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Activation of plant retrotransposons
under stress conditions Marie-Angèle Grandbastien
Activation of retrotransposons by stresses and external change is common in all eukaryotes,
including plants. The transcriptional activation of several well-characterized plant retrotransposons seems to be tightly linked to molecular pathways activated by stress, and activation
is under the control of cis-regulatory sequences strikingly similar to those of plant defence
genes. These regulatory sequences are highly variable, suggesting that retrotransposons
could evolve through modification of their regulatory features. As the molecular basis for
their regulation is becoming better understood, it is possible to begin to assess the putative
biological impact of this stress response.

R

etrotransposons were first characterized in animal and
yeast genomes, but evidence has accumulated in recent
years to show that they are present in all plant genomes and
can constitute a very large part of some of them. They are one of
the two classes of transposable elements, defined according to
their mode of propagation: retrotransposons (also termed class I

elements) transpose via an RNA intermediate; and class II elements only use DNA in movement. Representatives of all types
of retrotransposons have been detected in plant genomes1,2. A
growing body of evidence shows that the activity of these retrotransposons is, as in animal systems, tightly controlled, and that
abiotic and biotic stresses are major factors in their transcriptional
and transpositional activation.
Regulation of retrotransposition

Retrotransposons can be separated into two major subclasses that
differ in their structure and transposition cycle (Fig. 1). Elements
of subclass I are bounded by two long terminal repeats (LTRs)
and are termed LTR retrotransposons; elements of subclass II do
not possess LTRs and are therefore termed non-LTR retrotransposons. Both these subclasses form a DNA daughter copy by
reverse transcription of an RNA template, and their replication
cycle involves an intermediate cytoplasmic step. This replicative
transposition mechanism means that retrotransposons are potentially very invasive. To ensure the viability of their host, and
hence their own survival, retrotransposition is tightly controlled
(Fig. 2). This control involves element-encoded functions and
host factors. One of the major control steps is transcription, which
determines both the production of the RNA template and the
synthesis of mRNAs required for protein synthesis. In LTR

retrotransposons, transcriptional control involves cis-regulatory
sequences that are usually found in the element’s LTR, in particular the U3 region located upstream of the transcription start site
(Fig. 2), or in downstream, untranslated sequences.
A survey of active plant retrotransposons

Transpositional activity has been reported for only a few elements, mostly those belonging to the LTR retrotransposon subclass (Table 1). Most of these were isolated after transposition into
or next to a host gene (Table 1). A few mobile elements were first
characterized by PCR amplification of genomic DNA or cDNA,
but were subsequently shown to transpose. Evidence for transpositional activity can also be inferred from the analysis of LTR
sequences, which are identical in newly transposed copies: this is
the case of the Osser element, which is probably active, although
direct transposition has not been reported3. So far, however, functional copies able to transpose in foreign species have only been
characterized for Tnt1A (Ref. 4) and Tto1 (Ref. 5).

Homologous transcripts have been reported for a larger number
of elements (Table 1). In several cases, however, the nature of the
transcript has not been fully established and it could derive from
co-transcripts originating from external, upstream promoters (Box
1). Specific transcripts, starting in the element’s LTR or at its 5′extremity for non-LTR retrotransposons, were demonstrated for
Tnt1A, Tto1, BARE-1, Tos17, Huck (Z. Avramova, unpublished)

and possibly SIRE-1, as well as for the short interspersed nuclear
elements (SINEs) S1Bn and TS elements (see Table 1). Definitive
evidence of LTR transcriptional ability was further obtained for
Tnt1A, BARE-1 and Tto1 (Table 1) by using constructs in which reporter genes were placed under the control of the element’s LTR.
Regulation of the activity of plant retrotransposons
Developmental regulation

In animals and yeast, the expression of retrotransposons is under
the control of hormonal and developmental factors. A general picture of expression is difficult to establish for many plant retrotransposons, because comparative studies in different tissues have not
been done. However, the expression of the most well-characterized
plant retrotransposons is not constitutive. Developmental regulation has been shown for Tnt1, which is only expressed in roots
and then only at low levels6; for Tto1, Tos10 and Tos17, which are
not expressed in leaf tissues7,8; and for the mobile B5, Hopscotch,
Stonor and Magellan elements, which are not expressed in most
plant tissues (S. Wessler, unpublished). Expression of the maize
PREM-2 element was detected only in early microspores9. However, the expression of Opie, Huck and Cinful (Refs 10 and 11),
and of BARE-1 (Ref. 12), was detected in leaf tissues.
Stress activation: the in vitro track

A common feature of most retrotransposons is that they are

activated by stress and environmental factors. The most wellcharacterized plant retrotransposons are particularly affected by
protoplast isolation or in vitro cell or tissue culture (Table 1).
Insertion of retrotransposons into coding sequences after protoplast or cell culture was demonstrated in tobacco13, in Nicotiana
plumbaginifolia (C. Meyer, unpublished) and in rice7, indicating
that retrotransposition might make a significant contribution to
somaclonal variation. The first direct evidence of activation of a
plant retrotransposon by stress came from the discovery that the
expression of Tnt1A was highly induced in protoplasts isolated
from tobacco leaf tissue6. In accordance with this, Tnt1A transposition into the tobacco nitrate reductase (nia) gene was detected in
plants regenerated from protoplast-derived cell cultures13. However, Tnt1A transcription was not detectable in suspension cell
cultures, even though a weak increase in copy number was observed8. In contrast, both the expression and the transposition of

Copyright © 1998 Elsevier Science Ltd. All rights reserved. 1360 - 1385/98/$19.00 PII: S1360-1385(98)01232-1

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Retroviruses
5'-LTR

gag pr
gag

env

3'-LTR

RT endo
pol

Retrotransposons
Subclass I: LTR retrotransposons
Superfamily Ty1/copia

gag pr
endo

RT

Superfamily Ty3/gypsy

gag pr
RT endo
ORF3
Subclass II: Non-LTR retrotransposons
‘
gag

‘

Superfamily LINEs

N

RT

[A]n

Superfamily SINEs
[A]n

Fig. 1. Overall organization of the different types of retrotransposons in comparison with
retroviruses, with classification according to Ref. 30. Elements of the subclass I have an
overall organization similar to retroviruses. They are bounded by long terminal repeats
(LTRs) that contain signals for initiation and termination of transcription, and carry one or
several open reading frames (ORFs) with coding potential for the structural and enzymatic
proteins needed for the retrotransposition cycle: the gag domain, encoding proteins that form
the nucleocapsid core; the protease (pr) domain, encoding proteins that are involved in the
maturation of the different proteins; the reverse transcriptase (RT) domain, encoding the
enzymes responsible for the creation of a DNA copy from the genomic RNA template; and
the endonuclease (endo) domain, encoding proteins necessary for the integration of the DNA
copy into the host genome. LTR retrotransposons form cytoplasmic, virus-like particles
(VLPs) in which the RNA template is reverse transcribed into a DNA daughter copy. LTR
retrotransposons are further divided into two superfamilies: the Ty3/gypsy superfamily, in
which the organization of the coding domain is the same as that of retroviruses; and the
Ty1/copia superfamily, in which the endo domain is placed upstream of the RT domain. The

major difference between retroviruses and LTR retrotransposons is that the latter do not
encode the envelope (env) gene responsible for the formation of the extracellular infectious
virion. However, the boundaries between retroviruses and retrotransposons appear increasingly blurred, as several members of the Ty3/gypsy superfamily also contain an additional
open reading frame (ORF3), sometimes encoding an env-like gene, and might thus represent
intermediates between LTR retrotransposons and retroviruses. Non-LTR retrotransposons do
not contain LTRs and are terminated by an A-rich tail ([A]n). Long interspersed nuclear elements (LINEs) generally contain two ORFs, the second showing similarities to the RT
domain and the first encoding a putative gag-type nucleic-acid-binding protein (‘gag’).
Although LINEs do not contain a recognizable endo domain, some elements might contain a
putative nuclease domain (N ). The retrotransposition cycle of LINEs is not well understood,
but it has been proposed that they might form cytoplasmic particles able to carry the RNA
template into the nucleus, where reverse transcription would occur simultaneously with integration31. Short interspersed nuclear elements (SINEs) have no coding capacity and are
thought to use foreign RT domains to achieve their life cycle, through incorporation of their
RNA into the cytoplasmic particles of LINEs (Ref. 31).

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Tto1, Tos10 and Tos17 is activated in cell
cultures7,8. Tto1, but not Tos17, expression

is further increased by protoplast isolation.
The barley BARE-1 element is expressed
in callus tissues, as well as in leaf-derived
protoplasts, although protoplast expression
appears to derive from BARE-1 expression in the leaves12. Transcripts of the soybean S1Bn SINE elements were detected in
callus cultures14, and protoplast-specific
RNA sequences of LTR retrotransposons
were characterized in potato15.
A link with defence responses?

Protoplast isolation, as well as cell and callus culture, induces major modifications of
cell metabolism and gene expression16,17. In
leaf-derived protoplasts, the former metabolic activity of the leaf cell is replaced by
a new programme. This is characterized
by the activation of growth- and stressrelated genes (e.g. defence genes, which are
activated after pathogen attack). Growthrelated genes are probably involved in the
re-initiation of cell division; the activation
of stress-related genes might be a consequence of the original wounding. Protoplast isolation also involves enzymatic
degradation of the cell wall, using extracts
from phytopathogenic fungi. The activation of stress-related genes might thus

also result from cell wall hydrolysis or
from pathogenic compounds present in
fungal extracts. Growth- and defencerelated genes are also expressed in callus
and cell cultures, suggesting that the programmes of callus tissues are similar to
those induced after wounding and during
callus formation in the plant, involving
both a stress response and cell division16.
The activation of several plant retrotransposons in these particular conditions 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. Partial answers
were provided by further studies of the
expression of the tobacco Tnt1A and Tto1
elements. Tnt1A protoplast-specific expression results mostly from the effect of
fungal extracts, and the Tnt1A promoter is
also activated by other compounds of microbial origin, salicylic acid, wounding
(Fig. 3), and viral, bacterial or fungal attacks18. Similarly, the expression of the
Tto1 element is induced by viral attacks,
wounding, salicylic acid and jasmonate19,20.
The expression of the two best-characterized plant retrotransposons is thus induced
by different biotic or abiotic factors that

can elicit plant defence responses. Further
analysis suggested that Tnt1A expression
was tightly linked to the early steps of
the defence gene activation pathways18.

trends in plant science
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However, the expression of Tto1, but not
of Tnt1A, in suspension cell cultures shows
that the activating pathways differ for each
element and that Tto1 expression might be
under a dual control such as stress and cell
division, as observed for several genes activated after protoplast isolation17. Interestingly, expression of the rice Tos17 element
in cell cultures is not enhanced by protoplast isolation7, suggesting that the control
of Tos17 differs from that of Tnt1 and Tto1.
Whether the link between retrotransposon activation and plant defence responses
can be extended to elements other than Tnt1
and Tto1 is not yet known. However, other
transcribed retrotransposon sequences have
also been detected in stressed tissues, such
as Tto5 (detected in tobacco treated with
salicylic acid or after viral inoculation19) and
the Tpt sequences (detected in anaerobically stressed seedlings of loblolly pine; C.
Kinlaw, unpublished). Transposition of the
maize Bs1 element was also detected after
viral infection21, although a direct link between Bs1 movement and infection has not
been established.
Tnt1A and Tto1 regulatory sequences
and defence genes

5'-LTR
U3 RU5

Nucleus

3'-LTR

gag

pol

Cytoplasm

U3 RU5

5 Integration
endo
1 Transcription

AAA
Genomic RNA

mRNA(s)

DNA
RT
RNA
4 Reverse transcription

endo
2 Protein synthesis

RT

VLP

gag
3 RNA packaging and VLP assembly

Fig. 2. The major control points of the transposition cycle of long terminal repeat (LTR)
retrotransposons. (1) Transcription begins in the 5′-LTR, at the boundary between the U3
and R domains, and terminates in the 3′-LTR, at the boundary between the R and the U5
domains, to produce a full-length RNA bounded by the redundant R domain. The fulllength RNA will serve as a template for reverse transcription, as well as an mRNA template
for the production of the proteins. (2) Control of the gag : pol ratio during protein synthesis
is necessary, structural gag protein being required in large quantities for the assembly of
virus-like particles (VLPs). The gag and pol domains are usually found in different frames,
and the pol products are synthesized as a gag–pol fusion polyprotein resulting from a translational frame shift. Alternative strategies, such as transcript splicing or specific degradation of the pol domain of the gag–pol polyprotein, are used by elements in which the
gag–pol domains are in the same reading frame. (3) RNA packaging and VLP assembly are
tightly dependent on specific interactions between the genomic RNA and gag nucleic-acidbinding domains. A parameter essential to this step is the activation of the protease, which
catalyses the maturation of the gag–pol polyprotein during VLP assembly, thus ensuring
the incorporation of functional enzymatic proteins into the VLP. (4) Reverse transcription
is a very complex process that depends on the availability of a particular host tRNA and
leads to the generation of a linear extrachromosomal DNA form bounded by two identical
LTRs. (5) Integration involves the processing of the ends of the linear extrachromosomal
DNA form and the joining of the retrotransposon DNA to the cleaved host DNA. Both steps
are catalysed by the endo-encoded protein, but the mechanism by which the DNA daughter
copy is transported to the nucleus is not well understood.

Promoter structure and function have been
studied in detail for Tnt1A, Tto1 and BARE-1.
Tandemly repeated cis-regulatory sequences
were identified in the U3 region of Tnt1A
and Tto1: a 31 bp repeat, the BII box, present in three or four copies in transcriptionally active elements, is involved in Tnt1A
activation by protoplast isolation and fungal elicitins18,22; and a repeated 13-bp motif is involved in Tto1 expression in callus
and after wounding or jasmonate application (S. Takeda et al., unpublished). Putative regulatory motifs were also detected in
U3 regions of Tos17 (Ref. 7) and BARE-1
(Ref. 12). The specificity of expression of
BARE-1 in leaves or calli also involves the
alternative use of different LTR promoters12.
Interestingly, Tnt1A and Tto1 repeated cis-acting motifs share
similarities with a motif involved in the activation of several plant
defence genes, the H-box23,24. In addition, the Tnt1A U3 region
contains other sequences highly similar to regulatory motifs of
stress-induced plant genes. These similarities provide a plausible
explanation for the molecular basis of both Tnt1 and Tto1 activation by stresses and pathogen attacks. A MYB-related factor,
LBM1, involved in Tto1 transcriptional activation in protoplasts
through specific binding to the 13-bp motif, was recently characterized (K. Sugimoto, S. Takeda and H. Hirochika, unpublished).
However, LBM1 is not expressed in suspension cell cultures, indicating that other factors are involved in Tto1 activation in these
conditions.
These results illustrate the complexity of retrotransposon transcriptional regulation, and indicate that the subtle differences
between Tnt1 and Tto1 stress activation features might be linked
to the presence in their LTR of several cis-regulatory sequences
each capable of responding to a different stimulus. Although some

of the molecular transduction pathways involved in the expression
of Tnt1 and Tto1 are different, the overall result appears to be surprisingly similar – increased expression in response to a diverse
array of stress conditions that activate plant defence responses.
Evolution of retrotransposon transcriptional control

Reverse transcription is prone to error. As a consequence, a retrotransposon can generate a population of different but closely related daughter copies, and this variability might play a role in the
evolution of the control of retrotransposon activity. For example,
the generation of transcriptionally inactive Tnt1A copies, through
specific deletions of BII sequences, is a frequent event22. As also
demonstrated for SINE S1Bn elements14, only a limited number of
family members are responsible for the transcripts, possibly in
order to limit the hazardous effects of growing populations of
retrotransposons22. The variability of retrotransposon populations
might also constitute a reservoir of potentially useful genomes,
endowing retrotransposon populations with high adaptability22, as
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Table 1. Active or potentially active plant retrotransposonsa
Classification

Species

Evidence for recent mobility

Evidence for transcripts

Refs

In leaves and callus
None
None
None
In rootsb, leavesb and tasselsb
In rootsb, leavesb and tasselsb
None
In early microsporesb
In protoplastsb
In protoplastsb
In protoplastsb
In protoplastsb
In seedlingsb
In seedlings and leaf tissues
None
None

12
34
34
25
10, 11
10, 11
3
9
15
15
15
15
33
35, d
34, e
f

Tobacco
Tobacco
Tobacco
Wheat

None
Transposition into the waxy gene
Transposition into the waxy gene
Transposition into the waxy gene
None
None
Copy with identical LTRs
None
None
None
None
None
None
None
Transposition into the waxy gene
Transposition into the nia gene in protoplast
cultures
Transposition into the nia gene in protoplast
cultures; small increase in copy number in cell
cultures
Copy number increase in cell cultures
Copy number increase and active transposition
into coding sequences in cell and tissue cultures
Small increase in copy number in cell cultures
Copy number increase in cell and tissue
cultures; transposition into the nia gene in
protoplast cultures
Small increase in copy number in cell cultures
None
None
Polymorphism in regenerated plants

Maize
Maize
Maize
Tomato
Pinus taeda

None
None
Transposition into the waxy gene
None
None

Maize

Transposition near the zeinA gene in
somatic tissues

Subclass I: LTR retrotransposons
Superfamily Ty1/copia
BARE-1
Barley
B5
Maize
G
Maize
Hopscotch
Maize
Ji
Maize
Opie
Maize
Osser
Volvox carteri
PREM-2
Maize
Prt1c and Prt3c
Potato
Prt4c
Potato
Prt5c
Potato
Prt6c
Potato
R9c
Rye
SIRE-1
Soybean
Stonor
Maize
Tnp2/Tnt1B
Nicotiana
plumbaginifolia
Tnt1A
Tobacco

Tos10c
Tos17

Rice
Rice

Tos19c
Tto1

Rice
Tobacco

Tto2c
Tto3c
Tto5c
Wis-2
Superfamily Ty3/gypsy
Cinful
Huck
magellan
TCI-4
Tptb
Zeon-1

Atypical or not yet classified
Bs1
Maize
PREM-1
TOC1

Maize
Chlamydomonas
reinhardtii

Subclass II: Non-LTR retrotransposons
Superfamily SINEs
S1Bn
Rapeseed
TS
Tobacco

In roots, in protoplasts, and after 18
wounding and pathogen attacks
In cell cultures
In cell cultures

7
7

In cell culturesb
In protoplasts, cell and tissue
cultures, and after wounding
and viral attack
In protoplastsb
In protoplastsb
After viral attackb
In protoplasts

7
8, 20, g

In leavesb
In roots, leaves and tassels
None
In seedsb
In anaerobically stressed
seedlingsb
In endospermb

Transposition into the Adh gene after
None
viral infection
None
In early microsporesb
Transposition into the OEE1 gene and
Yes
increase in copy number during mitotic growth

None
None

In shoots, roots and callus
By in vitro transcription

8
8
19
32, h
10, 11
10, 11
37
38
i
36

21
39
40

14
41

a
The table includes all those plant retrotransposons for which transcripts or mobility have been reported. However, any transcripts only shown to be expressed
from foreign promoters are excluded; elements showing intervarietal or interallelic polymorphisms, indicative of fairly recent activity, are also excluded, because
this is a poor criterion for present activity. bThe possibility that transcripts are initiated from external upstream promoters has not been ruled out. cPartial
sequences isolated by PCR methods. dH. Laten, unpublished. eS. Marillonnet and S. Wessler, unpublished. fC. Meyer, unpublished. gP. Grappin and M-A.
Grandbastien, unpublished. hH. Lucas, unpublished. iC. Kinlaw, unpublished.

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Box 1. What is an active retrotransposon?
Retrotransposons encode for proteins involved in retrotransposition, and produce RNA both for protein production and for reverse
transcription [although short interspersed nuclear elements (SINEs)
are an exception]. As for class II elements, a defective retrotransposon can be trans-activated. Transposition has been shown for
Bs1, Zeon-1, G, Stonor and TOC1, which all lack important coding
regions or have coding domains interrupted by unsuitable stop
codons. The presence of functional gag–pol domains is thus not a
prerequisite for transposition, provided signals important for retrotransposition, such as priming sites or encapsidation signals, are
still present. However, a retrotransposon will not transpose in the
absence of the genomic RNA used as the template for reverse transcription. In terms of mutagenic impact on the host genome, the best
criterion for activity is thus the ability to produce a transcript, and
this criterion has been used, in addition to mobility, to establish the
list of plant retrotransposons that are active or potentially active.
For some elements, preliminary information on transcriptional
activation is provided by partial cDNA sequences. This information
should be taken cautiously, because transcripts containing retrotransposon sequences can derive from cotranscripts originating
from external, upstream promoters. Their expression pattern will reflect the activation of the foreign promoters only. This is also illustrated by the detection, in several cases, of multiple RNA species,
or even smears or RNA of variable sizes, which are best explained
by simultaneous cotranscription from different cellular promoters.
However, this does not exclude the possibility that a specific element transcript could also be expressed, as demonstrated for Tnt1,
TOC1, Tos10 and Tos17, and S1Bn. Different transcript sizes can
also be produced internally from modified members of a given family, such as deleted copies or copies carrying additional sequences.
For instance, the mobile G element of maize is a deleted derivative
of the B5 element. Similarly, the maize Cinful, Zeon-1 and Cin1
elements are very closely related, Zeon-1 being a copy in which
most coding domains, except gag, are deleted and replaced by sequences of unknown origin; Cin1 represents a single long terminal
repeat (LTR) of the same family. The transposition of G and B5,
in particular, has required two mRNA species of different sizes.
Cotranscripts originating from the transcriptional start site of the
element and proceeding into downstream cellular sequences also
cannot be excluded. It thus appears that, if retrotransposon sequences isolated from cDNA clones are by themselves not convincing evidence for the transcriptional activation of the element,
then transcripts of abberrant or multiple sizes could still provide
information on the expression conditions of a given element. Nevertheless, active retrotransposons have been successfully isolated
after preliminary characterization of partial sequences through
reverse transcription PCR (Tto1 and Tos17) or even genomic PCR
(Tos10). Tto1, Tos10 and Tos17 PCR sequences were also shown
to hybridize to an RNA of adequate size, thus increasing the probability that this transcript was specific. It thus appears that, provided some precautions are taken, active retrotransposons can
successfully be isolated via their transcription products.

Fig. 3. Transcriptional activation of the Tnt1A tobacco retrotransposon after wounding or treatment with compounds of
microbial origin. The expression of the retrotransposon promoter
is detected by dark-blue coloration resulting from GUS activity in
transgenic plants containing the long terminal repeat (LTR)–GUS
construct, in which the GUS reporter gene was placed under
control of the Tnt1A LTR. Experiments presented here were
performed on tomato plants, demonstrating that the Tnt1A regulation observed in tobacco is maintained in other species.
(a) Tnt1A expression after wounding of leaves with razor blades:
a dark-blue coloration resulting from a high level of LTR–GUS
expression is detected in the close vicinity of gashes. (b) Control
wounding experiment performed with tomato plants containing
the CaMV 35S–GUS contruct, in which the GUS reporter gene
was placed under the control of the constitutive CaMV 35S promoter. (c) Tnt1A expression after needle injection of extracts of
the fungus Trichoderma viride, used as a source of cellulases to
degrade cell walls during protoplast isolation. LTR–GUS expression is detected in the leaf region in which the fungal extract has
diffused. Photos courtesy of Corinne Mhiri.

proposed for retrovirus quasispecies. For instance, Tnt1A and S1Bn
transcript populations vary between different cellular contexts14,22,
and this variability affects mostly regulatory sequences in the case
of Tnt1A. Retrotransposon populations might thus evolve differently, in particular in their expression features, if maintained
under different environmental conditions.
Rapid evolution of retrotransposon regulatory sequences is also
illustrated by the observation that the Tnt1 family is composed of
three different subfamilies, Tnt1A, Tnt1B and Tnt1C, highly similar
in their coding domains, but completely different in their regulatory

U3 sequences22. The three subfamilies appeared after a period of
evolution in which regulatory sequences diverged widely while
flanking regions remained more constant. The stress-activated
Tnt1 elements cloned after transposition in tobacco are members
of the Tnt1A subfamily, which are predominant in tobacco. However, whether divergences in the regulatory sequences between
the three subfamilies are linked to differences in their expression
features remains to be determined. Interestingly, the three subfamilies were present before Nicotiana speciation, but are differently distributed in each Nicotiana species.
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Why do retrotransposons contain regulatory motifs similar to
those of cellular genes?

As with plant retrotransposons, many animal or yeast retroviral-like
elements contain motifs similar to regulatory sequences of cellular
genes, and several hypotheses have been advanced to explain this:
• Ancestral captures of cellular 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 genes25 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 drift26.
• Appearance of retrotransposon regulatory sequences by convergent evolution; for instance, the high U3 plasticity of the
Tnt1 family would by itself explain the appearance of stressrelated regulatory sequences in the Tnt1A subfamily.
None of the above hypotheses is completely satisfactory: the
first implies that evolutionary patterns should be different between
regulatory and coding sequences, which is not the case for Tnt1
(Ref. 22), and the presence in Tnt1A of several regulatory motifs
found associated with a diversity of defence genes in many plant
species appears incompatible with the two others. However, the
possibility that these and other mechanisms might have combined
with those of their host during retrotransposon co-evolution, creating the present situation, cannot be excluded. Characterization of
LTR evolutionary features in other retrotransposon families would
provide further information on the origin and fate of retrotransposon regulatory regions.
The putative biological impact of retrotransposon activation
by stress

Transposable elements are a major source of genetic variation that
ranges from gross chromosomal alterations up to very fine tuning
of the expression of cellular genes2. This, together with the observation that transposons are activated by stress and environmental
changes, led to the hypothesis that transposable elements are
involved in host adaptation to environmental changes27,28. In particular, through modifications of gene regulation, transposable elements have been proposed as major factors in macroevolution26.
However, no clear example of an important biological role for such
a modification in a natural population has yet been provided, and
the question as to whether transposable elements are selfish parasitic sequences or pacemakers of evolution is still controversial2.
In the light of this debate, the maintenance of retrotransposon
stress activation might be purely fortuitous, and could have been
driven by random choice within highly variable element populations. However, the transcriptional features and regulatory sequences of elements such as Tnt1A or Tto1 appear too specific to
endorse this hypothesis in full. The situation is perhaps best summarized by Freeling29:
‘For those of us who still trust our senses, the design and behavior of every living thing evidences an adaptedness born of
exploitation. I cannot imagine a living system not exploiting
transposons for something once they existed.’
Whatever the origin of Tnt1 and Tto1 regulatory sequences,
they might have been maintained because they confer a selective advantage, either to the host plant, such as through stress adaptation,
or to the element itself. In particular, stress activation might allow
elements to move only in rare situations, without major effects on
host viability, a prerequisite for their own survival. In addition,
the activation of retrotransposons during pathogen attack could
increase the possibility of horizontal transmission between plants
186

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and pathogens, allowing the element to colonize new hosts. The true
answer might lie between these extremes, and different elements
could have been maintained for very different reasons. Further research, in particular a demonstration that stress-induced transposition events can be transmitted to the progeny in natural situations,
will be necessary. One argument against any positive impact of
stress-activated retrotransposons on evolution derives from the
observation that the stress-activated Tnt1A subfamily is not the
predominant Tnt1 subfamily in all Nicotiana species22: the Tnt1B
and Tnt1C subfamilies are predominant in the related species
Nicotiana plumbaginifolia, for instance. However, although Tnt1B
and Tnt1C are apparently not expressed in tobacco protoplasts,
members of the Tnt1B subfamily were isolated after their transposition in protoplast-derived cell cultures of N. plumbaginifolia
(C. Meyer, unpublished). This suggests that other Tnt1 subfamilies might also be activated by stress, through pathways that might
differ and could be more precisely adapted to the particular species in which they predominate. All Tnt1 subfamilies might thus
have been maintained as a consequence of their similar overall
biological impact, each subfamily being preferentially amplified
in a different Nicotiana genome to enable a better adaptation to its
host and to its particular environmental history.
Conclusions and future prospects

A variety of retrotransposons has now been characterized in
higher plants, and a clearer picture of their genomic organization,
as well as of their evolution, is emerging. However, the control of
their transposition cycle, from transcription up to the integration of
the daughter copy, is not well understood. Further studies, in particular characterization of additional active plant retrotransposons,
will be necessary in order better to understand their impact on
plant genomes. The activity of class II elements has been easily
traced in plants partly because of their conservative mechanism of
transposition, which results in frequent somatic instabilities. As
retrotransposon insertions are stable, many plant retrotransposons
were obtained by chance as inactivated insertions. A fruitful strategy based on the high conservation of some retrotransposon coding
domains, originally developed in tobacco8, has allowed the isolation of retrotransposon sequences from their transcription products, thus increasing the probability that they represent sequences
from active copies (Box 1). Several studies have now been started
in different species to isolate retrotransposon sequences expressed
in specific stress conditions. This strategy could be applied to any
plant species and will undoubtedly allow the characterization of
new active elements, as well as provide important information on
their conditions of expression and mobility.
The regulatory features of the most well-characterized plant
retrotransposons indicate that these elements represent sensitive
markers of plant stress and could be used for different biotechnological applications. Their promoters could be used to drive transgene expression in strategies devised for the creation of plant
resistance to pathogens, and might also be interesting tools for
studying the plant defence response (e.g. in screening the stress
signals). In addition, the fusion of LTR regions to reporter genes
could provide sensitive indicators of the plant response to environmental stress or to agrochemicals and pollutants. A major
advantage of retrotransposon sequences over the plant defence
gene promoters usually exploited for these studies is that retrotransposon regulatory features are carried by compact regions.
Since transposable elements can remain active after insertion into
different regions of the genome, it would also be interesting to
determine whether their expression patterns are less sensitive to
the local genomic environment than transgenes driven by cellular
gene promoters.

trends in plant science
reviews
Many years have passed since Barbara McClintock’s seminal
analysis of variegated maize kernels and her description of regulated mobile DNA sequences. Transposon research has largely
been fuelled by the promise of new applications, such as genetagging and the creation of transformation vectors, but the natural
behaviour of these elements remains an intrinsically exciting
research theme. It has implications for a better understanding of
various areas: the control of gene expression; genome evolution
and speciation; the idea of the environment influencing genome
structure; and the possibility that the activity of plant retrotransposons is directly linked to defence responses.
Acknowledgements

We are very grateful to Z. Avramova, T. Brown, H. Hirochika,
C. Kinlaw, A. Kumar, H. Laten, H. Lucas, C. Meyer and S. Wessler
for preprints and unpublished information, and to H. Lucas for
critically reviewing the manuscript.
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Marie-Angèle Grandbastien is at the Laboratoire de Biologie
Cellulaire, Institut National de la Recherche Agronomique (INRA),
Centre de Versailles, 78026 Versailles cedex, France
(tel +33 1 30 83 30 24; fax +33 1 30 83 30 99;
e-mail gbastien@versailles.inra.fr).

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