NADH-Glutamate synthase in alfalfa root nodules. Immunocytochemical localization

Abstract

In root nodules of alfalfa (Medicago sativa L.), N2 is reduced to NH4+ in the bacteroid by the nitrogenase enzyme and then released into the plant cytosol. The NH4+ is then assimilated by the combined action of glutamine synthetase (EC 6.3.1.2) and NADH-dependent Glu synthase (NADH-GOGAT; EC 1.4.1.14) into glutamine and Glu. The alfalfa nodule NADH-GOGAT protein has a 101-amino acid presequence, but the subcellular location of the protein is unknown. Using immunocytochemical localization, we determined first that the NADH-GOGAT protein is found throughout the infected cell region of both 19- and 33-d-old nodules. Second, in alfalfa root nodules NADH-GOGAT is localized predominantly to the amyloplast of infected cells. This finding, together with earlier localization and fractionation studies, indicates that in alfalfa the infected cells are the main location for the initial assimilation of fixed N2.

Figure 1

A, Immunoblot analysis of the pGEX-NADH-GOGAT fusion protein. The blot of a 15% polyacrylamide gel was probed with polyclonal NADH-GOGAT antiserum and developed with alkaline-phosphatase-conjugated secondary antibodies. Lanes 1 through 3 contain 10 μg of protein each. Lane 1 contains total protein from E. coli carrying the uninduced pGEX-NADH-GOGAT construct; lane 2 contains total E. coli protein carrying the induced pGEX-NADH-GOGAT construct; and lane 3 contains the partially purified pGEX-NADH-GOGAT construct cleaved with thrombin. B, Immunoblot of total soluble nodule protein. The blot of a 6% polyacrylamide gel was probed with affinity-purified NADH-GOGAT antibodies and developed with alkaline-phosphatase-conjugated secondary antibodies. Lane 1 contains 100 μg of soluble protein of effective nodules.

Figure 2

Localization of the NADH-GOGAT protein in longitudinal sections of 19-d-old (A–C) and 33-d-old (E and F) root nodules. Nodule ultrastructure was classified based on the nomenclature of Vasse et al. (1990): the meristem (zone I), the invasion zone (zone II), the interzone (*), and the N₂-fixing zone (zone III). Sections A through E were probed with affinity-purified NADH-GOGAT antibodies and with alkaline-phosphatase-conjugated secondary antibodies. The signal, which reflects the localization of the NADH-GOGAT protein, is seen as blue spots. The black arrows point toward infected cells, and the red arrow points toward uninfected cells. Longitudinal section of a 19-d-old alfalfa root nodule is shown in A. Enlargement of the boxed regions in A shown in B and C includes part of the N₂-fixing zone (zone III) with infected and uninfected cells. D shows control, affinity-purified NADH-GOGAT antibodies that were incubated together with the partially purified NADH-GOGAT polypeptide on a longitudinal section through a 19-d-old root nodule. E shows a longitudinal section through a 33-d-old root nodule, and F shows an enlargement of the boxed region in E that is part of the proximal region of the nodule. Bars in A and E = 270 μm; bars in B, D, and F = 70 μm; and bar in C = 15 μm.

Figure 3

Immunocytochemical localization of NADH-GOGAT protein in root nodules of alfalfa. The section was probed with affinity-purified NADH-GOGAT antibodies and with secondary antibodies coupled to 18-nm gold particles. A shows the overview from which subsequent pictures were taken. Parts of an infected cell with bacteroids and cytosol are shown in B. C shows an amyloplast of an infected cell. D and E show an amyloplast of uninfected cells and intercellular space, respectively. ba, Bacteroid; cyt, cytosol; m, mitochondria; ap, amyloplast; cw, cell wall; is, intercellular space. Arrows point toward areas containing gold particles. Bar in A = 5 μm; bars in B through E = 1 μm.

Figure 4

Nitrogen assimilation in alfalfa root nodule and the cellular localization of the enzymes involved. Two linked Glu cycles are proposed to be involved in the production of Asn. Cycle 1, Cytosolic Glu cycle; cycle 2, amyloplast Glu cycle. Metabolites involved are NH₄⁺, Gln, Glu, 2-oxoglutarate, oxaloacetate, Asp, and Asn.