First Extensive Microscopic Study of Butternut Defense Mechanisms Following Inoculation with the Canker Pathogen Ophiognomonia clavigignenti-juglandacearum Reveals Compartmentalization of Tissue Damage.

Ophiognomonia clavigignenti-juglandacearum endangers the survival of butternut (Juglans cinerea) throughout its native range. While screening for disease resistance, we found that artificial inoculations of 48 butternut seedlings with O. clavigignenti-juglandacearum induced the expression of external symptoms, but only after a period of dormancy. Before dormancy, compartmentalized tissues such as necrophylactic periderms (NPs) and xylem reaction zones (RZs) contributed to limiting pathogen invasion. Phenols were regularly detected in RZs, often in continuity with NPs during wound closure, and confocal microscopy revealed their presence in parenchyma cells, vessel plugs and cell walls. Vessels were blocked with tyloses and gels, particularly those present in RZs. Suberin was also detected in cells formed over the affected xylem by the callus at the inoculation point, in a few tylosis walls, and in longitudinal tubes that formed near NPs. Following dormancy, in all inoculated seedlings but one, defensive barriers were breached by O. clavigignenti-juglandacearum and then additional ones were produced in response to this new invasion. The results of this histopathological study indicate that trees inoculated in selection programs to test butternut canker resistance should go through at least one period of dormancy and that asymptomatic individuals should be dissected to better assess how they defend themselves against O. clavigignenti-juglandacearum.

[The trichomes of pericarp in Juglans (Juglandaceae): scanning microscopy, fluorescent microscopy amd histochemistry].

Four types of glandular and non-glandular trichomes of pericarp in four Juglans species (J. ailanthifolia, J. cordiformis, J. mandshurica and J. regia) from Juglandaceae were studied by scanning electron microscopy, fluorescent light microscopy and histochemistry. The capitate trichomes on short stalk, the capitate trichomes on long stalk and the peltate trichomes belong to glandular trichomes; the simple hairs concern to non-glandular trichomes. The investigated species differ one from another in dimensions and distribution oftrichomes as well as the chemical content and the mechanism of secretion. The fluorescent markers and histochemical tests show the presence of flavonoids, tannins and polyphenols in trichomes on short and long stalk. In peltate trichomes the flavonoids and tannins were found in lesser quantity and the polyphenols are absent. In simple hairs the phenolic substances have not been recognized. It has been come out with the suggestion about a functional role of each type of trichomes.

Toward an improved model of maple sap exudation: the location and role of osmotic barriers in sugar maple, butternut and white birch.

Two theories have been proposed to explain how high positive pressures are developed in sugar maple stems when temperatures fluctuate around freezing. The Milburn-O'Malley theory proposes that pressure development is purely physical and does not require living cells or sucrose. The osmotic theory invokes the involvement of living cells and sucrose to generate an osmotic pressure difference between fibers and vessels, which are assumed to be separated by an osmotic barrier. We analyzed wood of Acer saccharum Marsh., Juglans cinerea L. and Betula papyrifera Marsh. (all generate positive pressures) examining three critical components of the osmotic model: pits in cell walls, selectivity of the osmotic barrier and stability of air bubbles under positive xylem pressure. We examined the distribution and type of pits directly by light and scanning electron microscopy (SEM), and indirectly by perfusion of branch segments with fluorescent dyes with molecular masses similar to sucrose. The latter approach allowed us to use osmotic surrogates for sucrose that could be tracked by epifluorescence. Infusion experiments were used to assess the compartmentalization of sucrose and to determine the behavior of gas bubbles as predicted by Fick's and Henry's laws. The SEM images of sugar maple revealed a lack of pitting between fibers and vessels but connections between fiber-tracheids and vessels were present. Fluorescein-perfusion experiments demonstrated that large molecules do not diffuse into libriform fibers but are confined within the domain of vessels, parenchyma and fiber-tracheids. Results of the infusion experiments were in agreement with those of the fluorescein perfusions and further indicated the necessity of a compartmentalized osmolyte to drive stem pressure, as well as the inability of air bubbles to maintain such pressure because of instability. These results support the osmotic model and demonstrate that the secondary cell wall is an effective osmotic barrier for molecules larger than 300 g mol(-1).

Evidence of the involvement of plant ligno-cellulosic structure in the sequestration of Pb: an X-ray spectroscopy-based analysis.

European walnut (Juglans regia) plants were grown in pots, on peat soil contaminated with lead (Pb), for four years. European walnut was chosen because it grows in Mediterranean climates, it yields a high biomass, and a fine quality wood. In the above ground parts Pb concentration was 1000 times lower than in roots: in 50 g roots there was 450 mg of Pb. Microanalysis of roots found in periderm more than 50% of the total root Pb. Pb L(III) EXAFS spectroscopy was performed on: root powder from Pb-exposed plants, Pb-impregnated cellulose and lignin. Comparison of plant material with lignin and cellulose helped to envisage a plant disposal strategy for Pb. This may consist in establishing links with large organic molecules, which are abundant constituents of cell walls. EXAFS spectroscopy evidenced the presence of Pb-O bindings within the ligno-cellulosic structure in roots. Lead was scantly conveyed to the shoots, giving to walnut plants an added asset in Pb phytostabilization.

Phloem unloading in developing walnut fruit is symplasmic in the seed pericarp and apoplasmic in the fleshy pericarp.

The sieve element-companion cell (SE-CC) complex of the sepal bundles feeding the fleshy pericarp of developing walnut (Juglans regia L.) fruit is structurally symplasmically isolated, but the SE-CC complex of the minor ventral carpellary bundles located in the seed pericarp and feeding the seed is structurally symplasmically connected to its adjacent parenchyma cells. 14C-autoradiography indicated that the phloem of both the sepal and carpellary bundles was functional for unloading. Confocal laser scanning microscopy imaging of carboxyfluorescein unloading showed that the dye is confined to the phloem strands of the sepal bundles in the fleshy pericarp, but released from the phloem strands of the minor ventral carpellary bundles into the surrounding parenchyma cells in the seed pericarp. A 60-kDa acid invertase was immunolocalized to the cell wall of SE-CC complex and parenchyma cells in both the fleshy and seed pericarp. These data provide clear evidence for an apoplasmic phloem unloading pathway in the fleshy pericarp and a predominant symplasmic phloem unloading pathway parallel with a possible apoplasmic path as suggested by the presence of the extracellular invertase in the seed pericarp. A model of complex phloem unloading pathways in developing walnut fruit has been proposed.