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Arabidopsis Gene Knockout: Phenotypes Wanted Nicolas Bouché* and David Bouchez†

Arabidopsis Gene Knockout: Phenotypes Wanted Nicolas Bouché* and David Bouchez†

111

Arabidopsis knockout: wanted Nicolas Bouché* and David Bouchez†

Gene knockout is considered to be a major component of the paradigm (i.e. finding biological functions behind ) toolbox, and is aimed at revealing the [2]. The challenge now is to attach functions to an function of genes discovered through large-scale sequencing unprecedented number of genes: out of more than programs. In the past few years, several Arabidopsis 25,000 genes identified in Arabidopsis, the function of only populations mutagenized with insertion elements, such as the a few thousand can be defined with great confidence. T-DNA of or transposons, have been produced. These large populations are routinely screened for insertions As part of the toolbox for functional genomics, reverse into specific genes, allowing mass-isolation of knockout lines. comprises a set of methods devised to interfere Although many Arabidopsis knockouts have already been with proper gene activity, in particular by abolishing gene obtained, few of them have been reported to present function (i.e. by knockout) [2]. Homologous recom- informative phenotypes that provide a direct clue to gene bination allows gene replacement in vivo, substituting a function. Although functional redundancy explains the lack of wild-type gene for a copy that has been modified in vitro. phenotypical alterations in some cases, it also appears that This approach is widely used in yeast genetics to assess or many are conditional and/or do not alter plant modify gene function and hundreds of knockouts have morphology even in the presence of severe physiological been obtained in mice. allows defects. Consequently, gene knockout per se is not sufficient construction of lines carrying not only null mutations, but to assess gene function and must be integrated into a more also site-specific mutations such as amino-acid replace- global approach for determining biological functions. ments, insertions, deletions and modification of regulatory elements. As far as plants are concerned, homologous Addresses recombination is only available in the moss Physcomitrella INRA, Laboratoire de Biologie Cellulaire, Route de Saint Cyr, 78026 patens [3]. Until now, and despite considerable efforts, targeted Versailles, France disruption by homologous recombination has been mostly *Present address: Centre for Plant Sciences, School of , unsuccessful in higher plants, or at least not feasible in University of Leeds, Leeds LS2 9JT, UK †e-mail: [email protected] routine experiments [4].

Current Opinion in Plant Biology 2001, 4:111–117 For several plant species in which homologous recombi- 1369-5266/01/$ — see front matter nation is not available, gene-disruption methods have © 2001 Elsevier Science Ltd. All rights reserved. been designed by making use of large populations mutag- Abbreviations enized with an insertion element – transposons or the AKT1 Arabidopsis K+ TRANSPORTER1 T-DNA of Agrobacterium [5–11]. These methods rely on CCA1 CIRCADIAN-CLOCK ASSOCIATED 1 the use of a polymerase chain reaction (PCR) to screen ORF open reading frame DNA pools prepared from the population for the desired PCR polymerase chain reaction insertions. PCR primers specific for both the target gene SEP1–3 SEPALLATA1–3 SHP1 SHATTERPROOF1 and the insertion element are used. The large populations SKOR Stellar outward-rectifying K+ channel (Table 1), DNA pools and protocols currently available already allow the isolation of knockout lines for virtually Introduction every Arabidopsis gene [12••]. Surprisingly, although The sequencing of the of the higher plant PCR-based screens have been available is now completed [1••], and large col- to Arabidopsis researchers for several years, relatively few lections of expressed sequence tags are available for a informative knockouts have been reported in the litera- number of other plants. Such a large amount of plant ture to date. In fact, the vast majority of the several sequencing data is expected to provide new ways of hundred knockouts obtained in Arabidopsis do not give addressing biological questions from an integrated per- rise to visible, directly informative phenotypes, and they spective, ranging from to evolution. By await further analysis. Several limitations are already themselves, however, DNA or sequences fail to apparent from the experience gained in the past few provide much biologically relevant information for the vast years. They essentially concern the difficulty in finding majority of genes that have been identified through large- and studying informative phenotypes, and in dealing with scale sequencing programs. Although more efficient the large amount of structural redundancy unravelled by bioinformatic tools are becoming available for the annotation the genomic sequence of Arabidopsis. Indeed, although of genome sequences most, if not all, such information Arabidopsis, Drosophila and Caenorhabditis elegans share a must be regarded as hypothetical in the absence of exper- similar number of gene types (about 11,000 in imental evidence. Thus, the previous paradigm of Arabidopsis), multigene families, present as clustered molecular genetics (i.e. finding genes behind biological and/or dispersed copies, are particularly frequent in this functions) is gradually being reversed into a post-genomic simple plant genome [1••]. 112 Genome studies and molecular genetics

Table 1

Populations available for reverse genetics in Arabidopsis thaliana.

Type of element Average copy Element mobility* Reporter gene/ Lines available from Reference(s) number per element design the stock centers genome

Two component transposon 1–3 Stable (transposase 47,450 ‘SLAT’ lines (50% are [7] removed system based on removed by negative are available as DNA pools)† En/Spm selection) Two component transposon 1 Stable (transposase GUS reporter (gene 931 ‘IMA’ lines† [6] system based on Ac/Ds removed by negative trap and enhancer trap) selection) Two component transposon 20–25 Stable (transposase ‘ITS’ lines available available [5] system based on En/Spm removed by genetic on request from A Pereira† segregation) T-DNA 1–3 Stable 15,400 [11] T-DNA 1–3 Stable GUS reporter 9,800 [43,44] T-DNA No data Stable GUS reporter 17,600 [9] (enhancer/promotor trap) En-1 (En/Spm family) 6 Mobile [10] autonomous transposon T-DNA No data Stable Activation tag 22,672 [8] composed by multi- merized 35S enhancers

Populations and DNA pools are available from Arabidopsis stock (URL http://nasc.nott.ac.uk/) and through the recently developed centers: URLs http://nasc.nott.ac.uk/ and http://aims.cps.msu.edu/aims/ Arabidopsis transposon insertion database (URL http://formaggio.cshl.org/ *Different trap systems developed in plants were recently reviewed [15•]. ~h-liu/attdb/index.html). Ac, Activator element; Ds, Dissociation † Systematic sequencing of insertion sites (‘flanking sequence tags’ element; En, Enhancer; GUS, β-glucuronidase; IMA, Institute of [FSTs]) was performed for several of these lines. Sequences are Molecular Agrobiology, Singapore; ITS, Inhibitor-tagged site; SLAT, available at the Nottingham Arabidopsis stock centre Sainsbury Laboratory Arabidopsis thaliana; Spm, Suppressor-mutator.

Several recent reviews describe various resources available based on homologous recombination. Modified copies of for gene knockout in Arabidopsis [12••,13,14•,15•]. In this genes under study can, however, be re-introduced into null review, we will give an update on the outcome of reverse lines for fine structure–function analyses. genetics approaches in this species so far, and discuss some limitations and perspectives that have emerged from a few Phenotypic characterisation of knockouts years experience. Since the first disruption of actin genes in Arabidopsis [17], only a few of the several hundred knockouts obtained in Isolation of knockouts in Arabidopsis different laboratories have been reported to exhibit infor- It is expected that about 150,000–200,000 insertions are neces- mative phenotypes (Table 2). Even fewer display sary to obtain a reasonable knockout saturation of the genome. morphological changes that provide a direct clue to gene To date, several hundred thousand T-DNA and transposon function. Out of about 200 knockout lines isolated by our insertion lines have been generated in many laboratories and, group, fewer than 2% display significant morphological at least in part, have gradually become available to the com- alterations. Nevertheless, unexpected phenotypes can be munity. Recently, several groups have initiated programs for found upon careful examination of mutant plants. DAG1 is the systematic sequencing of insertion sites in various popula- known to encode a transcription factor, but the sequence tions (Table 1) [5–7]. In the next few years, it can be expected and mRNA expression pattern of this gene do not give any that the combined size of these populations will allow the clue to its function at the whole-plant level. Analysis of a systematic disruption of all Arabidopsis genes, and that the knockout line, however, showed that DAG1 is involved in search for knockout mutants will be greatly facilitated by the the control of seed dormancy [18••]; dag1 plants have dis- use of knockout databases. In addition, recent reports on the torted siliques, with seeds that germinate earlier than double-stranded RNA-mediated interference (RNAi) approach normal and, in contrast to wild-type seeds, do not require in Arabidopsis have opened new perspectives for sequence- light for their germination. specific inhibition of gene function in this species (e.g. [16]). When compared with the wild type, however, most knockout Gene knockout allows the isolation of loss-of-function , lines do not exhibit visible changes in standard culture but does not have the versatility of gene replacement conditions. In order to circumvent this problem, it was Arabidopsis gene knockout: phenotypes wanted Bouché and Bouchez 113

Table 2

Arabidopsis mutants obtained by reverse genetics.

Gene /plant morphology /gene function Mutagen Reference(s)

AKT1 Growth of akt1 plants on media Inward rectifying K+ channel, T-DNA [23,26] containing ≤ 100 µM K+ is inhibited; shaker-like family reduced K+ uptake; wild-type morphology on many nutrient media SKOR 50% of the wild-type K+ content Shaker-like outward channel involved T-DNA [22] in shoot; wild-type morphology in K+ release into the xylem sap

– AtCLC-a Level of NO3 reduced by 53% in the Anion channel T-DNA [24] knockout; in shoot; morphology ACT2, ACT4, ACT7 Plants exhibit reduced fitness; Actins T-DNA [41,42] wild-type morphology AtDMC1 Low fertility; wild-type morphology Function in interhomolog T-DNA [29] recombination; homology with RecA and RAD51 AtTERT Reduced telomere size; wild-type Telomerase T-DNA [28•] morphology for 10 generations CCA1 Period of oscillation of several circadian Myb-related protein; controls the T-DNA [27•] genes modified, wild-type morphology circadian regulation of several genes

ADC2 Wild-type morphology Arginine decarboxylase En-1 transposon [45]

DAG1 Twisted siliques; seeds germinating Zinc-finger transcription factor involved T-DNA [18••] in darkness in the control of seed germination AtToc159 Albino Chloroplast biogenesis, import of T-DNA [46] photosynthetic in chloroplasts MUP24.4 REVOLUTA mutant phenotype Homeobox genes, function unknown T-DNA [47] SEP1, SEP2, SEP3 sep1 sep2 sep3 triple mutant AGL genes; part of the ABC genes En-1 transposon; [36] produces flowers with all organs controlling the four organs type of a T-DNA replaced by sepals. Single knockouts; typical dicotyledon flower wild-type phenotype SHP1 and SHP2 Fruits of the double mutant shp1 shp2 MADS-box genes Homologous [37••] are not dehiscent. Single knockouts; recombination; wild-type phenotype T-DNA CNGC1 Pb2+ tolerance, wild-type morphology Cyclic-nucleotide-gated channel T-DNA [25•] AtRAD50 Sterility, sensitivity to a DNA RAD51 homologue T-DNA [48] damaging agent AtPIN2 Roots shortened, no gravitropism Similarity with transmembrane En-1 transposon [49] transporters, auxine action regulation AtFLS No production of falvonol kaempferol, Flavonol synthase, gene isolated by En-1 transposon [10] wild-type morphology (including the homology with genes involved in seeds, which present a wild-type flavonoid biosynthesis colour) ICL Cotyledon expand but fail to green in Isocitrate lyase, one of the two enzymes En-1 transposon [50] short days; wild-type morphology of the glyoxylate cycle

A non-exhaustive survey of published Arabidopsis mutants obtained by reverse genetics from insertional mutagenesis populations. Several others have been reported, but no detailed phenotypic analyses have been reported so far. proposed that mutants should be tested under a wide range conditional and revealed only by a specific combination of of environmental conditions that would reveal conditional environmental parameters. It must be stressed, however, phenotypes [14•,19]. This proposition makes sense as it is that the systematic analysis of the effects of so many (inter- likely that plants, as sessile , have evolved many acting) factors (e.g. light quality, intensity and regime; adaptive traits that allow them to cope with profound mineral nutrition and temperature) on Arabidopsis mutants changes in their biotic and abiotic environment. Many loss- is, in practice, impossible for most laboratories. For such a of-function mutants are therefore expected to be systematic exploration of gene function, the moss P. patens 114 Genome studies and molecular genetics

provides a versatile experimental system that certainly data — was instrumental in guiding the analysis of knockout deserves more attention. It presumably shares many genes mutant plants. Another example of the importance of and functions with higher plants. The major part of the functional knowledge is given by a mutant of a CIRCADIAN- moss life cycle is haploid and its organogenesis is simple rel- CLOCK ASSOCIATED1 (CCA1) protein. In the cca1 ative to that of higher plants, although many environmental knockout [27•], various clock-controlled genes have a and hormonal controls seem to be conserved. It is easily modified period of oscillation but, to date, no particular grown in vitro under controlled conditions, which could morphological change has been detected in the mutant. allow automation and miniaturization of experiments. As for Arabidopsis systems, it will prove difficult to design Until now, therefore, most success stories in this field concern experiments at a reasonable cost, unless additional information genes on which precise functional hypotheses could be defined on gene function allows restriction of the number of envi- based on other pieces of information, for example, strong ronments imposed and measurements performed. sequence conservation with proteins that have been charac- terised in other organisms (e.g. telomerase [28•]; AtDMC, a Indeed, the situation changes remarkably when other RecA homolog involved in meiosis [29]), or biochemical pieces of information on gene activity are available before characterisation of the protein (e.g. as an ion channel). In these mutant analysis. For example, information about mRNA cases, and despite the fact that most mutants do not display morphological changes, the authors were able to detect alter- and/or protein expression pattern, protein localisation or ations in specific processes. Obviously, mutant phenotypes are biochemical activity often transforms a desperate hunt for a not restricted to morphological changes and probably only a phenotype into hypothesis-based research. In yeast, such small proportion of mutations will affect plant morphology. large-scale analysis of gene function, based on gene disrup- tion coupled with expression data and immunolocalisation, Finally, one must stress that the notion of a biological func- revealed that 25% of 2000 open reading frames (ORFs) tion is becoming a rather complex one that refers to many exhibit a phenotype [20]. In the next few years, a great levels of complexity in living organisms, and therefore can effort will be devoted to large-scale expression profiling of only be defined using a variety of complementary experi- all Arabidopsis genes in a wide range of developmental and mental approaches and skills. Ideally, functional studies environmental situations [21]. This will, undoubtedly, be a should include the elucidation of molecular and bioche- tremendous help for the phenotypic analysis of knockouts, mical mechanisms, should provide an understanding of especially for ‘orphan’ genes, which do not show similarity how these mechanisms are integrated into cells, tissues to any known genes. and organisms, not to mention populations, and should involve an evolutionary perspective. Gene knockout pro- Can sequence annotation provide functional information? vides a powerful way to approach many aspects of gene Such information, derived from sequence analysis, concerns function, but only if enriched with other information and about half of the detected genes and is generally restricted resources that guide the process of mutant analysis. to similarity with known proteins or motifs. It mostly concerns biochemical activities (e.g. protein kinase activity) Structural versus functional redundancy and is often of little help in guiding mutant analysis in the Another aspect that is important for the utility of gene absence of developmental alterations. knockouts, is the unexpected prevalence of structural redundancy that has emerged from the Arabidopsis genome Knockouts obtained in several ion channels in Arabidopsis sequence: gene duplication is frequent and most genes exemplify the need for additional information [22–24,25•]. belong to gene families. There is no unified definition of a The Stellar outward-rectifying K+ channel (SKOR) is a gene family and no consistency in the degree of similarity shaker-like channel, identified in Arabidopsis by sequence that is used to identify members of the same family. similarity with AKT1 (Arabidopsis K+ TRANSPORTER1) However, many highly similar genes are found either as and KAT1 (K+ Arabidopsis thaliana channel1). SKOR was tandem clusters or as dispersed copies in the genome; large shown to be involved in K+ release into the xylem sap [22]. duplications of chromosomal regions have also been iden- A T-DNA tagged mutant, skor-1, was found to have 50% of tified [30–33]. On chromosomes 2 and 4, 15% of the the wild-type K+ content in its shoots, but normal levels in predicted proteins are found in clusters that range from its roots. The total inorganic cation concentration in leaves two genes to tandem repetitions of 15 genes [31,32]. was, however, unchanged because of an increase in Ca2+. The disruption of an anion channel [24] leads to a reduction The significance of the high level of structural redundancy, of NO3 concentration of up to 50% in the mutant. The dis- a general feature of eukaryotic , is not clear at ruption of the AKT1 K+ channel [23] also demonstrated present [34]. A major question for the future is whether that this channel is an important component of K+ uptake structurally related genes have partially or totally over- [26]. In all three cases, and despite drastic physiological lapping functions, or whether the different isoforms have alterations, mutant plants do not display significant morpho- specialised, non-overlapping functions that are adapted logical changes. Prior knowledge of the biochemical to different physiological contexts? The distinction function of these proteins — obtained from sequence between these propositions is, of course, of crucial impor- analysis, expression patterns or (electro)physiological tance with respect to reverse genetics strategies. Previous Arabidopsis gene knockout: phenotypes wanted Bouché and Bouchez 115

Arabidopsis mutant analyses clearly show that both situa- SEPALLATA1–3 (SEP1–3) [37••]. The mutants were tions exist: many genes cloned from mutants belong to PCR-isolated from insertion collections. Single sep mutants gene families (e.g. the AGAMOUS and other MADS-box do not lead to a particular phenotype, but in the triple genes) and yet display strong phenotypes. On the other mutant, sep1; sep2; sep3, all flower organs are transformed hand, clear examples of functional redundancy are known into sepals, conducing the authors to include the SEP genes (e.g. for ABSCISIC-ACID INSENSITIVE1 [ABI1] and in the ABC model of flower development. Similarly, the ABI2 [35]). SHATTERPROOF1 (SHP1) and SHP2 MADS-box genes were shown to be required for fruit dehiscence, which is A few examples of reverse genetics screens for members of abolished only in the double mutant [38••]. In these cases, large gene families have been reported. For example, it can be concluded that these highly similar genes are, at 47 knockout lines in MYB transcription factors [19], least partly, functionally redundant. obtained from both T-DNA and transposon collections, have been subjected to various environmental conditions The strict definition of genetic redundancy is a situation in in order to identify phenotypical changes with little suc- which disruption of a gene is selectively neutral (i.e. it does cess to date. Similarly, genes implicated in the not affect fitness), so there would be no selection pressure phenylpropanoid biosynthesis pathway have been against the accumulation of deleterious mutations in truly knocked out. The resulting mutants were screened at redundant genes. Hence, it is difficult to imagine such redun- 109 loci and 55 transposon lines were recovered [10]. One dancy being evolutionarily stable and yet redundancy, at least mutant corresponding to a putative flavonol synthase was structurally speaking, seems to be the rule rather than the identified and shown to exhibit a reduced level of flavonol exception in eukaryotic genomes. Although some models kaempferol. Finally, 12 T-DNA knockouts were isolated have been proposed that deal with the maintenance of true from 70 genes of the large cytochrome-P450 multigene redundancy in evolution [39], they lack physiological support. family [11]. One knockout line exhibited a runt pheno- type, but the complementation of the mutant with the Experimentally, fitness measurements require the use of wild-type gene is yet to be reported. large populations through many generations. This is not always practicable with higher organisms such as plants, From these three large-scale surveys, only two mutant but in yeast there are indications that true functional lines were found to present morphological changes when redundancy may not be as widespread as suggested by compared with wild-type plants. Although these results sequence redundancy. In an experiment in which seem to indicate a great deal of functional redundancy in 29 knockout strains with no phenotype were cultivated in the gene families studied, a definite picture will only competition with the wild-type strain on rich medium, emerge when more information is available on individual 19 knockouts were shown to have significantly reduced genes and proteins, and when combinations of knockouts fitness [40]. Thus, the lack of visible phenotypes in knockout are available. mutants, which has also been noted in systems such as mice, should not be attributed systematically to redundancy, Because of the tight genetic linkage between tandemly but presumably more often to our inability to detect weak repeated genes it is difficult to combine knockouts, at physiological changes. This difficulty was addressed with least from insertion populations, by crossing. Hence, the Arabidopsis lines disrupted in actin genes, of which ten use of reverse genetics for the functional analysis of indi- copies are present in the Arabidopsis genome [41,42]. vidual genes within a tandemly repeated sequence may Individual act2-1 homozygous mutants grew like the wild- be precluded. The use of radiation-induced deletion type and had a comparable seed production, but the fre- stocks might prove useful for this purpose by allowing the quency of the mutant was significantly reduced in recovery of deletions covering gene clusters through PCR successive generations at a greater rate than would be screens. Moreover, the systematic analysis of all possible expected in the absence of selection. The authors estimated combinations of knockouts in a given family would be at that the mutated act2-1 allele would be lost in only best impractical, if not unfeasible (e.g. there are 31 combi- 20 generations. In the same line, a mutant disrupted in a nations of single, double, and multiple knockouts for a telomerase catalytic subunit is viable for 5–6 generations, family of five genes). Here again, the input of other infor- but gradually and slowly loses telomeric DNA [28•]. mation, such as expression patterns and detailed sequence Defects in cell proliferation, especially in the shoot meristem, data, is clearly needed. An interesting alternative to the start to be detected in subsequent generations, leading to combination of independent knockouts is virus-induced the death of plants 10 generations after loss of telomerase . The inactivation of genes by this method (T McKnight, personal communication). can such target whole gene families whose sequences diverge by up to 10–20% [36]. The concept of redundancy presumably hides a diversity of physiological and evolutionary situations, which need to The potential for combining independent knockouts in be approached on a case-by-case basis and using appropri- similar genes was illustrated by the disruption of three ately adapted experimental strategies. The assessment of AGAMOUS-LIKE MADS-box genes designated the contribution of a particular gene to global fitness is 116 Genome studies and molecular genetics

interesting per se, but will not provide much information 6. Parinov S, Sevugan M, De Y, Yang WC, Kumaran M, Sundaresan V: Analysis of flanking sequences from dissociation insertion lines. about its role in the physiology of the . A database for reverse genetics in Arabidopsis. Plant Cell 1999, 11:2263-2270. Conclusions 7. Tissier AF, Marillonnet S, Klimyuk V, Patel K, Torres MA, Murphy G, Reverse genetics methods are now well established in Jones JD: Multiple independent defective suppressor–mutator transposon insertions in Arabidopsis: a tool for functional Arabidopsis and are widely employed to identify knockouts genomics. Plant Cell 1999, 11:1841-1852. from populations mutagenised with an insertion element, 8. Weigel D, Ahn JH, Blazquez MA, Borevitz JO, Christensen SK, either the T-DNA of Agrobacterium or a transposon. The Fankhauser C, Ferrandiz C, Kardailsky I, Malancharuvil EJ, Neff MM et al.: Activation tagging in Arabidopsis. Plant Physiol 2000, technology itself is becoming both increasingly simple and 122:1003-1014. more accessible, and databases of insertion sites are becom- 9. Campisi L, Yang Y, Yi Y, Heilig E, Herman B, Cassista AJ, Allen DW, ing publicly available on the internet. However, only a Xiang H, Jack T: Generation of enhancer trap lines in Arabidopsis small proportion of the knockout lines obtained to date dis- and characterization of expression patterns in the inflorescence. Plant J 1999, 17:699-707. play directly informative phenotypes at the whole-plant 10. Wisman E, Hartmann U, Sagasser M, Baumann E, Palme K, level. Several reports have shown that this lack of pheno- Hahlbrock K, Saedler H, Weisshaar B: Knockout mutants from an types is presumably caused by the ability of higher plants to En-1 mutagenized Arabidopsis thaliana population generate phenylpropanoid biosynthesis phenotypes. Proc Natl Acad Sci adapt their physiology to various stresses and constraints USA 1998, 95:12432-12437. without undergoing morphological changes, and by our 11. Winkler RG, Frank MR, Galbraith DW, Feyereisen R, Feldmann KA: inability to detect slight physiological alterations and/or Systematic reverse genetics of transfer-DNA-tagged lines of weak reductions in fitness, and partial or complete func- Arabidopsis. Isolation of mutations in the cytochrome P450 gene superfamily. Plant Physiol 1998, 118:743-750. tional redundancy. 12. Parinov S, Sundaresan V: Functional genomics in Arabidopsis: •• large-scale insertional mutagenesis complements the genome Despite its limitations, especially in comparison with gene sequencing project. Curr Opin Biotechnol 2000, 11:157-161. replacement, which allows fine structure–function analyses, A review focusing on the techniques and resources available for reverse genetics in Arabidopsis. Different types of populations are described with a gene knockout remains a powerful technology. Obviously, special emphasis on the use of T-DNA versus transposons. The authors this tool alone is not sufficient and must be integrated into discuss the recent development of transposon-based collections containing stable transposon integrations. In addition, the different screening a more general approach to gene function. In particular, strategies that are currently available for finding knockouts are summarised. development of other functional genomics tools, such as 13. Martienssen RA: Functional genomics: probing plant gene function global transcriptome and proteome analyses and metabolic and expression with transposons. Proc Natl Acad Sci USA 1998, profiling, will be of crucial importance if we are to take full 95:2021-2026. benefit of reverse genetics in Arabidopsis. 14. Krysan PJ, Young JC, Sussman MR: T-DNA as an insertional • mutagen in Arabidopsis. Plant Cell 1999, 11:2283-2290. This review describes in detail the PCR methods used to isolate knockouts Acknowledgements from T-DNA populations. Various technical questions are discussed We are grateful to Herman Höfte, Hélène Lucas and Hillel Fromm for regarding the quality of the DNA pools and design of primers. The critically reviewing the manuscript. Work in David Bouchez’s laboratory Arabidopsis Gene Knockout Facility (URL http://www.biotech.wisc.edu/ Arabidopsis/) uses a PCR-based approach and, for a fee, provides the was supported by grants from the European Union (BIO4-CT95), Institut Arabidopsis community with access to 60,400 T-DNA lines. National de la Recherche Agronomique, the French Ministry for Research (ACC-SV4) and the French Embassy in Israel. Nicolas Bouché 15. Springer PS: Gene traps. Tools for plant development and was a recipient of a Dufrenoy fellowship from the Académie • genomics. Plant Cell 2000, 12:1007-1020. d’Agriculture de France. This review describes different types of ‘trapping systems’ available in plants (i.e. enhancer-trap, promoter-trap and gene-trap systems) and the reporter genes in use. The different systems are discussed in the context of References and recommended reading identifying new functions involved in plant development. Papers of particular interest, published within the annual period of review, have been highlighted as: 16. Chuang CF, Meyerowitz EM: Specific and heritable genetic interference by double-stranded RNA in Arabidopsis thaliana. • of special interest Proc Natl Acad Sci USA 2000, 97:4985-4990. •• of outstanding interest 17. McKinney EC, Ali N, Traut A, Feldmann KA, Belostotsky DA, 1. The Arabidopsis Genome Initiative: Analysis of the genome McDowell JM, Meagher RB: Sequence-based identification of •• sequence of the flowering plant Arabidopsis thaliana. Nature 2000, T-DNA insertion mutations in Arabidopsis: actin mutants act2-1 408:796-815. and act4-1. Plant J 1995, 8:613-622. The first complete sequence of a plant genome, which was established through an international consortium of laboratories. 115.4 megabases of 18. Papi M, Sabatini S, Bouchez D, Camilleri C, Costantino P, Vittorioso P: •• Identification and disruption of an Arabidopsis zinc finger gene DNA have been sequenced and 25,000 genes have been identified. A controlling seed germination. Genes Dev 2000, 14:28-33. landmark in biology, opening new avenues for plant research. The role of DAG1, which encodes a zinc-finger transcription factor, was 2. Bouchez D, Höfte H: Functional genomics in plants. Plant investigated by a reverse genetics approach. The homozygous knockout, Physiol 1998, 118:725-732. has twisted siliques and seeds that, unlike wild-type seeds, can germinate in total darkness. Hence, DAG1 is shown to be involved in the control of 3. Schaefer DG, Zryd JP: Efficient in the moss seed dormancy. The authors of this paper describe the only work to date in . Plant J 1997, 11:1195-1206. which a knockout phenotype provides a direct clue to gene function. 4. Mengiste T, Paszkowski J: Prospects for the precise engineering of 19. Meissner RC, Jin H, Cominelli E, Denekamp M, Fuertes A, Greco R, plant genomes by homologous recombination. Biol Chem 1999, Kranz HD, Penfield S, Petroni K, Urzainqui A et al.: Function search in 380:749-758. a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations 5. Speulman E, Metz PL, van Arkel G, te Lintel Hekkert B, Stiekema WJ, in R2R3 MYB genes. Plant Cell 1999, 11:1827-1840. Pereira A: A two-component enhancer–inhibitor system for functional analysis of the Arabidopsis 20. Ross-Macdonald P, Coelho PS, Roemer T, Agarwal S, Kumar A, genome. Plant Cell 1999, 11:1853-1866. Jansen R, Cheung KH, Sheehan A, Symoniatis D, Umansky L et al.: Arabidopsis gene knockout: phenotypes wanted Bouché and Bouchez 117

Large-scale analysis of the yeast genome by transposon tagging protein phosphatases 2C involved in abscisic acid signal and gene disruption. Nature 1999, 402:413-418. . Plant Cell 1997, 9:759-771. 21. Schaffer R, Landgraf J, Pérez-Amador M, Wisman E: Monitoring 36. Baulcombe DC: Fast forward genetics based on virus-induced genome-wide expression in plants. Curr Opin Biotechnol 2000, gene silencing. Curr Opin Plant Biol 1999, 2:109-113. 11:162-167. 37. Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF: B and 22. Gaymard F, Pilot G, Lacombe B, Bouchez D, Bruneau D, Boucherez J, •• C floral organ identity functions require SEPALLATA MADS-box Michaux-Ferriere N, Thibaud JB, Sentenac H: Identification and genes. 405 + Nature 2000, :200-203. disruption of a plant shaker-like outward channel involved in K This paper and [38••] present the first examples in which the combination of release into the xylem sap. Cell 1998, 94:647-655. individual knockouts that have no individual mutant phenotypes results in 23. Hirsch RE, Lewis BD, Spalding EP, Sussman MR: A role for the informative combined mutant phenotypes. In the work described in this AKT1 potassium channel in plant nutrition. Science 1998, paper, three knockouts in AGAMOUS-LIKE genes (SEP1–SEP3) were 280:918-921. recovered from T-DNA and transposon populations. The triple mutant presented a phenotype similar to that obtained when the B and C 24. Geelen D, Lurin C, Bouchez D, Frachisse JM, Lelievre F, Courtial B, components of the ABC system (such as AGAMOUS and APETALA3) are Disruption of putative anion channel Barbier-Brygoo H, Maurel C: mutated, leaving only the A activity. All of the flower organs were replaced gene AtCLC-a in Arabidopsis suggests a role in the regulation of by sepals. This work provides evidence that functional redundancy exists nitrate content. Plant J 2000, 21:259-267. among SEP1, SEP2 and SEP3, and, hence, explains why the SEP genes 25. Sunkar R, Kaplan K, Bouché N, Arazi T, Dolev D, Talke IN, Maathuis FJM, were not previously identified by forward genetics. • Sanders D, Bouchez D, Fromm H: Expression of a truncated NtCBP4 channel in transgenic plants, and disruption of 38. Liljegren SJ, Ditta GS, Eshed Y, Savidge B, Bowman JL, Yanofsky MF: the homologous Arabidopsis CNGC1 gene confer Pb++ tolerance. •• SHATTERPROOF MADS-box genes control seed dispersal in Plant J 2000, 24:533-542. Arabidopsis. Nature 2000, 404:766-770. A cyclic-nucleotide-gated channel (CNGC) was disrupted in Arabidopsis. The two SHATTERPROOF (SHP) genes are members of the MADS-box The homozygous knockout has improved tolerance to Pb2+ together with multigene family, sharing 87% identity at the amino-acid level. SHP1 was attenuated accumulation of this metal. A similar phenotype was observed in isolated from a T-DNA population and SHP2 was previously disrupted by tobacco in which a encoding a truncated version of the channel, homologous recombination. The single shp knockouts display no apparent lacking regulatory domains, was overexpressed. This work provides an phenotype, but fruits of the double mutant shp1; shp2 are unable to example of the complementarity between gene knockout and transgenic develop a dehiscent zone. This, together with the analysis of transgenic studies in revealing gene function. plants overexpressing the SHP genes, suggests that SHP1 and SHP2 26. Spalding EP, Hirsch RE, Lewis DR, Qi Z, Sussman MR, Lewis BD: encode a redundant function involved in the formation of the fruit-valve Potassium uptake supporting plant growth in the absence of margin. AKT1 channel activity: inhibition by ammonium and stimulation by 39. Nowak MA, Boerlijst MC, Cooke J, Smith JM: Evolution of genetic sodium. 113 J Gen Physiol 1999, :909-918. redundancy. Nature 1997, 388:167-171. 27. Green RM, Tobin EM: Loss of the circadian clock-associated 40. Thatcher JW, Shaw JM, Dickinson WJ: Marginal fitness • protein 1 in Arabidopsis results in altered clock-regulated gene contributions of nonessential genes in yeast. expression. Proc Natl Acad Sci USA 1999, 96:4176-4179. Proc Natl Acad Sci Disruption of a circadian-clock associated protein (CCA1) resulted in the USA 1998, 95:253-257. modified expression of five circadian genes, which displayed a shorter 41. Asmussen MA, Gilliland LU, Meagher RB: Detection of deleterious period of oscillation than is normal. In addition, CCA1 was shown to be genotypes in multigenerational studies. II. Theoretical and involved in light regulation of gene expression: in cca1-1 knockouts, the experimental dynamics with selfing and selection. Genetics 1998, expression level of a gene induced by phytochrome was reduced. 149:727-737. Surprisingly, no morphological changes were detected in the knockout mutant. 42. Gilliland LU, McKinney EC, Asmussen MA, Meagher RB: Detection of 28. Fitzgerald MS, Riha K, Gao F, Ren S, McKnight TD, Shippen DE: deleterious genotypes in multigenerational studies. I. Disruptions • Disruption of the telomerase catalytic subunit gene from in individual Arabidopsis actin genes. Genetics 1998, Arabidopsis inactivates telomerase and leads to a slow loss of 149:717-725. telomeric DNA. Proc Natl Acad Sci USA 1999, 96:14813-14818. 43. Bouchez D, Camilleri C, Caboche M: A binary vector based on The disruption of a telomerase gene, which results in shorter telomeres in the knockout plants, is reported. Interestingly, mutants are viable for at least Basta resistance for in planta transformation of Arabidopsis two generations, presenting no apparent modification, although telomere thaliana. Comptes Rendus de L’Academie des Sciences Serie III length decreases as the generations progress. Sciences de la Vie 1993, 316:1188-1193. 29. Couteau F, Belzile F, Horlow C, Grandjean O, Vezon D, Doutriaux MP: 44. Bechtold N, Ellis J, Pelletier G: In planta Agrobacterium mediated Random chromosome segregation without meiotic arrest in both gene transfer by infiltration of adult Arabidopsis thaliana plants. male and female meiocytes of a dmc1 mutant of Arabidopsis. Comptes Rendus de L’Academie des Sciences Serie III Sciences de Plant Cell 1999, 11:1623-1634. la Vie 1993, 316:1194-1199. 30. Blanc G, Barakat A, Guyot R, Cooke R, Delseny M: Extensive 45. Soyka S, Heyer AG: Arabidopsis knockout of ADC2 gene duplication and reshuffling in the Arabidopsis genome. Plant reveals inducibility by osmotic stress. FEBS Lett 1999, Cell 2000, 12:1093-1102. 458:219-223. 31. Mayer K, Schuller C, Wambutt R, Murphy G, Volckaert G, Pohl T, 46. Bauer J, Chen K, Hiltbunner A, Wehrli E, Eugster M, Schnell D, Dusterhoft A, Stiekema W, Entian KD, Terryn N et al.: Sequence and Kessler F: The major protein import receptor of plastids is analysis of chromosome 4 of the plant Arabidopsis thaliana. essential for chloroplast biogenesis. Nature 2000, 403:203-207. Nature 1999, 402:769-777. 47. Ratcliffe OJ, Riechmann JL, Zhang JZ: INTERFASCICULAR 32. Lin X, Kaul S, Rounsley S, Shea TP, Benito MI, Town CD, Fujii CY, FIBERLESS1 is the same gene as REVOLUTA. Plant Cell 2000, Mason T, Bowman CL, Barnstead M et al.: Sequence and analysis 12:315-317. of chromosome 2 of the plant Arabidopsis thaliana. Nature 1999, 402:761-768. 48. Allego ME, Jeanneau M, Granier F, Bouchez D, Bechtold N, White CI: 33. Terryn N, Heijnen L, De Keyser A, Van Asseldonck M, De Clercq R, Disruption of the Arabidopsis gene leads to plant sterility Verbakel H, Gielen J, Zabeau M, Villarroel R, Jesse T et al.: Evidence and MMS sensitivity. Plant J 2001, in press. for an ancient chromosomal duplication in Arabidopsis thaliana 49. Muller A, Guan C, Galweiler L, Tanzler P, Huijser P, Marchant A, Parry G, by sequencing and analyzing a 400-kb contig at the APETALA2 Bennett M, Wisman E, Palme K: AtPIN2 defines a locus of Arabidopsis locus on chromosome 4. FEBS Lett 1999, 445:237-245. for root gravitropism control. EMBO J 1998, 17:6903-6911. 34. Brookfield J: Genetic redundancy. Adv Genet 1997, 36:137-155. 50. Eastmond PJ, Germain V, Lange PR, Bryce JH, Smith SM, Graham IA: 35. Leung J, Merlot S, Giraudat J: The Arabidopsis ABSCISIC Postgerminative growth and lipid catabolism in oilseeds lacking ACID-INSENSITIVE2 (ABI2) and ABI1 genes encode homologous the glyoxylate cycle. Proc Natl Acad Sci USA 2000, 97:5669-5674.

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