E SSENTIAL M INERALS

To maintain a well functioning, healthy body, hu- mans require 17 different essential minerals in their

diet. Minerals are inorganic ions found in nature and cannot be made by living organisms. They can be divided into two classes: macronutrients and micro- nutrients. Macronutrients are the minerals that we need in large quantity, including calcium, phospho- rus, sodium, magnesium, chlorine, sulfur, and sili- con. Micronutrients, or trace minerals, are the min- erals that are required in small amounts, of which iron is the most prevalent, followed by fluorine, zinc, copper, cobalt, iodine, selenium, manganese, molyb- denum, and chromium. Although a balanced con- sumption of plant-based foods should naturally pro- vide these nutrients, mineral deficiency, especially of iron, is widespread among the world population.

Iron

Even though iron is required in trace amounts, it is the most widespread nutrient deficiency worldwide. It is believed that about 30% of the world population suffers from serious nutritional problems caused by insufficient intake of iron (WHO 1992). Iron is an important constituent of hemoglobin, the oxygen- carrying component of the blood, and is also a part of myoglobin, which helps muscle cells to store oxygen. Low iron levels can cause the development of iron deficiency anemia. In an anemic person the blood contains a low level of oxygen, which result in many health problems including infant retardation (Walter et al. 1986), pregnancy complications (Mur- phy et al. 1986), low immune function (Murakawa et al. 1987), and tiredness (Basta et al. 1979). Iron is present in food in both inorganic (ferric and ferrous) and organic (heme and nonheme) forms. Heme iron, which is highly bioavailable, is derived primarily from the hemoglobin and myoglobin of flesh foods such as meats, fish, and poultry (Taylor et al. 1986). In humans, reduced iron (ferrous) is taken up more readily than oxidized (ferric) iron. Several ap- proaches have been used in the fight against iron deficiency including nutraceutical supplementation, food fortification, and various methods of food prep- aration and processing (Maberly et al. 1994). So far, none of these approaches has been successful in eradicating iron deficiency, especially in developing countries. A new tool in the fight against nutrient deficiency is the use of biotechnology to improve essential mineral nutrition in staple crops.

At this time, there are basically two ways in which genetic engineering can be used for this pur-

pose: (1) by increasing the concentration of the iron- binding protein ferritin and (2) by reducing the amount of iron-absorption inhibitor phytic acid. Although iron intake is important for human health, it can be toxic, so the ability to store and release iron in a controlled manner is crucial. The 450 kDa fer- ritin protein, found in animals, plants, and bacteria, can accumulate up to 4500 atoms of iron (Andrews et al. 1992). This protein consists of 24 subunits assembled into a hollow spherical structure within which iron is stored as a hydrous ferric oxide miner- al core (Fig. 3.6). The two main functions of ferritin in living organisms are to supply iron for the synthe- sis of proteins such as ferredoxin and cytochromes and to prevent free radical damage to cells. Studies have shown that ferritin can be orally administrated and is effective for treatment of rat anemia (Beard et al. 1996), suggesting that increasing ferritin content of cereals may solve the problem of dietary iron deficiency in humans. Japanese researchers (Goto et al. 1999) introduced soybean ferritin cDNA into rice plants, under the control of a seed specific promoter, GluB-1, from the rice seed-storage protein gene encoding glutelin. The two advantages of this pro-

moter are the accumulation of iron specifically in the rice grain endosperm, and its ability to induce ferritin at a high level. The ferritin cDNA was isolat- ed from soybean cotyledons, inserted into the binary vector pGPTV-35S-bar, and transferred into rice using Agrobacterium. The iron content of the rice seed in the transgenic plants was three times greater than that of the untransformed wild-type plants.

Phytic acid, or phytate, is the major inhibitor of many essential minerals, including iron, zinc, and magnesium, and is believed to be directly responsi- ble for the problem of iron deficiency (Ravindran et al. 1995). In cereal grains, phytic acid is the primary phosphate storage, and it is deposited in the aleurone storage vacuoles (Lott 1984). During seed germina- tion, phytic acid is catalyzed into inorganic phos- phorous, by the action of the hydrolytic enzyme phytase (EC 3.1.3.8) (Fig. 3.7). There is little or no phytase activity in the dry seeds or in the digestive tract of monogastric animals (Gibson and Ullah 1990, Lantzsch et al. 1992). In a recent study, it has been shown that phytase activity can be reestab- lished in mature dry seeds under optimum pH and temperature conditions (Brinch-Pedersen et al. 2002).

A reduction in the amount of phytic acid in staple foods is likely to result in a much greater bioavail- ability of iron and other essential minerals. Lucca et al. (2002) inserted a fungal (Aspergillus fumigatus) phytase cDNA into rice to increase the degradation of phytic acid. Rice suspension cells, derived from immature zygotic embryos, were used for biolistic transformation with the A. fumigatus phytase gene. Phytase from A. fumigatus was the enzyme of choice because it is heat stable and thus can refold into an active form after heat denaturation (Wyss et al. 1998). The main purpose of this research was to increase phytase activity during seed germination and to retain the enzyme activity in the seed after food processing and in the human digestive tract. Although the researchers achieved high expression levels of phytase in the rice endosperm, by placing it under the control of the strong tissue-specific globu- lin promoter, the thermotolerance of the transgenic rice was not as high as expected. It has been specu- lated that the reason for this unexpected low ther- mostability of the A. fumigatus phytase in transgenic rice is due to the interference of the cellular environ- ment of the endosperm to maintain the enzyme in an active configuration (Holm et al. 2002). Further

studies are needed to develop an endogenous phy- tase enzyme that is thermostable and maintains high activity in plant tissues.