Golden Rice is a variety of rice that was created by inserting several genes that cause the rice grain to accumulate Beta-Carotene, a molecule that can be metabolised to produce Vitamin A. Vitamin A deficiency is a serious medical problem that is of particular importance for people who subsist on diets composed primarily of rice. Golden Rice is not currently used for production but it undergoing field trials and breeding in preparation for widespread use.
Vitamin A
Structure and metabolism
Beta-Carotene (top) can be metabolized to produce retinol (bottom), a form of Vitamin A. This is a major source of Vitamin A for humans who are unable to synthesize the molecule de novo.
The term vitamin A can refer to several similar molecules called the retinoids. A major source of vitamin A in the human diet comes from the metabolism of Beta-Carotene. Beta-Carotene is a orange pigment that is involved in photosynthesis and is the primary molecule responsible for giving carrots their orange color. The enzyme beta-carotene 15,15'-monooxygenase catalyzes the cleavage of beta-carotene and produces two molecules of retinal, a molecule form of vitamin A.
Function of vitamin A
Vitamin A has several important roles in humans including vision, gene regulation, skin health, the immune system, and several others. Not all of these processes are well understood and research into them is ongoing. Perhaps the best understood role for vitamin A is in vision. 11-cis-retinal, a form of vitamin a, is present in both rod and cone cells in the retina and undergoes a conformation change to the trans form upon absorption of light. This conformation change triggers a signal transduction cascade that leads to the firing of neurons, which the brain interprets as vision.
Vitamin A deficiency: symptoms and prevalence
It is estimated that 250 million children are deficient in vitamin A. Vitamin A deficiency can lead to a weakened immune system, night blindness, general blindness, and death. According the World Health Organization, between 250 to 500 thousand children lose their sight every year due to vitamin A deficiency. Half of these blind children go on to die within a year of losing their sight (WHO). The primary source of vitamin A for animals is through the consumption and metabolism of beta-carotene. High levels of beta-cartotene are found in a number of foods including carrots, squash, spinach, and butter. The RDA for vitamin A can be achieved with 50 g of these foods. Many people in the developing world do not have regular access to these foods and are therefore deficient in vitamin A. A number of programs are trying to alleviate vitamin A deficiency by providing supplements; however, these programs can be expensive and difficult to implement.
Mechanism of Beta-Carotene production in Golden Rice
Beta-Carotene pathway
The carotenoid biosynthetic pathway. The inserted genes were psy and crt1. crt1 is a bacterial gene that can perform the function of three different plant genes. The rice endosperm was found to naturally have lycopene cyclase activity. Consequently, this gene did not need to be inserted. (Figure from Mayer, 2007).
Efforts began in 1992 to engineer rice to have a greater vitamin A content. The endosperm of rice naturally contains geranylgeranyl-diphosphate, a compound that is only a few biosynthetic steps away from beta carotene (Figure 1) (Ye, 2000). To allow the rice grains to accumulate beta carotene, it was necessary to introduce introduce the enzymes for the remaining steps of the pathway in the same compartment as geranylgeranyl-diphosphate.
The first enzyme needed is phytoene synthase (psy). This enzyme catalyzes the reaction of geranylgeranyl diphosphate to make phytoene. The psy gene encoding for this enzyme was taken from daffodil and placed under the control of an endosperm-specific promoter. To get the enzyme into the plastid (the compartment where geranylgeranyl diphosphate is located), a plastid signaling sequence was placed onto the 5' end of the gene (Ye, 2000).
In plants, there are separate enzymes for the next three steps of the pathway. However, in certain bacteria there exists an enymze called bacterial carotene desaturase the performs the function of three enzymes. Using the gene for this enzyme reduced the amount of work needed to correctly transform and express each gene. The gene for the bacterial carotene desaturase was placed under the control of a constitutive cauliflower mosiac virus promoter and tagged with a 5' plastid signaling peptide to direct it into the plastid (Ye, 2000).
The plastid of the rice endosperm was found to have endogenous beta lycopene cyclase activity. As a result, a gene construct incorporating this gene, a promoter, and a signal peptide did not have to be inserted into the rice genome to for the final step of the pathway to make Beta-Carotene.
Optimization
The differences in carotenoid levels in these three strains are evident by the amount of orange pigmentation. On the left is wild type rice, in the middle is rice with the daffodil phytoene synthase (psy), and on the right is rice with the maize psy. (Figure from Paine, 2005).
A rice strain with the above gene constructs was able to produce Beta-Carotene, as seen by the orange color of the subsequent rice grains. However, the amount of Beta-Carotene produced was not enough to provide a substantial amount of vitamin A to people consuming typical amounts of the rice on a daily basis. Subsequent work following the initial success of this first Golden Rice variety set out to improve the accumulation of Beta-Carotene in Golden Rice. It was hypothesized the psy enzyme was the rate-limiting step of the pathway. The study tried several other psy genes including those from rice, tomato, corn and pepper and compared them to the daffodil gene that was already in use. It was found that the corn and rice variants of the psy gene were approximately 10 to 20 times better than the daffodil psy gene. An improved version of Golden Rice, dubbed Golden Rice 2, incorporated the psy gene from corn and was found to have a 23 fold increase in the accumulation of Beta-Carotene in the rice grains. The increase in Beta-Carotene accumulation is clearly visible by the coloration of the grains, seen in the figure to the right.