Involvement of peroxidases in the formation of the brown coloration of heartwood in Juglans nigra.

Oxidase activities were investigated within the cross-section of walnut trunk in relation to the brown staining of heartwood, especially in the transition zone where the colour change occurs. The distribution of peroxidase activity was investigated using 3,3'-diaminobenzidine (DAB) or guaiacol as a substrate. Generally, the highest activity was found in the cambial zone and in the middle sapwood. This activity was mainly vacuolar. However, during autumn a peak of activity was observed in the transition zone with DAB, but not with guaiacol. Immunohistolocalization of the peroxidase revealed that the protein was present in the transition zone even if the enzymatic activity was not detectable. Flavan-3-ols were abundantly localized in the transition zone and it is hypothesized that they are physiological substrates of peroxidases. By contrast, polyphenoloxidases do not seem to be implicated in heartwood formation.

Flavan-containing cells delimit Frankia-infected compartments in Casuarina glauca nodules.

We investigated the involvement of polyphenols in the Casuarina glauca-Frankia symbiosis. Histological analysis revealed a cell-specific accumulation of phenolics in C. glauca nodule lobes, creating a compartmentation in the cortex. Histochemical and biochemical analyses indicated that these phenolic compounds belong to the flavan class of flavonoids. We show that the same compounds were synthesized in nodules and uninfected roots. However, the amount of each flavan was dramatically increased in nodules compared with uninfected roots. The use of in situ hybridization established that chalcone synthase transcripts accumulate in flavan-containing cells at the apex of the nodule lobe. Our findings are discussed in view of the possible role of flavans in plant-microbe interactions.

Unfolding and refolding of active apple polyphenol oxidase.

For the first time, unfolding (6 M guanidine) and refolding of partially proteolysed purified polyphenol oxidase (PPOr) was achieved, with 88% of activity recovered. Optimal refolding conditions consisted in stepwise dialysis of guanidine treated extracts, the dialysis buffers containing 1 M (NH4)2SO4 and 100 microM CuSO4. However, CuSO4 had limited effect on the recovering of PPOr activity, whereas (NH4)2SO4 was essential. Concerning the PPO tertiary structure, denaturing conditions (combinations of boiling and reducing agent) used on SDS-PAGE have shown (i) a compact tertiary structure and (ii) the presence of disulfide bonds in PPOr, accounting for the shift between 27 and 41 kDa, and 41 and 42 kDa, respectively. Resistance to proteolytic cleavage was used to study the conformational changes induced by the denaturing treatments. Folded PPOr was resistant to further proteolysis whereas unfolded PPO was totally digested, indicating the role of tertiary structure of PPOr in the resistance to proteases.

Biosynthesis of phenolic compounds inVitis vinifera cell suspension cultures: Study on hydroxycinnamoyl CoA:ligase.

In cell suspensions cultures from grape berry pulp (Vitis vinifera cv. Gamay fréaux)hydroxycinnamoyl CoA ligase (CoAL) displayed maximum activity (100 %) forp-coumaric acid and then, in decreasing order, for ferulic acid (81.3 %) and caffeic acid (60.4 %). No activity was detected with sinapic and cinnamic acids. The changes in CoAL activity during the growth cycle of the culture displayed two peaks : the highest (6 h after subculturing) was linked with a strong increase in protein caused by dilution ; the second was weaker and occurred on the 7th day of culture.Grape cell suspension accumulated mainly peonidin (Pn) and cyanidin (Cy) glucosides (Pn 3-glucoside, Cy 3-glucoside, Pn 3-acetylglucoside, Pn 3-caffeylglucoside, Pn 3-p-coumarylglucoside, and Cy 3-p-coumarylglucoside). Maximum accumulation of anthocyanins was associated with the exponential growth phase of the culture and might be the result of the substantial increase in CoAL activity resulting from the effect of dilution. The second enzyme activity peak was probably oriented towards the acylation of anthocyanins since the percentage of acylated forms increased with time after subculturing.

[Relationship between anatomical structure and metabolism of plant tissues. I. Differences between the qualitative and quantitative composition of phenolic substances of apple explants and that of callus and cells produced by the culture]. / Relation entre la structure anatomique et le métabolisme des tissue végétaux. I. Différences entre la composition qualitative et quantitative des substances phénoliques d'explantats de pomme, et celles de cals et de cellules qui en sont issus par culture.

Relationship between anatomical structure and metabolism of plant tissues. I. Differences between the qualitative and quantitative composition of phenolic substances of apple-fruit explants and that of calli and cells cultured from these explants. Phenolic compounds of intact apple-fruits (CV. Golden delicious), fruit fragments cultured on agar medium, newly formed calli and cell suspensions prepared from these calli, were studied quantitatively and qualitatively. The phenol content of the tissues decreased during the first days of culture, then recovered practically the initial level just before the initiating calli became visible. This content is very low in the calli and in the cultured cells. But the most remarkable result is that the qualitative compositions of the phenols extracted from the fruit tissues, the calli and the cells were different : p-coumaryglucose, which is abundant in the fruit, disappeared almost completely in the calli, in which three compounds were formed de novo, X1 which was tentatively identified as ferulylquinic acid, X2, a glycoside of p-coumaric acid different from p-coumarylglucose, and X3 not yet identified; the cells synthesized also X1 and X3 but not X2, and contained no p-coumaryglucose while feruylquinic acid was abundant. The study on the processes of induction or regulation of the enzymes implied in these metabolic modifications is under way.