REVIEW
RETROTRANSPOSONS AND THEIR ROLE IN PLANT - GENOME
EVOLUTION
E. Todorovska
AgroBioInstitute, Sofia, Bulgaria
Correspondence to: Elena Todorovska
E-mail: [email protected]
ABSTRACT
A large amount of description data now exists for wide variety retrotransposons in higher plants, but much less is known for
molecular basis of the mechanisms of trancriptional activation and the frequency of their transposition activation during plant
species evolution. Because the transposition is a replicative cycle which can eventually produce many copies comprising a large
fraction of the plant genome this obviously has profound effects on the evolution of the structure of retrotransposon populations
and the overall genome structure of each species.
This review provides an insight on the structure, genomic organization, mechanisms involved in regulation of the expression,
transposition, and evolution of the most widely distributed LTR retrotransposons in plants and especially within the Triticeae
tribe, and discusses both their contributions to plant genome evolution and their use as genetic tools in plant biology.
Key words: Retrotransposons, Plants, Triticeae, Expression,
Tissue culture, Retrotransposon-based markers
Introduction
Retrotransposons were first characterized in animal and yeast
genomes, but evidence has accumulated in recent years to
show that they are present in high copy number in many plant
genomes and can constitute a very large part of some of them,
especially those with large genomes. As one of the most fluids
of genomic components, varying greatly in copy number over
relatively short evolutionary timescale they represent one of
the most important factors affecting the structural evolution
of plant genomes, especially those of the higher plants. The
basic parameters for these mobile elements varying during the
evolution of higher plant genomes are the population structure
(how many copies of which kinds of retrotransposons are
present) and transpositional activity (what proportion of which
transposons are active and what are their transposition rates).
Retroelements classification and regulation of
retrotransposition
Retrotransposons are one of the two major groups of
transposable elements detected both in animal and plant
genomes at the last decade and defined according to their mode
of propagation. They differ from other transposons (class II
elements that use DNA in movement such as Ac and En / Spm)
by their ability to transpose via an RNA intermediate (also
termed class I elements).
Plant retrotransposons are separated in two major subclasses
that differ in their structure and transposition cycle:
1. LTR retrotransposons (Ty1-copia group also known
as Pseudoviridae and gypsy group - Metaviridae) –
retrotransposons having long terminal repeats (LTRs)
and encoding products with structural homology to the
retroviral gag- and pol-encoded proteins (Fig. 1).
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2. non-LTR retrotransposons (LINE and SINE elements)
(9, 24, 44) – retrotransposons also encoding gag- and
pol-like proteins but having no long terminal repeats.
Both these subclasses derived from a reverse transcription
of an RNA template by forming a DNA daughter copy and their
replication cycle involves an intermediate cytoplasmic step
as it was mentioned for LTR retrotransposons and vertebrate
retroviruses forming the retrovirus-related transposon family
in animals. This replicative transposition mechanism within
host genomes means that retrotransposons are potentially very
invasive. To ensure the viability of their host, and hence their
own survival, retrotransposition is tightly controlled. This
control involves element-encoded functions and host factors.
One of the most important control steps is transcription, which
determines both the production of the RNA template and the
synthesis of mRNA required for protein synthesis.
In LTR retrotransposons, transcriptional control elements
involves cis – regulatory sequences that are usually found in
elements LTR (5’LTR), in particular the U3 region located
upstream of the transcription start site, or in downstream,
untranslated sequences (27), whereas 3’LTR provides
terminator and polyadenilation signals. The collected till now
data of genome sequencing efforts showed that the LTRs of
cereal retrotransposons can vary in length from few 100bp Tos17 (28) to over 5 kb – Sukkula (79), Grande (23). They have
at their termini small inverted repeats, which give the LTRs a
universal 5’TG….CA3’ structure. Immediately internal to the
5’ and 3’LTRs are the priming sites for reverse transcription
for generating the (-) and (+) strands respectively. An internal
domain specifying one or more open reading frames lies in
between the primer binding sites. Fig. 1 describes the structure
of the most abundant LTR retrotransposons in cereals - Ty1copia and gypsy. In copia-like elements the internal domain
encodes in sense orientation a capsid protein (GAG), aspartic
proteinase (AP) which cleaves the expressed polyprotein
into a functional components, integrase (IN) which carries
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out the insertion of the cDNA into the plant genome, reverse
transcriptase (RT) which is responsible for the creation of cDNA
copies, and RNase H (RH) which is important for replication.
In gypsy-like elements in contradiction to copia-like elements,
the integrase (IN) is at the end of the open reading frame.
Fig. 1. Organization of the two major classes of LTR retrotransposons.
LTR retrotransposons and closely related infectious and
endogenous retroviruses (97) have similar intracellular life
cycles and major encoded proteins (gag, which codes proteins for
virus particles, and pol, which encodes the enzymatic activities
for replication) (54), but retrotransposons lack a domain env,
encoding an envelope glycoprotein critical for infectivity. The
encoded by retroviruses envelope glycoproteins associate with
cell membranes and facilitate the budding of viral core particles
from infected cells. In addition, they also mediate infection by
recognizing cellular receptors (14). Recently, an env domain
encoding a putative envelope protein which is generally
considered to be a predictor of a retroelement’s infectious
nature (67) was found in few copia-like elements (SIRE-1)
and some plant gypsy-like retrotransposons (Athila, Cyclops,
Calypso, Bagy-2, Rigy-2) (13, 40, 49, 67, 89, 91, 99).
Interestingly, there are no reports on the presence of
env-like sequences in the copia-like retrotransposons in D.
melanogaster or any other invertebrate or vertebrate (16).
Therefore, the presence of env-like sequences in both copia
and gypsy groups suggests that the env gene was acquired
by these two groups of retrotransposons independently (46).
Alternatively, closely related relatives retroviral derivatives
of copia and gypsy retrotransposons invaded the genome of
plants and subsequently lost their env gene (47). Currently, it
is unknown which process proceeded first. Nevertheless, the
existence of plant retroviruses supports the hypothesis for an
apparent horizontal transfer of plant viruses in plants (1, 67).
Distribution
Representatives of all types of retrotransposons have been
detected in plant genomes (7, 44) (Table 1). Their ubiquity
in the plant kingdom suggests that they are of very ancient
origin (8).
Among them the LTR-retrotransposons appear to be widespread in plants (18, 30, 93), and represent the most abundant
class of transposable elements. Both major groups of LTR
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retrotransposons (Ty1-copia and gypsy groups) have been
found in a variety of angiosperms (flowering plants).
Until now the Ty1-copia group is the best-characterized
LTR retrotransposon group in plants (18, 43, 48, 76, 83, 93). In
at least some plant species Ty1-copia group retrotransposons
are so numerous that they comprise major fractions of the
genome (64, 65, 74). Highly heterogenous populations of Ty1copia group retrotransposons are found in many higher plant
genomes (18, 28, 42, 93). Phylogenetic comparisons among
these transposons, both within and between species, have been
carried out for dicot plant species and these suggest that Ty1copia group retrotransposons already existed early in plant
evolution and diverged into heterogeneous subgroups before
modern plant orders arose. The descendants of these subgroups
have been transmitted vertically to descendant species, with
horizontal transfer between species being either very rare
or non-existed (28). In monocots similarly complex sets of
interrelated Ty1-copia group retrotransposons were found (32,
57, 58, 59, 66, 74), some of which span species boundaries
(60, 66). All studies to date confirm that the amplification and
dispersion of Ty1-copia group retrotransposons as well as other
transposon groups play an important role in plant-genome
evolution (20, 28, 45).
The second large LTR retrotransposon group, gypsy–
like elements originally described in the artropods and few
taxonomically skattered plants was found to be widespread in
cereals and the plant kingdom (84). Vershinin and colaborators
(87) have found 4 families of gypsy– like elements based on
the cloned RT domains. Estimating number of hybridizing
representatives of the obtained four gypsy– like families to
gridded and blotted BAC clones it has been found that they
constitute ~3% of the barley genome.
Are retrotransposons active?
At the last decade transcriptional activity has been reported
for only few elements, mostly those belonging to the LTR
retrotransposon subclass. Most of these have been isolated
after transposition into or next to a host gene. Most of the
mobile elements have been characterized by PCR amplification
of genomic DNA or cDNA but were subsequently shown
to transpose. Evidence for retrotransposition activity have
been reported from analysis of LTR sequences which are
identical in newly transposed copies as it have been observed
for the Osser elements also in the green alga Volvox carterii.
Specific transcripts, starting in the element’s LTR have been
demonstrated for tobacco- Tnt1A and Tto1, barley- BARE1, rice- Tos17 and maize- Huck retrotransposons. Definitive
evidence of LTR transcriptional ability have been further
confirmed for Tnt1A, BARE-1 and Tto1 by using constructs
in which reporter genes were placed under the control of
element’s LTR.
Regulation of the activity of retrotransposons
The expression of retrotransposons in animal and yeast is under
the control of hormonal and developmental factors. A general
picture for the regulation of the expression of retrotransposons
in plants is not yet fully established because of the absence of
comparative studies in different plant tissues. Transcription of
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most of the active plant elements characterized to date is largely
quiescent during normal development but can be induced by
biotic and/or abiotic stresses, including cell culture, wounding,
and pathogen attack (27, 95).
A. Developmental regulation
The developmental regulation of the expression have been
showed for Tnt1, which is only expressed in roots at very low
levels (69); for Tto1, Tos10 and Tos17, which are not expressed
in leaf tissues and for mobile B5, Hopscotch, Stonor and
Magelan elements, which are not expressed in plant tissues.
Expression of the maize Prem-2 element has been detected
only in early microspores. The expression of maize Opie,
Huck and Cinful retroelements and barley BARE-1 has been
observed in leaf tissues.
B. Activation of retrotransposons by stress factors
Retrotransposons as other transposable elements are the
major source of genetic variation that can ranges from gross
chromosomal alterations up to very fine tuning of the expression
of cellular genes (7). Evaluation the potential of activating
retrotransposons through plant tissue culture to generate new
insertion mutations into the genome is of a great importance
for improving the present-day plant species. A common feature
of the most retrotransposons is their activation by stress and
environmental factors. The most well characterized plant
LTR-retrotransposons (Ty1-copia subgroup) are particularly
affected by protoplast isolation or in vitro cell or tissue culture
(reviewed by 27) (Table 1).
From Table 1 it is obvious that retrotransposons have been
major contributors to the structure of plant genomes but the
extent to which these sequences are transcriptionally active
in present-day species is not yet clear. Sequence analysis
has shown that a large proportion of plant Ty1-copia group
retrotransposons contain damaged reading frames, indicative
of defective elements (18, 19, 30, 93). However a number of
plant Ty1-copia group retrotransposons are transcribed in their
host species (32, 66, 69, 81). Several maize elements have
transposed at least once in the recent evolutionary past (70, 96)
and several tobacco and rice elements transposed in cultured
cells or protoplasts (25, 31, 32). Lastly, high levels of insertional
polymorphism within species for BARE-1 of barley and PDR1
of pea suggest strongly that they have been transposing at
least very recently on an evolutionary scale time (17, 37, 94).
An insertion of retrotransposons into coding sequences after
protoplast or cell tissue tobacco and rice cultures (25, 32) has
been observed, indicating that they might take a significant
contribution to somaclonal variation.
The fact that some retrotransposons can be activated in their
host species after protoplast isolation as well as cell and callus
culture leads to the question as to whether their expression is
linked to the activation of cell division programmes or to the
activation of stress responses or to both. Protoplast isolation and
cell or callus culture are the major inductors of modifications
of the cell metabolism and gene expression. In the leaf-derived
protoplasts, the former metabolic activity of the leaf cell is
replaced by a new programme which is characterized by
activation of growth- and stress-related genes (defense genes,
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which are activated after pathogen attack). Growth-related
genes probably involved in the re-initiation of cell division and
the activation of stress-related genes might be a consequence of
the original wounding. Protoplast isolation included enzymatic
degradation of cell wall from pathogenic compounds present in
the extracts of phytopathogenic fungi. The activation of stressrelated genes as a consequence of protoplast isolation might
be also a result from cell wall hydrolysis or from pathogenic
compounds of fungal extracts. Growth- and defense-related
genes are also expressed in callus and cell cultures, suggesting
that callus tissue culture programmes are similar to those
induced after wounding and during callus formation in the
plant, involving both a stress response and cell division. To
the present a partial answer has been provided from the studies
on the expression of the tobacco Tnt1A and Tto1 and rice Tos
17 retrotransposons. Tnt1A protoplast-specific expression
results mostly from the effect of fungal extracts, and the Tnt1A
promoter is also activated by the other compounds of microbial
origin, salicylic acid, wounding and viral, bacterial or fungal
attacks (26). Similarly, expression of Tto1 retrotransposon is
inducing by viral attacks, salicylic acid or jasmonate. This
suggests that the expression of the two best-characterized plant
retrotransposons (Tnt1A and Tto1) is induced by different biotic
and abiotic factors that can elicit plant deffence responses. The
expression of rice Tos 17 retrotransposon in cell culture is not
enhanced by protoplast isolation in comparison to tobacco
Tnt1A and Tto1 retrotransposons, suggesting that the control of
Tos 17 differs from that of Tnt1A and Tto1. Therefore, common
keys of mechanism of the activation of retrotransposons might
to be further elucidated. Several studies on retrotransposon’s
transcriptional control have revealed the structure and the
function of promoter regions in Tnt1A, Tto1 and BARE-1
elements. Tandemly repeated cis- regulatory sequences were
identified in the U3 region of Tnt1A and Tto. A 31 bp repeat motif,
the BII box, present in three or four copies in transcriptionally
active elements is involved in Tnt1A activation by protoplast
isolation and fungal elicitins (12, 26). A 13 bp repeated motif
is involved in Tto1 expression in callus culture after wounding
or jasmonate application. Putative regulatory motifs have been
also detected in U3 regions of Tos 17 (32) and BARE-1 (80)
but the specificity of the expression of BARE-1 in leaves or
calli involves the alternative use of different LTR promoters.
Tnt1A and Tto1 repeated cis-acting motifs share similarities
with H-box - the motif involved in the activation of several
plant growth- and stress-related genes (e.g. defense genes). In
addition the Tnt1A U3 region contains also sequences highly
similar to regulatory motifs of stress- induced plant genes. These
similarities provide a plausible explanation for the molecular
basis of Tnt1A and Tto1 activation by stress and pathogen
attacks. A MYB-related factor, involved in Tto1 transcriptional
activation in protoplast have been characterized by its specific
binding to the 13 bp repeated motif but its expression was
not detected in suspension cell cultures, indicating that other
factors are probably involved in Tto1 activation in these
conditions. These results are an illustration for the complexity
of retrotransposon’s transcriptional regulation, which probably
included different cis-regulatory elements each capable to
respond to different stimulus.
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TABLE 1
Plant LTR - retrotransposons - evidence for active or potentially active retrotransposons in some plant species anad especially
in Gramineae
Classification
Species
Osser
BARE -1
Wis - 2
B5
G
Hopscotch
Ji
Opie
Stonor
PREM-2
SIRE-1
Tos 10
Volvox carterii
Barley
Wheat
Maize
Maize
Maize
Maize
Maize
Maize
Maize
Soybean
Rice
Tos 17
Rice
Evidence for recent mobility
Subclass I: LTR retrotransposons
Superfamily Ty1-copia
Copy with identical LTRs
None
Polymorphism in regenerated plants
Transposition into the waxy gene
Transposition into the waxy gene
Transposition into the waxy gene
None
None
Transposition into the waxy gene
None
None
Copy number increase in cell cultures
Copy number increase and active transposition into
coding sequences of several genes in cell and tissue
cultures
Small increase in copy number in cell cultures
None
None
None
None
Rice
Potato
Potato
Potato
Potato
Nicotiana
Transposition into the nia gene in protoplast cultures
Tnp 2 / Tnt 1B
plumbagi-nifolia
Transposition into the nia gene in protoplast
Tobacco
Tnt 1A
cultures; small increase in copy number in cell
cultures
Copy number increase in cell and tissue cultures;
Tobacco
Tto 1
transposition into the nia gene in protoplast cultures
Tobacco
Small increase in copy number in cell cultures
Tto 2
Tobacco
None
Tto 3
Tobacco
None
Tto 5
Superfamily Ty3 - gypsy
Maize
None
Cinful
Maize
None
Huck
Maize
Transcription into the waxy gene
Magellan
Maize
Transposition near the zeinA gene in somatic tissues
Zeon -1
Tomato
None
TCI - 4
Superfamily Ty3 – gypsy-env
Barley
None
Bagy-2
Atypical or not yet classified
Maize
Transposition into the Adh gene after viral infection
Bs -1
Maize
None
PREM -1
Subclass I: Non - LTR retrotransposons
Superfamily SINEs
Rapeseed
None
S -1
Tobacco
None
TS
Tos 19
Prt 1 and Prt 3
Prt 4
Prt 5
Prt 6
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Evidence for transcripts
None
In leaves and callus
In protoplasts
None
None
None
In roots, leaves and tassels
In roots, leaves and tassels
None
In early microspores
In seedlings and leaf tissues
In cell cultures
In cell cultures
In cell cultures
In protoplast
In protoplast
In protoplast
In protoplast
None
In roots, in protoplast, and after
wounding and pathogen attacks
In protoplasts, cell and tissue cultures
and after wounding and viral attack
In protoplast
In protoplast
After viral attack
In leaves
In roots, leaves and tassels
None
In endosperm
In seeds
In leaves, cell culture, pollen, embryo
None
In early microspores
In shoots, roots and callus
By in vitro transcription
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The variability of U3 sequence correlates also with
differences in the pattern of expression of the detected Tnt1A,
Tnt1B Tnt1C subfamilies of the Tnt1 retrotransposon in the
genomes of most Nicotiana species (5). Each of the three Tnt1
subfamilies is induced by stress, but their promoters have a
different response to different stress-associated signaling
molecules. While the Tnt1A subfamily is particularly strongly
induced by elicitors and methyl jasmonate, the expression of
the Tnt1C subfamily is more sensitive to salicylic acid and
auxins. The direct relationship between U3 sequence variability
and differences in the stress-associated expression of the Tnt1
elements present in a single host species gives support to the
postulated by the authors that retrotransposons have adapted to
their host genomes through the evolution of highly regulated
promoters that mimic those of the stress-induced plant genes.
The analysis of the transcriptional control of a retrotransposon
population such as Tnt1 provides also new insights into the study
of the complex and still poorly understood network of defenseand stress-induced plant signal transduction pathways.
Transcriptionally active Ty1-copia LTR-retrotransposons
have bee also found in oat by RT-PCR of the reverse
transcriptase domain (41). Sequence analysis of the RT-PCR
clones suggested that oat LTR-retrotransposons consist of at
least seven groups, designated as Oatrt1 to Oatrt7. The sequence
analysis of the full length copy of Oatrt1 (OARE-1) isolated
from an oat genomic library, showed that it is a 8,665 bp long
member of the BARE-1 subgroup. The expression of OARE-1
is intensively induced by wounding, UV light, jasmonic acid
and salicylic acid, and its pattern is very similar to that of the
PAL (phenylalanin ammonia lyase) gene. Furthermore, OARE1 is highly activated by infection with an incompatible race
of the crown rust fungus, Puccinia coronata suggesting that
OARE-1 is highly sensitive to various abiotic and biotic stimuli
leading to plant defense responses.
Kalendar et al. (38) note a remarkable connection between
the presence within the BARE-1 promoter, of ABA (abscissic
acid)-response elements, found in water-stress induced genes
and BARE-1 copy number variation, suggesting that BARE-1
proliferation in wild barley populations may be stress-induced.
Several hypotheses have been proposed to explain the intriguing
feature of plant retrotransposon containing regulatory motifs
similar to those of cellular genes as it has been observed for
many animal and yeast retroviral-like elements.
• Ancestral captures of cell sequences by mechanisms
similar to retroviral transduction;
• Retrotransposon-mediated transfers of regulatory
sequences to cellular genes: data have accumulated on
the presence of transposable elements in the regulatory
regions of genes (96) and on their involvement in
changes in gene regulation, and it has been proposed that
new regulatory features could be acquired by cellular
genes after nearby transposable element insertions
and subsequent disappearance of the insertion through
random drift;
• Appearance of retrotransposon regulatory elements by
convergent evolution: a good explanation of this is the
higher U3 plasticity of Tnt1 family. Several regulatory
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motifs associated with a diversity of defense genes were
found in the Tnt1A subfamily.
None of the above mentioned hypotheses is completely
satisfactory but the possibility that these and other mechanisms
might have combined with those of their host during
retrotransposon co-evolution, creating present situation, cannot
be excluded.
Further characterization of LTR evolutionary features of
other retrotransposon families would provide information on
the origin and fate of retrotransposon regulatory sequences.
One of the main questions is the putative biological
impact of retrotransposon activation. Together with a role
in diversification of genetic material there is a proposition
that retrotransposon activation is one of the key factors
involved in the host adaptation to enviromental changes. The
retrotransposons can be inserted in the regulatory sequences of
plant cell genes thus changing their expression. As one of the
main modificators of gene expression regulation transposable
elements are proposed to be a major factor in macroevolution.
All mentioned above features of regulatory elements of the
most-well characterized plant retrotransposons indicate that
they represent sensitive markers of plant stress and could be
used for different biotechnological applications (27). Their
promoters can be used to drive transgene expression in strategies
directed towards development of plant resistance to pathogens,
and might also be interesting tool for studying plant defense
response. In addition, the fusion of LTR regions containing
promoter elements responsible for active transposition of
retrotransposons to reporter gene(s) could provide sensitive
indicators of plant response to environmental stress or to
agrochemicals and pollutants. Since retrotransposons can
remain active and stable inherited after insertion into different
regions of the host genome, it would be interesting to
determine whether their expression is less sensitive to the local
genomic enviroment than transgenes driven by cellular gene
promoters.
Characterization of representatives of Ty1-copia subgroup
retrotransposons
Several Ty1-copia subgroup retrotransposons have been well
characterized at the last decade in different plant species and
especially in Triticeae.
The BARE-1 retrotransposons is a transcribed 8.9 kb in
size, Ty-1 copia-like retroelement with LTRs of 1.829 kb, long
untranslated leader of 2kb and well-conserved coding domains
for GAG, AP, IN, RT and RH, and an active promoter (82,
83). The BARE-1 family is present on average in 14,000 fulllength copies dispersed on all chromosomes that constitutes
nearly 3% of the total size of barley genome (80, 81, 82, 88).
Solo LTRs of BARE–1 account for at least ~ 64,000 additional
elements in the genome and the genomes of Hordeum species
have 7-42 fold more BARE–1 LTRs than full length copies of
this retrotransposon. Both, full length and solo LTRs BARE–1
consitute about 5-10% of barley genome depending of the
accession analysed and contributes not only to size variation
over the entire genome but to local genome diversification and
size differences on a local scale (38, 88). BARE-1 elements are
BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/3
transcribed in barley tissues from promoters within the LTRs.
The BARE-1 LTR contains two promoters both of which are
active in vivo. For the downstream TATA box, both positive and
negative regulatory regions for transient expression in barley
protoplast were uncovered (80). The upstream segment that
contributes much more to expression is GARE element which
responds transcriptionaly to gibberelic acid by interaction with
nuclear factors. Reverse transcription follows transcription,
and depends upon a (–)-strand initiation site (PBS), which is
generally complementary to a tRNA in LTR retrotransposons,
and a (+) strand initiation site (PPT), which is purinerich. These are highly conserved both for BARE-1 (82) and
LARD elements (39) in barley. Evidence for production and
packaging of BARE-1 cDNAs has been found (36). Translation
of the LTR retrotransposons generates one single polyprotein,
or two separate gag and pol products. These are processed to
mature forms by retrotransposon- encoded proteinase (34).
The BARE-1 elements contain a single open reading frame,
as does yeast Ty5, and antibodies to both GAG and integrase
reveal that processing to the predicted mature sizes takes
place in a variety of tissues (36). Furthermore, BARE-1 GAG
antibodies also detect a full-length translation product and its
proteolytic processing products from barley and other cereals
including rice (90). Translation of a third encoded domain,
for the envelope protein, has not yet been directly shown in
barley for the Bagy-2 element, although the requisite RNA
splicing takes place (89). The GAG and integrase products of
BARE- 1 appear able to form virus-like particles, which can
be visualized from sucrose gradients, associated with reverse
transcriptase activity (36).
Most of the preferable integration sites BARE-1 elements
are other BARE–1 elements or other retrotransposons thus
indicating that nested insertion patterns may represent a basic
feature of plant retrotransposons. The higher number of solo
BARE -1 LTRs (up to 42 folds more) than full length copies
of this retrotransposon in barley (88) indicates that LTRLTR recombination may play a major role in counteracting
retrotransposon-based genome expansion. This form of
intrachromosomal recombination in a single retrotransposon
results in a loss of one LTR and the internal domain, leaving
behind a recombinant solo LTR.
In situ hybridization analysis to metaphase spreads of
barley (80) has shown that Ty1-copia group retrotransposons
are distributed throughout the genome on all chromosomes,
but are less prevalent or absent in certain heterochromatic
regions. Recent investigations have shown that BARE-1 is
unusual in that it is transcriptionaly active in leaf tissues of
barley under normal growing conditions, as well as in tissues
of in vitro grown cultures (80). Molecular markers partly based
on conserved BARE-1 sequence motifs have been tested on
different Hordeum species and H. vulgare varieties and proven
to reveal considerable polymorphism, suggesting ongoing,
active transposition of BARE-1 like elements in barley genome
(37). Thus, the observed transposition activity of BARE-1 like
retrotransposons principally should allow the introduction of
new insertional mutations into the barley genome. Pearce et
al. (66) have shown that a BARE-1 subgroup isolated from
BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/3
rye genome DNA is also transcriptionally active in other
monocotyledons as barley, wheat under normal growing
conditions. In comparison to BARE-1 retrotransposon all other
plant Ty1-copia retrotransposons studied to date are only
transcriptionally activated in the host tissues under abiotic
and biotic stress conditions (30, 32, 69). BARE-1 is highly
polymorphic not only among H. vulgare cultivars, indicating
its recent transposition (37) but also it is widely distributed
within the Triticeae tribe, especially wheat, rye and oat
(28). The observed significant level of BARE-1 insertional
polymorphism within T. aestivum, S. cereale, and A. sativa
is an indication for high transcriptional activity of Ty-1 copia
group retrotransposons in Triticeae species. The polymorphism
level is highest among the rye (S. cereale) in comparison
to oat (A. sativa) and wheat (T. aestivum) cultivars. BARE1 retrotransposons can be successfully used as molecular
markers not only for distinguishing of species and varieties but
in following plant genome evolution.
In wheat and its relatives the Ty-1 copia group
retrotransposon family called Wis-2 has been studied (62, 63).
Based on the nucleotide similarities of the reverse transcriptase
(RT) domain Matsuoka and Tsunewaki (58, 59) have identified
seven families in wheat genome. These families are common
to the genome of ancestral diploid species of wheat, evidence
that they have been established prior to diploid speciation.
Their presence into the genomes of rice and barley has been
suggested based on the sequence similarity of preliminary
determined Ty-1 copia elements in rice (Tos) and barley
(BARE-1).
Ty1-copia group retrotransposons have been extensively
studied in rice (32, 33). The total copy number of retrotransposons
in rice genome is estimated to be about 1000, showing that
retrotransposons are a major class of transposable elements in
rice. The restricted distribution, along with low copy number
suggested that copia-like retrotransposons in rice are relatively
inactive during evolution compared with those in other plants.
The distributional features of the copia -like retrotransposons
suggest the existence of possible lineages among the rice
chromosomes, which in turn suggest that chromosome
duplication and diversification may be a mechanism for the
origin and evolution of the rice chromosomes. Although Ty1copia group retrotransposons appear inactive during normal
growth conditions, an activation of a few members this group
retrotransposons designated as Tos families (Tos10, Tos17 and
Tos19) under tissue culture conditions has been shown (33).
Tos17 is the first retrotransposon of monocotyledonous plants
(rice) whose transcriptional and transpositional activities have
been demonstrated. The activity of Tos17 as the other studied
T1-copia retrotransposons is also regulated at transcriptional
level. A repetitive sequence of 16 bp found in the LTR of
Tos17 may be a regulatory element but no homology between
transcription regulatory elements of tobacco Tnt1A and Tto1 and
Tos17 has been observed. These differences provide important
material for studies on the mechanisms of transcriptional
regulation of retrotransposons. This proposed involvement of
non-homologous cis- regulatory elements in the transcription
of retrotransposons in different plant species or dependition on
299
the type of inductor (different stresses as protoplast formation,
induced by elicitor/s present in the protoplasting enzyme,
prepared from pathogenic fungus, rather than protoplast
formation itself or tissue culture). Increasing in the copy
number of Tos17 (5 to 30 transposed Tos 17 copies in all plants
regenerated from tissue cultures, including transgenic plants)
with prolonged culture period (from 3 to 24 months) has been
observed. The copy number of Tos17 ranges from one to four
among six closely related rice varieties examined and suggests
that it have a weak activity under the natural conditions.
The high transpositional activity of Tos17 could be due to
wounding, because cultured cells are quite similar to woundhealing tissues. Retrotransposons can also serve as agents to
induce tissue culture-induced mutations. At least five out of
nine target sites of Tos17-transposition induced during tissue
culture are the coding regions of genes. Regenerated plants
with Tos17 insertions in the phytochrome A gene, S-receptor
kinase- like gene and gene encoding a ring-finger protein have
been obtained. These results indicate that activation of Tos17
is an important cause of tissue culture-induced mutations and
may be useful tool for insertional mutagenesis and functional
analysis of genes. The mobility of rice retrotransposons can
be also successfully used for estimation of the transposition
events during the evolution of this plant species. The presence
or absence of transposable elements at particular sites on
the genome gives ambiguous information on the time of
transposition. Based on the comparison of the hybridization
patterns of the indica and japonica rice subspecies it has
been suggested that the time of transposition differ between
Tos retrotransposon families. Most members of Tos2 family
have been transposed and then been fixed before divergence
of the indica and japonica subspecies, whereas most members
of Tos1 and Tos3 may have transposed after their divergence.
Because no copy number changes have been observed with
the most rice retrotransposons among rice cultivars these
retrotransposons probably are inactive at present time and
they are poor marker candidates for cultivar discrimination in
comparison to BARE-1 retrotransposons which show a high
insertional polymorphism among barley cultivars.
High number of Ty-1 copia group retrotransposons - B5; G;
Hopscotch; Ji; Opie; Stonor; Prem-1; Fourf; Victum etc. have
been identified at the last decade in maize (3, 74). Each highly
repetitive retrotransposon family constitutes a substantial
portion of the maize genome (61). It has been estimated that ~
50% of the maize genome consists of retrotransposon sequences,
with five large families contributing up to 25% of the genomic
DNA (74). The 30 000-copy Opie element, for instance, should
constitute about 10 to 15% of the 2400-megabase maize genome
(2). In total Ty-1 copia group retrotransposons - Opie and Ji
as well as Ty-3 gypsy group LTR retrotransposons – Cinful,
Grande and Huck account for >25% of the maize genome.
Not all retrotransposon families, however, are present at high
copy numbers. Some representatives of Ty-1 copia group
retrotransposonsons in maize - Fourf, Victum are presented
in lower copy number. Hundreds or perhaps thousands of
families containing <100 elements is fought to be present in
maize (74). The preferable target sites for insertion of these
elements are often other retrotransposon elements (nested
300
retrotransposons) mostly located into intergenic regions and
extremely rarely genes themselves. Several studies (6, 15, 74)
have confirmed that the nested retroelement structure apparently
represents the standard organization of the intergenic regions
in maize. Regions around telomeres, centromeres, knobs,
ribosomal DNA repeats or other unusual structures would be
likely exceptions. Interestingly, the elements found to cause
mutations in maize are those that are present in the genome
at relatively low copy numbers. These include Magellan (four
to eight copies per haploid genome), (70), Hopscotch (two to
six copies), (96), and Bs1 (one to five copies). Members of
the B5 family, with only two to four copies are responsible
for three independent mutations (wxB5, wxG, and bm-3), (86,
92) while Magellan elements have been found in two mutant
alleles (wxM and pl-987), (70) and Stonor as mutant wxStonor
allele (55). The wx-K mutation results from the insertion of
a copia-like retrotransposon Hopscotch which has one long
open reading frame encoding all of the domains required for
transposition, into exon 12 of the maize waxy gene is reported
from Shawn et al. (77). Computer-assisted database searches
using Hopscotch and other plant copia-like retroelements
as query sequences have revealed that ancient, degenerate
retrotransposon insertions are found in close proximity to
21 previously sequenced plant genes. The data suggest that
these elements may be involved in gene duplication and the
regulation of gene expression.
Insertion of retrotransposons into gene sequences has
also been reported for low copy Tos17 (32) and Tnt1A
retrotransposons indicating that retrotransposition might take a
significant contribution to somaclonal variation (25).
No member of any of the larger families of maize elements,
such as Opie (30 000 copies), or Grande, Ji, or Huck (each
with >10 000 copies), has caused any of the characterized
maize mutations despite the fact that some appear to be largely
intact and capable of further retrotransposition (74). These
data led San-Miguel et al. (74) to suggest that there may be
a cause-and-effect relationship between element family size
and the propensity to insert into genes. That is, elements from
very large families may thrive because they display a target
site preference. This way, they avoid mutating maize genes.
Whether this trend holds true for other plant species cannot
be ascertained at this time because few spontaneous mutations
outside of maize have been characterized.
Characterization of representatives of gypsy-like env
subgroup retrotransposons
Two major subgroups of gypsy-like LTR retrotransposons have
been found:
- gypsy-like (Bagy-1; Cereba; Fatima; Kulkuri; Latidu;
Laura; Lolaog; Romani –Hv1, -Hv-2, -Hv-3) and
- gypsy-like env (Bagy-2; Sabrina; R173; Wham; Wobi,
Egug) which are widespread in the plant kingdom (84)
and especially in Triticeae.
The env- containing gypsy-like LTR retrotransposons are
not rare but form part of a separate and widespread clade found
througout the flowering plants (89). Among the gypsy-like env
retrotransposons, Bagy-2, in barley (79) and its homolog in
BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/3
rice Rigy-2 (91) were recently identified and characterized.
Sequences from two Bagy-2 clones (AF254799 and AJ279072)
reveal that the element is about 10 kb overall, containing
LTRs of 1520–1537 bp and an internal protein-coding region
organized similarly to other gypsy-like elements. These LTRs
are relatively long for plant retrotransposons, as are those of
another barley retrotransposon, BARE-1 (52, 82). Immediately
interior to the 5’ LTR is a putative primer binding site (PBS),
which is identical in 17 of 18 nucleotides at the 3’ end of a
tRNA-Glu from Schizosaccharomyces pombe (98). The use of
tRNA-Glu is fairly unusual among plant retrotransposons, but
is shared with legume gypsy–like env element Cyclops-2 (13).
One base upstream of the 3’ Bagy-2 LTR sequence a possible
polypurine tract (PPT), which is identical to that other gypsylike elements such as Athila and again very similar to Cyclops2 (12 of 14 bases), is found. The derived amino-acid sequence
of the Bagy-2 internal domain, encompassing GAG, RP, RT,
RH, and IT domains, are also most similar to those of Athila
(Arabidopsis thaliana) and Cyclops-2 (pea), and gypsy-like in
domain order. The prevalence of the Bagy-2 retrotransposon
appears comparable to that of BARE-1 with about 104 copies
in the genome of barley.
The rice homolog (Rigy-2) of barley Bagy-2 has also been
described (91). The four Rigy-2 copies described contain other
retrotransposons nested within them. The consensus element
contains LTRs of 1171 bp and a total size of 9753 bp.
Both Bagy-2 and Rigy-2 contain a region, the position
and structure of which match env at the 3’ end of the internal
domain. The presence env is confirmed by amplification of
barley genomic DNA or barley BAC clones using primers
flanking env domain. In barley, the env- containing gypsy-like
Bagy-2 element is transcribed in all tissue examined and the
Bagy-2 transcripts undergo splicing to generate subgenomic env
product in a manner parallel to what is found in the retroviruses
(89, 91). The putative envelope polypeptides of Bagy-2 and
Rigy-2 contain predicted N-glycosilation sites, leucine zipper
and transmembrane domains typical of retroviral ENVs. Bagy2 displays an insertional polymorphismin in closely related
barley cultivars, implying recent transpositional activity. In
other closely related to barley cereals, the env primers can also
be used directly to demonstrate env transcription.
Isolation of retrotransposons
Large genome sequencing projects have enhanced our
understanding of diversity and evolutionary trends among
retrotransposons (16). In this regard, plant retrotransposons
were identified through plant genome sequencing projects.
The basic method for isolation of LTR retrotransposons
includes few key steps:
1. Retrotransposon sequences might be amplified by
polymerase chain reaction (PCR) of genomic or cDNA
using primers complementary to highly conserved
sequences of LTRs or RT domain of retrotransposons.
2. Further the resulted PCR products are subjected to
cloning and sequencing or are directly sequenced.
3. Putative retrotransposon sequences are compared to
preliminary determined plant LTR retrotransposons.
BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/3
Recently some gypsy–like env retrotransposons such
as Gossypium, H. vulgare and O.sativa retroviruses, were
identified using a novel approach (1, 91). These elements were
isolated using specific oligonucleotides for the gypsy envgene, suggesting that env-like genes are ubiquitous in the plant
kingdom, and are evolutionary related to the Drosophila gypsy
env-gene. In addition, this approach offers a simple and universal
method for the isolation of env-like genes in plants (1).
Retrotransposon-based marker systems
The dispersion, ubiquity and prevalence of retrotransposons
in plant genomes provide an excellent basis for development
of marker systems. Their structure and replication give them
several advantages as DNA markers (37):
1. They contain long, defined, conserved sequences which
can be used for cloning of specific markers and flanking
sequences;
2. Replicationally active members of a retrotransposon
family will produce new insertions in the genome
leading to polymorphism. The new insertions may then
be detected and used to order the insertion events in a
lineage, thereby helping to establish phylogenies (78).
The fact that retrotransposons are transposed by an RNA
intermediate step, creating stable inserted into the host genome
cDNA daughter copies supposes that these insertions will behave
as Mendelian loci during crosses and segregate accordingly.
Hence, retrotransposon-based markers would be expected
to be co-dominant. On the other hand retrotransposons may
integrate in principle in either orientation into the host genome
leading to the fact that any two members of a retrotransposon
family may be found in different orientations: head-to-head,
tail-to-tail, or head-to-tail. This can increase the number of
detected polymorphisms and depends on the method and
primer combinations applied.
The inherent replicative mode of transposition endows
retrotransposons with considerable advantages as genetic tools
in plant genome analysis.
Several conceptually related methods have been reported.
One (71) examines internal differences in the sequence or
structure of retrotransposons themselves as diagnosed by
restriction site polymorphism within the transposon. The
other molecular marker techniques such as Sequence-Specific
Amplification Polymorphism (S-SAP) and the related IRAP
(Inter-Retrotransposon Amplified Polymorphism) and REMAP
(Retrotransposon-Microsatellite Amplified Polymorphism),
are based on retrotransposon activity, and are increasingly
widely used.
The Sequence-Specific Amplified Polymorphism (SSAP) is a modified AFLP (Amplified Fragment Length
Polymorphism) method based on BARE-1, (94) in which a
PCR primer facing outward from BARE-1 long terminal repeat
(LTR) is used in combination with an AFLP adapter primer.
Variability in the banding pattern is a result from variation in
the distance between a restriction site and a BARE-1 element.
Highly prevalent retrotransposons such as BARE -1,
BAGY-1, BAGY-2, Sabrina, Nikita and Sukkula require the
use of selective primers, either for the adapter primer or the
301
retrotransposon primer or both, in the S-SAP reaction for
efficient amplification and visualization in barley (50). In the
S-SAP technique, the selective bases added to the primers
reduce the complexity of the amplified DNA, depending on
the copy number of the retrotransposon targets.
The method gives several advantages over the AFLP:
more polymorphism, more codominance, and more even
chromosomal distribution. The main disadvantage of the
method is due to the necessity of restriction digestion of gDNA
to provide sites for adapter ligation as in AFLP method. The
sensitivity of commonly used restriction endonucleases (PstI,
EcoRI) to DNA methylation, combined with the high and
potentially variable degree of CG and CXG methylation in
plant DNA, means also that some apparent polymorphism may
neither be sequence-based nor heritable.
Recently Tang et al. (85) reported the development of a highthroughput sequence-specific amplification polymorphism
(S-SAP) method based on copia-like retrotransposons to
fulfill the increasing desire of screening large numbers of
samples in plants. In this method the classic approach for
digestion, ligation and pre-amplification has been combined
with optimized fluorescent multiplex PCR for simultaneously
selective amplifying S-SAP fragments, and multiple S-SAPs
were subsequently detected by capillary electrophoresis using
ABI PRISM 3700 capillary instruments. Comparisons of results
from multiplex PCR with simplex PCR, and from capillary
electrophoresis with slab-gel electrophoresis demonstrated that
this method is an efficient, economical, and accurate means for
high-throughput and large-scale genotyping retrotransposon
variation in plants.
The S-SAP has been used in numerous plant species where
terminal LTR sequences can be effectively cloned (75). The use
of S-SAP has been described for barley (94), wheat (Triticum
aestivum L.) and wild relatives (28, 73), pea (Pisum sativa L.)
(17), alfalfa (Medicago sativa L.) (68), etc. However, proper
use of the SSAP technique requires either radioactivity or
fluorescent labelling of primers and product detection. Both
these drawbacks could be overcome by the IRAP method.
IRAP (Inter-Retrotransposon Amplified Polymorphism)
and REMAP (Retrotransposon-Microsatellite Amplified
Polymorphism) are retrotransposon-based methods which
require no DNA digestion (37). Both IRAP and REMAP can
be used to examine the polymorphism in retrotransposon
insertion sites, IRAP between retrotransposons themselves and
REMAP between retrotransposons and microsatellites (SSRs).
The both methods used primers facing outward from the long
terminal repeats of the BARE-1 retrotransposon. The primers
are designed so to be complementary to essential for the
expression (promoter and processing signals) and integration
LTR sequences of retrotransposons that tend to be highly
conserved. Primers complementary to highly conserved (-)and (+)-strand reverse transcriptase gene can also be used. To
define highly conserved regions for designing of primers a set
of BARE-1 LTRs needs to be preliminary compared. Based on
analysis of a set of barley genomic BARE-1 LTRs, Suoniemi et
al. (82) defined, the regions 109 bp in from the 5’ terminus and
144 bp in from 3’ terminus of the LTR which also tend to be
302
highly conserved in the Wis-2 retrotransposon family of wheat
for primer design. The appearance of new polymorphic bands
can be explain with insertion of new BARE-1 copies into the
genome within amplifiable distance of another BARE-1 LTR
(IRAP) or a microsatellite (REMAP), or from an increase in
the repeat number of a microsatellite to a point sufficient for
amplification (REMAP). With the IRAP the increase in number
of polymorphic bands can be observed in comparison to S-SAP
method. This is due to the fact that with a single outwardfacing BARE-1 LTR primer in IRAP method is possible to
generate PCR product corresponding to the BARE-1 elements
in head-to-head orientation. Both the nested insertion of one
BARE-1 element into another as well as a nearby insertion
would generate an IRAP band. This is not surprising because
it is commonly known that most of retrotransposons can serve
as matrix for insertion of others. The IRAP method succeeds
because, rather than being dispersed at a predicted average
distance of 50 kb, retroelements are clustered in the barley
genome. Likewise, microsatellites and retrotransposons tend
to be clustered in the genome, increasing the utility of REMAP
(72). The methods are robust regarding the generation of solo
LTRs. Although LTR-LTR recombination may delete the
internal region of an element, the remaining solo LTR leaves
many such losses transparent for marker systems based on
LTRs. This is because recombination does not remove the
annealing site, generally near the outer edge of the LTR, for
primers used in polymorphism detection. Similarly, stacked
insertions of one retrotransposon into another, and subsequent
deletions within such nests, will not affect the products
generated from the LTRs of the original element because
these events will be “behind”, or 5’), to the position of the
primer (76). Retrotransposon marker systems such as RBIP
/Retrotranspososn-Based Insertion Polymorphisms (21) and
TAM (Tagged Microarray Marker) (22), which score the
presence or absence of individual insertions based on specific
amplification between the flanking sequences, have been also
described but till recently not yet been adapted to barley, but
are currently under development. In comparison to SSAP,
IRAP, and REMAP methods which are multiplex and generate
anonymous marker bands, RBIP scores individual loci, akin to
microsatellite systems.
The retrotransposon-based marker systems are extensively
used in fingerprinting, mapping, marker-assisted selection,
evolutionary studies, and retrotransposon activities in
different plant species (37, 94), Oryza (35), and Spartina (4).
Additionally, it proved to be suitable for studies of genome
evolution (38, 89), and in gene mapping of barley (53) and
wheat (10).
Phylogeny and transpositional activity of Ty1-copia and
gypsy–like subgroups retrotransposons in cereal genomes
The advantage of application of retrotransposon-based marker
systems is consists in the ability to track an insertion event
and its subsequent vertical radiation through a pedigree or
phylogeny (78). Few studies (28, 60) have revealed the extreme
complexity of the population structure of Ty1-copia subgroup
retrotransposons in Gramineae. The obtained barley and
wheat Ty1-copia retrotransposon sequences have been used
BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/3
to construct phylogenetic trees in barley and wheat and also
were compared to the other preliminary determined Ty1-copia
retrotransposons in these and other species from Gramineae.
The barley Ty1-copia subgroup retrotransposons population is
comprised of a highly heterogenous set of retrotransposons,
together with a collection of sequences that are closely related
to the BARE-1 element. This is not surprising because most of
the individual copies of plant Ty1-copia group retrotransposons
are probably generated early in the evolution of this species
and are not active, and can accumulate mutations (nucleotide
substitutions, deletions or insertions) at high levels in the
absence of selective pressure. The preferable target of some
of them are frequently other retrotransposons thus leading to
disruption of the coding potential and to creation of a highly
heterogeneous Ty1-copia retrotransposon group as it have
been observed for many Ty1-copia group retrotransposons
in maize. Wheat also contains a highly diverse Ty1-copia
retrotransposon populations (W family) most of them forming
new subgroups - the more related to BARE-1 subgroup Tta12
and the other Tta15 subgroup along with the retrotransposon
sequences showing no close homology to them and forming
other not well defined group in wheat. Comparison of all
determined till now Ty-1 copia group retrotransposons in the
species belonging to Gramineae reveals several interesting
results. Closely related to BARE-1-homologous sequences are
obtained also in rye and oat. This is an indication that close
homologous of BARE-1 are therefore broadly distributed
among the species of the tribe Triticeae. The rice Ty-1 copia
group retrotransposon sequences are more restricted. Some
representatives of this group retrotransposons are members
of supergroup Tta15 of wheat but no homology have been
observed with Ty-1 copia group retrotransposons of the other
species belonging to Gramineae.
As compared to Ty1-copia subgroup the gypsy- like
subgroup retrotransposons has shown to be also heterogenous in
Gramineae which is mostly likely due to the presence/abcence
of env domain. On the other hand the env sequences of gypsylike retrotransposons constitute a very heterogenous collection
(51, 54). The considerable size and sequence diversity among
retroviral envelope proteins, does not allow the use of primers
designed for amplification of env sequences in non-closely
related plant species (91). This sequence diversity supposes the
application of a different approach based on primers not for
ENV but amplifying RT domain to demonstrate the ubiquity
of env-containing, gypsy- like elements in plants. Based on
this approach and subsequent alignment of 328 gypsy-like
RT sequences from known retrotransposons and database
accessions of all organisms constructed on the basis of earlier
alignments Vicient et al., (91) demonstrated the presence of
this class retroelements in all angiosperm genomes examined
so far. The resulting phylogenetic tree based on these sequences
and subsets thereof is consistent with the reported earlier one
(56) and showed that the plant gypsy-like elements are resolved
into two lineages, one universally lacking env domains and the
other containing the sequences amplified together with the
other elements containing env-like or long 3’ regions. In this
tree the phylogenetic distinctness of the Athila clade of gypsylike env retrotransposons (Bagy-2, Rigy-2, Athila, Calipso,
BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/3
Cyclops) could be explained with their horisontal transmission
spread between species. This supports the supposition that
plant ENVs may have intracellular or intraplant rather than
infective roles, either in element replication or in cell-to-cell
movement within the plant (91).
This opens a range of new application of retrotransposons in
both animals and plants: germline–therapy of mammals using
retroviruses in which ENV proteins are modified to affect host
range and targeting (11) and improvement of recalcitrant to
conventional transformation crops (91).
Conclusions
Cellular genes comprise at most 5-10% of cereal genomes;
the rest is occupied primarily by retrotransposons which move
intracellularly by a replicative mechanism similar to that of
retroviruses. Recent sequencing of long contiguous segments
of cereal genomes have revealed the prevalence of full-length
retrotransposons and their solo LTR derivative indicating
that integration and recombinational loss of retrotransposons
are major factors shaping the genome. Because LTR
retrotransposons present a major share of the genome, make
easily detectable genetic changes having known ancestral and
derived states, and contain conserved regions for which PCR
primers may be designed, retrotransposon’s insertions can be
exploited as powerful molecular markers systems for mapping,
marker-assisted breeding, phylogenetic analyses, biodiversity
determinations, and evolutionary studies.
The fact that some of the plant retrotransposons are
structurally intact and transcriptionaly active (1, 67, 89, 90)
promotes the possibility of their use as potent vehicles for
interspecies gene flow in plants. In other words, a vector
based on intact plant retrotransposons could be an important
additional tool for the production of transgenic plants with
well-defined, foreign DNA inserts required for biosafety
approval and commercialization in next future. Furthermore,
the development of plant ratrotransposons as gene vectors is of
great advantage to transfer genes of interest and can overcome
some problems of the currently used transformation methods.
In addition unlike animals, plants do not sequester their germ
line and infected somatic plant cells can give rise to floral
organs and seeds.
However, before retrotransposons to be used as vectors in
routine genetic transformation for improvement of crops, it is
imperative to understand how plant retrotransposons naturally
contribute to interspecies gene flow, and thus rationally evaluate
recent concerns regarding the use of genetically modified crop
species. To do this, many aspects of the life cycle and the
mechanisms regulating retrotransposon expression and its link
to cellular processes in plants must be revealed and clarified.
Both classes are flanked by long terminal repeats (LTRs)
and usually encode all of the proteins required for their
transpozition, including a capsid (GAG), protease, integrase
reverse transcriptase, and RNase H. The LTRs contain
inverted repeats (triangles) at their termini. The primer binding
sites (PBS) and polypurine tract (PPT) are present in most
studied elements and are required for replication by reverse
transcriptase (RT). The protein-coding region is frequently
303
separated into two domains by a frame-shift (between GAG,
the capsid protein, and aspartic proteinase -AP). The two
groups can be distinguished by replacement of integrase (IN),
which in copia-like elements precedes the RT and ribonuclease
H (RH) but in gypsi-like.
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