Golden Rice: A Crop of the Future?

What is golden rice? Rice that has been genetically engineered to produce carotenoids (a group of plant pigments that are important to the human diet, because they serve as the only precursors of vitamin A). Among them the most important is β-carotene (also known as pro-vitamin A). The human body can change pro-vitamin A to vitamin A. Normal rice does not produce carotenoids in the endosperm (the edible part of the plant), because it does not perform photosynthesis. To make rice produce carotenoids, two genes have been introduced into the rice genome: phytoene synthase (psy) and phytoene desaturase (crt I). The more golden the rice, the more β-carotene that rice produces.

Figure 1: A picture of normal white rice vs. golden rice. Figure from Golden Rice, http://www.goldenrice.org/

Why make golden rice? Golden rice was made to help fight vitamin A deficiency (VAD). VAD is prevalent among people who are poor, and eat mainly rice or other carbohydrate-rich, micronutrient-poor food. VAD causes 250,000 to 500,000 children to go blind every year and each year more than half of those who go blind die. VAD is also responsible for compromising the immune systems of ~40% of the children under the age of five in the developing world. This deficiency is most pronounced in Southeast Asia and Africa.

How is β-carotene made? Figure 2: A simplified version of the β-carotene pathway in golden rice. Figure from Wikipedia, http://en.wikipedia.org/wiki/Image:Carotenoid.PNG

Known information about rice It was known that rice plants produced β-carotene in the green tissues, but not in the edible part of the plant (the endosperm). It was also known that the rice endosperm produce gernanylgernayl diphosphate (a precursor of β-carotene).

How was golden rice made? The original golden rice was created in 1999 by a collaboration between Peter Beyer and Ingo Potrykus . To do this, they added three genes to rice: phytoene synthase (psy) and lycopene β-cyclase from daffodil (Narcissus pseudonarcissus) and phytoene desaturase (crt I) from the soil bacterium Erwinia uredovora. Agrobacterium transformation was used to introduce three different plasmids into the rice genome.

How was golden rice made? The first plasmid contained the psy and crtI genes under the control of the endosperm specific glutelin (Gt1) and the constitutively active cauliflower mosaic virus (CaMV) promoters. This plasmid also contained an aphIV expression cassette that selected for hygromycin resistance. This plasmid also contained a sequence that directed the formation of lycopene to be made in the endosperm plastids, which is where gernanylgernayl diphosphate is made. Figure 3: Structure of the first plasmid used to make golden rice. Figure from Ye et. al, Science, 2000.

How was golden rice made? To complete the pathway, co-transformation was done using two more plasmids. The second plasmid was the same as the first, except that it lacked the aphIV expression cassette. The third plasmid contained the lycopene β-cyclase gene controlled by the rice glutelin promoter and the aphIV gene controlled by the CaMV promoter. This plasmid also contained a peptide that allowed it to be transported to the plastids. Figure 4: Structures of the second (top) plasmid and the third (bottom) plasmids used to make golden rice. Figure from Ye et. al, Science, 2000.

How was golden rice made? To determine which plants had taken up the appropriate plasmids, the plants were tested for hygromycin resistance, and were analyzed using southern hybridization and restriction digests. From these, plants showing all four introduced genes were planted, and the seeds were analyzed. The most successful plant produced ~1.6µg/g of β-carotene in the endosperm. This would not be enough to meet the vitamin A requirement in children (the recommended daily allowance is 300µg, but 150µg would be sufficient). Figure 5: Results of β-carotene production in the plant producing the largest amount. Results obtained by using 1g carotenoid extract and high- performance liquid chromatography. Figure from: Ye et. al, Science, 2000.

How was golden rice made? It was also discovered that none of the plants accumulated detectable amounts of lycopene, which meant that lycopene β-cyclase was either produced in the endosperm constitutively or was turned on when lycopene was present. This meant that this gene did not need to be added to the rice to form β-carotene.

The new golden rice Further experiments showed that the rate-limiting step in this reaction was the enzymatic activity of psy. In 2005, researchers (Paine et.al, 2005) discovered that using a psy gene and new promoter from maize instead of daffodil and using a seed-specific promoter, instead of a constitutive promoter for the crtI gene, produced ~31µg/g of β-carotene. They referred to this rice as Golden Rice 2. 72g of this rice would provide the 150µg of vitamin A that is needed daily in children. Most children in countries where rice is a staple food consume more than 72g of rice a day. Figure 6: Results showing wild-type rice, Golden Rice 1, and Golden Rice 2. Figure from The Golden Rice Project, http://www.goldenrice.org/Content2-How/how1_sci.html

Concerns with golden rice: Is it safe? Golden rice is safe, because it contains β-carotene, which is a precursor to vitamin A and not vitamin A itself. The body will only make vitamin A if it needs it. If it does not need it then the excess β-carotene will either be stored or excreted. There has been no information to show that carotenoids have any ill-effects on humans.

Concerns with golden rice: gene flow The chances of the introduced genes escaping are very low due to the fact that rice pollen is only viable for 3-5 minutes. Also, these genes confer no advantage to other plants, since other plants produce carotenoids. In order to lower the chances of the genes escaping even further, the crops could be planted by having staggered flowering days and by having a certain distance between fields. Since the two genes do not give a selective advantage, they will probably be diluted out of a population, instead of incorporated into one.

Concerns with golden rice: the antibiotic resistance marker Some people are wary of having a hygromycin resistance marker included in the transformed rice, but the marker was not included in the final product through the process of co-transformation. Also, it was found that hygromycin does not pose a threat to humans or the environment. An alternative technology has been created by Syngenta called Positech® that identifies transformants using a non-metabolizable sugar instead of antibiotic resistance.

Concerns with golden rice: farmers, rice varieties, and rice color. The cost of seed is another concern, but the project’s hope is that it will be able to provide seed to poor farmers for free. The trait of producing β-carotene can be made to occur in many varieties of rice, which means that local areas will not have to change their rice or farming habits. It is also hoped that since only the color of the rice has changed, that the public is willing to accept a rice that is not white.

Concerns with golden rice: unmilled rice, other methods to get vitamin A Although the aleurone layer in unmilled rice (brown rice) does contain vitamins such as vitamin B, it does not contain vitamin A. Also, the lipids in the aleurone layer cause it to go rancid quickly, especially in tropical climates. Vitamin A could also be obtained through capsules, but it is not possible to reach every child to give them these capsules, and the amount of money to do this annually in a country such as Nepal or Ghana is ~2 million dollars.

Concerns with golden rice: economics Disadvantages: -Some countries refuse to import transgenic crops. -People may be unwilling to by golden rice. Advantages: -Medical costs related to VAD would decrease. -Worker productivity would increase.

Where is golden rice at? In 2004: field trials were done in the United States and it was concluded that ~6µg/g of β-carotene were produced. In 2005: 100kg of polished rice grain was collected for further testing. In 2008: field trials are being conducted in the Philippines using golden rice.

References Golden Rice Project, http://www.goldenrice.org/index.html GMO Compass. Golden Rice: First field tests in the Philippines. http://www.gmo-compass.org/eng/news/358.docu.html Paine, J, Shipton, C, Chaggar, S, Howells, R, Kennedy, M, et al. Improving the nutritional value of Golden Rice through increased pro-vitamin A content. Nature Biotechnology, 2005. Ye, X, Al-Babili, S, Kloti, A, Zhang, J, Lucca, P, Beyer, P, Potrykus, I. Engineering the Provitamin A (β-carotene) Biosynthetic Pathway into (Carotenoid Free) Rice Endosperm. Science, 2000:287, 303-305.