Signaling by reactive molecules and antioxidants in legume nodules

Abstract

Legume nodules are symbiotic structures formed as a result of the interaction with rhizobia. Nodules fix atmospheric nitrogen into ammonia that is assimilated by the plant and this process requires strict metabolic regulation and signaling. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved as signal molecules at all stages of symbiosis, from rhizobial infection to nodule senescence. Also, reactive sulfur species (RSS) are emerging as important signals for an efficient symbiosis. Homeostasis of reactive molecules is mainly accomplished by antioxidant enzymes and metabolites and is essential to allow redox signaling while preventing oxidative damage. Here, we examine the metabolic pathways of reactive molecules and antioxidants with an emphasis on their functions in signaling and protection of symbiosis. In addition to providing an update of recent findings while paying tribute to original studies, we identify several key questions. These include the need of new methodologies to detect and quantify ROS, RNS, and RSS, avoiding potential artifacts due to their short lifetimes and tissue manipulation; the regulation of redox-active proteins by post-translational modification; the production and exchange of reactive molecules in plastids, peroxisomes, nuclei, and bacteroids; and the unknown but expected crosstalk between ROS, RNS, and RSS in nodules.

Keywords: antioxidants; legume-rhizobium symbiosis; nitrogen fixation; post-translational modifications; reactive nitrogen species; reactive oxygen species; reactive sulfur species; redox signaling.

Fig. 1

Legume nodule formation and development and distinctive structural features of indeterminate and determinate nodules. (a) Root hair of Lotus japonicus showing curling, entrapped bacteria, and incipient infection thread. The image was obtained 2 wk post inoculation (wpi) with Mesorhizobium loti strain MAFF303099 labeled with DsRed. (b) Same symbiotic system as (a) showing a long infection thread and cortical cell division. (c) Nodule primordium of L. japonicus expressing GUS driven by the leghemoglobin Lb2 promoter. Note blue staining in the infected cells. Image was taken at 5 d post inoculation (dpi). (d) Low‐magnification view of a determinate soybean nodule treated with nitrate showing bacteroids stained with SYTO 85 (nucleic acid stain; magenta). The image was taken at 4 wpi. (e) Similar view at high magnification showing infected cells replete of bacteroids and interstitial (uninfected) cells that appear ‘empty’. The image also shows that NO production (DAF‐2 DA; green) overlaps with bacteroids (SYTO 85; magenta). (f) Longitudinal section of an indeterminate nodule (Medicago truncatula) stained with toluidine blue and showing zone I (meristem), zone II (infection), interzone II–III, zone III (fixation; distal and proximal), and zone IV (senescence). Note some infection threads, especially in zone II and interzone II–III. (g) Section of a determinate nodule (L. japonicus) stained with toluidine blue. Note intense staining of infected cells. (h) Macroscopic sections of indeterminate (pea) and determinate (soybean) nodules. In the indeterminate nodule, the white, red, and green colors mark zones I–II, III, and IV, respectively. In the determinate nodule, the white and red colors mark the cortex and the infected zone. In both cases, the red color is due to the presence of millimolar concentrations of leghemoglobin. Bars: (a, b) 25 μm; (c, g) 100 μm; (d, f) 200 μm; (e) 50 μm. Credits and thanks: (a, b) Fukudome et al. (2016); (c, g) Wang et al. (2019); (d, e) Calvo‐Begueria et al. (2018); (f) Peter Mergaert (Institute des Sciences du Végétal, Gif‐sur‐Yvette, France); and (h) Larrainzar et al. (2020).

Fig. 2

Selected methods used to localize production of reactive molecules in legume nodules. (a) Hydrogen peroxide (H₂O₂) (cerium chloride method). Transmission electron micrograph of a pea nodule (zone II) marking the accumulation of H₂O₂ (Ce³⁺ is oxidized by H₂O₂ to produce electron dense deposits of cerium perhydroxides) at patches in the periphery of the infection thread (arrows), as well as surrounding the bacteria within the infection thread (double arrowhead). (b) The same method as (a) was applied to show H₂O₂ accumulation in the intercellular space (is) in the inner cortex of a mature cowpea nodule. The matrix of the space and the cell walls that surround them contain abundant H₂O₂ (arrows). (c) Superoxide radicals (nitroblue tetrazolium) in a Lotus japonicus nodule deficient in the three Lbs (lb123 mutant). Staining is observed in the infected zone. (d) Hydrogen peroxide (diaminobenzidine) in a nodule of the same mutant as (c). Staining is observed in the infected zone. (e) Detection of NO (4,5‐diaminofluorescein diacetate; DAF‐2 DA) in a soybean nodule produced by a Bradyrhizobium diazoefficiens strain lacking NorC. The green fluorescence is seen in the nodule parenchyma (cell layers of the cortex that surround the infected zone) and in the infected zone. (f) Detection by electron paramagnetic resonance spectroscopy of the nitrosyl‐leghemoglobin (Lb²⁺NO) complex. Arrows indicate the spectroscopic features marking the presence of Lb²⁺NO, and therefore NO production, in soybean nodules formed by the B. diazoefficiens nirK and norC mutants. Note that Lb²⁺NO was not detectable in nodules formed by the wild‐type strain or the napA mutant. (g, h) Detection of H₂S and polysulfides with fluorescent probes in a nodule of L. japonicus. (g) HSip‐1 DA stain for H₂S. (h) Merged image of calcofluor stain for cell walls (dark blue) and SSP4 stain for polysulfides (green); thus, in the merged image, polysulfides in the infected cells appear as light blue. Bars: (a, b) 150 nm; (c, d) 150 μm; (e) 200 μm; (g, h) 100 μm. Credits and thanks: (a, b) M. C. Rubio (CSIC, Zaragoza, Spain); (c) Rubio et al. (2004); (d) E. K. James (The James Hutton Institute, UK); (e, f) Calvo‐Begueria et al. (2018); (g, h) M. Fukudome and T. Uchiumi (Kagoshima University, Japan).

Fig. 3

Reactive oxygen species (ROS) metabolism in legume nodules. The scheme is based on biochemical studies and on transcriptomic and proteomic data of Lotus japonicus (https://lotus.au.dk; Wang et al., 2019, 2022) and Medicago truncatula (https://medicago.toulouse.inrae.fr/MtExpress; Wienkoop et al., 2012). Among other ROS metabolic pathways in the bacteroids, the figure shows the generation of ROS through the irreversible oxidation by O₂ of the Fe–S clusters of the nitrogenase enzyme complex. Dashed lines indicate potential signaling of ROS generated in the cytoplasm and conveyed to the nuclei. Blunt‐ended arrows indicate ROS scavenging. AQP, aquaporin; ASC, ascorbate; CAT, catalase; FTR, ferredoxin‐thioredoxin reductase; GalLDH, l‐galactono‐1,4‐lactone dehydrogenase; Gpx, glutathione peroxidase; GR, glutathione reductase; Grx, glutaredoxin; GSH/GSSG, reduced/oxidized glutathione; GSHS, glutathione synthetase; Lb²⁺/Lb²⁺O₂, deoxyferrous/oxyferrous leghemoglobin; mETC, mitochondrial electron transport chain; Nrx, nucleoredoxin; NTR, NADPH‐thioredoxin reductase; Prx, peroxiredoxin; RBOH, respiratory burst homolog (NADPH‐oxidase); SOD, superoxide dismutase; Trx, thioredoxin; UOX, urate oxidase; XOR, xanthine oxidoreductase; γEC, γ‐glutamylcysteine; γECS, γ‐glutamylcysteine synthetase.

Fig. 4

Reactive nitrogen species (RNS) metabolism in legume nodules. The scheme is based on biochemical studies and on transcriptomic and proteomic data of Lotus japonicus (https://lotus.au.dk; Wang et al., 2019, 2022) and Medicago truncatula (https://medicago.toulouse.inrae.fr/MtExpress; Wienkoop et al., 2012). Dashed lines indicate reactions that have been demonstrated only in vitro or that have been shown to occur in leaves. These reactions need therefore to be confirmed in nodules. Note the central role of NO, its interactions with GSH and hemoglobins, and as a precursor of peroxynitrite (ONOO⁻). Also note that the major source of NO in bacteroids is denitrification. In contrast, the assimilatory reduction of NO₃ ⁻ is involved in NO production only in the free‐living rhizobia but not in Sinorhizobium meliloti bacteroids and needs to be confirmed in bacteroids of other rhizobial species. ARC, amidoxime reducing component; Bjgb, single‐domain hemoglobin; Glb²⁺O₂/Glb³⁺, oxyferrous/ferric phytoglobin; GSNO, S‐nitrosoglutathione; Hmp, flavohemoglobin; Lb²⁺O₂/Lb³⁺, oxyferrous/ferric leghemoglobin; mETC, mitochondrial electron transport chain; Nap, respiratory nitrate reductase; NasC/NarB, bacteroid assimilatory NR; NiR, plant nitrite reductase; NirA/NirBD, bacteroid assimilatory nitrite reductase; NirK, respiratory nitrite reductase; NOD, NO dioxygenase; NorC, respiratory NO reductase; NOS‐like, NO synthase‐like activity (arginine‐dependent); NosZ, respiratory N₂O reductase; NR, plant nitrate reductase; NTR, NADPH‐thioredoxin reductase; SNOs, S‐nitrosothiols; Trx, thioredoxin; XOR, xanthine oxidoreductase.

Fig. 5

Reactive sulfur species (RSS) metabolism in legume nodules. The scheme is based on biochemical studies and on transcriptomic and proteomic data of Lotus japonicus (https://lotus.au.dk; Wang et al., 2019, 2022) and Medicago truncatula (https://medicago.toulouse.inrae.fr/MtExpress; Wienkoop et al., 2012). Some key sulfate transporters (SULTR1, SULTR3, SST1, and SulfABCD) are shown. For simplicity, oxidative reactions of thiols of protein Cys residues (P–SH) to sulfenyl (P–SOH), sulfinyl (P–SO₂H), and sulfonyl (P–SO₃H) groups are only indicated in the cytosol but may occur also in other cellular compartments. Similarly, the reaction of H₂S with S‐nitrosothiols (SNOs) to form polysulfides (RS _n H), or the reactions catalyzed by d‐CDES/l‐CDES, are shown only in the cytosol. Persulfidation of Cys residues (P–SSH) is thought to occur mainly through the reaction of P–SOH with H₂S (Gotor et al., 2019). The dashed line indicates that persulfuration of nitrogenase proteins needs to be confirmed in vivo. 3‐MP, 3‐mercaptopyruvate; 3‐MPST, 3‐mercaptopyruvate sulfurtransferase; APR, adenosine 5′‐phosphosulfate reductase; APS, adenosine‐5′‐phosphosulfate; ATPS, ATP sulfurylase; CAS, β‐cyanoalanine synthase; CSE, cystathionine γ‐lyase; d‐CDES, d‐cysteine desulfhydrase; l‐CDES, l‐cysteine desulfhydrase; OAS, O‐acetylserine; OASTL‐A1, OASTL‐B and OASTL‐C, O‐acetylserine(thiol)lyase isoforms A1 (cytosol), B (plastids), and C (mitochondria); PAPS, 3′‐phosphoadenosine‐5′‐phosphosulfate; SIR, sulfite reductase; SNOs, S‐nitrosothiols.