Three classes of hemoglobins are required for optimal vegetative and reproductive growth of Lotus japonicus: genetic and biochemical characterization of LjGlb2-1

Journal of Experimental Botany, Vol. 72, No. 22 pp. 7778–7791, 2021 https://doi.org/10.1093/jxb/erab376 Advance Access Publication 13 August 2021 © The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Experimental Biology. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. RESEARCH PAPER Three classes of hemoglobins are required for optimal vegetative and reproductive growth of Lotus japonicus: genetic and biochemical characterization of LjGlb2-1 IreneVillar1,†, MariaC.Rubio1,†, LauraCalvo-Begueria1, CarmenPérez-Rontomé1, EstibalizLarrainzar2, MichaelT.Wilson3, NielsSandal4, LuisA.Mur5, LonglongWang6, BrandonReeder3, DeqiangDuanmu6, ToshikiUchiumi7, JensStougaard4 and ManuelBecana1,*, 1 Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, Consejo Superior de Investigaciones Científicas, Apartado 13034, 50080 Zaragoza, Spain 2 Department of Sciences, Institute for Multidisciplinary Research in Applied Biology, Campus Arrosadía, Universidad Pública de Navarra, 31006 Pamplona, Spain 3 School of Life Sciences, Essex University, Wivenhoe Park, Colchester CO4 3SQ, UK 4 Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10, 8000 Aarhus C, Denmark 5 Aberystwyth University, Institute of Biological, Environmental and Rural Sciences, Aberystwyth, SY23 3DA, Wales, UK 6 State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan, 430070, China 7 Graduate School of Science and Engineering, Kagoshima University, 1-21-35 Korimoto, Kagoshima 890-0065, Japan † These authors contributed equally to this work. * Correspondence: [email protected] Received 15 July 2021; Editorial decision 27 July 2021; Accepted 11 August 2021 Editor: Richard Napier, University of Warwick,UK Abstract Legumes express two major types of hemoglobins, namely symbiotic (leghemoglobins) and non-symbiotic (phytoglobins), with the latter being categorized into three classes according to phylogeny and biochemistry. Using knockout mutants, we show that all three phytoglobin classes are required for optimal vegetative and reproductive development of Lotus japonicus. The mutants of two class 1 phytoglobins showed different phenotypes: Ljglb1-1 plants were smaller and had relatively more pods, whereas Ljglb1-2 plants had no distinctive vegetative phenotype and produced relatively fewer pods. Non-nodulated plants lacking LjGlb2-1 showed delayed growth and alterations in the leaf metabolome linked to amino acid processing, fermentative and respiratory pathways, and hormonal balance. The leaves of mutant plants accumulated salicylic acid and contained relatively less methyl jasmonic acid, suggesting crosstalk between LjGlb2-1 and the signaling pathways of both hormones. Based on the expression of LjGlb2-1 in leaves, the alterations of flowering and fruiting of nodulated Ljglb2-1 plants, the developmental and biochemical phenotypes of the mutant fed on ammonium nitrate, and the heme coordination and reactivity of the protein toward nitric oxide, we conclude that LjGlb2-1 is not a leghemoglobin but an unusual class 2 phytoglobin. For comparison, we have also characterized a close relative of LjGlb2-1 in Medicago truncatula, MtLb3, and conclude that this is an atypical leghemoglobin. Keywords: Hemoglobins, Lotus japonicus, Medicago truncatula, mutants, nitric oxide, symbiosis. applyparastyle "fig//caption/p[1]" parastyle "FigCapt" This paper is available online free of all access charges (see https://academic.oup.com/jxb/pages/openaccess for further details) Downloaded from https://academic.oup.com/jxb/article/72/22/7778/6350237 by CSIC user on 11 January 2022 Hemoglobins in plant vegetative and reproductive growth | 7779 Introduction Legumes contain two major types of hemoglobins, namely symbiotic or leghemoglobins (Lbs) and non-symbiotic or phytoglobins (Glbs). In general, Lbs are expressed in nodules at millimolar concentrations and are present as a mixture of components, the relative proportions of which vary with nodule age and stress conditions; however, the reason for the existence of these multiple components is uncertain (see review by Larrainzar et al., 2020). The major function of Lbs is to buffer O2 in the infected cells so that bacteroids can simultaneously sustain high respiration and N2 fixation rates (Appleby, 1984; Ott etal., 2005; Wang etal., 2019). In contrast, Glbs are present at low micromolar concentrations in most plant tissues (Hill, 2012). They are categorized into three phylogenetic classes, at least two of which are present in legumes. Class 1 Glbs have a very high O2 affinity and hence are unlikely to transport and deliver O2 for metabolic reactions. Among other functions, they are involved in plant responses and adaptations to hypoxia and in nitric oxide (NO) homeostasis (Igamberdiev and Hill, 2004). These Glbs are also crucial for the onset and functioning of the legume–Rhizobium symbiosis by modulating NO levels (Shimoda etal., 2005; Fukudome etal., 2016; Berger etal., 2020). Class 2 Glbs have homology with Lbs and show moderate O2 affinity and NO-scavenging activity, which is compatible with a role in modulating O2 and/or NO levels in vivo (Hebelstrup and Jensen, 2008; Smagghe et al., 2009). Class 3 Glbs share homology with the ‘truncated’ hemoglobins of prokaryotes and show low O2 affinity. They have unknown functions, although roles in the suppression of plant defense responses during rhizobial and mycorrhizal symbioses and in protection from nitrosative stress have been suggested (Vieweg etal., 2005). Among other factors, the functions of Lbs and Glbs are associated with protein structure and the heme reactivity with physiological ligands such as O2 and NO. Class 1 and class 2 Glbs are hexacoordinate because they have the fifth (proximal) and sixth (distal) positions of the heme iron coordinated to His or, more rarely, to other amino acid residues. In contrast, Lbs and class 3 Glbs are pentacoordinate because they have a proximal His residue but lack an amino acid residue at the distal position (Watts etal., 2001; Smagghe etal., 2009). The degree of hexaor pentacoordination of the hemes has a large effect on their reactivities, as illustrated by comparing the O2 affinities of Lbs and class 1 Glbs. The hemes of Glbs and Lbs are involved in several reactions with NO (see review by Becana etal., 2020). One of them is the oxidation of NO to NO3– by the NO dioxygenase (NOD) activity of oxyferrous hemoglobin (2+O2) (Igamberdiev and Hill, 2004), and another is the reduction of nitrite to NO by the nitrite reductase (NiR) activity of deoxyferrous hemoglobin (2+) (Sturms etal., 2011; Tiso etal., 2012). Both yield ferric hemoglobin (3+) that needs to be reduced back to the 2+ form in order to sustain the reactions. The genomes of the model legumes Medicago truncatula and Lotus japonicus contain multiple Lb and Glb genes. A search of the latest genome version of M. truncatula ecotype A17 (Mtv5.0) reveals that it may express up to twelve Lbs (MtLb1 to MtLb12), three class 1 Glbs (MtGlb1-1, MtGlb1-2, MtGlb1-3), and two class 3 Glbs (MtGlb3-1, MtGlb3-2) (Berger etal., 2020; Larrainzar etal., 2020). Similarly, a search of the latest genome version of L.japonicus ecotype Gifu (Ljv1.2; Kamal etal., 2020) shows that it encodes two class 1 Glbs (LjGlb1-1, LjGlb1-2), one putative class 2 Glb (LjGlb2-1), one uncharacterized hemoglobin (provisionally designated as LjGlb2-2), and two class 3 Glbs (LjGlb3-1, LjGlb3-2) (Larrainzar et al., 2020). The nodulation phenotypes of mutants deficient in LjGlb1-1 (Fukudome et al., 2016) or its homolog MtGlb1-1 (Berger etal., 2020) have been examined and both proteins have been found to be important for the onset of symbiosis. However, the phenotype of these mutants at the reproductive stage, as well as the phenotypes of other hemoglobin mutants, are unknown. In the first part of this study, we provide detailed information on the symbiotic performance, from seedling to seedproducing mature plants, of L. japonicus knockout mutants for LjGlb1-1, LjGlb1-2, LjGlb2-1, and LjGlb3-2. The mutants, which bear insertions of the retrotransposon LORE1, were generated in Denmark (Urbański etal., 2012; Małolepszy etal., 2016) or Japan (Fukai et al., 2010). Unfortunately, there are no insertional mutants available in either of the two LORE1 collections for LjGlb2-2 and LjGlb3-1. The second part of our study focuses on characterizing LjGlb2-1 because of its peculiarities. The protein shares high sequence identity with Lbs but, unlike them, it is hexacoordinate in the 3+ form (Calvo-Begueria etal., 2017) and is expressed also in the roots (Bustos-Sanmamed et al., 2011). Finally, because MtLb3 is phylogenetically related to LjGlb2-1 (Larrainzar etal., 2020), the tissue location and biochemical properties of both proteins are compared to help determine if they are functional homologs. Materials andmethods Biological materials and growth conditions Plants of Lotus japonicus ecotype Gifu B-129 mutated in Glb genes were obtained from the LORE1 mutant collection (Lotus Base, https://lotus. au.dk; Mun et al., 2016) except for P0494, which was obtained from Legume Base (National BioResource Project, https://www.legumebase. brc.miyazaki-u.ac.jp/). LORE1 is an endogenous 5-kb retrotransposon that inserts in the genome to create loss-of-function mutations. LORE1 mutants are stable, the plants are not transgenic, mutations are heritable, and mutant phenotypes are maintained through generations (Madsen et al., 2005; Urbański et al., 2012; Małolepszy et al., 2016). Plants of L.japonicus ecotype MG-20 mutated in the three Lb genes were generated using CRISPR/Cas9 (Wang etal., 2016). Seeds of L.japonicus were gently scarified with sand paper, disinfected for 20min with 2% sodium hypochlorite, washed, and imbibed overnight in the dark at room temperature. They were then transferred to 0.5 % (w/v) agar plates and kept in the dark at 4°C for 2 d.For germination, Downloaded from https://academic.oup.com/jxb/article/72/22/7778/6350237 by CSIC user on 11 January 2022 7780 | Villar etal. plates were placed vertically for 3 d in the dark at 23°C and for 2 d more with a 16/8-h light/dark photoperiod (140 μmol photons m–2 s–1) at 23/21°C. The seedlings were then transferred to plates with Jensen medium (Pajuelo and Stougaard, 2005) and grown with a 16-h photoperiod as indicatedabove. Seeds of Medicago truncatula ecotype Jemalong A17 were scarified with sulfuric acid for 8min, sterilized with 4% sodium hypochlorite for 2min, washed, and imbibed for 8h at 25°C. Seeds were then stratified for 2 d at 4°C on 0.5% (w/v) agar plates and germinated at 23°C for 1 d in the dark. Nodulation and growth conditions are described below, together with the protocol for producing hairy roots. Plant phenotyping The phenotypes of the L. japonicus Gifu mutant lines were examined under non-nodulating and nodulating conditions. To phenotype nonnodulated plants, 5-d-old seedlings were grown for 3 weeks on Jensen plates supplemented with 1.5 mM NH4NO3 and growth parameters were measured (shoot and root lengths and weights, leaf numbers and weights). To phenotype nodulated plants, 5-d-old seedlings were transferred to Jensen plates, inoculated with Mesorhizobium loti strain R7A (107 cells per root), and grown for 4 or 5 weeks. Shoot and root lengths and weights, and leaf and nodule numbers and weights were then measured. After this first phenotyping, nodulated plants were transferred to pots (12.5 cm diameter) containing vermiculite and were watered twice a week with B&D nutrient solution (Broughton and Dilworth, 1971) supplemented with 0.25mM NH4NO3. The growth phenotypes were examined again at 8 weeks post-germination. Finally, a set of nodulated plants was left to reach the reproductive stage in order to examine flowers and fruits. Plant material was harvested and immediately placed in liquid nitrogen, and then stored at –80°C until use. Complementation of the lb123mutant The genomic sequences of LjLb2 and LjGlb2-1 were amplified using gene-specific primers (LjLb2–BamHI–F, 5´-AAAGGATCCATGGGTTT CACTGCACAG-3´; LjLb2–AscI–R, 5´-GATGGCGCGCCTCAG CTCATTGCCTTCTTAA-3´; LjGlb2-1–BamHI–F, 5´-CATGGATC CATGGCTACATTCAGTGAGGAG-3´; LjGlb2-1–AscI–R, 5´-ACTG GCGCGCCTCAACTCATTCCCTTTTTAATCAC-3´), cloned into pC1300-sGFP coupled to the LjLb2 promoter, and the plasmids were introduced into Agrobacterium rhizogenes strain LBA1334. Hairy root transformation was then performed on the lb123 triple-mutant as described by Wang etal. (2019). Plants with positive transgenic roots were identified by GFP fluorescence, transferred to pots containing perlite/vermiculite (1:1), and inoculated with Mesorhizobium loti strain MAFF303099. Symbiotic phenotypes were examined at 5 weeks post-inoculation, which included measurement of nitrogenase activity using the acetylene reduction assay, as described by Wang etal. (2019). Cloning of the MtLb3 promoter, expression in hairy roots, and histochemical localization A DNA fragment encoding ~2 kb upstream of the predicted coding region of MtLb3 was amplified by PCR using the primers 5´-CACCTCTGCCTACAACTCTATTTGTCAG-3´ and 5´-GTGTTTGTGTTTTTTGCTTTTCTT-3´. The promoter region was cloned into the pENTR/D-TOPO vector (Invitrogen), sequenced, and subcloned into the binary vector pBGWFS7 (Karimi etal., 2002) using Gateway technology to make a transcriptional fusion with the uidA gene. The empty vector pBGWFS7 was used as a negative control for transformations. Plasmids were electroporated into Agrobacterium rhizogenes ARqua1, and transformed hairy roots were generated in M.truncatula Jemalong A17 as described by Boisson-Dernier etal. (2001). At 4 weeks old, transformed plantlets were transferred to pots containing a perlite/vermiculite mixture (1:1) and inoculated with Sinorhizobium meliloti 1021. The MtLb3 promoter activity in the M. truncatula roots and nodules was localized histochemically as previously described (Villar et al., 2020). Briefly, roots and nodules were pre-fixed in 0.3% (w/v) paraformaldehyde in 100 mM sodium phosphate buffer (pH 7.0) for 30min, and then washed in buffer. Samples were then vacuum-infiltrated for 20min in β-glucuronidase (GUS) staining solution containing 1mg ml−1 X-Gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronic acid), 2.5mM potassium ferricyanide, 2.5mM potassium ferrocyanide, 10mM EDTA, 0.1% (v/v) Triton X-100, and 100mM sodium phosphate (pH 7.0), and incubated for 2–4h at 37°C in the dark. Finally, samples were washed in 70% ethanol and phosphate buffer, mounted on glass slides in 50% glycerol (prepared in buffer), and visualized using a Leica M165 FC stereomicroscope with transmitted light. Some nodules were embedded in 5% (w/v) agarose and 60-µm sections made using a vibratome (Leica VT1000S). Metabolomics Leaf material (100mg) was ground and extracted in 1ml chloroform/ methanol/water (1:3:1) solution. The samples were then centrifuged at 6000 g for 1 min at 4 °C. The supernatant was collected and dried in a vacuum-concentrator without heating. After drying, 200 μl of 50% methanol was added and 70μl was transferred into a HPLC glass vial with a 0.2ml flat-bottom micro-insert. Samples were run randomly using an autosampler with a tray temperature of 15°C. The sample injected was set at a volume of 20μl into a flow volume of 60μl ml–1 water/methanol (70/30%) using a Surveyor liquid chromatography system (Thermo Scientific). Flow-infusion electrospray–high-resolution mass spectroscopy was performed using an Exactive Plus Orbitrap MS (Thermo Scientific). Mass ions (m/z) were generated in both positive and negative ionization modes over four scan ranges (15–110, 100–220, 210–510, and 500–1200) with an acquisition time of 5min. Individual ionization peak values were normalized as a percentage of the total ion count for each sample. Values of m/z that varied significantly between the experimental classes were identified by one-way ANOVA using Benjamini–Hochberg adjustment in order to control the false-discovery rates. The high-resolution accurate mass values of statistically significant m/z were used to interrogate the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http:// www.genome.jp/kegg/) and the Direct Infusion Metabolite database (https://dimedb.ibers.aber.ac.uk/). Identification was based on the ‘MS Peaks to Pathways’ algorithm (Barnes etal., 2016) (tolerance=5ppm; reference library, Arabidopsis thaliana). This involved metabolites being annotated using the KEGG database considering the following possible adducts: [M]+, [M+H]+, [M+NH4]+, [M+Na]+, [M+K]+, [M-NH2+H]+, [M-CO2H+H]+, [M-H2O+H]+, [M]−, [M−H]−, [M+Na−2H]−, [M+Cl]−, [M+K−2H]−. Correlations between multiple adducts of a metabolite were used in the identification process. Expression analyses Total RNA was extracted from nodules (20–40mg), roots (80–100mg), and leaves (50–60mg) using the RNAqueous isolation kit (ThermoFisher Scientific), treated with DNaseI (Roche), and cDNA was synthesized using MMLV-RT (Promega). Quantitative reverse-transcription (qRT-) PCR analyses were performed using a 7500 Real-Time PCR System (Applied Biosystems) as previously described (Rubio et al., 2019). Primer sequences and efficiencies are given in Supplementary Table S1. Normalized relative quantities (NRQs) were calculated using LjUbiquitin, LjATP synthase, and LjeIF4A as the reference genes. At least three biological replicates per treatment were used and reactions were carried out in triplicate. Statistical analyses were performed using log2(NRQ)- transformed data and one-tailed Student’s t-tests for comparisons of means. Downloaded from https://academic.oup.com/jxb/article/72/22/7778/6350237 by CSIC user on 11 January 2022 Hemoglobins in plant vegetative and reproductive growth | 7781 Protein expression and purification The ORFs encoding LjGlb2-1, MtLb3, and MtLb3-C135S were cloned into the Champion pET11a expression vector (Novagene) and expressed with an N-terminal Strep-tag in Escherichia coli C41(DE3) cells (Lucigen, Middleton, MI, USA). Cells were pre-cultured overnight in 100ml of LB medium with 100µg ml–1 ampicillin at 37°C with mild agitation (200 rpm). Then, 10 ml of the pre-culture was used to inoculate 1 l of Terrific Broth medium with 100µg ml–1 ampicillin, and cells were incubated at the same conditions as above until an OD at 600nm of 0.6–0.7 was reached. Isopropyl β-D-1-thiogalactopyranoside was then added to the medium up to a concentration of 0.5mM, and the cells were grown at 28°C for 16h. The cells were then washed in phosphate buffer and stored at –80ºC for no longer than 3 weeks. For the purification protocol, cells were resuspended in 20mM Tris (pH 8.0) containing 150mM NaCl, then sonicated (3 × 2 min), and cleared by centrifugation. The supernatant was loaded onto a StrepTactin Sepharose High Performance column (GE Healthcare), previously equilibrated with the same buffer. After loading the protein, the column was washed with at least five volumes of buffer, and the recombinant proteins were eluted with buffer containing 2.5mM desthiobiotin (Merck). The proteins were dialysed, concentrated, oxidized with ferricyanide, desalted through a NAP-5 mini-column (GE Healthcare) equilibrated with the same buffer without NaCl, and concentrated again. Purified proteins were quantified in the ferric form using an extinction coefficient of 150mM–1 cm–1 for the Soret band. UV-visiblespectra The UV-visible spectra of the 3+ and 2+ forms, as well as the spectra of their complexes, were obtained as previously described (Villar etal., 2020). Spectra were recorded immediately after production of the hemoglobin complexes using a 0.1-cm cuvette with a Lambda 25 spectrophotometer (Perkin-Elmer). Hemoglobin concentrations are indicated in the legends of Supplementary Figs S1, S2, and S3 for LjGlb2-1, MtLb3, and MtLb3-C135S, respectively. NO binding, NO dioxygenase activity, and NiR activity For measurement of NO binding, 2+ hemoglobins (5μM) in 50mM potassium phosphate buffer (pH 7.0) and 150mM NaCl were degassed in a glass tonometer connected to a supply of argon gas and a vacuum pump. Repeated degassing cycles were performed to remove O2. The NO donor, proli-NONOate (Cayman Chemical), was prepared in 25mM NaOH to a concentration of 45mM and then purged with argon gas. The concentration of proli-NONOate was checked by UV spectroscopy using an extinction coefficient at 252nm of 8400 M−1 cm−1 (Maragos etal., 1991). Solutions containing NO were prepared by injecting into the degassed buffer using a Hamilton syringe. The degassed proteins were transferred anaerobically to a 10-ml glass Hamilton syringe and mixed with the NO donor at a 1:1 ratio using an Applied Photophysics SX20 stopped-flow instrument fitted with a diode array spectrophotometer. The final concentrations of 2+ hemoglobin and NO were 2.5μM and 10–160μM, respectively. Kinetics were performed at 20°C. To measure NOD activity, 2+ hemoglobins (5μM) in the same buffer as above were oxygenated by passing them through Sephadex G-25 (NAP5) mini-columns. Complete oxygenation was checked using an Agilent 8453 diode array spectrophotomer. The buffer and NO donor were prepared as for the NO-binding experiments. Finally, 2+O2 hemoglobins were rapidly mixed with the NO donor (1:1) at final concentrations of 2.5μM and 5–160μM, respectively, using the SX20 stopped-flow instrument mentioned above pre-cooled to 10°C. To assay NiR activity, degassed 2+ hemoglobin was treated with NaNO2 dissolved in buffer with sodium dithionite. The final concentrations of 2+ hemoglobin and NaNO2 were 2.5μM and 0.05–1.0mM, respectively. Reactions were performed at room temperature. Optical spectra were recorded using an Agilent 8453 diode array spectrophotometer. Rate constants were determined from time-courses (0–200s) by following the conversion of 2+ to 2+NO hemoglobin in the Soret band and then by fitting the data to exponential functions using the leastsquare method in the Microsoft Excel solver program. Results Lotus japonicus plants deficient in Glbs display gene-specific alterations in the vegetative and reproductivestages The first objective of this study was to examine the phenotypes at the vegetative and reproductive stages of L.japonicus plants lacking Glbs under nodulating conditions. To do this, we obtained homozygous seeds of mutant lines bearing exonic insertions of the LORE1 retrotransposon. Supplementary Table S2 lists the gene names, their identification numbers in the Gifu genome (Kamal etal., 2020), the line names, and the tag insertion positions. It also shows the residual LjGlb transcript levels of the mutant plants, which were 1–7% of those of the wild-type (WT) plants, confirming that all the lines used in our study are virtually null-mutants. The Ljglb1-1 mutant has previously been described by Fukudome etal. (2016). For the Ljglb1-2 and Ljglb2-1 mutants, only one line was selected for each because they bear single exonic insertions; in contrast, two independent lines were required to ascertain the phenotype of the Ljglb3-2 plants because they contain multiple exonic insertions (LotusBase; Małolepszy etal., 2016). The mutant lines for all four genes were phenotyped under nodulating conditions. Young plants (4 weeks old) of the Ljglb1-1 and Ljglb2-1 mutants had smaller shoots and roots as well as fewer leaves and nodules than the WT plants (Fig. 1A). The older plants (8 weeks old) of both mutants had lower leaf and nodule numbers and shoot weights, and the Ljglb1-1 plants also had smaller shoots and reduced weights of roots, leaves, and nodules (Fig. 1B). In contrast, the Ljglb1-2 mutant showed a similar phenotype to the WT plants both at 4 and 8 weeks old. Nodulated young plants (5 weeks old) of the two Ljglb3-2 mutant lines had smaller shoots and fewer leaves than the WT (Fig. 1A), whereas the older plants (8 weeks old) had fewer leaves and reduced weights of shoots, roots, and leaves. Plants of line 30086451 also had shorter shoots (Fig. 1B). The young Ljglb3-2 plants had fewer nodules than the WT (Fig. 1A) but this difference was no longer present in the older plants; however, the nodules of line 30108411 weighed less than those of the WT in older plants (Fig. 1B). The phenotypes of nodulated plants were also examined at flowering and fruiting (Table 1). Compared with the WT, all the mutant lines showed a delay of 1 or 2 weeks until they reached the maximum numbers of flowers and pods, with the exception of Ljglb1-1, which was delayed by 1 week in reaching the flowering peak but not the pod peak. The two class 1 Glb mutants showed contrasting differences in pod number, which Downloaded from https://academic.oup.com/jxb/article/72/22/7778/6350237 by CSIC user on 11 January 2022 7782 | Villar etal. increased by 63% in Ljglb1-1 and decreased by 31% in Ljglb1-2 compared with the WT. In contrast, the Ljglb2-1 plants showed no differences in the number of flowers and pods. The Ljglb3-2 mutant lines had more flowers and pods than the WT plants. In addition, the Ljglb1-1, Ljglb2-1, and Ljglb3-2 (30086451) mutants produced smaller pods and fewer seeds than theWT. LjGlb2-1 is also functional in non-nodulated plants and its deficiency causes alterations in the metabolome The hemoglobin LjGlb2-1 is particularly interesting because of its unusual features, as recently discussed by Larrainzar etal. (2020). LjGlb2-1 shows high homology with typical Lbs but has a hexacoordinate heme (see below) and its gene expression is localized not only in nodules but also in the apex and vascular bundles of the roots (Bustos-Sanmamed etal., 2011). This prompted us to focus the rest of our study on LjGlb2-1. First, we quantified the expression of LjGlb2-1 in WT plants grown for 21 d in Jensen plates supplemented with 1.5mM NH4NO3. Under these non-nodulating conditions, the gene was expressed at a level ~80-fold lower in the roots than in the leaves (Fig. 2A). This finding was interesting because a previous study of nodulated plants grown in hydroponics had shown that LjGlb2-1 expression was highest in nodules, moderately high in roots, and very low in leaves (Bustos-Sanmamed etal., 2011). Next, we phenotyped the Ljglb2-1 mutant plants under the same non-nodulating conditions that were used for transcript quantification and found that they showed decreases of 32–43% in all the measured growth parameters compared to the WT plants (Fig. 2B). We then carried out a complementation study by producing hairy roots of the lb123 mutant, which is devoid of the three LjLbs (Wang etal., 2019). The roots of this mutant were transformed via A.rhizogenes with constructs bearing the LjGlb2-1 gene under the control of the LjLb2 promoter. As controls, we used constructs in which the LjLb2 promoter was driving the LjLb2 and GUS genes. The results indicated that LjGlb2-1 did not complement the lb123 mutant, based on the decreases in plant length Fig. 1. Growth phenotypes of nodulated Lotus japonicus mutants deficient in LjGlb1-1, LjGlb1-2, LjGlb2-1, or LjGlb3-2. Plants were phenotyped at (A) 4 weeks old except for Ljglb3-2, which was assessed at 5 weeks old, and (B) at 8 weeks old (all lines). Two independent lines were used to phenotype the Ljglb3-2 plants, 30086451 and 30108411. WT (4+4) indicates the wild-type used for Ljglb1-1, Ljglb1-2, and Ljglb2-1 (4 weeks in plates, 4 weeks in pots); WT (5+3) indicates the wild-type used for the Ljglb3-2 lines (5 weeks in plates, 3 weeks in pots). Data are means (±SE) of 10–25 plants representative of two or three sets of plants grown independently. Significant differences compared with the relevant WT were determined using Student’s t-test: *P<0.05. Downloaded from https://academic.oup.com/jxb/article/72/22/7778/6350237 by CSIC user on 11 January 2022 Hemoglobins in plant vegetative and reproductive growth | 7783 and weight, the white color of the nodules, and the very low acetylene reduction activity compared with the lb123 roots transformed with the LjLb2 gene (Fig. 3). Consequently, the results support a hypothesis that LjGlb2-1 is not a typical Lb but is instead a hemoglobin with at least some functions that are non-symbiotic. To further investigate the phenotype of the Ljglb2-1 mutant at the biochemical level, we performed a metabolomics Fig. 2. Expression of LjGlb2-1 and growth phenotypes of non-nodulated Lotus japonicus plants. Measurements were taken on 21-d-old plants grown on Jensen plates supplemented with 1.5mM NH4NO3. (A) Expression of LjGlb2-1 in non-nodulated wild-type (WT) plants, presented as normalized relative quantity (NRQ). Transcript levels were normalized using the geometric means of LjUbiquitin, LjATP synthase, and LjeIF4A as reference genes. Data are means (±SE) of three biological replicates. (B) Phenotypes of the WT and mutant deficient in LjGlb2-1. Data are means (±SE) of 55–60 plants from four sets of plants grown independently. Significant differences between means were determined using Student’s t-test: *P<0.05. Table 1. Flowering and fruiting phenotypes of nodulated Lotus japonicus plants of the wild-type and Ljglb1-1, Ljglb1-2, Ljglb2-1, and Ljglb3-2 mutants. Parameter WT (4+ 4) Ljglb1-1 Ljglb1-2 Ljglb2-1 WT (5+ 3) Ljglb3-2 (30086451) Ljglb3-2 (30108411) Flowering peak timea16 17 17 18 16 18 18 Flower numberb8.8±1.6 12.4±2.0 8.2±1.4 9.8±1.5 8.2±1.6 22.1±3.8* 13.0±2.5* Pod peak timea21 21 22 23 21 23 23 Pod numberb19.5±2.3 31.8±4.3* 13.5±1.6* 21.5±2.9 17.0±0.5 28.3±3.6* 22.7±4.8* Pod length (cm)c2.8±0.1 2.5±0.1* 2.8±0.1 2.5±0.1* 2.8±0.1 2.5±0.0* 2.9±0.1 Seed number per podc10.8±0.4 8.5±0.4* 11.9±0.7 8.9±0.4* 10.5±0.5 9.4±0.3* 10.7±0.8 a Weeks from germination. b Value at the peak time. Data are means (±SE) of 3–9 plants (Ljglb3-2) or 15–35 plants (all other lines). c Data are means (±SE) of 20–80 (Ljglb3-2) or 50-100 (all other lines) pods from one to four sets of plants grown independently. Data were obtained from one (Ljglb3-2 30108411), two (Ljglb1-2, Ljglb3-2 300846451), or four (Ljglb1-1 and Ljglb2-1) sets of plants grown independently. WT (4+4) indicates the wild-type used for Ljglb1-1, Ljglb1-2, and Ljglb2-1 (4 weeks in plates, 4 weeks in pots); WT (5+3) indicates the wild-type used for the Ljglb3-2 lines (5 weeks in plates, 3 weeks in pots). Significant differences compared with the corresponding WT were determined using Student’s t-test: *P<0.05. Downloaded from https://academic.oup.com/jxb/article/72/22/7778/6350237 by CSIC user on 11 January 2022 7784 | Villar etal. study in leaves of non-nodulated plants, where expression of LjGlb2-1 was maximal in the WT plants (Fig. 2A). This identified 1283 m/z features within the metabolite profile (Supplementary Dataset S1). Principal component analysis of these features indicated a clear separation between the WT and Ljglb2-1 plant metabolomes (Fig. 4A). The major sources of variation were identified based on one-way ANOVA, correcting for false discovery rates and using a significance level of P<0.05. Compounds found to show the highest variation between the two genotypes were identified using metabolite databases, and the relative magnitudes of these variations are indicated in the heatmap in Fig. 4B. The results suggested that most of the discriminatory metabolites were found at higher levels in Ljglb2-1. Considering all of the metabolites, it is worth noting the changes in the contents of several amino acids and intermediates of the tricarboxylic acid and glycolytic pathways. Thus, the leaves of Ljglb2-1 plants contained higher levels of valine, alanine, threonine, serine, glutamine, and glutamate compared with the WT (Fig. 5A). The mutant also had increased levels of sucrose, glyceraldehyde-3-phosphate, lactate, ribulose 1,5-bisphosphate, and pyruvate, and lower levels of citrate, cis-aconitate, oxaloacetate, and phosphoenolpyruvate (Fig. 5B). Metabolite profiling also revealed alterations in the hormone levels in the leaves of mutant plants, including an increase in salicylic acid (SA) and a decrease in methyl jasmonic acid (MeJA) (Fig. 6). It was also possible to identify m/z features linked to hormones in our metabolomic data that were not shown to be significantly different in our statistical pipeline. Thus, we were able to describe the levels of indoleacetic acid, abscisic acid, zeatin (and its glucoside), and polyamines in our plants. However, none of them appeared to change significantly in Ljglb2-1 compared to WT plants, except the Fig. 3. Complementation analysis of the Lotus japonicus lb123 mutant by LjGlb2-1. Phenotypes of (A) whole plants and (B) nodules of hairy root transformed plants at 6 weeks post-inoculation. Roots of the lb123 mutant were transformed with Agrobacterium rhizogenes strain LBA1334 harboring the LjLb2 promoter coupled to either LjLb2, GUS, or LjGlb2-1. The numbers in (B) indicate the proportion of plants bearing pink nodules. Scale bars are (A) 5cm and (B) 1mm. (C) Shoot fresh weight per plant. (D) Acetylene reduction activity (ARA) expressed per nodule fresh weight. Data are means (±SE) of 16–17 plants. Different letters indicate significant differences between means as determined by Duncan’s multiple range test (P<0.05). Downloaded from https://academic.oup.com/jxb/article/72/22/7778/6350237 by CSIC user on 11 January 2022 Hemoglobins in plant vegetative and reproductive growth | 7785 content of zeatin-glucoside, which decreased in the mutant plants (Fig. 6). LjGlb2-1 and MtLb3 are both located in roots and nodules and show different biochemical features The functions of hemoglobins are associated to their locations in plant tissues and their reactivities toward physiological ligands. This prompted us to characterize LjGlb2-1. The protein shares high sequence identity with MtLb3 (Medtr1g090810), and they cluster together and clearly away from other Lbs and Glbs of the model legumes (Larrainzar etal., 2020). These observations and the anomalous features of LjGlb2-1 led us to compare the expression patterns of LjGlb2-1 and MtLb3 in nodulated roots and the reactivities of their proteins to ascertain if they might be functional homologs. Because LjGlb2-1 activity has previously been localized in nodules and roots using promoter–GUS fusions (Bustos-Sanmamed etal., 2011), we used the same procedure for MtLb3. Our results showed that the MtLb3 promoter was active in the apex and vascular bundles of the primary and secondary roots (Supplementary Fig. S4). In nodules at ~2 weeks post-infection, MtLb3 activity was predominantly found in the apex (zones I+ II), and after a further 2 weeks it was also detected in the vascular bundles and, to a lesser extent, in zone III. The expression pattern of MtLb3 in the roots was therefore similar to that of LjGlb2-1 observed by Bustos-Sanmamed etal. (2011). We then set out to compare the two proteins, together with a MtLb3 mutant in which its single Cys was replaced by Ser (MtLb3–C135S). Unfortunately, we could not produce the corresponding derivative of LjGlb2-1 (LjGlb2-1–C65S) in sufficient quantities for characterization, which suggested that the Cys residue is critical for protein stability (Calvo-Begueria etal., 2017). Having produced purified proteins, we first obtained the UV-visible spectra of the 3+ and 2+ forms, as well as the spectra of the proteins bound to the heme ligands O2, NO, CO, and cyanide (Supplementary Figs S1–S3). In this study, LjGlb2-1 had a Strep-tag instead of a His-tag (Sainz etal., 2013), confirming that the protein is hexacoordinate in the 3+ form (Soret at 410nm, band at 535nm, and shoulder at 562nm). The 3+ protein produced the typical cyano complex with cyanide, whereas the 2+ protein was mostly pentacoordinate (Soret at 427nm and β band at 558nm) and yielded the expected complexes with O2, CO, and NO (Supplementary Fig. S1). However, a small proportion of hexacoordinate protein was also detectable in the 2+ form according to the shoulder observed at ~534nm. The spectra of MtLb3 (Supplementary Fig. S2) and MtLb3–C135S (Supplementary Fig. S3) were virtually identical (±2 nm), showing pentacoordination in the 3+ form (bands at 528 and 625nm, shoulder at 564nm) and 2+ form (β band at 559nm). Fig. 4. Metabolomic characterization of the Lotus japonicus Ljglb2-1 mutant. Metabolites extracted from leaves of 28-d-old wild-type (WT) and Ljglb2-1 plants were determined using flow-infusion electrospray–high resolution mass spectroscopy. (A) The derived matrix (Supplementary Dataset S1) was assessed by partial least-squares discriminant analysis. The major sources of variation were identified based on one-way ANOVA correcting for false-discovery rates and using a significance level of P<0.05. The discriminatory m/z were identified based on accurate mass (5ppm resolution) and correlations between multiple adducts of the targeted metabolite as indicated by DIMEdb (https://dimedb.ibers.aber.ac.uk). (B) Heatmap comparing the levels of the identified metabolites in WT and Ljglb2-1 plants. Downloaded from https://academic.oup.com/jxb/article/72/22/7778/6350237 by CSIC user on 11 January 2022 7786 | Villar etal. Next, we compared LjGlb2-1 and MtLb3 by measuring their reactivity toward NO, because this is a key signal molecule and the kinetics may give insights into the protein functions. To study the kinetics of NO binding, the 2+ hemoglobins were mixed with proli-NONOate and the appearance of the nitrosyl complex (2+NO) was followed under anaerobic conditions by stopped-flow spectroscopy (Fig. 7A, B). The reaction was fast for all three proteins, but the 2+ form was still visible after the dead-time of the instrument (1.2ms) and the rates of formation of the 2+NO hemoglobins could be measured. There were clear differences between the proteins. The time-course of MtLb3 fitted a double-exponential function whereas that of MtLb3–C135S fitted a single-exponential function, which suggested that a second, slower reaction occurred during the last step of the NO binding to MtLb3. The observed rate constant (kobs) exhibited a hyperbolic relationship with NO concentration (Fig. 7C). The initial change in kobs appeared to be at least 1×107 M–1 s–1 for both MtLb3 and MtLb3–C135S, and the rate constants approached maximum values of ~860s–1 and ~695s–1, respectively. These values represent the pseudo-firstorder rate constants that reflect the limitation of the rate of access of NO to the heme. In many cases they are the His off rates from the iron, but here they probably reflect the rate of movement of distal heme-pocket residues that sterically hinder the binding of NO. The NO-binding kinetics of LjGlb2-1 was slower than that of MtLb3 and was independent of NO concentration, with an estimated NO-binding rate constant (kNO) of 2.5s–1 (Fig. 7C, inset). This indicated that the entry of NO into the heme cavity of LjGlb2-1 was somewhat limited by the presence of the distal His. This finding was unexpected because, as indicated above, the 2+ protein showed a mainly pentacoordinate spectrum. Because all three hemoglobins were able to scavenge NO in vitro, we measured their NOD activities, which also allowed us to determine whether the Cys residue of MtLb3 plays a role in NO scavenging. To this end, the 2+O2 proteins were mixed with a solution of NO and the reaction kinetics were monitored by stopped-flow spectroscopy. The first spectrum of LjGlb2-1 (taken at 1.2ms) was almost the same as that of a 3+ protein, indicating that the reaction was already complete (Fig. 7D). It can therefore be concluded that the NOD rate constant (kNOD) must be >250s–1 even at very low NO concentrations (5μM), yielding a second-order rate constant of >5×107 M–1 s–1. In sharp contrast, the initial spectra (again Fig. 5. Comparison of metabolites discriminating between the Lotus japonicus wild-type (WT) and Ljglb2-1 mutant plants. Box-and-whisker plots of (A) amino acids and (B) metabolites associated with bioenergy that significantly discriminated between the two genotypes (P<0.05). Downloaded from https://academic.oup.com/jxb/article/72/22/7778/6350237 by CSIC user on 11 January 2022