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11 Plant Biology and Agriculture Plants Constitute the Only Renewable Source of Energy The enormous quantity of energy from the sun that is captured by the earth every day becomes available for life processes only through the photosynthetic activities of plants, algae, and a few kinds of bacteria. These activities have resulted in the characteristics of the atmosphere that we breathe and have altered our atmosphere's chemical makeup so that it is hospitable to animal life; pro- longed over hundreds of millions of years, these activities have given rise to the fossil fuels that power our civilization. At the same time, photosynthesis consti- tutes the only renewable source of energy that is available to us for the future: a source of energy that is clean and potentially inexhaustible. Since plants directly or indirectly provide for our fuel and fiber needs in addition to being our primary source of food, they are exceedingly important to us from every point of view. Understanding their characteristics is of vital importance for the advance of biological knowledge and for human prosperity as well. Vegetation plays a major role in maintaining the earth-atmosphere system in a habitable state. Except for the polar icecaps, snow- and ice-covered mountains, and certain of the earth's deserts, all land masses are covered with vegetation. This vegetation contributes to global energy and water budgets through modifica- tion of the solar energy, water, carbon dioxide, and nitrogen exchanges at the earth's surface. In short, not the atmosphere, nor the soil, nor any of the other conspicuous features of the earth's surface would exist in their present condition if it were not for the existence of photosynthesis, a process that we now believe to have evolved among the cyanobacteria (blue-green algae) at least 3.5 billion years ago-at least 2 billion years before the origin of any photosynthetic eukaryotes and more than 3 billion years before that of plants. 365

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366 OPPORTUNITIES IN BIOLOGY The solar energy metabolically fixed through photosynthesis constitutes about 0.3 percent of the total solar radiation that reaches the surface of the earth. In addition, a substantial fraction of the solar radiation that reaches the earth is converted into latent heat that leaves the earth's surface through plant transpira- tion. Some 75 trillion tons of water evaporate each year from the vegetation to the atmosphere. Agricultural vegetation is responsible for about one-third of this water flux and also, because of the coupling of these two processes, for one-third of the total photosynthetic energy fixation. Natural tree ecosystems in the tropical and subtropical zones constitute the major vegetation mediating global water and carbon dioxide exchanges. The greater part of our food is produced by a few species of annual crop plants, mostly in the temperate and semihumid to semiarid middle latitudes. In food production, accumulation of dry matter is the process of greatest importance. Most of the weight in dry matter comes from the 175 billion tons of carbon dioxide that agricultural plants fix annually through photosynthesis. In addition to the more obvious activities of plants that occur above ground, extensive activity in modifying the characteristics of the soil is a role of the roots. The amount of water passing through a plant in its transpiration stream is many times the amount required to supply its internal needs. All of this water, together with the inorganic nutrients that plants require for growth, enters plants by way of their roots. Plants accomplish this through physical forces and highly specific transport systems. Interactions between plants and soil microorganisms are of critical importance for certain assimilatory processes: for example, Rhizobium, Frankia, and certain free-living bacteria for obtaining nitrogen, and mycorrhizal fungi, regularly associated with the roots of approximately 80 percent of all plant species, for phosphorus uptake. In addition, roots produce hormones that are important in directing the characteristics of shoot growth. PLANTS AND THEIR ENVIRONMENT Through Evolution, Plants Have Developed Characteristics to Cope with Their Environment One group of unicellular eukaryotes, the green algae (Chlorophyta), consists of organisms that share a number of biochemical and structural characteristics with plants. The similarities are so great that it is generally agreed that plants were derived from green algae, and specifically from organisms that had many of the features of the multicellular, freshwater alga Coleochaete. The cellulose cell walls that are such an important feature of the adaptation of plants on land originated among the green algae, as did the ability to form starch granules within the chloroplasts rather than free in the cytoplasm, and as did certain unique features of cell division that are common to all plants. The ancestors of plants invaded the land at least 430 million years ago, already multicellular and thus

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PLANT BIOLOGY AND AGRICULTURE 367 protected from the environmental extremes that they were to encounter there. The earliest plants were evidently mycorrhizal, the adaptive features of their symbio- sis with fungi assisting them in growing on and eventually molding the features of the raw soils of those ancient times. With their rigid cellulose cell walls, the bodies of plants are put together as if from a series of bricks. The sorts of cellular movements characteristic of animal embryology are impossible among such organisms, as is the ability to move from place to place in search of more suitable habitats or mates. Consequently, plants have evolved features that suit them to a sessile existence. Their life processes are bathed by a continuous stream of water that moves steadily from their root hairs into their roots, up through specialized conducting cells called xylem through the stems and into their leaves, and then mostly dissipates through the leaves through specialized openings called stomata, which also admit the carbon dioxide that plants require for photosynthesis. A waxy cuticle, similar to the outer covering of may arthron~xl.s evolved among the earliest Plants and helps to protect them _ ~ ~ , ~ ~ from drying out. Rooted in one place, many kinds of plants must tolerate a wide variety of environmental extremes. The consequent selection pressures led to the evolution of plant species that can withstand temperatures ranging from that of liquid nitrogen (-195.8C) to 90C and that could grow between temperatures lower than 0C and higher than 60C. Some plants are able to grow in solutions as concentrated as saturated salt and to withstand desiccation to the air-dry state. An additional characteristic of plants not found in animals is the ability to grow endlessly from areas of cell division, or meristems, that occur at the tips of the roots and shoots. New plants can be propagated from such meristems, and, depending on their growth form, may grow through the soil into areas of favorable nutrient status. Each plant can be both embryonic and senescent simultaneously, and the entire history of a plant's development can often be traced in a single organ. Understanding Plant Characteristics Is Important to Agricultural Development In nature, the ability of plants to reproduce is of fundamental importance. When plants are grown as crops, it is often their seeds, fruits, or vegetative reproductive structures, such as tubers or fleshy roots, that people desire. The characteristics of crops have been modified by selection and hybridization for at least 11,000 years and are now being modified more precisely by the techniques of genetic engineering. Cultural practices are also important in promoting crop yield. In all types of agriculture, opportunities exist to improve yield in different areas of the world. The optimal yield of particular strains of pants is usually tested by growing them in nonlimiting conditions-ones in which they do not encounter stress from drought, lack of nutrients, pests, or for any other reason. Such conditions are

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368 OPPORTUNITIES IN BIOLOGY approximated when crops achieve their highest recorded yields. Record yields may be some three to seven times as high as average yields obtained under more usual conditions in the same year. In the United States, for example corn (maize) had a record yield of 19,300 kilograms per hectare in 1975, but yielded only 4,600 kg/ha on average. Average agricultural productivity, therefore, falls far short of the genetic potential present in today's crops. Major environmental pressures must affect plants in ways that prevent the expression of their full genetic potential. Thus, improvements in productivity need not rest solely on increases in genetic poten- tial. For this reason, both the identification of the environmental forces and the manipulation of crops to express their genetic potential more fully are important research areas in plant biology. What are some of the environmental pressures that decrease productivity in plants? Diseases and insects are important contributors: These pests depress U.S. crop yields by an estimated 5.1 and 3.0 percent, respectively, below their genetic potential. In addition, weeds, which compete with the crops, depress yields another 3.5 percent overall, despite the widespread and relatively efficient appli- cation of control measures. An additional large depression in yields must be attributed to the only other factor that can be unfavorable, the physicochemical environment. An unfavorable physicochemical environment is found in soils and climates that are ill suited for plants. Adverse physicochemical environments such as an insufficient supply of water or nutrients-caused yields to be far below their genetic potential. Sometimes the environments in which crops are grown are inherently unfavorable, and sometimes farmers choose not to improve these conditions or cannot afford to do so. At any rate, physiochemical limiting factors are the most important negative influence on U.S. agriculture. In many other parts of the world, crop losses caused by diseases, insects, and weeds are considerably more severe than in the United States, but physiochemical factors usually pre- dominate everywhere in limiting agricultural yields. The major physicochemical resources for plants are water, soil type, nutri- ents, carbon dioxide, oxygen, and solar radiation. Of these, water is generally the most limiting. Permanently dry and shallow (drought-prone) soils make up about 45 percent of the total U.S. land area. About 40 percent of insurance payments for crop losses are made to drought-stricken farmers. Cold and wet environments are also important limiting factors, followed by salinity, hail, and wind. To cope with water deficits, farmers have for thousands of years irrigated their fields, which has contributed significantly to higher yields. However, water has become increas- ingly scarce, and many alternative uses compete with agriculture for it. In addition, water of poor quality causes progressive soil degradation and a conse- quent loss of overall productivity. Irrigation, therefore, affords only an incom- plete solution to the water limitations encountered by plants. Plant nutrients will probably be less limiting than water in the immediate future because they are more abundant or can be produced in sufficient quantities at an acceptable cost. Supplies of nitrogen, phosphorus, and potassium are likely

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PLANT BIOLOGY AND AGRICULTURE 369 to be sufficient to support U.S. agriculture for the next 30 to 40 years. However, energy must be used in the manufacture of ammonia (the major source of nitro- gen), and this is the largest energy input to the nonirrigated farm. Thus, the cost of energy will be a major constraint on the availability of nitrogen. Similarly, the cost of pesticides will increase with energy costs. Thus, the use of water, certain nutrients, and pesticides will be increasingly restricted, either because resources are limited or for economic reasons. These limitations may be overcome, in part, by the use of plants with lower requirements for these resources, a key aspect of the potential of Genetic engineering in combination with other methods for crop ~ ~ i, %, improvement. The Mechanisms by Which Plants Cope with Adverse Environments Have Only Begun to Be Understood in Molecular Detail Unfavorable environmental conditions depress the rate of photosynthesis, and we are starting to understand the mechanisms by which this process occurs. Such knowledge will be highly applicable for the improvement of crop perform- ance. The expansion of cell walls during plant growth is also affected by unfavorable environmental conditions. These walls contain cellulose as reinforc- ing microfibrils embedded in a carbohydrate and protein matrix that can flow in a plastic manner. The large pressures inside the cell, which are generated by osmotic forces, can cause plastic deformation of the wall and cell enlargement. The orientation of the cellulose microf~brils determines the direction of growth. Plant cell enlargement is extremely sensitive to certain environmental conditions and is retarded by low temperatures and drought. Such adverse conditions, for example, can cause seeds to fail to germinate or flowers to fail to open. The changes in molecular architecture of the cell wall that take place under such circumstances are largely unknown, as are the roles of water transport and plant hormones. Understanding these factors more completely bears directly on our ability to improve agricultural yield and quality. The mechanisms of inorganic ion accumulation by plants also constitute a critical area for investigation. Plants differ genetically in their ability to accumu- late nutrients especially nitrogen, phosphorus, and iron from a given kind of soil, but the molecular bases of these differences are poorly understood. By manipulating these features, performance would be improved. Plants vary in their ability to withstand freezing temperatures; for example, some plants have developed a way to keep water unfrozen in cells at temperatures as low as 40C. This ability permits some kinds of trees and shrubs to survive the extreme freezing temperatures that occur seasonally at high latitudes and high altitudes, but we do not understand its structural and molecular underpinnings at all. The reproductive structures of plants are characteristically more susceptible to low temperatures than their stems and leaves, but, again, we do not understand the mechanisms involved.

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370 OPPORTUNITIES IN BIOLOGY Many tropical and subtropical plants die when temperatures drop below 12C, but some, such as cotton, can become acclimated to such chilling condi- tions. Acclimation is accompanied by a change in the phospholipid composition of the outer membranes of the cells and probably includes similar changes in the energy-transducing membranes of the mitochondria Energy metabolism seems to play an essential role in the breakdown of cellular functions in cold-sensitive plants. Many of the storage problems of fruits and vegetables can be traced to the breakdown of membranes and the derangement of energy metabolism that occur at these temperatures. The biochemical basis for chilling resistance and acclima- tion needs to be established much more firmly to form a basis for improving the ability of subtropical plants to resist cool temperatures. A better understanding of water transport in plants can likewise improve crop performance. The transport of water through the vascular system occurs under great tension (negative hydrostatic pressures), and the continuity of the water pathways is sometimes broken an abrupt event that seems to be caused by cavitation of water under tension. An embolism that forms in the vascular tissue blocks further transport in that section of the system. Modern methods of electronic analysis indicate that such events occur frequently and are influenced by vascular architecture. Knowledge of how to keep the vascular pathways intact and filled with water is an important need. Accurate studies of plant biology demand access to controlled environments. Growth chambers and similar facilities petit the efficient evaluation of factors affecting growth of plants throughout their entire life cycles. In addition, tissue culture and seedling culture systems provide convenient ways to study problems of plant growth. Such systems provide opportunities to explore how limiting water affects the growth of roots and shoots and allows the use of biophysical methods, growth regulators, genetic mutants, and molecular genetics to explore some of the reasons for altered development. Tissue culture systems likewise permit experimentation under controlled conditions. They have the additional advantages that metabolites can be supplied in the culture medium and that selection pressures can be created to identify desired genotypes at the cellular level. Taken together, these research areas illustrate some of the ways a better understanding of plant growth could improve agricultural productivity. In prin- ciple, most of the features we have discussed should have a genetic basis. Thus, selection for more efficient water use and nutrient acquisition, as well as for the ability to avoid toxic ions, should help produce plants able to withstand unfavor- able environments. The genetic and molecular mechanisms of plant resistance to disease and insect attack are also becoming known. Pest organisms are not only responsible for crop loss in the field, but also for a large amount of loss during storage. In these areas, as in many others, an improved understanding of the ways plants grow and develop will enhance our ability to produce better crops.

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PLANT BIOLOGY AlID AGRICUL7VRE PHOTOSYNTHESIS A Better Understanding of Photosynthesis Is Crucialfor Our Future 371 Plants, like all organisms, depend on the products of photosynthesis for their growth (Figure 11-1~. The accumulation of plant biomass is a measure of the plant's total photosynthesis less the respiratory losses Hat have occurred during its growth. Crop productivity is linked to the seasonal photosynthetic perform- ance of the crop canopies. For this reason, knowledge of the relation between productivity and photosynthesis has largely provided the incentive for the broadly based research effort into this elementary plant process. Advances in Photosynthesis Research Utilize the Full Range of Modern Biological Approaches from Biophysics to Molecular Biology Tremendous slides have been made in gathering information about the catalytic components of photosynthesis at the level of atomic structure. Wide- ranging discoveries have created the opportunity to understand photosynthetic mechanisms at a molecular level. The most significant of these breakthroughs has Light :~ Starch CO2 + H2O Sucrose F-~-1 Sucrose ~ -1 Sucrose Vascular tissue Starch CO2 + H2O Sucrose (Stored) Photosynthetic plant tissues (Sources: leaves stems, etc.) N on photosynthetic plant tissues: (Sinks: roots, seeds, tubers, etc.) FIGURE 11-1 Diagrammatic illustration of carbon processing in green plants. [B. B. Buchanan, University of Califomia, Berkeley]

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372 OPPORTUNITIES IN BIOLOGY been the recent crystallization of the photosynthetic reaction center of the purple bacterium Rhodopseudomonas viridis and the determination of its three-dimen- sional structure by x-ray diffraction analysis (see Figure 3-2~. The wealth of existing knowledge concerning the mechanistic features of these complexes, which lends significance to this structural information, calls for corresponding structural work on other catalytic components of photosynthesis. The new struc- tural information contributed immediately to our understanding of the molecular functioning of photosynthetic bacterial reaction centers. The three-dimensional structure along with the biochemical and biophysical information about the vari- ous catalytic and redox-active sites (sites of electron transfer) have focused attention on specific regions of the amino acid sequence, which seem to have special significance in light absorption and in charge-transfer processes. In this prokaryotic photosynthetic organism, designed alterations in the genes coding for polypeptides that make up the reaction center are possible and becoming routine. This sort of molecular engineering, coupled with the sophisticated capabilities of molecular spectroscopy and biochemistry, is certain to contribute much to our understanding of photosynthesis. The crystallization of the Rhodopseudomonas reaction center has signifi- cance beyond the information obtained from its x-ray structure since it represents a fundamental discovery pertaining to the crystallization of integral membrane proteins. An intensive effort is under way to crystallize other major polypeptide complexes of bacterial and plant photosynthetic membranes. The development and refinement of numerous other physical techniques are contributing to the revolution in structural information about the catalytic compo- nents of photosynthesis. In particular, dynamic information about structural transformations occurring during catalysis, which cannot be obtained from the static picture provided by x-ray analysis, is now becoming available through the use of powerful physical methods. The development of high-resolution nuclear magnetic resonance ~MR) techniques and their application to biology have been particularly successful. For instance, spin-echo NMR techniques allow the selec- tive detection of a small subset of highly mobile, charged amino acid side chains that extend from the protein into the surrounding aqueous environment. The focus of this technique can be narrowed further to those amino acid residues that respond during catalysis; in other words, attention can be focused on the catalytic site as has been done for the chloroplast's coupling-factor enzyme. Even greater detail about the identity and rearrangements of catalytic site groups can come from other NMR techniques, such as double resonance and two-dimensional methods. NMR is but one example of the array of physical techniques being used to analyze the structural basis of photosynthetic reaction mechanisms. Neutron scattering, electron scattering and electron microscopy, linear and circular di- chroism, resonance Raman spectroscopy, Fourier transform infrared spectros- copy, extended x-ray absorption fine structure, and electron paramagnetic reso

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PLAINT BIOLOGY AND AGRICULTURE 373 nance spectroscopy are all contributing to the accumulating wealth of information about the molecular structure of the photosynthetic apparatus. The amino acid sequence of photosynthetic membrane polypeptides has recently been determined from the nucleotide sequence of the corresponding genes. This advance, in tum, has permitted estimation of the two-dimensional folding patterns of these proteins by hydropathy analysis. This information has been taken into account in the most recent models of electron transfer through the complex. Much Has Been Learned About Regulatory Mechanisms in Photosynthesis, but Much Remains to Be Done With our current knowledge about the component processes of photosynthe- sis, it has become possible to investigate specific questions about their interde- pendence. The most important mechanism in the regulation of chloroplast pro- cesses is light activation, a central feature that coordinates the light-driven reac- tions with the so-called dark reactions of photosynthesis. Light is absorbed by chlorophyll and is converted to regulatory signals that modulate the activity of selected enzymes. Such regulation is essential because enzymes that degrade carbohydrates coexist in chloroplasts with enzymes of carbohydrate synthesis. Some biosynthetic enzymes are activated by light, whereas degradative enzymes are deactivated by light. In this way, the concurrent functioning of pathways that operate in opposing directions (futile cycling) is minimized and the efficiency of temporally disparate metabolic processes is maximized. A number of soluble enzymes of photosynthetic carbon dioxide assimilation and other biosynthetic pathways show a similar activation response to light. Light regulates specific enzymes through a number of complementary mechanisms that have been identified during the past few years. These include changes in the concentration of certain ions, the concentration of regulatory metabolites, and the oxidation state of thiol groups (-SH) on key regulatory enzymes. Important in such thiol changes is the ferredoxin-thioredoxin system, a system in which thiore- doxins small regulatory proteins are reduced in the light by the photosynthetic apparatus. The reduced thioredoxins, in turn, reduce and thereby activate selected target enzymes. In this way, the cell can adjust flux through the metabolic pathways associated with oxygenic photosynthesis in accordance with energy and metabolite status. The catalytic activity of ribulose bisphosphate carboxylase/oxygenase (ru- bisco) is also modulated by light. Rubisco, which is the most abundant enzyme in the biosphere, performs the carboxylation reaction, the basis for photosynthetic carbohydrate production. Studies on the mechanism of rubisco activation have taken an unexpected turn with the recent discovery of the involvement of a polypeptide dubbed "activase." In certain plants, a newly identified inhibitor, 2'- carboxyarabinitol-l-phosphate, turns off the enzyme at night. The mechanisms

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374 OPPORTUNITIES IN BIOLOGY controlling the formation of this inhibitor and the mode of regulation by the activase are currently under active investigation. During the past years, much progress has been made in understanding the regulation of sucrose production in plants. Sucrose is the mobile form of energy that most plants form for transport to photosynthetic sinks; for example, to storage organs such as tubers and seeds that are the source of most of the world's food. During photosynthesis, chloroplasts convert carbon dioxide, water, and phosphate to triose phosphates, which migrate to the cytosol and combine to form sucrose. Photosynthesis requires inorganic phosphate, which is released during sucrose synthesis; therefore, photosynthesis and sucrose synthesis must be closely coordi- nated. There is evidence that this coordination is provided in part by phosphate. Recently, a second compound specifically serving this function has been identi- fied. Fructose-2,6-bisphosphate coordinates the metabolism of sucrose and starch and, in so doing, links metabolic processes of the chloroplast with those of the cytosol. Recent results suggest that fructose-2,~bisphosphate may also coordi- nate cytosolic and amyloplast (a starch containing plastic) metabolism in sink tissues. Our Current Knowledge of the Biochemistry and Physiology of Photosynthesis Has Made It Possible to Study the Process in Whole Plants or Intact Tissues Studies on photosynthesis have important applications to the improvement of agricultural productivity. Low temperatures; drought; photoinhibition; the accu- mulations of herbicides, pesticides, or fertilizers; and pollution are examples of frequently encountered conditions that compromise the efficiency of production in crops. Impaired photosynthesis is a major contributor to these losses, and we need to understand the mechanisms by which it takes place, something we are now in a position to do. One area poised for major advances is the application of recently developed and adapted physical techniques to investigate component processes of photosyn- thesis in situ. Techniques such as kinetic absorption spectroscopy, delayed light imaging (Figure 11-2), Now spectroscopy, flash fluorescence, and photoacoustic spectroscopy are now applied to diagnostic studies of how particular environ- mental conditions may influence intersystem electron transfer, adenosine triphosphate formation and consumption, enzyme activation, light regulation, photosynthetic reaction center activity, and the transfer of light energy. The use- fulness of these techniques depends on an underlying experimental basis for inter- preting the often complex results obtained from in situ measurements. This fact points to the need for expanding this information base. The development of "model" plant systems will unquestionably contribute to the solution of problems in photosynthesis related to agriculture. A notable recent advance has been the development of vigorous photoautotrophic cultured cell lines. Because of the difficulty of growing plants to maturity under heterotrophic

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PLANT BIOLOGY AND AGRICULTURE 375 FIGURE 11-2 Delayed light imaging in herbicide-treated bean leaves. Leaves normally emit a tiny portion of the photosynthetically active radiation that they absorb. Defects in certain chloroplast processes increase the amount of light that is emitted. This feature of photosynthesis has been exploited by using the increase in light emission to reveal the photosynthetic performance of different portions of leaves under stress. The four phytoluminographs depict the spatial distribution of delayed light emission in a red kidney bean leaf after the lower half of the leaf blade was sprayed with an herbicide. The herbicide was awlied 5 minutes before illumination; the notations refer to the length of time the leaf had been illuminated. The herbicide used inhibits the enzyme glutamine synthetase, and its action results in the accumulation of ammonium ions, which is the most likely metabolic cause of delayed light emission. [Donald R. on, University of Illinois] conditions, the screening and selection of photosynthetic mutants is generally limited to positive selection methods. The advent of these photoautotrophic cell lines and the promise they hold for expanding the use of mutants in photosynthe- sis research emphasizes the need for substantial effort aimed at developing reli- able plant regeneration procedures. It is becoming increasingly evident that cyanobacteria represent a highly useful model system for chloroplasts. The pro- gress that has been made in developing a genetic transformation system for cyanobacteria highlights their potential for the investigation of photosynthetic processes and events generally. Among plants, Arabidopsis and Petunia are genetically tractable, and they lend themselves particularly well to being modified through genetic engineering. They offer many approaches to long-standing agri- cultural problems with new research strategies having potential far beyond what could be imagined just a few years ago.

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392 OPPORTUNITIES IN BIOLOGY plasmid, the vir region, contains six genes necessary for the events leading to the integration of the T-DNA into the plant genome. Genes in the vir region are activated by messenger molecules that are released by Be plant. In some characteristics, crown gall strikingly resembles a disease on olive and oleander plants caused by the bacterium Pseudomonas savastanoi. This bacterium induces tumorous growth by secreting high concentrations of indo- leacetic acid and cytokinin into the tissues surrounding the point of infection. The enzymes necessary for the production of these growth regulators in P. savastanoi are functionally identical to those encoded by A. tumefaciens T-DNA, although the genes in P. savastanoi are located on separate plasmids and expressed in the bacterium. No transformation of the plant genome occurs. The nucleotide sequences in the coding regions of the genes for cytokinin and indoleacetic acid synthesis show a high degree of homology with those of the corresponding genes from T-DNA. However, the promoter regions, which control the expression of these genes, are entirely different in A. tumefaciens and P. savastanoi. This difference in structure was expected since the Pseudomonas genes are designed for expression in a bacterial (prokaryotic) cell, whereas the T-DNA genes must function in a plant (euk~ryotic) cell. The similarities in the structural genes indicate that the growth-hormone genes in the two tumorigenic systems have a . . common origin. The Ti Plasmid Provides a Vehicle for Gene Transfer in Plants Once it was known that A. tumefaciens actually transforms its plant hosts, scientists recognized the potential usefulness of the Ti plasmid as a means of introducing foreign genes into plants. In a number of laboratories, they designed so-called disarmed versions of the Ti plasmid. The genes associated with growth- hormone production were removed from the T-DNA, thereby preventing tumor formation in transformed plants. An antibiotic resistance gene was introduced as a selectable marker in an existing T-DNA gene for opine synthesis, and gene- cloning sites were constructed within the disarmed T-DNA. The genes of the vir region necessary for the integration process must be retained on the Ti plasmid or placed into a second helper plasmid. Once the desired gene is spliced into the T- DNA of the vector plasmid, the recombinant plasmid is reintroduced into A. tumefaciens. The bacterial cells are incubated with plant protoplasts or with leaf disks to allow transformation to occur. The protoplasts or leaf disks are then freed of the bacterium and placed upon a medium favoring plant regeneration. This transformation procedure has become so standardized that it is now routine in the hands of trained scientists. The Ti vector system has been used to introduce DNA sequences that cause disease and insect resistance into plants. For example, genes responsible for the production of an insect toxin have been transferred from Bacillus thuringiensis into plants. These plants became resistant to certain insects as a result of this

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PLANT BIOLOGY AND AGRICULTURE 393 transformation. Disease resistance to some viruses has been introduced by genetically engineering tobacco and tomato plants to produce the coat protein of tobacco or alfalfa mosaic virus. These plants show resistance to virulent strains of these viruses. An alternative method to introduce virus resistance into plants makes use of small RNA molecules known to act as 'parasites" of some plant viruses. These entities, called satellite RNA, replicate only in plant tissues that are infected with a specific virus, frequently reducing the extent of replication of the virus and ameliorating the symptoms that the virus alone would induce. The satellite RNA becomes enrobed in the coat protein of the virus and thus may be co-transmitted from plant to plant with the virus. The protective effect of the satellite RNA continues even in the subsequently infected plants. Recently DNA copies of cucumber mosaic and tobacco ringspot virus satellite RNA have been introduced into tobacco plants. Plants transformed with either one of the satellite RNAs and then inoculated with the respective virus showed greatly reduced symptoms in comparison with similarly inoculated, untransformed plants. Virulence Factors of Certain Plant Pathogens Are Natural Herbicides Many pathogens produce secondary metabolites that are toxic to plants. These chemicals are side-products of amino acid, carbohydrate, nucleotide, and lipid metabolism. One such chemical is tabtoxinine-,B-lactam, which is produced by a bacterium, Pseudomonas syringae pv. tabaci. If secreted into the cells of its host, tobacco, tabtoxinine-,8-lactam specifically inhibits the plant's glutamine syn~etase, an enzyme essential for the production of precursors for protein and nucleic acid synthesis. The pathogen escapes the inhibitory action of its own toxin by several mechanisms, among them being the production of a glutamine synthetase that is less sensitive to the toxin. This phenomenon, called self- protection, is common among pathogens that produce toxins as a part of their repertoire of pathogenic determinants. Because toxins produced by plant pathogens kill or injure plant cells, they may be viewed as natural herbicides. The activity of such toxins in selective instances provides a conceptual basis for the use of chemicals for weed control. Indeed, there is an herbicide that imparts its weed-killing effects by inhibiting the glutamine synthetase of plants. An alternative to the use of synthetic chemicals for the control of weeds is seen in fungi that are selectively pathogenic for weedy plants. Since He basis for this host selectivity is the production of a host-selective toxin, this phenomenon is being explored as a way to control weeds. Recognition and Defense Molecules Function in Path~gen-Plant Interactions Specific molecules function in the maintenance of many kinds of order in biological systems. Enzymes recognize substrates, cells recognize other cells and pollen compatibility, and incompatibility determines fertility in plants. Recogni

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394 OPPORTUNITIES INBIOLOGY ~::~:~:~:~; ::: GENETICALLY EN~GIN~EERED~RESISTANG'E~ ~ ~:~ :~ ~ ~ ~::~AGAIN:ST~:~P~LA:NT~:VI~:RU~SES~ ~ ~:~ ~ ~ ~ ~ ~ ~ ~_, . ~, . ~: ~ ~ i, ~ ~., ~, ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~.~ ~ ~ : ::~: ~:~ ~:I/:;~n~e~lraalilonal~;memo~ot~prot~l~ng~crop~plants~aga:Inst~:~speci~:~vl~r~Us~:~: :~ :::::~: ::: :: ::~ ::::: :::::: ~ ::: i: ::::: ::~ :~ :: :~ : :::::::: ::::::: :~:: i: :::~::~:: ::::: : ~ : ::~::::~ ~ ::: ::: ~ i:: ::~ :: :~ :~: :: :~ ~i~d~iseases~has~ :been~1o :~sea~:~ for resistard:~s~ ns~6f~crop~or~its~ clos=e;i~:~ i: ~:~ :atNes in: theft wiW~and~then~td~;~intro~)he~enes~;:~responsi:ble~ f~r~this~ ~:~ ~res~lstance~into~ti~t~d~i:~:st~ra~l~ns~with~le~aa~roTn To~mic~Dro~les~:lifDI-~:~ ~,~ ~:t~ne~:~:pron~uct~ion~ot ~d~ls~ease-resistan~w~rth~ baa ~ro~no::mic~ ~ l To JO~years,~a:~r~d.i~Furthe~more,~suitable~ ~resi~gen~for~a~tmpor:ant d~ve~not; been~id:e:ntified~:~:rli~:~ ~;~ many ~cases. ~ l~e~m~anyr~ crop p;labts~ai~ vu~iln~erable~to~viial~attack.~ AL : ~;~::situad~lon: that h~as~n~eg~dtJ~e~eco~nom~i~c~conseg~uenes c ~:~ ~ ~:~ ~:~ ~:~ ~ :::::::: ~:~::~:::~:~:~::~:~:~:~:~:: ~:::~::~:::~::~:~:~::::~::~:~:::~:~:~:::~:~:~: :~: ~ ::::~:::~:~::~:: ~:~:~:~::~:~ ::: ~ ~:::~::~: ~ ~:~:~ ~ Over~the: past :20 y~ears,~1ant pathos gists: have also :~used:~:thbi~method~ ~ ~ ~ ~ ~ ~ :, ~ ~ ~ ~ ~ ~ .: ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ .~ ~ ~ ~ :~ ~ ~ ~ ~ ~ i, ~ ~ ~ ~ . ~ ~ ~ ~ . . ~ ~ ~;~OJ~ cOoS spr~r~ctio~n~:~to~ gee res~stance~4air~`,l::r!a Amman crop.: ~:~:~ His m~ethod~:~is ~based~on~:~i::a~n~ observation ~mad;e~more~ than:: 6 - act ago ~ ~ ~ . ~ it: ~ ~:th:at~lobacco;~p lants~cou~ld~ be ~p~d~ ~:against~: :virulent~strain:s ~:~toba~w~ :: ~ ~ ~ ~ . ~ I. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~rnosa~c~r~us~ ~ l~th~~:werrifirminyulated~w~;th~am~ildi~strain~of~the~virus. ~::~ :~ ~:~ ~ ,:ls~approac ~ ~ has its~::r~s~s~incel~m~ild~ v~iral~sitr~a~ns~ can~Ye~n~se:~:td~viruJ~ent~l~:~:~:~ ~ ~ a. ~ . ~ . ~ . :. ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ it. ~ ~ ~ ~ ~ ~ . ~ ~ :~:~ones;~ ~ ,= 1~ mews divas :ate~:rat pert ~an~or~em~t Get D ant. ARIA ~so. virus: strains: :: i: :::: ::~: : :::: :::: ::: ::::::::: :::: i::: :~ ::::: ~::~:~:~:~::~:~: ~ ~ ~:~::~:~ ~ ~:~:~ w l t n ~ ~ ~ m ~ ~ ~ e ff e : c t s ~ ~ ~ ~ ~ ~ a n ~ ~ ~ d e ~ e l o p ~ ~ ~ ~ ~ s ~ n e r g ~ i s b c ~ ~ ~ i n t e r ~ i o n ~ s ~ w ~ ~ h ~ o ~ ~ ~ ~ ~ v i ~ u s ~ ~ s ~ . ~Nonethelass~,~the~m~ethod~ has ~bee~n~use~l in~enh~ar~ino~ croD~resistar~ce~in:~ ~1 ~ ~ ~ ~ ~ _ ~::~:~some~::~instar~ce~s~: :~ ~ ~ ~; ~ ~ ~ : : ~llie phenom~eno~n~;~cross~pro~tion ~prov~id~ the~conceptual~basis~f~r~ ~ ~ . . . . . ~ ~ ~.: ~ ~ .. ~ ~ ~ ~ 9 e~n~elica y~e~ng~lne~e r Q a r esistance~agal~ns t two~ ~c ~nter e ~nt~t y pes~ 0 f~ p an t YI- ru~ses,~l ~v ~anc ~ a ta ta~mosalc~v~lrus~(A ~l ~).~Th~e~ A - hdc~n~transior-~ ~mation~ ~sys;te~m ~was~ used ~ to~ introduce~the ~ge~nes ~that~encodel~the~ co~at~ ~pratein of ~:l MV and AIMV i - ~tomato ;ar d~tobacco~plants.~ ~Coat ;protei~n~'s~ ::::: ::: : : :~ : : :~:: :: ~ : :::~ :: ~: ~:~: :: ::~::: ::::~:~ ~:~ ~:: ~ : ::: : :: : ::::: : :~:: ::: : ::::~ : :~ : :::: ~::: :::: : ::: :: :::~:: :::::: ~ :~normal;~; wrapped ~around~the ~viral~n~ucle~c acid~ to form~lthe~ virus~parti^.~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ :~ ~ ~ ~ants: reg:enerated ~from~the~transfo:rmed:ce}ls~(t~ransg:an:ic~plants)~ producdd~l ~TMV~and~Al~MV~co~protein, but appearedito be~normal~in~l~other~respects. :~Whe~n~prQg~enyl:::of~:the~transgenic~pldrits~were~:~inocu~:with~::viru~lent~strains~ ~:~:: ~f~71~1V~o~r~:Al~MV,:: the~plants:~:~her~esca~d ~:infection:~or~devel~ped~ ~a~ liss : :: : : : : :: : : ~ ~:~sever~form :of :the~d~isease~than~ did:~t~::~nontransformed~p~ts~:~(F~ure 1~1~-~:~: ~::~5)~. ~:~The~mol~ecular mechanism~:~:responsiblelfo:r~ - gineered'~cross-protectidn:~i~ ~ . . . . _ ~ :~ 1as ~:not ;~ye '~ ~es~n ~eluadated~. ~ Reent~ results::~ ind~icate~:~t~hat~fewer :~:infebtion ::~ ~sites~::~are~::established in:~:transpenic~plants,~probably::b~cause~Qf~somei~block ~: at en: early seage~::~6f:the~infect~ion:~process.::::~::U:~infedtio~n does~occu~r:~:the~::rates: :~::1 :: :: :: : . :: ~ ~ : : ~ : :::: ~ :: ::::::: ~ :::: :: :: ::: : :~ : : :: :: : :: :: :: : : : :: : :: : :: : :: :~ :: ~ : of v~ral~ ~replication:~:and~ ~spread:~:through~:~th:e :~plant~:~:are~: reduced.~: Stud~Ks in :: prog ress: :~aim:::::at:~::expl ain ing:~:th:e m echari ism of ~ p~rotection, ~ increasing: the:~ level~:~ ~ :: ot protect~on, and extending~ protection::::: to other:viruses :and other~:plant::: . ~ . . . . spec~es.: ~'t ~s ~expectea~:~ t~h at:~genetically~ engin 8 e~red :cross-proteh~n~::will~::: :relatively: soon beco:me a annQraliv :anal~c~bln mathnd tn intrmH:' '~:~ vira : res;isttance~:into plants.:: ::: ::: :~: :: :~:~ :~ :~:~:~:: :: ~:~:~ ::~ :: ::~:: :~: ~:~:~ ~:::~:~: ~:~ : ~:~ :: :: :: ~:~:~ :: :~ ~ : :~:: ~ ~:~:~ :: ~:~ ::: ~, - ~_ ~. ~. : :: :: :: :: :: :~ : ~ ~:: ~:~

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PLANT BIOLOGY AND AGRICULTURE 395 FIGURE 11-5 Genetically engineered cross-proteciion against tobacco mosaic virus (TMV). (heft) Control tobacco plant (VF36) inocallated with a severe strain of TMV (PV 230). (Right) Transgenic tobacco plant that expresses the TMV coat protein gene (VF36 +CP) also infected with TMV strain PV230. [Roger N. Beachy, Washington University] lion molecules also mediate the interactions between pathogens and their plant hosts. An example for this is the specific messenger function of small polysac- charide fragments of fungal cell walls, called elicitors, which induce plants to produce chemicals called phytoalexins, which in turn might confer disease resis- tance to plants because they are toxic to the microorganisms that induce phytoalexin production. A specific molecular configuration is recognized by the plant be- cause any rearrangement in elicitor structure either abolishes or greatly reduces its activity. The biochemical basis for disease resistance and susceptibility is particularly well investigated in the case of root rot in peas. When attacked by the pathogenic fungus Nectria haematococca, peas produce a phytoalexin called pisatin. Some strains of the fungus are sensitive and others tolerant to pisatin. The sensitive strains cause mild disease in peas, from which the plants recover, whereas pisatin- tolerant strains are highly virulent and kill the plants. Tolerant strains respond to pisatin by producing an enzyme, pisatin demethylase, which degrades pisatin to a nontoxic product. Thus, the fungus has developed a way to circumvent a defense mechanism of the plant. Pisatin demethylase is a cytochrome P4so monooxyge- nase, the same type of enzyme that functions in mammalian livers as a detoxifying

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396 OPPORTUNITIES IN BIOLOGY agent. The gene encoding pisatin demethylase has been isolated from N. haema- tococca. Study of the cloned gene will help us to understand how the fungus recognizes phytoalexins and how it has evolved the capacity to live in their presence. In solanaceous plants, such as tomato and potato, small cell-wall fragments called oligogalacturonides are released when plant tissue is injured by chewing insects or mechanical rupture. Such oligogalacturonides, or perhaps some other signal molecules, are transported throughout the plant and systemically induce the production of a powerful proteinase inhibitor that interferes with the digestion of proteins. In the initial act of feeding, chewing insects seem to activate a defense system that renders the plant less digestable and may discourage further feeding. The systemic production of proteinase inhibitors in response to injury also occurs in nonsolanaceous plants; it might represent a general plant defense against insect predation. In some pathogen-plant interactions, a plant's susceptibility or resistance to a particular pathogen is determined by a single gene in the host and another in the pathogen. This gene-for-gene relationship implies a high degree of specificity and recognition between plant and pathogen. Resistance may arise from a specific interaction between gene products of the pathogen and the host plant. Susceptibility would result from a lack of such an interaction. These hypotheses are being tested by isolating genes from bacteria that confer race specificity for their hosts. The products of the genes are being identified, and the structures responsible for specificity will be determined. With Today's Technology, Fundamental Problems of Plant-Pathogen Interactions Can Be Investigated at the Molecular Level The intimate relationship that has evolved between plant and pathogen is now the focus of attention by plant scientists. These studies have been greatly en- hanced by new techniques in cell culture, chemical analysis, and molecular biology. It should be possible now to obtain answers regarding the responses of plants to challenge by abiotic factors, pathogens, or pests. What is the nature of disease or insect resistance in plants? How are resistant responses induced? What controls the expression of these resistance genes? Why are these genes not expressed in certain host-parasite combinations? Central to much of this research is our current ability to study mechanisms of communication between organisms and between cells. Transmembrane signaling, second messenger activity, and long-distance communication will be areas of active research during the next decade. Other research areas with exceptional opportunity include the nature of pathogen genes that are essential for causing disease. Is specificity determined by the nature of the plant products that induce the expression of pathogenicity genes or is it determined by regulatory functions that modify the response of pathogens

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PLANT BIOLOGY AND AGRICULTURE 397 to these products? The recent success in conferring resistance to plants by introducing viral coat-protein genes suggests that, as we understand the mecha- nisms of cross protection, we will also be able to exploit this phenomenon to control virus disease. The excitement generated by our ability to transform plants by means of the Agrobacterium T-DNA should be tempered by our ignorance as to how this plant pathogen is able to transfer DNA or how this DNA is integrated in the plant chromosome. The search for other pathogens that can serve as sources of vectors to introduce useful genes in our major cereal crops will, in Me near future, greatly expand our ability to improve plants by genetic engineering. Rapidly expanding computer technology has increased our knowledge of how plant pathogens are disseminated Cooperation among mathematicians, computer experts, and plant pathologists will continue to lead to better prediction of epidemics and, thus, to more rational application of control procedures. There is now renewed interest in the ability of bacteria to colonize leaf surfaces, for example. What triggers the change from epiphytic to parasitic habit in certain bacteria? The recent interest in the possible use of epiphytic, non-ice nucleating bacteria (which do not act as ice-nucleation centers) to prevent frost damage to plants is an example of how answers to some fundamental questions in plant- pathogen interactions can help stimulate the plant biotechnology industry. GENETIC IMPROVEMENT OF PLANTS Plant-Breeding Programs Can Now Be Enhanced by Molecular Biology During this century, plant breeding has led to substantial increases in crop yield through the production of hybrids with increased vigor, the modification of plant chemical composition or morphology, and the genetic transfer of disease resistance, among others. The methods of molecular biology can now be applied to complement those of conventional genetics, especially where barriers of sexual incompatibilty or of sterility preclude the introduction of desirable traits through breeding. Genetic engineering has also made it possible to introduce into plants genes from other organisms, regardless of their genetic relationship. In addition, plants such as Arabidopszs thaliana are being used as models for the study of plant molecular genetics, which will provide basic insights into plant biology. Tissue Culture Plant Improvement Through Tissue Culture Is Feasible, but Remains Technically Difficult Plant cell and tissue culture is an important tool for improving plant charac- teristics. Calli originally derived from plant tissue can be subcultured indefinitely

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398 OPPORTUNITIES INBIOLOGY :~::~:~:::~:~ARA:B/DOF'SIS - ~TOO~L~FOR~PL:ANT:~MO~R BE - So: T~:~:;~ ~ 18~: m~tna/l~an~:~an~n~u~aL~:~::~ ~ ~ ~ ~ ~ ~: :. ~ ~ ~ ~ ~ ~: I. ~ ~ ~ . ~ ~ ~ ~ .~ ~ ~ ~ ~ ~ ~ ~ ,~ :~ :~ ~ :~m~u:stara owl Yin irass:~cac~ ~stu:oles~n~p ~a~nt~gen~ics~w" ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~: ~:more~tl~8Wyears~:ago bathe German botinist~Eduard:S~r - ~Ariioiig: ~ :~::~:: : :::::::: ::::::::: :::: ::: ::::::: :::: ~:~ ~:~:~: ::::::::::: All::::::: ::: I:: :~:::: ::::::: :::::~:::~::::: ::::::: :~:: ::: I:::: :~ ::::::: :~ :: :: :~: ::: I:::: :: the advantages are~i~e~sm~all:~size~short gene:r~ioin:~time~ flus set ~:~production~of~this common~pld~^ur9~ 1~6).i Save: ~ ~p~:ce~ntime0~:one~ptant yields:~thousands~of~sedds~, As Lyle: :~ ~ ~:~ may:: Scoop ~n~sb~6 ~weeks.~l - i: scrams ~DU~Q~ A; a: ~:~:~ I::: :: : I:: :: :: :::: : ::: ~::~mam~ as~ 1 u.~uuu seeas~an~ c erm~ln~ated:~in~ o~ne~Petrl~d~ ~ ~: ~ ~:~ ~ ~ ~ ~:~ ~::~:~:~:~ ~ent~sit~s~::~revealdd:~it:ion~hta m~ Arabi ~:~:: :~: ~:~::~:~:~::: ~:~::~:~:~ ::~:~:~:~:~: :~:~:~:~:~::: ::~::: ~ :::~:~:~:~:::~: ~:~:~: :~:::~:~ :~:~::~ ::~::~:::::::::~:~::::~:~: ::~::~::::::~:::::::~::: :~::~::~ ~:~ ~a~ pn~e~:for~studies~in~ plant ~ol~gene~s~:~: ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ :~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~: a s t ~ ~ ~ ~ s m a ~ ~ 9 e : n o m e : k n o w n : ~ : i n ~ ~ ~ p l a n t s , ~ ~ i ~ h a b o : l l t ~ ~ 0 ~ k R ~ b ~ ~ s : ~per~h ~c ~ 5~il~k~ ~g~no~simpl~yi~::O~e ~:~ ~cbni~g of part~u~enes~which~can~:~:~ther ~tor~is~a;ilrig~ : ~ : : : :: ::: : ::::: ::: :~::: :: :: : ::~:::::: : ::: :: :: : ::: ~:: :::: ~: : : : ::: :: : :: :: : ~:: ~ : : :: ::~::~:: :: :: ~ :: :::: : :::: :: ~ ~ :~ ~:~oorrespp~dIng~genFes~f~m~otli~sj~r~:n~t~nshr~n:bx~eots.~:~:: ~: ~:~:~ Vew:~i~mp~t~:~ intensifi~ed::~ research~with~A~:~arose:~m~:the~:~:~ ~ . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~rea Iz~on t ~at~nes~d mutations~can ~b~o~ained~with re:l~ e~lbili~is~:~:~ ::: ::: ::::::::: ~:: ~:: :::: ::~ : ::~ ::~ :~ :::: ~ :::: :: : ~ :: : :~ : :: . . . . . ~ . ~ :~process~, ~ : 1~ ~seeas~are~ ltreat~a~ ,~::~a~mutagen~:~ana~fed ~a~ 1e~ ~ , . . ~ . . , . _ : ::: r esu l~lng~ ~:~p :an :s :::~ are~ ~ a owec~ ~ to:: se t~ertl '~.~ :::~ ~ ~e ~:~p:ro~ny: ~of ~::these ~plar~ts ~:::~ ~ :~ ~ ~ ~ ~ , 4. . ~ . ca 1 e '~lne~ M2 gen~e~rallon,~:are~::~usea~tor screenlog~tor~:mutants.~:~::~ :Many:~mut~:: ~:~::~ ~:~ ::tions~with~ a~::loss:~in~some ~mefabol~ic~h:indtion have~bQan:~f~und~:~a~frequency::~::: ~: . ~ ~ ~ ~ ~ ~ ~ ~ ~ ~:: ot one~:~i~n~2,000:~:MI2:~p):ants.::: ~Specific sereens:~yieW~dd~:~:well-d~ir - ~bioche~m~ical~:~ ~:~ mu T ants,~w~n~Icn~perm~'tted~:spec'hc~m~.tabolic~pathways,~sUth as~thdt:invo~lved~ . , ~ . . . . ~ ~In DnotorQs~DIratic~n~ tn n~ tr~:cQd ~ C3thor m~l~lI::~nt,: t:h:::'t ~: h:~uc~ ~h~"n ~ic~lat^~ :: ~ ~: ~::::: :::: ~ ~:~ i nclu:de~some ~thaf ~ ar=e ~ter~d~: in :::~the~ fatty~;~ ac~:co~mplement~ of: :the~ir~ m:em-::: ~::~:: ~: ::branes,:~dthers~in ~whtch: btarch sy:nthesis~is~blocked, :arid stillbih:e~rs~ in: wh ch~:~::~ ~1 ~:~:~hormone~:::b~sythesTis~:is~:blodkdd~:or:~:holmfone~::~sansitivitir:~altered.~::~S6ch ~ :::~::muta~nts~ar~:: valu~able~tobts:~for:~the~study~6f~plant:~physiology:and~develop-~ :: :::: ~m~ent.~: :~::: ~::~:~ ::::~:::~ ~: ~ ~ ~ ~ ~ : _ .. . ... ::: ::::: ::::: :: :: ::: ::: ::~ ~:: ::~ ::::: :~ :: ~:: ~: ~::~;~:~:~:::~:::::~: ::: ~;:~:~n~ent~,~^ran'~ops~s~was~used in:~ths:~isolation ~:~heirbicide-res~stance::~:~ :~ ~:~mutants.~ ~:About~o - :~:~o~of:: 100,000~ ~M2~seeds~ :sown::~on:~an~:~:ag~ar~:m~i~um~:~:: ~ :~oolita~ning~ ~a~ suHonylurea~ herbicide prov~d:::~to ~:be~:resistant~ :to~:~th:is~compo~und.~::~;:~ _` ., . . . . . . . . .. , ~ ~ ~ ~ ~ ~:~:su~on~u~rea~ne~roicides::~'nh~bR::acetola~ate~ synt:hase,~ an~enzyme i:n~:the~: bb-:~:~ :: sYnth:et:ic::~Dathwav of :bra:nch~:~amino acid:s.:~:~ ThQ~m~:~tated~a:Q:nQ~:fnr :~f=t~l:~:: :: : ~:: ~lale symnas~e ~was Clonec:: trom Ar~ aops/s :and :::was~::used: to: transform : tobacco ce:lls.::::: : The: :~:::regenera ed: t:obacco: plants ::::showed stable: h~erbicide ~ ::resistance. ;Expenments~ ~ this:ki:nd un:derscore:the::practical ~and:theoretical:~ : . :::importance :of th:e :A:rabi~s~s system :and :::ds potential for future:;:contribu- ::: . t'^nc, : ,,~, , ~: :::: : ~::::

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PLANT BIOLOGY AND AGRICULTURE Ll\f ~YI ~: L ; - it ~ l ~61 l ;.-\.\ Ad; / A: ~ (-- ~_~l FIGURE 11~ Arabidopsis thaluana. [Chris Somerville, Michigan State University] 399 on suitable synthetic media and induced to regenerate roots or shoots by altering the proportion of auxin to cytokinin in the culture medium. The resulting plants can be grown to maturity. Altematively, plant cell walls can be digested and calli regenerated from single protoplasts. The fusion of protoplasts permits the recom- bination of genetic material, even from unrelated kinds of organisms. Some plants can also be regenerated by the induction of embryos in cells derived from tissue that is not normally embryonic. Despite these advances, however, we know virtually nothing about the principles that allow He regeneration of some kinds of plants from protoplasts and that seem to preclude such regeneration (at least by

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400 OPPORTUNITIES IN BIOLOGY the available techniques) in many others, such as most commercially grown cereals. A serious problem that limits the utilization of plant tissue cultures for various purposes, including the preservation of desirable strains, is the high frequency of genetic changes in such cultures, a phenomenon called somaclonal variation. Such changes include alterations of chromosome number, chromoso- mal breakage, genomic rearrangements, and point mutations. Some of these changes may be advantageous, conferring such features as disease resistance, increased sugar yields (sugar cane), tuber uniformity (potatoes), and high levels of fruit solids (tomatoes). In some widely used cultivars of agronomically important plants, in which infertility has precluded the introduction of new traits by breeding, genetic diversity provided by somaclonal variation can be exploited for the selection of desired phenotypes. We Need to Learn More About the Principles That Underlie Plant Regeneration How do nutritional and hormonal factors influence the developmental fate of plant cells? What biochemical pathways have to be activated for root and shoot differentiation to occur? How are these pathways regulated? Cultured cells should be particularly well suited for such investigations because their growth conditions can be controlled rigorously. Somaclonal variation raises some basic questions concerning genomic organization and stability in plants. What factors lead to the observed destabilization of the plant genome, and, conversely, what factors maintain stability? What precise changes occur in the genome as a result of culturing? Answers to these questions will have direct consequences for the application of tissue culture technology to plant improvement and for our basic understanding of the mechanisms of plant growth and development. Plant Cell Transformation Plant Cell Transformation in Agrobacterium Has Become an Important Research Tool in Plant Molecular Biology Such fundamental questions as the way gene expression is regulated in plants have been investigated through the use of Agrobacterium-induced transformation. Nuclear genes under phytochrome control for example, the gene encoding the small subunit of the chloroplast enzyme rubisco-have been transferred from one species into another in such a way that the regulation of gene expression by light can be studied against a background of precisely defined gene constructions. In particular, it has been possible to identify the promoter and enhancer sequences that regulate the expression of these genes. These achievements constitute the first steps in clarifying the mechanisms by which environmental signals are transduced in plants.

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PLANT BIOLOGY AND AGRICULTURE 401 The T-DNA of Agrobacterium has also been used successfully to transfer herbicide resistance into plants. The mode of action of three commonly used herbicides has recently been elucidated. Glyphosate and sulfonylureas inhibit specific enzymes in the biosynthetic pathways of aromatic and branched-chain amino acids, respectively. Triazines block the binding of plastoquinone to an electron transport protein of the photosynthetic apparatus. Mutant plants that are insensitive to sulfonylureas and triazines have now been characterized at the molecular level. In each instance, herbicide resistance was associated with a single nucleotide change in the genes encoding the two target proteins. Resis- tance to sulfonylureas and triazines has been conferred on susceptible plants by transformation, through the use of the respective genes from herbicide-resistant plants. Plants with increased resistance to glyphosate have also been obtained through genetic engineering. Creating herbicide-resistant plants is especially worthwhile in the case of medium- to low-acreage crops, which do not warrant the development of selective herbicides. The methods of weed control that become possible in such systems decrease production costs and increase yields. The use of the Agrobacterium transformation system is limited by the host range of the bacterium. Although most dicotyledonous plants are susceptible to crown-gall disease, most monocotyledonous plants, including cereals, are not. Therefore, only a few monocotyledonous species have been transformed with Agrobacterium until now. However, striking success has been achieved by transforming plant cells directly with DNA. With polyethylene glycol used to perturb the plasma membrane of protoplasts, an antibiotic resistance gene has been introduced into tobacco cells. Plants regenerated from such transformed cells and their sexual offspring express the antibiotic-resistance trait in a stable manner. This same technique was also successful in transforming the cells of a monocotyledonous plant, the ryegrass (Lolium perenne). A technique called electroporation offers another method to transform plants that do not become infected by Agrobacterium. Electrical pulses are used to temporarily perforate the cell membrane of protoplasts, permitting DNA to enter the cell. With electroporation, protoplasts of maize have recently been trans- formed with an antibiotic resistance gene. All that stands in the way of staWy transforming many species is our inability to regenerate whole plants from proto- plasts or call). To get around the problem of regeneration from protoplasts or call), it is possible to shoot DNA-coated microprojectiles directly into intact plant cells. Potential targets include meristems and pollen, which can be cultured in vitro or used in sexual crosses, respectively. The Techniques of Genetic Engineering Hold Enormous Promise for Agriculture Potential improvements in crop plants through genetic engineering include increased yield, lowered production costs, improved nutritional qualities, adapta- tions to unfavorable growing conditions, and new biosynthetic capacities. The

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402 OPPORTUNITIES IN BIOLOGY feasibility of obtaining a number of traits of these kinds by genetic engineering has already been demonstrated in laboratory trials. Other applications of gene transfer technology, such as the utilization of the plant's biosynthetic machinery for the production of foreign compounds with high commercial value, have not yet been realized. "Custom" crop plants capable of synthesizing specific proteins, valuable oils, or secondary metabolites for medical use could be the bases of new agricultural industries. To attain these goals, research in the plant sciences must proceed at all levels. Discoveries in plant physiology and biochemistry must keep pace with the rapidly progressing field of plant molecular biology. The metabolic functions of plants must be explored further. Enzymes involved in the synthesis of plant products must be characterized and regulatory mechanisms in plant metabolism elucidated. The mode of action of plant hormones requires intensive study as do the reactions that mediate compatibility and incompatibility between pollen and stigma, symbi- onts and roots, and pathogens and plants. A thorough knowledge of the processes that could be altered for the improvement of plants provides the basis for the application of recombinant DNA and transformation technologies.