Vol.:(0123456789)1 3 Symbiosis (2023) 90:321–343 https://doi.org/10.1007/s13199-023-00939-3 Metabolic and genetic responses to simulated overwintering conditions of alfalfa‑rhizobia associations contrasted in their freezing tolerance Emmanuelle D’Amours1,2 · Annick Bertrand1  · Jean Cloutier1 · Annie Claessens1 · Solen Rocher1 · Philippe Seguin2 Received: 7 April 2023 / Accepted: 8 September 2023 / Published online: 6 October 2023 © Crown 2023 Abstract The study of winter stress tolerance in perennial legumes needs to consider the complete symbiotic system including both plants and bacteria since these two partners are differentially affected by stress conditions. Here, we compared the regrowth after a freezing stress of four different associations of two alfalfa populations differing in freezing tolerance (A-TF0 and A-TF7) inoculated with two Sinorhizobium (Ensifer) meliloti strains (B399 and NRG34) of contrasted adaptation to cold. To understand the contribution of each partner to a better regrowth performance of an association after freezing, we identi- fied molecular traits having major roles in cold acclimation, freezing tolerance, and those involved in the crosstalk between alfalfa and its symbiotic partner. Regrowth after exposure to a freezing stress was 35% larger in the A-TF7 × NRG34 than in the A-TF0 × B399 association. The metabolomic study of roots, crowns and, more specifically, nodules, revealed profound changes in these organs, switching from a sink to support cold acclimation to a source of reserves enabling regrowth after deacclimation. Marked increases in concentrations of stachyose and raffinose, two sugars of the raffinose-family oligosac- charides (RFO), and in the expression level of a gene of the RFO synthetic pathway were observed in response to cold acclimation supporting the importance of a protective role for RFO in alfalfa. Both cold-adapted partners of the symbiotic association contributed to increases in arginine concentration in nodules in response to cold acclimation and deacclimation underscoring the importance of N storage and remobilization for a successful overwintering in alfalfa. Keywords Medicago sativa L. · Sinorhizobium meliloti · Freezing stress · Nodule · Metabolites · Cold acclimation Abbreviations Ala Alanine Arg Arginine Asn Asparagine Asp Aspartic acid DM Dry matter DW Dry weight Formo Formononetin FlaTot Total flavonoids Gln Glutamine Glu Glutamic acid Gly Glycine His Histidine Ile Isoleucine Leu Leucine Lys Lysine Met Methionine N Nitrogen NSC Non structural carbohydrates Phe Phenylalanine Pro Proline RFO Raffinose-family oligosaccharides Ser Serine Thr Threonine Tyr Tyrosine Val Valine Orn Ornithine AATot Total amino acids SSTot Total soluble sugars AABA α-Aminobutyric acid GABA γ-Aminobutyric acid * Annick Bertrand Annick.bertrand@agr.gc.ca 1 Quebec Research and Development Centre, Agriculture and Agri-Food Canada, Quebec City, QC G1V 2J3, Canada 2 Department of Plant Science, McGill University, Sainte-Anne-de-Bellevue, QC H9X 3V9, Canada http://orcid.org/0000-0001-7768-2992 http://crossmark.crossref.org/dialog/?doi=10.1007/s13199-023-00939-3&domain=pdf 322 E. D’Amours et al. 1 3 1 Introduction Alfalfa (Medicago sativa L.) is the most important forage crop species and the fourth largest crop by area in Canada with 3 million hectares of pure stands or alfalfa-based mix- tures (Statistics Canada 2022). Alfalfa ability to establish a symbiosis with the nitrogen (N) fixing bacterial partner Sinorhizobium (Ensifer) meliloti reduces the need for N ferti- lization of this crop as well as for subsequent crops making it a key contributor to the current efforts to reduce agriculture reliance on fossil fuels (Cummings 2005). In northern latitudes, winter survival, field persistence and yield of alfalfa depend on its ability to tolerate low freezing temperatures (Bélanger et al. 2006; Seppänen et al. 2018). Predicted climate change will increase the risks of winter injury to alfalfa due to higher temperatures in the fall allowing less favorable hardening conditions and to the diminution of insulating snow cover protection during winter due to a higher incidence of freezing rain and thawing events (Bélanger et al. 2002). Perennial plants such as alfalfa go through the process of cold acclimation that increases their tolerance to freez- ing temperatures and promotes the accumulation of reserve metabolites to support spring regrowth (Dhont et al. 2002). Cold acclimation elicits several molecular changes in plants (Bertrand et  al. 2020a). For instance, low temperature induces starch hydrolysis and modifies the concentration of cryoprotective sugars such as sucrose, and raffinose-family oligosaccharides (RFO) in overwintering roots and crowns of alfalfa (Castonguay et al. 2009). Amino acids like proline, arginine and histidine also accumulate during cold accli- mation (Dhont et al. 2006). Proline was shown to stabilize membranes and protect against oxidative stress (Szabados and Savoure 2009). Arginine is known as a precursor of pol- yamines which are involved in complex signaling networks that regulate stress responses (Alcázar et al. 2011; Menén- dez et al. 2019; Pál et al. 2015) and is also an important N reserve to support spring regrowth (Dhont et al. 2006). Cold acclimation elicits major changes in gene expression and several cold regulated (COR) genes have been identi- fied and characterized in alfalfa and other perennial species (Bertrand et al. 2017; Juurakko and Walker 2021). While the process of cold acclimation has been extensively studied in alfalfa, there is a knowledge gap regarding the deacclimation process and associated metabolic changes. Deacclimation occurs rapidly upon the return to warm temperatures in the spring. During that period, plants are more susceptible to damage by freeze–thaw cycles (Kalberer et al. 2006). The unpredictable effects of climate change on spring tempera- tures urge the need to better understand the deacclimation process since perennials will likely be exposed to more fre- quent and larger temperature fluctuations in springtime. Alfalfa has been shown to have a large genetic diver- sity for freezing tolerance which has been successfully leveraged to improve this complex trait using a recurrent selection approach under controlled conditions (Bertrand et al. 2017). A potential relationship between freezing tolerance and changes in gene expression and metabolite composition in cold-acclimated alfalfa is highlighted by the differential accumulation of COR gene products and of metabolites in alfalfa populations recurrently selected for superior freezing tolerance (Castonguay et al. 2006). Low temperature stress can not only reduce alfalfa sur- vival and productivity but can also negatively impact its rhizobial partner and hinder the establishment of effec- tive rhizobia-legume interactions (Prévost et al. 2003). The crosstalk between the symbiotic partners is initiated by the secretion of flavonoids by alfalfa (mainly formononetin, medicarpin and coumestrol) which concentrations have been shown to be modified by low temperature causing changes in nod genes activity in the symbiont (Zhang et al. 1996, 2009). Moreover, low temperatures has been shown to decrease Nod factors production by rhizobia while these lipo-chitooligosaccharides compounds are essential for the infection process (Duzan et al. 2006; Zhang et al. 1996). D’Amours et al. (2022) recently showed that choice of the rhizobial strain directly affects the level of nodule damage after a freezing stress. Different proportions of undam- aged and necrotic nodules were observed according to the rhizobial strain in symbiosis with alfalfa, after exposure to freezing. This observed in vivo differences in nodule freez- ing damages was linked with plant yield: alfalfa inoculated with a freezing-tolerant strain had a larger proportion of active nodules with no damage after the freezing stress along with a greater plant vigor and shoot regrowth as compared to symbiosis with freezing-sensitive strains. Varying responses of legume-rhizobia associations to environmental stresses were reported (Bertrand et al. 2020b; Sanz-Sáez et al. 2012). The underlying molecu- lar bases of differential tolerance between host-symbiont combinations remain to be elucidated. A feedback regu- lation between host-plants and nodules has been shown to depend on resources availability and environmental conditions (Bertrand et al. 2016; Marquez-Garcia et al. 2015) as well as on the symbiotic rhizobial strain. For instance, when comparing three Bradyrhizobium strains in association with soybean growing under contrasted levels of atmospheric CO2, the strain associated with the highest yield also induced the highest ureides concentra- tion in nodules under elevated CO2 along with the highest nitrogenase activity (Bertrand et al. 2011).This relation- ship indicated a positive-feedback stimulation: soybean mobilized energy reserves to support more nodules, and in return nodules synthesized more ureides to support plant growth. 323Metabolic and genetic responses to simulated overwintering conditions of alfalfa‑rhizobia… 1 3 Strains of S. meliloti have been shown to directly affect the level of freezing tolerance of alfalfa (Bertrand et al. 2007) and, recently, alfalfa regrowth after freezing was reported to differ depending on the associated S. meliloti strain (D’Amours et al. 2022). To better understand the mechanisms of tolerance of alfalfa-rhizobia associations, this study focused on metabolites and genes that were pre- viously reported to play major roles in cold acclimation and freezing tolerance of alfalfa and nodules. Flavonoids involved in the crosstalk between alfalfa and rhizobia were also quantified as well as the expression of genes of their biosynthetic pathway. 2 Materials and Methods Inoculum production, plant growth conditions and sampling procedures have been described in D’Amours et al. (2022) and are summarized below. 2.1 Sinorhizobium meliloti strains and plant material Based on the results of our previous study on the effect of S. meliloti strains on alfalfa yield after a freezing stress (D’Amours et al. 2022), two strains inducing contrasted responses in alfalfa, ‘B399’ and ‘NRG34’, were selected for the present study. Plants inoculated with the commercial strain ‘B399’ (provided by Instituto de Genética “Edwald Alfredo Favret”, INTA, Buenos Aires, Argentina) were com- pared to plants inoculated with strain ‘NRG34’ isolated from Northwestern Canada (Rice et al. 1995). Both strains were grown in yeast extract mannitol (YEM) broth (Vincent 1970) at 28 °C for 24 to 48 h and a viability count was performed to adjust inoculum at 108 cells mL−1. These strains were used to inoculate two populations of alfalfa contrasted in their levels of freezing tolerance (i.e., A-TF0 and A-TF7). The cultivar ‘Apica’ (A-TF0) was developed at the Quebec Research and Development Centre (QRDC) of Agriculture and Agri-Food Canada (Michaud et al. 1983) and has a freezing lethal temperature for 50% of the plants (LT50) of -20 °C, while population ‘A-TF7’ was obtained after seven cycles of recurrent selection for improved freezing tolerance from the original cultivar Apica and has a LT50 of -26 °C (Bertrand et al. 2020a; D’Amours et al. 2022). 2.2 Plant growth conditions Sterilized seeds of two alfalfa populations were individually seeded in Ray Leach Cone-tainers TM (SC-10 Super Cell. Stuewe & Sons Inc, Tangent, OR) filled with sterilized Tur- face® (Profile Products LLC, Buffalo Grove, IL). One week after seeding, plants were inoculated with 1 mL of either strain B399 or NRG34 containing 108 cells. Uninoculated controls for each alfalfa population (A-TF0 and A-TF7) were included to ensure that there was no uncontrolled sources of rhizobia contamination. These control plants failed to grow due to the lack of N input from fixation or nutrient solu- tion and were not included in the statistical analysis. Plants were grown and sampled under the experimental conditions illustrated in Fig. 1 and as described in details in D’Amours et al. (2022). Briefly, plants were grown in a growth cham- ber under a temperature regime of 21/17 °C day/night and a 16 h-photoperiod and fertilized three times a week with 0.50 N-free Hoagland solution (Hoagland and Arnon 1950). Plants were harvested at the four following sampling events. Fig. 1 Plants growth conditions and sampling events. Plants grown under controlled conditions were harvested at four sampling events: non-acclimated plants (NA) were sampled after being grown for 8 weeks under a 21/17°C, Day/Night (D/N) temperature regime. Plants were then cold acclimated for 4 weeks (CA) and sampled, and then exposed to a freezing stress of -11°C and transferred back to optimal regrowth conditions (21/17°C, D/N). Two days after freezing stress (AFS) and three weeks of regrowth after freezing (RAF) plants were sampled again 324 E. D’Amours et al. 1 3 1) Non-acclimated (NA) plants were sampled after eight weeks of growth (32 plants: 8 replicates × 2 alfalfa popula- tions × 2 strains). 2) Cold acclimated (CA) plants were sam- pled after an additional two weeks of growth at 2 °C under a 8 h-photoperiod, followed by two weeks at -2 °C in the dark (Fig. 1). The remaining plants were then exposed to a non-lethal freezing stress in a large programmable freezer in which temperature was gradually reduced from -2 °C to -11 °C (D’Amours et al. 2022), these frozen plants were then thawed for 24 h at 4 °C in darkness and exposed to the initial optimal regrowth conditions by progressively increas- ing the air temperature from 4 °C to 21/17 °C day/night. 3) Two days after this freezing stress (AFS), 32 plants were sampled to characterize the symbiosis immediately follow- ing the freezing stress. 4) The sampling of the remaining 32 plants was made after three weeks of regrowth after freezing (RAF) to study the effect of deacclimation. 2.3 Root exudates sampling and biochemical analyses 2.3.1 Root exudates sampling In a first step, root exudates were collected at each sampling event (i.e., NA, CA, AFS, and RAF). For this purpose, plants were carefully removed from their cone-tainers and gently shaken to remove the excess of turface and then washed three times in distilled water in order to remove any traces of turface and nutrient solution. Excess water was removed by gently pressing roots in absorbent paper towels. Each plant was then transferred into a 250-mL beaker contain- ing 100 mL of ultrapure water and soaked for 10 min to collect root exudates. Plants were then removed from water and the soaking water containing root exudates was filtered using filter papers with 25 µm particle retention (Cytivia, Whatman™ no.4, Marlborough, MA). The remaining filtrate (75 mL) was separated into three equal volumes of 25 mL and transferred in 50-mL screw cap tubes (Sarstedt Inc Nümbrecht, Germany) in order to proceed to the biochemi- cal analysis of three types of compounds: soluble sugars, amino acids and flavonoids. Tubes were kept on ice during sampling and then covered with perforated parafilm to allow freeze-drying. Root exudates were kept at -40 °C until being freeze-dried during 140 h to obtain dry-powered exudates (Labconco, Model Freezone12, Kansas City, MO). 2.3.2 Soluble sugars and amino acids extraction A first tube of dry-powered root exudates was diluted in 1 mL of ultrapure water to suspend soluble sugars and free amino acids into an aqueous phase. Tubes were immediately heated 20 min at 65 °C to stop enzymatic activity. Tubes were then cooled in an ice water bath before being vortexed and centrifuged 30 s at 2,150 × g and the suspension was transferred into a 1.5 mL-microtube and kept frozen at -80 °C. Samples were centrifuged for 3 min at 11,350 × g prior to chromatographic analyses for soluble sugars and amino acids as described below. 2.3.3 Flavonoids extraction A second tube of dry-powered root exudates was dissolved in 1 mL of MeOH 80% to extract flavonoids. Tubes were immediately heated 15 min at 65 °C then cooled in an ice water bath before being vortexed and centrifuged 30 s at 2,150 × g. The suspension was transferred to 1.5 mL micro- tubes and kept frozen at -80 °C. Samples were centrifuged for 3 min at 11,350 × g, prior to UPLC analysis. 2.4 Plant sampling for extraction of metabolites and RNA Plants were separated into three major sections: roots sys- tems, crowns and shoots that were kept on ice during sam- pling. For each plant, nodules were detached from roots using tweezers and transferred into 5-mL tubes. Nodules were freeze-dried prior to biochemical analysis and their total dry weight (DW) was recorded. The crowns, consid- ered as the 2-cm subsections between shoots and roots, were sampled and analyzed separately because of their reported key role in the cold acclimation of alfalfa (Castonguay et al. 2009). Crowns were cut into two longitudinal sub-sections, and their fresh weight was recorded. The first crown sub-sec- tion (approx. 0.1 g) was flash frozen and manually ground in liquid nitrogen and kept at − 80 °C for RNA extraction. The second sub-section of crowns was freeze-dried (Labconco, Model Freezone12), the DW was recorded and samples were ground using an OMNI Bead Ruptor 24 (PerkinElmer, Inc. Kennesaw, GA) before further biochemical analyses. A 8 cm-long subsample of roots containing a mixture of fine roots and tap roots was weighted (around 0.1 g) and flash frozen in liquid nitrogen for RNA extraction. A second root subsample was weighted (approx. 0.2), freeze-dried, and ground (OMNI Bead Ruptor) prior to biochemical analysis. The remaining part of the root system was dried separately at 55 °C for 72 h for the measurement of total root DW (includ- ing the calculated DW of all sub sections). Shoots were dried at 55 °C for 72 h for the measurement of total shoot dry weight (including the calculated DW of crown sub-sections). Grinded samples of crowns and roots were analyzed for their soluble sugars and amino acids contents after extraction in methanol-chloroform-water as described in Bertrand et al. (2020b). Grinded samples of nodules were analyzed for their content in soluble sugars, amino acids, and flavonoids. For this purpose, the total dry weight of each nodules samples (between 0.02 and 0.09 g) was extracted in 2 mL of MeOH 325Metabolic and genetic responses to simulated overwintering conditions of alfalfa‑rhizobia… 1 3 80%. Tubes were heated 20 min at 65 °C, rapidly cooled on ice and centrifuged (10 min at 1,200 × g at 4 °C). A first sub- sample of 0.9 mL was evaporated to dryness (Savant Speed- vac plus SC210A, Holbrook, NY), solubilized in 0.9 mL of ultrapure water, and kept at -20 °C until the analysis of soluble sugars (HPLC, Waters Inc. Milford, MA) and amino acids (UPLC, Acquity, Waters Inc, Milford, MA). A sec- ond subsample of 0.9 mL of the supernatant was transferred into 1.5 mL microtubes and frozen at -20 °C until flavonoid analysis concentration by UPLC. Subsamples of roots were also extracted in MeOH 80% and treated similarly to ana- lyze their flavonoids concentration. Prior to chromatographic separation by HPLC or UPLC, all samples were centrifuged for 3 min at 11,350 × g. 2.5 Metabolites quantification 2.5.1 Quantification of carbohydrates and amino acids Soluble sugars were separated and quantified on a chroma- tographic analytical system controlled by the Empower II software (Waters, Milford, MA) as described in Bertrand et al. (2020b). Peak identity and quantity were determined for raffinose, stachyose, sucrose, glucose, fructose and pini- tol by comparison to standards (Sigma–Aldrich, Oakville, ON, Canada). Starch was quantified in non-soluble residues following the extraction of soluble sugars. Starch in the resi- dues was hydrolyzed into glucose by adding 3 mL of diges- tion buffer (200 mM sodium acetate, pH 4.5) containing amyloglucosidase (15 U mL−1; Sigma-Aldrich, Oakville, ON, Canada), and incubation (60 min at 55 °C). After cen- trifugation, the supernatant was collected for quantification of glucose by HPLC, as described above. Starch concentra- tion was calculated by subtracting the amount of soluble glucose from the total amount of glucose measured fol- lowing digestion with amyloglucosidase. For crowns, roots and nodules, results of carbohydrates determinations were expressed as concentrations on a dry matter (DM) basis (mg g−1 DM). For root exudates, results were reported on a root DM basis by taking into account the DM of the root systems that were soaked in ultrapure water to extract exudates. Total soluble sugars (SSTot) is the sum of concentrations of indi- vidual sugars raffinose, stachyose, sucrose, pinitol, glucose and fructose and non-structural carbohydrates (NSC) is the sum of SSTot and starch. Twenty-one amino acids (alanine, arginine, asparagine, aspartate, glutamate, glutamine, glycine, γ-aminobutyric acid (GABA), α- aminobutyric acid (AABA), histidine, proline, methionine, lysine, serine, leucine, isoleucine, ornithine, phenylalanine, threonine, tyrosine and valine) were separated and quantities were determined by compar- ison to a standard mix containing the 21 amino acids. Each individual amino acid were provided by Sigma–Aldrich (Oakville, ON, Canada) and the standard mix was prepared in our laboratory. For crowns, roots and nodules, results were expressed as concentrations on a DM basis (µmol g−1 DM). For root exudates results were reported in µmol on root DM basis by taking into account the DM of the root systems that were soaked in ultrapure water to extract exudates. The total free amino acids (AATot) was the sum of concentrations of the 21 free amino acids. 2.5.2 Quantification of flavonoids Naringenin, luteolin, echinatin, coumestrol, formononetin and medicarpin and their conjugates were separated and quantified using Waters ACQUITY UPLC analytical sys- tem controlled by the Empower II software (WATERS, Milford, MA, USA). Flavonoids were separated using a BEH C8 column (2.1 mm × 100 mm, 1.7 µm, Waters), and detected using a Photodiode Array (PDA) Detector set at 287 nm. The chromatographic conditions were as follows: column temperature, 35  °C flow rate, 0.35 mL  min−1, mobile phase A, 0.2% formic acid in water, mobile phase B: methanol 100%. The gradient was of 65% A at 0.2 mL  min−1 and a gradual decrease until 2%, before to returning to initial condition of 65% A, for a total run of 8 min. Peak identity and quantity of each flavonoids were determined by comparison to standards. Results for flavo- noids determinations in roots were expressed as concen- trations on a dry matter (DM) basis (µg g-1DM). For root exudates, the calculation was as described in the above section. Total flavonoids (FlaTot) are the sum of each indi- vidual concentration of the seven flavonoids. 2.6 Analysis of gene expression 2.6.1 RNA extraction and cDNA synthesis Total RNA was extracted from 0.1 g of crowns and roots using CTAB-based protocol (Dubé et al. 2013). Total RNA was quantified using NanoDrop™ One/OneC Microvol- ume UV–Vis Spectrophotometer (Thermo Fisher Scientific Inc. Waltham, MA). Residual genomic DNA was removed by a treatment with DNaseI (Invitrogen, Burlington, ON, Canada) prior to cDNA synthesis. First strand cDNA was synthesized from 1 μg of total RNA and oligo(dT)18 prim- ers using the Transcriptor First Strand cDNA synthesis Kit (Roche Applied Science, Laval, QC, Canada) following the manufacturer instructions. cDNA synthesis reactions were performed for each sample considered in the subsequent RT-qPCR analyses. Thus, a total of 256 reactions were per- formed: 8 replicates × 2 alfalfa populations × 2 strains × 4 sampling dates × 2 organs (crowns and roots). 326 E. D’Amours et al. 1 3 2.6.2 RT‑ qPCR analysis of COR gene expression The expression of seven genes of interested (GOI) that are cold-regulated (COR) was measured and compared between the different treatments (Table 1). The five genes related to cold acclimation and freezing stress tolerance were selected from previous studies on alfalfa (Bertrand et al. 2017; Cas- tonguay et al. 2015; Dubé et al. 2013) and their expressions were analyzed in crown samples. Two genes of interest linked to the phenylpropanoid synthetic pathway leading to flavonoid synthesis were selected based on literature (Gifford et al. 2018) and their expressions were measured in alfalfa root samples. Specific qPCR primers were designed using the methodology described in Castonguay et al. (2020), to amplify these last two genes of interests in Medicago sativa samples. Primer design was based on homologous Medicago tranculata genes sequences retrieved from a BLASTn search on NCBI database [Medtr4g088190 (IOMT or 2,7,4′-trihy- droxyisoflavanone 4′-O-methyltransferase, EC 2.1.1.212) and Medtr5075450 (C4H or cinnamic acid 4-hydroxylase EC 1.14.13.11)] (Gifford et al. 2018). The sequences of all primers selected for RT-qPCR analysis of GOI as well as of the three reference genes described below are listed in Table 1. The expression of COR genes was measured in crowns as this tissue plays a key role in cold acclimation of alfalfa. The expression of genes involved in flavonoid bio- synthesis was measured in roots as this tissue is the synthetic site of flavonoids. The RT-qPCR was carried out in a Mastercycler® ep realplex system (Eppendorf Canada, Mississauga, ON, Canada) using the QuantiTect® SYBR Green PCR kit (QIAGEN, Toronto, ON, Canada) as described by Dubé et al. (2013). The 10-μl reaction mixture contained 3 μl of first-strand cDNA and 0.5 μM of each of the forward and reverse primers. The thermocycler program was set to: 15 min at 95 °C for denaturation; 40 cycles of 15 s at 95 °C, 15 s annealing at 60 °C; 60 s extension at 72 °C. Reactions with real-time PCR were carried out with con- trol water samples included as checks for potential con- tamination with genomic DNA. Efficiency of PCR was calculated from the linear regression of a seven fold dilu- tion of PCR products using the following equation: Effi- ciency % = (10(−1/slope)-1) × 100. The threshold cycle (Cq) values at which the PCR product fluorescence rises over the background fluorescence was determined by the instru- ment software which was set to default parameters. For the normalization of results, reference genes were selected by screening several candidates identified by Castonguay Table 1 List of genes of interest (GOI) and reference genes (Ref) selected for expression analysis in alfalfa crowns and/or roots, sequence homology based on Medicago trunculata genes retrieved using a BLAST search in the NCBI database, sequences of PCR primers, and PCR fragment sizes are also presented Plant Sample Gene Gene annotation Type Primer sequence (5’-3’) Primer reference Ampli- con size (bp) Crown GaS Galacticnol synthase GOI ACC CTC CTG AGA ACC AAA CC Bertrand et al. 2017 197 GGC CTC ACA GAA ACA GTC CA Crown SPS Sucrose phosphate synthase GOI TCC CAA GCC CTC AGA TAC C Castonguay et al. 2015 146 CTG CTT CCG ACT CCC TTC A Crown Susy Sucrose synthase GOI CCG ATT GAC ATC CTT CTA CCC Castonguay et al. 2015 235 GTC CTT TGA CTC CTT CCT CCT Crown K3-dehydrin K3-dehydrin GOI GGA GCT GTG GAC AAG ATC AAGG GTG TCC TTG TCC ATG TCC AGT ACA Dubé et al. 2013 345 Crown / Roots Aptase Vacuolar H +—ATPase A subunit Ref CTA CGA ACG TGC TGG GAA AG Castonguay et al. 2015 124 GAG GGT TGC AGA TGT CAC G Crown UBQ2 Ubiquitin 2 Ref GGA CTC AAG GTG GCC AAA C GAG GGT TGC AGA TGT CAC G Castonguay et al. 2015 197 Roots C4H Cinamic acid 4-hydoxylase GOI CCC CGG AAT CAT TCT TGC CT TTT GAT TGT CCG GGT GGA GG Designed for this study 93 Roots IOMT Isoflavone-O-methytransferase GOI Designed for this study 144 CGC ACA ACG GAT TCT TCG AG ACG AAC CCG AAA GAG TTG GA Roots eEF-1α Eukariotic elongation factor 1-alphas Ref GAG CCA AAG AGA CCC ACA GAC Castonguay et al. 2015 192 TCA GTG AGA GCC TCG TGG T 327Metabolic and genetic responses to simulated overwintering conditions of alfalfa‑rhizobia… 1 3 et al. (2015) for the stability of their expression using the geNormPlus M value program included in the qBasePlus software (Biogazelle, Ghent, Belgium). Results were nor- malized in each tissues with three reference genes: ubiqui- tin 5 (crown), eukariotic elongation factor 1-alphas (roots) and H-ATPase (crowns and roots). Relative quantification was calculated with the qBase software using the 2-ΔΔCq or comparative Cq method based on the differences in Cq between the target and the reference genes and corrected for PCR efficiency. 2.7 Statistical analysis Statistical analysis of biomass were made using a one-way analysis of variance (ANOVA) for a randomized complete block design with the SAS MIXED procedure (SAS® Stu- dio, 2020, Version 3.81, SAS Institute Inc., Cary, NC). The model was used to compare the effects of the associations between alfalfa populations and S. meliloti strains on shoot regrowth and nodules biomass. Concentrations of metabo- lites and gene expression were analyzed using a two-way analysis of variance (ANOVA) model for a randomized complete block design with the SAS MIXED procedure (SAS® Studio, 2020, Version 3.81, SAS Institute Inc., Cary, NC). The model was used to establish, in a first step, the effects of sampling events. ANOVA were then performed for each sampling events separately to estab- lish the effects of the two alfalfa populations, the two S. meliloti strains, and their interactions on the concentra- tions of carbohydrates, amino acids, and flavonoids and on genes expression, for each plant organs and root exudates. The Residual normality and variance homogeneity were verified using the UNIVARIATE procedure. The Shap- iro–Wilk’s and Kurtosis’s tests were used to verify the normality of the data distribution. Pairwise comparisons of means differences were made using a Fisher’s least sig- nificance difference test (LSD) at P ≤ 0.05. The log two- fold changes in metabolite concentrations that significantly differed between alfalfa populations and S. meliloti strains were calculated in non-acclimated (NA), cold acclimated (CA), 48 h after freezing stress (AFS) and regrowth after freezing (RAF) plants. The graphical representation of metabolite variations related to either alfalfa populations or S. meliloti strains based on this calculation shows the differential contribution of each alfalfa population (A-TF0 vs A-TF7) and each strain (B399 vs NRG34) to the meta- bolic changes at each sampling event. 3 Results 3.1 Comparative assessment of shoot and nodule biomass in alfalfa‑rhizobia associations under varying environmental conditions There was no difference in shoot DW between the non- acclimated (NA) four alfalfa-rhizobia associations tested (Table 2). In contrast, shoot regrowth three weeks after exposure to freezing stress (RAF) was significantly dif- ferent between the populations × strains associations. The shoot DW of the freezing tolerant alfalfa population A-TF7 inoculated with the freezing tolerant strain NRG34 was significantly higher (+ 35%) than the shoot DW observed with the combination of the less freezing tolerant popula- tion and freezing sensitive strain A-TF0 × B399 (Fig. 2a). The shoot DW of A-TF0 × NRG34 was also significantly higher (+ 17%) than that of A-TF0 × B399. It is noteworthy that the shoot DW of the A-TF7 × B399 was intermediate between those of A-TF0 × B399 and A-TF0 × NRG34 even though it did not significantly differ with from the shoot DW of these two associations. The four alfalfa-rhizobia associations also differed in nodules DW after a freezing stress (Table 2, Fig. 2b). While no difference in nodules DW was observed in NA and CA plants, the nodules DW of A-TF0 × NRG34 was significantly larger than both asso- ciations with strain B399 two days after the freezing stress (AFS). The significant difference in nodules DW between A-TF0 × NRG34 and the other three alfalfa-rhizobia asso- ciations was much larger three weeks after the exposure to a sublethal freezing stress (RAF). Table 2 P values of the analysis of variance comparing the effects of four alfalfa populations × rhizobia strains associations (A-TF0 × B399, A-TF0 × NRG34, A-TF7 × B399 and A-TF7 × NRG34) on shoot dry weight (g) and nodules dry weight (g) of alfalfa Measurements were made at four sampling events: in non-acclimated plants (NA), cold-acclimated (CA) plants, plants exposed to a freez- ing stress and transferred back to optimal regrowth conditions for 48 h (AFS), and after a 3-weeks regrowth (RAF). Shoot DW could not be measured in CA and AFS plants as the shoots were damaged by these treatments (not assessed, n.a.). Numbers in bold represent significant differences at P ≤ 0.05 Event sampling NA CA AFS RAF Shoot dry weight 0.480 n.a n.a < 0.001 Nodules dry weight 0.430 0.120 0.016 0.011 328 E. D’Amours et al. 1 3 3.2 Variations in metabolite concentrations between sampling events 3.2.1 Non‑structural carbohydrates Concentrations of starch and soluble sugars in roots, crowns and nodules significantly varied between sampling events (Table 3, Fig. 3). This observation was also true for root exu- dates except for glucose and fructose concentrations that did not vary between samplings. Starch concentration under NA conditions was much higher in roots (322 mg  g−1 DM) and crowns (272 mg  g−1 DM) than in nodules (1.6 mg  g−1 DM). After four weeks of CA, starch concentrations were reduced by one half in roots and crowns while its concentration dou- bled in nodules (Fig. 3). Starch concentrations decreased in subsequent AFS and RAF sampling points in nodules, roots and crowns to reach levels that were lower than those measured in NA plants. Concomitantly, a marked increase was observed in total soluble sugars in CA crowns (+ 222%), roots (+ 200%) and nodules (+ 136%) as compared to NA. More specifically, sucrose concentration in nodules, roots and crowns was 2.5 to 4 times higher in CA than in NA plants and then decreased gradually in subsequent AFS and RAF samplings. Concentration of glucose decreased in roots and crowns for CA, AFS and RAF when compared to NA plants while in nodules, a slightly higher glucose concentra- tion was observed in CA as compared to NA. Cold acclima- tion induced a marked increase of stachyose and raffinose in root exudates, roots, crowns and nodules. While stachyose and raffinose were undetectable in NA nodules, they reached concentrations as high as 9 and 17 mg  g−1, respectively, in CA nodules and then decreased progressively to 0 in subse- quent sampling events. Generally, the fructose concentration remained stable in nodules, roots, and crowns in response to CA but a significant increase in fructose concentration was observed in nodules of AFS and RAF plants as compared to NA and CA plants. The concentration of pinitol in nodules decreased progressively at each subsequent sampling events in exudates, nodules, roots and crowns as compared to NA. 3.2.2 Amino acids Concentrations of total free amino acids in NA samples were much higher in nodules (523.4 μmol  g−1 DM) comparatively to roots (143.4 μmol  g−1 DM), crowns (131.8 μmol  g−1 DM), and root exudates (2.5 µmol  g−1 root DM (Fig. 3). Total amino acids concentration decreased with CA in root exudates and in nodules while it remained stable between samplings in roots and crowns (Table 3, Fig. 3). In con- trast, individual free amino acids varied in roots, crowns and nodules between samplings except for proline in nod- ules and crowns, aspartate in nodules, and AABA in roots. Contrarily to the increase that was generally observed in nodules, roots and crowns between NA and the other sam- plings, individual free amino acids mostly decreased in root exudates at CA and AFS samplings with the exception of methionine and arginine that remained stable. Asparagine was by far the most abundant amino acid, representing 35% of total amino acids in root exudates, 80% in nodules, 58% in roots and 52% in crowns in NA samples. Cold acclimation Fig. 2 Shoot dry weight (DW) (a), and nodules DW (b) of four associations combining two alfalfa populations A-TF0 (in grey) and A-TF7 (in white) inoculated with two S. meliloti strains B399 (lined pattern) and NRG34 (dotted pattern). Alfalfa plants were grown under controlled conditions and sampled at four sampling events: non-acclimated plants (NA) were grown 8 weeks under a 21/17 °C, Day/Night (D/N) temperature regime. Plants were then cold accli- mated during 2 weeks at 2  °C followed by two weeks at -2  °C and sampled again (CA). After their exposure to a freezing stress of -11  °C, alfalfa plants were transferred back to optimal regrowth conditions (21/17  °C, D/N) and sampled after two days (AFS), and after three weeks (RAF). Shoots were only sampled for NA and RAF plants since CA and AFS shoots and leaves were killed by the 2 weeks exposure to subfreezing temperature in the dark while nodule dry weight was measured at the four sampling events. Error bars rep- resent the Standard Error of the Mean (SEM), n = 8. No letter means that there is no significant differences between treatments for that event sampling while different letters represent significant differences as determined by the Fisher’s least significant difference (LSD) test at P ≤ 0.05 329Metabolic and genetic responses to simulated overwintering conditions of alfalfa‑rhizobia… 1 3 Table 3 Analysis of variance (P values) comparing the effect of sampling events on the concentration of different metabolites (sug- ars, amino acids, and flavonoids) in root exudates, nodules, roots and crowns of four alfalfa populations-rhizobial strains associa- tions. Reported metabolic functions of sugars and amino acids related to cold acclimation and freezing stress tolerance in plants are pre- sented as well as the reported functions of flavonoids in the rhizos- phere with their corresponding references Metabolites Metabolic functions References Event sampling Alfalfa organs Exudates Nodules Roots Crowns P values Sugars   Sucrose Osmotic adjustment Dhont et al. 2002 < 0.001 < 0.001 < 0.001 < 0.001   Glucose Osmotic adjustment Dhont et al. 2002 0.0903 0.0049 0.0133 < 0.001   Stachyose Cryoprotectant Castonguay et al. 2011 < 0.001 < 0.001 < 0.001 < 0.001   Raffinose Cryoprotectant Castonguay et al. 2011 < 0.001 < 0.001 < 0.001 < 0.001   Fructose TCA cycle/ antioxidative protection under cold sterss Bogdanović et al. 2008 0.1533 < 0.001 < 0.001 < 0.001   Pinitol Osmoprotectant Castonguay et al. 2011 0.0068 < 0.001 < 0.001 < 0.001   SSTot < 0.001 < 0.001 < 0.001   Starch Carbon storage Gurusamy et al. 2000; Dhont et al. 2002; 0.015 < 0.001 < 0.001   NSC < 0.001 < 0.001 < 0.001 Amino Acids   Glu Nitrogen assimilation and transport Liu et al. 2018 0.508 0.00 < 0.001 < 0.001   Gln Nitrogen assimilation and transport Liu et al. 2018 < 0.001 0.00 < 0.001 < 0.001   Pro Osmoprotectant Castonguay et al. 2011 < 0.001 0.36 < 0.001 0.48   Orn Polyamine synthesis, Arg pathway Majumdar et al. 2016 < 0.001 < 0.001 < 0.001 < 0.001   Arg Cryoprotectant, chelator of nitrate to prevent damages to membranes and ice formation Alcázar et al. 2011 0.761 < 0.001 < 0.001 < 0.001   His Nitrogen storage Dhont et al. 2006 < 0.001 < 0.001 < 0.001 < 0.001   Asp Carbon skeleton, precursor leading to the biosynthesis of multiple biomolecules required for plant growth and defense Galili 2011 0.024 0.24 < 0.001 < 0.001   Asn Nitrogen assimilation and transport Sulieman et al. 2010 < 0.001 < 0.001 0.02 < 0.001   Ala First stress signal, accumulation induced by radical formation Ben-Izhak Monselise et al. 2003; Schulte et al. 2021 < 0.001 < 0.001 < 0.001 < 0.001   Thr Conjugated with metabolism of branched-chain amino acids Val and Leu Hildebrandt 2018 < 0.001 < 0.001 < 0.001 < 0.001   Lys Degradation to Val, Leu, Ile, alternative respiratory substrate under a stress Hildebrandt 2018 0.036 < 0.001 < 0.001 < 0.001   Met Conjugated with metabolism of branched-chain amino acids/essential for efficient nodulation by rhizobia Hildebrandt 2018; Barra et al. 2006 < 0.001 < 0.001 < 0.001 < 0.001   Ile Involved in fatty acid branching pat- tern for membrane adaptation to low temperatures Klein et al. 1999 < 0.001 < 0.001 < 0.001 < 0.001   Leu Conjugated with metabolism of branched-chain amino acids Hildebrandt 2018 < 0.001 < 0.001 < 0.001 < 0.001   Val Conjugated with metabolism of branched-chain amino acids Hildebrandt 2018 < 0.001 < 0.001 < 0.001 < 0.001   Ser Carbon skeleton, regulation of intracel- lular redox Igamberdiev and Kleczkowski 2018 < 0.001 < 0.001 < 0.001 < 0.001   Gly Osmotic adjustment Igamberdiev and Kleczkowski 2018 < 0.001 0.01 < 0.001 < 0.001   GABA Reactive oxygen scavenging Abd Elbar et al. 2021 < 0.001 < 0.001 < 0.001 0.05   AABA Isomere of GABA not well studied Bertrand et al. 2016 < 0.001 < 0.001 0.09 < 0.001 330 E. D’Amours et al. 1 3 induced a decline in asparagine concentration in all sam- ples. In crowns, asparagine concentration re-increased after a 3-wks regrowth (RAF) reaching a level higher than in NA samples. Glutamate concentrations slightly increased in CA and AFS roots and crowns compared to NA. An increase in concentrations of glutamine was observed in CA and AFS nodules while a decrease occurred in roots and crowns at those sampling events. Concentrations of ornithine, arginine and histidine increased significantly in CA roots and crowns (avg. of + 82, + 77, and + 42%, respectively) and even more so in nodules (+ 917, + 798, and + 129%, respectively). The concentration of these amino acids remained high in the AFS samples. Concentrations of alanine and glycine increased in CA nodules, remained stable in AFS samples and sub- sequently decreased to a level lower than that observed in NA nodules in RAF samples. Alanine and glycine concen- trations initially decreased in CA roots and crowns and re- increased in AFS samples. Serine concentrations increased with cold acclimation in nodules (+ 219%), roots (+ 70%) and crowns (+ 62%), whereas its concentration decreased in root exudates. An increase in threonine, lysine, isoleucine, leucine, valine, tyrosine and phenylalanine was observed in nodules, roots and crowns 48 h after a freezing stress (AFS) but their levels remained low. The GABA concentration was at a lower level in nodules and roots at RAF as compared to the other samplings. The AABA concentrations decreased in nodules at CA, AFS and RAF compared to NA, while it increased slightly in response to CA and AFS in crowns. 3.2.3 Flavonoids Total flavonoid concentrations were higher in roots (89 mg  g−1 DM) than in nodules (52 mg  g−1 nodules DM) and in root exudates (2.7 mg  g−1 root DM) of NA plants (Table 3, Fig. 3). In CA, AFS and RAF plants, concentra- tion of total and individual flavonoids differed between samplings. In general, total flavonoids increased at each subsequent sampling in nodules while it decreased in roots and root exudates (Fig. 3). Formononetin was the flavonoid with the highest concentration in all samples, representing slightly more than 75% of all flavonoids in root exudates and nodules, and 95% in roots. Formononetin concentra- tion decreased in response to CA and AFS in nodules and roots while it increased in root exudates in response to CA. Most of individual flavonoids decreased in root exudates with CA and AFS except for formononetin and medicarpin. In roots, concentrations of naringenin and medicarpin were not impacted by the cold acclimation and freezing stress. In nodules, all flavonoids except for fomononetin increased in CA samples and showed an additional increase 48 h after the freezing stress (AFS) with concentrations of naringenin (+ 48%), luteolin (+ 107%), echinatin (+ 367%), coumestrol Table 3 (continued) Metabolites Metabolic functions References Event sampling Alfalfa organs Exudates Nodules Roots Crowns P values   Tyr Precursor of phenolic compounds, act as antioxidants with mechanisms involv- ing both free radical scavenging and metal chelation Feduraev et al. 2020 < 0.001 < 0.001 < 0.001 < 0.001   Phe Precursor of phenolic compounds, act as antioxidants with mechanisms involv- ing both free radical scavenging and metal chelation Feduraev et al. 2020 0.001 < 0.001 < 0.001 < 0.001   AATot < 0.001 < 0.001 0.07 0.11 Favonoids   Naringenin nod gene inducer Peters et al. 1986 < 0.001 0.01 0.46   Luteolin nod gene inducer, ROS scavenging Peters et al. 1986; Chen et al. 2020 < 0.001 < 0.001 < 0.001   Echinatin nod gene inducer Hartwig et al. 1990 0.000 < 0.001 < 0.001   Coumestrol Phytoestrogen, plant defense response stress, nod gene repressing activity on S. meliloti Fields et al. 2018/ Zuanazzi et al. 1998 0.005 < 0.001 0.00   Formononetin nod gene inducer Dakora et al. 1993 0.018 0.00 < 0.001   Medicarpin Repress nod gene induction in alfalfa roots Zuanazzi et al. 1998 < 0.001 < 0.001 0.31   FlaTot 0.054 < 0.001 < 0.001 331Metabolic and genetic responses to simulated overwintering conditions of alfalfa‑rhizobia… 1 3 (+ 253%) showing as compared to NA (Fig. 3). In roots, only luteolin and echinatin increased with CA and freezing stress, while formononetin and coumestrol decreased when compared to NA. 3.3 Variations in metabolite concentrations related to alfalfa populations in cold acclimated (CA) and deacclimated (RAF) plants Concentrations of various metabolites differed signifi- cantly between alfalfa populations in CA and RAF sam- ples (Figs. 4 and 5 left panels, Supplemental Tables 1–4). In CA plants, the freezing-tolerant population A-TF7 had higher concentrations of total soluble sugars and sucrose in roots and crowns while pinitol and glucose were more abundant only in A-TF7 crowns as compared to the less freezing tolerant A-TF0 (Fig. 4, left panel). Starch was more abundant in roots of A-TF0 than A-TF7. For amino acids, ornithine was the amino acid showing the most striking difference between the two alfalfa populations with higher level in nodules, roots, and crowns of popula- tion A-TF7 when compared to A-TF0. Arginine was the second amino acid with highest level in nodules and in roots of population A-TF7 when compared to A-TF0, fol- lowed by histidine in nodules and lysine in both nodules and roots. GABA concentration was higher in nodules and roots of population A-TF0 compared to A-TF7. Methio- nine concentration was also higher in crowns of popula- tion A-TF0. We also observed higher concentrations of histidine in root exudates and in nodules of population A-TF7. For flavonoids, higher concentration of medicarpin was observed in root exudates and higher concentration of naringenin and luteolin in nodules associated with A-TF0 than those associated with population A-TF7. Fig. 3 Average concentration of metabolites measured in exudates, nodules, roots and crowns of the four alfalfa × strain associations at each sampling event (n = 32). Metabolite concentrations of sugars (mg/g DM), amino acids (µmol/g DM) and flavonoids (µg/g DM) were measured in non-acclimated plants (NA), cold-acclimated (CA) plants, plants exposed to a freezing stress and transferred back to optimal regrowth conditions for 48 h (AFS), and after a 3-weeks regrowth (RAF). Colors represent the log twofold change of either a decrease (red) or an increase (blue) of metabolite concentrations compared to NA plants. Color code is shown at the bottom of the last column of the Fig.  3. Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartic acid; Formo, formononetin, FlaTot, total flavonoids; Gln, glutamine; Glu, glutamic acid; Gly, glycine; His, histidine; Ile, isoleucine; Leu, leucine; Lys, leucine; Lys, lysine; Met, methionine; NSC, non structural carbohydrates; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Tyr, tyrosine; Val, valine; Orn, Ornith- ine; AATot, Total amino acid; SSTot, total soluble sugars; AABA, α-aminobutyric acid; GABA, γ-aminobutyric acid 332 E. D’Amours et al. 1 3 Three weeks after the freezing stress (RAF) concentra- tions of starch in both roots and crowns and raffinose and NSC in crowns were higher in population A-TF7 than in A-TF0 while glucose concentration was higher in A-TF0 roots (Fig. 5 left panel, Supplemental Table 4). Concentra- tions of arginine and ornithine were higher in both nodules and roots of A-TF7 than in A-TF0. Histidine concentra- tion was also higher in nodules associated with population A-TF7. Concentrations of glutamine in roots and aspar- tate and glutamate in both roots and crowns were higher in A-TF7 than in A-TF0. GABA concentration was higher in roots and crowns and alanine and serine in crowns of A-TF0. For flavonoids only a difference in naringenin con- centration in root exudates was found between the two alfalfa populations with more than twice the concentration in A-TF0 than in A-TF7 (Fig. 5). Variations in metabolite concentrations related to alfalfa populations in NA and AFS plants are presented in supplemental Figs. 1 and 2. 3.4 Variations in metabolite concentrations related to S. meliloti strains in cold acclimated (CA) and deacclimated (RAF) plants Differences in the metabolite profiles of alfalfa-rhizobia associations established with freezing-sensitive strain B399 and freezing-tolerant strain NRG34 were observed after cold acclimation (CA). More noticeable variations occurred in nodules than in roots and crowns (Fig. 4 right panel, Fig. 4 Graphic representation of log twofold changes in metabo- lites concentration showing the differential contribution of each alfalfa population (A-TF0 vs A-TF7) and each S. meliloti strain (B399 vs NRG34) to the metabolic changes in CA root exudates, nodules, roots and crowns. The left panel compares the two alfalfa populations with significant higher concentra- tion in A-TF7 on the right to zero line (white) and significant higher concentration in A-TF0 on the left (black). The right panel compares the two rhizo- bial strains with significant higher concentration in response to inoculation with NRG34 on the right to zero line (white) and significant higher concentra- tion in response to B399 on the left (black). Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartic acid; Formo, formon- onetin, FlaTot, total flavonoids, Gln, glutamine; Glu, glutamic acid; Gly, glycine; His, histi- dine; Ile, isoleucine; Leu, leu- cine; Lys, leucine; Lys, lysine; Met, methionine; NSC, non structural carbohydrates; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Tyr, tyrosine; Val, valine; Orn, orni- thine, AATot, Total amino acid; SSTot, total soluble sugars; AABA, α-aminobutyric acid; GABA, γ-aminobutyric acid 333Metabolic and genetic responses to simulated overwintering conditions of alfalfa‑rhizobia… 1 3 Supplemental Table 2). For non-structural carbohydrates, pinitol concentrations in nodules and crowns as well as glu- cose and fructose concentrations were higher in nodules of plants inoculated with strain B399 than with NRG34. Dif- ferences in cold-induced changes in concentrations of spe- cific free amino acids were also noticeable between strains. Higher concentrations of AABA, aspartate, histidine and threonine in root exudates, AABA and tyrosine in nodules, and GABA and glutamine in roots were detected in the symbiotic association with strain B399. Conversely, higher concentrations of methionine, arginine, and histidine were observed in nodules of alfalfa inoculated with strain NRG34 than in those inoculated with strain B399. As for flavonoid concentrations, luteolin and naringenin concentrations were higher in nodules of plants inoculated with strain NRG34 than in those associated with strain B399. After three weeks of regrowth after freezing stress (RAF), higher concentrations of pinitol were still detected in crowns and nodules of alfalfa inoculated with strain B399 (Fig. 5 right panel, Supplemental Table 4). For amino acids, con- centrations of proline, aspartate, and glycine were higher in nodules of alfalfa inoculated with strain B399 than in nodules of alfalfa inoculated with strain NRG34 (Fig. 5 right panel). Arginine and phenylalanine were alternatively more abundant in nodules of alfalfa inoculated with strain NRG34 than in nodules of alfalfa inoculated with strain B399. Total flavonoid and formononetin concentrations were higher in roots of alfalfa inoculated with strain NRG34 than in those inoculated with strain B399 as well as concentration of lute- olin in nodules (Fig. 5). Variations in metabolite concentra- tions related to S. meliloti strains in NA and AFS plants are presented in supplemental Figs. 1 and 2. Fig. 5 Graphic representation of log twofold changes in metabo- lites concentration showing the differential contribution of each alfalfa population (A-TF0 vs A-TF7) and each S. meliloti strain (B399 vs NRG34) to the metabolic changes in RAF root exudates, nodules, roots and crowns. The left panel compares the two alfalfa populations with significant higher concentra- tion in ATF-7 on the right to zero line (white) and significant higher concentration in ATF-0 on the left (black). The right panel compares the two rhizo- bial strains with significant higher concentration in response to inoculation with NRG34 on the right to zero line (white) and significant higher concentra- tion in response to B399 on the left (black). Ala, alanine; Arg, arginine; Asn, asparagine; Asp, aspartic acid; Formo, formon- onetin, FlaTot, total flavonoids, Gln, glutamine; Glu, glutamic acid; Gly, glycine; His, histi- dine; Ile, isoleucine; Leu, leu- cine; Lys, leucine; Lys, lysine; Met, methionine; NSC, non structural carbohydrates; Phe, phenylalanine; Pro, proline; Ser, serine; Thr, threonine; Tyr, tyrosine; Val, valine; Orn, orni- thine, AATot, Total amino acid; SSTot, total soluble sugars; AABA, α-aminobutyric acid; GABA, γ-aminobutyric acid 334 E. D’Amours et al. 1 3 Few interactions between the alfalfa population and the S. meliloti strain on metabolites concentrations were observed in nodules and crowns in response to CA, AFS and RAF. Higher concentrations of fructose, alanine, AABA and medi- carpin for the association A-TF0 × B399 were noticeable cases (Supplemental Tables 2–4) as well as variations in the concentration of several free amino acids in root exudates in AFS (Supplemental Table 3). 3.5 Gene expression in crowns and roots in response to sampling event, alfalfa populations and rhizobial strains The relative expression of all the genes of interest (GOI) measured in crowns was impacted by the sampling events (Table  4). When expression profiles were compared to those in NA plants, the most striking modifications of gene expression were observed in response to CA with the up- regulation of galactinol synthase (GaS) and K3-dehydrin, and the down-regulation of sucrose synthase (SuSy) expres- sions (Table 4). The expression levels of these three genes returned to a level comparable to NA plants at the sub- sequent samplings (AFS and RAF). As observed in AFS crowns, freezing stress induced the down-regulation of SPS gene (Table 4). When looking at the effect of alfalfa popu- lations on gene expression, the relative expression of SuSy was higher in A-TF0 than in A-TF7 at the CA and RAF samplings (Figs. 6a and b, Supplemental Table 5). Gene expression of SuSy in crowns did not differ according to strain inoculation (Fig. 6). In roots, the expression of genes coding for enzymes involved in the biosynthetic pathways of flavonoids and sec- ondary metabolites differed according to sampling events (cinnamic acid 4-hydroxylase (C4H; and 2,7,4′-trihydroxy- isoflavanone 4′-O-methyltransferase (IOMT; P = 0.021). CA and AFS roots show that cold acclimation and freezing stress induced slight but significant up-regulation of C4H gene and down-regulation of the IOMT gene (Table 4). The gene expression of both genes in roots were lower in RAF than in NA roots. The relative expression of C4H was higher in A-TF0 than in A-TF7 in the NA and RAF samplings (Figs. 7a and b, Supplemental Table 5) while the expres- sion of IOMT was higher in A-TF0 than in A-TF7 in CA plants (Fig. 7c, Supplemental Table 5). We also observed an interaction between alfalfa population and S. meliloti strains on the relative expression of IOMT in AFS sampling (Fig. 8d and Supplemental Table 5). Relative expression of IOMT was 77% higher for alfalfa population A-TF0 com- bined with strain B399 when compared to alfalfa population A-TF7 combined with the same strain and both association of alfalfa population inoculated with NRG34 were interme- diate and were not different (Fig. 7d). 4 Discussion The study of winter stress tolerance in legumes needs to consider the complete symbiotic system including both plants and bacteria since these two partners are differen- tially affected by stress conditions (Hawkins and Oresnik 2022). Here, we compared the regrowth after a freezing stress of four different associations of alfalfa populations and S. meliloti strains and observed up to 35% yield dif- ferences between the best (A-TF7 × NRG34) and the worst (A-TF0 × B399) association (Fig. 2a). Both partners were shown to contribute to the recovery after exposure to a freezing stress with a 12% difference that was explained by a population effect (A-TF0 vs A-TF7) and 17% that was attributable to a strain effect (B399 vs NRG34). To under- stand the contribution of each partner to the better regrowth performance of an association after freezing, a thorough investigation of the mechanisms at play was undertaken by monitoring metabolites and genes having major roles in cold acclimation, freezing tolerance, and those involved Table 4. Relative expression levels of seven gene of interest (GOI) in crowns (five GOI) and roots (two GOI) of four associations combining two alfalfa populations A-TF0 and A-TF7 inoculated with two S. meliloti strains, B399 and NRG34 Alfalfa plants grown under controlled conditions were sampled at four sampling events: before cold-accli- mated (NA), after cold-acclimation (CA), after freezing stress (AFS) and after three weeks of regrowth after freezing (RAF). Relative expression levels and analysis of variance (p-value for n = 32) of the effects of sampling event are presented Alfalfa organs Gene Gene annotation Relative expression levels by sampling event Crowns NA CA AFS RAF P value GaS Galactinol synthase 0.34 46.93 2.13 0.81 < 0.001 SPS Sucrose phosphate synthase 1.23 1.60 0.64 0.99 < 0.001 Susy Sucrose synthase 1.93 0.37 1.63 1.52 < 0.001 K3-dehydrin K3-dehydrin 0.20 33.79 0.97 0.87 < 0.001 Roots C4H Cinamic acid 4-hydoxylase 1.10 1.36 1.42 0.88 0.018 IOMT Isoflavone-O-methytransferase 1.92 0.88 1.20 0.93 0.021 335Metabolic and genetic responses to simulated overwintering conditions of alfalfa‑rhizobia… 1 3 in the crosstalk between alfalfa and its symbiotic partner. The metabolomic study of roots, crowns and, more spe- cifically, of nodules, revealed profound changes in these organs, switching from a sink to support cold acclimation to a source of reserves enabling regrowth after deacclima- tion. Our approach tracing molecular changes separately in alfalfa populations and rhizobial strains provided a novel perspective on their respective contribution to the vigor of spring regrowth of alfalfa. It also allowed the identification of molecular traits potentially conferring superior productiv- ity of alfalfa that was exposed to a sublethal freezing stress. 4.1 Effects of cold acclimation, freezing stress and deacclimation on metabolites and gene expression 4.1.1 Sugars In response to cold acclimation we noted an important increase of soluble sugars concentrations in nodules, roots and crowns of alfalfa. Those observations are concord- ant with similar results reported of the accumulation of soluble sugars in perennial organs of alfalfa in response to low temperature and photoperiod to provide energy for plant overwintering and regrowth (Fig. 3; Bertrand et al. 2017; Castonguay et al. 2011). The separate biochemical analysis of nodules allowed us to better understand the pivotal role of these belowground organs. For instance, our results highlight the importance of nodules as large carbon sinks during cold acclimation leading not only to the accumulation of sucrose, stachyose, raffinose, and starch in these organs, but also to a significant increase in their biomass after exposure to cold temperatures (CA) (Fig. 2). The allocation of photosynthates into nodules has been linked with the translocation of sugars from other plant organs such as leaves and roots in response to abi- otic stress (Bertrand et al. 2016). The large hydrolysis of starch in roots and crowns likely contributed to the new carbohydrates input, including starch, in nodules (Fig. 3). An accumulation of starch grains, amyloplast and ole- osomes have been observed in perennial beach pea nod- ules before winter (Chinnasamy and Bal 2003; Gurusamy et al. 2000) and these storage compounds were proposed to serve as energy source for metabolic activities dur- ing winter dormancy of nodules that lasts for prolonged periods under arctic/subarctic conditions. In our study, the concentration of total sugars in nodules decreased to levels below those observed in NA nodules upon the return of alfalfa to optimal regrowth conditions (RAF). This observation makes a compelling case for soluble sug- ars and starch as key contributors to the nodules survival during overwintering period and as a source of energy to resume N fixation and ultimately support alfalfa regrowth in the spring (D’Amours et al. 2022). An important accu- mulation of sucrose was observed in nodules, roots and crowns in response to cold acclimation while its concen- tration decreased in roots exudates with sampling events. Sucrose is a recognized cryoprotectant that can stabilize freeze-dehydrated cells by interacting with cell membrane (Tarkowski and Van den Ende 2015). In alfalfa, a close Fig. 6 Relative expression in alfalfa crowns of sucrose synthase (SuSy) in (a) cold-acclimated (CA) and (b) 48 h after freezing stress in two alfalfa populations contrasted in their freezing tolerance lev- els (A-TF0 and A-TF7) analyzed by RT-qPCR. Error bars represent the SEM, n = 16. Significant differences between populations at each event, determined by a t-test, are indicated with the following levels of probability: * P ≤ 0.05, ** P < 0.01, *** 336 E. D’Amours et al. 1 3 relationship between sucrose accumulation and superior freezing tolerance has been demonstrated (Castonguay et al. 2011). The decrease in sucrose at regrowth after freezing (RAF) could be partly explained by the quick up-regulation of sucrose synthase (Susy) in nodules after freezing stress to cleave sucrose into fructose and glu- cose and to provide succinate and malate to the bacte- roids, directly feeding into their tricarboxylic acid cycle (TCA) cycle (Geddes and Oresnik 2014; Liu et al. 2018). Interestingly, fructose concentration increased more than three fold in nodules 48 h after freezing stress. Fructose has a high capacity for scavenging superoxide and has been shown to be involved in antioxidative protection in pea under chilling stress (Bogdanović et al. 2008). The marked increase in RFO in root exudates, roots, crowns and nodules in response to CA confirm their importance in the freezing tolerance of alfalfa. Concentrations of RFO are intrinsically linked with the level of freezing toler- ance of alfalfa due to their cryoprotective actions (Ber- trand et al. 2017). Furthermore, RFO are known to support the growth and survival of symbiotic N-fixing bacteria (e.g. S. meliloti) in the rhizosphere of germinating seeds and alfalfa seedlings (Bringhurst et al. 2001). The larger concentration of RFO in nodules than in other perennial Fig. 7 Relative expression in roots of cinnamic acid 4-hydroxylase (C4H) in non-acclimated NA alfalfa (a) and in RAF alfalfa regrowth (b). Relative expression of isoflavone O-methyltransferase (IOMT) (c) in two CA alfalfa populations contrasted in their freezing toler- ance levels (A-TF0 and A-TF7) and of four associations combining two alfalfa populations (grey scale) A-TF0 (in grey) and A-TF7 (in white) inoculated two S. meliloti strains (patterns), B399 (lined) and NRG34 (dotted), sampled 48  h after freezing (AFS) (d) Error bars represent the SEM for n = 16. Significant differences between popu- lations at each event, determined by a t-test, are indicated with the following levels of probability: * P ≤ 0.05, ** P < 0.01, *** P < 0.001. Different letters indicate significant differences in relative expression of IOMT due to pop × strains interactions at P ≤ 0.05 (n = 8) 337Metabolic and genetic responses to simulated overwintering conditions of alfalfa‑rhizobia… 1 3 organs (crowns and roots) highlights the importance of the investment of host plant into nodules protection to maxi- mize their survival and allow for a quick return of N fixa- tion in the spring to support alfalfa regrowth. 4.1.2 Amino acids Amino acids constitute an important source of organic N for spring regrowth and several amino acids possess osmo- protectant and cryoprotectant attributes that help stabilize plant cells under stress (Bertrand et al. 2017, 2020a; Dhont et al. 2006). Cold acclimation has been frequently reported to induce an increase in total amino acids concentration in roots and crowns of alfalfa plants grown under non-limiting nitrogen conditions (Bertrand et al. 2020a; Castonguay et al. 2011; Dhont et al. 2006). In the present study, total amino acids (AATot) did not increase in nodules, roots and crowns in response to cold acclimation which could be due to the fact that the only source of N was provided by the symbi- otic N fixation. The conditions of cold acclimation that were used in this experiment (low temperature and short photo- period) were far from optimal for symbiotic N fixation which is most effective at 25 °C (Alexandre and Oliveira 2013). These conditions likely restricted amino acids synthesis and accumulation, as opposed to what is observed when non- limiting inorganic N source is used with cold acclimated alfalfa plants as in Castonguay et al. (2011). Amino acid concentrations were almost four fold higher in nodules than in roots and crowns, and this was a constant effect at all sampling events, showing the importance of resource invest- ment in nodules by host plants (Fig. 3). Asparagine, which is recognized as the primary N fixed compound as well as the principal amino acid transported in xylem in legume species with indeterminate nodules such as alfalfa, was the most abundant amino acid in all samples for non-acclimated plants and represented up to 80% of the pool of free amino acids (Table 3; Bertrand et al. 2016; Sulieman et al. 2010). Its progressive decrease in concentration at each sampling event (Fig. 3) confirms the slowdown of N fixation under CA as well as the transformation of this major pool of N compounds into other amino acids involved specifically in stress tolerance. For instance, ornithine and arginine as well as their precursor glutamate increased in response to CA and are known to be precursors of polyamines which confer plant resistance to various abiotic stresses (Anwar et al. 2018). Ornithine has also been reported as a signal and regulatory molecule (Majumdar et al. 2016), which could explain why its concentration, while changing significantly in response to CA and freezing, remained low as compared to arginine. Arginine, with its high N to C ratio is considered a stor- age compound from which N could be readily incorporated into other N-compounds essential for active regrowth, as previously reported in overwintering alfalfa (Castonguay et al. 2011; Dhont et al. 2006). While proline accumula- tion in responses to low temperatures has previously been observed in alfalfa (Bertrand et al. 2020a; Castonguay et al. 2011; Liu et al. 2019), its direct involvement in the acquisi- tion of freezing tolerance remains unclear. In the present study, proline concentration did not increase in response to decreased temperatures suggesting that proline synthesis is regulated independently of the glutamate-ornithine-arginine pathway (Majumdar et al. 2016). On the other hand, the lack of proline increase could indicate that low tempera- tures stress increased the degradation of proline to provide a source of carbon and nitrogen to the bacteroid, thereby supporting recent studies suggesting that proline metabo- lism may play an essential role of energy transfer in the legume-Rhizobium symbiosis under stress (Sabbioni and Forlani 2022). We also noted an increase of alanine, serine and glycine with cold acclimation in nodules and an increase of those three amino acids in roots and crowns 48 h after the freezing stress. Alanine accumulation is recognized as an universal first stress signal in a wide variety of organ- isms including plants (Table 3, Ben-Izhak Monselise et al. 2003). Moreover, it has been recently proposed that alanine could play an important role in the symbiosis by sustain- ing bacteroid metabolism under oxygen limitation (Schulte et al. 2021). Serine/glycine metabolism was shown to be an important key player in biochemical adaptation to envi- ronmental stress by regulation of intracellular redox, pH regulation and energy levels (Igamberdiev and Kleczkowski 2018). Another interesting observation is a sharp increase of methionine, tyrosine, phenylalanine, threonine, lysine, iso- leucine and leucine concentrations 48 h after freezing stress (Fig. 3, AFS). Methionine synthesis, which is provided by the host plant to the nodules, has been reported to be essen- tial for efficient nodulation by various rhizobia (Barra et al. 2006). Tyrosine and phenylalanine are aromatic amino acid produced by the shikimate pathway which require phospho- enolpyruvate as substrate (Dunn 2014). Tyrosine is essential for the nodule formation and can be used by the bacteroid as source of carbon and nitrogen (Saha et al. 2016). Both tyrosine and phenylalanine can act as substrates for the phenylpropanoids biosynthesis pathway and were found in root exudates (Feduraev et al. 2020). Other protein-bounded amino acids have been reported to increase in response to cold and frost stress in A. thaliana and Camellia sinensis and, while the authors suggested a role for these amino acids in freezing stress acquisition, the mechanism involved would need further investigations (Hildebrandt 2018; Samarina et al. 2020). 4.1.3 Flavonoids Flavonoids are crucial signaling molecules that play an essential role in the Rhizobium-legume symbiosis as 338 E. D’Amours et al. 1 3 chemoattractant and nod gene inducers. Flavonoids are excreted by the plant in the rhizosphere to modulate com- munications with microorganisms, either to ensure protec- tion against pathogens or to attract beneficial microbes (Fal- cone Ferreyra et al. 2012). Only few and specific flavonoids excreted by the legume-host will activate the expression of a group of bacterial nod genes, leading to the synthesis of the Nod factor (lipochitooligosaccharides), essential for initiat- ing nodules formation and to maintain symbiotic activity (Reviewed in Liu and Murray 2016). Following a stress, the crosstalk between the host plants and microorganisms is modified to face the new conditions and the measurement of flavonoid concentrations in root exudates could give infor- mation on the signals exchanged between the partners. An increase of formononetin and medicarpin was observed in root exudates after CA (Fig. 3). Formononetin is the most abundant flavonoid in alfalfa while its derivative medi- carpin is the second most abundant (Gifford et al. 2018). Formononetin has been reported to be involved in nodules organogenesis (Mathesius 2001) and in the activation of nod gene transcription in Rhizobium meliloti (Table 3, Dakora et al. 1993). Thus, the CA-induced increase of formononetin concentration in roots exudates could be linked to the de novo nodules synthesis necessary for the plant to acclimate to cold. On the other hand, formononetin could have been released directly from stressed roots to regulate germination of pathogenic fungi (Tsai and Philipps 1991). Formononetin is also a precursor of medicarpin which was shown to exert an inhibitory effect on incompatible bacterial strains and to repress nod gene transcription in alfalfa roots (Hartwig et al. 1990). The increase in medicarpin concentration in root exudates under CA indicates that protection mechanisms against pathogens are also in place. Those mechanisms play an important role in the host range rhizobia specificity of the symbiotic association since pathogenic bacteria can pro- duce similar signaling molecules to facilitate their invasion of the host plant (Wang et al. 2018). The triggering of pro- tection mechanisms is supported by the observation of large increases in echinatin, coumestrol and medicarpin concomi- tant with decrease in formononetin in nodules during the experiment. Echinatin is a potent antagonist of Gram + bac- teria with antifungal and antibacterial activity (Dong and Song 2020) while coumestrol has been shown to be induced by fungal infection in alfalfa (Table 3, Fields et al. 2018). Increase in medicarpin and decrease in formononetin in CA nodules could have had an inhibitory effect on the nod gene activity and the cellular division of S. meliloti (Zhang et al. 2009). Flavonoids have also been reported to provide stress protection against UV light, drought, salinity, freezing and to act as scavengers of free radicals such as reactive oxygen species (Baskar et al. 2018; Laoué et al. 2022; Schulz et al. 2016; Sharma et al. 2019). Recently, it has been reported that genes involved in the phenylpropanoids pathways are regulated by low temperatures in alfalfa (Liu et al. 2022). Echinatin could potently increase plant tolerance against several biotic and abiotic stresses (Sharma et al. 2019; Tri- pathi et al. 2016). Luteolin is essential for the nodulation process by controlling the expression of nodABC during the development of the symbiosis between rhizobia and alfalfa and has also been reported as an excellent free-radical scav- enger to protect again oxidative stress (Chen et al. 2020), to enhance starch hydrolysis and soluble sugars accumulation (El-Shafey and AbdElgawad 2012), and to be involved in enhanced salt stress tolerance (Song et al. 2022). 4.1.4 COR genes expression The expression of the following COR genes of carbohydrate synthetic pathways were affected by the exposure to cold temperature in alfalfa crowns: galactinol synthase (GaS), sucrose synthase (Susy), and sucrose phosphatase synthase (SPS) (Table 4). For instance, the transcript level of GaS, a key enzyme catalyzing the first step of RFO biosynthesis, increased markedly in response to CA, in accordance with the sharp increase in raffinose and stachyose concentra- tions that we observed in crowns, and in accordance with previous reports (Bertrand et al. 2016; Cunningham et al. 2003; Liu et al. 2019; Xu et al. 2020). Consistently with previous observations in alfalfa (Bertrand et al. 2017), the gene expression of Susy declined in response to CA and, since Susy is responsible for the cleavage of sucrose into glucose and fructose, its down-regulation along with the up regulation of SPS resulted in sucrose accumulation that we observed in storage organs in response to CA. The Susy progressively returned to the non-acclimated level at the next sampling events which is consistent with the progres- sive decrease in sucrose concentration in crowns, roots and nodules after the freezing stress. Furthermore, it was sug- gested that Susy activity might be essential for N fixation in root nodules (Gordon et al. 1999). The quick induction of Susy expression that we observed 48 h after the freezing stress concurs with an important role for Susy in the deac- climation process as it would positively activate the reprise of biological N fixation in post-freezing nodules (Gordon et al. 1999). A strong up-regulation of K3-dehydrin, coding for an osmotic-stress protein was also observed in response to cold acclimation. The K3-dehydrin is known to be asso- ciated with freezing tolerance in cold-acclimated alfalfa trough membrane-stabilizing effect (Bertrand et al. 2016; Dubé et al. 2013; Xu et al. 2020). The transcript levels of K3-dehydrin did not vary between alfalfa-rhizobia associa- tions and, as such, the up-regulation of K3-dehydrin seems to be part of a general protective process of cold acclimation in alfalfa. In general, our results confirmed previous studies showing that COR genes involved in carbohydrate metabolic pathways are crucial for cold acclimation and freezing stress 339Metabolic and genetic responses to simulated overwintering conditions of alfalfa‑rhizobia… 1 3 tolerance and that they are also key actors in the deacclima- tion process. It would be interesting to investigate the level of expression of those genes in nodules and in response to cold/freezing/deacclimation processes to better understand their role in the recovery of the symbiotic association after freezing. 4.2 Contribution of each partner to the increased freezing tolerance of the association 4.2.1 Sugars and amino acids We monitored the independent effects of populations and strains on metabolic changes that occurred during cold acclimation (Fig. 4) and deacclimation (Fig. 5) to better understand their respective contribution to the recovery of the symbiotic association after a freezing stress. In gen- eral, metabolites concentration in crowns significantly dif- fered mainly in response to alfalfa populations while strains affected mainly nodules metabolites. For instance, RFOs and sucrose increased in alfalfa crowns in response to CA (Fig. 3) but only sucrose was more abundant in freezing- tolerant population A-TF7 as compared to A-TF0, indicating that under conditions of the current study the accumulation of sucrose was more determinant for regrowth after freezing than RFO. Our observations concur with previous reports on cold-induced accumulation of cryoprotective sucrose and RFO in alfalfa populations recurrently selected for superior freezing tolerance (Castonguay et al. 2011). Interestingly, the accumulation of glucose and fructose in CA nodules in response to the inoculation with freeze-sensitive strain B399 seems to indicate that the strain has an effect on the hydrolysis of sucrose by the plant. In response to CA, both symbiotic partners of the most freezing tolerant associa- tion (A-TF7 × NRG34) induced an increase in arginine and histidine, confirming the importance of a strategy for N storage during cold acclimation of alfalfa. The response to deacclimation (RAF) also shows a greater remobilisation of N through arginine, ornithine, and histidine in A-TF7 as compared to A-TF0 as well as through arginine for NRG34. During cold acclimation and deacclimation, both symbiotic partners of the most freezing sensitive associa- tion (A-TF0 × B399) showed higher accumulation of osmo- protectants and scavenging reacting oxygen species amino acids like GABA, serine, alanine and proline in crowns and proline in nodules (Figs. 4 and 5). These reactions could indicate that these plants suffered more damage by freezing that triggered reparation mechanisms such as ROS scaveng- ing. On the other hand, higher contents of cryoprotective substances after freezing stress could be beneficial to reduce the risks of damages caused by abrupt freeze–thaw episode in early autumn or spring (Xu et al. 2020). 4.2.2 Flavonoids and regulation of C4H and IOMT genes Production of flavonoids by the phenylpropanoid pathway was influenced by both symbiotic partners at different sam- pling events. With cold acclimation we found a higher con- centration of the nodulation repressor medicarpin in nod- ules of the less freezing tolerant association (A-TF0 × B399) when compared to the most freezing tolerant association (A-TF7 × NRG34) (Supplemental Table  2). The inhibi- tory and antimicrobial effect of medicarpin could be linked with the higher incidence of freezing damage and necrosis of nodules of those plants as observed in D’Amours et al. (2022). The up-regulation of the cinnamic acid 4-hydroxy- lase (C4H) gene was observed in roots in response to CA, which is coherent with the observed concentration increases of the flavonoid precursor tyrosine as well as of total flavo- noids concentrations in roots and nodules observed at this sampling point. Low temperature has been reported as the main factor responsible for the accumulation of flavonoids with antioxidant properties, either through induced expres- sion of the encoding gene or increase enzymatic activity of C4H (He et al. 2022). However, as a major rate-limiting enzyme in the phenylpropanoid biosynthesis that separates the pinocembrin pathway from the lignin/monolignol syn- thesis pathway, the up-regulation of C4H could also be linked to the accumulation of other secondary metabolites including lignin (Gifford et al. 2018). In alfalfa, isoflavone O-methyltransferase (IOMT) catalyses the reaction leading to 7-O-methyl daidzein, the precursor of formononetin and, further down the pathway to medicarpin (He et al. 1998). The IOMT was down-regulated with CA and its expression was lower in freezing-tolerant population A-TF7 than in A-TF0. This is consistent with the decrease of formonon- etin observed in both roots and nodules at CA sampling and with the higher concentration of medicarpin in root exu- dates of A-TF0. A lesser repression of expression of the gene involved in synthesis of formononetin and medicarpin by cold and freezing stress could be associated with less freez- ing tolerance. Upon return to conditions allowing regrowth, at RAF, the expression of IOMT further increased. A larger formononetin concentration in roots of plants inoculated with NRG34 at RAF suggests that strain-induced signals are more active during regrowth after freezing in the specific A-TF7 × NRG34 association. However further investigations are necessary to better understand the role of flavonoids in plant stress tolerance (He et al. 2022). 5 Conclusion This study highlights the importance to consider the rhizo- bial symbiosis in strategies aimed at improving stress toler- ance in legumes. Rhizobia and plants play complementary 340 E. D’Amours et al. 1 3 roles to ensure enhanced regrowth after freezing. It was shown that root nodules, while accumulating a large pool of free amino acids and carbohydrates during cold acclima- tion, turned into a source of reserves that enable regrowth in spring after deacclimation. The study also identified meta- bolic and genetic traits that confer superior yields to alfalfa populations exposed to a sublethal freezing stress such as the accumulation and remobilization of N storage amino acids during cold acclimation and deacclimation, respec- tively. Choosing the right partners when applying rhizobial inoculants may contribute in improving the stress tolerance of alfalfa. Supplementary Information The online version contains supplemen- tary material available at https:// doi. org/ 10. 1007/ s13199- 023- 00939-3. Acknowledgements This work was supported by a grant program of Agriculture and Agri-Food Canada. The first author was financially supported by a scholarship from the Fonds de Recherche Québécois Nature et Technologies. The authors sincerely thank Josée Bourassa, Josée Michaud and Sandra Delaney for their technical assistance and Dr Yves Castonguay for his thorough revision of the manuscript. We also thank, Dr Nicolas Ayub from Instituto de Genética “Edwald Alfredo Favret”, INTA who kindly provided us the commercial S. meliloti strain, B399. Author contributions All authors contributed to the study conception and design. Material preparation, data collection and analysis were per- formed by Emmanuelle D’Amours, Annick Bertrand and Jean Cloutier. The first draft of the manuscript was written by Emmanuelle D’Amours and all authors revised the previous versions of the manuscript. All authors read and approved the final manuscript. Funding Open Access funding provided by Agriculture & Agri-Food Canada. This work was supported by a grant program of Agriculture and Agri-Food Canada. The first author was financially supported by scholarship from the Fonds de Recherche Québécois Nature et Technologies. Data availability The datasets generated during and analyzed during the current study are available from the corresponding author on rea- sonable request. Declarations Competing interests The authors have no relevant financial or non- financial interests to disclose. 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References Abd Elbar OH, Elkelish A, Niedbała G, Farag R, Wojciechowski T, Mukherjee S, Abou-Hadid AF, El-Hennawy HM, Abou El-Yazied A, Abd El-Gawad HG, Azab E, Gobouri AA, El-Sawy AM, Bon- dok A, Ibrahim MFM (2021) Protective effect of γ-aminobutyric acid against chilling stress during reproductive stage in tomato plants through modulation of sugar metabolism, chloroplast integ- rity, and antioxidative defense systems. Front Plant Sci 12:663750. https:// doi. org/ 10. 3389/ fpls. 2021. 663750 Alcázar R, Cuevas JC, Planas J, Zarza X, Bortolotti C, Carrasco P, Salinas J, Tiburcio AF, Altabella T (2011) Integration of poly- amines in the cold acclimation response. Plant Sci 180:31–38. https:// doi. org/ 10. 1016/j. plant sci. 2010. 07. 022 Alexandre A, Oliveira S (2013) Response to temperature stress in rhizobia. Crit Rev Microbiol 39(3):219–228. https:// doi. org/ 10. 3109/ 10408 41X. 2012. 702097 Anwar A, She M, Wang K, Riaz B, Ye X (2018) Biological roles of ornithine aminotransferase (OAT) in plant stress toler- ance: present progress and future perspectives. Int J Mol Sci 19(11):3681. https:// doi. org/ 10. 3390/ ijms1 911368 Barra L, Fontenelle C, Ermel G, Trautwetter A, Walker GC, Blanco C (2006) Interrelations between glycine betaine catabolism and methionine biosynthesis in Sinorhizobium meliloti strain 102F34. J Bacteriol 188(20):7195–7204. https:// doi. org/ 10. 1128/ JB. 00208- 06 Baskar V, Rajendran V, Ramalingam S (2018) Flavonoids (antioxi- dants systems) in higher plants and their response to stresses. In: Gupta D, Palma J, Corpas F (eds) Antioxidants and antioxidant enzymes in higher plants. Springer, Cham, pp 253–268. https:// doi. org/ 10. 1007/ 978-3- 319- 75088-0_ 12 Bélanger G, Rochette P, Castonguay Y, Bootsma A, Mongrain D, Ryan DAJ (2002) Climate change and winter survival of peren- nial forage crops in eastern Canada. Agron J 94:1120–1130. https:// doi. org/ 10. 2134/ agron j2002. 1120 Bélanger G, Castonguay Y, Bertrand A, Dhont C, Rochette P, Couture L, Drapeau R, Mongrains D, Chalifour FP, Michaud R (2006) Winter damage to perennial forage crops in eastern Canada: causes, mitigation, and prediction. Can J Plant Sci 86:33–47. https:// doi. org/ 10. 4141/ P04- 171 Ben-Izhak Monselise E, Parola AH, Kost D (2003) Low-frequency electromagnetic fields induce a stress effect upon higher plants, as evident by the universal stress signal, alanine. Biochem Bio- phys Res Commun 302(2):427–434. https:// doi. org/ 10. 1016/ S0006- 291X(03) 00194-3 Bertrand A, Prévost D, Bigras FJ, Castonguay Y (2007) Elevated atmospheric CO2 and strain of rhizobium alter freezing toler- ance and cold-induced molecular changes in alfalfa (Medicago sativa L.). Ann Bot 99:275–284. https:// doi. org/ 10. 1093/ aob/ mcl254 Bertrand A, Prévost D, Juge C, Chalifour FP (2011) Impact of elevated CO2 on carbohydrate and ureide concentrations in soybean inocu- lated with different strains of Bradyrhizobium japonicum. Botany 89(7):481–490. https:// doi. org/ 10. 1139/ b11- 034 Bertrand A, Bipfubusa M, Dhont C, Chalifour FP, Drouin P, Beau- champ CJ (2016) Rhizobial strains exert a major effect on the amino acid composition of alfalfa nodules under NaCl stress. Plant Physiol Biochem 108:344–352. https:// doi. org/ 10. 1016/j. plaphy. 2016. 08. 002 Bertrand A, Bipfubusa M, Claessens A, Rocher S, Castonguay Y (2017) Effect of photoperiod prior to cold acclimation on freez- ing tolerance and carbohydrate metabolism in alfalfa (Medicago sativa L.). Plant Sci 264:122–128. https:// doi. org/ 10. 1016/j. plant sci. 2017. 09. 003 https://doi.org/10.1007/s13199-023-00939-3 http://creativecommons.org/licenses/by/4.0/ https://doi.org/10.3389/fpls.2021.663750 https://doi.org/10.1016/j.plantsci.2010.07.022 https://doi.org/10.3109/1040841X.2012.702097 https://doi.org/10.3109/1040841X.2012.702097 https://doi.org/10.3390/ijms1911368 https://doi.org/10.1128/JB.00208-06 https://doi.org/10.1128/JB.00208-06 https://doi.org/10.1007/978-3-319-75088-0_12 https://doi.org/10.1007/978-3-319-75088-0_12 https://doi.org/10.2134/agronj2002.1120 https://doi.org/10.4141/P04-171 https://doi.org/10.1016/S0006-291X(03)00194-3 https://doi.org/10.1016/S0006-291X(03)00194-3 https://doi.org/10.1093/aob/mcl254 https://doi.org/10.1093/aob/mcl254 https://doi.org/10.1139/b11-034 https://doi.org/10.1016/j.plaphy.2016.08.002 https://doi.org/10.1016/j.plaphy.2016.08.002 https://doi.org/10.1016/j.plantsci.2017.09.003 https://doi.org/10.1016/j.plantsci.2017.09.003 341Metabolic and genetic responses to simulated overwintering conditions of alfalfa‑rhizobia… 1 3 Bertrand A, Gatzke C, Bipfubusa M, Lévesque V, Chalifour FP, Claes- sens A, Rocher S, Tremblay GF, Beauchamp CJ (2020b) Physi- ological and biochemical responses to salt stress of alfalfa popula- tions selected for salinity tolerance and grown in symbiosis with salt-tolerant rhizobium. Agronomy 10(4):569. https:// doi. org/ 10. 3390/ agron omy10 040569 Bertrand A, Claessens A, Bourassa J, Rocher S, Baron V (2020a) A whole-plant screening test to select freezing-tolerant and low- dormant genotypes. In: Hincha DK, Zuther E (Eds), Plant cold acclimation, methods and protocols. Methods in molecular biol- ogy 2156, 2nd edition, Humana Press, Springer Science+Business Media, LLC, part of Springer Nature 2020a, New York pp 53–60 Bogdanović J, Mojović M, Milosavić N, Mitrović A, Vucinić Z, Spasojević I (2008) Role of fructose in the adaptation of plants to cold-induced oxidative stress. Eur Biophys J 37(7):1241–1246. https:// doi. org/ 10. 1007/ s00249- 008- 0260-9 Bringhurst RM, Cardon ZG, Gage DJ (2001) Galactosides in the rhizo- sphere: utilization by Sinorhizobium meliloti and development of a biosensor. Proc Natl Acad Sci USA 98(8):4540–4545. https:// doi. org/ 10. 1073/ pnas. 07137 5898 Castonguay Y, Laberge S, Brummer EC, Volenec JJ (2006) Alfalfa winter hardiness: a research retrospective and integrated perspec- tive. Adv Agron 90:203–265. https:// doi. org/ 10. 1016/ S0065- 2113(06) 90006-6 Castonguay Y, Michaud R, Nadeau P, Bertrand A (2009) An indoor screening method for improvement of freezing tolerance in alfalfa. Crop Sci 49:809–818. https:// doi. org/ 10. 2135/ crops ci2008. 09. 0539 Castonguay Y, Bertrand A, Michaud R, Laberge S (2011) Cold-induced biochemical and molecular changes in alfalfa populations selec- tively improved for freezing tolerance. Crop Sci 51:2132–2144. https:// doi. org/ 10. 2135/ crops ci2011. 02. 0060 Castonguay Y, Michaud J, Dubé M (2015) Reference genes for RT- qPCR analysis of environmentally and developmentally regulated gene expression in alfalfa. Am J Plant Sci 06:132–143. https:// doi. org/ 10. 4236/ ajps. 2015. 61015 Castonguay Y, Rocher S, Bertrand A, Michaud J (2020) Identification of transcripts associated with the acquisition of superior freezing tolerance in recurrently-selected populations of alfalfa. Euphytica 216:27. https:// doi. org/ 10. 1007/ s10681- 020- 2559-2 Chen HI, Hu WS, Hung MY, Ou HC, Huang SH, Hsu PT, Day CH, Lin KH, Viswanadha VP, Kuo WW, Huang CY (2020) Protec- tive effects of luteolin against oxidative stress and mitochondrial dysfunction in endothelial cells. Nutr Metab Cardiovasc Dis 30(6):1032–1043. https:// doi. org/ 10. 1016/j. numecd. 2020. 02. 014 Chinnasamy G, Bal AK (2003) Seasonal changes in carbohy- drates of perennial root nodules of beach pea. J Plant Physiol 160(10):1185–1192. https:// doi. org/ 10. 1078/ 0176- 1617- 01090 Cummings SP (2005) The role and future potential of nitrogen fixing bacteria to boost productivity in organic and low-input sustainable farming systems. Environ Biotechnol 1(1):1–10 Cunningham SM, Nadeau P, Castonguay Y, Laberge S, Volenec JJ (2003) Raffinose and stachyose accumulation, galactinol synthase expression, and winter injury of contrasting alfalfa germplasms. Crop Sci 43:562–570. https:// doi. org/ 10. 2135/ crops ci2003. 5620 D’Amours E, Bertrand A, Cloutier J, Classens A, Rocher S, Seguin P (2022) Impact of Sinorhizobium meliloti strains and plant popula- tion on regrowth and nodule regeneration of alfalfa after a freezing event. Plant Soil. https:// doi. org/ 10. 1007/ s11104- 022- 05662-4 Dakora FD, Joseph CM, Phillips DA (1993) Alfalfa (Medicago sativa L.) Root exudates contain isoflavonoids in the presence of Rhizo- bium meliloti. Plant Physiol 101(3):819–824. https:// doi. org/ 10. 1104/ pp. 101.3. 819 Dhont C, Castonguay Y, Nadeau P, Bélanger G, Chalifour FP (2002) Alfalfa root carbohydrates and regrowth potential in response to fall harvests. Crop Sci 42(3):754–765. https:// doi. org/ 10. 2135/ crops ci2002. 7540 Dhont C, Castonguay Y, Nadeau P, Bélanger G, Drapeau R, Laberge S, Avice JC, Chalifour FP (2006) Nitrogen reserves, spring regrowth and winter survival of field-grown alfalfa (Medicago sativa) defo- liated in the autumn. Ann Bot 97(1):109–120. https:// doi. org/ 10. 1093/ aob/ mcj006 Dong W, Song Y (2020) The significance of flavonoids in the process of biological nitrogen fixation. Int J Mol Sci 21(16):5926. https:// doi. org/ 10. 3390/ ijms2 11659 26 Dubé MP, Castonguay Y, Cloutier J, Michaud J, Bertrand A (2013) Characterization of two novel cold-inducible K3 dehydrin genes from alfalfa (Medicago sativa spp. sativa L.). Theor Appl Genet 126:823–835. https:// doi. org/ 10. 1007/ s00122- 012- 2020-6 Dunn MF (2014) Key roles of microsymbiont amino acid metabolism in rhizobia-legume interactions. Crit Rev Microbiol 41(4):411– 451. https:// doi. org/ 10. 3109/ 10408 41X. 2013. 856854 Duzan HM, Mabood F, Souleimanov A, Smith DL (2006) Nod Bj-V (C18:1, MeFuc) production by Bradyrhizobium japonicum (USDA110, 532C) at suboptimal growth temperatures. J Plant Physiol 163(1):107–111. https:// doi. org/ 10. 1016/j. jplph. 2005. 04. 029 El-Shafey NM, AbdElgawad H (2012) Luteolin, a bioactive flavone compound extracted from Cichorium endivia L. subsp. divarica- tum alleviates the harmful effect of salinity on maize. Acta Physiol Plant 34:2165–2177. https:// doi. org/ 10. 1007/ s11738- 012- 1017-8 Falcone Ferreyra ML, Rius SP, Casati P (2012) Flavonoids: biosynthe- sis, biological functions, and biotechnological applications. Front Plant Sci 3:222. https:// doi. org/ 10. 3389/ fpls. 2012. 00222 Feduraev P, Skrypnik L, Riabova A, Pungin A, Tokupova E, Maslen- nikov P, Chupakhina G (2020) Phenylalanine and tyrosine as exogenous precursors of wheat (Triticum aestivum L.) secondary metabolism through PAL-associated pathways. Plants 9(4):476. https:// doi. org/ 10. 3390/ plant s9040 476 Fields RL, Barrell GK, Gash A, Zhao J, Moot DJ (2018) Alfalfa coumestrol content in response to development stage, fungi, aphids, and cultivar. Agron J 110:910–921. https:// doi. org/ 10. 2134/ agron j2017. 09. 0535 Galili G (2011) The aspartate-family pathway of plants: linking produc- tion of essential amino acids with energy and stress regulation. Plant Signal Behav 6(2):192–195. https:// doi. org/ 10. 4161/ psb.6. 2. 14425 Geddes BA, Oresnik IJ (2014) Physiology, genetics, and biochemistry of carbon metabolism in the alphaproteobacterium Sinorhizobium meliloti. Can J Microbiol 60(8):491–507. https:// doi. org/ 10. 1139/ cjm- 2014- 0306 Gifford I, Battenberg K, Vaniya A, Wilson A, Tian L, Fiehn O, Am B (2018) Distinctive patterns of flavonoid biosynthesis in roots and nodules of Datisca glomerata and medicago spp. Revealed by metabolomic and gene expression profiles. Front Plant Sci 9:1463. https:// doi. org/ 10. 3389/ fpls. 2018. 01463 Gordon AJ, Minchin FR, James CL, Komina O (1999) Sucrose syn- thase in legume nodules is essential for nitrogen fixation. Plant Phys 120(3):867–878. https:// doi. org/ 10. 1104/ pp. 120.3. 867 Gurusamy C, Davis PJ, Bal AK (2000) Seasonal changes in perennial nodules of beach pea (Lathyrus maritimus [L.] Bigel.) with special reference to oleosomes. Int J Plant Sci 161(4):631–638. https:// doi. org/ 10. 1086/ 314291 Hartwig UA, Maxwell CA, Joseph CM, Phillips DA (1990) Effects of alfalfa nod gene-inducing flavonoids on nodABC transcription in Rhizobium meliloti strains containing different nodD genes. J Bacteriol 172(5):2769–2773. https:// doi. org/ 10. 1128/ jb. 172.5. 2769- 2773. 1990 https://doi.org/10.3390/agronomy10040569 https://doi.org/10.3390/agronomy10040569 https://doi.org/10.1007/s00249-008-0260-9 https://doi.org/10.1073/pnas.071375898 https://doi.org/10.1073/pnas.071375898 https://doi.org/10.1016/S0065-2113(06)90006-6 https://doi.org/10.1016/S0065-2113(06)90006-6 https://doi.org/10.2135/cropsci2008.09.0539 https://doi.org/10.2135/cropsci2008.09.0539 https://doi.org/10.2135/cropsci2011.02.0060 https://doi.org/10.4236/ajps.2015.61015 https://doi.org/10.4236/ajps.2015.61015 https://doi.org/10.1007/s10681-020-2559