cell culture-induced gradual and frequent epigenetic … · cell culture-induced gradual and...

Cell Culture-Induced Gradual and Frequent EpigeneticReprogramming of Invertedly Repeated TobaccoTransgene Epialleles1[W]

Katerina Krizova, Miloslava Fojtova*, Ann Depicker, and Ales Kovarik

Institute of Biophysics, Academy of Sciences of the Czech Republic, 612 65 Brno, Czech Republic (K.K., M.F.,A.K.); and Department of Plant Systems Biology, Flanders Institute for Biotechnology, and Department ofMolecular Genetics, Ghent University, B–9052 Ghent, Belgium (A.D.)

Using a two-component transgene system involving two epiallelic variants of the invertedly repeated transgenes in locus1 (Lo1) and a homologous single-copy transgene locus 2 (Lo2), we have studied the stability of the methylation patterns andtrans-silencing interactions in cell culture and regenerated tobacco (Nicotiana tabacum) plants. The posttranscriptionallysilenced (PTGS) epiallele of the Lo1 trans-silences and trans-methylates the target Lo2 in a hybrid (Lo1/Lo2 line), while itstranscriptionally silenced variant (Lo1E) does not. This pattern was stable over several generations in plants. However, in earlyLo1E/Lo2 callus, decreased transgene expression and partial loss of Lo1E promoter methylation compared with leaf tissue inthe parental plant were observed. Analysis of small RNA species and coding region methylation suggested that the transgeneswere silenced by a PTGS mechanism. The Lo1/Lo2 line remained silenced, but the nonmethylated Lo1 promoter acquiredpartial methylation in later callus stages. These data indicate that a cell culture process has brought both epialleles to a similarepigenetic ground. Bisulfite sequencing of the 35S promoter within the Lo1 silencer revealed molecules with no, intermediate,and high levels of methylation, demonstrating, to our knowledge for the first time, cell-to-cell methylation diversity of callus.Regenerated plants showed high interindividual but low intraindividual epigenetic variability, indicating that the callus-induced epiallelic variants were transmitted to plants and became fixed. We propose that epigenetic changes associated withdedifferentiation might influence regulatory pathways mediated by trans-PTGS processes.

Plants regenerated from calli often display qualita-tive and quantitative phenotypic alterations, cytolog-ical abnormalities, sequence changes, and geneactivation and silencing. These cell culture-inducedchanges, collectively called somaclonal variation, maybe stable or unstable, irreversible or reversible, meiot-ically reset or transgenerationally transmitted (Karp,1991; Phillips et al., 1994; Kaeppler et al., 2000). Thevariation in gene expression is believed to be mediatedby epigenetic mechanisms involving methylation ofDNA and chromatin modifications. Shifts from paren-tal methylation states have been observed in many cellcultures and regenerated plants at both global andlocal levels (Kaeppler and Phillips, 1993; Smulderset al., 1995; Olhoft and Philips, 1999; Jaligot et al., 2000;Kubis et al., 2003; Schellenbaum et al., 2008). Allevia-

tion of silencing of ribosomal genes (Komarova et al.,2004; Koukalova et al., 2005), increased transposonactivity (Hirochika, 1993; Grandbastien, 1998), andactivation of cell cycle-controlling genes (Williamset al., 2003) were reported in studies linking changesin DNA methylation with changes in expression pat-terns. The important outcome of the cultivation ofplant cells under suboptimal conditions is the forma-tion of epialleles, epigenetically modified alleles of agene (for review, see Finnegan, 2002; Arnholdt-Schmitt, 2004). For example, epigenetic changes(mainly in DNA methylation levels) were reported inresponse to water deficiency (Labra et al., 2002), os-motic stress (Kovarik et al., 1997), and heavy metalpresence (Aina et al., 2004).

Stability of transgene expression is one of the keyrequirements for successful establishment of trans-genic lines in agriculture and biotechnology. Rou-tinely, stable epialleles are selected, ensuring highlevels of the recombinant protein product. However,even metastable epialleles could be of potential inter-est, since attenuated expressionmay provide favorablephenotypic traits. The common features of unstable ormetastable transgene loci are the repetitiveness ofDNA sequences (presence of direct and inverted re-peats), preexisting methylation, and/or distinct ge-netic environments (Kakutani, 2002; Richards, 2006).The influence of cell culture on gene expression andepigenetic modification has been studied in several

1 This work was supported by the Academy of Sciences (grantnos. A600040611, AV0Z50040702, and AVOZ50040507), the GrantAgency of the Czech Republic (grant nos. 204/05/H505 and 521/07/0116), and MSMT (grant no. LC06004). M.F. and A.K. acknowledgethe Research Foundation Flanders for regular visiting fellowships.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Miloslava Fojtova ([email protected]).

[W] The online version of this article contains Web-only data.www.plantphysiol.org/cgi/doi/10.1104/pp.108.133165

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reports, often with contrasting outcomes: some lociacquired epigenetic silencing (Meng et al., 2006), whilethe other, originally silenced loci tend to increaseexpression (Mitsuhara et al., 2002) in the cell cultureenvironment.

In plants, silencing may occur at transcriptional andposttranscriptional levels. Transcriptional gene silenc-ing (TGS) is characterized by repression of transcrip-tion and is correlated with cytosine methylation of thepromoter at symmetrical and nonsymmetrical motifs(Vaucheret and Fagard, 2001). The posttranscriptionalsilencing (PTGS) is defined as a mRNA degradationprocess triggered by double-stranded small RNA(smRNA) molecules (Vaucheret et al., 2001). In plants,it often associates with DNA methylation of codingsequences. Both PTGS and TGS may be influenced byenvironmental and developmental factors (De Neveet al., 1999). The factors underlying the direction ofexpression phenotype changes are largely unknown,but it is likely that T-DNA organization, preexistingepigenetic modifications, polyploidy, and type of si-lencing play a role. For example, reversion of PTGSinto TGS phenotype has been reported in tobacco(Nicotiana tabacum; Fojtova et al., 2003) and petunia(Petunia hybrida; Kanazawa et al., 2007) transgenescontaining inverted repeats.

To study the influences of cell culture stress onepiallelic stability, we designed a two-componenttransgene system containing a silencing trigger locusand a nonsilenced single-copy target locus serving as asensitive reporter for the trans-silencing capacity ofthe silencing locus. In these hybrids, the trigger locusorganized as an inverted repeat occurs in two meiot-ically stable epiallelic states, termed Lo1 and Lo1E(Van Houdt et al., 2000a; Fojtova et al., 2003). Theneomycin phosphotransferase II (nptII) reporter genewithin Lo1 is characterized by an active transcription,RNA degradation, and methylation of the codingregion but not the promoter. The epiallelic variantLo1E was previously obtained by regeneration ofplants from Lo1 line callus (Fojtova et al., 2003). Inthese regenerants, the nptII reporter gene was found tobe transcriptionally silenced and the linked 35S pro-moter contained heavy methylation of symmetricaland nonsymmetrical motifs. Both epiallelic variantsLo1 and Lo1E displayed high trans-generation stabil-ity of their methylation patterns. Only the Lo1 epiallelewas able to trans-silence homologous loci by a PTGSmechanism, while the Lo1E epiallele did not trans-silence the homologous locus (Fojtova et al., 2006).This demonstrated that epigenetic modification of asilencer locus can dramatically influence the expres-sion of unlinked genes. Here, we studied the stabilityof epiallelic variants of the silencer locus in callusculture. Evidence was obtained for diversification ofepiallelic variants in tissue culture and erasure ofparental methylation imprints in callus culture. Tran-sition to the other extreme of epialleles was observedin approximately one-fourth of regenerated plants inboth directions.

RESULTS

Description of the Transgenic Loci andExperimental Setup

Transgenic loci used for the construction of thetobacco hybrid lines are schematically depicted inFigures 1A and 2A. The transgenic line HeLo1 carriesLo1, which contains two copies of the GVchs287T-DNA in an inverted repeat arrangement. The bacte-rial nptII reporter transgene driven by the 35S pro-moter is silenced at the posttranscriptional level in Lo1(Van Houdt et al., 1997). The HeLo1E line containingepimutated transcriptionally silenced Lo1 (termedLo1E) was obtained as a regenerated individual fromthe HeLo1 cell culture (Fojtova et al., 2003). Lo2consists of a single-copy insertion of T-DNA and ischaracterized by high and stable nptII expression (VanHoudt et al., 2000a). Epialleles of the Lo1 were com-bined with the homologous transgenic Lo2 in hybridplants. The PTGS epiallele of the Lo1 was shown toinduce trans-silencing and trans-methylation of theLo2 in Lo1/Lo2 hybrids, while its TGS counterpart(Lo1E) was unable to trans-silence the Lo2 (Fojtovaet al., 2006).

The epiallelic stability of transgenes in hybrid linesduring dedifferentiation and differentiation processeswas investigated as shown in Figure 3; two indepen-dently established callus cultures from each hybridline were analyzed. A total of 28 Lo1/Lo2 and 12Lo1E/Lo2 regenerated plants were analyzed for re-porter transgene expression and silencing locus pro-moter methylation.

Changes in Transgene Expression during Callus Culture

and the Regeneration Process

The nptII transgene transcript levels in parentalhybrid plants, calli, and regenerants were analyzedby RNA gel blot hybridization and real-time PCRanalysis (Fig. 4). A strong hybridization signal wasobtained with RNA extracted from the parental hybridplant containing a TGS variant (Lo1E/Lo2) but not aPTGS variant (Lo1/Lo2) of Lo1. In the Lo1E/Lo2callus, expression started to progressively deteriorate,and fully developed 3-month-old callus already hadnegligible transcription (Fig. 4A). The nptII expressionin the Lo1/Lo2 line was not influenced by in vitroculture stress, and the nptII transcript levels were lowin both plant and calli.

Plants were regenerated from the 12-month-oldLo1/Lo2 and Lo1E/Lo2 calli. There was more than10-fold variation in nptII mRNA levels between theindividuals. About three-fourths of Lo1E/Lo2 plants(Fig. 4B) showed high expression of the nptII gene. Inthese individuals, expression at both RNA and protein(Supplemental Fig. S1) levels was comparable to thatof the parental plant. Most (about four-fifths) regen-erated plants from Lo1/Lo2 calli retained a silencedphenotype, while six individuals (R2, R6, R14, R16,R23, and R24) showed partial relief of trans-silencing.

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Analysis of the nptII-Derived smRNA Molecules

Next, we wished to investigate the nature of callus-induced expression changes. We have previouslyshown that communication between the PTGS Lo1and Lo2 is mediated by nptII-derived smRNA mole-cules in Lo1/Lo2 hybrid plants, whereas these mole-cules are absent in the Lo1E/Lo2 (Fojtova et al., 2006).Here, we analyzed smRNAs in Lo1/Lo2 and Lo1E/Lo2 parental and selected regenerated plants. RNAsamples enriched with a low molecular weight frac-tion were immobilized on the membrane and hybrid-ized against the radioactively labeled nptII RNA senseprobe (Fig. 5). The parental Lo1/Lo2 plant displayedthe strong hybridization signal in the low molecularweight region; the length of the fragment was about 21nucleotides. The signal was also detected in the 12-month-old Lo1/Lo2 callus and in the silenced regen-erated plants (R4 and R9), while it was absent in the

regenerants that expressed higher levels of the nptIImRNA (R2 and R6).

In the Lo1E/Lo2 hybrid line, the nptII hybridizationsignalwas observed in the callus sample and in silencedregenerant (r3) but not in thenonsilencedparental plantand the nonsilenced regenerants (r2 and r6). Theseresults indicate that the changes in expression patternsduring dedifferentiation and plant regeneration canbe correlated with changes in smRNA levels.

Analysis of the 35S Promoter Methylation bySouthern-Blot Hybridization

We tested the hypothesis that the cell culture-induced alterations of RNA silencing signals mightbe explained by destabilization of the epiallelic statesof the silencing trigger Lo1. It is known that strongmethylation of a promoter can influence transcription

Figure 1. Analysis of the 35S pro-moter methylation in the Lo1/Lo2and Lo1E/Lo2 parental hybridplants, derived calli, and regen-erated plants using methylation-sensitive restriction endonuclease.A, Genomic organization of trans-genic loci and scheme of the di-gestion. BglII (BII) enzyme wasused for the dissection of the par-ticular regions of T-DNA in hybridlines. Sau96I (S96I) restriction en-donuclease was applied to analyzethe methylation of cytosines inthe nonsymmetrical sequence con-text. Arrows schematically delimitlengths of fragments that resultedafter the digestion. P35S, Promoterfrom the Cauliflower mosaic virus;nptII, bacterial neomycin phospho-transferase II gene; 3#chs, transcrip-tion termination sequence from the3# untranslated region of the chal-cone synthase gene from snap-dragon (Antirrhinum majus); RB,T-DNA right border. B, Southern-blot analysis of 35S promoter meth-ylation. DNAs were predigested byBglII restriction enzyme to obtainlocus-specific bands (2 lines). Thesamples in + lines were digestedwith methylation-sensitive Sau96I.The 6.4-kb BglII/Sau96I band indi-cates complete methylation of allfour Sau96I sites within two 35Spromoters of the Lo1. The 4.8-kbBglII/Sau96I band originated frommethylation of one of the pro-moters on each site of the invertedrepeat.

Epigenetic Instability of Tobacco Transgene Epialleles

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of a linked gene. To study methylation changes ac-companying dedifferentiation and plant regeneration,we carried out Southern-blot hybridization using amethylation-sensitive restriction enzyme (Fig. 1). BglIIenzyme was used to separate the transgenic loci. Afterthe BglII digestion, Sau96I restriction enzyme, sensi-tive to themethylation of cytosine in a nonsymmetricalsequence context, was applied. There are two Sau96Isites within the core 35S promoter, at 2163 and 2175(with respect to the transcription start site, Fig. 1A).

The BglII digestion of DNA from hybrids yieldedthe 8.5- and 6.4-kb hybridization bands correspondingto Lo2 and Lo1 (E), respectively. This pattern is con-sistent with the absence of methylation at the BglII sitelocated distally to the 35S promoter close to the leftborder. In accordance with the previously reportedresults (Fojtova et al., 2006), promoters of both trans-genic loci in PTGS Lo1/Lo2 were free of methylation,as indicated by complete Sau96I digestion of both BglIIbands into a 0.8-kb Sau96I/BglII band (Fig. 1B). Highmolecular weight bands appeared in a 12-month-oldLo1/Lo2 callus, indicating slow hypermethylation ofthe Lo1 promoter upon callus culture. However, meth-ylation never reached saturation levels, since productsof partial digestion (hybridization signal in an approx-imately 4.8-kb region) were visible even in 48-month-old callus samples (Supplemental Fig. S2).

The Lo1E/Lo2 line showed heavy methylation ofpromoter within Lo1E in parental plants, as indicatedby high resistance of the 6.4-kb BglII band to the Sau96I

digestion (Fig. 1B). In the derived callus, increaseddigestibility of the 6.4-kb band appeared, resulting inincreased intensity of the signal in 0.8-kb regions andappearance of the approximately 4.8-kb band. Therestriction map (Fig. 1A) indicates that this bandrepresents products of the partial digestion of pro-moters on both sites of the inverted repeat. The diges-tion profiles were relatively stable over the 48-monthcultivation period (Supplemental Fig. S2), suggestingthat the loss of methylation did not reach complete-ness at any callus stage.

In Lo1/Lo2 plants regenerated from the 12-month-old callus, the methylated Sau96I fragments werefound (Fig. 1B), suggesting that methylation patternswere transmitted from calli into plants. Interestingly,the most heavily methylated sites were found amongthe R2 and R6 plants. Both individuals showed ele-vated levels of the nptII transcripts (Fig. 4), supportingthe hypothesis that inactivation of the Lo1 promoter isassociated with loss of RNA silencing signal. Thedigestibility of the Lo2-specific 8.5-kb band remainedunchanged (high), indicating the absence of methyla-tion of the 35S promoter within Lo2. Similar to theLo1/Lo2, the Lo1E/Lo2 regenerated plants showedinterpopulation variation in methylation profiles.While in most individuals (represented by the r2, r5,and r6 regenerants) the 6.4-kb fragment was largelyundigested (indicating heavy methylation of the Lo1promoter), the DNAs from r3 and r4 regenerantsshowed increased sensitivity toward Sau96I. Only

Figure 2. Analysis of the 3# nptIIcoding region methylation in theLo1/Lo2 and Lo1E/Lo2 parentalhybrid plants, derived calli, andregenerated plants using themethylation-sensitive restrictionendonuclease. A, Genomic organi-zation of transgenic loci andscheme of the digestion. EcoRV(EV) methylation-insensitive en-zyme was used for the dissectionof the particular regions of T-DNAin hybrid lines. SmaI (S) enzyme issensitive to methylated cytosines inthe CG sequence context. Arrowsschematically delimit lengths of re-striction fragments. Abbreviationsand symbols are as in Figure 1A.B, Southern-blot methylation anal-ysis of the genic region. The exper-imental strategy was analogous tothat described in Figure 1B. The1.8-kb EcoRV/SmaI band repre-sents a methylated Lo2 fragment.

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the r3 and r4 regenerants showed Lo2 silencing (Fig.4), confirming an inverse correlation between Lo1promoter methylation and trans-PTGS.

Methylation Patterns of the 35S Promoter Determined byBisulfite Sequencing

Next, we wished to examine the homogeneity ofmethylation patterns at the silencing locus (Lo1) bybisulfite sequencing analysis in a parental Lo1/Lo2hybrid, various stages of callus, and regeneratedplants (for the tissues, see Supplemental Fig. S3). Sinceit was impossible to distinguish between clones de-rived from individual loci due to sequence homology,we separated both loci by gel electrophoresis after theBglII digestion (there are two BglII sites within Lo1 butonly a single site within Lo2; Fig. 1A). The 35S pro-moter of Lo1 represented by the 6.4-kb band wasisolated and subjected to bisulfite conversion, cloning,and sequencing. The distribution of methylcytosineresidues along the individual clones is diagrammati-cally shown in Figure 6A, and the data are summa-rized in Figure 6B and Table I. The examined regioncontains 12, seven, and 66 cytosine residues at CG,CHG, and CHH sequence contexts, respectively. Inaccordance with Southern-blot hybridization (Fig. 1B),there was no or negligible methylation of the 35S

promoters in the clones derived from the parentalLo1/Lo2 plant (top). Similarly, the green leaf discsused for callus induction did not acquire significantmethylation. On the other hand, few clones derivedfrom early callus showed increases of methylation,especially in nonsymmetrical contexts. The fully de-veloped 12-month-old callus yielded clones with var-iable levels of methylation. There were clones with no(top lanes), intermediate (middle lanes), and high(bottom lanes) levels of methylation. The clones fromregenerated plant R2 (nonsilenced by PTGS) showedrelatively homogenous profiles with both CG and non-CG methylation. Clones derived from homologousLo2 were nonmethylated (data not shown).

DNA Methylation in the Coding Region

The coding region methylation is considered a hall-mark of PTGS (Ingelbrecht et al., 1994). We havestudied methylation of the nptII coding region ofboth loci in the Lo1E/Lo2 hybrid using SmaI cuttingbetween the nptII coding region and the 3# untrans-lated region. The enzyme is sensitive to methylation ofthe outer and inner C within the CCCGGG recognitionsequence. To separate transgenic loci, the DNAs weredigested with EcoRV, providing 3.0- and 1.8-kb bandscorresponding to Lo1 and Lo2 fragments, respectively(Fig. 2A). As shown in Figure 2B, the 1.8-kb band ofLo2 was efficiently digested in parental nonsilencedLo1E/Lo2 plants, 12-month-old derived callus, andregenerated r2, r5, and r6 plants. Late-stage callus andboth r3 and r4 regenerants that showed Lo2 silencingdisplayed an absence of SmaI digestion, indicating thepresence of cytosine methylation in the Lo2 codingregion in these individuals.

In Lo1/Lo2 hybrids, strong genic methylation ofboth transgenic loci observed in parental plants per-sisted during 24 months of the callus cultivation (Fig.2B). In regenerated plants, R4 and R9 individuals withsilenced Lo2 and low methylation of Lo1 promoterexhibited high levels of Lo2 trans-methylation at theSmaI restriction site. On the other hand, Lo2 methyl-ation was decreased in weakly silenced R2 and R6plants. The 3.0-kb Lo1-specific band remained meth-ylated irrespective of the cultivation conditions. Theseresults indicate that the methylation of the silencingLo1 promoter was inversely correlated with trans-methylation of the SmaI site in the Lo2.

DISCUSSION

In this work, we used a two-component transgenemodel system to study the stability of RNA-mediatedhomologous transgene interactions in cell culture. Weshow that cell culture resulted in blurring of theparental epigenetic expression and methylation pat-terns at the silencing locus associated with increasedepiallelic diversity. These changes influenced the pro-duction of smRNA molecules from a linked gene and

Figure 3. Scheme of the experimental strategy. Lo1, Plant line hemi-zygous for the transgenic locus 1, with expression of the nptII reportertransgene silenced posttranscriptionally; Lo1E, plant line hemizygousfor the transgenic locus 1E, with expression of the nptII reportertransgene silenced transcriptionally; Lo2, plant line hemizygous for thetransgenic locus 2, with active expression of the nptII reporter trans-gene; Lo1/Lo2, posttranscriptionally silenced hybrid plant line com-bining locus 1 and locus 2; Lo1E/Lo2, hybrid plant line combininglocus 1E and locus 2, with active expression of the locus 2 nptIItransgene.





silencing of a homologous unlinked locus. Despite adominant PTGS phenotype in callus culture, a highproportion of nonsilenced individuals were found

among regenerated plants. The regenerated plantshave partially but not completely reestablished origi-nal expression patterns.

Figure 4. Analysis of nptII expression in the parental plants, derived calli, and regenerants of the Lo1/Lo2 and Lo1E/Lo2 hybridlines. A, Northern-blot analysis of the nptII reporter transgene expression. About 5 mg of total RNA was loaded per line andhybridized against the nptII DNA probe. The hybridizationwith the tobacco 18S rDNA probe is shown as a control for loading. B,Quantification of the nptII expression by real-time PCR. The nptII RNA levels were normalized to actin mRNA. The expressionlevel of the nonsilenced Lo2 line was arbitrarily designated 100%. The transgenes whose expression exceeded 30% of that of Lo2were considered “nonsilenced” (threshold indicated by the thick horizontal line).

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Prevalent PTGS Phenotype in Hybrid Callus Cultures

The Lo1E/Lo2 line containing a TGS variant of thesilencer had high NPTII levels in parental leaf. How-ever, progressive silencing evolved in cell culture. Thehybrid combining a Lo1 PTGS epiallele was silenced inboth plants and calli. The presence of nptII gene-specific smRNAs andmethylation of the coding regionwithin the target indicated that silencing occurred atthe posttranscriptional levels. This seems contradic-tory to previous reports demonstrating the loss ofPTGS in calli and rapidly dividing cells (Mitsuharaet al., 2002; Correa et al., 2004). There might be severalexplanations. First, differences in the genomic organi-zation of silencing loci need to be considered. It isknown that trans-silencing elicited by hairpin con-structs and viruses (Dalmay et al., 2000) does notrequire some components of RNA-processing machin-ery, including RNA-dependent RNA polymerase, thatare needed for trans-PTGS. Perhaps there is a variationin the sensitivity of PTGS to environmental factorsamong the systems. Furthermore, unlike virus-induced PTGS (Correa et al., 2004), we did not detectany specific high molecular weight RNA species thatwould potentially indicate aberrant processing of thenptII-specific RNA signals (data not shown). We alsofound no evidence for the influence of the genomiclocation of target loci (Fojtova et al., 2006) recentlyreported for trans-TGS systems (Fischer et al., 2008).These data support the hypothesis that the trans-silencing RNA signals are produced only from thetranscriptionally active Lo1, regardless of the devel-opmental status and cultivation conditions. In conclu-sion, tobacco callus cultures probably contain allnecessary components able to execute trans-PTGStriggered by invertedly repeated loci.

Cell Culture-Induced Methylation Mosaicism at theSilencing Locus Accounts for the Dominant PTGSPhenotype in Hybrids

Upon callus culturing in the Lo1E/Lo2 line, themethylated and inactive promoter epiallele (Lo1E)

underwent hypomethylation. Previous experimentsshowed that the TGS 35S promoter is sensitive tohypomethylation, since the 5-azadeoxycytidine treat-ment resulted in elevated transcription of the linkednptII gene (Fojtova et al., 2006). It is likely that cellculture-induced hypomethylation activated the Lo1Epromoter, as a result of which the production of trans-silencing RNA signal has been restored. In contrast toLo1E, the active 35S promoter within Lo1 underwentprogressive methylation in cell culture. After 12months, the methylation profiles of promoters in theLo1/Lo2 and Lo1E/Lo2 lines were almost indistin-guishable, suggesting that cell culture had blurreddifferences in methylation imprints between the lines.The partial digestion of promoter fragments withmethylation-sensitive enzymes indicated that interme-diate methylation phenotypes have been established.This assumption has been further confirmed by bisul-fite sequencing of the 35S promoter, which revealedastonishing clone-to-clone variation in methylationstates of the callus clones but not those of parentalleaf (Fig. 6). There were clones with no or marginalmethylation, while some clones acquired full methyl-ation of the 35S promoter.

Possible Mechanism of Callus-Induced

Methylation Changes

The contrasting trends in methylation dynamics ofthe TGS and PTGS epialleles of Lo1 suggest that theremight be two opposing forces acting on the promoterexposed to the cell culture environment. The reducedmethylation in Lo1E may reflect a more general trendtoward genome hypomethylation in plant cell cultures(Kaeppler and Phillips, 1993; Kubis et al., 2003). On theother hand, the proximity of inverted repeats occupy-ing distinct chromatin structure (Ebbs et al., 2005) andintensive transcription over the methylated codingregion (van Blokland et al., 1997) could promotemethylation spreading from the coding region intothe promoter. Inverted repeat structure could poten-tially generate read-through transcripts, giving rise to

Figure 5. Detection of nptII-derived smRNA mole-cules in parental hybrid plants, derived calli, andregenerated plants. The fraction of RNA enriched forlow molecular weight fragments (about 50 mg perline) was hybridized against sense-specific nptIIriboprobe. End-labeled 21- and 25-nucleotide (nt)oligomers were used as size markers (M). Cuttings ofethidium bromide (EtBr)-stained gels are shown as acontrol for loading.





Figure 6. Bisulfite methylation analysis of the Lo1 in a Lo1/Lo2 plant, derived callus, and regenerated plants. Approximately 300bp of the 35S promoter was sequenced. A, CyMATE output of the distribution of C methylation along the sequenced clones.

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methylation-inducing smRNA molecules (Mette et al.,2000). Although we failed to identify promoter-specific smRNAs, the initial nonsymmetrical methyl-ation imposed on a “naive” Lo1 promoter duringcallus induction (Fig. 6A; Table I) suggests that sometype of RNA-directed process, acting in cis, mightbe involved. The symmetrical CG sites seem to bemethylated later at more advanced stages of calluspropagation. This mode of methylation dynamics isconsistent with a gradual and perhaps stochastic pro-cess leading to denser methylation in advanced stages.In this context, methylation changes were more pro-nounced in plants regenerated from advanced stagesof callus growth compared with those of earlier pas-sages (Peredo et al., 2006).Recently, a subset of endogenous promoters was

reported to undergo hypermethylation in Arabidopsis(Arabidopsis thaliana) callus and suspension cultures(Berdasco et al., 2008). As in our system, the epigeneticchanges were associated with transcriptional silencingof linked genes. However, endogenous promoters ofArabidopsis were methylated exclusively at CG dinu-cleotides, while the Lo1 35S promoters were methyl-ated at both CG and non-CG motifs. Perhaps theremight be differences in the mechanism of hyper-methylation of endogenous genes and transgenes incallus cultures. It is necessary to stress that methyla-tion changes affected 35S promoters within the PTGSLo1 only, since a homologous promoter within thenon-silenced Lo2 remained unmethylated (and active)throughout callus cultivation (Fig. 1B; Fojtova et al.,2003).

High Interindividual and Low IntraindividualEpigenetic Variability in Regenerated Plants

About three-fourths of regenerants from silent 12-month-old Lo1E/Lo2 callus showed expression levels

of the nptII reporter gene comparable to that of thenonsilenced parental plant. Similarly, about one-fifth ofregenerated plants from Lo1/Lo2 callus (establishedfrom a silenced Lo1/Lo2 plant) showed elevated re-porter gene expression, although in this case its levelwas lower than that of the Lo1E/Lo2 regenerants.Thus, there was considerable interpopulation vari-ability in expression patterns among regenerated plants(Fig. 4), likely reflecting epigenetic mosaicism of aparental callus. Indeed, bisulfite sequencing revealedlarge clone-to-clone methylation variability in callus(Fig. 6), indicating that callus is a mixture of cells withdifferent epigenetic phenotypes at the silencing triggerLo1. The silenced phenotype was accompanied bycoding region methylation and production of nptII-specific smRNAs, indicating a PTGS mechanism. Onthe other hand, these attributes were absent in non-silenced regenerants, which had heavy methylation ofthe TGS 35S promoter at the silencing Lo1. Theseresults support the hypothesis that the promoter ac-tivity of the Lo1 is a key player in the trans-silencingprocess in this system. Bisulfite analysis (Fig. 6)showed that all clones derived from a nonsilencedregenerant had nearly complete methylation of an as-1regulatory element (containing two CG motifs at 267and279; Kanazawa et al., 2006) at the silencing triggerLo1. Since a factor binding to as-1 was shown to bemethylation sensitive, we can speculate that the meth-ylation of the as-1 element accounts for promoterinactivity in regenerated plants. Interestingly, theintraindividual methylation variability among the se-quenced clones was negligible, indicating that plantstend to maintain accurate setting of epigenetic statesthroughout mitosis. The uniform methylation profilesacross the sequenced clones further support a single-cell rather than a multiple-cell origin of regenerants.

The increased frequency of methylated epialleles inregenerated plants possibly reflects the trend toward

Figure 6. (Continued.)Filled and empty symbols indicate the presence and absence of C methylation, respectively. The positions of Sau96I sites(previously analyzed for C methylation by blot hybridization) and the as-1 element are indicated. Note the increasednonsymmetrical methylation in three clones from the early callus. B, Percentage of total cytosine methylation in samples.Variation in methylation density between the clones is indicated by error bars (SD).

Table I. Summary of bisulfite sequencing analysis of the 35S promoter within the silencing locus (Lo1)in Lo1/Lo2 parental plant, derived calli, and a regenerated plant

Sample/Methylation MotifmCG mCHG mCHH

Meana Rangeb Mean Range Mean Range

%

Parental PTGS Lo1/Lo2 plant 1 0–8 0 0–0 1 0–1Disc of leaf (green tissue) 0 0–0 2 0–14 0 0–3Early callus (4 weeks) 0 0–0 0 0–0 5 0–11Advanced callus (12 months) 48 0–83 51 0–88 34 22–44Nonsilenced regenerant (no. 2) 90 83–100 75 57–100 34 22–44

aPercentage methylation at CG, CHG, and CHH contexts was calculated from the clones (Fig. 6A) andexpressed as a mean. bMethylation homogeneity between the clones is indicated by a range.





increased genome methylation in regenerated plants.For example, rDNA (Koukalova et al., 2005), sometransposons (Grandbastien, 1998), and other repeats(Jaligot et al., 2000) appeared to be less methylated incallus compared with regenerated plants. Also, lessdifferentiated cells contain a lower degree of methyl-ation than fully differentiated mature leaf (Mathieuet al., 2003). It is not certain whether cells with highlymethylated DNA are favored for regeneration andselected or whether hormonal factors (LoSchiavo et al.,1989) associated with the regeneration process in-crease overall genomic methylation.

It is becoming apparent that in eukaryotic genomesmany genes occur in multiple families that are sub-jected to an orchestrated epigenetic regulation (Melquistet al., 1999) and that no gene exists in isolation fromother genes (Matzke and Matzke, 1990). Our resultsshow that epigenetic change in a single locus couldpotentially influence the expression state ofmany otherhomologous loci through RNA signals. We proposethat “hidden” epigenetic variability unraveled by adedifferentiation process might have its molecularbackground in promoters containing inverted and tan-dem repeats. It will be interesting to determine ifphenotypes modified by cell culture (Meins andThomas, 2003) originate from loci containing theseDNA structures.

MATERIALS AND METHODS

Plant Material and Callus Culture Conditions

All transgenic tobacco (Nicotiana tabacum) SR1 plants were generated by

Agrobacterium tumefaciens-mediated transformation (Ingelbrecht et al., 1994).

The plants hemizygous for the PTGS Lo1 (HeLo1; Figs. 1A and 2A) were

obtained by crossing a plant homozygous for Lo1 with an untransformed SR1

tobacco plant (Van Houdt et al., 2000b). The line hemizygous for the TGS Lo1E

was obtained by plant regeneration from long-term Lo1 callus cultures

(Fojtova et al., 2003). The line hemizygous for Lo2 (HeLo2; Figs. 1A and 2A)

was obtained by crossing a plant homozygous for Lo2 with an untransformed

SR1 tobacco plant (Van Houdt et al., 2000a). The hybrids hemizygous for Lo1

and Lo2 (Lo1/Lo2) and hemizygous for Lo1E and Lo2 (Lo1E/Lo2) were

obtained by crossing the respective parental plants. In all crosses, the Lo1 or

Lo1E plant served as the mother donor. Progeny plants were screened for the

presence of the two transgenic loci by DNA gel-blot hybridization.

Calli were established from leaf explants by hormonal treatment and

grown in 0.8% agar containing B5 salts supplemented with Suc (30 g L21),

a-naphthaleneacetic acid (2.0 mg L21), and 6-benzylaminopurine (0.2 mg L21).

Callus was transferred onto fresh agar medium every 3 to 4 weeks.

To obtain regenerated plants, calli were transferred onto shoot-inducing

medium containing a-naphthaleneacetic acid (0.2 mg L21) and 6-benzyl-

aminopurine (2.0 mg L21). About eight to 10 plantlets were obtained after

two subcultures (1 month each). After the rooting phase on growth medium

without hormones, the plantlets were transferred into greenhouse conditions.

DNA Isolation and Southern-Blot Hybridization

Total genomic DNA was isolated from lyophilized leaves or calli by the

cetyltrimethylammonium bromide method as described previously (Kovarik

et al., 2000).

DNA methylation was analyzed with methylation-sensitive restriction

endonucleases. Approximately 20 mg of genomic DNAwas digested with an

enzyme excess (5 units per 1 mg of DNA). After digestion, the DNA was

separated by electrophoresis on a 1% (w/v) agarose gel. The gels were alkali

blotted onto a Hybond XL membrane (GE Healthcare) and hybridized against

32P-labeled DNA probes (DekaLabel kit; MBI Fermentas) for at least 16 h at

65�C. The nptII-coding sequence and the 35S promoter probes were prepared

from the approximately 830- and 980-bp inserts of the pGEMnptII and

pGSJ290 plasmids, respectively (Van Houdt et al., 1997). After washing under

high-stringency conditions (twice for 5 min in 23 standard saline citrate

[SSC] + 0.1% [w/v] SDS and twice for 20 min in 0.23 SSC + 0.1% [w/v] SDS at

65�C; 13 SSC = 150mMNaCl and 15mM Na3-citrate, pH 7.0), the hybridization

bands were visualized with a STORM PhosphorImager (GE Healthcare) and

the data were processed with ImageQuant software (GE Healthcare).

RNA Isolation, Northern-Blot Hybridization, and

Real-Time PCR Analysis

Total RNAwas isolated from young leaves with the RNeasy Plant Mini Kit

(Qiagen) and extensively treated by DNaseI (TURBO DNA-free; Applied

Biosystems/Ambion) according to the manufacturer’s instructions. After

electrophoresis on a 1.2% (w/v) formaldehyde-agarose gel, the gel was

washed for 10 min in sterile water to remove the formaldehyde. The RNA

was denatured in 0.05 M NaOH and blotted onto a Hybond XLmembrane (GE

Healthcare) in 203 SSC. Radioactively labeled nptII DNA probes were

hybridized in ULTRAhyb buffer (Applied Biosystems/Ambion) for 24 h at

35�C. After washing under low-stringency conditions (twice for 15 min in 23SSC + 0.1% [w/v] SDS at 50�C), the hybridization bands were visualized with

a STORM PhosphorImager. To evaluate the nptII expression levels, the

intensities of the nptII hybridization signals were normalized to the 18S

ribosomal RNA bands (Lim et al., 2000).

cDNAs were prepared by reverse transcription of RNAs using the Super-

Script II Reverse Trancriptase (Invitrogen) and Random Nonamers (Sigma).

Quantification of the nptII level related to the actin transcript was done using

the Power SYBR Green PCR Master Mix (Applied Biosystems/Ambion) by

the 7300 Real-Time PCR System (Applied Biosystems/Ambion). nptII was

amplified with the forward primer 5#-CGTTACAAGAGAGAAATCGCC-3#and the reverse primer 5#-TTCTATCGCCTTCTTGACGAG-3#; actin was

amplified with the forward primer 5#-CTGGATTTGCTGGTGATGAT-3# andthe reverse primer 5#-CYCTCTTGGATTGAGCTT-3# in the same PCR cycle

(initial denaturation at 94�C for 10 min followed by 35 cycles of 20 s at 94�C, 20s at 56�C, and 30 s at 72�C). The SYBR Green I fluorescence was monitored

consecutively after the extension step. The amount of nptII transcript was

determined in three independent reactions for regenerated plants; for parental

hybrid plants (Lo1/Lo2 and Lo1E/Lo2) and PTGS Lo1 plant, RNAs isolated

from three individuals were analyzed in triplicate. nptII relative levels were

expressed as percentage of the Lo2 nonsilenced transgenic plant expression.

smRNA Molecule Isolation and Northern-Blot Hybridization

Fractions of smRNA molecules were isolated as described previously

(Hamilton and Baulcombe, 1999) with minor modifications (Fojtova et al.,

2006).

The smRNAs were separated by electrophoresis (15% polyacrylamide, 7 M

urea in 0.53 TBE; 13 TBE = 90 mM Tris-borate and 2 mM EDTA, pH 8.0),

blotted onto a nylon Hybond XL membrane (GE Healthcare) with a semi-dry

blot instrument (trans-Blot SD Semi-Dry Transfer Cell; Bio-Rad) in 0.53 TBE,

and fixed by UV cross-linking. To estimate the size of the RNA bands, 21- and

25-nucleotide oligonucleotides were used as markers. The single-stranded

RNA probe was transcribed in the sense orientation from a linearized

pGem-3Zf(+)npt plasmid with [32P]UTP using the RNAMaxx High-Yield

Transcription Kit (Stratagene). Hybridization was performed as described

(Hamilton and Baulcombe, 1999; Mette et al., 2000) in ULTRAhyb buffer

(Applied Biosystems/Ambion) at 35�C for 48 h. The membrane was washed

twice in 23 SSC + 0.1% (w/v) SDS for 30 min at 35�C, then in 20 mM Tris-HCl,

5 mM EDTA (pH 8.0), 60 mM NaCl, and 10 mg mL21 RNaseA (for 1 h at 37�C) toremove unspecific background, and exposed to the PhosphorImager screen.

Bisulfite Genomic Sequencing

Bisulfite treatments were carried out on purified genomic DNA using the

EpiTect Bisulfite Kit (Qiagen). The primers for the amplification of the 35S

promoter and 5# nptII region are as follows: forward primer 5#-CATTACAT-

CACCCATAATAAATACTTTCTC-3#, the first reverse primer 5#-GAATA-

GAGAGAAAGATATATTTTTTAAGAT-3#, and the second reverse primer

Krizova et al.




5#-TAATAGAGATTGGAGTTTTTAAGAAAGTAG-3#. The PCR program con-

sisted of 2 min of initial denaturation at 94�C followed by 35 cycles of 0.5 min

at 94�C, 1.5 min at 45�C, and 1 min at 72�C. The program was ended with an

extension step for 10 min at 72�C. The PCR products were cloned into a TA

vector (pDrive; Qiagen), and five to 18 clones from each sample were

sequenced (Eurofins MWG Operon). The data were processed and methyla-

tion density was calculated using CyMATE software (Hetzl et al., 2007).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Analysis of the NPTII protein level in the Lo1E/

Lo2 parental plant, callus, and regenerants.

Supplemental Figure S2. Southern-blot methylation analysis of the 35S

promoter in 48-month-old Lo1/Lo2 and Lo1E/Lo2 calli.

Supplemental Figure S3. Description of tissues used for methylation

analysis by bisulfite sequencing.

Received November 25, 2008; accepted December 22, 2008; published January

7, 2009.

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