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A genomic perspective on plant transcription factors José Luis Riechmann* and Oliver J Ratcliffe Data from the Arabidopsis genome project suggest that more than 5% of the genes of this plant encode transcription factors. The necessity for the use of genomic analytical approaches becomes clear when it is considered that less than 10% of these factors have been genetically characterized. A variety of tools for functional genomic analyses in plants have been developed over the past few years. The availability of the full complement of Arabidopsis transcription factors, together with the results of recent studies that illustrate some of the challenges to their functional characterization, now provides the basic framework for future analyses of transcriptional regulation in plants. Addresses Mendel Biotechnology, 21375 Cabot Boulevard, Hayward, California 94545, USA; *e-mail: jriechmann@mendelbio.com Current Opinion in Plant Biology 2000, 3:423–434 1369-5266/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviations AG AGAMOUS ANL2 ANTHOCYANINLESS2 AP2 APETALA2 bZIP basic-region leucine-zipper CAL CAULIFLOWER DAG1 Dof AFFECTING GERMINATION 1 DEF DEFICIENS FUL FRUITFULL FT FLOWERING LOCUS T GLO GLOBOSA Mbp megabase pairs/million base pairs NPR1 NONEXPRESSER OF PR GENES 1 PAN PERIANTHIA PLE PLENA RNAi RNA interference SEP1–3 SEPALLATA1–3 SHP1 SHATTERPROOF1 SQUA SQUAMOSA TRAB1 TRANSCRIPTION FACTOR RESPONSIVE FOR ABA REGULATION 1 YPD Yeast Proteome Database

Introduction With the completion of the Arabidopsis thaliana genome sequence, the full complement of transcription-factorencoding genes from a plant can be characterized and functionally analyzed for the first time. The recent shift from a ‘gene-centric’ to a ‘genome-centric’ perspective in biology is particularly appropriate for the study of transcription factors. Transcription-factor genes comprise a substantial fraction of all eukaryotic genomes, and the majority can be grouped into a handful of different, often large, gene families according to the type of DNA-binding domain that they encode. Functional redundancy is not unusual within these families; therefore the proper characterization of particular transcription-factor genes often

requires their study in the context of a whole family. Transcription factors form intricate networks, both through protein–protein interactions (among themselves and with proteins of other classes) and at the transcriptional level. Thus, because they control the expression of the genome, ultimately, their functions cannot be understood without considering their activities at a genome-wide scale. In this review, we intend to provide a genomic perspective on plant transcription factors, and for that purpose we have focused mainly on results from Arabidopsis. We begin with an overview of the transcription-factor-gene content of the Arabidopsis genome, and discuss particular challenges to progress in understanding these genes that have been illustrated by recent studies. We then consider techniques for characterizing the regulatory networks that are formed by transcription factors. More general reviews on the concepts, methodologies and prospects of plant genomics, and on plant transcription factors have been published recently [1•,2].

Transcription-factor-gene content of the Arabidopsis genome The Arabidopsis genome consists of approximately 130 megabase pairs (or million base pairs [Mbp]) of DNA. The determination and analysis of the sequence of chromosomes 2 and 4 have provided the first detailed description of a higher plant genome [3••,4••]. The data obtained so far suggest that Arabidopsis contains close to 30,000 genes, a relatively large number compared to the 18,424 and 13,601 genes predicted to form the genomes of Caenorhabditis elegans and Drosophila melanogaster, respectively (Table 1). The scope of genomic studies is highlighted by the fact that approximately 50% of the Arabidopsis genes have no proposed function [3••,4••]. Importantly, the Arabidopsis genome, like those of Saccharomyces cerevisiae, C. elegans and Drosophila, also contains extensive duplications. These include many tandem gene duplications as well as large-scale duplications on different chromosomes [3••,4••], and might affect about 40% of the total number of genes. The prevalence of gene duplication in Arabidopsis suggests that redundancy will present an obstacle to the functional analysis of genes. How does the Arabidopsis transcription-factor-gene content compare to that of other eukaryotic organisms? The estimated 700 and 500 transcription factors encoded by the Drosophila and C. elegans genomes, respectively, account for 5% and 2.7% of their total number of genes (Table 1). The analysis of approximately 110 Mbp of sequence, including the annotated sequences of chromosomes 2 and 4 [3••,4••], suggests that there might be more than 1700 transcriptionfactor genes in Arabidopsis. This represents more than 5% of the estimated 30,000 genes in this plant species. The difference in transcription-factor-gene content between


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Table 1 Transcription-factor gene content of multicellular eukaryotes. Organism

Genome size

Total number of predicted genes

Total number of predicted TF genes

% of genes that encode TFs

% of TFs genetically characterized

References

A. thaliana*

~130 Mbp (120 Mbp of euchromatin)

28,000–30,000

~1700

5.6%

~7%

[3••,4••,65]

C. elegans

97 Mbp

18,425

~500

2.7%

<20%

[7••,66–68]

~180 Mbp (120 Mbp of euchromatin)

13,601

694

5%

~25%

[5••,7••]

D. melanogaster

*Estimations based on the analysis of approximately 110 Mbp of genomic sequence and a genome size of 130 Mbp. TF, transcription factor.

the Drosophila and the C. elegans genomes has been proposed to reflect the substantial regulatory complexity of Drosophila [5••]. From that point of view, Arabidopsis is certainly not simpler than Drosophila. It has been noted before that many transcription-factorgene families exhibit great disparities in abundance among different eukaryotic organisms, and that some families are lineage specific [5••,6,7••]. The major transcription-factor families of Arabidopsis are listed in Table 2. Interestingly, many of them are specific to plants (e.g. APETALA2 [AP2]/ethylene-responsive-element binding protein [EREBP], NAC, WRKY, AUXIN RESPONSE FACTOR [ARF]-Aux/IAA and Dof [for DNA-binding with one finger]). Some of the other groups (e.g. MYB, MADS and basic-region leucine-zipper [bZIP]), which are not especially numerous in animals or yeast, have been significantly amplified in the plant lineage. The extent to which the Arabidopsis complement of transcription factors represents a canon for other plants is an open question. The largest transcription-factor family in Arabidopsis is the MYB group, with approximately 180 members (Table 2). A phylogenetic comparison of a subset of maize and Arabidopsis MYB sequences shows that the amplification of this group occurred prior to the separation of monocots and dicots [8]. However, it has been predicted that maize contains more than 200 MYB genes, and several subgroups appear to have originated recently or undergone duplication [8]. At least two clades of MADS-box genes also appear to have been amplified in the phylogenetic lineage that led to grasses [9]. These recent expansions could have allowed a functional diversification that might not be present in Arabidopsis.

Searching for the function of genes: reverse genetics Only about 7% of the Arabidopsis transcription factors have been genetically and functionally characterized, many of those through the traditional genetic approach (now known as ‘forward genetics’) whereby genes are first defined by a mutant phenotype and then isolated. Clearly, this approach will continue to be extremely fruitful, and will be greatly facilitated by the information and reagents derived from

genomics programs (see Table 3). The recent abundance of sequence data has, however, made ‘reverse genetics’ (i.e. from sequence to function) methods essential. A variety of strategies have been devised to generate and isolate mutants in known genes of Arabidopsis and other plant species (such as maize) by T-DNA- or transposoninsertional mutagenesis [10,11•,12••,13•,14,15] (see also Update). In these methods, large populations of tagged mutants are generated, which can then be screened for insertions in specific genes. Alternatively, the insertion tags can be individually sequenced and compiled in databases that can be searched for a gene-disruption event of interest. The MYB gene family has been the subject of a coordinated effort for characterization using genomic approaches [16]. The first step in this project, which was initiated before a sizeable fraction of the Arabidopsis genome had been sequenced, consisted of cloning more than 90 family members, and determining their map positions and expression profiles [17,18]. A reverse screen for a total of 73 genes was then undertaken using a variety of transposon- and T-DNA-tagged populations. This resulted in the identification of 47 insertions in 36 different MYB genes [19•]. Homozygous lines for insertions in 26 of those genes were isolated, but these exhibited no obvious morphological alterations. Additional greenhouse and plate-based assays later revealed phenotypic changes in only a minority of these lines [19•]. The lack of phenotypic alterations in these mutants is probably due to functional redundancy among family members as well as by the limited number of assays that were performed. It is worth noting, however, that only 22 of the 47 insertions were actually within the corresponding open reading frames, and that many of the insertions reduced, but did not abolish, gene expression [19•]. The study illustrates two of the problems associated with this technique: insertions in particular genes might not be found, and the alleles generated might not be null. Nevertheless, the functions of several Arabidopsis transcription factors have been recently elucidated using insertion mutants that were isolated by reverse screening. These include the zinc-finger Dof AFFECTING GERMINATION 1 protein (DAG1) [20•], the MADS-domain


A genomic perspective on plant transcription factors Riechmann and Ratcliffe

425

Table 2 Major families of Arabidopsis transcription factors. Gene family

Estimated number Gene family functions† of genes in the Arabidopsis genome*

Predicted number of proteins‡

Genetically characterized Arabidopsis factors

D. melanogaster C. elegans S. cerevisiae

MYB

180

Secondary metabolism, cellular morphogenesis, signal transduction in plant growth, abiotic and biotic stress responses, circadian rhythm, and dorsoventrality

AtMYB2, ATR1, CCA1, CPC, GL1, LHY, WER

35

16

19

AP2/EREBP

150

Flower development, cell proliferation, secondary metabolism, abiotic and biotic stress responses, ABA response, and ethylene response

ABI4, ANT, AP2, CBF1-3/DREB1A-C, DREB2A, ERF1

0

0

0

NAC

105

Development, pattern formation, and organ separation

CUC2, NAP

0

0

0

bHLH/MYC

100

Anthocyanin biosynthesis, light response, flower development and abiotic stress

PIF3

61

38

8

bZIP

100

Seed-storage gene expression, photomorphogenesis, leaf development, flower development defense response, ABA response, and gibberellin biosynthesis

ABI5, HY5, PAN

24

18

15

HB

90

Development (leaf, root, internode, and ovule), stem cell identity, cell differentiation, growth responses, anthocyanin accumulation, and cell death

ANL2, ATHB-2, BEL1, GL2, KNAT1, REV, STM, WUS

113

88

10

Z-C2H2

85

Flower development, flowering time, seed development, and root nodule development

FIS2, SUP

352

138

47

MADS

80

Flower development, fruit development, flowering time, and root development

AG, AGL15, ANR1, AP1 AP3, CAL, FLC, FUL, PI, SEP1, SEP2, SEP3, SHP1, SHP2, SOC1, SVP

2

2

4

WRKY

75

Defense response

0

0

0

ARF-Aux/IAA

42

Auxin responses, development, and floral meristem patterning

0

0

0

Dof

41

Seed germination, endosperm-specific DAG1 expression, and carbon metabolism

0

0

0

AXR2, AXR3, ETT, MP, NPH4, SHY2

*Estimation based on the analysis of approximately 110 Mbp of genomic sequence (including chromosomes 2 and 4) and a genome size of 130 Mbp. †Includes data from species other than Arabidopsis. ‡Compilation of data from [7••] (URL: http://www.sciencemag.org/ feature/data/1049664.shl), and our own analysis. (JL Riechmann, OJ Ratcliff, unpublished data.) ABA, abscisic acid. Gene families: ARF, AUXIN RESPONSE FACTOR; bHLH, basic helix-loop-helix; EREBP, ethylene-responsive-binding element protein; HB, homeobox; IAA, indoleacetic acid; and Z-C2H2, zinc-finger protein of the C2H2 type. Arabidopsis factors: ABI4, ABSCISIC ACID INSENSITIVE 4; AGL, AGAMOUS-LIKE; ANT, AINTEGUMENTA; ATR1, ALTERED TRYPTOPHAN REGULATION 1; AXR, AUXIN RESISTANT; BEL1, BELL 1; CBF1–3, CRT/DRE BINDING FACTOR 1–3; CCA1,

CIRCADIAN CLOCK ASSOCIATED 1; CUC2, CUP-SHAPED COTYLEDON 2; CPC, CAPRICE; DREB1A–C, DRE BINDING PROTEIN 1A–C; ERF1, ETHYLENE RESPONSE FACTOR 1; ETT, ETTIN; FIS2, FERTILIZATION-INDEPENDENT SEED 2; FLC, FLOWERING LOCUS C; GL1, GLABROUS 1; HY5, HYPOCOTYL ELONGATED 5; KNAT1, KN1-LIKE Arabidopsis thaliana 1; LHY, LATE ELONGATED HYPOCOTYL; MP, MONOPTEROS; NAP, NAC ACTIVATED BY AP3/PI; NPH4, NONPHOTOTROPIC HYPOCOTYL 4; PI, PISTILLATA; PIF3, PHYTOCHROME INTERACTING FACTOR 3; REV, REVOLUTA; SHY2, SHORT HYPOCOTYL2; SOC1, SUPPRESSOR OF OVEREXPRESSION OF CO 1; STM, SHOOTMERISTEMLESS; SUP, SUPERMAN; SVP, SHORT VEGETATIVE PHASE; WER, WEREWOLF; and WUS, WUSCHEL.

proteins SEPALLATA1–3 (SEP1–3) and SHATTERPROOF1 (SHP1) [21••,22••], and the homeodomain-leucine zipper protein MUP24.4, which was found to correspond to REVOLUTA [23•] (Table 3).

The stochastic nature of insertional mutagenesis implies that additional, directed, techniques for reverse genetics are required. An alternative method, designed not to disrupt a gene but to interfere with its expression, is based on RNA


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Table 3 Genetically characterized plant transcription factors.* Gene(s)

Family

Species

Method

Function

References

ABI5

bZIP

Arabidopsis

Positional cloning, and genome sequence

Abscisic acid response

[69]

ANL2

HB

Arabidopsis

Transposon tagging

Root development, anthocyanin accumulation

[43•]

ATHB-2

HB

Arabidopsis

Ectopic overexpression

Shade-induced growth responses

[39]

CRC

YABBY

Arabidopsis

Positional cloning

Carpel and nectary development

[70•]

DAG1

DOF

Arabidopsis

Reverse genetics

Seed dormancy

[20•]

DIC

TCP

Antirrhinum

Sequence homology, and transposon mutagenesis

Flower development, asymmetry

[71••]

FAR

MADS

Antirrhinum

Sequence homology, and reverse genetics

Flower development, C-function gene

[33•]

FIL

YABBY

Arabidopsis

Positional cloning

Development, abaxial–adaxial patterning

[72]

INO

YABBY

Arabidopsis

Positional cloning

Ovule development, abaxial–adaxial patterning

[73]

Mszpt2-1

Z-C2H2

Medicago sativa

Antisense RNA, ectopic overexpression

Root nodule organogenesis

[74•]

Nodule inception (Nin)

Lotus japonicus

Transposon tagging

Root nodule organogenesis

[75••]

NZZ

Arabidopsis

Transposon tagging, and genome sequence

Ovule and anther development

[76•]

PIF3

bHLH

Arabidopsis

T-DNA tagging

Light response

[77]

REV/IFL1

HB

Arabidopsis

Reverse genetics, and positional cloning

Apical meristem development, fiber differentiation

Rht-B1a, Rhtt-D1a, d8

GRAS

Wheat, maize

Sequence homology

Gibberellin response

[79••]

SEP1, SEP2, SEP3

MADS

Arabidopsis

Reverse genetics

Flower development, organ identity

[21••]

SHP1, SHP2

MADS

Arabidopsis

Reverse genetics

Fruit dehiscence

[22••]

SHI

Arabidopsis

Activation tagging

Gibberellin response

[42•]

SVP

MADS

Arabidopsis

Transposon tagging

Flowering time

[80]

SI1

MADS

Maize

Transposon tagging

Flower development, B-function gene

SPL

Arabidopsis

Transposon tagging

Sporocyte development

SAP

Arabidopsis

Transposon tagging

Flower development

[83]

WER

MYB

Arabidopsis

Positional cloning

Epidermal cell patterning

[84]

[23•,78]

[81••] [82]

*Only examples of plant transcription factors genetically characterized during the period of this review are included. † Indicates that the gene does not belong to any of the major families. CRC, CRABS CLAW; DIC, DICHOTOMA; FAR, FARINELLI; FIL, FILAMENTOUS FLOWER; IFL1, INTERFASCICULAR FIBERLESS1; INO, INNER

NO OUTER; Mszpt2-1, Medicago sativa zinc-finger protein 1; Nin, Nodule inception; NZZ, NOZZLE; REV, REVOLUTA; Rht-B1, Reduced height B1; SAP, STERILE APETALA; SHI, SHORT INTERNODES; SI1, Silky1; SPL, SPOROCYTELESS. See footnote to Table 2 for definition of additional abbreviations.

interference (RNAi) [24]. RNAi, which was first described in C. elegans, refers to the block in gene expression that is caused by double-stranded RNA. It has been shown that the simultaneous expression of sense and antisense RNA can silence an endogenous reporter gene in transgenic rice [25]. The potential of RNAi for sequence-specific inhibition of gene function in Arabidopsis has been investigated with four different genes, three of which encode transcription factors that are involved in flower development: the MADS-box genes AP1 and AGAMOUS (AG), and the bZIP gene PERIANTHIA (PAN) [26•]. Specific and heritable phenocopies of ag and ap1 loss-of-function mutations were obtained by this

method, although only a small percentage of the lines showed a phenotype comparable to those of null ag or ap1 mutants. Significant interference with the activity of the third gene, PAN, was only detected in a mutant background known to enhance pan phenotypes. The relatively low penetrance of strong phenotypes currently represents a problem for the use of RNAi in high-throughput analysis. A related approach for inhibiting gene function, virus-induced gene silencing, has been reviewed recently [27]. Efficient homologous recombination systems, similar to those developed for the generation of gene knock-outs in


A genomic perspective on plant transcription factors Riechmann and Ratcliffe

yeast, mouse, or the moss Physcomitrella patens, are still not available for higher plants. However, the recently described DNA/RNA oligonucleotide-mediated sitedirected mutagenesis of maize and tobacco represents a step in that direction, although substantial improvements in efficiency are still needed [28,29].

Functional redundancy among transcription factors The extent of functional redundancy among transcription factors is illustrated by several recent studies on MADS-box genes. MADS-box genes are numerous in plants, and they control many aspects of plant development — most notably flower development, but also flowering time, and fruit and root development (Table 2) [9,30]. Redundancy within this class was first demonstrated between the Arabidopsis floral-meristem-identity genes CAULIFLOWER (CAL) and AP1 [31], but many other examples are now coming to light. SHP1 and SHP2 are MADS-box genes that are required for fruit dehiscence in Arabidopsis [22••]. SHP1 and SHP2 show 87% identity at the amino acid sequence level, and have similar expression patterns. Single mutants for either of these genes are phenotypically wild-type, but the shp1; shp2 double mutant bears fruit that fail to dehisce. Thus, the SHATTERPROOF genes encode redundant proteins that are required for the proper development of the fruit-valve margin [22••]. Four Arabidopsis MADS-box genes, AP1, AP3, PISTILLATA (PI), and AG, determine floral-organ identity following the postulates of the combinatorial ABC model [32]. (Genes of the A-, B-, and C-classes function combinatorially in whorls 1 and 2, 2 and 3, and 3 and 4, respectively, to specify the identities of sepals [A function, whorl 1], petals [A and B functions, whorl 2], stamens [B and C functions, whorl 3], and carpels [C function, whorl 4].) It has now been shown, however, that the organ-identity function of some of these genes requires the activity of three other members of the MADS-box family, SEP1, SEP2 and SEP3, which are closely related to each other and functionally redundant [21••]. Insertion mutants of SEP1, SEP2, and SEP3 have been obtained but these showed no marked phenotypic alteration, either singly or in double mutant combinations. The triple mutant, however, displayed a remarkable phenotype: all of the floral organs were converted to sepals. This implies that the B- and C-function genes (AP3 and PI, and AG, respectively) require SEP1–3 to specify petal, stamen, and carpel identities [21••]. Partially redundant functions have also been recently described for Antirrhinum FARINELLI (FAR), a C-function organ-identity gene that is closely related to PLENA (PLE) [33•], and for Arabidopsis FRUITFULL (FUL) [34•]. In addition to regulating fruit development, FUL was found to act redundantly with AP1 and CAL (to

427

which it is related in sequence and phylogeny) to control inflorescence architecture [34•,35]. All of these examples should not be regarded as exceptions. In fact, sequence analysis of the Arabidopsis MADS-box genes suggests that more than 40% might have (partially) redundant functions. A similar scenario will probably be encountered for the other plant transcriptionfactor families.

Gain-of-function approaches As the previous examples illustrate, a problem with the use of loss-of-function alleles to determine gene function is that that redundantly acting genes cannot be easily characterized. In those circumstances, the standard routine has been to generate transgenic plants in which those genes are constitutively expressed. There are many recent instances in which this approach has provided a useful means for revealing genetic function, especially when the function of the gene was already partially understood [36•–38•,39]. Furthermore, screens for dominant gain-of-function mutations, performed on special T-DNA or transposon insertion collections, can lead to the identification of novel genes. Lately, the utility of such screens has been demonstrated in Arabidopsis [40•] (see also Update). FLOWERING LOCUS T (FT), which acts to promote flowering, was recently cloned by activation tagging. The role of FT was confirmed as the gain-of-function mutants produced by activation tagging were early flowering, whereas ft loss-offunction alleles cause a late-flowering phenotype [41•]. The Arabidopsis transcription factor SHORT INTERNODES, which affects gibberellin responses, was also isolated as a result of an overexpression mutation [42•]. Despite such successes, gain-of-function approaches are subject to a number of difficulties. Most importantly, it can be unclear whether a phenotype reflects the true function of a gene or whether it is simply caused by interference with unrelated processes. Such problems could be particularly prevalent for transcription factors. This is illustrated by studies of mutations in the homeobox gene, ANTHOCYANINLESS2 (ANL2). A late-flowering dominant mutant of ANL2 (lab1-1D) was uncovered by Weigel et al. [40•]. This does not correlate with the anl2 loss-of-function phenotype, which shows no effects on flowering but influences anthocyanin accumulation and the cellular organization of the root [43•]. Further insights concerning gene function can be obtained using variations of the gain-of-function approach that permit regulated spatial or temporal mis-expression of a transgene, as exemplified by recent studies in which the Arabidopsis homeodomain protein WUSCHEL was mis-expressed in a specific region of the meristem [44••]. Modified versions of transcription factors can be prepared that are constitutively or conditionally active. Constitutively active forms can be obtained by fusion to a strong activation domain. Such constructs can afford a useful tool in analyzing genetic pathways. This has been


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demonstrated in Arabidopsis when the LEAFY protein fused to a VP16 activation domain was used to dissect interactions between LEAFY and its targets during floral patterning [45,46•]. Conditionally active factors can be generated by fusion to the heterologous glucocorticoid receptor. Using such variants, the specific effects and direct targets of a transcription factor can be more easily recognized. Examples of Arabidopsis transcription factors to which this method has already been applied include CONSTANS (CO), AP3, and LEAFY [47,48,49•] (see also Update). It is likely that the most powerful application of these and other inducible systems will be in expressionprofiling experiments that are used to uncover the networks of genes regulated by transcription factors.

Unraveling the networks: gene-expression profiling A centerpiece of functional genomic studies is DNA microarray technology, which allows the parallel monitoring of the expression of thousands of genes. This method and alternative techniques, along with their applications in plants, have been reviewed extensively [50,51]. Only preliminary reports of plant DNA microarray experiments have been published [52] (see also Update). It is obvious, however, that genome-wide expression-profiling experiments will be a cornerstone in the functional analysis of transcription factors, and that they will have a profound impact on plant biology research [1•]. DNA microarrays have already been used for a variety of purposes in several organisms including yeast, Drosophila, C. elegans and humans. These uses include: the characterization of cellular responses to various stimuli, the comprehensive description of gene regulation by signal transduction pathways, the analysis of mutants and developmental processes, and the molecular study and classification of disease states. The first genome-wide expression profiling study to be carried out on plants made use of a gel-based mRNA-profiling technology to characterize the scope of gene expression controlled by maize C1/R and P proteins [53••] (see also Update). The MYB transcription factor C1 and the basic helix-loop-helix (bHLH) protein R cooperate to activate the entire anthocyanin pathway, part of which is also independently activated by the MYB protein P [54]. The mRNA profiles of 6000–8000 genes were examined in maize cell-suspension lines following the induced expression of either a C1-R fusion protein or P. These profiles revealed that the regulatory functions of C1/R and P might be more extensive than originally expected, both because of the increased number of genes that they might regulate and the possibility that they might also act as repressors. The method described above and DNA microarray experiments of similar design (which can be further refined to distinguish between direct and indirect effects on gene expression) will ultimately be used to characterize the

plant transcriptome in all of its dynamic nature. Further illumination will be provided by the comparative analysis of promoter sequences of genes that share similar expression profiles [55•]. In addition, genome-wide searches for characterized transcription-factor binding sites are possible [56•]. The integration of these types of wide-ranging analyses will be used to define the networks by which transcription factors act.

Combinatorial control: protein–protein interactions The combinatorial nature of transcriptional control in eukaryotic cells allows for the generation of regulatory diversity by a limited number of factors [57]. This mode of regulation involves the formation of multi-protein complexes and results in the existence of networks in which different activators and/or repressors cooperate to the regulate multiple targets. Several examples of direct interactions between different plant transcription factors have been described [57]. Lately, most of the newly identified interactions have been discovered using the yeast-based two-hybrid system. For instance, NONEXPRESSER OF PR GENES 1 (NPR1), a key regulator of gene expression in the salicylic-acid-mediated plant defense response, interacts with several members of the TGA subgroup of bZIP proteins [58•,59•]. The NPR1–TGA interaction enhances the DNA-binding activity of the bZIP proteins, which, in turn, appears to be critical for the activation of defense genes. Using similar approaches, the rice bZIP protein TRANSCRIPTION FACTOR RESPONSIVE FOR ABA REGULATION 1 (TRAB1) has been found to interact with the transcription factor VIVIPAROUS1 (VP1), thus providing a mechanism for VP1-mediated, abscisic acid (ABA)-inducible gene expression [60•]. The Antirrhinum MADS-domain proteins SQUAMOSA (SQUA), DEFICIENS (DEF) and GLOBOSA (GLO) have various meristem and organ-identity functions in flower development [9,30], and also cooperate in establishing the whorled pattern of the flower in a partially redundant manner [61•]. DEF and GLO bind to DNA in the form of DEF–GLO heterodimers, whereas SQUA can do so as a SQUA–SQUA homodimer. A modified twohybrid system was used to show that DEF and GLO form ternary complexes with SQUA in yeast [61•]. Additional in vitro analyses indicated that DEF–GLO–SQUA ternary complexes bind to DNA with increased affinity [61•]. These observations expand what is known of the scope of potential regulatory interactions among MADS-domain proteins. Despite these and other examples, the number of reported interactions among plant transcription factors is small. It has been shown, however, that the two-hybrid system is amenable to a high-throughput genomic approach. A screen to identify interactions among all of the 6000 proteins encoded by the yeast genome was recently


A genomic perspective on plant transcription factors Riechmann and Ratcliffe

429

Table 4 Genome-wide analysis of protein–protein interactions in yeast.*† Interactor

YPD description‡

Interactor

YPD description

DAL82

Transcriptional activator

DAL82

Transcriptional activator

HAP2

Component of CCAAT-binding factor

HAP3

Component of CCAAT-binding factor

HAP2

Component of CCAAT-binding factor

HAP5

Component of CCAAT-binding factor

HAP3

Component of CCAAT-binding factor

HAP5

Component of CCAAT-binding factor

INO4

bHLH transcription factor

INO2

bHLH transcription factor

MET31

C2H2-type zinc-finger protein

GCN4

bZIP transcription factor

RIM101

Transcription factor

ZAP1

C2H2-type zinc-finger transcriptional activator

YAP5

bZIP transcription factor

RCS1

Transcription factor

CUP2

Transcription factor

RPB10

RNA polymerase subunit

FZF1

C2H2-type zinc-finger transcription factor

TFC4

TFIIIC component

CSE2

RNA polymerase subunit

MED4

RNA polymerase subunit

MED11

RNA polymerase subunit

SRB6

RNA polymerase subunit

MED11

RNA polymerase subunit

MET18

RNA pol II transcription and DNA repair

MED7

RNA polymerase subunit

MED4

RNA polymerase subunit

SRB5

RNA polymerase subunit

MED8

RNA polymerase subunit

SRB7

RNA polymerase subunit

MED4

RNA polymerase subunit

SRB7

RNA polymerase subunit

MED7

RNA polymerase subunit

TAF40

TFIID component

TAF25

RNA polymerase subunit

TAF60

TFIID component

TAF17

TFIID component

TFA1

TFIIE component

TFA2

TFIIE component

TFA1

TFIIE component

TFB1

TFIIH component

RRN7

Component of RNA pol I core factor

RRN6

Component of RNA pol I core factor

RRN9

Component of the UAF complex

RRN10

Component of the UAF complex

NIF3

RNA pol II transcription

NIF3

RNA pol II transcription

GCN5

Component of HAT complexes

ADA2

Component of HAT complexes

*Results from [62••]. URL: http://portal.curagen.com. †Only interactions among proteins in the 'Transcription Factor' or 'RNA pol II' categories of YPD [64] are included. ‡Descriptions from YPD [64]. URL: http://www.proteome.com

attempted, and similar experiments are being pursued in C. elegans [62••,63•]. The Yeast Proteome Database (YPD) [64] classifies proteins according to their functional roles: 372 different proteins are grouped under the ‘Transcription Factor’ and/or ‘Pol II transcription’ categories, which include transcription factors as well as components of the basal transcription machinery. The screen detected 25 ‘interactor pairs’ (involving 39 different proteins) in which both partners are among those 372 proteins (Table 4) [62••]. However, many of those protein pairs consist of components of the general transcription apparatus, and the number of interactions detected specifically among transcription factors was low (Table 4). Thus, it is clear that two-hybrid analyses will reveal only a subset of the interactions that occur in a cell, underscoring the necessity for the development of alternative techniques.

Conclusions A holistic view (in which whole entities, as fundamental components of reality, have an existence other than as the mere sum of their parts) of the regulation of transcription will ultimately emerge from the genomic analyses of transcription factors. For Arabidopsis, as for any other organism, progress toward this goal will begin with comprehensive analyses of the genome sequence to identify transcription-factor genes as well as the sequences on which they act and the genes that they regulate. Extensive DNA microarray experiments, the genomewide mapping of protein–protein interactions (and the use of proteomics approaches that are beyond the scope of this review), and the comprehensive phenotypic characterization of mutants for all transcription-factor genes will then be necessary. Finally, the plethora of data generated must be correlated and integrated. As the work summarized in


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this review illustrates, the first steps of what promises to be a long journey have already been taken.

Update Research in plant genomics and transcription factors progresses at a rapid pace. A reverse genetics strategy to screen for chemically induced mutations in target sequences (known as Targeting Induced Local Lesions IN Genomes or TILLING) has recently been described for Arabidopsis [85•,86]. Because chemical mutagenesis can generate a wide variety of mutant alleles, this method represents a valuable alternative to insertional mutagenesis. A T-DNA tagging approach has recently been used to identify regulators of the terpenoid indole alkaloid metabolic pathway in Catharanthus roseus (the Madagascar periwinkle) [87••]. The approach led to the isolation of a jasmonate-responsive transcription factor of the AP2/EREBP family, ORCA3, which was shown to regulate the genes of primary and secondary metabolic pathways. This transcription factor might link plant stress responses to changes in metabolism [87••]. A steroid-inducible version of CO, created by a protein fusion to the glucocorticoid receptor, has recently been used, in combination with suppression subtraction hybridization, to isolate early targets of CO regulation [88••]. One of the genes identified among those early targets is a member of the MADS-box family, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1). Finally, DNA microarray studies have recently been used to explore the developmental process of fruit ripening in strawberry, which identified a novel strawberry alcohol acetyltransferase (SAAT) gene involved in flavor biogenesis [89••], and to examine the wounding response of Arabidopsis [90•].

Acknowledgements We thank our colleagues at Mendel Biotechnology for discussions, insight and comments. We are grateful to Marty Yanofsky and Elliot Meyerowitz for providing manuscripts before publication. Finally, we apologize to those whose work we could not cite because of space constraints.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • of special interest •• of outstanding interest 1. Somerville C, Somerville S: Plant functional genomics. Science • 1999, 285:380-383. A concise and excellent review that encompasses information on the content of plant genomes and their functional analyses. The review also describes how genomics is influencing and will continue to influence plant biology research. 2.

3. ••

Liu L, White MJ, MacRae T: Transcription factors and their genes in higher plants. Functional domains, evolution and regulation. Eur J Biochem 1999, 262:247-257.

Lin X, Kaul S, Rounsley S, Shea TP, Benito M-I, Town CD, Fujii CY, Mason T, Bowman CL, Barnstead M et al.: Sequence and analysis of chromosome 2 of the plant Arabidopsis thaliana. Nature 1999, 402:761-768. This landmark paper and [4••] report the sequence and organization of Arabidopsis chromosomes 2 and 4, respectively. These articles afford the first

detailed view of a higher plant genome. Chromosome 2 consists of 19.6 Mbp of DNA and is predicted to contain 4037 genes. Chromosome 4 (17.38 Mbp) is predicted to encode 3744 genes. Extensive gene and large-segment duplications were detected. Approximately half of the genes in these two chromosomes have been classified according to a definite or putative function. 4. ••

The European Union Arabidopsis Genome Sequencing Consortium and The Cold Spring Harbor, Washington University in St Louis and PE Biosystems Arabidopsis Sequencing Consortium: Sequence and analysis of chromosome 4 of the plant Arabidopsis thaliana. Nature 1999, 402:769-777. See annotation for [3••]. 5. ••

Adams MD, Celniker SE, Holt RA, Evans CA, Gocayne JD, Amanatides PG, Scherer SE, Li PW, Hoskins RA, Galle RF et al.: The genome sequence of Drosophila melanogaster. Science 2000, 287:2185-2195. The rapid sequencing, using a whole-genome ‘shotgun’ strategy, of the 120 Mbp euchromatic region of the Drosophila genome, and its analysis, is reported. Drosophila was found to contain only approximately 13,600 genes, fewer than C. elegans. This is the first report of the use of the ‘shotgun’ strategy in a complex eukaryotic genome. 6.

Chervitz SA, Aravind L, Sherlock G, Ball CA, Koonin EV, Dwight SS, Harris MD, Dolinski K, Mohr S, Smith T et al.: Comparison of the complete protein sets of worm and yeast: orthology and divergence. Science 1998, 282:2022-2028.

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Rubin GM, Yandell MD, Wortman JR, Gabor Miklos GL, Nelson CR, Hariharan IK, Fortini ME, Li PW, Apweiler R, Fleischmann W et al.: Comparative genomics of the eukaryotes. Science 2000, 287:2204-2215. The gene content of the three available eukaryotic genomes, those of C. elegans, Saccharomyces cerevisiae, and D. melanogaster, is reviewed in the context of cellular, developmental and evolutionary processes. The authors point out that C. elegans and D. melanogaster have similarly sized sets of non-redundant proteins that are each approximately twice the size of that of yeast. This suggests that apparent developmental complexity is not necessarily related to absolute gene number. 8.

Rabinowicz PD, Braun EL, Wolfe AD, Bowen B, Grotewold E: Maize R2R3 Myb genes: sequence analysis reveals amplification in the higher plants. Genetics 1999, 153:427-444.

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14. Walbot V: Saturation mutagenesis using maize transposons. Curr Opin Plant Biol 2000, 3:103-107.

be identical to those of INTERFASCICULAR FIBERLESS1 (IFL1) [78], revealing that IFL1 and REVOLUTA are the same gene.

15. Krysan PJ, Young JC, Sussman MR: T-DNA as an insertional mutagen in Arabidopsis. Plant Cell 1999, 11:2283-2290.

24. Bosher JM, Labouesse M: RNA interference: genetic wand and genetic watchdog. Nat Cell Biol 2000, 2:E31-E36.

16. Jin H, Martin C: Multifunctionality and diversity within the plant MYB-gene family. Plant Mol Biol 1999, 41:577-585.

25. Waterhouse PM, Graham MW, Wang M-B: Virus resistance and gene silencing in plants can be induced by simultaneous expression of sense and antisense RNA. Proc Natl Acad Sci USA 1998, 95:13959-13964.

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Romero I, Fuertes A, Benito MJ, Malpica JM, Leyva A, Paz-Ares J: More than 80 R2R3–MYB regulatory genes in the genome of Arabidopsis thaliana. Plant J 1998, 14:273-284.

18. Kranz HD, Denekamp M, Greco R, Jin H, Leyva A, Meissner RC, Petroni K, Urzainqui A, Bevan M, Martin C et al.: Towards functional characterisation of the members of the R2R3–MYB gene family from Arabidopsis thaliana. Plant J 1998, 16:263-276. 19. Meissner RC, Jin H, Cominelli E, Denekamp M, Fuertes A, Greco R, • Kranz HD, Penfield S, Petroni K, Urzainqui A et al.: Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes. Plant Cell 1999, 11:1827-1840. This is the first report in plants of a large-scale functional analysis across a whole transcription-factor family by gene disruption. Collections of both transposon and T-DNA insertion lines were screened for 73 different genes, leading to the identification of insertions in 36 genes. A preliminary examination of plants carrying these disruptions revealed few novel phenotypes, suggesting that a significant number of the disrupted transcription-factor genes are conditionally active or partially redundant. The study also allows a direct comparison of different transposon and T-DNA insertion collections, and illustrates the difficulties encountered with these techniques. 20. Papi M, Sabatini S, Bouchez D, Camilleri C, Costantino P, • Vittorioso P: Identification and disruption of an Arabidopsis zinc finger gene controlling seed germination. Genes Dev 2000, 14:28-33. This paper details how the function of a zinc-finger gene, DAG1, was uncovered by a reverse genetic approach. The Arabidopsis DAG1 gene was isolated by its homology to the tobacco gene Nicotiana tabacum rolB domain B Factor 1 (NtBBf1), and a dag1 mutant was obtained by screening a Y-DNA insertion collection. Mutant and expression analyses revealed that DAG1 is a maternally acting factor that promotes seed dormancy. 21. Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF: B and C •• floral organ identity functions require SEPALLATA MADS-box genes. Nature 2000, 405:200-203. The function of three closely related MADS-box genes, SEP1–3 (previously known as AGAMOUS-LIKE 2 (AGL2), AGL4 and AGL9), is characterized. These genes were originally identified by their homology to AG, and they exhibit expression patterns that implicate them in the regulation of floral development. The authors adopted a reverse genetic approach, screening transposon and T-DNA insertion collections to identify mutants in each of the three genes. Singly, the mutants showed only subtle phenotypes. When the triple mutant was produced, however, it displayed a novel feature: all four floral whorls contained sepals. This demonstrated that the SEP genes act redundantly and are required in combination with the B and C organ-identity genes to specify the differences in organ types between floral whorls. These results provide a remarkable example of functional redundancy within the MADS-box family, and further develop the ABC model of floral-organ-identity determination. SEP1–3 could represent cofactors that are required by the floral homeotic genes for the conversion of leaves (the proposed ‘ground sate’ of floral organs) into flower parts. Additional experimentation, making use of the many loss- and gain-of-function alleles that are available for a variety of genes, will position SEP1–3 more precisely in the current framework of flower development in the near future. 22. Liljegren S, Ditta GS, Eshed Y, Savidge B, Bowman JL, Yanofsky MF: •• SHATTERPROOF MADS-box genes control seed dispersal in Arabidopsis. Nature 2000, 404:766-770. This paper provides a further example of functional redundancy between closely related MADS-box genes. SHP1 and SHP2 (previously known as AGL1 and AGL5) were initially identified by their homology to AG. A shp1 mutant was obtained by screening T-DNA insertion lines and combined with a shp2 mutant (which was previously generated via homologous recombination). Single mutants were phenotypically wild-type but the double mutant produced fruits that failed to dehisce. This, along with constitutive expression results, demonstrates that the SHP genes promote the development of valve margins and fruit dehiscence. 23. Ratcliffe OJ, Riechmann JL, Zhang JZ: INTERFASCICULAR • FIBERLESS1 is the same gene as REVOLUTA. Plant Cell 2000, 12:315-317. The authors screened a T-DNA insertion collection to isolate a mutant for a novel homeobox gene of unknown function. The insertion mutant had an identical phenotype to revoluta mutants; complementation and allele-sequencing analysis demonstrated that the novel gene was REVOLUTA. The sequence, mutant phenotype, and map position of this gene were subsequently found to

26. Chuang C-F, Meyerowitz EM: Specific and heritable genetic • interference by double-stranded RNA in Arabidopsis thaliana. Proc Natl Acad Sci USA 2000, 97:4985-4990. RNA interference (RNAi) is used to reduce the activity of selected genes in Arabidopsis. Constructs co-expressing sense and antisense sequences from four different genes AG, AP1, PAN (which are transcription factors), and CLAVATA3 were transformed into Arabidopsis. Transformants showed the known mutant phenotypes at a much higher frequency than plants singly expressing either the antisense or sense version of a transgene alone. Different lines exhibited heritable phenotypes of varying severity, mimicking an allelic series; this was found to correlate with the level of endogenous mRNA present. Interestingly, the use of AP1 did not result in an ap1 ; cal double mutant phenotype in any of the lines analyzed, although AP1 and CAL are 76% identical in the segment used in the RNAi constructs. The study suggests that RNAi might be applicable to genomics programs as a reverse genetic approach, although the relatively low penetrance of strong phenotypes currently represents a problem for its use in high-throughput analysis. It should also be noted that this method might not provide information about genes for which low expression levels are sufficient for normal gene function, as exemplified by PAN. 27.

Baulcombe DC: Fast forward genetics based on virus-induced gene silencing. Curr Opin Plant Biol 1999, 2:109-113.

28. Zhu T, Peterson DJ, Tagliani L, St Clair G, Baszczynski CL, Bowen B: Targeted manipulation of maize genes in vivo using chimeric RNA/DNA oligonucleotides. Proc Natl Acad Sci USA 1999, 96:8768-8773. 29. Beetham PR, Kipp PB, Sawycky XL, Arntzen CJ, May GD: A tool for functional plant genomics: chimeric RNA/DNA oligonucleotides cause in vivo gene-specific mutations. Proc Natl Acad Sci USA 1999, 96:8774-8778. 30. Riechmann JL, Meyerowitz EM: MADS domain proteins in plant development. J Biol Chem 1997, 378:1079-1101. 31. Kempin SA, Savidge B, Yanofsky MF: Molecular basis of the cauliflower phenotype in Arabidopsis. Science 1995, 267:522-525. 32. Bowman JL, Smyth DR, Meyerowitz EM: Genetic interactions among floral homeotic genes of Arabidopsis. Development 1991, 112:1-20. 33. Davies B, Motte P, Keck E, Saedler H, Sommer H, Schwarz• Sommer Z: PLENA and FARINELLI: redundancy and regulatory interactions between two Antirrhinum MADS-box factors controlling flower development. EMBO J 1999, 18:4023-4034. An Antirrhinum MADS-box gene, FAR, that is closely related to PLE was identified. The authors then screened a transposon insertion collection to obtain far mutants. Genetic, expression, and protein-interaction studies of FAR, PLE, and other genes involved in floral patterning, were used to analyze the function of FAR. It is shown that FAR and PLE, the two partially redundant Antirrhinum C-function genes, play complementary but somewhat distinct roles in flower development. 34. Ferrándiz C, Gu Q, Martienssen R, Yanofsky MF: Redundant regulation • of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER. Development 2000, 127:725-734. This paper describes a case of partial redundancy within the MADS-box gene family. By analyzing mutants for FUL in combination with mutants for the related genes AP1 and CAL, FUL was discovered to have a previously unrecognized role in inflorescence development. FUL affords a good example of a gene co-opted during evolution for two unrelated functions. By phylogeny, FUL belongs to the AP1 clade, yet it also participates in fruit development together with members of the AG clade. 35. Gu Q, Ferrándiz C, Yanofsky MF, Martienssen R: The FRUITFULL MADS-box gene mediates cell differentiation during Arabidopsis fruit development. Development 1998, 125:1509-1517. 36. Fernandez DE, Heck GR, Perry SE, Patterson SE, Bleecker AB, • Fang S-C: The embryo MADS domain factor AGL15 acts postembryonically: inhibition of perianth senescence and abscission via constitutive expression. Plant Cell 2000, 12:183-197. A detailed analysis of expression patterns combined with the effects of constitutive AGL15 activity, reveals that this gene could regulate age-dependent developmental programs in Arabidopsis.


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37. •

Mizukami Y, Fischer RL: Plant size organ control: AINTEGUMENTA regulates growth and cell numbers during organogenesis. Proc Natl Acad Sci USA 2000, 97:942-947. In this paper and [38•], the authors compare the effects of loss- and gain-offunction of the APETALA2/ethylene-responsive-binding-protein (EREBP) gene AINTEGUMENTA. It is shown that the transcription factor that it encodes regulates plant organ size by controlling the extent of cell proliferation within organs. Together with the findings described in [36•,38•], these studies illustrate how gain-of-function mutants can uncover gene activities not revealed by loss-of-function alleles.

46. Busch MA, Bomblies K, Weigel D: Activation of a floral homeotic • gene in Arabidopsis. Science 1999, 285:585-587. This painstaking study illuminates interactions that occur between transcription factors that are involved in floral patterning. A series of reporter constructs were generated carrying various genomic fragments of the AG gene in front of a minimal promoter GUS fusion. The constructs were then introduced into wild-type plants, leafy mutants and plants carrying an activated form of LEAFY. The results show that LEAFY is a direct activator of AG, and that this direct activation relies upon the interaction of LEAFY with an enhancer element within the AG gene.

38. Krizek BA: Ectopic expression of AINTEGUMENTA in Arabidopsis • plants results in increased growth of floral organs. Dev Genetics 1999, 25:224-236. In this paper and [37•], a gain-of-function approach is used to show that the AP2/EREBP gene AINTEGUMENTA regulates plant organ size by controlling the extent of cell proliferation and/or cell expansion within organs.

47.

39. Steindler C, Matteucci A, Sessa G, Weimar T, Ohgishi M, Aoyama T, Morelli G, Ruberti I: Shade avoidance responses are mediated by the ATHB-2 HD-Zip protein, a negative regulator of gene expression. Development 1999, 126:4235-4245.

49. Wagner D, Sablowski RWM, Meyerowitz EM: Transcriptional • activation of AP1 by LEAFY. Science 1999, 285:582-584. The authors used a steroid-inducible system to demonstrate that LEAFY is a transcriptional activator of the APETALA1 gene in vivo.

40. Weigel D, Ahn JH, Blazquez MA, Borevitz JO, Christensen SK, • Fankhauser C, Ferrandiz C, Kardailsky I, Malancharuvil EJ, Neff MM et al.: Activation tagging in Arabidopsis. Plant Physiol 2000, 122:1003-1014. The authors discuss the results of screening tens of thousands of Arabidopsis plants that had been transformed with activation-tagging constructs that contain transcriptional enhancers derived from the cauliflower mosaic virus 35S promoter. More than 30 dominant mutations affecting a variety of processes were isolated. The transformation vector was also engineered with a bacterial selection marker and replication origin to allow the rapid isolation of tagged genes by plasmid rescue.

50. Richmond T, Somerville S: Chasing the dream: plant EST microarrays. Curr Opin Plant Biol 2000, 3:108-116.

41. Kardailsky I, Shukla VK, Ahn JH, Dagenais N, Christensen SK, • Nguyen JT, Chory J, Harrison MJ, Weigel D: Activation tagging of the floral inducer FT. Science 1999, 286:1962-1965. This paper illustrates the potential of activation tagging. In a random screen of activation-tagged lines, the authors found a gain-of-function early-flowering mutant with terminal flowers. The tagged gene was then cloned by plasmid rescue. The insertion mapped close to FT, a gene known to promote flowering; the authors sequenced a range of ft alleles and determined that the cloned gene was FT. This gene encoded a homolog of TERMINAL FLOWER1, a protein that acts to repress the floral transition. The work is of particular interest as it reveals that closely related proteins can perform opposite functions within biological control networks. 42. Fridborg I, Kuusk S, Moritz T, Sundeberg E: The Arabidopsis dwarf • mutant shi exhibits reduced gibberellin responses conferred by overexpression of a new putative zinc finger protein. Plant Cell 1999, 11:1019-1031. The authors discovered a transposon insertion mutant, short internodes, with characteristics similar to those of mutants with altered biosynthesis of, or responses to, gibberellic acid (GA). The tagged gene was identified by an inverse polymerase chain reaction and found to encode a new putative zincfinger transcription factor. Expression data then revealed shi to be a gain-offunction mutant, in which the gene is constitutively expressed from a 35S promoter within the transposon insertion. The authors suggest that SHI might be a transcriptional repressor of GA responses. 43. Kubo H, Peeters AJM, Aarts MGM, Pereira A, Koornneef M: • ANTHOCYANINLESS2, a homeobox gene affecting anthocyanin distribution and root development in Arabidopsis. Plant Cell 1999, 11:1217-1226. The isolation of ANTHOCYANINLESS2, an Arabidopsis homeobox gene of the GLABRA2 group, by transposon tagging is presented. Studies on the mutant revealed that this gene influences anthocyanin accumulation in the leaf as well as affecting cellular organization in the root. Interestingly, it has been reported that an activation allele of ANTHOCYANINLESS2 delays flowering [40•]. 44. Schoof H, Lenhard M, Haecker A, Mayer KFX, Jurgens G, Laux T: The •• stem cell population of Arabidopsis shoot meristems is maintained by a regulatory loop between the CLAVATA and WUSCHEL genes. Cell 2000, 100:635-644. This paper provides an elegant example of the utility of directed mis-expression to reveal gene function. The authors used a two-component system to direct expression of the homeobox gene WUSCHEL from the CLAVATA1 and AINTEGUMENTA promoters. The work demonstrates that WUSCHEL is both necessary and sufficient to induce shoot meristem stem-cell identity. The data support a model in which a balance between stem-cell proliferation and organ initiation is achieved in the meristem via a self-regulatory loop between the WUSCHEL and CLAVATA genes. 45. Parcy F, Nilsson O, Busch MA, Lee I, Weigel D: A genetic framework for floral patterning. Nature 1998, 395:561-566.

Simon R, Igeno MI, Coupland G: Activation of floral meristem identity genes in Arabidopsis. Nature 1996, 384:59-62.

48. Sablowski RWM, Meyerowitz EM: A homolog of NO APICAL MERISTEM is an immediate target of the floral homeotic genes APETALA3/PISTILLATA. Cell 1998, 92:93-103.

51. Schaffer R, Landgraf J, Perez-Amador M, Wisman E: Monitoring genome-wide expression in plants. Curr Opin Biotech 2000, 11:162-167. 52. Ruan Y, Gilmore J, Conner T: Towards Arabidopsis genome analysis: monitoring expression profiles of 1400 genes using cDNA microarrays. Plant J 1998, 15:821-833. 53. Bruce W, Folkerts O, Garnaat C, Crasta O, Roth B, Bowen B: •• Expression profiling of the maize flavonoid pathway genes controlled by estradiol-inducible transcription factors CRC and P. Plant Cell 2000, 12:65-79. An example, the first of its kind, of the use of a genome-wide mRNA-profiling technique to characterize the activities of plant transcription factors. The experiments detected differential expression of genes known to be regulated by maize C1/R and P, as well as of many other genes, indicating that these transcription factors play more complex roles than was originally expected. 54. Mol J, Grotewold E, Koes R: How genes paint flowers and seeds. Trends Plant Sci 1998, 3:212-217. 55. Wolfsberg TG, Gabrielian AE, Campbell MJ, Cho RJ, Spouge JL, • Landsman D: Candidate regulatory sequence elements for cell cycle-dependent transcription in Saccharomyces cerevisiae. Genome Res 1999, 9:775-792. A statistical method is developed to scan upstream regions of genes, which had been shown by microarray experiments to be cell-cycle regulated, for short elements that might be involved in the regulation of co-expressed groups. 56. Zhong H, McCord R, Vershon AK: Identification of target sites of • the α2-Mcm1 repressor complex in the yeast genome. Genome Res 1999, 9:1040-1047. This study demonstrates that if the binding sites of a transcription factor are well characterized, then novel target genes can potentially be identified by searching the genome sequence for those sites. 57.

Singh KB: Transcriptional regulation in plants: the importance of combinatorial control. Plant Physiol 1998, 118:1111-1120.

58. Zhang Y, Fan W, Kinkema M, Li X, Dong X: Interaction of NPR1 with • basic leucine zipper protein transcription factors that bind sequences required for salicylic acid induction of the PR-1 gene. Proc Natl Acad Sci USA 1999, 96:6523-6528. The mechanism by which NPR1 transduces salicylic acid signals to upregulate pathogenesis-related (PR) genes is investigated. Using the yeast two-hybrid system, it is shown that NPR1 specifically interacts with a subclass of bZIP transcription factors. The authors then demonstrate that these factors bind an element in the PR1 promoter. 59. Després C, DeLong C, Glaze S, Liu E, Fobert PR: The Arabidopsis • NPR1/NIM1 protein enhances the DNA binding activity of a subgroup of the TGA family of bZIP transcription factors. Plant Cell 2000, 12:279-290. The authors demonstrate, by a combination of in vivo and in vitro assays, that the Arabidopsis NPR1 protein (an important factor required for induced and acquired systemic disease resistance) differentially interacts with members of the TGA family of bZIP transcription factors. The relevance of those interactions in vivo is deduced from the observation that point mutations that abolish NPR1 function also disrupt the interaction with the TGA proteins.


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60. Hobo T, Kowyama Y, Hattori T: A bZIP factor, TRAB1, interacts with • VP1 and mediates abscisic acid-induced transcription. Proc Natl Acad Sci USA 1999, 96:15348-15353. Using a yeast two-hybrid screen, TRAB1 is shown to interact with VP1. VP1 mediates gene activation in seeds in response to abscisic acid (ABA), although VP1 does not directly bind to ABA-responsive elements (ABREs). ABREs are recognized by TRAB1. The authors propose that VP1 acts upon abscisic-acid-response elements via the interaction with TRAB1. 61. Egea-Cortines M, Saedler H, Sommer H: Ternary complex formation • between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus. EMBO J 1999, 18:5370-5379. Genetic analysis of double mutants between SQUA, and DEF or GLO indicated that these MADS-box genes have a (previously unknown) partially redundant function in establishing the whorled pattern of the Antirrhinum flower. To investigate this hypothesis, the authors tested interactions between the proteins; it is shown that the three proteins form a ternary complex (via their carboxy termini) in yeast. The ternary complex (composed of a DEF–GLO heterodimer and a SQUA–SQUA homodimer) exhibits a significant increase in DNA-binding affinity compared to either of the two dimers. This work emphasizes that such interactions at the protein level between transcription factors have expanded the potential for regulating biological networks. Thus, studies of this kind will become increasingly important in providing a complete understanding of transcription-factor activity. These results also help to explain, in part, the previous observations that determination of floral-organ identity by Arabidopsis MADS-domain proteins is independent of their individual DNA-binding specificity. 62. Uetz P, Giot L, Cagney G, Mansfield TA, Judson RS, Knight JR, •• Lockshon D, Narayan V, Srinivasa M, Pochart P et al.: A comprehensive analysis of protein–protein interactions in Saccharomyces cerevisiae. Nature 2000, 403:623-627. The authors performed and compared two large-scale two-hybrid assays to examine the potential interactions among the approximately 6000 gene products encoded by the yeast genome. In the first study, a panel of colonies was assembled, each containing one of the 6000 gene products fused to an activation domain. To reveal interactions, colonies harboring one of 192 proteins fused to a DNA-binding domain were each mated to the whole panel of activation domain fusions. In a second, high-throughput approach, cells expressing the 6000 different activation-domain fusions were pooled together into a library. This was screened against a panel containing 6000 proteins fused to DNA-binding domains. A total of 957 putative interactions were identified in these experiments, a significant number of which occurred between proteins of no functional classification. This indicates that there could be numerous pathways and complexes that have not yet been discovered by conventional approaches. It is noted, however, that this study revealed only a subset of previously described interactions; it does not, therefore, represent a complete description of all of the potential protein interactions in yeast. 63. Walhout AJM, Sordella R, Lu X, Hartley JL, Temple GF, Brasch MA, • Thierry-Mieg N, Vidal M: Protein interaction mapping in C. elegans using proteins involved in vulval development. Science 2000, 287:116-122. This paper describes a pilot study to assess the potential of the two-hybrid approach for large-scale mapping of protein interactions in C. elegans. Using recombinational cloning, open reading frames of 29 genes involved in vulval development were inserted into ‘bait’ and ‘prey’ vectors, and their interactions were tested in yeast. The authors conclude that genome-wide protein-interaction mapping is feasible and report that such a project has been initiated for C. elegans. 64. Hodges PE, McKee AHZ, Davis BP, Payne WE, Garrels JI: The Yeast Proteome Database (YPD): a model for the organization and presentation of genome-wide functional data. Nucleic Acids Res 1999, 27:69-73. 65. Carnegie Institution of Washington Department of Plant Biology and the National Center for Genome Resources (NCGR): The Arabidopsis Information Resource, TAIR. URL http://www.arabidopsis.org/ 66. The C. elegans Sequencing Consortium: Genome sequence of the nematode C. elegans: a platform for investigating biology. Science 1998, 282:2012-2018. 67.

Ruvkun G, Hobert O: The taxonomy of developmental control in Caenorhabditis elegans. Science 1998, 282:2033-2041.

68. Clarke ND, Berg JM: Zinc fingers in Caenorhabditis elegans: finding families and probing pathways. Science 1998, 282:2018-2022. 69. Finkelstein RR, Lynch TJ: The Arabidopsis abscisic acid response gene ABI5 encodes a basic leucine zipper transcription factor. Plant Cell 2000, 12:599-609.

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70. Bowman JL, Smyth DR: CRABS CLAW, a gene that regulates carpel • and nectary development in Arabidopsis, encodes a novel protein with zinc finger and helix-loop-helix domains. Development 1999, 126:2387-2396. The map-based cloning of CRABS CLAW is presented, along with extensive expression and phenotypic analyses that define the role of the gene in gynoecium and nectary development. The encoded protein defines a novel class (i.e. YABBY) of plant-specific transcription factors. It is worth noting that FILAMENTOUS FLOWER (FIL) [72] and INNER NO OUTER (INO) [73] have subsequently been placed within this class. 71. Luo D, Carpenter R, Copsey L, Vincent C, Clark J, Coen E: Control of •• organ asymmetry in flowers of Antirrhinum. Cell 1999, 99:367-376. The authors present the cloning of the DICHOTOMA (DICH) gene, along with analysis of the relative roles of DICH and CYCLOIDEA (both of which are TCP-domain transcription factors) in establishing asymmetry within the flowers and floral organs of Antirrhinum. This work provides the first indication that asymmetry within a plant organ can be regulated by subdomains of gene activity. It also affords another powerful illustration of the utility of transposon mutagenesis as a tool for uncovering gene function. 72. Sawa S, Watanabe K, Goto K, Kanaya E, Morita EH, Okada K: FILAMENTOUS FLOWER, a meristem and organ identity gene of Arabidopsis, encodes a protein with a zinc finger and HMGrelated domains. Genes Dev 1999, 13:1079-1099. 73. Villanueva JM, Broadhvest J, Hauser BA, Meister RJ, Schneitz K, Gasser CS: INNER NO OUTER regulates abaxial–adaxial patterning in Arabidopsis ovules. Genes Dev 1999, 13:3160-3169. 74. Frugier F, Poirier S, Satiat-Jeunemaitre B, Kondorosi A, Crespi M: A • Krüppel-like zinc finger protein is involved in nitrogen-fixing root nodule organogenesis. Genes Dev 2000, 14:475-482. The authors combine analysis of expression patterns and antisense plants to show that Mszpt2-1, a putative transcription factor of the TFIIIA zincfinger class, is required for bacterial invasion and differentiation of root nodules in alfalfa. 75. Schauser L, Roussis A, Stiller J, Stougaard J: A plant regulator •• controlling development of symbiotic root nodules. Nature 1999, 402:191-195. The authors screened an Ac-element insertion collection of the model legume Lotus japonicus to identify a mutant, nin, that was deficient in nodule inception. NIN was cloned via the transposon tag and was found to encode a factor with regional similarity to transcription factors and membrane-spanning domains. The authors suggest that NIN might be membrane-bound, and proteolytically cleaved on infection by the bacterial symbiont to release an intercellular domain, which then enters the nucleus as a transcriptional co-regulator. This paper shows the feasibility of using sophisticated molecular-genetic techniques in leguminous plants, and is of great significance because little is known about how plants regulate root-nodule organogenesis. 76. Schiefthaler U, Balasubramanian S, Sieber P, Chevalier D, Wisman E, • Schneitz K: Molecular analysis of NOZZLE, a gene involved in pattern formation and early sporogenesis during sex organ development in Arabidopsis thaliana. Proc Natl Acad Sci USA 1999, 96:11664-11669. This paper provides a good example of how genomics programs can ease the cloning of genes identified by forward genetics. The authors found alleles of nozzle in a transposon-induced mutant population. This allowed the direct identification of the gene because sequences flanking the insertions corresponded to a clone already sequenced by the Arabidopsis genome project. The cellular function of NOZZLE is unclear but various lines of evidence suggest it may act as a transcription factor. 77.

Halliday KJ, Hudson M, Ni M, Qin M, Quail PH: poc1: an Arabidopsis mutant perturbed in phytochrome signaling because of a T DNA insertion in the promoter of PIF3, a gene encoding a phytochrome-interacting bHLH protein. Proc Natl Acad Sci USA 1999, 96:5832-5837.

78. Zhong R, Ye Z-H: IFL1, a gene regulating interfascicular fiber differentiation in Arabidopsis, encodes a homeodomain-leucine zipper protein. Plant Cell 1999, 11:2139-2152. 79. Peng J, Richards DE, Hartley NM, Murphy GP, Devos KM, Flintham JE, •• Beales J, Fish LJ, Worland JE, Pelica F et al.: ‘Green revolution’ genes encode mutant gibberellin response regulators. Nature 1999, 400:256-261. The authors show that the phenotypes of high-yielding dwarf wheat varieties, which facilitated the ‘green-revolution’ in the 1970s, are caused by mutations in orthologs of Arabidopsis GIBBERELLIN INSENSITIVE (GAI), a gene encoding a transcription factor of the GRAS family. An expressed sequence tag from rice with homology to GAI was identified in database searches and used to isolate a wheat cDNA. DNA-blot analysis then demonstrated that this clone corresponded to the Reduced height-1 loci, which regulates


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dwarfing in wheat, and the Dwarf-8 gene from maize. In common with the Arabidopsis gai alleles, the reduced height-1 and dwarf-8 alleles that cause dwarfing are semi-dominant; these alleles were found to encode products with amino-terminal defects. The wild-type proteins were proposed to act as growth repressors that are inhibited by gibberellins through their amino termini. When the amino terminus is defective, the proteins inhibit growth in a dominant manner. To demonstrate this, the authors overexpressed mutant gai protein in rice and demonstrated that it caused dwarfing. This work is of exceptional significance, not only because it revealed a transcription factor with a highly conserved function, but because it also proved that a single dominant version of a dwarfing gene could be introduced into any transformable crop, thereby rapidly generating yield-enhanced strains. 80. Hartmann U, Höhmann S, Netteshein K, Wisman E, Saedler H, Huiser P: Molecular cloning of SVP: a negative regulator of the floral transition in Arabidopsis. Plant J 2000, 21:351-360. 81. Ambrose BA, Lerner DR, Ciceri P, Padilla CM, Yanofsky MF, •• Schmidt RJ: Molecular and genetic analyses of the Silky1 gene reveal conservation in floral organ specification between eudicots and monocots. Mol Cell 2000, 5:569-579. This paper describes the cloning of the maize Silky1 (SI1) gene by directed transposon tagging and demonstrates that this gene is an ortholog of the B-function organ-identity genes DEF and AP3. The flower phenotype of double mutants between si1 and zag1 (for Zea ag1), a homolog of the C-function gene AG, provides evidence that an ABC mechanism determines floral-organ identity in monocots. Furthermore, this study provides molecular evidence for the relationships between monocot and dicot floral organs. 82. Yang W-C, Ye D, Xu J, Sundaresan V: The SPOROCYTELESS gene of Arabidopsis is required for initiation of sporogenesis and encodes a novel nuclear protein. Genes Dev 1999, 13:2108-2117. 83. Byzova MV, Franken J, Aarts MGM, Almeida-Engler JD, Engler G, Mariani C, Lookeren Campagne MMV, Angenent GC: Arabidopsis STERILE APETALA, a multifunctional gene regulating inflorescence, flower, and ovule development. Genes Dev 1999, 13:1002-1014. 84. Lee MM, Schiefelbein J: WEREWOLF, a MYB-related protein in Arabidopsis, is a position-dependent regulator of epidermal cell patterning. Cell 1999, 99:473-483. 85. McCallum CM, Comai L, Greene EA, Henikoff S: Targeted screening • for induced mutations. Nature Biotechnol 2000, 18:455-457. A method to screen for chemically induced mutations in target sequences of Arabidopsis is described. The strategy combines ethyl methanesulfonate (EMS)-induced mutagenesis with denaturing high-performance liquid chromatography (DHPLC) to detect base-pair changes by heteroduplex analyses. In contrast to the reverse genetics approaches that are based on insertional mutagenesis, this new method can generate a wide variety of mutant alleles and is, in principle, applicable to any species that can be chemically mutagenized. Increases in throughput are, however, still needed. Because of the many background mutations that can be induced by EMS treatments, back-crossing to wild-type plants (or crosses between different mutant alleles) might be required before phenotypic analyses are performed.

86. McCallum CM, Comai L, Greene EA, Henikoff S: Targeting induced local lesions in genomes (TILLING) for plant functional genomics. Plant Physiol 2000, 123:439-442. 87. ••

Van Der Fits L, Memelink J: ORCA3, a jasmonate-responsive transcriptional regulator of plant primary and secondary metabolism. Science 2000, 289:295-297. A T-DNA activation tagging approach in Catharanthus roseus cell suspensions is used to screen for regulators of the terpenoid indole alkaloid metabolic pathway. An AP2/EREBP transcription factor, ORCA3, was identified, and it is further shown that the coordinated regulation of primary and secondary metabolic-pathway genes can be mediated in plants by a single transcription factor. The study is also remarkable in showing the feasibility of using activation tagging in plant cell lines of non-model plant species. 88. Samach A, Onouchi H, Gold SE, Ditta GS, Schwarz-Sommer Z, •• Yanofsky MF, Coupland G: Distinct roles of CONSTANS target genes in reproductive development in Arabidopsis. Science 2000, 288:1613-1616. This study affords a powerful demonstration of the use of glucocorticoid receptor (GR) fusions to identify early targets of a transcription factor. CONSTANS is a zinc-finger transcription factor that promotes flowering in Arabidopsis in response to daylength. A CO::GR protein fusion was used to isolate direct downstream targets of CO, as well as to help position known genes within the regulatory network of Arabidopsis flowering-time pathways. The variety of early CO targets identified reflects the intricacy of the flowering-time regulatory network. 89. Aharoni A, Keizer LCP, Bouwmeester HJ, Sun Z, Alvarez-Huerta M, •• Verhoeven HA, Blaas J, van Houwelingen AMML, De Vos RCH, van der Voet H et al.: Identification of the SAAT gene involved in strawberry flavor biogenesis by use of DNA microarrays. Plant Cell 2000, 12:647-661. DNA microarrays (consisting of ~1700 cDNA clones derived from strawberry fruit) were used to characterize differential gene expression throughout fruit development. Among the genes found to be expressed at significantly higher levels in ripe than in unripe berries was a novel strawberry alcohol acetyltransferase (SAAT). Expression of SAAT in E. coli was used to confirm its biochemical activity. SAAT exhibits low sequence similarity to other known AAT enzymes, thus highlighting the value of the expression-profiling experiments in identifying this gene. The study also shows the feasibility of using microarray technology with non-model plant species for which extensive sequence data are not available, as well as some of the problems for such studies. 90. Reymond P, Weber H, Damond M, Farmer EE: Differential gene • expression in response to mechanical wounding and insect feeding in Arabidopsis. Plant Cell 2000, 12:707-719. The authors use a small cDNA microarray (~150 elements) to begin characterizing the responses to mechanical wounding and insect feeding in Arabidopsis. This study reflects the power of combining gene-expression profiling technology and genetically characterized mutants to dissect plant responses to environmental changes.

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requires their study in the context of a whole family. Transcription factors form intricate networks, both through protein–protein interacti...

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