Nicotianamine Synthase 2 Is Required for Symbiotic Nitrogen Fixation in Medicago truncatula Nodules.

Viviana Escudero,Isidro Abreu,Jiangqi Wen,Camille Larue,Manuel González-Guerrero,Kirankumar S Mysore,José M Argüello,Lorena Novoa-Aponte,Juan Imperial,Jorge Castillo-González,Javier Abadía,Manuel Tejada-Jiménez,Eric Del Sastre,Hiram Castillo-Michel,Ana Álvarez-Fernández

doi:10.3389/fpls.2019.01780

Abstract

Symbiotic nitrogen fixation carried out by the interaction between legumes and diazotrophic bacteria known as rhizobia requires relatively large levels of transition metals. These elements are cofactors of many key enzymes involved in this process. Metallic micronutrients are obtained from soil by the roots and directed to sink organs by the vasculature, in a process mediated by a number of metal transporters and small organic molecules that facilitate metal delivery in the plant fluids. Among the later, nicotianamine is one of the most important. Synthesized by nicotianamine synthases (NAS), this molecule forms metal complexes participating in intracellular metal homeostasis and long-distance metal trafficking. Here we characterized the NAS2 gene from model legume Medicago truncatula. MtNAS2 is located in the root vasculature and in all nodule tissues in the infection and fixation zones. Symbiotic nitrogen fixation requires of MtNAS2 function, as indicated by the loss of nitrogenase activity in the insertional mutant nas2-1, phenotype reverted by reintroduction of a wild-type copy of MtNAS2. This would result from the altered iron distribution in nas2-1 nodules shown with X-ray fluorescence. Moreover, iron speciation is also affected in these nodules. These data suggest a role of nicotianamine in iron delivery for symbiotic nitrogen fixation.

Highlights

Nitrogen is one of the main limiting nutrients in the biosphere, in spite of N2 abundance (Smil, 1999; Hoffman et al, 2014)
Mutant Database (Sun et al, 2019; https://medicago-mutant. noble.org/mutant/index.php) showed NF15101 as one of the available mutants with a nitrogen fixation deficient phenotype. This line has 22 Tnt1 insertions, 10 of which interrupted different M. truncatula genes (Supplementary Table 1), Medtr2g070310 among them. This gene encodes a protein with 52% identity and 67% similarity to Arabidopsis thaliana NAS2 protein, and was renamed MtNAS2
Homozygous plants from a R2 seed population were identified by a PCR reverse screening (Cheng et al, 2014). These homozygous plants were considered as null mutants for MtNAS2, since MtNAS2 expression levels were below our detection limit (Figure 1C)

Summary

Introduction

Nitrogen is one of the main limiting nutrients in the biosphere, in spite of N2 abundance (Smil, 1999; Hoffman et al, 2014). Symbiotic nitrogen fixation by the legume-rhizobia system is carried out in root nodules (Downie, 2014) These are differentiated organs that develop after a complex exchange of chemical signals between the symbionts (Oldroyd, 2013). Nodule development follows either an indeterminate or a determinate growth pattern, based on whether they maintain an apical meristem to sustain growth (Vasse et al, 1990) As this meristem allows for sustained growth in indeterminate nodules, four developmental zones appear: the meristematic region or zone I; the infectiondifferentiation zone or zone II, where rhizobia are released in the cell and start differentiating; the fixation zone or zone III, where nitrogenase is active; and the senescent zone or zone IV, where symbiosomes are degraded and nutrients recycled (Burton et al, 1998). Some authors define a transition interzone between zones II and III (Roux et al, 2014)

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Conclusion