September 1999: (ll)S13-S18
`
`Agricultural Approaches to Improving Phytonutrient Content
`in Plants: An Overview
`Leon V. Kochian, Ph.D., and David F. Garvin, Ph.D.
`
`Recent advances in plant molecular biology, func-
`tional genomics, and biochemistry have opened
`up a number of new avenues of research that will
`enable plant biologists to characterize, increase
`and modify plant content of a wide range of es-
`sential minerals and vitamins, as well as a number
`of secondary plant compounds that appear,to play
`a role in improving human health and nutrition. In
`this review, several examples of exciting new re-
`search applying plant genomic and molecular ge-
`netic approaches to the improvement of phyto-
`nutrient content and composition in plants are
`presented. Research focusing on the elucidation
`of many of these complex biosynthetic and trans-
`port pathways in plants will require considerable
`resources in terms of funding, time, and person-
`nel. As plant biologists move into interdisciplinary
`collaborations with nutritionists and food scientists,
`attention must be paid to a more complete identi-
`fication and characterization of specific bioactive
`phytonutrients. Also, a more detailed assessment
`of the health-promoting properties of these com-
`pounds is needed, particularly for many of the
`secondary plant compounds for which clear epi-
`demiologic and clinical data are still lacking. Fi-
`nally, in order for significant progress to be made
`in modifying the nutrient composition of crops, a
`major investment must be made by funding agen-
`cies.
`
`Introduction
`There is considerable current interest in the role that plants
`and more specifically, plant-based foods, play in human
`nutrition and health. Recent shifts in dietary guidelines
`
`Dr. Kochian is a Plant Physiologist and Dr. Garvin
`is a Plant Molecular Biologist at U.S. Plant, Soil and
`Nutrition Laboratory, USDA-ARS, Cornell University,
`Ithaca, NY 14853, USA.
`
`Nutrition Reviews”, Vol. 57, No. 9
`
`indicate that the typical American diet should contain a
`larger proportion of plant-based foods, i.e. vegetables,
`fruits, nuts, legumes, and grains.[ While the preponder-
`ance of attention is currently focused on the secondary
`products in plant foods, attention must be focused as well
`on proteins; lipids; carbohydrates; and vitamins and min-
`erals, particularly iron, zinc, and calcium, nurients in which
`certain population groups in both the United States and
`worldwide are commonly deficient.
`Plants synthesize and accumulate an astonishingly
`diverse array of organic secondary products, and data are
`beginning to accumulate indicating that a number of these
`organic cofipounds have health-promoting properties.
`Much of the current interest in phytonutrients has fo-
`cused on these secondary plant products. At this time,
`little is known about the biosynthesis and accumulation
`of many of these compounds in plant tissues, and the
`specific bioactive forms within broad categories of sec-
`ondary compounds have yet to be identified. However,
`recent technologic advances in plant genomics and bio-
`technology as well as the focused application of tradi-
`tional and molecular-assisted plant breeding practices now
`provide plant biologists with an array of powerful tools to
`elucidate the mechanisms and regulation of the complex
`biosynthetic pathways for many of these compounds, and
`to begin to design strategies to alter the amount, distribu-
`tion, and forms of those phytochemicals with health-pro-
`moting properties.
`
`What Do We Mean by the Term Phytonutrient?
`A literal definition for the term phytonutrient is “a nutrient
`derived from plants.” Thus, we would be talking about a
`plant-based substance essential for proper metabolism and
`function in humans. This would include proteins, lipids,
`and carbohydrates as well as essential minerals and vita-
`mins. However, the term is generally used much more
`loosely to include any organic or inorganic compound in
`plant foods that has a positive impact on human health or
`nutrition. This would include the true essential nutrients
`and vitamins such as vitamin E, pro-vitamin A carotenoids,
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`iron, zinc, calcium, and selenium for which there is solid
`evidence as to their importance in human health and nutri-
`tion from research in animal model systems as well as from
`clinical and epidemiologic ~tudies.~,~ Also included under
`this umbrella term would be the broad classes of second-
`ary compounds such as phytoestrogens, isoflavonoids,
`anthocyanins, polyphenols, and glucosinolates that can
`improve human nutrition and health by mechanisms that
`are not, or are only partially, defined. A better term for
`these secondary plant products might be “accessory health
`factors” (GF Combs, personal communication). This term
`indicates that although not essential for humans, these
`compounds could play an important role in improving
`human health by reducing the impact of certain chronic
`diseases (e.g., heart disease, cancer) and the effects of
`aging.
`
`Phytocompounds Within Plant Systems
`The essential and non-essential phytonutrients are ac-
`quired, synthesized, translocated, and stored in specific
`plant tissues by a variety of processes and pathways.
`The essential minerals enter the plant primarily by absorp-
`tion from the soil solution into plant roots. Mineral nutri-
`ents such as potassium, magnesium, calcium, iron, zinc,
`manganese, copper, and nickel are absorbed as metal ions,
`whereas nitrogen (N), phosphorus, and sulfur (S) are ab-
`sorbed as oxyanions that are subsequently modified by
`reductive assimilatory pathways (for N and S) and incor-
`porated into proteins, lipids and carbohydrates.5,6 Vita-
`mins such as E, C, folate, and the pro-vitamin A caro-
`tenoids-as well as numerous secondary plant com-
`pounds-are
`synthesized via a number of different pri-
`mary and secondary biochemical pathways. There are ap-
`proximately 100,000 secondary compounds synthesized
`by different plant species that do not appear to play a
`direct role in plant growth and development.’ These com-
`pounds play roles in a diverse range of processes, includ-
`ing defense against herbivore and pathogen attack, pro-
`tection against abiotic environmental stresses such as UV
`damage, and as attractants for pollinators.’ There are four
`general categories of secondary compounds:
`
`. Phenolics, which are aromatic compounds that include
`
`the flavonoids, the largest group of phenolics. The fla-
`vonoids, which are synthesized via the shikimic acid
`pathway, can be sub-classified into different groups
`that include potentially important phytonuitrients in-
`cluding isoflavonoids, flavones, flavonols, anthocya-
`nins, tannins, and lignin.
`Terpenes, which include the carotenoids, steroids, and
`limonoids, are lipids that are made via the mevalonic
`acid pathway.
`Alkaloids, a diverse group of N-containing secondary
`compounds biosynthesized primarily from amino acids
`which include toxic and psychoactive plant compounds
`
`such as nicotine, caffeine, cocaine, morphine, strych-
`nine, and atropine.
`Non-alkaloid nitrogenous compounds, which include
`the glucosinolates and glycosides. The biosynthetic
`pathways for these different secondary compounds are
`usually quite complex, with numerous steps, branch
`points, and many closely related compounds in any
`particular group of compounds.
`
`Experimental Framework for Engineering Plant
`Foods to Improve Human Health
`Plant researchers moving into this field must be highly
`selective in choosing which plant compounds they ma-
`nipulate, because of both the complexities of plant biol-
`ogy and the obvious demand for careful and methodologic
`alterations of plant nutrition value. However, at present‘,
`the roles and benefits for many of the secondary com-
`pounds of interest in plants are still poorly defined. In
`many of the clinical and epidemiologic studies, particu-
`larly for organic phytonutrients, a broad category repre-
`senting a mixture of many related plant-based compounds
`has been shown to have potential for improving human
`health. Moreover, the specific bioactive species has often
`not been identified. Additionally, some of the relevant bio-
`synthetic pathways have not been fully characterized at
`the biochemical and molecular levels, which limits the im-
`mediate pos3ibilities for altering levels of intermediates of
`these pathways. Finally, many secondary compounds are
`found in specific plant species or plant families, some of
`which are not readily amenable to manipulation by mo-
`lecular approaches.
`An example of successful cooperation between plant
`biologists and human nutritionists comes from the excit-
`ing recent findings from the Jean Mayer USDA-ARS Hu-
`man Nutrition Research Center on Aging at Tufts Univer-
`sity. Researchers there have developed an assay to rap-
`idly quantify antioxidant activity in different foods, and
`have found that highly colored fruits such as blueberries
`and strawberries had the highest antioxidant activity of
`any plant foods.g Subsequent work strongly suggests that
`the antioxidant compounds in these fruits are anthocya-
`nins, which are pigmented flavonoids belonging to the
`largest class of phenolic compounds in plants. Very re-
`cently, these same researchers showed that in long-term
`feeding studies of rats, supplementation of diets with ex-
`tracts from antioxidant-rich fruits and vegetables appeared
`to retard age-associated declines in the central nervous
`systems as well as cognitive behavioral deficits.’O These
`findings clearly point out a potentially productive and
`important avenue of research for plant biologists. How-
`ever, the specific antioxidant compound has not yet been
`identified, and it could be one or more members of a large
`number of anthocyanins produced by a pathway that is
`presently not clearly delineated. Thus, rapid progress in
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`terms of increasing the content of specific anthocyanins
`in order to enhance the anti-aging properties of a particu-
`lar plant food is not immediately forthcoming, simply be-
`cause plant biologists cannot yet target a specific com-
`pound.
`In contrast to the difficulties encountered by plant
`biologists in modifying anthocyanins in fruits, a success-
`ful genomics-based approach was recently used to meta-
`bolically engineer the vitamin E (a-tocopherol) biosyn-
`thetic pathway in plant seeds." This was considered mean-
`ingful to human health because of the tendency for many
`Americans to consume less vitamin E than is recom-
`mended. These research findings hold great promise for
`increasing the vitamin E content of agriculturally impor-
`tant oil crops and are a good example of the potential that
`exists for manipulating phytonutrients in plants.
`When research priorities are developed for determin-
`ing which phytonutrients should be targeted, the essen-
`tial minerals and vitamins listed above should be given
`careful consideration. For example, only 21% ofthe diets
`of teenage girls in the U.S. meet the Recommeded Di-
`etary Allowances (RDAs) for iron, 16% meet the RDAs for
`calcium, and many do not meet RDAs for magnesium, zinc,
`vitaminA and vitamin E.I Iron is an excellent example of an
`essential phytonutrient that could have a great positive
`impact on human health if more iron was available from
`plant foods. There are significant segments of the US.
`population that are iron deficient. Up to 20% of all pre-
`menopausal women in the U.S. have low hemoglobin lev-
`els, and this increases to 40% for pregnant, low-income
`African-American women.' This has important implica-
`tions for the United States, as recent research indicates
`that iron deficiency during pregnancy may retard the cog-
`nitive development in the young child of an iron-deficient
`mother. This problem is even more severe in developing
`countries as iron deficiency is the most prevalent mineral
`nutrient deficiency in the world, affecting more than 2
`billion people worldwide? The global extent and severity
`of this problem indicates that research aimed at enhanc-
`ing the iron fortification of staple foods such as cereal
`grains should have a high priority both for developing
`nations and for developed countries such as the United
`States.
`It is clear that if phytonutrient research is to develop
`and succeed, better interdisciplinary linkages are needed
`between experts in plant biology, human nutrition, and
`food science. Plant biologists need to be continuously
`informed about new evidence on the biologic roles of plant
`foods in the human system. Similarly, nutritionists need to
`know more about the possibilities for biochemical and
`molecular manipulation of the phytonutrient content of
`plants, as well as about the obstacles. We can envision an
`iterative process in which nutritionists identify specific
`phytonutrients to be targeted by plant biologists, who
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`Nutrition Reviews", Vol. 57, No. 9
`
`alter the content and/or form of the compound, and the
`modified plant food is then tested with animal or in vitro
`models, or in clinical trials. The findings from these trials
`would feed back to the plant biologists for additional modi-
`fication in plants. Several ARS laboratories already exist
`that are models for such interdisciplinary links. For 60
`years, the U.S. Plant, Soil and Nutrition Laboratory at
`Cornell University has brought together soils scientists,
`plant biologists and nutritionists to focus on a lab charter
`to investigate plant and soil factors that have an impact
`on human nutrition and health. More recently, the USDA-
`ARS Children's Human Nutrition Center in Houston has
`added several experts in plant biology to their laboratory
`staff of medical doctors, nutrition scientists, dietitians,
`and chemists to investigate problems in maternal, infant,
`and child health and nutrition.
`
`Agricultural Approaches for Improving
`Phytonutrients in Plants
`The current and ongoing revolution in biology regarding
`structural and functional genomics has provided plant
`biologists with a powerful new array of approaches and
`tools that should allow them not only to elucidate the
`complex transport and biosynthetic pathways for specific
`phytonutrients, but also to modify the amount, form and
`location of these compounds in specific plant tissues and
` organ^.'^.^^ Additionally, the focusing of plant breeding
`approaches on the identification and exploitation of ge-
`netic variation for phytonutrient-related traits is already
`yielding progress.
`
`Plant Breeding Approaches
`As plant breeders and plant molecular geneticists begin
`to direct research toward phytonutrients in crop plants,
`they are finding significant genetic variation in the con-
`tent of specific phytonutrients. This variation is currently
`being exploited to enhance phytonutrient levels through
`both traditional and marker-assisted plant breeding.'"I6 A
`good example of this is the breeding effort that has yielded
`carrot germplasm with increased levels of provitamin A
`carotenoids.I6J7 This approach allows plant breeders to
`implement phytonutrient modification (for those com-
`pounds for which significant variation exists) and is cov-
`ered in more detail in several of the other reviews in this
`publicati~n.*~J~
`Another approach used by plant breeders is the use
`of induced mutagenesis to create new variability for a trait
`when existing natural variation is not adequate. Because
`of its inherent inefficiency, mutation breeding has histori-
`cally played a minor role in crop improvement. However,
`now that we are armed with a sophisticated understand-
`ing of plant biochemistry and with methods and protocols
`for rapid analysis of chemical composition, it is sensible
`to revisit the use of induced mutagenesis as an approach
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`to altering phytonutrient composition in crops. One major
`factor that will aid in the successful implementation of
`mutagenesis to favorably alter phytonutrient composition
`is that in many instances, target phytonutrients may be
`non-essential to plant productivity because they are prod-
`ucts of secondary metabolism. As such, it should be pos-
`sible to induce dramatic shifts in their abundance without
`detrimentally affecting crop performance or quality.
`One group of compounds for which mutation breed-
`ing may find an application is the flavonoids. Following
`the first committed step of flavonoid synthesis (the con-
`densation ofmalonyl-CoA and coumaryl-CoA to form chal-
`cone), the different classes of flavonoids-including an-
`thocyanins, flavones, and proanthocyanidins-are
`syn-
`thesized. As discussed previously, it has been suggested
`that some of these flavonoids have health-promoting prop-
`erties. In contrast, other flavonoids have been found to
`have a deleterious impact on nutrition, at least in animals.
`For example, high proanthocyanidin content is associated
`with poor feed quality in sorghum.
`A useful example of how mutagenesis can be ffsed to
`favorably alter flavonoid composition comes from research
`conducted more than 20 years ago at the Carlsberg Labo-
`ratories in Denmark. Mutagenesis with compounds such
`as EMS has been used to identify barley mutants with
`altered flavonoid composition. These mutants represent
`at least 29 complementation groups (and thus, 29 distinct
`loci).2o From a basic standpoint, the mutants have been
`invaluable in deciphering the biosynthetic interrelation-
`ships of the different flavonoids in barley. This has been a
`difficult issue to address because some steps in flavonoid
`biosynthesis are reversible and because certain flavonoids
`may be synthesized from more than one point in the path-
`way. Biochemical analyses of the different mutants made
`it possible to determine the different steps in flavonoid
`biosynthesis that are genetically controlled, and the se-
`quence in which they occur.2o This was accomplished by
`examining the flavonoid composition of the different mu-
`tants for the preferential accumulation of certain com-
`pounds and the loss of others.
`There is also practical significance to some of the
`barley mutants that have an altered flavonoid composi-
`tion. First, those mutations that specifically eliminate
`proanthocyanidin accumulation are of great interest to
`the beer brewing industry. Barley proanthocyanidins
`strongly react with proteins in the beer and form a precipi-
`tate that has to be removed by chemical methods. Brew-
`ing beer with grain from barley mutants lacking
`proanthocyanidins sidesteps the formation of these pre-
`cipitates, thus providing a simple means of eliminating
`certain chemical steps from the brewing process.2i Sec-
`ond, one of the classes of barley flavonoid mutants lacks
`dihydroflavonol reductase activity, and thus accumulates
`the substrate for this enzyme, dihydroquercetin. Height-
`
`ened accumulation of this compound has been found to
`inhibit infection by certain pathogenic fungi?* While these
`two examples do not directly relate to human health, they
`nonetheless demonstrate that the spectrum of flavonoids
`in a plant can be specifically altered by mutagenesis breed-
`ing. As such, it should be possible to apply this approach
`in crops to enhance levels of flavonoids or other com-
`pounds that act as phytonutrients for humans, or to elimi-
`nate compounds deleterious to human health.
`
`Bioengineering of Selected Biosynthetic
`Path ways
`The tools becoming available from plant genomics and for
`tissue specific gene transformation of many plant species
`provide new avenues for plant biologists to dissect out
`the complex biosynthetic pathways for the synthesis of
`many phytonutrients. They can also use these tools to
`selectively modify specific intermediates in these path-
`ways. A good example of this is the recent success by
`Shintani and DellaPenna in using a genomics-based ap-
`proach to clone a key enzyme in the synthesis of vitamin
`E in plants, and then to overexpress this enzyme in seeds
`in order to elevate seed vitamin E content." Vitamin E, or
`a-tocopherol, is a lipid-soluble phenolic compound syn-
`thesized only in higher plants and some photosynthetic
`bacteria. Thers are four tocopherols made in plants-a-,
`p-, y-, and 6-tocopherol-with
`a-tocopherol having the
`highest vitamin E activity and thus being the most impor-
`tant for human health. Much of the vitamin E in the human
`diet comes from vegetable oils, derived from the seeds of
`soybean, maize, cottonseed, and the rape plant. In all of
`the major oilseed crops, a-tocopherol is found in rela-
`tively low levels, while its immediate biosynthetic precur-
`sor, y-tocopherol is found at much higher concentrations
`(10- to 20-fold higher). These findings suggest that the
`final step in a-tocopherol synthesis, which is catalyzed
`by y-tocopherol methyltransferase (y-TMT), is limiting in
`the seeds of the major oil crops.
`The a-tocopherol biosynthetic pathway has been de-
`scribed and one of the enzymes of the pathway, p-hydroxy-
`phenylpyruvate dioxygenase (HPPDase), has recently
`
`been cloned from Arabidopsis and ~ a r r o t . ~ ~ , * ~ However, it
`has been difficult to clone the genes encoding the other
`pathway enzymes, as they are all membrane-bound. There-
`fore, these researchers used a genomics-based approach
`to clone the y-TMT gene by using sequence similarities
`between Arabidosis and the photosynthetic bacteria
`Synechocystis PCC6803, for which the entire genome has
`been recently sequenced. The gene encoding the
`Arabidopsis HPPDase was used to search the synecho-
`cystis genome database, and in this way the homologous
`synechocystis HPPDase gene was cloned and found to
`lie within a predicted 10 gene operon. As bacterial biosyn-
`thetic genes are often organized in operons, the authors
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`speculated that the gene encoding y-TMT might be nearby
`to the cloned BPPDase gene. A candidate gene that pre-
`dicted a protein with high amino acid sequence similarity
`to plant methyl transferases was found nearby and cloned.
`A null mutant for this gene was created, and biochemical
`analysis of tocopherol synthesis in the mutant as well as
`enzyme activity studies with the recombinant protein
`showed that indeed, the gene encoded y-tocopherol
`methyltransferase.
`This allowed the researchers to use the synechocystis
`y-TMT protein sequence to search the Arabidopsis ex-
`pressed sequence tag (EST) data base, which enabled them
`to clone the homologous gene from Arabidopsis. Subse-
`quently, the Arabidopsis y-TMT was overexpressed in
`transgenic Arabidosis plants behind a seed specific pro-
`moter and the resulting plants showed a dramatic shift in
`their tocopherol accumulation. In seeds of wildtype
`Arabidopsis, as in the oilseed crops, >95% of the total
`tocopherol exists as y-tocopherol. In the transgenic plants,
`there was a greater than 80-fold increase in seed a-toco-
`pherol content, and >95% of the total tocopherol was a-
`tocopherol. If, as expected, y-TMT activity in the com-
`mercially important oilseed crops is also limiting, this ap-
`proach should significantly increase seed a-tocopherol
`levels and will most likely enhance the nutrition value of
`these crops as sources of vitamin E. This is an elegant
`example of the potential that exists to modify phytonutrient
`content and composition in plant foods.
`
`Expression of Phytonutrient Genes in Plants
`It may be possible to use plant biotechnology methodolo-
`gies to enhance phytonutrient content in specific plant
`tissues without having a complete or detailed understand-
`ing of the complex transport or biosynthetic pathways for
`the phytonutrients. For example, there is considerable in-
`terest in elevating the levels of iron in staple foods such
`as the seeds of rice and maize, in order to begin to address
`the widespread iron deficiency in developing countries.2
`Normally, plant seeds are poor sources of iron. The path-
`way for iron from the soil to the developing seed is quite
`complex and poorly understood, involving a number of
`cell types and both membrane and long-distance trans-
`port systems. Genes encoding the plant iron storage pro-
`tein (phytoferritin) have been cloned,25 and a number of
`labs are actively pursuing the overexpression of the
`phytoferritin genes in seeds of cereal crops with the hope
`of providing a biotechnological solution to iron fortifica-
`tion of staple plant foods.
`One recent example of this biotechnology approach,
`which holds promise for increasing the pro-vitamin A caro-
`tenoid content of staple foods such as rice, comes from
`the recent work of Burkhardt and colleagues.26 Rice en-
`dosperm contains neither p-carotene nor any of the imme-
`diate precursors for the biosynthesis of p-carotene. In
`this study, however, biochemical analysis of immature rice
`
`Nutrition Reviews@, Vol. 57, No. 9
`
`endosperm revealed the presence of geranyl diphosphate,
`which is a 20-carbon isoprenoid precursor necessary for
`carotenoid synthesis. Therefore, in a effort to improve the
`nutrient quality of rice, the gene encoding the enzyme
`phytoene synthase from daffodil was expressed in rice
`using an endosperin specific promoter. Phytoene synthase
`is the first of 4 specific enzymes needed for the synthesis
`of p-carotene in plants. The researchers were successful
`in expressing the active phytoene synthase enzyme and
`in synthesizing phytoene in rice endosperm. This research
`demonstrated that it is possible to engineer a critical step
`for carotenoid synthesis in a non-photosynthetic tissue
`that normally is devoid of carotenoids. These findings
`have opened the way for transforming rice endosperm
`with the remaining three enzymes needed for p-carotene
`synthesis, which may have a large impact in lessening’
`vitamin A deficiency in developing countries that depend
`on rice as a staple food.
`
`1.
`
`2.
`
`3.
`
`4.
`
`5.
`
`6.
`
`7.
`
`8.
`
`9.
`
`10.
`
`11.
`
`12.
`
`13.
`
`Welch RM. Fashioning healthful agricultural sys-
`tems. In: Combs GF, Welch RM, eds. Creating a
`Healthful Food System: Linking Agriculture to Hu-
`man Needs, lthaca NY. Cornell University Press;
`199817-14
`Combs GF, Welch RM, Duxbury JM, Uphoff NT.
`Food-Based Approaches to Preventing Micronutri-
`ent Malnutrition: An International Agenda. lthaca
`NY. Cocpell University Press; 1996
`Yates AA. Overview of key nutrients: energy and
`macronutrient aspects. Nutr Rev 1998, 56: S29-
`s33
`Lachance PA. Overview of key nutrients: micronu-
`trient aspects. Nutr Rev 1998, 56: S34-S39
`Marschner H. Mineral nutrition of higher plants, 2nd
`ed. San Diego CA; Academic Press: 1995
`Kochian LV. Mechanisms of micronutrient uptake
`and translocation in plants. In: Mortvedt JJ, Cox
`FR, Shuman LM, Welch RM, eds. Micronutrients in
`Agriculture, 2nd ed. Madison WI: Soil Science So-
`ciety of America; 1991 :229-96
`Conn E. The world of phytochemicals. In: Gustine
`DL, Flores HE, eds. Phytochemicals and health,
`Rockville MD. American Society of Plant Physiolo-
`gists; 1995:l-15
`Taiz L, Zeiger E. Plant Physiology. Redwood City
`CA: Benjamin Cummings Publishing Co., Inc;.
`1991 131 8-45
`Hong W, Cao G, Prior RL. Total antioxidant capac-
`ity of fruits. J Agric Food Chem 1996:44:701-5
`Joseph JA, Skukitt-Hale B, Denisova NA, Prior RL,
`Cao G, Martin A, Taglialatela G, Bickford PC. Long-
`term dietary strawberry, spinach or vitamin E supple-
`mentation retards the onset of age-related neuronal
`signal transduction and cognitive behavioral defi-
`cits. J Neuroscience 1998;18:8047-55
`Shintani D, DellaPenna D. Elevating the vitamin E
`content of plants through metabolic engineering.
`Science, 1998;282:2098-2100
`Saier MH Jr. Genome sequencing and informatics:
`new tools for biochemical discoveries. Plant Physiol
`1998;49: 151 -71
`Bouchez D, Hofte H. Functional genomics in plants.
`
`RIMFROST EXHIBIT 1018 page 0005
`
`S17
`
`

`

`14.
`
`15.
`
`16.
`
`17.
`
`18.
`
`19.
`
`20.
`
`21.
`
`Plant Physiol 1998:118:725-32
`Wang M, Goldman IL. Phenotypic variation in free
`folic acid content among F, hybrids and inbreds of
`red beet. J Amer SOC Hort Sci 1996;121:104-42
`Patil BS, Pike LM, Yo0 KS. Variation in the quercitin
`content in different colored onions. J Amer SOC Hort
`Sci 1 995; 1 20:909-13
`Simon PW, Wolff XV, Peterson CE, et al. High caro-
`tene mass carrot population. Hort Sci 1989;24:174-
`5
`Tigchelaar EC, Tomes ML. Caro-Rich tomato. Hort
`Sci 1974;9:82
`Farnham MW, Simon PW, Stommel JR. Improved
`phytonutrient content through plant genetic im-
`provement. Nutr Rev 1999;57(9):S19-S26
`Goldman IL, Kader AA, Heinz C. Influence of pro-
`duction, handling and storage on phytonutrient
`content of foods. Nutr Rev 1999;57(9):S47-S53
`Jende-Strid B. Genetic control of flavonoid biosyn-
`thesis in barley. Hereditas 1993; 1 1 9:187-204
`Von Wettstein D. Breeding of value added barley
`by mutation and protein engineering. In: Induced
`mutations and molecular techniques for crop im-
`provement. Vienna AU. International AtQmic En-
`
`22.
`
`23.
`
`24.
`
`25.
`
`26.
`
`ergy Agency 1995; 65-75
`Skadhauge B., Thomsen KK, Von Wettslein D. The
`role of the barley testa layer and its flwonoid con-
`tent in resistance to Fusarium. infections. Heredi-
`tas 1 997: 126: 147-60
`Garcia I, Rodgers M, Lenne C, Rolland A, Sailand
`A, Matringe M. Subcellular localization and purifi-
`cation of a p-hydroxyphenylpyruvate dioxygenase
`from cultured carrot cells and characterization of
`the corresponding cDNA. Biochem J 1997;325:
`761-9
`Norris SR, Shen X, DellaPenna D. Complementa-
`tion of the Arabidopsis pds l mutation with the gene
`encoding p-hydroxyphenylpyruvate dioxygenase.
`Plant Physiol 1998; 1 17: 131 7-23
`Deak M, Horvath GV, Davletova S, et al. Plants
`ectopically expressing the iron-binding protein fer-
`ritin, are tolerant to oxidative damage and pathQ-
`gens. Nature Biotech 1999; 17:192-196
`Burkhardt PK, Beyer P, Wuenn J, et al. Transgenic
`rice (Oryza sativa) endosperm expressing daffodil
`(Narcissus pseudonarcissus) phytoene synthase
`accumulates phytoene, a key intermediate of pro-
`vitamin A biosynthesis. Plant J 1997;11:1071-78
`
`S18
`
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`
`RIMFROST EXHIBIT 1018 page 0006
`
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