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4 Plant Science There have been remarkable advances in the molecular understanding of a few of the key processes in plants during the past decade. An important component of these advancements has been the application of new technologies for isolating, cloning, and characterizing genes. me ability to apply these techniques is based on fundamental knowledge in physiology and biochemistry that has accumu- lated over the years. Their application has opened new frontiers in the study of plant growth and development. Molecular genetic approaches have been applied to nearly 50 plant genes, primarily those associated with seed storage proteins, chloroplasts, photosynthesis, and biological nitrogen fixation. Notably, a number of the genes that code for important enzymes in photosynthesis and nitrogen fixation have been identified, cloned, and sequenced. Several of the genes for storage proteins in crop plants have been cloned and characterized. In addi- tion, the ability to regulate the expression of genes controlling the biosynthesis of the key enzymes in photo- synthetic carbon dioxide fixation is under active study. Studies are also being initiated to clone the genes for phytochrome as well as those for some of the known plant hormones. Comparable progress is needed in other areas of plant science research. A lack of basic information on the biochemistry of many metabolic and regulatory steps is delaying progress in using molecular genetics to establish the mechanisms employed in controlling plant growth. This chapter suggests ways of strengthening research that emphasizes integration of traditional biochemical and physiological 54

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55 research with molecular genetic approaches. Three gen- eral areas of research, all of which are basic to plant growth, are discussed: (1) the interaction of carbon and nitrogen metabolism in supporting optimal plant growth; (2) the role of plant hormones and phytochrome in regu- lating plant growth and development; (3) and the limita- tions to growth imposed by physicochemical stresses such as cold, heat, drought, and salinity. Carbon and Nitrogen Input for Plant Growth Both the carbon and nitrogen that are essential com- ponents for all forms of life ultimately cycle through the atmosphere. Plants take up these elements, in the form of carbon dioxide and nitrogen gas, from the atmo- sphere via photosynthesis and nitrogen fixation. Both processes involve reduction reactions, which require energy. Solar energy drives the photosynthetic reduction of carbon dioxide into sugars. me chemical energy stored in these products of photosynthesis is then used for nitrogen fixation. me relative availability of these two key constituents, reduced carbon and reduced nitrogen, can greatly regulate plant growth. Photosynthesis Photosynthesis encompasses the most important reac- tions on earth; life depends directly upon solar energy captured and stored photosynthetically. Not only do plants, through photosynthesis, reduce carbon dioxide to the food and fuel products that sustain life, but photo- synthesis also produces the oxygen required to deoxidize these products to release their energy. Light capture, carbon dioxide fixation, and oxygen evolution were recognized as components of the photosyn- thetic process more than 200 years ago. These partial reactions have now been described in considerable detail. Initial light capture and energy conversion occurs within a few nanoseconds after a photon is absorbed by chloro- phyll. Subsequent electron and proton transfers, occurring within a few milliseconds, generate chemical energy in the form of adenosine triphosphate (ATP) and reduced pyridine nucleotide (NADPH). These compounds in turn power the reduction of carbon dioxide.

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56 Chloroplast Functions The chloroplast is a remarkable organelle. It con- tains all the units essential for photosynthesis--the light-gathering pigments, the membrane components and cofactors that mediate electron transfer, the enzymes involved in ATP and NADPH production, the enzymes for carboxylation and reduction of substrates, and the system that liberates oxygen from water. Strong oxidizing and reducing agents are maintained in close proximity, but their interactions are controlled. For example, the chlorophylls that capture light and drive the energy-coupling steps of photophosphorylation (the process that produces ATP) as well as pyridine nucleotide reduction are organized within the thylakoid membranes in the chloroplast. The enzymes that catalyze the reduction of carbon dioxide for the synthesis of phosphorylated sugar intermediates occur in the stroma surrounding these membranes. Chloroplasts contain DNA, ribosomes, and the other components needed for protein synthesis. Major advances in identifying the genes that encode structural compo- nents of the chloroplast have been made in the last 10 years, largely through integrated studies using genetics, molecular biology, and biochemical analysis. Chloroplast DNA accounts for only about 10 percent of the genetic in- formation needed for chloroplast structure and function. Most of the structural proteins of the chloroplast are encoded in the DNA of the cell nucleus, synthesized in the cytoplasm, and then imported into the chloroplast. The information now available concerning chloroplast inheritance is spawning research toward practical appli- cations. Investigations include manipulating chloroplast genes that confer selective resistance against specific herbicides in crop plants and designing genes that pro- duce the enzymes involved in carbon dioxide fixation to increase the overall efficiency of the carboxylation reaction. Carbon Fixation The path of carbon in photosynthesis has been clearly defined. Carbon dioxide is fixed to yield the three- carbon molecule phosphoglyceric acid, which is then converted to sugars. This process characterizes the so- called C3 plants. In other plants, atmospheric carbon

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57 dioxide is initially fixed to yield a four-carbon mole- cule that is translocated to neighboring cells where it is decarboxylated to give up the carbon dioxide. These are known as C4 plants. This released carbon dioxide is subsequently fixed to yield phosphoglyceric acid via the C3 pathway. The formation of phosphoglyceric acid in both C3 and C4 plants is accomplished by the enzyme ribulose-1, 5-bisphosphate carboxylase-oxygenase, often called Rubisco. It can react with either carbon dioxide to car- boxylate ribulose-1,5-bisphosphate to two molecules of phosphoglyceric acid or with oxygen to oxidize ribulose-1,5-bisphosphate to one molecule of phospho- glyceric acid plus the two-carbon molecule, called phosphoglycolic acid, which is involved in photores- piration. This oxidation reaction is wasteful since energy must be consumed to resynthesize the ribulose-l, 5-bisphosphate. It would be advantageous, therefore, to cause Rubisco to decrease or lose its oxidative reaction function. Experimental elevation of carbon dioxide in the air produces major increases in yield of most field crops by increasing the carboxylation reaction. Although this technique demonstrates great potential, carbon dioxide enhancement of large areas is impractical. Rubisco is inefficient in catalyzing the carboxylation reaction. Attempts have been made to improve its effi- ciency, but the changes induced by genetic manipulation using both mutational selection and site-specific changes in the DNA sequence have only decreased its activity. In addition, attempts to specifically inhibit the oxygenate function without disturbing the carboxylase have been unsuccessful. If the catalytic sites are different, theoretically the oxygenase activity of Rubisco could be blocked. Selective pressures to accelerate the activity of Rubisco have existed for millions of years; it is hardly remarkable that a decade of research has not brought improvement in the carboxylation reaction, which theoretically might be possible. The problems in bioengineering an improved Rubisco focus on the fact that it is a large enzyme consisting of eight small subunits with molecular weights of 14,000 each and eight large subunits with molecular weights of 56,000 each. me larger subunits are coded for by chlo- roplast genes, and the small units are coded for by nuclear genes. The amino acid sequences of the large and small sub- units of Rubisco have been completed for some plants.

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58 Research is focusing, in particular, on the large subunit which contains the catalytic sites for carboxlyation and oxygenation. Several laboratories are concentrating on genetically engineering changes in the chloroplast genes that encode for the large subunits. Their objective is to locate a form of this enzyme that has an increased overall activity or a selectively decreased oxygenate activity. A great technical limitation to this work exists: mere is no gene transfer system yet available for inserting recombinant genes into chloroplasts. Photosynthetic Efficiency On the average, only one quarter of 1 percent of the radiant energy reaching the earth is captured by photo- synthetic organisms. m is largely is a measure of the density of photosynthetic plants on the earth's surface and the efficiency with which they can absorb light and photosynthesize throughout all the seasons. In contrast, a highly efficient C4 plant, such as corn, may utilize 5 percent of incident radiant energy during its most rapid period of growth. What is the potential efficiency of photosynthesis? One answer is provided by measurements of quantum effi- ciency. Efficiencies of one carbon dioxide molecule fixed per 8 to 10 quanta absorbed have been measured using single-cell algae. m ese efficiencies are high, in terms of conversion of incident light, and probably can only be achieved under the optimal experimental conditions--very low light intensities in comparison to average sunlight. For this and other reasons, it is impractical to extrapolate these measurements to field crops. Many factors such as the developmental stage of the plant and the presence of biological and physicochemical stresses can reduce photosynthetic efficiency. In addi- tion, each step in the photosynthetic process, from the absorption of light energy to the conversion and storage of energy in the synthesis of sugar molecules, can be affected differently by various limiting factors. Thus, to improve overall photosynthetic efficiency, researchers must first understand the steps in photosynthesis and the factors limiting their efficiency. Although it is doubtful that quantum efficiency in the field can approach that achieved with algae in the laboratory, record yields in field plots are a reasonable

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59 goal. A record corn yield in the United States can be more than 300 bushels per acre, compared with an average yield of about 100 bushels per acre. Comparisons indicate that full photosynthetic growth potential is seldom realized. How can photosynthetic efficiency be improved? One possible approach is suggested by the difference in photosynthetic efficiency between C3 and C4 plants. Major crops, such as wheat, rice, and the seed legumes, are C3 plants. It may be possible to convert them to a C4-type metabolism. C4 plants are more efficient, primarily because the C4 pathway serves as a metabolic carbon dioxide pump that raises the carbon dioxide concentration, by ribulose-1,5-bisphosphate carboxylase-oxygenase, at the site of carbon dioxide -fixation. This increases the rate of carboxylation and at the same time suppresses the rate of oxygenation. m is process is carried out at a level near carbon dioxide saturation, resulting in an enhanced rate of net photosynthesis. There are significant anatomical differences between the leaves of C3 and C4 plants. Specialized bundle sheath cells in C4 plants contain many chloroplasts; C3 plants have few if any chloroplasts in their bundle sheath cells. It is in the bundle sheath cells of C4 plants that the carbon dioxide concentration is elevated and increases the rate of carboxylation by ribulose-l, 5-bisphosphate carboxylase-oxygenase. C4 plants may have evolved from C3 plants. Nature has provided some plants with intermediate ana- tomical and biochemical properties. m ese plants serve as encouraging models. They include the composite Flaveria and the grasses Panicum and Neurachne. These ~- intermediate plants are tools useful in studying the inheritance and relative advantages of photosynthetic efficiency in the C3 and C4 pathways, under different limiting factors. Some of the leading research on photo- synthesis in C3 and C4 plants has been conducted in ARS laboratories. The photosynthetic potential of a plant cannot be achieved if its growth is limited by physicochemical stresses or by nutrient deficiencies. Efforts to improve photosynthetic efficiency can be enhanced by research focused on resistance to physicochemical stress and utilization of nutrients from the soil. Although outlines of the basic steps of photosynthesis appear clear, virtually every aspect of this complex

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60 process requires continued investigation. A thorough understanding of the basic mechanisms of photosynthesis may reveal new information that will permit researchers to increase photosynthetic efficiency and productivity and direct it toward the generation of the most desirable plant products. Harvest Index m e most effective way to improve the harvest index (the ratio of harvested part of the plant to total plant) may be to improve the accumulation of photosynthate in the desired plant part. Traditional breeding methods have selected for an improved harvest index in many crops and have led to substantial gains in crop yields. Such successes through plant selection have been achieved in the absence of a clear understanding of the factors under genetic as well as environmental control that determine crop yield. Additional improvements in yield and harvest index may depend on a full understanding of these factors and their interactions at the molecular and genetic level. With this information, scientists may now be able to take advantage of recombinant gene transfer methods to further improve crop quality and yields. Plants have often been selected as crops based on their parts that accumulate the products of photosyn- thesis. Plants with lush vegetative growth are used for feed or fodder while they are undergoing rapid photosyn- thetic growth. Alternatively, plants that deposit their photosynthate in stems, roots, fruits, or seeds may be selected for these attributes and harvested after their storage organs have achieved maximal size. In these plants the investigator attempts to redirect photo- synthate to the portion of the plant that will be used for feed or food. Researchers are currently attempting to improve the harvest index in soybean, for example. Before harvest the soybean plant undergoes senescence and mobilizes a high percentage of its nitrogen from roots, nodules, stems, and leaves for deposition in the seeds as protein. In a sense the plant destroys itself to produce a viable, energy-rich seed to preserve the plant line for the next season. If the soybean plant's delicate control mechanisms can be manipulated to prolong the period of active photo- synthesis in the plant without destroying its ability to

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61 go through senescence at the proper time, the harvest of seed will be greater. Foliar application of plant growth substances or nutrients can artificially prolong active vegetative growth. Subsequently, total crop yield may be increased. Integrated research programs that focus on both photosynthesis and developmental biology will con- tribute to a future understanding of the factors that link photosynthetic productivity and storage mobilization capacities. Photosynthesis in the chloroplast produces hexoses and hexose phosphates that are either converted to and im- mobilized as chloroplast starch or converted to sucrose. Sucrose is the major form of carbohydrate transported from the site of photosynthesis in the leaf to other parts of the plant. It is readily converted to starch and other storage products in seeds and storage organs. Control of the transfer of sucrose to various parts of the plant and optimization of deposition of carbohydrate, protein, and fat reserves in seeds and storage organs determines the harvestable yield of a crop. Using empir- ical methods in plant breeding and selection, researchers have successfully increased the harvest index of many plants. Little is known, however, about the processes regulating the translocation and metabolism of photo- synthetically fixed carbon. Species vary in their rates of accumulation of starch and sucrose in leaves. Wheat, barley, and spinach accumulate more sucrose than starch in leaf mesophyll cells in contrast to species such as peanuts, soybeans, and tobacco, which accumulate more starch than sucrose. Studies on the enzymatic steps involved in the biosyn- thesis of starch and sucrose indicate that inorganic phosphate and triose-phosphate have profound effects on regulating the rates of these interconnected biosynthetic pathways. Research focusing on a full understanding of the regulation of the storage and transport of carbo- hydrates has been modest. The partitioning of photosynthate is a major factor determining harvest index. Research on the metabolism of the hexose products of photosynthesis and the regulation of their conversion to carbohydrate, protein, and lipid storage products should be increased. Nitrogen Metabolism Nitrogen is a key element required by plants, and it is commonly the limiting element in plant productivity.

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62 me world capacity for commercial fixation of nitrogen is about 60 million metric tons per year, the bulk of which is used as fertilizer. Dependence on this source for agricultural use has created problems. High cost precludes its use in many areas; natural gas as the feed- stock for chemical fixation is a limited, nonrenewable resource that will increase in cost; and the use of too much fertilizer may be accompanied by excessive losses through leaching, erosion, and denitrification. Leaching into water supplies may raise nitrate concentrations to harmful levels. When biological fixation is substituted for chemical fixation of nitrogen, the energy of sunlight is substi- tuted for the energy of natural gas. Sunlight is captured through photosynthesis, and its use preserves fossil fuels. The nitrogen fixed biologically in the root nodules of leguminous plants is quickly assimilated into organic nitrogen compounds in the plant and is sub- ject to very low levels of leaching and denitrification. Although some major seed and forage legumes such as soy- beans and alfalfa take advantage of biological nitrogen fixation, there is potential for improving and extending the advantages of biological nitrogen fixation to other crops. Biological Nitrogen Fixation The association between leguminous plants and their root nodule bacteria is the preeminent system for biolog- ical nitrogen fixation (BNF) in agricultural crop plants. There are, in addition, certain free-living bacteria and bacteria in loose association with plants whose nitrogen- fixing capabilities warrant further investigation. Substantial advances have been made in studies of the biochemistry and genetics of nitrogen fixation. The enzymes and the electron transfer sequence involved in the steps that reduce nitrogen gas to ammonium can be described in some detail. Study of the genetics of the nitrogenase system in the free-living, nitrogen-fixing bacterium Klebsiella pneumonias has shown that 17 genes are involved. The function of most of these genes has been defined. Now it is necessary to establish comparable detailed information on the genetics of nitro- genase in other nitrogen-fixing organisms, including the symbiotic bacteria Rhizobium spp., blue-green algal species, photosynthetic bacterial species, and the azotobacter and the clostridia.

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63 In symbiotic biological nitrogen fixation, detailed information on the contribution of both the bacterium and the plant must be determined. For example, studies of the rhizobia-legume association have shown that the genetic information for production of the globin in hemoglobin, found in the leguminous nodules, is con- tributed by the plant. Scientists may eventually manipu- late genes to improve nitrogen fixation or to introduce it into other bacteria or higher plants not now capable of fixing nitrogen. A thorough understanding of the genetic systems of both the bacterium and the plant will enhance the chances of success in transferring genetic elements. There are marked differences in the effectiveness of symbiotic nitrogen-fixing systems, but the character- istics governing good or poor associations have not been defined. Until these factors are defined, genetic manip- ulations will remain empirical. In addition, symbiotic nitrogen-fixing systems require large amounts of photosynthate--10 to 12 grams of photosynthate are utilized in fixing 1 gram of nitrogen. Decreasing this energy requirement is a major research challenge. Nitrogen-fixing systems dissipate 25 percent or more of their energy in producing hydrogen rather than in reducing nitrogen. Hydrogen production is apparently inherent in nitrogenase action. The only way known currently to decrease this energy loss is to recycle the hydrogen to recapture its energy. Oxidation of hydrogen via a hydrogenate enzyme can be coupled to ATP formation and to reductant formation. ATP and reductant can then be used to support nitrogenase activity. The gene for hydrogenate, Age+, has been transferred to Rhizobium japonicum. Soybeans inoculated with Age+ rhizobia produce higher yields of protein than those infected with comparable t~e~ rhizobia, which lack this enzyme. Sim- ilar improvements through manipulation of other genes or modification of the nitrogenase genes for increased efficiency can be made when other factors limiting nitrogenase activity are defined. Improving Symbiotic Nitrogen Fixation About 85 percent of legume inoculant used in the United States is applied to soybeans. Indigenous rhi- zobia are so dominant in most soybean fields, however, that improved rhizobia strains that are added to the soil

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64 do not compete effectively in the process of Modulation To improve legume-bacterial symbiosis, the competi- tiveness of added, improved rhizobia strains must be increased. A superior nitrogen-fixing strain developed under controlled conditions in the greenhouse will be of little use in the field unless it can form nodules on the soybean plants in competition with indigenous bacteria. Processes such as primary attraction, binding, and in- fection may be important aspects of this competition. Such processes may be influenced by special glycoprotein molecules, (called lectins) on the root surface, but this must be established clearly and it must be controlled. Mutants of rhizobia have been produced that appear to enhance the early growth of soybeans. m is optimal growth, however, has not been maintained until harvest. Improvements under field conditions are of economic significance; however, few have been verified. Host Plant Improvement Carbon and nitrogen metabolism share an intimate relationship. Biological nitrogen fixation requires great amounts of energy, supplied primarily by photosynthesis. Progress in the under- standing of photosynthesis has been impressive, but further research is needed to define its interactions with major limiting factors in plant growth. With advances in experimental techniques and a better under- standing of the fundamental metabolic steps in both photosynthesis and biological nitrogen fixation, researchers are better equipped to study the feedback relationships between these two processes. An improved understanding of the interactions among nitrogen metabo- lism, photosynthetic carbon fixation, and the distribution of fixed carbon throughout the plant will contribute to eventual increases in the harvest index. Recent studies suggest that transformations of carbon compounds at the site of nitrogen fixation in the plant may be important in nitrogen fixation. Research con- ducted by ARS scientists has demonstrated the ability of root systems of certain leguminous plants to fix carbon dioxide. These transformations may be a part of the conversion of photosynthate to compounds especially useful as acceptors for newly fixed nitrogen. The transfer of the genes for nitrogen fixation to nonleguminous plants, such as corn, is appealing and should be studied on a long-term basis. Genes for nitrogen fixation have been transferred from the free- living bacterium Klebsiella pneumonias to the bacterium ,

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70 different stages of development. Ultimately, character- ization of enzymes may lead to the development of genetic probes that will assist in identifying and cloning genes that code for and regulate endogenous levels of plant hormones. Tools, such as monoclonal antibodies that are specific for certain enzymes, are needed to identify and localize hormone biosynthesis within tissues or cells. Plant hor- mones are low-molecular-weight compounds that do not, by themselves, have antigenic properties. Monoclonal anti- body probes for the plant hormones are now being devel- oped, however, by covalently linking them to the surface of a carrier protein macromolecule. The carrier protein serves as the antigen to stimulate antibody production. Because the attached low-molecular-weight plant hormone has become a surface characteristic of the carrier pro- tein, some of the antibodies produced might recognize and have affinity for free, unlinked hormone molecules. This approach, using antibodies against plant hor- mones, is in its early stages. It does offer, however, a level of sensitivity for both chemical identification and quantitation that may match the physiologically active concentrations of the hormones in plant tissues. A dis- advantage of this method is that tannins and other phen- olic substances, often found in plant extracts, can denature proteins, including antibody proteins, and might obfuscate the sensitivity of the analysis. Chemical Analysis Sensitive chemical analyses are greatly aiding studies in plant hormone biosynthesis. High-resolution analytical instruments are now available for the chemical identification and quantification of hormones in the plant. This analytical capability is based on the use of high-performance liquid chroma- tography (HPLC) followed by gas chromatography, coupled with mass spectrometry (GCMS) or nuclear magnetic resonance or both. m ese methods provide accurate separation, identification, and quantitation of the minute amounts of hormones present in plant tissues. The instrumentation is costly and its operation and maintenance demand special analytical skills. m e accuracy and sensitivity provided by these methods, however, are often required. In addition, the biosynthetic origin and metabolic fate of these hormones in plants are being studied using radioactive and atomic mass labeling techniques.

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71 Genetic Variants Single gene mutants have also been important in studies on the biosynthetic pathways of hor- mones in plants. For example, dwarf mutants of corn have been successfully used to study gibberellin biosynthesis. Several derivatives of the basic gibberellin chemical structure are synthesized in plants. For example, only one gibberellin, gibberellin A1, is active in ~~ ~ ~ ~ Other gibberellins in controlling shoot growth in corn. the plant are important intermediates in its biosynthesis. Dwarf mutants of corn are unable to synthesize gibberellin A1; they are unable to carry out one or more of the steps in the interconversion of one gibberellin to another. Use of those m'~t~nP~ h== h^^n _ _ . . . . . . ~ ~ ~ _ ~ ~ Critical toot in defining the sequences of conversions of the many qibberellins to the Final - Act iv" an - ~'l-' ~ , ~ ~ ~ _ , gibberellin A1. In addition, mutants will be important experimental models for understanding the regulation of hormone levels during appropriate stages of development. For example, in viviparous mutants of corn, the maturing seed does not become dormant but instead continues to grow and ger- minate while still on the ear of corn. me dormancy of normal seed is associated with a relatively high concentration of abscisic acid. Research indicates that insufficient levels of abscisic acid are present during the maturation of the viviparous seed to impose dormancy. Gene Expression Scientists have searched for specific cures that recognize and interact with a hormone or phytochrome in studies of their mechanisms of action. Radioisotopically labeled hormone molecules with high specific activity are used in attempting to locate and identify the receptor sites that bind the hormone. Thus far, this method has not succeeded in plants as it has in the case of identification of the receptor sites of steroid hormones in animals. Thus far, scientists have not succeeded in identifying a specific binding site with characteristics that correlate exactly with the physio- logical response induced by the plant hormone. An alternative approach for studying the molecular mechanisms involved in hormone-related responses is to study enzymes and other gene products that appear in response to hormone application. The effect of receptor mole- ~ ~ ~ & ~ ~ 8~

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72 gibberellins on the synthesis of the starch-hydrolyzing enzyme alpha-amylase in the aleurone cells of germinating cereal grains is a classic example. Gibberellin regulates the expression of these hydrolytic genes in aleurone cells, as demonstrated by the increased levels of alpha-amylase messenger RNA s in response to the gibberellin. Similarly, the level of mRNAs coding for seed storage proteins in developing seeds has been shown to be regulated by abscisic acid. While it is difficult to determine whether the reg- ulation of gene expression is a primary or secondary response to the specific hormone, it should be possible to locate and clone the genes and determine how the hormone triggers the regulatory sequence. Photomorphogenesis Light serves an important regula- tory role in plant growth and development in addition to providing the energy source for photosynthesis. Photomorphogenesis, the light-regulated developmental changes of a plant, is primarily under the control of a pigment called phytochrome. Phytochrome regulates such diverse effects as internode elongation, leaf unfurling, flowering, seed germination, and chloroplast movement. This high-molecular-weight pigment consists of a tetrapyrrole chromophore attached to a specific protein. Phytochrome exists in two molecular configurations that are reversibly interconver ted by light. One config- uration, Pfr, is the active form, while the other, Pr' is inactive. Red light converts Pr to Pfr; far red light converts Pfr to Pr. Thus, changes in light quality (the amount of red versus far red light) serve as a reversible biological switch in plant development. Research on phytochrome has focused on the chemical and physical characteristics of phytochrome and on its location in the cell as well as changes in gene expres- sion regulated by phytochrome. Specific genes regulated by phytochrome have now been cloned. Several of these are for proteins involved in photosynthesis, such as the small subunit of Rubisco, and the chlorophyll a/b pro- teins. These phytochrome-regulated genes are useful in the study of transcriptional regulation of the individual genes and can be used to study the gene regulatory sequences that respond to phytochrome. Chemical approaches that further explore the molecular structure of the phytochrome protein, immunological

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73 approaches to localize phytochrome within the cell, and study of the regulation of gene expression by phytochrome are contributing to an understanding of photomorphogen- esis and the relationships that exist between phyto- chrome and the phenomena it regulates. The recent obser- vation that phytochrome controls the transcription of its own gene will have a profound effect on our understanding of photomorphogenesis. Cell Culture and Plant Regeneration Two classes of plant hormones, the auxins and the cytokinins, must be added to culture media to support plant cell proliferation in vitro. While it is rela- tively easy to fulfill the requirements for meristematic plant cells to continue unorganized cell proliferation in tissue culture, it is far more difficult to obtain organ- ized growth and regeneration of plants. Only certain genotypes of a species will readily regenerate from tissue culture. The factors necessary for regeneration of intact plants from tissue culture remain largely unknown. It appears, however, that plant hormones are involved, because the relative concentrations of auxin and cytokinin added to media can promote or inhibit regeneration. The ability to regenerate plants from cell cultures at will is important to progress with gene transfer in plants. In a useful gene transfer system, DNA is intro- duced into a cell of the species of interest, and that cell is regenerated into a functioning plant that has been altered only by the introduced DNA. Plant organ and tissue culture is a well-established technology that originated in the early part of the twentieth century. In certain horticultural species, use of tissue culture is a small but important industry. Progress in manipulating cultures of some major food crops, including the cereals and legumes, to achieve plant regeneration has been much slower than with other crops such as potato, tomato, and tobacco. His major deficiency in the fundamental knowledge of plant devel- opment will become an even greater constraint to research in the future unless it is closed by a major commitment to the study of plant regeneration from tissue culture. Understanding the role of plant hormones in organogenesis and growth is an important aspect of this research.

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74 Research Status New and sophisticated techniques, including instru- mentation for high-resolution chemical analyses, monoclonal antibodies, and methods for identifying, cloning, and sequencing genes are rapidly advancing the understanding of the regulation of plant growth and development. There is a wealth of descriptive infor- mation on the roles of plant hormones and the photomorphogenic pigment phytochrome as regulators in coordinating the development of form and function in plants. Increasing evidence points to phytochrome and plant hormones as major factors in gene expression. As the molecular understanding of gene expression in plants increases, so will the opportunities for identifying the mechanisms of action that plant hormones and phytochrome use to regulate gene expression. Alternatively, regu- latory sequences of genes that respond to a plant hormone or phytochrome can be used as powerful tools in genetic engineering. 'The original discovery of phytochrome and much of the outstanding early basic research conducted on this photo- morphogenic pigment was accomplished by ARS scientists. The ARS should strive to reestablish its leadership role in basic research on plant growth and development. The focus of future research efforts within the ARS should include: Biosynthesis and degradation of plant hormones and phytochrome, with an emphasis on the regulation of genes coding for enzymes that synthesize or inactivate these substances; A molecular understanding of the role of phytochrome and plant hormones in regulating gene expression, particularly on their effects on the regulatory sequences of genes; and o The role of regulatory substances in major yield- controlling processes such as flowering, fertilization, germination, and senescence. Physicochemical Stress Physicochemical stresses such as drought, cold, heat, salt, and toxic ions cause extensive crop losses in the United States and throughout the world. These stresses are the main factors limiting expansion of food, feed,

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75 and fiber production. They are the basis for unrealized production potential. This is indicated by the fact that the average yield for eight major U.S. crops including corn, wheat, soybeans, sorghum, oats, barley, potatoes, and sugar beets is estimated to be only some 20 percent of the record yield for the same crops. Of the unrealized 80 percent of the potential yield, physi- cochemical stress accounts for about 70 percent with the remaining 10 percent attributable to insects and diseases.] The effects of physicochemical stress may be dramatic, killing or severely injuring whole crops. Factors causing such dramatic effects include extremes of temper- atures and severe drought, such as occurred in the midwestern United States in 1983 when the average bushel per acre yield for corn in Illinois dropped 40 percent compared to the average yield for the state in the previous year. Less apparent are stress conditions that cause no visible injury but still retard plant growth and reduce crop yield. Factors causing these more subtle effects are limited water supply, unfavorable tempera- tures, saline soils, and the presence in the soil of toxic ions such as aluminum. Increasing the tolerance of major crops to physicochemical stress could produce enormous benefits by increasing or stabilizing produc- tivity with little additional cost to the farmer. The major obstacle to increasing the tolerance of crop plants to physical and chemical stresses is the lack of fundamental knowledge of the basic mechanisms of stress injury and stress tolerance. Past breeding programs for increased stress tolerance have used a trial-and-error approach based on the ability of a genotype to survive a particular physicochemical stress. Determining only survival, however, gives little indication of the specific stress effects that contribute to the dramatic drop in productivity potential noted previously. A dearth of knowledge of specific mechanisms that confer increased stress tolerance has precluded combination of compatible traits into desirable stress-resistant genotypes. In addition, conventional breeding methods allow gene transfer between closely related plants only, which restricts the available gene pool. Recent advances in plant molecular genetics, however, have opened up the 1J- S. Boyer, 1982. Plant productivity and environment. Science 281:443-448.

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76 possibility of transferring specific traits between dif- ferent, genetically incompatible species. But progress in conventional breeding and genetic engineering ap- proaches will remain extremely limited until researchers have a better understanding of the effects of specific stress factors on plant growth and the effects of the genetic traits that mitigate the impact of stress factors Plant Responses to Stress Factors The effects of drought, salinity, heat, and cold on plants are usually multiple and often interrelated. Studies of the responses of plants to physicochemical stresses show that many biophysical and biochemical steps in metabolism can be affected. From this complex of multiple responses, it is often difficult to identify the primary site of damage. Thus, many response factors must be studied to determine the threshold of damage for dif- ferent responses and to understand the relationships that might exist between the primary effect and the cascade of processes that are, in turn, affected. Drought Water stress reduces or arrests plant growth because of a variety of effects. The carbon fixation steps of photosynthesis stop because carbon dioxide exchange into the leaf is blocked as the stomata! pores close to halt further water loss from the plant via transpiration. Heat energy from solar radiation is no longer effectively dissipated through the evaporation of water, and leaf temperatures may rise to damaging levels. Solar energy, absorbed by photosynthetic pigments, is no longer channeled to carbon fixation, and the photosyn- thetic system can be inactivated as energy is diverted to harmful reactions. Without sufficient turgor pressure the cells at the growing tips of plants cannot expand and elongate. Growth is also arrested because the products of photosynthesis needed for cell wall and protein biosynthesis are inireduced supply. Plant growth substances involved in regulating the opening and closing of stomates are also likely mediators of plant growth changes brought about by water stress. me number of possible effects in the cascade of responses to drought will depend on the severity and duration of the water stress. Some responses, such as stomata! closure, are quickly reversed when water is

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77 added. The kinds and degree of damage and the rates of repair or replacement are unknown. Also, drought may trigger long-term developmental changes in morphology and growth pattern that limit flowering, pollination, and seed development, which can greatly reduce crop yield. Salinity The osmotic properties of high concentrations of salt ions in soil water produce the same effects as those resulting from drought. In addition to creating this state of so-called physiological drought, the excess of salt ions can cause ionic imbalances across plant membranes and in the cytoplasm that lead to impairment of metabolism. Low Temperature Chilling temperatures lower the rate of enzyme activity and retard plant metabolism, including the processes of photosynthesis. Low temperature can also cause phase transitions that alter the molecular configuration of lipid components in membranes. This can result in leakage of ions and other solutes and impairment of water uptake. Such phase transitions may adversely affect the integrity and function of other important cell membranes such as the vacuolar membrane, or tonoplast, and mitochondrial and chloroplast membranes. Freezing temperatures just below O C disrupt membranes, especially the external plasma membrane. me formation of ice crystals causes - mechanical injury to cells and produces severe water stress and excessive solute concentration. High Temperature As temperatures in a plant are raised, water loss increases, thereby causing and exacerbating the effects of water stress during drought. Excess heat energy can cause metabolic imbalance by denaturing enzymes. Photosynthesis and other key processes in chloroplasts are reduced partly through loss of the integrity of chloroplast membranes. For example, increased temperature can result in increased fluidity of chloroplast membrane lipids. This may be responsible for decreases in the activity of the photosynthetic photosystems organized on those membranes within the chloroplast . Stress-Tolerance Mechanisms Through genetic change and natural selection, many plants have been able to adapt their physiology and

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78 morphology to tolerate climatic and environmental extremes. Mangrove trees grow in sea water, and certain other wild species can tolerate highly saline soils. Cacti and other desert plants survive wide temperature shifts between night and day as well as periods of prolonged drought. Some plants have adapted to the frequent freezing and thawing conditions of the tundra. Others manage to tolerate otherwise toxic levels of ions found in mine spoils and in serpentine or acid soils. Many plants survive and grow in poor soils with limited nutrients. Certain plants also exhibit tolerance to atmospheric pollutants such as ozone and sulfur dioxide. Most of the plants that have adapted to survive in truly extreme conditions are wild and not considered to be important to U.S. agriculture. Such wild plants, native to contrasting environments that are extreme in exhibiting one or a combination of stress factors, how- ever, can provide invaluable experimental material for the identification of stress-tolerant mechanisms and genetic manipulation. A comparison of physiological responses to stress in both stress-sensitive and stress-tolerant species is a valuable experimental approach. For example, tolerance of water stress by the photosynthetic system may in part depend on the ability of the plant to minimize the accu- mulation of excess excitation energy during periods of high irradiance. Plant-water relationships might also affect repair processes that may be operating both during and following exposure of the leaves to high irradiance levels. The plant's ability to maintain an adequate rate of repair, even during periods of low water potentials, may be an important stress-tolerance mechanism. Tolerance to salinity stress may depend on the ability to accommodate osmotic changes by concentrating ions and other solutes in leaves, roots, and specialized cells. Small molecules such as praline, glycine-betaine, and polyols accumulate in some species when they are subjected to water or salinity stress. In addition to their role in osmotic adjustment, these small molecules are possible factors in stabilizing supramolecular complexes in the cytoplasm during water, salinity, and temperature stress. Research Status Research programs on physicochemical stress must be considered long range. Because the potential impact on

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79 agriculture is enormous, this research should receive high priority within the ARS. Attention must be focused toward research approaches that characterize basic mechanisms used by plants in responding and adapting to the stresses of drought, excessive solar radiation, low and high temperatures, salinity, and toxic ions. Research approaches should also be designed to determine interactive relationships of the effects of major stress factors. The work must be conducted at varying levels of organizational complexity to yield an understanding of the function at the whole-plant level. Research must include major plant processes such as photosynthesis, nitrogen metabolism, protein synthesis, and the transport of water, ions, and other solutes that are either excluded from or concentrated in intracellular compart- ments such as the vacuole and other organelles. Comparative studies on plants that exhibit marked differences in their tolerance to a given stress factor provide a powerful approach toward uncovering the basic mechanisms of tolerance to physicochemical stresses. It is important, therefore, that the investigator be free to choose experimental plants best suited for the problem under investigation. The three major areas of research that should be emphasized in ARS programs on physical and chemical stress are: (1) the primary sites of damage to the plant caused by a specific stress factor; (2) the mechanisms-- morphological, physiological, biophysical, and biochemical--employed by stress-resistant plants to avoid and tolerate stress; and (3) the genetic bases of these tolerance mechanisms. More specifically, the ARS must intensify research efforts in the following areas: Mechanisms of water and solute transport, espe- cially into and within the roots, and the design of innovative approaches for detecting and measuring changes in the metabolism and membrane permeability of roots; The role of small molecules such as praline, glycine-betaine, and polyols, not only in osmotic adjustment but also in stabilizing molecular complexes during stress-induced dehydration; The role of excessive light as a destructive agent when photosynthesis is limited under conditions of water, salinity, and temperature stress;

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80 Identification of genes and gene products associated with stress tolerance in stress-adapted genotypes and species; and O Aspects of membrane properties such as changes in the biosynthesis of major membrane constituents; temperature-related changes in lipid fluidity and membrane protein stability that affect the functional integrity of the chloroplast, mitochondrial, vacuolar, and plasma membranes; and related aspects including dehydration-induced phase transitions, freeze-induced electrical perturbations, and changes in thermomechanical properties.