ScienceWeek

4. EXPERIMENTAL PROGRESS IN GM FOODS

ON RICE GENOMES

The following points are made by K. Livingstone and L.H. Rieseberg (Current Biology 2002 12:R470):

1) Although completion of the heavily anticipated human genome sequence project will provide information needed to combat inherited maladies, the recent completion of two sequences of the rice genome [1,2] may be a far greater gift to humanity. After all, as the Byzantine proverb states, "He who has bread has many problems, he who has no bread has only one problem". Because of the importance of rice and its status as a model for all grasses, these sequences will provide a basis for future genetic improvement of all the cereal grains, our most important food resource. Beyond the obvious agricultural benefit, these sequences may also provide unparalleled views of the processes operating on DNA sequences that change the function and organization of genes, leading to the formation of new species.

2) The two rice sequences are from subspecies that represent the major cultivated gene pools of rice, Oryza sativa L. ssp. indica and O. sativa ssp. japonica. The indica type is primarily grown in China, and the Beijing Genomics Institute (BGI) determined its sequence [1]. The japonica subspecies is preferred in Japan, and Syngenta AG's Torrey Mesa Research Institute (TMRI) determined its sequence [2]. These are both draft sequences, produced by randomly sequencing small genomic bits and relying on multiple, offset sequences to assemble the larger pieces.

3) From a functional standpoint, while each draft should contain nearly all the genes in rice, many of the sequences identified as genes are only predicted on the basis of different gene detection algorithms. It will take a long time to validate the expression of the putative genes biologically and take full advantage of these efforts. Structurally, both drafts cover the majority of the rice genome, but many of the intergenic regions are missing. Consequently, each draft resembles a puzzle with tens of thousands of pieces on the table, but only a few joined to start to form a picture of the twelve rice chromosomes.

4) Even in a draft state, however, these sequences provide enormous agricultural benefits. Rice is a crucial staple for much of the world's population, and rice is also the compact key to other grass genomes [3] . The main differences between rice and maize, wheat, barley, and so on are that, while the same genes are found in each species, they are in different arrangements, amid various amounts of species-specific "junk" DNA. The compactness of the rice genome, coupled with a known sequence, will make identification of important genes easier. In addition, the rice sequence provides a means for directing searches in other grasses to the genes in a particular chromosomal region. The TMRI group [2] has already demonstrated the power of a focused search approach to define candidates for a subset of agronomically important traits mapped in maize [4,5].

References (abridged):

1. Yu J., Hu S., Wang J., Wong G.K.-S., Li S., Liu B., Deng Y., Dai L., Zhou Y. and Zhang X. et al. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. Indica) Science, 296:79-92

2. Goff S.A., Ricke D., Lan T.H., Presting G., Wang R., Dunn M., Glazebrook J., Sessions A., Oeller P. and Varma H. et al. (2002) A draft sequence of the rice genome (Oryza sativa L. ssp. Japonica) Science, 296:92-100

3. Devos K.M. and Gale M.D. (2000) Genome relationships: the grass model in current research. Plant Cell, 12:637-646

4. The Arabidopsis Genome Initiative (2000) Analysis of the genome sequence of the flowering plant Arabidopsis thaliana Nature, 408:796-815

5. Brendel V., Kurtz S. and Walbot V. (2002) Comparative genomics of Arabidopsis and maize: prospects and limitations. Genome Biol, 3:1005

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GENETICALLY MODIFIED PLANTS FOR IMPROVED TRACE ELEMENT NUTRITION

The following points are made by B.Lonnerdal (J Nutr. 2003 May 133:1490S):

1) Deficiencies of iron and zinc are common worldwide. Various strategies have been used to combat these deficiencies including supplementation, food fortification and modification of food preparation and processing methods. A new possible strategy is to use biotechnology to improve trace element nutrition. Genetic engineering can be used in several ways; the most obvious is to increase the trace element content of staple foods such as cereals and legumes. This may be achieved by introduction of genes that code for trace element-binding proteins, overexpression of storage proteins already present, and/or increased expression of proteins that are responsible for trace element uptake into plants.

2) However, even very high levels of expression may not substantially increase the iron and zinc contents unless many atoms of trace elements are bound per protein molecule. Another possibility is to introduce a protein that specifically enhances trace element absorption even in the presence of naturally occurring inhibitors, thus improving bioavailability. Genetically modifying plants so that their contents of inhibitors of trace element absorption such as phytate are substantially reduced is another approach. Increasing the expression of compounds that enhance trace element absorption such as ascorbic acid is also a possibility, although this has received limited attention so far. Iron absorption may be increased by higher ascorbic or citric acid content but require overexpression of enzymes that are involved in the synthetic pathways. Finally, a combination of all of these approaches perhaps complemented with conventional breeding techniques may prove successful.

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GENE DISCOVERY AND PRODUCT DEVELOPMENT FOR GRAIN QUALITY TRAITS

The following points are made by B. Mazur et al (Science 1999 285:372):

The composition of oils, proteins, and carbohydrates in seeds of corn, soybean, and other crops has been modified to produce grains with enhanced value. Both plant breeding and molecular technologies have been used to produce plants carrying the desired traits. Genomics-based strategies for gene discovery, coupled with high-throughput transformation processes and miniaturized, automated analytical and functionality assays, have accelerated the identification of product candidates. Molecular marker-based breeding strategies have been used to accelerate the process of moving trait genes into high-yielding germplasm for commercialization. These products are being tested for applications in food, feed, and industrial markets.

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EXPLOITING THE FULL POTENTIAL OF DISEASE-RESISTANCE GENES FOR AGRICULTURAL USE

The following points are made by C.M. Rommens and G.M. Kishore (Curr Opin Biotechnol. 2000 11:120):

Effective and sustained control of fungal pathogens and nematodes is an important issue for all agricultural systems. Global losses caused by pathogens are estimated to be 12% of the potential crop production, despite the continued release of new resistant cultivars and pesticides. Furthermore, fungi are continually becoming resistant to existing resistance genes and fungicides, and a few of the pesticides are being withdrawn from the market for environmental reasons. In addition to reducing crop yield, fungal diseases often lower crop quality by producing toxins that affect humans and human health. Additional methods of disease control are therefore highly desirable. Breeding programs based on plant disease-resistance genes are being optimized by incorporating molecular marker techniques and biotechnology. These efforts can be expected to result in the first launches of new disease-resistant crops within the next five years.

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MOLECULAR STRATEGIES FOR GENE CONTAINMENT IN TRANSGENIC CROPS

The following points are made by H. Daniell (Nature Biotechnol. 2002 20:581):

1) The potential of genetically modified (GM) crops to transfer foreign genes through pollen to related plant species has been cited as an environmental concern. Until more is known concerning the environmental impact of novel genes on indigenous crops and weeds, practical and regulatory considerations will likely require the adoption of gene-containment approaches for future generations of GM crops.

2) Most molecular approaches with potential for controlling gene flow among crops and weeds have thus far focused on maternal inheritance, male sterility, and seed sterility. Several other containment strategies may also prove useful in restricting gene flow, including apomixis (vegetative propagation and asexual seed formation), cleistogamy (self-fertilization without opening of the flower), genome incompatibility, chemical induction/deletion of transgenes, fruit-specific excision of transgenes, and transgenic mitigation (transgenes that compromise fitness in the hybrid).

3) As yet, however, no strategy has proved broadly applicable to all crop species, and a combination of approaches may prove most effective for engineering the next generation of GM crops.

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ENGINEERING THE PROVITAMIN A (-CAROTENE) BIOSYNTHETIC PATHWAY INTO (CAROTENOID-FREE) RICE ENDOSPERM

The following points are made by X. Ye et (Science 2000 287:303):

1) Vitamin A deficiency causes symptoms ranging from night blindness to those of xerophthalmia and keratomalacia, leading to total blindness. In Southeast Asia, it is estimated that a quarter of a million children go blind each year because of this nutritional deficiency (1). Furthermore, vitamin A deficiency exacerbates afflictions such as diarrhea, respiratory diseases, and childhood diseases such as measles (2,3). It is estimated that 124 million children worldwide are deficient in vitamin A (4) and that improved nutrition could prevent 1 million to 2 million deaths annually among children (3). Oral delivery of vitamin A is problematic (5), mainly due to the lack of infrastructure, so alternatives are urgently required. Success might be found in supplementation of a major staple food, rice, with provitamin A. Because no rice cultivars produce this provitamin in the endosperm, recombinant technologies rather than conventional breeding are required.

2) Immature rice endosperm is capable of synthesizing the early intermediate geranylgeranyl diphosphate, which can be used to produce the uncolored carotene phytoene by expressing the enzyme phytoene synthase in rice endosperm. The synthesis of beta-carotene requires the complementation with three additional plant enzymes: phytoene desaturase and beta-carotene desaturase, each catalyzing the introduction of two double bonds, and lycopene cyclase, encoded by the /lcy/ gene. To reduce the transformation effort, a bacterial carotene desaturase, capable of introducing all four double bonds required, can be used.

3) In summary: Rice (Oryza sativa), a major staple food, is usually milled to remove the oil-rich aleurone layer that turns rancid upon storage, especially in tropical areas. The remaining edible part of rice grains, the endosperm, lacks several essential nutrients, such as provitamin A. Thus, predominant rice consumption promotes vitamin A deficiency, a serious public health problem in at least 26 countries, including highly populated areas of Asia, Africa, and Latin America. The authors report that recombinant DNA technology was used to improve rice nutritional value in this respect. A combination of transgenes enabled biosynthesis of provitamin A in the rice endosperm.

References (abridged):

1. A. Sommer, J. Nutr. 119, 96 (1988)

2. J. P. Grant, The State of the World's Children (Oxford Univ. Press, Oxford, 1991)

3. K. P. West Jr., G. R. Howard, A. Sommer, Annu. Rev. Nutr. 9, 63 (1989)

4. J. H. Humphrey, K. P. West Jr., A. Sommer, WHO Bull. 70, 225 (1992)

5. A. Pirie, Proc. Nutr. Soc. 42, 53 (1983)

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NUTRITIONAL GENOMICS: MANIPULATING PLANT MICRONUTRIENTS TO IMPROVE HUMAN HEALTH

The following points are made by Dean DellaPenna (Science 1999 285:375):

1) Humans require a diverse, well-balanced diet containing a complex mixture of both macronutrients and micronutrients in order to maintain optimal health. Macronutrients--carbohydrates, lipids, and proteins (amino acids) -- make up the bulk of foodstuff and are used primarily as an energy supply. Micronutrients are organic or inorganic compounds present in small amounts and are not used for energy, but are nonetheless needed for good health. Essential micronutrients in the human diet include 17 minerals and 13 vitamins required at minimum levels to alleviate nutritional disorders. Nonessential micronutrients encompass a vast group of unique organic phytochemicals that are not strictly required in the diet, but when present at sufficient levels are linked to the promotion of good health.

2) Modifying the nutritional composition of plant foods is an urgent worldwide health issue as basic nutritional needs for much of the world's population are still unmet. Large numbers of people in developing countries exist on simple diets composed primarily of a few staple foods (cassava, wheat, rice, and corn) that are poor sources of some macronutrients and many essential micronutrients. Consequently, the diet of over 800 million people does not contain sufficient macronutrients, and micronutrient deficiencies are even more prevalent (1). As examples of the magnitude of micronutrient deficiencies, estimates place 250 million children at risk for vitamin A deficiency (in up to 500,000 annually, this deficiency will cause irreversible blindness), 2 billion people at risk for iron deficiency (with children and women of reproductive age particularly vulnerable), and 1.5 billion people at risk for iodine deficiency (2). Even in industrialized nations, where both food abundance and variety are excellent and daily caloric intake is often excessive, micronutrient deficiencies are surprisingly common owing to poor eating habits.

3) While a plant-based diet can, in theory, ensure the adequate nutrition of humans at all stages of life, in practice, plant micronutrient levels vary widely and dietary micronutrient intake varies depending on the primary plant food source. Major staple crops contain insufficient concentrations of many essential vitamins and minerals to meet the U.S. recommended dietary allowance (RDA); therefore, nutrient fortification of the food supply is a necessary practice (3). RDAs can also be somewhat misleading because they are not the levels needed for optimal health, but rather the minimum levels needed to alleviate specific nutritional disorders (4). As such, RDAs do not reflect the growing knowledge base indicating that the elevated intake of specific vitamins and minerals (for example, vitamins E and C, carotenoids, and selenium) significantly reduces the risk of diseases such as certain cancers, cardiovascular diseases, and chronic degenerative diseases associated with aging (5). In order to obtain such therapeutic levels in the diet, additional fortification of the food supply will be required as well as modification of dietary preferences, or direct modification of micronutrient levels in food crops.

4) In addition to essential vitamins and minerals, plants also synthesize 80,000 of the 100,000 characterized secondary metabolites on the planet. This myriad of phytochemicals can be separated into several groups, with some containing several thousand chemically distinct compounds. Unlike the ubiquitous vitamins and minerals, specific phytochemicals are often unique to certain plant species or genera where they have evolved to play roles in development, stress responses, defense, or central and secondary metabolism. Many phytochemicals also have significant consequences for human health and are thought to be a major reason that plant-rich diets are associated with lower morbidity and mortality in adult life. Unfortunately, many of the best-characterized health-promoting phytochemicals are only present in plants or plant-derived products that are consumed at low levels in the American diet. Thus, because of dietary preferences, the health benefits associated with intake of specific phytochemicals are not fully realized in most American populations.

5) In summary: The nutritional health and well-being of humans are entirely dependent on plant foods either directly or indirectly when plants are consumed by animals. Plant foods provide almost all essential vitamins and minerals and a number of other health-promoting phytochemicals. Because micronutrient concentrations are often low in staple crops, research is under way to understand and manipulate synthesis of micronutrients in order to improve crop nutritional quality. Genome sequencing projects are providing novel approaches for identifying plant biosynthetic genes of nutritional importance. The authors use the term "nutritional genomics" to describe work at the interface of plant biochemistry, genomics, and human nutrition.

References (abridged):

1. D. H. Calloway, Human Nutrition: Food and Micronutrient Relationships (International Food Policy Research Institute, Washington, DC, 1995)

2. B. A. Underwood, Nutr. Today 33, 121 (1998)

3. W. Mertz, Nutr. Rev. 55, 44 (1997)

4. A. E. Harper, Annu. Rev. Nutr. 7, 509 (1987)

5. J. E. Buring and C. H. Hennekens, Nutr. Rev. 55, S53 (1997)

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ELEVATING VITAMIN E CONTENT OF PLANTS VIA GENETIC ENGINEERING

The *chloroplasts of higher plants produce numerous compounds important for human agriculture and nutrition. *Tocopherols, the lipid-soluble *antioxidants sometimes known collectively as vitamin E, are one such group of compounds and are synthesized only by photosynthetic organisms. The 4 naturally occurring tocopherols, alpha-, beta-, gamma-, and delta-tocopherol, differ only in the number and position of methyl substituents on the aromatic ring. In addition to their role as antioxidants, tocopherols stabilize polyunsaturated fatty acids within *lipid bilayers by protecting them from *lipoxygenase attack. Of tocopherol species present in foods, alpha-tocopherol is the most important to human health and has the highest vitamin E activity. Although all tocopherols are absorbed equally during digestion, only alpha-tocopherol is preferentially retained and distributed throughout the body. Alpha-tocopherol is an essential component of mammalian diets, and intakes in excess of the US recommended daily allowances are apparently correlated with decreased incidence of a number of degenerative human diseases. Plant oils, the main dietary source of tocopherols, typically contain alpha-tocopherol as a minor component, but with high levels of biosynthetic precursor, gamma-tocopherol.

D. Shintani and D. DellaPenna (Science 1998 282:2098) now report the use of genetic engineering to clone the final enzyme in alpha-tocopherol synthesis, gamma-tocopherol methyltransferase. The authors report that *overexpression of gamma-tocopherol methyltransferase in *Arabidopsis seeds shifted oil composition in favor of alpha-tocopherol. The authors suggest that similar increases in agricultural oil crops would increase vitamin E levels in the average US diet.

Notes:

*chloroplasts: Chloroplasts are cell organelles involved in photosynthesis, and are found in all photosynthetic plant cells. The typical higher plant chloroplast is lens-shaped and approximately 5 microns in diameter. The number per cell can vary from 1 to over 100, depending on the organism. There is some evidence that chloroplasts may have originated from photosynthetic bacteria that became *endosymbiotic with plant cells.

*endosymbiotic: Endosymbiosis is an arrangement in which one organism lives inside another organism, but the term is usually restricted to arrangements of mutual benefit, thus not including parasite-host relationships.

*Tocopherols: "Tocopherol" is a generic term for di- and trimethyltocols. Alpha-tocopherol is 5,7,8-trimethyltocol. Although the tocopherols are sometimes known collectively as "vitamin E", the usual referent for vitamin E is alpha-tocopherol.

*antioxidants: In general, an antioxidant is any substance that opposes oxidation or inhibits reactions produced by dioxygen or peroxides. An antioxidant is usually effective because it can itself be more easily oxidized than the substance protected. The term is often applied to substances that can trap free radicals, thereby breaking a chain reaction that normally leads to extensive biological damage.

*lipid bilayers: Lipid bilayers are spontaneously forming self-organizing bimolecular layers of certain molecules (lipids) with long nonpolar chains terminated by a polar group. Such molecules are found in cell membranes, and also in soaps. A variety of artificial lipid bilayer membrane systems can be investigated in the laboratory. The cell membrane itself is basically a lipid-bilayer structure.

*lipoxygenase: In general, any member of a group of enzymes that catalyze the oxidation of polyunsaturated fatty acids to a particular corresponding hydroperoxide. Such enzymes are found widely distributed in plants and animals (including humans).

*overexpression: In general, the term "expression" refers to any gene activity, but particularly to activity that results in the production of the specific protein encoded by the gene. The expression of genes is closely regulated in the cell, so that underexpression and overexpression are potential pathological abnormalities of cell function. In the context of this report, however, overexpression is genetically engineered in a plant to produce a result of potential human benefit.

*Arabidopsis: (Arabidopsis thaliana) (thale cress) A weed of the mustard family with a small genome of 120 million base pairs. Arabidopsis is now an important laboratory species, and it is presently the model for physiological, biochemical, cell biological, and developmental studies of over 250,000 plant species.

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