Callose in sporogenesis: novel composition of the inner spore wall in hornworts

Sporogenesis is a developmental process that defines embryophytes and involves callose, especially in the production of the highly protective and recalcitrant spore/pollen wall. Until now, hornworts, leptosporangiate ferns and homosporous lycophytes are the only major plant groups in which the involvement of callose in spore development is equivocal. Through aniline blue fluorescence and immunogold labeling in the transmission electron microscope, we provide indisputable evidence for the presence of callose in the spore wall of five hornwort genera, but not in the derived Dendroceros , an epiphyte that produces multicellular spores. We present evidence that callose appears in the developing spore wall and is retained throughout development as a wall constituent of the intine or inner spore wall, a novel location for this polysaccharide in embryophytes. In endosporic and multicellular spores/pollen of Dendroceros , the liverwort Pellia , and Arabidopsis , callose appears in the newly formed cell walls only following the first mitotic division. Further probing for other wall polymers in hornworts reveals the presence of cellulose (Calcofluor fluorescence) in the spore intine, aperture and around the equatorial girdle. Further immunogold labeling with monoclonal antibodies identifies pectin and hemicellulose in hornwort intines. The persistence of callose, a typically transient cell wall constituent, with cellulose, pectins and hemicellulose in the intine, supports specialized functions of callose in spores of hornworts that include reduced water loss when spores are dry and mechanical flexibility to withstand desiccation.