International Journal of Molecular Sciences Review Iron Transport across Symbiotic Membranes of Nitrogen-Fixing Legumes David A. Day 1,* and Penelope M. C. Smith 2 1 College of Science and Engineering, Flinders University, GPO Box 2100, Adelaide, SA 5001, Australia 2 School of Life Sciences, La Trobe University, Melbourne, VIC 3083, Australia; [email protected] * Correspondence: David.Day@flinders.edu.au Abstract: Iron is an essential nutrient for the legume-rhizobia symbiosis and nitrogen-fixing bac- teroids within root nodules of legumes have a very high demand for the metal. Within the infected cells of nodules, the bacteroids are surrounded by a plant membrane to form an organelle-like structure called the symbiosome. In this review, we focus on how iron is transported across the symbiosome membrane and accessed by the bacteroids. Keywords: legumes; nitrogen fixation; symbiosomes; iron 1. Introduction Iron is an essential nutrient for cell function and plants have developed specialised mechanisms for mobilising, absorbing and storing the metal. Cellular iron homeostasis is important because iron is toxic when present in excess. Symbiotically grown legumes have a particularly high requirement for iron and low iron severely retards their growth and their ability to fix atmospheric nitrogen [1,2]. Legumes form a symbiosis with nitrogen-fixing soil bacteria (rhizobia) that enables the plants to utilize atmospheric nitrogen for growth. Infection of the legume root by rhizobia Citation: Day, D.A.; Smith, P.M.C. results in the formation of specialized organs called nodules that provide the microaerobic Iron Transport across Symbiotic conditions required for operation of the bacterium’s nitrogenase enzyme. The establishment Membranes of Nitrogen-Fixing of the nodule begins with signaling between the plant root and the rhizobia, and results Legumes. Int. J. Mol. Sci. 2021, 22, 432. https://doi.org/10.3390/ in the bacteria colonizing root hairs. An infection thread is formed from invagination of ijms22010432 the root cell wall and the bacteria travel through the infection thread into the root cortex where nodule formation is initiated. Eventually, in response to unknown signals, the wall Received: 15 December 2020 of the infection thread and that of root cortex cells are degraded and the rhizobia released Accepted: 31 December 2020 into the cells. Within the nodule infected cells, the rhizobia are enclosed in a plant-derived Published: 4 January 2021 membrane to form an organelle-like compartment called the symbiosome (Figure1). Within this symbiosome the rhizobia differentiate into their symbiotic form, the bacteroid, which Publisher’s Note: MDPI stays neu- begin to fix nitrogen to ammonia via the enzyme nitrogenase. The symbiosome membrane tral with regard to jurisdictional clai- (SM) effectively controls the exchange of metabolites between the symbiotic partners. The ms in published maps and institutio- SM, although originating from the plasma membrane of infected nodule cells, evolves nal affiliations. over the course of nodule organogenesis to become a new and specialized membrane containing symbiosis-specific proteins synthesized from plant nuclear genes (see [3–5] for recent reviews). Mature nodules are composed of an infection zone, which contains both infected and Copyright: © 2021 by the authors. Li- censee MDPI, Basel, Switzerland. uninfected cells, surrounded by layers of cortical cells (3,5). Nutrients are transported This article is an open access article to the nodule via the vasculature, which terminates in the cortex. Nodules are generally distributed under the terms and con- considered as two distinct types, determinate and indeterminate. Determinate nodules, ditions of the Creative Commons At- such as those on soybean and Lotus japonicus, are spherical and clearly divided into the tribution (CC BY) license (https:// central infected zone and the outer cortex. Cells in the infected zone contain bacteroids creativecommons.org/licenses/by/ all at approximately the same developmental stage. Indeterminate nodules, on the other 4.0/). hand, have an elongated or branched shape and have a persistent meristem that remains Int. J. Mol. Sci. 2021, 22, 432. https://doi.org/10.3390/ijms22010432 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2021, 22, 432 2 of 8 throughout the life of the nodule. Indeterminate nodules are segmented into distinct developmental zones: a meristematic zone at the tip, an invasion zone where the invading Int. J. Mol. Sci. 2021, 22, x FOR PEER REVIEW 2 of 8 rhizobia are released, a transition zone where the rhizobia differentiate into bacteroids, a nitrogen fixation zone, and a zone of senescence closest to the root. Figure 1. (A) nodules on the root of a soybean plant. (B) detail of infected cells. V = vacuole; Figure 1. (A) nodules on the root of a soybean plant. (B) detail of infected cells. V = vacuole; as = air as = air space; uc = uninfected cell; ic = infected cell; pbm = peribacteroid membrane, now known as space; uc = uninfected cell; ic = infected cell; pbm = peribacteroid membrane, now known as the the symbiosome membrane; bact = bacteroid. symbiosome membrane; bact = bacteroid. Nitrogen fixing legume nodules have a very high requirement for iron and nodule ironMature content isnodules higher are than composed any of the of other an plantinfection organs zone, [4,6 –which8]. Iron contains is an essential both infected cofactor andfor nitrogenase,uninfected cells, the nitrogen-fixingsurrounded by enzyme,layers of as cortical well as cells respiratory (3,5). Nutrients chain components are trans- portedand other to the essential nodule enzymes via the vasculature, in bacteroids which [9]. It terminates is also required in the forcortex. the infectedNodules cells’are generallymitochondrial considered respiratory as two chain distinct components types, determinate and for the synthesisand indeterminate. of leghemoglobin Determinate in the nodules,infected cells,such as which those is on essential soybean for and the transportLotus japonicus of oxygen, are tospherical the bacteroids and clearly [8–10 divided]. Given intothat the leghemoglobin central infected is present zone and in mM the concentrationsouter cortex. Cells in the in infectedthe infected cell cytosolzone contain [10,11] bacteroidsand that infected all at approximately cells in a mature the same soybean developmental nodule contain stage. up Indeterminate to 12,000 mitochondria nodules, peron thecell other [12] andhand, as have many an as elongated 50,000 bacteroids or branched [13,14 shape], then and the have requirement a persistent for meristem iron in nodule that remainscells is enormous. throughout the life of the nodule. Indeterminate nodules are segmented into distinctNitrogen developmental fixation zones: has been a meristematic studied extensively zone at the in tip, a number an invasion of different zone where legumes, the invadingincluding rhizobia the model are plants released,Medicago a transition truncatula zoneand whereLotus the japonicus rhizobia, and differentiate important cropsinto bacteroids,such as soybean a nitrogen and pea.fixation Iron zone, is most and likely a zone imported of senescence into the closest nodule to asthe ferric root. citrate via the xylemNitrogen [15 ,16fixing] and legume subsequently nodules must have cross a very a number high requirement of cell layers for to reachiron and the infectednodule ironcells content [17]. Both is higher symplastic than any and of apoplastic the other routesplant organs are utilized [4,6–8]. for Iron this is transport an essential [15 ].co- A factormember for ofnitrogenase, the NRAMP the family nitrogen-fixing in M. truncatula enzyme,, MtNRAMP1, as well as catalyses respiratory ferrous chain iron compo- uptake nentsinto infectedand other cells essential [18], possibly enzymes acting in bacteroids in concert [9]. with It Multidrugis also required and Toxic for the Compound infected cells’Extrusion mitochondrial protein 67 respiratory (MtMATE67), chain which compon catalyzesents and citrate for effluxthe synthesis and appears of leghemoglo- to enhance biniron in uptake the infected by infected cells, which cells [19 is]. essential In this review, for the wetransport focus on of theoxygen subsequent to the bacteroids events during [8– 10].which Given iron that within leghemoglobin nodule infected is present cells is in transported mM concentrations into the symbiosomes in the infected for cell delivery cytosol to [10,11]the bacteroids. and that infected cells in a mature soybean nodule contain up to 12,000 mito- chondria per cell [12] and as many as 50,000 bacteroids [13,14], then the requirement for iron2. Studies in nodule with cells Isolated is enormous. Symbiosomes NitrogenMost studies fixation have has utilised been soybean studied nodules extensively because in ofa number the relative of easedifferent of symbiosome legumes, includingpurification the frommodel this plants species. Medicago Initial truncatula experiments and Lotus showed japonicus that isolated, and important symbiosomes crops suchaccumulate as soybean ferric and iron pea. when Iron presented is most likely with ferricimported citrate into [20 the,21 ],nodule and that as ferric this involves citrate via the theconcomitant xylem [15,16] uptake and of subsequently citrate [21]. However, must cross the a ironnumber was notof cell taken layers up byto thereach bacteroids the in- fectedwithin cells and [17]. remained Both symplastic in the symbiosome and apoplastic space [routes20]. Another