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Soybean nodulin 26 is expressed and targeted to the symbiosome membrane of nitrogen-fixing nodules, where it forms an aquaporin channel with a modest water transport rate. In this study, we show that the phosphorylation of nodulin 26 on Ser-262, which is catalyzed by a symbiosome membrane-associated calcium-dependent protein kinase, stimulates its intrinsic water transport rate. Furthermore, using a phosphospecific antibody, we have elucidated the developmental appearance and regulation of nodulin 26 phosphorylation in vivo. Although nodulin 26 protein is detected first in differentiating infected cells (16 days), phosphorylated nodulin 26 does not become pronounced until infected cell maturation (25 days). Phosphorylation is sustained at steady state levels until entry into senescence. Nodulin 26 phosphorylation is enhanced further by osmotic stresses (water deprivation and salinity). Thus, the phosphorylation of nodulin 26 coincides with the establishment of mature nitrogen-fixing symbiosomes, is regulated by osmotic stresses that induce calcium-signaling pathways, and appears to be part of the adaptive responses of infected cells to osmotic challenge.
INTRODUCTION
Under conditions of limiting soil nitrogen, legumes can be infected by bacteria of the Rhizobiaceae family, resulting in the development of a symbiotic nitrogen-fixing organ, the nodule (reviewed by Gualtieri and Bisseling, 2000; Stougaard, 2000; Hirsch et al., 2001). During this process, the bacteria become enclosed within specialized infected cells within the core of the nodule. Most of the cytosolic volume of these cells is occupied by large numbers of organelles referred to as symbiosomes (Roth et al., 1988). The symbiosome consists of three major components: the nitrogen-fixing rhizobia bacteroids, the external symbiosome space, and a membrane of plant origin known as the symbiosome membrane.
The symbiosome membrane is highly specialized and participates in the exchange of metabolites (the efflux of fixed nitrogen and the uptake of a carbon source in the form of dicarboxylates) between the endosymbiont and the plant host as well as serving to protect the bacteroid from plant host defense responses (Udvardi and Day, 1997; Day et al., 2001). During the biogenesis of the symbiosome, a number of plant-encoded nodule-specific gene products (nodulins) are targeted to the symbiosome membrane, where they are presumed to aid in the establishment and maintenance of symbiosis (Fortin et al., 1985; Panter et al., 2000). Nodulin 26 is a symbiosome membrane-specific protein...