Renée Goldberg

Isolation and characterization of Populus isoperoxidases involved in the last step of lignin formation

Peroxidases (EC 1.11.1.7) from Populus x euramericana were investigated during the dormant and growing seasons using histochemical and biochemical methods. The activities of syringaldazine oxidase and p-paraphenylenediamine-pyrocatechol oxidase in sections of branches were maximal during spring in both phloem and young xylem. Cytoplasmic and cell-wall peroxidase activities from different lignified tissues were estimated in vitro. Pronounced differences were noticed between fractions isolated during spring and winter. Gel electrophoresis showed the presence of an anionic fast-migrating isoperoxidase group with a high syringaldazine-oxidase activity. The isoenzymes of this group were different in winter and in spring. The properties of these isoperoxidases (kinetic constants, pH optimum, resistance to heat) were investigated after isolation by ion-exchange chromatography.

Some Properties of Syringaldazine Oxidase, a Peroxidase Specifically Involved in the Lignification Processes

Summary Cell wall peroxidases from poplar stem were investigated through parallel biochemical and histochemical techniques. Oxidation of syringaldazine was obtained only in lignifying cells. That it was a true peroxidasic activity was demonstrated by its absolute requirement for exogeneous H 2 O 2 for in vitro assays. However syringaldazine oxidation could be obtained in situ in the absence of exogeneous hydrogen peroxide probably because of the production of H 2 O 2 by the lignifying cell walls themselves. Syringaldazine oxidase activity was strongly bound to the cell walls and fairly resistant to heat inactivation. It exhibited a very high affinity towards its substrate. The K M value was 100 to 1000 times higher with syringaldazine than with guaiacol.

Tetramethylbenzidine and p-phenylenediamine-pyrocatechol for peroxidase histochemistry and biochemistry: Two new, non-carcinogenic chromogens for investigating lignification process

Tetramethylbenzidine (TMB) and p-phenylenediamine-pyrocatechol (PPD-PC) are oxidized by peroxidases in the presence of hydrogen peroxide. On tobacco stem sections TMB gives a blue reaction product at pH 4.5 in lignifying cell walls, when PPD-PC gives a violet one at pH 7.6. The reaction products are water soluble and oxidation time course can be recorded. The maximal absorbance was obtained at 654 nm for the TMB oxidation and at 557 nm for the PPD-PC one. The chromogens are also suitable for electrophoresis gel staining. Inhibition sensitivity and isoperoxidase patterns carried on tobacco peroxidases demonstrated the involvement of several different isoperoxidases in the oxidation of TMB and PPD-PC.

Ratio of free to bound polyamines during maturation in mung-bean hypocotyl cells

Free- and bound-polyamine levels were estimated in successive segments of the mung-bean hypocotyl. Three aliphatic polyamines (putrescine, spermidine and spermine) were found in proportions which depended on the state of maturation. In young cells, most of the polyamines were located in the protoplasm whereas in older cells they were mostly bound to the cell walls. Spermidine was always the main bound polyamine, and putrescine, the main free polyamine.

AN EFFICIENT PROCEDURE FOR STUDYING PECTIN STRUCTURE WHICH COMBINES LIMITED DEPOLYMERIZATION AND 13C NMR

Abstract A protocol for partial thermally-induced depolymerization of differently methoxylated pectin samples is described. The resulting macromolecules have been fully characterized with various complementary techniques, such as size exclusion chromatography (SEC), potentiometry, viscometry and 13C NMR. Optimum conditions afford samples at 50–80% yield with weight-average molecular weights in the 4 to 20 kDa range. The major fraction of these polysaccharides adopts the random-coil conformation and such samples are suitable for 13C NMR structural studies at room temperature. The methoxyl distributions of two apple pectin samples with a degree of esterification (DE) between 54 and 74% and a citrus pectin (DE, 72%) were shown to be random in nature, whereas that of a lightly methoxylated apple pectin (DE 39%) was partially blockwise. The carbon relaxation parameters of the depolymerized pectins attain asymptotic values for MW > 4 kDa. The MW values estimated from intrinsic viscosity data with the Mark-Houwink relationship reported for native pectins are in good agreement with those obtained by either end-group analysis (NMR) or SEC. Thus, all the physicochemical data indicate that the secondary structure of the isolated chains of depolymerized pectin is closely related to that of the parent polymers. Finally, pectinmethylesterase activity towards the depolymerized pectins was similar to that of the untreated samples.

Characterization of the pectin methylesterase-like gene AtPME3: a new member of a gene family comprising at least 12 genes in Arabidopsis thaliana

Pectin demethylesterification appears to be catalysed by a number of pectin methylesterase (PME) isoenzymes in higher plant species. In order to better define the biological role of these isoenzymes in plant cell growth and differentiation, we undertook molecular studies on the PME-encoding genes in Arabidopsis thaliana. In this paper, we report the characterization of AtPME3, a new PME-related gene of 4kb in length that we have mapped on Chromosome III. AtPME3 encodes a putative mature PME-related isoenzyme of 34kDa with a basic isoelectric point. Since the extent of the gene family encoding PME in higher plant species is still unknown, we resorted to the use of degenerate primers designed from several well-known consensus regions to identify new PME-related genes in the genome of Arabidopsis. Our results, in combination with several known expressed sequences tags (ESTs), indicate that the Arabidopsis genome contains at least 12 PME-related genes. Consequently, a method of systematic gene expression analysis has been applied in order to discern the expression pattern of these 12 genes throughout the plant at the floral stage. Whereas most of these genes appeared to be more or less ubiquitously expressed throughout the plant, several genes are distinguishable by their strikingly specific expression in certain organs. The present data bring a new insight into the role of specific PME-related genes in flower and root development.

Pectin methylesterases from poplar cambium and inner bark : localization, properties and seasonal changes

Abstract. In the course of a study on the early events of cambial derivative differentiation in Populus × euramericana, seasonal changes in the pattern of pectin methylesterase (PME, EC 3.1.1.11) isoforms were followed. During the resting season, cell wall extracts contained mainly alkaline isoforms with an Mr around 55 kDa and optimal pH between 5.6 and 6.0. Neutral isoforms with an Mr around 35 kDa and optimal pH between 6.0 and 6.6 predominated in the extracts during the period of high meristematic activity. In the active cambial initials and in their immediate derivatives, the enzymes were immunolocalized exclusively in the dictyosomes. In older cells, they were present both in dictyosomes and in wall junctions. These results indicate that exportation of neutral PMEs towards the walls might be considered as an early marker of differentiation in cambial derivatives.

Purification and characterization of pectin methylesterases from mung bean hypocotyl cell walls

Abstract A saline eluate of Mung bean hypocotyl cell walls was submitted to carboxy-methyl Sepharose chromatography. Detection of pectin methylesterase (PME) activities after IEF of the active fractions revealed the occurrence of at least four PME isoforms. These isoforms were further purified by cation exchange chromatography, and their Mr pH sensitivity and substrate specificity determined.

Development of arabinans and galactans during the maturation of hypocotyl cells of mung bean (Vigna radiata Wilczek)

Abstract Hypocotyl cell walls contain galactans and arabinans that are soluble in boiling water. During maturation, the Ara/Gal ratio remains unchanged but high-molecular-weight galactans are replaced by smaller polymers. On the basis of the 1 H-n.m.r. 2D-COSY(δ-δ, 1 H- 1 H)n.m.r., and 13 C-n.m.r. spectra, a (1→5)-α-Ara f structure can be proposed for the arabinans in both young and nature cell walls. However, the galanctan(s) changed from a probably highly branched to an unbranched (1→4)-β-Gal p structure during maturation.

Development of isoperoxidases along the growth gradient in the mung bean hypocotyl

Abstract Cytoplasmic and wall bound peroxidases were extracted from successive segments of decreasing growth potential along the mung bean hypocotyl. Active wall bound peroxidases were present in the epidermis and external parenchyma layers at the end of the elongation phase. Two fast migrating anionic isoperoxidases covalently bound to the cell walls increased when the cell walls lost their plasticity. These isoenzymes were characterized by a high affinity for several peroxidase substrates and high thermal stability.