Environmental stress activation of plant long-terminalrepeat retrotransposons

Ahmed M. AlzohairyA, Jamal S. M. SabirB, Gábor GyulaiC, Rania A. A. YounisD,Robert K. JansenB,E and Ahmed BahieldinB,D,F

AGenetics Department, Faculty of Agriculture, Zagazig University, Zagazig 44511, Egypt.BKing Abdulaziz University, Faculty of Science, Department of Biological Sciences, Genomics and BiotechnologySection, Jeddah 21589, Saudi Arabia.

CInstitute of Genetics and Biotechnology, St. Stephanus University, Gödöllo�� H-2103, Hungary.DGenetics Department, Faculty of Agriculture, Ain Shams University, Cairo 11241, Egypt.EDepartment of Integrative Biology, University of Texas at Austin, Austin, TX 78712, USA.FCorresponding author. Email: bahieldin55@gmail.com

Abstract. Genomic retrotransposons (RTs) are major components of most plant genomes. They spread throughout thegenomes by a process termed retrotransposition,which consists of reverse transcription and reinsertion of the copied elementinto a new genomic location (a copy-and-paste system). Abiotic and biotic stresses activate long-terminal repeat (LTR) RTsin photosynthetic eukaryotes from algae to angiosperms. LTR RTs could represent a threat to the integrity of host genomesbecause of their activity and mutagenic potential by epigenetic regulation. Host genomes have developed mechanisms tocontrol 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 includepathogen infection, mechanical damage, in vitro tissue culturing, heat, drought and salt stress, generation of doubledhaploids, X-ray irradiation and many others. Reactivation of LTR RTs differs between different plant genomes. Theexpression levels of reactivated RTs are influenced by the transcriptional and post-transcriptional gene silencingmechanisms (e.g. DNA methylation, heterochromatin formation and RNA interference). Moreover, the insertion ofRTs (e.g. Triticum aestivum L. Wis2–1A) into or next to coding regions of the host genome can generate changes in theexpression of adjacent host genes of the host. In this paper, we review the ways that plant genomic LTR RTs are activatedby 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 repeatDNA 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-pastemechanism) transpose through reverse transcription of anRNA intermediate (Kumar and Bennetzen 1999; Wicker andKeller 2007), and Class II elements (a cut-and-paste mechanism)transpose via a DNA intermediate (Le et al. 2000). Both classesbecome reactivated under different biotic and abiotic stresses(reviewed byMansour 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-repeatin miniature, TRIM) and parasitic (large retrotransposonderivatives, LARD) forms. Copia and Gypsy elements includetwo genes. The first is Gag, which codes for the group-specificantigens (GAGs) that form the virus-like particle (VLP) wherereverse transcription takes place. The second is Pol (encodingpolymerase), which comprises four domains: pr/ap-pr (aspartic

protease), rt (reverse transcriptase; synonymous with DNA-directed RNA polymerase or DdRpol), rt (ribonuclease-H) andint (integrase). These structural proteins and enzymes areresponsible for the replication and integration of LTR RTsback into the genome at a new locus (Mansour 2007). Therecombinant, nonautonomous LTR RTs of LARD, TRIM andsolo have lost their coding genes through recombinationduring a series of transpositions over a long time (Alzohairyet al. 2013). The non-LTRRTs (i.e. RTswithout LTRs) comprisethree subgroups of long interspersed nuclear elements, shortinterspersed nuclear elements and the Type II introns ofmitochondrial DNA. The latter subgroup is beyond the scopeof the present review. The similarity and dissimilarity of theconserved structure and modifications between those groupscan be traced back using bioinformatic software such asGene Tracer (Issa et al. 2012). Below, we provide a briefreview of how plant genomic LTR RTs are activated byenvironmental stimuli to affect the host genome restructuringand diversification.

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Biotic and abiotic activation of LTR RTsThe LTR RTs of transposable elements (TEs) are the mostabundant class of TEs in plant genomes. They comprise 15%of the nuclearDNAofArabidopsis thaliana (L.)Heynh., 50–80%of Poaceae genomes, and 90% of some Liliaceae species(reviewed by Feschotte et al. 2002; Sabot and Schulman2006). LTR RTs are flanked by direct repeats of 50 and 30 LTRsequences, andGag and Pol are encoded between the two LTRs.LTRs carry gene promoters and regulatory sequences of theCAAT box (e.g. a sequence of CCATT), the TATA box (e.g.TGGCTATAAATAG), transcription start (e.g. CCCATGG),

polyadenylation signal (e.g. AATAAG) and polyadenylationstart (e.g. TAGT) (Ramallo et al. 2008). Depending on thegene position of int and rh, the LTR RTs of plants andanimals are classified into two main types of elements: Copiaand Gypsy (Xiong and Eickbush 1990), although this groupingmay be an oversimplification (Havecker et al. 2004; Jurka et al.2007).

Biotic and abiotic stresses act as epigenetic reactivators tostimulate plant LTR RTs in algae and plants (Table 1) (Flavellet al. 1992; Voytas et al. 1992; Suoniemi et al. 1998; Mansour2007, 2008, 2009; Alzohairy et al. 2012, 2013). The

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 BARE1 Copia (Kalendar et al. 2000)Adenine starvation Ty1 Copia (Todeschini et al. 2005)Cell wall hydrolases Tnt1 Copia (Pouteau et al. 1991)Chilling Tos17 Copia (Hirochika 1995)Cytosine demethylation Tos17 Copia (Liu et al. 2004)Fungal infection Erika Gypsy (Ansari et al. 2007)Heat shock MAGGY Gypsy-like (Ikeda et al. 2001)High salt concentrations TLC1 Copia (Tapia et al. 2005)In vitro regeneration Tnt1 Copia (Grandbastien 1998)Interspecific hybridisation Wis2–1A Copia (Kashkush et al. 2002, 2003).Mechanical damage Tnt1 Copia (Grandbastien et al. 1997)Microbial factors Tnt1 Copia (Grandbastien 2004)Protoplast and tissue culture Tnt1 Copia (d’Erfurth et al. 2003)Protoplast preparation TLC1 Copia (Tapia et al. 2005)Resistance to bacterial blight

and plant developmentTos17 Copia (Sha et al. 2005)

Tissue culture Tto1, Tos17 Copia (Hirochika et al. 1996; Liu et al. 2004)LORE1 Gypsy (Madsen et al. 2005)

Trichothecene mycotoxindeoxynivalenol

Retrotransposon-like NA (Ansari et al. 2007)

UV light Reme1 Copia (Ramallo et al. 2008)Viral infection Tos17 HERV-W Copia LTR RT (Hirochika 1995; Nellaker et al. 2006;

Dellaporta et al. 1984)Wounding TLC1 Copia (Tapia et al. 2005)

5′ LTR 5′ UTR gag ap int rt-rh 3′ UTR 3′ LTR

PBS, DIS, TPPISPCoding Region

Internal Domain

Copia Family

Internal Domain

Coding Region

5′ LTR 5′ UTR gag ap intrt-rh 3′ UTR 3′ LTR

PBS, DIS, TPPISPGypsy Family

Fig. 1. Schematic structure differences between long-terminal repeat (LTR) retrotransposons (RTs) of Copia and Gypsyfamilies. 50 gag, group-specific antigen or capsid protein gene; ap, aspartic protease gene; int, integrase gene; rt, reversetranscriptase gene; rh, ribonuclease-H gene; 30 UTR, 30 untranslated region; PBS, primer binding site; DIS, dimerisation signal;PSI, packaging signal; PPT, polypurine tract.

B Functional Plant Biology A. M. Alzohairy et al.

environmental stimuli can be pathogen infection; mechanicaldamage; in vitro tissue culturing; heat, drought and salt stress;hybridisation; generationof doubledhaploids orX-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 (Kashkushet al. 2003; Jiang et al. 2011; Alzohairy et al. 2012), genomerestructuring and diversification (Kumar and Bennetzen 1999;Alzohairy et al. 2013). This variation in activation levelsbetween organisms and RT families within species is believedto be associated with differences in promoter activities(Alzohairy et al. 2012). Promoter activation level couldbe tested using promoter expression vectors (Alzohairy et al.2013).

Life cycles of LTR RTs, spreading vertically andhorizontally, result in large pools of RNA transcripts thatresemble those of lentiviruses of retroviridae (such as HIV(Feschotte et al. 2002)). One of the main differences betweenRTs and the infectious retroviruses is the presence of an activeand intact envelope (env) gene in retroviruses, which allowsthe virus particle to infect another cell. RTs proceed throughreverse transcription, resulting in cDNA copies being reinserted(‘jumped’) into a new location of the genome. This movementmay interrupt a gene (Kobayashi et al. 2004) and can changegene expression (Kashkush et al. 2002, 2003) and genomeorganisation (Grandbastien et al. 2005). Consequently, LTR

RTs are a major source of the insertions associated withnatural mutations (Kashkush et al. 2002, 2003; Baskaev andBuzdin 2012).

Examples of reactivation of LTR RTs in eudicots

Biotic and abiotic activation of Tnt1Ain Nicotiana tabacum

The Tnt1A element in Nicotiana tabacum L. protoplasts wasshown to be reactivated in response to wounding, biotic elicitors,pathogens and fungal extracts (Melayah et al. 2001). Exposure tocell-wall hydrolase enzymes also activates Tntl expression inNicotiana tabacum (Pouteau et al. 1991). These same authorsindicated that Tntl transcription represented the first exampleof tissue culture-induced mutagenesis in plants and provideda molecular basis for somaclonal variation. Furthermore, theyshowed that reactivation was promoter-dependent. Transcriptionof the Tnt1 retroelement under environmental stimuli starts attwo initiation sites within the LTR. No CAAT boxes wereobserved within the promoter of Tntl (Grandbastien et al.2005), whereas two TATA boxes are located at nucleotidepositions 234 and 464 upstream from the two initiation sites(Fig. 2).

Most expression features of Tnt1A can be deduced fromthe structure of its regulatory regions, which are located in theLTR that contains several cis-acting elements that are similarto the well characterised motifs involved in activation ofdefense 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,

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Fig. 2. Consensus nucleotide sequence of the Tnt1A 50 long-terminal repeat (LTR). The boundaries of the U3 andU5 regions are indicated below the sequence, the repetitive (R) region (black) and TATA boxes (blue) are indicatedabove the sequence. The BI, BII, BIII and BIIId boxes (red), representing cis-acting elements (transcriptionalactivators), are indicated above the sequence. The position of the different consensus motifs (green) found in otherstress-inducible genes is indicated below the sequence. The TGAGG or TGACG element is a responsive element toreactive oxygen species and oxidative stress. Onemissing nucleotide is indicated by a dash. The TCAmotif is highlyconserved among stress-inducible genes and binds salicylic acid-inducible tobacco nuclear proteins (Goldsbroughet al. 1993). Dehydration-responsive element (DRE) is a responsive element with a core motif (CCGAC) that isinvolved 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).

Activation of plant LTR retrotransposons Functional Plant Biology C

several repeats of GCC or GGC, an ethylene-responsiveelement (ERE), are also found dispersed on the Tnt1A U3region within the BII repeats for increased binding of proteinfactors in ethylene-treated plants (Hart et al. 1993). EREboxes also mediate ozone-induced gene expression inN. tabacum (Grimmig et al. 2003). In the U5 region, thereare 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 alsoa sequence in the U5 region homologous to a droughtresponse element core motif (CCGAC) that is involved inactivation by drought, high salt and cold (Shinozaki et al.2003). In addition, the region surrounding the U3–repetitiveregion–U5 boundaries shows homology to the TCA motif,which is highly conserved among stress-inducible genes andbinds salicylic acid-inducible nuclear proteins in N. tabacum(Goldsbrough et al. 1993). BI and BII boxes behave astranscriptional activators and BII boxes bind specific proteinfactors in N. tabacum protoplasts (Casacuberta andGrandbastien 1993). BII boxes are also involved in Tnt1Atranscriptional induction by cryptogein, a fungal elicitin(Vernhettes et al. 1997) and by salicylic acid (Grandbastienet al. 1997).

The activation of TLC1.1 in Lycopersicon chilense

The TLC1.1 RT family of Lycopersicon chilense Dun. isreactivated by wounding, protoplast preparation, high saltconcentration and stress-associated signalling moleculesincluding ethylene, methyl jasmonate, salicylic acid and 2,4-dichlorophenoxyacetic acid (Tapia et al. 2005). Ethylene-dependent signalling may be the main pathway involved in theregulation of TLC1.1 expression. A 270-bp fragment of thepromoter of TLC1.1 was necessary to activate the transcriptionof the GUS reporter gene in transgenic plants in response to thetreatment with salicylic acid, ABA, methyl jasmonate, hydrogenperoxide and the synthetic auxin 2,4-D (Tapia et al. 2005; Salazaret al. 2007). This response requires two 57-bp tandem repeats(TRS1 and TRS2) with the core 8-bp sequence ATTTCAAAbeing present in the U3 domain. These two boxes are termedEREs 1 and 2. The 57-bp tandem repeats are located withinthe 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 inGag particles undermethyljasmonate-treated leavesofN. tabacum (Takeda et al.2001). In an

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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-specificantigen 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 consensusnucleotide sequence of the TLC1.1 50 LTR region showing the first 270 nucleotides comprising the U3 domain (Nucleotides1–226), the repetitive domain (Nucleotides 227–232) and part of the U5 domain (Nucleotides 233–270). The boundaries ofthe U3 and partial U5 regions are indicated below the sequence; the repetitive region (black) and TATA boxes (blue) areindicated above the sequence. The 57-bp tandem repeated sequences of the TRS1 and TRS2 motifs (red) are indicated abovethe sequence, and the two core 8-bp sequences ATTTCAAA of the ethylene-responsive element (ERE) box motifs (green) areindicated below the sequence. The response requires the two 57-bp tandem repeats (TRS1 and TRS2) with their core EREmotifs present in the U3 domain.

D Functional Plant Biology A. M. Alzohairy et al.

earlier report, Takeda et al. (1999) showed that the Tto1 LTRpromoter is responsible for thehigh level of expression in culturedtissues, which was further enhanced by protoplast formation oftransgenic plants. They demonstrated that two 13-bp repeatmotifs (TGGTAGGTGAGAT) in the LTR function as cis-regulatory elements to confer the required response. The 13-bpmotif contains a conserved motif called Box L (also called theAC-I, H-box or H-box like sequence), which is involved in theexpression of downstream phenylpropanoid synthetic genes inNicotiana tabacum, Hordeum vulgare L., Asparagus officinalisL. 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 duringtissue 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 heritablealteration in the DNA methylation pattern of flanking genomicregions. This RT is regulated mainly at the transcriptional leveldue to the presence of 16-bp repeats upstream of the TATA box(Fig. 5). The Tos17 sequence has two identical LTRs of 138 bp.TheLTRsof all plantRTsbeginwithTGand endwithCA, exceptfor Tos17, which ends with GA. The sequence is flanked by 5-bpdirect repeats (GTCTC), which results from the duplication of thetarget sequence during the insertion into the genome (Fig. 5).

Evolutionary role of Wis2–1A RTs in Triticum aestivumdevelopment

ReactivationofWis2–1ARTswasdetected inanewly synthesisedTriticum aestivum L. amphiploid derived from interspecifichybridisation followed by chromosome doubling (Kashkushet al. 2002, 2003). Sequence analysis of the 1755-bp 50 LTRrevealed that Wis2–1A differs from standard RTs by six

nucleotide substitutions. Wis2–1A RTs contain a few tandemdirect repeats caused by possible slippage mechanisms duringreplication.Less thanhalf of the length of theLTRs is occupied byhairpin structures. The origin of these inverted repeats can beexplained by the insertion and imprecise excision of TEs anderrors when the DNA replication intermediate switches the RNAtemplate during replication. Wis2–1A activation has played animportant evolutionary role in T. aestivum’s development by cis-or trans-regulation (or both) of the neighbouring genes in theallopolyploid genomes (Wittkopp et al. 2004). Differentiallyexpressed transcripts showed silencing of genes adjacent toLTRs that were in opposite orientation relative to the LTR’sreadout activity and the position of the LTR in the 30 untranslatedregion downstream of the coding genes. The high expression ofantisense transcripts might be followed by formation of double-strandedRNAand 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 ofchoice to explain the regulation of retroelements duringtransposition and reintegration in the host genome. Thecanonical BARE1 element is predicted to be around 8.9 kblong (Vicient et al. 1999a). Upon activation of BARE1, as inother LTR RTs, transcription of its mRNA starts but is thenfollowed by translation and processing of polyproteins (e.g.capsid protein (Gag), aspartic proteinase, integrase and reversetranscriptase (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 backinto the nuclear genome. Proteinase cleaves the polyproteininto the functional proteins. The reverse transcriptase copiesthe RNA into cDNA, and the integrase inserts the cDNA backinto 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. Theactivation is controlled by the cell using several mechanisms,

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Fig. 4. (a) Schematic structure of the Tto1 retrotransposon with the first 199-bp consensus nucleotide sequence of the50 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 thesequence. The 13-bp motif functions as a cis-regulatory element to induce gene expression during tissue culturing andprotoplast formation.

Activation of plant LTR retrotransposons Functional Plant Biology E

such as RNA interference or antisense RNA, the cell cycle andepigenetic silencing by DNA methylation (Liu et al. 2004; Shiet al. 2007;Matsuda and Garfinkel 2009;Mirouze et al. 2009; Laet al. 2011).

Regulation for excess production of Gag

VLP (or Gag) formation is a critical step in the RTs’ lifecycleupon activation and requires higher amounts of Gag proteincompared with the other enzymatic components of theRT. There are several alternative mechanisms for this:

* Deficiency of programmed frame-shifting at the Gag–Poljunction, resulting in the Gag–Pol polyprotein beingexpressed in lower amounts than GAG (Voytas and Boeke1993; Haoudi et al. 1997). This is generally achieved bytranslational frame-shifting of –1 or +1 nucleotides by theribosome;

* Deletion of the entire pol region from the transcript bydifferential (alternative) splicing, which has been reportedin Drosophila’s Copia superfamily (Brierley and Flavell1990);

* A slow rate of Gag–Pol transcript translation as comparedwith that of the alternatively spliced RNA for Gag production(Brierley and Flavell 1990); and

* Post-translational degradation of the Pol proteins (Irwin andVoytas 2001).

H.vulgareBARE1 encodes its proteins in a single open readingframe (Tanskanen et al. 2007). It contains two TATA boxes inwhich RNA is expressed in 10 types, five from each TATA box(Chang and Schulman 2008). The first box, TATA1, producesunprocessed (unspliced, uncapped and nonpolyadenylated)transcripts (gRNA) whose fate is packaging into the VLP. Thegenomic 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. Thedownstream, second box TATA2 serves to initiate a shortertranscript dedicated to translation that cannot be reversetranscribed due to the lack of a repetitive region at its ends(Fig. 6). This transcript, however, possesses a typical cap andpoly(A) tail required for translation.Someof theTATA2products

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 wasfound to be two nucleotides after the end of the predicted Gagcoding region, creating a stop codon three amino acids beyondthe predicted end of the Gag. Thus the spliced RNA (104 bpshorter) expresses Gag only , whereas unspliced RNA expressesGag 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 productsrepresent gRNA as being dimerised, packaged and transportedto 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. aestivuminflorescences in response to fungal mycotoxin (trichothecenemycotoxin deoxynivalenol). Heritable alteration of cytosinemethylation patterns in the flanking regions of Tos17 RTs wasdetected in three O. sativa lines (the parental line O. sativa. cv.Matsumae and two introgression lines, RZ2 and RZ35) inresponse to tissue culture (Liu et al. 2004). As a result, theactivity of the retroelement was also different due to the factthat each line harbours different copies of Tos17. Theretroelement was transcriptionally reactivated and temporarilymobilised only in O. sativa line RZ35 during tissue culture, andwas repressed upon plant regeneration (Liu et al. 2004). InMedicago sativa L., repetitive elements were found to carryLTR RTs and other retroelement-like sequences. They showeddifferent expression levels in M. sativa varieties under lowtemperatures (Ivashuta et al. 2002). Different levels ofsequence variability in the Tnt1 of different Solanaceaespecies 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 oftissue-specific activities. Using the model legume Lotus

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Fig. 5. (a) Schematic structure of theTos17–1 retrotransposon (RT). (b) Nucleotide sequence of theTos17–1 50LTR region(138 bp) comprising the TATA box and two 16-bp motifs, as indicated above the sequence. The 16-bp motifs are identifiedupstream of the TATA box. These motifs regulate transcriptional levels during tissue culture, cytosine demethylation andpathogen induction (Hirochika et al. 1996; Liu et al. 2004; Sha et al. 2005; Long et al. 2009; La et al. 2011). The 50 long-terminal repeat (LTR) region beginswithTG (green), like otherRTs, but endswithGA(green), rather thanCA like otherRTs,and is flanked by two 5-bp repeats (GTCTC). These repeats are named the target sequences (TS) as a consequence of theduplication 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 bindingsite; PPT, polypurine tract.

F Functional Plant Biology A. M. Alzohairy et al.

japonicus, (Regel)K.Larsen itwas found that theGypsyLORE1a,a member of the chromovirus LORE1 family, was epigeneticallyderepressed via tissue culture and transposes in themale germline(Fukai et al. 2010). This chromovirus has potential for generatinggenetic as well as epigenetic diversity in the host plant. It isspeculated that the tissue specificity of TEs should be taken intoaccount when considering their impact on the host genome’sdynamics and evolution.

In A. thaliana, nearly all TEs are transcriptionally silenced(Lippman et al. 2004). Unlike animals, plants do not erase theDNA methylation patterns during meiosis (Law and Jacobsen2010); hence DNA methylation is inherited and acts tomaintain TE silencing across generations (e.g. trans-generational epigenetic inheritance) (Becker et al. 2011). Thisprocess is continually reinforced through RNA-directed DNAmethylation (Law and Jacobsen 2010; Haag and Pikaard 2011).This process requires the action of RNA Pol IV, a plant-specificDNA-dependent RNA polymerase that transcribes theheterochromatic regions of TEs into noncoding transcripts.These transcripts are converted into double-stranded RNA andcleaved into small interfering RNAs 24 nucleotides long. Thelatter targets nascent TE transcripts in the nucleus produced byRNA Pol V (Nuthikattu et al. 2013).

Retrotransposon insertions lead to changes in expressionof host plant genes, phenotypic diversity andreorganisation of genomes

The insertion of retroelements in or next to coding regions of thehost genome can generate changes in host gene expression. TntlAtransposition preferentially targets coding regions, resulting in a

natural source of phenotypic diversity (Kumar and Bennetzen1999; Grandbastien et al. 2005). The activation of T. aestivumWis2–1A in both sense and antisense forms was associated withsilencing and activation of the corresponding genes, respectively(Kashkush et al. 2002, 2003). The genomic shock model ofMcClintock (1984) indicated that mobile genetic elements playa crucial role in the reorganisation of plant genomes induced byenvironmental challenges. Based on this theory, promoters ofactive RTs should contain cis-regulatory elements in thepromoter of the U3 region of 50 LTR and the ability toassociate with the signal transduction pathways necessary fordefense responses (Salazar et al. 2007). Some promoter elementsof active RTs are similar in sequence to those of several plantdefense genes (White et al. 1994; Casacuberta and Santiago2003; Dunn et al. 2006). Tissue culture might be a usefulapproach to activate endogenous TEs epigenetically(Alzohairy et al. 2012; Fukai et al. 2013). If the promoters inthese TEs are active, the newly transposed copies could bedetected directly in regenerated plants. However, if thepromoters are active in tissues producing germline, the newtranspositions could also be detected in R1 generation.

Genomic control of RT activities

Expression levels of RTs, even after reactivation, are generallylower than those of the genes of host plants (Wessler 1996). Thislow level of expressionmight be due to theweakness of upstreampromoters, or transcriptional or post-transcriptional genesilencing, including mechanisms of DNA methylation,heterochromatin formation and RNA interference or antisenseRNAs (Okamoto and Hirohiko 2001; Matsuda and Garfinkel

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Fig. 6. (a) Schematic structure of theHordeumvulgareBARE1 retrotransposonwith the 50 long-terminal repeat (LTR) region toindicate the repetitive region (R, black) and the twoTATAboxes (1 and2).BARE1 activation is controlledby severalmechanisms,such as RNA interference or antisense RNA, cell cycles and epigenetic silencing by DNAmethylation (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 atNucleotides (nt) 1293 and 1656, respectively. The first box produces unprocessed transcripts (e.g. gRNA) to be reverse-transcribed as induced by the R region then packed into the virus-like particle (VLP). The second box results in shorter post-transcriptionally processed transcripts dedicated to translation. The latter products are either spliced (shorter than 104 bp) toproduce 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; Vicientet al. 2005; Reddy 2007; Tanskanen et al. 2007; Chang and Schulman 2008). Splicing creates a stop codon of three amino acids atthegag–pol junction.Reverse transcriptase (rt-rh) reverse-transcribes theRNAtranscript starting from the tRNAiMet as a primer atthe primer binding site (PBS) into cDNAand through formation of the second primer at the polypurine tract (PPT) by the RNaseHmoiety. ap, aspartic protease gene; int, integrase gene.

Activation of plant LTR retrotransposons Functional Plant Biology G

2009). Cheng et al. (2006) indicated epigenetic silencing byDNA methylation of Tos17 RTs. Matsuda and Garfinkel(2009) provided evidence that antisense RNAs from theretrovirus-like element Ty1 inhibit retrotransposition post-translationally in yeast. These antisense transcripts overlap theTy1 sequence (Lesage and Todeschini 2005) necessary for copynumber control and inhibit transposition in trans orientation.As a consequence, altering Ty1’s copy number or deletingsequences in the copy number control region significantlyaffects Ty1’s movement. This phenomenon is associated withthe inhibition of Pol processing and, consequently, the dramaticdecrease of integrase and reverse transcriptase levels, and theinhibition of tRNA-Meti annealing and the inability to synthesiseTy1 cDNA (as described by Matsuda and Garfinkel (2009) inFig. 4). However, some RTs can escape these silencingmechanisms, and maintain their ability to transpose and bereactivated by stress stimuli (Kumar and Bennetzen 1999).Therefore, TEs represent a major threat to the host genomesbecause of their potential to edit plant and animal genomes(Jensen et al. 1999; Hirochika et al. 2000; Kidwell and Lisch2000; Mansour 2007).

Conclusions

LTR RTs are mostly dormant in plant genomes but sometimesbecome 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 Liliaceaegenomes. The response of LTR RTs is promoter-dependentand differs due to differences in environmental stimuli,resulting in various levels and means of transpositionactivity, genome structuring and diversification. Theseenvironmental stimuli include wounding, biotic elicitors,pathogens and fungal extracts, and many other effectors suchas exposure to cell-wall hydrolase enzymes and somaclonalvariation during tissue culture in eudicots. LTRs contain severaltypes of cis-acting elements similar to the well-characterisedmotifs involved in activation of defense genes. Activation ofmonocot LTR RTs are mainly regulated at the transcriptionallevel; however, some LTR RTs are also regulated bydownstream biological processes. Some stresses were shownto induce transposition, whereas others inhibit it. Although theexpression levels of LT -RTs are generally lower than those ofthe genes of host plants, they have a major influence on genomesize expansion and shrinkage. This low level of expressionmight be due to the weakness of upstream promoters, or thetranscriptional gene silencing (TGS)/post-transcriptional genesilencing (PTGS) gene silencing, including mechanisms ofDNA methylation, heterochromatin formation and RNAinterference or antisense RNAs. Silencing of someretroelements results in the silencing of adjacent genes in theopposite orientation. However, some RTs can escape silencingmechanisms, and maintain their ability to transpose and bereactivated by stress stimuli. Attempts to detect RTs and theiractivation under abiotic stress in other plant families (e.g.,Apocynaceae, including the evergreen dwarf shrub Rhazyastricta) based on RNA-Seq data are under way. We speculatethat these attempts will give a deeper view on the patterns of RTactivation under abiotic stress in plants.

Acknowledgements

Support was provided by the USA National Science Foundation to RKJ(IOS-1027259). The authors gratefully acknowledge the financial supportfrom the Deanship of Scientific Research at King Abdulaziz University,Jeddah, Saudi Arabia, represented by the Unit of Strategic TechnologiesResearch through project Number 431/008-D for the project entitled:‘Environmental meta-genomics and biotechnology of Rhazya stricta andits 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’.

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