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In this study, a correlation is described between low cytoplasmic pH, measured with the fluorescent probes 2',7'-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 produdion 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 i s discussed.Some plant tissues are able to produce substantial amounts of secondary metabolites. However, the production of these compounds in plant cell and tissue cultures is generally low. A number of treatments have been described that lead to an increased production; these treatments often stimulate the defense response in the cell (Scheel and Parker, 1990).In a previous paper (Hagendoom et al., 1991a), we ana- 723lyzed the effect of elicitors on the plasma membrane of Petunia hybrida cell suspensions. It appeared that changes in the activity of the plasma membrane ATPase, and subsequently of ApH, were sufficient to increase PAL activity and product formation in these cells. This conclusion was based on two different experimental approaches. First, we tested a number of plasma membrane ATPase inhibitors and ionophores that specifically influence ApH, A*, or both for their ability to activate secondary metabolism. Second, we directly showed changes in cytoplasmic pH upon activation of a secondary metabolic pathway with the aid of the fluorescent probe oxonol VI.In this paper we show that this relation between low cytoplasmic pH and secondary metabolite production is not limited to P. hybrida and the products of the PPP but can also be observed in different model systems. We investigated the production of secondary metabolites by cell suspensions derived from three plant species belonging to three different fa...

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Primary processes during elicitation of the phenylpropanoid pathway (PPP) were studied in Petunia hybrida cell suspensions. We tested the hypothesis that decrease of the proton gradient across the plasma membrane activates the PPP. Induction of the PPP was determined by measuring phenylalanine ammonia Iyase activity. A variety of ATPase inhibitors and ionophores were tested for the ability to elicit the PPP. The ATPase inhibitors orthovanadate and NN'-dicyclohexylcarbodiimide and the ionophores carbonyl cyanide-4-trifluoromethoxyphenylhydrazone and nigericin were all effective elicitors. Carbonyl cyanide-4-trifluoromethoxyphenylhydrazone and nigericin elicit also when used in combination with NN'-dicyclohexylcarbodiimide. Valinomycin had little effect on phenylalanine ammonia Iyase activity. Treatment with orthovanadate or nigericin led to the formation of lignin. Alkalinization of the external medium by NN'-dicyclohexylcarbodiimide, carbonyl cyanide-4-trifluoromethoxyphenylhydrazone, and nigericin was observed directly with the use of a sensitive pH electrode and internal acidification was deduced from the changes in emission intensity of the fluorescent probe bis [3-propyl-5-oxoisoxazol-4-yl] pentamethineoxonol. These data indicate that changes in the activity of the plasmamembrane H+-ATPase, and subsequent decrease of the proton gradient (particularly of the pH gradient) by itself are sufficient to influence phenylalanine ammonia Iyase activity of P. hybrida cells and are therefore important intermediates in signal transduction.In plant cell and tissue cultures, generally no significant production of secondary metabolites is observed. To initiate this production, many strategies have been followed, such as treatment with so-called elicitors (see ref. 8 and references therein). For example, biotic elicitors, isolated from plant pathogens, induced the production of isoflavonoid phytoalexins in soybean (7). Also abiotic elicitors, such as high medium osmolarity (15), are reported to induce the production of secondary metabolites.

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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 valinomytin-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. Proteoliposome Membrane potential Diffusion potentialCarotenoid shifr Oxonol VI

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