Hemoglobin LjGlb1-1 is involved in nodulation and regulates the level of nitric oxide in the<i>Lotus japonicus–Mesorhizobium loti</i>symbiosis

Fukudome, Mitsutaka; Calvo-Beguería, Laura; Tomohiro, Kado; Osuki, Ken-ichi; Rubio, Maria C.; Murakami, Eiichi; Nagata, Masaki; Kucho, Ken-ichi; Sandal, Niels; Stougaard, Jens; Becana, Manuel; Uchiumi, Toshiki

doi:10.1093/jxb/erw290

Cited by 40 publications

(58 citation statements)

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“…Specifically, Glb1-1 is induced by NO, whereas Glb1-2 and Glb2 are not (Shimoda et al, 2005;Bustos-Sanmamed et al, 2011). After only 4 h of inoculation with Mesorhizobium loti, the roots of L. japonicus produce a transient increase in NO concentration along with the induction of Glb1-1, whereas after infection with pathogenic bacteria the NO concentration remains high for at least 24 h (Shimoda et al, 2005;Nagata et al, 2008;Fukudome et al, 2016). These and other observations led the authors to propose that Glb1-1 acts as an NO scavenger at the early stage of rhizobial infection, thereby preventing the plant's defense response.…”

Section: Introductionmentioning

confidence: 98%

CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation

et al. 2019

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Summary Legume nodules contain high concentrations of leghemoglobins (Lbs) encoded by several genes. The reason for this multiplicity is unknown. CRISPR/Cas9 technology was used to generate stable mutants of the three Lbs of Lotus japonicus. The phenotypes were characterized at the physiological, biochemical and molecular levels. Nodules of the triple mutants were examined by electron microscopy and subjected to RNA‐sequencing (RNA‐seq) analysis. Complementation studies revealed that Lbs function synergistically to maintain optimal N2 fixation. The nodules of the triple mutants overproduced superoxide radicals and hydrogen peroxide, which was probably linked to activation of NADPH oxidases and changes in superoxide dismutase isoforms expression. The mutant nodules showed major ultrastructural alterations, including vacuolization, accumulation of poly‐β‐hydroxybutyrate and disruption of mitochondria. RNA‐seq of c. 20 000 genes revealed significant changes in expression of carbon and nitrogen metabolism genes, transcription factors, and proteinases. Lb‐deficient nodules had c. 30–50‐fold less heme but similar transcript levels of heme biosynthetic genes, suggesting a post‐translational regulatory mechanism of heme synthesis. We conclude that Lbs act additively in nodules and that the lack of Lbs results in early nodule senescence. Our observations also provide insight into the reprogramming of the gene expression network associated with Lb deficiency, probably as a result of uncontrolled intracellular free O2 concentration.

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Section: Introductionmentioning

confidence: 98%

CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation

et al. 2019

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“…The differences in CO binding kinetics found in this work, along with the large variations in O 2 affinities and expression profiles reported earlier (Sainz et al, 2013), strongly suggest that the three proteins perform non-redundant functions. Overexpression of LjGlb1-1 increases the symbiotic performance of L. japonicus (Shimoda et al, 2009) and, conversely, the knocked-out line shows alterations in the infection process and produces fewer nodules than the wild-type line (Fukudome et al, 2016). Consequently, transgenic approaches aimed at increasing the content of each of the three nsHbs, or of a combination of them, in the model legume L. japonicus (first stage) and in a crop legume with comparable nsHbs, such as soybean or common bean (second stage), are likely to result in outperformance of plants, at least under symbiotic conditions.…”

Section: Discussionmentioning

confidence: 99%

“…Besides leghemoglobins, whose expression is restricted to nodules, legumes contain nsHbs in leaves, roots, and nodules (Andersson et al, 1996; Bustos-Sanmamed et al, 2011). In fact, some of these nsHbs have important functions in the onset of symbiosis (Fukudome et al, 2016) and exhibit high expression in nodules compared with other plant organs (Bustos-Sanmamed et al, 2011). In this work, we have studied the nsHbs of Lotus japonicus , a model legume for classical and molecular genetics (Handberg and Stougaard, 1992).…”

Section: Introductionmentioning

confidence: 99%

Characterization of the Heme Pocket Structure and Ligand Binding Kinetics of Non-symbiotic Hemoglobins from the Model Legume Lotus japonicus

et al. 2017

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Plant hemoglobins (Hbs) are found in nodules of legumes and actinorhizal plants but also in non-symbiotic organs of monocots and dicots. Non-symbiotic Hbs (nsHbs) have been classified into two phylogenetic groups. Class 1 nsHbs show an extremely high O2 affinity and are induced by hypoxia and nitric oxide (NO), whereas class 2 nsHbs have moderate O2 affinity and are induced by cold and cytokinins. The functions of nsHbs are still unclear, but some of them rely on the capacity of hemes to bind diatomic ligands and catalyze the NO dioxygenase (NOD) reaction (oxyferrous Hb + NO → ferric Hb + nitrate). Moreover, NO may nitrosylate Cys residues of proteins. It is therefore important to determine the ligand binding properties of the hemes and the role of Cys residues. Here, we have addressed these issues with the two class 1 nsHbs (LjGlb1-1 and LjGlb1-2) and the single class 2 nsHb (LjGlb2) of Lotus japonicus, which is a model legume used to facilitate the transfer of genetic and biochemical information into crops. We have employed carbon monoxide (CO) as a model ligand and resonance Raman, laser flash photolysis, and stopped-flow spectroscopies to unveil major differences in the heme environments and ligand binding kinetics of the three proteins, which suggest non-redundant functions. In the deoxyferrous state, LjGlb1-1 is partially hexacoordinate, whereas LjGlb1-2 shows complete hexacoordination (behaving like class 2 nsHbs) and LjGlb2 is mostly pentacoordinate (unlike other class 2 nsHbs). LjGlb1-1 binds CO very strongly by stabilizing it through hydrogen bonding, but LjGlb1-2 and LjGlb2 show lower CO stabilization. The changes in CO stabilization would explain the different affinities of the three proteins for gaseous ligands. These affinities are determined by the dissociation rates and follow the order LjGlb1-1 > LjGlb1-2 > LjGlb2. Mutations LjGlb1-1 C78S and LjGlb1-2 C79S caused important alterations in protein dynamics and stability, indicating a structural role of those Cys residues, whereas mutation LjGlb1-1 C8S had a smaller effect. The three proteins and their mutant derivatives exhibited similarly high rates of NO consumption, which were due to NOD activity of the hemes and not to nitrosylation of Cys residues.

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“…Thus, treatment of M. truncatula roots in interaction with S. meliloti with 1‐mM cPTIO inhibits nodulation as long as cPTIO is present in the medium (del Giudice et al, ), suggesting that NO is needed for symbiosis. In contrast, in the L. japonicus – Mesorhizobium loti symbiosis, the overexpression of Pgb LjHb1 (LjGlb1) or AfHb1 results in a decrease in NO level and an increase in nodule number (Shimoda et al, ), whereas L. japonicus lines mutated for LjGlb1 show higher NO production and lower infection and nodule number (Fukudome et al, ). Similarly, experiments carried out in peas ( Pisum sativum ) in symbiosis with Rhizobium leguminosarum show that 2‐mM sodium nitroprusside, a NO donor, affects the adhesion and the penetration of rhizobia in roots and that this effect is reversed by the addition of 2‐μM equine erythrocyte haemoglobin (Glyan'ko, Mitanova, & Vasil'eva, ).…”

Section: Does Soil N Status and No Influence Nodulation?mentioning

confidence: 99%

“…Cytochrome c oxidase (COX; Complex IV) and Complex III (bc 1 ) are the sites of NO production in ETC ( Figure 1). Inhibition of the Mitochondrial Complex III with myxothiazol and inhibition of Complex IV with KCN indeed lead to the abolishment of NO production in mitochondria (reviewed in Fukudome et al, 2016 Another NO biosynthetic pathway that has been shown to be involved in nodules is the NOS pathway. To date, no NOS-like gene or protein homologue for NOS have been identified in higher plants, but a gene named AtNOA1 (NO associated) and encoding a GTPase was characterized in Arabidopsis thaliana.…”

Section: No Sources During Symbiosis: Current Statusmentioning

confidence: 99%

Pathways of nitric oxide metabolism and operation of phytoglobins in legume nodules: Missing links and future directions

et al. 2018

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The interaction between legumes and rhizobia leads to the establishment of a beneficial symbiotic relationship. Recent advances in legume - rhizobium symbiosis revealed that various reactive oxygen and nitrogen species including nitric oxide (NO) play important roles during this process. Nodule development occurs with a transition from a normoxic environment during the establishment of symbiosis to a microoxic environment in functional nodules. Such oxygen dynamics are required for activation and repression of various NO production and scavenging pathways. Both the plant and bacterial partners participate in the synthesis and degradation of NO. However, the pathways of NO production and degradation as well as their cross-talk and involvement in the metabolism are still a matter of debate. The plant-originated reductive pathways are known to contribute to the NO production in nodules under hypoxic conditions. Non-symbiotic hemoglobin (phytoglobin) (Pgb) possesses high NO oxygenation capacity, buffers and scavenges NO. Its operation, through a respiratory cycle called Pgb-NO cycle, leads to the maintenance of redox and energy balance in nodules. The role of Pgb/NO cycle under fluctuating NO production from soil needs further investigation for complete understanding of NO regulatory mechanism governing nodule development to attain optimal food security under changing environment.

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Hemoglobin LjGlb1-1 is involved in nodulation and regulates the level of nitric oxide in theLotus japonicus–Mesorhizobium lotisymbiosis

Abstract: HighlightMissense and null Lotus japonicus mutants allowed us to demonstrate that hemoglobin LjGlb1-1 is required for infection thread elongation and nodule formation, probably by regulating nitric oxide production in the roots.

Cited by 40 publications

References 44 publications

CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation

CRISPR/Cas9 knockout of leghemoglobin genes in Lotus japonicus uncovers their synergistic roles in symbiotic nitrogen fixation

Characterization of the Heme Pocket Structure and Ligand Binding Kinetics of Non-symbiotic Hemoglobins from the Model Legume Lotus japonicus

Pathways of nitric oxide metabolism and operation of phytoglobins in legume nodules: Missing links and future directions

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