10 pages, 6 figures.-- This work is part of the Ph.D. thesis of J.L. (supervised by M.A.M. and M.B.).Sequence data from this article can be found in the GenBank data library under accession number DQ455608.Ascorbic acid (vitamin C) is a major antioxidant and redox buffer, but is also involved in other critical processes of plants. Recently, the hypothesis has been proposed that legume nodules are unable to synthesize ascorbate and have to import it from the shoot or root, thus providing a means by which the plant regulates nodule senescence. The last step of ascorbate biosynthesis in plants is catalyzed by L-galactono-1,4-lactone dehydrogenase (GalLDH). The mRNAs encoding GalLDH and three other enzymes involved in ascorbate biosynthesis are clearly detectable in nodules. Furthermore, an active membrane-bound GalLDH enzyme is present in nodule mitochondria. Biochemical assays on dissected nodules reveal that GalLDH activity and ascorbate are correlated in nodule tissues and predominantly localized in the infected zone, with lower levels of both parameters (relative to the infected tissues) in the apex (87%) and senescent region (43%) of indeterminate nodules and in the peripheral tissues (65%) of determinate nodules. In situ RNA hybridization showed that the GalLDH mRNA is particularly abundant in the infected zone of indeterminate and determinate nodules. Thus, our results refute the hypothesis that ascorbate is not synthesized in nodules and lend support to a previous conclusion that ascorbate in the infected zone is primarily involved in the protection of host cells against peroxide damage. Likewise, the high ascorbate and GalLDH activity levels found in the apex of indeterminate nodules strongly suggest a participation of ascorbate in additional functions during symbiosis, possibly related to cell growth and division and to molecular signaling.This work was supported by the Ministerio de Educación y Ciencia-Fondos Europeos de Desarrollo Regional (grant no. AGL2005-01404) and by Gobierno de Aragón-Fondo Social Europeo (group E33 and grant no. PIP137/2005). We also acknowledge postdoctoral contracts of the "Ramón y Cajal" (M.A.M.) and "Juan de la Cierva" (J.R., M.J.C.) programs from the Ministerio de Educación y Ciencia, and a predoctoral fellowship (J.L.) from Gobierno de Aragón.Peer reviewed