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Salt-Induced Tissue-Specific Cytosine MethylationDownregulates Expression of
 HKT 
 Genes in ContrastingWheat (
Triticum aestivum 
 L.) Genotypes
Suresh Kumar, Ananda Sankara Beena, Monika Awana, and Archana Singh
Plants have evolved several strategies, including regulation of genes through epigenetic modifications, to copewith environmental stresses. DNA methylation is dynamically regulated through the methylation and demethy-lation of cytosine in response to environmental perturbations. High-affinity potassium transporters (HKTs) haveaccounted for the homeostasis of sodium and potassium ions in plants under salt stress. Wheat (
Triticum aestivum
L.) is sensitive to soil salinity, which impedes its growth and development, resulting in decreased productivity.The differential expression of 
 HKTs
 has been reported to confer tolerance to salt stress in plants. In this study, weinvestigated variations in cytosine methylation and their effects on the expression of 
 HKT 
 genes in contrastingwheat genotypes under salt stress. We observed a genotype- and tissue-specific increase in cytosine methylationinduced by NaCl stress that downregulated the expression of 
 TaHKT2;1
 and
 TaHKT2;3
 in the shoot and roottissues of Kharchia-65, thereby contributing to its improved salt-tolerance ability. Although
 TaHKT1;4
 wasexpressed only in roots and was downregulated under the stress in salt-tolerant genotypes, it was not regulatedthrough variations in cytosine methylation. Thus, understanding epigenetic regulation and the function of HKTswould enable an improvement in salt tolerance and the development of salt-tolerant crops.
Keywords:
 DNA methylation, epigenetics, gene regulation, salt stress,
 Triticum aestivum
Introduction
P
lants
,
 being sessile
, have developed several strategiesto cope with environmental stresses, including alter-ations in the expression level of genes through epigeneticmodifications such as DNA methylation. DNA methylationplays a key role in gene expression through the RNA-directed DNA methylation (RdDM) of genes and the in-duction of histone modifications. Cytosine methylation hasbeen reported to be involved in many vital biological pro-cesses, including transposon movement, genome imprinting,and regulation of gene expression (Yan
 et al.
, 2010). Abioticstresses have direct, negative effects on the biochemicaland physiological processes that are associated with plantgrowth and development, which results in a significant re-duction in crop yield.One of the detrimental effects of salinity is the accumu-lation of sodium ion (Na
+
) in plant tissues, which inhibitsthe uptake of the potassium ion (K 
+
) from soil. Na
+
and K 
+
have similar chemical properties and content ratio in non-saline soils; however, the physiological effects of these ionson the metabolism and growth of plants are quite different.Maintaining a high K 
+
 /Na
+
ratio has been suggested to be amajor strategy for plants to cope with salt stress (Hamamoto
et al.
, 2015).At the cellular level, the mechanisms for salt tolerancefunction to reduce Na
+
accumulation in the cytoplasm bylimiting the entry of Na
+
into cells, actively transporting Na
+
out of cells, and compartmentalizing Na
+
into vacuoles (Shi
et al.
, 2003). K 
+
is preferred for uptake into roots from thesoil, and most plants exhibit a high degree of K 
+
 /Na
+
dis-crimination for uptake. High-affinity potassium transporters(HKTs) have been reported to be active at the plasmamembrane level and function as Na
+
 /K 
+
symporters as wellas selective Na
+
uniporters (Horie
 et al.
, 2009). HKTs mayhave two major functions, namely to take up Na
+
from thesoil to reduce the requirement of K 
+
when K 
+
is a limitingfactor and to reduce the accumulation of Na
+
in the leaf byremoving Na
+
from the xylem sap and loading Na
+
into thephloem sap (Brini
 et al.
, 2009). HKTs belong to the HKT/ Trk/Ktr-type
+
transporter superfamily that is found inmicroorganisms and plants (Yamaguchi
 et al.
, 2013).
Division of Biochemistry, Indian Agricultural Research Institute, New Delhi, India.
ª
 Suresh Kumar
 et al.
, 2017; Published by Mary Ann Liebert, Inc. This Open Access article is distributed under the terms of the CreativeCommons Attribution Noncommercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits any noncommercial use,distribution, and reproduction in any medium, provided the original author(s) and the source are credited.
DNA AND CELL BIOLOGYVolume 36, Number 4, 2017Mary Ann Liebert, Inc.Pp. 283–294DOI: 10.1089/dna.2016.3505
283
 
Loss-of-function analysis of 
 HKT1
 in Arabidopsis andwheat established that the primary role of AtHKT1 is toretrieve Na
+
from the xylem in the roots to reduce thetransport of Na
+
from the root to the shoot. However, therole of HKT1 in the leaves, if any, remains elusive. A pri-mary role of HKT2 is the mediation of nutritional Na
+
ab-sorption and Na
+
uptake from soil into the roots of
+
-starved plants to compensate for the deficiency of K 
+
(Horie
et al.
, 2009). The downregulation of 
 TaHKT2
 in salt-tolerantwheat genotypes has been reported to confer tolerance tosalt stress (Singh
 et al.
, 2015). Although some reportschallenge the assumption that Na
+
exclusion leads to im-proved salinity tolerance,
 HKTs
 have emerged as crucialcomponents of salt stress tolerance.DNA methylation is one of the most studied epigeneticprocesses, because it results in a direct and heritable cova-lent modification triggered by external stimuli. Such modi-fications may be reversible and can be associated with theinactivation and activation of genes (Zemach
 et al.
, 2010).Demethylation of functionally inactive genes due to expo-sure to abiotic stresses may initiate their expression, and thestress may also cause heritable changes in cytosine meth-ylation to form epialleles (Kou
 et al.
, 2011). The importanceof epigenetic variations due to stressful condition arisesfrom the fact that these epigenetic modulations can be in-herited in the form of epigenetic memory (Boyko and Ko-valchuk, 2010). Understanding the molecular mechanismsunderlying stress-induced epigenetic regulation of geneexpression may facilitate breeding programs in improvingcrop plants without excessive genetic modification.Genome-wide high-resolution analysis of DNA methylationin rice revealed that 8% of the genes were methylated withintheir promoters, whereas 31% of the genes were methylatedwithin their coding regions (Yan
 et al.
, 2010). Responses toenvironmental factors may vary among plant species; some of them can modulate the physiological and developmental ma-chinery of plants to mitigate the effect of the stress. Mangrovesgrowing in contrasting natural habitats (riversides or salt mar-shes) differed with respect to cytosine methylation despite thesmall genetic variation (Lira-Medeiros
 et al.
, 2010) betweenthem. Thus, epigenetic variations need to be investigated so thatepialleles can be identified and exploited in crop breedingprograms to improve the adaptability of plants under changingclimatic conditions (Kou
 et al.
, 2011).Plants contain relatively high levels of 5-methylcytosine (5-mC), ranging from 6% to 25% of the total cytosine, dependingon the species (Steward
 et al.
, 2002). Unlike DNA methylationin mammals, wherein it predominantly occurs in the CGcontext, DNA methylation in plants occurs in all three cytosinecontexts: CG, CHG, and CHH (H
=
A, T, or C) (Wang
 et al.
,2016). In Arabidopsis, symmetric CG and CHG methylation ismaintained by DNA methyl transferase1 (MET1) andChromomethylase3 (CMT3) during DNA replication,whereas asymmetric CHH methylation is established
 denovo
 by domains rearranged methylase2 (DRM2) via theRdDM pathway (Law and Jacobsen, 2010). The passiveor active demethylation process may be used to remove 5-mCs. In plants, the active DNA demethylation pathway isinitiated by a subfamily of typical HhH-GPD enzymes, in-cluding Repressor of Silencing-1 (ROS1), Demeter (DME),Demeter-like2 (DML2), and Demeter-like3 (DML3). Re-cently, Wang
 et al.
 (2016) reported MET18 to be a com-ponent of the active DNA demethylation pathway in plantsand demonstrated that it plays an epigenetic role in theregulation of gene expression in Arabidopsis.Wheat (
Triticum aestivum
 L.) is one of the most widelycultivated cereals in the world. It is sensitive to soil salinity,which impedes its growth and development, resulting in re-duced crop productivity or failure of the crop. Some wheatgenotypes possess a unique ability to rapidly adapt to salt stress,whereas others are highly sensitive because of their geneticmakeup and regulatory architecture. For instance, Kharchia-65and KRL-210 are well-known salt-tolerant wheat geno-types (Sairam
 et al.
, 2005). On the basis of a multivariable(biochemical and physiological parameters) comprehensiveanalysis of wheat genotypes under salt stress, we identifiedKharchia-65andHD-2329asthemostcontrastingpairoflocallyavailable wheat genotypes with regard to salt tolerance (Beena
et al.
, unpublished data). However, differences in the methyla-tion patterns and the epigenetic responses of these contrastingwheat genotypes under salt stress have been underexplored.The present study examined the effects of salt stress on theextentandpatternofcytosinemethylationandtheireffectsonthe expression of 
 HKT 
 genes in the two contrasting wheatgenotypes, Kharchia-65 and HD-2329. We addressed thefollowing two basic queries: (i) whether epigenetic changes,if any, are triggered by salt stress in bread wheat and (ii)whether epigenetic responses of the salt-tolerant (Kharchia-65) and salt-sensitive (HD-2329) genotypes are similar. Ourinvestigation revealed that cytosine methylation was inducedbysaltstressinagenotype-andtissue-specificmanner,whichdownregulated the expression of 
 TaHKT2;1
 and
 TaHKT2;3
in the shoots and roots of salt-tolerant and salt-sensitive ge-notypes. However, the root-specific downregulation of the
TaHKT1;4
 gene was not found to be controlled through themodulation in DNA methylation.
Materials and Methods
Plant materials and salt treatment 
Two locally available, highly contrasting bread wheatgenotypes (Kharchia-65, salt tolerant and HD-2329, saltsensitive) were used in the present investigation. The seedsof the contrasting wheat genotypes were surface sterilizedby using 0.1% mercuric chloride for 2min, followed bywashing three times with sterilized distilled water. Six seedswere sown at equal intervals in 15-cm pots that were filledwith agro-coir peat. Six pots for each genotype were grownunder controlled conditions in a glasshouse at the NationalPhytotron Facility, IARI, New Delhi. On the basis of theresults of our preliminary experiment (Beena
 et al.
, un-published data), 14-day-old seedlings (in three pots) of eachgenotype were treated with 200mM NaCl that was dis-solved in half-strength Hoagland solution. The remainingthree pots of each genotype were maintained untreated ascontrols. Salt stress treatment was continued for 14 daysuntil the effects of salt stress were visible on the sensitivegenotype. Fourteen days after the treatment (DAT), shootand root samples were collected for molecular analyses.
Isolation of nucleic acids from plant tissues 
Genomic DNA from plant tissues was isolated by usingthe DNeasy Plant Mini Kit (Qiagen). The shoot and root
284 KUMAR ET AL.
 
samples were first mechanically disrupted by using liquidnitrogen and then chemically lysed. RNAs were removed byusing RNase A treatment during the lysis step by followingthe protocol prescribed by the manufacturer of the kit. Thepurified genomic DNA was eluted in low-salt buffer (AEbuffer) and stored at
 -
20
C for downstream use.Total RNAs were isolated from 100mg root and shoottissue samples by using the RNeasy Plant Mini Kit (Qiagen)by following the manufacturer’s instructions. The isolatedRNAs were treated with RNase-Free DNase Set (Qiagen)for on-column digestion of DNA during RNA isolation. Thequality of the isolated RNA samples was assessed throughdenaturing agarose (1%) gel electrophoresis. The quantifi-cation of isolated RNAs was performed by using the Nano-Drop spectrophotometer (ND-1000), and the A
260/280
, A
260/230
ratios were used to assess purity of the RNA.
PCR cloning of 
 HKT1;4
 gene 
The genomic DNA (100ng) isolated from the shoot tis-sues was used as a template for the amplification of 
 HKT1;4
by using a primer pair (Table 1) that was designed for thelast quarter of 
 HKT1;4–2
 (KF443079.1) CDS of 
 Triticumdurum
. The following conditions were used for amplifica-tion of the gene: initial denaturation at 94
C for 5min,followed by 36 cycles at 94
C for 1min, 56
C for 1min,72
C for 1min, and final extension at 72
C for 5min. ThePCR products were visualized on a 1.5% agarose gel, andthe amplicons from Kharchia-65 and HD-2329 were clonedin pGEM-T Easy vector (Kumar and Saxena, 2016). Thecloned fragments were outsourced for 5
·
sequencing bySanger’s dideoxy method. The sequences were analyzed atNCBI and EMBL databases for homology search with the
 HKTs
 from other plant species. The partial
 HKT1;4
 se-quences were submitted to the EMBL database.
Semi-quantitative expression analysis of 
 TaHKT1;4
 gene 
RNA samples isolated from root and shoot tissues showingA
260/280
 between 1.8–2.0 and A
260/230
 >
 2.0 were used forcDNA synthesis. First-strand cDNA was synthesized by usingan equal amount (0.5
m
g) of total RNA as the template and2.0
m
mol oligo-dT primer in a 20
m
L reaction volume at 37
Cfor 1h by using the Revert Aid Premium first-strand cDNAsynthesis kit (Fermentas), as per the manufacturer’s instruc-tions, by using a Triple Master PCR system (Eppendorf). Thefirst-strand cDNA (2.0
m
L) was used for expression analysisof 
 TaHKT1;4
 gene by using the gene-specific primers. PCRconditions were as previously mentioned, and the number of PCR cycles for semi-quantitative analysis was optimized byassessing the amplification products after 20, 24, 28, 32, and36 cycles on 1.5% agarose gel. Actin and Ferredoxin-NADP(H) oxidoreductase were used as reference genes. Toensure the reproducibility of the results, the experiment wasrepeated three times.
Estimation of genome-wide DNA methylation 
The global DNA methylation status of shoot and root tis-sues of the contrasting wheat genotypes under salt stress andcontrolled conditions was estimated by using the MethylFlashMethylated DNA Quantification (Colorimetric) kit (Epigentek).
Table
 1.
 Primers Used for Cloning, RT-PCR, QPCR, and Bisulfite Sequencing Analysesof High-Affinity Potassium Transporter Genes in Bread Wheat
S. No. Sequence Annealingtemp. (
C)Product size (bp) Usage HKT1;4
 gene1 Forward primer: ATTCAGGCAACACCTAATCATGC 56 473 RT-PCRReverse primer: GCATCACAAGAATGAGGATGAGC 581 Cloning2 Forward primer: TTTCTGTTCCAGGTACCTGCCTCCATACA 49 384 Bisulte sequencingReverse primer: ARAARCCCCCATTTCCATCCRCACTRC3 Forward primer: ACCTCGCCATCTTCATCATC 56 199 qPCRReverse primer: GCTTCCATGAAGGAAACCAAActin gene4 Forward primer: TGGGATGCCACCAAAGAC 56 380 RT-PCR and qPCRReverse primer: TGATACGCAAATGTTGAGCFerredoxin-NADP(H) oxidoreductase (
TaFNRII 
) gene5 Forward primer: CAGTGATCTTCACTTCTGAAC 56 200 RT-PCR and qPCRReverse primer: CGAGGACAAGAACGGGAAG
 HKT2;1
 gene6 Forward primer: TATGTGATGAGTCGCAGCTTGAA 56 316 qPCRReverse primer: GCAACAAGAGGCCTGAATTCTTT7 Forward primer: TTYAATTYAGYYAAGAATGTAYAGAG 49 254 Bisulte sequencingReverse primer: AARAACCATARTTTCATTTARARRCAC
 HKT2;3
 gene8 Forward primer: TGAAGCCAAGCAACCCTAAC 56 178 qPCRReverse primer: CCAAGCAGGGAAACAAACAT9 Forward primer: GAATTATTTGGTGTTTTATTTTTYGGTTT 51 369 Bisulte sequencingReverse primer: ACACRATAACCRATATAACTCTACTATC
 HKT 
, high-affinity potassium transporter; RT-PCR, reverse transcription-polymerase chain reaction; qPCR, quantitative PCR.
SALT-INDUCED EPIGENETIC VARIATIONS IN BREAD WHEAT 285

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