Anatomy of a Revolution

WITH this volume, GENETICS announces that Arabidopsis has joined the Security Council of Model Genetic Organisms. These favored few form the standard to which all other organisms are compared. Like the Security Council of the United Nations, where there is broad geographical representation, the Security Council of Genetic Organisms seeks broad phylogenetic representation. Therefore, the current membership also includes a virus (lambda), a gram positive bacterium (Bacillus subtilis), a gram negative bacterium (Escherichia coli), a fungus (Saccharomyces cerevisiae), a unicellular alga (Chlamydomonas reinhardtii), a worm (Caenorhabditis elegans), an arthropod (Drosophila melanogaster), a vertebrate (Mus musculus), and humans. The idea is that an intense concentration on the genetics of one of the representatives provides a window on the biology of all the other species in that phylum. Arabidopsis has emerged as the flowering plant delegate, presaging dramatic changes in agriculture and the topsy-turvy world of nutritional sciences.

To be elected to the Security Council, a delegate organism must meet objective criteria. It must be amenable to all the classical genetic moves—tests of dominance, complementation, and recombination. Its genome should be sequenced or in the process of being sequenced, and it must be readily transformed with DNA. With all of these techniques in place, geneticists can perform their sacred task: connecting genes with their function. Having said all this, elevation to membership on the Security Council (as with the permanent members of the U.N.) has a strong historical component. Buttressed by 90 years of study, Drosophila is unlikely to be replaced by another Arthropod. And the inclusion of humans smacks of self-interest on the part of the selection committee.

While Arabidopsis fulfills the criteria for membership, its meteoric rise to council rank is still surprising. As few as 10 years ago, most botanists shunned this tiny weed despite the fact that its small size, short generation time, and small genome with little repeated DNA were well advertised (REDEI 1975 ; PRUITT and MEYEROWITZ 1986 ). Yet, today there is virtually no major academic institution or agrotech company that does not have an Arabidopsis group; indeed, many formerly disdainful botanists are now among Arabidopsis' most avid proponents. So, what is responsible for this abrupt turnaround? I believe that the rapid rise of Arabidopsis as a premier genetic system is a consequence of the fortuitous confluence of three factors—scientific, social, and economic.

The crucial scientific factors were the concurrent development of Arabidopsis genomics¹ and the technical toolbox of modern molecular genetics. Together, these have made it possible to make mutants, to clone the corresponding gene, and to decipher the function of that gene. Genomic advances include the development of high-resolution physical and genetic maps and the attendant YAC, BAC, and lambda libraries. This genomic infrastructure has accelerated all genetic work and forms the backbone of the global Arabidopsis Sequencing Project. The goal is to have the complete sequence of the roughly 100 Mb in the Arabidopsis genome by the turn of the century.

In addition to these advances in genomics, we also have a panoply of new genetic techniques. There is a convenient transposon tagging system (HEHL 1994 ; AZPIROZ-LEEHAN and FELDMANN 1997 ) that permits not only the rapid isolation of mutants, but also the easy acquisition of the corresponding genes. There are also useful enhancer trap and gene trap constructs that facilitate cell and tissue localization for developmental and regulatory analysis (SUNDARESAN et al. 1995 ). But it is the development of a rapid transformation protocol that has propelled Arabidopsis to center stage.

Remarkably, Arabidopsis can be transformed simply by dipping whole plants into a solution of Agrobacterium containing a T-DNA plasmid (BECHTOLD et al. 1993 ; CHANG et al. 1994 ). Plants are inverted and immersed in Agrobacterium, and the combo is placed under vacuum for a brief period. The Agrobacterium infiltrates the tissues and delivers the T-DNA plasmid containing the DNA of interest into cells, where it becomes stably integrated into the genome. As some of the transformed cells develop into gametes, the seeds of these plants give rise to stable transformants in the next generation. Prior to this discovery, transformation in Arabidopsis was a nightmare, requiring a skilled technician to establish cell cultures from plants, transform the cells in culture, and then regenerate them into plants. This painstaking process was slow (4–6 months), cumbersome, and inefficient. More often than not, the few transformants obtained had genetic abnormalities (sterility, somaclonal variation) that made them useless for further analysis.

The in planta transformation protocol is easy and avoids all the problems of the cell culture approach. Without much supervision, even a high school student can generate large numbers of fertile transformants. This procedure is now used routinely to identify genes by functional complementation. Once the relevant mutation is localized on the physical map (1 cM = ~200 kb), DNA segments contained within that region can be readily transformed into the plant to identify the one that suppresses the mutant defect. The current protocol makes it practical to scan across a segment of 50 kb (10–15 genes) and to obtain unambiguous gene identification in 2–3 months.

The Arabidopsis transformation system still lacks a reliable method for obtaining homologous integration. This system would permit the simple construction of knockouts and the integration of virtually any DNA segment at a predetermined chromosomal location without rearranging the rest of the genome. This shortcoming has not precluded the election of Arabidopsis to the Security Council, especially since other members, notably Drosophila and the worm, currently suffer from this limitation. However, there are many fascinating plant phenomena that will be understood only when a homologous integration system is developed.

A good example is gene silencing, a phenomenon in which the presence of extra copies of a gene introduced by transformation inactivates both the transgene and the resident homologous gene (FLAVELL 1994 ; MARTIENSSEN 1996 ). Silencing is not simply a consequence of transformation because genes that have duplicated spontaneously can also be silenced (BENDER and FINK 1995 ). This homology-dependent gene inactivation may hold the secret as to how the genome defends itself against rampant gene duplication and the invasion of alien DNAs (viruses and transposons). At least two different types of silencing exist, one operating at the transcriptional and the other at the post-transcriptional level (DEPICKER and VAN MONTAGU 1997 ). Elucidation of the mechanism of silencing is hampered by the inability to direct a series of diagnostic constructs to the same chromosomal location. Currently, each construct ends up at a different site, making the results subject to the interpretation that some of the phenotypes may be due to chromosomal position effects. The recent report of a homologous integration event (KEMPIN et al. 1997 ) holds out the hope that this impasse may soon be surmounted.

In addition to these scientific developments, there have also been "extrascientific factors," both social and economic, that were critical to the transcendence of Arabidopsis. A key social factor was the excellent mentoring of students by the scientists who initially populated the field (among them, FRED AUSUBEL, GLORIA CORUZZI, RON DAVIS, ELLIOT MEYEROWITZ, and CHRIS SOMERVILLE). This founding group of molecular biologists transmitted to their students the community spirit that emerged from the lambda, E. coli, yeast, worm, and Drosophila traditions. Equally important, these mentors not only allowed but encouraged their students to take their successful projects with them when they embarked upon their own independent careers, creating a web of interacting laboratories. This amicable spirit fostered the rapid dissemination of strains, DNA libraries, techniques, and projects with the consequent rapid acceleration of research.

Another social factor was Cold Spring Harbor's adoption of Arabidopsis as the organism for the plant molecular biology course. The plant's rapid growth and short generation time make it a natural for a brief summer course program. The Cold Spring Harbor imprimatur served simultaneously to advertise the organism and to recruit young scientists to this new and burgeoning field. With the publication of the book, Arabidopsis (MEYEROWITZ and SOMERVILLE 1994 ), Cold Spring Harbor also promoted the codification and dissemination of useful lore and techniques.

Notwithstanding these scientific and social factors, this abrupt turnabout could not have happened without timely financial backing. The church regrettably no longer supports plant science as it did in MENDEL's time. Fortunately, the National Science Foundation (NSF) stepped up to promote Arabidopsis. There are many program directors at NSF who deserve thanks, but it was Dr. DELILL NASSIR who first sensed the zeitgeist and championed Arabidopsis when it was not fashionable to do so. She used discretionary funds, her wise counsel, and influence within the federal funding agencies to get the ball rolling—supporting meetings, identifying the movers and the shakers, and making sure that the best Arabidopsis proposals were supported. We may praise the organism and prize our geniuses, but the role of federal support in the emergence of Arabidopsis cannot be overemphasized.

All of these factors have combined to make Arabidopsis a premier organism with which to study problems of interest to all modern biologists. Many of these topics are covered in this commemorative issue. The central themes of modern biology—development, transcription, cell biology—all can be studied elegantly in Arabidopsis. Some of them, specifically the response to gases, are arguably studied best in Arabidopsis. Moreover, the plant-specific problems of phototropism, gravitropism, photomorphogenesis, and disease resistance, formerly studied by classical genetics or physiological experiments, are now being unraveled at the cellular level by the application of genomics and the molecular genetic techniques discussed earlier. Many of these findings are directly applicable to the improvement of crops, which probably explains why this inedible weed has suddenly become the darling of the agricultural schools.

Advances in the genomics and molecular genetics of Arabidopsis will permit scientists to attack problems that formerly seemed completely beyond their grasp. I would like to address two of these, the first theoretical and the second practical.

	Plant movement

TOP Plant movement The genetics of nutrition LITERATURE CITED

One of the key unanswered theoretical questions in biology is: What is the basis for concerted plant cell movements? We know that these dramatic movements are induced by external stimuli (light, gravity, and touch), but we do not have a conceptual framework that permits an understanding of the resulting behaviors. This problem was addressed eloquently by Charles Darwin more than a hundred years ago:

We believe that there is no structure in plants more wonderful, as far as its functions are concerned, than the tip of the radicle... It is hardly an exaggeration to say that the tip of the radicle thus endowed, and having the power of directing the movements of the adjoining parts, acts like the brain of one of the lower animals; the brain being seated within the anterior end of the body, receiving impressions from the sense organs and directing the several movements.

CHARLES DARWIN, The Power of Movement

in Plants (1896)

DARWIN's comparison of the tip of the stem to the "brain of the lower animals" focuses squarely on the issue, but his metaphor is inappropriate. Plants have no single organ, like the brain, that can be construed as directing movement. Moreover, movement in animals can be ascribed to the position and connectivity of the underlying anatomical structures—muscles innervated by axons linked to the central nervous system. But plant anatomy has not revealed the equivalent of a neural network that could account for the concerted behavior of some cells but not others.

Although plants do not have the equivalent of neural networks, they have plasmodesmata, channels through the cell walls that provide a continuous cytoplasmic connection between adjacent cells (LUCAS 1995 ; DING 1997 ). Plasmodesmata are portals for intercellular communication as shown by their role in the spread of plant viruses; these viruses encode proteins that dilate the channels to enhance their movement throughout the plant (WAIGMANN and ZAMBRYSKI 1995 ; HEINLEIN et al. 1995 ). Perhaps cells that respond together for plant movement are united by an underlying network of connectivity via plasmodesmata. This speculation raises many questions. Are there cellular proteins, analogous to the viral movement proteins, that regulate the flow of information through the plasmodesmata in response to external signals? Are there transcellular cytoskeletal structures that extend through plasmodesmata from one cell to another? How do these hypothetical regulatory and cytoskeletal elements coordinate the cell wall expansion that is thought to be necessary for motion?

If plasmodesmata provide the connections for concerted plant movements, then there must be some as yet undiscovered factor(s) that impose the selectivity that permits one group of cells to respond to a stimulus and another to ignore it. Group identity could be conferred by a restricted pattern of intercellular connectivity (only a subset of cells is connected by the same plasmodesmal highway) and specific intercellular molecular signals. This possibility could be revealed by the analysis of mutants defective in response to the stimulus coupled with improved biochemical techniques for the identification of plasmodesmal constituents. Once the relevant molecules have been identified, new imaging techniques applied to wild-type and movement mutants might reveal a structural or chemical network shared by those cells that respond in concert. Thus, the solution to the problem of plant movement raised by DARWIN may not come from a unifying theoretical insight, but rather by stitching together the answers to many diverse puzzles.

	The genetics of nutrition

TOP Plant movement The genetics of nutrition LITERATURE CITED

The remarkable developments in Arabidopsis research will form the vanguard of the third revolution in agriculture. As JARED DIAMOND has noted, "Human history's most important event since the last Ice Age was the rise of agriculture in southwest Asia's Fertile Crescent" (DIAMOND 1997 ). This first tsunami, which occurred in 9000 BC, was followed by another wave that occurred early in this century: the engineering of domesticated plants by the application of Mendelian genetics in the '20s and '30s (see Figure 1). The third wave is building now, with the application of new DNA technologies. Already, introductory botanical texts instruct today's college students that genetic engineering will yield plants that make vitamins and drugs; resist drought, disease, and insects; and "make better tasting food, clean the environment, recycle wastes, prevent tooth decay, and produce antibiotics, biodegradable plastics, and fragrances" (MOORE et al. 1998 ). A few of the more sober predictions already have been realized.

View larger version (166K): In this window In a new window Download PPT slide   Figure 1. —The revolution in agriculture. The photograph illustrates the result of genetic engineering in maize. Teosinte, shown on the left, is the wild grass thought to be the progentor of modern corn. The first revolution involved domestication and selection by natives of the new world. The second depended upon Mendelian genetics to produce the cob on the right. In the center are the F1 progeny of a cross between teosinte and modern corn. The third revolution may produce plants that look dramatically different from those of the 20th century. The photograph was kindly provided by JOHN DOEBLEY of the University of Minnesota.

The scientific and social factors are clearly in place for Arabidopsis to lead the third phase of the agricultural revolution, but the economic factor is not. The difficulty is that agricultural research is largely funded by the U.S. Department of Agriculture (USDA), whose policies have remained conservative and unimaginative in the face of the momentous changes that could enable the agency to carry out its mission. Even with well-meaning staff, the USDA has been unable to counteract the political forces that historically have stymied its effectiveness. Its largest competitive grant program, the National Research Initiative Competitive Grants Program, has a woefully inadequate budget of only 97.2 million dollars (FY 1998). Moreover, with an overhead rate of 14% it does not pay the true costs of research, making its grants noncompetitive with those from other federal agencies.

One solution to this problem would be to place the funding of plant engineering within the National Institutes of Health. The creation of a National Institute of Nutrition would house Arabidopsis in the appropriate pantheon of Institutes dedicated to the health of the nation. Such an act would not only provide support to accelerate the revolution, it would bring a breath of fresh air to nutrition, a field fundamental to public health that has so far failed to benefit from the advances in molecular genetics. Despite the enormous interest in diet, there is little critical information about it; the professional journals and the popular press abound with contradictory claims bewildering to both scientists and nonscientists.

The confusion caused by the disarray in nutrition is captured poignantly in WOODY ALLEN's (1973) classic movie Sleeper, in which Miles Monroe, the owner of the Happy Carrot Health Food Club, emerges from a 200-year cryogenic immersion in the year 2173. The following is the discussion between two nutritionists observing this modern Rip van Winkle:

• Scientist 1: Has he asked for anything special?

• Scientist 2: Yes, this morning for breakfast he requested something called wheat germ, organic honey, and tiger's milk.

• Scientist 1: Oh yes! Those were the charmed substances that some years ago were thought to contain life-preserving properties.

• Scientist 2: You mean there was no deep fat, steak, cream pies, hot fudge?

• Scientist 1: Those were thought to be unhealthy. Precisely the opposite of what we now know to be true.

• Scientist 2: Incredible!

But it is no joking matter. The problem with research in nutrition is that there is insufficient information about the two components of the system: the plants that are the source of food and the animals that eat them. The ability to alter genetically both parts of the equation would provide a powerful antidote to the current chaos. Although plants are key to our diet, we know little about how they synthesize, metabolize, and deposit the nutrients that are essential to our survival and longevity. Much of this confusion could be remedied by using Arabidopsis as a model for nutritional studies. At the outset, it would be important to unravel the pathways by which fatty acids, vitamins, and other metabolites are produced, how they are regulated, and how their deposition in the various tissues of the plant is controlled.

On the other side of the equation, there is a dearth of knowledge about how we metabolize plant compounds. Superimposed on this shortcoming is the problem that the human population is genetically heterogeneous for the ability to metabolize plant compounds. What's good to eat for one person may be life threatening for another. The inability of people with phenylketonuria to metabolize phenylalanine is emblematic of thousands of other genetically controlled metabolic differences in the human population. This genetic heterogeneity confounds current studies.

It is worth contemplating how the field of nutrition would be changed by genomics and molecular genetics. Think of the power of testing a battery of mouse mutants consuming a diet of well-defined plant mutants. Such experiments would define the critical nutrients that promote good health, longevity, and resistance to infection. But, the benefits go much deeper. For example, one might find that a particular mouse mutant always dies of coronary artery disease when fed a wild-type plant, but survives without complications when fed a mutant plant unable to produce a particular lipid. The ability to perform such experiments would turn nutrition into an exact science.

One objection to this scenario is that Arabidopsis is not an edible plant. But, here the spirit of the Security Council should prevail: What we learn in Arabidopsis is directly applicable to the engineering of crop plants. Once we learn to make knockouts and homologous replacements in Arabidopsis these technologies will be quickly adapted to other plants. There seems to be no resistance to applying this idea to rapeseed, a close relative of Arabidopsis that is a source of cooking oil. But, many balk at the idea that Arabidopsis, a dicot, is a good model for the monocots (corn, rice, wheat, and sorghum), which constitute the major source of protein for animals and humans.

However, the new field of genomics has provided data suggesting that perhaps too much has been made of the monocot/dicot distinction (PATERSON et al. 1996 ). Although monocots and dicots are thought to have diverged from each other 130–200 million years ago, there is considerable synteny between the two groups: large tracts of genes in Sorghum are in the same order as they are in Arabidopsis. This colinearity has direct consequences for the engineering of crop plants. Synteny between Arabidopsis and the monocots means that genes in Arabidopsis have functions similar to their orthologues in monocots, and knowing their position in Arabidopsis will aid in their isolation and manipulation in crop plants.

A second objection is that unlike mice, we are not inbred and our genetic diversity will preclude the establishment of a universally applicable diet. True enough. However, with the completion of the human genome project and the attendant technologies for genotyping individuals, it will be possible to identify the alleles associated with many metabolic differences. Once we understand these human differences, it should be possible to design transgenic plants that better meet our needs.

The inclusion of Arabidopsis in the Security Council is noteworthy because it heralds a new era. Although there will be many surprises along the way, one thing is clear: The emergence of a reliable and facile model plant that instructs both basic research and agriculture will alter the course of science and, ultimately, the course of history. And I predict that when Sleeper's Miles Monroe awakens in the future, the nutritionists will want to know his genotype before he dines on the extraordinary plants of the 22nd century.

	FOOTNOTES

¹ Arabidopsis World Wide Web electronic resources: http://genome.www.stanford.edu/Arabidopsis, http://nasc.life.nott.ac.uk/centre.html,Arabidopsis@net.bio.net.

	LITERATURE CITED

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PATERSON, A. H., T.-H. LAN, K. P. REISCHMANN, C. CHANG, and Y.-R. LIN et al., 1996  Toward a unified genetic map of higher plants, transcending the monocot-dicot divergence. Nature Genet. 14:380-382[Medline].

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