Carbon and nitrogen transport across the legume symbiosome membrane

Author: Nick Booth

Booth, Nick, 2023 Carbon and nitrogen transport across the legume symbiosome membrane, Flinders University, College of Science and Engineering

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Abstract

Nitrogen is one of the most limiting factors for plant growth. The application of synthetic nitrogen fertilisers can alleviate this issue, but they are expensive and can leach into the surrounding environment. Legumes have developed a symbiotic relationship with soil bacteria known collectively as rhizobia, which fix atmospheric nitrogen into a form usable by the plant. During this symbiosis, legumes develop highly specialised organs on their roots, called nodules, which are infected by the rhizobia. Inside the infected nodule cells, the rhizobia differentiate into nitrogen fixing bacteroids that are excluded from the plant cytosol by a membrane of plant origin to form an organelle-like structure. This is the functioning unit of symbiotic nitrogen fixation and is referred to as the symbiosome. The Symbiosome Membrane (SM) regulates metabolite exchange between the symbionts via transport proteins synthesised by the plant. While the bacteroids are dependent on the plant for many nutrients, the principal exchange of nutrients across the SM is the uptake of reduced carbon to support nitrogen fixation in the bacteroids and the efflux of fixed nitrogen that is supplied to the plant. However, the identity of the transporters involved in this exchange remains elusive.

Previous studies have implicated a non-selective cation channel in the efflux of fixed nitrogen from the symbiosome, which is inwardly rectified by cytosolic Mg2+ and has a preference for ammonium. The present work has shown that when the aquaporin Nodulin 26 (NOD26), an abundant protein on the soybean symbiosome membrane, is heterologously expressed in Xenopus laevis oocytes, monovalent cation induced currents are observed. These currents are selective for ammonium and inhibited by divalent cations. GmNOD26 is regulated by phosphorylation of a specific serine residue, S262. Using phosphomimetics I demonstrate a switch in the permeability of GmNOD26 to water and cations. Through expression in X. laevis oocytes and complementation of mutant Saccharomyces cerevisiae strains, ammonia and ammonium are confirmed as the only nitrogen species transported through GmNOD26. This study provides strong evidence that GmNOD26 is a multifunctional channel that facilitates both ammonia and ammonium efflux from nitrogen-fixing nodules and that this transport is regulated by phosphorylation of S262.

Previous work has established that uptake of reduced carbon, in the form of organic acids like malate, by symbiosomes occurs through a dicarboxylate carrier energised by the electrochemical potential across the SM and inhibited by phthalonic acid. However, proteomic studies of isolated SM have failed to identify typical plant dicarboxylate transporters. Transcriptomic studies have shown that Aluminium activated Malate Transporters (ALMT) are highly expressed in nodules. I identified 13 ALMT genes in the Lotus japonicus genome, but only LjALMT1 was highly expressed in nodules, but it appears to localise to the root and nodule vascular tissues, rather than the infected region of nodules. I therefore turned to other possible candidates in the proteome of isolated soybean SM, in particular eight members of the Nitrate Peptide transporter Family (NPF) of proteins. I analysed the dicarboxylate transport activity of these proteins in X. laevis oocytes and identified GmNPF1.2 as a dicarboxylate carrier. In GmNPF1.2-expressing X. laevis oocytes, ionic currents were activated by both malate and succinate. The transport of malate by GmNPF1.2 was confirmed through [14C] measurements and shown to be inhibited by phthalonic acid.

These data suggest that ammonium/malate exchange across the soybean SM is facilitated by GmNPF1.2, as the SM dicarboxylate carrier, and GmNOD26, as the SM non-selective monovalent cation channel. However, confirmation requires further experimentation with soybean, using mutagenesis techniques to investigate symbiotic phenotypes.

Keywords: Nitrogen fixation, Malic acid, Ammonium, Symbiosome, Legume

Subject: Biology thesis

Thesis type: Doctor of Philosophy
Completed: 2023
School: College of Science and Engineering
Supervisor: Kathleen Soole