Arabidopsis thaliana as an Experimental Organism 1. 2.

Giovanna Serino1, Giuliana Gus

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 eu,aryotic processes. "he reasons for this success are diverse, and are based on a combination

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 uni ue 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.

of uni ue features, including- $1% Arabidopsis has one of the smallest genomes in the plant ,ingdom. $2% its genome has been completely se uenced 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 $1% both mutations and transgenic plants can be efficiently produced ma,ing Arabidopsis an ideal system for gene2s 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

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.

sea level up to *.20+ m in both open or disturbed habitats, including roadsides, riverban,s and roc,y 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 $:AS5http-77Arabidopsis.info7% in the 9nited ;ingdom, provide seed stoc,s 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 se uence

Arabidopsis was first suggested as a suitable model for plant biological studies in the 1)*+s. Seventy years

along with gene structure, gene product information, metabolism, gene e#pression, <:A and seed stoc,s,

genome maps, genetic and physical mar,ers, 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 A&65 :AS5

http-77www.genevestigator.com

"he Genevestigator database includes Arabidopsis gene e#pression data

http-77www.biosci.ohio8state.edu http-77www.Arabidopsis.info

"he Arabidopsis &iological 6esource 5enter "he :ottingham Arabidopsis Stoc, 5enter manage <:A and seed stoc,s

&A6

http-77www.bar.utoronto.ca7efp7cgi8 bin7efp'eb.cgi

"he &io8Array 6esource for Arabidopsis (unctional Genomics provides tissue and organ gene e#pression data "he Seattle "I??I:G Pro>ect provides series of induced point mutations in genes of interest

S"P

http-77tilling.fhcrc.org7

AG6I;3?A

http-77www.agri,ola.org

"he Arabidopsis genomic 6:Ai ,noc,8out 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 sili ua 0@ 2+ mm long, containing 2+@/+ seeds. Arabidopsis

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 *8wee,8old plant of the fre uently 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 wee,s after germination of the seeds and fertilisation can ta,e place / wee,s after germination in early genotypes. Arabidopsis produces progeny almost e#clusively by self8pollination and the seeds develop from the Aygote within the ovule. "his process ta,es 2@/ wee,s, resulting in a total minimum generation time of appro#imately 1 wee,s. 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 Aygote 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

&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 Aygote 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 wal,ing stic, stages, occurs mainly through cell e#pansion.

accumulate tannin8li,e 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 re uire 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

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

organs and structures throughout the lifespan of the plant. See also Seed Germination and 6eserve BobiliAation

"he above8ground shoot apical meristem consists of a 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. central Aone with slowly dividing cells that replenish the cells leaving the neighbouring peripheral Aone. In the peripheral Aone, 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 "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 (lowering starts with a change in the shape of the shoot apical meristem from flat to more rounded. "his Shoots and &uds

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

reference se uence from which results in Arabidopsis could be e#tended to other plants $Arabidopsis Genome Initiative, 2+++%. "he siAe of the Arabidopsis genome is appro#imately 120 megabases, and the se uenced 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

9nderneath the soil, the embryonic $FprimaryG% 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 upta,e, develop above the elongation Aone 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

24 *+1 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 uni ue se uences 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 14I 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++/%.

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

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

Arabidopsis genome "he Arabidopsis genome se uence was completed in 2+++, providing a valuable resource for furthering the ,nowledge of Arabidopsis biology, as well as a

genomic resources and technologies that will be described in detail later in this article. See also !u,aryotic 5hromosomes, and Plant :uclear Genome 5omposition

Arabidopsis &unctional (enomics

"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 se uenced c<:A e#pressed se uence 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 se uencing techni ues, 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 web8 ueryable 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 Se uence Analysis, :e#t Generation Se uencing "echnologies and "heir Applications, and "ranscriptional Profiling in Plants

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 mar,ers in planta and then they are purified by fluorescence8activated cell sorting $(A5S%, followed by transcriptome analysis $&irnbaum et al., 2++/%. See also Genetic !ngineering6eporter 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 siAe, 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 se uenced at single8base resolution in developing floral tissues, as well as in adult plants $?ister et al., 2++H. 5o,us et al., 2++H%. 5ombining these results with transcriptome data from the same tissues revealed a stri,ing correlation between small 6:As

Gene e#pression studies have also been greatly facilitated by the employment of proteins with an easily uantified or imaged activity, such as 8glucuronidase

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

$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

"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 ac uire 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+I of all predicted Arabidopsis gene models $&aerenfaller et al., 2++H%.

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 mar,ers. In the past two decades various methods for Arabidopsis transformation have been developed. :owadays, however, the Ffloral dipG method is the most widely used protocol for producing transgenic Arabidopsis plants in most laboratories $5lough and &ent, 1))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

Arabidopsis transformation is now highly reproducible and reliable, ma,ing possible a variety of genome8wide functional studies re uiring 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+ $or ard genetics In molecular genetics, the function of a gene is typically inferred by analysing the phenotype of the organism when that gene2s activity is altered or disrupted. =istorically, a wide variety of physical and

(ene trans$er )trans$ormation* in Arabidopsis+ a po er$ul tool to stud% gene $unction A ma>or brea,through 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++*%.

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

Arabidopsis can be transformed relatively easily using Agrobacterium tumefaciens $Gelvin, 1))H, 2++)%,

under or respond to specific compounds or signals $e.g. the hormone8resistant phenotype of different groups of hormone insensitive mutants% $'ei and <eng, 1))1. Stepanova and !c,er, 2+++%. Butagenised collections can also be generated in specific mutant bac,grounds and employed in genetic screens adapted to isolate enhancers or suppressors of specific phenotypes, leading to the identification of genes wor,ing together within the same pathway $Page and Grossni,laus, 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

'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 than,s to multiple collaborative efforts, a catalogue of completely se uenced 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 Agronomi ue $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 $li,e 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 nonhouse,eeping genes that are e#pressed in a tissue8specific manner or in response to developmental7environmental cues, stresses or signals. Similarly, strong transcriptional enhancers can

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%.

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

collections databases, searching for insertion line7s in 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 se uenced genes and the terrific opportunity of studying gene function through new powerful reverse genetics tools $Alonso and !c,er, 2++1. Parinov and Sundaresan, 2+++%. 3nce the <:A se uence of a gene is ,nown, it can be used as the starting point to browse the available insertion which that particular gene is disrupted $(igure /%. "hese mutant lines can be re uested from the corresponding seed stoc, databases and the presence of the insertion can be confirmed by P56 analyses. 3nce homoAygous 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 lin,ed 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 geneticsG 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:1, 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:1, 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 siAe of mutant collections available to date, about 12.2I of the annotated Arabidopsis genes lac, an insert and H.2I 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 ,noc,8out alleles are lethal% or duplicated family members. In recent years, few alternative approaches, among which 6:A8 mediated gene silencing, have been successfully 3riginally described in plants, 6:A8mediated gene silencing is an ancient defence mechanism, conserved among most eu,aryotes, 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 bloc,s the translation of the m6:As transcribed from the targeted employed for targeted mutagenesis of genes whose functional analyses cannot be performed using insertion alleles.

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 se uences to which they are only partially complementary $reviewed by &artel, 2++*%. In plant, genome wide studies suggest that the mi6:As have fewer targets, ma,ing their use possible in the targeted mutagenesis of selected genes $Schwab et al., 2++1%. 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 se uencing methods and other technologies, will allow the se uencing of a significant number of Arabidopsis accession2s 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 ac uisition of geographic adaptations among ecotypes. See also :atural Eariation as a "ool for Gene Identification in Plants

Examples o$ Plant Research

here the (enetic ere

Arabidopsis research has significantly contributed to our understanding of the molecular details of flower development, which re uires 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 BeyerowitA, 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 0I 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,

and -olecular Approaches using Arabidopsis 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 brea,throughs in modern science that have greatly refined our understanding of ,ey biological processes. "hese include the genetic dissection of doAens of plant8specific developmental7environmental pathways li,e 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, li,e 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

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

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 e uivalences between orthologous mutant phenotypes $phenologs% in different species $BcGary et al., 2+1+%, suggests Arabidopsis as a model for the study of human neural

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.

crest disorders. "hese phenologs reveal the conservation of deeply homologous modular subnetwor,s among eu,aryotes, 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

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 uni ue perspective to our understanding of more universal cellular processes, from those that are conserved among most eu,aryotes li,e cell8cycle progression, transcriptional regulation, gene silencing, chromatin remodelling, signal transduction and regulated protein ubi uitylation and degradations to those associated with human genetic diseases li,e for e#ample neurons development, cancer progression, telomere regulation and cellular aging.

/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 eu,aryotic 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.

(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 se uences during evolution, which ma,e them not suited for functional genetic and genomics, mainly due to the many technical difficulties associated with plant transformation, genome se uencing, 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 siAe 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 upta,e 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 delta81 desaturase gene from Mortierella alpina along with an omega8/ desaturase resulted in an stearidonic acid $S<A% content of J2+I, and when a borage delta81 desaturase gene was e#pressed with an Arabidopsis omega8/ desaturase gene in soybean seed, S<A contents of as high as /+I were observed $!c,ert et al., 2++1%. 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 than,s 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 techni ues 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

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