Module 4: Nutrient Composition

Objectives - Nutrient Composition

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• Define each of the biotechnology techniques used to make nutritional changes in plants
• Describe how protein, fatty acid, and carbohydrate contents in food have been altered
• Identify specific food products that have had their nutrient content altered
• Identify micronutrients that have been modified
• Recognize phytonutrients that have been modified in foods
• Explain why antinutrients are monitored

Techniques

This first section is intended to provide a general background about what techniques are used to make nutritional changes in plants.

When genetic changes are made in crop plants, there are associated changes that occur in the plants’proteins, which in turn produce the new phenotype. These proteins arise from translation of DNA into RNA into amino acid sequences of polypeptide chains. Often, the proteins that are changed are ones that act as enzymes in the plant. These enzymes direct the synthesis of cell components that then determine the cell’s characteristics (e.g. what type of cell it is). Cells then make up tissues, organs and finally, the plant itself.

In order to make these proteins, and thereby the desired plant characteristic, several techniques are used, with the end result being addition of the desired foreign DNA into the plant.

Pathogen

One technique for introducing DNA changes into a plant involves the use of a plant pathogen, Agrobacterium tumefaciens.

In nature, this bacterium invades tissue wounds in plants(1). During this process the bacterium introduces a portion of its own DNA into the cell nuclei of the tissue it has invaded. That DNA becomes a part of the plant cell’s DNA makeup. This incorporation usually results in tumor formation within the tissue.

In the laboratory setting, the bacteria is introduced to the plant cell with its tumor causing DNA inactivated. When it invades the plant cell, it doesn’t cause tumor formation, however foreign DNA can be spliced into the bacteria’s DNA. The foreign DNA will be carried into the plant’s cell nuclei and become part of the plant’s chromosomes (1).

As part of the chromosome, promoters can then be used to express the genetic information from the foreign DNA. An important step in alteration involving nutrients is then expressing the genetic information at the correct stage of the plant’s development (e.g. leaves, flowers, fruits or seeds)(1).

Agrobacterium that contain a plasmid recombinant with the gene of interest are added to a solution containing callus cells	DNA, including the gene of interest, is inserted by the Agrobacterium into the nucleus of some of the callus cells where it may insert into a chromosome.

Particles

Not all plants are susceptible to the pathogen in the agrobacterium-based method of transformation. Therefore, for some plants another technique is used.

Microscopic gold or tungsten particles are used in this transformation method. The particles are accelerated toward the plant cells using an explosive charge, high-pressure helium, or electric discharge. A small number of the cells will be penetrated by the particles, resulting in transformation(1).

Gene gun transformation begins by growing cells in tissue culture, bombarding the cells with gene coated gold particles in the gene gun, selecting out transgenic cells on selection media, and regenerating the transgenic cells into plants.	Once inside the nucleus of a cell, the genes dissolve off of the gold particle and can potentially insert into a chromosome. A major limitation of this method is that several copies of the transgene will often insert into a chromosomal position. These high copy insertion events can be detected as DNA that should not be expressed by the plant cell and the transgene copies are silenced.

Electroporation

The third transformation method uses cells. These cells have had their outer protection, the cell wall, removed by an enzyme. The foreign DNA is then introduced via electroporation or by the natural uptake of DNA into the nucleus of the cell(1).

Antisense RNA Technology

The final technique involves using genetic information within the plant to decrease gene expression.

Techniques - Review Question

Question : Changes in a plant’s proteins translate into changes in the plant’s phenotype? True False

Macronutrients

Proteins

Plants naturally have an incomplete amino acid composition as it relates to human amino acid requirements. This can be overcome by including foods containing the amino acids that are low in the other food, thus creating a complete source of amino acids. This technique is often used in the traditional meals from vegetarian cultures (2).

 In most developed countries it is not difficult for individuals to attain complete sources of amino acids required for human health. However, in developing countries there are more challenges to overcome such as production costs, climatic restrictions on what crops can be grown, some traditional eating habits, and an overall lack of food (2).

Modifying Amino Acid Content

Research in this area has been focused on creating a complete amino acid source in plants. This is a difficult task, because plants are complex organisms. Protein expression is tissue specific, meaning that the functional requirements of each tissue dictate what proteins are manufactured there. Even within the tissue, proteins in individual cells are separated based on their function. This separation makes it essential to develop transformation systems that not only introduce the genetic information of interest, but that also work within the control mechanisms, such as tissue-specific expression (2). The introduced gene will only create the desired result when it is expressed in the correct tissue.

A. Amino acids are made from simple atoms and always have an amino end and an acid end	B. Proteins or polypeptides are made by connecting amino acids together	C. The parts of the gene; the promoter and termination sequences control transcription while the coding region encodes the protein..

In order to plan the correct addition of genetic information into the plant, several factors have to be considered. The most important consideration is the tissue or tissues in which the new or modified protein will be expressed and the proteins that will be modified. Plant protein in the diet comes primarily from plant seeds and to a lesser extent from the leaves. The function of protein in seeds is to provide nitrogen to the developing plant. Since that is their only function, modification of these proteins has less potential for disrupting other processes in the plant than modification of proteins in other plant tissues (2). For that reason, the seed is often the target for amino acid profile improvements in plants.

There are several approaches to enhancing amino acid content in plants. The first approach is to simply increase the amount of amino acids made in the plant to provide more protein per serving.

A second approach focuses on a change in the amount of high quality proteins in the plant. This involves making the amino acid components of the protein more available. Techniques for accomplishing this include increasing the transcription rate of the associated amino acid genes, increasing the stability or the rate of translation of the mRNA that encodes for the protein, or by making the protein product less susceptible to degradation after it is made.

The third approach is to increase the nutritional quality of the proteins made in the plant seeds. Proteins that are already made in a particular plant can have new amino acids introduced into them or new proteins that have the desired amino acid composition can be expressed (2).

In summary, most of the transgenic plants that are being developed are of three major types (2): 1. Genes of normally occurring protein within the plant is modified to add additional essential amino acids to its composition 2. A gene for a naturally occurring protein from another species is transformed into the plant to provide the complementary essential amino acids 3. A new gene for a designed protein (de novo) with a more desirable amino acid composition is introduced into the plant

Example

The synthesis of lysine, which is derived from asparate, is mainly controlled by two enzymes: aspartate kinase (AK) and dihydrodipicolinate synthetase (DHDPS). These enzymes are regulated by end-product feedback inhibition by lysine. In other words, when lysine accumulates the pathway by which it is created slows down. If the feedback inhibition can be overcome, production of lysine above normal amounts will occur. (2).

When bacterial genes were used to decrease the inhibition feedback system in transgenic plants, phenotypic abnormalities occurred in the plant. It was determined that promoters were needed to control when the amino acid was produced during the development of the plant (2). This illustrates the need for the desired amino acid to be produced in not only the correct tissue, but also at the correct stage of the plant’s development to minimize negative effects on the plant.

Protein Modified Foods

This picture shows a purple soybean flower.

This picture shows a wheat spikelet.

Carbohydrates

Research into the modification of fiber and complex carbohydrates in foods has increased. This increase has occurred due to the affects they have been found to have on lipid metabolism and diabetes control (3). The foods in this section have had their carbohydrate content modified, at least in the laboratory.

This picture shows a grain truck filled with harvested ears of corn.

Maize

The research into carbohydrate modification of maize began with investigation into naturally occurring mutants that were found to have altered seed starch composition. These plants helped researchers understand the biochemistry and genetics surrounding starch accumulation in maize. They then used this knowledge and antisense technology to change carbohydrate composition in other plants, such as tomatoes (3).

Potatoes

As was discussed in an earlier section, new protein expression results in changes in the plant. A new protein, ADP glucose pyrophosphorylase from Escherichia coli, was introduced into potatoes to change starch composition. Specifically, more starch accumulated in the potato (1). The increased starch results in less fat absorption during cooking and thus a significantly lower-fat product.

Carbohydrates - Review Question

Question : How has a change in potato to carbohydrate composition created a healthier product? less simple sugars to benefit diabetes more starch for less fat absorption during cooking less starch to decrease cooking time, resulting in lower fat absorption more carbohydrates to increase caloric density

Fatty acids

The fatty acid composition of plants depends on climatic conditions and the genetics of the plant. These fatty acids are found as both storage oils and membrane lipids, such as glycolipids, phospholipids and lipoproteins. This section will discuss genetic changes that have been made to change the fatty acid profile in various plants.

Polyunsaturated fatty acids

Polyunsaturated fatty acids (PUFAs) have been found to be important to human nutrition. One of the reasons for their importance is their hypocholesterolemic property related to heart disease. They are also involved in many biological processes in both structural and functional roles as parts of cell membranes, regulators of membrane permeability as well as proteins within the membrane. PUFAs are also involved in gene expression regulation, including genes that provide the information for fatty-acid synthesis, sodium-channel proteins, and cholesterol-7-alpha-hydroxylase. As a result of their part in gene expression, PUFAs impact cellular biochemical activities, transport processes involving lipid metabolism, immune responses, cold adaptation, and conditions such as carcinogenesis and cardiovascular disease (6)

Polyunsaturated fatty acids can be obtained from plant seeds, marine fish and certain mammals. It has been reported that the market for PUFAs may increase, resulting in the need for alternative sources of fatty acids (4). Genetic modifications could help provide alternative sources of these fatty acids and to provide a desirable PUFA profile to meet specific nutritional demands (6).

Trans fatty acids

Trans fatty acid consumption has been regarded as a possible risk factor for coronary heart disease. While most fatty acids are present in nature in a cis configuration (changes the shape of the molecule), trans fatty acids are created when plant oils are hydrogenated.

The concern about trans fatty acids can be overcome by the use of desaturase genes from plants (6). One example of such a gene is the Omega 6 desaturase gene from soybeans. This desaturase inserts a second double bond into oleic acid, making it polyunsaturated. Researchers have used this gene’s activity to suppress the existing gene’s activity. In other words, the addition of the second double bond is blocked. When the second double bond is not added, the result is a monounsaturated oil with no trans fatty acids created during hydrogenation (7).

Canola Oil

Canola oil developed from rapeseed was one of the first examples of nutrition concerns influencing oil seed crop developments (3). That modification was accomplished with traditional plant breeding, however now genetic methods provide a technique to make more specific modifications to plants’ fatty acid biosynthesis pathways (3). These biosynthesis pathways are controlled by multiple genes in plants, with some of the genes controlling fatty acids in specific tissues, which allows for changes that can be made in seed storage lipids (3).

Replacement of high saturated fat oils with less saturated ones is an area of research with human health implications. One example is high laurate canola oil (lauric acid is a medium chain fatty acid) created by inserting the 12:0 thioesterase gene from Umbellaria californica into canola. The benefit of the high amount of lauric acid is that the product can replace the high saturated fat tropical oils that are used in nondairy coffee whiteners and whipped toppings, as well as in confectionery products (1).

Vegetable Oil

There have been several developments used to change the fatty acid profile of vegetable oil. The first genetic modification used cyanobacterial desaturase introduction into the plant. The desaturase introduces a double bond into 16-chain and 18-chain saturated fatty acids which, if expressed in the seeds, the saturated fatty acid content of the oil could be reduced (7). The second modification uses delta 6 desaturase that introduces a double bond into linoleic acid. Linoleic acid is then converted into gamma-linoleic acid, the beneficial compound found in fish oils (7).

Micronutrients

Deficiencies of iron, vitamins A, E and C have lead to research into increasing the amounts of these micronutrients in staple foods. It has been estimated that two billion of the world’s population are iron deficient, while 400 million of the world’s population are vitamin A deficient. Many Americans don’t consume the recommend amount of vitamin E. This section discusses these micronutrients that have been modified in food.

Vitamin E

Most of the vitamin E in the human diet comes from vegetable oils made from soybeans, maize, cottonseed and rapeseed. In these crops, alpha-tocopherol, which has the highest vitamin E activity, is found in low levels. Gamma-tocopherol, the biosynthetic precursor to alpha-tocopherol, is found in these same plants at higher levels (8). Since the precursor is available in these plants, it suggests that the synthesis of alpha-tocopherol could be increased in oil crop seeds.

The enzyme that creates the final product in the pathway, alpha-tocopherol, has been overexpressed in transgenic Arabidopsis plants. The overexpression of this gene was paired with promoters for its expression in the seed, with the result being increased tocopherol accumulation.

Vitamin A

Rice

Rice grains do not naturally contain beta-carotene, the precursor to vitamin A. However, they do contain a substance, geranylgeranyl pyrophosphate that can be converted to beta-carotene. This conversion requires four enzymes in the biosynthetic pathway. The four genes for these enzymes have been isolated from daffodils and bacteria. These genes were then transferred into rice via Agrobacterium mediated transformation (9). The result of this transfer was several transgenic plants that had all four genes expressed, with a visible yellow endosperm that indicates the presence of carotenoids (9). A meal-sized portion of the transgenic rice contains enough vitamin A to meet the daily requirements for the vitamin (10).

Tomatoes

Research into carotenoid biosynthesis in tomatoes has lead to knowledge about genes such as Psy1, Pds, beta-Lcy and Epsilon-Lcy. Psy1 and Pds, which control lycopene synthesis, are up-regulated during the ripening process, while beta-Lcy and Epsilon-Lcy, which control lycopene cyclisation, are down-regulated. The result of this regulation is beta-carotene (the major dietary precursor of vitamin A) levels in ripe tomatoes less than 15% of the total carotenoids present.

Beta-carotene has been increased in tomatoes with overexpression of Arabidopsis beta-Lcy cDNA in the plant, which is fused to the Pds promoter. The Pds promoter, as mentioned before, is up-regulated during ripening. The result was increased beta-carotene in the tomato (11). The level of beta-carotene was 6 mg (the equivalent of the vitamin A RDA).

Iron

Rice

The accumulation of iron and ferritin in seeds occurs naturally. The seed ferritin is a target trait since it is a natural source of iron for development in plants and animals, it is already a normal seed component, it appears to be under accessible genetic control, and it is bioavailable (12).

The gene encoding the plant iron storage protein, phytoferritin, was cloned. Researchers hope that this gene can be used via overexpression in cereal crops to fortify them with bioavailable iron (8).

Another technique to fortify rice has been the transfer of the soybean ferritin gene via Agrobactrium-mediated transformation (9). A promoter for seed storage protein was used as a reference to express the soybean gene in the endosperm of the rice. The result was a threefold increase in iron content compared to untransformed controls (9).

The elimination of phytate, a sugar-like molecule that binds dietary iron, has been another focus of improving iron content in rice. A fungal gene for phytase, an enzyme that breaks down phytate, has been introduced into rice. The result of this addition was an increase in the bioavailability of the iron (9).

Vitamin C

Fruits and vegetables supply approximately 91% of the vitamin C in the U.S. food supply (3). The substantial variation of the amount of vitamin C in crops suggests that there is a potential for genetic modification.

Tomatoes

Currently, tomatoes with increased vitamin C content have been developed with traditional cross breeding between cultivated and wild species. However, poor yield often co-exists with the increased vitamin C. Until the interaction is better understood, modification of vitamin C in tomatoes has been discontinued (3).

Phytonutrients

Flavonoids

Flavonoids have been defined as polyphenolic compounds found in plants. These compounds are involved in plant growth and development. There has also been research that suggests flavonoids are beneficial to human health, possibly due to their antioxidant properties (5). Currently, over 400 individual flavonoids have been discovered.

The benefits include protection against chronic diseases, such as cardiovascular disease, that can be obtained by consuming foods containing these compounds. Their antioxidant properties towards free radicals have potential in anticancer activity, such as antiproliferation and promotion of differentiation and apoptosis (13).

There is some difficulty in obtaining good sources of flavonoids since many food sources have only small amounts in their edible parts. Foods may also contain flavonoids with suboptimal antioxidant characteristics, making them less than ideal sources (5).

Genetic engineering could play a role in improving these sources. Research in this area has lead to identification of enzymes needed to synthesize flavonoids, genes that encode for these enzymes, and genes that regulate the production of flavonoids. These developments puts researchers closer to being able to upregulate the flavonoid biosynthesis as well as to direct synthesis of desirable flavonoids in crop plants (5).

Isoflavonoids

Isoflavonoids are a class of flavonoids found mainly in legumes. They have been found to have oestrogenic and anticancer activity. This leads to the possibility of using isoflavones to treat or prevent hormone-related disorders.

The gene that encodes the main enzyme in isoflavonoid formation, cytochrome P450 mono-oxygenase isoflavone synthase (IFS) has been cloned. When this gene was expressed in plants, such as tobacco and maize, more isoflavonoids were found in the plant where the general flavonoid pathway was already active (5).

Experiments have also been done with non-legume plants. A transfer of the IFS gene found in soybeans to Arabidopsis (a flowering plant) resulted in conversion of narigenin to an isoflavone, genistein, a phytoestrogen of high medical interest (13). Success in studies such as this give researchers hope that they will be able to increase isoflavonoid production in crops such as tomato, maize, wheat or rice (5).

Here are some common questions about diet and cancer, including phytonutrients from the American Cancer Society.

Flavonols

Another class of flavonoids, flavonols, has been found to be good antioxidants and protective against cardiovascular disease. Tomatoes naturally contain this compound in the peel of the fruit in the form of quercetin. The peels also contain another flavonoid, chalcone narichalcone. Research data has suggested that flavonol biosynthesis in the peel is limited in part by low levels of gene expression of chalcone isomerase (CHI). Increasing synthesis of the CHI in the tomato was accomplished by using the petunia CHI gene to develop transgenic tomatoes. The result was a large increase in the amount of quercetin (70-fold) and flavonol levels within the tomato comparable to onions, which are naturally high in flavonols (5).

Here is some information about phytonutrients and cardiovascular disease from the American Heart Association.

Antinutrients

Evaluation of transgenic plants has also included a study of levels of inherent toxic and antinutritive substances. This has been done since genetic modification has the potential to affect expression of genes not directly involved in the nutritional (or other type) of change being made (14).

Antinutrients have been defined as substances that inhibit or block important pathways in metabolism, especially digestion (14). These substances reduce utilization of nutrients by the body, such as proteins, vitamins and minerals. The result is a decrease in the body’s ability to use the nutrients, even though they are present in the food.

Secondary, or unintentional, effects on the plant can occur when genetic material is introduced. With the transfer of one gene, multiple changes in the plant can result. Some of these effects could include increased synthesis activity of already present biochemical metabolism pathways, augmented synthesis caused by increased activation of other genes, decreased production of catabolic enzymes, or reduced degradation of substances (14).

The possibility of these effects being present in genetically engineered foods has lead to review of several products (14). The table below outlines food crops that have been tested for increased levels of antinutrients following genetic modification. The net result was a lack of significantly increased antinutrient levels in these foods, however it is important to recognize that these substances could exist and we need to be aware of them.

Rapeseed oil
Naturally occurring antinutrients

• Glucosinolates (thyroid gland increase, thyroxin synthesis decrease, metabolism impairments, iodine absorption decreases, protein digestion decreases)
• Phytic Acid (bioavailability of Ca, Mg, Fe, Zn, Cu, Mn decreased, protein and starch utilization decreased)

• Some cases of significant differences between glucosinate content of one modified line and non-transgenic plants
• Genetically modified lines fit within established range for glucosinate

• Phytic Acid (bioavailability of Ca, Mg, Fe, Zn, Cu, Mn decreased, protein and starch utilization decreased)

• In plant modified to control European corn borer, gene sequence found that could encode for protease inhibitor (Inhibition of trypsin and chymotrypsin, decreased digestion of protein)
• No protease inhibitor found in modified maize lines

• Glycoalkaloids (Gastrointestinal symptoms, inflammation of kidney, binds metals, decreased iron absorption, inhibits enzymes)
• Alpha-tomatine (Decreases during maturation so that red ripe tomatoes loose almost all tomatine when left on plant for 2-3 days)

• Shorter ripening times created via genetic modification could lead to increased tomatine
• GM tomatoes created for processing were tested
• Glycoalkaloid levels found to be within levels of non-GM tomato pastes

• Solanine (Gastrointestinal symptoms, inflammation of kidney, binds metals, decreased iron absorption, inhibits enzymes)
• Protease inhibitors (Inhibition of trypsin and chymotrypsin, decreased digestion of protein)
• Phenols (Decrease availability of trace elements)

• Overall, no increased contents of any inherent plant toxins or antinutrients examined

• Protease inhibitors (Inhibition of trypsin and chymotrypsin, decreased digestion of protein)
• Phytate (bioavailability of Ca, Mg, Fe, Zn, Cu, Mn decreased, protein and starch utilization decreased)

• No significant differences between modified varieties and control soybeans

Discussion Question : Do you think it is important to keep testing genetically modified foods for antinutrients?

Sources - Nutrient Composition

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12. Rosati C, Aquilani R, Dharmapuri S. Metabolic engineering of beta-carotene and lycopene content in tomato fruit. The Plant Journal: for cell and molecular biology. 2000;24(3):413-419.

13. Ruf S, Hermann M, Berger IJ. Stable genetic transformation of tomato plastids and expression of a foreign protein in fruit. Nature Biotechnology. 2001;19(9): 870-875.

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