Arabidopsis thaliana as an Introductory article

Experimental Organism . Introduction

Article Contents

Maarten Koornneef, Wageningen Agricultural University, Wageningen, The Netherlands . Description of the Development

Ben Scheres, Utrecht University, Utrecht, The Netherlands . Embryo and Seed Development
. Life Cycle
. Genetic Characteristics
Arabidopsis thaliana has become the model for plant genetics and plant development
. Genetic and Physical Maps
because of its genetic characteristics, such as short generation time, the small size of its
. Gene Transfer (Transformation) in Arabidopsis
genome and the availability of its complete DNA sequence.
. Recovery of Mutations and Functional Analysis of
Genes

Introduction . Gene Cloning in Arabidopsis

. Examples of Plant Research where the Genetic and
Molecular Approaches using Arabidopsis were
Arabidopsis thaliana (L.) Heyhn. is a small weed plant Important
belonging to the mustard family (Brassicaceae or Crucifer-
ae). The species can be found in nature almost everywhere
in the northern hemisphere in ruderal sites, such as sandy
patches along roads etc. Arabidopsis has been found from
sea level up to high in the Himalayas and from northern Description of the Development
Scandinavia to North Africa, including the Cape Verde
Islands at 168 latitude. It also grows in North America, Arabidopsis seeds are small (0.5 mm), oval-shaped and
probably following introduction from Europe. This might produced in large numbers (up to a few thousand per
also have been the origin for plants found in Australia and plant). Seeds can be germinated easily, although freshly
maybe even in Japan. harvested seeds may need a cold treatment of a few days
Arabidopsis was first suggested as a suitable model for and/or a period of storage to germinate fully, because they
plant biological studies, and especially genetics, in the are dormant. The seeds usually require light for germina-
1940s. The reasons for this were its small size, the ease with tion. The degree of dormancy can differ between geno-
which it can be grown, its self-fertilizing habit and the short types. Seedlings are small, with two cotyledons at opposite
generation time of many accessions (isolates), which are positions. They do not contain the small unicellular but
often called ecotypes in Arabidopsis. In greenhouse or in branched hairs or trichomes, which appear on the surface
climate chambers 6–8 weeks is sufficient time to complete of the true leaves. The latter are present on a nonelongating
the entire life cycle from germination until seed set. stem and thereby form a rosette of leaves. The first true
Furthermore, Arabidopsis has one of the smallest genomes leaves have trichomes only on the adaxial (upper) side;
among higher plants. These factors and the ability to subsequent leaves have them on both sides. In general, the
transform the plant, have made it the favourite plant model leaves are oval-shaped, but variations exist between
for molecular genetic studies to date. The research accessions in petiole morphology, the degree to which
advantages of Arabidopsis became clear in the early 1980s leaves are round or elongated and the serration of the leaf
and led to an upsurge in the international research effort on margins. The number of rosette leaves that are formed
it. The large research community provides additional depends on the genotype and environmental conditions
advantages for research nowadays; important resources and is strongly correlated with the time from germination
are available and their distribution is well organized. Stock to bolting and flowering.
centres in the UK and USA provide seed stocks of mutants Flowering starts with a change in the shape of the shoot
and wild accessions and DNA materials for research. apical meristem from flat to more rounded. Instead of leaf
Information is provided by the Arabidopsis database primordia, the meristem starts producing floral primordia
(TAIR) (see Further Reading for details). The presence of (Figure 1). Soon thereafter the main stem elongates (bolt),
the complete genomic DNA sequence by the end of the year which results in an inflorescence with a main stem that
2000 is another unique research resource. Until recently, carries a number of (cauline) leaves with a reduced number
Arabidopsis was considered to be less suitable for of trichomes on the adaxial sides and having axillary buds
cytogenetic studies. However, when the large pachytene that develop into secondary inflorescences. Higher on the
chromosomes are studied, in combination with in situ inflorescence stems the typical crucifer flowers arise with
hybridization, detailed cytogenetic analysis can be per- four whorls of floral organs. The first whorl has four sepals,
formed. the second one has four white petals, the third whorl has six
stamens and the fourth whorl or centre has two carpels,

ENCYCLOPEDIA OF LIFE SCIENCES © 2001, John Wiley & Sons, Ltd. www.els.net 1
 Arabidopsis thaliana as an Experimental Organism

Figure 2 Establishment of the Arabidopsis body plan in the embryo and

the structure of a primary root. A, apical region; C, central region; B, basal
region; HY, hypophyseal cell; SAM, shoot apical meristem; COT,
cotyledon; H, hypocotyl; ER, embryonic root; RM, root meristem; RMI, root
meristem initials.

greatly from that in many other plant species. A number of

male sterile mutants has been shown to be affected in
anther and especially tapetum development. After fertili-
zation the zygote follows a regular pattern of cell divisions,
which correlate with morphologically defined stages. Fate
mapping and cell ablation studies, however, show that
throughout development these cell divisions do not cause
the segregation of cell fates. Rather, cell identity is based on
continuous positional information in the embryo and in
the developing regions after embryogenesis, the meristems.
The first division of the zygote results in an embryo with a
small apical cell and a longer basal cell, from which the
filamentous suspensor, supporting the embryo, will be
Figure 1 An approximately 4-week-old plant of the frequently used
formed. Specific stages that are distinguished thereafter are
laboratory accession Landsberg erecta. Total plant height is at this stage 15 the octant stage (when the apical cell has given rise to two
cm. The insert shows a single flower. tiers of four cells), the dermatogen stage (when the
protoderm is formed by periclinal cell divisions), the
which are fused into the pistil. The genetic control of globular stage, the heart stage (when cotyledon primordia
flowering and especially the formation of floral organs has become visible) and the torpedo stage. At this stage, cell
been studied extensively and resulted in the so-called ABC division is arrested and further growth, during the so-called
model for flower formation. bend cotyledon and walking stick stages, occurs mainly
through cell expansion. Hereafter the seed-maturation
phase starts. During this period the seeds further accumu-
late food reserves (lipids, sugars, proteins), develop
Embryo and Seed Development desiccation tolerance and become dormant.
In the mature seed the embryo consists of two cotyledons
Embryo development (Figure 2) starts with the fusion of the derived from the upper tier, from which the shoot apical
egg cell present in the embryo sac of the polygonum type meristem (SAM) is also formed. Below this are present the
with a sperm cell (male gamete) deposited by the cotyledon shoulders derived from the upper-lower tier and
germinating pollen grain. Pollen develops from micro- below these, the hypocotyl derived from the lower-lower
spores within the two-lobed anther, which contains four tier from which the root meristem also originates. The tip
locules surrounded by a tapetum layer. This does not differ of the root meristem contains the root cap and quiescent

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 Arabidopsis thaliana as an Experimental Organism

centre which originate from the upper cell of the basal cell, and repeat the pattern of the primary roots, although the
called the hypophysis. cell number in the various layers is more variable.
In addition to the embryo proper, the seed consists of a Root development has been studied extensively by
seed coat or testa and endosperm. The latter develops from careful microscopy, cell lineage and cell-ablation analysis
the fertilization of the central cell, which contains two and a large number of mutants defective in root develop-
haploid nuclei, with the second male gamete and results in a ment have been described and are now being analysed at
triploid endosperm. This endosperm initially develops as a the molecular level.
syncytium which thereafter undergoes cellularization.
Later in seed development the endosperm dies, except the
outermost layer, and is replaced by the growing embryo.
The testa consists of two layers derived from the outer Life Cycle
and inner integument of the ovule. Early in seed develop-
ment, the outer integument consists of two layers. Later on, Depending on genotype and conditions, flower primordia
the cells in the outer layer of this integument produce become visible as early as 2 weeks after germination of the
mucilage, which is secreted by the seeds when they imbibe seeds and fertilization can take place 3 weeks after
water. The second layer of the outer integument, as well as germination in early genotypes. Arabidopsis produces
the two outer layers of the inner integument, are progeny almost exclusively by self-pollination and the
compressed in mature seeds and the remnants of the cell seeds develop from the zygote within the ovule. This
walls are impregnated with flavonoid-derived polymers. process takes 2–3 weeks, resulting in a total minimum
The innermost layer of the inner integument, called generation time of approximately 6 weeks. Although in
endothelium, is clearly distinct in structure from the other laboratory conditions six generations can be obtained per
layers but is also compressed at maturity and contains the year, Arabidopsis in nature probably produces only one
brown tannin-like pigments that give Arabidopsis seeds generation per year. In Europe, most Arabidopsis can be
their brown colour. seen flowering in spring and early summer. These plants
Upon germination the seedling grows and develops from might have germinated in spring (summer annuals) or
its shoot apical and root meristems. The activities of these during the previous fall (winter annuals). The latter are
meristems shape the structure of the mature plant, and they probably those genotypes that are late flowering in
are able to give rise to new structures throughout the greenhouse conditions, but which can respond strongly
lifespan of the plant. to a vernalization treatment that induces flowering. In
The above-ground shoot apical meristem consists of a addition to flowering, the presence of seed dormancy
central zone with slowly dividing cells that replenish the prevents several generations occurring in one year. There
cells leaving the neighbouring peripheral zone. In the are large genetic variations in nature for both flowering
peripheral zone, organ primordia arise. These primordia time and seed dormancy. However, the number of field
can give rise to leaves but also to modified leaf structures observations on the ecology of Arabidopsis is still limited.
such as, for example, the different floral organs. In the axils Very little is known about the distribution of the many
of peripheral organs, new meristems are formed and the small seeds produced by Arabidopsis. In nature, most seeds
time at which these are activated is important in determin- probably remain in the vicinity of the mother plant.
ing the architecture of the plant. Many genes have recently
been identified that play a role in the continuous allocation
of cells from the shoot apical meristem to newly formed
organs. Genetic Characteristics
Underneath the soil, the embryonic (‘primary’) root
meristem gives rise to various root tissues and, at a distance The size of the Arabidopsis genome is estimated to be
behind the meristem to new meristems that will form lateral approximately 130 megabases, organized into five chro-
roots. The structure of the primary root in Arabidopsis mosomes. The estimated total gene number is approxi-
seedlings is very regular and consists above the root cap of mately 25 000, which is close to the number for the worm
an outer layer of epidermis cells, which share a common Caenorhabditis elegans. The chromosomes are built up of
precursor cell with the lateral root cap. A single cortex and mainly unique sequences with, on average, one gene per
a single endodermis each with eight cells derive from 5 kb. Repeat sequences are clustered around the centro-
another precursor cell. Within the endodermis, a single meres and constitute the centromeric heterochromatin,
layer of pericycle cells surrounds the vascular bundle. In observed as condensed regions in chromosome prepara-
longitudinal section these layers form long and regular cell tions. The location of the centromeres is metacentric in
files. Root hairs, involved in solute uptake, develop above chromosomes 1, 3 and 5 and submetacentric in chromo-
the elongation zone as outgrowths of those epidermal cells somes 2 and 4. The latter two chromosomes contain large
that are located in the clefts between adjacent cortical cells. arrays of repeated ribosomal DNA (rDNA) genes at the
Lateral roots are initiated from cells in the pericycle layer end of their short arms. The telomeres consist of specific 7-

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 Arabidopsis thaliana as an Experimental Organism

bp repeats (TTTAGGG) characteristic for many plant tion method in most laboratories. To apply this procedure
species. agrobacteria are infiltrated (under vacuum) into plants
that have just started bolting. Apparently the bacteria can
infect cells that will go on to form gametes. This will result
in the formation of seeds that are transformed and which
Genetic and Physical Maps can be selected for at the seedling stage using the selection
markers mentioned above.
The first consistent map of the five linkage groups was
published in 1983 and was constructed on the basis of
extensive linkage analysis with 76 morphological markers
(mutants). Restriction fragment length polymorphism Recovery of Mutations and Functional
(RFLP) maps became available in 1988 and 1989. Analysis of Genes
Subsequently these RFLP markers and additional markers
based on polymerase chain reaction (PCR) technology As in other model species, the function of genes in
were mapped in a set of recombinant inbred lines (RILs) Arabidopsis is deduced from mutant versions of the genes
derived from a cross between the two most commonly used and the DNA sequences. Mutants are used to clone the
laboratory strains, Landsberg erecta (Ler) and Columbia respective genes (forward genetics) and to connect the
(Col). For the mapping of newly identified mutations sets function (altered in the mutant) with the protein encoded
of polymorphic PCR markers such as simple sequence by the gene. With the availability of the almost complete
length polymorphism (SSLPs or microsatellites) and sequence of genes, sequence data can also become the
cleaved amplified polymorphic sequences (CAPSs) and starting material for the analysis of gene function (reverse
so-called amplified fragment length polymorphism genetics). Searches are done for plants in which a particular
(AFLPTM) markers are available. gene is disrupted. Thereafter these plants are analysed for
The many molecular markers that were mapped served their phenotype. For this procedure DNA is isolated from
as anchors to construct the physical map of Arabidopsis a collection of plants with inserts introduced in plants by
based on genomic DNA cloned in yeast and bacterial transformation or transposable elements. Pooling strate-
artificial chromosomes (YACs and BACs). At present, gies are used to avoid the need to isolate DNA from
almost complete contigs are available for all five chromo- thousands of plants. In these DNA preparations, specific
somes. Clones that make up these contigs are used for the DNA is amplified using PCR in which one primer is specific
internationally coordinated sequencing of the complete for the target gene and the other one is specific for the
genome of Arabidopsis. The complete sequence of chromo- insert. Only those pools containing plants with an insert in
somes 2 and 4 was published in 1999 and the sequence of that specific gene allow the amplification of a fragment.
the three remaining chromosomes is expected to be When the plant with the insert is identified it can be
available on the internet by the end of 2000. analysed for its phenotype.
In addition to the sequencing of genomic clones, many Forward genetics requires the isolation of mutants. The
cDNAs have been partially sequenced and provide a large self-fertilizing character of Arabidopsis means that the
collection of ESTs (expressed sequence tags). starting material for most mutagenesis experiments is
homozygous and therefore recessive mutants are not
observed in the first generation of mutagenized plants.
This so-called M1 generation derives from seeds treated
Gene Transfer (Transformation) in with either classical mutagens such as irradiation and
Arabidopsis chemicals (e.g. ethylmethane sulfonate, EMS). Selfing of
these M1 plants produces the M2 generation, in which
Arabidopsis can be transformed relatively easy using the recessive mutants (the majority of all mutants) will
bacterium Agrobacterium tumefaciens containing the Ti segregate. The selection of mutants depends on the type
plasmid from which a specific region (T-DNA) is of mutants the study is interested in and can involve visible
transferred to the plant chromosome. Modern Agrobac- selection for aberrant phenotypes or the ability/inability to
terium vectors, called binary vectors, have a separate grow under specific selection conditions or even large-scale
plasmid containing the virulence genes (necessary for the chemical or biochemical screens. Presently, insertion
transfer of T-DNA) and a second plasmid with the T-DNA mutagenesis, where a gene is mutated by the insertion of
itself (containing the genes to be transferred). Antibiotic- DNA sequences, is frequently used because such mutants
and herbicide-resistance genes are suitable selection facilitate gene cloning. For this, plants transformed with
markers, which allow the identification of transformed Agrobacterium tumefaciens vectors (T-DNAs) or transpo-
plants. Although initially tissue culture methods were used sable elements are used. Transposable elements (TEs) are
to transform Arabidopsis, these procedures have been introduced only once into the host genome and mutate
replaced by the so-called in-planta or vacuum transforma- genes by their ability to move from one site in the genome

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 Arabidopsis thaliana as an Experimental Organism

to another. Mutations generated by TEs often are unstable Examples of Plant Research where the
because the element may excise from the gene in which it
was located, thereby restoring the function of the gene.
Genetic and Molecular Approaches
Modified Ac/Ds or En/I (= Spm/dSpm) elements from using Arabidopsis were Important
maize have been introduced into Arabidopsis and were
shown to be active. An active endogenous Arabidopsis The isolation of mutants and the subsequent cloning and
transposon, called Tag1, has been found in some Arabi- study of the genes have led to important new insights in
dopsis genotypes but is less frequently used in tagging plant developmental biology. This includes the genetic
experiments than maize TEs. dissection of the pathways of flowering, flower develop-
In addition to induced mutants the genetic variation ment, root development, trichome (hair) formation, etc.
present in different Arabidopsis accessions can be analysed Interestingly, a number of developmental principles
and will allow the identification of specific genes, as has appear to be shared with the animal kingdom, although
been demonstrated for genes conferring disease resistance, plants and animals evolved independently from different
flowering time, etc. unicellular ancestors. Recently, evidence has surfaced that
plants also deploy new developmental strategies; this is not
surprising given the fact that plant cells do not move
relative to one another and that they have to cope with
Gene Cloning in Arabidopsis environmental stresses during their continuous develop-
ment.
When it is demonstrated, using co-segregation analysis, In addition, Arabidopsis has been used for biochemical
that a specific T-DNA or TE causes a mutation, then part genetics. Pathways that have not been genetically studied
of the genomic DNA flanking the insertion can be isolated. before, such as photorespiration, cell wall synthesis, lipid
This flanking DNA represents part of the mutated gene synthesis, etc., have been analysed. Important contribu-
and can be used to clone the complete genome or tions have also been made to the field of hormone research
complementary DNA (cDNA) sequence of that gene from and have led to the isolation of many genes controlling the
genomic or cDNA libraries. The final proof that the gene biosynthesis and modes of action of various plant
has been cloned can be obtained by the demonstration of hormones. A fascinating challenge that is now ahead is to
complementation of the mutant by the complete gene and/ combine our emerging understanding of plant hormones
or by expression and sequence analysis of several mutants with that of the genes that control development. The signal
in that same gene. In the case of TEs, the DNA sequence of transduction of ethylene has been analysed almost
revertants will show specific target site duplications at the exclusively by using Arabidopsis mutants defective in
position where the original element was excised. This can ethylene action or those showing constitutive ethylene
also be used as proof that the gene for which the mutant responses. Although some plant growth regulatory proper-
was found has been isolated. ties were known before, the essential role of brassinoster-
In situations where only mutants induced by classical oids recently became fully appreciated when it was shown
mutagens are available, the respective genes can be isolated that some extreme dwarf mutants of Arabidopsis were
by map-based cloning. This procedure requires that the brassinosteroid deficient. These mutants were subse-
map position of the locus is determined in detail in relation quently used to clone the genes of the various biosynthetic
to molecular markers that are related with the physical steps.
map. This map position then allows the identification of Photomorphogenesis research has also benefited from
specific (YAC, BAC or P1) clones that should contain the the genetic approach. This has made it possible to analyse
gene. The confirmation that the right gene has been cloned the functions of the various phytochrome types and also to
can be obtained from the complementation, by transfor- clone the blue light photoreceptors called cryptochromes
mation, of the mutant phenotype with specific subclones which mediate hypocotyl inhibition and flowering. Re-
that contain only one specific open reading frame. cently, it has been shown that mammals and Drosophila
Additional arguments come from the analysis of the also possess cryptochromes which entrain the circadian
expression of the candidate gene in mutant and wild-type clock in these animals, probably using a mechanism similar
phenotypes and from the sequence of mutant alleles. to that used by plants, for instance to perceive daylength
Irradiation-induced mutants are useful in this respect signals.
because this mutagen, more often than chemical mutagens, Within a period of less than 20 years Arabidopsis has
produces deletions, which are easier to detect in molecular become the favourite model plant because of its amen-
analyses than single base pair changes. ability to genetics and molecular biology. It will undoubt-
edly remain the favourite model plant in future years
because of the vast amount of basic knowledge that has
accumulated. The availability of the complete DNA
sequence does not only greatly facilitate gene cloning and

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 Arabidopsis thaliana as an Experimental Organism

analysis, but it can also be used to construct microarrays or Martinez-Zapater JM and Salinas J (eds) (1998) Arabidopsis Protocols.
DNA chips with all genes of Arabidopsis. These can then be Totowa, NJ: Humana Press.
used to study the expression of these genes in various Meinke DW, Cherry JM, Dean C, Rounsley SD and Koornneef M
(1998) Arabidopsis thaliana: a model plant for genome analysis.
conditions, tissues, mutants, etc. This combination of
Science 282: 662–682.
advantages will not be easily surpassed by other plants and Meyerowitz EM and Somerville CR (1994) Arabidopsis. Cold Spring
therefore the status of ‘model plant’ is likely to be Harbor: Cold Spring Harbor Laboratory Press.
appropriate for Arabidopsis for a long time, as is the case Westhoff P, Jeske H, Jürgens G, Kloppstech K and Link G (1998)
for Drosophila melanogaster and Caenorhabditis elegans, Molecular Plant Development from Gene to Plant. Oxford: Oxford
the flies and worms that continue to serve in unravelling University Press.
many mysteries of development in the animal kingdom. Wolpert L, Beddington R, Brockes J, Jessell T, Lawrence P and
Meyerowitz E (1998) Principles of Development. Oxford: Current
Biology Ltd, Oxford University Press.
Further Reading
The Arabidopsis Information Resource (TAIR) (1997) [http://www.ar-
abidopsis.org]