Calcium/Calmodulin Modulates Salt Responses through Binding a Novel Interacting Protein SAMS1 in Peanut ( Arachis hypogaea L.)

Ca 2+ /CaM signal transduction pathway is well known to help plants to adapt to the environmental stress. However, our knowledge on the function proteins of Ca 2+ /CaM pathway in peanut remains limited. Here, using yeast two hybrid methods, we identified a novel calmodulin4 (CaM4) binding protein S-adenosyl-methionine synthetase1 (SAMS1) in peanut. The expressions of AhSAMS1 were obviously induced by Ca 2+, ABA and salt stress. To elucidate the function of AhSAMS1, physiological and phenotypic analyses were applied between wild type and transgenic materials. Overexpression of AhSAMS1 significantly increased Spd and Spm synthesis while decreased the contents of ethylene, thus eliminating excessive ROS in the transgenic lines under salt stress. Consistent with the induced expressions of SOS and NHX genes, AhSAMS1 reduced the uptake of Na + and the leakage of K + from mesophyll cells, and was less sensitive to salt stress during early seedling growth. Transcriptomics combined with epigenetic regulation uncovered the relationships between DEGs and DMRs, which raised the salt tolerance and plants growth. Together, our findings support a model in which the role of AhSAMS1 on ROS-dependent regulation of ion homeostasis was enhanced by Ca 2+ /CaM while AhSAMS1-induced methylation was regulated by CaM, thus providing a novel strategy to enhance plant salt tolerance. 1 INTRODUCTION Salt stress is a major environmental factor affecting photosynthesis, plant growth and productivity (Zhao et al., 2021). The increasing salinity, especially in arable land, may result in severe agricultural yield losses worldwide. Given this fact, understanding the mechanisms improving crop tolerance to high salt content in soil has become the critical task (Yang et al., 2018). Excessive salt ions can cause damage to plants mainly by increasing osmotic pressure, ion toxicity and oxidative stresses (Zhu, 2002). Osmotic stress is the first injury when plants are exposed to saline soil, which can be adjusted by modulating stomatal opening and accumulation of compatible solutes in the cell (Fàbregas et al., 2020). Salt ions absorbed by roots are transported long distances to shoots by transpiration streams and accumulate in the leaves, eventually leading to toxic effects. Thus, maintaining a suitable Na + :K + ratio in the cytoplasm is an important adaptive trait of salt-tolerant plants to excessive ions. Na + :K + homeostasis is mainly modulate by Salt Overly Sensitive (SOS) signal transduction pathway, which is a Ca 2+ -dependent activated signal transduction pathway. The mechanism of Ca 2+ -dependent Na + leakage pathway is well known in Arabidopsis, where the expression and activity of SOS1 ion transporters were controlled by Ca 2+ responsive SOS3-SOS2 protein kinase complex (Monihan et al., 2016). Ma et al., (2012) have reported the role of NADPH oxidase AtrbohD and AtrbohF on ROS-dependent regulation of K + homeostasis in Arabidopsis thaliana under salt stress while NADPH oxidase depends on Ca 2+ content and NAD kinase which was regulated by calmodulin (CaM). As a Ca 2+ sensor, CaMs play an important role in Ca 2+ signal transduction pathway. However, CaM has no enzymatic activity itself. Only after binding and activating its target protein, CaMs can regulate plant cell division, elongation, growth, development and stress resistance (Dell’Aglio et al., 2019; Reddy et al., 2011). In the process of signal transduction, CaM mainly transfers signals in the following two ways: first, activated Ca 2+ /CaM interacts with DNA-binding protein (transcription factor, TF) to regulate TF activity and then affects DNA transcription (Reddy et al., 2011). More than 2000 DNA-binding TFs were divided into 58 families based on DNA-binding domains and other conserved motifs, half of which were vegetation-specific families (Riechmann et al., 2000). Second, Ca 2+ -CaM complex bind and regulate downstream target enzymes. For example, Ca 2+ /CaM binding protein catalase can down-regulate H 2 O 2 and reduce reactive oxygen species (ROS) content in plants, thus improving the tolerance to oxidative stress (Yang & Poovaiah, 2002). The isolation of more target proteins will help to explore the molecular mechanism of Ca 2+ /CaM regulatory pathway. CaM-targeted proteins are currently known in yeast, animals, and model plants (Kim et al., 2015, 2016). However, CaM-targeted proteins in peanut are rarely reported. When plants cannot maintain ion balance, a series of secondary reactions such as oxidative stress occur. Reactive oxygen species (ROS) at high concentrations such as superoxide radicals, hydroxyl radicals and hydrogen peroxide can oxidize and damage cytoplasmic membrane and macromolecules (DNA, lipids, and proteins) severely and disrupt cellular metabolism (Genisel et al., 2015; Golldack et al., 2014). When these defenses are supersaturated, they cause genetic changes, and ROS can also cause epigenetic changes in DNA methylation (Silva et al., 2015). DNA methylation is one of the most important epigenetic phenomena. It is closely related to the growth and development of plants, and plays an important regulatory role in genomic imprinting, transposon silencing and transgenerational epigenetic inheritance (He et al., 2011; Law & Jacobsen, 2010). The main sequences contexts of DNA methylation in plants include CG, CHG, and CHH (where H is A, C or T) and are modified by different DNA methyltransferases. S-adenosyl-methionine (SAM), as a generic methyl donor, plays a key role in determining the degree of methylation. S-adenosyl-methionine synthetase (SAMS) catalyzes the formation of SAM from S-methionine and ATP. A previous study indicates that SAMS RNAi transgenic rice ( Oryza sativa ) lines with down-regulation of OsSAMS1, 2 and 3 show reduced DNA methylation (Li et al., 2011). DNA methylation of the expression of some salt-stress-responsive genes is a regulatory mechanism for plant responses to salt stress (Karan et al., 2012). Here, a CaM4 gene were cloned from peanut ( Arachis hypogaea L.) which can be induced by salt stress. In order to further explore the regulatory mechanism of CaM4 for salt tolerance, AhSAMS1 was screened as a novel Ca 2+ /CaM-interacting protein and heterologous overexpression in tobacco and Arabidopsis improved the salt tolerance of plants. This effect appears to be achieved by modulating polyamine synthesis and methylation signaling pathways and is mediated by Ca 2+ /CaM signaling pathway. 2 MATERIALS AND METHODS 2.1 Yeast two-hybrid assays The sequence of CaM4 gene was obtained from Arachis hypogeae using RACE method in our previous experiments. Yeast two-hybrid assays were performed according to the Matchmaker TM Gold Yeast Two-Hybrid System User Manual. The CaM4 open reading frame was amplified from peanut leaves, then ligated to the pGADT7 (Clontech) vector digested by Nde I/ BamH I. The SAMS1 coding region was amplified from a plasmid and then ligated to the pGBKT7 (Clontech) vector digested by Nde I/ BamH I. According to the lithium acetate transformation method, the expression vector pGADT7-AhCaM4 and pGBKT7-AhSAMS1 was co-transformed into yeast strain Y187 (Clontech). Cells were plated onto selective medium without DDO. Putative transformants were screened by the medium containting X-a-Gal and aureobasidin (QDO/X/A) without Leu, Trp, His, and adenine. The interactions between 53 and T proteins were used as positive control as well as Lam and T proteins were used as negative control. Autoactivation was analyzed by growth experiment when the detected proteins were co-transformed with the pGADT7 or pGBKT7 empty vector. Bimolecular fluorescence complementation Assay Luciferase complementation imaging assays were performed as described by Cui et al., (2012). The CaM4 and SAMS1 genes were inserted into pCambia-NLuc and pCambia-CLuc vectors separately. All the constructs were transformed into the A. tumefaciens strain, EHA105. An equal volume of A. tumefaciens harboring pCambia-NLuc and pCambia-CLuc (or their derivative constructs) was mixed to a final concentration of OD 600 = 1.5. Epidermal cell layers of leaves from N. benthamiana were assayed for four A. tumefaciens infiltration. Plants with stable transformation were grown at 23°C and allowed to recover for 3 d. Fluorescence signal was captured using a low-light cooled charge-coupled device imaging apparatus (NightOWL II LB983 with indiGO software) and the images were deal with the Adobe Photoshop. 2.2 Pull-down analysis The full length of CaM4 was inserted between EcoR I and Xho I of pGEX-4T-1 vector while the cDNA of SAMS1 was fused between Nde I and Xba I of pCzn1. The relevant plasmids were transformed into the expressing strain. The target proteins were purified by Ni Column Affinity Purification. Then GST labeled protein was used as negative control. CaM4-GSY and SAMS1-HIS recombinant protein were mixed in the same reaction system to fully interact and GST Resin was used to collect the complex protein. The products were separated and analyzed by using GST and His antibodies, respectively. 2.3 Localization assay The PCR products were cloned into the Spe I site of pCAMBIA1302 vector, and the plasmid containing pCAMBIA1302-SAMS1 was transformed into cloning strain and A. tumefaciens strain. Tobacco was infected by A. tumefaciens mediated method with pCambia1302 as control and PCAMBIA1302-SAMS1-GFP as experimental group. Images were obtained using laser confocal microscopy three days later. 2.4 Quantitative Real-Time PCR (QRT-PCR) Total RNA was isolated from peanut leaves using TRIZOL reagent (Tiangen) and subjected to DNAase treatment using TURBO DNA-free (Ambion). 2 μg of total RNA were used to synthesize cDNA using random primers and Superscript II reverse transcriptase (Invitrogen) according to the manufacturer´s protocol. TAKARA TB green Real-Time PCR System was used in the Quantitative real-time PCR assay. QRT-PCR reactions were carried out with the following oligonucleotides: S1 (5´-ACCCAACCAAGGTAGACAG-3´)/S2 (5´-CCAGTTCCATAGGTATCAAC-3´) for AhSAMS1, TUA1 (5´-CTGATGTCGCTGTGCTCTTGG-3´)/TUA2 (5´-CTGTTGAGGTTGGTGTAGGTAGG-3´) for AhTUA5 as an internal standard. Relative expression levels were calculated according to the formula described previously (de la Torre et al., 2013). pCAMBIA1381Z was used as the GUS staining vector. The sequence of AhSAMS1 promoter was connected to the vector by double enzyme digestion. Different tissues of tobacco were transformed with the expression vector and empty vector respectively. Then cut the corresponding plant tissues into appropriate sizes and placed in a 2 ml centrifuge tube. The GUS staining solution was used to submerge the plant tissue samples completely. It was incubated at 37℃ for 5-8 h, decolorized once with anhydrous ethanol, and then decolorized three times with 75% ethanol. The images were observed and photographed under stereomicroscope. 2.5 Determination of polyamine contents by HPLC Free polyamines were detected according to the method described by Liu et al., (2002). Leaves (1.0 g) were homogenized in 4 ml of 5% (v/v) perchloric acid and centrifuged at 15,000 × g for 30 min at 4˚C. Then 0.5 ml supernatant was added with 7 μl benzoic chloride and 1 ml sodium carbonate (2 mol•l -1 ), and the mixture was incubated at 37˚C for 20 min. The product was extracted with 2 ml of saturated sodium carbonate and 2 ml of ether, and centrifuged at 1,500 × g for 5 min. 1 ml solution of ether phase was dried in vacuum. Polyamines were quantified via HPLC after redissolving with 100 μl of methanol. 2.6 Physiological and Biochemical Determination The contents of MDA and relative electrolytic leakage were measured according to the procedure described by Yang et al., (2015). The assay for H 2 O 2 content was performed according to Sairam and Srivastava’s method (Sairam & Srivastava, 2002). The O 2 •− content was determined as described by Wang & Luo, (1990). Three-week-old Arabidopsis plants were used in the straining experiment. O 2 •− and H 2 O 2 were visually detected by treating leaves with NBT, as described by Rao & Davis, (1999) and with DAB, as described by Thordal-Christensen et al., (1997). 2.7 Ion content determination Before measurement, 7-d-old Arabidopsis seedlings including AhSAMS1 transgenic lines, WT and Atsams1 mutant lines were transferred to MS medium supplemented with 150 mM NaCl and treated for 3 d at 22°C in the greenhouse. Net fluxes of Ca 2+, Na + and K + measurements were conducted using roots and were measured noninvasively by the scanning ion-selective electrode technique (SIET) at the Xuyue (Beijing) Science and Technology Co., Ltd. The samples were calibrated in a set of correction solutions (0.1 mM NaCl, 0.5 mM KCl, 0.1 mM CaCl 2, 0.1 mM MgCl 2, 0.3 mM MES, pH 6.0) before use. The experiment was conducted in six replicates and the error bars indicated the SD. 2.8 Transcriptome analysis Leaf samples from WT and Arabidopsis thaliana overexpressed AhSAMS1 lines were harvested and subjected to RNA sequencing using BGISEQ-500 platform (BGI, Beijing, china). Total RNA from each sample was then used to enrich messenger RNA and to construct complementary DNA libraries. The sequencing reads which containing adaptor-polluted, low-quality and high content of unknown base (N) reads, should be removed before downstream analyses. All sequence data was uploaded into the BioProject database hosted by the National Center for Biotechnology Information (NCBI) under the BioProject ID: PRJNA503682. The clean reads were mapped to reference genome using HISAT after reads filtering and then gene expression level for each sample was calculated by applying the fragments per kilobase per million reads (FPKM) method with RSEM. Based on the gene expression level, we can identify the DEG (Differentially expression genes) between samples or groups. We performed Gene Ontology (GO) and KEGG pathway classification and functional enrichment to annotate gene function. 2.9 Whole Genome Bisulfite Sequencing The genomic DNA isolated from Arabidopsis thaliana overexpressed AhSAMS1 lines OE2-10, WT and CPZ-treated lines (pooled in equal quantity from the three independent biological replicates) were processed for bisulphite sequencing. The genomic DNA samples were fragmented to an average size of 250 bp via bioruptor. End repaired of DNA fragments, base A and TruSeq-methylated adapters were ligated to 3’ end and the DNA fragments, respectively. Approximately, 500 ng of adapter-ligated DNA fragments were used for bisulphite conversion using EZ DNA Methylation-Gold kit（ZYMO）. After desalting, size selection and PCR amplification, library quality was sequenced. The qualified libraries were sequenced using the HiSeq 2000 system (Illumina). For screening of Differentially Methylated Regions (DMRs), we look for windows containing at least 5 CGs (CHG or CHH) at the same location in both sample genomes. DMR is the region where methylation is significantly different between the two samples (a two-fold difference, and Fisher’s test Pvalue <= 0.05). The correlation analysis between methylation and transcriptome was determined by comparing methylation status of DMR-associated genes with their expression level measured by RNA-seq. 2.10 Southern and western blot Restriction enzymes BamH I, EcoR I, EcoR Ⅴ, and Nde I were used in this study to digest the DNA extracted from peanut leaves and the fragments were separated by agarose gel electrophoresis. DNA denaturation on the gel and in situ transfer of single stranded DNA fragments to nylon membranes, hybridization with DIG-labeled probes, and staining with autoradiography. Total proteins were mixed with boiling SDS-sample buffer supplemented with 4 M urea, and then fractionated through sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% polyacrylamide gradient gel. The protein blot was hybridized with the antibodies raised in rabbits against the full-length SAMS protein. The secondary antibody was peroxidase-conjugated goat anti-rabbit IgG. 3 RESULTS 3.1 Identification of SAMS1 from peanut In our previous study, a calmodulin ( CaM ) gene was isolated from peanut using RACE method. The full length of CaM gene was 976 bp and has been deposited in GenBank (No. KF431828.1), designated as AhCaM4 . The expression of AhCaM4 could be strongly induced by NaCl (Figure S1). In order to explore the AhCaM4 binding protein in the salt-resistant regulatory pathway, yeast two-hybrid assay was carried out in this study. S-adenosylmethionine synthase was identified as one of the potential CaM-interacting proteins. The coding gene was named AhSAMS1 because it was homologous to the Arabidopsis gene, AtSAMS1 . Sequence analysis indicated that its 1182 bp open reading frame encoded a polypeptide of 394 amino acids, with a predicted molecular mass of 42.9 kDa. Based on the phylogenetic tree analysis, SAMS1 in peanut had the highest homology with SAMS1 from Medicago truncatula (Figure S2). The genomic DNA of peanut was digested by four restriction endonucleases and the copy number of SAMS in peanut genome was analyzed by Southern blot. The results showed that four hybridization signals could be detected for each enzyme product, suggesting that there may be four SAMSs homologous sequences in peanut genome (Figure S3a). 3.2 Protein interaction between AhCaM4 and AhSAMS1 Yeast two-hybrid assay was performed to confirm the interaction between AhCaM4 and AhSAMS1. All the yeast transformants grew normally on SD/-Leu/-Trp (DDO) medium. The AH109 competent yeast cells transformed with the AhSAMS1 and pGADT7 vector or AhCaM4 and pGBKT7 vector can’t grow on the SD/-Leu/-Trp/-His/-Ade/X-gal/AbA (QDO/X/A) culture medium (Figure 1a), indicating that neither AhSAMS1 nor AhCaM4 had self-activated. Only yeast cells transferred with both PGBKT7-AhSAMS1 and PGADT7-AhCAM4 grew normally and turned blue on QDO/A/X medium. Positive and negative control was used to make the experimental results more credible. Truncating the gene sequence analyses identified that the amino acids 197 to 264 of AhSAMS1 and the C-terminal domain of AhCaM4 (amino acids 53 to 149) were responsible for their interaction (Figure S4). Bimolecular fluorescence complementation (BiFC) and Glutathione-S-transferase (GST) pull-down assay were also conducted to verify that the AhSAMS1 could interact with AhCaM4. As shown in Figure 1b, SAMS1-nLUC, CaM4-cLUC, cLUC and nLUC constructs were combined in pairs and transferred to the A. tumefaciens strains respectively, then injected into four different parts at the same leaf of Nicotiana benthamiana . LUC luminescence could be detected only in leaves where co-injected with SAMS1-nLUC and CaM4-cLUC, which suggested that AhSAMS1 interacts with AhCaM4. Pull-down analysis was conducted with GST-AhCaM4 and His-AhSAMS1 proteins that were expressed and purified from Escherichia coli Arctic Express. The results showed that GST-AhCaM4 but not GST alone was pulled down by His-AhSAMS1, also indicating that AhCaM4 could interact with the AhSAMS1 protein (Figure 1c). Subcellular localization of AhSAMS1 and AhCaM4 were assayed in vivo using Arabidopsis protoplasts derived from leaves. In the protoplasts transfected with p35S-AhSAMS1-GFP or p35S-AhCaM4-GFP, which expressed AhSAMS1-GFP or AhCaM4-GFP fusion protein, the green fluorescence was obviously correlated with chloroplasts and overlapped with red self-fluorescence in chloroplast (Figure 1d). The results of onion epidermis localization showed that AhSAMS1 also functioned in both the nucleus and the cytoplasm (Figure 1d). This nuclear SAMS may contribute to the formation of SAM which is more efficient for the methylation of DNA. 3.3 The expression pattern of AhSAMS1 Semi-quantitative RT-PCR and fluorescence quantitative PCR analysis were conducted to understand the endogenous expression of AhSAMS1 gene in various organs and stresses. The results exhibited that the expression level of AhSAMS1 gene was the highest in stems, followed by flowers and leaves (Figure 2a). Accordingly, transient and precise organization expressions of AhSAMS1 were evaluated. At least 20 independent transgenic tobacco lines were created for the constructs pCAMBIA1381-AhSAMS1 promoter and pCAMBIA1381. Stronger GUS staining was observed in roots, axillary buds and leaves (Figure 2a). The expression of AhSAMS1 was induced by salt stress, ABA and Ca 2+ . Its transcription level reached the peak after treated with NaCl for 1 day and then gradually decreased (Figure 2b). ABA induced the increasing of its transcript within 24 h, and remained high within 48 h (Figure 2c). Exogenous Ca 2+ also induced AhSAMS1 expression no matter in normal growth environment or under salt stress (Figure 2d). To further confirm our results, we used western blotting to detect the abundance of AhSAMS1. The AhSAMS1 band was prominent when the peanut plants were treated with NaCl for 1 day, however after salt addition for 2 days combined with CPZ treatment, the protein content strongly decreased, and the band became weaker with the increase of processing period. Similarly, when the peanut seedlings treated with CPZ alone for 0, 1, or 3 days, the protein abundance of AhSAMS1 decreased, but the AhSAMS1 expression level treated with CPZ alone were lower than corresponding CPZ combined salt stress treatment (Figure S3b). These findings illustrated that AhCaM4 positively modulated the function of binding protein AhSAMS1 under salt stress. 3.4 Overexpression of AhSAMS increases salt tolerance The full CDS of the AhSAMS1 were cloned into the PBI121 vector and the construct were then transformed into Arabidopsis and tobacco, respectively. A total of 5 (denoted OE-2, OE-26, OE-32 in tobacco, OE1-6, OE2-10 in Arabidopsis ) transgenic lines with kanamycin-resistance (T0) were selected for further analyses according to the expression levels of AhSAMS1 in homozygous transgenic plants using semi-quantitative RT-PCR or qRT-PCR (Figure S5a). After salt stress for 5 d, the contents of spermidine (spd) and spermidine (spm) in OE1-6 and OE2-10 increased significantly. While adding calmodulin inhibitor CPZ at the same time of salt stress it was found that the content of spd and spm decreased significantly in the AhSAMS1 overexpressing lines than that in the WT and mutant lines (Figure 3a). The germination rate of AhSAMS1 -overexpressing tobacco lines OE-2, OE-26, OE-32 was significantly higher than that of WT lines (Figure 3b), which was cultured on the 1/2MS medium treated with 150 mM NaCl for 7 days. The root length of transgenic Arabidopsis thaliana and WT plants treated with 150 mM NaCl for 15 days was measured to evaluate the salt stress tolerance of AhSAMS1 transgenic plants. The results showed that root growth of the WT plants was more severely impeded compared with that of the transgenic plants (Figure 3c). The seedlings of Arabidopsis thaliana with normal growth were transplanted to the substrate soil and then cultured continuously for 45 days. After 300 mM NaCl treatment nearly all the Arabidopsis WT seedlings and atsams1 mutants were dead, while AhSAMS1 overexpression lines showed salt resistance which could continue to grow and develop (Figure 3d). In addition, relative electrical conductivity (REC) and malondialdehyde (MDA) content accumulated more in the WT plants than transgenic tobacco plants when the plants were subjected to salt stress. The WT plants engendered obviously high levels of hydrogen peroxide (H 2 O 2 ) and superoxide anion (O 2•– ) content compared with those of transgenic plants under salt conditions (Figure 3e). 3,3’-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining assay were used to observe the accumulation of H 2 O 2 and O 2•– in Arabidopsis thaliana, WT and sams mutant plants visually. After salt stress, WT plants and atsams1 mutants exhibited darker color marks than the AhSAMS transgenic lines (Figure 3f). These results indicated that AhSAMS1 protected cell membranes from peroxidation by reducing intracellular ROS accumulation. 3.5 AhSAMS1 increased the expression of salt-resistant genes The SOS pathway which composed of SOS1, SOS2 and SOS3 is responsible for ion homeostasis and salt tolerance in plants. SOS1 encodes a plasma membrane Na + /H + antiporter and SOS3 is a Ca 2+ -binding protein by activating a protein phosphatase or inhibiting a protein kinase (or by doing both) that regulates K + and Na + transport systems (Gong et al., 2020). Regulation of plants by transcription factors is of great significance to enhance plant salt tolerance. MYB, WRKY, NAC and other transcription factors in plants have been extensively demonstrated to cross-integrate with ROS, Sn RK2, ABA and H 2 O 2 signaling pathways in response to high salt stress. The expression of these salt-related genes was analyzed in this study (Figure 4a). AhSAMS1 abundance enhances and activates the expression of these salt response genes ( SOS1, SOS2, SOS3, MYB1, MYB3, NHX2, NAC1 ). 3.6 Overexpression of AhSAMS1 affected the flux of net ion under salt stress Lower cytosolic Na + content is known to be a critical determinant of salt adaptation in plants. Na + efflux mainly relies on plasma membrane Na + /H + antiporters (NHXs) to transport Na + from cell membrane, thus reducing the toxicity of Na + to organelles. Vacuolar membrane NHXs compartment Na + into vacuolar driving by the transmembrane proton gradient. Microelectrode ion flux estimation (MIFE) assay was carried out to measure the Na + and K + efflux in AhSAMS1 transgenic, WT and atsams1 mutants. After 150 mM NaCl treatment, the net Na + efflux were observed in all tested seedlings, and the net Na + efflux from transgenic roots was greater than those of control plants (Figure 4b, c). For K + flux, it was noteworthy that a significantly induced K + was measured in the transgenic line OE1-6. On the contrary, WT and mutant lines showed much smaller K + influx than transgenic plants (Figure 4d, e). These observations suggested that overexpression of AhSAMS1 in Arabidopsis increased the capacity to discharge Na + and mediated K + influx under salt stress. This could help maintain Na + :K + homeostasis and improve the salt resistance of plants. To confirm whether Ca 2+ signaling pathway is involved in functions of AhSAMS1 upon salt stress, another set of Ca 2+ flux assays was performed (Figure 4f, g). The result demonstrated that more significant Ca 2+ influx in the root of transgenic plants may coordinate with AhSAMS1 to regulate plant salt tolerance. 3.7 Ca 2+ /CaM signal modulates polyamine and ethylene synthesis To confirm the regulation of Ca 2+ /CaM signal pathway on polyamine and ethylene synthesis, AhCaM4 was transformed in tobacco and generated more than 5 transgenic lines. Two lines S5 and S8 were selected according to the expression level. Compared with the WT tobacco, the ethylene production accumulated less in S5 and S8 (Figure 5a). Accordingly, the expression of genes encoding key enzymes in ethylene synthesis pathway ACC synthase (ACS) and ACC oxidase (ACO) were inhibited in the AhCaM4 overexpressed lines. Two key enzyme genes SAMS and SAMDC in polyamine synthesis pathway expressed in the opposite manner (Figure 5b). These results indicated that Ca 2+ /CaM signaling system can promote polyamine synthesis while reduce ethylene production in the seedling stage of peanut. 3.8 Transcription analysis We determined the transcript abundance of all the genes in transgenic Arabidopsis OE2-10, WT and CPZ treated lines using RNA-seq approach. The transcript genes presenting at least two-fold change with Q-value ≤0.001 were considered as differentially expressed genes (DEGs). A total of 586 DEGs (475 upregulated and 111 downregulated) between OE2-10 and WT (OE2-10/WT), and 1,691 DEGs (1603 upregulated and 88 downregulated) between OE2-10 and CPZ (OE2-10/CPZ) could be identified (Figure 6a). Molecular process and cellular process were primarily concentrated in the biological process category, which suggested that metabolic activity is higher in transgenic plants. In the cellular component issue, cell and organelle were concentrated while binding and catalytic activity mainly enriched in the molecular function category (Figure 6c). Further analysis of the DEGs revealed that there were several genes related to peroxidase family, methylation, calcium signaling pathway and transcription factors. Among these genes, 12 genes encoding peroxidase and 10 calcium signaling related genes were up-regulated (Table 1). The expressions of genes encoding auxin responsive proteins were up-regulated in transgenic Arabidopsis plants. In addition, the up-regulated genes also include osmoregulation proteins, ion transporters, LEA proteins, Hsp20 proteins and other salt resistance related proteins. Transcription factors are essential for a range of critical cellular processes. They bind to specific DNA sequences and control the expression of a series of functional genes. In our study, at least 174 transcription factors belonging to 30 families were found to be differentially expressed. Among them, NAC, MYB/MYB-related, AP2-EREBP and WRKY families which were widely reported as salt-resistant transcription factor were expressed differently in transgenic plants compared to WT and CPZ treated lines (Figure 6b). 3.9 Differential methylation and correlation analysis of transcription level of genes related to salt stress Genome-wide profiling of DNA methylation using bisulphite sequencing was performed in OE2-10, WT and CPZ lines. A total of 2,866 differentially methylated regions (DMRs) in OE2-10/CPZ and 6,069 DMRs in OE2-10/WT were identified (Figure 7a). The total reads were mapped to the genome of TAIR 10. We then obtained the methylation level of CG, CHG, and CHH by calculating the ratio of C to C+C/T using the tool of Bismark (Krueger and Andrews 2011). The methylation levels of CG (29.9%), CHG (11.1%), and CHH (3.5%) in OE2-10 were higher than the levels of CG (27.0%), CHG (9.2%), and CHH (2.9%) in WT while CG (26.5%), CHG (9.1%), and CHH (2.9%) in CPZ. Significant difference was not identified between WT and CPZ treated lines. It is worth noting that compared with CHG and CHH, the number of mCs in CG gene body was higher than that in the flanking sequence (Figure 7b, c). The differential transcript abundance and differential methylation level between OE2-10 and CPZ, OE2-10 and WT were shown respectively in Figure 8a. Increased evidences suggested that gene expression response to environmental stress was regulated by DNA methylation. DNA methyltransferase can enhance plant stress resistance by modulating methylation level of a few typical stress resistance genes (Le et al., 2014; Song et al., 2013). In this study, association analysis identified DMR related genes involved in salt stress response and epigenetic regulation (Figure 8b). The transcript abundance of NAC062, hydrolases and RAP2.6 exhibited negatively correlated with their methylation status when exogenously sprayed with calmodulin inhibitors (OE2-10/CPZ). However, the correlation between transcription level and methylation was inconsistent in OE2/WT, most of them such as genes encoding hop3 (heat shock protein 70-heat shock protein 90 organizing proteins), F-box protein, sucrose-proton symporter with negatively regulated between methylation status and transcript abundance while ATPase and arginase showed positively correlated (Figure 8b). These results demonstrated that the methylation level of genes in plants was altered when CaM was inhibited. The regulatory mechanisms of these altered genes in response to salt stress have already been demonstrated (Fernández-Bautista et al., 2017; Liu et al., 2020; Sharma et al., 2009), their changes in DNA methylation have not been reported. 4 DISCUSSION Peanut ( Arachis hypogaea L.) is one of the important oil crops which plays a critical role in ensuring the food security and economic development. With the global climate changing, the area of arid, semi-arid and saline-alkaline land is increasing (Morton et al., 2019). Expanding peanut cultivation in saline-alkali land is one of the important ways to avoid competing with grain crops for land. It is of great significance to clarify the mechanism of peanut salt resistance and improve peanut salt resistance for increasing total peanut yield. Ca 2+ is a universal second messenger in plant response to abiotic stress. On the one hand Ca 2+ depends on the oscillation of concentration to transmit signals, on the other hand can be decoded by downstream proteins such as CaM to complete the signal transduction process and alters a number of cell responses (Ma et al., 2020). Salt stress elicit rapid increases in the expression of AhCaM4 (Figure S1), however, CaM itself has no enzyme activity, but only binds Ca 2+ and further interacts to regulate the activity of downstream target proteins (Luan et al., 2002). Here, we describe that AhSAMS1 is a novel CaM binding protein in Arachis hypogaea L. which was encoded by AhSAMS1 gene. The same localization further confirmed the interaction between AhSAMS1 and AhCaM4 (Figure 1d). SAMS, as an important stress response gene, has been cloned and functionally analyzed in a variety of plant species. In cucumber, two members of SAMS in leaves obviously induced by salt stress, changed the mRNA abundance under NaCl treatment (He et al., 2019). It has also been reported that although four HvSAMS genes in barley leaves belong to the same gene family, there were differences in the response to NaCl and ABA (Kim, 2013). In our study, peanut SAMS1 mainly expresses in axillary buds and stems compared with leaves and roots (Figure 2a). Exogenous ABA and salt stress treatment induced AhSAMS1 expression in leaves (Figure 2), suggesting that AhSAMS1 may be involved in abiotic stresses response in an ABA-dependent manner. Overexpression of peanut SAMS1 gene also promotes the synthesis of polyamines (Spd and Spm) (Figure 3a), because SAM catalyzed by SAMS is the precursor of polyamine synthesis. The accumulation of polyamines in plants contributes to stress tolerance largely through reducing ROS accumulation (Alcázar et al., 2010; Helliwell et al., 2013; Nahar et al., 2016). Here, AhSAMS1 overexpressing in tobacco decreased the contents of H 2 O 2 and O 2•– (Figure 3e, f), which was beneficial to reduce the extent of ROS-induced lipid peroxidation reflected by MDA and REC (Nankivell et al., 1994). Thus the decrease in MDA contents and REC in AhSAMS1 overexpressing lines (Figure 3e) indicates that AhSAMS1 certainly alleviates ROS damage and maintains the integrity of membrane integrity. Polyamines have been widely accepted to be a ubiquitous molecule as they regulate plenty of metabolic pathways (Miller-Fleming et al., 2015). Accordingly, the expression levels of the key genes responding to salt stress SOS1, SOS2, SOS3, NHX2, MYB3 and NAC1 were up-regulated in the AhSAMS1 overexpressing lines (Figure 4a). The SOS and NHX genes are described to be essential for regulating Na + efflux and Na + /H + transport in the cytosol of cells (Zhu, 2002). In this study, salt stress aggravate the net Na + and K + efflux, however, the ability to extrude Na + from the root was stronger in the overexpressed plants than that in the WT and mutant plants, while the K + flux in the roots was opposite in AhSAMS1 overexpression lines (Figure 4b, d). Our results demonstrated that AhSAMS1 improved salt tolerance through maintaining ion homeostasis. These findings may be associated with more accumulation of polyamine in AhSAMS1 overexpressing lines，as it has been wildly reported that polyamines mediate the expression of ion channel genes, reducing the uptake of Na + and the leakage of K + from mesophyll cells (Shabala et al., 2007). In addition, SAM also acts as a precursor of ethylene metabolism so that there is competition between polyamine and ethylene synthesis (Sauter et al., 2013; Serrano et al., 2016; Tiburcio et al., 2014). To further proven whether Ca 2+ /CaM transduction pathway plays a role in the regulation of ethylene and polyamine synthesis, tobacco plants overexpressing AhCaM4 were used in our study (Figure 5a). At the seedling stage, the expression of key enzyme genes for polyamine synthesis is increased, which promotes polyamine synthesis (Figure 5b). Conversely, down-regulated expression of ethylene synthesis gene leads to a decrease in ethylene content. These data suggested that Ca 2+ /CaM signal pathway promotes the polyamine synthesis and negatively regulated the accumulation of ethylene (Figure 5a, b). Compared with OE2-10/WT, the number of up-regulated DEGs increased significantly in OE2-10/CPZ, and the number of these genes enriched in transcription factors was much higher than that in OE2-10/WT (Figure 6a, b). These results demonstrated that the transcription levels of some genes in AhSAMS1 overexpressing plants were inhibited after exogenous spraying of CaM inhibitor CPZ. Overall, the Ca 2+ /CaM signal transduction pathway could promote the role of polyamines in plant salt resistance. SAM is not only a biosynthetic precursor of ethylene and polyamines, but also participates in DNA methylation and its abundance provides methyl donors for the methylation of DNA, proteins, and lipids in eukaryotes (Shyh-Chang et al., 2013). DNA methylation has received considerable attention for regulating the expression of important functional genes in plant abiotic stress tolerance (Chinnusamy & Zhu, 2009; Wang et al., 2011). Whole genome sequencing is helpful for us to explore the regulation mechanism of DNA methylation on salt stress in plants. There exists difference in the methylation levels of C bases in CG, CHG and CHH among various species. In AhSAMS1 overexpression plants, the proportion of mCs in CG, CHG and CHH was higher than that in the WT and CPZ-treated Arabidopsis, meanwhile the proportion of mCs mainly accumulated at CG and CHG sites in different treated lines (Figure 7b). These three types of methylation play different roles in the process of genome regulation, which will have different effects on the characteristics of the phenotype (Cokus et al., 2008; Lister et al., 2008). Combined the differential methylation patterns and gene expression analysis, the results showed that the methylation levels of some DEGs in AhSAMS1 overexpressed plants were altered under salt stress, indicating plant tolerance can be enhanced by regulating methylation levels. These observations were identical to previous reports that linking DNA methylation status to transcriptional regulation at specific sites in different plant species (Dowen et al., 2012; Zilberman et al., 2007). Although we found significant enrichment of up-regulated and down-regulated genes corresponding to hypo and hyper-DMRs respectively (Figure 8b), a few DEGs exhibit consistent trend to their methylation. Thus, any or a combination of all the differences might contribute to salt tolerance and plants growth phenotypes. At the posttranslational level, AhCaM4 enhanced the abundance of AhSAMS1 (Figure S3b). As a result, more genes responding to salt stress were up-regulated under the CaM sufficiency condition (Figure 8b). Taken together, our working model (Figure 9) helps to guide future work to identify Ca 2+ /CaM signaling mechanisms. It was illustrated that the role of AhSAMS1 on ROS-dependent regulation of ion homeostasis was enhanced by Ca 2+ /CaM while AhSAMS1-induced methylation was regulated by CaM, thus providing a novel strategy to enhance plant salt tolerance.

This study is supported by National Key R&D Program of China (2018YFD1000900), Special Funds for Local Science and Technology Development Guided by the Central Committee (YDZX20203700001861), Youth fund of Shandong Natural Science Foundation (ZR2021QC163). CONFLICT OF INTERESTS The authors declare that they have no competing interests. DATA AVAILABILITY STATEMENT RNA sequencing data has been uploaded in NCBI Sequence Read Archive with accession number SRP167756. AUTHOR CONTRIBUTIONS X.L. and S.W. conceived and designed the experiments. S.Y., J.W. and Z.T. carried out most of the experiments, S.Y. analyzed the data. Y.L. and J.Z. performed part of experiments and F.G. tended the plants. S.Y., X.L. and S.W. wrote and revised the manuscript. All authors read and approved the final manuscript. SUPPORTING INFORMATION Additional Supporting Information may be found online in the Supporting Information section at the end of the article. Figure S1. Expression analysis of CaM4 in peanut leaves treated with 150 mM NaCl for 1, 2, 6 hours. The data presented are the mean values ± standard deviation (SD) of three individual experiments. Figure S2. Alignment of deduced amino acid sequences of SAMS1 from five plant species. (a) The accession numbers in GenBank of SAMS1 are as follows: AtSAMS1 (NP_171751.1), Arabidopsis thaliana ; RcSAMS1 (XP_002512570.1), Ricinus communis ; MtSAMS1 (XP_013463713.1), Medicago truncatula ; GmSAMS1 (XP_014633684), Glycine max ; AhSAMS1, Arachis hypogaea Huayu 22. The alignment was done using DNAman. (b) Evolutionary analysis of SAMS1 in different species. Figure S3. (a) Southern blot hybridization of SAMS1 gene in peanut leaves. (b) Western blot analyses of SAMS1 encoded by AhSAMS1 gene in peanut. N1+C0: Peanut leaves were treated with NaCl for 1 day without CPZ treatment; N2+C1: Peanut leaves were treated with NaCl for 2 day and CPZ for 1day; N4+C3: Peanut leaves were treated with NaCl for 4 day and CPZ for 3 day. C1: Peanut leaves were treated only with CPZ for 1 day. Figure S4. Different regions of SAMS1 and CaM4 were fused with AD and BD vectors, respectively, and cotransformed into the yeast strain AH109. The transformants were plated on SD/-Leu-Trp-His-Ade plus X-a-Gal and aureobasidin. Figure S5. Identification of transgenic plants by semi-quantitative RT-PCR and qRT-PCR analysis. (a) The expression level analysis of AhSAMS1 gene in different Arabidopsis thaliana lines. (b) Mutant line screening of sams gene family in Arabidopsis thaliana . (c) Determination of SAM concentration in Arabidopsis thaliana overexpressed and mutant lines (d) The expression level analysis of AhSAMS1 gene in different transgenic tobacco lines. Figure S6. The DEGs belong to the different transcription factor families.

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Yeast strains harboring the indicated plasmids were grown on selective medium without Leu and Trp (DDO; left panel) or selective medium without Leu, Trp, His, and adenine and with X-a-Gal and aureobasidin (QDO/A/X; right panel). (b) BiFC visualization of Agrobacterium -infiltrated tobacco ( Nicotiana benthamiana ) leaves containing different combination of constructs. The pseudocolor bar shows the range of luminescence intensity in the image. (c) Pull-down assays with E. coli -expressed His-AhSAMS1 proteins resulted in the precipitation of GST-AhCaM4; GST alone was used as the control. Blots were first probed with the anti-GST antibody and then stripped and probed with the anti-His antibody. (d) Subcellular localization of AhSAMS1 and AhCaM4 using Arabidopsis thaliana protoplasts and onion epidermal cells. Figure 2. Expression analyses of SAMS1 in wild-type peanut lines. (a) Expression of the AhSAMS1 gene in different tissues of peanut plants. The total RNA was isolated from roots, stems, leaves, axillary bud, and gynophores of wild-type peanut plants. The responses of AhSAMS1 gene to 200 mM NaCl (b), 100 μM ABA (c) and exogenous calcium treatment (d). The transcript level of AhSAMS1 was normalized to AhTUA5 expression. Error bars represent the SDs of triplicate reactions. R: roots, S: stems, L: leaves, A: axillary bud, G: gynophores. Figure 3. AhAMS1 contributes to the salinity tolerance of tobacco and Arabidopsis. (a) The Contents determination of the Put, Spd and Spm among WT, transgenic plants and mutants under salt stress and CPZ treatment. (b) Germination rate of the selected 15-day-old WT and AhSAMS1-overexpressing tobacco plants treated with salt stress. (c) Arabidopsis seedling response to a 15 d exposure to salinity stress. OE1-6 and OE2-10, Transgenic Arabidopsis lines expressing AhAMS1 heterologously; S1-3, atsams1 mutant line. (d) Seedling phenotypes of wild-type (WT) Arabidopsis, OE1-6 and OE2-10 (Transgenic Arabidopsis lines overexpressing AhAMS1 ), S1-3 and S1-6 ( atsams1 mutant line) in response to 300 mM NaCl for 45 d. (e) Effect of salt stress on the membrane damage and the activities of O 2 •− and H 2 O 2 in WT and transgenic plants. The data presented are the mean values ± SD of three individual experiments. (f) O 2 •− and H 2 O 2 were visually detected by staining of leaves after NaCl treatment with nitroblue tetrazolium (NBT) and with 3,3-diaminobenzidine (DAB) respectively. Figure 4. Na +, K + and Ca 2+ flux from the root epidermis of WT Arabidopsis, transgenic lines (OE1-6 and OE2-10) and atsams1 mutants (S1-3 and S1-6). (a) The expression of salt tolerance-related genes was analyzed by qRT-PCR. Each column is the mean of three biologically independent samples. (b) Dynamic changes of the net Na + flux within 10 min. Seeds were germinated for 7 days in a vertical manner on 1/2MS medium and transferred to the 1/2MS medium containing 150 mM NaCl. (c) Mean rate of Na + flux within the recording period. (d) Dynamic changes of the net K + flux within 10 min. (e) Mean rate of K + flux within the recording period. (f) Dynamic changes of the net Ca 2+ flux within 10 min. (g) Mean rate of Ca 2+ flux within the recording period. Figure 5. Ca 2+ /CaM pathway regulates the synthesis of polyamines and ethylene. (a) Identification of AhCaM4 transgenic tobacco plants by semi-quantitative RT-PCR or qRT-PCR analysis and compared the ethylene content between transgenic and wild-type lines. (b) Comparision of the key genes expression in the synthesis pathways of polyamine and ethylene between WT and AhCaM4 transgenic tobacco lines. Figure 6. Differential gene expression and gene ontology (GO) enrichment analysis among the WT Arabidopsis, transgenic lines and CPZ treated lines. (a) Numbers of differently expressed genes in OE2-10/WT and OE2-10/CPZ. Numbers of upregulated and downregulated genes are shown with different colors. (b) Number of genes from top 15 transcription factor families represented in the differentially expressed genes in OE2-10/CPZ and OE2-10/WT. (c) GO enrichment analysis of genes showing differential expression in OE2-10/CPZ and OE2-10/WT, respectively. Biological process, cellular component and molecular function were three main categories. The x-axis indicates the number of genes in a category, and the y-axis indicated the GO terms. Figure 7. Differential methylation in WT, transgenic Arabidopsis lines (OE2-10) and CPZ treated lines. (a) Number of differentially (hyper and hypo) methylated regions (DMRs) between OE2-10 and CPZ (OE2-10/CPZ) and OE2-10 and WT (OE2-10/WT). (b) Whole-genome DNA methylation levels and relative changes in the DNA methylation levels of CG, CHG, and CHH in WT Arabidopsis, transgenic lines and CPZ treated lines. (c) Frequency distribution histograms of significant methylation differences ( P < 0.01). Figure 8. Correlation analysis between differential methylation and transcript abundance. (a) Heatmap representation of the differential methylation levels and differential expression of DMR-associated genes showing negative correlation. Color scales at the bottom represent status of methylation and transcript abundance. (b) Heatmap representation of the differential methylation (M) and differential expression (E) of DMR-associated genes known to be involved in salt stress response and epigenetic regulation of gene expression. Gene identifiers and gene descriptions are given on the left and right sides of the heatmap, respectively. Colors at the bottom represents status of differential methylation (hypo/hyper) and differential expression (up/down). Figure 9. A working model for AhSAMS1 mediated salt tolerance controlled by Ca 2+ /CaM signaling pathway. Figure 1. AhSAMS1 interacts with the AhCaM4 both in vitro and in vivo. (a) Yeast two-hybrid analysis of interaction between AhSAMS1 and AhCaM4. Interaction between 53 and T proteins was used as positive control and interaction between Lam and T proteins was used as negative control. Yeast strains harboring the indicated plasmids were grown on selective medium without Leu and Trp (DDO; left panel) or selective medium without Leu, Trp, His, and adenine and with X-a-Gal and aureobasidin (QDO/A/X; right panel). (b) BiFC visualization of Agrobacterium -infiltrated tobacco ( Nicotiana benthamiana ) leaves containing different combination of constructs. The pseudocolor bar shows the range of luminescence intensity in the image. (c) Pull-down assays with E. coli -expressed His-AhSAMS1 proteins resulted in the precipitation of GST-AhCaM4; GST alone was used as the control. Blots were first probed with the anti-GST antibody and then stripped and probed with the anti-His antibody. (d) Subcellular localization of AhSAMS1 and AhCaM4 using Arabidopsis thaliana protoplasts and onion epidermal cells. Figure 2. Expression analyses of SAMS1 in wild-type peanut lines. (a) Expression of the AhSAMS1 gene in different tissues of peanut plants. The total RNA was isolated from roots, stems, leaves, axillary bud, and gynophores of wild-type peanut plants. The responses of AhSAMS1 gene to 200 mM NaCl (b), 100 μM ABA (c) and exogenous calcium treatment (d). The transcript level of AhSAMS1 was normalized to AhTUA5 expression. Error bars represent the SDs of triplicate reactions. R: roots, S: stems, L: leaves, A: axillary bud, G: gynophores. Figure 3. AhAMS1 contributes to the salinity tolerance of tobacco and Arabidopsis. (a) The Contents determination of the Put, Spd and Spm among WT, transgenic plants and mutants under salt stress and CPZ treatment. (b) Germination rate of the selected 15-day-old WT and AhSAMS1-overexpressing tobacco plants treated with salt stress. (c) Arabidopsis seedling response to a 15 d exposure to salinity stress. OE1-6 and OE2-10, Transgenic Arabidopsis lines expressing AhAMS1 heterologously; S1-3, atsams1 mutant line. (d) Seedling phenotypes of wild-type (WT) Arabidopsis, OE1-6 and OE2-10 (Transgenic Arabidopsis lines overexpressing AhAMS1 ), S1-3 and S1-6 ( atsams1 mutant line) in response to 300 mM NaCl for 45 d. (e) Effect of salt stress on the membrane damage and the activities of O 2 •− and H 2 O 2 in WT and transgenic plants. The data presented are the mean values ± SD of three individual experiments. (f) O 2 •− and H 2 O 2 were visually detected by staining of leaves after NaCl treatment with nitroblue tetrazolium (NBT) and with 3,3-diaminobenzidine (DAB) respectively. Figure 4. Na +, K + and Ca 2+ flux from the root epidermis of WT Arabidopsis, transgenic lines (OE1-6 and OE2-10) and atsams1 mutants (S1-3 and S1-6). (a) The expression of salt tolerance-related genes was analyzed by qRT-PCR. Each column is the mean of three biologically independent samples. (b) Dynamic changes of the net Na + flux within 10 min. Seeds were germinated for 7 days in a vertical manner on 1/2MS medium and transferred to the 1/2MS medium containing 150 mM NaCl. (c) Mean rate of Na + flux within the recording period. (d) Dynamic changes of the net K + flux within 10 min. (e) Mean rate of K + flux within the recording period. (f) Dynamic changes of the net Ca 2+ flux within 10 min. (g) Mean rate of Ca 2+ flux within the recording period. Figure 5. Ca 2+ /CaM pathway regulates the synthesis of polyamines and ethylene. (a) Identification of AhCaM4 transgenic tobacco plants by semi-quantitative RT-PCR or qRT-PCR analysis and compared the ethylene content between transgenic and wild-type lines. (b) Comparision of the key genes expression in the synthesis pathways of polyamine and ethylene between WT and AhCaM4 transgenic tobacco lines. Figure 6. Differential gene expression and gene ontology (GO) enrichment analysis among the WT Arabidopsis, transgenic lines and CPZ treated lines. (a) Numbers of differently expressed genes in OE2-10/WT and OE2-10/CPZ. Numbers of upregulated and downregulated genes are shown with different colors. (b) Number of genes from top 15 transcription factor families represented in the differentially expressed genes in OE2-10/CPZ and OE2-10/WT. (c) GO enrichment analysis of genes showing differential expression in OE2-10/CPZ and OE2-10/WT, respectively. Biological process, cellular component and molecular function were three main categories. The x-axis indicates the number of genes in a category, and the y-axis indicated the GO terms. Figure 7. Differential methylation in WT, transgenic Arabidopsis lines (OE2-10) and CPZ treated lines. (a) Number of differentially (hyper and hypo) methylated regions (DMRs) between OE2-10 and CPZ (OE2-10/CPZ) and OE2-10 and WT (OE2-10/WT). (b) Whole-genome DNA methylation levels and relative changes in the DNA methylation levels of CG, CHG, and CHH in WT Arabidopsis, transgenic lines and CPZ treated lines. (c) Frequency distribution histograms of significant methylation differences ( P < 0.01). Figure 8. Correlation analysis between differential methylation and transcript abundance. (a) Heatmap representation of the differential methylation levels and differential expression of DMR-associated genes showing negative correlation. Color scales at the bottom represent status of methylation and transcript abundance. (b) Heatmap representation of the differential methylation (M) and differential expression (E) of DMR-associated genes known to be involved in salt stress response and epigenetic regulation of gene expression. Gene identifiers and gene descriptions are given on the left and right sides of the heatmap, respectively. Colors at the bottom represents status of differential methylation (hypo/hyper) and differential expression (up/down). Figure 9. A working model for AhSAMS1 mediated salt tolerance controlled by Ca 2+ /CaM signaling pathway. Figure S1. Expression analysis of CaM4 in peanut leaves treated with 150 mM NaCl for 1, 2, 6 hours. The data presented are the mean values ± standard deviation (SD) of three individual experiments. Figure S2. Alignment of deduced amino acid sequences of SAMS1 from five plant species. (a) The accession numbers in GenBank of SAMS1 are as follows: AtSAMS1 (NP_171751.1), Arabidopsis thaliana ; RcSAMS1 (XP_002512570.1), Ricinus communis ; MtSAMS1 (XP_013463713.1), Medicago truncatula ; GmSAMS1 (XP_014633684), Glycine max ; AhSAMS1, Arachis hypogaea Huayu 22. The alignment was done using DNAman. (b) Evolutionary analysis of SAMS1 in different species. Figure S3. (a) Southern blot hybridization of SAMS1 gene in peanut leaves. (b) Western blot analyses of SAMS1 encoded by AhSAMS1 gene in peanut. N1+C0: Peanut leaves were treated with NaCl for 1 day without CPZ treatment; N2+C1: Peanut leaves were treated with NaCl for 2 day and CPZ for 1day; N4+C3: Peanut leaves were treated with NaCl for 4 day and CPZ for 3 day. C1: Peanut leaves were treated only with CPZ for 1 day. Figure S4. Different regions of SAMS1 and CaM4 were fused with AD and BD vectors, respectively, and cotransformed into the yeast strain AH109. The transformants were plated on SD/-Leu-Trp-His-Ade plus X-a-Gal and aureobasidin. Figure S5. Identification of transgenic plants by semi-quantitative RT-PCR and qRT-PCR analysis. (a) The expression level analysis of AhSAMS1 gene in different Arabidopsis thaliana lines. (b) Mutant line screening of sams gene family in Arabidopsis thaliana . (c) Determination of SAM concentration in Arabidopsis thaliana overexpressed and mutant lines (d) The expression level analysis of AhSAMS1 gene in different transgenic tobacco lines. Figure S6. The DEGs belong to the different transcription factor families. Supplementary Material File (table 1.docx) - Download - 23.24 KB Information & Authors Information Version history Copyright This work is licensed under a Non Exclusive No Reuse License.

Authors Metrics & Citations Metrics Article Usage 227views 135downloads Citations Download citation Xin-Guo Li, Sha Yang, Jianguo Wang, et al. Calcium/Calmodulin Modulates Salt Responses through Binding a Novel Interacting Protein SAMS1 in Peanut ( Arachis hypogaea L.). Authorea. 31 January 2024. DOI: https://doi.org/10.22541/au.170670221.10024320/v1 DOI: https://doi.org/10.22541/au.170670221.10024320/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu.