suspension cells closely resemble mesophyll cells as to cell membrane transport properties.Abbreviations V _m membrane potential(mV) - R _o input resistance () - R _m membrane input resistance () - r _m specific resistance (resistivity) of the membrane (m²)     

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.     

Net fluxes and pools of nitrogenous compounds during suspension culture of photoautotrophic Chenopodium rubrum cells

Abstract. Suspension cultured cells of Chenopodium rubrum were grown photoautotrophically under a diurnal light-dark cycle of 16-8h. The following phases of the batch culture were differentiated: a short lag, a cell division phase terminated by a pronounced transition to stationary maintenance which finally gradually passed into senescence. Nitrogen fluxes typical of these stages were followed by measuring uptake of NO₃ and NH₄⁺ from the medium and their incorporation into the cellular fractions of nitrogenous compounds. Activities of seven N-metabolizing enzymes were determined. Compartmentation of enzymes and nitrogenous compounds was analysed after isolation of intact chloroplasts and vacuoles from protoplasts. Eighty-two per cent of the N originally present in the medium was taken up and incorporated to an extent of 80% into protein until the end of the division phase. Net protein synthesis ceased upon transition to the stationary phase. During the division phase a vacuolar pool of NO₃ was established and then maintained throughout the resting phase. Free cellular NH₄⁺ was not localized within the vacuole and responded to the ammonium content of the medium. Amino acids accumulated in the cells especially during the stationary phase, during which they were present in the vacuole. Typical nitrogen relations are portrayed as flux diagrams for one day of each of the essential developmental phases. The enzyme activities were easily sufficient to account for the observed flow rates of the corresponding nitrogenous compounds. Hence, uptake of NO₃ and NH₄⁺ must be considered as steps limiting N metabolism in Chenopodium rubrum cell suspensions.     

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

[³H]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 [³H]dihydrozeatin-O-glucoside and [³H]dihydrozeatin riboside-O-glucoside. Both these compounds could be shown to be compartmented within the vacuole, whereas [³H]dihydrozeatin and [³H]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 [³H]dihydrozeatin metabolites out of the cells. Whereas [³H]dihydrozeatin riboside was found to be the major extracellular [³H]dihydrozeatin metabolite, the O-glucosides of neither this compound nor [³H]dihydrozeatin could be detected in the medium. The differential compartmentation of [³H]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.Abbreviations (diH)Z dihydrozeatin - (diH) [9R]Z 9--D-ribofuranosyl dihydrozeatin - HPLC high-performance liquid chromatography - ODS octododecyl silica - PEP phosphoenolyruvate     

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.Abbreviation PCR polymerase chain reaction We would like to thank Michaela Bittner, Rainer Ehneß and Monika Kammerer for skillful technical assistance and S. Buchhauser and H. Hallmer for photographic work. This work was supported by the Deutsche Forschungsgemeinschaft (SFB 43) and by Fonds der Chemischen Industrie.     

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.     

Charybdotoxin blocks cation-channels in the vacuolar membrane of suspension cells of Chenopodium rubrum L.

Using the patch-clamp technique, we studied the action of charybdotoxin which blocks Ca(2+)-activated large-conductance K+ channels in animal tissue on the slow-activating (SV), Ca(2+)-activated cation channel in the vacuolar membrane of suspension-cells of Chenopodium rubrum L. The toxin reversibly reduced the vacuolar current with EC50 approximately 20 nM suggesting structural similarities between ion channels in animal and plant membranes.     

Alterations of the chlorophyll-protein pattern in chronically photoinhibited Chenopodium rubrum cells

When photoautotrophic Chenopodium rubrum L. culture cells were exposed to high photon flux densities for seven consecutive light periods a marked reduction in photochemical efficiency, chlorophyll (Chl) content and Chl a/b ratio occurred. These alterations were accompanied by distinct changes in the pigment and protein composition of the thylakoid membranes. In photosystem II (PSII) a reduction in the relative contents of proteins from the reaction center (D1 protein, D2 protein and Cyt b₅₅₉) and the inner antenna (CP43 and CP47) was observed. In agreement with the reduction in the Chl a/b ratio an increase in the relative content of the major light-harvesting complex of PSII (LHCII) could be demonstrated. The minor chlorophyll-proteins of PSII were only slightly affected but PSI (quantified as total complex) showed a reduction upon chronic photoinhibition. The changes in protein composition were accompanied by a drastic increase in the contents of lutein and the xanthophyll-cycle pigments and by a reduction in the β-carotene content. The effects on lutein and xanthophyll-cycle pigment content were most pronounced in stroma thylakoids. Here, an increase in LHCII (which harbours these pigments) was clearly detectable. Considering the pigment content of LHCII, the change in its apoprotein content was not large enough to explain the pigment changes.     

Ethylene regulation of apoplastic invertase expression in autotrophic cells of Chenopodium rubrum

Physiological concentrations of ethylene in the growth medium of autotropic suspension culture Chenopodium rubrum L. cells reduced the activity of cell wall bound invertase by 25 – 47%, compared to controls. Northern blot analysis using homologous probe binding to total RNA preparations revealed that reduction in specific activity was paralleled by repression of the corresponding gene.     

Fatty acid composition of Leptospira lipids as a taxonomic criterion

The fatty-acid composition of microbial cells in 17 pathogenic and saprophytic Leptospira strains, comprising 14 serovars and 10 serogroups, has been studied. The strains under investigation have proved to fall into 3 groups differing by this characteristic. The group of saprophytic strains is characterized by a comparatively high level of myristic acid and, consequently, by the ratio of saturated and unsaturated fatty acids with 14 carbon atoms approaching 1:1; besides, it is also characterized by a lower, in comparison with the pathogenic Leptospira strains belonging to the serogroups Icterohaemorrhagiae, Canicola, Ballum has a higher level of unsaturated fatty acids. The second group of pathogenic Leptospira strains including the serogroups Grippotyphosa, Hebdomadis, Pomona, Tarassovi, Pyrogenes, Australia has been found to occupy an intermediate position between the first group of pathogenic Leptospira strains and the group of saprophytic ones. As the difference in the content of myristic acid in pathogenic and saprophytic Leptospira strains is a stable characteristic, it can be used for the differentiation of these strains. The present investigation has revealed that the distribution of the main fatty acids in Leptospira phospholipids is similar to their distribution in Leptospira neutral lipids with the exception of unsaturated fatty acid with 14 carbon atoms, occurring mainly in phospholipids.     

Fatty acid composition of total lipids and phospholipids of membrane preparations of transport ATPases

The fatty acid composition of total lipids and phospholipids of duck salt gland Na,K-ATPase (outer plasma membrane) and of rabbit skeletal muscle Ca-ATPase (intracellular membrane) was investigated. The bulk of Na,K-ATPase fatty acids is represented by palmitic (16:0), oleic (18:1), stearic (18:0) and arachidonic (20:4) acids. The duck salt gland is characterized by rather a high content of unsaturated fatty acids, especially of arachidonic acid. The unsaturation index of total-lipid fatty acids increases during purification of these preparations in the following order: homogenate greater than microsomal fraction greater than purified enzyme. The fatty acid composition of Na,K-ATPase total lipids and phospholipids reveals certain differences. Phospholipids contain more stearic and liholeic (18:2) acids than total lipids, but the level of arachidonic acid in them is twice as low. Besides, phospholipids were found to contain polyunsaturated docosohexaenic (22:6) acid. The bulk of fatty acids of rabbit skeletal muscle Ca-ATPase total lipids and phospholipids is represented by 16:0, 18:0, 18:1 and 18:2 acids. The content of polyunsaturated fatty acids in this preparation is much lower than in duck salt gland Na,K-ATPase. The fatty acid composition of total lipids and phospholipids in rabbit skeletal muscle Ca-ATPase differ insignificantly. The differences in the fatty acid composition of membrane preparations under study is conditioned mainly by the fractional composition of their lipids.     

Rhythms as photoperiodic timers in the control of flowring in Chenopodium rubrum L.

Summary In C. rubrum, the amount of flowering that is induced by a single dark period interrupting continuous light depends upon the duration of darkness. A rhythmic oscillation in sensitivity to the time that light terminates darkness regulates the level of flowering. The period length of this oscillation is close to 30 hours, peaks of the rhythm occurring at about 13, 43 and 73 h of darkness.Phasing of the rhythm by 6-, 12- and 18-h photoperiods was studied by exposing plants to a given photoperiod at different phases of the free-running oscillation in darkness. The shift in phase of the rhythm was then determined by varying the length of the dark period following the photoperiod; this dark period was terminated by continuous light.With a 6-h photoperiod the timing of both the light-on and light-off signals is shown to control rhythm phasing. However, when the photoperiod is increased to 12 or 18 h, only the light-off signal determines phasing of the rhythm. In prolonged periods of irradiation-12 to 62 h light—a durational response to light overrides any interaction between the timing of the light period and the position of the oscillation at which light is administered. Such prolonged periods of irradiation apparently suspend or otherwise interact with the rhythm so that, in a following dark period, it is reinitiated at a fixed phase relative to the time of the light-off signal to give a peak of the rhythm 13 h after the dusk signal.In daily photoperiodic cycles rhythm phasing by a 6-h photocycle was also estimated by progressively increasing the number of cycles given prior to a single dark period of varied duration.In confirmation of Bünning's (1936) hypothesis, calculated and observed phasing of the rhythm controlling flowering in c. rubrum accounts for the photoperiodic response of this species. Evidence is also discussed which indicates that the timing of disappearance of phytochrome P_fr may limit flowering over the early hours of darkness.     

Turgor pressure and water transport properties of suspension-cultured cells of Chenopodium rubrum L.

The turgor pressure and water relation parameters were determined in single photoautotrophically grown suspension cells and in individual cells of intact leaves of Chenopodium rubrum using the miniaturized pressure probe. The stationary turgor pressure in suspension-cultured cells was in the range of betwen 3 and 5 bar. From the turgor pressure relaxation process, induced either hydrostatically (by means of the pressure probe) or osmotically, the halftime of water exchange was estimated to be 20±10 s. No polarity was observed for both ex- and endosmotic water flow. The volumetric elastic modulus, , determined from measurements of turgor pressure changes, and the corresponding changes in the fractional cell volume was determined to be in the range of between 20 and 50 bar. increases with increasing turgor pressure as observed for other higher plant and algal cells. The hydraulic conductivity, Lp, is calculated to be about 0,5–2·10^–6 cm s^–1 bar^–1. Similar results were obtained for individual leaf cells of Ch. rubrum. Suspension cells immobilized in a cross-linked matrix of alginate (6 to 8% w/w) revealed the same values for the half-time of water exchange and for the hydraulic conductivity, Lp, provided that the turgor pressure relaxation process was generated hydrostatically by means of the pressure probe. Thus, it can be concluded that the unstirred layer from the immobilized matrix has no effect on the calculation of Lp from the turgor pressure relaxation process, using the water transport equation derived for a single cell surrounded by a large external volume. By analogy, this also holds true for Lp-values derived from turgor pressure changes generated by the pressure probe in a single cell within the leaf tissue. The fair similarity between the Lp-values measured in mesophyll cells in situ and mesophyll-like suspension cells suggests that the water transport relations of a cell within a leaf are not fundamentally different from those measured in a single cell.     

Photoperiodic control of cytokinin transport and metabolism in Chenopodium rubrum

Cytokinin (CK) levels in the short-day plant Chenopodium rubrum L. are known to fluctuate diurnally. The aim of this work was to investigate if the diurnal changes are brought about by changes in transport and/or metabolism of CKs. The effect of photo-period on cytokinin transport was studied by analysing CK concentrations in root, leaf and apical exudates, respectively, under constant light (CL), a 12-h photoperiod (DL) inductive for flowering, DL in which darkness was interrupted at the end of hour 6 by 15 min red light (R), or by 15 min R followed by 30 min far-red irradiation (R/FR). The concentrations of cytokinins (zeatin, zeatin riboside, isopentenyladenine, isopen-tenyladenosine) in all three types of exudates were significantly higher in the first 12-h period after the end of 12 h darkness than in CL. The R break almost fully negated the effect of darkness and its effect was reversed by FR, showing the involvement of phytochrome in the regulation of CK transport. In the next 12-h interval, i.e. 12–24 h after the end of darkness, the CK level remained high in the leaf exudate only, but to a much lower extent than in the previous 12 h. The highest CK concentration (increase by 108%) was observed in apical exudates during inductive darkness. A comparison of the CKs present in the individual exudates indicates that those arriving at the apical part are derived mostly from leaves with varying contributions by the xylem. The metabolism of applied [³H]-zeatin riboside (ZR) was studied using HPLC separation of the metabolites. Metabolism was found to be very rapid and different glucosides, adenine and adenosine were the main metabolites after 12 h incubation with labelled ZR in all regimes tested. The only metabolite that seems to be under photoperiodic control is ZR-5′-monophosphate. It is as yet not clear if photoperiod controls the phosphorylation or dephosphorylation reaction. The activity of the main cytokinin degradative enzyme, cytokinin oxidase, did not change during the photoperiodic regimes tested.