Mechanisms of primordium formation during adventitious root development from walnut cotyledon explants.

In walnut (Juglans regia L.), an otherwise difficult-to-root species, explants of cotyledons have been shown to generate complete roots in the absence of exogenous growth regulators. In the present study, this process of root formation was shown to follow a pattern of adventitious, rather than primary or lateral, ontogeny: (i) the arrangement of vascular bundles in the region of root formation was of the petiole type; (ii) a typical root primordium was formed at the side of the procambium within a meristematic ring of actively dividing cells located around each vascular bundle; (iii) the developing root apical meristem was connected in a lateral way with the vascular bundle of the petiole. This adventitious root formation occurred in three main stages of cell division, primordium formation and organization of apical meristem. These stages were characterized by expression of LATERAL ROOT PRIMORDIUM-1 and CHALCONE SYNTHASE genes, which were found to be sequentially expressed during the formation of the primordium. Activation of genes related to root cell differentiation started at the early stage of primordium formation prior to organization of the root apical meristem. The systematic development of adventitious root primordia at a precise site gave indications on the positional and biochemical cues that are necessary for adventitious root formation.

Differential localization of arabinan and galactan side chains of rhamnogalacturonan 1 in cambial derivatives.

The development of pectin structural features during the differentiation of cambial derivatives was investigated in aspen (Populus tremula L. x P. tremuloides Michx.) using biochemical and immunocytochemical methods. Comparisons were also made between active and resting tissues. Active tissues, in particular cambial cells and phloem derivatives, were characterized by a high pectin content. Use of antibodies raised against arabinan side chains of rhamnogalacturonan 1 (LM6), as well as biochemical analysis, revealed an obvious decrease from the cortex to the differentiating xylem. Galactan side chains, detected with LM5 antibodies, were present mainly in the cambial zone and enlarging xylem cells. In contrast, they were totally absent from sieve-tube cell walls. Image analysis of LM5 immunogold labelling in the cambial zone showed a clustered distribution of galactan epitopes in the radial walls, a distribution which might result from the association of two different periodic processes, namely the exocytosis of galactan and wall expansion. Cessation of cambial activity was characterized by cell wall thickening accompanied by a sharp decrease in the relative amount of pectin and a lowering of the degree of methylesterification. The data provide evidence that the walls of phloem and xylem cells differ in their pectin composition even at a very early stage of commitment. These differences offer useful tools for identifying the initial cells among their immediate neighbours.

Structure and functions of the vascular cambium.

Following a general outline on the functioning of the cambium, the authors review the data acquired over the last 20 years. The interest is focused on: i) the ultrastructural characteristics of the two kinds of initials; ii) the early structural, metabolic and molecular modifications occurring during the first stages of daughter cell differentiation into either xylem or phloem; iii) the complex rhythmic changes of structure, metabolism and activity undergone by cambial cells during the seasonal cycle; iv) the characteristics and control of the cessation of cambial activity in autumn and of its reactivation in spring; v) the main research approaches in cell and molecular biology presently open to the students of the cambial meristem.

Localized graft incompatibility in pear/quince (Pyrus communis/Cydonia oblonga) combinations: multivariate analysis of histological data from 5-month-old grafts.

To characterize the structural events associated with incompatibility of graft development, we conducted a histological study of compatible and incompatible pear/pear and pear/quince grafts that had been grown for five months in a greenhouse. Multivariate analysis of histological data describing the structure of the graft union allowed us to discriminate between compatible and incompatible combinations before either macroscopic examination or qualitative microscopic examination differences between graft combinations became evident. The histological variables responsible for the discrimination between incompatible and compatible unions were related to three typical symptoms of graft incompatibility: bark discontinuity, which was the main feature; cambial dysfunction; and accumulation of starch in the scion. Little cell necrosis was observed at the interface of incompatible grafts at the 5-month stage of graft development. Multivariate analysis of histological data provides a new tool for studying early structural events resulting from the graft incompatibility response and for diagnosing early graft incompatibility.

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

In the course of a study on the early events of cambial derivative differentiation in Populus x 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 M(r) around 55 kDa and optimal pH between 5.6 and 6.0. Neutral isoforms with an M(r) 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.

Cell-wall polysaccharides in growing poplar bark tissue.

In order to study changes in the cell-wall composition of growing poplar cambium during the seasonal cycle, cell walls were isolated from the cambium and newly formed vascular tissues of poplar branches at three times during the year (winter, beginning of spring, end of summer). Polysaccharide material was isolated by sequential extraction of the cell walls and analysed. The principal polysaccharides identified were pectins and xylose- and glucose-containing polysaccharides, possibly xylans and (xylo)glucans. Our results indicate changes in the relative quantities of these polysaccharides during the seasonal cycle.

Characterization and Localization of a Phenoloxidase in Mung Bean Hypocotyl Cell Walls.

The occurrence of proteins able to oxidize polyphenols even in the absence of H2O2 was recently reported in mung bean (Vigna radiata L.) hypocotyl cell wall extracts (R. Goldberg, A. Chabanet, A.M. Catesson [1993] In K.G. Welinder, S.K. Rasmussen, C. Penel, H. Greppin, eds, Plant Peroxidases: Biochemistry and Physiology, pp. 296-300). Therefore, the possible presence of a laccase in the extracts was investigated using immunocytological and biochemical approaches. An enzyme catalyzing phenol oxidation in the presence of molecular O2 was extracted and purified from the cell walls. This 38-kD cationic protein, like o-diphenoloxidases, was unable to oxidize p-diphenols or p-diamines. However, it crossreacted with an anti-laccase antiserum and, like laccases, its activity was inhibited by N-cetyl-N,N,N-trimethylammonium bromide but not by ferulic acid salts. Immunolabeling data showed that the 38-kD oxidase was absent from all cellulosic cell walls. It was localized only in lignifying and lignified cell walls. This restricted localization suggests that this laccase-like phenoloxidase could participate in the lignification process but not in the primary wall stiffening, which develops in the epidermal and cortical tissues along the mung bean hypocotyl.

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.