Christina C Vinson

Ana P Z Mota

Brenda N Porto

Thais N Oliveira

Iracyara Sampaio

Ana L Lacerda

Etienne G J Danchin

Patricia M Guimaraes

Thomas C R Williams

Ana C M Brasileiro
email: ana.brasileiro@embrapa.br

Characterization of raffinose metabolism genes uncovers a wild Arachis galactinol synthase conferring tolerance to abiotic stresses

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

In silico expression profiling of A. duranensis genes. Illumina RNA-seq data previously obtained by our group were used to determine the in silico expression profiles of GolS, RS, STS, AGAL and BFLUCT genes in Arachis spp. This data comprises: (1) Transcripts expressed in roots of A. duranensis and A. stenosperma plants submitted to dehydration treatment, by the withdrawal of hydroponic nutrient solution from 25 to 150 min [START_REF] Vinson | Early responses to dehydration in contrasting wild Arachis species[END_REF] and pooled in equal amounts, a treatment which we previously showed to induce major alterations in the transcriptome of these species. (2) Transcripts expressed in A. duranensis and A. stenosperma plants subjected to a decreased in soil availability with withholding of irrigation for four days, a treatment which induced proteomic and transcriptomic alterations [START_REF] Carmo | Comparative proteomics and gene expression analysis in Arachis duranensis reveal stress response proteins associated to drought tolerance[END_REF] . The differential expression values (log2 of fold-change) between stressed and control samples in A. duranensis and A. stenosperma roots were plotted in a heatmap graph using the heatmap2 from ggplot R package, as previously described [START_REF] Guimaraes | Genome-wide analysis of expansin superfamily in wild Arachis discloses a stress-responsive expansin-like B gene[END_REF] .

Arabidopsis thaliana lines overexpressing AdGolS3 gene. The coding sequence of the AdGolS3 gene was identified by the alignment of the Aradu.ZK8VV gene model (https ://peanu tbase .org) with the four best BLASTn hits of A. duranensis at NCBI (https ://www.ncbi.nlm.nih.gov). The obtained AdGolS3 consensus sequence (981 bp) was synthetized and cloned under the control of the A. thaliana actin 2 promoter in the binary vector pPZP_201BK_EGFP [START_REF] Chu | A technique to study Meloidogyne arenaria resistance in Agrobacterium rhizogenes-transformed peanut[END_REF] by Epoch Life Science Inc. (TX, USA). The resulting vector, pPZP-AdGolS3, was transferred to the Agrobacterium disarmed strain 'GV3101' and the transformed colonies selected by PCR with specific eGFP and AdGolS3 primers (Table S1), using standard protocols.

Wild-type (WT) A. thaliana plants (ecotype Columbia; Col-0) were transformed with the GV3101-pPZP-AdGolS3 Agrobacterium strain by the floral dip immersion method [START_REF] Clough | Floral dip: A simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana[END_REF] . The eGFP-positive and hygromycin-resistant T0 transformants were grown in a controlled growth chamber (21 °C with a 12 h photoperiod and light intensity of 120 µmols.m -2 .s -1 ) to obtain transgenic AdGolS3 overexpressing (OE) lines, as described previously [START_REF] Mota | Contrasting effects of wild Arachis dehydrin under abiotic and biotic stresses[END_REF] . T1 seeds of each transgenic line obtained by self-pollination of T0 plants were germinated on hygromycin selective medium and T1 plants grown to maturity. Self-pollinated T2 seeds derived from each T1 plant were maintained separate and 24 T2 seeds tested for homozygosity through germination on hygromycin selective medium. If all T2 seeds germinated in hygromycin selective medium, we considered that they derived from a T1 plant homozygote producing T2 homozygous seeds. All the subsequent stress treatments and analyses were conducted with homozygous AdGolS3-OE plants of the T2 generation.

Transgene expression and sugar content in AdGolS3-OE lines. The expression of AdGolS3

transgene in 13 independent Arabidopsis OE lines was confirmed through qRT-PCR analysis as described below (2.10), using specific AdGolS3 primers (Table S1). The content of four sugars (glucose, fructose, sucrose and raffinose) was determined in leaves of one-month-old WT and transgenic plants (five individuals per genotype) as described previously [START_REF] Moreira | A genome-scale metabolic model of soybean (Glycine max) highlights metabolic fluxes in seedlings[END_REF] . Sugars were extracted using 80% (v/v) ethanol at 80 °C, and the extracts dried and resuspended in water for analysis. Samples were analyzed using a High Performance Anion Exchange (HPAE) chromatography system (Dionex, ICS 3,000, Sunnyvale, CA, USA) equipped with a pulsed amperometric detector and Carbopak PA-10 column. Sugars were separated using an isocratic method with 52 mM NaOH and a column flow of 0.2 mL.min -1 over 35 min and quantified using standard curves.

Dry-down assay. Based on the HPAE analysis, three AdGolS3-OE lines (GolS17, GolS20 and GolS22) that showed significantly higher levels of raffinose compared to WT were selected for the subsequent abiotic stress assays. Seeds from WT and transgenic plants were sown in 250 mL pots containing the same amount of substrate (Carolina Soil, CSC, Brazil) and maintained under the growth conditions described above. The dry-down assay started when the plants were 30 days old and lasted for 20 days. Plants were divided into three treatments: (1) Control (CTR) group maintained under irrigated conditions, i.e., around 70% of field capacity (FC); (2) Stressed (STR) group where irrigation was suspended; and (3) Rehydrated (REH) group where STR plants were irrigated 24 h before collection. CTR, STR and REH treatments started at the same time and were carried out in parallel, each group with its own set of plants. At the end of the assay, ten individuals from each treatment (CTR, STR and REH) were collected (at 9 am) for each genotype, weighed and stored at -80 °C for later biochemical and molecular analyses.

The leaf disc submersion methods were used for the determination of the relative water content (RWC) and electrolyte leakage (EL), as described previously [START_REF] De Brito | Physiological traits for drought phenotyping in cotton[END_REF][START_REF] Whitlow | An improved method for using electrolyte leakage to assess membrane competence in plant tissues[END_REF] . For RWC and EL measurements, three leaf discs of 0.4 cm 2 were used per individual for each treatment (CTR, STR and REH).

Soluble sugar analysis and metabolic profiling. Soluble sugars (glucose, sucrose and raffinose) were separated and quantified in the three selected AdGolS3-OE lines and in WT plants under CTR, STR and REH conditions using the HPAE chromatography system, as described above. Metabolic profiling of these four genotypes was carried out according to [START_REF] Lisec | Gas chromatography mass spectrometry-based metabolite profiling in plants[END_REF] . Samples of lyophilized tissue were extracted using the methanol:chloroform:water method with ribitol as an internal standard. Aliquots of the polar phase were dried and derivatized using methyoxyamine hydrochloride in pyridine followed by MSTFA. Samples were analyzed using an Agilent 7820A GC coupled to an Agilent 5,975 MSD equipped with a 30 m HP5-ms column. Metabolites were identified by comparison with a custom mass spectral library and chromatograms were aligned using MetaAlign [START_REF] Lommen | Interface-driven, versatile metabolomics tool for hyphenated full-scan mass spectrometry data preprocessing[END_REF] .

NaCl and PEG treatments. Based on the results of the dry-down experiment, the GolS22 OE line was selected for evaluation of its performance in response to two additional abiotic stress treatments: NaCl (salt stress) and polyethylene glycol (PEG; osmotic stress). WT and GolS22 plants were grown as described above for 30 days then divided between three groups: (1) Control group maintained under irrigated conditions (maintained at 70% FC); (2) Salt stressed group irrigated with a 150 mM NaCl solution instead of water; and (3) Osmotic stressed group irrigated with a PEG 6,000 20% (w/v) solution instead of water. Plants from each group were maintained under these conditions for 15 days. Ten individuals from each treatment were collected (at 9 am) per genotype, weighed and analyzed for RWC and EL, as described above.

qRT-PCR analysis. The relative expression of the AdGolS3 transgene and of a subset of Arabidopsis genes was determined in WT plants and OE lines by qRT-PCR analysis, as previously described [START_REF] Mota | Contrasting effects of wild Arachis dehydrin under abiotic and biotic stresses[END_REF] . This gene subset comprises five Arabidopsis genes selected based on their putative interaction with AtGolS2 in Arabidopsis, as predicted by geneMANIA [START_REF]The GeneMANIA prediction server: biological network integration for gene prioritization and predicting gene function[END_REF] . It includes (Table S1): Glutathione S-transferase (AtGSTU24), Stress-Associated Protein (AtSAP13), Ascorbate Peroxidase (AtAPX1), Peroxisomal Catalase (AtCAT2) and Alpha amylase family protein (AtEMB2729). AtGolS2 is the orthologous of the AdGolS3 gene in Arabidopsis and was included in the qRT-PCR analysis. Specific primers were designed (Table S1) using the software Primer3Plus, following the parameters described previously [START_REF] Morgante | Reference genes for quantitative reverse transcription-polymerase chain reaction expression studies in wild and cultivated peanut[END_REF] . The qRT-PCR reactions were performed on a StepOne Plus Real-Time PCR System (Applied Biosystems, Foster City, USA) in technical triplicates for each sample, using No Template (NTC) and No Amplification (NAC) samples as negative controls. The relative quantification (RQ) of mRNA levels was normalized with AtACT2 and AtEF-1α reference genes (Table S1).

Results

Genome-wide identification of wild Arachis genes involved in RFO metabolism. Galactinol synthase (GolS). We identified six plant proteins with experimentally verified biochemical function in the CAZy database as GolS proteins that shared the common conserved PFAM domain (PF01501) of the GT8 family. The two belonging to the Fabaceae family (GLYMA Q7XZ08 and MEDSA Q84MZ5) were then used as references for HMM profile construction.

Using this HMM profile, 30 proteins from 11 Fabaceae species were identified in UniProt as putatively belonging to the GolS family (Table S2), together with five proteins each for A. duranensis and A. ipaënsis genomes. These 10 Arachis putative GolS proteins ranged from 311 to 341 amino acids in length (average of 328) without signal peptides (Table 1). Phylogenetic analysis performed with the 40 putative GolS proteins revealed two distinct clusters: cluster 1, with 15 proteins, and cluster 2 with 25 (Fig. S1a). Arachis GolS proteins had representatives in both clusters, with six proteins in cluster 1 and four in cluster 2 (Fig. S1a).

Phylogenetic clustering was associated with the number of exons, with all genes coding the six proteins from cluster 1 consistently having three exons and those from cluster 2 having three to five exons (Fig. S1b). Three highly-conserved protein motifs were identified in the in the Arachis GolS family (Fig. S1c).

Raffinose synthase (RS) and stachyose synthase (STS).

Although the RS and the STS families have distinct EC codes, they are characterized by the presence of the same conserved GH36 family domain (PFAM domain PF05691). We found two plant proteins functionally characterized as belonging to the RS family and only one from the STS family in the CAZy database that were then used for the construction of separate RS and STS HMM profiles.

In total, 62 putative RS proteins were identified in 11 Fabaceae species in UniProt (Table S2). Another seven RS proteins were found in A. duranensis and six in A. ipaënsis, with an average length of 773 amino acids (ranging between 546 and 1,035) and lack of signal peptides (Table 1). Phylogenetic analysis of the 75 putative Fabaceae RS separated these proteins into seven distinct clusters (Fig. S2a). The Arachis RS proteins were evenly distributed among six clusters, with one protein per cluster, except for cluster 1, with two A. duranensis proteins (AdRS1 and AdRS2). As for GolS, RS protein clustering is related to its gene organization in Arachis, with genes belonging to the same cluster presenting a similar number of exons (Fig. S2b), which ranges from three exons in cluster 2 to more than 12 exons in clusters 1 and 3. The analysis of proteins sequences showed the presence of three conserved motifs in the 13 Arachis RS (Fig. S2c).

Fourteen putative STS proteins were retrieved from nine Fabaceae species (Table S2), including three from A. ipaënsis and only one from A. duranensis. These four putative STS Arachis proteins ranged from 359 to 1,696 amino acids in length (average of 945), without signal peptides (Table 1). Phylogenetic analysis revealed a highly conserved STS family in Fabaceae, forming two clusters: the first one with all proteins exclusive to the genus Arachis (AdSTS1, AiSTS1, AiSTS2 and AiSTS3) and the second with 10 proteins from other Fabaceae species (Fig. S3a). Exceptionally, no relationship was observed between this phylogenetic clustering and the intron/exon organization of the STS Arachis genes (Fig. S3b). The sequences of the four Arachis STS proteins showed at least three conserved motifs (Fig. S3c).

Alpha-galactosidase (AGAL).

AGAL is part of the GH27 enzyme family and is characterized by the presence of the conserved PFAM domain of melibiase_2 (PF16499). We retrieved three functionally characterized AGAL plant proteins from the CAZy database that were used to construct the HMM profile.

This profile revealed 56 putative AGAL proteins from 14 Fabaceae species (Table S2), from which four proteins belonged to A. duranensis and four to A. ipaënsis, with an average length of 374 amino acids, ranging from 178 to 437. Phylogenetic analysis divided these 56 putative AGAL proteins into three clusters (Fig. S4a). Clusters 2 and 3 each contain a single representative from A. duranensis and A. ipaënsis, whereas cluster 1 has two representatives from each species. Interestingly, the two A. duranensis (AdAGAL1 and AdAGAL2) and A. ipaënsis (AdAGAL1 and AdAGAL2) AGAL proteins that contained signal peptides shared the same cluster 1 (Table 1 and Fig. S4a). The Arachis proteins within the same cluster showed a similar intron/exon structure, except for those in cluster 3, where the A. duranensis AdAGAL4 gene had 15 exons while A. ipaënsis AiAGAL4 contained only seven (Fig. S4b). The protein sequence analysis showed that all eight Arachis AGAL proteins share at least three conserved common motifs (Fig. S4c).

Beta-fructofuranosidase (BFLUCT).

The BFLUCT enzyme family belongs to CAZy family GH32 and is also defined by the presence of two glycosyl hydrolase PFAM domains: Glyco_Hydro32N (PF00251) and Glyco_ Hydro32C (PF08244). In the CAZy database, 78 plant proteins were functionally characterized as BFLUCT and we used the 11 Fabaceae proteins as references for the construction of an HMM profile.

A total of 114 putative BFLUCT proteins was retrieved from 13 Fabaceae species (Table S2 (Table 1). Phylogenetic analysis produced five clusters, from which, four contained at least one representative of each Arachis species (Fig. S5a). Five A. duranensis and four A. ipaënsis BFLUCT proteins harbored signal peptides, but, unlike the AGAL proteins, these proteins did not share the same clusters (Fig. S5a). In general, the intron/exon organization of genes belonging to the same protein cluster was similar (Fig. S5b). Three protein motifs were identified and conserved in all Arachis BFLUCT, except for AdBFLUCT6 (Fig. S5c).

Genomic distribution and duplication patterns of RFO metabolism genes in wild Arachis.

Genome-wide analysis of the two wild Arachis species with genome sequences so far available identified 28 RFO metabolism genes in A. duranensis and 31 in A. ipaënsis. In both species, these genes were unevenly distributed in the ten chromosomes regardless of the enzyme family (Fig. 1). The majority of the 59 genes were restricted to the distal chromosomal regions, in accordance with previous studies showing the gene-rich characteristic of these hot recombination hotspot regions in wild Arachis genomes [START_REF] Guimaraes | Genome-wide analysis of expansin superfamily in wild Arachis discloses a stress-responsive expansin-like B gene[END_REF][START_REF] Bertioli | The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut[END_REF][START_REF] Mota | Comparative root transcriptome of wild Arachis reveals NBS-LRR genes related to nematode resistance[END_REF] .

All the genes identified as involved in raffinose metabolism were duplicated in both Arachis species. The majority of gene copies (50.8%) resulted from dispersed duplications, 35.6% originated from whole-genome (WGD)/segmental duplication, 8.5% from proximal duplication and 5.1% from tandem duplications (Table 1). In the GolS family, specifically, the gene copies resulted mostly from WGD/segmental duplications (80%), while Figure 1. Genomic distribution of RFO metabolism Arachis genes. Distribution of galactinol synthase (GolS), raffinose synthase (RS), stachyose synthase (STS), alpha-galactosidase (AGAL) and beta-fructofuranosidase (BFLUCT) genes in the ten chromosomes of Arachis duranensis (A01-A10) and A. ipaënsis (B01-B10). Synteny between the two genomes is represented by lines. GolS (purple), STS (yellow), RS (green), AGAL (blue) and BFLUCT (red). The figure was generated by Circa software (https ://omgen omics .com).

in the RS and AGAL families, the dispersed gene duplications represented the majority (92.3% and 75%, respectively; Table 1). Expansion of STS family genes similarly resulted from tandem and dispersed duplications (50% each) and the BFLUCT family from WGD/segmental and dispersed duplications (41.7% each) (Table 1).

Expression profiling of Arachis RFO metabolism genes in response to water deficit. The expression patterns of the 28 A. duranensis RFO metabolism genes in response to two types of drought imposition (dehydration and dry-down) were analyzed using our previous transcriptome data obtained from the drought-tolerant accession K7988 of A. duranensis and the drought-sensitive accession V10309 of A. stenosperma [START_REF] Vinson | Early responses to dehydration in contrasting wild Arachis species[END_REF] . This analysis revealed that the expression of most of the genes involved in RFO metabolism were modulated in response to water deficit, with distinct patterns and expression levels depending on the type of stress imposed and, to a lesser extent, on the Arachis genotype (Fig. 2).

The Arachis RS genes exhibited small variations in their expression levels in response to the two types of drought imposition in both genotypes. The exception was AdRS6 with an high upregulation (fold change > 3) under dry-down in A. stenosperma (Fig. 2). Concerning the single representative of the STS family, AdSTS1 was moderately downregulated in both Arachis genotypes in response to the two stresses.

The expression profile of the four representatives of the AGAL family, the initial enzyme responsible for raffinose breakdown, was different in the two treatments, regardless of the Arachis genotype (Fig. 2). Under dehydration stress, AGAL genes exhibited an overall downregulation pattern, with low expression levels, whereas under the dry-down stress, they did not seem to be modulated. Likewise, most of the 11 A. duranensis BFLUCT genes were downregulated in response to the dehydration treatment whilst only being slightly affected by drydown (Fig. 2). The exception was AdBFLUCT3, which was strongly induced (fold change > 10) in A. duranensis plants submitted to dry-down.

Genes coding for the five GolS showed contrasting expression behaviors in response to the two types of drought imposition, regardless of the Arachis genotype, with a general upregulation under dehydration and downregulation under dry-down (Fig. 2). This was especially evident for AdGolS4 and AdGolS5, which exhibited strong upregulation (fold change > 4) in response to dehydration. AdGolS2 was the only GolS gene downregulated in response to dehydration.

However, AdGolS3 drew particular attention as it exhibited the greatest difference in expression between the dehydration (upregulation of 3.15-fold) and dry-down (downregulation of -8.16-fold) treatments in the droughttolerant A. duranensis (Fig. 2). Conversely, in the more drought-sensitive A. stenosperma, the difference in the expression magnitude between dehydration (upregulation of 2.19-fold) and the dry-down (downregulation of -1.75-fold) was much smaller (Fig. 2). AdGolS3 is also the orthologue of Arabidopsis AtGolS2, which is known to be responsive to diverse abiotic stresses, and confers enhanced drought tolerance when overexpressed in transgenic plants [START_REF] Taji | Important roles of drought and cold inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana[END_REF] . Given its differential regulation in drought tolerant and sensitive Arachis species, and orthologous relationship to AtGolS2, we therefore selected AdGolS3 for further in planta functional studies, to better understand the role of GolS genes in the molecular response underlying the process of water loss in wild Arachis.

Screening of A. thaliana lines overexpressing

AdGolS3. The AdGolS3 coding sequence was predicted by the alignment of five sequences: Aradu.ZK8VV (https ://peanu tbase .org); GW944818.1; XM_016113210.2; HP005973.1 TSA and GW952716.1 (https ://www.ncbi.nlm.nih.gov). The consensus sequence of 981 bp was cloned into pPZP-AdGolS3 and used to produce T0 primary Arabidopsis transformants. A total of 13 independent homozygous OE lines at T2 generation were obtained and AdGolS3 overexpression was confirmed by qRT-PCR analysis in all of these OE lines, with the expression levels relative to the two reference genes varying among individual lines (Fig. S6). AdGolS3 expression was not detected in WT plants.

Given the putative involvement of AdGolS3 in the synthesis of raffinose series sugars, the leaf sugar content in the 13 OE lines was also analyzed and compared to WT plants. Leaves from four OE lines (GolS10; GolS17; GolS20 and GolS22) showed a significantly higher level of raffinose compared to WT plants, whereas four (GolS2; GolS4; GolS6, and GolS8) unexpectedly had a significant decrease (p < 0.05, Fig. S7). There was no clear relationship between the transgene expression levels (Fig. S6) and raffinose contents (Fig. S7) in the 13 OE lines, consistent with the complex relationship between transcript abundance and accumulation of metabolites that lie downstream of the encoded enzyme. Overall, the content of other sugars also involved in RFO metabolism (glucose, fructose and sucrose) was not affected by AdGolS3 overexpression (Fig. S7). The exception was the GolS20 OE line, which presented an indirect, and specific, effect in its overall sugar content, with a significant increase in the concentration of all four sugars (glucose, fructose, sucrose and raffinose) compared to WT (Fig. S7). Interestingly, this OE line showed one of the lowest levels of ectopic AdGolS3 expression (Fig. S6). Based on these findings, and given previous reports of correlations between accumulation of RFOs and tolerance to stress [START_REF] Elsayed | Physiological aspects of raffinose family oligosaccharides in plants: Protection against abiotic stress[END_REF][START_REF] Sengupta | Significance of galactinol and raffinose family oligosaccharide synthesis in plants[END_REF] , the three promising OE lines that showed the highest levels of raffinose accumulation with variable levels of transgene expression (GolS17, GolS20 and GolS22) were selected for further stress assays and physiological and biochemical analyses.

Analysis of Arabidopsis plants overexpressing AdGolS3. Plant growth, relative water content (RWC)

and electrolyte leakage (EL). Over the 20 days of the dry-down assay, the morphology of the aerial part of the AdGolS3 OE lines remained similar to that of WT plants under normal irrigation conditions (CTR group) (Fig. 3). In the STR and REH groups, WT plants displayed severe morphological damage (leaf wilt) after 20 days without irrigation, whereas OE lines exhibited fewer symptoms of water deficiency (Fig. 3). In the REH group, one day after rehydration, transgenic plants recovered faster than WT, indicating that the AdGolS3 overexpression increased the ability of plants to recover their normal phenotype, as that observed in CTR group. Plants submitted to dehydration for 20 days (STR and REH groups) had less shoot biomass when compared to the CTR group, with no significant differences between WT and OE lines (Fig. 4a). The analysis of the leaf relative water content (RWC) in plants maintained under CTR conditions showed values of 85-86%, typical of turgid leaves, with no differences between WT and OE lines (Fig. 4b). An overall reduction in RWC was observed when plants were submitted to STR conditions when compared to CTR. Under dehydration imposition, the three OE lines displayed RWC values higher than WT plants, with the GolS22 OE line showing a significant difference of 25%. Following 24 h of rehydration, both GolS17 and GolS22 OE lines reached higher RWC values than WT plants, comparable to those found for plants under CTR conditions, indicating their ability to rapidly recover to a high leaf water status in response to water availability (Fig. 4b). Accordingly, the opposite behavior was observed for leaf electrolyte leakage (EL) measurements at the end of the dry-down assay, with a significantly lower leakage in the GolS22 line under stressed conditions and in the GolS17 and GolS22 lines following rehydration, as compared to the WT plants (Fig. 4c), representing good evidence of cell membrane stability under stress in these OE lines. Interestingly, GolS17 and GolS22 lines showed significant differences in EL values compared to WT plants, even when plants were maintained under CTR conditions (Fig. 4c). RWC and EL measurements have been widely used to reflect, respectively, water loss control and membrane stability in plants submitted to www.nature.com/scientificreports/ water-limited conditions, and therefore an improvement in drought tolerance. These results could thus indicate that AdGolS3 overexpression increases plant tolerance to water deficit stress.

Soluble sugar content and metabolic profiling. Soluble sugar (glucose, sucrose and raffinose) abundance in leaves of WT and OE lines (GolS17, GolS20 and GolS22) under CTR, STR and REH conditions were also analyzed. Despite the highly variable absolute concentration of these sugars, previously determined for the 13 OE lines under CTR condition only (Fig. S7), the relative percentage abundance was generally stable in the GolS17, GolS20 and GolS22 lines, regardless of the treatment applied (Fig. S8). The abundance of raffinose relative to glucose and sucrose was indeed higher in the GolS17 and GolS22 OE lines compared to WT plants under CTR conditions. These results corroborated the previous analysis of the absolute raffinose concentration (Figs. S7 andS8). Also, the relative abundance of raffinose did not differ between GolS20 and WT plants under CTR conditions, but it significantly increased from 2.5 to 12.9% when GolS20 plants was submitted to STR conditions (Fig. S8). Conversely, the relative abundance of raffinose in the GolS17 OE line significantly decreased from 7.6% of total sugars under CTR conditions to 3.9% when plants were rehydrated. Interestingly, the ratio between glucose and sucrose relative abundance was almost stable (around 1:1), and apparently independent of the treatment (CRT, STR or REH). In contrast, the ratio between raffinose and these sugars was more variable (Fig. S8).

To further characterize the metabolic response of the AdGolS3 OE lines under STR and REH conditions, we carried out GC-MS based metabolite profiling of leaf tissues (Table 2 and Fig. S9). Overall, few differences between the WT and OE lines were detected in the metabolic profile regardless of the treatment. Under CTR conditions, GolS17 contained less sucrose whilst GolS22 contained more raffinose and galactinol than WT, mirroring the results obtained by HPAE (Table 2; Figs. S7, S8 and S9a). When expressed relative to all detected metabolites abundances of both raffinose and galactinol were also greater in GolS17 than in WT (Table 2 and Fig. S9b), again reflecting a shift in metabolite profile towards raffinose accumulation. As well as these alterations in Table 2. Metabolic profiling in AdGolS3-OE lines. Relative abundance of metabolites extracted from leaves of wild-type (WT) untransformed plants and three OE lines (GolS17, GolS20 and GolS22) under control (CTR), stressed (STR) and rehydrated (REH) conditions. Values indicate the abundance of each metabolite relative to that detected in WT plants [average (AVG) and standard deviation (SD) of four to five individuals]. Values in bold are significantly different from WT plants for a given treatment, whilst underlined values are significantly different from the control treatment for a given genotype (WT or each OE line) (t-test, p < 0.05). Two chromatographic peaks were detected for fructose and glucose.

Control (CTR)

Stressed (STR) Rehydrated (REH) sugars, citrate concentrations were also higher in GolS20 and threonate concentrations lower in GolS22 under CTR conditions. Under STR condition, the only difference was an increased concentration of putrescine in the GolS22 line compared to WT. Under the REH, when expressed relative to all detected metabolites, raffinose and galactinol in GolS22 remained greater than in WT (Table 2 and Fig. S9).

As anticipated, greater differences were observed between stressed and non-stressed control plants of the same genotype (WT and each OE line) (Table 2 and Fig. S9a). In stressed WT plants, for example, 13 out 26 metabolites showed increased concentrations, including alanine, beta-alanine, aspartate, citrate, fructose, galactinol, glucose, glycerol, myo-inositol, leucine, raffinose, threonate, and valine (Table 2 and Fig. S9a). GolS17 metabolism was similarly affected, with a total of 17 different metabolites showing significant alterations under stressed conditions. Fewer changes, however, were detected in both GolS20 and GolS22 OE lines, as only three (galactinol, myo-inositol and raffinose), and six (beta-alanine, fructose, gluconate, myo-inositol, leucine and valine) metabolites were significantly perturbed by the drought imposition, respectively. Myo-inositol was the only metabolite with increased abundance under stress in all genotypes (Table 2). Whilst large increases in proline were detected in individual samples, the only significant increase was in stressed GolS17 plants relative to their non-stressed controls. Rehydration generally led to metabolite concentrations returning to the levels observed in control conditions. One notable exception was that of myo-inositol, which remained at elevated concentrations relative to control plants for WT, GolS17 and GolS20 (Table 2).

NaCl and PEG treatments. Additional analysis of GolS22 and WT plants grown under well-irrigated CTR, salt stress (irrigated with NaCl 150 mM) and osmotic stress (irrigated with PEG 20%) conditions was conducted after 15 days of treatment. The phenotype of the GolS22 transgenic plants was similar to that of WT under CTR conditions, with RWC values (78% and 74%, respectively) typical of turgid leaves (Fig. 5a). Similar RWC values for WT and GolS22 plants (65% and 61%, respectively) were maintained even when salt stress treatment was applied; however, RWC was significantly greater in GolS22 (62%) under osmotic stress (Fig. 5a). EL values in the GolS22 line were significantly lower than in WT plants under both stress conditions (NaCl and PEG) (Fig. 5b), as previously observed for drought and rehydration (Fig. 4c) treatments. Together, these results indicate that overexpression of AdGolS3 led to better water retention and membrane stability not only during drought imposition and rehydration recovery but also under both salt and osmotic stresses. qRT-PCR expression analysis. Considering that AdGolS3 overexpression enhanced drought tolerance in transgenic Arabidopsis, altering sugar and metabolic profiles, the expression levels of a subset of six genes was evaluated. This subset comprises the AtGolS2 gene, which is the AdGolS3 ortholog in Arabidopsis, and five other Arabidopsis genes coding for proteins that putatively interact with AtGolS2 based on a predicted protein-protein interaction network (Fig. S10). These genes were also selected based on their known involvement in drought and osmotic stress responses. The expression analysis of the six genes was carried out by qRT-PCR in roots from three OE lines (GolS17, GolS20 and GolS22) and WT plants submitted to CTR, STR and REH conditions. As a whole, AdGolS3 overexpression by itself was sufficient to induce the expression of five (AtAPX1, AtCAT2, AtGSTU24, AtEMB2729 and AtGolS2) of the six Arabidopsis genes, given that their expression was higher in OE lines than in WT plants under CTR conditions. (Fig. 6a). Interestingly, under drought stress imposition, the expression behavior of four (AtAPX1, AtCAT2, AtGSTU24 and AtEMB2729) of these genes changed drastically with a general pattern of downregulation, and an average decrease of almost threefold in transcript levels when compared to CTR conditions (Fig. 6b).

After subsequent plant rehydration, the expression levels of these four genes increased, almost reaching the basal levels observed under CTR conditions (Fig. 6c). Conversely, the endogenous Arabidopsis GolS gene (AtGolS2) was the only gene for which the expression increased (4.12-fold) under drought imposition as compared to CTR conditions, with significant upregulation in all OE lines (Fig. 6b). However, despite its remarkable upregulation under STR conditions, the expression of the AtGolS2 gene rapidly dropped and returned to the basal CTR levels just 24 h after rehydration, as also observed for the other four genes (Fig. 6c). It is interesting that the expression of AtSAP13 was negatively affected by AdGolS3 overexpression, but not by dry-down or rehydration treatments, since its downregulation was maintained (average RQ of 0.81) in the three OE lines under all of the studied conditions (Fig. 6). These findings suggest that AdGolS3 may play an important role in drought-associated pathways by modulating the transcriptional dynamics of downstream genes.

Discussion

The majority of RFO metabolism genes have undergone dispersed duplications in wild Arachis. RFOs are part of the molecular network activated by plants in response to a range of environmental stresses and currently emerge as key components in stress tolerance, acting as osmoprotectants, antioxidants and signaling molecules [START_REF] Guerra | Post-transcriptional and post-translational regulations of drought and heat response in plants: A spider's web of mechanisms[END_REF][START_REF] Sami | Role of sugars under abiotic stress[END_REF] . Due to their importance, the principal enzymes involved in the first steps of RFO biosynthesis (GolS, RS and STS) have been thoroughly studied at the genome-wide scale in many plant species [START_REF] Zhou | Genome-wide identification of genes involved in raffinose metabolism in Maize[END_REF][START_REF] Li | Genome-wide identification and expression profiling analysis of the galactinol synthase gene family in cassava (Manihot esculenta Crantz)[END_REF][START_REF] You | Genome-wide identification and expression analyses of genes involved in raffinose accumulation in sesame[END_REF][START_REF] Filiz | Genome-wide identification of galactinol synthase (GolS) genes in Solanum lycopersicum and Brachypodium distachyon[END_REF][START_REF] Falavigna | Evolutionary diversification of galactinol synthases in Rosaceae: Adaptive roles of galactinol and raffinose during apple bud dormancy[END_REF][START_REF] Ferreira-Neto | Inositol phosphates and Raffinose family oligosaccharides pathways: Structural genomics and transcriptomics in soybean under root dehydration[END_REF] . However, little attention has been given to the enzymes involved in RFO catabolism, such as AGAL and BFLUCT. These enzymes are equally important for the accumulation of RFOs in plants, but only few reports provide a comprehensive analysis of genes involved in both RFO biosynthesis and catabolism [START_REF] Sengupta | Significance of galactinol and raffinose family oligosaccharide synthesis in plants[END_REF][START_REF] Zhou | Genome-wide identification of genes involved in raffinose metabolism in Maize[END_REF][START_REF] Ferreira-Neto | Inositol phosphates and Raffinose family oligosaccharides pathways: Structural genomics and transcriptomics in soybean under root dehydration[END_REF] .

The availability of A. duranensis and A. ipaënsis genomes [START_REF] Bertioli | The genome sequences of Arachis duranensis and Arachis ipaensis, the diploid ancestors of cultivated peanut[END_REF] , the progenitors of peanut (A. hypogaea), enabled the genome-wide search for gene families and the assessment of their evolutionary history in these wild species. Here, a comprehensive analysis of five gene families (GolS, RS, STS, AGAL and BFLUCT) was conducted, which lead to the identification of 28 genes related to both RFO biosynthesis and catabolism in A. duranensis and 31 in A. ipaënsis. This is in accordance with the 35 RFO-related genes described for Z. mays [START_REF] Zhou | Genome-wide identification of genes involved in raffinose metabolism in Maize[END_REF] and the 58 RFO-related loci identified in G. max [START_REF] Ferreira-Neto | Inositol phosphates and Raffinose family oligosaccharides pathways: Structural genomics and transcriptomics in soybean under root dehydration[END_REF] .

The phylogenetic analysis of putative RFO-related proteins from Fabaceae, including these five enzyme families identified in wild Arachis, showed a clear subdivision within each family, supported by high bootstrap values. This clustering, based on phylogenetic proximity, was related to the intron/exon organization of the corresponding Arachis genes, as previously observed for other plant species [START_REF] Zhou | Genome-wide identification of genes involved in raffinose metabolism in Maize[END_REF][START_REF] Li | Genome-wide identification and expression profiling analysis of the galactinol synthase gene family in cassava (Manihot esculenta Crantz)[END_REF][START_REF] You | Genome-wide identification and expression analyses of genes involved in raffinose accumulation in sesame[END_REF] . Similarly to the conservation of gene structure, amino acid sequences were highly conserved too, for example, the hydrophobic pentapeptide (APSAA), characteristic of the GolS family [START_REF] Peters | Protection mechanisms in the resurrection plant Xerophyta viscosa (Baker): Both sucrose and raffinose family oligosaccharides (RFOs) accumulate in leaves in response to water deficit[END_REF] , was found for all the proteins from A. duranensis and A. ipaënsis, as well as other conserved motifs in the different enzyme families.

The highly duplicated state of the genes involved in raffinose metabolism is consistent with previous descriptions for other gene families in wild Arachis, such as expansin and NBS-LRR families [START_REF] Guimaraes | Genome-wide analysis of expansin superfamily in wild Arachis discloses a stress-responsive expansin-like B gene[END_REF][START_REF] Mota | Comparative root transcriptome of wild Arachis reveals NBS-LRR genes related to nematode resistance[END_REF] , however, this was not observed for the dehydrin family [START_REF] Mota | Contrasting effects of wild Arachis dehydrin under abiotic and biotic stresses[END_REF] . The majority of the wild Arachis RFO metabolism genes have undergone dispersed duplications. Conversely, the genes from the GolS family have undergone duplication mostly by WGD and can be observed in gene blocks in two different chromosomes. Other studies also found that segmental duplications were the driving force for the expansion of GolS genes in S. indicum and Rosaceae genomes, being associated with a possible subfunctionalization [START_REF] You | Genome-wide identification and expression analyses of genes involved in raffinose accumulation in sesame[END_REF][START_REF] Falavigna | Evolutionary diversification of galactinol synthases in Rosaceae: Adaptive roles of galactinol and raffinose during apple bud dormancy[END_REF] .

The expression profiling of wild Arachis genes differed according to the water-deficit treatment. Wild and cultivated Arachis genotypes exhibit contrasting transpiration behaviors under water-limited conditions, with variable levels of water stress tolerance among the wild species [START_REF] Leal-Bertioli | The effect of tetraploidization of wild Arachis on leaf morphology and other drought-related traits[END_REF] . Accordingly, the accessions K7988 of A. duranensis and V10309 of A. stenosperma have been selected as the drought-tolerant and the drought-sensitive genotypes, respectively, in our functional genomics studies [START_REF] Guimaraes | Functional genomics in peanut wild relatives[END_REF][START_REF] Brasileiro | Transcriptome profiling of wild Arachis from water-limited environments uncovers drought tolerance candidate genes[END_REF][START_REF] Vinson | Early responses to dehydration in contrasting wild Arachis species[END_REF][START_REF] Guimaraes | Genome-wide analysis of expansin superfamily in wild Arachis discloses a stress-responsive expansin-like B gene[END_REF][START_REF] Carmo | Comparative proteomics and gene expression analysis in Arachis duranensis reveal stress response proteins associated to drought tolerance[END_REF] . Here, the comprehensive expression analysis conducted in these genotypes revealed that most of the 28 A. duranensis genes involved in RFO metabolism, and their orthologs in A. stenosperma, are responsive to drought imposition.

Overall, the expression profiling of these genes indicated that, in wild Arachis, the molecular responses necessary to trigger RFO biosynthesis, accumulation, and eventually catabolism, differed according to the severity of the water loss process, as demonstrated for resurrection plants [START_REF] Zhang | Molecular responses to dehydration and desiccation in desiccation-tolerant angiosperm plants[END_REF] . As GolS is the first committed enzyme and the key regulator of the RFO pathway, its general induction in Arachis roots submitted to dehydration could be a rapid transcriptional response to the severe process of water loss during air-drying. However, over the 150 min time frame of the experiment, increased levels of GolS transcripts seem to be insufficient to allow the observation of the expected increase in the expression of the downstream genes involved in the subsequent steps of RFO biosynthesis (RS and STS). Similar comprehensive expression profiles of GolS, RS and STS genes were observed in response to water-deficit treatments in S. indicum, M. esculenta and G. max [START_REF] Li | Genome-wide identification and expression profiling analysis of the galactinol synthase gene family in cassava (Manihot esculenta Crantz)[END_REF][START_REF] You | Genome-wide identification and expression analyses of genes involved in raffinose accumulation in sesame[END_REF][START_REF] Ferreira-Neto | Inositol phosphates and Raffinose family oligosaccharides pathways: Structural genomics and transcriptomics in soybean under root dehydration[END_REF] . Also, genes involved in RFO catabolism (AGAL and BFLUCT ) were downregulated in response to this severe water-limited condition. It suggested the role of some members of these two multigene families in the transcriptional regulatory networks of drought tolerance in Arachis. Conversely, the moderate drought process imposed by four days of soil drying did not appear to affect the expression of GolS genes in Arachis. Under this moderate stress condition, the subsequent steps of the RFO metabolic pathway were also not yet induced, and accordingly, the transcript levels of genes coding for the enzymes that participate in RFO biosynthesis (RS and STS) and catabolism (AGAL and BFLUCT ) were kept rather constant or slightly repressed.

AdGolS3 showed opposite expression behavior between drought-tolerant and drought-sensitive wild Arachis. The overexpression of GolS genes leads to enhanced tolerance to abiotic stresses (drought, salt, heavy metal, cold and heat), by increasing galactinol and RFO contents in transgenic dicots and monocot species [START_REF] Taji | Important roles of drought and cold inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana[END_REF][START_REF] Liu | Expression of galactinol synthase from Ammopiptanthus nanus in tomato improves tolerance to cold stress[END_REF][START_REF] Selvaraj | Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field[END_REF][START_REF] Wang | Overexpression of a common wheat gene Galactinol synthase3 enhances tolerance to zinc in Arabidopsis and rice through the modulation of reactive oxygen species production[END_REF][START_REF] Himuro | Arabidopsis galactinol synthase AtGolS2 improves drought tolerance in the monocot model Brachypodium distachyon[END_REF][START_REF] Yu | Enhancement of abiotic stress tolerance in poplar by overexpression of key Arabidopsis stress response genes, AtSRK2C and AtGolS2[END_REF][START_REF] Song | Cloning of galactinol synthase gene from Ammopiptanthus mongolicus and its expression in transgenic Photinia serrulata plants[END_REF][START_REF] Zhuo | A cold responsive galactinol synthase gene from Medicago falcata (MfGolS1) is induced by myo-inositol and confers multiple tolerances to abiotic stresses[END_REF][START_REF] Honna | Molecular, physiological, and agronomical characterization, in greenhouse and in field conditions, of soybean plants genetically modified with AtGolS2 gene for drought tolerance[END_REF] . These transgenes were isolated from a number of plant species being the AtGolS2 gene from Arabidopsis, the most commonly used. An AtGolS2 ortholog has been isolated from Thellungiella salsuginea, and its overexpression also resulted in improved tolerance to abiotic stress in transgenic plants [START_REF] Sun | Overexpression of TsGOLS2, a galactinol synthase, in Arabidopsis thaliana enhances tolerance to high salinity and osmotic stresses[END_REF] . Besides being the ortholog of AtGolS2 in A. duranensis, the AdGolS3 gene was selected for further functional analysis since it exhibited differential expression behavior between the drought-tolerant A. duranensis and the drought-sensitive A. stenosperma, in both severe and moderate water-limited conditions. Moreover, our previous qRT-PCR expression analysis [START_REF] Vinson | Early responses to dehydration in contrasting wild Arachis species[END_REF] showed that AdGolS3 had a higher magnitude of expression in the tolerant A. duranensis throughout the dehydration treatment. Together, these findings indicate AdGolS3 as a putative regulator gene of the RFO pathway in the A. duranensis drought tolerance mechanisms that could be involved in the early and differential responses to severe and moderate processes of water loss.

Overexpression of AdGolS3 in Arabidopsis increased water retention, maintained membrane integrity and altered metabolic profile. Under water deficit and PEG-mediated osmotic stress, and following rehydration, Arabidopsis AdGolS3 OE lines exhibited higher RWC values than WT plants, indicating less water loss. The capacity of transgenic plants to maintain high leaf water status under abiotic stresses, as expressed in higher RWC, was also observed in some crop species overexpressing GolS genes, such as rice, poplar and chickpea [START_REF] Selvaraj | Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field[END_REF][START_REF] Yu | Enhancement of abiotic stress tolerance in poplar by overexpression of key Arabidopsis stress response genes, AtSRK2C and AtGolS2[END_REF][START_REF] Kamble | Ectopic over-expression of ABA-responsive Chickpea galactinol synthase (CaGolS) gene results in improved tolerance to dehydration stress by modulating ROS scavenging[END_REF] . This greater water retention in transgenic plants due to GolS overexpression may reflect a better uptake of soil water by roots and/or lower transpiration. It could increase their efficiency in the control of stomatal opening under water deficit conditions, as previously shown in transgenic Brachypodium distachyon plants [START_REF] Himuro | Arabidopsis galactinol synthase AtGolS2 improves drought tolerance in the monocot model Brachypodium distachyon[END_REF] . We also observed a reduced EL when OE lines were submitted to drought, salinity and osmotic stresses, and following rehydration, indicating that AdGolS3 overexpression attenuated the damage of cell membranes. The electrolyte leakage results from loss of cell membrane integrity caused by the generation of ROS in plants submitted to stress conditions and is commonly used to estimate the degree of the membrane injury. Our findings suggested that the overexpression of AdGolS3 led to a common mechanism to respond to, at least, three different types of abiotic stresses (drought, salinity and osmotic), which involves better water retention and less damage to the plasma membrane, as previously suggested by [START_REF] Himuro | Arabidopsis galactinol synthase AtGolS2 improves drought tolerance in the monocot model Brachypodium distachyon[END_REF][START_REF] Song | Cloning of galactinol synthase gene from Ammopiptanthus mongolicus and its expression in transgenic Photinia serrulata plants[END_REF][START_REF] Sun | Overexpression of TsGOLS2, a galactinol synthase, in Arabidopsis thaliana enhances tolerance to high salinity and osmotic stresses[END_REF] . The maintenance of plant water relations and cell membrane integrity have been considered important factors contributing to abiotic stress tolerance.

In addition, overexpression of AdGolS3 resulted in Arabidopsis plants with increased concentrations of few metabolites, including galactinol, product of GolS enzyme activity, and a direct precursor of raffinose. Accordingly, leaf raffinose concentrations were also higher in OE lines than in WT plants as well as raffinose representing a greater proportion of leaf sugar. As expected, when drought stress was imposed the metabolic profile in WT plants changed drastically, with increased levels of most metabolites. Nevertheless, few metabolites were altered in GolS20 and GolS22 OE lines in response to drought stress, which exhibited an overall similar metabolic profile to non-stressed CTR conditions. This suggests that AdGolS3 overexpression in these two lines may have led to a reduction in the metabolic perturbation caused by water deficit. Interestingly, qRT-PCR analysis showed that the expression of the endogenous Arabidopsis AtGolS2 gene was also highly induced in response to drought stress in all OE lines compared to WT. It is likely to have contributed to increased galactinol accumulation with minor alterations in the metabolite profile of transgenic plants. The AtGolS2 gene is known to be induced in Arabidopsis by drought and salinity stresses and is directly regulated by the heat shock transcription factors [START_REF] Taji | Important roles of drought and cold inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana[END_REF][START_REF] Song | Overexpression of heat shock factor gene HsfA3 increases galactinol levels and oxidative stress tolerance in Arabidopsis[END_REF] .

Following rehydration, most metabolite concentrations in WT plants and OE lines returned roughly to levels found in non-stressed plants. However, myo-inositol concentrations remained elevated in WT plants and two of the OE lines, possibly due to its role as one of the principal metabolites, together with galactinol, of the classical RFO pathway [START_REF] Elsayed | Physiological aspects of raffinose family oligosaccharides in plants: Protection against abiotic stress[END_REF] .

The ability of AdGolS3 to improve the tolerance to three different types of abiotic stresses corroborates previous studies that signal GolS as a regulator of the synthesis of galactinol and raffinose, and indirectly other sugars, in stress-tolerant transgenic plants [START_REF] Taji | Important roles of drought and cold inducible genes for galactinol synthase in stress tolerance in Arabidopsis thaliana[END_REF][START_REF] Selvaraj | Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field[END_REF][START_REF] Himuro | Arabidopsis galactinol synthase AtGolS2 improves drought tolerance in the monocot model Brachypodium distachyon[END_REF][START_REF] Yu | Enhancement of abiotic stress tolerance in poplar by overexpression of key Arabidopsis stress response genes, AtSRK2C and AtGolS2[END_REF][START_REF] Song | Cloning of galactinol synthase gene from Ammopiptanthus mongolicus and its expression in transgenic Photinia serrulata plants[END_REF][START_REF] Zhuo | A cold responsive galactinol synthase gene from Medicago falcata (MfGolS1) is induced by myo-inositol and confers multiple tolerances to abiotic stresses[END_REF][START_REF] Honna | Molecular, physiological, and agronomical characterization, in greenhouse and in field conditions, of soybean plants genetically modified with AtGolS2 gene for drought tolerance[END_REF][START_REF] Sun | Overexpression of TsGOLS2, a galactinol synthase, in Arabidopsis thaliana enhances tolerance to high salinity and osmotic stresses[END_REF] . This may be related to the multiple putative functions of these oligosaccharides in plants. Raffinose may act to stabilize sensitive macromolecular structures and membranes under stress as well as act as an osmolyte [START_REF] Himuro | Arabidopsis galactinol synthase AtGolS2 improves drought tolerance in the monocot model Brachypodium distachyon[END_REF] . Besides, such sugars represent also essential sources of energy, not only during germination but during recovery from a variety of abiotic stresses. Furthermore, galactinol and raffinose may also scavenge hydroxyl radicals, leading to oxidative stress defense [START_REF] Nishizawa | Galactinol and raffinose constitute a novel function to protect plants from oxidative damage[END_REF][START_REF] Stoyanova | The food additives inulin and stevioside counteract oxidative stress[END_REF][START_REF] Hernandez-Marin | Carbohydrates and their free radical scavenging capability: A theoretical study[END_REF] .

Overexpression of AdGolS3 in Arabidopsis modulated the expression of genes involved in plant protection against oxidative damage. Whilst ROS have important signaling roles in plant defense mechanisms, their increased production in response to stress can damage cellular components and is often accompanied by harmful effects on basic cellular processes [START_REF] Sami | Role of sugars under abiotic stress[END_REF][START_REF] Mhamdi | Reactive oxygen species in plant development[END_REF] . Plants have evolved detoxification mecha-nisms to control the excess accumulation of ROS that include antioxidant enzymes, such as catalase (CAT), ascorbate peroxidase (APX), and glutathione S-transferase (GST). CAT converts H 2 O 2 into H 2 O and O 2 and is induced by multiple abiotic stresses [START_REF] Alam | Comprehensive analysis and transcript profiling of Arabidopsis thaliana and Oryza sativa catalase gene family suggests their specific roles in development and stress responses[END_REF] . Likewise, APX is responsible for the removal of H 2 O 2 and its activity increases in response to stress exposure whereas the knockout of its cytosolic isoforms reduced tolerance to a variety of abiotic stresses [START_REF] Shigeoka | Cellular redox regulation, signaling, and stress response in plants[END_REF] . GSTs, such as that encoded by AtGSTU24, have both catalytic and non-catalytic activities that allow them to act in a variety of plant defense strategies, such as antioxidant regulation, adaptation and tolerance to abiotic constraints and pathogen resistance [START_REF] Nianiou-Obeidat | Plant glutathione transferase-mediated stress tolerance: Functions and biotechnological applications[END_REF][START_REF] Abuqamar | A mutation in the expansin-like A 2 gene enhances resistance to necrotrophic fungi and hypersensitivity to abiotic stress in Arabidopsis thaliana[END_REF] .

Given that the AdGolS3 OE lines exhibited increased stress tolerance, we analyzed the expression of genes coding for these three antioxidant enzymes in Arabidopsis. The expression behavior of AtCAT2; AtGSTU24 and AtAPX1 was similar, with increased transcript levels as a result of AdGolS3 overexpression, which dropped to a pattern of downregulation when plants were submitted to STR and REH conditions.

The overexpression of AdGolS3 therefore coincided with accumulation of transcripts of at least the three antioxidant enzyme-coding genes that could potentially increase plant capacity to detoxify ROS, as indicated by their expression profile under CTR conditions, though the mechanistic link between these observations is unclear. Moreover, and consistent with our analysis of electrolyte leakage, lower antioxidant enzyme transcript levels under drought stress suggests that the AdGolS3 overexpression plants suffer from less oxidative than WT plants. This could be due to the action of RFOs as non-enzymatic antioxidants [START_REF] Nishizawa | Galactinol and raffinose constitute a novel function to protect plants from oxidative damage[END_REF][START_REF] Jing | Functional characterization of galactinol synthase and raffinose synthase in desiccation tolerance acquisition in developing Arabidopsis seeds[END_REF] , or due to their action in maintaining plant water content and protecting against the negative effects of desiccation. Additional experiments, including measurement of antioxidant enzyme activities and ROS quantification, will be required to determine how RFOs act in the context of AdGolS3 overexpression.

The potential roles of AtSAP13 and AtEMB2729, genes with altered expression in Arabidopsis OE lines. Following identification of proteins that putatively interact with AtGolS2, we carried out expression analysis of a member of the "Stress-Associated Protein" family (AtSAP13) and a member of the alpha-amylase protein family (AtEMB2729). These genes displayed opposite expression behaviors, with AtSAP13 consistently being downregulated in OE lines regardless of the conditions studied (CTR, STR and REH), and AtEMB2729 being induced under these conditions. Whilst the mechanism of action of SAP family, a class of zinc-finger proteins with A20/AN1 domains, remains unknown, they have been considered as novel regulators (positive or negative) of stress signaling mediated by abscisic acid (ABA) and some representatives of the SAP family displayed a negative role in stress tolerance, by increasing plant sensitivity to drought, cold and salinity [START_REF] Zhou | Genome-wide identification and characterization of stress-associated protein (SAP) gene family encoding A20/AN1 zinc-finger proteins in Medicago truncatula[END_REF][START_REF] He | Genome-wide identification of stress-associated proteins (SAP) with A20/AN1 zinc finger domains associated with abiotic stresses responses in Brassica napus[END_REF] . Likewise, little is known about the contribution of the AtEMB2729 gene (also designated as branching enzyme 1, BE1) to abiotic stress responses [START_REF] Wang | The Arabidopsis BE1 gene, encoding a putative glycoside hydrolase localized in plastids, plays crucial roles during embryogenesis and carbohydrate metabolism[END_REF] . Besides playing a critical function in embryogenesis and maintenance of carbohydrate homeostasis, BE1 genes may also act in the regulation of auxin and cytokinin metabolism [START_REF] Wang | The Arabidopsis BE1 gene, encoding a putative glycoside hydrolase localized in plastids, plays crucial roles during embryogenesis and carbohydrate metabolism[END_REF][START_REF] Wang | The BRANCHING ENZYME1 gene, encoding a glycoside hydrolase family 13 protein, is required for in vitro plant regeneration in Arabidopsis[END_REF] . The induction of AtEMB2729 in AdGolS3 OE lines was consistent with a potential role in regulating sugar metabolism under abiotic stress conditions. Elucidation of the function of these genes in stress tolerance and their functional relationship with AtGolS2 and RFO metabolism will, however, require further investigation including phytohormone profiling.

Improved tolerance to abiotic stresses and accumulation of antinutritional effects.

In the present study, the overexpression of the AdGolS3 gene increased the abundance of galactinol and raffinose in transgenic Arabidopsis plants and conferred tolerance to drought, salt and osmotic stresses. These results therefore corroborate previous reports demonstrating that the overexpression of GolS genes can successfully impart tolerance to multiple abiotic stresses in the models Arabidopsis and tobacco [START_REF] Sengupta | Significance of galactinol and raffinose family oligosaccharide synthesis in plants[END_REF] and also in crop species such as rice, soybean, tomato, and poplar [START_REF] Liu | Expression of galactinol synthase from Ammopiptanthus nanus in tomato improves tolerance to cold stress[END_REF][START_REF] Selvaraj | Overexpression of an Arabidopsis thaliana galactinol synthase gene improves drought tolerance in transgenic rice and increased grain yield in the field[END_REF][START_REF] Yu | Enhancement of abiotic stress tolerance in poplar by overexpression of key Arabidopsis stress response genes, AtSRK2C and AtGolS2[END_REF][START_REF] Honna | Molecular, physiological, and agronomical characterization, in greenhouse and in field conditions, of soybean plants genetically modified with AtGolS2 gene for drought tolerance[END_REF] . It is worth noting, however, that the natural accumulation of RFOs in mature seeds to protect the embryo from desiccation is considered an antinutritional factor for many grain legumes [START_REF] Obendorf | Soluble carbohydrates in legume seeds[END_REF][START_REF] Toomer | Nutritional chemistry of the peanut (Arachis hypogaea)[END_REF] . Thus, the reduction of RFO content, or even their removal, to improve digestibility and nutritional quality of seeds, has been the focus of many legume breeding programs [START_REF] Silva | Effect of a mutation in Raffinose Synthase 2 (GmRS2) on soybean quality traits[END_REF] . It includes the development of transgenic plants by the antisense/RNAi suppression of RFO-synthesizing genes or by the overexpression of RFOdegrading genes [START_REF] Polowick | Over-expression of α-galactosidase in pea seeds to reduce raffinose oligosaccharide content[END_REF] . Therefore, the consistent benefits regarding abiotic stress tolerance of transgenic plants overexpressing GolS genes could come with the potential drawback: higher contents of RFOs in their mature seeds.

Conclusions

In the present work, we studied five key enzymes (GolS; RS; STS; AGAL and BFLUCT) involved in the biosynthesis and catabolism of RFOs in legumes, with an emphasis on wild relatives of peanut, A. duranensis and A. ipaënsis. This represents the first genome-wide survey of genes associated with RFO metabolism in wild Arachis, including those responsive to drought. Furthermore, the overexpression of AdGolS3 gene, encoding a galactinol synthase isolated from A. duranensis, led to increased raffinose production and tolerance to drought, salt and osmotic stresses in transgenic Arabidopsis. In addition to alterations in metabolite profile, the overexpression of AdGolS3 modulated the expression of antioxidant genes, suggesting a protective effect in preventing oxidative damage of plant cells. AdGolS3 is, therefore, a promising candidate gene for introduction in transgenic crops to increase their tolerance to abiotic stresses, though potential impacts on seed nutritional quality must be considered, particularly in legume crop engineering.

Figure 2 .

 2 Figure 2. Expression profiling of RFO metabolism Arachis genes. Heatmap of the 28 transcripts identified as involved in RFO metabolism (GolS; RS; STS; AGAL and BFLUCT) in Arachis spp. Expression patterns (log2based values from RNA-seq data) of Arachis duranensis genes and A. stenosperma orthologs were determined in plants submitted to drought (dry-down) imposed by the gradual decrease in soil water availability and to dehydration by withdrawal of hydroponic nutrient solution. The heatmap was generated by ggplot2 version 3.1 in Rstudio.

Figure 3 .

 3 Figure 3. Phenotype of A. thaliana plants submitted to three treatments. Wild-type (WT) plants and three AdGolS3 OE lines (GolS17, GolS20 and GolS22) submitted to three treatments: Control group maintained under irrigation (70% FC); Stressed group where irrigation was suspended for 20 days; and Rehydrated group where irrigation was suspended for 20 days followed by irrigation for 24 h. Each plant is a distinct individual and is representative of each treatment group.

Figure 4 .

 4 Figure 4. Performance of A. thaliana plants under the dry-down treatment. Wild-type (WT) plants and three AdGolS3 OE lines (GolS17, GolS20 and GolS22) submitted to a dry-down treatment for 20 days (Stressed group), followed by 24 h of rehydration (Rehydrated group) and the corresponding irrigated (70% FC) control (Control group). (a) Shoot biomass (grams of fresh weight per plant). (b) Percentage of leaf relative water content (RWC). (c) Percentage of leaf electrolyte leakage (EL). Bars represent value means and standard errors. * and + indicate statistically significant differences compared to control WT plants for each OE line (T-test, n = 7-10, p < 0.05 and p < 0.1 respectively).

Figure 5 .

 5 Figure 5. Performance of A. thaliana plants under the NaCl and PEG treatments. Wild-type (WT) plants and the AdGolS3 OE line GolS22 submitted to salt stress (irrigated with 150 mM NaCl) and osmotic stress (irrigated with PEG 6,000 20%) treatments for 15 days and the corresponding irrigated (70% FC) Control. (a) Percentage of leaf relative water content (RWC). (b) Percentage of leaf electrolyte leakage (EL). Bars represent value means and standard errors. * indicate statistically significant differences compared to control WT plants for GolS22 OE line (T-test, n = 11 or 12, p < 0.05).

Figure 6 .

 6 Figure 6. qRT-PCR expression analysis in A. thaliana plants. Relative quantification of mRNA levels of six Arabidopsis genes (AtAPX1, AtCAT2, AtGSTU24, AtSAP13; AtEMB2729 and AtGolS2) in the three OE lines (GolS17; GolS20 and GolS22) relative to the wild-type (WT) plants, under (a) control, (b) stressed and (c) rehydrated conditions. Values are means ± SD of three biological replicates and the significant (p < 0.05) differences between WT and OE lines are marked with *.

to 676 Family Name Species Gene name Chromosome Start End Duplication type Signal peptide

  ), besides 11 proteins in A. duranensis and 13 in A. ipaënsis, with an average length of 552 amino acids, ranging from 140

								Duplication	Signal
	Family	Name	Species	Gene name	Chromosome Start	End	type	peptide
		Aradu.EQ3HU Aradu.XWB1G	Arachis duranensis Arachis duranensis	AdGolS1 AdAGAL1	Aradu.A09 Aradu.A10	22,459,116 16,753,344	22,460,518 16,757,269	WGD WGD	NO YES
		Aradu.P39C1 Aradu.8WX2Z	Arachis duranensis Arachis duranensis	AdGolS2 AdAGAL2	Aradu.A06 Aradu.A04	6,944,440 103,979,917 103,983,868 WGD 6,947,461 WGD	NO YES
		Aradu.ZK8VV Aradu.47BJJ	Arachis duranensis Arachis duranensis	AdGolS3 AdAGAL3	Aradu.A06 Aradu.A10	5,706,973 70,967,137	5,708,738 70,973,272	WGD Dispersed	NO NO
	AGAL GolS	Aradu.Z0EIQ Aradu.9K3NU Aradu.01PEQ Araip.WG870 Araip.L3NWH Araip.L2HQR	Arachis duranensis Arachis duranensis Arachis duranensis Arachis ipaënsis Arachis ipaënsis Arachis ipaënsis	AdGolS4 AdGolS5 AdAGAL4 AiAGAL1 AiGolS1 AiAGAL2	Aradu.A09 Aradu.A09 Aradu.A05 Araip.B10 Araip.B09 Araip.B04	18,124,933 18,134,426 84,051,791 23,922,271 28,388,070 112,413,967 112,417,806 Dispersed 18,127,295 WGD 18,138,923 Tandem 84,059,234 Dispersed 23,926,161 Dispersed 28,389,727 WGD	NO NO NO YES NO YES
		Araip.M48BY Araip.EXU6F	Arachis ipaënsis Arachis ipaënsis	AiGolS2 AiAGAL3	Araip.B06 Araip.B10	9,687,845 98,873,071	9,689,970 98,879,240	WGD Dispersed	NO NO
		Araip.NEM14 Araip.VT0TG	Arachis ipaënsis Arachis ipaënsis	AiGolS3 AiAGAL4	Araip.B06 Araip.B05	11,622,775 144,126,670 144,129,965 Dispersed 11,624,454 WGD	NO NO
		Araip.SRA93 Aradu.8MB2V	Arachis ipaënsis Arachis duranensis	AiGolS4 AdBFLUCT1 Aradu.A09 Araip.B09	23,495,394 6,611,685	23,497,841 6,619,003	WGD WGD	NO YES
		Araip.40N3F Aradu.27C7S	Arachis ipaënsis Arachis duranensis	AiGolS5 AdBFLUCT2 Aradu.A08 Araip.B09	23,506,965 22,961,400	23,510,107 22,964,409	Proximal WGD	NO YES
		Aradu.HC115 Aradu.23E3L	Arachis duranensis Arachis duranensis	AdRS1 AdBFLUCT3 Aradu.A09 Aradu.A06	11,295,903 7,652,756	11,300,129 7,660,363	Dispersed Dispersed	NO NO
		Aradu.G4PX6 Aradu.W64DR	Arachis duranensis Arachis duranensis	AdRS2 AdBFLUCT4 Aradu.A08 Aradu.A07	1,275,877 16,812,196	1,279,624 16,815,218	Dispersed WGD	NO YES
		Aradu.BR5RX Aradu.ET296	Arachis duranensis Arachis duranensis	AdRS3 AdBFLUCT5 Aradu.A06 Aradu.A01	93,996,626 108,864,036 108,867,587 WGD 93,999,785 Dispersed	NO YES
		Aradu.J3GMZ Aradu.T5FHF	Arachis duranensis Arachis duranensis	AdRS4 AdBFLUCT6 Aradu.A06 Aradu.A03	39,901,809 108,878,603 108,881,468 Proximal 39,907,567 Dispersed	NO NO
		Aradu.F2QXB Aradu.YZR0Z	Arachis duranensis Arachis duranensis	AdRS5 AdBFLUCT7 Aradu.A02 Aradu.A05	86,738,383 92,641,782	86,738,756 92,647,101	Dispersed Dispersed	NO NO
		Aradu.Q8JL3 Aradu.RZZ9E	Arachis duranensis Arachis duranensis	AdRS6 AdBFLUCT8 Aradu.A06 Aradu.A03	44,328,240 70,531,598	44,341,051 70,535,609	Dispersed Dispersed	NO YES
	RS	Aradu.YX91W Aradu.NB8XZ	Arachis duranensis Arachis duranensis	AdRS7 AdBFLUCT9 Aradu.A03 Aradu.A03	40,458,135 11,428,507	40,464,544 11,436,420	Dispersed Dispersed	NO NO
		Araip.KNT6A Aradu.1B80H	Arachis ipaënsis Arachis duranensis	AiRS1 AdBFLUCT10 Aradu.A01 Araip.B07	972,692 41,923,639	976,495 41,930,127	Dispersed WGD	NO NO
		Araip.57L7Q Aradu.7N2H0	Arachis ipaënsis Arachis duranensis	AiRS2 AdBFLUCT11 Aradu.A05 Araip.B01	133,866,829 133,869,985 Dispersed 13,499,316 13,503,397 WGD	NO NO
	BFLUCT	Araip.5E5Q0 Araip.EW2ZU Araip.C8IBG Araip.XJD82	Arachis ipaënsis Arachis ipaënsis Arachis ipaënsis Arachis ipaënsis	AiRS3 AiRS4 AiBFLUCT1 Araip.B09 Araip.B03 Araip.B05 AiBFLUCT2 Araip.B08	1,783,130 141,696,277 141,702,037 Dispersed 1,791,977 WGD 8,212,457 8,219,686 WGD 1,317,638 1,321,036 WGD	NO NO YES YES
		Araip.LBU7J Araip.DD0BF	Arachis ipaënsis Arachis ipaënsis	AiRS5 AiBFLUCT3 Araip.B09 Araip.B03	46,915,043 9,475,352	46,927,158 9,484,989	Dispersed Dispersed	NO NO
		Araip.87RQD Araip.ET6IJ	Arachis ipaënsis Arachis ipaënsis	AiRS6 AiBFLUCT4 Araip.B07 Araip.B07	160,342 124,769,441 124,772,982 WGD 163,931 Dispersed	NO NO
		Aradu.K6F4E Araip.9LD3P	Arachis duranensis Arachis ipaënsis	AdSTS1 AiBFLUCT5 Araip.B06 Aradu.A06	7,161,722 133,132,276 133,136,581 WGD 7,173,062 Dispersed	NO YES
	STS	Araip.371ZH Araip.HL8V4 Araip.KD23G Araip.0311L	Arachis ipaënsis Arachis ipaënsis Arachis ipaënsis Arachis ipaënsis	AiSTS1 AiBFLUCT6 Araip.B06 Araip.B06 AiSTS2 Araip.B06 AiBFLUCT7 Araip.B02	9,408,043 133,147,400 133,150,417 Proximal 9,411,003 Tandem 9,392,860 9,396,289 Tandem 107,331,957 107,337,444 Dispersed	NO NO NO NO
		Araip.V1U21 Araip.6800 W	Arachis ipaënsis Arachis ipaënsis	AiSTS3 AiBFLUCT8 Araip.B06 Araip.B03	41,723,282 88,870,223	41,724,718 88,874,024	Dispersed Dispersed	NO YES
	Continued	Araip.571BV	Arachis ipaënsis	AiBFLUCT9 Araip.B08	8,176,140	8,183,448	Proximal	NO
		Araip.Q5XJH	Arachis ipaënsis	AiBFLUCT10 Araip.B08	8,243,124	8,244,014	Proximal	NO
		Araip.KLH8I	Arachis ipaënsis	AiBFLUCT11 Araip.B03	14,010,059	14,019,188	Dispersed	NO
		Araip.RKE1H	Arachis ipaënsis	AiBFLUCT12 Araip.B01	49,729,438	49,734,903	Dispersed	NO
		Araip.28YBL	Arachis ipaënsis	AiBFLUCT13 Araip.B05	14,535,718	14,540,357	Dispersed	NO

Table 1 .

 1 Raffinose

metabolism genes in Arachis spp. Genes coding for galactinol synthase (GolS), raffinose synthase (RS), stachyose synthase (STS), alpha-galactosidase (AGAL) and beta-fructofuranosidase (BFLUCT) identified in Arachis duranensis and Arachis ipaënsis.

  This work was supported by grants from EMBRAPA (Brazilian Agricultural Research Corporation), INCT PlantStress Biotech (project number 465480/2014-4) and FAPDF (Distrito Federal Research Foundation; project number 0193.001565/2017). CCV, APZM, BNP and ALL received fellowships from CAPES (Brazilian Federal Agency for Support and Evaluation of Graduate Education) and CNPq (Brazilian National Council for Scientific and Technological Development). EGJD was supported by a visiting scientist grant from the Science without Boarders CNPq program.

Scientific RepoRtS | (2020) 10:15258 | https://doi.org/10.1038/s41598-020-72191-4

Scientific RepoRtS| (2020) 10:15258 | https://doi.org/10.1038/s41598-020-72191-4

Acknowledgements

We are grateful to Mário Alfredo Passos Saraiva (Embrapa Cenargen, Brazil) for valuable contributions to the qRT-PCR experiments.

Funding

Author contributions

Competing interests

The authors declare no competing interests.