In most parts of the world
especially in Asia and Africa the nutritional deficiency in diet is a serious
problem. This is due a huge increase in the population and poverty elevation in
the world particularly in the third world. Malnutrition is a source of various
diseases especially in children and in women. The blindness of the children is
also due to this nutritional deficiency. The blindness in children is caused by
the vitamin A deficiency in most parts of the developing world. It is estimated
that about 250,000-500,000 children are suffering from vitamin A deficiency
(West and Darnton-Hill, 2001). It also weakens the immune system, thus
increasing the occurrence and severity of the disease. Vitamin A deficiency also
causes some serious problems in adults especially in the pregnant and lactating
women about 600,000 women died each year (Sommer and West, 1996). The deficiency
can be reduced in the developing countries by educating people about the food
fortification and supplementation. The need is to produce enriched beta-carotene
(which is a building block of Vitamin A) staple food through plant breeding.
Some crop species like sweet potato and maize are enriched in vitamin A but
these are not staple foods. Rice is a staple food in most of the countries of
the Asia but its endosperm doesn’t contain the β-carotene. So, the genetic
engineering is done to make it enriched with vitamin A (Bouis, 2000). Now-a-days
the genetically engineered rice is known as “Golden Rice” which is developed by
Drs. Ingo Potrykus and Peter Beyer at Swiss Federal Institute of Technology in
1999 (Ye et al., 2000). The golden rice also contains snippets of DNA taken from
bacteria and daffodils. The amount of rice in a third world diet could provide
about 15% of the recommended daily allowance of vitamin A, sufficient to prevent
blindness. Two genes are inserted in the rice genome, which complete the
biochemical pathway needed for beta-carotene production in the grain.
These two transgenes turned Golden Rice into a reality. The first transgenes
encodes phytoene synthase (PSY), which utilizes the endogenously synthesized
geranylgeranyl-diphosphate to form phytoene, a colorless carotene with a triene
chromophore (Burkhardt et al., 1997). The second encodes a bacterial carotene
desaturase (CRTI) that introduces conjugation by adding four double bonds. The
combined activity of PSY and CRTI leads to the formation of lycopene, which is a
red compound due to its undecaene chromophore. Lycopene has never been observed
in any rice transformant and different genetic backgrounds. Instead, alpha- and
beta-carotene are found together with variable amounts of oxygenated carotenoid
such as lutein and zeaxanthin. The carotenoid pattern observed in the endosperm
revealed that the pathway proceeded beyond the end point expected from the
enzymatic action of the two transgenes alone. A detail analysis of the
underlying mechanism was recently published by co-workers from Peter Beyer's lab
in the journal Plant Physiology (Schaub et al., 2005). Their findings are
explained in some detail below.
(Schaub et al., 2005)
The precursor molecule for carotenoid biosynthesis is geranylgeranyl diphosphate
(GGDP). Horizontal bars delimit the steps of the carotenoid biosynthetic pathway
that were overcome using the two transgenes phytoene synthase (PSY) and the
multifunctional bacterial carotene desaturase (CRTI), rather than the two plant
desaturases PDS and ZDS.
One explanation is that enzymes downstream along the pathway, such as lycopene
cyclases (LCYs) and alpha and beta-carotene hydroxylases (HYDs) are being
produced in wild-type rice endosperm, while PSY and one or both of the plant
carotene desaturases phytoene desaturase (PDS) and ζ-carotene desaturase (ZDS)—are
not. Synthesis of lycopene by PSY and CRTI in transgenic plants provides the
substrate for these downstream enzymes and consequently enables the formation of
observed products. The fact that a PSY transgenes alone led to phytoene
accumulation but not to desaturated products (Burkhardt et al., 1997) is
evidence for the absence of at least one active desaturase, namely PDS.
Similarly, the expression of CRTI alone did not produce any color in rice
endosperm, because of the lack of PSY activity.
First Generation of Golden Rice
The first breakthrough in the development of Golden Rice was the result of a
collaboration between Peter Beyer and Ingo Potrykus and was obtained around
Easter 1999 (Ye et al., Science 287:303-5, 2000). This paper provided the proof
that beta-carotene could be produced in the rice grain. At the time it was still
believed that, beside phytoene synthase and carotene desaturase, a third enzyme,
lycopene cyclases, was needed to complement the biosynthetic pathway.
With the proof of concept in hands, the scientists immediately proceeded to
develop ways of improving the production and accumulation of carotenoid in the
seed, as it was recognized that at the levels attainable at the time (1.6 µg/g)
Golden Rice would not be able to fully cover the daily pro-vitamin A
requirements of the target population in the absence of a more varied diet.
While some population strata in SE Asia do consume more varied diets, many of
the poorest do not, in fact in some rural population rice makes up more than 80%
of their daily caloric intake.
These efforts led to the development of what we could call the first generation
of Golden Rice (after the proof of concept), also known as SGR1. This version
only contained the phytoene synthase (psy) gene from daffodil and the carotene
desaturase (crtI) gene from the bacterium Erwinia uredovora. Further, in this
version both genes were expressed only in the rice endosperm. The levels of
carotenoids obtained in the greenhouse were not very unlike the precursor
version (1-2 µg/g) but in the field production and accumulation amounted to an
average of 6 µg/g. This might have been due to improved growth conditions and to
the selection process that the plants underwent. This level of carotenoids
contents is expected to be able to cover the recommended daily intake values for
children when taking into consideration a modest intake of vegetables and fish
or other animal sources.
Gene construct used to generate Golden Rice. RB, T-DNA right border sequence;
Glu, rice endosperm-specific glutelin promoter; tpSSU, pea ribulose bis-phosphate
carboxylase small subunit transit peptide for chloroplast localisation; nos,
nopaline synthase terminator; Psy, phytoene synthase gene from Narcissus
pseudonarcissus (GR1) or Zea mays (GR2); Ubi1, maize polyubiquitin promoter; Pmi,
phosphomannose isomerase gene from E. coli for positive selection (GR2); LB,
T-DNA left border sequence.
The first generation of Golden Rice was a valuable proof of concept, but it was
recognised that to combat vitamin A deficiency more efficiently higher
β-carotene accumulation levels would be required. As only two transgenes are
required in the process, the logical approach was to identify the bottleneck of
the biosynthetic pathway and fine-tune the enzymatic activities of the two gene
products involved, phytoene synthase (PSY) and carotene desaturase (CRTI). This
can be done by replacing the genes with homologues from other sources or
modifying their regulatory regions.
In most multi-step biosynthetic pathways there is a rate-limiting step. Making a
long story short, the bottleneck in this case was the enzymatic activity of PSY.
After trying with PSY genes from different sources it turned out that the maize
and rice genes gave the best results (Paine et al., 2005). In the process Golden
Rice lines were obtained that accumulated up to 37 µg/g carotenoid of which 31––
µg/g is β-carotene (as compared to the first generation Golden Rice where only
1.6 µg/g were obtained.
The recommended daily allowance (RDA) of vitamin A for 1-3 year-old children is
300 µg (half the RDA is enough to maintain vitamin A at a normal, healthy
level). Based on a retinol equivalency ratio for β-carotene of 12:1, half the
RDA would be provided in 72 g of the new-generation Golden Rice. This is
perfectly compatible with rice consumption levels in target countries, which lie
at 100-200 g of rice per child per day. The new generation, also known as GR2
contains β-carotene levels that will allow providing an adequate amount of pro-
vitamin A in normal children's diets in SE Asia.
REFERENCE:
1. Ye X, Al-Babili S, Klöti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000)
Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free)
rice endosperm. Science 287:303-305.
2. Burkhardt P, Beyer P, Wunn J, Kloti A, Armstrong G, Schledz M, von Lintig J,
Potrykus I (1997) Transgenic rice (Oryza sativa)endosperm expressing daffodil
(Narcissus pseudonarcissus) phytoene synthase accumulates phytoene, a key
intermediate of provitamin A biosynthesis. Plant J 11:1071-1078.
3. Beyer P, Mayer M, Kleinig H (1989) Molecular oxygen and the state of
geometric isomerism of intermediates are essential in the carotene desaturation
and cyclization reactions in daffodil chromoplasts. Eur J Biochem 184:141-150.
4. Bartley GE, Scolnik PA, Beyer P (1999) Two Arabidopsis thaliana carotene
desaturases, phytoene desaturase and zeta-carotene desaturase, expressed in
Escherichia coli, catalyze a poly-cis pathway to yield pro-lycopene. Eur J
Biochem 259:396-403.
5. Al-Babili S, Ye X, Lucca P, Potrykus I, Beyer P (2001) Biosynthesis of
beta-carotene (provitamin A) in rice endosperm achieved by genetic engineering.
Novartis Found Symp 236:219-228; discussion 228-232.
AUTHOR:
M. Ibrahim khan and Muhammad Noor Chan Department of Plant Breeding and
Genetics, University of Agriculture Faisalabad.