Effect of Elicitors on the Plasmamembrane of Petunia hybrida Cell Suspensions : Role of DeltapH in Signal Transduction.

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 lyase activity. A variety of ATPase inhibitors and ionophores were tested for the ability to elicit the PPP. The ATPase inhibitors orthovanadate and N,N'-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 N,N'-dicyclohexylcarbodiimide. Valinomycin had little effect on phenylalanine ammonia lyase activity. Treatment with orthovanadate or nigericin led to the formation of lignin. Alkalinization of the external medium by N,N'-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 lyase activity of P. hybrida cells and are therefore important intermediates in signal transduction.

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 wavelength 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.

Lipid specificity for the reconstitution of well-coupled ATPase proteoliposomes and a new method for lipid isolation from photosynthetic membranes.

The lipid specificity for the enzymatic and proton-translocating functions of a reconstituted thermophilic ATPase complex has been investigated. The proteoliposomes were prepared from the ATPase complex of the thermophilic cyanobacterium Synechococcus 6716 and various lipids and lipid mixtures extracted from this organism and from a related mesophilic strain. Some commercial lipids were used as well. An improved method of lipid extraction from chlorophyll-containing membranes is presented. This method is based on acetone extraction and additional chlorophyll separation and results in higher yields, less chlorophyll contamination and a simpler procedure than the conventional methods based on chloroform/methanol extraction. The lipids of Synechococcus 6716 thus extracted were fractionated by thin-layer chromatography. The fatty acyl chain composition of the separated lipids was analyzed by gas chromatography. The coupling quality of the reconstituted ATPase proteoliposomes made of different lipids was tested by a membrane-bound fluorescent probe and uncoupler stimulation of ATP hydrolysis. None of the separated lipids alone was able to produce a well-coupled system. The best results were obtained with the native lipid mixture. The minimum requirement was the combination of a typical bilayer-forming lipid and the non-bilayer (hexagonal II structure)-forming monogalactosyldiacylglycerol. Lipids from the mesophilic Synechococcus 6301 and commercial lipids (also mesophilic) produced poorly coupled vesicles but significant improvement was obtained when thermophilic monogalactosyldiacylglycerol was included. Both the reconstituted and solubilized ATPase complex have a sharp temperature optimum at 50 degrees C. The effect of reconstitution and measurement temperatures on the yield of well-coupled vesicles from different lipid sources was also studied.

Structural comparison of yeast ribosomal protein genes.

The primary structure of the genes encoding the yeast ribosomal proteins L17a and L25 was determined, as well as the positions of the 5'- and 3'-termini of the corresponding mRNAs. Comparison of the gene sequences to those obtained for various other yeast ribosomal protein genes revealed several similarities. In all split genes the intron is located near the 5'-side of the amino acid coding region. Among the introns a clear pattern of sequence conservation can be observed. In particular the intron-exon boundaries and a region close to the 3'-splice site show sequence homology. Conserved sequences were also found in the leader and trailer regions of the ribosomal protein mRNAs. The 5'-flanking regions of the yeast ribosomal protein genes appeared to contain sequence elements that many but not all ribosomal protein genes have in common, and therefore may be implicated in the coordinate expression of these genes. The amino acid coding sequences of the ribosomal protein genes show a biased codon usage. Like most yeast ribosomal protein molecules, L17a and L25 are particularly basic at their N-terminus.