M.J.M. Hagendoorn

Relation between primary and secondary metabolism in plant cell suspensions

Cell suspensions ofMorinda citrifolia are able to produce large amounts of anthraquinones (AQ) when they are cultivated on a B5-medium containing 1 mg 1-1 naphtyl acetic acid (NAA); this production is inhibited by addition of 2,4-dichloro-phenoxyacetic acid (2,4-d). Also during cultivation on 1 mg 1-1 2,4-d AQ-production is absent.It appeared that in the presence of NAA a kind of ‘AQ-production’ program is switched on: cell division rate is low as well as metabolic activity, while endogenous sugar levels are high. The same properties develop in the presence of auxins like indolyl-acetic acid and p-chloro-phenylacetic acid. With 2,4-d and related auxins (like p-chloro-phenoxyacetic acid) AQ production is absent and emphasis is laid on a developmental program characterized by high cell division rates, high metabolic activity and low endogenous sugar contents. Independent of the type of auxin applied, the cells grow as a suspension consisting of finely dispersed cells. The ‘AQ-producing differentiation program’ cannot be maintained during a consecutive series of subculturings: with increasing AQ-contents the viability of the cells and the cell division rate decrease.The possible mechanisms of regulation of AQ-production by auxins are discussed as well as the advantages of the use of theMorinda model system in the study of the relation between growth, primary and secondary metabolism.

Cytoplasmic Acidification and Secondary Metabolite Production in Different Plant Cell Suspensions (A Comparative Study)

In this study, a correlation is described between low cytoplasmic pH, measured with the fluorescent probes 2[prime],7[prime]-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (acetoxymethyl ester) and bis- [3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol, and the production of secondary metabolites for several plant cell-suspension systems. Anthraquinone production in Morinda citrifolia suspensions is negligible in the presence of 2,4-dichlorophenoxyacetic acid (2,4-D), whereas with naphthalene acetic acid (NAA) a significant accumulation is realized. NAA-grown cells showed a lower cytoplasmic pH than did 2,4-D-grown cells. Addition of 2,4-D or parachlorophenoxy acetic acid to NAA-grown cells resulted in an inhibition of anthraquinone production and an increase of the cytoplasmic pH, whereas addition of parachlorophenyl acetic acid had no effect on either parameter. Lignin production in Petunia hybrida cells could be induced by subculturing them in a medium without iron. These cells showed a lower cytoplasmic pH than control cells. Addition of Fe3+ led to a decreased lignin content and an increased cytoplasmic pH. Two cell lines of Linum flavum showed a different level of coniferin and lignin concentration in their cells. Cells that accumulated coniferin and lignin had a lower cytoplasmic pH than cells that did not accumulate these secondary metabolites. Apparently, in different species and after different kinds of treatment there is a correlation between acidification of the cytoplasm and the production of different secondary metabolites. The possible role of this acidification in secondary metabolite production is discussed.

Orthovanadate induced lignin production, in batch and continuous cultures of Petunia hybrida

Summary In Petunia hybrida cv. Violet 30 cell suspensions, phenylalanine ammonia lyase (PAL), the first enzyme of the phenylpropanoid pathway (PPP), can be induced by various treatments, e.g. biotic elicitors (sterilized microorganisms, nigeran) or abiotic elicitors (orthovanadate, pH-shift). Orthovanadate activated the branch of the PPP, leading to production of lignin-like material. Significant production especially occurred after elicitation in the early growth phase. Six days after elicitation an increase was observed in the amount of substances, coloured by phloroglucinol, as well as in the amount of insoluble cell constituents. The product could be identified as a guaiacyl type lignin by pyrolysis mass spectrometry and pyrolysis gas chromatography mass spectrometry. In contrast, both guaiacyl and syringyl type lignin was found by the same techniques in xylem of the fully grown plants. Increase of the pH in continuous cultures of Petunia hybrida cells also led to production of lignin-like material. Interference with the pH gradient across the plasma membrane (pH-shift or inhibition of H + -ATPases by orthovanadate) apparently activates the branch of the PPP leading to lignin production.

ATP synthesis by ATPase proteoliposomes from the thermophilic cyanobacterium Synechococcus 6716 by ionophore-induced electric potentials and proton gradients

Abstract ATP synthesis driven by low pre-established electric potentials and pH gradients is studied in large ATPase proteoliposomes, prepared from the ATPase complex and native lipids from the thermophilic cyanobacterium Synechococcus 6716. Electric potentials and pH gradients were achieved by valinomycin and nigericin, respectively, in the presence of a K + gradient across the membrane. External base-pulses were also applied. In this system ATP synthesis driven by valinomycin-induced K + influx, nigericin-induced internal acidification and by external base-pulses is demonstrated. Electric potentials and pH gradients of equivalent size lead to roughly similar ATP synthesis activities. ATP synthesis is optimal at 80–100 nM valinomycin and at 0.75−1 μM nigericin at the proper pre-set ion gradients. Uncoupler and DCCD inhibit ATP synthesis. Prior activation of the complex by thiol agents or trypsin was not required for synthesis activity. The ATP synthesis rate increases with the size of electric potential or pH gradient. The threshold value of the electrochemical gradient for significant ATP synthesis is about 30 mV. ATP production proceeds for more than 60 min. The generation of ionophore-induced electric potentials and pH gradients have been followed by oxonol VI and intraliposomal Neutral red, respectively. The extent of the absorbance changes of both probes is proportional to the size of electric potential or pH gradient. Ionophore-induced oxonol VI and Neutral red responses are stable for at least 30 min. The results are discussed in terms of membrane permeability and vesicle size.

The use of carotenoids and oxonol VI as probes for membrane potential in proteoliposomes

Carotenoids present in lipids extracted from the cyanobacterium Synechococcus 6716 indicate trans‐membrane potential in proteoliposomes reconstituted from these lipids and the ATPase complex isolated from the same organism. A carotenoid absorbance band shift to a longer wavelenght is obtained with valinomycin‐induced potassium ion diffusion potentials, irrespective of the polarity of the potassium gradient. In contrast to this, the (externally added) probe oxonol VI only shows an absorbance band shift when the external potassium ion concentration is higher than the internal one. In liposomes without ATPase complex, no carotenoid absorbance band shifts were observed.

Anthraquinone glycosylation and hydrolysis in Morinda citrifolia cell suspensions: Regulation and function

Summary Morinda citrifolia cells grown in suspension are able to accumulate significant amounts of anthraqui-nones (AQ) when grown in a low-auxin medium. As most of these anthraquinones are stored as glycosides, we studied the nature of the sugar moiety and several aspects of the glycosylation and hydrolysis reactions involved in the synthesis and degradation of these secondary metabolites. The sugar moiety of anthraquinones produced by Morinda cell suspensions appeared to be the disaccharide primverose (6-O-β-D-Xylopyranosyl-D-glucose). Extracts of AQ-producing cells contained also free primverose that was probably formed during the extraction procedure by the action of hydrolytic enzymes. This hydrolytic capacity was also present in non-AQ-producing, 2,4-D grown Morinda cells. These non-producing cells were also used for feeding experiments in which the capacity of Morinda cells to process added AQ (aglucons and glycosides) was tested. Addition of the aglucon alizarin (in the presence of DMSO) resulted in uptake and glycosylation; extracts of these cells also contained primverose, indicating that the normal glycosylation machinery was also active in these non-producing cells. Addition of AQ glycosides (a mixture of alizarin-primveroside and lucidin-primveroside) also resulted in uptake and processing of the added metabolites. Successive hydrolysis and glycosylation reactions lead to cells which contain a mixture of AQ aglucons and AQ glycosides after a few days of incubation. Apparently the mechanism for hydrolysis and glycosylation of AQ is also present or rapidly induced in cells that do not produce these compounds themselves, indicating that glycosylation of AQ and subsequent storage in the vacuoles probably is not a crucial step in the regulation of the production of these compounds. The significance of the presence of this hydrolysis and glycosylation machinery for the handling of e.g. xenobiotics is discussed.

ABA reduces respiration and sugar metabolism in developing carrot (Daucus carota L.) embryoids.

Summary Addition of abscisic acid (ABA) to developing carrot embryoids affects respiration and carbohydrate metabolism. Non-treated embryoids have a high level of respiration expressed per gram protein and consume al most all of their endogenous carbohydrates during the ten day culture period. In contrast, embryoids grown with either 1.9 or 38 µM ABA, have a lowered respiration rate and maintain their carbohydrate contents at 20 % of the DW. Embryoids acquire complete desiccation tolerance, when they are treated with 38 µM ABA, whereas only 65 % of the embryoids survive desiccation with 1.9 µM ABA. The reduced respiration of the developing embryoids might result in lower free radical levels after dehydration, in this way preventing a subsequent viability loss. We suggest that there is a relation between viability loss due to desiccation and respiration rate, although the latter is not the only factor involved.

Cell division versus secondary metabolite production in Morinda citrifolia cell suspensions

Summary Morinda citrifolia cell suspension cultures are capable of accumulating high levels of anthraquinones, but appear to display an inverse relationship between the rate of cell division and anthraquinone production: low auxin concentrations lead to a high anthraquinone production and a low growth rate while at high auxin concentrations the reverse was observed. NAA and 2,4-D both showed this effect although there was a difference with respect to the optimal concentration, 2,4-D being active at approximately a hundred-fold lower concentration than NAA. We studied the inverse relation between cell division and anthraquinone accumulation in two ways. First, batch cultures of Morinda citrifolia were treated with hydroxyurea, which almost completely inhibited cell division. The 2,4-D cultures still did not accumulate anthraquinones. Second, we compared continuous cultures grown in the presence of NAA or 2,4-D at the same cell division rates. Only the NAA culture accumulated anthraquinones. The overall high sugar content of the cells under the various cultivation conditions indicated that sufficient energy and carbon substrates were always present. The possible direct effect of auxins on the induction and activity of the pathways leading to anthraquinone production is discussed.

Accumulation of anthraquinones in Morinda citrifolia cell suspensions

Cell suspensions of Morinda citrifolia were cultivated in a B5-medium containing 4% sucrose as the sole carbon source and 1 mg l-1 naphthyl acetic acid (NAA) or 1 mg l-1 2,4-dichloro-phenoxyacetic acid (2,4-D). Both auxins were able to support growth but only in the presence of NAA anthraquinone production was observed. 2,4-D inhibited the production in NAA cultures. Anthraquinone synthesis took place in the growth and the stationary phase and amounts of 0.2-0.4 mmol (about 100–200 mg) g-1 dry weight could be reached.

Comparison of ATP synthesis efficiencies in ATPase proteoliposomes of different complexities

Abstract ATP synthesis by the ATPase complex of the thermophilic cyanobacterium Synechococcus 6716 has been studied in each of three reconstituted proteoliposome systems: • ATPase complex reconstituted with the native lipids of Synechococcus 6716 by detergent dialysis. ATP synthesis of about 100 nmol min−1 (mg protein)−1 was observed at 50 ° C after a valinomycin-induced K+ diffusion potential (inside positive), nigericin-induced K+-H+ exchange (internal pH decrease) and an external base pulse, equivalent to about 65 mV. • ATPase complex co-reconstituted with bacteriorhodopsin of Halobacterium halobium and soybean phospholipids by sonication and detergent removal by gel filtration. Light-induced ATP synthesis at 40 ° C of 70 nmol min−1 (mg ATPase protein)−1 was obtained. Absorbance changes of the electric potential-indicating probe oxonol VI induced by flashed and continuous light were observed. In this preparation, with the pH indicator neutral red, only light-driven external alkalinization could be detected. • ATPase complex co-reconstituted by detergent dialysis with bovine-heart ubiquinol: cytochrome c oxidoreductase and photosynthetic reaction centres of Rhodopseudomonas sphaeroides and soybean phospholipids, with intraliposomal cytochrome c and additional Q-10. Light-driven synthesis of about 1.5 nmol min−1 (mg ATPase protein)−1 was obtained at 40°C. Flash-induced internal cytochrome c reduction was also demonstrated in this preparation.