Calcium/calmodulin modulates salt responses by binding a novel interacting protein SAMS1 in peanut(Arachis hypogaea L.)

Sh Yng,Jinguo Wng,Zhohui Tng,Yn Li,Jilei Zhng,Feng Guo,Jingjing Meng,Feng Cui,Xinguo Li,*,Shuo Wn

a Institute of Crop Germplasm Resources,Shandong Academy of Agricultural Sciences,Jinan 250100,Shandong,China

b Institute of Agricultural Resources and Environment,Shandong Academy of Agricultural Sciences,Jinan 250100,Shandong,China

c Shandong Academy of Agricultural Sciences/Shandong Provincial Key Laboratory of Crop Genetic Improvement,Ecology and Physiology,Jinan 250100,Shandong,China

Keywords:AhCaM4 AhSAMS1 Protein interaction Polyamines Salt tolerance

ABSTRACT The Ca2+/CaM signal transduction pathway helps plants adapt to environmental stress.However,our knowledge on the functional proteins of Ca2+/CaM pathway in peanut(Arachis hypogeae L.)remains limited.In the present study,a novel calmodulin 4(CaM4)-binding protein S-adenosyl-methionine synthetase 1(SAMS1)in peanut was identified using a yeast two-hybrid assay.Expression of AhSAMS1 was induced by Ca2+,ABA,and salt stress.To elucidate the function of AhSAMS1,physiological and phenotypic analyses were performed with wild-type and transgenic materials.Overexpression of AhSAMS1 increased spermidine and spermidine synthesis while decreased the contents of ethylene,thereby eliminating excessive reactive oxygen species(ROS)in transgenic lines under salt stress.AhSAMS1 reduced uptake of Na+and leakage of K+from mesophyll cells,and was less sensitive to salt stress during early seedling growth,in agreement with the induction of SOS and NHX genes Transcriptomics combined with epigenetic regulation uncovered relationships between differentially expressed genes and differentially methylated regions,which raised the salt tolerance and plants growth.Our findings support a model in which the role of AhSAMS1 in the ROS-dependent regulation of ion homeostasis was enhanced by Ca2+/CaM while AhSAMS1-induced methylation was regulated by CaM,thus providing a new strategy for increasing the tolerance of plants to salt stress.

1.Introduction

Salt stress impairs photosynthesis,plant growth,and productivity[1].The increasing salinity,particularly in arable land,results in severe agricultural yield losses worldwide.Given this fact,understanding the mechanisms to improve the tolerance of crops to high salt content in the soil has become a critical endeavor[2].

Excessive salt ions damage plants primarily by increasing osmotic pressure,ion toxicity,and oxidative stresses[3].Osmotic stress is the first injury when plants are exposed to saline soil.It can be adjusted by modulation of stomatal opening and accumulation of compatible solutes in the cells[4].Salt ions absorbed by roots are transported long distances to shoots by transpiration streams and accumulate in leaves,eventually leading to toxic effects.Maintaining a suitable Na+:K+ratio in the cytoplasm is an adaptive trait of salt-tolerant plants in the presence of excessive ions.Na+:K+homeostasis is modulated primarily by the Salt Overly Sensitive(SOS)signal transduction pathway,which is a Ca2+-dependent activated signal transduction pathway.The mechanism of Ca2+-dependent Na+leakage is well known in Arabidopsis,where the expression and activity of SOS1 ion transporters are controlled by the Ca2+responsive SOS3-SOS2 protein kinase complex[5].Ma et al.[6]have reported the role of NADPH oxidases AtrbohD and AtrbohF in the ROS-dependent regulation of K+homeostasis in Arabidopsis under salt stress.NADPH oxidase depends on the content of Ca2+and NAD kinase,which is regulated by calmodulin(CaM).

As Ca2+sensors,CaM functions in the Ca2+signal transduction pathway.However,CaM has no enzymatic activity itself.CaM can regulate plant cell division,elongation,growth,development,and stress resistance only after binding and activating its target proteins[7,8].During signal transduction,CaM transfers signals mainly in the following two manners.First,activated Ca2+/CaM interact with a DNA-binding protein to regulate its activity and then affect DNA transcription[8].More than 2000 DNA-binding proteins are divided into 58 families based on their DNA-binding domains and other conserved motifs.Half of these are specific to plants[9].Second,the Ca2+-CaM complex binds to and regulates downstream target enzymes.The Ca2+/CaM binding protein catalase down-regulates the production of hydrogen peroxide(H2O2)and the content of ROS in plants,increasing tolerance to oxidative stress[10].Isolation of more target proteins will shed light on the mechanism of the Ca2+/CaM regulatory pathway.CaM-targeted proteins are currently known in yeast,animals,and model plants[11,12].However,CaM-targeted proteins in peanut have rarely been reported.

When plants cannot maintain their ion balance,a series of secondary reactions including oxidative stress occur.High concentrations of ROS,such as superoxide radicals,hydroxyl radicals,and hydrogen peroxide(H2O2)can oxidize and severely damage the cytoplasmic membrane and macromolecules(DNA,lipids,and proteins)and disrupt cellular metabolism[13,14].When these defenses are supersaturated,they cause genetic changes,and ROS can also cause epigenetic changes in DNA methylation[15].DNA methylation is closely associated with the growth and development of plants,and regulates genomic imprinting,transposon silencing,and transgenerational epigenetic inheritance[16,17].The main sequence contexts of DNA methylation in plants include CG,CHG,and CHH(where H is A,C or T,respectively)and are modified by multiple DNA methyltransferases.S-adenosyl-methionine(SAM),as a generic methyl donor,influences the degree of methylation.S-adenosyl-methionine synthetase(SAMS)catalyzes the formation of SAM from S-methionine and ATP.In a previous study[18],SAMS RNAi transgenic rice(Oryza sativa)lines in which OsSAMS1,2 and 3 were downregulated displayed a reduction in DNA methylation.Salt stress can induce demethylation of stressrelated genes coding regions and increase gene expression to cope with environmental stress[19].

In the present study,a CaM4 gene that could be induced by salt stress was cloned from peanut(Arachis hypogaea L.).To further investigate the regulatory mechanism of CaM4 for salt tolerance,AhSAMS1 was identified as a novel Ca2+/CaM-interacting protein.Heterologous overexpression of AhSAMS1 in tobacco and Arabidopsis increased their salt tolerance.This effect appears to be achieved by modulation of polyamine synthesis and methylation signaling pathways and is mediated by the Ca2+/CaM signaling pathway.

2.Materials and methods

2.1.Yeast two-hybrid assays

The sequence of the CaM4 gene was obtained from cultivated peanut using the rapid amplification of cDNA ends(RACE)method in our previous experiments.Yeast two-hybrid assays were performed according to the Matchmaker Gold Yeast Two-Hybrid System user manual.The CaM4 open reading frame was amplified from peanut leaves and ligated to the pGBKT7(Clontech)vector digested by NdeI/BamHI.A cDNA library was constructed using peanut leaves treated with salt stress.The cDNA library was combined with pGADT7 and transformed into the yeast strain Y187.CaM-interacting proteins were identified by pGBKT7-AhCAM4 and PGADT7-cDNA cotransformation.

To further confirm the interaction between AhCAM4 and AhSAMS1,the SAMS1 coding region was amplified from a plasmid and then ligated to the pGADT7(Clontech)vector digested by NdeI/BamHI.The lithium acetate transformation method was used to cotransform the expression vector pGBKT7-AhCaM4 and pGADT7-AhSAMS1 into strain Y187(Clontech).The cells were plated onto selective medium without SD/-Leu/-Trp(DDO).Putative transformants were screened on medium that contained X-a-Gal and aureobasidin(QDO/X/A)without Leu,Trp,His,or adenine.The interactions between 53 and T proteins were used as positive control and Lam and T proteins as negative control.Autoactivation was tested with a growth experiment when the detected proteins were co-transformed with the pGADT7 or pGBKT7 empty vector.

2.2.Firefly luciferase complementation imaging assay

Luciferase complementation imaging assays were performed as described by Cui et al.[20].The CaM4 and SAMS1 genes were separately inserted into pCAMBIA-NLuc and pCAMBIA-CLuc vectors.All the constructs were transformed into Agrobacterium tumefaciens strain EHA105.A.tumefaciens suspension containing equal volumes of pCAMBIA-NLuc and pCAMBIA-CLuc(or their derivative constructs)was mixed to a final concentration of OD600=1.5.Epidermal cell layers of leaves from N.benthamiana were used for four A.tumefaciens infiltrations.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 Adobe Photoshop.

2.3.Pull-down analysis

The full length of CaM4 was inserted between EcoRI and XhoI sites in the pGEX-4 T-1 vector to produce a Glutathione Stransferase(GST)-tagged recombinant protein.The cDNA of SAMS1 was fused between NdeI and XbaI sites of pCzn1 to produce a Histagged recombinant protein.The plasmids were transformed into the expression strain.His-AhCaM4 proteins were eluted from glutathione-agarose beads before incubation with GST-AhSAMS1,which remained attached to the tetradentate-chelated nickel resin for a pulldown analysis with the GST-and His-tagged proteins.In general,proteins were incubated for at least 4 h at 4°C with shaking before centrifugation.The precipitates were washed at least three times to remove nonspecific binding and boiled for 10 min.His-AhCaM4 and GST-AhSAMS1 recombinant proteins were mixed in the same reaction system to fully interact and GST resin was used to collect the complex protein.

2.4.Subcellular localization assay

Three DNA constructs(p35S-GFP,p35S-AhCaM4-GFP,and p35S-AhSAMS1-GFP)were created to investigate the intracellular targeting of AhCaM4 and AhSAMS1 using transient expression in Arabidopsis mesophyll protoplasts.The complete coding regions of AhCaM4 and AhSAMS1 were subcloned into the p35S-GFP vector,upstream to and in frame with the green fluorescence protein(GFP)coding region.Arabidopsis mesophyll protoplasts were isolated,transfected with the three constructs,and examined by laser confocal microscopy.

2.5.Detection of the expression of AhSAMS1

Total RNA was isolated from peanut leaves using TRIZOL reagent(Tiangen)and subjected to DNase treatment using TURBO DNA-free(Ambion).Two μg of total RNA were used to synthesize cDNA using random primers and Superscript II reverse transcriptase(Invitrogen)according to the manufacturer’s protocol.A TaKaRa(Dalian,Liaoning,China)TB green Real-Time PCR system was used for real-time quantitative reverse transcription PCR(qRT-PCR).qRT-PCR reactions were performed with the following oligonucleotides:S1(5′-ACCCAACCAAGGTAGACAG-3′)/S2(5′-CCA GTTCCATAGGTATCAAC-3′)for AhSAMS1,TUA1(5′-CTGATGTCGCT GTGCTCTTGG-3′)/TUA2 (5′-CTGTTGAGGTTGGTGTAGGTAGG-3′)with AhTUA5 as an internal standard.The relative levels of expression were calculated as described previously[21].For ABA treatment,the plant leaves were sprayed with 100 μmol L-1ABA.

Semiquantitative reverse transcription polymerase chain reaction(RT-PCR)was carried out using total RNAs.The amount of RNA absorbed in reverse transcription was determined based on the RNA concentration.Primers TUA5-F,TUA5-R,Actin-F,and Actin-R were used to adjust the cDNA contents of templates to the same concentration,and the RT-PCR experiments were repeated three times.

pCAMBIA1381Z was used as the β-glucuronidase(GUS)staining vector.The sequence of the AhSAMS1 promoter was connected to the vector by double enzyme digestion.Multiple tobacco tissues were transformed with the expression vector and empty vector separately.All these plant tissues were cut into small pieces and placed in 2 mL centrifuge tubes.The GUS staining solution was used to completely submerge the plant tissue samples.It was incubated at 37°C for 5–8 h and then decolorized once with anhydrous ethanol and three times with 75% ethanol.The images were observed and photographed under a stereomicroscope.

2.6.Determination of polyamine contents

Free polyamines were detected following Liu et al.[22].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.An 0.5 mL volume of supernatant was added to 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 1500×g for 5 min.A 1 mL aliquot of the ether phase was dried under vacuum.Polyamines were quantified by high-performance liquid chromatography(HPLC)after redissolution in 100 μL of methanol.

2.7.Physiological and biochemical determination

The contents of malondialdehyde(MDA)and relative electrical conductivity(REC)were measured following Yang et al.[23].The assay for the content of H2O2was performed as described by Sairam and Srivastava[24].The contents of superoxide anions(O2-)were determined following Wang and Luo[25].Threeweek-old Arabidopsis plants were used in the staining experiment.O2-and H2O2were visually detected by treating leaves with nitroblue tetrazolium(NBT),as described by Rao and Davis[26]and with 3,3′-diaminobenzidine(DAB),as described by Thordal-Christensen et al.[27].

2.8.Ion content determination

Before measurement,7-d-old Arabidopsis seedlings including the AhSAMS1 transgenic lines,wild type(WT),and Atsams1 mutant lines were transferred to MS medium supplemented with 150 mmol L-1NaCl and treated for 3 d at 22°C in the greenhouse.Net fluxes of Ca2+,Na+and K+were measured noninvasively in roots by the scanning ion-selective electrode technique(SIET)at Xuyue Science and Technology Co.,Ltd.(Beijing,China).The samples were calibrated with a set of correction solutions(0.1 mmol L-1NaCl,0.5 mmol L-1KCl,0.1 mmol L-1CaCl2,0.1 mmol L-1MgCl2,and 0.3 mmol L-1MES,pH 6.0)prior to use.The experiment was conducted with six replicates.

2.9.Transcriptome analysis

Leaf samples from WT and Arabidopsis lines that overexpressed AhSAMS1 were harvested and subjected to RNA sequencing using the BGISEQ-500 platform(BGI,Beijing,China).Total amounts and integrity of RNA were measured with a Thermo NanoDrop 2000 spectrophotometer(Thermo Fisher Scientific,Waltham,MA,USA).Total RNA from each sample was then used to enrich messenger RNA and construct complementary DNA libraries.Sequence reads containing adapter-polluted,low-quality reads,and reads with a high content of unknown bases(N)were removed.All the 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 the reference genome using HISAT after read filtering,and the level of gene expression for each sample was calculated by applying the fragments per kilobase per million reads(FPKM)method with RSEM.Based on the level of gene expression,differentially expressed genes(DEGs)in samples or groups were identified.DEG,Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)were analyzed as previously described[28].TBtools was used to visualize the gene expression[29].

2.10.Whole-genome bisulfite sequencing

Genomic DNA isolated from Arabidopsis lines overexpressing AhSAMS1(OE2-10),WT,and chlorpromazine(CPZ)-treated lines(pooled in equal quantity from three independent biological replicates)were processed for bisulfite sequencing.The genomic DNA samples were fragmented to an average size of 250 bp with Bioruptor.End-repaired DNA fragments,base A and TruSeqmethylated adapters were ligated to the 3′end and the DNA fragments,respectively.Approximately 500 ng of adapter-ligated DNA fragments were used for bisulfite conversion using EZ DNA Methylation-Gold Kit(ZYMO).After desalting,size selection,and PCR amplification,the library was sequenced on a HiSeq 2000 system(Illumina).Differentially methylated regions(DMRs)were identified as windows that contained at least five CGs(CHG or CHH)at the same location in both sample genomes.The DMR was the region where methylation differed between the two samples by at least twofold by Fisher’s test P≤0.05).The correlation between methylation and transcriptome was determined by comparing the methylation status of DMR-associated genes with their level of expression measured by RNA-seq.

2.11.Southern and Western blotting

The restriction enzymes BamHI,EcoRI,EcoRV,and NdeI were used to digest DNA extracted from peanut leaves and the fragments were separated by agarose gel electrophoresis.The DNA was denatured on the gel and the single-stranded DNA fragments were transferred to nylon membranes in situ,hybridized with digoxigenin(DIG)-labeled probes,and stained for autoradiography.

Total proteins were mixed with boiling SDS-sample buffer supplemented with 4 mol L-1urea,and then fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE)using a 12.5 % polyacrylamide gradient gel.The protein blot was hybridized with the antibodies raised in rabbits against the fulllength SAMS protein.The secondary antibody was peroxidaseconjugated goat anti-rabbit IgG.

3.Results

3.1.Identification of SAMS1 from peanut

A calmodulin(CaM)gene was isolated from peanut using the RACE method.The full length of the CaM gene was 976 bp and the sequence was deposited in GenBank(No.KF431828.1)as AhCaM4.The expression of AhCaM4 was strongly induced by NaCl(Fig.S1A).Overexpressing AhCaM4 in tobacco plants increased germination percentage and reduced ROS accumulation under salt stress(Fig.S1B;Table S1).To investigate the role of the AhCaM4-binding protein in the salt-resistant regulatory pathway,a yeast two-hybrid assay was performed.S-adenosyl methionine synthase was identified as one of the potential AhCaM4-interacting proteins.The coding gene was designated AhSAMS1 because it was homologous to the Arabidopsis gene AtSAMS1.Its 1182-bp open reading frame encoded a polypeptide of 394 amino acids,with a predicted molecular weight of 42.9 kDa.Based on a phylogenetic tree,SAMS1 in peanut had the highest similarity to SAMS1 from Medicago truncatula(Fig.S2).The genomic DNA of peanut was digested with four restriction endonucleases and the copy number of SAMS in the peanut genome was determined by Southern blotting.Four hybridization signals could be detected for each enzyme product,suggesting that there were four homologous SAMSs sequences in the peanut genome(Fig.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.AH109 competent yeast cells transformed with the pGADT7-AhSAMS1 and pGBKT7 vectors or the pGBKT7-AhCAM4 and pGADT7 vectors could not grow on SD/-Leu/-Trp/-His/-Ade/X-gal/AbA(QDO/X/A)culture medium(Fig.1A),indicating that neither AhSAMS1 nor AhCaM4 had self-activated.Only yeast cells transformed with both pGADT7-AhSAMS1 and pGBKT7-AhCAM4 grew normally and turned blue on QDO/A/X medium.Positive and negative controls were used to ensure that the experimental results were credible.Sequence truncation analyses showed 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(Fig.S4).

Firefly luciferase complementation imaging assay(LCI)and GST pulldown assay were also performed to verify that AhSAMS1 could interact with AhCaM4.As shown in Fig.1B,SAMS1-nLUC,CaM4-cLUC,cLUC and nLUC constructs were combined in pairs,transferred separately to the A.tumefaciens strains,and injected into four different parts of the same N.benthamiana leaf.LUC luminescence was detected only in leaves that had been co-injected with SAMS1-nLUC and CaM4-cLUC,suggesting that AhSAMS1 interacts with AhCaM4.Pulldown analysis was performed with GSTAhCaM4 and His-AhSAMS1 proteins that were expressed and purified from E.coli.GST-AhCaM4 but not GST alone was pulled down by His-AhSAMS1,also indicating that AhCaM4 could interact with the AhSAMS1 protein(Fig.1C).

Subcellular localization of AhSAMS1 and AhCaM4 was performed in vivo using Arabidopsis protoplasts derived from leaves.The recombinant fusion was transiently expressed in Arabidopsis leaf protoplasts.The location of green fluorescence corresponded with those of the nucleus,cytoplasm,and plasma membrane in protoplasts transfected with p35S-AhSAMS1-GFP or p35SAhCaM4-GFP,which expressed respectively AhSAMS1-GFP or AhCaM4-GFP fusion proteins(Fig.1D).

3.3.The expression pattern of AhSAMS1

Semi-quantitative RT-PCR and fluorescence quantitative PCR analysis were performed to investigate the endogenous expression of AhSAMS1 in various organs and under various stresses.Expression of AhSAMS1 gene was highest in stems,followed by flowers and leaves(Fig.2A).Accordingly,the transient and precise organization expression of AhSAMS1 was evaluated.At least 20 independent transgenic tobacco lines were created for the constructs pCAMBIA1381-AhSAMS1 promoter and pCAMBIA1381.Stronger GUS staining was also observed in stems,axillary buds,and leaves(Fig.2A).The expression of AhSAMS1 was induced by salt stress,ABA,and Ca2+.Its transcription level reached a peak after treatment with NaCl for 1 d and then gradually decreased(Fig.2B).ABA induced the increase of its transcript within 24 h,and the level of expression remained high for 48 h(Fig.2C).Exogenous Ca2+also induced the expression of AhSAMS1 in either normal growth or salt stress environments(Fig.2D).

Fig.1.AhSAMS1 interacts with AhCaM4 both in vitro and in vivo.(A)Yeast twohybrid 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)LCI visualization of Agrobacteriuminfiltrated tobacco(Nicotiana benthamiana)leaves containing combinations of constructs.The pseudocolor bar shows the range of luminescence intensity in the image.(C)Pulldown 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 anti-GST antibody and then stripped and probed with anti-His antibody.(D)Subcellular localization of AhSAMS1 and AhCaM4 using Arabidopsis thaliana protoplasts.

To further confirm our results,we used Western blotting to detect the abundance of AhSAMS1.The AhSAMS1 band was prominent when plants were treated with NaCl for 1 d.However,after salt treatment for 2 d combined with CPZ treatment,the protein content was greatly decreased,and the band became weaker after salt treatment for 4 d combined with CPZ treatment.Similarly,when the seedlings were treated solely with CPZ for 0,1,or 3 d,the protein abundance of AhSAMS1 decreased,but the expression of AhSAMS1 treated with CPZ alone was lower than that of the corresponding combination of CPZ with salt-stress treatment(Fig.S3B).These findings showed that AhCaM4 positively modulated the function of binding protein AhSAMS1 under salt stress.

Fig.2.Expression analyses of SAMS1 in wild-type peanut lines.(A)Expression of the AhSAMS1 gene in tissues of peanut plants.Total RNA was isolated from roots,stems,leaves,axillary buds,and gynophores of wild-type peanut plants.Responses of AhSAMS1 to 200 mmol L-1 NaCl(B),100 μmol L-1 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 buds;G,gynophores.Values are means±standard deviation(SD)of three individual experiments.Different letters indicate differences among treatments(P＜0.05).

3.4.Overexpression of AhSAMS1 increased salt tolerance

The full coding sequence(CDS)of AhSAMS1 was cloned into the pBI121 vector and the construct was then transformed separately into Arabidopsis and tobacco.Five(named OE-2,OE-26,and OE-32 in tobacco and OE1-6 and OE2-10 in Arabidopsis)transgenic plants with kanamycin resistance(T0)were selected for further analyses according to the levels of expression of AhSAMS1 in homozygous transgenic plants using semi-quantitative RT-PCR or qRT-PCR(Fig.S5A).The atsams1 mutants were purchased from the TAIR database and screened with the primers LP and RP(Fig.S5B).After salt stress for 5 d,the contents of spermidine(spd)and spermidine(spm)in OE1-6 and OE2-10 were significantly increased.When the calmodulin inhibitor CPZ was added at the time of salt stress,the contents of spd and spm decreased significantly in the AhSAMS1-overexpressing lines relative to those in the wild type(WT)and mutant lines(Fig.3A).

The germination rate of AhSAMS1-overexpressing tobacco lines OE-2,OE-26,and OE-32 was significantly higher than that of the WT lines(Fig.3B).The root length of transgenic Arabidopsis and WT plants treated with 150 mmol L-1NaCl for 15 d was measured to evaluate the salt-stress tolerance of AhSAMS1 transgenic plants.The root growth of the WT plants was more severely impeded than that of the transgenic plants(Fig.3C).Seedlings of Arabidopsis with normal growth were transplanted to the substrate soil and cultured continuously for 45 d.Nearly all the WT Arabidopsis seedlings and atsams1 mutants died after treatment with 300 mmol L-1NaCl,whereas the AhSAMS1-overexpressing lines were resistant to salt and continued to grow and develop(Fig.3D).Contents of REC and MDA accumulated more in WT than in transgenic tobacco plants when the plants were subjected to salt stress.WT plants generated high levels of H2O2and O2-compared with those of transgenic plants under salt treatment(Fig.3E).DAB and NBT staining assays were used to visually observe the accumulation of H2O2and O2-in Arabidopsis,WT,and sams mutant plants.After salt stress,WT plants and atsams1 mutants showed darker color than the AhSAMS1 transgenic lines(Fig.3F).These results indicated that AhSAMS1 protected cell membranes from peroxidation by reducing the accumulation of intracellular ROS.

3.5.AhSAMS1 increased the expression of salt-resistant genes

The plant SOS pathway,which is composed of SOS1,SOS2,and SOS3,is responsible for ion homeostasis and salt tolerance.SOS1 encodes a plasma membrane Na+/H+antiporter,and SOS3 is a Ca2+-binding protein that activates a protein phosphatase or inhibits a protein kinase(or does both)that regulates the K+and Na+transport systems[30].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 be as important as ROS,SnRK2,ABA,and H2O2signaling pathways in response to high salt stress.The expression of these salt-responsive genes was measured in this study(Fig.4A).Abundant AhSAMS1 activates the expression of these salt response genes(SOS1,SOS2,SOS3,MYB1,MYB3,NHX2,and NAC1).

3.6.Overexpression of AhSAMS1 affected the net ion flux under salt stress

Fig.3.AhAMS1 contributes to the salinity tolerance of tobacco and Arabidopsis.(A)The Contents determination of Put,Spd,and Spm among WT,transgenic plants and mutants under salt stress and CPZ treatment.(B)Germination rate of the selected 15-d-old WT and AhSAMS1-overexpressing tobacco plants exposed to 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 exposure to 300 mmol L-1 NaCl for 45 d.(E)Effect of salt stress on membrane damage and activities of O2-and H2O2 in WT and transgenic plants.Values are means±standard deviation(SD)of three experiments.(F)O2-and H2O2 were visually detected by staining of leaves after NaCl treatment with nitroblue tetrazolium(NBT)and 3,3-diaminobenzidine(DAB)separately.Values are means±standard deviation(SD)of three experiments.Asterisks indicate differences from WT exposed to the same treatment(*,P＜0.05).

Fig.4.Na+,K+and Ca2+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-responsive genes was measured by qRT-PCR.Each column represents the mean of three biologically independent samples.(B)Dynamic changes of net Na+flux within 10 min.Seeds were germinated for 7 d and were placed vertically on 1/2 MS medium and transferred to 1/2 MS medium containing 150 mmol L-1 NaCl.(C)Mean rate of Na+flux during the recording period.(D)Dynamic changes of net K+flux during 10 min.(E)Mean rate of K+flux during the recording period.(F)Dynamic changes of net Ca2+flux during 10 min.(G)Mean rate of Ca2+flux during the recording period.Values are means±standard deviation(SD)of six plants.Asterisks indicate differences from WT of the same treatment(*,P＜0.05).

A lower content of cytosolic Na+is known to be a critical determinant of salt adaptation in plants.Na+efflux relies mainly on plasma membrane Na+/H+antiporters(NHXs)to transport Na+out of the cell membrane,thus reducing the toxicity of Na+to organelles.Vacuolar membrane NHXs confine Na+in vacuoles,driven by the transmembrane proton gradient.Microelectrode ion flux estimation(MIFE)assay was performed to measure the Na+and K+efflux in AhSAMS1 transgenic,WT,and atsams1 mutants.After treatment with 150 mmol L-1NaCl,a net efflux of Na+was observed in all the seedlings tested,and the net Na+efflux from transgenic roots was higher than those of control plants(Fig.4B,C).For K+flux,it was noteworthy that a significant K+flow was measured in the transgenic line OE1-6.In contrast,WT and mutant lines showed much smaller K+influx than transgenic plants(Fig.4D,E).These observations suggested that the overexpression of AhSAMS1 in Arabidopsis increased the capacity to discharge Na+and mediated K+influx under salt stress.This could help to maintain Na+:K+homeostasis and improve the salt resistance of plants.To confirm whether the Ca2+signaling pathway is involved in functions of AhSAMS1 upon salt stress,another set of Ca2+flux assays was performed(Fig.4F,G).The results suggested that more significant Ca2+influx in the roots of transgenic plants than that in WT plants may coordinate with AhSAMS1 to regulate plant salt tolerance of plants.

3.7.Ca2+/CaM signal modulates polyamine and ethylene synthesis

To confirm the regulation by the Ca2+/CaM signal pathway of polyamine and ethylene synthesis,AhCaM4 was transformed in tobacco and more than five transgenic lines were generated.Two lines(S5 and S8)were selected according to their level of expression.In comparison with WT tobacco,less ethylene accumulated in S5 and S8(Fig.5A).The expression of genes that encode key enzymes in ethylene synthesis pathway,such as ACC synthase(ACS)and ACC oxidase(ACO)was correspondingly inhibited in the AhCaM4ioverexpressing lines.Two key enzyme-encoding genes,SAMS and SAMDC,in the polyamine synthesis pathway were expressed in the opposite manner(Fig.5B).

3.8.Transcription analysis

We determined the transcript abundance of all the genes in transgenic Arabidopsis OE2-10,WT and CPZ-treated lines using a RNA-seq approach.Transcripts presenting at least twofold change with Q-value≤0.001 were considered differentially expressed genes(DEGs).A total of 586 DEGs(475 upregulated and 111 downregulated)differentiating OE2-10 and WT(OE2-10/WT),and 1691 DEGs(1603 upregulated and 88 downregulated)differentiating OE2-10 and CPZ(OE2-10/CPZ)were identified(Fig.6A).Molecular process and cellular process were concentrated in the biological process category,which suggested that metabolic activity was higher in transgenic plants.In the cellular component analysis,cells and organelles were concentrated,whereas binding and catalytic activity was enriched mainly in the molecular function category(Fig.6C).There were several genes associated with peroxidase family,methylation,calcium signaling pathway,and transcription factors.Among them,12 genes encoding peroxidase and 10 calcium signaling-associated genes were up-regulated(Table 1).Expression of genes encoding auxin-responsive proteins was upregulated in transgenic Arabidopsis plants.Up-regulated genes also included osmoregulation proteins,ion transporters,late embryogenesis abundant(LEA)proteins,Hsp20 proteins,and other salt resistance-associated 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.At least 174 transcription factors belonging to 30 families were differentially expressed.Among them,NAC,MYB/MYBrelated,AP2-EREBP and WRKY families that are widely reported to be salt-resistance transcription factors were expressed differently in transgenic plants than in the WT and CPZ-treated lines(Fig.6B).

Table 1 Genes differentially expressed between OE2-10 and WT.

Fig.5.The Ca2+/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 comparison of ethylene content between transgenic and wild-type lines.(B)Comparison of expression of key genes in the synthesis pathways of polyamine and ethylene between WT and AhCaM4 transgenic tobacco lines.Asterisks indicate significant differences from WT of the same treatment(*,P＜0.05).

Fig.6.Differential gene expression and Gene Ontology(GO)enrichment analysis in 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 in 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 the three main categories.The x-axis indicates the number of genes in a category and the y-axis indicates the GO terms.

3.9.Differential methylation and correlation analysis of transcription levels of genes responding to salt stress

Genome-wide profiling of DNA methylation using bisulfite sequencing was performed in OE2-10,WT,and CPZ lines.A total of 2866 DMRs in OE2-10/CPZ and 6069 DMRs in OE2-10/WT were identified(Fig.7A).The reads were mapped to the genome of TAIR 10.We obtained the methylation level of CG,CHG,and CHH by calculating the ratio of C to C+C/T using the Bismark methylation tool[31].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 and of CG(26.5%),CHG(9.1%),and CHH(2.9%)in CPZ.There was no significant difference between WT and CPZ-treated lines.In contrast to CHG and CHH,the number of mCs in the CG gene body was higher than that in the flanking sequence(Fig.7B,C).

The differential transcript abundances and differential methylation levels between OE2-10 and CPZ and OE2-10 and WT are shown in Fig.8A.The response of gene expression to environmental stress appeared to be regulated by DNA methylation.DNA methyltransferase can increase plant stress resistance by modulating methylation levels of a few typical stress-resistance genes[32,33].In this study,association analysis identified genes associated with DMR that were involved in salt stress response and epigenetic regulation(Fig.8B).The transcript abundances of NAC062,hydrolases,and RAP2.6 were negatively correlated with their methylation status following exogenous application of 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 proteins,and sucroseproton symporter displayed opposite regulation trends between their methylation status and transcript abundance,whereas ATPase and arginase showed the same regulation trends(Fig.8B).These results demonstrated that the level of methylation of the 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[34–36].However,to our knowledge,their changes in DNA methylation have not been reported.

4.Discussion

Peanut is an oil crop that supports food security and economic development.With changes in the global climate,the areas of arid,semi-arid and saline–alkaline land are increasing[37].Expanding the cultivation of peanut in saline–alkaline land is an important way to avoid competing with grain crops for land.Thus,it is desirable to clarify the mechanism of peanut salt resistance and improve it for increasing peanut yield.

Fig.7.Differential methylation in WT,transgenic Arabidopsis lines(OE2-10),and CPZ-treated lines.(A)Numbers 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).

Fig.8.Correlation between differential methylation and transcript abundance.(A)Heat map 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)Heat map 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 shown on respectively the left and right sides of the heat map.Colors at the bottom represents status of differential methylation(hypo-or hyper-)and differential expression(up/down).

Ca2+is a universal second messenger in the response of plants to abiotic stress.On the one hand Ca2+depends on the oscillation of concentration to transmit signals,on the other hand it can be decoded by downstream proteins such as CaM to complete the signal transduction process and alter cell responses[6].Salt stress elicits rapid increases in the expression of AhCaM4,which can improve the salt resistance of transgenic tobacco(Fig.S1;Table S1).However,CaM itself has no enzymatic activity;it only binds Ca2+and further interacts to regulate the activity of target proteins[38].In this study,we have shown that AhSAMS1 is a novel CaM-binding protein in peanut that is encoded by AhSAMS1.The same localization further confirmed the interaction between AhSAMS1 and AhCaM4(Fig.1D).

SAMS,a stress-responsive gene,has been cloned and functionally analyzed in a variety of plant species.In cucumber,two members of SAMS in leaves were induced[39].It has also been reported[40]that although four HvSAMS genes in barley leaves belong to the same gene family,there were differences in their responses to NaCl and ABA.In our study,peanut SAMS1 was expressed more highly in stems and axillary buds than in leaves and roots(Fig.2A).Exogenous ABA and salt stress treatment induced the expression of AhSAMS1 in leaves(Fig.2).Its elevated level of expression could contribute to the increased protection of plants from the damaging effects of salt stress in an ABA-dependent manner.The overexpression of the peanut SAMS1 gene also promotes the synthesis of polyamines(Spd and Spm)(Fig.3A),because SAM catalyzed by SAMS is a precursor for polyamine synthesis.The accumulation of polyamines in plants contributes to their tolerance to stress by reducing the accumulation of ROS[41–43].In the present study,overexpressing AhSAMS1 in tobacco reduced the contents of H2O2and O2-(Fig.3E,F).As a result,fewer ROS were transferred to the cytoplasm,resulting in less damage to the plasma membrane in AhSAMS1-overexpressing lines than in WT seedlings(Fig.3E)[44].These results suggest that AhSAMS1 catalysis of polyamine synthesis acts to alleviate oxidative stress and damage to cellular components,such as membrane lipids,under salt stress.

Polyamines regulate numerous metabolic pathways[45].Expression of the key genes SOS1,SOS2,SOS3,NHX2,MYB3 and NAC1 that respond to salt stress were up-regulated in lines that overexpressed AhSAMS1(Fig.4A).SOS3,a myristoylated calciumbinding protein,interacts with and activates SOS2.The SOS2/SOS3 kinase complex phosphorylates and activates the SOS1 protein,which is assumed[3]to mediate the efflux of Na+at the root epidermis and long-distance transport from roots to shoots in Arabidopsis while protecting individual cells from Na+toxicity under severe salt stress.In the present study,salt stress increased the net efflux of Na+and K+;however,the ability to extrude Na+from the root was stronger in the overexpressing plants than in the WT and mutant plants,while the K+flux in roots showed the opposite behavior in AhSAMS1-overexpressing lines(Fig.4B,D).Our results suggest that AhSAMS1 is involved in Na+and/or K+homeostasis.These findings may be associated with the increased accumulation of polyamines in AhSAMS1-overexpressing lines,given that it has been reported[46]that polyamines mediate the expression of ion-channel genes,reducing uptake of Na+and leakage of K+from mesophyll cells.

SAM also acts as a precursor in ethylene metabolism,so that there is competition between polyamine and ethylene synthesis[47–49].In the CaM4-overexpressing lines,the expression of key enzyme genes for polyamine synthesis is increased,which promotes polyamine synthesis(Fig.5B).In contrast,down-regulated expression of ACS and ACO led to decreased ethylene content.These observations suggested that the Ca2+/CaM signal pathway promotes the synthesis of polyamines and negatively regulates the accumulation of ethylene at the seedling stage.In the transcriptome experiment,the number of up-regulated DEGs increased significantly in OE2-10/CPZ in comparison with OE2-10/WT,and the number of these genes enriched in transcription factors was much higher than that in OE2-10/WT(Fig.6A,B).These results also show that the transcription levels of some genes in plants that overexpressed AhSAMS1 were inhibited after the CaM inhibitor CPZ was exogenously applied.Overall,the Ca2+/CaM signal transduction pathway could promote the synthesis of polyamines in plant salt resistance.

Fig.9.A working model for AhSAMS1-mediated salt tolerance controlled by the Ca2+/CaM signaling pathway.

SAM not only is 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[50].DNA methylation has received considerable attention for regulating the expression of important genes in the tolerance of plants to abiotic stress[51,52].Whole-genome sequencing is helpful for investigatingof the regulatory mechanism of DNA methylation on salt stress in plants.There are differences among species in the levels of methylation of the C bases in CG,CHG and CHH.In AhSAMS1-overexpressing plants,the proportion of mCs in CG,CHG and CHH was higher than that in the WT and CPZ-treated Arabidopsis,and the proportion of mCs accumulated mainly at CG and CHG sites in treated lines(Fig.7B).These three types of methylation play different roles in genome regulation,with differing effects on phenotype[53,54].Combining the analyses of differential methylation patterns and gene expression showed that the methylation levels of some DEGs in plants that overexpressed AhSAMS1 were altered under salt stress,indicating that tolerance can be increased by regulation of methylation levels.These observations were identical to those of previous studies that linked DNA methylation status to transcriptional regulation at specific sites in multiple plant species[55,56].Although we found significant enrichment of up-regulated and down-regulated genes corresponding to hypo and hyper-DMRs respectively(Fig.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 the phenotypes of plant growth.At the posttranslational level,AhCaM4 increased the abundance of AhSAMS1(Fig.S3B).As a result,more genes that responded to salt stress were upregulated under the CaM sufficiency condition.Taken together,our working model(Fig.9)may guide future work to identify Ca2+/CaM signaling mechanisms.In it,the role of AhSAMS1 in ROS-dependent regulation of ion homeostasis is enhanced by Ca2+/CaM,while AhSAMS1-induced methylation is regulated by CaM,providing a novel strategy for increasing the tolerance of plants to salt stress.

Accession numbers

RNA sequencing data has been uploaded to the NCBI Sequence Read Archive under accession ID SRP167756.

CRediT authorship contribution statement

Sha Yang:Writing–review & editing,Writing–original draft,Data curation.Jianguo Wang:Writing–original draft.Zhaohui Tang:Data curation.Yan Li:Methodology.Jialei Zhang:Methodology.Feng Guo:Methodology.Jingjing Meng:Resources.Feng Cui:Resources.Xinguo Li:Writing–review & editing.Shubo Wan:Writing–review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This study is supported by the National Key Technology Research and Development Program of China(2018YFD1000900),the Natural Science Foundation of Shandong Province(ZR2020MC094),the Natural Science Foundation of Shandong Province(ZR2021QC163),Special Funds for Local Science and Technology Development Guided by the Central Committee(YDZX20203700001861).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.06.007.