Review

Transposons as tools for functional genomics

Srinivasan Ramachandran, Venkatesan Sundaresan*
Institute of Molecular Agrobiology, National University of Singapore, 1, Research Link, Singapore 117 604, Singapore
Received 19 December 2000; accepted 25 January 2001
Abstract Transposons have been used extensively for insertional mutagenesis in several plant species. These include species
where highly active endogenous systems are available such as maize and Antirrhinum majus, as well as species where
heterologous transposons have been introduced through transformation, such as Arabidopsis thaliana and tomato. Much of the
past use of transposons has been in traditional forward genetics approaches, to isolate and molecularly characterize genes
identied by mutant phenotypes. With the rapid progress in the genome projects of different plants, large-scale transposon
mutagenesis has become an important component of functional genomics, permitting assignment of functions to sequenced
genes through reverse genetics. Different strategies can be pursued, depending upon the properties of the transposon such as the
mechanism and control of transposition, and those of the host plant such as transformation efficiency. The successful use of these
strategies in A. thaliana has made it possible to develop databases for reverse genetics, where screening for the knockout of a
gene of interest can be performed by computer searches. The extension of these technologies to other plants, particularly
agronomically important crops such as rice, is now feasible. 2001 ditions scientiques et mdicales Elsevier SAS
gene tagging / reverse genetics / transposons
Ac, Activator element / Ds, Dissociation element / dSpm, defective Suppressor-mutator element / En, Enhancer / FST,
anking sequence tag / I, Inhibitor / Mu, Mutator / Spm, Suppressor-mutator
1. INTRODUCTION
The advent of large-scale sequencing projects in
several plants, together with the anticipated comple-
tion of whole genome sequences for Arabidopsis
thaliana and possibly rice as well, has resulted in an
explosion of gene sequence information in plants. As
more and more sequences are available in the data-
bases, it becomes critical to assign functions to the
thousands of new genes identied. There are several
approaches being attempted to tackle this problem.
The most direct approach to determine the functions
of the sequenced genes in an organism is to disrupt or
generate mutations in the genes and analyse the
consequences. Methods that have been developed in
plants for this purpose include gene replacement, sense
and anti-sense suppression, and insertional mutagen-
esis. Although recently it has been shown that targeted
gene replacement by homologous recombination is
possible in A. thaliana, the frequency is very low [39],
which makes the method laborious for generating large
numbers of gene knockouts. Recently, a nuclear gene
of Nicotiana tobaccum has been mutated by site-
specic base substitution using self-complementary
chimeric oligonucleotides [9] but this method is also
too labour intensive for use in functional genomics.
Strategies for sense and anti-sense suppression have
been developed for the inactivation of known genes [8,
37] but as these strategies require generation of several
independent transgenic lines for each gene they are
currently limited to the study of single genes. Cur-
rently the most widely used approach for large-scale
gene function analysis in plants is random insertional
mutagenesis. Either T-DNA (reviewed in [2, 42]) or
transposons (reviewed in [50, 77, 84]) can be used as
insertional mutagens in plants.
Insertional mutagenesis using Agrobacterium medi-
ated T-DNA integration into plant genomes (primarily
in A. thaliana, and more recently in rice) has proved to
be very successful [2, 34, 42]. This approach has the
advantage of simplicity as each transformant yields a
stable insertion in the genome and does not need
additional steps to stabilize the insert. Several groups
have used this approach in A. thaliana to generate tens
of thousands of independent lines that can be used for
reverse genetics. Recently, a National A. thaliana
Knockout Facility has been established at the Univer-
*Correspondence and reprints: fax +65 872 7007.
E-mail address: director@ima.org.sg (V. Sundaresan).
Plant Physiol. Biochem. 39 (2001) 243252
2001 ditions scientiques et mdicales Elsevier SAS. All rights reserved
S0981942801012438/REV
sity of Wisconsin, USA, with access to 60 480 inser-
tion lines [42]. Modied T-DNA insertions have been
used in A. thaliana as gene [3], promoter traps [46]
and in activation tagging [85]. Recently, Jeon et al.
[34] have also been using T-DNA insertions for
functional genomics in rice. Despite the extremely
successful use of T-DNA in A. thaliana, there are
however a few disadvantages to this approach. The
integration of the T-DNA is generally complex, result-
ing in tandem direct and inverted repeats and deletions
in one or more borders. Such rearrangements can make
the subsequent molecular analysis difficult in many
cases, and adversely affect the success of large-scale
strategies such as anking sequence databases
(described later). Secondly, complex and multiple
insertions are more likely to lead to artefactual patterns
of reporter gene expression when using entrapment
vectors such as gene and enhancer traps. Finally, while
the T-DNA approach is extremely useful for plant
species where quick and efficient transformation meth-
ods are available, it may not be feasible in those plant
species where the transformation methods are slow or
labour intensive.
For these reasons, insertional mutagenesis using
transposable elements offers some advantages over
T-DNAmutagenesis. The insertions generated by trans-
posons are generally single intact elements, which lend
themselves easily to molecular analysis. Such inser-
tions are also less likely to result in artefactual patterns
of expression if the transposon is being used as a gene
trap or enhancer trap. An additional advantage is that
many transposons can be excised from the disrupted
gene in the presence of transposase. Such excisions
can result in phenotypic reversion to the wild type or
give rise to alleles with weaker phenotypes. This
property of many transposons provides ready conr-
mation that the mutation was really tagged by the
transposon, as well as the possibility of generating an
allelic series. In addition, another property of several
transposons to preferentially insert into genetically
linked sites [7, 33, 36], can be used to perform local
mutagenesis in a particular region of interest by
re-mobilizing the transposon [31, 35, 69]. Finally,
transposons can be used for insertional mutagenesis in
plant species where transformation is inefficient, since
the generation of new insertions occurs through cross-
ing or propagation rather than through transformation.
In this review, we discuss the most widely used
transposons and howthese elements have been exploited
successfully in heterologous plant species as an inser-
tional mutagen and as a tool for reverse genetics for
functional genomics.
2. ENDOGENOUS TRANSPOSABLE
ELEMENTS IN DIFFERENT PLANT SPECIES
Transposable elements like Activator (Ac),
Suppressor-mutator/Enhancer (Spm/En) and Mutator
(Mu) were originally discovered and molecularly char-
acterized in maize (table I [15, 16, 52, 53, 63, 64, 67]).
Subsequently, a number of endogenous transposable
elements in other plant species that are similar or
distantly related to the maize Ac element have been
identied including Tam3 in Antirrhinum majus [14,
28], Tag1 in A. thaliana ecotype Landsberg [79] and
slide1 in tobacco [25]. Also Spm/En-like transposable
elements have been reported in A. majus (Tam1 [58]),
rice (Tnr3 [56]) and in Petunia (Psl [72]).
Endogenous transposable elements have been used
to clone genes in maize, A. majus and Petunia hybrida.
In maize, all three families (Ac, Spm and Mu) of
transposable elements have been widely used as tags
to isolate genes (reviewed in [24, 83]). The Tam
elements from A. majus (reviewed in [24]) and the
dTph elements from P. hybrida [73] have proved
similarly valuable in their host species. Some families
of endogenous elements are present in high-copy
numbers in their hosts. For example elements of the
Mu family in maize and dTph family in P. hybrida
exist in more than 100 copies per genome. This is very
advantageous for large-scale mutagenesis, as a few
thousand lines will be sufficient to cover the whole
genome. On the other hand, there are a few disadvan-
tages in using high-copy number endogenous trans-
posons. First, since there is a number of insertions per
line, there will be a continuous transposition of ele-
ments. If there was a signicant frequency of germinal
excisions, this would result in mutations that are due to
the excision footprints, and therefore not tagged.
Table I. Endogenous transposons in different plants.
Element Plant Similar to Reference
Activator, Ac Maize [15, 16, 52]
Tam3 Antirrhinum
majus
Ac of maize [14, 28]
Tph1 Petunia hybrida Ac of maize [23]
Tag1 Arabidopsis
thaliana
Distantly related
to Ac of maize
[19, 79]
Slide Tobacco Ac of maize [25]
Spm/En Maize [53, 63, 64, 65]
Tam1 A. majus Spm/En [58]
Tnr3 Rice Spm/En [56]
Psl P. hybrida Spm/En [72]
Mutator Maize [67]
244 S. Ramachandran, V. Sundaresan / Plant Physiol. Biochem. 39 (2001) 243252
Secondly, conrmation that a mutation is indeed caused
by a particular copy of the transposon may require
several out-crosses to remove other elements, which is
a time consuming process. Finally, the use of gene/
enhancer trap elements is not feasible, as the patterns
of expression due to several insertions of the elements
per plant will be additive and therefore inaccurate.
One of the most successfully used high-copy num-
ber transposon families is the Mu transposable element
family consisting of the autonomous element MuDR
and the non-autonomous elements Mu1 to Mu8
(reviewed in [83]). These elements have been used to
clone several genes in maize [10]. The Mu elements
have been particularly effective tool for gene tagging
for the following reasons: (a) higher forward mutation
frequency when compared to Ac or Spm element (20 to
50 % higher) (reviewed in [83, 84]); (b) equal trans-
position to linked and unlinked sites when compared
to other transposable elements [47]; (c) characteristi-
cally late excisions which stabilizes new alleles in
sibling progeny and reduces the probability of foot-
print mutations. However, transfer of the Mu system
into heterologous plant species has not yet been
successfully accomplished.
Retrotransposons, which transpose through a RNA
intermediate, have been shown to generate spontane-
ous mutations in maize [82] and are the major class of
transposable elements in most plants (reviewed in
[11]). Retrotransposons are potentially very useful for
gene tagging as these elements generate stable inser-
tions, and unlike many DNA transposons they inte-
grate into unlinked sites. However, the relatively low
frequencies of transpositions of most plant retrotrans-
posons have restricted their uses for large-scale gene
tagging. Recently however, the Tos17 retrotransposon
has been developed as a promising system for rice
[29]. The endogenous rice Tos17 retrotransposons
appear to be inactive during normal growth conditions,
but they are reactivated by tissue culture, resulting in
high transposition frequencies suitable for insertional
mutagenesis [29].
3. TRANSPOSONS
IN HETEROLOGOUS SYSTEMS
The rst report that maize transposable element
Ac/Ds can be active in a heterologous system came
from studies with transgenic tobacco (Nicotiana tobac-
cum) by Baker et al. [4, 5]. Since then, the maize
Ac/Ds elements have been shown to transpose actively
and been exploited in tagging studies in a number of
heterologous species including A. thaliana, rice, tomato,
P. hybrida, ax, carrot, lettuce, potato, etc. (table II).
Similarly maize Spm/dSpm (En/I) has been utilized
successfully in tobacco, potato and A. thaliana (table
II). Apart from maize transposable elements, the A.
thaliana transposon Tag1 which is distantly related to
the maize Ac, has been shown to be active in tobacco
[19] and in rice [48].
In order to better regulate the transpositional events
and obtain stable insertions, two-component systems
are preferred. In a typical two-component system, a
transgenic line will be generated which harbours an
immobilized (wings clipped) autonomous element
(Ac or Spm/En) that will provide the transposase
source. Second line transgenic for the non-autonomous
element (Ds or dSpm/I), which cannot transpose unless
the transposase source is available, will be generated.
Selectable markers such as antibiotic resistance or
herbicide resistance markers can be engineered in the
non-autonomous elements to select for the presence of
transposed elements. In order to monitor the transpo-
sition events, the non-autonomous transposon can be
inserted between a promoter and the marker gene so
that excision results in expression of the selectable
marker gene (reviewed in [76]). Plants homozygous
for transposase and for the dependent element are used
as starter lines and crossed to facilitate transposition.
Lines carrying stable single insertions can be obtained
subsequently by segregation of the transposase source,
which can be simplied by the use of negative
selection markers [76] (gure 1B).
The ability of Spm/En elements to amplify through
continued propagation in A. thaliana has been exploited
to increase the number of insertions per plant [74, 88].
Table II. Transposons activity in heterologous plants.
Plant of origin Transposon
type
Activity in
heterologous
plants
Reference
Maize Ac/Ds A. thaliana [7, 81]
Rice [32, 57, 70]
Tomato [35, 54, 90]
Petunia [13, 26, 66]
Flax [17, 44]
Tobacco [5, 18, 86]
Carrot [81]
Lettuce [89]
Potato [40]
Maize Spm/En A. thaliana [1, 12]
Potato [21]
Tobacco [51, 62]
Arabidopsis Tag1 Rice [48]
S. Ramachandran, V. Sundaresan / Plant Physiol. Biochem. 39 (2001) 243252 245
In one strategy, the autonomous Spm/En element was
transformed into A. thaliana and propagated for ve
generations by a single seed descent. This yielded
15 000 insertions in 3 000 lines [88]. Alternatively, a
two component (in-cis) En-I (Spm/dSpm) system has
been introduced into A. thaliana, where a mobile I
(dSpm) element and an immobilized trans-active En
(Spm) transposase are contained in the same T-DNA.
This in-cis element system was used to generate
2 592 A. thaliana lines containing multiple insertions
(approx. twenty inserts/line) of mobile I element [74].
This approach of amplifying the number of insertions
per plant has the very signicant advantage that it
permits near-saturation mutagenesis of a plant genome
with a relatively small number of plants. However,
there are also some disadvantages in using this
approach, arising from the continuous nature of the
transposition events. First, the PCR strategies to iden-
tify a gene knockout are complicated by somatic
insertion events that will result in the detection of
insertions which are not transmitted through the germ
line. Second, the presence of footprints in genes due to
imprecise excisions will lead to mutations that are not
tagged.
Most transposable elements, including Ac/Ds and
En/Spm, have a tendency to preferentially transpose to
genetically linked sites [7, 33, 36]. This feature can be
advantageous for directed tagging of a specic target
gene or for performing local (regional) insertional
mutagenesis in a selected region of a chromosome
when a transposable element is inserted close to the
target gene or within the derived chromosomal region
(gure 1A) [31, 35, 69]. In another instance, Ac/Ds
transposons and cDNA scanning methods were used
together to perform regional insertional mutagenesis
on genes from CIC7E11/8B11 and 5CIC5F11/CIC2B9
loci on A. thaliana chromosome V [31, 69]. This
allows cloning of cDNAs from a small region in the
genome. The anking sequences of insertions showed
that 1420 % of the transpositions were located in
about 1 Mb of genomic DNA surrounding Ds donor
sites.
When random saturation mutagenesis is desired, the
high fraction of closely linked site transpositions poses
a serious limitation. In principle, this limitation can be
overcome by using many starter lines, with Ds/dSpm
insertions distributed evenly over different chromo-
somes. Alternately, in the Ac-Ds system of Sundaresan
et al. [77], the propensity of Ds elements to transpose
to linked sites is overcome by selection against the
donor site using a negative selection marker. By
simultaneous selection against the transposase source
and the donor site, a population of stable, unlinked,
transposon insertion lines can be generated (gure 1B)
[61, 77]. In the modied Spm/En system designed by
Tissier et al. [78], the Spm transposase and the mobile
dSpm elements are contained within the same T-DNA
transformed into A. thaliana. This system eliminates
the need for crossing, and also reduces the number of
progeny required for the selection by a factor of four,
as the negative selection is applied against only a
single locus as opposed to two loci when the trans-
posase is introduced separately. However, the mainte-
nance of starter lines becomes problematic, as the
dSpm elements will continually transpose in the pres-
Figure 1. Schematic diagram of different approaches to transposon
tagging. In both A and B, the Ds element is mobilized in the presence
of Ac transposase. A, Directed tagging; B, random tagging. 1, 2 and 3
represent different chromosomes, and X, Y, Z different genes. C,
Insertion of gene trap or enhancer trap element. SA, splice acceptor;
TA, minimal promoter; E, enhancer sequence in the genome; X,
unknown gene and GUS, glucuronidase. D, Insertion of activation
tagging element. X denotes an unknown gene. TE, transposable
element; LB, left border of T-DNA; RB, right border of T-DNA; PSM,
positive selection marker; NSM, negative selection marker.
246 S. Ramachandran, V. Sundaresan / Plant Physiol. Biochem. 39 (2001) 243252
ence of the Spm transposase, so that this system
requires a relatively large number of independent
starter lines obtained through transformation.
4. SPECIALIZED TAGGING SYSTEMS
4.1. Gene and enhancer trap elements
Insertional mutagenesis by transposon tagging is
useful when disruption of a gene leads to an obvious
phenotype. But in eukaryotic systems, disruptions of
genes frequently do not result in visible phenotypes
due to functional redundancy, or they may result in
early lethality that obscures late-acting functions when
the same gene has multiple functions in development.
To overcome these difficulties, modied transposons
called gene trap or enhancer trap transposable ele-
ments have been developed (reviewed in [43, 75, 76]).
Such modied elements were rst used in Drosophila
melanogaster and mouse [71, 87], and were subse-
quently extended to plant transposon systems [7577].
Gene/enhancer trap elements carry a reporter gene
whose expression pattern reects the activity and
regulation of the disrupted genes (gure 1C). The
enhancer traps contain a reporter gene driven by a
minimal or weak promoter in the dependent element.
The reporter gene expression is achieved (by utilizing
the endogenous enhancer sequences) only when the
transposition occurs within or close to a gene (gure
1C). A variation of this vector called a promoter trap
contains a promoterless reporter gene, which will
express only when the transposon is inserted down-
stream of an active endogenous plant promoter. In
contrast, gene trap vectors are designed to contain one
or more splice acceptor sequences preceding the reporter
gene. This allows expression of the reporter only when
it inserts in a transcribed region. The splice donor sites
from the intron of the endogenous gene and the splice
acceptor sequences, which precede the reporter gene,
will be spliced to generate a fusion transcript (reviewed
in [50, 75, 76]). Thus far only the Ac/Ds transposon
system has been modied for enhancer and gene trap
approaches.
4.2. Activation tagging elements
Gene disruptions through insertional mutagenesis
nearly always generate recessive loss-of-function muta-
tions. Such mutations do not always produce an
obvious phenotype due to various factors such as
functional redundancy (see next section). In such
cases, increasing the expression level or ectopically
expressing a gene can provide dominant gain-of-
function mutations that produce informative mutant
phenotypes. A strategy to generate mutants as a con-
sequence of increased or expanded expression of a
tagged gene is known as activation tagging. The
principle involves using strong enhancers in T-DNAor
a transposon resulting in ectopic expression or over-
expression of nearby genes through transcriptional
activation. The rst successful activation tagging in
plants was reported by Hayashi et al. [27], who
introduced four copies of enhancer elements from the
cauliower mosaic virus 35S promoter (CaMV 35S)
into T-DNA, generating gain-of-function mutations
and novel phenotypes. This approach has been used to
generate a large-scale tagging population by Wiegel et
al. [85]. The principle of activation tagging has also
been adapted for transposable elements by Wilson et
al. [87]. They used the Ds element with a complete
CaMV 35S promoter pointing outwards from the
element (gure 1D), and generated a population of
insertions through crosses with Ac transposase. This
approach was used to identify dominant mutations at
various loci in A. thaliana, including TINY, Late
Elongated Hypocotyl (LHY), and Short Internodes
(SHI) [22, 68, 87]. A limitation of this approach is that
the observed frequency of dominant mutations through
activation tagging has been signicantly lower than
that of recessive mutations arising from insertional
inactivation [85], suggesting that many genes may be
over-expressed without resulting in observable pheno-
types. Nevertheless, activation tagging has proven to
be a valuable complementary approach for the identi-
cation of gene functions.
5. FORWARD VS. REVERSE GENETICS
Cloning of genes that produce mutant phenotype or
function forms the basis of forward genetics (i.e.
phenotype or function to gene). On the other hand, if
the gene sequence is known and the biological func-
tion of that gene is not known, a knockout mutant can
be generated and analysed to determine its function.
This forms the basis for reverse genetics, i.e. from
gene to phenotype or function (reviewed in [42, 50,
60]). Since genome sequencing projects for various
plant species are progressing rapidly, more and more
sequences encoding predicted genes are available in
the databases. Reverse genetics strategies will be of
great importance for the purpose of assigning func-
tions to predicted genes. As discussed earlier, gene
disruption by transposons constitutes a powerful tool
for reverse genetics.
S. Ramachandran, V. Sundaresan / Plant Physiol. Biochem. 39 (2001) 243252 247
6. STRATEGIES
FOR REVERSE GENETICS SCREENING
In order to perform reverse genetic screens effi-
ciently, it is necessary to generate a large population of
transposon tagged mutants. The number of lines to be
screened is dependent on the genome size and the
number of genes of a given plant species, the type of
transposon used (single or multi-copy) etc. A popula-
tion containing insertions with a reasonable probabil-
ity of nding at least one insertion in any given gene
is important for the success of this approach.
In order to screen for an insertion in a particular
gene, a PCR-based strategy has been applied in D.
melanogaster for site-specic selection of insertions
using P elements [6, 38]. In this approach, a gene-
specic primer and an insertion-specic primer are
used for PCR amplication. Several insertion lines are
pooled together and the DNA extracted is then used as
a template for PCR reactions. Samples of 20 to
100 insertion lines are pooled to extract genomic
DNA, and a gene-specic primer and an insertion-
specic primer were used for PCR [55, 78]. Any pool
showing a positive signal is re-screened using DNA
from individual lines, to identify the line carrying the
insertion of interest. Several pools can be combined to
form a super pool if the number of insertion lines in
the population is very high [78]. Alternatively a
three-dimensional matrix pooling strategy has been
tested for P. hybrida lines carrying the multi-copy
dTph1 transposon [41]. The leaf material from a
population of 1 000 plants were pooled according to a
three-dimensional matrix (columns, rows and blocks).
DNA samples were extracted from ten blocks of
100 lines. Similarly DNA is extracted from ten col-
umns and ten rows each of them containing 100 lines.
Altogether thirty DNA samples were used to perform
PCR, which can identify a single plant that contains
the insertion in the specic gene. This method is
advantageous as it requires fewer amplications, and
directly identies the single plant with insertion (gure
2 [41]).
An alternate approach to identify the genes that
have been tagged in a population of insertion lines
involves random amplication of the DNA anking
the insertions. Several different methods can be fol-
lowed within this approach. Transposon display [80]
and amplication of insertion mutagenized sites (AIMS)
[20] are techniques used to identify tagged genes in
families with multi-copy transposons like dTph1 in
Petunia and Mu in maize. For example, the maize Bx1
gene and the Fbp1 gene fragment from Petunia were
isolated by this method [20, 80]. The transposon
display and AIMS techniques utilize adapters that are
ligated to the genomic DNA, which has been digested
with restriction enzyme(s). The appropriate restriction
enzyme(s) are selected which recognizes one site in
the insertion element and the other in the anking
region. A chimeric adapter and transposon primer is
used from one end and an adapter primer from the
other end to amplify the anking region in the trans-
poson display technique. On the other hand, adapter
primers are used as forward and reverse primers for
amplication in AIMS approach. Flanking regions
obtained by amplications are displayed which can be
cloned and sequenced subsequently ([20, 80] and
reviewed in [50]).
A method called inverse display of insertions (IDI)
has been developed by Tissier et al. [78] to isolate the
anking sequences spanning the dSpm/I element. The
inverse PCR (iPCR) method [59] has been adapted for
Figure 2. Flow chart for the use of insertional mutants in a reverse
genetics approach to functional genomics. The steps involved from the
identication of an insertion in a gene of interest to the phenotypic
characterization of the mutant are detailed.
248 S. Ramachandran, V. Sundaresan / Plant Physiol. Biochem. 39 (2001) 243252
this strategy. Since the selection of restriction sites (not
too close or too far from the insertion site) is a critical
factor for a successful iPCR, three different restriction
enzymes were used to greatly increase the probability
of amplifying the anking region. DNA pools from
insertion lines were used as template and iPCR reac-
tions were performed. These products were mixed and
spotted in grid arrays on a nylon membrane, and
hybridized with labelled probes from the genes of
interest [78].
Alternatively, the DNA anking the insertions can
be amplied and sequenced individually to catalogue
the insertions by chromosomal location [61, 78]. This
strategy is especially useful when substantial genome
sequence information is available, as in the case of A.
thaliana. A protocol such as iPCR, ligation-mediated
PCR or thermal asymmetric interlaced PCR (TAIL-
PCR; [49]) can be used to amplify and sequence the
anking region of single- or low-copy insertion lines.
With an appropriately constructed database of such
anking sequences, it will be feasible to identify
insertions in a particular gene by simple computer
searches, removing the necessity for the more tedious
pooling and hybridization protocols. In addition, even
if no hit is found, the availability of a transposon
insertion close to a gene of interest can be quickly
ascertained by a computer search. As described previ-
ously, transposons from the Ac/Ds and Spm/En fami-
lies are very useful for mutagenesis of closely linked
genes, and a collection of sequenced insertions pro-
vides launch pads for tagging most of the genes within
the genome.
7. GENE SEQUENCE
TO FUNCTIONAL ANALYSIS
Once a knockout in a desired gene is identied
through reverse genetics strategies among the popula-
tion of insertional mutants, it becomes necessary to
functionally characterize the mutant (gure 2). The
rst step in the characterization process is to obtain a
mutant that has a single insertion in the gene of
interest. If a two-component transposable element
system (Ac/Ds for example) has been used in generat-
ing a population of insertion mutants, then obtaining
single insertion line is rather straight forward. On the
other hand, if multi-copy transposable elements are
used to perform the reverse genetics screen, then it is
necessary to do several out-crosses to make sure that
only a single insertion is in the gene. The next step for
characterization is the identication of phenotypes
caused by gene knockout. If there is an observable
phenotype caused by the single stable insertion, then
the gene functions may be deduced through detailed
analysis of the phenotype (gure 2). However, there
are many instances, where a gene knockout does not
show an observable phenotype. This could be due to
the fact that several genes may be required and
expressed only under specic conditions, such as
pathogen infection or environmental stresses. For muta-
tions in such genes, in order to detect a phenotype it
will be necessary to subject the plants to conditions in
which the gene is required. These conditions may
include challenges with pathogens and the use of
specic growth media or growth conditions. For
example, a T-DNA insertion in the AKT-1 potassium
channel gene of A. thaliana was identied by a reverse
genetics approach using pooled PCR and hybridiza-
tion. On most nutritional media, the akt-1 mutant
plants were indistinguishable from wild type plants,
but on media containing a low potassium concentra-
tion of 100 M or less, the growth of the mutant plants
was defective [30].
Another reason for not observing a clear phenotype
when a gene is knocked out is functional redundancy
with other genes. In such cases, creation of double or
triple mutants of the functionally redundant genes will
uncover the phenotype and permit characterization of
their functions (gure 2). For example, A. thaliana
genes SHATTERPROOF1 (SHP1) and SHATTER-
PROOF2 (SHP2) regulate fruit dehiscence or pod
shatter. Both genes are functionally redundant, as
neither single mutant produces a novel phenotype.
However, shp1 and shp2 double mutants produce fruits
or pods that do not shatter [45]. Since the A. thaliana
genome-sequencing project is almost complete and the
sequences are available, it is feasible to identify all the
closely related members of a gene family in this
species. Together with computer searches of anking
sequence databases, it will soon be possible to con-
struct combinations of mutants to reveal novel func-
tions that have gone undetected using the current
genetics methodologies.
8. CONCLUSION
In the post-genome era, sequences of many plant
genomes will be available and functional assessment
of the genes identied will depend on many approaches
including insertional mutagenesis, polymorphism
analysis, expression microarrays, and bioinformatics.
A large population of insertional mutants generated by
transposon mutagenesis can be used to dissect out
S. Ramachandran, V. Sundaresan / Plant Physiol. Biochem. 39 (2001) 243252 249
gene functions in combination with the sequences
obtained fromthe ESTand genome sequencing projects
in many plant species, through various strategies such
as pooled PCR screening and the construction of
anking sequence databases. Recent advances demon-
strate the feasibility of using transposon-based func-
tional genomics approaches for the study of any
species of owering plant.
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