Mdoryco1-1, a bidirectionally transcriptional Ty1-copia retrotransposon from Malus × domestica

Scientia Horticulturae 220 (2017) 283–290

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Mdoryco1-1, a bidirectionally transcriptional Ty1-copia retrotransposon from Malus × domestica Lulu Wang a,c,1 , Yuying He a,1 , Huarong Qiu a , Jing Guo a , Mengxue Han a , Junyong Zhou a,b , Qibao Sun b , Jun Sun a,c,∗ a

College of Horticulture, Anhui Agricultural University, 130 West Changjiang Road, Hefei City 230006, Anhui Province, People’s Republic of China Horticulture of Research Institute, Anhui Academy of Agricultural Sciences, 40 South Nongke Road, Hefei City 230031, Anhui Province, People’s Republic of China c State Key Laboratory of Tea Plant Biology and Utilization, Anhui Agricultural University, 130 West Changjiang Road, Hefei City 230006, Anhui Province, People’s Republic of China b

a r t i c l e

i n f o

Article history: Received 3 January 2017 Received in revised form 28 March 2017 Accepted 28 March 2017 Available online 23 April 2017 Keywords: Ty1-copia-like retrotransposon Malus × domestica Bidirectionally transcription Stresses Spur mutation

a b s t r a c t Most retrotransposons from plants are believed to be either transcriptionally inactive or transcriptionally silent in somatic tissues but active during particular stages of development or under certain conditions. We have identified a bidirectionally transcriptional apple Ty1-copia-like retrotransposon called Mdoryco1-1, which was associated with apple spur mutation. Mdoryco1-1 belongs to a low-copy-number family in the sequenced apple genome. These copies were highly similar. The sense and antisense strands of the 5 -LTR (long terminal repeat) and 3 -LTR drove expression of GUS in transiently transformed tobacco leaves, activity which was dependent on the presence of the promoter function in the Mdoryco11 LTR. The transcriptional activity of Mdoryco1-1 was increased after ABA (abscisic acid), wounding and 2,4-D (2,4-dichlorophenoxyacetic acid) treatments, but not SA (salicylic acid) treatment, despite the predicted TCA-element (cis-acting element involved in salicylic acid responsiveness) in the Mdoryco1-1 LTR sense strand. The analysis of flanking sequences indicates that the Mdoryco1element is adjacent to genes in apple genome. The results suggested that the expression of Mdoryco1 could affect adjacent gene transcription. © 2017 Elsevier B.V. All rights reserved.

1. Introduction Transposable elements are mobile genetic elements that are present, often in high copy number, in the plant genome. Transposable elements are divided into two classes according to their transposition mechanism and mode of propagation (Kumar and Bennetzen, 1999). Those that replicate via an RNA intermediate are termed Class I elements or retrotransposons, while those that transpose as a DNA segment by a “cut-and-paste” mechanism are termed Class II elements or DNA transposons. Retrotransposons with long terminal repeat (LTR) at their ends in direct orientation are further divided into two major super families, Ty1-copia and Ty3-gypsy,

∗ Corresponding author at: College of Horticulture, Anhui Agricultural University, 130 West Changjiang Road, Hefei City 230006, Anhui Province, People’s Republic of China. Tel.: +86 13866765720. E-mail address: [email protected] (J. Sun). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.scienta.2017.03.049 0304-4238/© 2017 Elsevier B.V. All rights reserved.

depending on the relative order of the integrase (int) and reverse transcriptase (rt) domains. Most retrotransposons are believed to be either transcriptionally inactive (Kumar and Bennetzen, 1999) or transcriptionally silent in somatic tissues but active during particular stages of development or under certain conditions, such as after wounding or when ABA, 2,4-D, or SA are elevated (Rico-Cabanas and MartínezIzquierdo 2007; He et al., 2010; Du et al., 2015). The molecular basis of the LTR retrotransposon stress response is attributed to the LTR sequences located at both ends that contain transcription start sites and regulatory motifs strikingly similar to those of cellular gene promoters (Grandbastien et al., 1997; Ito et al., 2013; Anca et al., 2014; Cao et al., 2015). The presence of the promoters and regulatory sequences at both extremities could affect transcription of adjacent genes, by producing sense or antisense transcripts of the genes (Kashkush et al., 2003; Huettel et al., 2006; Kashkush and Khasdan, 2007; Butelli et al., 2012). In blood oranges, the transcriptional start site and cold-responsive elements within the LTR of the retrotransposon Tcs when situated by the Ruby gene, an activator of anthocyanin synthesis-confer the fruit-

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specific and cold-dependent regulation of expression, whether the LTR is inserted in the same or opposite orientation relative to the Ruby gene (Butelli et al., 2012). In wheat and rice, silencing of genes adjacent to LTRs was associated with higher levels of antisense transcripts originating from the LTR (Kashkush et al., 2003; Kashkush and Khasdan, 2007). An increasing number of phenotypic changes associated with adjacent LTR retrotransposons has been reported in plants (Kashkush et al., 2003; Kobayashi et al., 2004; Kashkush and Khasdan, 2007; Xiao et al., 2008; Butelli et al., 2012). Retrotransposons, based on their abundance in plant genomes, their transcription orientation, and their sensitivity to stress, are believed to play important roles in the modulation of host gene expression. However, the mechanisms controlling retrotransposon expression are not clearly understood. DNA methylation does seem to be associated with retrotransposon expression in plants (Martienssen and Colot, 2001; Liu et al., 2004), because high levels of cytosine methylation resulted in transcriptional inactivity. Apple is among the major fruit crops in the world. Propagated apple plants arising from buds are prone to be variable, an effect termed bud mutation. One of the mechanisms proposed to underlie bud mutation is retrotransposon activation. In the apple genome, retrotransposons represent the most abundant transposable element fraction, comprising 38% of the total genome and 89% of all transposable elements (Velasco et al., 2010). The polymorphism between the cultivar ‘Red Delicious’ and its spur mutations were detected using IRAP (Inter-retrotransposon amplified polymorphism) (Sun et al., 2010b), a technique using the retrotransposon ARL-12 (Sun et al., 2010a). Here, we report the isolation of the complete genomic sequence of ARL-12 from the cultivar ‘Red Delicious’ using genome walking. The retrotransposon was named Mdoryco1-1 (Malus × domestica oryco-like), according to the structure of Ty1-copia retrotransposons (Llorens et al. 2009). Mdoryco1-1 expression was analyzed by testing the ability of both the sense and antisense strands of its two LTRs to direct expression of a reporter gus gene in a transient assay. We tested for inducible expression in apple of Mdoryco1-1 in response to wounding as well as exogenous applications of ABA, 2,4-D, or SA. The methylation levels of the Mdoryco1-1 LTR sense strand was analyzed under stresses.

2. Materials and methods 2.1. Plant material Three-year-old potted apple trees (Malus × domestica ‘Red Delicious’) were used as experimental material. Nicotiana benthamiana leaves were used for promoter functional studies. Genomic DNA was extracted using the method of Murray and Thompson (Murray and Thompson, 1980).

2.2. Isolation of a full-length apple Ty1-copia-like retrotransposon Mdoryco1-1 The RNaseH-LTR section named ARL-12 was initially obtained from the apple variety Gala (Sun et al., 2010a). Starting from ARL-12 LTR, IPCR (inverse PCR) was performed on DNA from the apple variety ‘Red Delicious’. DNA from leaves was digested with HindIII and self-ligated. IPCR was used to obtain a fragment of the retrotransposon using primers IP1-F and IP1-R (Table S1), designed based on ARL-12 (Sun et al., 2010a). The majority of the Mdoryco1-1sequence was obtained by conventional PCR using primers Mdoryco1-1-F and Mdoryco1-1-R (Table S1). DNA was digested with VspI and selfligated. The full-length retrotransposon Mdoryco1-1 was obtained by IPCR using primers IP2-F and IP2-R (Table S1).

2.3. Bioinformatic analysis of Mdoryco1-1 in apple The genome sequence of apple was scanned for Mdoryco11 sequence using the BLASTN program with either the 4996-bp Mdoryco1-1 retrotransposon or the 5 -LTR as the query sequence. For Mdoryco1, only retrotransposons predicted to have the complete structure were selected. For truncated Mdoryco1, only sequences with >80% identity to Mdoryco1-1 LTR sequence were used. Because transcription activation of retrotransposons like Mdoryco1-1 LTR could cause the expression of adjacent genes in the apple genome, we used approximately 2000 bp of flanking sequences on both strands out from each LTR to search the Malus × domestica genome for identification of closest genes. 2.4. Ectopic expression of the sense or antisense strand of the 5 -LTR or 3 -LTR of the retrotransposon Mdoryco1-1 To determine if the LTRs of the retrotransposon Mdoryco1-1 have any promoter function, the promoter 35S on pBI121 vectors was substituted by either the sense strand of the 5 -LTR, the antisense strand of the 5 -LTR, the sense strand of the 3 LTR or the antisense strand of the 3 -LTR using In-Fusion HD Cloning Kits (Takara) to form the respective recombinant constructs (pB5 -sense-LTR:GUS, pB5 -antisense-LTR:GUS, pB3 -sense-LTR:GUS and pB3 -antisense-LTR:GUS). The fusion constructs and the positive control CaMV 35S-GUS (pBI121) were separately transferred into Agrobacterium tumefaciens strain EHA105 by heat shock. A. tumufaciens-mediated transformation was used for transient GUS expression in Nicotiana benthamiana leaf. A. tumufaciens transformation of Nicotiana benthamiana leaf was used as negative controls. 2.5. Treatment with abiotic stresses Young leaves of three-year-old potted apple trees were sprayed with either 1 mM salicylic acid (SA) in sterile water/0.01% ethanol, 50 ␮M 2,4-dichlorophenoxyacetic acid (2,4-D) in sterile water/0.01% ethanol, or 100 ␮M abscisic acid (ABA) in sterile water/0.01% ethanol, or were treated with wounding by making 40–50 pinholes per leaf with a needle (Wn). Control plants were sprayed with sterile water/0.01% ethanol. After 0–24 h of treatments with ABA, 2,4-D, or water, or 9 h after treatment with SA or wounding, leaves were collected and subjected to qRT-PCR as described below. Three trees were employed in each treatment. Samples after 9 h were subjected to methylation analysis. 2.6. LTR expression analysis by real-time qPCR After abiotic stress treatments, total RNA was extracted and purified from 1 g of leaves using RNeasy Plant Mini kit (Qiagen, Courtabeuf, France) following the manufacturer’s instructions. DNase-treated total RNA was further purified and retrotranscribed into cDNA using the PrimeScriptTM RT reagent kit with gDNA Eraser (Perfect Real Time; TaKaRa). ® Quantitative real-time RT-PCR was performed using SYBR Premix ExTaqTM (TliRNaseH Plus; TaKaRa) following manufacturer’s instructions. Real-time RT-PCR reactions were carried out in a final ® volume of 20 ␮L containing 10 ␮L of 2 × SYBR Premix Ex Taq, 0.4 ␮M of each primer (Table S1), ROX Reference Dye (50×) and 2 ␮L of cDNA. The following thermal profile was used: 1 cycle of 95 ◦ C for 30 s, 40 cycles of 95 ◦ C for 5 s and 60 ◦ C for 30 s in optical 48-well plates with a StepOneTM Real-Time PCR System (Applied Biosystems). The primers used for qPCR experiments are given in Table S1. Quantitative real-time PCR reactions were carried out in three biological replicates. No-template negative controls (water controls) were performed. After the amplification steps, the melting curve was determined to verify that only one specific product

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had been amplified. The relative expression level changes for 5 LTR and 3 -LTR in response to the stresses was determined with the 2−Ct method (Livak and Schmittgen, 2001). The data were expressed as mean ± standard deviation. The expected amplicon size from the LTR was 128 bp. Apple GAPDH was used as a reference for the mRNA expression (Table S1). 2.7. DNA methylation analysis Genomic DNA was modified using the EZ DNA MethylationGoldTM kit (ZYMO-RESEARCH, USA) according to the manufacturer’s instructions. Briefly, 500 ng of DNA was first treated by bisulfate containing C-T conversion reagent and then incubated at 98 ◦ C for 10 min followed by 64 ◦ C for 2.5 h. Modified DNA was purified using Zymo-Spin IC TM Columns, and stored at −20 ◦ C until use. Primers for Mdoryco1-1 LTR for bisulfate sequencing were designed by the Meth-Primer Program (http://www.urogene.org/ methprimer; Table S1). For each PCR reaction, 2.5 ␮L of bisulfatetreated DNA was used in a 50 ␮L reaction system. The PCR products were cloned into the pMD18-T vector (Takara, Dalian, China) and sequenced. At least ten clones were sequenced for each sample. The methylation levels for each of the three types of cytosines, CG, CHG and CHH, were calculated by dividing the number of non-converted (methylated) cytosines into the total number of cytosines and were expressed as percentage (%) per site. Analyses of the bisulfate sequencing results were conducted at the Kismeth website (http:// Katahdin.mssm.edu/kismeth). 3. Results 3.1. Structure of Mdoryco1-1 retrotransposon The known sequence (ARL-12) was used to isolate the full-length sequence of the retrotransposon Mdoryco1-1 by inverse-PCR. The retrotransposon Mdoryco1-1 (GenBank accession no. KF955541) is 4996 nucleotides in length and flanked by 5-bp target site (ATTGG) duplications in the host DNA. The deduced amino acid sequence of Mdoryco1-1 shows that the retrotransposon Mdoryco1-1 is a typical Ty1-copia-like retrotransposon (Llorens et al., 2009), containing two LTRs of 388 bp in length that flank the element internal region, which sequentially encoded the proteins GAG, proteinase, integrase, reverse transcriptase and RNaseH (Fig. 1A). The alignment of different domains of Mdoryco1-1 with other plant retrotransposons are shown in Fig. 1A, in which similarities included several highly conserved functional motifs found in retrotransposons. On the basis of amino acid sequences of putative polyproteins, Mdoryco1-1 was clearly clustered within the class of Ty1-copia retrotransposons using the neighbor-joining method (Saitou and Nei, 1987) (Fig. 1B). In Mdoryco1-1, there was a potential site for minus strand priming (PBS) at the 3 end of the 5 -LTR, a sequence complementary to the 3 sequence of methionyl tRNA (Fig. 1A). A polypurine tract (PPT), which is involved in the priming of RTN plus-strand cDNA synthesis, was present upstream of the 5 end of the 3 -LTR (Fig. 1A). The two 388-bp LTRs share 99.23% identity (3 bp differences) (Fig. 2), which suggests that Mdoryco1-1 corresponds to a recent insertion (Fig. 2A). Each LTR contains 4-bp, perfect, inverted repeats at their ends (5 -TGTT...AACA-3 ) (Fig. 2A). The Mdoryco1-1 sequence contains 3 stop codons at positions 1718, 3284 and 3626. A blastn search against the Malus × domestica genome sequence identified four complete Mdoryco1 elements, numbered Mdoryco1-2 to Mdoryco1-5. The five Mdoryco1 elements (Mdoryco1-1 to Mdoryco1-5) showed 98.59% identity, which suggested that the amplification of the Mdoryco1-like elements in the apple genome was a recent event. The blastn search also revealed the presence of at least 36 complete, 388-bp LTRs of the truncated

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Mdoryco1-like element in the apple genome, which showed 94.75% identity. The analysis also indicated that 37 of the 43 truncated LTRs in the apple genome retained approximately 197 bp of the 5 -end of the LTR (Table S2), which showed 74.73% identity. 3.2. Prediction of promoter and regulatory elements of Mdoryco1-1 LTR Because transcription of LTR retrotransposons can be driven by a promoter in the LTRs and can be regulated by elements within the LTRs (Kashkush et al., 2003; Ramallo et al., 2008; He et al., 2010; Butelli et al., 2012), a search for eukaryotic promoter functional motifs and regulatory elements was performed in the 5 -LTR and 3 -LTR of Mdoryco1-1 using the PlantCARE Database (http://bioinformatics.psb.ugent.be/webtools/plantcare/ html/) (Lescot et al., 2002). In the sense strands of the LTRs, we identified two putative TATA boxes (nts 9–12 and nts 31–35), a core promoter element, around −30 of transcription start; a CAAT box (nts 254–257), a common cis-acting element in promoter and enhancer regions; an AT-rich element (nts 366–376), for binding ATBP-1; a TCA-element (nts 283–292), a cis-acting element involved in salicylic acid responsiveness; and, in only the 5 -LTR, two 10-bp tandem TC-rich repeats (nts 304–313 and 348–357), cis-acting elements involved in defense and stress responsiveness (Fig. 2A,). The 3 -LTR differed from 5 -LTR by 3 bp (Fig. 2). The 3 nucleotide transitions were located at nt 151 (A to G), nt 304 (A to G) and nt 350 (T to C) in the sense strand (Fig. 2A). The first nucleotide mutation (nt 151) created a putative GAG-motif, part of a light −responsive element, in the sense strand of 3 -LTR, which was lacking in the 5 -LTR (Fig. 2A). The latter two nucleotide divergences (nt 304 and nt 350) resulted in loss of the two putative tandem TC-rich repeats in the sense strand of 3 -LTR (Fig. 2A). Because the LTRs of retroelements can provide bidirectional activator sequences (Kashkush et al., 2003; Butelli et al., 2012), we also searched for promoter and regulatory elements within the antisense strands of the Mdoryco1-1 LTRs using the PlantCARE Databases (Fig. 2B). In the antisense strands of both 5 -LTR and 3 -LTR,we identified three putative TATA boxes (nts 143–147, 283–289 and 280–284), four CAAT boxes (nts 20–23, 332–336, 333–337 and 369–373), a 5 -UTR Py-rich stretch (nts 98–107), a cis-acting element conferring high transcription levels, a Box III (nts 295–304), a protein binding site, a G-box (nts 261–267), a cisacting regulatory element involved in light responsiveness, an HSE (nts 355–364), a cis-acting element involved in heat stress responsiveness, and an MBS (nts 176–181), a MYB binding site involved in drought-inducibility (Fig. 2B,). There were no differences in the identified cis-acting elements between the 5 -LTR and 3 -LTR antisense strands (Fig. 2B). 3.3. Functionality of the Mdoryco1-1 LTRs In order to see if the putative promoters found in the sense and antisense strands of both the 5 -LTR and 3 -LTR of the retrotransposon Mdoryco1-1 are functional in plant cells, transient gene expression experiments were undertaken in tobacco leaves transformed by Agrobacterium infiltration. Transcriptional fusion constructs of the sense or antisense strand of the 5 -LTR or 3 LTR were made containing putative promotors and GUS gene (pB5 -sense-LTR:GUS, pB5 -antisense-LTR:GUS, pB3 -sense-LTR:GUS and pB3 -antisense-LTR:GUS). The results of ectopic expression showed that the above four Mdoryco1-1 LTR constructs, 5 -senseLTR, 5 -antisense-LTR, 3 -sense-LTR and 3 -antisense-LTR, could drive GUS expression (Fig. 3). The stronger histochemical GUS staining under 5 -sense-LTR was observed in leaves (Fig. 3C). The intensity of GUS staining was stronger for the 5 -sense-LTR compared to 3 -sense-LTR, 3 -antisense-LTR and 5 -antisense-LTR

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Fig. 1. Mdoryco1-1, a transcriptionally active Ty1-copia retrotransposon from Malus × domestica. A. The structure of the Mdoryco1-1 element is schematically shown at the top of the figure. LTR, long terminal repeat sequence; PBS, primer binding site; GAG, genes encoding group-specific antigen; PR, protease; INT, integrase; RT, reverse transcriptase; RH, RNaseH; PPT, polypurine tract. The accession number for Mdoryco1-1 is KF955541. Alignment of conserved functional motifs corresponding to GAG, PR, INT, RT, RH from copia AL137898.1 (Arabidopsis thaliana), and Osser X69522.1 (Volvoxcarteri f. nagariensis), and the sequenced Mdoryco1-1 element. B. Dendrogram of conceptually translated amino acid sequences from internal domain of Mdoryco1-1 and other retrotransposons. The tree was obtained by the neighbor-joining method. Horizontal distances are proportional to evolutionary distance according to the scale shown on the bottom. The tree was displayed with MEGA4.0 program showing bootstrap values (from 1000 replicates) higher than 50%. Accession numbers: Tnt1 × 13777 (Nicotiana tabacum), Tto1 D83003 (Nicotiana tabacum), RIRE1 D85597 (Oryza australiensis), CIRE1 AM040263 (Citrus sinensis), AtRE1 AB021265.1 (Arabidopsis thaliana), FaRE1 FJ871121 (Fragaria ananassa), Gypsy P10401 (Drosophila melanogaster).

(Fig. 3C–F). These results suggest that the 5 -LTR and 3 -LTR functions as bidirectional promoters in an independent manner. 3.4. Activity of Mdoryco1-1 retrotransposon in apple The cis-acting elements related to stress response in the LTRs suggested that Mdoryco1-1 expression could be mediated by different stresses. Thus, we investigated the effects of ABA, 2,4-D, SA and wounding on the expression of the LTRs of Mdoryco11. At first, the response to ABA and 2,4-D was tracked over the course of 24 h by qRT-PCR. The result showed similar timing of the induction in response to ABA and 2,4-D (Fig. 4A; B). The transcriptional activation in response to ABA and 2,4-D peaked at 9 h. Here after, the LTR expression levels in response to stresses were compared at 9 h. Results from qRT-PCR analysis showed that expression of the Mdoryco1-1 LTR was increased after ABA, wounding and 2,4-D treatments. However, expression did not increase after SA treatment (Fig. 4C), despite the predicted TCA element, a cis-acting element involved in salicylic acid responsiveness, in

the sense strands of both 5 -LTR and 3 -LTR. Expression after ABA, 2,4-D and wounding treatments increased by 3.2-fold, 2.4-fold and 2-fold (compared with control), respectively, although direct motifs related to ABA, 2,4-D and injury signaling pathways were not predicted in Mdoryco1-1 LTR. These results suggest the presence of cis-acting elements that mediate activation of the LTRs in Mdoryco1-1 in response to different signaling pathways. 3.5. Genes adjacent to Mdoryco1-1 in the apple genome The above analyses of Mdoryco1-1 LTR showed that the sense and antisense strands of both LTRs could promote transcription and that their transcription levels increased by stresses, including wounding or treatments with ABA or 2,4-D. Together these results suggested that the promoter and regulatory elements of the Mdoryco1-1 LTRs could alter the expression activity of flanking genes. Approximately 2000 bp of sequences adjacent to Mdoryco1, truncated Mdoryco1, solo LTR or 197 bp of truncated LTR were used to search the Malus × domestica genome for identification of

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Fig. 2. Comparative alignment of the 5 -LTR and 3 -LTR of Mdoryco1-1. A. Comparative alignment of the sense strand of 5 -LTR and the sense strand of 3 -LTR. B. Comparative alignment of the antisense strand of 5 -LTR and the antisense strand of 3 -LTR. Nucleotide differences are colored red. Primers for qRT-PCR are marked by red arrows under the primers. TATA-box, core promoter element around −30 of transcription start; CAAT-box, common cis-acting element in promoter and enhancer regions; GAG-motif, part of a light responsive element; TC-rich repeats, cis-acting element involved in defense and stress responsiveness; TCA-element, cis-acting element involved in salicylic acid responsiveness; AT-rich element, binding site of AT-rich DNA binding protein (ATBP-1); 5UTR Py-rich stretch, cis-acting element conferring high transcription levels; MBS, MYB binding site involved in drought-inducibility; G-box, cis-acting regulatory element involved in light responsiveness; Box III, protein binding site; HSE, cis-acting element involved in heat stress responsiveness. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

adjacent genes. The flanking sequences were classified in three classes: CDS (coding sequences), repetitive (repetitive elements), and uncharacterized DNA (when a target sequence could not clearly be predicted). A total of 83 flanking sequences found by BLASTx (E value of 10−11 ) were selected, of which 43 sequences were CDS and 33 were repetitive elements (Table S2). These results are in agreement with previous reports that showed that retrotransposons are adjacent to genes (Tadege et al., 2008; Rakocevic et al., 2009; Jia et al., 2014).

3.6. Epigenetic regulation of Mdoryco1-1 LTR DNA methylation has been shown to regulate the transcriptional activity of retrotransposons. The cytosine methylation pattern in Mdoryco1-1 LTR was determined using the bisulfite methylation profiling method (Reinders et al., 2008). With this technique, unmethylated cytosines are converted to uracil, but 5methylcytosine located in symmetrical CG and CHG (N = A or T) or nonsymmetrical CHH (H = A, T, or C) sequences are not (Frommer et al., 1992; Vanyushin, 2006). The bisulfite-mediated cytosine conversion and subsequent PCR create C-to-T transitions, which

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Fig. 3. Mdoryco1-1 LTR transient expression in tobacco leaves. The constructs, GUS driven by CaMV 35S (positive control) (B), GUS driven by 5 -LTR sense strand (C), GUS driven by 5 -LTR antisense strand (D), GUS driven by 3 -LTR sense strand (E), or GUS driven by 3 -LTR antisense strand (F), were separately transformed into tobacco leaves. Tobacco leaves transformed with A. tumefaciens EHA105 served as negative control (A).

Table 1 The methylation levels of the sense strand of the 5 -LTR of Mdoryco1-1 under different treatments. Treatment

Methylation level of CGN (%)

Methylation level of CHG (%)

Methylation level of CHH (%)

Total methylation level (%)

Control SA. Wounding 2,4-D ABA

100 90 100 100 100

77.56 77.78 81.11 78.89 76.67

7.97 16.52 10.65 7.61 14.78

22.11 28.77 24.91 22.11 27.54

can be detected by sequencing. In order to detect modification of the Mdoryco1-1 LTR methylation status under stress, gDNA was extracted from ‘Red Delicious’ leaves treated with water, ABA, SA, 2,4-D, or wounding 9 h after treatments. The sense strand of the 5 -LTR was selected to study the methylation status of the Mdoryco1 family due to the close connection between this LTR and the retrotransposon transcriptional activity. The 5 -LTR sense strand of Mdoryco1-1 contained 2 CG sites, 9 CHG sites, and 46 CHH sites (Fig. S1). The genomic copies were distinguished by specific primers in the flanking genomic regions (Table S1). After stress treatments, most of the CHG and CHH sites throughout the 5 -LTR sense strand were more methylated, although some treatments did cause decreased methylation at certain types of sites (Table 1). The percentage of m CG methylation did not change after wounding, 2,4D, or ABA treatments, but did decrease after SA treatment (Table 1). The large number and wide distribution of the Mdoryco1 retrotransposons and related LTR copies in the genome make it difficult to analyze the methylation level of Mdoryco1-1 LTR under stress. The sense and antisense strands of Mdoryco1-1 LTR have been individually transformed into tobacco for future analysis of methylation changes of the element under stress.

4. Discussion According to the current paradigm, most plant LTR retrotransposons are inactive under normal development, but are frequently activated under stress conditions. This model conflicts with our results for the apple retrotransposon Mdoryco1. Our transient expression study showed that both the 5 -LTR and 3 -LTR of Mdoryco1-1 function as bidirectional promoters in tobacco leaves, as is consistent with some LTR retrotransposons (Neumann et al., 2003; Gómez et al., 2006; Vukich et al., 2009). Transcriptional activity of the Ogre elements in the pea genome has been detected in leaves, roots, and flowers (Neumann et al., 2003). The Gypsy-like retrotransposon Grande is transcribed in leaves in the genera Zea and Tripsacum (Gómez et al., 2006). In sunflower, retrotransposon transcription occurs in embryos, leaves, roots, and flowers (Vukich et al., 2009). Retrotransposon LTRs can drive synthesis of new transcripts from flanking sequences, including the antisense or sense strands of known genes (Kashkush et al., 2003; Kashkush and Khasdan, 2007; Butelli et al., 2012). Our results showed that Mdoryco1-1 LTRs were bidirectional promoters and adjacent to genes. Using data at PlantCARE, regulatory elements were predicted in both sense and antisense strands of the Mdoryco1-1 LTRs (Ito et al., 2013; Cao et al.,

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Fig. 4. Expression analysis of Mdoryco1-1 LTR in apple under stress conditions. A. Time course of the Mdoryco1-1 LTR expression under ABA treatment. B. Time course of the Mdoryco1-1 LTR expression under 2,4-D treatment. C. Expression of Mdoryco11 LTR under abscisic acid (ABA), wounding (Wn), 2,4-dichlorophenoxyacetic acid (2,4-D) and salicylic acid (SA) treatment 9 h after treatment. Water-treatment was used as control. At least three independent determinations were performed for each sample. qRT-PCR was used to monitor Mdoryco1-1 LTR transcript abundance in apple. Data represent mean ± SD from one biological experiment. Three biological replicates were repeated.

2015). From this study, questions arise concerning how the LTRs drive the synthesis of transcripts from adjacent sequences of either the sense or antisense strands in the apple genome. In Arabidopsis, the 1329-bp intergenic fragment is a heat-inducible bidirectional promoter and the region governing the heat induciblity is possibly shared between two genes heat shock protein 100 protein (AtClpBC) and choline kinase (AtCK2) (Mishra and Grover, 2014). In wheat and rice, the LTR drove synthesis of adjacent genes in the opposite orientation to their native promoters, which resulted in silencing of these genes (Kashkush et al., 2003; Kashkush and Khasdan, 2007). The blood orange fruit color trait arises from expression of the Ruby gene (a Myb activator of anthyocyanin synthesis) driven and regu-

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lated by promoter and regulatory sequences contained within the LTR of the retrotransposon Tcs1, which is inserted in its promoter (Butelli et al., 2012). Another blood orange variety of Chinese origin contains an upstream insertion of Tcs2, another copy of the same retroelement, in reverse orientation to Ruby. However, Ruby expression in the Chinese blood orange accession is initiated downstream from the Tcs2 insertion, indicating that the LTR supplies regulatory sequences. In this study, although an SA-responsive element lies in the LTR sense strand (Fig. 2A), the transcriptional level of Mdoryco1-1 was not increased after treatment with salicylic acid (Fig. 4C), which suggests that the elements within the sense strand of the Mdoryco11 LTR may not respond to SA. However, the transcriptional activation of the retrotransposon Mdoryco1-1 was significantly upregulated after ABA, 2,4-D, and wounding treatments. Although the ABRE element, which responds to the stress hormone ABA, is not present in the ABA-responsive Mdoryco1-1 LTR, an MBS-motif, a MYB binding site involved in drought-inducibility, is located in the antisense strand of the Mdoryco1-1 LTRs. The importance of ABA in regulating flower development under water stress in Arabidopsis (Fitzpatrick et al., 2011) and its accumulation in spikes of drought-sensitive wheat (Ji et al., 2011) may help explain this increased expression of the Mdoryco1-1 LTR under ABA stress. Our results indicated that the elements within the antisense strand of the Mdoryco1-1 LTRs are associated with signal transduction related to the apple stress response. In this study, the high transcriptional level of the Mdoryco1-1 LTR under stresses ABA, 2,4-D, and wounding could be the expression of the antisense strand of the LTR. The cis-acting elements were predicted in both the first 197 bp at the 5 -end of sense strand and the last 197 bp at the 3 -end of antisense strand of Mdoryco1-1 LTR (Fig. 2A, B). Only two TATA-boxes were present in the sense strand of the first 197 bp at the 5 -end of Mdoryco1-1 LTR (Fig. 2A). However, three CAAT-boxes, two TATAboxes, a Box III, a G-box, and an HSE-motif were predicted in the antisense strand at the 3 -end of Mdoryco1-1 LTR (Fig. 2B). These results illustrate the complexity of the transcriptional regulation of the retrotransposon Mdoryco1-1 at the LTRs and indicate that there are differences between the stress-responsive regulatory features of the sense strand and the antisense strand. Certainly, the relationships or possible interactions between the stressors ABA, 2,4-D, and wounding and the elements within the antisense strand of Mdoryco1-1 LTR should be tested in future studies. In our study, the methylation level of the LTR sense strand under stress was higher, which seemed to be consistent with the expression of LTR under SA treatment. Since transposition of retrotransposons could produce fatal mutation to the host genome, plants, as hosts of retrotransposons, have evolved mechanisms to repress their transposition. The higher methylation level of the Mdoryco1-1 LTR sense strand under stress could be attributed to the necessary maintenance of host genome stability. However, the increased transcription of the Mdoryco1-1 LTR under stress might be from the activation of the Mdoryco1-1 LTR antisense strand and linked to the adjacent gene’s transcriptional regulation under stress conditions, which fits McClintock’s view of transposons as controlling elements that modulate gene expression and function. Certainly, the role of the antisense transcripts originating from the LTR remains to be established. In both wheat and rice studies, most readout transcripts were produced in the LTR antisense orientation, with opposite orientation to the gene, which results in the silencing of adjacent genes (Kashkush et al., 2003; Kashkush and Khasdan, 2007). Further questions arising from this study concern why and how the transcription level increases from the antisense strand of Mdoryco1-1 LTR under stress conditions.

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