Involvement of ligninlike compounds in toxicity of dietary alder leaf litter against mosquito larvae.

The toxicological characteristics of dietary decomposed alder leaf litter against mosquito larvae were further investigated through enzymatic and chemical purification of a phenoliclike cell-wall fraction isolated from crude litter. The toxicity of the subfractions obtained was controlled by standard bioassays on third instars of Aedes aegypti chosen as a reference target species. Enzymatic hydrolyses of the cell-wall fraction were performed with caylase, pectolyase, esterase, and beta-glycosidase, in order to release, respectively, cellulose material and phenolic compounds bound to lignins. These treatments did not affect the larvicidal activity and the phenolic activity of the cell-wall fraction. Chemical alkaline and acid hydrolyses were carried out to break ester and glycosidic bonds of the cell-wall fraction. Comparison of HPLC profiles of the hydrolysates from both toxic and nontoxic fractions did not reveal differences between the phenolic acids released. Aluminum chloride, known for its phenolic complexing activity, counteracted the larvicidal activity of the cell-wall fraction. Altogether, these results suggest the involvement of ligninlike compounds in the toxicity of dietary alder leaf litter against larval mosquitoes. The toxicity of this fraction, which was very sensitive to drastic and smooth oxidations, seemed to be associated with a strong oxidative potential. These results are discussed in relation to a possible mode of action of lignins in the plant-mosquito interactions.

Role of vegetable tannins in habitat selection among mosquito communities from the Alpine hydrosystems.

The role of vegetable tannins in habitat selection among mosquito communities in Alpine hydrosystems was investigated through ecotoxicological comparison of 19 arthropod species characteristic of 12 breeding sites known for their abiotic environmental factors and their different riparian vegetation. The toxicity of tannins was experimentally compared among species representative of both the dipteran fauna and the crustacean fauna associated with the mosquito breeding sites. Bioassays using tannic acid solutions at concentrations from 0.1 to 11 mM separated the dipteran taxa into five groups of differential sensitivity and the crustacean taxa into four groups. The different levels of sensitivity among taxa were correlated with the various amounts of total phenolics and tannins found in the most prominent plant types associated with the different breeding sites. This suggested that tannins and, more generally, phenolic compounds may be involved in plant-arthropod interactions in Alpine hydrosystems.

Identification of an essential histidine residue at the active site of the tonoplast malate carrier in Catharanthus roseus cells.

The involvement of a histidyl residue in the binding or translocation step was investigated in the malate carrier at the tonoplast of Catharanthus roseus cells. The transport rate was strongly stimulated when the pH of the incubation medium was decreased from pH 7.0 to 5.0. The histidine-specific reagent diethylpyrocarbonate (DEPC) efficiently inhibited the activity of the malate carrier. Inhibition developed rapidly and was completed after 5 min at a concentration of 2 mM DEPC. The original substrate, malate, partially protected the carrier from inactivation by DEPC. Other organic acids (citrate, quinate) which are known to affect the malate transport of isolated vacuoles or tonoplast vesicles also showed protective properties. Inhibition of malate transport on tonoplast vesicles can also be achieved by photooxidation in the presence of the dye Rose Bengal. Malate also proved to protect against inactivation. The results strongly support the notion that a histidyl residue(s) is involved either in the binding or translocation of malate and that the protonation of the histidyl residue is essential to provide a high rate of malate transport.

Vacuolar Release of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid, the Conjugated Form of the Ethylene Precursor.

The mechanisms underlying the vacuolar retention or release of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, has been studied in grape (Vitis vinifera) cells grown in vitro using the technique of compartmental analysis of radioisotope elution. Following its accumulation in the vacuole, M[2,3-(14)C]ACC could be released from cells when the vacuolar pH was artificially lowered by external buffers from its initial value of 6.2 to below the critical pH of 5.5. Successive release and retention of vacuolar MACC could be achieved by switching the vacuolar pH from values lower and higher than 5.5. The rate constant of efflux was highly correlated with the vacuolar pH. In plant tissues having low vacuolar pH under natural conditions, e.g. apple fruits (pH 4.2) and mung bean hypocotyls (pH 5.3), an efflux of M[2,3-(14)C]ACC also occurred. Its rate constant closely corresponded to the theorical values derived from the correlation established for grape cells. Evidence is presented that the efflux proceeded by passive lipophilic membrane diffusion only when MACC was in the protonated form. In contrast to other organic anions like malic acid, the mono and diionic species could not permeate the tonoplast, thus indicating the strict dependence of MACC retention upon the ionic status of the molecule and the absence of carrier-mediated efflux.

Preparation of sealed tonoplast and plasma-membrane vesicles from Catharanthus roseus (L.) G. Don. cells by free-flow electrophoresis.

Highly purified tonoplast and plasmamembrane vesicles were isolated from microsomes of Catharanthus roseus (L.) G. Don. by preparative free-flow electrophoresis. The relative amounts of tonoplast and plasma-membrane vesicles in the total microsomes varied with the pH of the grinding medium. The most electronegative fractions were identified as tonoplast using nitrate-inhibited, azide-resistant Mg(2+)-ATPase and pyrophosphatase activities as enzyme markers. The least electronegative fractions were identified as plasma membrane using glucan-synthase-II and UDPG:sterolglucosyl-transferase activities as enzyme markers. Other membrane markers, latent inosine-5'-diphosphatase (Golgi), NADPH-cytochrome-c reductase (ER) and cytochrome-c oxidase (mitochondria) were recovered in the fractions intermediate between tonoplast and plasma membrane and did not contaminate either the tonoplast or the plasma-membrane fractions. In the course of searching for a reliable marker for tonoplast, the pyrophosphatase activity was found to be essentially associated with the tonoplast fractions purified by free-flow electrophoresis from C. roseus and other plant materials. The degree of sealing of the tonoplast and plasmamembrane vesicles was probed by their ability to pump protons (measurements of quinacrine quenching) and to generate a membrane potential (absorption spectroscopy of Oxonol VI). A critical evaluation of vesicles sidedness is presented.

A reversible carrier mediates the transport of malate at the tonoplast of Catharanthus roseus cells.

The conditions of malate transport were defined in tonoplast vesicles purified from a microsomal homogenate of Catharanthus roseus cells by preparative free-flow electrophoresis. Isolated vesicles exhibited malate transport when the membranes were prepared by grinding the cells in a homogenisation medium only buffered in the acidic pH range. By using vesicles energized artificially by an imposed pH gradient (acid interior), it was shown that malate is actively accumulated in response to the generation of a proton-motive force. Several lines of evidence (saturation kinetics, action of malate analogs and protein modifiers) support the concept that malate transport is mediated by a protein carrier which could be implicated in the uptake process as its protonated form. The malate transported in the vesicles was released by lowering the external malate concentration. The release was prevented by the anion transport inhibitor DIDS indicating the reversibility of the carrier.

Carrier-Mediated Uptake of 1-(Malonylamino)cyclopropane-1-Carboxylic Acid in Vacuoles Isolated from Catharanthus roseus Cells.

The uptake of 1-(malonylamino)cyclopropane-1-carboxylic acid (MACC), the conjugated form of the ethylene precursor, into vacuoles isolated from Catharanthus roseus cells has been studied by silicone layer floatation filtering. The transport across the tonoplast of MACC is stimulated fourfold by 5 millimolar MgATP, has a K(m) of about 2 millimolar, an optimum pH around 7, and an optimum temperature at 30 degrees C. Several effectors known to inhibit ATPase (N,N'-dicyclohexylcarbodiimide) and to collapse the transtonoplastic H(+) electrochemical gradient (carbonylcyanide m-chlorophenylhydrazone, gramicidin, and benzylamine) all reduced MACC uptake. Abolishing the membrane potential with SCN(-) and valinomycin also greatly inhibited MACC transport. Our data demonstrate that MACC accumulates in the vacuole against a concentration gradient by means of a proton motive force generated by a tonoplastic ATPase. The involvement of a protein carrier is suggested by the strong inhibition of uptake by compounds known to block SH-, OH-, and NH(2)- groups. MACC uptake is antagonized competitively by malonyl-d-tryptophan, indicating that the carrier also accepts malonyl-d-amino acids. Neither the moities of these compounds taken separately [1-aminocyclopropane-1-carboxylic acid, malonate, d-tryptophan or d-phenylalanine] nor malate act as inhibitors of MACC transport. The absence of inhibition of malate uptake by MACC suggests that MACC and malate are taken up by two different carriers. We propose that the carrier identified here plays an important physiological role in withdrawing from the cytosol MACC and malonyl-d-amino acids generated under stress conditions.

Day-night changes in the levels of adenine nucleotides, phosphoenolpyruvate and inorganic pyrophosphate in leaves of plants having Crassulacean acid metabolism.

The levels of phosphorylated compounds studied during the dark period of Crassulacean acid metabolism (CAM) in Kalanchoë leaves showed increases for ATP and pyrophosphate and decreases for ADP, AMP and phosphenolpyruvate; levels of inorganic phosphate remained constant. Changes in adenylate levels and the correlated nocturnal increase in adenylate-energycharge were closely related to changes in malate levels. The increase in ATP levels was much inhibited in CO2-free air and stimulated after induction of CAM in short-day-treated plants of K. blossfeldiana cv. Tom Thumb. Changes in levels of phosphoenolpyruvate and pyrophosphate were independent of the presence of CO2. The results show the operation of complex regulatory mechanisms in the energy metabolism of CAM plants during nocturnal malic-acid accumulation.

The proton-pumps at the plasmalemma of Catharanthus roseus cells.

Cultured Catharanthus roseus cells exhibit transmembrane ferricyanide (FIC) reduction which is associated with a proton translocation and a decrease in the ATP content of the cells. The H+ efflux and the ATP consumption may be counteracted by vanadate, a specific inhibitor of the ATPase activity, and by Na2WO4 which prevents FIC reduction. From these data it is concluded that the redox chain could be coupled with ATP hydrolysis for electrogenic proton extrusion which may involve a redox control mechanism for the plasmamembrane ATPase.

Cation stimulation of the proton-translocating redox activity at the plasmalemma of Catharanthus roseus cells.

Cultured Catharanthus roseus cells exhibit transmembrane ferricyanide reduction through a plasma membrane redox system which may be associated with proton translocation. Evidence shows that endogenous pyridine nucleotides serve as hydrogen donors for the reaction. The proton translocating function of the redox system is confirmed, in intact cells and isolated protoplasts, by the ability of Ca(2+) and other cations to increase both the redox activity and the efflux of protons. The role of the cations is seen to be not a simple general charge screening phenomenon as already described. By using ionic surfactants (CP(+), SDS(-)) it was shown that the net surface charge of the membrane can interact in the activation process via a cation attraction effect. It is proposed that specific binding of cations to the plasma membrane could alter the conformation of the redox system facilitating its interaction with NADH.

Phenolic metabolism in petunia tissues. I. Characteristic responses of enzymes involved in different steps of polyphenol synthesis to different hormonal influences.

Pronounced changes in enzymatic patterns occur in petunia tissues when calluses are subcultured on media containing different growth substances. As judged by variations of enzymes related to primary metabolism (6-phosphogluconate and malate dehydrogenases) there are individual responses for each metabolic pathway. Concerning the enzymes of aromatic metabolism: (a) Phenylalanine ammonia-lyase, cinnamate and p-coumarate hydroxylases and the enzyme(s) activating phenylpropanoid units vary in the same manner. (b) Chalcone-flavanone isomerase, a key enzyme in the synthesis of flavonoids, and coniferyl alcohol dehydrogenase, which leads to the monomers of lignins, have, on the other hand, an independent behaviour. These responses show that the enzymes involved in the synthesis and activation of phenylpropanoid units seem to act coordinately in plants. Moreover, the data suggest that the common pathway leading to the activated cinnamic acids and the specific metabolic steps of lignin and flavonoid synthesis are regulated in a different way.