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November 2, 1999 22:53 Annual Reviews AR095-15

?Annu. Rev. Genet. 1999. 33:479–532

Copyright c© 1999 by Annual Reviews. All rights reserved

PLANT RETROTRANSPOSONS

Amar Kumar and Jeffrey L. BennetzenScottish Crop Research Institute, Invergowrie, Dundee, DD2 5DA, Scotland andDepartment of Biological Sciences, Purdue University, West Lafayette, Indiana47907-1392; e-mail: [email protected]

Key Words DNA markers, evolution, genome organization, mutation, regulation,retroelements

■ Abstract Retrotransposons are mobile genetic elements that transpose throughreverse transcription of an RNA intermediate. Retrotransposons are ubiquitous in plantsand play a major role in plant gene and genome evolution. In many cases, retrotrans-posons comprise over 50% of nuclear DNA content, a situation that can arise in just afew million years. Plant retrotransposons are structurally and functionally similar to theretrotransposons and retroviruses that are found in other eukaryotic organisms. How-ever, there are important differences in the genomic organization of retrotransposonsin plants compared to some other eukaryotes, including their often-high copy num-bers, their extensively heterogeneous populations, and their chromosomal dispersionpatterns. Recent studies are providing valuable insights into the mechanisms involvedin regulating the expression and transposition of retrotransposons. This review de-scribes the structure, genomic organization, expression, regulation, and evolution ofretrotransposons, and discusses both their contributions to plant genome evolution andtheir use as genetic tools in plant biology.

CONTENTS

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480Types, Structures, and Distributions of Retrotransposons in Plants. . . . . . . . . 481

LTR Retrotransposons of the Ty1-copiaGroup . . . . . . . . . . . . . . . . . . . . . . . . . . 482LTR Retrotransposons of the Ty3-gypsyGroup . . . . . . . . . . . . . . . . . . . . . . . . . . 489LINEs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 489SINEs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490

Retrotransposon Dispersal and Organization in The Plant Genome. . . . . . . . 490Chromosomal Locations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 490Retrotransposon Abundance in Plants Relative to Other Eukaryotes. . . . . . . . . . . 493Local Structures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 495Insertion Site Preferences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 496In Organellar Genomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497

Regulation of Expression and Transposition. . . . . . . . . . . . . . . . . . . . . . . . . . 497Transcriptional Regulation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 497

0066-4197/99/1215-0479$08.00 479



?480 KUMAR ■ BENNETZEN

Activation by Biotic and Abiotic Stresses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 498Regulatory Sequences. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 499Epigenetic and Posttranscriptional Regulations. . . . . . . . . . . . . . . . . . . . . . . . . 500Retrotransposon Regulation as a Form of Host Defense. . . . . . . . . . . . . . . . . . . . 502

Plant Retrotransposon Origins and Evolution. . . . . . . . . . . . . . . . . . . . . . . . . 503The Frequency and Timing of Retrotransposition During Evolution. . . . . . . . . . . 503Heterogeneity. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 505Internal Mutation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 506Retrotransposons and Retroviruses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 507Horizontal Transmission?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 508

Contributions of Retrotransposons to the Evolution of Plant Genesand Genomes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510Gene Mutation. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 510Genome Size. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 511Genome Rearrangement. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 516Roles for Retrotransposons?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 517

Retrotransposons as Genetic Tools. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518Molecular Markers for Phylogenetic, Biodiversity, and Genetic

Linkage Analyses. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 518Gene Tagging and Functional Analysis of Genes. . . . . . . . . . . . . . . . . . . . . . . . . 519Evaluation of Somaclonal Variations. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 519

Conclusions and Future Prospects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 520

INTRODUCTION

The mobile genetic elements called retrotransposons are ubiquitous in the plantkingdom. They are present in high copy numbers in most plants, making themmajor constituents of plant genomes. Retrotransposons move to new chromosomallocations via an RNA intermediate that is converted into extrachromosomal DNAby the encoded reverse transcriptase/RNaseH enzymes prior to reinsertion intothe genome (13, 16). This replicative mode of transposition can rapidly increasethe copy numbers of elements and can thereby greatly increase plant genome size(93, 156). The DNA transposable elements (for example,Ac, Tam1, andEn/Spm)transpose by an excision/repair mechanism and usually do not greatly increaseplant genome size (97). Retrotransposons, like DNA transposable elements, cangenerate mutations by inserting within or near genes. Moreover, retrotransposon-induced mutations are relatively stable; because they transpose via replication, thesequence at the insertion site is retained.

Many of the properties of transposable elements suggest that they are parasiticor selfish DNAs (34, 134). Because they are expressed in the genome and subjectto many of the same rules of inheritance as are the genes of their genomic “host,”a retrotransposon can be co-opted for use in a biological process that benefitsthe organism. For instance, the preferential insertion of some retrotransposons inDrosophilaat telomeric locations has removed the need for a telomerase function inthese insects (137). However, discussion of plant/transposable element interactions



?PLANT RETROTRANSPOSONS 481

are often most informative from a plant/parasite perspective, and we often employthis approach of looking at the genome as an ecosystem in which different DNAcomponents vie for influence and survival (7, 34, 134).

Because retrotransposons have the potential to dramatically alter gene func-tion and host genome structure, it is not surprising that their transpositional ac-tivities are regulated both by retrotransposon- and host-encoded factors, possiblyto avoid deleterious effects on host and retrotransposon survival. The intimaterelationship between retrotransposons and their plant hosts has existed for manymillions of years. We are just beginning to understand how retrotransposons andtheir hosts’ genomes have co-evolved mechanisms to regulate transposition, in-sertion specificities, and mutational outcomes in order to optimize each other’ssurvival. Here, we review the current status of research on plant retrotransposonswith respect to their structure, genomic organization, expression, regulation, trans-positional activities, and evolution. We also discuss their contributions to plantgenome evolution and their applications as tools for phylogenetic studies, bio-diversity assessments, and gene tagging. Since the first publication on a plantretrotransposon in 1984 (165), many publications on this subject have appeared,and it is not possible to refer to all of them in this review. For additional back-ground, we suggest any of several previous reviews on plant retrotransposons(5, 48, 54, 55, 95, 97, 169, 202).

TYPES, STRUCTURES, AND DISTRIBUTIONSOF RETROTRANSPOSONS IN PLANTS

Retrotransposons are the most abundant and widespread class of eukaryotic trans-posable element, consisting of the long terminal repeat (LTR) and the non-LTRretrotransposons (Figure 1) (5, 54, 93, 97). LTR retrotransposons are further sub-classified into the Ty1-copia and the Ty3-gypsygroups that differ from eachother in both their degree of sequence similarity (207) and the order of en-coded gene products (Figure 1). Ty1-copiaretrotransposons are present through-out the plant kingdom, in species ranging from single-cell algae to bryophytes,gymnosperms, and angiosperms (44, 195). Ty3-gypsyretrotransposons also havebeen found to be widely distributed in the plant kingdom, including both gym-nosperms and angiosperms (90, 96, 131, 164, 176, 181). Furthermore, both thesegroups of retrotransposons are commonly found in high copy number (up to afew million copies per haploid nucleus) in plants with large genomes (Table 1)(5, 95, 97, 101, 147, 158). The non-LTR retrotransposons, LINEs (long interspersedrepetitive elements) and SINEs (short interspersed repetitive elements) can alsobe found in high copy numbers (up to 250,000) in the plant species studied to date(5, 97, 101, 169). Like the LTR retrotransposons, LINEs are present throughoutthe plant kingdom (129, 130). SINEs have been found in several angiosperms(32, 126, 130, 142, 210) and may be ubiquitous in plants. Table 1 shows a list ofsome well-characterized retrotransposons in plants.




LTR Retrotransposons of the Ty1-copia Group

The LTR retrotransposons have direct long terminal repeats (LTRs) that can rangefrom a few 100 bp to over 5 kb in size (Figure 1; Table 1). The LTRs do notencode any known proteins, but they do contain the promoters and terminatorsassociated with the transcription of LTR retrotransposons. LTRs terminate in shortinverted repeats, usually 5′—TG–3′ and 5′—CA–3′. The LTR retrotransposonsencode a number of proteins, specified by three major genes calledgag, pol, andint. These genes and proteins are all specified by a single mRNA molecule thathas the structure 5′—R–U5—PBS–coding region–PPT–U3—R–3′, where R, U5,PBS, PPT, and U3 stand for repeated RNA, unique 5′ RNA, primer binding site,polypurine tract, and unique 3′ RNA, respectively (Figure 1). Hence, transcriptioninitiates at the 5′ end of R in the 5′ LTR and terminates in the 3′ end of R inthe 3′ LTR. As this result suggests, the 3′ LTR also contains a promoter thatcan lead to readthrough transcription of sequences downstream from the insertedelement. The proteins encoded bygag, pol, andint are synthesized as a polyproteinthat is cleaved into functional peptides by apol-encoded protease (PR). Thegaggene encodes proteins involved in maturation and packaging of retrotransposonRNA and proteins into a form suitable for integration into the genome. Thepolgene encodes reverse transcriptase and RNase H activities that are required forreplication/transposition of the retrotransposon, whereasint encodes the integrasethat allows the DNA form of the retrotransposon to insert at a new chromosomallocation. In some cases,gag, pol, and int proteins are encoded within a singletranslational reading frame, but in other cases, two or more reading frames arepresent. This leads to a requirement for a frameshift or translational reinitiation to

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−Figure 1 General structures of the Ty1-copia, Ty3-gypsy, LINE, and SINE retrotrans-posons. The LTR retrotransposons have long terminal repeats in direct orientation ateach end. Within the LTRs are U3, R, and U5 regions that contain signals for initiationand termination of transcription. The transcript (thin arrow) is shown below each block;it initiates at the 5′ end of R within the 5′ LTR and terminates at the 3′ end of R withinthe 3′ LTR. The genes within the retrotransposons encode capsid-like proteins (CP),endonuclease (EN), integrase (INT), protease (PR), reverse transcriptase (RT), andRNAase-H. Other sequences featured are PBS (primer binding sites), PPT (polypurinetracts), NA (nucleic acid binding moiety), IR (inverted terminal repeats), DR (flankingtarget direct repeat), 5′ UTR (5′ untranslated region), 3′ UTR (3′ untranslated region),and Pol III A and B-promoter recognition sites for RNA polymerase III. These figuresare not drawn to scale, as the LTR retrotransposons range from a few kb up to 15 kbin size. LINEs usually range in size from less than 1 kb to maybe 8 kb, while SINEsare normally 100 bp to 300 bp in size. The envelope (env) gene-like sequence in theposition of ORF 3, where a functionalenvgene is present in the animal retroviruses,has been found in both Ty1-copiaand Ty3-gypsygroups (not shown). The function oftheenvgene-like sequence in plant retrotransposons is unknown.



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make appropriate peptides downstream of the change in frame, and thereby canlead to a lower level of these downstream proteins relative to those upstream.

LTR retrotransposons undergo intracellular replication by a complex processthat first requires synthesis of a retrotransposon mRNA molecule (16). This mRNAspecifies the proteins needed for replication, and also serves as the template forreplication. The primer binding site on the mRNA molecule is complementary to acellular RNA, usually the 3′ end of a host tRNA. In vivo hybridization between theretrotransposon mRNA and the appropriate tRNA provides a short double-strandedRNA region with a free 3′ hydroxyl from the tRNA. Reverse transcriptase can usethis 3′ end as a primer, then synthesizing a DNA complement to the R and U5portions of the 5′ LTR. Reverse transcriptase cannot initially synthesize any addi-tional DNA, however, because it has come to the end of the template, the 5′ endof the retrotransposon mRNA. However, the RNase H molecule encoded by theretrotransposon specifically digests the RNA in any RNA:DNA hybrid, thus free-ing up a single-stranded DNA with homology to the R sequence that is also foundat the 3′ end of retrotransposon mRNAs. Hybridization between these sequencesleads to a circular structure that allows a continuation of the reverse transcrip-tion until a single-stranded DNA complementary to all of the element-internalsequences is synthesized to generate a single-stranded DNA circle. Second-strandDNA synthesis requires the action of RNase H and a polypurine tract (PPT) thatis just 5′ to the 3′ LTR. Once the double-stranded linear DNA molecule is synthe-sized, it can then be incorporated into the target genome by the action of integrase,which appears to cut both donor and target molecules. The DNA is apparentlycut with nicks that are staggered by 3 to 5 bp (a size that is consistent for anygiven integrase), thereby creating a flanking target direct repeat that is 3 to 5 bpin size.

In plants, only a few groups have attempted to study the replication cycle ofLTR retrotransposons. Initially, it was shown that tobaccoTnt1 transposes via anRNA-intermediate in the genome ofArabidopsis thaliana(108). Later, this groupdemonstrated that the replication cycle of the element involves generation of extra-chromosomal double-stranded DNA linear intermediates ofTnt1(43). They havealso shown that the primer binding site (PBS) and the PPT of theTnt1 used asprimers for the initiation of minus- and plus-strand DNAs are separated by twonucleotides from the 5′ and 3′ LTRs, respectively, giving rise to extrachromosomallinear intermediates that have two more base pairs at each end than the integratedcopies, as was observed for retroviruses and the yeast Ty3 retrotransposons (43).Prior to integration into the host genome, the two terminal nucleotides at the 3′

end of these linear intermediates are removed, as was seen with yeast Ty3 retro-transposons and retroviruses. Extrachromosomal circular DNA intermediates ofanother tobaccoTto1retrotransposon have also been identified and characterized(68). Interestingly, analyses on generated models for the secondary and tertiarystructure of the integrase (INT) core domain ofBARE-1retrotransposons, com-prising 80 clones from 28Hordeumaccessions, have revealed that they are similarto structures of human immunodeficiency virus and avian sarcoma virus INs and




thatHordeumretrotransposon INT has been under purifying selection to maintaina structure typical of retroviral INTs (175). However, further research is neededto determine whether the replication cycle of LTR retrotransposons is the same inplants as in other eukaryotes.

LTR Retrotransposons of the Ty3-gypsy Group

Ty3-gypsyretrotransposons encode the same functions as do Ty1-copiaretrotrans-posons, and they have a very similar genomic organization (Figure 1; Table 1).A significant difference is the order ofint andpol genes within the retrotranspo-son. Comparisons of reverse transcriptase sequences indicate that the Ty3-gypsyand Ty1-copia retrotransposons do form different lineages, so this difference ingene order makes good cladistic sense (207). By sequence and cladistic criteria,it appears that animal retroviruses are derived from Ty3-gypsyretrotransposons,presumably by the acquisition of an envelope (env) function that allowed themto be packaged into viral particles that permit intercellular infectivity (180, 207).When present,envis usually the last protein coding sequence upstream of the 3′

LTR. In some cases, it can be unclear whether a Ty3-gypsy-like element is actuallya retrotransposon or a retrovirus (5, 94). Based on present knowledge, the mechan-ics of intracellular element transcription, replication, and integration are the samefor Ty1-copiaand Ty3-gypsyretrotransposons, and for retroviruses.

LINEs

The LINE elements are simpler than LTR retrotransposons, but still encode manyof the same proteins (Figure 1; Table 1). LINEs havegag and pol genes, butlack an identified integrase. Thegag loci of LINEs usually specify a protein withendonucleolytic activity (EN), and this may be involved in integration of DNAversions of LINEs into chromosomal DNA (26). Alternatively, or additionally,LINEs may integrate into chromosomal DNA via the action of a low-frequencyDNA repair activity (123). By sequence diversity criteria, LINE elements appearto be the oldest class of eukaryotic retrotransposons, and cladistic studies suggestthat the first LTR retrotransposons may have arisen by acquisition of LTRs by aLINE (207).

In plants, little is known about the mechanisms of LINE transcription or inte-gration. Most of the LINEs that have been seen in plants are 5′ truncated, as areabout 95% of human L1 elements (160), suggesting similar transcription, replica-tion, and integration processes. In humans, active L1 elements are transcribed afterrecognition of an internal promoter, perhaps involving participation of both RNApolymerase II and RNA polymerase III (98). The bicistronic L1 mRNA is thentranslated, producinggagandpol proteins. Apparently, the endonuclease protein(EN) encoded bypol then cuts the target site, and homology at this target siteserves to prime reverse transcription of the L1 element bypol (107), subsequentlyleading to integration, presumably via nonhomologous end joining (123).




SINEs

The small retrotransposons called SINEs are very different from the other classesof retrotransposons in that they do not encode anytrans-acting transposition func-tions (Figure 1; Table 1). All known SINEs are derived from RNA polymerase IIIproducts (e.g. tRNAs) that appear to have evolved an ability to be efficiently repli-cated and integrated by proteins encoded by LINEs and/or LTR retrotransposons(15, 160). In this regard, SINEs are most like intronless pseudogenes, the mRNAmolecules that are occasionally reverse-transcribed and then inserted into eukary-otic genomes (37, 106). However, unlike the mRNAs that are RNA polymerase IIproducts, genes transcribed by RNA polymerase III usually contain their promotersequences within the RNA-coding region. Hence, if an RNA polymerase III prod-uct is rarely (perhaps inadvertently) utilized as a template to make DNA and thenintegrates into a genome, it will have a promoter that allows essentially normaltranscription. The simplest interpretation of natural selection says that evolutionof such sequences will lead to the origin of SINEs with better and better affinityfor the reverse transcription and integration processes (34, 134). Although it is notclear how SINEs replicate or integrate, the similarity in the varying sizes of theirflanking target DNAs, the shared presence of integrated polyA tails, and their fre-quent correlative abundance with LINEs and pseudogenes suggest that they mayuse LINE-specified functions (15, 39, 133, 179).

RETROTRANSPOSON DISPERSAL AND ORGANIZATIONIN THE PLANT GENOME

Major efforts have been made recently to study the genomic and physical organiza-tion of retrotransposons in gymnosperms and angiosperms. Their replicative modeof transposition and amplification has made retrotransposons highly successful ininvading almost all parts of plant genomes, where they can account for over 50%of the nuclear genome (90, 101, 124, 128, 141, 142, 156, 158, 210).

Chromosomal Locations

In situ hybridization data on metaphase chromosomes and prophase nuclei haverevealed that Ty1-copia retrotransposon sequences are dispersed throughout theeuchromatin, sometimes evenly and sometimes unevenly, depending on the plantspecies and the particular element employed (Figure 2) (17, 61, 124, 140–142).However, many elements are present in lower amounts or absent in certain regions(e.g. centromeres, interstitial and terminal heterochromatic regions, and ribosomalDNA sites) (Figure 2, Figure 3a,b) (17, 61, 124, 140, 141). There are exceptions tothis general observation, though, including elements that are preferentially abun-dant at the terminal heterochromatic regions ofAllium cepa(Figure 2) (142) orthe paracentromeric heterochromatic region ofA. thalianaandCicer arietinum(Figure 2) (17, 61). For example,Athila, which consists of LTRs flanking two




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?Figure 3 Physical locations of the Ty1-copia (a, b), Ty3-gypsy(c), LINE(d, e), and SINE (f, g) retroelements along metaphase chromosomes ofplants have been shown using in situ hybridization.




non-retrotransposon open reading frames (ORFs), is concentrated in the paracen-tromeric heterochromatin ofArabidopsis, where it is associated with the major180-bp centromeric satellite DNA (144).

Recently, several groups have identified retrotransposon sequences in the cen-tromeric regions of several cereal species (Figure 2) (1, 121, 150). Probes withsequence similarity to Ty3-gypsywere isolated from a BAC clone of a sorghumcentromere. The probes hybridized exclusively to centromeric regions of sorghumchromosomes and those of allGramineaetested (121). A barley homologue of thissorghum Ty3-gypsyintegrase sequence was used to clone a barley retrotransposon(cereba) that was subsequently found to be almost exclusively localized withinthe centromeric regions of several cereal species, including barley, wheat and rye(Figure 3c) (150).

The presence of Ty3-gypsyretrotransposon sequences in the centromeric re-gions of all investigated species ofGramineaeis intriguing. This suggests thatthese retrotransposons either are ancient insertions that amplified before the di-vergence of theGramineaeor have independently arrived and/or amplified, plusbecome preferentially located in centromeric regions, in all of these species. Thehigh degree of conservation ofcerebaretrotransposon sequences within the cen-tromeric regions of theGramineaespecies indicates a possible association withcentromeric function. Earlier studies have shown that someDrosophilaretrotrans-posons are stable components of the telomeric and centromeric heterochromatin(146). However, it remains to be determined whether plant retrotransposon se-quences have any direct roles in centromere and/or telomere functions.

Recently, individual full-size copies ofGrande, Zeon-1, PREM-2, RE-10, andRE-15 retrotransposons have been found to interrupt tandemly arranged 180-bpunits of knob DNA associated with cytologically detectable heterochromatic com-ponents of pachytene maize chromosomes (1). Knob DNA in maize is associatedwith several genetic effects, including segregation distortion in the female game-tophyte and late flowering time. Previously, genetic effects associated with knobheterochromatin have been attributed only to the 180-bp repeats. However, the oc-casional retrotransposon insertions could also be involved in attenuation or controlof the multiple genetic effects associated with knob DNA.

LINEs and SINEs, the non-LTR retrotransposons, also have been studied fortheir chromosomal distributions using in situ hybridization. Elements of both typesshow dispersed chromosomal patterns of distribution, but some elements show dis-tinct clustering in specific regions of chromosomes. For example, some LINEs insugar beets are preferentially clustered in the subtelomeric regions of most chromo-some arms (Figure 3d,e) (91). Like the Ty1-copia retrotransposons, these LINEsexhibited almost no hybridization signal in the region of the tandem 18S-5.8S-25SrDNA gene repeats (Figure 3e) (91, 161). In situ hybridization analyses with theSINE element S1Bn of Brassica napusyielded a dispersed pattern of chromoso-mal localization, but also showed hybridizational signal in the centromeric regionsand a tendency to co-localize with rDNA sites (Figure 3f, g) (53). Although S1Bnelements are methylated at a level twice that of the average methylation level of




theB. napusnuclear DNA, their chromosomal localization was not predominantlyassociated with some extensively methylated regions of chromosomes, like theheterochromatin associated with centromeres and telomeres (53).

From many in situ and genomic sequencing analyses, it initially appeared thatthe percentage of retrotransposons in euchromatic regions as compared to hete-rochromatic regions was generally high. However, these studies were biased byboth the common selection of retrotransposons originally found near genes asthe in situ hybridization probe (38), and by the fact that genomic sequencing ef-forts were focused on genic regions (11, 182). Recent data from the sequencing ofArabidopsiscentromeric regions suggest that retrotransposons are highly enrichednear centromeres, and often arranged as nested series like those seen in intergenicregions of the more complex maize genome (158; R Martienssen, R McCombie& R Wilson, personal communication; http://www.cshl.org/protarab).

Database searches have shown that retrotransposon sequences are present ei-ther within, or close to, many genes. These retrotransposon legacy sequences areoften fragmentary and apparently ancient (19, 202, 203). However, analysis of thedistribution of retrotransposons within a contiguous 240-kb stretch of the maizegenome revealed that most retrotransposons are located between genes (158). Itmay be that many retrotransposons have evolved to transpose primarily into rela-tively inactive regions of plant chromosomes, such as intergenic spacers and otherrepetitive sequences, to avoid mutating genes at a high frequency (158). Otherwise,the host cell would accumulate a lethal level of mutations. In this way, retrotrans-posons could proliferate as dispersed sequences without being deleterious to thehost genome. By minimizing the generally negative (e.g. mutational) effects ofindividual retrotransposons, these elements could actively increase host genomesize.

Retrotransposon Abundance in Plants Relativeto Other Eukaryotes

Genomic sequencing of the 80–90% of theArabidopsisgenome that does not in-clude centromeric heterochromatin, telomeres, or satellite repeats has revealed arelatively low density and simple arrangement of repetitive DNAs. In a contiguousregion of approximately 1.9 Mb on chromosome 4 ofA. thaliana(genome sizeof ca 130 Mbp) (11) a retrotransposon was found about every 130 kb, suggestingthat about 1000 of these elements will be found in the genic regions of the entiregenome. These would comprise about 4% of theArabidopsisgenome, with themore concentrated retrotransposons in the centromeric regions accounting foranother 4–6%. The 4-Mbp genome of the yeastSaccharomyces cerevisiaecon-tains 331 retrotransposon sequences that constitute about 377 kb, or 3.1%, of theyeast genome. These elements belong to five families of LTR retrotransposons;217 Ty1, 34 Ty2, 41 Ty3, 32 Ty4, and 7 Ty5 (87). The non-LTR retrotrans-posons, LINEs and SINEs, appear to be absent from theS. cerevisiaegenome.In contrast, the filamentous fungusMagnaporthe grisea(with a genome size of




about 40 Mbp) contains both LTR and non-LTR retrotransposons at copy num-bers varying from a few to 100 (84). InCaenorhabditis elegans, which has agenome size of about 100 Mbp, most of the retroelements appear to be non-LTRretrotransposons, including 59Rte-1family members, of which 9 are full-length(3298 bp) (209). A few Ty3-gypsygroup retrotransposons have been identifiedin this nematode, but it appears that the Ty1-copia group is either absent orrare. Furthermore, there have been no reports of either retrotransposon activity orretrotransposon-induced mutations inC. elegans. Most of the identified retrotrans-posons inC. eleganswere found to be defective (E Berezikov, personal communi-cation). InDrosophila melanogaster, which has a genome size of about 165 Mbp,both LTR-and non-LTR retrotransposons are present. Unlike yeast,C. elegans,andArabidopsis, Drosophilaalso contains active retroviruses (170). To date, thereare at least 23 families of LTR retrotransposons and retroviruses, and at least11 families of non-LTR retrotransposons, that have been identified inDrosophila(http://fly.ebi.ac.uk:7081/transposons/lk/melanogaster-transposon.html). Together,these retrotransposons constitute 5–10% of theDrosophilagenome. In stark con-trast toC. elegans, many of theDrosophilaretrotransposons are active and fre-quently generate insertional mutations.

Interestingly, SINEs are either absent or rare in the genomes ofArabidopsis,C. elegans, andDrosophila. These are likely to be exceptional cases, because closerelatives of these species do contain SINEs. For example,Brassica napus, whichbelongs to the sameCruciferaefamily as doesArabidopsis, contains numerousSINEs (32). It is not clear why these small genome eukaryotes (Arabidopsis, about130 Mbp;C. elegans, about 100 Mbp;Drosophila, about 165 Mbp) are deficientin SINEs. One possibility is that these organisms may have an effective process forremoval of retrotransposons and pseudogenes from their genomes. Alternatively,the relatively low activity of LINEs in these species (exceptDrosophila) mayhave led to a very low frequency of new SINE insertions (39). Further research isrequired to resolve this enigma.

Genome size variation is correlated both with the total mass of retrotrans-posons that are present and with the number of different retrotransposon families.As mentioned above, the 4 MbpS. cerevisiaegenome contains five different LTRretrotransposon families that account for about 3% of the genome (87). InAra-bidopsis, both LTR- and non-LTR retrotransposons have been identified, includingTy1-copia, Ty3-gypsy, and LINE elements, with 1–100 families of each group that,in total, probably account for 4–10% of total nuclear DNA, whereas in maize, thereare thousands of different families of LTR and non-LTR retrotransposons that to-gether account for 70–85% of the nuclear genome (156, 158).

In most mammalian genomes, including the human genome, LINEs and SINEs(e.g. L1 and ALU elements) appear to be the major mobile sequences, representingabout 35% of the total DNA, with up to 100,000 copies of each element type(167). Interestingly, although retroviruses and their remnants, including retroviralelements with deletedenvgene, (i.e. Ty3-gypsy-like elements), are widespread inmammals, LTR retrotransposons of the Ty1-copiagroups appear to be absent or




rare. In contrast, LINEs, SINEs, Ty3-gypsy, and Ty1-copiaretroelements appear tobe abundant in plants. All in all, plant genomes contain an exceptionally abundantand diverse set of retrotransposons compared to most other eukaryotic genomes,with the exception that functional retroviruses are either absent or present in verysmall numbers.

Local Structures

In most cases, plant retrotransposons have been studied initially as single insertions(12, 20, 56, 69, 70, 72, 76, 78, 91, 101, 128, 131, 155, 187, 188) or as PCR ampli-fied fragments of a population of elements (45, 46, 82, 83, 92, 141, 142, 172, 194).Hence, these studies did not provide much information regarding the local orga-nization of elements within a small chromosomal region. Similarly, standard insitu hybridization experiments only reveal gross general patterns. Recent genomicsequencing studies have begun to provide information on the local organization ofretrotransposons and their interspersion profiles relative to genes.

The first studies of the interspersion patterns of genes with repetitive DNAsin plants were initiated over 25 years ago, utilizing genomic DNA renaturationanalyses (44). It was not known then that most of the repetitive sequences underinvestigation would turn out to be retroelements, primarily LTR retrotransposons.However, these Cot analyses routinely indicated that most of the repetitive DNAswere interspersed with low copy-number, presumably gene-containing, sequences(44, 59). These studies had the disadvantage that they primarily provided resultsthat averaged the actual individual situations across an entire genome.

Genomic sequence analyses have largely confirmed the predictions from Cotstudies. The interspersed repetitive DNAs, mostly retrotransposons and a class ofsmall DNA transposable elements called miniature inverted repeat transposableelements (MITEs) (203), are intermixed with each other and with genes in domainsthat are usually 100 kb or less in size. Many transposable elements, commonlyincluding retrotransposon fragments (203), are actually found within functioninggenes. In small genome plants, these retroelements have usually been observed assingle elements or solo LTRs (11, 24). This simple pattern has also been observedin some gene-rich regions of large plant genomes, like those of maize and barley(105, 136). However, the largest segment of a complex plant genome sequenced todate, an approximate 225-kb region around maizeadh1, has shown a very differ-ent pattern of retroelement arrangement (158, 182). In this region, LTR retrotrans-posons were very abundant, and were found mainly as elements inserted withineach other. The insertions showed some other biases, including a fivefold prefer-ence for insertion into the LTRs rather than the internal retrotransposon domainsand the fact that 17 of 22 were in the same potential transcriptional orientation(158, 182). These nested LTR retrotransposons were found in six blocks, with oneof these blocks possibly interrupting one of the nine putative genes identified inthe area. In contrast, the three LINEs detected in this region were all found withinputative introns of genes (182). Although further genomic sequencing studies will




be needed to determine whether this organizational pattern is common in plants,or an exceptional property of the maize genome, it is interesting that the first se-quencing of DNA nearArabidopsiscentromeres has also uncovered a gene-poorregion that is rich in nested retrotransposons (R Martienssen, R McCombie &R Wilson, personal communication; http://www. cshl.org/protarab).

Insertion Site Preferences

All mobile DNAs exhibit some level of insertion site preference. Among theDNA transposable elements, the degree of specificity can vary tremendously.Even the most random insertion mutagens, like Tn5 ofE. coli, P elements ofD. melanogaster, or Mutator of maize, will insert into some genes at frequenciesover tenfold higher than into other genes. Many of these elements exhibit a pref-erence for insertion into genic regions of a genome, and into specific locationswithin or near a gene (e.g. promoters) (10, 86). Moreover, some elements, likeAcof maize, prefer to insert at sites linked to the location of the starting element (35).

Insertion specificities for retroviruses and retrotransposons have been reportedin many different eukaryotes (27, 99), but this question has not been extensivelyinvestigated in plants. In some cases, the preferential integration sites are locatedin actively transcribed chromosomal regions, particularly in or near promoters. Inother cases, preferential integration sites are located in repeated heterochromaticregions. In yeast, most or all retrotransposons show target specificity. For in-stance, the Ty1, Ty2, and Ty3 elements integrate preferentially upstream of genestranscribed by RNA polymerase III, including tRNA, 5S, and U6 genes, whileTy5 primarily inserts in silenced regions of the yeast genome, including silencedmating-type cassettes and telomeric regions (50, 211).

It is not entirely clear how and why retrotransposons target specific regions orsequences of the host genome. Selection of the insertion site may be made by anintegration complex, consisting of retrotransposon-encoded integrase, a reversetranscribed cDNA copy of the element, and various host-encoded factors (27, 99).The most precise information regarding preferential insertions has come fromresearch on yeast retrotransposons. For example, the Ty3 integration complex wasfound to have an affinity with transcription factors that interact with all tRNAgenes (88). Similarly, there is preferential insertion into silent chromatin by Ty5.This specificity can be abolished by a single amino acid change in the integraseprotein (50) or by mutations in nuclear proteins that specifically interact withsilent/heterochromatic regions of the yeast genome (211).

In plants, there is evidence that some retrotransposons show preferential inser-tion into some genomic regions. For example, many Ty1-copia retrotransposonsare found preferentially within euchromatic regions in those plant species studied todate (51, 69, 70). In contrast, some of the cereal Ty3-gypsygroup retrotransposonsaccumulate primarily within heterochromatic regions, such as near centromeresand the knob DNA in maize. Also, Zepp, a LINE-like retrotransposon inChlorella,which accumulates in the telomeric region, integrates preferentially into itself and




another Zepp sequence (62). It is not clear whether the biased accumulation inany of these cases is due to preferential insertion, or perhaps to a less active pro-cess for removal from these regions and/or less fitness selection acting againstretrotransposons in these regions.

When specificities are seen, some may be shared by many different retrotrans-poson families. For instance, theGrande, Zeon-1, and RE-15 retrotransposonseach were found inserted at the same site in the 180-bp repeat sequences of knobDNA (1). Both Ty1-copia and Ty3-gypsyretrotransposons have been found inintergenic locations within euchromatic regions of the maize genome (158). Incontrast, the low-copy-number Ty1-copiaretrotransposonsTos17of rice andTpv2of common bean,Phaseolus vulgaris, are mostly targeted near and within genes(51, 70). Therefore, it appears that each retrotransposon has evolved its own patternof insertion within a plant’s genome.

In Organellar Genomes

The completed sequences of both chloroplast and mitochondrial genomes fromplants (109, 185) have allowed investigation of whether these additional cellulargenomes contain any retrotransposons or retrotransposon legacies. There have notbeen any reports of retrotransposons or retrotransposon fragments within the smalland gene-dense chloroplast genome of plants. In contrast, mitochondrial genomesappear to contain many fragments of Ty1-copia, Ty3-gypsy, and LINE-like retro-transposons. No intact elements have been seen, and there is no evidence of mu-tations caused by retrotransposon insertion. About 4% or more of theArabidopsismitochondrial genome consists of these retrotransposon fragments, through 9 ormore independent acquisition events (185). It is possible that these fragments ac-cumulated within the relatively large and repeat-rich plant mitochondrial genomesas a passive outcome of DNA acquisition from the nuclear genome (14, 162).

REGULATION OF EXPRESSION AND TRANSPOSITION

The expression of retrotransposons in plants and in other eukaryotic organismsis regulated, thereby regulating their transposition frequency in the host genome.The evolution of control mechanisms for the transcription and transposition ofretrotransposons in the host genome may be crucial to minimize their possibledeleterious effects on the host. Thus, it is not surprising that plant retrotransposonsare transcriptionally silent in most plant tissues during development.

Transcriptional Regulation

Because retrotransposons cannot transpose without the presence of an RNA tem-plate available for reverse transcription, the simplest way to control their activitywould be via regulation of transcriptional initiation. Many retrotransposons showunique patterns of developmental and/or environmental regulation. For instance,




transcripts of tobaccoTnt1, barleyBARE-1, and maize PREM-2 have been detectedprimarily in roots, leaves, or young microspores, respectively (148, 172, 173, 184).Likewise, inS. cerevisiaeandDrosophila, the expression of retrotransposons isinduced by hormonal and developmental factors (16, 33).

There are only a few reports on the expression and regulation of non-LTR retro-transposons in plants. For example, the SIBn family of SINEs has been shown tobe expressed in shoots, roots, and callus tissues (31). Moreover, transcriptionalanalysis of SIBn has revealed that the major SIBn RNAs result from cotranscriptionderived from an element present in a POL II transcriptional unit. Only rarely dotissue-specific SIBn transcripts result from the expression of a small number of ge-nomic elements by POL III (31). It appears either that the SIBn POL III promoteris highly repressed inB. napuscells or that transcriptional signals external to theSINE element are necessary in vivo to obtain transcription. Our knowledge of theregulation of LINE expression in plants is limited. In the mouse genome, it has beendemonstrated that LINE-1 expression is under the control of a tightly regulated tem-poral and spatial program of events during development and differentiation (183).

A correlation between transcription and transposition of retrotransposons hasbeen demonstrated for the tobaccoTto1and riceTos17retrotransposons (64, 70).For example, transposition ofTto1andTos17was concomitant with an increase inthe levels of their RNAs, suggesting that transposition of these retrotransposonsis regulated mainly at the transcriptional level. For example, a tenfold increase inthe copy numbers ofTto1 elements was observed in cultured cells, whereTto1transcripts are very abundant (64). A similar pattern of increase in copy number forthe riceTos17elements during tissue culture conditions also has been demonstrated(70). However, this is not the situation for several other retrotransposons. Forexample, barleyBARE-1(an active element in the recent past) is highly transcribedin leaves, but its transposition has not been observed (172). This reminds us that,although transcription is a prerequisite for transposition of an element, other stepsare also required and may be regulated.

Activation by Biotic and Abiotic Stresses

Many of the plant retrotransposons studied to date are transcriptionally activatedby various biotic and abiotic stress factors (55). The expression of the tobaccoTnt1 and Tto1 retrotransposons is greatly increased by several abiotic stresses,including protoplast isolation, cell culture, wounding, methyl jasmonate, CuCl2,and salicylic acid (64, 120, 125, 147, 148, 177, 178). Similarly, various biotic stressfactors such as fungal extracts fromTrichoderma viride(148, 178) or inoculationwith various viral, bacterial, or fungal pathogens (119, 147) have been shownto activate transcription of these retrotransposons. In contrast toTnt1 andTto1,transcription ofTos17is induced only by tissue culture. TobaccoTto5was isolatedas a salicylic acid-inducible retrotransposons by the RT-PCR method (177). Atpresent, it is not known whether abiotic stresses such as cold and heat treatment canalso induce transcription in these retrotransposons. Thus, these results indicate that




retrotransposons carrycis-acting sequences that control their expression patternsin the host.

Regulatory Sequences

Transcription of LTR retrotransposons is controlled bycis-acting elements in the5′ LTR and adjacent untranslated regions. The structure and function of promotersfromTnt1A,Tto1, andBARE-1have been extensively studied using LTR-GUS tran-scriptional fusions in transformed plants. Transient and stable expression assays ofLTR-GUS fusions containing various lengths of 5′ LTR regulatory sequences haveshown that the majorcis-acting sequences controllingTnt1AandTto1expressionare located within the U3 region of the 5′ LTR (147, 148, 177, 178). In the case ofTnt1, the U3 region contains severalcis-acting sequences, including a short palin-dromic sequence named BI, and a segment of 31 bp tandemly repeated in three orfour copies, named BII. Both BI and BII behave as transcriptional activators. TheBII box binds specific protein factors in tobacco protoplasts (22), and has beenshown to be involved in the transcriptional activation ofTnt1by several biotic andabiotic factors (55, 189).

A similar study on the LTR promoter ofTto1using LTR-GUS constructs hasshown that the 5′ LTR containscis-acting sequences involved in the induction ofTto1expression by abiotic and biotic stress factors (177, 178). Deletion analysison the LTR promoter has shown that enhancer-like sequences upstream from theTATA box are required for a high level of expression in the callus. Recently, bygain-of-function analysis, a 13-bp repeated motif (TGGTAGGTGAGAT) has beenidentified as a positivecis-acting regulatory sequence in the U3 region. This motifis involved in expression in protoplasts and callus, as well as in wound- and methyljasmonate–induced expression in leaves (178). Furthermore, gene expression me-diated by the 13-bp motif was found to be associated with genes that conditionplant defense against microbial attack (178). For example, the complementarysequence of the 13-bp motif contains the box L sequence (also called AC-I) andthe core sequence of the H-box elements, which are highly conserved among thepromoters of phenylpropanoid biosynthesis genes (178).

To gain further insight into the regulation ofTto1 and defense-related genes,cDNAs encoding four different proteins that bind to the 13-bp motif have beenisolated (178). One protein (LBM1, LTR binding MYB) is identical to the pre-viously reported MYB-1, which is induced by virus infection. LBM1 contains aDNA binding domain conserved among the MYB class of transcription factors.Accumulation of LBM1 mRNA was induced rapidly and transiently by cuttingleaf tissues and by elicitors. LBM1 cantrans-activate reporter gene expressiondependent on its binding to the 13-bp motif. A 10-bp sequence (TTGGTAGGTG)containing a conserved motif between box L and the H-box has been shown tobe essential for the binding of LBM1 protein to the 13-bp motif. LBM1 also ac-tivated transcription from the promoter of the phenylalanine ammonia lyase gene(Pv-PAL2) in tobacco protoplasts through binding to the box L and the H-box-like




motif (box P). These results suggest that the LBM1 protein may be involved inthe stress response ofTto1and defense-related genes (178; H Hirochika, personalcommunication).

The activation of the expression of tobacco retrotransposonsTnt1 and Tto1by pathogen-derived factors that are involved in the activation of plant defenseresponses is intriguing. Moreover, thecis-acting regulatory motifs of both retro-transposons share similarities with the H-box motif involved in the activation ofseveral plant defense genes (55). The finding that the 13-bp motif contains theL/Ac-I/H-box motif, conserved among promoters of the phenylpropanoid biosyn-thetic genes, raises the question of whether an ancientTto1-like retrotransposoncontributed to acquisition ofcis-regulatory sequences in promoters of some de-fense genes (178). Alternatively,Tto1 may have acquired this sequence from adefense gene, or they could have evolved independently in defense genes andretrotransposons by convergent selection. Takeda and coworkers have analyzedthe promoter of the defense-activated asparagusAoPR1gene (200) and have foundthat it contains an H-box-like motif and several other regions homologous to thecomplementary sequence of theTto1LTR promoter. Interestingly, theAoPR1pro-moter can be activated in a similar fashion to theTto1LTR promoter by abioticand biotic stresses, including tissue culture, wounding, pathogen infection, andsalicylic acid. However, the biological significance of this, as well the origin ofthese regulatory sequences in retrotransposons and host genes, is unclear at presentand warrants further investigation.

LTR-GUS construct analyses have shown that theTnt1andTto1LTR promotersare active in heterologous systems, including transgenicArabidopsis, tomato, orrice and that the expression patterns are similar to those seen in the natural host,tobacco. Moreover, activation of theTnt1andTto1elements by abiotic and bioticstresses is maintained in heterologous hosts (69, 125). However, theTnt1promoter,with expression confined to roots in the homologous tobacco system, was alsoexpressed in the flowers of transgenicArabidopsisand tomato (125), and wasfound to be induced by treatment with auxins inArabidopsis(139). These resultssuggest that, beyond the conservedcis-acting regulators involved in abiotic andbiotic stress induction of these retrotransposons, there are also some host-specificregulators of the expression of theTnt1promoter.

Epigenetic and Posttranscriptional Regulations

Host cells, and possibly the retrotransposons themselves, have evolved mecha-nisms to minimize the negative effects of element retention and transposition. It hasbeen proposed that DNA methylation evolved as a means of containing the spreadof transposable elements and viruses in host genomes (208). This defense modelhas been supported recently by analysis of an interspecific mammalian hybridbetweenMacropus eugeniiandWallabia bicolor in which genome-wide under-methylation of the hybrid genome and a high level of amplification of retroelementsare correlated (132).




In plants, analyses of repetitive DNAs have routinely shown that they are cy-tosine methylated in the sequences 5′-CG-3′ and 5′-CNG-3′. These methylatedDNAs include those flanking standard plant genes in species like maize (9), andthese methylated sequences have now been shown to be mostly LTR retrotrans-posons (158). Cytosine methylation has been associated with genetic inactivityand a heterochromatic state in many higher eukaryotes, including plants where denovo methylation was first detected during the inactivation of DNA transposableelements of theMutator andAc/Ds families (10, 97). This cytosine methylationhas been associated with both transcriptional inactivity and a higher rate of C to Ttransitions, giving rise to enhanced transcriptional and mutational silencing (157).It is not clear, though, despite many years of study in many species, whether themethylation itself establishes the inactive state of a retrotransposon, or whether itis a secondary (possibly maintenance) outcome of inactivation initially caused bya change in chromatin structure.

The most recent evidence on the role of DNA methylation in the regulation ofretrotransposition comes from a study onTto1by the Hirochika group in Japan (67).It was observed that after the initial active retrotransposition in theArabidopsisgenome,Tto1 became silent. However, the silencing ofTto1 was only observedin those transgenicArabidopsislines whereTto1copy number was increased byactive transposition during culture-mediated transformation process. Thus, thissilencing appears to mimic repeat-induced gene silencing (115). This silencingcorrelated with a reduced level of theTto1 transcript and with methylation of theinactivatedTto1. To investigate the causal relationship between DNA methyla-tion and silencing ofTto1, the hypomethylation mutation,ddm1, was introducedinto the line carrying silencedTto1 copies through conventional crossing. In ahomozygousddm1background, the silencedTto1 became hypomethylated andits transcription was activated. This result suggests that DNA methylation may beresponsible for silencingTto1 in wild-typeArabidopsis(67).

Interestingly, in theDrosophilaandS. cerevisiaegenomes, which lack cytosinemethylation of DNA in general, an inactive chromatin state can be established andmaintained without the need for DNA methylation (36, 58). An even more drasticcontrast is shown by the exceptional case of the chordateCione intestinalis, wheretransposable elements are generally unmethylated and most genes are methylated(166). Hence, DNA methylation is not obligatory for genetic silencing in all eu-karyotes, although a standard structure for inactive chromatin with deacetylatedhistones may be universal (154).

It is not clear how an epigenetic silenced state is either initiated or maintainedfor retrotransposons. In many cases, epigenetic inactivation of an element may oc-cur because of its insertion in or near an already heterochomatic block of DNA, asis the likely case for the insertion of the most abundant maize retrotransposons intomethylated, intergenic blocks (9, 158, 182). Silencing in this case could occur by aprocess analogous to position effect variegation inDrosophila, where movement ofnormally euchromatic genes into a heterochromatic region leads to a progressive ra-diation of the silenced heterochromatic state into the translocated gene(s) (196). In




other cases, retrotransposons may be epigenetically modified and/or posttranscrip-tionally suppressed by a process similar to homology-based gene silencing, firstdetected in plants but now also identified in some animal systems (75, 135). Someforms of paramutation (71), the coordinate inactivation of a transposable elementfamily, and homology-based gene silencing all involve correlated DNA methyla-tion and transcriptional inactivation of repeated sequences scattered throughout aplant genome (112, 115). How homologous sequences widely dispersed through agenome are found and inactivated is not clear, nor is it completely clear why repeatslike transposable elements, transgenes, and their chromosomal homologues aretargeted for silencing while normal gene families largely escape this inactivation.Numerous studies are under way in this area (60), and the insights obtained will betremendously valuable to understanding the regulation of plant retrotransposons.

In the yeastS. cerevisiae, several studies have shown that the transpositionof Ty1 elements is controlled at posttranscriptional steps (99). A recent work il-lustrates the concept that conserved cellular functions, such as those involved indifferentiation and DNA repair/recombination, have been adapted by the host forpreventing rampant proliferation of retrotransposons (reviewed in 29). Host genesthat encode Fus3p (a MAP kinase involved in the mating pheromone signal trans-duction pathway) and TFIIH (an RNA polymerase II general transcription factorinvolved in initiation and promoter clearance, and in transcription-coupled repair)have been shown to regulate a posttranscriptional step in retroviral replication inyeast. It appears that Fus3p inhibits the accumulation of Ty1 proteins and thatTFIIH promotes degradation of Ty1 cDNAs. It is not known whether similar hostgene-mediated or other types of posttranscriptional control mechanisms exist forthe regulation of retrotransposition in plants.

Retrotransposon Regulation as a Form of Host Defense

Some retrotransposons might have beneficial effects on a plant, through mutationsthat provide new regulatory properties to a gene (202) or perhaps by contributingto DNA repair (123) or centromere function (121, 150). However, many of theexpression, mutation, and insertion properties of these elements suggest that theireffects are being minimized. With any highly adapted host/parasite interaction,the parasite will contribute as little as possible to decreased host fitness. The hostshould also evolve such minimization/defense processes, and several seem to beacting on plant retrotransposons.

Most of the known retrotransposons appear to be defective in their ability toencode all necessary transposition functions, owing to insertions, deletions, andother mutations (45, 46, 72, 76, 114, 141, 186, 194, 203). The epigenetic regulationof plant retrotransposons associated with DNA methylation and presumed hete-rochromatization may also be involved in keeping retrotransposon transcriptionat a low level (208). In maize, for example, the retrotransposons that make up50–80% of the DNA contribute only 10% or less of the mRNA in most tissuesunder most circumstances (B Bowen, personal communication). Moreover, thisDNA methylation is associated with a two- to threefold higher transition mutation




rate in these elements, thus causing them to decay to a nonfunctioning form morerapidly than other sequences.

Retrotransposons can inactivate other retrotransposons by inserting within thema structure often seen in the intergenic regions of the maize genome (158). In acompetition between retrotransposons for use of host resources, this inactivation ofone retrotransposon by insertion of another would be an ingenious approach (95).Moreover, the host might benefit from such an insertion pattern, because theseelements would not be generating mutations as often, and thus natural selectioncould act on host factors that regulate this insertion specificity.

PLANT RETROTRANSPOSON ORIGINS AND EVOLUTION

Very little is known about the timing or particular steps in the origin of retro-transposons overall, or about their arrival within a particular kingdom or species.Sequence comparisons of conserved gene products encoded by LINEs and LTRretrotransposons suggest that the first retrotransposons were LINE elements, andthat LTR retrotransposons evolved from them by the acquisition of terminal directrepeats (207). InDrosophila, tandem insertion by theHeT-Aelement suggests howthis process could simply create two flanking LTRs (30, 40). Retroviruses appearto have evolved in animals from a subclass of Ty3-gypsyretrotransposons by ac-quisition of an envelope function (180, 207). Some believe that retrotransposonsoriginated at the beginning of the transition from a putative RNA-based cellulargenome to the DNA-based genome now shared by all living cells (52).

The ubiquity of plant retrotransposons and their extreme sequence heterogeneitysuggests that they were present in the first plants (46, 95, 129, 176, 194). Alterna-tively, retrotransposons may have originated after the creation of the first eukary-otes, and have reached their current wide dispersal by a combination of verticaland horizontal transmission. Nonetheless, they have clearly been highly successfulin proliferating within all higher eukaryotic genomes. Their contributions to plantgenome evolution are many and significant (5, 48, 55, 93, 95, 97, 202). As with anyother DNA sequence present in a plant genome, selective pressures can act to uti-lize that retrotransposon sequence for the benefit of the entire organism, if anysuch benefit can be acquired by chance mutational alterations. Hence, we expectthat retrotransposons or retrotransposon components will occasionally be found tocontribute important factors to plant fitness, as has been seen with the contributionsof these elements to the promoters of many “wild-type” plant genes (203).

The Frequency and Timing of RetrotranspositionDuring Evolution

Although a great deal of information exists regarding the genomic organizationof retrotransposons, little is known concerning the frequency and possible period-icity of element transposition during plant evolution. These questions have beenmore deeply investigated with the DNA transposable elements.Mutatorof maize,




for instance, commonly transposes more than once per plant generation, therebyincreasing copy numbers several fold and making many mutations in a single grow-ing season (10, 97). Since the survival and evolution of retrotransposons and thelarge-scale structure of plant genomes are greatly influenced by the transpositionalactivity of retrotransposons, it is important to understand this aspect of retrotrans-poson behavior. Recently, element-anchored PCR analyses have been used to studythe insertional polymorphism of the Ty1-copia group retrotransposons in plants(41, 57, 143, 201). Those studies have shown thatBARE-1insertions, for example,are highly polymorphic in barley, wheat, rye, and oat, suggesting that the trans-positional activity of this retrotransposon has persisted for millions of years inseveral cereal species (57, 201). Recently, a similar study has shown that a fewretrotransposons have been transpositionally active in the recent past in severalPisumspecies (41, 143).

The transpositional activity of plant retrotransposons varies considerably onthe evolutionary time-scale, with some retrotransposons having been active in thedistant past whereas others have been active more recently. A Ty1-copiaelement,R9of rye [about 50,000 copies per genome (140)], is similar toBARE-1of barley[about 70,000 copies per genome (172)]. Gel blot hybridization analyses indicatethat a close relative of theR9subgroup is present in high copy numbers in barley,oat, and wheat. Although Northern analysis on RNAs from seedlings shows thatthe R9/BARE-1subgroup is transcribed in all these cereal plants, the amount oftranscript varies from high in barley, to moderate in wheat and rye, to extremely lowin oat (140). It will be interesting to determine how these retrotransposons havebecome different in transcriptional activity between these closely related cerealhosts, or whether differences in the host transcriptional machinery account for thevariable expression.

A different approach has been used to demonstrate that most retrotransposonsin the maize genome have transposed within the last 2–6 million years (157). Thisstudy was based on calculating the rate of mutations in the LTR sequences ofseveral retrotransposons in theadh1region of maize. As pointed out earlier, thetwo LTRs of a retrotransposon are synthesized from a single template during theirreplication cycle. Thus, the 5′ LTR is usually identical to the 3′ LTR at the time ofa retrotransposon’s insertion into the genome. Therefore, the date of insertion canbe estimated from sequence divergence between the two LTRs, assuming a fairlyconstant rate of nucleotide substitution since the insertion date.

Some retrotransposon families are essentially specific to one genome in plantsof a hybrid origin. For example, in Triticale, an allopolyploid with two wheatgenomes and one rye genome, a rye-origin retrotransposon probe hybridizesmuch more strongly to the rye-origin chromosomes than to those of the wheatgenomes (140). Likewise, in hexaploid oat,Avena sativa(2n = 6x = 42, withA, C, and D genomes), a retrotransposon probe isolated from a diploid A genomespecies hybridizes only weakly to C-genome chromosomes (83). This indicates thatretrotransposition of these elements has been relatively infrequent since the poly-ploid was created. Otherwise the retrotransposons would now be homogeneously




distributed across all genomes within the same nucleus. Alternatively, some retro-transposons may have evolved to a degree that they are compatible only withinsertion sites within one particular genome, as appears to be the case for retro-transposon amplification in an interspecific wallaby hybrid (132).

Heterogeneity

Fortuitous discovery by genomic sequencing, identification of retrotransposonsas the causal agents in various mutations, and the directed acquisition of retro-transposons by PCR techniques have all yielded information indicating there isa great variety of retrotransposons both within a single plant and between plantspecies. Both LTR and non-LTR retrotransposons, including the Ty1-copiagroup,the Ty3-gypsygroup, LINE, and SINE, are usually present in a plant genome(Table 1) (5, 55, 95, 97). Among these retrotransposons, the Ty1-copiaretrotrans-posons have been studied in most detail, and this has revealed that they are presentas highly heterogeneous populations in all higher plant genomes. For example,when 31 subcloned fragments of the reverse transcriptase genes of the Ty1-copiagroup retrotransposons from potato were sequenced, each was found to be differ-ent, with predicted amino acid similarities between individual fragments varyingfrom 5% to 75% (45). Such extreme heterogeneity has subsequently been observedamongst the reverse transcriptase genes of Ty1-copiagroup retrotransposons fromseveral monocotyledonous and dicotyledonous plant species (45, 46, 89, 114, 128,140–142, 186). BothD. melanogasterand S. cerevisiaepossess a small num-ber of homogeneous elements, with less than 5% divergence within the families(87, 127). These results indicated that plant Ty1-copiagroup retrotransposons aremore heterogeneous than those in many animals and lower eukaryotes.

There could be several reasons for differences in the degree of heterogeneityof Ty1-copiagroup retrotransposons between plants and other eukaryotes. Retro-transposons proliferate by a replicative mode of transposition, with each replicationcycle capable of generating defective elements. The mutation rate during a sin-gle transposition cycle of yeast Ty1 retrotransposons has been estimated to be2.5× 10−5 (49). Additionally, retrotransposons are capable of generating largepopulations in relatively short evolutionary times. In yeast, it has been found thathigh homogeneity in both sequence and size variation of Ty1 elements is due to ahigh level of genomic turnover of retrotransposons in the genome (80, 87). Thus,the majority of retrotransposons in the yeast genome are derived from transposedevents over a relatively recent evolutionary time. In contrast, a plant genome cantolerate accumulation of large amounts of DNA and does not appear to removethese sequences rapidly. It is possible, therefore, that early plants possessed onlya few active populations of retrotransposons that subsequently proliferated intolarge populations in the host genomes. The mixed populations of active and de-fective retrotransposons then accumulated mutations over time, and eventuallygave rise to highly heterogeneous populations. If, as appears likely (45, 46, 114,187, 191), the majority of plant retrotransposons have been transmitted vertically




into subsequent generations, with horizontal transfer between species being veryrare, then heterogeneous lineages should predominate. Another feature, whichmay contribute to the high level of heterogeneity in plant retrotransposons, is thedistinctive strategy for determination of germline in plants. Unlike animals, plantsdo not have a sequestered germline; they have meristematic cells that are capableof differentiating into somatic tissues or reproductive organs throughout the lifecycle. This means that somatic tissues, with mutations accumulated in them duringplant growth, can give rise to germ cells, as long as mutations are not lethal. In ani-mals, because the germ cells are selected early in embryogenesis, it may be that theanimal germ cells are better protected against mutation. All of these phenomenaare likely to be responsible for the current heterogeneity of plant retrotransposons.

Heterogeneity generated in the 5′ LTR sequences and adjacent leader sequencescan have effects on the evolution of retrotransposons. Indeed, extensive sequencevariation has been found among the LTRs and adjacent leader sequences ofcopiaretrotransposons inD. melanogasterpopulations (28). However, the functional sig-nificance of the sequence variation has not yet been demonstrated. In the humanretrovirus-like element, RTVL-H, it has been shown that the LTRs are functionallydiverse and can promote the expression of a linked gene (42). In plants, the tobaccoTnt1 retrotransposons members have been extensively studied for sequence het-erogeneity of both coding domains and U3 regulatory sequences from a number ofNicotianaspecies (23, 190). This study has revealed that there are three differentsubfamilies ofTnt1 elements,Tnt1A, Tnt1B, andTnt1C, that differ completelyin their U3 regions but share conserved flanking regions (190). U3 divergencebetween the three subfamilies is found in the region that contains the regulatorysequences of an activeTnt1-94retrotransposon. On the basis of these data, the au-thors have proposed that this high sequence variability could allow these elementsto evolve regulatory mechanisms in order to optimize their coexistence with theirhost genome. LINEs, SINEs, and Ty3-gypsyelements are also found as heteroge-neous populations in plant genomes, but detailed investigations of their possibleorigins and evolution have not been performed yet.

Internal Mutation

In most plants, the majority of the retrotransposons in the genome have structuressuggesting that they are defective. Many members of a retrotransposon familydiffer by large internal insertions or deletions (72, 111). In some cases, as with the5′ ends of LINE elements, truncated insertions suggest that the internal variationwas generated during transposition. The relatively low replication accuracy ofreverse transcriptase will also increase the rate of internal mutation.

In many cases, defective retrotransposons will continue to persist and evenamplify within a genome. By sequence criteria, many LTR retrotransposons appearto be lacking in some essential component for transpositon, such as the absenceof apol coding potential inBs1(76). The absence of complete coding capacity forone of these elements does not necessarily mean that it will not transpose.Bs1itselfwas first detected as a de novo insertion mutagen, indicating that it had utilized




POL and any other possible missing functions from some other retrotransposon(76, 78). Similarly, SINEs do not appear to encode any proteins, therefore requiringtrans-encoded factors for every enzymatic or packaging step in their transpositionprocess (133). It is not clear how broad the specificity is for an enzyme encodedby one retrotransposon to act on another retrotransposon of a different family.The similar structure of the target repeats flanking LINEs, SINEs, and intronlesspseudogenes suggests that they use the same mechanism for integration into thegenome, presumably the endonuclease encoded by LINEs (39), although they mayalso be inserted as repair templates at sites of double-stranded DNA breaks (123).The observation that LINEs, SINEs, and intronless pseudogenes all seem to becommon in some species (e.g. humans), while all are relatively rare in other species(e.g. maize) concurs with a LINE-endonuclease model. LTR retrotransposons mayshow some preference for use of their own integrase, but it is unlikely that thisbias is absolute. However, the knowledge that retrotransposons do not need to befully intact in order to transpose does not indicate how or why they are so variablein sequence and so often defective.

A second class of mutation seen for several LTR retrotransposons has beenan apparent deletion of some standard retrotransposon genes, often accompaniedby the presence of additional internal sequences (18, 72, 76, 77, 111, 182, 191).In many cases, these defective retrotransposons have been identified in multiplecopies or as de novo insertions, indicating that they can retrotranspose by the useof trans-acting factors. One class of internal mutation is observed in the case ofsolo LTRs, where an apparent unequal exchange has led to the loss of one LTRand all internal sequences (25, 158, 165, 191). It is not known whether other lossesof internal sequences might be due to ectopic recombination or through a standardDNA breakage/repair mechanism. Sequence acquisitions may occur by ectopicgene conversions like those proposed for theMutator elements of maize (10), orby recombination between an mRNA-derived template and the retrotransposonwithin the replication/integration complex (16).

Regardless of their origin(s), internal variation within retrotransposons canprovide the raw material for either new retrotransposon properties or new capabil-ities acquired by the host genome. Such changes could explain sudden bursts inretrotransposon activity, perhaps by random mutation, to create a retrotransposonpromoter with a new tissue specificity (e.g. in “germline” cells) and/or a higherexpression level. The relative inaccuracies of reverse transcriptases provides anindication that internal variation is programmed into the system, and many host“defense” activities (e.g. cytosine methylation) also will increase variability. Thesealtered elements should be selected for their ability to amplify and persist withina genome, while natural selection acting on the host will utilize this variabilitywhenever possible.

Retrotransposons and Retroviruses

Although LTR retrotransposons are strikingly similar to retroviruses in both ge-nomic organization and their coding sequences, they lack sequences encoding the




envelope (ENV) protein (Figure 1). Thus, the retrotransposon replication cycle isrestricted to the cell of the host genome. Theenvgene is required by retrovirusesfor cell-to-cell transfer, thereby allowing infectivity. Until recently, retroviruseswere thought to be restricted to vertebrates. The first conclusive evidence for thepresence of a retrovirus in an invertebrate,Drosophila melanogaster, was reportedin 1994. A LTR retrotransposon of the Ty3-gypsygroup (mdg-4) was shown topossess a functionalenvgene, and has now been reclassified as a retrovirus orendogenous retrovirus (170). As pointed out earlier, it is believed that retroviruseshave evolved from the Ty3-gypsygroup retrotransposons by acquiring theenvgene or vice versa (94, 177, 204). In plants, Ty3-gypsyretrotransposons have beenfound with additional sequences in the region usually occupied byenv, but it is notknown whether they have anyenv-like gene function (24, 72, 76, 206). Recently,a Ty1-copiagroup retrotransposon has been found in soybean that has novelenv-like sequences in the usualenvregion of the retrotransposon (100). This suggeststhat the acquisition ofenv-like and/orenv-localized sequences in Ty3-gypsyandTy1-copiagroups has occurred independently or occurred once in one element andthen was transferred to the others by recombination (94). Recombination betweenretrotransposons appears to be a more common occurrence than was consideredpreviously (79).

The presence of putativeenv-like sequences in plant LTR retrotransposonsis intriguing. In animals, theenvgene is involved in transferring virus particlesfrom one cell to another through the plasma membrane by a receptor-mediatedendocytosis. In plants, a scenario similar to that seen in animals is unlikely to occurbecause of the plant cell wall (94). Enveloped viruses might move from cell to cell inplants via the intercellular channels called plasmodesmata. Virus-encoded proteinsthat modify the molecular size exclusion limit of plasmodesmata (21) usually assistthe movement of plant viruses through plasmodesmata. Such transfer proteins arenot known to be encoded by retrotransposons. Perhaps some of theenv-localizedfunctions that appear to be encoded by some LTR retrotransposons in plants couldperform such a transfer function.

It is possible that the putativeenv-like genes in plants have evolved along dif-ferent lines, leading to ENV-like proteins with either a different function or nocurrent specific role in plants. Future work on this subject should reveal the pre-cise function, if any, of the putativeenv-like gene in plants. In animals, retrovirusesare responsible for some of most deadly diseases known, but such pathogenicityof retroviruses is not known in plants. It may be important to determine what it isabout the biology of plants that has restricted (or even eliminated) the pathogenic-ity of retroviruses in the absence of an identified humoral or cellular immunesystem.

Horizontal Transmission?

Phylogenetic analyses based on sequences of the reverse transcriptase and inte-grase genes from both Ty1-copiaand Ty3-gypsyretrotransposons in plants have




provided strong evidence, with some exceptions, for the vertical transmission ofretrotransposons (45, 46, 114, 187, 190). Simple inheritance thus appears to be themost frequent mode of transmission. In contrast, the horizontal transmission ofplant retrotransposons has not been demonstrated. This is not surprising, becausemost or all plant retrotransposons lack the envelope (env) gene that is required byretroviruses for cell-to-cell transmission.

Nonetheless,env-like gene sequences have been found in some plant retrotrans-posons. One possible explanation for this result would be that these sequences arelegacies ofenv genes that functioned in another species, perhaps in an insect(170). If an insect retrovirus, for instance, were transferred to a plant cell duringthe feeding process, it is possible that it would prove to be a competent intracel-lular retrotransposon. The success of some human-assisted horizontal transfers ofretrotransposons from one plant species to another indicates that the host factorsneeded for retrotransposition are often conserved in higher plants (69, 108, 139),so natural horizontal transfers should have an equal chance of success. A possibleorigin from insect retroviruses is suggested for many plant retrotransposons be-cause of their transcriptional activation by stress (55). This activation makes sensefor a virus, because damage-associated stress (e.g. insect feeding) suggests that avector is available for transfer to the next possible host.

Similarly, it is possible that retrotransposons could be passively hijacked, in rareevents, by true retroviruses or by some other invading plant pathogen, pest, or en-doparasite (7, 94). Given that eukaryotic cells, including those of plant protoplasts,have the capacity to take up foreign nucleic acids into their genomes, perhaps thechief limitation to horizontal transmissions will be the small number of cells thatcontribute to the next generation (i.e. the germline). However, horizontal transfersof retrotransposons that occur as rarely as once every few million years can betremendously important, particularly if the acquired element has the potential toamplify up to thousands of copies per nucleus (141, 156). Conclusive evidencefor a recent horizontal transfer ofcopia retrotransposon betweenDrosophilamelanogasterandD. willistoni has been demonstrated (80a). Although, the modeof this horizontal transfer is unknown since thecopiaelement lacks the envelopegene and therefore thought to be not infectious. The circumstantial evidence sug-gests that parasitic mites may be involved in vectoring DNA betweenDrosophilaspecies (80a).

One likely mode for the horizontal transfer of retrotransposons from one plantspecies into another would be by wide crosses. Such crosses can lead to at leasttransient loss of epigenetic control (i.e. suppression) of transposable elements(86, 132). Because many plant species use relatively nonspecific gametic inter-change processes (e.g. wind-assisted or insect-assisted pollination), usually unpro-ductive crosses between different species may be particularly common in plants.Rarely, such a wide cross may give rise to an allopolyploid (102), thereby bringingthe retrotransposon populations from two species into the same resultant species.More commonly in chance matings between two different species, the fertiliza-tion is not productive and/or embryogenesis aborts. In some exceptional cases,




however, the two genomes of very different species (e.g. maize and oats) may co-exist in the same nucleus for a few plant generations (2). Eventually, one parentalset of chromosomes may be eliminated, but the transfer of a retrotransposon fromone genome to another may occur in the interim.

CONTRIBUTIONS OF RETROTRANSPOSONS TO THEEVOLUTION OF PLANT GENES AND GENOMES

Gene Mutation

Retrotransposons are involved in generating mutations through insertions near orwithin genes. Both LTR and non-LTR retrotransposons have generated mutationsin plants: some examples for the Ty1-copia, Ty3-gypsy, LINE, and SINE retro-transposons are listed in Table 2. Many retrotransposon insertions within and inclose proximity to genes affect their expression in a negative fashion by decreasingor abolishing transcription of the gene or by detrimental alterations in transcriptprocessing and/or stability. In other cases, however, insertion of retrotransposonsequences within or near a gene has more complex effects on gene expression, in-cluding alterations of temporal and spatial patterns of transcription or the structureof the resultant protein (48, 97, 202).

The splicing of retrotransposon sequences can sometimes restore proper synthe-sis of a protein (187) or a truncated protein (60, 110). Insertion of a retrotransposoncan also result in gross changes in the prematuration of a transcript. Intron removalby splicing, using a cryptic splice site or sites within the element, will sometimesyield a protein product that differs from the wild type, usually having reducedfunctionality. For example, one of three retrotransposon-mutated maizewaxyalle-les,wxG, has an altered pattern of tissue-specific expression, with thirtyfold moreenzymatic activity in pollen than in the endosperm (110). It was demonstrated thatthe increase in Waxy enzymatic activity in pollen as compared to the endospermwas due to tissue-specific differences in RNA processing (i.e. there is approx-imately 30-fold more correctly spliced RNA in pollen than in the endosperm).Therefore, thewxGallele provides a good example of a retrotransposon insertionthat has resulted in tissue-specific alternative splicing.

Many plant genes currently considered wild type contain legacies of previ-ous retrotransposon insertion (Table 2) (202, 203). For example, over 30 genesfrom both dicotyledenous and monocotyledenous plants were found to have an-cient, degenerate retrotransposon insertions in 5′ or 3′ flanking regions, basedon computer-assisted database searches using Ty1-copia group retrotransposonsas query sequences (202, 203). This is probably a major underestimate, partlybecause elements that have greatly diverged will not be recognized. As databasesgrow and search programs become more sophisticated, most (perhaps all) plantgenes will likely be found to contain some transposable element legacy withintheir regulatory and/or structural components.

Three of four genes in the pearbcSfamily have the same retrotransposon se-quence in the promoter region, suggesting that the insertion predated the gene




duplication events. The combined expression level of three element-associatedrbcSgenes is 30–50% lower than that of the gene that does not contain the retro-transposon insertion, strongly suggesting that the 829-bp insertion sequence har-bors a negative regulator of transcription (203). Thus, it appears that retrotrans-poson insertions have been important contributors to the establishment of novelpatterns of transcription for a wide range of plant genes, over 100 reported sofar. The presence of remnants of retrotransposon sequences within and in closeproximity to genes suggests that these sequences have evolved and have been fixedunder selective pressures for regulatory roles (117).

Compared to the relatively intact retrotransposons found in recent insertion mu-tations or in the intergenic regions between maize genes (56, 65, 76, 158), manyof the retrotransposon sequences found within or close to wild-type genes appearto be truncated or rearranged (202, 203). Older retrotransposon-induced mutationsin maize often contain defective elements (151, 187, 193, 203). The degeneratestructure of these elements is probably due to a combination of their age and theprocess of natural selection. Once inserted near or within a gene, the retrotrans-poson sequences would be selected for a possible positive effect on that gene’sfunction.

Interestingly, the retrotransposons that are found to cause insertional mutationsin plants are usually present in the host genome at relatively low copy numbers.In maize, for example, theBs1retrotransposon is found in one to five copies (78),Hopscotchin two to six copies (203), andMagellanin four to eight copies (151).Similarly, the mutationalTnt1andTto1 families in tobacco andTos17family inrice are also present in relatively low copy numbers [about 1–100 (56, 64, 65, 70)].On the other hand, the abundant maize retrotransposons such asOpie, Grande,Ji, andHuck (each with copy numbers of 10,000 or more) have not been asso-ciated with any characterized maize mutations. Thus, it has been postulated thatthere might be a cause-and-effect relationship between retrotransposon familycopy number and the propensity to insert into genes (158). Very abundant fam-ilies of retrotransposons in maize andBARE-1(about 50,000 copies) in barleyappear to display a target site preference for intergenic regions and for previouslytransposed retrotransposons (158, 174). Alternatively, less fitness selection actingagainst retrotransposons in these regions may be responsible for such distributionpatterns of these high copy retrotransposons. The target site preference and activeselection hypotheses are not mutually exclusive.

Genome Size

Retrotransposons play a major role in determining the size of plant genomes (93).For example, small genomes in plants likeArabidopsis(1C equal to about 130Mbp) might be the consequence of a lack of retrotransposon proliferation (205).On the other hand, large genomes in plants like faba bean and maize (1C equal toabout 128,000 Mbp and 3200 Mbp, respectively) might be the result of success-ful colonization and amplification of retrotransposons (141, 158). Indeed, severaldifferent families of retrotransposons have amplified and attained copy numbers




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greater than 10,000 in the maize genome. The total copy number of all types ofmaize retrotransposons is estimated to be approximately 300,000, thereby compris-ing about 50–80% of the maize nuclear genome (156). An estimated divergencetime between maize and sorghum is about 15–20 million years ago, and bothhave 10 chromosome pairs, but the maize nuclear genome is 3–4 times longer thanthe sorghum genome (3). Molecular hybridization and sequence analysis data haverevealed that these two grass genomes are very similar in the content and order oftheir genes (6, 73, 122). However, the nearly two dozen retrotransposons that makeup over 70% of the region surrounding the maizeadh1gene are all absent fromthe orthologousadh region in sorghum (4, 156) and most are not detected in thesorghum genome by gel blot hybridization (9). Hence, these results indicate thatthe increase in maize genome size was largely due to an extensive proliferationof retrotransposons. This result also suggests that these elements inserted afterthe divergence of maize and sorghum, a suggestion that was confirmed by datingexperiments that indicated these retrotransposons had all arrived and/or amplifiedin the maize genome within the last 2 to 6 million years (157).

It is possible that analogous patterns of retrotransposon distribution and orga-nization will be found in the genomes of other plant and animal species that havehigh retrotransposon copy numbers. Genomic sequencing in large genome eukary-otes continues to be limited primarily to gene-rich regions, so we do not know thearrangement of DNAs in the largest genomic component of any large genome.However, preliminary sequence analysis of the DNA adjacent toArabidopsiscen-tromeres has uncovered regions rich in LTR retrotransposons, arranged in a nestedorganization like that observed in maize (R Martienssen, R McCombie & R Wilson,personal communication, http://www. cshl.org/protarab).

Although retrotransposons have played a major role in the expansion of plantgenomes (8, 93), it is also likely that many retrotransposon sequences have beeneliminated by various mechanisms, such as unequal recombination between theLTRs of an LTR retrotransposon (to generate solo LTRs) (25, 158, 165, 191) orother types of deletion (145). Nonetheless, the dispersed organization of retro-transposons makes it unlikely that known genomic elimination processes suchas large deletions can effectively remove a significant amount of the genomicsequences within a relatively short evolutionary time period without having dele-terious affects on the survival of the host by concurrent elimination of the genesthat are interspersed with retrotransposons (8, 95).

Genome Rearrangement

Retrotransposons can rearrange genomes with or without transpositional compe-tence. At least one retrotransposon,Bs1, has acquired a portion of another gene, andthen amplified it by transposition throughout the maize genome (18, 77, 78). Thisis highly significant, as it shows how genes or parts of genes might be amplifiedand dispersed in genomes. Similarly, the rare action of retrotransposon reversetranscription and integration functions on normal cellular mRNAs has created




intronless pseudogenes in plants (37, 106). These amplified gene sequences canserve as the raw material for the evolution of new genes.

Because of their high copy numbers and dispersal throughout plant genomes,retrotransposons could serve as sites of unequal or ectopic recombination. Thelegacies of such unequal recombinations between the LTRs of a single retro-transposon have been noted many times in plants by the existence of solo LTRs(25, 158, 165, 191). Even in methylated, presumably heterochromatic regionswhere these elements often accumulate, rare solo LTR are observed (158). Ectopicrecombination between retrotransposons of the same family that are in direct ori-entation on the same chromosome can cause reciprocal duplications and deletions,whereas unequal recombinations between elements on the same chromosome thatare in opposite orientation will cause a chromosomal inversion of the sequencesbetween the two retrotransposons. Ectopic recombination between two elementson different chromosomes can cause reciprocal translocations. These types ofretrotransposon-mediated genome rearrangement have been observed in yeast andDrosophilaas products of unequal recombination (104, 204). In plants, however,the structural and mechanistic foundations of such chromosomal rearrangementshave not yet been studied in great numbers, so we do not yet know how frequentlythey can be attributed to retrotransposon involvement.

Roles for Retrotransposons?

In most eukaryotic organisms, centromeres, telomeres, and other heterochromaticregions of chromosomes contain large numbers of retrotransposons, making itpossible that retrotransposons have acquired some structural and/or functionalroles in these heterochromatic regions in plants. Indeed, a role ofHeT-A andTART retrotransposons in buffering the shortening of chromosome ends has beenclearly demonstrated inDrosophila(137).

The exceptional conservation of a particular family of retrotransposons in allgrass centromeric regions suggests that these elements are involved in centromerefunction (121, 150). Similarly, the presence of retrotransposon fragments withinthe regulatory regions of genes indicates that they have evolved to a form that isinvolved in specific gene regulation. Because LTR retrotransposons, for instance,carry a promoter within each LTR, it is not surprising that their insertions shouldsometimes cause new patterns of gene expression or at least have the potential toevolve into new patterns of gene expression (48, 202). It was partly this capacity ofmobile DNAs to serve as controlling elements that caused McClintock to believethat their major role in evolution was to rearrange genomes under stresses so severethat only a rare individual with a totally changed genome might survive (116).Although this model does not appear to make evolutionary sense, it is certain thatthe presence of transposable elements within a genome will allow them to be usedby their hosts in whatever way might be beneficial, even if the elements themselvesare present primarily for selfish or parasitic reasons (34, 134). Future studies willindicate how plant genomes have evolved to utilize retrotransposon sequences.




RETROTRANSPOSONS AS GENETIC TOOLS

Molecular Markers for Phylogenetic, Biodiversity,and Genetic Linkage Analyses

There are several advantages in using retrotransposon sequences as molecularmarkers. They are ubiquitous, present in high copy numbers as highly heteroge-neous populations, are widely dispersed on chromosomes, and show insertionalpolymorphism both within and between species in plants (95). Furthermore, activeretrotransposons will produce new insertions in the genome, leading to polymor-phism. The new insertions may then be detected and used to temporally orderinsertion events in a lineage, helping to establish phylogenies. These genetic prop-erties have recently allowed retrotransposons to be exploited as DNA markersto study biodiversity in maize, pea, and barley (41, 81, 152) and to generate ge-netic linkage maps in barley and pea (41, 95, 198, 199, 202). Furthermore, becausemany retrotransposons are widely distributed within the euchromatin domains ofchromosomes, it should be possible to generate markers linked to agronomicallyimportant genes. Indeed, retrotransposonTnd-1 has provided a marker that islinked to black root resistance in tobacco (85).

One of the most useful retrotransposon-based marker systems in revealing largenumbers of highly polymorphic markers is sequence-specific amplification poly-morphism (SSAP). SSAP is a multiplex amplified fragment length polymorphisms(AFLP)-like technique that displays individual retrotransposon insertion as bandson a sequencing gel. Moreover, SSAP-based markers appear to be better for esti-mating phylogenetic relationship in plants compared with the conventional AFLP-based makers, because one of the primers is based on specific retrotransposonsequences (41). Furthermore, a multi-retrotransposon approach has been recentlyused to estimate phylogenetic relationships within species and between speciesin legume (143) and cereal (57) plants. This approach is particularly informativebecause each element has a unique transpositional history. For example, retroele-ments that have transposed in the recent past should be extremely polymorphicwithin a species, and these can be used for genetic linkage and intraspecific geneticdiversity studies. Some that were active only a million years ago or more shouldbe useful for determining relationships between species or even between genera.

In the past, a major hurdle in using the retrotransposon-based SSAP method wasthe limited numbers of LTR sequences available. The recent development of a newtechnique for rapid isolation of plant Ty1-copia retrotransposon LTR sequencesfor molecular studies (143) has made the application of a retrotransposon-basedSSAP method highly accessible to all plant species. The SSAP method should alsobe applicable for determining the integration patterns of specific elements in thehost genome by subcloning, sequencing, and analyzing SSAP bands.

A PCR-based approach to detect individual retrotransposon insertions has beendeveloped using primers derived from the element and its flanking DNA (47, 51).This method produces a dominant marker system, where the different allelic states




(i.e. presence and absence of the retrotransposon insertion) at a locus can be re-vealed. A major advantage of this retrotransposon-based insertion polymorphism(RBIP) method is that it could be fully automated for high throughput markeranalysis (47). Sequence analysis of clones from an enriched barley microsatellitelibrary has revealed that retrotransposons sequences are intimately associated withmicrosatellites (153). This property has been exploited to develop a molecularmarker method, REMAP (Retrotransposon-Microsatellite Amplified Polymorp-phism), that can be used to distinguish between barley varieties and to producefingerprint patterns for species across a genus (81).

Non-LTR retrotransposons also have been used as molecular markers to studygenetic diversity at the species and genus levels (63, 126). For example, ricep-SINE1elements have been used to demonstrate thatpSINE-1-r2transposed tothewx locus after the divergence ofOryza rufipogonandO. sativafrom theOryzaancestor that carried the AA genome. In contrast,pSINE-1-r1transposed at anearlier time to another position in thewx locus before the divergence of variousrice species (63). Also,BrassicaSINE elements have been used to study geneticdiversity inCruciferaespecies and to determine the extent to which introgressionhas occurred between cultivated and wild species inBrassica(103; JM Deragon,unpublished data).

Gene Tagging and Functional Analysis of Genes

Some retrotransposons have features that make them ideal genetic tools for genetagging in plants (65). These include (a) retrotransposon-mediated insertion muta-tions that are stable because they transpose by a replicative mode, (b) transpositiontarget sites that are unlinked to the site of the original copy, making it relativelyeasy to generate a large collection of random insertions for saturation mutagenesis,(c) transposition that can be regulated by abiotic and biotic stress conditions,(d) high mutagenicity due to preferential transposition into genes, (e) low copynumbers, thereby facilitating the identification of the retrotransposon insertion re-sponsible for a specific mutation, and (f ) endogenous retrotransposons that areubiquitous in plants and may be active in many species. In rice, the endogenousTos17retrotransposon meets all the conditions mentioned above for its use in tag-ging genes.Tos17is highly active under tissue culture conditions and preferentiallyinserts into or near genes (65). It has been used to generate a large collection ofrandom insertions for saturation mutagenesis and has been used to clone a numberof genes in rice (65; H Hirochika, unpublished data). Additionally,Tos17has beenused in a reverse genetic approach to identify null mutant lines for theOSH15gene(Oryza sativahomeobox; aknotted-type homeobox gene), thereby demonstratingthatOSH15has a role in rice internode development (159).

Evaluation of Somaclonal Variations

Several techniques for genetic manipulation and vegetative propagation of plantsare dependent upon in vitro plant regeneration systems. However, in vitro




manipulation processes are prone to somaclonal variations, which may be highlyundesirable (70). Somaclonal variation is the collective term given to the highfrequency of mutations encountered among plants regenerated from cell and tis-sue cultures under in vitro conditions. There is now good evidence to show thata significant percentage of mutations among in vitro manipulated plants are dueto the induced activation of retrotransposons during cell and tissue culture condi-tions. For example, over 10% of regenerated rice plants possessed retrotransposon-induced mutations (70; H Hirochika, unpublished data). The retrotransposon-based molecular markers described earlier provide an excellent tool for evaluatingretrotransposon-induced mutations among in vitro manipulated plants. Addition-ally, a better understanding of the regulation of expression and transposition ofretrotransposons should give some clues to avoiding somaclonal variation amongin vitro manipulated plants.

CONCLUSIONS AND FUTURE PROSPECTS

We now know that plant retrotransposons are responsible not only for generatingmutations in plant genomes but also for significantly increasing the size of manyplant genomes. Their replicative modes of transposition have equipped them withthe ability to rapidly proliferate within their hosts, allowing them to become amajor component of higher plant genomes. Retrotransposons are also one of themost fluid of genomic components, varying greatly in copy number, genomiclocalization, and sequence structure over relatively short evolutionary times. Theseproperties make retrotransposons one of the major forces influencing the structureof plant genomes. Although their initial and major continuing motivation appearsto be selfish and/or parasitic, retrotransposons will continue to be used wheneverpossible by their hosts.

Mutations caused by retrotransposon insertions within or in close proximity togenes can result in gene inactivation or alterations of the expression patterns ofthe genes, or the structure of the encoded proteins. Many gene promoters con-tain fragments of retrotransposons that now contribute to that gene’s regulation.Ectopic recombination between these mobile sites of homology could amplify ordecrease genome size and gene family number. However, it remains to be provenwhether any retrotransposon-induced changes have been positively selected innatural populations.

For the future, many questions remain to be answered. These can be conve-niently separated into four categories: origins, specificities, effects, and uses.There is no definitive information on the origin of any plant retrotransposon fam-ily. Are some LTR retrotransposons actually defective insect retroviruses that havearrived in plant genomes via horizontal transfer? Or, do horizontal transfers ofintracellular elements occur through wide crosses or by rare incorporation of se-quences from a pathogen or parasite? Perhaps horizontal transfers do not happen atall, or at least do not account for many of the elements. Their differential presenceand/or copy numbers may be determined by whether they have amplified or have




been lost in one species or another. At a more detailed level, how do retroelementswithin a family change; how fast and by what mechanisms? These will be diffi-cult questions to answer, partly because their great diversity, high copy numbers,unpredictable amplification potential, and rapid evolutionary change can easilyobscure simple patterns of element relatedness or origin. However, these prob-lems will be partly offset by the huge amounts of genome sequence data that willbecome available in upcoming years.

Numerous studies have documented specificities for retrotransposon expres-sion and insertion. Several groups have begun characterizing the regulation ofLTR promoters by transacting factors associated with host developmental andstress response pathways. Studies of epigenetic gene regulation, including thosepertinent to mobile DNAs and transgenes, also will expand our understanding ofretrotransposon biology. We have not yet identified any factors in plants that mightdetermine the different insertion specificities of different retrotransposon families.We also do not know the degree to which a retrotransposon can use transacting fac-tors from other retroelements for replication and insertion. What is the relationshipbetween an element’s transcription and its potential for transposition?

The effects of retrotransposons on gene and genome structure, including genomesize, have been well documented. However, how often do retrotransposons am-plify to high copy numbers? What are the processes that remove retrotransposonsfrom a genome, and how rapidly do they act? How often do retrotransposonsinsert into or near genes in standard plant populations, and how often/rapidly dothese sequences evolve into a form that is utilized by the affected gene? Howoften have retrotransposons been used for non-genic tasks, such as contributinga functional component to centromeres? If retrotransposons are used for variousroles in genome structure or function, how did the elements change to permit thisuse, and how does their use differ between species?

Finally, we are just beginning to see some of the ways that plant retrotransposonscan be used for the study and improvement of plants. Their outstanding potentialas genetic tools for plant genome analysis, linkage mapping, phylogeny and ge-netic biodiversity studies, gene tagging, functional analysis of genes, evaluationof somaclonal variants, and in gene transfer processes appear to be most promis-ing. Understanding the possible horizontal transfer of retrotransposons could alsobe very valuable, perhaps to set an upper limit for the rate at which transgenesmight escape from genetically engineered crops. Despite comprising the majorityof many plant genomes and their many roles in the mutation and evolution of genesand genomes, only a handful of laboratories investigate these elements. Much fu-ture work is needed, and the tools are all in hand, so we can expect continuing greatgains in our understanding of plant retrotransposons for the foreseeable future.

ACKNOWLEDGMENTS

We thank all of our colleagues for providing their published and unpublishedpapers, especially Michael Ashburner, Andreas Bachmir, Eugene Berezikov, BenBowen, Jean-Marc Deragon, Tom Eickbush, Noel Ellis, David Finnegan, Andy




Flavell, Marie-Angele Grandbastien, Pat Heslop-Harrison, Hirohiko Hirochika,Howard Laten, Rob Martienssen, John McDonald, Norihiro Okada, Steve Pearce,Ingo Schubert, Alan Schulman, Daniel Voytas, and Susan Wessler. We are gratefulto Sue and Hiro for their comments on the manuscript, and to Pat and Ingo forthe LINE, SINE, andgypsypictures, respectively. AK acknowledges the ScottishOffice Agricultural, Environmental and Food Department and EC BiotechnologyFramework IV for financial support. JLB acknowledges financial support from theUSDA CSREES program (97-35300-4594).

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