4 Golden Rice: a case study
4.2 The science behind Golden Rice
Modifying crops to produce the Bt toxin (Section 3.1) was, in some ways, relatively simple. The toxin is a single protein and can therefore be produced as a result of the insertion of a single gene into the plant's genome. Similarly, introducing herbicide tolerance (Section 3.2) typically involves modifying the action of a single enzyme, and therefore modification again involves the insertion of a single gene.
β-carotene is not a protein. It is a hydrocarbon, i.e. a compound containing only hydrogen and carbon atoms.
Not directly; genes generally code for proteins. However, β-carotene is produced by a series of biochemical reactions, each of which is catalysed by a specific enzyme. Each of these enzymes (which are proteins) will be coded for by a specific gene.
The series of reactions that produces β-carotene in plants begins with the compound isopentenyl diphosphate (abbreviated as IPP). A common intermediate in many of the biochemical pathways from IPP, geranylgeranyl diphosphate (GGPP), is present in rice endosperm, but conversion to β-carotene was expected to require a four-stage process, involving four separate enzymes (Figure 9).
Figure 9: Carotenoid biosynthesis in plants. Carotenoids are produced in a series of interlinked steps within plastids, a type of organelle found in plant cells (not to be confused with plasmids which we met earlier). They are derived from a common precursor, isopentenyl diphosphate (IPP). The first step in the carotenoid pathway is the combination of two molecules of geranylgeranyl diphosphate (GGPP) to produce phytoene. (The symbol ‘ζ’ in ζ-carotene is the Greek letter ‘zeta’.)
Given that GGPP is already present in the cells of the rice endosperm, how many genes have to be introduced to allow its conversion into β-carotene?
The process involves four stages, each catalysed by its own enzyme. In order to produce these four enzymes, four genes would have to be introduced.
The development of β-carotene-enriched rice was first proposed in 1992, by German and Swiss scientists, Peter Beyer and Ingo Potrykus respectively. At the time, the work seemed almost ludicrously ambitious. To attempt to introduce a single protein via insertion of a single gene was difficult enough, but to introduce four at once was surely too difficult. Potrykus had approached Nestlé, one of the world's largest food corporations, to fund the work, but was turned down. Eventually, he persuaded the Rockefeller Foundation, a charitable institution, to provide the funding to start the work.
Potrykus’ team planned to introduce each gene separately into individual rice plants, and then perform conventional crossing experiments in an attempt to produce a plant with all four enzymes active in the endosperm. Their method of choice was to use microprojectile bombardment (Box 1) on cells from immature rice embryos. The initial results were encouraging, and introduction of phytoene synthase was unproblematic. Phytoene was shown to accumulate in the endosperm, and the plants were healthy and fertile. However, repeated attempts to introduce the second enzyme in the sequence, phytoene desaturase, failed to produce healthy plants.
The project appeared to have reached a dead end, but a new member of the project team came up with some radical new ideas. Xudong Ye had just finished his doctoral research in a related area, and was eager to continue his studies with Potrykus. Unfortunately his time with the group was limited, and he could devote only one year to the work, as he planned to go to America. In order to have any prospect of success within the timescale, and after discussion with his colleagues, he proposed restarting the work, using a new approach. His plan was:
- To introduce the genes using Agrobacterium-mediated transformation.
- To insert a bacterial gene encoding an enzyme that would convert phytoene directly to lycopene, in effect performing two steps of the sequence in a single transformation.
- To introduce the genes for all three enzymes that were needed at once.
The proposed simplified pathway is summarised in Figure 10.
Figure 10: Proposed simplified route to β-carotene. What was proposed was that β-carotene would be produced in the rice endosperm by a three-step sequence: (1) GGPP would be converted to phytoene in the normal way, catalysed by phytoene synthase produced by a gene from a daffodil. (2) Phytoene would be converted directly to lycopene, catalysed by bacterial phytoene desaturase. (3) Lycopene would be converted to β-carotene, catalysed by lycopene β-cyclase, again produced by a daffodil gene.
Introducing sequences for three enzymes would be easier than introducing four, but despite using the generally more effective Agrobacterium-mediated Ti plasmid method, this would still be attempting to do a great deal of transformation all at once.
Why do you think that Potrykus and his co-workers initially used the less effective biolistic transformation method?
Rice is a monocot, and you may recall that, until relatively recently, the A. tumefaciens method was restricted for use with dicots (Box 1).
The necessary genes had to be isolated, cloned and spliced into the T-DNA of a Ti plasmid, using the techniques we have discussed in Section 2. Remember that each gene sequence requires a promoter as well as the gene itself.
The promoter ‘turns on’ the gene, i.e. it causes the cell's machinery to start transcribing the sequence of DNA.
In this case, the promoter needs to be one that is specific to the endosperm, so that the gene will be expressed in the endosperm and not in any other part of the plant. Further sequences are also required, including antibiotic resistance genes or other selection markers, sequences that allow some of the enzymes to be bound to a membrane within the cell, and sequences that produce proteins facilitating transport of the enzymes from the cytoplasm of the cell into specific organelles. The details of these sequences do not really concern us here, but it is important to appreciate that in order to introduce a gene for each enzyme, a whole series of sequences have to be introduced.
The team undertook two experiments, in each case using A. tumefaciens and the binary vector system described in Section 2.2. The technique involved the infection of immature rice embryos, rather than fragments of mature plants.
- Experiment 1: The team produced A. tumefaciens with an artificial Ti plasmid containing the series of sequences necessary to introduce active phytoene synthase and the bacterial phytoene desaturase. They attempted to infect around 800 immature rice embryos, of which 50 were found to have taken up the sequences. These embryos would be expected to produce only the first two of the enzymes required, those needed to convert GGPP to lycopene.
- Experiment 2: The team produced two types of modified A. tumefaciens. Type A contained all the sequences necessary for active phytoene synthase and the bacterial phytoene desaturase enzyme, as previously. Type B contained the series of sequences necessary to introduce the final enzyme in the biosynthesis, lycopene β-cyclase. 500 immature rice embryos were infected with both types of A. tumefaciens at once. Sixty embryos could be shown to have been infected by type A, but only 12 to have been infected by both types of A. tumefaciens.
The team was able to grow the 50 rice embryos from Experiment 1 and the 12 doubly infected embryos from Experiment 2 into mature rice plants. They allowed the plants to self-fertilise, and go on to produce a crop of rice (Figure 11).
Figure 11: Polished rice grains derived from Experiments 1 and 2. (a) Panel 1 shows unmodified rice, the control; panels 2, 3 and 4 show rice derived from three different plants from Experiment 1. (b) Panels 1 to 4 show rice derived from four different plants from Experiment 2. Note that at lower concentrations, β-carotene will give a yellow rather than an orange colouration.
Look again at Figure 9 and Figure 10. Assuming the enzymes are expressed and active in both cases, what intermediates from the β-carotene pathway would you expect to see produced in the rice grains from each experiment?
From Experiment 1 we might expect to see increased levels of lycopene, compared to unmodified grains. In Experiment 2 we would expect to see increased levels of β-carotene.
As lycopene is red and carotene is yellow-orange, if significant amounts of the products were present we might expect Experiment 1 to produce red rice grains, while Experiment 2 would produce the expected ‘golden’ rice. In fact, both experiments produced grains that showed a more or less intense yellow colour (Figure 11). Both lines could be shown to contain β-carotene, along with lutein and zeaxanthin, which are also products of the carotene biosynthetic pathway.
The rice unexpectedly showed a yellow colouration, strongly suggesting that any (red) lycopene produced had been converted to yellow β-carotene. It appears that the rice grains are able to produce their own lycopene β-cyclase. It may be that at high concentrations of lycopene, the production of this enzyme is induced, or it may be that the enzyme is already present.
The fact that only two of the three genes had to be introduced was an unexpected bonus, and remember that initially the expectation was that four genes would be necessary. Subsequent work in a number of research teams has concentrated on introducing the genes for phytoene synthase and phytoene desaturase.
A great deal of work remained to be done before anyone could imagine the rice being grown for human consumption, but this genetically modified rice represented a huge technical breakthrough. Whatever your opinions about genetic manipulation, it is hard not to admire the ingenuity of the work.