SPOROGENESIS AND CALLOSE LOCALIZATION IN ANTHOCERATOPHYTA

Spores are fundamental to the reproductive success of all land plants. The success of a spore lies in its recalcitrant multi-layered spore wall commonly made of sporopollenin, cellulose, and pectin. However, other polysaccharides may be associated with the intine of spores, and their patterns of deposition vary across taxa. Callose, a plant 1-3-β-glucan polysaccharide, has unsubstantiated accounts of its presence and absence in association with the spore mother cell wall of hornworts for more than a century. To address this conundrum, I used aniline blue, a fluorochrome that has high specificity of binding to beta glucans and when excited with ultraviolet light, it will fluoresce yellow-green. However, due to the limited resolution power of that technique, I also used transmission electron microscopy (TEM) with immunogold labeling to observe the ultrastructural localization of callose using anticallose, a monoclonal antibody. I also bioinformatically probed the genomes and transcriptomes of hornworts to elucidate the callose synthase genes and enzymes which may be responsible for the putative callose deposition. Because of the asynchronous spore development, each sporophyte has a continuum of spores at various developmental stages. Subsequently, the sporophyte of many hornworts makes an ideal system to study all aspects of sporogenesis. For the first time, I provide unequivocal, correlative evidence for callose involvement in spore wall development in hornworts. Here we report on the spatiotemporal deposition of callose in sporogenesis of Phaeoceros carolinianus, and we show that callose is a common wall constituent of the spore intine in three other genera (Anthoceros, Notothylas, and Nothoceros). Callose deposition in hornworts is post-meiotic and begins during early spore wall development after a white lined lemella is formed and during expansion of the exine. As sporopollenin is deposited and the spore wall thickens, callose remains localized in the intine during the remainder of sporogenesis. The occurrence of callose in hornwort spores is a first record of this polysaccharide in the inner spore wall (intine or endospore) of any embryophyte. This suggests that callose may serve the same or similar roles in hornwort intine development and function as pectin-cellulose does in later diverging taxa. Bioinformatic tblastn techniques and molecular high through put Illumina genome sequencing combined with blast techniques for orthologs to callose synthase genes from Arabidopsis thaliana and Physcomitrella patens did not provide any evidence as to if callose synthase genes are present in hornworts This was due to database contamination, sample contamination, and sample quality