Bacterial strains and growth conditions
Bacterial strains used in this work are listed in Table 1. E. coli S17.1 cells were routinely grown in LB (Luria-Bertani) medium (Miller 1972) at 37ºC and 170 rpm, and was used as donor in plasmid pGUS3 conjugative transfer. Antibiotics were added to E. coli cultures at the following concentrations: streptomycin (Sm), 20 µg∙ml− 1; spectinomycin (Spc), 20 µg∙ml− 1; kanamycin (Km), 25 µg∙ml− 1. E. meliloti cells were routinely cultured in TY (Tryptone-Yeast) medium (Beringer 1974) at 30 ºC and 170 rpm. Then, cells were grown microaerobically in minimal medium (Robertsen 1981) supplemented with 10 mM KNO3. To achieve microaerobic conditions, 100-ml flasks were sparged with a gas mixture consisting of 2% O2 and 98% N2 into cultures previously inoculated at an initial OD600 of 0.1–0.15, maintaining a relation of 1:5 between liquid and gas phases. Antibiotics were added to E. meliloti cultures at the following concentrations: Sm, 200 µg∙ml− 1; Km, 200 µg∙ml− 1. For competitivity assays, X-Gluc (5-bromo-4-chloro-3-indolyl β-D-glucuronide) was added to the E. meliloti cultures at a final concentration of 50 µg∙ml− 1.
Table 1
Bacterial strains and plasmids used in this study; Tp, trimethoprim
Strains or plasmids | Relevant description | Source of reference |
Escherichia coli | | |
S17.1 | thi, pro, recA, hsdR, hsdM, RP4Tc::Mu, Km::Tn7; Tpr Smr Spcr | Simon et al. (1983) |
Ensifer meliloti | | |
1021 | WT, Smr | Meade et al. (1982) |
4002 | nap overexpressing strain (WT containing pDS4002); Smr Kmr | Torres et al. (2018) |
4004 | WT containing pDS4004 (empty plasmid derived from pDS4002); Smr Kmr | Torres et al. (2018) |
4004-GUS3 | WT 4004 containing pGUS3; Smr Kmr | This work |
2011mTn5STM.3.02.F08 | napA::mini-Tn5; Smr Kmr | Pobigaylo et al. (2006) |
2011mTn5STM.5.07.B03 | nosZ::mini-Tn5; Smr Kmr | Pobigaylo et al. (2006) |
Plasmids | | |
pDS4002 | Fragment of 4709 bp corresponding to E. meliloti napEFDABC operon cloned in pBBR-MCS2; Kmr | Torres et al. (2018) |
pDS4004 | Empty pBBR-MCS2 derived from pDS4002; Kmr | Torres et al. (2018) |
pGUS3 | Translational fusion between pnfeD (coordinates 2993–3345) and gusA in pBI101; Kmr | García-Rodríguez and Toro (2000) |
Plant growth conditions
Alfalfa (Medicago sativa, cv. Victoria) seeds were surface-sterilized by immersion in 2.5% HgCl2 for 9 minutes. Then, seeds were washed with sterile distilled water and germinated on filter paper discs in Petri dishes in darkness for 2–3 days at 30 ºC. Alfalfa plants were grown using a modified Rigaud and Puppo nutrient solution (1975) (referred as NS throughout the manuscript): K2SO4, 174 mg∙l− 1; KH2PO4, 68 mg∙l− 1; K2HPO4, 44 mg∙l− 1; MgSO4∙7H2O, 123 mg∙l− 1; H3BO3, 2.85 mg∙l− 1; ZnSO4∙7H2O, 0.55 mg∙l− 1; MnSO4∙4H2O, 3.07 mg∙l− 1; Na2MoO4∙2H2O, 0.11 mg∙l− 1; CaSO4∙2H2O, 150 mg∙l− 1; Sequestrene® 138 G100 (6.2 mg chelated Fe), 25 mg∙l− 1. The NS was supplemented with 1, 2, 3, 4 or 10 mM KNO3. The standard Cu concentration of NS was 0.2 mg∙l− 1 (0.8 µM). For studies of the effect of Cu on symbiosis, NS was supplemented with a higher Cu concentration of 5 mg∙l− 1 (20 µM), used in previous studies from our group (Tortosa et al. 2020). Plants in tubes or pots were placed into growth chambers from the Greenhouse and Growth Chamber Service (GGCS) (EEZ, Granada, Spain) with the following parameters: night/day temperature, 24/20 ºC; photoperiod, 16/8 h; photosynthesis photon flux density of 403 µmol photons∙m− 2∙s− 1.
Plant experimental setting
For nodulation kinetics assays, a methodology described by Torres et al. (2013) was used. Basically, germinated seeds were transferred into 43-ml autoclaved glass tubes containing 10 ml water and kept in darkness for approximately 24 h. Then, these tubes were placed into the growth chamber and, after 5 days, water was replaced with 10 ml of NS containing a cell suspension of approximately 108 CFU∙ml− 1. NS was supplemented with 1, 2, 3 or 4 mM KNO3. Anoxic conditions were achieved sparging NS with N2 gas before adding the inoculum. Finally, tubes were incubated for 30 days, and nodule number was daily counted.
For competitivity assays, seeds were germinated as for nodulation kinetics experiments. After 5 days, water was replaced with 10 ml of NS supplemented with 3 mM KNO3 and containing the respective inoculum [nap overexpressing strain 4002 (denoted as nap+ throughout the manuscript), the WT strain 4004-GUS3 or a mix in 1:1 proportion] at a cellular density of approximately 105 CFU∙ml− 1. Tubes were incubated for 30 days in the controlled environmental chamber with the parameters enumerated above and nodules were revealed with X-Gluc (0.53 mg∙ml− 1) according to Nogales et al. (2006).
For plant assays in pots, germinated seeds were transferred into 250-ml autoclaved pots filled with perlite as substrate. Different experiments were carried out regarding the aim of the assay (Supplentary Fig. S1).
Experiment 1 (Supplementary Fig. S1a): in order to study the influence of nitrate and flooding in N2O emissions from alfalfa nodules, these pots were placed on 1-l glass jars containing 500 ml of N-free NS. Eight seedlings per pot were inoculated at sowing with a cell suspension of the WT 1021 strain of about 108 CFU∙ml− 1. Three sets were established: the first set, with 20 pots, was watered with N-free NS, and the second and third sets, with 10 pots each, were watered with NS supplemented with 1 mM or 3 mM KNO3, respectively. Plants were watered every two weeks under sterile conditions. Seven days before harvesting (i. e., after 43 days), a nitrate shock of 10 mM KNO3 was applied to 10 pots from the first set. Additionally, at the same time, half of each set was also subjected to flooding conditions, which were achieved by removing alfalfa plants from the pots and transferring them into a glass jar filled with 900 ml NS, thus nodulated roots were completely submerged. After 50 days growth, plants and nodules were harvested.
Experiment 2 (Supplementary Fig. S1b): to investigate the role of Nap in N2O emissions from alfalfa nodules, pots were prepared as described above, and 10 pots with 8 seedlings each were inoculated at sowing with a cell suspension of about 108 CFU∙ml− 1 of the WT 1021, the nap+ strain, or the napA mutant strain (denoted as nap− throughout the manuscript). Plants were grown for 50 days, and watered every two weeks under sterile conditions. Seven days before harvesting, the pots were watered with NS supplemented with 10 mM KNO3 and subjected to flooding conditions as indicated above. After plant growth, the nodules harvested from 5 pots from each treatment were immediately used for N2O emission measurements, whereas the nodules harvested from the remaining 5 pots from each treatment were frozen in liquid nitrogen and stored at -80 ºC for further determinations.
Experiment 3 (Supplementary Fig. S1c): this experiment was performed to study the involvement of Cu in plant and nodule physiology as well as in N2O emissions from alfalfa nodules. For this goal, pots were placed on glass jars containing 500 ml of N-free NS. Eight seedlings per pot were inoculated at sowing with the WT 1021 or the nosZ mutant strain at a cellular density of about 108 CFU∙ml− 1. For pots inoculated with the WT 1021 strain, three different sets of 10 pots each were established: the first set was watered with NS without Cu added, the second set, with NS supplemented with 0.8 µM CuSO4∙5H2O, and the third set, with NS containing 20 µM CuSO4∙5H2O. The 5 pots inoculated with the nosZ mutant were watered only with 0.8 µM Cu NS. Plants were grown for 43 days and plants and nodules from 5 pots from each set inoculated with the WT were harvested. To induce N2O production, the remaining 5 pots from each WT set were treated with 10 mM KNO3 and subjected to flooding for 7 days. Then, nodules were harvested and N2O emissions from detached nodules were measured.
Plant physiological analyses
Physiological data such as nodule number (NN), nodule fresh weight (NFW), shoot dry weight (SDW) and plant dry weight (PDW) were recorded and expressed per plant. SDW and PDW were determined after 3 days in an oven at 70 ºC.
Prior to analytical determinations (nitrogen and copper content), dry shoots and roots were ground further using an IKA A11 mill to less than 0.5 mm according to Tortosa et al. (2020). Seeds and nodules were dried in an oven at 70 ºC for only one day.
Nitrogen content was analysed in dried and ground shoots of alfalfa plants by the N/C Analysis Service of Estación Experimental del Zaidín (EEZ, Granada, Spain) by using an elemental analyser LECO TruSpec® CN (LECO, St Joseph, MI, USA). Briefly, the sample was subjected to complete combustion at 950 ºC in the presence of O2. Then, all the nitrogen oxides formed were converted into N2, and this gas was detected by a thermal conductivity detector. Data were expressed as mg N∙g− 1 of dry sample.
Cu concentration was analysed in dry alfalfa seeds and nodules, as well as in dry and ground roots and shoots, using the Inductively Coupled Plasma Optical Emission Spectrometer (ICP-OES) available at the Instrumental Technical Service of EEZ (Granada, Spain), model PlasmaQuant® PQ 9000 (Analytik Jena, Jena, Germany). Data were expressed as mg Cu∙kg− 1 of dry sample.
Determination of leghemoglobin content in nodules
Leghemoglobin (Lb) concentration in nodules was determined by fluorimetry according to a method described by Tortosa et al. (2020), which was based on the standard method established by Larue and Child (1979), but was adapted to alfalfa nodules in the present work. Briefly, 0.125–0.13 g of NFW were crushed and homogenised with a pestle in a cold porcelain mortar by adding 6 ml of buffer solution (50 mM Na2HPO4∙2H2O/NaH2PO4∙H2O, pH 7.4; 0.02% w/v K3Fe(CN)6; 0.1% w/v NaHCO3) and 0.1 g of polyvinyl polypyrrolidone (PVPP). The extract was centrifuged at 12,000x g at 4 ºC for 20 min and 200 µl from the supernatant were transferred into glass tubes containing 3.15 ml of a saturated calcium oxalate solution (66 g∙l− 1) and subsequently autoclaved at 120 ºC during 30 min. Then, samples were cooled down and measured in a Shimadzu spectrofluorometer (Shimadzu Scientific Instruments, Kyoto, Japan), setting λ = 405 nm and 600 nm as excitation and emission wavelengths, respectively, and comparing to non-autoclaved samples as control. Lb concentrations were expressed as mg Lb∙(g NFW)−1 and obtained after extrapolation of the data using a human hemoglobin standard curve built from a stock of 300 mg∙l− 1 and including the following concentrations (mg∙l− 1): 0, 60, 120, 180, 240 and 300.
Nitrous oxide determinations
Detection of N2O emissions was performed according to Tortosa et al. (2020), including certain modifications for alfalfa detached nodules. Briefly, harvested nodules from the same pot (0.2–0.3 g) were immediately transferred to a 10-ml glass vial (SUPELCO®) and a volume of 1 ml or 100 µl NS (with the corresponding nitrate and Cu concentration) was added depending if nodules were isolated from flooded or non-flooded plants, respectively. The vials containing nodules were incubated at 30 ºC. N2O was detected by an HP 4890 gas chromatography instrument provided with an electron capture detector (ECD) as essentially described by Torres et al. (2014). The column was packed with Porapak Q 80/100 mesh. N2 was used as the carrier gas at a flow rate of 23 ml∙min− 1. The injector, column and detector temperatures were 125, 60 and 375°C, respectively. Gas samples were taken from the headspace of the vials after 3 and 6 h incubation and injected manually by using luer-lock gas-tight syringes BD Microlance™ 3. Peaks corresponding to N2O were integrated by using GC ChemStation Software (Agilent Technologies, Santa Clara, CA, USA) and the values obtained were used to calculate N2O concentration in each sample by extrapolation from a standard curve, performed by using 2% (v/v) N2O standard (Air Liquid, Paris, France) and including the following gas volumes: 0, 0.2, 0.4, 0.6, 0.8 and 1 ml. Total N2O concentration was determined by taking into account both N2O in headspace, and dissolved N2O applying Bunsen solubility coefficient (47.2% at 30°C). N2O fluxes from alfalfa detached nodules were expressed as nmol N2O∙(g NFW∙h)−1.
Bacteroids isolation
For bacteroids isolation, a method described by Mesa et al. (2004) was used. Basically, bacteroids were isolated by homogenizing 0.25 g of alfalfa nodules with a pestle in a cold porcelain mortar with 7.5 mL of extraction buffer (45.5 g∙l− 1 D-mannitol dissolved in 50 mM Tris-HCl, pH 7.5) previously added. Then, the extract obtained was filtered through a sterile cheesecloth filter and centrifuged at 250x g at 4 ºC for 5 min to remove nodule debris. Subsequently, the supernatant was centrifuged at 12,000x g at 4 ºC during 10 min and pellets were washed twice and resuspended in 0.5 ml of wash buffer (50 mM Tris-HCl, pH 7.5) prior to biochemical determinations.
Protein and nitrite determinations
Total protein concentration was estimated colorimetrically after alkaline lysis (1N NaOH at 100°C during 20 min) by using the Bradford reagent (Bio-Rad, Hercules, CA, USA) and extrapolating from a standard curve including 0, 4, 8, 12, 16 and 20 µg∙ml− 1 of bovine serum albumin (BSA) from a stock solution of 100 µg∙ml− 1 (Bradford 1976).
The nitrite concentration present in the bacteroids extract was estimated colorimetrically after diazotisation by adding the sulphanilamide/naphtylethylene diamino dihydrochloride reagent (Hageman and Hucklesby 1971) and extrapolating from a standard curve including 0, 20, 40, 60, 80 and 100 µM NaNO2 from a stock solution of 100 µM.
Determination of nitrate reductase (NR, EC 1.7.99.4), nitrite reductase (NIR, EC 1.7.2.1) and nitrous oxide reductase (N2OR, EC 1.7.2.4) activities
Methyl viologen (MV+)-dependent nitrate reductase (MV+-NR) and nitrite reductase (MV+-NIR) activities were performed as essentially described by Delgado et al. (2003). The reaction mixtures contained 200 µM methyl viologen, 20–30 µg protein from the cell suspension, 50 µl distilled water, and 10 mM KNO3 for MV+-NR or 100 µM NaNO2 for MV+-NIR assays, adding 50 mM Tris-HCl buffer up to reach a final volume of 450 µl in each reaction tube. Before measurements, a 46 mM sodium dithionite solution was prepared freshly (8 mg∙ml− 1 in 50 mM Tris-HCl buffer, pH 7.5), transferring 50 µl from it to each reaction tube. After incubation for 20–30 min at 30°C, the reaction was stopped by vigorous shaking until disappearance of blue color from the samples. Control tubes were prepared as the reaction tubes, but these tubes were shaken vigorously immediately after the addition of dithionite. MV+-NR activity was expressed as nmol NO2− produced∙(mg protein∙min)−1. MV+-NIR activity was expressed as nmol NO2− consumed∙(mg protein∙min)−1. Two biological replicates for each treatment were assayed.
Nitrous oxide reductase (N2OR) activity was measured as essentially described by Tortosa et al. (2020), setting some modifications for alfalfa nodules. The assay was performed in 10-ml glass SUPELCO® vials, adding 0.15–0.2 mg protein and 60 mM sodium succinate as electron donor. Then, a mixture of 2% (v/v) N2O and 98% (v/v) N2 (Air Liquid) was injected to reach a final concentration of 0.1% (v/v). To achieve anoxic conditions, the vials were sparged with N2 gas during 7 min. All the vials were incubated at 30°C for 1 h. Next, 0.5-ml aliquots were taken from the headspace of each vial. N2O measurements and concentration calculations were performed as described above. N2OR activity was expressed as nmol N2O consumed∙ (mg protein∙h)−1. Two biological replicates for each Cu condition were used.
Statistical analysis
Data were checked for normal distribution according to Kolmogorov-Smirnov and Shapiro-Wilk tests. For data obtained from plant assays in tubes, inferential statistics were performed by applying parametric ANOVA and a post-hoc Tukey HSD test at p ≤ 0.05 with SPSS software. For data obtained from plant assays in pots, inferential statistics to test null hypothesis were performed by applying non-parametric Kruskal-Wallis test for more than two unpaired treatments. Next, a post-hoc U Mann-Whitney test at p ≤ 0.05 with SPSS software was performed.