Physical properties of the cell wall of photoautotrophic suspension cells fromChenopodium rubrum L.

Photoautotrophic suspension cells ofChenopodium rubrum were used to determine Donnan potential, charge density and pore-radius distribution in the cell wall. Experiments were done either with turgescent cells or with isolated cell walls. Titration of a cell-wall-generated 9-aminoacridine fluorescence quench with salts of mono- and divalent cations was used to determine Donnan potential and charge density. The experiments and theory were adapted from measurements of membrane surface charges. A tenfold increase in ionic strength, which decreases the repellant forces between charges of the same sign, led to an approximately threefold increase in the measured charge density, thus resulting in a much smaller decrease of the Donnan potential than would be expected if the charge density remained fixed. This decreased influence of ionic strength on the Donnan potential, resulting from the elasticity of the cell wall, was also measurable but less pronounced when the wall of intact cells was stretched by turgor. The porosity of the cell wall was determined by longterm uptake of polyethylene glycols of different molecular weights, and by gel filtration of polyethylene glycols and dextrans as well as mono- and disaccharides using intact suspension cells as matrix. Both methods gave a mean pore diameter of about 4.5 nm and a maximum pore size of 5.5 nm. The resulting pores-size distribution was slightly broader with the latter method.Abbreviations 9-AA 9-aminoacridine - DMBr₂ decamethoniumbromide=N,N,N,N,N,N hexamethyldecane-1,10-diaminebromide - DW dry weight after lyophilization - EDTA ethylene diaminetetra acetic acid - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FW fresh weight - Mops 3-(N-morpholino)propanesulfonic acid - MW molecular weight - PEG polyethylene glycol     

Production of betalains by suspension cultures of Chenopodium rubrum L.

Red-violet cell suspension cultures of Chenopodium rubrum were found to accumulate the betacyanins amaranthin, celosianin and betanin and the betaxanthins vulgaxanthin I and vulgaxanthin II. Under a 16-h daylight regime the cells accumulated 0.3–0.4% betacyanins on a dry mass basis after 2–3 weeks of cultivation on the growth medium. Experiments to define a production medium for betacyanins failed with this habituated line. The accumulation could however be increased up to 1% or 100 mg betacyanins/1 by feeding tyrosine and by adaptation of the inoculum size to the nutrient concentration.     

Floral and growth responses in Chenopodium rubrum L. to an extract from flowering Nicotiana tabacum L.

Flowering of Chenopodium rubrum seedling plants was obtained in continuous light after application of fractions of a partially purified extract from leaves of flowering Maryland Mammoth tobacco (Nicotiana tabacum). The stage of flowal differentiation was dependent on the age of the Chenopodium plants used for the bioassay. Apices of plants treated with the extract at the age of four or seven days showed an advanced branching of the meristem or the beginning of formation of a terminal flower; treatment with the extract of plants 12 d old resulted in rapid formation of flower buds in all assay plants. Non-treated control plants kept in continuous light remained fully vegetative. The effects of the extract on flowering were associated with pronounced growth effects. Floral differentiation was preceeded by elongation of the shoot apex. Extension of all axial organs occurred, while growth of leaves, including leaf primordia, was inhibited. The pattern of growth after application of the flower-inducing substance(s) did not resemble the effects of the known phytohormones, but showed some similarities to growth changes resulting from photoperiodic induction of flowering.     

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.     

Light quantity and quality interactions in the control of elongation growth in light-grown Chenopodium rubrum L. seedlings

The spectral control of hypocotyl elongation in light-grown Chenopodium rubrum L. seedlings has been studied. The results showed that although the seedlings responded to changes in the quantity of combined red and far-red radiation, they were also very sensitive to changes in the quantity of blue radiation reaching the plant. Altering the proportion of red: far-red radiation in broad waveband white light caused marked differences in hypocotyl extension. Comparison of the responses of green and chlorophyll-free seedlings indicated no qualitative difference in the response to any of the light sources used, although photosynthetically incompetent plants were more sensitive to all wavelengths. Blue light was found to act primarily of a photoreceptor which is different from phytochrome. It is concluded that hypocotyl extension rate in vegetation shade is photoregulated by the quantity of blue light and the proportion of red: far-red radiation. In neutral shade, such as that caused by stones or overlying soil, hypocotyl extension appears to be regulated primarily by the quantity of light in the blue waveband and secondarily by the quantity of light in the red and far-red wavebands.Abbreviations B blue - FR far-red - k ₁, k ₂ rate constants for photoconverison of Pr to Pfr and Pfr to Pr, respective - k ₁/k ₁ +k ₂= phytochrome photoequilibrium - k ₁ +k ₂= phytochrome cycling rate - Pr=R absorbing form of phytochrome - Pfr=FR absorbing form of phytochrome - P_tot Pr+Pfr - PAR photosynthetically active radiation = 400–700 nm - R red - WL white light     

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)     

Crop/weed gene flow:Chenopodium quinoa Willd. andC. berlandieri Moq.

Introduction of the Andean grain chenopod (Chenopodium quinoa) into North America placed this crop within the distributional range of a related wild species,C. berlandieri. This wild species, native to the North American flora, is cross-compatible withC. Quinoa. Isozyme analysis of progeny fromC. berlandieri plants growing within and at the periphery of theC. Quinoa fields, combined with fertility assessment and phenetic comparison among putative hybrids and parental types, indicates that over 30% of progeny from wild plants growing as weeds withC. quinoa in 1987 were crop/weed hybrids. This high incidence of interspecific gene flow from crop to weed appears to be the result of asymmetric pollen flow to free-living plants from high-density cultivated populations. The observed level of crop/weed hybridization, combined with heterosis and partial fertility of F₁ crop/weed hybrids, suggests that repeated annual cycles ofC. quinoa cultivation within the North American range ofC. berlandieri could produce introgressive change among sympatric wild populations. In terms of risk assessment for biotechnology, these results suggest that the breeding system may not provide an accurate indication of the potential for genetic interaction among predominately self-pollinating grain crops and their free-living relatives.     

Calmodulin regulates the Ca2+-dependent slow-vacuolar ion channel in the tonoplast of Chenopodium rubrum suspension cells

The patch-clamp technique was applied to vacuoles isolated from a photoautotrophic suspension cell culture of Chenopodium rubrum L. and vacuolar clamp currents, which are predominantly carried by the previously identified Ca²⁺-dependent slow vacuolar (SV) ion channels, were recorded. These currents, which were activated by 1-s voltage pulses of -100 mV (vacuolar interior negative) in the presence of 100 M Ca²⁺ (cytosolic side), could be blocked completely and reversibly by the calmodulin antagonist W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide] and its chlorine-deficient analogue W-5; half-maximum inhibition was found at approx. 6 M for W-7 and 70 M for W-5. Inhibition was reversed by addition of 1 g · ml^–1 calmodulin purified from Chenopodium cell suspensions; reversal by bovine brain calmodulin was scarcely appreciable. We conclude that cytosolic calmodulin mediates the Ca²⁺ dependence of the SV-channel in the Chenopodium tonoplast.Abbreviations SV-channel slowly activated, vacuolar ion channel - W-5 N-(6-aminohexyl)-1-naphthalenesulfonamide - W-7 N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide We acknowledge support by the Deutsche Forschungsgemeinschaft and the Bundesminister für Forschung und Technologie, Bonn, and by the Justus-Liebig-Universität Giessen (to W.B.)     

Phytochrome and internode elongation in Chenopodium polyspermum L. sites of photoreception

The elongation of the fourth internode of fully green Chenopodium polyspermum L. is modulated by far-red light (FR) given in addition to the main light period. Two different types of organs are responsible for the photoreception of FR producing the end-of-day effect; the stem and the leaves situated just above and below the reacting internode. Photoreversibility can be obtained within certain limits in the two organs. Evidence is presented which shows that in the fully green plant there is an interorgan reaction whose primary reaction is the photoconversion of phytochrome.Abbreviations and Symbols D darkness - FR far red light - R red light - P phytochrome - P_FR phytochrome in the FR absorbing form - 9+15 D (or light treatment) photoperiod of a 9 h main light period followed by 15 h of D (or light treatment)     

The relationship between phytochrome-photoequilibrium and Development in light grown Chenopodium album L.

Chenopodium album seedlings were grown in light environments in which supplementary far-red light was mixed with white fluorescent light during various parts of the photoperiod. Both the logarithmic rate constant of stem extension and the leaf dry weight: stem dry weight ratio were linearly related to estimated phytochrome photoequilibrium () in each treatment regime. These data are taken to be indicative of a functional link between phytochrome and development in the green plant. A layer of chlorophyllous tissue only affected the linearity between calculated and the logarithmic stem extension rate at high chlorophyll concentrations, whilst even low concentrations-equivalent to the levels found in stem tissue-caused a significant shift in measured . End-of-day supplementary far-red (FR) light induced between 0–35 per cent of the response elicited by all-day supplementary FR, whilst daytime supplementary FR (with a white fluorescent light end-of-day treatment) induced approximately 90 per cent. The ecological significance of this difference is discussed with respect to shade detection.Paper 7 in the series The function of phytochrome in the natural environment [for paper 6 see McLaren, J.S., Smith, H., Plant, Cell and Environment 1, 61–67, 1978]     

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.     

The serine carboxypeptidase like gene family of rice (Oryza sativa L. ssp. japonica)

Serine carboxypeptidases (SCPs) comprise a large family of protein hydrolyzing enzymes and have roles ranging from protein turnover and C-terminal processing to wound responses and xenobiotic metabolism. The proteins can be classified into three groups, namely carboxypeptidase I, II and III, based on their coding protein sequences and the fact that each family is characterized by a central catalytic domain of unique topology designated as the “α/β hydrolase fold”. The available SCP protein sequences have been utilized as datasets to build a HMM (hidden Markov model) profile, which is used to search the rice (Oryza sativa L. ssp. japonica) proteome. A total of 71 SCP and serine carboxypeptidase-like (SCPL) protein-coding genes exist in rice. The intron-exon structure, chromosome localization, expression and characteristics of encoded protein sequences of the 71 putative genes are reviewed.     

Synthesis of hydroxycinnamic acid esters of betacyanins via 1-O-acylglucosides of hydroxycinnamic acids by protein preparations from cell suspension cultures of Chenopodium rubrum and petals of Lampranthus sociorum

Protein preparations from cell suspension cultures of Chenopodium rubrum L. and petals of Lampranthus sociorum (L.Bol.) N.E.Br. (Mes.C.L.Bol.) catalyzed the formation of acylated betacyanins, i.e. celosianin I and II (p-coumaroyl-and feruloylamaranthins) and lampranthin I and II (p-coumaroyl- and feruloylbetanins), from 1-O-(p-coumaroyl)-and 1-O-feruloyl--glucoses as acyldonors and the respective acceptor molecules amaranthin (betanidin 5-O-sophorobiuronic acid = betanidin 5-O--[12]-glucuronosyl--glucoside) and betanin (betanidin 5-O--glucoside). The enzymes involved could generally be classified as 1-O-hydroxycinnamoyl--glucose:betanidinglycoside O-hydroxycinnamoyltransferases (EC 2.3.1.-).Abbreviations HCA hydroxycinnamic acid - HCA hydroxycinnamoyl (=hydroxycinnamic acid-ester moiety) - HPLC high-performance liquid chromatography - TLC thin-layer chromatography     

Pharmacology of the SV channel in the vacuolar membrane of chenopodium rubrum suspension cells

Single channel performance and deactivation currents have been analyzed in the presence of cation channel blockers to reveal pharmacological properties of the slow-activating (SV) cation-selective ion channel in the vacuolar membrane (tonoplast) isolated from suspension cells of Chenopodium rubrum L. At a holding potential of –100 mV, the SV channel showed half-maximal inhibition with 20mm tetraethylammonium (TEA), 7 m 9amino-acridine, 6 m (+)-tubocurarine, 300nm quinacrine, and 35 m quinine, respectively. The SV channel is also blocked by charybdotoxin (20nm at –80 mV) but not by apamine. 9-Amino-acridine, (+)-tubocurarine and quinacrine act in a voltage-dependent fashion, binding to the open channel and to different sites along the transmembrane voltage profile according to Woodhull (J. Gen. Physiol. 61:687–708, 1973). No binding site could be specified for charybdotoxin, which binds to the closed channel, and for quinine. Except for quinine, all tested blockers were effective only if added to the cytoplasmic side of the tonoplast. A structural relationship between the SV channel and Maxi-K channels in animal systems is inferred.We are grateful to Prof. F. Dreyer and Dr. J. Beise from the Pharmacology Department of the Justus-Liebig-Universität Giessen for continuous interest and helpful suggestions. This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Be 466/21-5) and the Bundesminister für Forschung und Technologie, Bonn.     

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.