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Functional genomics in Arabidopsis: large-scale insertional
mutagenesis complements the genome sequencing project
Serguei Parinov and Venkatesan Sundaresan*
The ultimate goal of genome research on the model flowering
plant Arabidopsis thaliana is the identification of all of the
genes and understanding their functions. A major step towards
this goal, the genome sequencing project, is nearing
completion; however, functional studies of newly discovered
genes have not yet kept up to this pace. Recent progress in
large-scale insertional mutagenesis opens new possibilities for
functional genomics in Arabidopsis. The number of T-DNA and
transposon insertion lines from different laboratories will soon
represent insertions into most Arabidopsis genes. Vast
resources of gene knockouts are becoming available that can
be subjected to different types of reverse genetics screens to
deduce the functions of the sequenced genes.
Addresses
Institute of Molecular Agrobiology, 1 Research Link, The National
University of Singapore, Singapore 117604
*e-mail: director@ima.org.sg
Current Opinion in Biotechnology 2000, 11:157–161
0958-1669/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
Ac/Ds Activator/Dissociation
En/Spm Enhancer/Suppressor-mutator
I
Inhibitor
Introduction
Arabidopsis thaliana has been the subject of intense
research for over a decade as a model organism for the molecular genetics of flowering plants because of its short
generation time, small genome and high gene density. A
very important step of this research is the Arabidopsis
Genome Initiative (AGI) to determine the genome
sequence of Arabidopsis. Currently, over 80% of the predicted 130 Mb of Arabidopsis genome has been sequenced,
including complete sequences for two out of five
Arabidopsis chromosomes, 2 and 4 [1,2]. It is expected that
the entire genome sequencing will be completed before
the end of the year 2000, thus making this the first genome
of a flowering plant to be sequenced.
The utility of the genome-sequencing project relies significantly on the prediction of coding regions in the genome.
Much of the genomic sequences of bacterial artificial chromosome (BAC) and P1 clones submitted to GenBank are
annotated with the results of gene prediction, produced by
a combination of different computer algorithms. Such
annotation is often a short summary and it does not always
unambiguously reflect the real gene sequences.
Nevertheless, half of the predicted genes match expressed
sequence tag (EST) and cDNA sequences [3]. Alternative
sources of gene annotation are also being developed. For
example, the Arabidopsis Genome Displayer (KAOS;
http://www.kazusa.or.jp/kaos) and the Arabidopsis Genome
Annotation Database at the Institute for Genomic
Research (http://www.tigr.org/tdb/ath1/htmls/ath1/html)
present recent updates of the annotation for sequenced
Arabidopsis clones. The convenient graphical outputs provided by these sites help users to make independent
decisions about the gene structure in a selected region of
genome by comparison of the predictions made with different gene prediction algorithms.
According to the analysis of sequenced genomic regions on
chromosomes 2, 4 and 5 [1–3], ~50% of the Arabidopsis
genome encodes genes, with an average density of about
one gene per 4.5 kb. Therefore, the complete ~130 Mb
Arabidopsis genome should carry about 25,000–30,000
genes, most of which have not been studied yet. The completion of the genome project, therefore, opens a new
frontier for functional genomics. The accumulation of
sequence and functional information about Arabidopsis
genes will promote the application of reverse genetics
methodologies to decipher the functions of novel genes
that are homologous to known genes, or to study unknown
genes that interact biochemically (e.g. in a yeast twohybrid assay) with a gene of known function. This review
covers recent progress in the application of large-scale
insertional mutagenesis strategies for functional genomics
in Arabidopsis.
Gene inactivation: site-specific versus random
insertional mutagenesis
The complete inactivation of a gene is generally the most
direct way to understand its function. Unfortunately, there
are no effective methods for targeted gene replacement in
flowering plants. Nevertheless, development of novel
tools for site-specific mutagenesis continues. Beetham
et al. [4], for example, successfully used self-complementary chimeric oligonucleotides to introduce site-specific
base substitution in a nuclear gene of Nicotiana tabacum.
This approach, however, has to be improved significantly
in terms of efficiency for use in functional genomics.
Approaches such as antisense and co-suppression have
been used effectively to inactivate genes of known
sequence [5]. These strategies are also not efficient
enough to be used on a large scale, and presently are mostly limited to the study of single genes.
Currently, the most effective alternative strategy is random
large-scale insertional mutagenesis. The high gene density
[3] in the Arabidopsis genome is particularly favorable for
random mutagenesis, as every second insertion will disrupt
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a gene sequence. The insertion of large T-DNA or transposon constructs that are several kb in length most often
leads to complete inactivation of a gene. Because the
sequence of the insertion is known, primers complementary to the end of insertion and to the gene of interest can
be used for PCR screening of insertion collections to identify the plants carrying an insertion within the gene of
interest. Alternatively, the disrupted gene can be identified directly by sequencing of the DNA flanking each
insertion [6]. The completion of the genome-sequencing
project will enable any short flanking sequence to be used
to find the exact position of the insert within sequenced
genomic DNA, which then can be analyzed to identify the
disrupted gene.
Systems for insertional mutagenesis
T-DNA as a mutagen
Agrobacterium-mediated T-DNA transformation has been
used for insertional mutagenesis in different plant species.
Plant transformation by Agrobacterium results in the integration into the nuclear genome of a sequence called
T-DNA, which is carried on a bacterial plasmid. As the use
of T-DNA as a mutagen in Arabidopsis has recently been
reviewed in detail elsewhere [7••], only a brief summary is
presented here. Large collections of T-DNA transformants
of Arabidopsis have been collected independently by several groups and have been used extensively for reverse
genetics [7••,8,9]. One advantage of using T-DNA as an
insertional mutagen is that it directly generates stable
insertions into genomic DNA and does not require additional steps to stabilize the insert. A second advantage is
that T-DNA insertion appears to be completely random, as
no T-DNA integration hot spots or integration preferences
have been reported. On the other hand complex patterns
of T-DNA integration, including transfer of vector
sequences adjacent to T-DNA borders and the large frequency of concatemeric T-DNA insertions, can complicate
further PCR analysis for reverse genetics [10,11]. Small
and major chromosomal rearrangements induced by
T-DNA integration have been frequently observed, leading to difficulties in the genetic analysis of the insertion,
such as mutant phenotypes that are not correlated with the
T-DNA insertion [12,13]. Nevertheless, the development
of highly efficient methods of T-DNA transformation for
Arabidopsis [7••,14] has made it feasible for laboratories to
generate thousands of transformants relatively rapidly, and
as a result T-DNA is now a widely used insertional mutagen in Arabidopsis.
Transposon mutagenesis
Two systems of heterologous transposable elements, both
originating in maize, have been successfully used for insertional mutagenesis in Arabidopsis: Activator/Dissociation
(Ac/Ds) and Enhancer/Suppressor-mutator (En/Spm). There
are two advantages of using the transposons for insertional
mutagenesis. First, unlike T-DNA, a transposon can be
excised from the disrupted gene in the presence of transposase, with the resulting reversion of the mutation. This
provides a simple means to confirm that an observed mutation is tagged by the transposon [15,16••,17••].
Second, most transposition events occur preferentially to
linked sites [18,19]. A transposon insertion library generated from any donor transposon will therefore be enriched for
insertions into the adjacent genes. If there is a transposable
element near to the gene of interest, it can be efficiently
mobilized to reinsert within the gene. This strategy has
been successfully applied by Ito et al. [19] and Seki et al.
[20] for regional Ac/Ds insertional mutagenesis of the genes
from CIC7E11/8B11 and 5CIC5F11/CIC2B9 loci on
Chromosome 5. These authors focused their attention on
the cDNAs specific to these chromosomal regions, which
were selected using a cDNA-scanning method that allows
one to selectively clone cDNAs from any small region of
genome. PCR analysis of DNA flanking the insertions
showed that 14–20% of transpositions were actually located
within the target regions, each representing about 1 Mb of
genomic DNA surrounding the Ds donor sites.
On the other hand, preferential short-range transposition is
an obvious drawback if the target goal is to achieve random
saturation of genome with insertions. To address this problem, a two-element Ac/Ds system was developed in which
negative selection against closely linked transpositions was
employed [21••]. Simultaneous negative selection against
the Ac transposase permitted selection for plants that carry
stable Ds insertions. Using this system, two major Ds transposition hot spots located in the narrow regions adjacent to
nucleolar organizer regions on chromosome 2 and 4 were
found, indicating that these regions of the genome are
preferential targets for Ds insertions. It has been demonstrated that Ds elements transpose preferentially to 5′
regions of genes, therefore increasing the chances of
obtaining complete loss-of-function mutations, which are
simpler to interpret.
A similar system that utilizes En/Spm transposable elements was designed by Tissier et al. [22••] in order to
simplify the process of generating transposants. In this system, selectable markers that allow selection of soil-grown
plants were employed to reduce the labor associated with
growing plants on media and transplanting. Another modification is that in contrast to the Ac/Ds system described
above in which the initial transformants carrying Ac and Ds
are maintained as separate lines [21••], the mobile dSpm is
combined together with the Spm transposase in the same
T-DNA so that negative selection has to be applied against
only a single locus, thereby reducing the number of progeny to be screened by a factor of four. In this system, it is
not possible to maintain the primary transformants as
defined starter lines, because the dSpm elements will be
continually transposing in the presence of Spm transposase. Instead of maintaining and using several starter
lines, a large number of independent primary T-DNA
transformants were generated as sources of transpositions.
Availability of simple T-DNA transformation methods
Functional genomics in Arabidopsis Parinov and Sundaresan
makes this strategy feasible in Arabidopsis, though not in
most other plants.
The two systems described above were designed to select
predominantly stable single insertions. Hence further
mutant analysis of these lines is relatively straightforward.
Also, each individual insertion line can be rapidly characterized by direct sequencing and analysis of DNA flanking
the insertion [21••,22••]. Notwithstanding the above
advantages, it is necessary to generate more than 100,000
individual insertion lines for saturation of the genome.
An alternative approach for insertional mutagenesis using
plants carrying multiple insertions of a mobile Inhibitor I
(dSpm) element has been used, which permits genome
saturation using a relatively low number of lines [16••].
Seven ‘founder’ lines were used as starters each carrying
7–10 independent inserts of I (dSpm) and a source of En
(Spm) transposase that is required for transposition of the I
(dSpm) element. After several generations, a ‘multipletransposon population’ of 2,592 lines, each carrying
20–25 I elements, was generated, corresponding to
50,000–65,000 insertions in total. Because of the high gene
density in Arabidopsis, 20 insertions in a plant will probably
result in inactivation of multiple genes, which could result
in complex mutant phenotypes. Nevertheless, due to the
extensive functional redundancy in the Arabidopsis
genome [7••,17••,23], this method can be effectively utilized for studies of genes that produce unambiguous
phenotypes when knocked out, as demonstrated by
Speulman et al. [16••]. The presence of the En transposase
in these lines allows the identification of revertants within
the same generation. Characterization of lines that carry
multiple insertions of mobile transposons frequently
requires many extra steps compared to single-copy insertion lines, especially when the phenotype of a gene
knockout is not clear. For stabilization of the insertion, as
well as for reduction of transposon copy number, these
lines would have to be subjected to subsequent crosses to
wild-type plants. The genetic analysis of the observed
mutants is also complicated by the presence of multiple
insertions and transposon footprints that result in gene
inactivation with no associated transposon. In addition, a
background of somatic insertions complicates PCR screening strategies to identify gene knockouts, as some of these
insertions can generate the correct PCR products, but will
not be germinally transmitted. Some of these drawbacks
could be overcome by generating a much larger collection
of multiple transposon insertion lines, which would
increase the probability of having several independent
insertions into the same gene, which can be used for confirmation of the mutant phenotypes.
Screening strategies for reverse genetics
For effective reverse genetics, it is preferable to have as
large a collection of insertions as possible. Assuming a
130 Mb genome size, it is estimated that at least 120,000
random insertions are required to tag any 5 kb Arabidopsis
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gene with 99% probability. The screening of such collections in order to obtain an insertion in a particular gene of
interest is usually performed using PCR-based approaches.
Two basic strategies have been applied for the screening of
insertion lines.
The first so-called ‘pooling strategy’ is to combine 20–100
insertion lines together to form a pool. DNA extracted from
these pools is then used to perform the PCR screening
using gene- and insert-specific primers [8,17••,22••]. It
reduces the work associated with DNA preparation and
PCR screening by a factor equal to number of lines in a
pool. Individual lines from the PCR-positive pool can be
subsequently examined to identify the line carrying the
required insertion. As the number of pools required for
genome saturation is also high (for example 1000 pools of
100 lines each for a collection of 100,000 insertions), DNA
from pools can also be combined into superpools and the
PCR screening can be performed in stages [7••,22••].
Alternatively, plants can be pooled according to a threedimensional gridding strategy that further reduces the
number of DNA pools for the PCR screen [16••]. As an
example, a collection of 1000 insertion lines can be pooled
into a grid consisting of 10 rows, 10 columns and 10 blocks,
so that an individual line is identified by an address of one
row, one column and one block. Thus only 30 DNA samples have to be prepared and subjected to PCR to identify
the exact line carrying the desired insertion within the population of 1000. A modified format of the pooling strategy
called ‘inverse display of insertions’, designed to compact
and simplify distribution of the DNA libraries, has also
been tested [17••,22••]. In this method, the multiple fragments flanking the insertions from the DNA pools are
amplified by PCR, and the products immobilized on membranes. The screening is then performed by hybridization
of the DNA of interest with the membranes carrying the
array of DNA samples. The advantage of pooling strategies
is that they are not very labor intensive and they provide a
fast short-term resource for researchers planning to perform
their own PCR screening of available collections.
An alternative strategy is for sequences flanking every individual insert to be amplified and sequenced and placed in a
catalogue [21••,22••]. In this case, screening for a mutant
line is simplified to a search for a corresponding insertion
site in the catalogue. Obviously, this strategy can only be
used for insertions that can be stably propagated. The largescale application of this strategy requires much more time
and effort than the pooling strategy. Random sequencing of
every insertion line can be convenient and economically
attractive over the long run, however, because it provides
precise information and eliminates the subsequent labor
associated with DNA distribution and PCR screening. The
detailed analysis of every insertion site can be omitted if all
flanking sequences are organized into a basic local alignment search tool (BLAST) dataset, so that the actual
screening operation is a BLAST search of such a dataset for
a sequence identical to the sequence of a given gene.
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Current availability of insertions covers at
least half of all Arabidopsis genes
Large collections of T-DNA and transposon insertion
lines have recently been produced independently by different laboratories. An important point is that the number
of DNA samples extracted from these lines is also substantial. For example, DNA pools of 48 from 35,000
T-DNA insertion lines (55,000 inserts) [8], 960 DNA
pools (also combined into large superpools) representing
48,000 independent stable dSpm transposants [22••], and
pools of DNA from 2592 multiple insertion lines (representing 50,000 to 65,000 inserions of I[dSpm]) [16••] have
been generated. Many of these lines have been sent to
The Arabidopsis Biological Resource Center (ABRC) and
Nottingham Arabidopsis Stock Center (NASC).
Additionally, an Arabidopsis knockout facility has recently
been established at the University of Wisconsin to provide access for the PCR screening of 60,480 T-DNA
insertion lines [7••]. In parallel, identification of the stable insertion positions by sequencing and analysis of
DNA flanking the inserts has also progressed
[16••,20,21••,22••]. For example, a database of 1200 dSpm
Sequenced Insertions Sites (SINS) from pools of transposants [22••], and a database of 500 individual Ds insertion lines with identified positions [21••] have been
established. Both databases are expected to expand significantly in the near future.
A practical evaluation of some of the available collections
of DNA samples for reverse genetics screening has been
performed by Meissner et al. [17••]. They used PCR- and
hybridization-based methods to identify insertions in
Arabidopsis genes of the MYB family of transcription factors. The collection of insertion lines they used for
screening included 13,264 T-DNA lines, 28,800 stable
dSpm insertion lines, 8000 lines containing ~50,000
autonomous En/Spm insertions, and 2592 En/dSpm lines
containing 20–25 mobile dSpm insertions. In total, the
screened lines represent roughly 150,000 insertion events
that are estimated to hit 85% of the ~1.6 kb long MYB
genes, assuming a random distribution of inserts. In fact,
47 insertions into 36 genes out of a total of 73 MYB members (50%) were isolated. Assuming the median size of an
Arabidopsis gene is 2.1 kb [7••] these results indicate that
effective saturation of more than 50% of genes was
achieved in the experimental population.
Conclusions
The intensive efforts in the past few years to undertake
insertional mutagenesis in Arabidopsis are beginning to make
a major impact on the functional genomics of this plant.
Large numbers of T-DNA and transposon insertion lines
have been generated by different groups, and if combined
together these collections may already represent insertions
into most Arabidopsis genes [7••,8,9,16••,21••,22••,24,25].
The Arabidopsis Biological Resource Center (ABRC) of the
Ohio State University (http://aims.cps.msu.edu/aims) [24] in
cooperation with Nottingham Arabidopsis Stock Center
(NASC; http://nasc.nott.ac.uk) [25] are organizing the collection, preservation and distribution of transgenic seeds
acquired from these sources. DNA sample collection from
some of these lines has also been initiated in order to enable
screening for insertions in particular genes of interest by
PCR [7••,8,16••,22••] or hybridization [22••]. As an alternative to PCR screening, databases of insertion sites have been
established by laboratories that are sequencing DNA flanking the insertions [21••,22••].
Because of the high gene density, insertional mutagenesis
is a powerful tool in Arabidopsis — every second insertion
can be found within an Arabidopsis gene. Despite growing
collections of insertions, saturation insertional mutagenesis
may remain an elusive goal, especially for small targetgene sizes. For example, to find an insertion in a 1 kb gene
with 99% probability, 600,000 insertion lines would need
to be generated and screened [7••].
In cases where the desired insertion is not found by
screening the available collections of insertions, a gene can
still be tagged effectively using a closely linked transposon
insertion [19,20,21••]. The donor line containing the closely linked transposable element can be selected from the
collections of mapped transposons using the sequence
databases [21••,22••] and crossed with plants expressing
the transposase to remobilize the element.
The above resources build a strong foundation for further functional characterization of inactivated genes.
Many Arabidopsis genes demonstrate different levels of
functional redundancy, whereas others are only needed
for survival in specific conditions, such as resistance to
specific biological or environmental stresses [7••]. As a
result, most of the insertion lines do not demonstrate
clear morphological phenotypes when grown and examined under standard conditions [7••,8,17••,23]. The
redundancy problem can be addressed by constructing
double mutants with closely related genes. Identification
of knockouts in such genes by either the pooling strategy or the flanking sequence database strategy will be an
important step in this approach. In the case of conditional phenotypes, examination of mutant plants under a
variety of experimental conditions will be required to
decipher gene function [7••]. In this case, homology of
the disrupted gene to known genes can help to predict
the proper experimental conditions. In either case, functional studies of the genes will be greatly facilitated by
knowledge of their expression patterns and regulation.
The use of gene/enhancer trap elements can provide this
information for individual genes [23,26]. On a larger
scale, this information will be considerably enhanced by
the use of new technologies such as microarrays (see
Schaffer et al., this issue pp 162–167).
Acknowledgements
We thank the National Science and Technology Board of Singapore for
supporting our research.
Functional genomics in Arabidopsis Parinov and Sundaresan
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Functional genomics in Arabidopsis: large-scale insertional
mutagenesis complements the genome sequencing project
Serguei Parinov and Venkatesan Sundaresan*
The ultimate goal of genome research on the model flowering
plant Arabidopsis thaliana is the identification of all of the
genes and understanding their functions. A major step towards
this goal, the genome sequencing project, is nearing
completion; however, functional studies of newly discovered
genes have not yet kept up to this pace. Recent progress in
large-scale insertional mutagenesis opens new possibilities for
functional genomics in Arabidopsis. The number of T-DNA and
transposon insertion lines from different laboratories will soon
represent insertions into most Arabidopsis genes. Vast
resources of gene knockouts are becoming available that can
be subjected to different types of reverse genetics screens to
deduce the functions of the sequenced genes.
Addresses
Institute of Molecular Agrobiology, 1 Research Link, The National
University of Singapore, Singapore 117604
*e-mail: director@ima.org.sg
Current Opinion in Biotechnology 2000, 11:157–161
0958-1669/00/$ — see front matter
© 2000 Elsevier Science Ltd. All rights reserved.
Abbreviations
Ac/Ds Activator/Dissociation
En/Spm Enhancer/Suppressor-mutator
I
Inhibitor
Introduction
Arabidopsis thaliana has been the subject of intense
research for over a decade as a model organism for the molecular genetics of flowering plants because of its short
generation time, small genome and high gene density. A
very important step of this research is the Arabidopsis
Genome Initiative (AGI) to determine the genome
sequence of Arabidopsis. Currently, over 80% of the predicted 130 Mb of Arabidopsis genome has been sequenced,
including complete sequences for two out of five
Arabidopsis chromosomes, 2 and 4 [1,2]. It is expected that
the entire genome sequencing will be completed before
the end of the year 2000, thus making this the first genome
of a flowering plant to be sequenced.
The utility of the genome-sequencing project relies significantly on the prediction of coding regions in the genome.
Much of the genomic sequences of bacterial artificial chromosome (BAC) and P1 clones submitted to GenBank are
annotated with the results of gene prediction, produced by
a combination of different computer algorithms. Such
annotation is often a short summary and it does not always
unambiguously reflect the real gene sequences.
Nevertheless, half of the predicted genes match expressed
sequence tag (EST) and cDNA sequences [3]. Alternative
sources of gene annotation are also being developed. For
example, the Arabidopsis Genome Displayer (KAOS;
http://www.kazusa.or.jp/kaos) and the Arabidopsis Genome
Annotation Database at the Institute for Genomic
Research (http://www.tigr.org/tdb/ath1/htmls/ath1/html)
present recent updates of the annotation for sequenced
Arabidopsis clones. The convenient graphical outputs provided by these sites help users to make independent
decisions about the gene structure in a selected region of
genome by comparison of the predictions made with different gene prediction algorithms.
According to the analysis of sequenced genomic regions on
chromosomes 2, 4 and 5 [1–3], ~50% of the Arabidopsis
genome encodes genes, with an average density of about
one gene per 4.5 kb. Therefore, the complete ~130 Mb
Arabidopsis genome should carry about 25,000–30,000
genes, most of which have not been studied yet. The completion of the genome project, therefore, opens a new
frontier for functional genomics. The accumulation of
sequence and functional information about Arabidopsis
genes will promote the application of reverse genetics
methodologies to decipher the functions of novel genes
that are homologous to known genes, or to study unknown
genes that interact biochemically (e.g. in a yeast twohybrid assay) with a gene of known function. This review
covers recent progress in the application of large-scale
insertional mutagenesis strategies for functional genomics
in Arabidopsis.
Gene inactivation: site-specific versus random
insertional mutagenesis
The complete inactivation of a gene is generally the most
direct way to understand its function. Unfortunately, there
are no effective methods for targeted gene replacement in
flowering plants. Nevertheless, development of novel
tools for site-specific mutagenesis continues. Beetham
et al. [4], for example, successfully used self-complementary chimeric oligonucleotides to introduce site-specific
base substitution in a nuclear gene of Nicotiana tabacum.
This approach, however, has to be improved significantly
in terms of efficiency for use in functional genomics.
Approaches such as antisense and co-suppression have
been used effectively to inactivate genes of known
sequence [5]. These strategies are also not efficient
enough to be used on a large scale, and presently are mostly limited to the study of single genes.
Currently, the most effective alternative strategy is random
large-scale insertional mutagenesis. The high gene density
[3] in the Arabidopsis genome is particularly favorable for
random mutagenesis, as every second insertion will disrupt
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a gene sequence. The insertion of large T-DNA or transposon constructs that are several kb in length most often
leads to complete inactivation of a gene. Because the
sequence of the insertion is known, primers complementary to the end of insertion and to the gene of interest can
be used for PCR screening of insertion collections to identify the plants carrying an insertion within the gene of
interest. Alternatively, the disrupted gene can be identified directly by sequencing of the DNA flanking each
insertion [6]. The completion of the genome-sequencing
project will enable any short flanking sequence to be used
to find the exact position of the insert within sequenced
genomic DNA, which then can be analyzed to identify the
disrupted gene.
Systems for insertional mutagenesis
T-DNA as a mutagen
Agrobacterium-mediated T-DNA transformation has been
used for insertional mutagenesis in different plant species.
Plant transformation by Agrobacterium results in the integration into the nuclear genome of a sequence called
T-DNA, which is carried on a bacterial plasmid. As the use
of T-DNA as a mutagen in Arabidopsis has recently been
reviewed in detail elsewhere [7••], only a brief summary is
presented here. Large collections of T-DNA transformants
of Arabidopsis have been collected independently by several groups and have been used extensively for reverse
genetics [7••,8,9]. One advantage of using T-DNA as an
insertional mutagen is that it directly generates stable
insertions into genomic DNA and does not require additional steps to stabilize the insert. A second advantage is
that T-DNA insertion appears to be completely random, as
no T-DNA integration hot spots or integration preferences
have been reported. On the other hand complex patterns
of T-DNA integration, including transfer of vector
sequences adjacent to T-DNA borders and the large frequency of concatemeric T-DNA insertions, can complicate
further PCR analysis for reverse genetics [10,11]. Small
and major chromosomal rearrangements induced by
T-DNA integration have been frequently observed, leading to difficulties in the genetic analysis of the insertion,
such as mutant phenotypes that are not correlated with the
T-DNA insertion [12,13]. Nevertheless, the development
of highly efficient methods of T-DNA transformation for
Arabidopsis [7••,14] has made it feasible for laboratories to
generate thousands of transformants relatively rapidly, and
as a result T-DNA is now a widely used insertional mutagen in Arabidopsis.
Transposon mutagenesis
Two systems of heterologous transposable elements, both
originating in maize, have been successfully used for insertional mutagenesis in Arabidopsis: Activator/Dissociation
(Ac/Ds) and Enhancer/Suppressor-mutator (En/Spm). There
are two advantages of using the transposons for insertional
mutagenesis. First, unlike T-DNA, a transposon can be
excised from the disrupted gene in the presence of transposase, with the resulting reversion of the mutation. This
provides a simple means to confirm that an observed mutation is tagged by the transposon [15,16••,17••].
Second, most transposition events occur preferentially to
linked sites [18,19]. A transposon insertion library generated from any donor transposon will therefore be enriched for
insertions into the adjacent genes. If there is a transposable
element near to the gene of interest, it can be efficiently
mobilized to reinsert within the gene. This strategy has
been successfully applied by Ito et al. [19] and Seki et al.
[20] for regional Ac/Ds insertional mutagenesis of the genes
from CIC7E11/8B11 and 5CIC5F11/CIC2B9 loci on
Chromosome 5. These authors focused their attention on
the cDNAs specific to these chromosomal regions, which
were selected using a cDNA-scanning method that allows
one to selectively clone cDNAs from any small region of
genome. PCR analysis of DNA flanking the insertions
showed that 14–20% of transpositions were actually located
within the target regions, each representing about 1 Mb of
genomic DNA surrounding the Ds donor sites.
On the other hand, preferential short-range transposition is
an obvious drawback if the target goal is to achieve random
saturation of genome with insertions. To address this problem, a two-element Ac/Ds system was developed in which
negative selection against closely linked transpositions was
employed [21••]. Simultaneous negative selection against
the Ac transposase permitted selection for plants that carry
stable Ds insertions. Using this system, two major Ds transposition hot spots located in the narrow regions adjacent to
nucleolar organizer regions on chromosome 2 and 4 were
found, indicating that these regions of the genome are
preferential targets for Ds insertions. It has been demonstrated that Ds elements transpose preferentially to 5′
regions of genes, therefore increasing the chances of
obtaining complete loss-of-function mutations, which are
simpler to interpret.
A similar system that utilizes En/Spm transposable elements was designed by Tissier et al. [22••] in order to
simplify the process of generating transposants. In this system, selectable markers that allow selection of soil-grown
plants were employed to reduce the labor associated with
growing plants on media and transplanting. Another modification is that in contrast to the Ac/Ds system described
above in which the initial transformants carrying Ac and Ds
are maintained as separate lines [21••], the mobile dSpm is
combined together with the Spm transposase in the same
T-DNA so that negative selection has to be applied against
only a single locus, thereby reducing the number of progeny to be screened by a factor of four. In this system, it is
not possible to maintain the primary transformants as
defined starter lines, because the dSpm elements will be
continually transposing in the presence of Spm transposase. Instead of maintaining and using several starter
lines, a large number of independent primary T-DNA
transformants were generated as sources of transpositions.
Availability of simple T-DNA transformation methods
Functional genomics in Arabidopsis Parinov and Sundaresan
makes this strategy feasible in Arabidopsis, though not in
most other plants.
The two systems described above were designed to select
predominantly stable single insertions. Hence further
mutant analysis of these lines is relatively straightforward.
Also, each individual insertion line can be rapidly characterized by direct sequencing and analysis of DNA flanking
the insertion [21••,22••]. Notwithstanding the above
advantages, it is necessary to generate more than 100,000
individual insertion lines for saturation of the genome.
An alternative approach for insertional mutagenesis using
plants carrying multiple insertions of a mobile Inhibitor I
(dSpm) element has been used, which permits genome
saturation using a relatively low number of lines [16••].
Seven ‘founder’ lines were used as starters each carrying
7–10 independent inserts of I (dSpm) and a source of En
(Spm) transposase that is required for transposition of the I
(dSpm) element. After several generations, a ‘multipletransposon population’ of 2,592 lines, each carrying
20–25 I elements, was generated, corresponding to
50,000–65,000 insertions in total. Because of the high gene
density in Arabidopsis, 20 insertions in a plant will probably
result in inactivation of multiple genes, which could result
in complex mutant phenotypes. Nevertheless, due to the
extensive functional redundancy in the Arabidopsis
genome [7••,17••,23], this method can be effectively utilized for studies of genes that produce unambiguous
phenotypes when knocked out, as demonstrated by
Speulman et al. [16••]. The presence of the En transposase
in these lines allows the identification of revertants within
the same generation. Characterization of lines that carry
multiple insertions of mobile transposons frequently
requires many extra steps compared to single-copy insertion lines, especially when the phenotype of a gene
knockout is not clear. For stabilization of the insertion, as
well as for reduction of transposon copy number, these
lines would have to be subjected to subsequent crosses to
wild-type plants. The genetic analysis of the observed
mutants is also complicated by the presence of multiple
insertions and transposon footprints that result in gene
inactivation with no associated transposon. In addition, a
background of somatic insertions complicates PCR screening strategies to identify gene knockouts, as some of these
insertions can generate the correct PCR products, but will
not be germinally transmitted. Some of these drawbacks
could be overcome by generating a much larger collection
of multiple transposon insertion lines, which would
increase the probability of having several independent
insertions into the same gene, which can be used for confirmation of the mutant phenotypes.
Screening strategies for reverse genetics
For effective reverse genetics, it is preferable to have as
large a collection of insertions as possible. Assuming a
130 Mb genome size, it is estimated that at least 120,000
random insertions are required to tag any 5 kb Arabidopsis
159
gene with 99% probability. The screening of such collections in order to obtain an insertion in a particular gene of
interest is usually performed using PCR-based approaches.
Two basic strategies have been applied for the screening of
insertion lines.
The first so-called ‘pooling strategy’ is to combine 20–100
insertion lines together to form a pool. DNA extracted from
these pools is then used to perform the PCR screening
using gene- and insert-specific primers [8,17••,22••]. It
reduces the work associated with DNA preparation and
PCR screening by a factor equal to number of lines in a
pool. Individual lines from the PCR-positive pool can be
subsequently examined to identify the line carrying the
required insertion. As the number of pools required for
genome saturation is also high (for example 1000 pools of
100 lines each for a collection of 100,000 insertions), DNA
from pools can also be combined into superpools and the
PCR screening can be performed in stages [7••,22••].
Alternatively, plants can be pooled according to a threedimensional gridding strategy that further reduces the
number of DNA pools for the PCR screen [16••]. As an
example, a collection of 1000 insertion lines can be pooled
into a grid consisting of 10 rows, 10 columns and 10 blocks,
so that an individual line is identified by an address of one
row, one column and one block. Thus only 30 DNA samples have to be prepared and subjected to PCR to identify
the exact line carrying the desired insertion within the population of 1000. A modified format of the pooling strategy
called ‘inverse display of insertions’, designed to compact
and simplify distribution of the DNA libraries, has also
been tested [17••,22••]. In this method, the multiple fragments flanking the insertions from the DNA pools are
amplified by PCR, and the products immobilized on membranes. The screening is then performed by hybridization
of the DNA of interest with the membranes carrying the
array of DNA samples. The advantage of pooling strategies
is that they are not very labor intensive and they provide a
fast short-term resource for researchers planning to perform
their own PCR screening of available collections.
An alternative strategy is for sequences flanking every individual insert to be amplified and sequenced and placed in a
catalogue [21••,22••]. In this case, screening for a mutant
line is simplified to a search for a corresponding insertion
site in the catalogue. Obviously, this strategy can only be
used for insertions that can be stably propagated. The largescale application of this strategy requires much more time
and effort than the pooling strategy. Random sequencing of
every insertion line can be convenient and economically
attractive over the long run, however, because it provides
precise information and eliminates the subsequent labor
associated with DNA distribution and PCR screening. The
detailed analysis of every insertion site can be omitted if all
flanking sequences are organized into a basic local alignment search tool (BLAST) dataset, so that the actual
screening operation is a BLAST search of such a dataset for
a sequence identical to the sequence of a given gene.
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Current availability of insertions covers at
least half of all Arabidopsis genes
Large collections of T-DNA and transposon insertion
lines have recently been produced independently by different laboratories. An important point is that the number
of DNA samples extracted from these lines is also substantial. For example, DNA pools of 48 from 35,000
T-DNA insertion lines (55,000 inserts) [8], 960 DNA
pools (also combined into large superpools) representing
48,000 independent stable dSpm transposants [22••], and
pools of DNA from 2592 multiple insertion lines (representing 50,000 to 65,000 inserions of I[dSpm]) [16••] have
been generated. Many of these lines have been sent to
The Arabidopsis Biological Resource Center (ABRC) and
Nottingham Arabidopsis Stock Center (NASC).
Additionally, an Arabidopsis knockout facility has recently
been established at the University of Wisconsin to provide access for the PCR screening of 60,480 T-DNA
insertion lines [7••]. In parallel, identification of the stable insertion positions by sequencing and analysis of
DNA flanking the inserts has also progressed
[16••,20,21••,22••]. For example, a database of 1200 dSpm
Sequenced Insertions Sites (SINS) from pools of transposants [22••], and a database of 500 individual Ds insertion lines with identified positions [21••] have been
established. Both databases are expected to expand significantly in the near future.
A practical evaluation of some of the available collections
of DNA samples for reverse genetics screening has been
performed by Meissner et al. [17••]. They used PCR- and
hybridization-based methods to identify insertions in
Arabidopsis genes of the MYB family of transcription factors. The collection of insertion lines they used for
screening included 13,264 T-DNA lines, 28,800 stable
dSpm insertion lines, 8000 lines containing ~50,000
autonomous En/Spm insertions, and 2592 En/dSpm lines
containing 20–25 mobile dSpm insertions. In total, the
screened lines represent roughly 150,000 insertion events
that are estimated to hit 85% of the ~1.6 kb long MYB
genes, assuming a random distribution of inserts. In fact,
47 insertions into 36 genes out of a total of 73 MYB members (50%) were isolated. Assuming the median size of an
Arabidopsis gene is 2.1 kb [7••] these results indicate that
effective saturation of more than 50% of genes was
achieved in the experimental population.
Conclusions
The intensive efforts in the past few years to undertake
insertional mutagenesis in Arabidopsis are beginning to make
a major impact on the functional genomics of this plant.
Large numbers of T-DNA and transposon insertion lines
have been generated by different groups, and if combined
together these collections may already represent insertions
into most Arabidopsis genes [7••,8,9,16••,21••,22••,24,25].
The Arabidopsis Biological Resource Center (ABRC) of the
Ohio State University (http://aims.cps.msu.edu/aims) [24] in
cooperation with Nottingham Arabidopsis Stock Center
(NASC; http://nasc.nott.ac.uk) [25] are organizing the collection, preservation and distribution of transgenic seeds
acquired from these sources. DNA sample collection from
some of these lines has also been initiated in order to enable
screening for insertions in particular genes of interest by
PCR [7••,8,16••,22••] or hybridization [22••]. As an alternative to PCR screening, databases of insertion sites have been
established by laboratories that are sequencing DNA flanking the insertions [21••,22••].
Because of the high gene density, insertional mutagenesis
is a powerful tool in Arabidopsis — every second insertion
can be found within an Arabidopsis gene. Despite growing
collections of insertions, saturation insertional mutagenesis
may remain an elusive goal, especially for small targetgene sizes. For example, to find an insertion in a 1 kb gene
with 99% probability, 600,000 insertion lines would need
to be generated and screened [7••].
In cases where the desired insertion is not found by
screening the available collections of insertions, a gene can
still be tagged effectively using a closely linked transposon
insertion [19,20,21••]. The donor line containing the closely linked transposable element can be selected from the
collections of mapped transposons using the sequence
databases [21••,22••] and crossed with plants expressing
the transposase to remobilize the element.
The above resources build a strong foundation for further functional characterization of inactivated genes.
Many Arabidopsis genes demonstrate different levels of
functional redundancy, whereas others are only needed
for survival in specific conditions, such as resistance to
specific biological or environmental stresses [7••]. As a
result, most of the insertion lines do not demonstrate
clear morphological phenotypes when grown and examined under standard conditions [7••,8,17••,23]. The
redundancy problem can be addressed by constructing
double mutants with closely related genes. Identification
of knockouts in such genes by either the pooling strategy or the flanking sequence database strategy will be an
important step in this approach. In the case of conditional phenotypes, examination of mutant plants under a
variety of experimental conditions will be required to
decipher gene function [7••]. In this case, homology of
the disrupted gene to known genes can help to predict
the proper experimental conditions. In either case, functional studies of the genes will be greatly facilitated by
knowledge of their expression patterns and regulation.
The use of gene/enhancer trap elements can provide this
information for individual genes [23,26]. On a larger
scale, this information will be considerably enhanced by
the use of new technologies such as microarrays (see
Schaffer et al., this issue pp 162–167).
Acknowledgements
We thank the National Science and Technology Board of Singapore for
supporting our research.
Functional genomics in Arabidopsis Parinov and Sundaresan
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