1-s2.0-S1673852711001305-main

DNA methylation changes detected by methylation-sensitive amplified polymorphism in two contrasting rice genotypes under salt stressWensheng Wang a ,b ,1,Xiuqin Zhao a ,1,Yajiao Pan a ,Linghua Zhu a ,Binying Fu a ,*,Zhikang Li a ,baInstitute of Crop Sciences,National Key Facility for Crop Gene Resources and Genetic Improvement,Chinese Academy of Agricultural Sciences,Beijing 100081,ChinabInternational Rice Research Institute,DAPO Box 7777,Metro Manila,PhilippinesReceived 8April 2011;revised 19July 2011;accepted 20July 2011AbstractDNA methylation,one of the most important epigenetic phenomena,plays a vital role in tuning gene expression during plant development as well as in response to environmental stimuli.In the present study,a methylation-sensitive amplified polymorphism (MSAP)analysis was performed to profile DNA methylation changes in two contrasting rice genotypes under salt stress.Consistent with visibly different phenotypes in response to salt stress,epigenetic markers classified as stable inter-cultivar DNA methylation differences were determined between salt-tolerant FL478and salt-sensitive IR29.In addition,most tissue-specific DNA methylation loci were conserved,while many of the growth stage-dependent DNA methylation loci were dynamic between the two genotypes.Strikingly,salt stress induced a decrease in DNA methylation specifically in roots at the seedling stage that was more profound in IR29than in the FL478.This result may indicate that demethylation of genes is an active epigenetic response to salt stress in roots at the seedling stage,and helps to further elucidate the implications of DNA methylation in crop growth and development.Keywords:DNA methylation;MSAP;Salt tolerance;Rice1.IntroductionCrop growth and development is greatly influenced by various abiotic stresses,such as salinity,cold,heat and drought.Rice is an agronomically important cereal crop and it is considered to be salt-sensitive,with expressing different responses at different growth stages to salt stress.At the seedling stage,salt stress can cause a significant reduction in the germination index and seedling vigor (Shannon et al.,1998).The different responses of rice varieties have been described (Lutts et al.,1995),and previous efforts to dissect the genetic basis of salt tolerance in rice indicate that it is a quantitative trait with generally low heritability and expres-sivity (Ammar et al.,2007).A number of quantitative trait loci(QTLs)have been mapped (Takehisa et al.,2004;Hu et al.,2010),and several genes were identified to be related to salt tolerance (Ren et al.,2005;Hu et al.,2008).However,there have been only a few studies aimed at understanding the relationship between epigenetics and the regulation of gene expression under salt stress in rice.DNA methylation is an important epigenetic modification that may contribute to environmentally-induced phenotypic variations by modifying gene expression in a tissue-specific or a developmental stage-dependent manner (Angers et al.,2010;Zhang et al.,2010).Abiotic stress is one such example that results in altered gene expression by DNA hypo-methylation or hyper-methylation.In maize roots,it was reported that cold stress treatment induced DNA hypo-methylation in the nucle-osome core,resulting in up-regulation of ZmM11gene (Steward et al.,2002).Studies on leaves show that aluminum,salt,cold and oxidative stresses induced DNA demethylation in the coding sequence of NtGPDL gene within 1h,concomitant with NtGDPL gene expression (Choi and Sano,2007).There is*Corresponding author.Tel:þ861082106698,fax:þ861082108559.E-mail address:fuby@ (B.Fu).1These authors contributed equally to thiswork.Available online at Journal of Genetics and Genomics 38(2011)419e4241673-8527/$-see front matter Copyright Ó2011,Institute of Genetics and Developmental Biology,Chinese Academy of Sciences,and Genetics Society of China.Published by Elsevier Limited and Science Press.All rights reserved.doi:10.1016/j.jgg.2011.07.006an evidence to suggest that alterations in DNA methylation are required for environmental stress protection.High increases in CNG methylation level were observed in Mesembryan-themum crystallinum genome under high salinity conditions (Dyachenko et al.,2006).Furthermore,DNA methylation may endow plants with a better ability to respond to envi-ronmental stresses.For example,Wada et al.(2004)describes the activation of a set of specific stress response genes following the down-regulation of Met1-induced genomic hypo-methylation.This study sought to investigate DNA methylation changes in salt-stressed roots and leaves at various developmental stages using two rice genotypes,FL478and IR29,which share approximately50%of their genome yet have different degrees of salt tolerance(Gregorio et al.,1997).The effect of salt stress on the spatial and temporal DNA methylation changes revealed in this study will provide information for further dissecting the molecular mechanism of rice adaptation to salt stress.2.Materials and methods2.1.Plant materials and growth conditionsTwo indica rice genotypes,FL478and IR29,were used in this study.FL478is a recombinant inbred line developed at the International Rice Research Institute(IRRI)using Pokkali (salt-tolerant donor)and IR29.IR29is an improved indica cultivar currently used as a salt-sensitive standard(Bonilla et al.,2002).Previous studies showed the contrasting morphological and physiological changes of those two culti-vars under salt stress(Gregorio et al.,2002;Suriya-arunroj et al.,2004;Walia et al.,2005).All seeds were sterilized in0.1%NaClO(v/v)and then germinated at37 C in an incubator.The germinated seeds were planted in a seedling nursery.2.2.Salt stress treatment and morphological analysis at seedling stageRice plants with four leaves were transplanted into tanks,as described by Gregorio et al.(1997)and watered with Yoshida solution(Yoshida et al.,1976)in the greenhouse.Salt stress was applied by adding NaCl to afinal electrical conductivity (EC)of12dS mÀ1.The EC of control tanks was around 1.0dS mÀ1.The pH of the nutrient solution was adjusted daily to5.5by adding sulfuric acid and was refreshed every week. Rice plants were exposed to salt stress for13days.Roots and leaves under stress and control plants were collected and snap-frozen for DNA extraction.Three replicates were prepared from each sample for DNA methylation analysis.After13days of salt stress,the Modified Standard Evalua-tion System for rice was used to rate the visual symptoms of salt injury,as described by Gregorio et al.(1997)(Supplementary Table1).The plant height(cm)was measured from the base of the stem to the tip of the topmost leaf of the plants.Root length(cm)was measured for each plant.2.3.Salt stress treatment and morphological analysis at tillering and booting stagesRice plants were grown in large potsfilled with mixed soil (50N,25P and25K mg KgÀ1)and irrigated with fresh water. Salinity stress treatment was performed by adding NaCl to the stress tank until the EC was8dS mÀ1,and adjusting the EC with fresh water or NaCl every two days;the EC of the control tank was1dS mÀ1by adding fresh water.Roots and leaves were harvested and snap-frozen for DNA extraction after15 days of stress treatment.Three replicates were prepared from each sample for DNA methylation analysis.After15days of salt stress,the plant height(cm)was measured,and the tiller number was counted for each plant. Each treatment was replicated three times.The SAS program GLM was used to perform ANOV A analysis(SAS Institute, 1999).2.4.Methylation-sensitive amplified polymorphism (MSAP)analysisTotal genomic DNA was extracted using the improved CTAB method.MSAP(methylation-sensitive amplified poly-morphism)analysis was performed as described by Xiong et al.(1999),with minor modifications.First,we used several primers to identify the robustness of the three repli-cates for each sample;the results showed identical MSAP bands among three replicates(data not shown).These repli-cates were then mixed to provide a pool sample for the following MSAP analysis.Two enzyme combinations were used:Eco R I/Msp I and Eco R I/Hpa II;the sequence infor-mation for the adapters and primers of pre-amplification and selective amplification are provided in Supplementary Table2. The MSAP PCR products were separated on the6% sequencing gels and visualized with silver staining.The MSAP pattern for displaying the DNA fragments resulting from digestions with the isoschizomers were divided into the following four types(Supplementary Table3):Type I bands, present for both enzyme combinations;Type II bands,present only for Eco R I/Hpa II;Type III bands,present for Eco R I/ Msp I;and Type IV bands,absent from both enzyme combi-nations.Here,Type II represents semi-methylated bands, whereas Type III and IV bands represent semi-or full meth-ylation,respectively(Chen et al.,2009).2.5.Cloning and characterization of the differentiallyamplified DNA fragmentsDifferentially amplified fragments were selected,isolated, reamplified and purified with Wizard SV gel and the PCR clean-up system(Promega,Madison,USA).Briefly,the polymorphic bands were excised from the gel using a steril-ized surgical blade,hydrated in20m L of TE buffer(pH8.0)in an eppendorf tube and incubated in boiling water for5min. The supernatant was recovered by centrifugation and used for the re-amplification.The reamplified DNA fragments were purified and cloned with T-vector(TaKaRa,Dalian,China)for420W.Wang et al./Journal of Genetics and Genomics38(2011)419e424sequencing,and the sequences obtained were analyzed by NCBI BLAST (/Blast.cgi )and EMBL BLAST (/Tools/blast/).3.Results3.1.Phenotypic differences between FL478and IR29under salt stressThree developmental stages were used to test the various effects of salinity on rice growth:seedling,tillering and booting stages.The two rice genotypes,FL478and IR29,showed extreme variability in their reaction to salt stress at the seedling stage (Supplementary Fig.1).While FL478had a high degree of salinity tolerance (mean SES score of 2),IR29was very sensitive to salt stress (mean SES score of 7).In both genotypes,salt stress significantly reduced the plant height at the seedling stage,but the root lengths were less affected (Table 1).The plant height and tiller number of both lines were also comparatively analyzed at the tillering stage under salt stress and control conditions.The plant height of IR29was significantly reduced by salt stress treatment,but there was no significant reduction of plant height detected in FL478under salt stress (Table 1and Supplementary Fig.2).However,the tiller numbers of FL478and IR29were not affected by salinity (Table 1and Supplementary Fig.2).At the booting stage,no significant difference in plant height or tiller number were detected between salt-stressed and control plants in both lines (Table 1).3.2.MSAP analysis of DNA methylation in FL478and IR29Thirty-eight primer combinations were used to detect cytosine methylation at CCGG sequences in the two rice genotypes.During the three different developmental stages,971e 996clear and reproducible fragments were amplified in at least one sample of leaves or roots of FL478and IR29under high salinity conditions.Based on the MSAP profiles,the number of methylated (hemi-methylated and fully-methylated)DNA bands was determined.The results showed that 212e 269bands were polymorphic (Type II þType III þType IV bands),accounting for 21.8%e 27.0%of the total bands.Thesefindings indicate distinct DNA methylation levels in the different samples (Table 2).3.3.DNA methylation status in leaves or roots of FL478and IR29under normal growth conditions at three developmental stagesThe DNA methylation levels were assessed in two tissues of the FL478and IR29rice genotypes.Overall,the number of methylated DNA bands observed in the leaves or roots of FL478(230e 268)was higher than that of IR29(212e 249),comprising 23.1%e 26.9%and 21.8%e 25.6%of the total bands,respec-tively (Table 2).Among these,28bands (2.8%of the total MSAP bands)were found to be polymorphic between FL478and IR29,with 15and 10bands specific to FL478and IR29,respectively;this indicated an epigenetic difference between these two lines.All 28of these polymorphic bands were stably independent of tissue,growth stage and environmental condi-tions (Supplementary Table 4).We detected 17bands (1.7%of the total MSAP bands)that were differentially amplified between the leaf and root tissues.Of these,16were commonly tissue-specific in both FL478and IR29,with only one band specific to the leaves of IR29(Supplementary Table 5).At each of the developmental stages,a number of DNA-methylated bands were detected to be stage-specific.DNA methylation levels in leaves at the seedling and tillering stages of FL478(26.4%)and IR29(24.9%)were relatively higher than those in leaves at the booting stage (24.4%and 24.5%,respectively)under control conditions.However,in the roots,the DNA methylation level at the tillering stage was higher than the levels observed at the seedling and booting stages (Table 2).In FL478,54DNA-methylated bands were identi-fied to be stage-specific.Among them,19and 8bands were present in leaves and roots at the booting stage,respectively;but only 21and 4bands were present in roots at the seedling stage and tillering stage,respectively.As for IR29,33stage-specific DNA-methylated bands were identified.At the seed-ling,tillering and booting stages,14,4and 9bands were amplified in roots,respectively,whereas 5bands were present in leaves at the booting stage.The full data are provided as supplementary material (Supplementary Table 6).Table 1The mean performances of FL478and IR29for phenotypic traits under salinity stress and control conditions evaluated at different development stages.Stage Genotype Treatment Plant height a Root length a Tiller number a SeedlingFL478Control 52.77Æ2.8011.06Æ0.80e Stress 39.72Æ0.80***12.96Æ1.50e IR29Control 39.15Æ1.7012.81Æ0.70e Stress 26.33Æ1.30***14.41Æ0.70eTilleringFL478Control 98.23Æ4.60e 10.67Æ1.50Stress 94.33Æ4.50e 9.67Æ2.10IR29Control 87.87Æ2.38e 12Æ1.00Stress 77.27Æ2.91**e 12.67Æ1.20BootingFL478Control 114Æ2.60e 8.67Æ0.60Stress 108.33Æ6.50e 9.33Æ1.50IR29Control 100.00Æ1.00e 9.67Æ1.20Stress98.67Æ1.50e10.00Æ1.00**,***denote P ＜0.01and P ＜0.001,respectively.aResults are mean values of three replicates ÆSD.Each replicate was derived from three plants.421W.Wang et al./Journal of Genetics and Genomics 38(2011)419e 4243.4.DNA methylation changes of FL478and IR29induced by salt stressDNA methylation levels of FL478and IR29under salt-stressed and control conditions were compared by MSAP profiles(Supplementary Fig.3).As shown in Table2,FL478 leaves at the booting stage showed a slightly enhanced proportion of DNA methylation after salt stress,from244 (control)to251(stressed)bands.However,no obvious changes were detected in the FL478leaves at the seedling and tillering stages under salt stress.However,in FL478roots,salt stress decreased DNA methylation levels at the seedling stage, with the total DNA methylation bands under control and stress conditions were252and230,respectively(25.3%and23.1% of the total bands),whilst no obvious differences were found at either the tillering or booting stages.The IR29variety also showed a tendency toward the changes in DNA methylation by salt stress.Strikingly,in IR29 roots,DNA methylation levels decreased from24.6%to 21.8%at the seedling stage under salt stress,a far greater decrease than we observed in the FL478.However,at the tillering and booting stages and in both leaves and roots,DNA methylation was unaffected by salt stress.The changes in DNA methylation banding patterns(hemi-or full methylation)induced by salt stress were further analyzed.The results showed that,in both rice lines,all of these changed bands were from type IV fully-methylated DNA fragments(Table2).This indicates that salt stress induced the decrease in DNA methylation in seedling roots in both lines, and the changes resulted from the full-methylated cytosine on both DNA strands.3.5.Sequence analysis of the differentially methylatedDNA bandsTwenty-seven differentially methylated DNA bands were cloned and sequenced.The resulting sequences were blasted against the databases at NCBI and EBI websites.As shown in Table3,the range of fragment sizes was82e202bp,with an average of150bp.Among these27DNA sequences,three (S1,S2and S3)were specifically methylated during the booting stage in both lines and unaffected by salt stress.Their sequences are homologous to genes encoding GYF domain-containing proteins,histone-lysine N-methyltransferases and OsWRKY transcription factors with WRKY and zincfinger domains.Five sequences(T1to T5)were present only in leaf or root tissue and not affected by salt stress.Of them,T1and T2are leaf-specifically methylated and highly homologous to cytochrome b6-f complex subunit4,Ser/Thr phosphatase family of proteins and NB-ARC domain-containing proteins.In contrast,T3,T4and T5were root tissue-specific and homolo-gous to photosystem I assembly protein,retrotransposon and NADPH quinine oxidoreductase.The remaining nineteen DNA sequences,coded as P1e P19, were de-methylated or methylated in response to salt stress in FL478and IR29.Three of these(P1,P2and P10)were identified as genes encoding putative proteins.The rest of the sequences were found to be homologous to genes encoding hydrolases(P3,P13and P14),cytochrome subunits(P4and P6),transcription factors(P8and P15),kinases(P9and P12), and genes involved in redox regulation,such as Acetyl-CoA, peroxidases and ABC transporters.Together these results confirm that salt stress affected a large amount of diverse genes by DNA methylation changes.4.DiscussionEpigenetic processes,such as DNA methylation,can be influenced by environmental stimuli(Angers et al.,2010).In this study,the MSAP approach was used to characterize the spatial and temporal DNA methylation changes in rice caused by salt stress.The two genotypes,FL478and IR29,share at least50%of their genome,as IR29is one of the parents used to develop the RIL population,including FL478(Gregorio et al.,1997);however,these two varieties were previously identified as differing in salt tolerance.The phenotypic results in this study confirmed that these two lines have strikingly different responses to salt stress,especially at the seedlingTable2DNA methylation changes in leaves and roots at three stages under control and stress conditions.Genotype FL478IR29Tissue Leaves Roots Leaves RootsStage Seedling Tillering Booting Seedling Tillering Booting Seedling Tillering Booting Seedling Tillering Booting MSAP band types C S C S C S C S C S C S C S C S C S C S C S C SI733734733733752745744766728727732733729729729729734733732759725722729729 II777777777977777777777877III160159159159159159157157157157157157159159159159159159156156156156156156 IV969697977885886610410310099767676767172764983857979 Total methylatedbands a263262263263244251252230268269264263242242242242237238239212246249242242MSAP(%)26.426.326.426.424.525.225.323.126.92726.526.424.924.924.924.924.424.524.621.825.325.624.924.9 Fully-methylatedbands b256255256256237244245223261260257256235235235235230231232205239241235235Full methylationratio(%)25.725.625.725.723.824.524.622.426.226.125.825.724.224.224.224.223.723.823.921.124.624.824.224.2C and S indicate the control and stress conditions,respectively.a Total methylated bands¼IIþIIIþIV;b Fully-methylated bands¼IIIþIV.422W.Wang et al./Journal of Genetics and Genomics38(2011)419e424stage.Correspondingly,we detected a number of salt stress-induced DNA methylation changes that were genotype-and organ/tissue-dependent.Previous results indicated that DNA methylation differences might occur among distinct cultivars (Ashikawa,2001;Wang et al.,2004;Takata et al.,2005).In this study,we found that small but distinct changes to DNA methylation in FL478and IR29rice varieties.These stable DNA methylation differences may be epigenetic markers that are potentially linked to phenotypic variations between closely related strains (Takata et al.,2005).Previously,studies showed that tissue-specific DNA methylation was related to the differential expression of genes that constitute transcription networks regulating tissue specificity (Aceituno et al.,2008;Lu et al.,2008).We identi-fied a few common leaf-or root-specific DNA methylation bands in both FL478and IR29.These conserved,inter-cultivar,tissue-specific DNA methylation loci could be related to diverse functional differentiations of leaves and roots in rice.Additionally,a large amount of differentially methylated DNA bands was confirmed to be dependent on the growth stage in FL478and IR29,indicating the dynamic nature of cytosine methylation in different developmental stages of rice plants.Strikingly,in both lines,we found distinct decreases in DNA methylation in roots at the seedling stage induced by salt stress,but the salt-sensitive genotype IR29showed a greater decrease in DNA methylation under high-salt conditions.Further analysis indicated that the majority of salt-induced cytosine methylation changes occurred symmetrically on both DNA strands.Our results are consistent with the previ-ously reported induction of a large number of genes by salinity stress in sensitive genotypes (Walia et al.,2005,2007),con-firming that DNA methylation can fine-tune gene expression in response to salt stress.Although DNA demethylation may be either an indirect effect of salt stress or a protectiveTable 3BLAST results of 27polymorphic methylated DNA fragments.MSAP fragment Accession no./locus nameNucl.Id (%)Sequence homologyName Primer (E/HM)Size (bp)S19/3883AT5G08430.166SWIB complex BAF60b domain-containing protein/GYF domain-containing protein S210/312139LOC_Os11g3890066Histone-lysine N-methyltransferas,H3lysine-9specific SUVH1,putative,expressed S38/32115LOC_Os02g26430.163OsWRKY42e Super family of TFs having WRKY and zinc finger domains,expressed T110/312193LOC_Os09g3629079Ser/Thr protein phosphatase family protein,putative,expressed T29/32145LOC_Os05g1504065NB-ARC domain-containing protein,expressed T36/31196LOC_Os10g3821695Photosystem I assembly protein ycf3,putativeT46/315137LOC_Os12g2907072Retrotransposon protein,putative,Ty3-gypsy subclass LOC_Os01g72430j 65NADPH quinone oxidoreductase,putative,expressed T57/3890LOC_Os01g3142074Retrotransposon protein,putative,Ty3-gypsy subclass P15/35110LOC_Os04g1437074Hypothetical proteinP21/32105AC116369.5100OSJNBb0113I20genomic sequenceP39/315147AT3G60480.169Hydrolase,acting on carbon-nitrogen (but not peptide)bonds LOC_Os07g0971069OsFBX220-F-box domain-containing protein,expressedP48/313202CD718353.193An expressed sequence tag for abiotic stressed leaves of Vitis vinifera var.Chardonnay Vitis viniferaLOC_Os04g1684496Cytochrome b 6-f complex subunit 4,putative LOC_Os09g28740100Gibberellin receptor GID1L2,putative,expressedLOC_Os09g3629072Ser/Thr protein phosphatase family protein,putative,expressed P53/33128LOC_Os11g3922066Acyl-coenzyme A oxidase,putative,expressed P62/31199LOC_Os10g3703480Cytochrome P450,putativeLOC_Os07g3636082OsFBO19w F-box and other domain-containing protein,expressed LOC_Os02g2601483Terpene synthase,putative,expressed LOC_Os07g4449975Peroxidase,putative,expressedCB096663.180IRRI Drought Stress Panicle Library,Oryza sativa L Indica Group cDNA clone P710/38145LOC_Os03g2240067Ulp1protease family protein,putative,expressedP85/33143LOC_Os02g4045063DEAD/DEAH box helicase domain-containing protein,expressed P95/33198LOC_Os03g5549061Casein kinase II subunit alpha-2,putative,expressed P105/33143LOC_Os11g2538070Retrotransposon protein,putative,Ty3-gypsy subclassP115/33148LOC_Os05g1459065MCM6-Putative minichromosome maintenance MCM complex subunit 6,expressed P125/33116LOC_Os08g1029074SHR5-receptor-like kinase,putative,expressed P133/3898AT2G42990.165GDSL-motif lipase/hydrolase family protein AT4G33520.364HMA6,metal-transporting P-type ATPase 1P148/3282LOC_Os02g0962088GDSL-like lipase/acylhydrolase,putative,expressedP158/32160LOC_Os02g5296068PHD-finger domain-containing protein,putative,expressed LOC_Os04g0547062Protein kinase,putativeP168/36129LOC_Os04g5493067ABC transporter,ATP-binding protein,putative,expressedP177/38132LOC_Os09g3252666Peptidyl-prolyl cis-trans isomerase,FKBP-type,putative,expressed P186/31187LOC_Os10g3821699Photosystem I assembly protein ycf3,putativeCI435299.1100O.sativa L leaf of seedling 50ppm CuSO4for 3days,Japonica Group cDNA P1910/31291AT4G39680.172SAP domain-containing proteinLOC_Os04g54360.168Glycosyl transferase 8domain-containing protein,putative,expressed423W.Wang et al./Journal of Genetics and Genomics 38(2011)419e 424mechanism by tuning gene expression must be further eluci-dated.Most importantly,this result may suggest that deme-thylation of genes in roots at the rice seedling stage could be an active epigenetic response to salt stress.Many differential changes in DNA methylation were found in this study,and salt stress has induced extensive alterations, such as hypo-methylation or hyper-methylation,at multiple genomic loci across the rice genome.The BLAST results of the differentially methylated DNA sequences showed a similar frequency of methylation in the coding and non-coding regions of the genome,suggesting that DNA methylation changes occur throughout the whole rice genome in response to salt stress.In addition,several genes related to biotic and abiotic stress responses were differentially methylated,sug-gesting that DNA methylation is widely involved in salt stress response by regulating these genes at the transcription level.Our results may point to gene demethylation in seedling roots as an active epigenetic response to salt stress,providing further clues for unraveling the molecular mechanism of rice adaptation to salt stress.AcknowledgementsThis work was supported by the grants from the Central Public-interest Scientific Institution Basal Research Fund of China and The National High Technology Research and Development Program of China(863Program,No. 2007AA10Z191).Supplementary dataSupplementary data related to this article can be found 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