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This article was downloaded by: [Universita' Milano Bicocca] On: 26 November 2012, At: 03:20 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tplb20 Methylation changes in specific sequences in response to water deficit c a a Massimo Labra , Candida Vannini , Marcella Bracale & Francesco Sala b a Dipartimento di Biologia Strutturale e Funzionale, Università degli Studi dell'Insubria, Via J.H. Dunant 3, Varese, Italy b Dipartimento di Biologia, Università degli Studi di Milano, Via Celoria 26, Milano, Italy c Dipartimento di Scienze dell'Ambiente e del Territorio, Piazza della Scienza 1, Universitd degli Studi di Milano, Bicocca, 20126, Milano, Italy Version of record first published: 05 Aug 2006. To cite this article: Massimo Labra, Candida Vannini, Marcella Bracale & Francesco Sala (2002): Methylation changes in specific sequences in response to water deficit, Plant Biosystems - An International Journal Dealing with all Aspects of Plant Biology: Official Journal of the Societa Botanica Italiana, 136:3, 269-275 To link to this article: http://dx.doi.org/10.1080/11263500212331351179 PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: http://www.tandfonline.com/page/terms-and-conditions This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material. PLANTBIOSYSTEMS,136 (3) 269-276, 2002 Methylation changes in specific sequences in response water deficit to Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012 M. LABRA,C. VANNINI,E SALAand M. BRACALE received 20 June 2001; revised version accepted 18 February 2002 ABSTRACT - Conditions of environmental stress may lead to epigenetic variability in plants. In particular, variation in genome methylation may be a mechanism of plant adaptation to abiotic stresses, We have investigated the relationship between water deficit and DNA changes in specific sequences by using the CRED-RA (Coupled Restriction Enzyme Digestion and Random Amplification) approach. Three polymorphic bands have been detected in dehydrated and rehydrated pea root tips. One of these bands is a zinc-finger sequence with three non canonical motifs, that could be involved in signal transduction pathways. KEY WORDS - Pisum sativum L., Pea, CRED-RA, Zinc finger, DNA methylation,RAPD An increasing body of experimental evidence indicates that conditions of environmental stress may lead to genotypic variability in plants. Heritable changes induced by the environment have been shown in flax (CULLIS, 1986), in Mesembryanthemum crystallinum L. (BOHNERT et aI., 1995), in Brassica nigra L. (WATERS& SCHAaL, 1996), and in Ustilago rnaydis L. (CULL~S, 1990). Moreover, genomic changes have been observed frequently in plants regenerated from tissue or cell cultures (KAEPPLER& PHILLIPS, 1993). Recently, epigenetic changes have also been associated to abiotic stress. Hypermethylation of heterochromatic loci has been reported in tobacco, either in response to osmotic stress (KOVARIKet al., 1997) or in silenced genes in transgenic plants (MEYERet al., 1992; MEYERet al., 1994). On the contrary, hypomethylation has been documented in chicory root tips (DEMEULEMEESTERet al., 1999) and in Arabidopsis lhaliana L. (FINNEGAN et al, 1998) when exposed to low temperature. Epigenetic changes have been observed in tissue cultures, while methylation polymorphisms have been frequently observed during the propagation of tissue cultures at the level of repeated sequences (SMULDERSet al., 1995) and may contribute to somaclonal variation (KAEPPLERet al., 2000). All these variations in genome methylation might be part of the plant's adaptation mechanisms to abiotic stresses (KOVARIKet al., 1997; MARTINESSEN& R~CHARDS,1995). However, more work is needed in order to clarify the metabolic processes, and the role of genes involved in these types of stress response. Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012 270 M. LABRa et al. In the present study we faced the problem of understanding the correlation between environmental stress and DNA methylation. Using the CRED-RA (Coupled Restriction Enzyme Digestion and Random Amplification) technique (CaI et al., 1996) we wanted to identify the genes involved in DNA methytation change in response to water stress. The CRED-RA technique is based upon the coupled restriction enzyme digestion and random amplification of DNA. It uses restriction endonucleases sensitive and insensitive to cytosine methylation in their targeting sites, so that DNA digestion is methylation-dependent (CAt et al., 1996). Results reveal the presence of three polymorphic bands, out of a total of 222 bands detected, one of which is a zinc-finger-like sequence induced in response to water stress. MATERIALS AND METHODS Elmer Gene Amp PCR System 9600. RAPD analysis requires only the presence of a single randomly chosen oligonucleotide (KARP et al., 1998). The PCR reaction was performed in 20 Ill reaction mixture containing 2 ml 10x-Dynazyme buffer (Celbio), 200 I~M of each dNTP, 20 ng DNA primer, 0.5 U Dynazyme II (Celbio) and 20 ng template DNA. An annealing step (1 rain at 36°C for 10-bp primers) or at 45°C (for 20-bp primers) and an extension step (72°C for 1 rain) followed an initial denaturation step (1 rain at 94°C). Primers, listed in Table 1, were obtained from Life Technologies. Amplification products were separated by electrophoresis on 2% ultrapure agarose gel, stained with ethidium bromide and visualised under UV light (Gel Doc 2000 Biorad). All bands detected were reproducible as assessed by three independent repetitions of the analysis. Plants and DNA extraction PoIymorphic bands analysis Pea (Pisum sativum L. cv. Lincoln) seeds were surface- Polymorphic bands were excised and gd-purified using sterilised in 10% NaC10 and germinated in moistened the QIAEX II agarose gd extraction kit (Qiagen) as agriperlite at 25°C in the dark. After 72 h, the seedlings described by the supplier. The purified fragments were were transferred to glass dissectors on top of 25 % glyc- cloned in the pUC57 plasmid by the T-doning kit (MBI erol (approx. 90% relative humidity) for 3 days in the Fermentas) and sequenced (MWG). Sequence analysis dark. In these conditions, the water content is reduced was performed by using the 2.0 version of the up to 82 %. These seedlings represented the dehydrated Washington University BLAST (http://www.ncbi.nlm. sample (D). Previous data showed that this treatment nih.gov/BLAST) software for gene and protein identifiinduces a drought stress able to promote a significant cation through sensitive, selective and rapid similarity cellular response, and that primary root tips always sur- search of protein and nucleotide sequence database. vive and resume growth upon rewatering (BRACALEet Sequence analysis was also performed by MOTIF dataal., 1997). To obtain rehydrated samples (R), some of the base (http://motiLgenome.AD.JP). dehydrated seedlings were transferred in water for 5 h and subsequently in moistened agriperlite at 25 °C in the Northern analysis dark. Control seedlings (C) were grown in agriperlite in Total RNA was extracted by TRIZOL (GibcoBRL) from the dark at 25°C for the same period of time. whole pea seedlings. Samples containing 10 lag of total Twenty-five root tips (2 mm in length) were excised RNA were electrophoresed on formaldehyde 1.2% from C, D and R seedlings and were ground to a fine (w/v) agarose gels and blotted onto Hybond-N+ mempowder in liquid nitrogen with a mortar and pestle. brane (Amersham). Filter hybridisation was performed Total genomic DNA was extracted and purified by using with the probes of the three clones of interest, labelled the DNeasy Plant mini kit (Qiagen). by using the Rediprime kit (Amersham), under high stringency wash conditions. Filters were finally exposed Genomic DNA digestion and CRED-RA to Kodak X-Omat AR X-ray film. The methylation status of the genome was analysed using the restriction enzymes MspI and HpaII. One pg Southern blotting of genomic DNA from each sample was digested with Ten pg of DNA were digested with Hind III and HhaI 15 U of each enzyme. Both enzymes recognise the restriction enzymes and separated on 0.8% (w/v) CCGG sequence, but HpaII is active only if both agarose gel in 1% (v/v) TBE buffer. The DNA was cytosines are unmethylated (CCGG), whereas MspI transferred by capillary blot onto Hybond-N+ memdoes not cleave the DNA when the external cytosine is branes (Amersham) according to the manufacturer's methylated (5=CCGG) (KovaRZKet al., 1997). instructions. Prehybridisation and hybridisation were Restricted DNA was amplified by the RAPD (Random performed at 65°C with constant agitation. Probes were Amplified Polymorphic DNA) method using a Perkin- generated from pUC57-CHL24-3 (accession number Methylation changes and water deficit 271 EMBLY14837, MARKAUSAKAS& DREGUNIENE, unpublished) plasmid by double digestion with EcoRI and BamHI. The insert was then labelled by using the Gene Images Alkphos direct labelling and detection system (Amersham). Autoradiography was performed at room temperature with Kodak X-Omat AR X-ray film and an intensifying screen. MCDR RESULTS Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012 D N A polymorphism detection by the C R E D - R A technique To determine the sequences involved in methylation change by water stress, DNA from 72-h dehydrated (D), rehydrated (R) and control (C) samples was fractionated with HpalI or MspI restriction enzymes and analysed 3500bp-.~ lO00bp-~ TABLE1 DNA primers for RM~D analysis Name FS-5 FS-6 FS-7 FS-8 FS-9 FS-10 FS-11 FS-I2 FS-13 FS-I4 FS-15 FS-16 FS-25 FS-26 FS-27 FR-27 FS-28 CHL9 CHL12 CHL16 CHL17 CHL18 CHL19 CHL20 CHL21 CHL22 CHL24 CHL26 CHL30 CHL34 CHL35 CHL24-3 CHL24,2 CHL24-I DNA sequence 5'-GGGATCCGGC-3' 5'-GCTTCGATAC -3' 5'-CCGAATTCGC-3' 5'-TGAGTCACCG-3' 5'-GGAGAGACTC-3' 5'.ACCAGGTTGG-3 ' 5'-GACGCCACAC-3' 5'-TGTAGCTGGG-3' 5'-GGAAGTCGCC-3' 5'-GTGGCAAGCC-3' 5'-ATCGGCTGGG-3' 5'-CTCCCGGTAC-3' 5'-CTTGCCCACG-3' 5'-ATCGGCTGGG-3' 5'-AGCCGGCCTT-3 ' 5'-ACGCGCGGGA-3 ' 5'-TGGCCCCGGT-3' 5'-GGCATGTCAAGCCCTGGTAA -3' 5'- CGGAAGCAATTTGCTTGGCT-3' 5'-CTCCCTCATGATTCTTGGGA -3' 5'-AAACTCAAGGCCACCCTTCC -3' 5'- CCTTTGCCTTAGGATTCGGT-3' 5'- GAGGATATGTTCGCCGTCCT-3' 5'- ACTAATGGCAACCCTTCGAG-3' 5'- AGAAGCATCCCAAAAGCGTC-3' 5'- TTGTTCCTGACCCTGGTTCA-3' 5 '-CATCCGCCAATATKFTCCCG-3 ' 5'-CAATTCGAGGATCCAGAGAC4' 5'-CAGGCAATCCTAAACTCTCT-3' 5'-ATCGGGAAAGACTCCGCAAA-3' 5'-GAGTACAGGTATGACAGGCA-3' FIGURE1 - CRED-RAanalysisperformedby usingCHL24 primer on DNA from control (C), dehydrated (D) and rehydrated (R) root tips. CHL24-1, CHL24-2 and CHL24-3 indicate bands isolated from the gd and sequenced. by CRED-RA. Since HpaII is active only if both cytosines are unmethylated (CCGG), whereas MspI cleaves the DNA when only the second cytosine is methylated (CsnlCGG), polymorphic bands detected were originated from a different methylation status in CCGG sequences localised in the amplification region. When cytosines in the CCGG sequence are methylated, HpaII and MspI are inactive, and consequently the amplification product can be obtained. On the contrary; in demethylation status, enzymes cut the DNA in a specific CCGG sequence, resulting in the absence of amplification fragments. Different RAPD primers were tested to detect scorable bands on the analysing gel. Bands on the gels were consistent and reproducible. The 32 primers listed in Table 1 produced a total of 222 bands, three of which were polymorphic. Polymorphic bands were obtained by 0 o 0 d') 0 9 N 0 o~ ! it- Io ffl IC~ I1i~- 0 H I-I t~ ~ 0 Inii_il o r0t i~i.~ H r~ ffl di~. t-I q r',t t~ o a" P_, 0 rl. ":::1 b,l ff o n, 0 u~ H H Q q Q ;~ Q ~ q © q~ ~ Q H ~ ~ H H ~ Q C~ ~ H H Q ~ H q q~ Q Q Q Q Q d~ Q q~ H H Q © Q q H Q H ~ ~ ~ H ~ H Q H ~ ~ H H H Q H Q ~ H Q Q q q H ~ ~ Q © H ~ q ~ Q Q ~ ~ © Q ~ ~ ~ ~ 0 H q Q Q ~ Q H q ~ q ~ ~ Q Q ~ ~ 0 Q Q H ~ H Q ~ q ~ H ~ H Q O ~ H H q~ Q H ~ Q © q H Q 0 ~ Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012 Q H H © ~ ~ q q~ Q ~ ~ © ~' Q q H d~ ~ Q d~ ~ q Q ~ q Q Q Q H H Q Q Q H ~ © © Q © q q H H H q q Q H q q ~ ~ Q Q Q Q ~ Q ~ q © q Q H ~ q Q ~ q~ H Q Q H Q H q Q Q Q MethyIation changes and water deficit 273 amplifying HpaII digested DNA with the CHL24 primer. Electrophoresis of the amplification products showed two bands (indicated as CHL24-1 and CIcLL243) present only in D and R samples, and one band (indicated as CHL24-2) detected only in C sample (Figure 1). D R C CHCL 24-1 Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012 Sequences analysis CHL24d and CHL24-3 (from D and R samples) and CHL24-2 (from C sample) bands were excised from the gel, cloned in the pUC57 plasmid and sequenced. Sequences of CHL24-1 and CHL24-3 are identical in bands purified from D and R samples. The sequences were submitted to the EMBL database and have received the following accession numbers: AJ311167 for CHL24-1, AJ312062 for CHL24-2 and AJ312063 for CHL24-3. The HpaII target sequence was detected in all three fragments by restriction analysis conducted using the Restriction Enzymes Map Database. This confirmed that polymorphic bands can be ascribed to methylation changes in CCGG sequences. CHL24-1 is 719-bp long and does not reveal significant similarity with sequences present in GenBank or EMBL database, while CHL24-2 (744 bp) shows a high sequence similarity (97% overlap) with mitochondrial DNA of Arabidopsis thaliana L. CHL24-3 is 807-bp long with an ORF of 612 bp (Figure 2A), and does not show similarity with other genes or sequences present in GenBank or EMBL. MOTIF analysis inside this clone reveals three non canonical zinc-finger motifs, two of the C-Xs-C-X;C-X;H type and a third of the C-XT-C-Xs-C-X;H type (Figure 2B). These data may suggest a relationship between CHL243 and a transcription factor containing the zinc-finger motif. Further analysis is needed in order to ascertain whether or not CHL24-3 is a putative zinc-finger transcription factor involved in methylation metabolisms. To establish a relationship between CHL24-3 and other zinc-finger sequences, a MOTIF - Genome Net database was used. The results shown in figure 2B describe the zinc-finger motif and the similarity between the zinc-finger motif of Arabidopsis thaliana (Athb-1) and our sequence. Athb-1 recognised a 9-bp symmetric sequence of DNA, and specific analysis showed that this gene could be involved in the signal transduction pathway necessary for plant growth and development (SESSA et al., t993). ,Northern and Southern blot analysis" As shown in Figure 3, northern analysis on the total RNA from control and treated seedlings confirmed' the results obtained using the CRED-RA technique. D R C CHCL 24-2 D R C CHCL 24-3 FIGURE 3 - Expression of CHL24-1, CHL24-2 and CHL24-3 mRNAs.Eachlanewas loadedwith 10 pg total t~NApreparedfrom C (control),D (dehydrated)and R (rehydrated)pea seedlings. Gel stainingwas used to verifythat equal amountsof RNA werepresent in eachlane. Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012 274 M. LaBr,a et al. Drought stress induces the appearance of CHCL24-1 and CHCL24-3 clones: their mRNA, absent in control conditions, accumulated upon drought treatment and slightly decreased after rehydration. On the contrary, CHCL24-2 mRNA levels remained high as long as the seedlings were kept under control conditions, and rapidly declined after drought treatment. To characterise the putative zinc-finger transcription factor, Southern blot analysis was performed by using as probe the CHL24-3 sequence. The analysis was performed on genomic DNA extracted from C, D and R samples digested with HindIII and HpaII. CHL24-3 sequence did not show a target sequence for Hind III, but the target sequence of HpaII was present in position 286 of the CtlL24-3 fragment. HpaII recognises the GCGC sequence, but this enzyme is active only if both cytosines are unmethylated. Figure 4 shows the results of the Southern blot. Using CHL24-3 as a probe, a 1200-bp fragment was detected in D and R samples, while it was absent in control sample. M CDR ",ql"--800bp FIGURE4 - Southern blot analysis of genomic DNA isolated from pea DISCUSSION In the present study, a relationship is established between water deficit and DNA methylation changes in specific sequences. Indeed, on the basis of the CREDRA approach, three polymorphic bands have been detected. This means that these sequences are regulated by specific DNA changes in response to water deficit. On the basis of these results, we may consider epigenetic change as an important mechanism in response to abiotic stress in plants. This consideration is in agreement with several papers describing a relationship between methylation changes and environmental stress (SABBAtI et al., 1995; KOVAPdKet J., 1997; FINNEGANet al., 1998; DEMEULEMEESTZR,1999). To define the role of the three isolated sequences, a BLAST analysis was performed. Analysis of the CHL24-3 sequence revealed three zincfinger domains. This type of zinc-finger domain is found in a number of proteins in a wide range of organisms; it is present in many regulatory proteins, including viral transcription factors and oncoproteins, and in plants it is involved in DNA repair and recombination (FREEMONTet aL, 1991). Much work has been done on the protein-DNA interaction of the zinc-finger protein. Crystallographic (PAVLETICH& PABO, 1991) and nuclear magnetic resonance studies (OMICHINSKI et al., 1993) have revealed DNA-protein interaction between a single zinc-finger motif and its target DNA sequence. The amino acids responsible for base recognition have been found by statistical sequence analysis. By domain swapping studies, root. Ten l~g of DNA were digested with HindIII and HpaII restriction enzymes, electrophoresed on a 0.8% agarose gel, transferred to nylon membranes and probed with CHL24-3. (C) Control root tips; (D) Dehydrated root tips; (R) Rehydrated root tips; (M) Molecular marker. it was shown that each zinc finger in the clustered zincfinger motifs spans a unit of three bases in the target sequences (NARDELLIet al., 1991). CHL24-3 is a zinc-finger sequence with three non canonical motifs. The repetition for three times of this motif, inside a sequence of only 807 bp, suggests that these amino acid combinations are not casual, but result from the translation of nucleotidic sequences related to specific zinc-finger functional domains. On the basis of our results and from database analysis, it is difficult to define a functional role for CHL24-3 zinc-finger sequences; however, we have shown a relationship between water stress, DNA methylation change and zinc-finger sequences. Further investigation will be necessary to characterise this sequence and to describe its metabolic rote in response to water stress. Finally, on the basis of our data, we consider CRED-RA to be a powerful tool in understanding whether methylation changes are directed to specific sequences or are a random response to stress found equally in all sequences. In addition, CRED-RA can be used to isolate genes or sequences involved in plant response to abiotic stress. Methylation changesand waterdeficit 275 Downloaded by [Universita' Milano Bicocca] at 03:20 26 November 2012 ACKNOWLEDGEMENTS KOVaRIK A., KOUKALOVAB., BEZDEK M., OPKINNY Z., i997 - Hypermethylation of tobacco heterochromatictoci in response to osmotic stress. Theor. Appl. Genet. 95: 301-306. W e would like to t h a n k Cristian Savini a n d Valentina MaRTINESSENR.A. & R~CHaRDSE.J., 1995 - DNA methylation in H a u p t for technical assistance. eukaryotes. Curr. Op. Genetics Development. 5: 234-242. 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Applied KaEPPLER S.M., KAEPPLER H.E, RttEE Y., 2000 - Epigenetic Genet. 91:1257-1264. aspects of somadonal variation in plants. Plant Mol. Biol. 43: WATERSE.R. & SC14AALB.A., 1996 - Heat shock induces a loss of 179-188. rRNA-encoding DNA repeats in Brassica nigra. Proc. Nail KA~ A., ISAACRG., INGRAMD.S. (Eds), 1999 - Molecular tools Acad. Sci., USA. 93: 1449-1452. for screening biodiversity. Chapman and Hall, London. AUTHORS Massimo Labra (corresponding author), Dipartimento di Scienze dell'Ambiente e del Territorio, Piazza della Scienza 1, Universitd degh" Studi di Milano - Bicocca, 20126 Milano, Italy Candida Vannini, MarcellaBraeale, Dipartimento di Biologia Strutturale e Funzionale, Via J.H.Dunant 3, Universit~ degli Studi delHnsubria, Varese, Italy Francesco SaIa, Dipartimento di Biologia, Via Celoria 26, Universitd degli Studi di Milano, Milano, Italy