Nonsymbiotic hemoglobins in rice are synthesized during germination and indifferentiating cell types

Nonsymbiotic hemoglobins (ns-Hbs) previously have been found in monocots an d dicots however, very little is known about the tissue and cell type local ization as well as the physiological function(s) of these oxygen-binding pr oteins. We report the immunodetection and immunolocalization of ns-Hbs in r ice (Oryza sativa L.) by Western blotting and in situ confocal laser scanni ng techniques. Ns-Hbs were detected in soluble extracts of different tissue s from the developing rice seedling by immunoblotting. Levels of ns-Hbs inc reased in the germinating seed for the first six days following imbibition and remained relatively constant thereafter. In contrast, ns-Hb levels decr eased during leaf maturation. Roots and mesocotyls contained detectable, bu t low levels of ns-Hbs. Split-seed experiments revealed that ns-Hbs are syn thesized de novo during seed germination and are expressed in the absence o f any signal originating from the embryo. Immunolocalization of ns-Hbs by c onfocal microscopy indicated the presence of ns-Hbs primarily in differenti ated and differentiating cell types of the developing seedling, such as the aleurone, scutellum, root cap cells, sclerenchyma, and tracheary elements. To our knowledge, this is the first report of the specific cellular locali zation of these proteins during seedling development.