Analysis of the DNA methylation of maize in response to cold stress based on MSAP

J. Plant Biol. (2013) 56:32-38

DOI 10.1007/s12374-012-0251-3

Analysis of the DNA Methylation of Maize (Zea mays L.) in Response to Cold Stress Based on Methylation-sensitive Amplified Polymorphisms

Xiaohui Shan ?, Xiaoyu Wang ?, Guang Y ang, Ying Wu, Shengzhong Su, Shipeng Li, Hongkui Liu and Y aping Y uan *

College of Plant Science, Jilin University, Changchun 130062, China

Received: July 3, 2012 / Accepted: December 13, 2012? Korean Society of Plant Biologists 2012

Abstract DNA methylation plays a vital role in tuning gene expression in response to environmental stimuli. Here,methylation-sensitive amplified polymorphisms (MSAP)were used to assess the effect of cold stress on the extent and patterns of DNA methylation in maize seedlings. Overall,cold-induced genome-wide DNA methylation polymorphisms accounted for 32.6 to 34.8% of the total bands at the different treatment time-points. It was demonstrated that the extent and pattern of DNA methylation was induced by cold stress through the cold treatment process and that the demethylation of fully methylated fragments was the main contributor of the DNA methylation alterations. The sequences of 28 differentially amplified fragments relevant to stress were successfully obtained. Under the cold stress, demethylation was detected in most fragments. BLAST results indicate that the homologues of these fragments are involved in many processes, including hormone regulation, cold response,photosynthesis, and transposon activation. The expression analysis demonstrated an increase in the transcription of five demethylated genes. Despite the fact that DNA methylation changes and cold acclimation are not directly associated, our results may indicate that the specific demethylation of genes is an active and rapid epigenetic response to cold in maize during the seedling stage, further elucidating the mechanism of maize adaptation to cold stress.

Key words: Cold stress, DNA methylation, Gene expression,Maize, MSAP

Introduction

DNA methylation, especially cytosine methylation, is an important epigenetic mechanism involved in many biological processes, including transposon proliferation, genomic imprinting, and the regulation of gene expression (Bird 2002; Y an et al. 2010). Growing evidence implicates DNA methylation in the regulation of plant gene expression in response to environmental stresses, including cold (Steward et al. 2002), drought (Labra et al. 2002; Tan 2010; Wang et al. 2011a), salt (Tan 2010; Wang et al. 2011b; Zhao et al.2010) , and metals (Aina et al. 2004; Kimatu et al. 2011). In most cases, a global demethylation of genomic DNA occurs in response to abiotic stress. There might be a close correlation between demethylation and gene expression,although few studies provide powerful evidence. Contrastingly,sometimes hypermethylation occurs in response to stress,such as the response of tobacco to NaCl (Kovarik et al.1997). These results imply that the DNA methylation pattern and alteration are related to the abiotic stress response in plants but that the association is obviously different between plant species.

The use of methylation-sensitive amplified polymorphisms (MSAP) is an effective technique for studying the genome methylation status (Xiong et al. 1999). It is a modification of the AFLP (amplified fragment length polymorphism) technique in which the isoschizomers Hpa II and Msp I are employed as ‘frequent-cutter’ enzymes. Both Hpa II and Msp I recognise 5’-CCGG-3’ but display differential sensitivity to DNA methylation. This technique has been applied to evaluate the level and pattern of cytosine methylation in plants (Ashikawa 2001; Dong et al. 2006; Li et al. 2008). The effect of abiotic stresses on cytosine methylation variation has been studied in crops (Karan et al. 2012; Kou et al. 2011; Tan 2010; Wang et al. 2011b; Zhong et al. 2009). The epigenetic variation needs to be explored in more genotypes so that a useful variability can be identified and exploited in crop

ORIGINAL ARTICLE

?

These authors contributed equally to this work.

*Corresponding author; Yaping Y uan Tel : +86-431-87835712

E-mail : yapingyuan@https://www.360docs.net/doc/c610667151.html,

breeding programs to enhance crop adaptation in unfavourable environments.

Low temperature is one of the most general abiotic stress factors limiting the growth, development, distribution, and yield of crops (Zhu et al. 2007). Maize (Zea mays L.), a very important crop, originates from subtropical regions and is known to be sensitive to low growth temperatures. Low temperature affects germination, seedling growth, early leaf development and overall maize crop growth and productivity.In northeast China, maize is often exposed to low temperatures during its seedling stage, resulting in yield loss. Some maize lines possess the unique ability to rapidly adapt to cold stress,indicating their unique genetic makeup and regulatory architecture. However, differences in methylation patterns and epigenetic responses in the maize lines under cold stress have been underexplored. In this study, we have investigated the extent and pattern of cytosine methylation under cold stress in a cold-tolerant line (W9816) of maize using the MSAP technique. MSAP fragments showed homology with stress-responsive genes, (retro)transposons, and genes involved in the regulation of development. The differential expression patterns of the cold-induced genes influenced by the methylation or demethylation status in the maize line revealed a possible role for epigenetic mechanisms in cold adaptation.

Results

Extent and Pattern of DNA Methylation under Control and Cold Stress Conditions

Sixty-four primer combinations were used to detect cytosine

methylation at CCGG sequences in the maize inbred line at seedling stage after 2 h, 6 h, 12 h, 24 h, and 48 h of exposure to cold stress and control (non-stress) conditions. Under 6°C conditions, 2,074 to 2,161 clear and reproducible methylated bands were amplified in at least one time-point sample

Table 1. MSAP-based cytosine methylation levels at different time-points under cold stress

Type

Cold stress time-point

0 h 2 h 6 h 12 h 24 h 48 h I 142014451441145214601468II 296295291278293304III 358355364361358359IV

1048382876747Total sites

217821782178217821782178Total amplified bands 207420952096209121612131Total methylated bands 758733737726718710MSAP (%)a

34.8033.6533.8433.3332.9732.60Fully methylated bands 462438446448425406Fully methylated ratio (%)b 21.2120.1120.4820.5719.5118.64Hemi-methylated ratio (%)c 13.5913.5413.6312.7613.4613.96Non-methylated ratio (%)d

65.20

66.35

66.16

66.67

67.03

67.40

a MSAP(%) = [(II + III + IV)/(I + II + III + IV)] × 100

b

Fully methylated ratio(%) = [(III + IV)/(I + II + III + IV)] × 100c

Hemi-methylated ratio(%) = [(II)/(I + II + III + IV)] × 100d

Non-methylated ratio(%) = [(I)/(I + II + III + IV)] × 100

Fig. 1. Examples of changing MSAP patterns detected in the different cold treatment samples (2 h, 6 h, 12 h, 24 h, and 48 h) compared to non-treatment samples (0 h). The DNA of different time-point samples was digested by Eco RI/Hpa II or Eco RI/Msp I. Typical demethylated loci are marked by arrowheads, and methylated loci are marked by hollow triangles.

(Table 1, Fig. 1). Table 1 shows the DNA methylation levels in the different samples. The number of methylated (hemi-methylated and fully methylated) DNA bands was determined, demonstrating that 710 to 758 bands were polymorphic (Type II+Type III+Type IV bands), accounting for 32.6% to 34.8% of all bands. Full-methylation (Type III) was detected more frequently than hemi-methylation (Type II), but the total number of these two kinds of methylated fragments was stable in each sample at all time-points. We found that Type IV fragments were the main source of the changes in the DNA methylation banding pattern induced by cold. At the 2 h, 6 h and 12 h time-points, the number of Type IV fragments was approximately 82 to 87; at the 24 h time-point, the number was 67; and at the last time-point (48 h), the number was down to 47 (Table 1). These findings indicate that the cold stress-induced demethylation in maize seedlings and the associated changes resulted from the fully-methylated cytosine on both DNA strands (Type IV). Cold-induced Methylation and Demethylation Changes

All possible banding patterns between control and cold stress samples of five time-points were compared to investigate the changes in cytosine methylation patterns under cold stress. Fifteen different banding patterns between the control and cold stress conditions were observed in the MSAP gels (Table 2). The patterns A-C represented the monomorphic class in which the methylation pattern was the same in the control and cold stress treatments. The patterns D-I indicated cytosine demethylation patterns, whereas possible cytosine methylation events induced by cold stress were represented by the patterns J-O. Approximately 88.06% to 90.13% of the CCGG sites remained unchanged under cold stress (Table 2). The percentage of the demethylated bands under cold stress was 6.29%, 6.06%, 6.52%, 6.38%, and 6.38%, while the percentage of methylated bands was 5.42%, 5.19%, 5.42%, 4.32%, and 3.49% in samples of the 2 h, 6 h, 12 h, 24 h, and 48 h time-points, respectively. In general, the cold stress induced more DNA demethylation than DNA methylation in this maize line (Table 2). The fully methylated loci (F, H and I) under non-stress conditions were demethylated after the low-temperature treatment, while the amount of methylated loci (Table 2) (M) at most time-points was stable and even decreased in the samples of the 48 h time-point. These data can explain why the number of Type IV bands decreased under cold stress conditions (Table 1).

Table 2. Analysis of DNA methylation patterns under cold stress with respect to control conditions in maize seedlings

Description of

Pattern Class

Banding Pattern

Different Treatment

Control Cold

Hpa II Msp I Hpa II Msp I 2 h 6 h12 h 24 h48 h

No change

A111113451350134413591373 B1010255254247258258 C0101323329327328332 Total19231933191819451963

88.29%88.75%88.06%89.30%90.13%

Demethylation

D10118591211 E01111920221622 F00117366777362 G011033136 H00102829252531 I0001698107 Total137132142139139

6.29% 6.06% 6.52% 6.38% 6.38%

Methylation

J111095579 K11012423231816 L100123324 M11004745515126 N10002833301318 O010084633 Total1181131189476

5.42% 5.19% 5.42% 4.32% 3.49%

A score of 1 and 0 represents the presence and absence of bands, respectively. V alues in parentheses indicate the percentage of bands in each pat-tern determined by dividing the number of bands in each pattern by the total number of bands in all three patterns.

Analysis of the Differentially Methylated DNA Sequences To identify the nature of the DNA sequences involved in (de)methylation under cold stress, 211 differentially methylated DNA bands were sequenced. The resulting sequences were BLAST searched against the databases at the NCBI (http:// https://www.360docs.net/doc/c610667151.html,) and MaizeGDB (http://blast.maizegdb. org) websites. Only 28 sequences returned BLAST results with the annotated sequences, and 7 of these 28 sequenced cytosine loci were methylated (Table 3). The positions of the cytosine alterations due to cold stress were located in the exon and UTR regions of these annotated sequences, suggesting gene body-specific methylation. The BLAST analysis of sequenced fragments revealed that the sequences are homologous to genes involved in numerous processes, including hormone regulation, cold response, plant development, photosynthesis, and transposon activation (Table 3). These results demonstrate that the genes involved in a wide range of cellular functions are affected mostly by gene body-specific (de)methylation alterations induced by cold stress in maize.

Expression Analysis of Polymorphic Fragments

The expression analysis of five MSAP polymorphic genes was performed using quantitative RT-PCR. Fig. 2 shows the expression results of the three time-points (12 h, 24 h, and 48 h) under cold stress. DM96, DM113, and DM177 were demethylated at the 2 h cold stress time-point; DM8 and DM11 were demethylated at the 24 h and 6 h cold stress time-points, respectively. The expression changes of some of the demethylated genes, including DM11, DM113, and DM177, were continually upregulated. The other demethylated genes (DM8 and DM96) showed the highest expression levels at the 24 h cold stress time-point, after which the expression level decreased slightly but still remained higher than that of the control.

Table 3. BLAST results of polymorphic methylated fragments

Fragment Methylation status

under stress

Accession Description E-value Position

DM8Demethylated NM_001153998Receptor kinase4e-18Exon

DM 11Demethylated NM_001157237Calcium-activated outward-rectifying potassium

channel 1

1e-72Exon

DM 14Demethylated AAK98713Putative transposable element1e-14Exon DM 27Demethylated NM_001157879Pectinesterase-1 3e-435’UTR DM 30Methylated NM_001158241Auxin-responsive SAUR family member 7e-273’UTR DM 47Demethylated X05422Chloroplast psbB-psbF-petB-petD gene cluster1e-30Exon DM 63Demethylated BAD81343MADS-box protein AGL357e-06Exon DM 81Demethylated NM_001157879Pectinesterase-1 1e-375’UTR DM 92Methylated AAG13527Kinesin-like protein 5e-10Exon DM 96Demethylated AF187822Transposon Doppia4 transposase 2e-76Exon DM 102Methylated ABQ44355Polyprotein1e-07Exon DM 103Demethylated AAL59229gag-pol8e-10Exon DM 109Methylated AY574035Rust resistance protein rp3-1 9e-68Exon DM 110Demethylated AAL76001Putative gag-pol polyprotein 1e-09Exon DM 111Demethylated NP_043003Ribosomal protein S12 7e-05Exon DM 113Demethylated NM_001154597CBS domain-containing protein 6e-141Exon DM 118Demethylated NP_001148053CBL-interacting serine/threonine protein kinase 155e-26Exon DM 137Methylated XP_003563758Receptor-like protein kinase HSL1-like4e-22Exon DM 154Demethylated NP_001105930Respiratory burst oxidase protein A4e-08Exon DM 156Demethylated NM_001154025Protein binding protein 2e-71Exon DM 168Demethylated XP_002878591Binding protein 1e-28Exon DM 176Demethylated AB298185P450, putative ankyrin repeat protein 2e-121Exon DM 177Demethylated NM_002467841TPR domain-containing protein 4e-61Exon DM 179Methylated NM_002440690Hexokinase-1 2e-109Exon DM 184Demethylated AAL76006Putative retrotransposon protein 2e-14Exon DM 196Methylated EU969358Gibberellin 20 oxidase 1 7e-76Exon DM 202Demethylated EU968954MADS-box transcription factor 29 1e-07Exon DM 220Demethylated HQ138672Mu transposon insertion mu1015801 flanking sequence 8e-13Exon

Discussion

Some studies have demonstrated that environmental stimuli can alter cytosine methylation patterns throughout the genome (Cao et al. 2011; Johnston et al. 2009; Kimatu et al.2011; Lu et al. 2007; Lukens and Zhan 2007; Wang et al.2011a; Wang et al. 2011b; Zhao et al. 2006; Zhao et al. 2010;Zhong et al. 2009). However, the relationship between DNA methylation and abiotic stress tolerance has rarely been studied in maize (Tan 2010). We used the MSAP technique to assess the extent and pattern of DNA methylation in response to cold stress. Overall, a significant association between the cold treatment and the level of methylation was seen in our study. The amount of DNA methylation was obviously decreased after 24 h of cold stress treatment. The cytosine methylation, especially the Type IV full methylation (Table 1), of maize seedlings decreased obviously at the genome level. This methylation showed a negative correlation with the period of cold treatment (Table 2). Further analysis indicated that the majority of cold-induced cytosine methylation changes occurred symmetrically on both DNA strands. In most situations, DNA demethylation is able to fine-tune gene expression. In this study, the decrease of cytosine methylation levels in maize seedlings may be an indirect effect of cold stress, but the decreased amount of Type IV (fully methylated)bands could provide further clues for unravelling the epigenetic regulation of maize adaptation to cold stress. The BLAST results of the MSAP fragments indicated that the search results from only 28 fragments contained the homologues and demonstrated a higher frequency of methylation in exons than in non-coding (UTR) regions,suggesting that DNA methylation changes were unbalanced in response to cold stress. This result is different from research conducted in rice (Wang et al. 2011a; Wang et al.2011b) and indicates that the methylation alteration could be induced by stress via distinct mechanisms in maize and rice.Despite the limiting BLAST results of the MSAP fragments,

we were able to identify different categories of genes, such as transposons, abiotic stress-responsive genes, and genes involved in plant development (Table 3). Two fragments (DM113 and DM118) were homologous to genes encoding the CBS domain-containing protein and CBL-interacting serine/threonine protein kinase (CIPK), which are involved in the abiotic stress response in plants (Piao et al. 2010;Singh et al. 2012; Tripathi et al. 2009). The genes pectinesterase-1, MADS-box protein, kinesin-like protein,and hexokinase are reported to be involved in ABA signalling, cell wall formation, flowering time and meiosis (Ausin et al. 2004; Chen et al. 2002; Ciereszko et al. 2001;Richard et al. 1994). The identification of a diverse category of genes with altered DNA methylation patterns in our study demonstrates that epigenetic modification may play a critical role in plant adaptation to environmental changes.

Transposons or retrotransposons are activated in response to abiotic stress and may generate true genetic or epigenetic changes, thus increasing plant adaptation to abiotic stresses (Reinders et al. 2009). Plant transposons are usually hypermethylated compared with host genes and are said to be epigenetically silenced (Rabinowicz et al. 2003). Seven fragments (DM14, DM96, DM102, DM103, DM110,DM184 and DM220) are homologous to genes related to transposons. Six of these genes (all except DM102) were demethylated under the cold stress conditions. Because the expression level of transposase is an important factor in the activation of TEs, we tested the expression of one gene (DM96,Transposon Doppia4 transposase) by qRT -PCR. The results demonstrated that the gene expression increased after the occurrence of demethylation (Fig. 2). The transposase expression always is able to induce the activation of transposons, and the transcriptional activation of transposons has the potential to alter the expression of adjacent genes (Kashkush et al. 2003). In rice, DNA affects the transposition activity of T os 17 and modulates the activity of neighbouring genes (Cheng et al. 2006). Therefore, cold-induced demethylation in maize and the activity of the genes neighbouring TEs may provide a new strategy for maize molecular breeding in the future. Five homologues of MSAP loci were selected to study the transcriptional changes: receptor kinase (DM8), calcium-activated outward-rectifying potassium channel 1 (DM11),transposon Doppia4 transposase (DM96), CBS domain-containing protein (DM113), and TPR domain-containing protein (DM177). The transcription of each of these genes increased, although two of them were expressed at slightly lower levels at the 48 h time-point than at the 24 h time-point. Despite the consistency of the expression results with the widely held belief that DNA methylation is associated with transcription inhibition, we still cannot exactly conclude that the methylation pattern alterations in maize correspond to gene expression

changes under cold stress because each MSAP fragment only

Fig. 2. Relative transcription levels of selected genes under cold stress.

provides information regarding the methylation change of one CCGG site. W e need more proof, such as bisulphite sequencing results, to back up our conclusions in the future.

Materials and Methods

Plant Materials and Cold Stress Treatment

A maize inbred line, W9816, which showed tolerance to cold stress, was used in this study. All seeds were sterilised in 75% ethanol (v/v) and then germinated at 25°C in the dark for 3 days. The uniformly germinated seeds were planted in pots filled with a transplanting medium containing peat, vermiculite and perlite (10:1:1, v:v:v). Maize seedlings were grown in an incubator at 25°C/20°C (light/ dark) with a photosynthetic photon flux density of approximately 450 l mol m?2 s?1 and 14 h/10 h (light/dark) cycles until the third leaves were fully expanded. For the cold stress treatment, the plants were kept in a 6°C growth chamber for durations of 2 h, 6 h, 12 h, 24 h and 48 h. The leaf tissues were harvested after the treatment and frozen in liquid nitrogen. The untreated seedlings (25°C) were used as controls and harvested at the same time as the stressed plants. Five replicates were prepared from each time-point for DNA methylation analysis. Methylation-sensitive Amplified Polymorphism (MSAP) Analysis Total genomic DNA was extracted using the improved CTA

B method. The MSAP analysis was performed as described (Dong et al. 2006; Xiong et al. 1999) with minor modifications. First, we identified the robustness of the five replicates using several primers (data not shown). These replicates were then mixed to provide a pool sample for the following MSAP analysis. Two enzyme combinations were used: Eco RI/Msp I and Eco RI/Hpa II; the sequence information for the adapters and primers of pre-amplification and selective amplification are provided in Table S1. The MSAP PCR products were separated on 6% sequencing gels and visualised via silver staining. The MSAP patterns for displaying the DNA fragments resulting from the digestions with the isoschizomers were divided into the following four types: Type I bands, present for both enzyme combinations; Type II bands, present only for Eco RI/Hpa II; Type III bands, present only for Eco RI/Msp; and Type I V bands, absent from both enzyme combinations. Here, Type II bands represent the hemimethylated state of 5’-CCGG sites due to methylation in one DNA strand but not in its complementary strand. Type III bands represent the case of full CG (internal cytosine of 5’-CCGG) methylation, whereas Type IV bands represent the case of full methylation at both cytosines (Karan et al. 2012; Wang et al. 2011b). The percentage of polymorphic MSAP bands in Table 1 was calculated using the following formula: MSAP (%) = [(II + III + IV)/(I + II + III + IV)] × 100. Isolation and Characterisation of the Differentially Amplified DNA Fragments

The differentially amplified fragments were excised from the gel using a sterilised surgical blade and incubated for 8 h at 37°C for further investigation. The supernatant was recovered by centrifugation and used for the re-amplification. The re-amplified DNA fragments were purified and cloned with the pMD18-T vector (Takara) for sequencing, and the sequences obtained were analysed by BLAST. Real-time Quantitative RT-PCR (qRT-PCR) Analysis

RNA was prepared by the RNAiso reagent (Takara) in accordance with the manufacturer’s instructions. Total RNA isolated was treated with DNa seI to remove genomic DNA contamination. The first-strand cDNA synthesis and the qRT-PCR were conducted using the SuperScript? III First-Strand Synthesis SuperMix (Invitrogen) and the SYBR Green JumpStartTM T aq ReadyMixTM (Sigma-Aldrich), respectively. The qRT-PCR was conducted in the ABI PRISM 7500 sequence detection system according to the manufacturer’s instructions. Each PCR reaction (20 μL) contained 10 μL of 2x real-time PCR Mix, 0.2 μM of each primer and cDNA at appropriate dilutions. The thermal cycling conditions were 95°C for 1 min, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. All reactions were performed in triplicate. Maize actin1 and GAPDH were amplified as endogenous controls. The primers used in this study are listed in Table S2.

Acknowledgements

This study was supported by the National High Technology Research and Development Program of China (863 Program) (No. 2011AA10A103), the Science Development Planning of Jilin Province (No. 20126030), and the National Natural Science Foundation of China (No.31100192).

Supporting Information

Additional Supporting nformation may be found in the online version of this article:

Table S1. Adapter and primer sequences for MSAP analysis. Table S2. Real-time RT-PCR primer.

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