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Sulfate Is Transported at Significant Rates Through the Symbiosome

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Sulfate Is Transported at Significant Rates Through the Symbiosome Plant Cell and Environment 42 (4): 1180-1189 (2019) 1 ORIGINAL ARTICLE 2 3 Sulfate is transported at significant rates through the symbiosome 4 membrane and is crucial for nitrogenase biosynthesis 5 6 7 Sebastian Schneider1 Arno Schintlmeister2,3 Manuel Becana4 Michael Wagner2,3 8 Dagmar Woebken2 Stefanie Wienkoop1* 9 10 1Division of Molecular Systems Biology, Department of Ecogenomics and Systems Biology, 11 University of Vienna, Austria 12 13 2Division of Microbial Ecology, Department of Microbiology and Ecosystem Science, 14 Research Network ‘Chemistry meets Microbiology’ University of Vienna, Austria 15 16 3Large-Instrument Facility for Advanced Isotope Research, University of Vienna, Austria 17 18 4Estación Experimental de Aula Dei, CSIC, Apartado 13034, 50080 Zaragoza, Spain 19 20 Correspondence 21 Stefanie Wienkoop, Division of Molecular Systems Biology, Department of Ecogenomics and 22 Systems Biology, University of Vienna, Austria. 23 E -mail: stefanie.wienkoop@ univie.ac.at 24 25 1 Plant Cell and Environment 42 (4): 1180-1189 (2019) 26 27 Abstract 28 Legume-rhizobia symbioses play a major role in food production for an ever growing human 29 population. In this symbiosis, dinitrogen is reduced ('fixed') to ammonia by the rhizobial 30 nitrogenase enzyme complex and is secreted to the plant host cells, while dicarboxylic acids 31 derived from photosynthetically-produced sucrose are transported into the symbiosomes and 32 serve as respiratory substrates for the bacteroids. The symbiosome membrane contains high 33 levels of SST1 protein, a sulfate transporter. Sulfate is an essential nutrient for all living 34 organisms, but its importance for symbiotic nitrogen fixation and nodule metabolism has long 35 been underestimated. Using chemical imaging, we demonstrate that the bacteroids take up 20- 36 fold more sulfate than the nodule host cells. Furthermore, we show that nitrogenase 37 biosynthesis relies on high levels of imported sulfate, making sulfur as essential as carbon for 38 the regulation and functioning of symbiotic nitrogen fixation. Our findings thus establish the 39 importance of sulfate and its active transport for the plant-microbe interaction that is most 40 relevant for agriculture and soil fertility. 41 42 KEYWORDS 43 legume nodules, nanoSIMS, nitrogen fixation, stable isotope labeling, sulfur deficiency, 44 symbiotic sulfate transporter (SST1) 45 46 2 Plant Cell and Environment 42 (4): 1180-1189 (2019) 47 1 INTRODUCTION 48 Nitrogen (N) is an essential plant nutrient and the most limiting factor for plant productivity 49 and agricultural production worldwide. However, the symbiosis established between 50 leguminous plants and certain soil bacteria, collectively known as rhizobia, is able to 51 overcome N limitation by providing nutritional benefits for both partners (Lugtenberg & 52 Kamilova, 2009). Symbiotic nitrogen fixation (SNF) is the largest natural source of N in 53 agricultural systems (Smil, 1999). This process enables plants to trap atmospheric N2 to 54 satisfy their N demand and is important for sustainable agriculture that faces the necessity of 55 reducing the input of N-fertilizers because their negative eutrophication effects (Peoples, 56 Herridge, & Ladha,1995) and their contribution to nitrous oxide emissions from soil that 57 accelerate global warming (Smith, Mosier, Crutzen, & Winiwarter, 2012). The legume- 58 rhizobia interaction leads to the formation of a new specialized plant organ, the nodule, where 59 SNF takes place. In many legumes, rhizobia colonize roots through formation of infection 60 threads near the tip of the epidermal root hair cells and are released into cortical cells via 61 endocytosis (Oldroyd, Murray, Poole, & Downie, 2011). The developing nodule cells 62 continue to proliferate and rhizobia become enclosed individually or in small groups within a 63 new organelle, the symbiosome. This consists of the bacteroids (differentiated rhizobia that 64 fix N2), a plant-derived symbiosome membrane (SM), and a symbiosome space (SS) between 65 the bacteroids and the membrane (Mellor, 1989). The SM is an interface for metabolic 66 exchange between the two symbiotic partners. Essentially, this exchange includes ammonium 67 produced by the bacteroid and photosynthetically-derived organic acids produced by the plant 68 (Udvardi & Poole, 2013). 69 During nodule development, both partners undergo coordinated differentiation that 70 involves global changes in gene expression (Colebatch et al., 2002; Fedorova et al., 2002; 71 Colebatch et al., 2004). The major rhizobial proteins induced through symbiosis and enabling 3 Plant Cell and Environment 42 (4): 1180-1189 (2019) 72 SNF are the nitrogenase components (Child, 1975; Scowcroft & Gibson, 1975). The 73 nitrogenase complex consists of an Fe-protein (dinitrogenase reductase) and a MoFe-protein 74 (dinitrogenase). The Fe-protein is a homodimer (2) containing a Fe4S4 cluster, whereas the 75 MoFe-protein is a heterotetramer (22) comprising Fe8S7 clusters (P-clusters) at the - 76 interface and FeMo-cofactors (MoFe7S9C-homocitrate) within the subunits (Rubio & 77 Ludden, 2008). Consequently, the synthesis of nitrogenase requires a considerable supply of 78 sulfur (S) and an insufficiency in this element drastically affects the symbiotic interaction 79 (DeBoer & Duke, 1982; Udvardi & Poole, 2013). 80 Sulfur is an indispensable and limiting nutrient for all living organisms (Zhao, Wood, & 81 McGrath, 1999). Sulfate is actively taken up and assimilated by plants and many 82 microorganisms via specific sulfate transporters. It is converted to the nutritionally important 83 S-containing amino acids cysteine (Cys) and methionine (Met), which are necessary for 84 protein biosynthesis (Leustek & Saito, 1999; Kopriva & Rennenberg, 2004). Furthermore, S 85 is used for the formation of coenzymes, ligands, and FeS clusters of enzymes (Davidian & 86 Kopriva, 2010). Hence, S malnutrition in plants leads to perturbations in amino acid pools and 87 causes reduction of biomass production and chlorophyll content (Nikiforova et al., 2005). 88 Sulfur deficiency of soils has gained increased attention over the past three decades on a 89 worldwide scale (Scherer, 2009). However, although this nutrient deficiency reduces crop 90 yield and quality, its crucial effects on SNF have been overlooked. The physiology of plant 91 sulfate transport has been extensively studied (Smith, Ealing, Hawkesfordt & Clarkson, 1995; 92 Takahashi et al., 2000; Buchner, Takahashi, & Hawkesford, 2004), and several genes 93 encoding high-affinity sulfate transporters have been isolated and characterized (Smith et al., 94 1997; Takahashi et al., 1997; Yoshimoto, Takahashi, Smith, Yamaya & Saito, 2002). It was 95 shown that plant organs have different affinities towards sulfate transport, which enables an 96 efficient uptake throughout the whole plant (Kreuzwieser, Herschbach, & Rennenberg, 1996; 4 Plant Cell and Environment 42 (4): 1180-1189 (2019) 97 Takahashi et al., 2000; Hawkesford, 2003). In early work, we provided evidence that sulfate 98 transporters play a role in SNF (Wienkoop & Saalbach, 2003). Based on proteomic analyses 99 of isolated SM from nodules of the model legume Lotus japonicus, the sulfate transporter 100 SST1 was identified and shown to be specifically expressed in the nodules (Krusell et al., 101 2005). Subsequently, SST1 was localized to the SM (Wienkoop & Saalbach, 2003) and 102 suggested to be responsible for the transport of sulfate from the plant to the bacteroids 103 (Krusell et al., 2005). These authors proposed that SST1 is also able to transport molybdate, 104 but this seems not to be the case because, very recently, molybdate-specific transporters have 105 been detected in Medicago truncatula nodules (Tejada-Jiménez et al., 2017; Gil-Díez et al., 106 2018). 107 There is increasing evidence that SST1 is imperative for nodule activity, but the role of S 108 in SNF is poorly defined (Krusell et al., 2005; Kalloniati et al., 2015). In this study, we 109 provide evidence for the crucial role of active transport of high levels of S through the SM 110 and its significant accumulation in the bacteroids, which is essential for nitrogenase 111 biosynthesis. 112 113 2 MATERIALS AND METHODS 114 2.1 Plant growth 115 Seeds of Lotus japonicus Gifu B-129 and its mutant derivative sst1-1 (sym13) were kindly 116 provided by Niels Sandal and Jens Stougaard (Aarhus University, Denmark). Surface- 117 sterilized seeds were germinated on agar plates in B&D nutrient solution (Broughton & 118 Dilworth, 1971) solidified with 0.8% plant agar. After 7 days, seedlings were inoculated with 119 Mesorhizobium loti strain R7A and transferred to pots containing sterilized clay granules 120 (Seramis, Westland Horticulture, Dungannon, UK) soaked in B&D nutrient solution 121 supplemented with 1 mM KNO3. Plants were then grown in N-free B&D solution in a climate 5 Plant Cell and Environment 42 (4): 1180-1189 (2019) 122 chamber with a 16-h photoperiod and 22°C/18°C (day/night) regime and a relative humidity 123 of 70% as described (Krusell et al., 2005; Kalloniati et al., 2015). 124 125 2.2 34S-Sulfate metabolic labeling in planta 126 Three weeks after infection, plants were checked for sufficient nodulation, irrigated for two 127 days with water to wash out the nutrients from the substrate, and divided into two subsets: 128 control plants and plants for 34S uptake. For stable isotope labeling in planta (34S SILIP), 32 . 32 32 . 32 . 32 . 32 . 129 Mg SO4 7H2O, K2 SO4, Mn SO4 H2O, Zn SO4 7H2O, Cu SO4 5H2O, and Co SO4 7H2O . 130 in the B&D solution were replaced by MgCl2, K2HPO4, MnCl2 2H2O, ZnCl2, CuCl2 2H2O, 131 and CoCl2, respectively. This was necessary to maintain exactly the same nutrient 34 34 132 concentrations as in the original solution. Likewise, Na2 SO4 [90 at% S, 98% (CP); Sigma- 133 Aldrich] was included as the only S source to maintain a concentration of 0.5 mM sulfate in 134 the B&D solution. Plants were watered with the 34S-nutrient solution and nodules were 135 harvested after 96 hr of labeling and used for protein extraction. Control plants (without 34S 32 136 labeling) were supplied with the same nutrient solution, but using unlabeled Na2 SO4.
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