Callose and homogalacturonan epitope distribution in stomatal complexes of Zea mays and Vigna sinensis.

This article deals with the distribution of callose and of the homogalacturonan (HG) epitopes recognized by LM20, JIM5, and 2F4 antibodies in cell walls of differentiating and functioning stomatal complexes of the monocotyledon Zea mays and the dicotyledon Vigna sinensis. The findings revealed that, during stomatal development, in these plant species, callose appears in an accurately spatially and timely controlled manner in cell walls of the guard cells (GCs). In functioning stomata of both plants, callose constitutes a dominant cell wall matrix material of the polar ventral cell wall ends and of the local GC cell wall thickenings. In Zea mays, the LM20, JIM5, or 2F4 antibody-recognized HG epitopes were mainly located in the expanding cell wall regions of the stomatal complexes, while in Vigna sinensis, they were deposited in the local cell wall thickenings of the GCs as well as at the ledges of the stomatal pore. Consideration of the presented data favors the view that in the stomatal complexes of the monocotyledon Z. mays and the dicotyledon V. sinensis, the esterified HGs contribute to the cell wall expansion taking place during GC morphogenesis and the opening of the stomatal pore. Besides, callose and the highly de-esterified HGs allow to GC cell wall regions to withstand the mechanical stresses exerted during stomatal function.

Callose implication in stomatal opening and closure in the fern Asplenium nidus.

The involvement of callose in the mechanism of stomatal pore opening and closing in the fern Asplenium nidus was investigated by examination of the pattern of callose deposition in open and closed stomata, and by examination of the effects of callose degradation and inhibition or induction of callose synthesis in stomatal movement. Callose was identified with aniline blue staining and a callose antibody and degraded via beta-1,3-D-glucanase. Callose synthesis was inhibited with 2-deoxy-D-glucose and induced by coumarin or dichlobenil. Stomatal pore opening and closing were assessed by estimation of the stomatal pore width. The open stomata entirely lacked callose, while the closed ones displayed distinct radial fibrillar callose arrays in the external periclinal walls. The latter displayed local bending at the region of callose deposition, a deformation that was absent in the open stomata. Both callose degradation and inhibition of callose synthesis reduced the stomatal ability to open in white light and close in darkness. By contrast, callose synthesis induction considerably improved stomatal pore opening and reduced stomatal closure in same conditions. The present data revealed that: during stomatal closure the external periclinal guard cell walls experience a strong mechanical stress, probably triggering callose synthesis; and that callose participates in stomatal movement.

The role of callose in guard-cell wall differentiation and stomatal pore formation in the fern Asplenium nidus.

BACKGROUND AND AIMS: The pattern of callose deposition was followed in developing stomata of the fern Asplenium nidus to investigate the role of this polysaccharide in guard cell (GC) wall differentiation and stomatal pore formation. METHODS: Callose was localized by aniline blue staining and immunolabelling using an antibody against (1 --> 3)-beta-d-glucan. The study was carried out in stomata of untreated material as well as of material treated with: (1) 2-deoxy-d-glucose (2-DDG) or tunicamycin, which inhibit callose synthesis; (2) coumarin or 2,6-dichlorobenzonitrile (dichlobenil), which block cellulose synthesis; (3) cyclopiazonic acid (CPA), which disturbs cytoplasmic Ca(2+) homeostasis; and (d) cytochalasin B or oryzalin, which disintegrate actin filaments and microtubules, respectively. RESULTS: In post-cytokinetic stomata significant amounts of callose persisted in the nascent ventral wall. Callose then began degrading from the mid-region of the ventral wall towards its periphery, a process which kept pace with the formation of an 'internal stomatal pore' by local separation of the partner plasmalemmata. In differentiating GCs, callose was consistently localized in the developing cell-wall thickenings. In 2-DDG-, tunicamycin- and CPA-affected stomata, callose deposition and internal stomatal pore formation were inhibited. The affected ventral walls and GC wall thickenings contained membranous elements. Stomata recovering from the above treatments formed a stomatal pore by a mechanism different from that in untreated stomata. After coumarin or dichlobenil treatment, callose was retained in the nascent ventral wall for longer than in control stomata, while internal stomatal pore formation was blocked. Actin filament disintegration inhibited internal stomatal pore formation, without any effect on callose deposition. CONCLUSIONS: In A. nidus stomata the time and pattern of callose deposition and degradation play an essential role in internal stomatal pore formation, and callose participates in deposition of the local GC wall thickenings.

Gentamicin resistance genes in environmental bacteria: prevalence and transfer.

A comprehensive multiphasic survey of the prevalence and transfer of gentamicin resistance (Gm(r)) genes in different non-clinical environments has been performed. We were interested to find out whether Gm(r) genes described from clinical isolates can be detected in different environmental habitats and whether hot spots can be identified. Furthermore, this study aimed to evaluate the impact of selective pressure on the abundance and mobility of resistance genes. The study included samples from soils, rhizospheres, piggery manure, faeces from cattle, laying and broiler chickens, municipal and hospital sewage water, and coastal water. Six clusters of genes coding for Gm-modifying enzymes (aac(3)-I, aac(3)-II/VI, aac(3)-III/IV, aac(6')-II/Ib, ant(2'')-I, aph(2'')-I) were identified based on a database comparison and primer systems for each gene cluster were developed. Gm-resistant bacteria isolated from the different environments had a different taxonomic composition. In only 34 of 207 isolates, mainly originating from sewage, faeces and coastal water polluted with wastewater, were known Gm(r) genes corresponding to five of the six clusters detected. The strains belonged to genera in which the genes had previously been detected (Enterobacteriaceae, Pseudomonas, Acinetobacter) but also to phylogenetically distant bacteria, such as members of the CFB group, alpha- and beta-Proteobacteria. Gm(r) genes located on mobile genetic elements (MGE) could be captured in exogenous isolations into recipients belonging to alpha-, beta- and gamma-Proteobacteria from all environments except for soil. A high proportion of the MGE, conferring Gm resistance isolated from sewage, were identified as IncPbeta plasmids. Molecular detection of Gm(r) genes, and broad host range plasmid-specific sequences (IncP-1, IncN, IncW and IncQ) in environmental DNA indicated a habitat-specific dissemination. A high abundance and diversity of Gm(r) genes could be shown for samples from faeces (broilers, layers, cattle), from sewage, from seawater, collected close to a wastewater outflow, and from piggery manure. In the latter samples all six clusters of Gm(r) genes could be detected. The different kinds of selective pressure studied here seemed to enhance the abundance of MGE, while an effect on Gm(r) genes was not obvious.