The Arabidopsis mutant pho1 is deficient in the transfer of Pi from root epidermal and cortical cells to the xylem. The PHO1 gene was identified by a map-based cloning strategy. The N-terminal half of PHO1 is mainly hydrophilic, whereas the C-terminal half has six potential membrane-spanning domains. PHO1 shows no homology with any characterized solute transporter, including the family of H ؉ -Pi cotransporters identified in plants and fungi. PHO1 shows highest homology with the Rcm1 mammalian receptor for xenotropic murine leukemia retroviruses and with the Saccharomyces cerevisiae Syg1 protein involved in the mating pheromone signal transduction pathway. PHO1 is expressed predominantly in the roots and is upregulated weakly under Pi stress. Studies with PHO1 promoter-␤ -glucuronidase constructs reveal predominant expression of the PHO1 promoter in the stelar cells of the root and the lower part of the hypocotyl. There also is ␤ -glucuronidase staining of endodermal cells that are adjacent to the protoxylem vessels. The Arabidopsis genome contains 10 additional genes showing homology with PHO1 . Thus, PHO1 defines a novel class of proteins involved in ion transport in plants. INTRODUCTIONThe radial movement of ions from root epidermal and cortical cells to the xylem can be mediated by two major pathways. In the apoplastic pathway, ions move radially toward the stele through the extracellular space, whereas in the symplastic pathway, ions move intracellularly from cell to cell via plasmodesmata (Bowling, 1981;Clarkson, 1993). Although a third pathway is possible, namely, one in which ions move from cell to cell through a successive uptake and release of ions from and into the extracellular space, the high energy requirement of this pathway makes it unlikely to play a major role in ion transport to the xylem.The movement of ions and water through the apoplast of the root is blocked at the level of the endoderm by the Casparian strip, a zone in which the cell wall is impregnated with hydrophobic compounds such as suberin and lignin. Thus, passage of ions beyond the Casparian strip and toward the stele must proceed via the symplasm. Once in the cells of the stele, the release of ions into the xylem requires their efflux out of the stelar cells. Thus, radial transport of ions from the external solution to the xylem requires a minimum of two passages across the plasma membrane, once for the uptake of ions into the epidermal, cortical, or outer surface of the endodermal cells, and then again for the efflux of ions out of the stelar cells before entering the xylem vessel (Bowling, 1981;Clarkson, 1993).The uptake of anions such as Pi into a cell is an energyrequiring process. The negatively charged phosphate ion (HPO 4 Ϫ 2 or H 2 PO 4 Ϫ ) must move against an electrical gradient, the interior of the cell being negatively charged ( ‫ف‬ Ϫ 100 mV), as well as against a concentration gradient, the intracellular concentration of Pi being 1000 to 10,000 times higher than the extracellular concentration (the concentration of P...

Abstract. Anchoring of proteins to membranes by glycosylphosphatidylinositols (GPIs) is ubiquitous among all eukaryotes and heavily used by parasitic protozoa. GPI is synthesized and transferred en bloc to form GPI-anchored proteins. The key enzyme in this process is a putative GPI:protein transamidase that would cleave a peptide bond near the COOH terminus of the protein and attach the GPI by an amide linkage. We have identified a gene, GAA/, encoding an essential ER protein required for GPI anchoring, gaa/ mutant cells synthesize the complete GPI anchor precursor at nonpermissive temperatures, but do not attach it to proteins. Overexpression of GAA/improves the ability of cells to attach anchors to a GPIanchored protein with a mutant anchor attachment site. Therefore, Gaalp is required for a terminal step of GPI anchor attachment and could be part of the putative GPI:protein transamidase.

A detailed microscopical analysis of the morphological features that distinguish different developmental stages of nodule organogenesis in wild-type Lotus japonicus ecotype Gifu B-129-S9 plants was performed, to provide the necessary framework for the evaluation of altered phenotypes of L. japonicus symbiotic mutants. Subsequently, chemical ethyl methanesulfonate (EMS) mutagenesis of L. japonicus was carried out. The analysis of approximately 3,000 M1 plants and their progeny yielded 20 stable L. japonicus symbiotic variants, consisting of at least 14 different symbiosis-associated loci or complementation groups. Moreover, a mutation affecting L. japonicus root development was identified that also conferred a hypernodulation response when a line carrying the corresponding allele (LjEMS102) was inoculated with rhizobia. The phenotype of the LjEMS102 line was characterized by the presence of nodule structures covering almost the entire root length (Nod++), and by a concomitant inhibition of both root and stem growth. A mutation in a single nuclear gene was shown to be responsible for both root and symbiotic phenotypes observed in the L. japonicus LjEMS102 line, suggesting that (a) common mechanism(s) regulating root development and nodule formation exists in legumes.

A range of novel expressed sequence tags (ESTs) associated with late developmental events during nodule organogenesis in the legume Lotus japonicus were identified using mRNA differential display; 11 O differentially displayed polymerase chain reaction products were cloned and analyzed. O f 88 unique cDNAs obtained, 22 shared significant homology to DNA/protein sequences in the respective databases. This group comprises, among others, a nodulespecific homolog of protein phosphatase 2C, a peptide transporter protein, and a nodule-specific form of cytochrome P450. RNA gel-blot analysis of 16 differentially displayed ESTs confirmed their nodule-specific expression pattern. The kinetics of mRNA accumulation of the majority of the ESTs analyzed were found to resemble the expression pattern observed for the L. japonicus leghemoglobin gene. These results indicate that the newly isolated molecular markers correspond to genes induced during late developmental stages of L. japonicus nodule organogenesis and provide important, novel tools for the study of nodulation.

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