Previous investigations suggested that specific auxin spatial distribution due to auxin movements to particular embryonic regions was important for normal embryonic pattern formation. To gain information on the molecular mechanism(s) by which auxin acts to direct pattern formation in specific embryonic regions, the role of a plasma membrane (PM) ATPase was evaluated as downstream target of auxin in the present study. Western-blot analysis revealed that the PM H ϩ -ATPase expression level was significantly increased by auxin in wheat (Triticum aestivum) embryos (two-three times increase). In bilaterally symmetrical embryos, the spatial expression pattern of the PM H ϩ -ATPase correlates with the distribution pattern of the auxin analog, tritiated 5-azidoindole-3-acetic acid. A strong immunosignal was observed in the abaxial epidermis of the scutellum and in the epidermal cells at the distal tip of this organ. Pseudoratiometric analysis using a fluorescent pH indicator showed that the pH in the apoplast of the cells expressing the PM H ϩ -ATPase was in average more acidic than the apoplastic pH of nonexpressing cells. Cellulose staining of living embryos revealed that cells of the scutellum abaxial epidermis expressing the ATPase were longer than the scutellum adaxial epidermal cells, where the protein was not expressed. Our data indicate that auxin activates the proton pump resulting in apoplastic acidification, a process contributing to cell wall loosening and elongation of the scutellum. Therefore, we suggest that the PM H ϩ -ATPase is a component of the auxin-signaling cascade that may direct pattern formation in embryos.
The stress-70 protein family has previously been shown to be a useful tool for molecular phylogeny at the kingdom to family levels. Although sequences of many members of the stress-70 family are available, few genes from the Protoctista have been sequenced to date. Phylogenetic analyses of algae based on various molecules have not, as yet, provided clear results concerning relationships between major divisions. We cloned and sequenced several algal stress-70 genes in order to provide additional data and to further analyse phylogenetic relationships among algal divisions. New nuclear sequences were obtained from Guillardia theta (Cryptophyta), Ascophyllum nodosum (Heterokontophyta) and Cyanophora paradoxa (Glaucocystophyta). Phylogenetic trees of the stress-70 protein family calculated using different methods are presented. In our trees, the heterokont alga Ascophyllum nodosum is closely related to the slime mould Dictyostelium discoideum, while the nucleomorph (eukaryotic endosymbiont) of the cryptophyte Rhodomonas salina seems to be related to the chlorobiont lineage. The glaucocystophyte Cyanophora paradoxa and the nuclear sequence (host) of the cryptomonad alga Guillardia theta also seem to be closely related. The Cryptophyta and the heterokont algae have evolved from different secondary endosymbiotic events involving different hosts and probably different endosymbionts. However, until more stress-70 sequences of algal divisions become available no definitive conclusions can be drawn concerning branching of the major divisions.
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