Interaction of Cytosolic Glutamine Synthetase of Soybean Root Nodules with the C-terminal Domain of the Symbiosome Membrane Nodulin 26 Aquaglyceroporin

Abstract

Nodulin 26 (nod26) is a major intrinsic protein that constitutes the major protein component on the symbiosome membrane (SM) of N 2 -fixing soybean nodules. Functionally, nod26 forms a low energy transport pathway for water, osmolytes, and NH 3 across the SM. Besides their transport functions, emerging evidence suggests that high concentrations of major intrinsic proteins on membranes provide interaction and docking targets for various cytosolic proteins. Here it is shown that the C-terminal domain peptide of nod26 interacts with a 40-kDa protein from soybean nodule extracts, which was identified as soybean cytosolic glutamine synthetase GS 1 ␤1 by mass spectrometry. Fluorescence spectroscopy assays show that recombinant soybean GS 1 ␤1 binds the nod26 C-terminal domain with a 1:1 stoichiometry (K d ‫؍‬ 266 nM). GS 1 ␤1 also binds to isolated SMs, and this binding can be blocked by preincubation with the C-terminal peptide of nod26. In vivo experiments using either a split ubiquitin yeast two-hybrid system or bimolecular fluorescence complementation show that the four cytosolic GS isoforms expressed in soybean nodules interact with full-length nod26. The binding of GS, the principal ammonia assimilatory enzyme, to the conserved C-terminal domain of nod26, a transporter of NH 3 , is proposed to promote efficient assimilation of fixed nitrogen, as well as prevent potential ammonia toxicity, by localizing the enzyme to the cytosolic side of the symbiosome membrane.The formation of legume-rhizobia N 2 -fixing symbioses in root nodules leads to the development of a novel organelle known as the "symbiosome," which houses the endosymbiotic rhizobia bacteroids. In mature nodules, the host infected cells are occupied by thousands of symbiosomes, which constitute the major organelle within this specialized cell type. The symbiosome is delimited by the symbiosome membrane (SM), 2 which controls the transport of all metabolites between the symbiont and the plant host. These transport activities include the efflux of the primary metabolic product of nitrogen fixation (NH 3 /NH 4 ϩ ) and the uptake of dicarboxylates as an energy source to support bacterial N 2 fixation (1, 2). Because of the unique role of the SM, a number of host nodulin ("noduleenhanced") proteins are synthesized and localized to this membrane (3, 4), where they serve transport and regulatory functions in the symbiosis.Early studies revealed that nodulin 26 (nod26), a member of the major intrinsic protein (MIP)/aquaporin superfamily, is among the ensemble of soybean SM nodulins produced during symbiosome biogenesis (4 -7). Since the original identification of nod26 over 20 years ago (4), the role of this channel in the symbiosis has been debated. Functional analyses show that nod26 is a multifunctional "aquaglyceroporin" that transports multiple substrates including water, formamide, and glycerol (6, 8). As a symbiosome-specific aquaglyceroporin, nod26 has been proposed to serve as a low energy transport pathway for water as well as osmolytes within the inf...

Matching the supply of bacterial nutrients to the nutritional demand of the animal host

“…These processes would tend to reduce feedback inhibition in the symbiont and are known to contribute to high efflux in certain other symbioses, e.g. of ammonia from nitrogen-fixing rhizobia to the host cell in leguminous plants [28][29][30]. However, our finding that methionine levels are elevated in bacteriocytes from aphids reared on methionine-free diet (figure 3b) is not readily compatible with host-mediated alleviation of feedback inhibition.…”

An RNA Sequencing Transcriptome Analysis Reveals Novel Insights into Molecular Aspects of the Nitrate Impact on the Nodule Activity of Medicago truncatula

“…There is convincing, albeit indirect, evidence for their at least partial involvement in ammonium transport. This is indicated by the fact that the cytosolic form of glutamine synthetase in soybean nodules interacts with a domain at the C-terminal end of nodulin 26 (Masalkar et al, 2010). While at 4 h, only one Plasma membrane Intrinsic Protein aquaporin was down-regulated and two Tonoplast Intrinsic Protein aquaporins were upregulated, a total of 15 aquaporin-related genes were differentially expressed at 28 h. Two genes (Medtr8g087710.1, Medtr8g087720.1) for a Nodule-26-Like Intrinsic Protein aquaporin on chromosome 8 are implicated to be involved in ammonium transport, judging from their level of expression and extent of their down-regulation between 4 and 28 h.…”

RNA Interference-Mediated Repression of S6 Kinase 1 Impairs Root Nodule Development in Soybean

“…It will be intriguing to find out if the activator of GS in plants such as a Dof factor (Reuda-Lopéz et al, 2008) would be functional in GmS6K1s-RNAi root nodules. Recently, GS was found to interact with the symbiosome membrane nodulin 26, a NH 3 transporter in soybean nodule (Masalkar et al, 2010). The binding of GS to nodulin 26 was proposed to promote efficient assimilation of fixed nitrogen.…”

“…Intermolecular signaling between two symbiotic partners promotes the entry of rhizobia into root cells and the formation of nodule primordia by cell proliferation in root cortex (Oldroyd and Downie, 2008;Oldroyd et al, 2011). This stage of nodule development is characterized by vast synthesis of membrane lipids to enclose endosymbiotic bacteria and synthesis of proteins including nodule-specific proteins (nodulins) such as leghemoglobins (Ott et al, 2005), nodulin-24 (Cheon et al, 1994), nodulin-26 (Masalkar et al, 2010) and so on. Successful symbiosis results in the nitrogen fixation and assimilation into ureides in tropical legumes such as soybean.…”