Functional Analysis of Nodulin 26, an Aquaporin in Soybean Root Nodule Symbiosomes

Abstract

Upon infection of soybean roots, nitrogen-fixing bacteria become enclosed in a specific organelle known as the symbiosome. The symbiosome membrane (SM) is a selectively permeable barrier that controls metabolite flux between the plant cytosol and the symbiotic bacterium inside. Nodulin 26 (NOD 26), a member of the aquaporin (AQP) water channel family, is a major protein component of the SM. Expression of NOD 26 in Xenopus oocytes gave a mercury-sensitive increase in osmotic water permeability (P f ). To define the biophysical properties of NOD 26 water channels in their native membranes, symbiosomes were isolated from soybean root nodules and the SM separated as vesicles from the bacteria. Permeabilities were measured using stopped-flow fluorimetry in SM vesicles with entrapped carboxyfluorescein. Osmotic water permeability (P f ) of SM was high, with a value of 0.05 ؎ 0.003 cm/s observed at 20°C (mean ؎ S.E.; n ‫؍‬ 15). Water flow exhibited a low activation energy, was inhibited by HgCl 2 (0.1 mM), and exhibited a unit conductance of 3.2 ؎ 1.3 ؋ 10 ؊15 cm 3 /s, a value 30-fold lower than that of AQP 1, the red blood cell water channel. Diffusive water permeability (P d ) was 0.0024 ؎ 0.0002 cm/s, and the resulting P f to P d ratio was 18.3, indicating that water crosses the SM in single file fashion via the NOD 26 water channel. In addition to high water permeability, SM vesicles also show high mercury-sensitive permeability to glycerol and formamide, but not urea, suggesting that NOD 26 also fluxes these solutes. Overall, we conclude that NOD 26 acts as a water channel with a single channel conductance that is 30-fold lower than AQP 1. Because the solutes that permeate NOD 26 are far larger than water, and water appears to cross the channel via a single file pathway, solute flux across NOD 26 appears to occur by a pathway that is distinct from that for water.Soil bacteria of the Rhizobium and Bradyrhizobium genera invade the roots of specific leguminous plants and establish a nitrogen fixing symbiosis. During this process, a developmental program is triggered resulting in the formation of root nodules. The bacterium becomes enclosed in a specific organelle, the symbiosome (1), within the infected cells of the nodule. This organelle is delimited by a membrane of plant origin, known as the symbiosome membrane (SM).1 This membrane regulates the efflux of fixed nitrogen from the endosymbiont to the plant cytosol as well as the influx of dicarboxylates (e.g. malate) from the plant, providing energy for nitrogen fixation (2-4).During nodule development, the expression of a number of nodule-specific proteins, known as nodulins, is induced. One of these proteins, nodulin 26 (NOD 26), is a major protein component of the SM (5). NOD 26 is a member of an ancient family of membrane channel proteins that include bacterial glycerol transporters, aquaporin (AQP) water channels, and various other intrinsic membrane proteins (6, 7). Structural similarities shared by family members include the presence of six putative transmemb...

Phosphorylation of Soybean Nodulin 26 on Serine 262 Enhances Water Permeability and Is Regulated Developmentally and by Osmotic Signals

“…The findings that nodulin 26 is produced specifically during the formation of the symbiosome membrane, is a major component of this symbiotic interface, and is responsible for the high water permeability of this membrane suggest a specialized role in symbiosis, perhaps in osmoregulation (Rivers et al, 1997;Dean et al, 1999). Furthermore, the finding that nodulin 26 is phosphorylated by a CDPK suggests that these potential activities are subject to regulation by calcium signal transduction.…”

“…(B) Comparison of water permeabilities of control oocytes or oocytes expressing wild-type nodulin 26 (WT) or human aquaporin 1 (AQP1). nel rates of these two MIP proteins (Rivers et al, 1997;Dean et al, 1999).…”

“…Nodulin 26 is a symbiosome membrane-specific protein that is the major protein component of this membrane (Fortin et al, 1987;Weaver et al, 1991). Nodulin 26 is a member of the major intrinsic protein (MIP) superfamily of water and solute channels, and it confers high water permeability on the soybean symbiosome membrane (Rivers et al, 1997;Dean et al, 1999). In addition, nodulin 26 is the major phosphorylation target for a calcium-dependent protein kinase (CDPK), which also resides on the symbiosome membrane (Weaver et al, 1991).…”

Expression and functional analysis of the rice plasma-membrane intrinsic protein gene family

“…Aquaporins have been found in bacteria, fungi, animals and plants. Plant aquaporins are classified into four major groups: the tonoplast intrinsic proteins (TIPs) localized to the vacuolar membranes, the plasma membrane intrinsic proteins (PIPs) localized to the plasma membranes, the NOD-26-like MIPs (NIPs), and the recently characterized small basic intrinsic proteins (SIPs) [2][3][4][5].…”

“…The structures of aquaporins have been intensively studied, revealing the functions of their conserved motifs and post-translational modification sites, in water transport [3][4][5][6][7][8][9][10][11][12][13][14][15]. The expression patterns of aquaporin genes in response to salt, drought, cold, submergence, light, and phytohormones have also been characterized in various plants [16][17][18][19][20][21][22][23][24].…”