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Arabidopsis thaliana as an ExperimentalOrganism

1. Giovanna Serino1,

2. Giuliana Gus

Introduction

Plant research plays a central role in broadening our

understanding of the natural world. As phototrophic

organisms, plants are invaluable components of

terrestrial ecosystems. In addition, due to their

phenotypic plasticity, they offer uniue opportunities to

study the basic mechanisms responsible for survival,

growth and adaptation. !lucidating how plants respond

to disturbed environments is critical in predicting the

threats posed by the growing human population. "he

need for renewable form of energy, the escalating

pressures for food and habitat preservation are all

modern challenges whose resolution will depend on

our understanding of plant physiology, genetics and

ecology.

In this conte#t, Arabidopsis thaliana, a flowering plant

$Angiosperm% belonging to the mustard family

$&rassicaceae% has undoubtedly emerged as the

primary e#perimental organism for the study of plant

biology. 'hereas it has no agronomic value,

 Arabidopsis has proven to be an ideal organism for

studying plant development at the molecular and

organismal level. In addition, because of the close

evolutionary relationships among all flowering plants,

findings in Arabidopsis can lead to applications in other

plant species, including economically important crops.

(inally, discoveries made in Arabidopsis have a strong

impact on research in animal systems including the

study of comple# human genetic diseases such as

cancer.

 Arabidopsis was first suggested as a suitable model

for plant biological studies in the 1)*+s. Seventy years

later, not only it continues to be the elective model

organism for plant research, but also it is now widely

accepted as a laboratory system for the study of most

basic euaryotic processes. "he reasons for this

success are diverse, and are based on a combination

of uniue features, including- $1% Arabidopsis has one

of the smallest genomes in the plant ingdom $2% its

genome has been completely seuenced and a

tremendous amount of resources is currently available

$/% the adult plant can be easily grown in controlled

conditions $*% it has a short generation time $0% it can

be manually pollinated and it is a prolific producer of

seeds and $% both mutations and transgenic plants

can be efficiently produced maing Arabidopsis an

ideal system for genes functional studies.

3ver 40+ Arabidopsis natural accessions $also

referred to as ecotypes% have been collected from

around the world. "hese accessions are uite different

in terms of anatomy, physiology and ecology, and their

distribution encompasses five 5ontinents, ranging from

sea level up to *.20+ m in both open or disturbed

habitats, including roadsides, riverbans and rocy

slopes.

"wo stoc centres, the Arabidopsis &iological

6esource 5enter $A&65- http-77www.biosci.ohio8

state.edu% in the 9nited States, and the :ottingham

 Arabidopsis Stoc 5enter $:AS5-

http-77Arabidopsis.info7% in the 9nited ;ingdom, provide

seed stocs of mutants and wild accessions, as well as

deo#yribonucleic acid $<:A% materials for research. A

comprehensive array of information is also provided by

the Arabidopsis Information 6esource $"AI6,

http-77www.Arabidopsis.org7%, which maintains an

updated database of genetic and molecular biology

data. "hese include the complete genome seuence

along with gene structure, gene product information,

metabolism, gene e#pression, <:A and seed stocs,

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genome maps, genetic and physical marers,

publications, and information about the Arabidopsis

research community $see "able 1 and (urther 6eading

for details%. See also !#perimental 3rganisms 9sed in

Genetics, =istory of Plant Sciences, and Plant

Genome Pro>ects 

Table 1. Programs and URLs described in this revie

!ame URL "omments

"AI6 http-77www.Arabidopsis.org "he Arabidopsis Information 6esource contains comprehensive

information resources for the Arabidopsis community

SIG:A? http-77Signal.sal.edu "he Sal Institute Genomic Analysis ?aboratory contains a wealth

of information including a comprehensive listing of "8<:A

insertions in Arabidopsis

Genevestigato

r 

http-77www.genevestigator.com "he Genevestigator database includes Arabidopsis gene

e#pression data

 A&65 http-77www.biosci.ohio8state.edu "he Arabidopsis &iological 6esource 5enter 

:AS5 http-77www.Arabidopsis.info "he :ottingham Arabidopsis Stoc 5enter manage <:A and seed

stocs

&A6 http-77www.bar.utoronto.ca7efp7cgi8

bin7efp'eb.cgi

"he &io8Array 6esource for Arabidopsis (unctional Genomics

provides tissue and organ gene e#pression data

S"P http-77tilling.fhcrc.org7 "he Seattle "I??I:G Pro>ect provides series of induced point

mutations in genes of interest

 AG6I;3?A http-77www.agriola.org "he Arabidopsis genomic 6:Ai noc8out line analysis contains a

collection of 6:Ai silencing gene8specific vectors for Arabidopsis

#escription o$ Anatom%

 Arabidopsis is a small annual $rarely biennial% plant

usually growing to 2+@20 cm tall. In general, leaves

are oval8shaped and are present on a nonelongating

stem, thereby forming a rosette at the base of the

plant. (ew, smaller $cauline% leaves are also on the

flowering stem and have a#illary buds that develop into

secondary inflorescences $(igure 1%. ?eaves are

covered with small unicellular hairs $called trichomes%.

"he number of rosette leaves that are formed depends

on the genotype and environmental conditions and is

strongly correlated with the time from germination to

flowering. "he flowers are / mm in diameter, arranged

in a corymb $a hallmar feature of the typical

Brassicaceae% and contain four whorls of floral organs.

"he first whorl has four sepals, the second one has

four white petals, the third whorl has si# stamens and

the fourth whorl or centre has two carpels, which are

fused together into the pistil. "he fruit is a si liua 0@

2+ mm long, containing 2+@/+ seeds.  Arabidopsis 

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seeds are small $+.0 mm%, oval8shaped and produced

in large numbers $up to a few thousand per plant%.

6oots are simple in structure, with a single primary

root that grows vertically downwards, later producing

smaller lateral roots. See also (lowers, ?eaf and

Internode, and "richomes 

&igure 1. An appro#imately *8wee8old plant of the freuently used laboratory

accession ?andsberg erecta. "otal plant height is at this stage 10 cm. "he

inset shows a single flower.

Li$e "%cle

<epending on genotype and conditions, flower

primordia become visible as early as 2 wees after

germination of the seeds and fertilisation can tae

place / wees after germination in early genotypes.

 Arabidopsis produces progeny almost e#clusively by

self8pollination and the seeds develop from the ygote

within the ovule. "his process taes 2@/ wees,

resulting in a total minimum generation time of

appro#imately wees. Although in laboratory

conditions four to si# generations can be obtained per

year, Arabidopsis in nature probably produces only

one generation per year. In !urope, most Arabidopsis 

can be seen flowering in spring and early summer.

"hese plants might have germinated in spring

$summer annuals% or during the previous fall $winter

annuals%. "he latter are probably those genotypes that

are late flowering in greenhouse conditions, but which

can respond strongly to a vernalisation treatment that

induces flowering. In addition to flowering, the

presence of seed dormancy prevents several

generations occurring in 1 year. "here are large

genetic variations in nature for both flowering time and

seed dormancy. =owever, the number of field

observations on the ecology of Arabidopsis is still

limited.

Arabidopsis #evelopment

!mbryo development $(igure 2% starts with the fusion of the egg cell with a sperm cell $male gamete% deposited by the

germinating pollen grain. Pollen develops from microspores within the two8lobed anther, which contains four locules

surrounded by a tapetum layer. "his does not differ greatly from that in many other plant species. After fertilisation the

ygote follows a regular pattern of cell divisions, which correlate with morphologically defined stages. (ate mapping and

cell ablation studies, however, show that throughout development these cell divisions do not cause the segregation of

cell fates. 6ather, cell identity is based on continuous positional information in the embryo and in the developing regions

after embryogenesis, the shoot and root meristems. See also Beristems, Plant !mbryogenesis $Cygotic and Somatic%, 

Pollen- Structure, <evelopment and (unction, and Positional Information in Plant <evelopment 

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&igure '. !stablishment of the Arabidopsis body plan in the embryo and the

structure of a primary root. A, apical region 5, central region &, basal region

=D, hypophyseal cell SAB, shoot apical meristem 53", cotyledon =,

hypocotyl !6, embryonic root 6B, root meristem 6BI, root meristem initials.

"he first division of the ygote results in an embryo

with a small apical cell and a longer basal cell, from

which the filamentous suspensor, supporting the

embryo, will be formed. Specific stages that are

distinguished thereafter are the octant stage $when the

apical cell has given rise to two tiers of four cells%, the

dermatogen stage $when the protoderm is formed by

periclinal cell divisions%, the globular stage, the heart

stage $when cotyledon primordia become visible% and

the torpedo stage. At this stage, cell division is

arrested and further growth, during the so8called bend

cotyledon and waling stic stages, occurs mainly

through cell e#pansion.

In the mature seed the embryo consists of two

meristems, the shoot apical meristem and the root

apical meristem, two cotyledons, and the hypocotyl.

See also  Apical Beristems, and 6oot Apical Beristems

In addition to the embryo proper, the seed consists of

a seed coat or testa and endosperm. "he latter

develops from the fertilisation of the central cell, which

contains two haploid nuclei, with the second male

gamete. "his endosperm initially develops as a

syncytium, which thereafter undergoes cellularisation.

?ater in seed development the endosperm dies, e#cept

the outermost layer, and is replaced by the growing

embryo.

"he testa consists of two layers derived from the outer

and inner integument of the ovule and protects the

embryo against adverse environmental conditions.

<uring the seed8maturation phase, the seeds

accumulate tannin8lie pigments and food reserves

$lipids, sugars and proteins%, develop desiccation

tolerance and become dormant. See also Ploidy

Eariation in Plants, and Seeds

Seeds can be germinated easily, although freshly

harvested seeds may need a cold treatment of a few

days and7or a period of storage to germinate fully,

because they are dormant. "he seeds usually reuire

light for germination. 9pon germination the seedling

grows and develops from its shoot and root apical

meristems. "he activities of these meristems shape

the structure of the mature plant, as they are able to

continuously generate cells that will give rise to new

organs and structures throughout the lifespan of the

plant. See also Seed Germination and 6eserve

Bobiliation

"he above8ground shoot apical meristem consists of a

central one with slowly dividing cells that replenish

the cells leaving the neighbouring peripheral one. In

the peripheral one, organ primordia arise. In the a#ils

of peripheral organs, new meristems are formed and

the time at which these are activated is important in

determining the architecture of the plant. Bany genes

have recently been identified that play a role in the

continuous allocation of cells from the shoot apical

meristem to newly formed organs. See also Plant 5ell

<ifferentiation, Plant 5ell Growth and !longation, , and

Shoots and &uds

(lowering starts with a change in the shape of the

shoot apical meristem from flat to more rounded. "his

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change is accompanied at molecular level by the

activation of genes involved in the regulation of the

transition from vegetative to reproductive growth.

Instead of leaf primordia, the meristem starts

producing floral primordia $(igure 1%. Soon thereafter

the main stem elongates $bolt%, which results in an

inflorescence. =igher on the inflorescence stems the

typical crucifer flowers arise. See also (loral

Beristems

9nderneath the soil, the embryonic $Fprimary% root

meristem gives rise to various root tissues and, at a

distance behind the root apical meristem to new

meristems that will form lateral roots. "he structure of

the primary root in Arabidopsis seedlings is very

regular and consists above the root cap of an outer

layer of epidermis cells, a single corte# and a single

endodermis. 'ithin the endodermis, a single layer of

pericycle cells surrounds the vascular bundle. In

longitudinal section these layers form long and regular

cell files. 6oot hairs, involved in solute uptae, develop

above the elongation one as outgrowths of those

epidermal cells that are located in the clefts between

ad>acent cortical cells. See also ?ateral7Secondary

6oots, Primary 6oot, and 6oots and 6oot Systems

6oot development has been studied e#tensively by

careful microscopy, cell lineage and cell8ablation

analysis and a large number of mutants defective in

root development have been described and are now

being analysed at the molecular level. See also Plant

Genetics and <evelopment

 

 Arabidopsis genome

"he Arabidopsis genome seuence was completed in

2+++, providing a valuable resource for furthering the

nowledge of Arabidopsis biology, as well as a

reference seuence from which results in Arabidopsis 

could be e#tended to other plants $Arabidopsis

Genome Initiative, 2+++%. "he sie of the Arabidopsis 

genome is appro#imately 120 megabases, and the

seuenced portion of the Arabidopsis genome now

stands at appro#imately 11) megabases. Immediately

after the initial data release, an annotation effort was

initiated, with the goal of refining gene structure and

gene function assignments. "he last annotation

release, "AI61+, contains annotations for about

24 *+ protein8coding genes $a surprisingly high

number, considering that humans have about /+ +++

genes%, *H24 pseudogenes or transposable elements

and 1/0) noncoding ribonucleic acids $6:As%

$http-77www.arabidopsis.org%. "he Arabidopsis genome

is organised in five chromosomes, which are built up of

mainly uniue seuences with, on average, one gene

per 0 b. "he latter two chromosomes contain large

arrays of repeated ribosomal <:A $r<:A% genes at the

end of their short arms. Interestingly, about 14 of all

 Arabidopsis gene family members are arranged in

tandem repeats of duplicated genes. Indeed, gene

duplication and retention in plants has been e#tensive

and gene families are generally larger in plants than in

animals. (urthermore, the Arabidopsis genome has

e#perienced three rounds of duplications during the

past 10+@2++ million years $&owers et al., 2++/%.

In 2+++, the Arabidopsis community proposed an

ambitious programme to determine the function of

every gene by 2+1+. "his pro>ect @ called the

 Arabidopsis 2+1+ Program @ has funded the

generation of a broad range of powerful genetic and

genomic resources and technologies that will be

described in detail later in this article. See also

!uaryotic 5hromosomes, and Plant :uclear Genome

5omposition

Arabidopsis &unctional (enomics

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"ogether with the decoding of Arabidopsis genome, an

international effort has been made to uncover the

 Arabidopsis transcriptome. Although originally,

genome8wide e#pression measurements were limited

to the use of collections of seuenced c<:A

e#pressed seuence tags $!S"s%, Arabidopsis 

researchers can now choose from a variety of methods

that allow the uantification of thousands of transcripts

in a single e#periment. "hese approaches, such as

ne#t8generation seuencing techniues, microarray

and real8time polymerase chain reaction $P56%

analysis, have allowed scientists for the first time to

capture the transcriptional programs that are active

during plant development, as well as responses to

environmental stimuli, such as biotic or abiotic stress.

<ata generated using these approaches have been

deposited in several public available databases and

these e#tensive resources can now be mined to

generate hypotheses. "wo web8ueryable databases

have incorporated most of these e#pression data sets

and are commonly used within the Arabidopsis 

community due to their user8friendly interfaces and

data mining capabilities- the &io8Array 6esource for

 Arabidopsis (unctional Genomics $&A6

http-77www.bar.utoronto.ca7efp7cgi8bin7efp'eb.cgi% and

Genevestigator $http-77www.genevestigator.com%

$&rady and Provart, 2++) &usch and ?ohmann, 2++4%

$"able 1%. See also (unctional Genomics in Plants,

Gene !#pression in Plants, Genome Seuence

 Analysis, :e#t Generation Seuencing "echnologies

and "heir Applications, and "ranscriptional Profiling in

Plants

Gene e#pression studies have also been greatly

facilitated by the employment of proteins with an easily

uantified or imaged activity, such as 8glucuronidase

$G9S%, green fluorescent protein $G(P% and firefly

luciferase $?95% as reporters of promoter or gene

activity $de 6ui>ter et al., 2++/%. "hese reporter genes

are now efficiently used not only to uantify gene

e#pression in tissue samples, but also for real8time

imaging of plant promoters and protein dynamics.

(luorescent proteins have been also used to profile the

transcriptome of single cell types or of single tissue

types. In this approach, specific cells or tissues are

first stably labelled with fluorescent marers in planta 

and then they are purified by fluorescence8activated

cell sorting $(A5S%, followed by transcriptome analysis

$&irnbaum et al., 2++/%. See also Genetic !ngineering-

6eporter Genes

6ecet efforts are also aimed at unravelling the

 Arabidopsis epigenome. Similar to animals, the

 Arabidopsis genome is sub>ected to epigenetic

modifications $such as histone modifications and <:A

methylation% and because of its compact sie, it has

triggered the development of a series of novel

epigenomics technologies. &ecause the epigenome,

similar to the transcriptome, is not static and can be

shaped by developmental signals and environmental

perturbations, many Arabidopsis epigenomes will need

to be unravelled. In one such study, Arabidopsis 

epigenomics has been used to uery patterns of <:A

methylation, transposable element e#pression and

small 6:A accumulation in different organs and

developmental stages of Arabidopsis. (or e#ample,

the Arabidopsis <:A methylome has been recently

seuenced at single8base resolution in developing

floral tissues, as well as in adult plants $?ister et al.,

2++H 5ous et al., 2++H%. 5ombining these results

with transcriptome data from the same tissues

revealed a striing correlation between small 6:As

and <:A methylation and suggested the intriguing

possibility that the supporting $endosperm% cells are

used as a source of small 6:As that can move into the

germline and reconfigure the epigenome. See also

&iological 5omple#ity- &eyond the Genome

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"he availability of new, powerful mass spectrometry

instruments with increased detection sensitivity,

together with protein and peptide fractionation

technologies and data analysis tools, have also

facilitated cataloguing of Arabidopsis proteomes to

acuire information about functional properties and

activities of the Arabidopsis genome. In a recent study,

a high8density Arabidopsis proteome map has been

assembled from different plant organs as well as from

undifferentiated culture cells which led to the

identification of 1/ +2) proteins, corresponding to

nearly 0+ of all predicted Arabidopsis gene models

$&aerenfaller et al., 2++H%.

6ecently, the word metabolome has been coined to

describe the total complement of small molecules in a

cell, tissue, organ or organism $&ino et al., 2++*%.

&ecause plants collectively produce a huge array of

chemicals, far more than are produced by most other

organisms, the study of metabolism and phenotype

often provides a more direct route to investigate gene

function in Arabidopsis, as well as for the identification

of genes involved in specific pathways in crops and

medicinal plants. Several metabolomics resources for

 Arabidopsis are available at the "AI6 website. See

also <:A Bethylation in <evelopment, and Betabolite

Profiling in Plants

 

(ene trans$er )trans$ormation* in  Arabidopsis+ a

poer$ul tool to stud% gene $unction

 A ma>or breathrough for the emergence of

 Arabidopsis as a model organism has been the

developmental of efficient transformation procedures,

which made possible the study of gene function

through random disruption of endogenous genes

$?orence and Eerpoorte, 2++*%.

 Arabidopsis can be transformed relatively easily using

 Agrobacterium tumefaciens $Gelvin, 1))H, 2++)%,

which contains a large endogenous plasmid $"i8

plasmid%, from which a specific region $nown as the

"8<:A% is transferred into the plant genome. Bodern

 Agrobacterium vectors, called binary vectors, have a

separate plasmid containing the virulence genes and a

second plasmid carrying the "8<:A itself $containing

the genes to be transferred%. Antibiotic8 and herbicide8

resistance genes are also present on the same "8<:A

and used as selection marers. In the past two

decades various methods for Arabidopsis 

transformation have been developed. :owadays,

however, the Ffloral dip method is the most widely

used protocol for producing transgenic Arabidopsis 

plants in most laboratories $5lough and &ent, 1))H%.

 Arabidopsis transformation is now highly reproducible

and reliable, maing possible a variety of genome8wide

functional studies reuiring the generation of large8

scale collections of thousands of transformants. See

also  Agrobacterium tumefaciens8mediated

"ransformation of Plant 5ells, Plant 5ell 5ulture, Plant

"ransformation, "i Plasmids, and "ransgenic Plants

 

Recover% o$ mutations and $unctional anal%sis o$

genes+ $orard genetics

In molecular genetics, the function of a gene is

typically inferred by analysing the phenotype of the

organism when that genes activity is altered or

disrupted. =istorically, a wide variety of physical and

chemical mutagens have been employed to generate

large8scale collections of mutants. "hese collections

have been e#tensively employed in genetic screens

aimed at identifying genes involved in specific

biological processes. "o this end, the type of mutants

isolated depends on the nature of the genetic screen

and can involve visible selection for aberrant

phenotypes in particular growth conditions $e.g. the

constitutive photomorphogenic mutant phenotype of

dar grown seedlings% or the ability7inability to grow

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under or respond to specific compounds or signals

$e.g. the hormone8resistant phenotype of different

groups of hormone insensitive mutants% $'ei and

<eng, 1)) Stepanova and !cer, 2+++%.

Butagenised collections can also be generated in

specific mutant bacgrounds and employed in genetic

screens adapted to isolate enhancers or suppressors

of specific phenotypes, leading to the identification of

genes woring together within the same pathway

$Page and Grossnilaus, 2++2%. =istorically, these

methods have been particularly suitable in generating

high rate of mutations, which saturated the genome

with unbiased distribution, leading to the identification

of hundreds of $sometimes spectacular% mutants.

:evertheless, the ma>or drawbac of these forward

genetics approaches is the laborious identification,

within a large genome, of the <:A alteration

responsible for the mutant phenotype. "he

improvement of recently developed methods for the

detection of single point mutations on a genome scale

could potentially facilitate the identification of mutations

induced by physical and chemical agents. &ecause of

their robustness and sensitivity, forward genetic

screens are still widely used in the investigation of

several signalling processes in Arabidopsis, and

collections of mutagenised seeds are available at the

 A&65 or at the :AS5. In addition, the Seattle

 Arabidopsis "I??I:G Pro>ect $http-77tilling.fhcrc.org7% 

discovers mutants with allelic series of mutations

induced by the chemical mutagen ethylmethan

sulfonate $!BS% in target genomic loci $"ill et al.,

2++/%. See also Plant Genetics and <evelopment, and

Plant Butagenesis and Butant Screening

In the late 1))+s, the development of efficient

transformation methods has provided the most

significant advance in the design of mutant screening,

by offering the possibility to saturate the genome with

random "8<:A and transposon insertions $see below%.

'ith this approach, a gene can be disrupted by

insertion of the "8<:A $or transposon% within or near

its coding or regulatory regions. After a mutant of

interest has been identified, the gene responsible for

the altered phenotype can be isolated by P568based

methods $Sessions et al., 2++2 Alonso et al., 2++/%.

&y the late 1))+s and thans to multiple collaborative

efforts, a catalogue of completely seuenced and

inde#ed "8<:A and transposon insertion collections

has become publicly available. "his catalogue includes

the GA&I8;A", SAI?, Sal, 'IS5, (?AG and more

recently the S; lines, which together amount to over

/20 +++ independent "8<:A insertion lines. "hese

lines may be browsed at several web sites, including

the "AI6 $http-77www.Arabidopsis.org7% and the Sal "8

<:A !#press database $http-77signal.sal.edu7cgi8

bin7tdnae#press%, and about two third of them are

maintained and available for order from the A&65 or

the :AS5, whereas the (?AG collections is available

directly from the Institute :ational de la 6echerche

 Agronomiue $I:6A%. See also Polymerase 5hain

6eaction $P56%, and "ransposons as :atural and

!#perimental Butagens

&esides disrupting gene function, random insertional

mutagenesis with promoterless reporter genes $lie

G(P or ?95% can be used to identify the regulatory

elements $promoters and enhancers% of genes of

interest $!nhancer7promoter "6AP lines%. "hese lines

can be e#tremely useful in identifying the regulatory

regions of nonhouseeeping genes that are e#pressed

in a tissue8specific manner or in response to

developmental7environmental cues, stresses or

signals. Similarly, strong transcriptional enhancers can

be delivered into the plant genome via "8<:A insertion

causing the deregulated over8e#pression of a gene,

which might alter specific cellular7developmental

processes, resulting in a mutant phenotype. See also

Genetic !ngineering- 6eporter Genes

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Recover% o$ mutations and $unctional anal%sis o$

genes+ reverse genetics

In the post8genomic era, the Arabidopsis community is

left with both the tremendous challenge of assigning

biological functions to all the seuenced genes and the

terrific opportunity of studying gene function through

new powerful reverse genetics tools $Alonso and

!cer, 2++ Parinov and Sundaresan, 2+++%. 3nce

the <:A seuence of a gene is nown, it can be used

as the starting point to browse the available insertion

collections databases, searching for insertion line7s in

which that particular gene is disrupted $(igure /%.

"hese mutant lines can be reuested from the

corresponding seed stoc databases and the presence

of the insertion can be confirmed by P56 analyses.

3nce homoygous mutants have been identified, they

can be used for detailed phenotypic analysis, aimed at

inferring the function of the gene of interest, by

investigating the defects lined to its altered or lost

function. See also (unctional Genomics in Plants, and

Polymerase 5hain 6eaction $P56% 

&igure ,. Gene functional analysis using reverse genetics tools. $a% 3utline of

the Freverse genetics strategy used for the functional analysis of genes of

interest. $b% !#amples of the application of reverse genetics tools to the

functional study of small gene families regulating crucial developmental

pathways in Arabidopsis. In Arabidopsis, two 53P) signalosome comple#

$5S:% subunits, 5S:0 and 5S:, are both encoded by two highly conserved

genes, named CSN5A and CSN5B, and CSN6A and CSN6B, respectively. "he

availability of "8<:A insertion lines in each member of these small gene

families has allowed the generation of the csn5a csn5b and csn6a cns6b 

double null mutants. In both cases, as shown in $b%, complete loss of function

of 5S:0 or 5S:, results in severe developmental defects causing post8

embryonic arrest at the seedling stage $ reproduced from Gusmaroli et al.,

2++4, http-77www.plantcell.org. 5opyright American Society of Plant &iologists%.

<espite the large sie of mutant collections available to

date, about 12.2 of the annotated Arabidopsis genes

lac an insert and H.2 are represented by only one

insertion. (urthermore, in several cases these

insertions are confined to regulatory regions and might

result in hypomorphic alleles, which might complicate

the functional analysis of the gene of interested. In

addition, insertional mutagenesis cannot be applied to

the study of essential genes $whose noc8out alleles

are lethal% or duplicated family members. In recent

years, few alternative approaches, among which 6:A8

mediated gene silencing, have been successfully

employed for targeted mutagenesis of genes whose

functional analyses cannot be performed using

insertion alleles.

3riginally described in plants, 6:A8mediated gene

silencing is an ancient defence mechanism, conserved

among most euaryotes, which possibly evolved as a

protective measure against viruses and to eep in

chec the activity of genes and transposons. "he

distinctive feature of 6:A8mediated gene silencing is

the production of 218nucleotide 6:As, nown as small

interfering 6:As $si6:As%, which blocs the

translation of the m6:As transcribed from the targeted

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genes. In recent years, this mechanism has been

successfully employed for targeted mutagenesis of

selected genes in several model organisms including

 Arabidopsis $reviewed by 'aterhouse and =elliwell,

2++/ BcGinnis, 2+1+%. A possible alternative to 6:A8

mediated gene silencing is the use of artificial mi6:A

$ami6:As%. In animals mi6:A can regulate the

e#pression of several target seuences to which they

are only partially complementary $reviewed by &artel,

2++*%. In plant, genome wide studies suggest that the

mi6:As have fewer targets, maing their use possible

in the targeted mutagenesis of selected genes

$Schwab et al., 2++%. See also Gene Silencing in

Plants, and 6:A Interference $6:Ai% and Bicro6:As

(inally, natural allelic variation among Arabidopsis 

accessions could be a useful tool to investigate gene

function. It is reasonable to predict that the

improvement of robust ultra high8throughput

seuencing methods and other technologies, will allow

the seuencing of a significant number of Arabidopsis 

accessions genomes. <ifferences in physiological

traits among accessions can therefore be used for

genome wide association mapping $'eigel and

:ordborg, 2++0%, potentially enabling the identification

of allelic variants that underlie the acuisition of

geographic adaptations among ecotypes. See also

:atural Eariation as a "ool for Gene Identification in

Plants

 Examples o$ Plant Research here the (enetic

and -olecular Approaches using  Arabidopsis ere

Important

"he availability of a considerable amount of forward

and reverse genetics tools in Arabidopsis has made

possible the molecular and biochemical dissections of

a wide array of metabolic and developmental

pathways, providing many breathroughs in modern

science that have greatly refined our understanding of

ey biological processes. "hese include the genetic

dissection of doens of plant8specific

developmental7environmental pathways lie for

e#ample those regulating- $1% seeds, roots, leaves,

trichomes, flowers, ovules and pollen formation $2% the

perception, response and adaptation to endogenous

and environmental signals or stresses, lie hormones

and light perception, photomorphogenesis,

photoperiodism, phototropism, gravitropism, circadian

rhythms, defence salinity, temperature and drought

$/% a variety of plants@insects@pathogens interactions.

Bore recently, also the pathways potentially involved

in biofuel production or improved food nutritional

values have been characterised. See also Plant

Genetics and <evelopment

 Arabidopsis research has significantly contributed to

our understanding of the molecular details of flower

development, which reuires the coordinated functions

of several homeobo# genes, similar to many other

developmental processes in animals. Studies on the

genetic control of flowering and the formation of floral

organs have shown that Arabidopsis homeotic mutants

can be found in which one flower organ can be

converted into another. "he study of these mutants

has led to the so8called A&5 model for flower

formation, according to which the combined

e#pression of three different classes $A, & and 5% of

homeotic genes can give rise to the different flower

organs $5oen and Beyerowit, 1))1%. Similarly,

 Arabidopsis has greatly contributed to our nowledge

of the circadian cloc, which is an internal biological

cloc that evolved to co8ordinate

molecular7behavioural processes that occur once per

day with the day@night cycle $Song et al., 2+1+%. In

 Arabidopsis, the circadian cloc regulates about 0 of

the genome. "he rhythmic functions of these genes

control many processes, including leaf and petal

movements, the opening of stomata, the discharge of

floral fragrances and many metabolic activities,

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especially those associated with seedling

development, photosynthesis and growth. "he

circadian cloc also regulates seasonal changes that

depend on day8length, including the transition to

flowering. 3ver the last two decades many circadian

mutants have been identified through genetic

screening in Arabidopsis. "he molecular and

biochemical analysis of these lines has tremendously

contributed to refine our understanding of the circadian

rhythms and the environmental cues responsible for

the entrainment of the cloc, such as light and

temperature. See also Arabidopsis- (lower

<evelopment and Patterning, (lowers, (loral

Beristems, and Plant 5ircadian 6hythms

 Arabidopsis has been also successfully used in

biochemical genetics. Pathways that have not been

genetically studied before, such as photorespiration,

cell wall synthesis, lipid synthesis, etc., have been

analysed. Important contributions have also been

made to the field of hormone research and have led to

the isolation of many genes controlling the

biosynthesis, perception and modes of action of

various plant hormones.

Interestingly, a number of Arabidopsis developmental

principles appear to be shared with the animal

ingdom, and an increasing amount of discoveries in

 Arabidopsis has also provided a uniue perspective to

our understanding of more universal cellular

processes, from those that are conserved among most

euaryotes lie cell8cycle progression, transcriptional

regulation, gene silencing, chromatin remodelling,

signal transduction and regulated protein ubiuitylation

and degradations to those associated with human

genetic diseases lie for e#ample neurons

development, cancer progression, telomere regulation

and cellular aging.

6ecently, for e#ample, an Arabidopsis mutant

defective in telomere maintenance has led to the

discovery of a new protein involved in telomere

metabolisms in human cells $Surovtseva et al., 2++)%.

Similarly, recent studies suggest that neurons and

plant root cells may actually grow using a similar

mechanism, a finding that could potentially lead to the

use of Arabidopsis as a model for neuronal

microtubule associated disorders $reviewed by

Gardiner and Barc, 2+11%. Another recent study based

on the systematic discovery of human disease models

through the identification of nonobvious euivalences

between orthologous mutant phenotypes $phenologs%

in different species $BcGary et al., 2+1+%, suggests

 Arabidopsis as a model for the study of human neural

crest disorders. "hese phenologs reveal the

conservation of deeply homologous modular

subnetwors among euaryotes, although they might

have been recruited to different functions at the

organism8level.

 Another e#ample of the critical role of Arabidopsis in

the study of fundamental cellular processes comes

from genetic screening aimed at identifying plant

mutants impaired in photomorphogenesis $see (igure

/b%, the light8induced developmental programme.

"hese screenings have led to the identification of the

53P) signalosome comple# $5S:%, a pivotal

molecular machine involved in the repression of light

inducible genes, among other essential pathways

$reviewed by 'ei et al., 2++H%. Since its original

discovery in plants, the 5S: has been identified in a

variety of euaryotic organisms including human and a

growing amount of genetic and molecular data suggest

its involvement in the regulation of a wide array of

signalling and developmental processes, including

embryogenesis, cell8cycle progression, <:A repair,

chromatin remodelling, circadian rhythms,

microenvironmental homeostasis and angiogenesis.

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(inally, basic research in Arabidopsis not only can

offer comprehensive insight into the integrated biology

of a higher plant, but also creates the opportunity for

crop improvement. Bost crop species have very large

genomes, often as a result of one or more round of

entire genome duplication events and accumulation of

noncoding seuences during evolution, which mae

them not suited for functional genetic and genomics,

mainly due to the many technical difficulties associated

with plant transformation, genome seuencing, gene

cloning and generation and isolation of mutant loci.

6ecent findings from the rice genome pro>ect revealed

that most classes of rice genes have homologs in

 Arabidopsis and that therefore the obvious difference

in gene number and genome sie between these two

species is due to rice polyploidy, rather than to the

presence of large families of rice genes that are not

present in Arabidopsis. In this conte#t, Arabidopsis 

molecular genetic research can lead to the

identification of candidate genes responsible for the

regulation of agronomical useful traits such as drought

tolerance, heavy metal or poisons uptae and

deto#ification, pathogen resistance, seed nutritional

value and so on. 3nce these candidate genes have

been identified in Arabidopsis and their molecular

pathways dissected with the contribution of in silico 

analyses and data mining of the available resources

and databases, they can be used- $1% as a starting

point for the identification and study of their crop

counterparts, or $2% they can be directly transferred

into relevant crop species by transformation, with the

intent of modulating and7or modifying agronomically

relevant traits. (or e#ample, the recent transformation

of Brassica juncea with ADS1, the Arabidopsis 

homolog of yeast and mammalian acyl85oA delta8)

desaturases has led to a significant decrease in the

level of seed saturated fatty acids, showing how the

manipulation of a single gene can successfully alter

the seed nutritional value $Dao et al., 2++/%. Similarly,

e#pression in soybean seeds of a delta8 desaturase

gene from Mortierella alpina along with an omega8/

desaturase resulted in an stearidonic acid $S<A%

content of J2+, and when a borage delta8

desaturase gene was e#pressed with an Arabidopsis 

omega8/ desaturase gene in soybean seed, S<A

contents of as high as /+ were observed $!cert et

al., 2++%. Arabidopsis has also been used as a

recipient for the heterologous reconstitution of omega8

/ very long8chain polyunsaturated fatty acids $Ki et al.,

2++*%, which play a ey role in brain function and in

the prevention of cardiovascular diseases and other

western pathology such as type 2 diabetes and

obesity, but are unfortunately absent in higher plant

seeds. In light of these and other pioneer studies,

 Arabidopsis still remains a valuable model for studying

molecular pathways that could lead to more

sustainable agricultural practices, improved plant

nutritional value, or to the large8scale production of

biofuels or biodegradable polymers as environmentally

friendly alternatives to plastic and other industrial

materials.

"onclusions

 As described in this review, the last decades have

witnessed an incredible development in plant

nowledge thans to studies on Arabidopsis. =owever,

much wor remains to be done- for e#ample the

function of most of the 24 +++ Arabidopsis genes

remained to be determined, as well as their molecular

and functional interactions. "he future development of

powerful genomic, microscopy and genetic techniues

will help us to reach these goals, the final ob>ective

being to obtain a complete nowledge of the integrated

biology of Arabidopsis, as a model for higher

multicellular organisms. 5learly, Arabidopsis will

remain an ideal species of choice for basic research

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and to study plant biology and beyond also in the ne#t

decades.