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.