Gilbert Alibert

Changes in biochemical composition of vacuoles isolated from Acer pseudoplatanus L. during cell culture

Abstract Vacuoles were isolated from Acer pseudoplatanus cell suspension culture using a one-step procedure involving the lysis of the protoplast plasmalemma through a gradient of Ficoll containing DEAE-Dextran. The vacuole suspensions were slightly contaminated by other organelles (less than 5%) and the isolated vacuoles readily accumulated neutral red. Since α-mannosidase was located exclusively in the vacuoles it was used as a convenient marker. It was shown that the number of vacuoles per protoplast decreased as the cell aged. Studies on the biochemical composition of the isolated vacuoles indicated that amino acids, organic acids and protein contents varied with the cell culture cycle, emphasizing the dynamic status of the vacuolar system in cell suspension cultures of Acer pseudoplatanus.

Intracellular transport and vacuolar accumulation of o-coumaric acid glucoside in Melitolus alba mesophyll cell protoplasts

Abstract Sweet clover leaves accumulate o-coumaric acid glucoside in the vacuole of the mesophyll cells. Protoplasts isolated from these cells are able to synthesize o-coumaric acid glucoside from exogeneous [14C]phenylalanine. Using this simplified system, kinetic analyses have shown that the glucoside is synthesized in the cytoplasm and thereafter transferred into the vacuole. No further degradation of the glucoside occurs in the isolated protoplasts after this transfer. Under our conditions, the vacuolar pH is reduced by light/dark transition and by inhibitors of photosynthesis. These treatments also depressed synthesis and transport of o-coumaric acid glucoside. The data support a relationship between the functioning of photosynthesis and pH cytoplasm/vacuole-dependent transport of o-coumaric acid glucoside into the vacuole. However as o-[ 14 C ] coumaric acid glucoside is taken up by isolated vacuoles without any energy requirement, no final conclusions can be drawn as to the coupling of o-coumaric acid glucoside transport with the acidification process. In intact plant or isolated protoplasts o-coumaric acid glucoside is synthesized as the trans isomer but accumulated in the vacuole in the cis form. When loaded with trans o-[ 14 C ] coumaric acid glucoside, reconstituted tonoplastic vesicles released this compound during molecular sieving on a Sepharose 4B column. In contrast, the labelled cis form was completely retained inside the vesicular lumen in a parallel experiment. These results suggest a selective permeability of the tonoplast to the two isomers which could be involved, in vivo, in the trapping of the glucoside inside the vacuole and its irreversible accumulation.

General scheme of analysis of phenolic compound in plant extracts by reversed-phase high-performance liquid chromatography

Abstract A general prcedure of analysis of phenolic compounds by reversed-phase high-performance liquid chromatography with ternary elution gradients has been designed. The method was applied to the separation and estimation of phenolic acids, phenolic aldehydes and coumarins from standard mixtures or from plant extracts. Examples of its application to the variations in phenolic acids during grape juice storage, to studies of the monomeric composition of poplar lignins and to estimation of the coumarin contents of sweet clover are given.

Recherches sur les enzymes catalysant la formation des acides phénoliques chez Quercus pedunculata (EHRH.) II. Localisation intracellulaire de la phenylalanine ammoniaque-lyase, de la cinnamate 4-hydroxylase, et de la “benzoate synthase”

Abstract This paper deals with some properties of enzymes involved in the synthesis of phenolic acids, with special reference to their intracellular location. The results obtained can be summarized as follows: 1. 1. Two phenylalanine ammonia-lyase isoenzymes coexist in Quercus roots as has been previously described for oak leaves. The cinnamate-sensitive isoenzyme is associated with the microsomal fraction whereas the benzoate-sensitive isoenzyme is located in the “F 10 000” fraction including mitochondria and microbodies. 2. 2. The cinnamate 4-hydroxylase, catalyzing p-coumarate formation, is tightly bound to microsomes and the “benzoate synthase”, an emzyme involved in the benzoic acid formation from t-cinnamate, is confined to the “F 10 000” fraction, presumably in microbodies. These data show that phenolic acids biosynthesis pathways are closely compartmented in organelles so that phenylpropanoid compounds are mainly formed in microsomes whereas C6−C1 acids are formed in microbodies. The regulation of these pathways seems to involve sophisticated mechanisms, associating isoenzymes with tight compartments.

Metabolisme des composes phenoliques chez le petunia v. utilisation de la phenylalanine par des chloroplastes isoles

Abstract The involvement of plastids in phenolic metabolism has been studied in Petunia after an isolation method of pure and physiologically intact chloroplasts has been adapted. Feeding experiments with [ 14 C] phenylalanine demonstrated in these isolated organelles: the synthesis of cinnamate and benzoate derivatives which, with the exception of the first term of each serie, are o -hydroxylated ( o -coumaric acid, coumarin and salicylic and gentisic acids); the lack of formation of labelled p -hydroxylated derivatives and flavanoids. The results are in agreement with the characterization, in the plastids of specific enzymes (PAL, cinnamate-2-hydroxylase) and the absence of cinnamate-4-hydroxylase. All these data suggest that the formation of p -hydroxylated derivatives, esters and flavonoids often characterized in chloroplasts may involve a cooperation between different organelles.

Reversible phosphorylation of tonoplast proteins involves tonoplast-bound calcium-calmodulin-dependent protein kinase(s) and protein phosphatase(s)

In highly purified tonoplast fractions from Acer pseudoplatanus cells, the in vitro reversible phosphorylation of proteins affected only a restricted set of polypeptides. The phosphorylation process has been shown to be dramatically stimulated by calcium via the mediation of calmodulin as the transducer. The protein kinase(s) was totally inhibited by micromolar concentrations of a calmodulin antagonist. Tonoplast appears to be potentially a good experimental system for the evaluation of the effects of protein phosphorylation on membrane properties in plants.

Metabolisme des composes phenoliques chez le petunia VI. Intervention des chloroplastes dans la biosynthese de la naringenine et de l'acide chlorogenique

Abstract Enzymatic extract from Petunia leaves catalyze different reactions involved in the synthesis of hydroxycinnamoyl esters and flavonoids. Attempts to localize the individual enzymes within isolated chloroplasts show that these organelles: contain hydroxycinnamoyl-CoA—quinate—hydroxycinnamoyl transferase and flovanone-synthetase involved respectively in the biosynthesis of chlorogenic acid and naringenin; catalyze the isomerisation of chalcone to flavanone; are not able to activate the C6—C3 units to their CoA esters. These results are confirmed by feeding experiments on isolated chloroplasts using quinate or acetate 14C in combination (or not) with exogenously supplied “cinnamoyl-CoA”. These data seem to indicate that the activated cinnamic units, involved in the formation of chloroplastic phenols, are synthetized in other cellular compartment(s).

Photosynthesis in Protoplasts From Melilotus alba: Distribution of Products Between Vacuole and Cytosol

Summary Vacuoles were isolated from mesophyll protoplasts from Melilotus alba (Desr.) after photosynthesis in 14 CO 2 . The soluble labelled products in protoplasts and vacuoles were analyzed. Appearance of label in the vacuoles proceeded only slowly. After 60 min of photosynthesis, it reached about 30% of the total soluble label in the protoplasts. The products were separated in acidic, basic and neutral fractions. Label appeared rapidly in the vacuolar acidic fraction, but slowly in the vacuolar basic and neutral fractions. These results indicate that newly synthesized organic acids are rapidly transferred to the vacuoles while the bulk of photosynthetic products, consisting of sugars and amino acids, remain predominantly cytosolic.

Photoregulation of the incorporation of guaiacyl units into lignins

When fed with [14C] phenylalanine in the light, xylem tissues isolated from poplar stems were able to incorporate part of the radioactivity into both the guaiacyl and the syringyl residues of lignins. In the dark, only syringyl units were integrated into the polymer whereas the guaiacyl residues remained unlabeled.When a membrane perturber (isopropanol) was added to the incubation mixture, the label was incorporated into the guaiacyl units either in the light or in the dark. Conversely, a membrane stabilizer (CaCl2) prevented the labeling of the guaiacyl units even when the tissues were illuminated. These results suggest that light acts through the modification of membrane permeabilities, altering specifically the synthesis and the transport or the polymerization of guaiacyl-type units during the process of lignification.

Photoperiodic control of phenolic metabolism in Kalanchoe blossfeldiana

Abstract When grown in conditions of long day length, the leaves of Kalanchoe blossfeldiana contain high levels of soluble phenolic compounds, mainly present as tannins. A decreasing concentration gradient is observed in the leaves from the apex to the base. When transferred to short day conditions, the ability of leaves of the same physiological age to accumulate phenolics decreases with time. The very low phenolic content after 25 short days indicates pronounced changes in the metabolism of the plant induced by new photoperiodic conditions. Moreover, during development in short days the amount of tannins per leaf reaches a maximum then decreases suggesting an over-polymerization or even a degradation of the substances. A similar lag time is required for the depressing effect of short days on phenolies and for their stimulating effect on CAM.