Vith NATO Advanced Study Institute Plant Genomes

Report of Meeting

VIth NATO Advanced Study Institute Plant Genomes

T he first NATO/FEBS Advanced Research Workshop on plant molecular biology was held in Strasbourg in 1976. Most of the world's plant molecular biologists could be found among the 140

participants. During those early years, the NATO/FEBS Workshops and Gordon Conferences, equally limited in numbers of participants, were virtually the only forums for plant molecular biologists. By 1982 the exponential growth in the field inspired the founding during the third NATO/FEBS Workshop held in Portese, Italy, of a mass organization, the International Society for Plant Molecular Biology. NATO/FEBS Workshops thus carry a special significance for the ISPMB.

Distinct from congresses and symposia, NATO/FEBS Workshops have a specific purpose of instructing and encouraging new researchers entering a field. A scan of the age distribution of participants and of their poster presentations at last May's Workshop at Schlot~ Elmau in Bavaria is clear evidence of vigorous new blood in the field of plant molecular biology.

The youthfulness and excitement at Elmau in 1990 was very much the same as in Strasbourg in 1976, but the current presentations would have astounded any participant emerging from a 14-year time warp. As superbly crafted by the organizers, Reinhold H e m n a n n and Brian Larkins, the theme of the workshop was that plant molecular biology has become a boulevard for the exploration of fundamental processes in every area of the plant sciences: biochemistry, physiology, genetics, pathology, morphogenesis, systematics, agr icul ture . . . No field is any longer resistant to analysis by molecular methods, and the animating principles of many of them are being revealed. Some specific examples follow.

Virology. Genes involved in cell-to-cell movement of viral infections have been recognized for about five years, but Paul Ahlquist (Madison) has recently identified the 3a gene of brome mosaic virus and cowpea chlorotic mottle virus as the element responsible. In an analysis of the

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features that control compatibility between the three species of bromo- virus genomic RNA, Ahlquist showed the importance of the noncoding regions, and of parts of the RNA-2 coding sequence. In contrast, switching the coat protein genes of brome mosaic and cowpea chlorotic mottle viruses had little effect, but the correct 3a gene was needed for each virus to invade its normal host systemically. The work of Ther6se Godefroy-Colburn's (Strasbourg) showed that the product of the 3a gene of alfalfa mosaic virus accumulates in the middle lamella and probably facilitates cell-to cell movement of the virus. Such a mechanism would be consistent with the known ability of the analogous TMV product to bind to single-stranded nucleic acid.

William Dawson (Riverside) has investigated the necrosis which appears as an early sign of infection of N' tobacco plants by tobacco mosaic virus. Mutations in parts of the gene for the coat protein of TMV that seem to be involved in subunit interactions also modulate a chlorotic response. The role of the coat protein in inducing necrosis is mysterious: although transformation of plants with the gene for the coat protein is sufficient to induce degradation of the chloroplast, the protein itself appears not to enter the chloroplast.

Simon Covey (Norwich) related symptom severity in cauliflower mosaic virusinfections to the ratio between virus 35S RNA and "dormant" supercoiled viral DNA.

A new genome strategy for a plant virus was described by Rob Goldbach (Wageningen) who showed that the smallest genome segment of tomato spotted wilt virus is ambisense RNA, as in some animal- infecting bunyaviruses.

The first reports of DI (defective interfering) RNA's associated with plant viruses appeared approximately two years ago. David A. Knorr (Berkeley) reported that these subviral RNAs are showing promise for the protection of plants against viral infections. Large-scale field trials with tomatoes are currently underway.

Another novel development is the apparent frequency with which ribozyme activities are associated with subvira! RNAs. These structures are now being exploited by Wayne Gerlach (Canberra) both as tools for scientific investigation and as a possible means of plant protection.

Small viral RNA's and viroids may interact directly with nucleic acids of the host. While not accou nting for all of the properties o f viroids, Heinz S/inger (Martinsried) has ga thered further examples of vi roids which can hybridize with the 7S RNA's of signal recognition particles.

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RFLPs rampant. Restriction fragment length polymorphisms have found wide application in the large-scale physical mapping of plant genomes, as fingerprints for plant varieties, and as markers for genetic characters. Michael Murray (Madison) described how RFLPs are used by Agrigenetics to monitor the introgression of a desirable character, such as disease resistance, from an otherwise inferior line into an elite variety (one with proven agricultural merit, such as the Rutgers tomato). The traditional procedure is to backcross the F~ with the original elite line, selecting at each step for the desirable character. At least six such backcrosses are usually considered to be sufficient to recover a significant portion of the elite genotype, but recombination is a stochastic process and, apart from a few markers, breeders have had no way of monitoring the progress of recovering the elite genotype. RFLP maps that cover every chromosome and that can distinguish among lines can be used as objective indicators of introgression and can be employed early in the life cycle of the plant.

Brian M. Hauge of Howard M. Goodman's group (Boston) described progress in generating an "integrated physical/RFLP map" of the Ara- bidopsis genome, in other words a library of cloned DNA which is not only complete but whose volumes comprise an ordered set. They now have 17,000 cosmid clones which fall into 750 contigs* and comprise 91 Mb. Since the cosmid clones correspond to an eight-fold sampling redundancy, it appears that a different strategy will be required to achieve the goal of single contigs for each of the five chromosomes. One possibility is to span the gaps between contigs with YAC's (yeast artificial chromosomes).

Scott V. Tingey (Wilmington) has developed a system called RAPD (random amplification of polymorphic DNA) for mapping RFLPs. Ran- dom oligomeric sequences serve as primers in a reaction similar to PCR (polymerase chain reaction). Each primer, normally a 10-mer in which each base is critical, identifies an average of four loci in soybean DNA. Since the oligonucleotides are simple to synthesize, it becomes possible to generate an indefinitely large number of probes inexpensively and by automation.

Perhaps the most significant development reported at the meeting was three-dimensional images of chromosomes using a field-emission

*Contig is laboratory jargon for a sequence derived from a series of overlapping clones. In sequencing a single, continuous piece o f DNA, whether of 5 kb or 5 Mb, the object would be to obtain a single contig.


source for scanning electron microscopy (Reinhold He,rmann, Mfinchen). Using the new $4000 microscope, sensational resolu tion between 1.5 and 3 nm was achieved permitting visualization of centromeres, spindle apparatus, and nucleolar organizers. Even more remarkable, specific sequences could be located on the chromosome by means of hybridization probes. Higher resolutions appear possible with a still newer instrument.

Transgenic plants in agriculture. Although the techniques of transformation and regeneration have made the genetic engineering of plants commonplace, the use of transgenic plants in agriculture has been slowed by real difficulties in introducing characters with clear advantages over established varieties or established agricultural practices, and by public anxieties which translate into regulatory barriers. Robert Fraley (St. Louis) described field tests by Monsanto of tomatoes transformed with genes for Bacillus thuringensis control proteins which protect against catapillar pests such as the pinworm to an extent competitive with chemical control. He also counted over 200 field trials around the world with transgenic plants; comments from other participants indicated that this tally is conservative.

Nitrogen fixation. The genetics of nitrogen-fixing organisms and that of the host plants developed quite separately, but are now coming together. Work of the group of Jean D6nari6, presented by David Barker (Castanet- Tolosan), has led to the identification of the molecules of nitrogen-fixing bacteria that interact with the host plant; the signal is an oligosaccharide reminiscent of oligosaccharins. Ton Bisseling (Wageningen) reported that an oligosaccharide of Rhizobium switches on an early nodulin gene and Desh Pal Verma (Columbus) described the role of Nod24 and Nod26, nodulins that function as components of the peribacterial membrane, providing communication between bacterioid and host. The sequence of Nod26 is similar to that of GlpF, a glycerol transporter in E. coli.

Phytopathology. Rapid changes are taking place in studies on fungal pathogens, especially with the new weapon of physical mapping. Stud- ies of Richard Oliver (Norwich) and Forrest Chumley (Wilmington) indicate that transposable elements in the pathogen may account for genetic instability. Although pathologists remain largely committed to the hypothesiso f single-gene determinants of susceptibility and resistance, demonstrations at the molecular level of the host remain elusive. Vlado

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Macko (Ithaca) has isolated a protein from oats that binds victorin, a host- selective toxin produced by Cochliobolus victoriae, but the gene is present in both susceptible and resistant lines. Richard Michelmore (Davis) may be close to isolating a resistance gene, having identified multiple RFLPs in lettuce that segregate close to resistance to downey mildew. Klaus Hahlbrock (K61n) has found a protein which acts as an elicitor* in a non- host to Phytophtora, but does not act as an elicitor in the host. Infection is followed by a massive ion flux, perhaps due to the collapse of the cytoskeleton.

Stress. Dorothea Bar tels (K61n) presen ted studies of Francesco Salamini's group on the resurrection plant, Craterostigmaplantagineum, which shows the most spectacular resistance to desiccation of any higher plant. All parts of the plant respond to stress by producing amphipathic proteins similar to those coded by lea (late embryo abundant) and rab (responsive to ABA) genes. The genes in each of these cases are responsive to ABA. Montserrat Paghs (Barcelona) also described Lea proteins in maize, one of which appears to be an RNA-binding protein. A conserved promoter element, GTGACTGGC, in rab genes was described by John Mundy (Copenhagen); constructs containing catenated copies of the motif were sufficient to confer responsiveness to ABA.

Mark Olive (Canberra) summarized the elegant studies of W. J. Peacock's group on ARE's (anaerobic responsive elements). There is a core consensus of GGXTT, response is proportional to the number of copies of the complete ARE, and there is an optimal distance between the sub-regions.

Fritz Sch6ffl (Bielefeld) described an artificial gene composed of upstream heat-shock elements fused to the a sequence coding for hygromycin resistance; transformants could be selected by hygromycin resistance of heat-shocked plants.

Transposons. Barbara McClintock, of course, discovered transposons in maize, and their study for many years was wholly in the realm of genetics. It was only a few years ago that transposons were isolated from plants, but they have now become objects on which real biochemistry can be done. Reinhard Kunze (K61n) from Peter Starlinger's group described a putative transposase identified by western blots of nuclear extracts of

*Elicitor is a substance that induces a hypersensitive defense reaction in a host.


Ac-containing strains. It is coded by ORFa of Ac and binds specifically to sub-terminal sequence motifs in Ac. According to Patrick Masson (Bal- timore) of Nina Federoff's group and Alfons Gierl (K61n) of Heinz Saedler's group, the transposase of En/Spm is more complicated; synthesis of a functional transposase requires the cooperation of two transcripts which appear to arise by an alternative splicing mechanism. The product of tnpA corresponds to the suppressor function; the product of tnpB, which is 100-fold less abundant, may be the specific transposase.

Marie-Angble Grandbastien (Versailles) described a pair of novel retrotransposons in tobacco, which show strong similarities to Copia.

Agrobacterium and transformation. For a long time the Ri (root-inducing) plasmid of Agrobacterium rhizogenes has been the less-studied member of the Agrobacterium family, but Paolo Costantino (Rome) and Jeff Schell (K61n) obtained novel information on the physiology of development by studying the four rol genes of the Ri plasmid. Plants transformed with roIB alone exhibit the hairy-root phenotype, including a 300-fold enhanced sensitivity to auxin. The promoter of roIB contains three specific regions, one of which directs expression in root meristems and a second in the phloem. But roIB by itself is often lethal or at least produces chlorosis; roIC by itself produces multiple shoots (see cover photo), but with rolB protects against the effects of hypersensitivity to auxin.

Schell's group has also come across some surprising twists in growth physiology as engineered by the Ti plasmid. Gene 5 of the T region has been found to code for an enzyme that synthesizes indoi-lactic acid, which in turn presumably binds to either an auxin receptor or an auxin carrier, because it protects against toxic effects of auxin. The surprising aspect is that indol-lactic acid apparently affects only responses to exogenous auxin. Gene 6B has a similar action through an unknown mechanism in balancing effects of excess cytokinin. It is not known if the roles seen for rolC, gene 5, and gene 6B have counterparts in normal physiology.

Transformation remains a realm with numerous prized objectives. Seeking stable transformation of maize, Michael Fromm (Albany) used "the gun*" to bombard suspension cultures with the gene for acetolactate synthase and selected for resistance to chlorsulfuron or Basra. Stable

*A ballistic device in which tissues are transformed by shooting them with pellets coated with DNA.

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transformants were obtained at a rate of I in 80 cm 2 of tissue bombarded, a yield that was 1/4000 that of transient transformants. Peter van den Elzen (Leiden) seeks site-directed mutagenesis in higher plants. One transformation introduced a defective kan gene and a second attempted to repair it. The success rate was 1 in 500.

Transport physiology. Lothar Willmitzer (Berlin) looking for sink- and source-specific genes, identified a source-specific member of the family of genes that code for patatin. He used the promoter to engineer a gene for invertase which would be secreted from mature mesophyll cells. As a consequence, sucrose was hydrolyzed in mature leaves of tobacco transformed with this gene. An astonishing result was precocious senescence of the leaves. In the question period Martin Chrispeels (La Jolla) noted that pathogens, which typically cause chlorosis, can also cause invertase to accumulate in the apoplast*.

Antisense RNA. From the experiences of Joseph Mol (Amsterdam) and Don Grierson (Nottingham), nearly all antisense constructs have the effect of decreasing expression of the target gene, but the mechanism of this inhibition appears to be elusive: the amounts of mRNA's are lowered, but no KNA-RNA hybrids are found.

Enzymology. Ganesh Kishore (St. Louis) described the engineeringof 5- enolpyruvyl-shikimate-3-phosphate synthase to obtain enzymes resistant to glyphosate in transgenic plants. Through sequential substitutions of amino acids, the Monsanto group has recently produced enzymes with high resistance to glyphosate and with essentially unaltered enzyme kinetics.

Storage proteins. Genes for seed storage proteins were the first to be isolated from plant nuclear genomes. The new themes in this field are the identification of structural elements not susceptible to analysis by traditional biochemical methods, and the engineering for structure and accumulation. Storage proteins of cereals are typically composed of repeating peptides, which may be related to requirements for folding (Brian Larkins, Tucson; Peter R. Shewry, Long Ashton). Niels C. Nielsen (West Lafayette) found that modifications of certain regions of glycinin

*The apoplast is a region of the plant outside the plasmalemma which is accessible to dissolved solutes by free diffusion.

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resulted in subunits that failed to assemble, whereas a hypervariable region near the COOH terminus was quite tolerant of insertions and modifications. The potential of this discovery for improving food quality of glycinin proteins is obvious.

T.J.V. Higgins (Canberra) has sought to engineer pasture plants that would accumulate ovalbumin and vicilin in their leaves. Levels of expression of these foreign genes were typically low in transgenic Medicago sativa (lucerne), but accumulation to 1% of total leaf protein was achieved when the gene products were directed to accumulate in the endoplasmic reticulum.

Storage proteins are known to be under the control of powerful regulatory genes. Brian Larkins and the team of Natale Di Fonzo (Bergamo) and Francesco Salamini (K61n) are studying how Opaque-2, Opaque-7, and Floury-2 affect rates of transcription of zein genes.

Interesting pathways. Jacques-Henri Weil (Strasbourg), who is also the President of the ISPMB, organized this session around the molecular analysis of several groups of enzymes and metabolic pa thways of intrin- sic interest to the plant sciences.

Studies of mutants of Arabidopsis by John Browse (Pullman) and Chris Somerville (East Lansing) have shown that the pathway of fatty acid biosynthesis is an amalgam of prokaryotic-like enzymes, mostly in the plastid, and eukaryotic-like enzymes, mostly in the cytoplasm.

Gloria Comzzi (New York) has identified two genes for glutamine synthetase; one is expressed only in photosynthetic cells, the other only in phloem companion cells.

Athanasios Theologis (Albany, California) has isolated two genes from zucchini for ACC synthase, the usual limiting factor for ethylene biosynthesis. Expression of ACC1 is induced by wounding, or treatment of tissue with auxin, LiCl, or cytokinin. The action of cytokinin, however, appears to occur at the level of the mRNA.

Isolation of a gene for DAHP synthase, the first committed step in the shikimic acid pathway, has led to some unexpected wrinkles. Klaus Herrmann (West Lafayette) finds the gene product directed to plastids, where aromatic amino acid biosynthesis is indeed known to occur, but most of the enzyme is located in the cell wall, where lignin biosynthesis occurs. Herrmann suspects that the existence of multiple isozymes of DAI-LP synthase is the most likely explanation for this phenomenon.

The pathway of anthocyanin biosynthesis in plants was one of the earliest examples of biochemical genetics, predating that of Neurospora,

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but an understanding on the molecular level has come together only recently. Udo Wienand and Heinz Saedler (K61n) have isolated C1, a regulatory gene in maize, that controls the structural genes of anthocyanin biosynthesis. The predicted product of C1 shares partial homology with that of the myb proto-oncogenes including features characteristic of transcription factors.

Alkaloids are small molecules, often containing nitrogen, o f bewilder- ing complexity, and of pervasive importance to pharmacology. Al- though the chemistry of alkaloids was well advanced in the 19th century, understanding of their biochemistry, especially their pathways of biosynthesis in the plant, is based largely on speculation. Toni Kutchan (MLinchen) described a major step forward in cloning the gene for strictosidine synthase from Rauwolfia serpentina and demonstrating its expression in E. coil StrSctosidine, an indo|e compound, is an important component in the family of rauwolfia alkaloids.

Chris Bowler (Gent) of Marc Van Montagu's group employed the classic technique of rescue of a mutant of E. coli lacking superoxide dismutase for the isolation of a gene for the prokaryotic type of SOD in chloroplasts.

Mitochondria. If the biggest news in the 80's concerning plant mitochondria was cytoplasmic male sterility (CMS), in 1990 it is surely editing. The CMS story is by no means complete: it is still not known why such chimeric genes as urfl3 in CMS-T maize and urfS in Petunia become lethal only during microsporogenesis, although Maureen Hanson (Ithaca) finds qualitative and quantitative changes with development in the sizes of polycistronic transcripts in the urfS region, and Charles S. Levings III (Raleigh) attributes the toxicity of Urf13 exclusively to a single amino acid at position 83.

Because of the great advantage of CMS to plant breeding, there have been various strategies to introduce CMS into agriculturally important species, so far without success. Inaningeniousengineeringofnuclearmale sterility, however, Jan Leemans (Gent) described an anther-specific cDNA named TA29 which corresponds to transcripts that occur specifically in the tapetum of microsporophyte tissue. The promoter of TA29 was fused to the coding sequence of ribonuclease T1 of Aspergil!us oryza, and the chimeric gene used to transform tobacco and subsequently Brassica rapa. The tapetum of microsporophyte tissue of transformed plants was observed to disintegrate and the plants were male-sterile. A stable line of heterozygotes was maintained by the ingenious introduc-


tion of sensitivity to a herbicide for segregants that are homozygous for the absence of the sterility gene.

By comparing DNA, RNA, and protein sequences, Jean-Michel Grienenberger (Strasbourg) has identified some consistent characteris- tics of the editing of mitochondrial transcripts: the codon that is edited is almost invariably CGG (Arg in the universal code); when conserved, it codes for Arg; when edited, it is changed to UGG (Trp). When they occur, changes invariably result in transcripts that follow the universal code and lead to protein sequences that are conserved with respect to those found in mitochondria from other organisms.

The gross structure of plant mitochondrial DNAs does not become simpler with the passage of time, but Christiane M.-R. Fauron (Salt Lake City) has identified repetitive elements in mtDNA's, some of which are normally active in recombination and others which can become activated and result in reversions from CMS.

Chloroplasts. Reinhold G. Herrmann (M/inchen) reported that 50 of the 55 or so major polypeptides comprising the thylakoid membrane have now been cloned. About half are coded in the nucleus and half in the plastid itself. As is the case with plastids from many plants, transcrip- tional regulation of nuclear-coded plastid genes may occur, but the limiting factor in the synthesis of the corresponding proteins occurs post- transcriptionally. HeHmann described a nuclear-coded protein in plastids with the structure of Zn fingers, and suggested that it may play a role in regulating transcription in the plastid. From comparisons of plastid- and nucleus-encoded genes in races of Euoenothera, Herrmann proposes that the classic problem of incompatibility between nuclear and plastid genomes in this plant may hinge on physical compatibility of subunits of multiprotein complexes.

At what level is the light-induced expression of plastid genes regu- lated: transcription, transcript stability, or translation? The most general answer to this question now appears to be, that depends! John E. Mullet (College Station) and Peter Westhoff (D6sseldorf) find that the accumulation of transcripts of many plastid genes is coupled to leaf development. In plants such as barley, where cell division and leaf development are completed in the dark, plastid-encoded transcripts accumulate in the dark. Where leaf development is dependent on light, however, as in maize and sorghum, the accumulation of transcripts is light dependent. Mullet was also able to demonstrate for the first time in an in-vitro system a requirement for chlorophyll in the synthesis of chlorophyll a apoproteins.

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Specifically, lysed chloroplasts produced the apoproteins only when chlorophyllide a and phytylpyrophosphate were added so that chlorophyll accumulated.

Westhoff found that immature plastids of sorghum, a C4 plant, synthesize transcripts for photosystem II, but that these are lost during the differentiation of bundle-sheath chloroplasts.

Jean-David Rochaix (Geneva) has found both nuclear and chloroplast mutants that affect the trans-splicingofpsaA in ChIamydomonas. Two kinds of nuclear mutants affect splicing of specific exons. The product of the novel chloroplast locus, named tscA (trans-splicing chloroplast), appears not to be a protein but an RNA. Rochaix also described a chloroplast expression vector selectable for spectinomycin resistance.

Tony Cashmore (Philadelphia) sounded a theme concerning promoter elements that is becoming more and more familiar: multiple, conserved elements responsible for tissue- and signal-specific expression bind to proteins which show strong similarities across evolutionary time. Spe- cifically, a protein that binds to the I box of cab and rbcS genes shows homology to GF-I, the erythroid transcription factor.

Transcription factors in plants are the new "growth industry".

Photoreceptors. Elaine M. Tobin (Los Angeles) has devised a suicide cassette for selecting mutants that are defective in their response to phytochrome. Specifically, a cab promoter, which is normally responsive to phytochrome, is fused to the coding region for tins2, which codes for the enzyme that converts indolacetamide or naphthaleneacetamide to IAA or NAA, respectively. Normal plants transformed with this casettebecome hypersensitive to naphthaleneacetamide, whereas plants defective in their response to phytochrome will survive.

Peter Quail (Albany, California) has localized the lesion in hyl and by2, a pair of phytochrome-deficient mutants of Arabidopsis. The mutants make normal phytochrome polypeptides and can be rescued with exog- enously supplied biliverdin, a likely precursor to the chromophore of phytochrome. It seems likely that these mutants are defective in the modification of tetrapyrroles. Quail has also attacked the problem of the very rapid repression by phytochrome of transcription of its own phyA genes. He found an upstream region of phyA to be sufficient to confer reversible repression of a reporter gene by red light. A putative transcription factor, GT-2, binds to this region.

Ferenc Nagy (Basel) has identified a promoter element in a cab gene of wheat which is necessary for circadian fluctuations in transcript levels.


Although causal connections have not been established, the isolation of this element, along with the per locus in Drosophila, provides a molecular handle to a fascinating biological phenomenon.

Protein transport. Maarten Chrispeels (La Jolla) has cloned a gene coding for a putative tonoplast metabolite transporter, which has similarities to Nod26 and to GI pF (cf. Nitrogen fixation). Chrispeels has also identified sequence elements near the N-terminus of phytohem- agglutinin which direct the protein to protein bodies or, to use a term Chrispeels would prefer, protein storage vacuoles.

Sufficient numbers of signal sequences for secretory proteins and of transit sequences of mitochondrial proteins have accumulated to pro- vide models for these sequences with predictive capacity. The number of known transit peptides for chloroplast proteins has only recently become large enough to allow meaningful comparisons. Gunnar von Heijne (Huddinge, Sweden) perceives a system of three domains: an uncharged N-terminal domain, a central domain lacking acidic residues, and a C-terminal domain that contains a conserved cleavage motif. He also noted a striking similarity between sequences that target to the intermembrane spaces of chloroplasts and mitochondria.

For all of the differences in size and complexity between plant mitochondrial genomes and those of other organisms, there is a surprising conservation of transport mechanisms. Marc Boutry (Louvain-la- Neuve) found that the precursors of the ]3-subunit of the F 1 ATPase of yeast and Nicotiana plumbaginifolia were appropriately imported into the heterologous mitochondria. Experiments with fusion proteins indicated that as few as 23 residues of the transit sequence were sufficient for correct targeting.

Colin Robinson (Coventry) detects the transport and processing of proteins into thylakoid membranes using an in-vitro system of stroma, ATP, and isolated thylakoids. The import system shows striking similarities to that of transport in E. coli into the periplasmic space.

Plant development. Ten years ago the isolation of nuclear genes seemed possible only for those which produced abundant products and usually involved the same productsand processes for which we already had good biochemical data. Growth, development, and morphogenesis might be at the very heart of biology, but the central domains of these phenomena were largely beyond the reach of biochemistry and con- sequently beyond the reach of molecular genetics. The construction of

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tissue- and stage-specific cDNA libraries has made possible the isolation of rarely and weakly expressed genes. In addition transposon tagging has rather suddenly changed the odds: any gene locus that results in a visible or selectable phenotype is likely to show up in a large concerted program, and such programs have been underway for several years with maize, Antirrhinum, and Arabidopsis.

The most dramatic results have been obtained in the isolation of genes regulating floral differentiation. Rosemary Carpenter (Norwich) and Hans Sornrner (K61n) summarized work on a number of homeotic genes identified in Antirrhinum. In each case mutations resulted in the conver- sion of one flower part to another or to changes in the number or structure of floral elements. Similar sets of genes have been isolated from Arabi- dopsis by Elliott Meyerowitz (Pasadena).

Transposon tagging is, of course, part of the more general strategy of insertional mutagenesis. Kenneth A. Feldmann (Wilmington) has been mutagenizing Arabidopsis with T-DNA's selected by resistance to kana- mycin. Feldmann estimates that 25,000 transformants will have a 95% probability of having an insert every five to six kb. The set is now about 10% complete.

Adrienne Clarke (Melbourne) was one of the first to obtain molecular probes in an area commonly thought to be beyond reach, self-incompatibility. That work has now progressed to the recognition of conserved and variable regions among different incompatibility alleles and, perhaps most significantly, homologies of the predicted gene products with certain ribonucleases. Clarke has evidence furthermore that rRNA's of incompatible pollen are degraded within the pollen tube.

The Knotted mutation in maize results in abnormal morphology. Sarah Hake (Albany, California) has isolated several knotted mutants of maize by transposon tagging. The molecular basis for the altered morphology remains unknown.

Perhaps the best news is that the proceedings of this extraordinary meeting will be published before the end of 1990. The next workshop in this series is scheduled for 1993 in Barcelona with Pedro Puigdomenech and Gloria Coruzzi as organizers.

---C.A.P

Vith NATO Advanced Study Institute Plant Genomes

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