MEETING REPORT · November 1995 1739 MEETING REPORT verse genetics is fast approaching. Nevertheless, standard "forward genetics" continues to be a mainstay of Arabidop-sis research

November 1995 1737

MEETING REPORT

Whetherthis indicatesagenuine mechanistic difference has long been debated; it may well be that the center of the petu- niafloral meristem is indeed carpel tissue. In any event, comparative study of additional aspects of flower development should help to show what aspects of flower development are truly ancient, which ones are more derived, and how changes in regulation might have occurred.

Rebecca Chasan

REFERENCES

Angenent, G.C., Franken, J., Busscher, M., Weiss, D., and van Tunen, A.J. (1994). Co- suppression of the petunia homeotic gene fbp2 affects the identity of the generative meristem. Plant J. 5, 33-44.

Angenent, G.C., Franken, J., Busscher, M., van Dijken, A,, van Went, J.L., Dons,

H. J.M., and van Tunen, A.J. (1995). A nove1 class of MADS box genes is involved in ovule development in petunia. Plant Cell 7,

Gasser, C.S., and Robinson-Beers, K. (1993). Pistil development. Plant Cell 5, 1231-1239.

Halder, G., Callaerts, P., and Gehring, W.J. (1995). lnduction of ectopic eyes by targeted expression of the eyeless gene in Drosophi- /a. Science 267, 1788-1792.

Mandel, M.A., Bowman, J.L., Kempin, S.A., Ma, H., Meyerowitz, E.M., and Yanofsky, M.F. (1992). Manipulation of flower structure in transgenic tobacco. Cell 71, 133-143.

Meyerowitz, E.M. (1994). Flower development and evolution: New answers and new ques- tions. Proc. Natl. Acad. Sci. USA 91, 5735-5737.

Modrusan, Z., Reiser, L., Feldmann, K.A., Fischer, R.L., and Haughn, G.W. (1994). Homeotic transformation of ovules into carpel- like structures in Arabidopsis. Plant Cell 6, 333-349.

1569-1582.

Ray, A., Robinson-Beers, K., Ray, S., Baker, S.C., Lang, J.D., Preuss, D., Milligan, S.B., and Gasser, C.S. (1994). Arabidopsis floral homeotic gene BELL (BEL1) controls ovule development through negative regulation of AGAMOUS gene (AG). Proc. Natl. Acad. Sci.

Robinson-Beers, K., Pruitt, R.E., and Gasser, C.S. (1992). Ovule development in wild-type Arabidopsis and two female-sterile mutants. Plant Cell 4, 1237-1249.

Rounsley, S.D., Ditta, G.S., and Yanofsky, M.F. (1995). Diverse roles for MADS box genes in Arabidopsis development. Plant Cell 7, 1259-1269.

Schneitz, K., Hülskamp, M., and Pruitt, R.E. (1995). Wild-type ovule development in Arabidopsis thaliana: A light microscope study of cleared whole-mount tissue. Plant

Tsuchimoto, S., van der Krol, A.R., and Chua, N.-H. (1993). Ectopic expression of pMADS3 in transgenic petunia phenocopies the petunia blind mutant. Plant Cell 5. 843-853.

USA 91, 5761-5765.

J. 7, 731-749.

Arabidopsis in Madison: Genes and Phenotypes Spread iike Weeds

Arabidopsis is one of just a handful of model organisms for which the prospect of mapping and sequencing the entire genome has seemed an attainable, relatively short-term goal. The reasons for this are very familiar: the small genome ( 4 0 0 Mb), low content of repetitive DNA and consequent high gene density, and rapid rate of reproduction. Once a complete physical map is available, of course, then a gene identified by its mutant phenotype can be cloned simply by locating it on the genetic map; this is a day to which Arabidopsis researchers have long looked forward! Although there is still a great deal of work to be done (and money to be spent) before that day arrives, impres- Sive progress has already been made, much of which was detailed at the most recent lnternational conference on Ara-

bidopsis Research. This meeting, the sixth such conference, brought over 600 participants to Madison, Wisconsin, from June 7 to 11,1995. The topics discussed included not only genome analysis but also development, biochemical genetics, growth regulators, stress and pathogen responses, and cell biology, the study of all of which will undoubtedly be enhanced by the availability of a complete physical map. In this report, I summarize a small selection of the many interesting presentations.

Gathering the Genes

Physical mapping requires the construc- tion of genome libraries containing the largest possible DNA fragments; yeast ar-

tificial chromosomes (YACs) are currently the best way to clone large fragments (i.e., of 400-500 kb). Severa1 different Arabidopsis YAC libraries have been con- structed; David Bouchez (Laboratoire de Biologie Cellulaire, INRA) described the CIC library, which is the product of a French interagency collaboration. The library, which was prepared from partia1 EcoRl digests of DNA isolated from pro- toplasts in agarose plugs, contains ~ 1 2 0 0 clones, -800 of which are “useful” (i.e., contain nuclear DNA in large fragments). The library also contains a very low proportion of chimeric clones. To test coverage of the genome, Bouchez and colleagues hybridized 370 markers to the library and found that 90% of them are contained within it. These markers fall into -100 contigs, of mean size 4000 kb.

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Frequency ofrecombination

| high

| average

low

YAC contig map RFLP map

Figure 1. Comparison of Physical and GeneticDistance on Chromosome 4.

The dashes on the YAC contig map indicate thebreaks between the three contigs. Provided byRenate Schmidt and Caroline Dean.

Using the CIC library and also addition-al YAC libraries (yUR EG, and EW).Renate Schmidt, Caroline Dean, and theircolleagues at the John Innes Centre haveconstructed YAC contigs around RFLPmarkers on chromosomes 4 and 5. Chro-mosome walking allowed them to link 13chromosome 4 YAC contigs (of averagesize 1.3 Mb) into just three contigs (of av-erage size 5.5 Mb) covering at least 90%of the genetic map of this chromosome(Schmidt et al., 1995). A comparison ofphysical and genetic distances on chro-mosome 4 indicates that the frequencyof recombination varies greatly along thechromosome, from 30 kb/cM to 1.6 Mb/cM,with an average of 175 kb/cM. This analy-sis also showed that regions in whichrecombination is suppressed turn out tobe scattered along the chromosome ratherthan restricted to the centromere region(Figure 1).

Genomic clones can be used not onlyfor physical mapping, of course, but alsofor sequencing. As Michael Bevan (JohnInnes Centre) pointed out, it is not yet prac-tical to sequence YAC clones directly;instead, the "European Scientists Se-quencing Arabidopsis" (ESSA) group is

sequencing cosmids and YAC subclones.The goal of the ESSA project is to se-quence 2 Mb of chromosome 4 and 500kb of other regions by the end of 1996,to sequence all 16.5 Mb of chromosome4 by the end of 1999, and finally to con-tribute to completing the sequence ofthe entire genome by the year 2004. TheESSA group has so far sequenced 350 kb,with fivefold redundancy resulting in anerror rate of about 1 in 5000 bases. Bevanspeculated that it may be possible in thefuture to relax these stringent standardsso as to obtain more total sequence.

Preliminary analysis of a 25-kb regionusing the neural network programGeneFinder indicates the presence of anumber of open reading frames (ORFs)(Figure 2). Some of these correspondto known proteins, some resemble knownsequences of unknown function, and stillothers look like nothing in the data bases.Some of these last are likely to beGeneFinder artifacts, but Bevan noted thatfurther "training" of the program shouldhelp eliminate them. Nevertheless, ~25%of the GeneFinder ORFs correspond topreviously identified expressed sequencetags (ESTs). GeneFinder also predictedthat some regions are transcribed on bothstrands; again, whether this is actually thecase is not yet clear. The challenge is todetermine the function of GeneFinder-identified ORFs, and Bevan pointed outthat a systematic function search can becarried out by screening for insertion al-leles of any ORF. That is, using theappropriate primer sets (one in the ORF ofinterest, one in a mobilized transposableelement), those plants in which a mobi-lized element has inserted into the ORFcan be detected by polymerase chainreaction (PCR).

A complementary approach to genom-ic sequencing is sequencing ESTs. TomNewman (MSU-DOE Plant Research Lab-oratory) provided an update on the MSUEST project, noting that as of June 1995,15,000 ESTs had been sequenced (out ofa goal of 36,000), with nearly 1000 beingadded each month. The redundancy rateis fairly low, with 9000 ESTs of 12,000 ex-amined being unique. In addition to

yielding information about the sequencesof expressed genes, ESTs can be usedto "anchor" YACs, as Frances Agyare (YorkUniversity) described. PCR primers cor-responding to ESTs are used first to testwhether the EST is unique and then toscreen a group of YAC clones (^3400 to-tal, from which chloroplast and repeatedsequences have been removed). Agyareand coworkers have used a pooling strate-gy (Balding, 1994) in which it should takejust 72 PCR reactions to identify the YACthat contains a particular EST.

With the large-scale identification ofnew genes, particularly those of unknownfunction, it will be necessary to devise ef-ficient techniques to examine function.One approach is to identify insertion al-leles, as Bevan discussed; another is tocharacterize expression patterns at vari-ous developmental stages and in variousenvironmental conditions. Mark Schena(Stanford University) described an alter-native to gel- and filter-based methods,which are slow and cumbersome to runon a large scale. The "DNA chip" (Schenaet al., 1995) is a microscope slide on whichPCR-amplified cDNAs are spotted in veryhigh density arrays (i.e., 1000 cDNAs/cm2). The expression of all of the corre-sponding genes in a single plant can beexamined by isolating mRNA from theplant, reverse transcribing it into cDNA inthe presence of fluorescent nucleotides,hybridizing the fluorescent probe to thechip, and measuring the fluorescence ateach spot. A two-color fluorescence label-ing and detection scheme makes itpossible to compare mRNA levels fromtwo differentially treated plants, allowingrapid identification of genes that differ inexpression pattern in the two plants. Be-cause the hybridization is done in a smallvolume, the technique is very sensitive,with all but the least-expressed genes be-ing detectable.

Developing Insights intoDevelopment

The genomics session provided ampleevidence that the day of large-scale re-

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MEETING REPORT

verse genetics is fast approaching.Nevertheless, standard "forward genetics"continues to be a mainstay of Arabidop-sis research. Mutant analysis not onlyenables the identification of genes thatparticipate in various processes but alsoreveals details of the process itself—thatis, it shows what kinds of phenotypic al-terations are possible and makes itpossible to assess the interactions amonggenes that appear to be involved in a com-mon process. Genetic analysis of embryodevelopment has, for example, shown thatthe Arabidopsis embryo consists of sev-eral independently patterned regionsalong both the apical-basal and radialaxes (Mayer et al., 1991). Several geneswhose mutant phenotypes suggest a rolein embryo patterning have now beencloned, and their characterization shouldultimately lead to an understanding of thebiochemical underpinnings of regionalspecification processes

Diane Shevell (Rockefeller University)discussed one of these genes, EMB30(also known as GNOM [GN]), mutationsin which disrupt apical-basal patterning.All alleles result in a variety of phenotypes,with some embryos lacking essentially allapical-basal polarity and others showingless severe defects (Shevell et al., 1994).In addition to these large-scale patterndefects, emb30 mutant embryos showdefects in cell shape, organization, andadhesion. Undifferentiated callus cells arealso defective in adhesion. Shevell sug-gested that EMB30 may therefore benecessary for proper control of cell divi-sion, expansion, and adhesion.

The predicted EMB30 protein is 27%similar to the yeast Sec7 protein, whichis thought to be part of a nonclathrin coaton vesicles transporting from the ER tothe Golgi. The highest similarity lies in a200-amino acid region whose function isnot known but that has turned up in a num-ber of animal proteins. One emb30 allelehas a missense mutation that alters anabsolutely conserved amino acid of this"Sec7 domain," which is consistent withthe possibility that this domain is impor-tant to EMB30 function. The gene appearsto be expressed throughout the plant, so

PHY D 1,3 GLUCANASE RNA POL SUBUNIT

STEROID BINDING PROTEIN

RNA BINDING PROTEIN S I PROTEINEST MATCH

SOAA ORF

PREDICTED GENES

Figure 2. Preliminary GeneFinder Analysis of a 25-kb Region of Chromosome 4.

Provided by Michael Bevan.

it is not clear why embSO mutations seemto have a preferential effect on apical-basal patterning. Perhaps EMB30 is re-quired for the secretion of a number ofproteins, a subset of which play a role inapical-basal patterning; alternatively, theeffects on patterning might be the second-ary result of the cellular defects.

The product of the KNOLLE (KN) gene,mutations in which alter radial patterning,also resembles a secretory pathway pro-tein. As Wolfgang Lukowitz (UniversitatTubingen) discussed, the KN proteinresembles animal and yeast syntaxins,which are thought to act as transport vesi-cle receptors. The outer cell layer of knembryos contains large, irregularly ar-ranged cells; to investigate whether thesecells are epidermal or are just located onthe outside of the embryo, Lukowitz tookadvantage of the fact that rusca (rus) mu-tants normally accumulate anthocyaninsin inner cell layers but not in epidermalcells. Some but not all of the outer cellsof kn fus double mutant embryos accu-mulated anthocyanins, suggesting that thekn mutation disrupts cell identity along theradial axis. Casper Vroemen (Wagenin-gen Agricultural University) and hiscolleagues provided confirmation of thisresult with their finding that a lipid trans-fer protein gene that is normally expressed

only in the protoderm is expressed in in-ternal cells of the kn mutant. Lukowitzsuggested that the apparent mixing of nor-mally distinct cell layers might result fromdefects in cell wall positioning, becausethe walls of the mutant are incomplete. KNis expressed in small patches throughoutthe embryo, an expression pattern simi-lar to that of cyclins, which raises thepossibility that it is involved in cell plateformation.

Another embryo development mutantwas identified by Ryuji Tsugeki (CarnegieInstitution of Washington) in a line contain-ing a transposed Ds element. Defectiveembryos are white and abort at the glob-ular-to-heart transition. After confirmingby analysis of revertants that the insertedDs was in fact responsible for the mutantphenotype, Tsugeki amplified DNA flank-ing the insertion site and found that thesequences are identical to an EST thatencodes a ribosomal protein S16 (RPS16)homolog. An interesting question is howthe embryo can develop as far as the heartstage without RPS16; one possibility isthat maternal supplies get the embryothrough the earlier stages of development.

An intriguing, and not yet understood,aspect of plant embryo development is theinteractions between the embryo proper,which arises from the upper cell of the two-

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cell embryo, and the suspensor, which alises from the lower cell. The suspensor promotes development of the embryo proper, whereas the developing embryo inhibits suspensor cell proliferation. In ab- normalsuspensor (sus) mutants, the first morphological defects are apparent in the gobular stage embryo proper; the suspensor subsequently proliferates and comes to resemble an abnormal embryo (Schwartz et al., 1994). Brian Schwartz (Oklahoma State University) used a T-DNA insertion allele to clone SUS2, whose product closely resembles the yeast spliceosome component PRP8 (although complementation experiments remain to be done). An interesting question will be whether the sus2 mutant actually accumulates unspliced RNAs. As- suming that SUS2 is in fact a PRP8 homolog, it could be a general splicing factor that is required for normal embryogenesis, with the suspensor abnormalities of sus2 mutants being a byproduct of the disruption of embryo development. An alternative, but perhaps less likely, possibility is that SUS2 is involved in the processing of one or more specific transcripts, such as one whose product is involved in embryo-suspensor signaling.

Unlike the suspensor, which normally degenerates as the embryo develops, the endosperm develops in parallel with the embryo. Several groups have isolated mutants with altered regulation ofendo- sperm development-specifically, mutants in which endosperm develops in the absence of the normal trigger, fertilization. Marilyn West (University of California, Davis) described the isolation by Bob Fischer’s (University of California, Berke- ley) group of a fertilization-independent endosperm (fie) mutant, which was obtained by screening for silique development in plants with a genetic block to fertilization. Abed Chaudhury (CSIRO) isolated three unlinked fertilization- independent seed(fis) mutants by screening for pseudorevertants of the pistillata (pr) mutant, which is stamenless and therefore self-sterile. Females heterozy- gous for the fie mutation or one of the fis

mutations produce 50% mutant embryo sacs that, in the absence of fertilization, not only form endosperm (that in the case of fis7 and fis2 has been shown to be diploid) but also induce surrounding sporophytic tissues to differentiate into seed coat and silique. The mutations also cause embryo lethality as a female gametophytic effect. Chaudhury has ob- served embryo-like structures in fis mutants in the absence of pollination, so these structures must be maternal in origin.

Because transmission of the fie and fis mutations through the female gametophyte results in abnormal embryos, these genes are presumably involved in embryo sac development or function (in addition to their apparent roles in suppressing endosperm development). As Animesh Ray (University of Rochester) pointed out, the female gametophyte is a good model system for studying plant development because it is a relatively simple structure whose formation nevertheless involves processes such as lineage-dependent morphogenesis, positional information, and programmed cell death. Several genes have already been identified that are involved in embryo sac development, and Ray described severa1 new sporophytic mutants in which this process is affected, extra cells and ardhagarva. The first has an alteration in the cell devision pattern early in ovule development, and in the second the megaspore fails to divide and then degenerates.

A structure of comparable simplicity and, presumably, mechanistic richness develops late in embryogenesis-the shoot apical meristem (SAM). As Kathy Barton (University of Wisconsin) emphasized, the SAM, whose formation and activity are proving amenable to study at the genetic level, is a very organized en- tity and nota random cluster of cells. The SAM alises from three cell layers of the torpedo stage embryo; the outer two layers divide anticlinally and the inner layer divides in multiple directions to form a broad and histologically distinct structure. In the shoot meristemless (stm) mutant,

these three cell layers are present, but they do not divide to form a SAM, nor do they become histologically distinct (Barton and Poethig, 1993). stm mutant seedlings often produce a mass of abnormal leaves from the hypocotyl, and seedlings in which STM is weakly suppressed (dueto genetic background) sometimes also produce a shoot that then terminates again in a m a s of abnormal leaves. This “stop-and-start” behavior of the residual SAM suggests a role for STM in SAM maintenance as well as initiation. Mapping and sequence data indicate that STM corresponds to a homolog of the maize KN7 gene, which when overexpressed causes extensive SAM formation (Sinhaet al., 1993). During embryogenesis, STM is expressed in cells that are the progenitors of the SAM. Dur- ing postembryonic development, STM is expressed in meristems but, like KN7, is excluded from cells that will differentiate, e.g., as leaves.

In contrast to STM, which is necessary for meristem formation, the CLAVATA (CLV) genes are required to prevent overpro- liferation of meristems. Double mutant analyses described by Steve Clark (University of Michigan) indicate that two of the CLVgenes, CLV7 and CLV3, appear to act in the same pathway. Clark also showed that the CLVgenes compete with STM to regulate cell differentiation, with STM inhibiting differentiation and the CLV genes promoting it. The lack of meristem activity in stm plants is thus the result of the activity of the CLVgenes, whereas the proliferation of undifferentiated cells in clv mutants results from STM activity. It does not appear that either of these genes acts by preventing the activity of the other; instead, their activities are balanced in some way. The CLV7 gene appears to encode a transmembrane receptor kinase, so it may be involved in the perception of an intercellular signal that causes meristem cells to stop dividing.

A number of additional mutants that affect SAM formation have also been isolated. Barton described the topless, pin- head@nh), and sam4 mutants, all of which are defective in SAM initiation and/or main-

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MEETING REPORT

tenance in the embryo. In addition, PNH seems to be involved in lateral meristem initiation. Thomas Laux (Universitat Tübin- gen) has identified three mutants-zwille, wuschel(wus), and pregnant-all of which lack avisible SAM in the embryo. Double mutants between wus and clvl resemble the wus single mutant, which suggests that CLV7 may act to downregulate WUS expression.

Trapping Genes in the Act

New genes can be identified not only by their sequences (e.g., as ESTs) or by their mutant phenotypes but also by their expression patterns. Arabidopsis researchers are beginning to exploit gene trapping techniques pioneered in Drosophila, in which reporter genes are used to identify sequences conferring "interesting" expression patterns. These approaches make it possible to identify genes that lack an obvious mutant phenotype, either because they are redundant or because they tunction both early and late in development, resulting in an unin- formative early lethality that effectively masks detection of a function later in development. Patty Springer (Cold Spring Harbor Laboratory) described an example of the latter type of gene, a member of the MCM2-3-5 family of yeast genes necessary for initiation of DNA replication. This gene, PROLFERA (PRL), was identified based on the expression pattern of a DsG "exon trap" that consists of a Ds element harboring a P-glucuronidase (GUS) reporter gene preceded by splicing signals (Springer et al., 1995). The PRL line expresses GUS in leaf primordia, lateral root primordia, and other groups of pro- liferating cells. The prl mutation shows reduced transmission through female, but not male, gametophytes, and homozygous embryos abort early in development. PRL thus appears to function in dividing cells both during megagametophyte and embryo development as well as post- embryonically.

Exon trapping is just one gene trapping technique; another is promoter trapping, in which a promoterless reporter gene is used to identify promoters that are expressed in interesting patterns. Patrick Gallois (University of Perpignan) has used a promoterless GUS construct to look for markers of early embryogenesis. One line expresses GUS in the root apex of late heart stage embryos, another in the suspensor. Gallois pointed out that such promoters might be useful for targeting the specific expression of introduced genes.

Enhancer trapping, a third gene trapping technique, uses a reporter gene under control of a minimal promoter to detect enhancers that drive specific expression patterns. Ueli Grossniklaus (Cold Spring Harbor Laboratory) used the enhancer trapping approach developed by Sundaresan et al. (1995) to identify enhancers that confer expression in ovules and female gametophytes. Of 500 F3 families examined for ovule staining, 15% showed staining specific to certain regions of the ovule andlor megagametophyte. Megagametophyte-specific staining patterns included the entire gametophye, the egg apparatus only, the synergids only, the oocyte only, and the four chalazal nuclei only. Sporophyte staining patterns included highly polar staining at either or both ends of the megagametophyte. In one line, just a single cell of the funiculus expressed GUS activity. Grossniklaus is also taking advantage of the fact that gene trapping has the potential to result in mutant phenotypes by screening the Fp generation for semisteriles (i.e., for lines in which the mutant allele fails to be trans- mitted through the megagametophyte) and the F3 generation for steriles.

The Making of a Flower

Flowering provides a paradigm of a process whose study at the genetic leve1 has already begun to yield the molecular tools necessary to explore the mechanistic details of the process, and severa1 talks

showed how these tools are being put to use. One important question concerns the mechanism by which vernalization, which is sensed in the apex, and photoperiod, which is sensed in the leaves, induce the vegetative-to-floral transition. A number of genes are involved in this process, some of which promote flowering constitutively (i.e., under both long and short days) and some of which promote flowering under long days specifically.

The product of the LD gene, which acts in the constitutive floral promotion pathway, has sequence features typical of nuclear proteins (Lee et al., 1994). Mito Aukerman and his collaborators in Rick Amasino's lab (University of Wisconsin) have now shown that LD sequences are able to target a GUS fusion protein to the nucleus of transgenic tobacco and that LD binds DNA. The LD transcript accumulates in the shoot apex and root of the seedling and in the apex, floral stems, and buds (but not leaves) of the mature plant, although the LD protein accumulates in the mature apex only. Aukerman hypothe- sized that the LD protein helps generate a meristem that is competent to respond to a floral signal. This role would be consistent with the fact that mutations in LD cause late flowering under all light re- gimes. By contrast, mutations in the CONSTANS(C0) gene delay flowering under long-day conditions only, making CO a member of the photoperiod-dependent class of floral promotion genes. The groups of George Coupland (John lnnes Centre) and Jo Putterill (University of Auckland) reported that CO encodes a protein with similarity to zinc finger- containing transcription factors (Putterill et al., 1995). The regulatory role of CO is emphasized by the Coupland group's find-, ing that overexpression of CO causes plants to flower early.

Another candidate regulator of the vegetative-to-floral transition is SPL3, which was described by Guillermo Cardon (Max-Planck-lnstitut für Züchtungsfor- shung). The Arabidopsis SPL genes are homologs of the Antirrhinum SPB genes, whose products bind to the promoter of

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the SQUAMOSA (SQUA) gene. SQUA is the homolog of the Arabidopsis meristem identity gene APETALA7 (AP7), which along with LEAFY (LFY) and other meristem identity genes imparts a floral identity to a meristem that has received a floral induction signal; such genes are good candidates to be regulated by genes that promote flowering. The AP7 promoter contains a sequence similar to the SQUA promoter element that binds SBP1, and Cardon showed that SPL3, the SPL protein that is most similar to SBP1, binds to theAP7 promoter. SPLB transcripts begin to accumulate well before AP7 transcripts do, which is consistent with a role in AP7 regulation. Overexpression of SPLB (under control of the cauliflower mo- saic virus [CaMV] 35s promoter) results in plants that flower early under both long and short days. This finding raises the possibility that SPLB is the product of one of the flowering time genes. It is not known whether SPL3 corresponds to a known gene, but a different SPL gene, SPL4, maps close to the FTflowering time locus, mutations in which cause late flowering under long-day conditions.

As Detlef Weigel (Salk Institute) discussed, the meristem identity genes show developmental and environmentally regulated redundancy, which makes assessing the precise function of individual meristem identity genes difficult. Weigel and his collaborators are thus using the alternative approach of asking about the effects on “naive” meristems of overexpressing single meristem identity genes. LFY overexpression (using a CaMV 35s promoter) has no effect on flower patterning per se, but it causes shoots to be transformed to flowers and therefore results in early flowering (Weigel and Nilsson, 1995). Overexpression of Arabidopsis LFY causes even more profound changes in flowering time in other plant species. Hy- brid aspen trees of the genus Populus normally flower after eight to 20 years, but transgenic 35S::LFY trees flower in less than 6 months. Alejandra Mandel and Marty Yanofsky showed that AP7 overexpression produces a similar acceleration

’

of flowering time in Arabidopsis (Mandel and Yanofsky, 1995).

The formation of floral meristems involves, in addition to AP7 and LFY, the CAULlFLOWER (CAL) gene. AP7 and CAL are partially redundant, with calmutations having an effect only in an ap7 mutant background. As Beth Savidge (Universi- ty of California, San Diego) discussed, double mutant plants undergo a normal flowering transition, but the inflorescence meristems then begin to generate additional inflorescence meristems, with the result that the plants resemble cultivated cauliflower. Transcripts of both AP7 and CAL, which encode highly related MADS DNA binding domain-containing proteins (Kempin et al., 1995), accumulate throughout the floral meristem initially and then become restricted to the outer two whorls. CAL expression then diminishes whereas that of AP1 stays high; Savidge suggested that this might account for the only par- tia1 rescue of ap7 mutants by CAL. When expressed under the control of the CaMV 35s promoter, both AP7 (Mandel and Yanofsky, 1995) and CAL are able to con- vert lateral and apical inflorescences to flowers; thus, either gene is sufficient to give a meristem a floral identity. Savidge showed that the cauliflower-like phenotype of Arabidopsis ap7 cal double mutants may be more than coincidental, because the CAL orthologs from 27 different varie- ties of cultivated cauliflower (Brassica oleracea var botrytis) all have a nonsense mutation in the coding region (all alleles have the same mutation). Their AP7 orthologs, however, appear to encode functional proteins.

Once floral meristem identity has been specified, organ identity genes act to define the identity of each whorl of floral organs. Two of these genes, AP3 and Pl, are required for the second and third whorl organs to develop into petals and stamens, respectively; mutations in either gene cause the second whorl organs to develop as sepals and the third whorl organs to develop as carpels. Like many other organ identity genes, AP3 and Pl are MADS domain proteins; their Antirrhinum

homologs, DEFA and GLO, have been shown to bind as heterodimers to a yeast MADS domain binding site (Trobner et al., 1992), and AP3 and PI have been demonstrated to bind to one another (Goto and Meyerowitz, 1994).

If AP3 and PI do act to regulate the expression of downstream genes, then they should contain nuclear localization signals. To define these signals, Vivian lrish (Yale University) bombarded onion cells with constructs in which the CaMV 35s promoter drives the expression of trans- lational fusions of portions of AP3 or PI to GUS. Surprisingly, neither AP3 nor PI was able to localize GUS to the nucleus, but when the AP3 and PI constructs were bombarded together, GUS activity was nuclear. The colocalization signal in AP3 maps to within a 69-amino acid domain at the N terminus. These results provide a plausible explanation for the observa- tion that plants that ectopically express the AP3 gene accumulate nuclear AP3 protein only in those cells that also express endogenousPl(Jacketal., 1994). Indeed, Beth Krizek (Caltech) showed that when both Pl and AP3 are expressed from a CaMV 35s promoter, nuclear AP3 protein accumulates throughout the plant. lrish has carried out similar experiments with the organ identity gene AGAMOUS (AG) that indicate that the AG protein is also unable to localize to onion cell nuclei on its own and may require a partner protein. lrish and her collaborators are now re- peating the experiments in transformed Arabidopsis plants to verify that the onion results accurately represent the situation in Arabidopsis.

Lighting the Way

Many presentations dealt with the question of how Arabidopsis (and of course, by extension, other plants) perceives and responds to various environmental signals and stresses. One environmental signal whose perception and transduction have been particularly well studied is light. The phytochromes, which are the photorecep-

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tors for red and far-red light, are dimers with covalently attached chromophores. Arabidopsis possesses five phytochrome genes, at least some of which have distinct functions. PhyA, for example, mediates deetiolation in response to far-red light, whereas deetiolation in response to red light requires phyB. By analyzing the light responses of transgenic Arabidopsis plants expressing hybrid phytochromes, Doris Wagner (USDA Plant Gene Expres- sion Center) and her coworkers showed that the N-terminal domains of the two phytochromes are responsible for their different photosensory specificities, whereas their C-terminal domains carry out a common function, presumably signaling to downstream components.

To identify regions within the C-terminal domain that are necessary for phytochrome signaling, Wagner and colleagues mutagenized PHYB-overexpressing lines (which have an increased sensitivity to red light) and screened for revertants that are insensitive to red light specifically. A large-scale mutagenesis experiment yielded 101 such revertants, four of which produced normal amounts of spectrally active, dimeric phyB and were linked to the transgene. The mutations, which re- duce phyB activity by 60- to lOOO-fold, all result in single amino acid alterations within a 62-amino acid region of the C-terminal domain of PHYB.

A possible downstream component with which the C-terminal domain of phytochrome might interact was described by Tedd Elich (Salk Institute), who identified a phytochrome-interacting protein, PIP, by using the C-terminal fragment of PHYB as a “bait” in a yeast two-hybrid system. PIP, which interacts with phyB holoprotein as well as the bait fragment, is a nove1 soluble, hydrophilic protein that contains severa1 intriguing motifs-a segment similar to a region of the dopamine receptor that interacts with G proteins and a possible serine protease catalytic site. PIP overexpression has no obvious phenotype, but antisense plants, despite their fairly modest reduction in PIP transcript

have rounder, more serrated leaves, and flower late, all phenotypes opposite to those of phy8 mutants. These phenotypes suggest that the activities of PIP and phyB are antagonistic and suggest that PIP may be a negative regulator of some phyB sig- na1 i ng pat hways.

The blue light photoreceptor has proven far more elusive than the phytochromes, but as Margaret Ahmad (University of Pennsylvania) discussed, one is now in hand. HY4, which is required for blue light responses (e.g., blue light inhibition of hypocotyl elongation), was proposed to encode a blue light photoreceptor (Ahmad and Cashmore, 1993) based on the strik- ing sequence similarities of its product to bacterial photolyases, which are flavo- proteins that are activated by absorption of bluelUV-A light. The HY4 protein does indeed bind a flavin chromophore (Lin et al., 1995b), and overexpression of the HY4 gene in tobacco confers hypersensitivity to bluelUV-A light (Lin et al., 1995a). The protein encoded by HY4 has thus been named cryptochromel (CRYl), the name commonly given to the plant bluelUV-A light receptor.

In addition to their defects in the response to blue and UV-A light, hy4 mutants are impaired in the response to green light. The absorption properties of CRYl’s non- covalently bound flavin chromophore, which shows stability in the semiquinone form, may account for this green light response (Lin et al., 1995b). Ahmad and her colleagues in the lab of Tony Cashmore have now shown that a mutant allele of HY4 that confers a normal green light response but an impaired blue light response retains flavin chromophore binding when expressed as a fusion protein in Escheri- chia coli. However, a second light- harvesting chromophore, which has been identified as a pterin (Malhotra et al., 1995) and that in photolyases contributes pri- marily to absorption of blue light, does not bind to the mutant fusion protein. The in vivo absorption properties of this mutant hy4 allele can therefore be explained by the known absorption properties of the

levels, are greener than wild-type plants, chromophores that bind to CRYl, provid-

ing strong evidence that CRYl is indeed a photoreceptor.

Although a simple model for light responses would invoke distinct photoreceptors independently mediating the response to different regions of the spec- trum, Ahmad noted that a double mutant with a severe phytochrome deficiency (i.e., carrying strong alleles of both HY7, a gene involved in chromophore biosynthesis, and PHYB) has a reduced response to blue light, both in inhibition of hypocotyl elongation and anthocyanin production. This reduced blue light response is almost assevere as that seen in the hy4 mutant, which suggests that CRYl photoreceptor function may involve an interaction with phytochrome.

However CRYl functions, one of the most rapid responses to blue light treat- ment is membrane depolarization, as Myeon Cho (University of Wisconsin) showed. This depolarization, which appears to result from activation of CI-- selective channels, is blocked by the anion channel blocker NPPB, which also reduces the extent to which blue light inhibits hypocotyl elongation. In hy4 null mutants, this blue light-induced depolarization is dramatically reduced (i.e., to less than 30°/o of wild-type levels). However, the significant residual response indicates that an additional blue light receptor@) ex- ists that mediates anion channel opening.

Biochemical analyses of phytochrome signaling events are still in their nascent stages, but genetic analyses have yielded a number of potential components of the phytochrome signaling pathway. Loss- of-function mutations in the COP/DET/FUS genes cause dark-grown seedlings to have some or all of the phenotypic characteristics of light-grown seedlings (e.g., a short hypocotyl) (for review, see McNellis and Deng, 1995, this issue). These genes act downstream of phytochrome and CRY7, which indicates that they are light-inactivated repressors of photomor- phogenesis. Ning Wei reviewed recent work carried out by her and her colleagues in Xing-Wang Deng’s lab (Yale University) that indicates that nuclear localization of

1744 The Plant Cell

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COPl, which has features of transcriptional regulators, is light regulated, with the protein nuclear in the dark but cytoplas- mic in the light (von Arnim and Deng, 1994). The COP9 protein is part of a large multiprotein complex in the nucleus; Wei reported that this localization is constitutive, unlike that of COP1, but that the COP9-containing complex of dark-grown plants is larger than that of light-grown plants. 60th the nuclear localization of COP9 and the stability of the COP9 complex (which may of course be related) require COP8 and COP77.

One obvious possibility is that COP8 and COPll , and perhaps the products of some other COPlDET loci, are part of the COP9 complex. Danny Chamovitz, also of the Deng group, is taking a biochemical approach to characterize the COP9 complex from cauliflower. (COP9 is very abundant in cauliflower heads.) The complex, which appears to contain a number of different proteins in equimolar amounts, remains stable during a 400-fold purification. Chamovitz now plans to microsequence the interacting proteins and then clone them.

Phytohormones and More

Although many genes have been identified whose mutant phenotypes indicate that they are involved in a phytohormone transduction pathway, phytohormone receptors have proven notoriously recal- citrant, in part because it has been difficult to connect biochemical observations with genetic data. Eric Schaller’s and Tony Bleecker’s (University of Wisconsin) finding that ETR1, whose genetics have long suggested it might be an ethylene receptor (dominant mutations in ETR7 result in ethylene insensitivity and a concomitant reduction in ethylene binding), actually binds to ethylene is thus an exciting breakthrough.

The C-terminal portion of ETRl resembles a class of prokaryotic signal transduction proteins known as two-component regulators. The two components-

a histidine kinase “sensor” and a response regulator that is phosphorylated by the sensor-are typically separate proteins, but ETRl possesses both domains. The N-terminal portion of ETR1 contains three transmembrane domains and two cysteine residues that mediate covalent homodimerization (Schaller et al., 1995). Some sensor proteins bind the stimuli that they sense directly, whereas others do not, so the two-component similarity does not in itself imply that ETRl is an ethylene receptor. To investigate this question directly, Schaller examined ethylene binding in yeast overexpressing ETRI. Whereas control yeast exhibited no saturable binding, ETR7-expressing yeast showed strong and saturable ethylene binding. Yeast expressing the errl-1 allele, which has a missense mutation in the second transmembrane domain, did not bind ethylene, whereas a similar alteration in the third transmembrane domain and alterations of the cysteine residues involved in dimerization had no effect on binding. The N-terminal region on its own is also able to bind significant levels of ethylene. It will be interesting to know how these alleles function in vivo; presumably the last allele will have no biological activity.

These results.firmly establish ETRl as an ethylene receptor, but it may not be the only ethylene receptor: Jian Hua and colleagues in the Elliot Meyerowitz lab (Caltech) showed that ERS, a gene identified by cross-hybridization with E TR7, encodes a highly similar protein that lacks a response regulator domain. When the Same point mutations that are found in err7 alleles are engineered into ERS, the resulting ers alleles cause dominant ethylene insensitivity in transgenic plants (Hua et al., 1995). The existence of ERS and the possibility that it is redundant with ETR7 may account for the fact that no recessive alleles of ETR7 have ever been identified.

Whatever the reason for the lack of recessive loss-of-function alleles of ETR7, this lack has made it difficult to asses whether ETR7 normally acts to activate ethylene responses (i.e., in the presence

of ethylene) or to prevent inappropriate ethylene responses (i.e., in the absence of ethylene). Either of these possibilities is consistent with the finding that ETRl binds ethylene and with the phenotype of dominant eb7 alleles, which could encode either dominant negative proteins (;.e., if E TR7 is an activator of ethylene responses) or gain-of-function proteins that constitutively prevent the ethylene response (i.e., if E TR7 normally inactivates ethylene responses).

ETR7 is not the only gene for which dominant alleles have been identified that confer phytohormone insensitivity but for which no recessive alleles have been found. Dominant mutations in the ABI7 phosphatase cause abscisic acid (ABA) insensitivity, but the effect of loss of ABI7 function is not known; similarly, a semi- dominant mutation in the GAIgene causes insensitivity to gibberellic acid (GA). To investigate the possible loss-of-function phenotype of GAI, Nick Harberd (John lnnes Centre) reverted the gaiphenotype with y-rays. Plants homozygous for the revertant alleles are wild type in appear- ance, which suggests that GAI function is not essential for normal development or that GAI shares redundant functions with another gene. It also cannot be ruled out that the gai mutation is a neomorphic allele of a gene that is not normally involved in GA signaling. This may be sorted out once GAI is cloned, which Harberd and colleagues are doing by recovering gai revertants under conditions in which Ds elements are mobilized.

Harberd and colleagues are also investigating the role of GAI by identifying second-site suppressors. Neither of two nonallelic suppressors, gas7 and gas2, restores GA sensitivity to gai mutants, but both double mutants are taller than the gaisingle mutant, and a triple mutant containing both suppressors is taller than even the wild type. Harberd pointed out that this phenotype is similar to that of slen- der pea mutants, whose growth is also independent of GA. An interesting possibility is that GAS7 and GAS2 act to repress plant growth (although neither mutation

November 1995 1745

MEETING REPORT

has a phenotype in a GAI+ background) and that GA acts, possibly via GAI, to re- lesse this repression.

Another gene that may act to repress GA responses is SPINDLY (SPY), which was identified by screening for mutants that are insensitive to an inhibitor of GA biosynthesis (Jacobsen and Olszewski, 1993). Mutations at SPY, all of which are recessive, also suppress a GA biosynthet- ic mutant, so they presumably result in a constitutive (;.e., GA-independent) GA response. In the absence of GA, homozygous spy mutant plants flower early and are pale, like wild-type plants sprayed daily with GA. Steve Jacobsen (University of Minnesota) reported that mutations in spy are also largely epistatic to mutations in gai, which is consistent with the hypothe- sis that SPY acts downstream of GA and GAI to negatively regulate GA responses. SPY has now been been cloned, and all spy alleles have alterations in the coding region of the gene, whose product is a tetratricopeptide repeat (TPR)-containing protein. The TPR, a 34-amino acid repeat found in a number of regulatory proteins, including transcriptional regulators, cell cycle regulators, and splicing proteins, is thought to mediate protein-protein interactions.

The ability to obtain loss-of-function constitutive response mutants such as spy and ctr l (which results in a constitutive ethylene response) highlights the importance of negative regulation in hormone response pathways (see Bowler and Chua, 1994). Negative regulation also seems to be involved in ABA responses, as Sean Cutler (University of Toronto) discussed. All ABA response mutants yet characterized have been identified on the basis of their ABA insensitivity, but Cutler has isolated several new response mutants by screening for the opposite phenotype, namely an enhanced ABA response. One of these mutants, eral, has a lesion in a gene whose product is similar to the subunit of farnesyltransferases. Farnesyl modifications have been impli- cated in membrane localization and protein-protein interactions; one way a far-

nesylation defect could increase hormone sensitivity is if the protein-protein or protein-membrane interactions it mediates are necessary for a negative regulator of hormone responses to become activated. A mutation that disrupts farnesylation might be expected to affect multiple signaling pathways, and it will be interesting to see whether era mutants have altered responses to other hormones.

However their presence or absence is transduced to the nucleus, phytohormones and other environmental effectors generally produce both transient and sus- tained changes in gene expression. In some cases, the sequences of genes whose expression levels are altered in response to a given stimulus may give important insights into how the plant responds or adapts to the stimulus. An example was provided by Janet Braam (Rice University), who is investigating the functions of genes upregulated by touch and other mechanical stresses. One of these genes, TCH4, encodes a xyloglucan endotransglycosylase (XET) (Xu et al., 1995). XETs cleave and rejoin the xyloglucan chains linking cellulose microfibrils and have therefore been suggested to play a role in the cell wall loosening that would accompany cell expansion; Braam suggested that such enzymes could also be involved in wall strengthening or xyloglucan incorporation. TCH4 is expressed in young, expanding tissues, which is in- dicative of a role in allowing or mediating cell expansion; in addition, TCH4 is induced not only by mechanical stimulation but also by other stimuli that promote cellexpansion, such as auxin and brassinosteroids.

Results presented by Mike Thomashow (Michigan State University) provided another example of the information provided by genes induced by particular stimuli. Cold acclimation involves extensive phys- iological changes, but it is not clear which changes actually provide protection from the cold. A number of genes are induced in cold-acclimated plants, several of which encode proteins that have long been sus- pected of being cryoprotectants based on

their solubility after boiling and on their presence in cold-acclimated plants specifically. Thomashow has found that one of these genes, COR75a, encodes a chloroplast stromal protein that when constitutively expressed displaces the chloroplast damage curve by 2O (in contrast to cold acclimation, which displaces the curve by SO). COR15a also seems to suppress cold-induced membrane fusion, a major problem for cold-treated cells. The mechanisms underlying these effects of COR15a remain to be determined.

Picking a Partner

Many proteins act not on their own, but rather in complexes with one or more additional proteins that form either transiently or more permanently. As the potential regulatory importance of such interactions has become clear, a great deal of atten- tion has focused on the development of techniques to identify interacting proteins (for review, see Phizicky and Fields, 1995). Finding interacting proteins can also be of immense help in trying to understand the biological and biochemical functions of proteins identified through genetic analysis or simply through their sequences, as is the case for more and more Arabidopsis proteins. When interactions are relatively stable and a fairly abundant source of protein is available, protein complexes can be purified by "classic" techniques such as column chromatog- raphy and the components of the purified complexes separated and identified, as Danny Chamovitz is doing with the cauliflower COP9 complex (see above). But to identify transient interactions involving low-abundance proteins, a number of library-based methods have been de- vised, and the use of some of these techniques was discussed at a well- attended workshop.

One library-based method is the yeast two-hybrid system used by Tedd Elich to identify a phytochrome-interacting protein. In this method, a DNA binding domain such as that of the yeast Ga14 protein is

1746 The Plant Cell

MEETING REPORT

fused to the protein under study (the “bait”) and transformed into yeast along with a reporter gene construct containing aGa14 binding site. This strain is then transformed with a cDNA library in which each cDNA is fused to a region coding for a transcriptional activation domain. If a cDNA encodes a protein that interacts with the bait protein, this interaction will bring the Ga14 DNA binding domain close to the transcriptional activation domain, and the reporter gene will be expressed. Despite, or perhaps because of, the power of this method, there are a number of caveats. Susanne Kohalmi (NRC Plant Biotechnol- ogy Institute) noted that some bait proteins themselves will function as transcriptional activators in yeast even if they are not authentic transcription factors. Some proteins will fold incorrectly in yeast, and yeast proteins may interfere with certain protein-protein interactions. The two-hybrid system will also not yield interacting proteins in cases in which more than two components are necessary for the interaction. It is also possible that proteins that do not interact in the plant will do so in yeast, and John Walker (Universityof Mis- souri) cautioned that once a potential interaction is identified, it is essential to demonstrate its biological relevance.

Despite these caveats, the technique is yielding impressive results. Using as a bait the catalytic subunit of acetolactate synthase (ALS), Kohalmi recovered a number of interacting cDNAs that encode a protein with homology to the small regulatory subunit of prokaryotic ALS, providing direct evidence that the plant enzyme actually possesses such a subunit. In a screen for proteins that interact with a type 1 protein phosphatase, Walker has identified a protein that shows similarity to the receiver domain of bac- teria1 response regulators.

Alon Samach (University of British Columbia) is screening Bill Crosby’s (NRC Plant Biotechnology Institute) two-hybrid library to find proteins that interact with BELL7 (BEL7), a gene reguired for normal ovule development. The groups of Bob Fischer (University of California,

Berkeley) and George Haughn (Universi- ty of British Columbia) showed that BEL1 is a homeodomain protein that possesses an N-terminal amphipathic helix that could mediate dimerization (Reiser et al., 1995). Samach and Zora Modrusan have now cloned severa1 BEL family members (BELH) from Arabidopsis, all of which share this predicted amphipathic helix. Using this region as a bait, Samach and his colleagues identified a number of interacting sequences, many of which correspond to one of the Arabidopsis KN1 homologs (KNAT proteins). Samach speculated that specific BELH and KNAT combinations might interact to regulate gene expression by binding DNA as heterodimers. He also described a possible way to test whether these proteins actually interact in vivo. Plants transformed with a KNAT-Gal4 DNA binding domain fusion protein would be crossed with plants in which a Gal4-responsive promoter drives reporter gene expression. If the KNAT protein interacts with one or more BELH family members, the reporter gene should be expressed in the same pattern as the BELH gene.

lnteracting proteins can also be identified by a direct protein probing approach, in which an expression library is screened with a labeled version of the protein of interest. Julie Stone (University of Missouri) described the use of a refinement of this approach, in which the protein is synthe- sized as a fusion with a protein kinase A substrate site that allows the protein to be labeled with 32P to high specific activity. Using this technique, Stone identified a protein that interacts with the receptor-like kinase RLK5. The interacting protein is a type 2C protein phosphatase whose cen- tral “kinase interaction” domain interacts not only with RLK5 but also with other kinases, including some from maize. Moreover, the kinase domain interacts only with the phosphorylated form of RLK5 (Stone et al., 1994).

The identification of interacting proteins or motifs can be used not just as an end in itself but as a means to a different end,

(University of Wisconsin) discussed. The idea is to fuse a ligand for the target protein to the ubiquitin-conjugating enzyme E2; this should then cause the target protein to be ubiquitinated and ultimately degraded by ubiquitin- dependent proteol- ysis. One way to identify such a ligand is by phage display, a technique in which random DNA sequences are inserted in- to the coat protein gene of a filamentous phage such as M13. The phage then “display” foreign peptides on their surfaces, and a phage expressing a peptide that interacts with the protein of interest can be identified by repeatedly passing the library over a substrate containing that protein. Gosink demonstrated that a fusion between E2 and a phage display peptide known to interact with S-protein, a fragment of bovine pancreatic RNase (Smith et al., 1993), ubiquitinates S-protein in vitro. Similarly, he showed that a chimeric E2 containing a calmodulin binding peptide (Dedman et al., 1993) is able to ubiquitinate calmodulin. Although degradation of these particular ubiquitinated proteins has yet to be demonstrated, Gosink has found that IgG that has been ubiquitinated by a chimeric E2 containing the IgG binding domains from protein A is indeed degraded in vitro. This technique could someday be a valuable adjunct to sense and antisense suppression of gene expression.

Dealing with the Data

As is the case for many other model organisms, particularly those whose genome analysis is being approached in an organized and systematic fashion, computers and the lnternet play a particularly important role in Arabidopsis research. A great deal of effort has focused on the development of data base resources that will help Arabidopsis researchers gain access to the enormous quantities of data being generated by various mapping and sequencing projects. The AAtDB (“An Arabidopsis thaliana

targeted degradation, as Mark Gosink Database,” which can be run on a Unix

November 1995 1747

MEETING REPORT

workstation or over the world-wide web at the URL http://probe.nalusda.gov:83OO/) provides access to such information as mapping data, sequence data, contact information, stock information, and biblio- graphic information from the Agricola and Medline data bases. And researchers can actually order stocks, as well as access a host of other Arabidopsis information servers, via AIMS (“Arabidopsis Informa- tion Management System”), which can be accessed at the URL http://genesys.cps. msu.edu:3333/).

Together with Weeds World, the offi- cial online electronic newsletter of the Multinational Coordinated Arabidopsis thaliana Genome Research Project (whose URL is http:/hyeeds.mgh.harvard.edu/ww/ home.html and whose August 1995 issue contains a summary of the meeting discussed in this report) and the usenet newsgroup bionet.genome.arabidopsis (whose contents can be accessed via AAtDB or AIMS), these data bases facili- tate communication between Arabidopsis researchers worldwide, helping to main- tain the community spirit that has always been a hallmark of Arabidopsis research. Given this community spirit, and the rec- ognition of funding agencies around the world of the importance of Arabidopsis research, Arabidopsis is likely to continue to yield dramatic new insights into the ba- sic biology of not only this plant but of all plants and, indeed, of eukaryotes in general.

Rebecca Chasan

ACKNOWLEDGMENTS

I would like to thank the many Arabidopsis researchers who generously took the time to reacl parts of my report and offer suggestions for im- provement. Any errors that remain are entirely my own.

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DOI 10.1105/tpc.7.11.1737 1995;7;1737-1748Plant CellR Chasan

Arabidopsis in Madison: genes and phenotypes spread like weeds.

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MEETING REPORT · November 1995 1739 MEETING REPORT verse genetics is fast approaching. Nevertheless, standard "forward genetics" continues to be a mainstay of Arabidop-sis research