Environmental Stress Activation of Plant LTR-Retrotransposons

CSIRO PUBLISHING Functional Plant Biology Review http://dx.doi.org/10.1071/FP13339 Environmental stress activation of plant long-terminal repeat retrotransposons Ahmed M. Alzohairy A, Jamal S. M. Sabir B, Gábor Gyulai C, Rania A. A. Younis D, Robert K. Jansen B,E and Ahmed Bahieldin B,D,F A Genetics Department, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt. King Abdulaziz University, Faculty of Science, Department of Biological Sciences, Genomics and Biotechnology Section, Jeddah 21589, Saudi Arabia. C  H-2103, Hungary. Institute of Genetics and Biotechnology, St. Stephanus University, Gödöllo D Genetics Department, Faculty of Agriculture, Ain Shams University, Cairo 11241, Egypt. E Department of Integrative Biology, University of Texas at Austin, Austin, TX 78712, USA. F Corresponding author. Email: [email protected] B Abstract. Genomic retrotransposons (RTs) are major components of most plant genomes. They spread throughout the genomes by a process termed retrotransposition, which consists of reverse transcription and reinsertion of the copied element into a new genomic location (a copy-and-paste system). Abiotic and biotic stresses activate long-terminal repeat (LTR) RTs in photosynthetic eukaryotes from algae to angiosperms. LTR RTs could represent a threat to the integrity of host genomes because of their activity and mutagenic potential by epigenetic regulation. Host genomes have developed mechanisms to control the activity of the retroelements and their mutagenic potential. Some LTR RTs escape these defense mechanisms, and maintain their ability to be activated and transpose as a result of biotic or abiotic stress stimuli. These stimuli include pathogen infection, mechanical damage, in vitro tissue culturing, heat, drought and salt stress, generation of doubled haploids, X-ray irradiation and many others. Reactivation of LTR RTs differs between different plant genomes. The expression levels of reactivated RTs are influenced by the transcriptional and post-transcriptional gene silencing mechanisms (e.g. DNA methylation, heterochromatin formation and RNA interference). Moreover, the insertion of RTs (e.g. Triticum aestivum L. Wis2–1A) into or next to coding regions of the host genome can generate changes in the expression of adjacent host genes of the host. In this paper, we review the ways that plant genomic LTR RTs are activated by environmental stimuli to affect restructuring and diversification of the host genome. Additional keywords: genome dynamics, transposition, retroelement. Received 22 November 2013, accepted 23 January 2014, published online 5 March 2014 Introduction Retrotransposons (RTs) with and without long-terminal repeat DNA sequences (LTRs) are mostly dormant in plant genomes. There are two major classes of these mobile genetic elements (Alzohairy et al. 2013). Class I elements (a copy-and-paste mechanism) transpose through reverse transcription of an RNA intermediate (Kumar and Bennetzen 1999; Wicker and Keller 2007), and Class II elements (a cut-and-paste mechanism) transpose via a DNA intermediate (Le et al. 2000). Both classes become reactivated under different biotic and abiotic stresses (reviewed by Mansour 2007, 2008, 2009; Alzohairy et al. 2012). LTR RTs are divided into two superfamilies: Copia and Gypsy (Fig. 1), and they exist in intact, defective (terminal-repeat in miniature, TRIM) and parasitic (large retrotransposon derivatives, LARD) forms. Copia and Gypsy elements include two genes. The first is Gag, which codes for the group-specific antigens (GAGs) that form the virus-like particle (VLP) where reverse transcription takes place. The second is Pol (encoding polymerase), which comprises four domains: pr/ap-pr (aspartic Journal compilation
CSIRO 2014 protease), rt (reverse transcriptase; synonymous with DNAdirected RNA polymerase or DdRpol), rt (ribonuclease-H) and int (integrase). These structural proteins and enzymes are responsible for the replication and integration of LTR RTs back into the genome at a new locus (Mansour 2007). The recombinant, nonautonomous LTR RTs of LARD, TRIM and solo have lost their coding genes through recombination during a series of transpositions over a long time (Alzohairy et al. 2013). The non-LTR RTs (i.e. RTs without LTRs) comprise three subgroups of long interspersed nuclear elements, short interspersed nuclear elements and the Type II introns of mitochondrial DNA. The latter subgroup is beyond the scope of the present review. The similarity and dissimilarity of the conserved structure and modifications between those groups can be traced back using bioinformatic software such as Gene Tracer (Issa et al. 2012). Below, we provide a brief review of how plant genomic LTR RTs are activated by environmental stimuli to affect the host genome restructuring and diversification. www.publish.csiro.au/journals/fpb B Functional Plant Biology A. M. Alzohairy et al. Biotic and abiotic activation of LTR RTs The LTR RTs of transposable elements (TEs) are the most abundant class of TEs in plant genomes. They comprise 15% of the nuclear DNA of Arabidopsis thaliana (L.) Heynh., 50–80% of Poaceae genomes, and 90% of some Liliaceae species (reviewed by Feschotte et al. 2002; Sabot and Schulman 2006). LTR RTs are flanked by direct repeats of 50 and 30 LTR sequences, and Gag and Pol are encoded between the two LTRs. LTRs carry gene promoters and regulatory sequences of the CAAT box (e.g. a sequence of CCATT), the TATA box (e.g. TGGCTATAAATAG), transcription start (e.g. CCCATGG), polyadenylation signal (e.g. AATAAG) and polyadenylation start (e.g. TAGT) (Ramallo et al. 2008). Depending on the gene position of int and rh, the LTR RTs of plants and animals are classified into two main types of elements: Copia and Gypsy (Xiong and Eickbush 1990), although this grouping may be an oversimplification (Havecker et al. 2004; Jurka et al. 2007). Biotic and abiotic stresses act as epigenetic reactivators to stimulate plant LTR RTs in algae and plants (Table 1) (Flavell et al. 1992; Voytas et al. 1992; Suoniemi et al. 1998; Mansour 2007, 2008, 2009; Alzohairy et al. 2012, 2013). The Internal Domain 5′ LTR 5′ UTR gag ap int rt-rh 3′ UTR 3′ LTR Copia Family PBS, DIS, PSI PP T Coding Region Internal Domain 5′ LTR 5′ UTR gag ap rt-rh int 3′ UTR 3′ LTR Gypsy Family PBS, DIS, PSI PP T Coding Region Fig. 1. Schematic structure differences between long-terminal repeat (LTR) retrotransposons (RTs) of Copia and Gypsy families. 50 gag, group-specific antigen or capsid protein gene; ap, aspartic protease gene; int, integrase gene; rt, reverse transcriptase gene; rh, ribonuclease-H gene; 30 UTR, 30 untranslated region; PBS, primer binding site; DIS, dimerisation signal; PSI, packaging signal; PPT, polypurine tract. Table 1. List and types of transposable elements (TEs) of stress activated long-terminal repeat (LTR) retrotransposons (RTs) in plants NA, not analysed Stress types RTs TE types References Adaptation to moisture Adenine starvation Cell wall hydrolases Chilling Cytosine demethylation Fungal infection Heat shock High salt concentrations In vitro regeneration Interspecific hybridisation Mechanical damage Microbial factors Protoplast and tissue culture Protoplast preparation Resistance to bacterial blight and plant development Tissue culture BARE1 Ty1 Tnt1 Tos17 Tos17 Erika MAGGY TLC1 Tnt1 Wis2–1A Tnt1 Tnt1 Tnt1 TLC1 Tos17 Copia Copia Copia Copia Copia Gypsy Gypsy-like Copia Copia Copia Copia Copia Copia Copia Copia (Kalendar et al. 2000) (Todeschini et al. 2005) (Pouteau et al. 1991) (Hirochika 1995) (Liu et al. 2004) (Ansari et al. 2007) (Ikeda et al. 2001) (Tapia et al. 2005) (Grandbastien 1998) (Kashkush et al. 2002, 2003). (Grandbastien et al. 1997) (Grandbastien 2004) (d’Erfurth et al. 2003) (Tapia et al. 2005) (Sha et al. 2005) Tto1, Tos17 LORE1 Retrotransposon-like Copia Gypsy NA (Hirochika et al. 1996; Liu et al. 2004) (Madsen et al. 2005) (Ansari et al. 2007) Reme1 Tos17 HERV-W Copia Copia LTR RT TLC1 Copia (Ramallo et al. 2008) (Hirochika 1995; Nellaker et al. 2006; Dellaporta et al. 1984) (Tapia et al. 2005) Trichothecene mycotoxin deoxynivalenol UV light Viral infection Wounding Activation of plant LTR retrotransposons Functional Plant Biology RTs are a major source of the insertions associated with natural mutations (Kashkush et al. 2002, 2003; Baskaev and Buzdin 2012). environmental stimuli can be pathogen infection; mechanical damage; in vitro tissue culturing; heat, drought and salt stress; hybridisation; generation of doubled haploids or X-ray irradiation (Hirochika 1995; Hagan and Rudin 2002; Hagan et al. 2003; Grandbastien 2004; Grandbastien et al. 2005; Cheng et al. 2006; Mansour 2007, 2008, 2009; Salazar et al. 2007; Alzohairy et al. 2012; Butelli et al. 2012; Karan et al. 2012; Novikov et al. 2012). LTR RTs can respond differently to separate stresses (Beguiristain et al. 2001; Sánchez-Luque et al. 2012), resulting in variable levels of transposition activity (Kashkush et al. 2003; Jiang et al. 2011; Alzohairy et al. 2012), genome restructuring and diversification (Kumar and Bennetzen 1999; Alzohairy et al. 2013). This variation in activation levels between organisms and RT families within species is believed to be associated with differences in promoter activities (Alzohairy et al. 2012). Promoter activation level could be tested using promoter expression vectors (Alzohairy et al. 2013). Life cycles of LTR RTs, spreading vertically and horizontally, result in large pools of RNA transcripts that resemble those of lentiviruses of retroviridae (such as HIV (Feschotte et al. 2002)). One of the main differences between RTs and the infectious retroviruses is the presence of an active and intact envelope (env) gene in retroviruses, which allows the virus particle to infect another cell. RTs proceed through reverse transcription, resulting in cDNA copies being reinserted (‘jumped’) into a new location of the genome. This movement may interrupt a gene (Kobayashi et al. 2004) and can change gene expression (Kashkush et al. 2002, 2003) and genome organisation (Grandbastien et al. 2005). Consequently, LTR Examples of reactivation of LTR RTs in eudicots Biotic and abiotic activation of Tnt1A in Nicotiana tabacum The Tnt1A element in Nicotiana tabacum L. protoplasts was shown to be reactivated in response to wounding, biotic elicitors, pathogens and fungal extracts (Melayah et al. 2001). Exposure to cell-wall hydrolase enzymes also activates Tntl expression in Nicotiana tabacum (Pouteau et al. 1991). These same authors indicated that Tntl transcription represented the first example of tissue culture-induced mutagenesis in plants and provided a molecular basis for somaclonal variation. Furthermore, they showed that reactivation was promoter-dependent. Transcription of the Tnt1 retroelement under environmental stimuli starts at two initiation sites within the LTR. No CAAT boxes were observed within the promoter of Tntl (Grandbastien et al. 2005), whereas two TATA boxes are located at nucleotide positions 234 and 464 upstream from the two initiation sites (Fig. 2). Most expression features of Tnt1A can be deduced from the structure of its regulatory regions, which are located in the LTR that contains several cis-acting elements that are similar to the well characterised motifs involved in activation of defense genes mostly found in the U3 region (Fig. 2). These motifs are short palindromic sequences (e.g. the BI box) and 31-bp tandem repeats (e.g. the BII box). Additionally, ′ ′ C ′ ′ 0 Fig. 2. Consensus nucleotide sequence of the Tnt1A 5 long-terminal repeat (LTR). The boundaries of the U3 and U5 regions are indicated below the sequence, the repetitive (R) region (black) and TATA boxes (blue) are indicated above the sequence. The BI, BII, BIII and BIIId boxes (red), representing cis-acting elements (transcriptional activators), are indicated above the sequence. The position of the different consensus motifs (green) found in other stress-inducible genes is indicated below the sequence. The TGAGG or TGACG element is a responsive element to reactive oxygen species and oxidative stress. One missing nucleotide is indicated by a dash. The TCA motif is highly conserved among stress-inducible genes and binds salicylic acid-inducible tobacco nuclear proteins (Goldsbrough et al. 1993). Dehydration-responsive element (DRE) is a responsive element with a core motif (CCGAC) that is involved in activation by drought, high salt and cold (Shinozaki et al. 2003). Ethylene-responsive element (ERE) boxes mediate ozone-induced gene expression (Grimmig et al. 2003). D Functional Plant Biology A. M. Alzohairy et al. several repeats of GCC or GGC, an ethylene-responsive element (ERE), are also found dispersed on the Tnt1A U3 region within the BII repeats for increased binding of protein factors in ethylene-treated plants (Hart et al. 1993). ERE boxes also mediate ozone-induced gene expression in N. tabacum (Grimmig et al. 2003). In the U5 region, there are two 16-bp tandemly repeated motifs (e.g. BIII boxes) as well as two 10-bp repeated motifs (e.g. BIIId boxes) overlapping with a responsive element (TGAGG or TGACG) to reactive oxygen species and oxidative stress. There is also a sequence in the U5 region homologous to a drought response element core motif (CCGAC) that is involved in activation by drought, high salt and cold (Shinozaki et al. 2003). In addition, the region surrounding the U3–repetitive region–U5 boundaries shows homology to the TCA motif, which is highly conserved among stress-inducible genes and binds salicylic acid-inducible nuclear proteins in N. tabacum (Goldsbrough et al. 1993). BI and BII boxes behave as transcriptional activators and BII boxes bind specific protein factors in N. tabacum protoplasts (Casacuberta and Grandbastien 1993). BII boxes are also involved in Tnt1A transcriptional induction by cryptogein, a fungal elicitin (Vernhettes et al. 1997) and by salicylic acid (Grandbastien et al. 1997). The activation of TLC1.1 in Lycopersicon chilense The TLC1.1 RT family of Lycopersicon chilense Dun. is reactivated by wounding, protoplast preparation, high salt concentration and stress-associated signalling molecules including ethylene, methyl jasmonate, salicylic acid and 2,4dichlorophenoxyacetic acid (Tapia et al. 2005). Ethylenedependent signalling may be the main pathway involved in the regulation of TLC1.1 expression. A 270-bp fragment of the promoter of TLC1.1 was necessary to activate the transcription of the GUS reporter gene in transgenic plants in response to the treatment with salicylic acid, ABA, methyl jasmonate, hydrogen peroxide and the synthetic auxin 2,4-D (Tapia et al. 2005; Salazar et al. 2007). This response requires two 57-bp tandem repeats (TRS1 and TRS2) with the core 8-bp sequence ATTTCAAA being present in the U3 domain. These two boxes are termed EREs 1 and 2. The 57-bp tandem repeats are located within the 270-bp fragment responsible for full promoter activity. These two boxes are upstream of the TATA (TATAAAA) box (Fig. 3). The Tto1 active promoter in Solanaceae In Solanaceae, Tto1 is accumulated in Gag particles under methyl jasmonate-treated leaves of N. tabacum (Takeda et al. 2001). In an (a) ′ (b) ′ ′ ′ ′ ′ ′ ′ ′ ′ Fig. 3. (a) Schematic structure of the Lycopersicon chilense TLC1.1 retrotransposon with the 50 long-terminal repeat (LTR) region stretched. The endo gene stands for endochitinase, which is induced by drought (Chen et al. 1994). gag, group-specific antigen or capsid protein gene; ap, aspartic protease gene; rt, reverse transcriptase gene; rh, ribonuclease-H gene; 30 UTR, 30 untranslated region; PBS, primer binding site; PPT, polypurine tract; ORF, open reading frame. (b) The partial consensus nucleotide sequence of the TLC1.1 50 LTR region showing the first 270 nucleotides comprising the U3 domain (Nucleotides 1–226), the repetitive domain (Nucleotides 227–232) and part of the U5 domain (Nucleotides 233–270). The boundaries of the U3 and partial U5 regions are indicated below the sequence; the repetitive region (black) and TATA boxes (blue) are indicated above the sequence. The 57-bp tandem repeated sequences of the TRS1 and TRS2 motifs (red) are indicated above the sequence, and the two core 8-bp sequences ATTTCAAA of the ethylene-responsive element (ERE) box motifs (green) are indicated below the sequence. The response requires the two 57-bp tandem repeats (TRS1 and TRS2) with their core ERE motifs present in the U3 domain. Activation of plant LTR retrotransposons Functional Plant Biology earlier report, Takeda et al. (1999) showed that the Tto1 LTR promoter is responsible for the high level of expression in cultured tissues, which was further enhanced by protoplast formation of transgenic plants. They demonstrated that two 13-bp repeat motifs (TGGTAGGTGAGAT) in the LTR function as cisregulatory elements to confer the required response. The 13-bp motif contains a conserved motif called Box L (also called the AC-I, H-box or H-box like sequence), which is involved in the expression of downstream phenylpropanoid synthetic genes in Nicotiana tabacum, Hordeum vulgare L., Asparagus officinalis L. and Phaseolus vulgaris L. (Sablowski et al. 1994; Seki et al. 1997; Takeda et al. 1999) (Fig. 4). Examples of reactivation of LTR RTs in Poaceae Variation in stress activation of Tos17 in Oryza sativa Tos17, a Copia-like RT of Oryza sativa L., is activated during tissue culture, cytosine demethylation and pathogen induction, but is not activated by protoplast formation (Hirochika et al. 1996; Liu et al. 2004; Sha et al. 2005; Long et al. 2009; La et al. 2011). The activation of Tos17 is also accompanied by heritable alteration in the DNA methylation pattern of flanking genomic regions. This RT is regulated mainly at the transcriptional level due to the presence of 16-bp repeats upstream of the TATA box (Fig. 5). The Tos17 sequence has two identical LTRs of 138 bp. The LTRs of all plant RTs begin with TG and end with CA, except for Tos17, which ends with GA. The sequence is flanked by 5-bp direct repeats (GTCTC), which results from the duplication of the target sequence during the insertion into the genome (Fig. 5). Evolutionary role of Wis2–1A RTs in Triticum aestivum development Reactivation of Wis2–1A RTs was detected in a newly synthesised Triticum aestivum L. amphiploid derived from interspecific hybridisation followed by chromosome doubling (Kashkush et al. 2002, 2003). Sequence analysis of the 1755-bp 50 LTR revealed that Wis2–1A differs from standard RTs by six nucleotide substitutions. Wis2–1A RTs contain a few tandem direct repeats caused by possible slippage mechanisms during replication. Less than half of the length of the LTRs is occupied by hairpin structures. The origin of these inverted repeats can be explained by the insertion and imprecise excision of TEs and errors when the DNA replication intermediate switches the RNA template during replication. Wis2–1A activation has played an important evolutionary role in T. aestivum’s development by cisor trans-regulation (or both) of the neighbouring genes in the allopolyploid genomes (Wittkopp et al. 2004). Differentially expressed transcripts showed silencing of genes adjacent to LTRs that were in opposite orientation relative to the LTR’s readout activity and the position of the LTR in the 30 untranslated region downstream of the coding genes. The high expression of antisense transcripts might be followed by formation of doublestranded RNA and subsequent post-transcriptional gene silencing (Kashkush et al. 2003). The H. vulgare BARE1 RT is the model of choice BARE1, a Copia-like RT, is introduced here as the model of choice to explain the regulation of retroelements during transposition and reintegration in the host genome. The canonical BARE1 element is predicted to be around 8.9 kb long (Vicient et al. 1999a). Upon activation of BARE1, as in other LTR RTs, transcription of its mRNA starts but is then followed by translation and processing of polyproteins (e.g. capsid protein (Gag), aspartic proteinase, integrase and reverse transcriptase (Wicker and Keller 2007)). Similar to retroviruses, GAG forms a shell, the VLP, which packages the nucleic acids (genomic RNAs) of the retroelement and transports them back into the nuclear genome. Proteinase cleaves the polyprotein into the functional proteins. The reverse transcriptase copies the RNA into cDNA, and the integrase inserts the cDNA back into the host genome. Various stresses were shown to induce (Grandbastien 1998; Kalendar et al. 2000; Mansour 2008, 2009; Alzohairy et al. 2012) or inhibit BARE1 transposition. The activation is controlled by the cell using several mechanisms, ′ (a) ′ – (b) – – E – – – (c) Fig. 4. (a) Schematic structure of the Tto1 retrotransposon with the first 199-bp consensus nucleotide sequence of the 50 long-terminal repeat (LTR) promoter. gag, group-specific antigen or capsid protein gene; ap, aspartic protease gene; int, integrase gene; rt, reverse transcriptase gene; rh, ribonuclease-H gene; PBS, primer binding site; PPT, polypurine tract. (b) The 13-bp (TGGTAGGTGAGAT) and 15-bp repeated motifs (black) and the TAT box (blue) are indicated below the sequence. The 13-bp motif functions as a cis-regulatory element to induce gene expression during tissue culturing and protoplast formation. F Functional Plant Biology (a) A. M. Alzohairy et al. ′ ′ (b) Fig. 5. (a) Schematic structure of the Tos17–1 retrotransposon (RT). (b) Nucleotide sequence of the Tos17–1 50 LTR region (138 bp) comprising the TATA box and two 16-bp motifs, as indicated above the sequence. The 16-bp motifs are identified upstream of the TATA box. These motifs regulate transcriptional levels during tissue culture, cytosine demethylation and pathogen induction (Hirochika et al. 1996; Liu et al. 2004; Sha et al. 2005; Long et al. 2009; La et al. 2011). The 50 longterminal repeat (LTR) region begins with TG (green), like other RTs, but ends with GA (green), rather than CA like other RTs, and is flanked by two 5-bp repeats (GTCTC). These repeats are named the target sequences (TS) as a consequence of the duplication of the target sequence during insertion into the host genome. gag, group-specific antigen or capsid protein gene; ap, aspartic protease gene; int, integrase gene; rt, reverse transcriptase gene; rh, ribonuclease-H gene; PBS, primer binding site; PPT, polypurine tract. such as RNA interference or antisense RNA, the cell cycle and epigenetic silencing by DNA methylation (Liu et al. 2004; Shi et al. 2007; Matsuda and Garfinkel 2009; Mirouze et al. 2009; La et al. 2011). Regulation for excess production of Gag VLP (or Gag) formation is a critical step in the RTs’ lifecycle upon activation and requires higher amounts of Gag protein compared with the other enzymatic components of the RT. There are several alternative mechanisms for this: * * * * Deficiency of programmed frame-shifting at the Gag–Pol junction, resulting in the Gag–Pol polyprotein being expressed in lower amounts than GAG (Voytas and Boeke 1993; Haoudi et al. 1997). This is generally achieved by translational frame-shifting of –1 or +1 nucleotides by the ribosome; Deletion of the entire pol region from the transcript by differential (alternative) splicing, which has been reported in Drosophila’s Copia superfamily (Brierley and Flavell 1990); A slow rate of Gag–Pol transcript translation as compared with that of the alternatively spliced RNA for Gag production (Brierley and Flavell 1990); and Post-translational degradation of the Pol proteins (Irwin and Voytas 2001). H. vulgare BARE1 encodes its proteins in a single open reading frame (Tanskanen et al. 2007). It contains two TATA boxes in which RNA is expressed in 10 types, five from each TATA box (Chang and Schulman 2008). The first box, TATA1, produces unprocessed (unspliced, uncapped and nonpolyadenylated) transcripts (gRNA) whose fate is packaging into the VLP. The genomic gRNA is dedicated to reverse transcription into cDNA, as induced by the repetitive region within the two TAT boxes, and ultimate insertion back into the host genome. The downstream, second box TATA2 serves to initiate a shorter transcript dedicated to translation that cannot be reverse transcribed due to the lack of a repetitive region at its ends (Fig. 6). This transcript, however, possesses a typical cap and poly(A) tail required for translation. Some of the TATA2 products are spliced to produce a subgenomic RNA encoding only Gag, thus enabling greater Gag production for VLP assembly (Manninen and Schulman 1993; Suoniemi et al. 1996a, 1996b; Vicient et al. 1999a, 1996b; Kalendar et al. 2000; Vicient et al. 2005; Reddy 2007; Tanskanen et al. 2007; Chang and Schulman 2008). The Gag–Pol splice junction was found to be two nucleotides after the end of the predicted Gag coding region, creating a stop codon three amino acids beyond the predicted end of the Gag. Thus the spliced RNA (104 bp shorter) expresses Gag only , whereas unspliced RNA expresses Gag and other polyproteins (Pol). The high U content (33.6–37.5%) in the spliced part is typical for plant introns (Ko et al. 1998). However, TATA1’s unspliced products represent gRNA as being dimerised, packaged and transported to the nucleus, and inserted into a new genomic location. The RTs’ response depends on the host genomes of plants The reactivation of RTs may differ among plant genotypes. Gypsy-like LTR RTs of Erika were upregulated in T. aestivum inflorescences in response to fungal mycotoxin (trichothecene mycotoxin deoxynivalenol). Heritable alteration of cytosine methylation patterns in the flanking regions of Tos17 RTs was detected in three O. sativa lines (the parental line O. sativa. cv. Matsumae and two introgression lines, RZ2 and RZ35) in response to tissue culture (Liu et al. 2004). As a result, the activity of the retroelement was also different due to the fact that each line harbours different copies of Tos17. The retroelement was transcriptionally reactivated and temporarily mobilised only in O. sativa line RZ35 during tissue culture, and was repressed upon plant regeneration (Liu et al. 2004). In Medicago sativa L., repetitive elements were found to carry LTR RTs and other retroelement-like sequences. They showed different expression levels in M. sativa varieties under low temperatures (Ivashuta et al. 2002). Different levels of sequence variability in the Tnt1 of different Solanaceae species indicated different levels of Tnt1 activity (Grandbastien et al. 2005). Tissue-specific activities of RTs in plants Plant genomes were found to contain TEs exhibiting a variety of tissue-specific activities. Using the model legume Lotus Activation of plant LTR retrotransposons Functional Plant Biology G (a) ′ ′ (b) Fig. 6. (a) Schematic structure of the Hordeum vulgare BARE1 retrotransposon with the 50 long-terminal repeat (LTR) region to indicate the repetitive region (R, black) and the two TATA boxes (1 and 2). BARE1 activation is controlled by several mechanisms, such as RNA interference or antisense RNA, cell cycles and epigenetic silencing by DNA methylation (Liu et al. 2004; Shi et al. 2007; Matsuda and Garfinkel 2009; Mirouze et al. 2009; La et al. 2011). (b) The two TATA boxes (1 and 2) are located at Nucleotides (nt) 1293 and 1656, respectively. The first box produces unprocessed transcripts (e.g. gRNA) to be reversetranscribed as induced by the R region then packed into the virus-like particle (VLP). The second box results in shorter posttranscriptionally processed transcripts dedicated to translation. The latter products are either spliced (shorter than 104 bp) to produce a subgenomic RNA encoding only group-specific antigens (GAG) or unspliced to express GAG and other polyproteins (Pol) (Manninen and Schulman 1993; Suoniemi et al. 1996a, 1996b; Vicient et al. 1999a, 1999b; Kalendar et al. 2000; Vicient et al. 2005; Reddy 2007; Tanskanen et al. 2007; Chang and Schulman 2008). Splicing creates a stop codon of three amino acids at the gag–pol junction. Reverse transcriptase (rt-rh) reverse-transcribes the RNA transcript starting from the tRNAiMet as a primer at the primer binding site (PBS) into cDNA and through formation of the second primer at the polypurine tract (PPT) by the RNaseH moiety. ap, aspartic protease gene; int, integrase gene. japonicus, (Regel) K.Larsen it was found that the Gypsy LORE1a, a member of the chromovirus LORE1 family, was epigenetically derepressed via tissue culture and transposes in the male germline (Fukai et al. 2010). This chromovirus has potential for generating genetic as well as epigenetic diversity in the host plant. It is speculated that the tissue specificity of TEs should be taken into account when considering their impact on the host genome’s dynamics and evolution. In A. thaliana, nearly all TEs are transcriptionally silenced (Lippman et al. 2004). Unlike animals, plants do not erase the DNA methylation patterns during meiosis (Law and Jacobsen 2010); hence DNA methylation is inherited and acts to maintain TE silencing across generations (e.g. transgenerational epigenetic inheritance) (Becker et al. 2011). This process is continually reinforced through RNA-directed DNA methylation (Law and Jacobsen 2010; Haag and Pikaard 2011). This process requires the action of RNA Pol IV, a plant-specific DNA-dependent RNA polymerase that transcribes the heterochromatic regions of TEs into noncoding transcripts. These transcripts are converted into double-stranded RNA and cleaved into small interfering RNAs 24 nucleotides long. The latter targets nascent TE transcripts in the nucleus produced by RNA Pol V (Nuthikattu et al. 2013). Retrotransposon insertions lead to changes in expression of host plant genes, phenotypic diversity and reorganisation of genomes The insertion of retroelements in or next to coding regions of the host genome can generate changes in host gene expression. TntlA transposition preferentially targets coding regions, resulting in a natural source of phenotypic diversity (Kumar and Bennetzen 1999; Grandbastien et al. 2005). The activation of T. aestivum Wis2–1A in both sense and antisense forms was associated with silencing and activation of the corresponding genes, respectively (Kashkush et al. 2002, 2003). The genomic shock model of McClintock (1984) indicated that mobile genetic elements play a crucial role in the reorganisation of plant genomes induced by environmental challenges. Based on this theory, promoters of active RTs should contain cis-regulatory elements in the promoter of the U3 region of 50 LTR and the ability to associate with the signal transduction pathways necessary for defense responses (Salazar et al. 2007). Some promoter elements of active RTs are similar in sequence to those of several plant defense genes (White et al. 1994; Casacuberta and Santiago 2003; Dunn et al. 2006). Tissue culture might be a useful approach to activate endogenous TEs epigenetically (Alzohairy et al. 2012; Fukai et al. 2013). If the promoters in these TEs are active, the newly transposed copies could be detected directly in regenerated plants. However, if the promoters are active in tissues producing germline, the new transpositions could also be detected in R1 generation. Genomic control of RT activities Expression levels of RTs, even after reactivation, are generally lower than those of the genes of host plants (Wessler 1996). This low level of expression might be due to the weakness of upstream promoters, or transcriptional or post-transcriptional gene silencing, including mechanisms of DNA methylation, heterochromatin formation and RNA interference or antisense RNAs (Okamoto and Hirohiko 2001; Matsuda and Garfinkel H Functional Plant Biology 2009). Cheng et al. (2006) indicated epigenetic silencing by DNA methylation of Tos17 RTs. Matsuda and Garfinkel (2009) provided evidence that antisense RNAs from the retrovirus-like element Ty1 inhibit retrotransposition posttranslationally in yeast. These antisense transcripts overlap the Ty1 sequence (Lesage and Todeschini 2005) necessary for copy number control and inhibit transposition in trans orientation. As a consequence, altering Ty1’s copy number or deleting sequences in the copy number control region significantly affects Ty1’s movement. This phenomenon is associated with the inhibition of Pol processing and, consequently, the dramatic decrease of integrase and reverse transcriptase levels, and the inhibition of tRNA-Meti annealing and the inability to synthesise Ty1 cDNA (as described by Matsuda and Garfinkel (2009) in Fig. 4). However, some RTs can escape these silencing mechanisms, and maintain their ability to transpose and be reactivated by stress stimuli (Kumar and Bennetzen 1999). Therefore, TEs represent a major threat to the host genomes because of their potential to edit plant and animal genomes (Jensen et al. 1999; Hirochika et al. 2000; Kidwell and Lisch 2000; Mansour 2007). Conclusions LTR RTs are mostly dormant in plant genomes but sometimes become reactivated under different biotic and abiotic stresses. LTR RTs are the most abundant class of TEs of plant genomes, as they comprise 80% or more of Poaceae and Liliaceae genomes. The response of LTR RTs is promoter-dependent and differs due to differences in environmental stimuli, resulting in various levels and means of transposition activity, genome structuring and diversification. These environmental stimuli include wounding, biotic elicitors, pathogens and fungal extracts, and many other effectors such as exposure to cell-wall hydrolase enzymes and somaclonal variation during tissue culture in eudicots. LTRs contain several types of cis-acting elements similar to the well-characterised motifs involved in activation of defense genes. Activation of monocot LTR RTs are mainly regulated at the transcriptional level; however, some LTR RTs are also regulated by downstream biological processes. Some stresses were shown to induce transposition, whereas others inhibit it. Although the expression levels of LT -RTs are generally lower than those of the genes of host plants, they have a major influence on genome size expansion and shrinkage. This low level of expression might be due to the weakness of upstream promoters, or the transcriptional gene silencing (TGS)/post-transcriptional gene silencing (PTGS) gene silencing, including mechanisms of DNA methylation, heterochromatin formation and RNA interference or antisense RNAs. Silencing of some retroelements results in the silencing of adjacent genes in the opposite orientation. However, some RTs can escape silencing mechanisms, and maintain their ability to transpose and be reactivated by stress stimuli. Attempts to detect RTs and their activation under abiotic stress in other plant families (e.g., Apocynaceae, including the evergreen dwarf shrub Rhazya stricta) based on RNA-Seq data are under way. We speculate that these attempts will give a deeper view on the patterns of RT activation under abiotic stress in plants. A. M. Alzohairy et al. Acknowledgements Support was provided by the USA National Science Foundation to RKJ (IOS-1027259). The authors gratefully acknowledge the financial support from the Deanship of Scientific Research at King Abdulaziz University, Jeddah, Saudi Arabia, represented by the Unit of Strategic Technologies Research through project Number 431/008-D for the project entitled: ‘Environmental meta-genomics and biotechnology of Rhazya stricta and its associated microbiota’. The research was funded in part by the project of ‘Excellence in Faculty Support-Research, Centre of Excellence 17586-4/ 2013/TUDPOL, Hungary’. References Alzohairy AM, Yousef MA, Edris S, Kerti B, Gyulai G, Sabir JSM, Radwan NA, Baeshen MN, Baeshen NA, Bahieldin A (2012) Detection of LTR retrotransposons reactivation induced by in vitro environmental stresses in barley (Hordeum vulgare) via RT-qPCR. Life Science Journal 9, 5019–5026. Alzohairy AM, Gyulai G, Jansen RK, Bahieldin A (2013) Transposable elements domesticated and neofunctionalized by eukaryotic genomes. Plasmid 69, 1–15. doi:10.1016/j.plasmid.2012.08.001 Ansari IK, Walter S, Brennan MJ, Lemmens M, Kessans S, McGahern A, Egan D, Doohan MF (2007) Retrotransposon and gene activation in wheat in response to mycotoxigenic and non-mycotoxigenic-associated Fusarium stress. Theoretical and Applied Genetics 114, 927–937. doi:10.1007/s00122-006-0490-0 Baskaev KK, Buzdin AA (2012) Evolutionary recent insertions of mobile elements and their contribution to the structure of human genome. Zhurnal Obshchei Biologii 73, 3–20. [In Russian] Becker C, Hagmann J, Müller J, Koenig D, Stegle O, Borgwardt K, Weigel D (2011) Spontaneous epigenetic variation in the Arabidopsis thaliana methylome. Nature 480, 245–249. doi:10.1038/nature10555 Beguiristain T, Grandbastien MA, Puigdomènech P, Casacuberta JM (2001) Three Tnt1 subfamilies show different stress-associated patterns of expression in tobacco. Consequences for retrotransposon control and evolution in plants. Plant Physiology 127, 212–221. doi:10.1104/ pp.127.1.212 Brierley C, Flavell AJ (1990) The retrotransposon Copia controls the relative levels of its gene products post-transcriptionally by differential expression from its two major mRNAs. Nucleic Acids Research 18, 2947–2951. doi:10.1093/nar/18.10.2947 Butelli E, Licciardello C, Zhang Y, Liu J, Mackay S, Bailey P, ReforgiatoRecupero G, Martin C (2012) Retrotransposons control fruit-specific, cold-dependent accumulation of anthocyanins in blood oranges. The Plant Cell 24, 1242–1255. doi:10.1105/tpc.111.095232 Casacuberta JM, Grandbastien M-A (1993) Characterization of LTR sequences involved in the protoplast specific expression of the tobacco Tnt1 retrotransposon. Nucleic Acids Research 21, 2087–2093. doi:10.1093/nar/21.9.2087 Casacuberta JM, Santiago N (2003) Plant LTR-retrotransposons and MITEs: control of transposition and impact on the evolution of plant genes and genomes. Gene 311, 1–11. doi:10.1016/S0378-1119(03) 00557-2 Chang W, Schulman AH (2008) BARE retrotransposons produce multiple groups of rarely polyadenylated transcripts from two differentially regulated promoters. The Plant Journal 56, 40–50. doi:10.1111/j.1365313X.2008.03572.x Chen RD, Yu LX, Greer AF, Cheriti H, Tabaeizadeh Z (1994) Isolation of an osmotic stress- and abscisic acid-induced gene encoding an acidic endochitinase from Lycopersicon chilense. Molecular & General Genetics 245, 195–202. doi:10.1007/BF00283267 Cheng C, Daigen M, Hirochika H (2006) Epigenetic regulation of the rice retrotransposon Tos17. Molecular Genetics and Genomics 276, 378–390. doi:10.1007/s00438-006-0141-9 Activation of plant LTR retrotransposons d’Erfurth I, Cosson V, Eschstruth A, Lucas H, Kondorosi A, Ratet P (2003) Efficient transposition of the Tnt1 tobacco retrotransposon in the model legume Medicago truncatula. The Plant Journal 34, 95–106. doi:10.1046/j.1365-313X.2003.01701.x Dellaporta SL, Chomet PS, Mottinger JP, Wood JA, Yu SM, Hicks JB (1984) Endogenous transposable element associated with virus infection in maize. Cold Spring Harbor Symposia on Quantitative Biology 49, 321–328. doi:10.1101/SQB.1984.049.01.038 Dunn CA, Romanish MT, Gutierrez LE, van de Lagemaat LN, Mager DL (2006) Transcription of two human genes from a bidirectional endogenous retrovirus promoter. Gene 366, 335–342. doi:10.1016/j. gene.2005.09.003 Feschotte C, Jiang N, Wessler SR (2002) Plant transposable elements: where genetics meets genomics. Nature Reviews. Genetics 3, 329–341. doi:10.1038/nrg793 Flavell AJ, Dunbar E, Anderson R, Pearce SR, Hartley R, Kumar A (1992) Ty1-copia group retrotransposons are ubiquitous and heterogeneous in higher plants. Nucleic Acids Research 20, 3639–3644. doi:10.1093/nar/ 20.14.3639 Fukai E, Umehara Y, Sato S, Endo M, Kouchi H, Hayashi M, Stougaard J, Hirochika H (2010) Derepression of the plant chromovirus LORE1 induces germline transposition in regenerated plants. PLOS Genetics 6, e1000868. doi:10.1371/journal.pgen.1000868 Fukai E, Stougaard J, Hayashi M (2013) Activation of an endogenous retrotransposon associated with epigenetic changes in Lotus japonicus: a tool for functional genomics in legumes. The Plant Genome 6, doi:10.3835/plantgenome2013.04.0009 Goldsbrough AP, Albrecht H, Stratford R (1993) Salicylic acid inducible binding of a tobacco nuclear protein to a 10 bp sequence which is highly conserved among stress-inducible genes. The Plant Journal 3, 563–571. doi:10.1046/j.1365-313X.1993.03040563.x Grandbastien M-A (1998) Activation of plant retrotransposons under stress conditions. Trends in Plant Science 3, 181–187. doi:10.1016/S1360-1385 (98)01232-1 Grandbastien M-A (2004) Stress activation and genomic impact of plant retrotransposons. Journal de la Societe de Biologie 198, 425–432. Grandbastien M-A, Lucas H, Mhiri C, Morel J-B, Vernhettes S, Casacuberta JM (1997) The expression of the tobacco Tnt1 retrotransposon is linked to the plant defense response. Genetica 100, 241–252. doi:10.1023/ A:1018302216927 Grandbastien M-A, Audeon C, Bonnivard E, Casacuberta JM, Chalhoub B, Costa A-PP, Le QH, Melayah D, Petit M, Poncet C, Tam SM, Van Sluys M-A, Mhiri C (2005) Stress activation and genomic impact of Tnt1 retrotransposons in Solanaceae. Cytogenetic and Genome Research 110, 229–241. doi:10.1159/000084957 Grimmig B, Gonzalez-Perez MN, Leubner-Metzger G, Vogeli-Lange R, Meins F, Hain R, Penuelas J, Heidenreich B, Langebartels C, Ernst D, Sandermann H (2003) Ozone-induced gene expression occurs via ethylene-dependent and -independent signaling. Plant Molecular Biology 51, 599–607. doi:10.1023/A:1022385104386 Haag JR, Pikaard CS (2011) Multisubunit RNA polymerases IV and V: purveyors of non-coding RNA for plant gene silencing. Nature Reviews. Molecular Cell Biology 12, 483–492. doi:10.1038/nrm3152 Hagan CR, Rudin CM (2002) Mobile genetic element activation and genotoxic cancer therapy: potential clinical implications. American Journal of Pharmacogenomics 2, 25–35. doi:10.2165/00129785-20020 2010-00003 Hagan CR, Sheffield RF, Rudin CM (2003) Human Alu elements retrotransposition induced by genotoxic stress. Nature Genetics 35, 219–220. doi:10.1038/ng1259 Haoudi A, Rachidi M, Kim MH, Champion S, Best-Belpomme M, Maisonhaute C (1997) Developmental expression analysis of the 1731 retrotransposon reveals an enhancement of Gag–Pol frameshifting in Functional Plant Biology I males of Drosophila melanogaster. Gene 196, 83–93. doi:10.1016/ S0378-1119(97)00203-5 Hart CM, Nagym F, Meins FJ (1993) A 61 bp enhancer element of the tobacco b-1,3-glucanase B gene interacts with one or more regulated nuclear proteins. Plant Molecular Biology 21, 121–131. doi:10.1007/ BF00039623 Havecker ER, Gao X, Voytas DF (2004) The diversity of LTR retrotransposons. Genome Biology 5, 225. doi:10.1186/gb-2004-5-6-225 Hirochika H (1995) Activation of plant retrotransposons by stress. In ‘Modification of gene expression and non-Mendelian inheritance’. (Eds K Oono, F Takaiwa) pp. 15–21. (National Institute of Agrobiological Resources: Tsukuba) Hirochika H, Sugimoto K, Otsuki Y, Tsugawa H, Kanda M (1996) Retrotransposons of rice involved in mutations induced by tissue culture. Proceedings of the National Academy of Sciences of the United States of America 93, 7783–7788. doi:10.1073/pnas.93.15.7783 Hirochika H, Okamoto H, Kakutani T (2000) Silencing of retrotransposons in Arabidopsis and reactivation by the ddm1 mutation. The Plant Cell 12, 357–369. Ikeda K, Nakayashiki H, Takagi M, Tosa Y, Mayama S (2001) Heat shock, copper sulfate and oxidative stress activate the retrotransposon MAGGY resident in the plant pathogenic fungus Magnaporthe grisea. Molecular Genetics and Genomics 266, 318–325. doi:10.1007/s0043 80100560 Irwin PA, Voytas DF (2001) Expression and processing of proteins encoded by the Saccharomyces retrotransposon Ty5. Journal of Virology 75, 1790–1797. doi:10.1128/JVI.75.4.1790-1797.2001 Issa M, Bakr HA, Alzohairy AM, Zeidan I (2012) Gene-Tracer: algorithm tracing genes modification from ancestors through offsprings. International Journal of Computers and Applications 52, 11–14. doi:10.5120/8308-1772 Ivashuta S, Naumkina M, Gau M, Uchiyama K, Isobe S, Mizukami Y, Shimamoto Y (2002) Genotype-dependent transcriptional activation of novel repetitive elements during cold acclimation of alfalfa (Medicago sativa). The Plant Journal 31, 615–627. doi:10.1046/j.1365-313X.2002. 01383.x Jensen L, Friis C, Ussery D (1999) Three views of microbial genomes. Research in Microbiology 150, 773–777. doi:10.1016/S0923-2508(99) 00116-3 Jiang B, Lou Q, Wu Z, Zhang W, Wang D, Mbira KG, Weng Y, Chen J (2011) Retrotransposon- and microsatellite sequence-associated genomic changes in early generations of a newly synthesized allotetraploid Cucumis  hytivus Chen & Kirkbride. Plant Molecular Biology 77, 225–233. doi:10.1007/s11103-011-9804-y Jurka J, Kapitonov V, Kohany O, Jurka MV (2007) Repetitive sequences in complex genomes: structure and evolution. Annual Review of Genomics and Human Genetics 8, 241–259. doi:10.1146/annurev. genom.8.080706.092416 Kalendar R, Tanskanen J, Immonen S, Nevo E, Schulman AH (2000) Genome evolution of wild barley (Hordeum spontaneum) by BARE-1 retrotransposon dynamics in response to sharp microclimatic divergence. Proceedings of the National Academy of Sciences of the United States of America 97, 6603–6607. doi:10.1073/ pnas.110587497 Karan R, DeLeon T, Biradar H, Subudhi PK (2012) Salt stress induced variation in DNA methylation pattern and its influence on gene expression in contrasting rice genotypes. PLoS ONE 7, e40203. doi:10.1371/journal.pone.0040203 Kashkush K, Feldman M, Levy AA (2002) Gene loss, silencing and activation in a newly synthesized wheat allotetraploid. Genetics 160, 1651–1659. Kashkush K, Feldman M, Levy AA (2003) Transcriptional activation of retrotransposons alters the expression of adjacent genes in wheat. Nature Genetics 33, 102–106. doi:10.1038/ng1063 J Functional Plant Biology Kidwell MG, Lisch DR (2000) Transposable elements and host genome evolution. Trends in Ecology & Evolution 15, 95–99. doi:10.1016/S01695347(99)01817-0 Ko CH, Brendel V, Taylor RD, Walbot V (1998) U-richness is a defining feature of plant introns and may function as an intron recognition signal in maize. Plant Molecular Biology 36, 573–583. doi:10.1023/A:1005 932620374 Kobayashi S, Goto-Yamamoto N, Hirochika H (2004) Retrotransposoninduced mutations in grape skin color. Science 304, 982. doi:10.1126/ science.1095011 Kumar A, Bennetzen JL (1999) Plant retrotransposons. Annual Review of Genetics 33, 479–532. doi:10.1146/annurev.genet.33.1.479 La H, Ding B, Mishra GP, Zhou B, Yang H, del Rosario Bellizzi M, Chen S, Meyers BC, Peng Z, Zhu J-K, Wang G-L (2011) A 5-methylcytosine DNA glycosylase/lyase demethylates the retrotransposon Tos17 and promotes its transposition in rice. Proceedings of the National Academy of Sciences of the United States of America 108, 15498–15503. doi:10.1073/pnas.1112704108 Law JA, Jacobsen SE (2010) Establishing, maintaining and modifying DNA methylation patterns in plants and animals. Nature Reviews. Genetics 11, 204–220. doi:10.1038/nrg2719 Le QH, Wright S, Yu Z, Bureau T (2000) Transposon diversity in Arabidopsis thaliana. Proceedings of the National Academy of Sciences of the United States of America 97, 7376–7381. doi:10.1073/ pnas.97.13.7376 Lesage P, Todeschini AL (2005) Happy together: the life and times of Ty retrotransposons and their hosts. Cytogenetic and Genome Research 110, 70–90. doi:10.1159/000084940 Lippman Z, Gendrel A-V, Black M, Vaughn MW, Dedhia N, McCombie WR, Lavine K, Mittal V, May B, Kasschau KD, Carrington JC, Doerge RW, Colot V, Martienssen R (2004) Role of transposable elements in heterochromatin and epigenetic control. Nature 430, 471–476. doi:10.1038/nature02651 Liu ZL, Han FP, Tan M, Shan XH, Dong YZ, Wang XZ, Fedak G, Hao S, Liu B (2004) Activation of a rice endogenous retrotransposon Tos17 in tissue culture is accompanied by cytosine demethylation and causes heritable alteration in methylation pattern of flanking genomic regions. Theoretical and Applied Genetics 109, 200–209. doi:10.1007/s00122004-1618-8 Long L, Ou X, Liu J, Lin X, Sheng L, Liu B (2009) The spaceflight environment can induce transpositional activation of multiple endogenous transposable elements in a genotype-dependent manner in rice. Journal of Plant Physiology 166, 2035–2045. doi:10.1016/j. jplph.2009.06.007 Madsen LH, Fukai E, Radutoiu S, Yost CK, Sandal N, Schauser L, Stougaard J (2005) LORE1, an active low-copy-number TY3-Gypsy retrotransposon family in the model legume Lotus japonicus. Plant J. 44, 372–381. doi:10.1111/j.1365-313X.2005.02534.x Manninen I, Schulman AH (1993) BARE-1, a Copia-like retroelement in barley (Hordeum vulgare L.). Plant Molecular Biology 22, 829–846. doi:10.1007/BF00027369 Mansour A (2007) Epigenetic activation of genomic retrotransposon. Journal of Cell and Molecular Biology 6, 99–107. Mansour A (2008) Utilization of genomic retrotransposon as cladistic molecular markers. Journal of Cell and Molecular Biology 7, 17–28. Mansour A (2009) Water deficit induction of Copia and Gypsy genomic retrotransposons. Plant Stress 3, 33–39. Matsuda E, Garfinkel DJ (2009) Posttranslational interference of Ty1 retrotransposition by antisense RNAs. Proceedings of the National Academy of Sciences of the United States of America 106, 15657–15662. doi:10.1073/pnas.0908305106 McClintock B (1984) The significance of responses of the genome to challenge. Science 226, 792–801. doi:10.1126/science.15739260 A. M. Alzohairy et al. Melayah D, Bonnivard E, Chalhoub B, Audeon C, Grandbastien M-A (2001) The mobility of the tobacco Tnt1 retrotransposon correlates with its transcriptional activation by fungal factors. The Plant Journal 28, 159–168. doi:10.1046/j.1365-313X.2001.01141.x Mirouze M, Reinders J, Bucher E, Nishimura T, Schneeberger K, Ossowski S, Cao J, Weigel D, Paszkowski J, Mathieu O (2009) Selective epigenetic control of retrotransposition in Arabidopsis. Nature 461, 427–430. doi:10.1038/nature08328 Nellaker C, Yao Y, Jones-Brando L, Mallet F, Yolken RH, Karlsson H (2006) Transactivation of elements in the human endogenous retrovirus W family by viral infection. Retrovirology 6, 30–44. Novikov A, Smyshlyaev G, Novikova O (2012) Evolutionary history of LTR retrotransposon chromodomains in plants. International Journal of Plant Genomics 2012, Article ID 874743. doi:10.1155/2012/874743 Nuthikattu S, McCue AD, Panda K, Fultz D, DeFraia C, Thomas EN, Slotkin RK (2013) The initiation of epigenetic silencing of active transposable elements is triggered by RDR6 and 21–22 nucleotide small interfering RNAs. Plant Physiology 162, 116–131. doi:10.1104/pp.113.216481 Okamoto H, Hirohiko H (2001) Silencing of transposable elements in plants. Trends in Plant Science 6, 527–534. doi:10.1016/S1360-1385(01) 02105-7 Pouteau S, Huttner E, Grandbastien M-A, Caboche M (1991) Specific expression of the tobacco Tnt1 retrotransposon in protoplasts. The EMBO Journal 10, 1911–1918. Ramallo E, Kalendar R, Schulman AH, Martinez-Izquierdo JA (2008) Reme1, a Copia retrotransposon in melon, is transcriptionally induced by UV light. Plant Molecular Biology 66, 137–150. doi:10.1007/s11103-0079258-4 Reddy ASN (2007) Alternative splicing of pre-messenger RNAs in plants in the genomic era. Annual Review of Plant Biology 58, 267–294. doi:10.1146/annurev.arplant.58.032806.103754 Sablowski RWM, Moyano E, Culianez-Macia FA, Schuch W, Martin C, Bevan M (1994) A flower-specific Myb protein activates transcription of phenylpropanoid biosynthetic genes. The EMBO Journal 13, 128–137. Sabot F, Schulman AH (2006) Parasitism and the retrotransposon life cycle in plants: a hitchhiker’s guide to the genome. Heredity 97, 381–388. doi:10.1038/sj.hdy.6800903 Salazar M, González E, Casaretto JA, Casacuberta JM, Ruiz-Lara S (2007) The promoter of the TLC1.1 retrotransposon from Solanum chilense is activated by multiple stress-related signaling molecules. Plant Cell Reports 26, 1861–1868. doi:10.1007/s00299-007-0375-y Sánchez-Luque F, López MC, Macias F, Alonso C, Thomas MC (2012) Pr77 and L1TcRz: a dual system within the 50 -end of L1Tc retrotransposon, internal promoter and HDV-like ribozyme. Mobile Genetic Elements 2, 1–7. doi:10.4161/mge.19233 Seki H, Ichinose Y, Ito M, Shiraishi T, Yamada T (1997) Combined effects of multiple cis-acting elements in elicitor-mediated activation of PSCHS1 gene. Plant & Cell Physiology 38, 96–100. doi:10.1093/ oxfordjournals.pcp.a029092 Sha AH, Huang JB, Zhang DP (2005) Relationship of activation of Tos17 and rice adult plant resistance to bacterial blight. Yi Chuan 27, 181–184. Shi X, Seluanov A, Gorbunova V (2007) Cell divisions are required for L1 retrotransposition. Molecular and Cellular Biology 27, 1264–1270. doi:10.1128/MCB.01888-06 Shinozaki K, Yamaguchi-Shinozaki K, Seki M (2003) Regulatory network of gene expression in the drought and cold stress responses. Current Opinion in Plant Biology 6, 410–417. doi:10.1016/S1369-5266(03) 00092-X Suoniemi A, Anamthawat-Jónsson K, Arna T, Schulman AH (1996a) Retrotransposon BARE-1 is a major, dispersed component of the barley (Hordeum vulgare L.) genome. Plant Molecular Biology 30, 1321–1329. doi:10.1007/BF00019563 Activation of plant LTR retrotransposons Functional Plant Biology Suoniemi A, Narvanto A, Schulman AH (1996b) The BARE-1 retrotransposon is transcribed in barley from an LTR promoter active in transient assays. Plant Molecular Biology 31, 295–306. doi:10.1007/ BF00021791 Suoniemi A, Tanskanen J, Schulman AH (1998) Gypsy-like retrotransposons are widespread in the plant kingdom. The Plant Journal 13, 699–705. doi:10.1046/j.1365-313X.1998.00071.x Takeda S, Sugimoto K, Otsuki H, Hirochika H (1999) A 13-bp cis-regulatory element in the LTR promoter of the tobacco retrotransposon Tto1 is involved in responsiveness to tissue culture, wounding, methyl jasmonate and fungal elicitors. The Plant Journal 18, 383–393. doi:10.1046/j.1365313X.1999.00460.x Takeda S, Sugimoto K, Kakutani T, Hirochika H (2001) Linear DNA intermediates of the Tto1 retrotransposon in Gag particles accumulated in stressed tobacco and Arabidopsis thaliana. The Plant Journal 28, 307–317. doi:10.1046/j.1365-313X.2001.01151.x Tanskanen JA, Sabot F, Vicient C, Schulman AH (2007) Life without GAG: the BARE-2 retrotransposon as a parasite’s parasite. Gene 390, 166–174. doi:10.1016/j.gene.2006.09.009 Tapia G, Verdugo I, Yanez M, Ahumada I, Theoduloz C, Cordero C, Poblete F, Gonzalez E, Ruiz-Lara S (2005) Involvement of ethylene in stressinduced expression of the TLC1.1 retrotransposon from Lycopersicon chilense Dun. Plant Physiology 138, 2075–2086. doi:10.1104/pp.105. 059766 Todeschini AL, Morillon A, Springer M, Lesage P (2005) Severe adenine starvation activates Ty1 transcription and retrotransposition in Saccharomyces cerevisiae. Molecular and Cellular Biology 25, 7459–7472. doi:10.1128/MCB.25.17.7459-7472.2005 Vernhettes S, Grandbastien M-A, Casacuberta JM (1997) In vivo characterization of transcriptional regulatory sequences involved in the defense-associated expression of the tobacco retrotransposon Tnt1. Plant Molecular Biology 35, 673–679. doi:10.1023/A:100582 6605598 K Vicient CM, Kalendar R, Anamthawat-Jonsson K, Suoniemi A, Schulman AH (1999a) Structure, functionality, and evolution of the BARE-1 retrotransposon of barley. Genetica 107, 53–63. doi:10.1023/ A:1003929913398 Vicient CM, Suoniemi A, Anamthawat-Jónsson K, Tanskanen J, Beharav A, Nevo E, Schulman AH (1999b) Retrotransposon BARE-1 and its role in genome evolution in the genus Hordeum. The Plant Cell 11, 1769–1784. Vicient CM, Kalendar R, Schulman AH (2005) Variability, recombination, and mosaic evolution of the barley BARE-1 retrotransposon. Journal of Molecular Evolution 61, 275–291. doi:10.1007/s00239-004-0168-7 Voytas DF, Boeke JD (1993) Yeast retrotransposons and tRNAs. Trends in Genetics 9, 421–427. doi:10.1016/0168-9525(93)90105-Q Voytas DF, Cummings MP, Konieczny AK, Ausubel FM, Rodermel SR (1992) Copia-like retrotransposons are ubiquitous among plants. Proceedings of the National Academy of Sciences of the United States of America 89, 7124–7128. doi:10.1073/pnas.89.15.7124 Wessler SR (1996) Plant retrotransposons: turned on by stress. Current Biology 6, 959–961. doi:10.1016/S0960-9822(02)00638-3 White SE, Habera LF, Wessler SR (1994) Retrotransposons in the flanking regions of normal plant genes: a role for Copia-like elements in the evolution of gene structure and expression. Proceedings of the National Academy of Sciences of the United States of America 91, 11792–11796. doi:10.1073/pnas.91.25.11792 Wicker T, Keller B (2007) Genome-wide comparative analysis of Copia retrotransposons in Triticeae, rice, and Arabidopsis reveals conserved ancient evolutionary lineages and distinct dynamics of individual Copia families. Genome Research 17, 1072–1081. doi:10.1101/gr.6214107 Wittkopp PJ, Haerum BK, Clark AG (2004) Evolutionary changes in cis and trans gene regulation. Nature 430, 85–88. doi:10.1038/nature02698 Xiong Y, Eickbush TH (1990) Origin and evolution of retroelements based upon their reverse transcriptase sequences. The EMBO Journal 9, 3353–3362. www.publish.csiro.au/journals/fpb