Functional divergence and symbiotic significance of nitrate reductase isoforms in Medicago truncatula
Abstract
Nitrate reductase (NR) is a key enzyme in nitrate assimilation, yet its function within nodules remains poorly understood. In Medicago truncatula, 3 NR genes, MtNR1, MtNR2, and MtNR3, exhibit distinct evolutionary origins and regulatory features. Phylogenetic analyses indicate that NR3-type genes, originated from a duplication of NR1 within the inverted repeat-lacking clade (IRLC) legumes, have lost the conserved phosphorylation sites critical for post-translational regulation. To assess the functional significance of these isoforms, we characterized single and double nr mutants obtained through Tnt1 transposon insertion under nitrate nutrition and during symbiosis. MtNR1 is the primary contributor to total NR activity, with nr1 and nr2 mutants retaining around 10% and 30% of wild-type levels, respectively. The nr1/nr2 double mutant shows an almost complete loss of NR activity and fails to survive under nitrate supply, demonstrating the essential and non-redundant roles of both isoforms. Under symbiotic conditions, single mutants displayed normal nodulation, whereas nodule development was nearly abolished in the double mutant despite continued MtNR3 expression. In addition to its role in nitrogen assimilation, single nr mutants showed increased sensitivity to hypoxic stress and impaired recovery of nitrogen fixation, revealing a role for NR in nodule energy metabolism through the phytoglobin-NO respiration pathway. We propose that the combined loss of NR1 and NR2 disrupts NO cycling linked to mitochondrial electron transport, thereby compromising the energy balance required for symbiosis under microoxic conditions. This work provides a framework to investigate NR diversification in legumes and opens perspectives for improving nitrogen fixation under environmental constraints.
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BibTeX
@article{bosseno2026functional,
title = {{Functional divergence and symbiotic significance of nitrate reductase isoforms in Medicago truncatula}},
author = {Marc Bosseno and Alexandre Demba and Natasha Horta Araújo and Dominique Colinet and Abir Israel and Marie Pacoud and Mickael Maucourt and Yassine El Fazaa and Daniel Jacob and Marc Lepetit and Dominique Rolin and Renaud Brouquisse and Alexandre Boscari},
journal = {Plant Physiology},
year = {2026},
month = jul,
doi = {10.1093/plphys/kiag377},
url = {https://hal.science/hal-05745992v1},
note = {CC BY},
}
RIS
TY - JOUR
TI - Functional divergence and symbiotic significance of nitrate reductase isoforms in Medicago truncatula
AU - Marc Bosseno
AU - Alexandre Demba
AU - Natasha Horta Araújo
AU - Dominique Colinet
AU - Abir Israel
AU - Marie Pacoud
AU - Mickael Maucourt
AU - Yassine El Fazaa
AU - Daniel Jacob
AU - Marc Lepetit
AU - Dominique Rolin
AU - Renaud Brouquisse
AU - Alexandre Boscari
PY - 2026
DA - 2026/07/02
JO - Plant Physiology
DO - 10.1093/plphys/kiag377
UR - https://hal.science/hal-05745992v1
AB - Nitrate reductase (NR) is a key enzyme in nitrate assimilation, yet its function within nodules remains poorly understood. In Medicago truncatula, 3 NR genes, MtNR1, MtNR2, and MtNR3, exhibit distinct evolutionary origins and regulatory features. Phylogenetic analyses indicate that NR3-type genes, originated from a duplication of NR1 within the inverted repeat-lacking clade (IRLC) legumes, have lost the conserved phosphorylation sites critical for post-translational regulation. To assess the functional significance of these isoforms, we characterized single and double nr mutants obtained through Tnt1 transposon insertion under nitrate nutrition and during symbiosis. MtNR1 is the primary contributor to total NR activity, with nr1 and nr2 mutants retaining around 10% and 30% of wild-type levels, respectively. The nr1/nr2 double mutant shows an almost complete loss of NR activity and fails to survive under nitrate supply, demonstrating the essential and non-redundant roles of both isoforms. Under symbiotic conditions, single mutants displayed normal nodulation, whereas nodule development was nearly abolished in the double mutant despite continued MtNR3 expression. In addition to its role in nitrogen assimilation, single nr mutants showed increased sensitivity to hypoxic stress and impaired recovery of nitrogen fixation, revealing a role for NR in nodule energy metabolism through the phytoglobin-NO respiration pathway. We propose that the combined loss of NR1 and NR2 disrupts NO cycling linked to mitochondrial electron transport, thereby compromising the energy balance required for symbiosis under microoxic conditions. This work provides a framework to investigate NR diversification in legumes and opens perspectives for improving nitrogen fixation under environmental constraints.
SN - 0032-0889
SN - 1532-2548
ER -