Designing a Biofortified Crop
This section introduces an idea for creating a rice variety with increased iron content in order to counteract the widespread iron deficiency and prevent the severe health conditions related to it. While it is not a complete research plan, it could give you a glimpse of how one could approach designing a new crop variety with useful features. With the right management and a bit of luck, those crops could help to solve some of the world's malnutrition issues and the resulting socioeconomic problems.
Last update: Sep 2020
Iron deficiency is an omnipresent nutritional disorder, affecting developing and developed countries alike. In fact, according to WHO, it is the most prevalent and largest-scale nutritional disorder in the world. It is estimated that 30% of the world’s population, which amounts to 2 billion people, are anemic. Anemia occurs due to iron deficiency, though the condition is often aggravated by infectious diseases such as malaria, tuberculosis, and HIV/AIDS. Iron deficiency can contribute to impaired development, poor work performance, deteriorating health, and premature death. It is especially prevalent in preschool children (Fig.1) and pregnant women. In the case of developing countries, as much as one in two pregnant women, and 40% of preschool children, suffer from anemia. It is estimated to contribute to 20% of all maternal deaths. Therefore, increasing iron intake is a major goal in improving population health. This can be achieved in several ways, such as the consumption of foods rich in iron, food fortification, and intake of iron supplements.
Figure 1. Prevalence of anemia by country
There are two forms of dietary iron: heme and non-heme iron. For humans, absorption of heme iron usually reaches around 15%–35%, while the absorption of non-heme iron is 2%–20% only. Heme iron and non-heme iron are commonly found in meat, poultry, fish, and seafood. On the other hand, plants contain predominantly non-heme iron only. Heme accounts for 95% of functional iron in the human body. It is located in the center of hemoglobin and myoglobin proteins, which are present in large quantities in meat. Therefore, meat is thought to be the richest source of heme iron. However, depending on household income, health conditions, diet, ethical values, and other lifestyle choices, numerous individuals cannot or will not consume meat or other heme-rich products. While for some of those individuals, fortified foods or iron supplements are a viable alternative to meat intake, part of the world’s population cannot access those as easily.
To prevent iron deficiency in the individuals without access to fortified foods or iron supplements, and to augment the existing methods of counteracting it in wealthier households, I would like to propose genetically engineering a crop that would be able to partially or fully satisfy the daily iron intake requirement. As non-heme iron is not absorbed into the body as readily as heme-iron, I would like to create a biofortified plant that could supply the latter, in the form of myoglobin proteins. In 2020, Carlsson et al. created Nicotiana benthamiana (tobacco) that could express functional human myoglobin (Mb) in its leaves, demonstrating this plant’s potential for the production of heme proteins for pharmaceutical development purposes. This study suggests that some plants, perhaps including some crops, could be able to produce heme-containing myoglobin, which could be potentially used for supplying dietary iron.
Choice of Crop
Rice (Oryza sativa) is the staple crop in many regions, feeding over two billion people in Asia and hundreds of millions in Africa and Latin America. It is said to be the most important food crop in developing countries. It is grown in over 100 countries, and 90% of its production comes from Asia. On a global scale, rice supplies around 19 % of dietary calories and 13 % of proteins. Therefore, it is a good candidate for heme iron biofortification. In this specific experiment, I would like to use japonica rice (Oryza sativa L.) cultivar Tsukinohikari, which has been shown to readily undergo the Agrobacterium-mediated gene transfer (Hiei et al., 1994).
The gene I would like to focus on is a pig (Sus scrofa) Mb gene coding for 154 amino acid long myoglobin, which I would like to express predominantly in rice endosperm (seeds). For this purpose, the Mb gene needs to be placed under the control of an endosperm-specific (or preferential) glutelin gene promoter, for example, OsGluC, OsGluB-5, or OsGluA-2 promoter. In 2008, Qu et al. showed that the OsGluC promoter exhibits the highest activity out of six (including the ones listed) promoters tested, driving even expression of the GUS gene throughout the whole endosperm. Therefore I would like to use the OsGluC promoter to drive the expression of SsMb in rice seeds.
Furthermore, in order to produce an even more nutritious rice variety, I would like to increase the concentration of myoglobin in endosperm by facilitating iron transport within the plant and into the endosperm. Iron transportation within the plant can be improved by overexpressing nicotinamine synthase (NAS) genes, as nicotinamine (NA) has been shown to chelate metal cations, including Fe(II) and Zn(II). The upregulation of the barley NAS gene (HvNAS1) was shown to increase iron concentration in polished (white) rice seeds. Therefore I will use HvNAS1 under the OsActin1 promoter in this study. Moreover, iron accumulation in the endosperm can be augmented by the expression of rice NA-Fe(II) transporter gene OsYSL2 under the control of the rice sucrose transporter promoter OsSUT1. This combination of HvNAS1 and OsYSL2, in addition to soybean ferritin gene expression, has been shown to increase iron (ferritin) content in rice before (Masuda et al., 2012). Therefore I would like to employ this approach, substituting ferritin (non-heme iron source) under OsGlb1 and OsGluB1 promoters with SsMb (heme iron source) under the stronger OsGluC promoter. Besides, I would like to test the biofortification results using SsMb under the OsGluC promoter alone, in order to assess whether the additional iron enrichment strategies can indeed increase iron content.
I will clone SsMb under the OsGluC promoter into a viral vector, as well as create a gene cassette containing SsMb under the OsGluC promoter, HvNAS1 under the OsActin1 promoter and OsYSL2 under the OsSUT1 promoter. Notably, the 3-gene construct will be very large. Therefore, I will use pBIGRZ1 as a binary vector since it has been demonstrated to introduce large sequences into rice plants before (Masuda et al., 2012). Hygromycin phosphotransferase (HPT) gene that contributes to hygromycin resistance will serve as a selectable marker in this experiment.
The 2 constructs will be introduced into rice by co-cultivation of Agrobacterium tumefaciens and scutellum-derived rice calli. The infected calli will then be cultured on selective medium containing hygromycin. The successfully transformed tissues will proliferate. Thus, Mb and Mb-NAS-YSL2 transgenic rice lines (T0 transgenic plants) will be obtained. The calli will then be transferred to a regeneration medium containing hygromycin and cultivated further. Next, the rice plants will be grown in a greenhouse, and self-pollinated, giving rise to T1 plants, which will be tested for hygromycin resistance, Mb, NAS, and YSL2 transcription, and heme iron content. T2 plants will be made and evaluated in the same way.
SsMb, HvNAS1, and OsYSL2 transcription levels will be assessed by performing qRT-PCR on RNA extracted from crushed rice seeds, with transcription levels normalized to alpha-tubulin expression. The presence of myoglobin protein can be detected with SDS-PAGE using the extract from ground rice endosperms. Iron content in polished rice seeds can be assessed using inductively coupled plasma atomic emission spectroscopy (Masuda et al., 2012).
If the heme iron content in the endosperm of transgenic lines will be significantly (several times) higher than that of non-transgenic Oryza sativa L., the next stage of experiments (field study) can commence. Also, assessing the differences in iron content between Mb and Mb-NAS-YSL2 rice lines will provide hints as to whether improving iron transportation could increase the amount of Mb in the endosperm, thus increasing the heme iron content. It is also possible that iron content would increase without a peak in Mb concentration, which would suggest that NAS and YSL2 contribute to iron accumulation in the endosperm via mechanisms other than Mb synthesis promotion. In this case, an additional study comparing Mb-NAS-YSL2 and NAS-YSL2 transgenic lines would be recommended. In the current experiment, however, the natural progression is to conduct a field study of the performance and yields of the two biofortified crops.
If possible, the T2 Mb and Mb-NAS-YSL2 rice lines, together with control, could be planted in an isolated rice paddy, and grown under similar conditions to regular crop rice, in regions that customarily grow rice, such as Niigata Prefecture in Japan. The yield, iron content, and appearance should be assessed.
Furthermore, experiments assessing whether myoglobin produced by the plant can withstand thermal processing of rice during cooking are necessary. Also, as NA has been shown to chelate other metal cations beside Fe(II), it is essential to assess whether the Mb-NAS-YSL2 rice seeds accumulate more toxic heavy metal cadmium than the non-transgenic control.
Moreover, to assess the safety and nutritional value of the crop, in vivo experiments on mammals are recommended. The two rice varieties could be fed to rats with and without anemia, and their influence on hemoglobin levels (using hemoglobin repletion bioassay) and the general health of the animals could be evaluated. Finally, a pilot study, which evaluates markers such as serum ferritin and total body iron in consenting adults fed the iron-rich rice, should be conducted before this modified crop makes its way into everyday use. If possible, preschool children should also be included in the study, provided parental consent is granted.
Overall, these results would provide preliminary information on the potential of the new rice variant in agriculture, and its promise of becoming a safe source of dietary iron for humans.
The study outlined above could result in the creation of myoglobin-producing rice variety, which could be used as a source of heme iron that is readily absorbed into the human body. Thus, the new crop could prevent iron deficiency in populations relying on rice as the staple food, and thus significantly improve population health. This study should be possibly most rigorous and exhaustive, without inspiring concerns questioning its ethical clearances. It should readily address the public’s doubts regarding this genetically modified crop, thus facilitating its possibly fastest introduction into the fields of developing and developed countries alike. However, as shown by the story of golden rice, public trust, and quick industrial success cannot be guaranteed (Dubock, 2014).
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