Solutions
A transgenic strand of maize that can lift millions out of poverty
Designing maize to be more efficient and resilient to external stresses to tackle food shortages and malnutrition.
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Maize is one of the most important food crops in the world, together with rice and wheat, providing at least 30% of the food calories to more than 4.5 billion people in 94 developing countries. [1] Today, maize is considered the staple crop for most of Sub-Saharan Africa, consumed regularly by more than 50% of the population. For over 300 million people in SSA, maize is their main food source, and its popularity is increasing. From 2007 to 2017, the area in which maize was grown in sub-Saharan Africa increased by almost 60%. [2] As of 2018, maize covered 25 million ha of land in sub-Saharan Africa, largely in smallholder systems that produce 38 million metric tons of maize per year. [3]
The graph below on the left, depicts the demand for maize between 1961 and 2009 in developing countries (pink), developed countries (green), and the world (orange). The graph below on the right clearly shows that the demand for maize (orange), is much higher than the demand for wheat (blue) and rice (green), the two other major staple crops in the world. [4]
Maize tastes great, it provides farmers with a source of income and feeds millions of people in developing countries. So what’s the issue?
There are many huge issues with maize, five of which I will outline for you here:
- Maize contains some vitamins A and E, but lacks the lower B vitamins that characterize other grains like sorghum or wheat. Maize is low in usable protein and lacks 82% of the vitamins that humans need to survive. [5] On top of the lack of important vitamins, maize also contains leucine and phytic acid which are antioxidants; substances that prevent the human body from absorbing nutrients, leading to nutrient deficiencies.
- Maize is a “thirsty” plant that requires up to 30% more water than wheat. [6] With climate change pushing average temperatures higher and higher, and droughts becoming more and more frequent, maize yields are decreasing at an alarming pace. Maize yields decrease by 70% when available water decreases by 10%. [7] Current estimates suggest that 25% of maize production is threatened by frequent drought, these stresses will increase with climate change, resulting in a potential loss of up to 10% in maize production in Africa and Latin America by 2055.
- Pests in developing countries cause yield losses up to 50% — the equivalent of annual revenue losses up to $0.9–1.1 billion for small-scale farmers across Africa. [8] Most African farmers cannot afford the expensive pesticides that would protect their crops from these pests, so instead, they are forced to suffer the consequences of major crop losses. Notorious maize pests in Africa include the maize stalk borer, fall armyworms, bore worms, and pollen beetles.
- Maize has a very long grow rate compared to other crops like tomatoes and lettuce. On average maize takes 3X more time from planting to harvest than most plants. [9]
- Maize is a crop that is very sensitive to weather changes and hot temperatures. Each degree day that is spent above 30°C reduces the final crop yield by 1%. Under drought conditions, crop yields are reduced by 1.7% a day. [10] Maize is also very sensitive to changes in soil composition and disruption near plant roots, which results in a 17% reduction in crop yields per year. [11]
All of this leads to a maize efficiency rate in SSA which is one-fifth less than the North American average.
All of these variables that limit maize yields in Africa are easily preventable. Pesticides and herbicides can fight off insects and pests, fertilizer can provide roots with the necessary nutrients to keep the plant strong and healthy, frequent watering and irrigation can prevent the plant from dehydration and modern farming equipment can turn soil preventing desertification.
Investing in the infrastructure needed to simultaneously deliver all of these solutions to crops is untenable for most small scale rural farmers. More importantly, these solutions don’t actually tackle the root problem: that maize is inefficient and extremely sensitive to external stresses.
My plan to improve maize.
Introducing my “new-and-improved” transgenic strand of maize which produces 40% more fruit, is resistant to pests, is drought tolerant, heat tolerant, contains more nutrients, and contains no antinutrients. This strand of maize is targeted specifically for Lesotho, Malawi, Zambia, and South Africa; four countries in Sub-Saharan Africa that are the primary growers of maize for Africa.
Overexpressing the Rieske FeS subunit to produce 40% more fruit per plant than average.
Photosynthesis is the primary determinant of crop yields. The efficiency in which a plant converts sunlight into ATP is the key factor that impacts final crop yield. (AKA: increasing photosynthetic efficiency = increasing crop yield.) [12]
Early work to improve photosynthetic efficiency through transgenic manipulation focused on the overexpression of a single individual enzyme in the photosynthesis cycle. This approach, however, has downregulated other important enzymes responsible for photosynthesis, which leads to a reduction in photosynthesis efficiency, and consequently in crop yield. [13]
My approach is to overexpress the Cytochrome (Cyt) b6f complex, a series of enzymes in mesophyll and bundle sheath cells which is important in photosynthesis.
The cyt b6f complex is responsible for regulating the electron transport capacity in plants, allowing more CO2 to enter into the leaves of the plant. An increase in CO2 concentration in plant leaves allows for better light conversion efficiency in the plant. In other words: the cyt b6f complex is responsible for allowing CO2 to enter into the plant, causing less energy to be wasted in the photosynthesis process, allowing the photosynthesis cycle to happen at a faster rate. An increase in cyt b6f has been shown to increase photosynthesis without changing concentrations of Rubisco or chlorophyll content, other important compounds in plants. [14]
The Cytochrome b6f complex consists of 4 large subunits:
- 32 kDa cytochrome f with a c-type cytochrome
- 25 kDa cytochrome b6 with a low- and high-potential heme group
- 17 kDa subunit IV
- 19 kDa Rieske iron-sulfur protein containing a 2Fe-2S cluster (usually just called Rieske FeS)
The Rieske FeS subunit of the cyt b6f complex has been shown to increase photosynthesis in C4 plants. [15] It is estimated that increasing the Rieske FeS subunit could double crop yields if done correctly.
Resistant to the maize stock borer and fall armyworms which destroy 10–15% of annual maize yields in Africa.
Lesotho, Malawi, Zambia, and South Africa have two primary pests which are notorious for destroying entire fields of maize: the maize stock borer and fall armyworm. The maize stalk borer accounts for more than 10% of annual crop loss in South Africa, and the fall armyworm causes up to 15% of annual crop losses in Zambia. [16] [17]
Most maize plants are genetically engineered to be resistant to pests already…but to North American pests, not pests in Africa. For example, BT corn is genetically engineered to overexpress the Cry1Ab protein, which is an insecticidal pesticide that is lethal to insects but harmless to humans. Another major issue with BT corn is that since it has been mass-produced throughout North America, pests are now developing resistance to BT.
Instead of overexpressing the very common Cry1Ab protein, my plan is to overexpress the Cry1F protein which is also derived from BT, but is much less commonly used. A study completed in South Africa proved that overexpressing the Cry1F protein in maize resulted in a plant that is 34% more resistant to armyworms than regular BT maize. [18]
Increasing drought tolerance by 40% by overexpressing ABA.
In southern Africa, drought during 2018 resulted in a food deficit of 3.3 million tons, putting an estimated 14 million people at risk of starvation. Current estimates suggest that 25% of maize production is threatened by frequent drought, resulting in annual crop losses of 10% in Africa. [19]
Maize is especially vulnerable to drought since it is a ‘thirsty’ plant that requires 30% more water than tomatoes. [20] Yield losses due to drought are responsible for 16% of total maize losses in Africa.
Abscisic acid (ABA) is a plant hormone that is responsible for many different development processes in plants, as well as the environmental stress response. Under drought stress, plants increase the production of ABA, which initiates stomatal closure reducing water loss, helping the plant avoid drought and conserve water. (AKA: plants produce more ABA under drought conditions which prevent water loss and keep the plant hydrated.) [21]
Plants overexpressing ABA have been shown to survive with 40% less water. [22] By genetically modifying maize to overexpress ABA, maize will need 40% less water on average.
There are three important genes involved in the ABA pathway:
- ZmPYL8
- ZmPYL9
- ZmPYL12.
These three genes are critical to the amount of ABA produced in plants. By overexpressing these three genes, plants will need an estimated 40% less water. [23]
Increasing heat tolerance in maize by overexpressing ICARUS1.
In the map of Africa below, you can see the temperature and climate of each country.
Rising global temperatures are expected to reduce yields by 30% by 2050. [24] This will put millions of people at risk of starvation. It is vital that maize, the world’s most important crop, can grow in the highest temperatures.
There is a gene in all plants called ICARUS1 (ICA1), which encodes a protein of the tRNAHis guanylyl transferase (Thg1) family. [25] The Thg1 family is responsible for plant growth in high temperatures. Overexpression of the ICA1 gene has been shown to aid plants in surviving at hot temperatures and increase their growing rate in warm temperatures. ICA1 has been nicknamed the “miracle gene” since overexpression of this single gene allows plant to grow at temperatures up to 30°C higher than usual. [26]
Contains more nutrients.
Globally one in five deaths is associated with a poor diet, the equivalent of 11 million deaths. [27] This number gets even higher in Africa where people turn to starchy/carbohydrate-dense foods that will sustain them longer than nutrient-dense fruits and vegetables. Maize is very high in fiber and carbohydrates, but lacks essential nutrients and minerals like iron, zinc, Vitamin A, C, B, K, and E. [28]
Iron deficiency anemia (IDA) is one of the most common micronutrient deficiencies which effects 2 billion people worldwide. Maternal and neonatal mortality due to IDA are significant contributors to the global mortality rate, especially in the developing world, affecting 43% of children, 38% of pregnant women, and 29% of non-pregnant women in Africa. [29]
Around 250 million preschool children suffer from vitamin A deficiency and are at a higher risk of death as a result of measles, diarrhea, and malaria. On a yearly basis, between 250,000 to 500,000 vitamin A-deficient children become blind, with half of them dying within a year of losing their vision.[30]
Iron
Nicotianamine synthase (NAS) is an enzyme that regulates iron concentration and production in plants. NAS also plays an important role in transporting nutrients throughout the body and helping the blood to absorb nutrients. [31]
It has recently been discovered that the enhancement of NAS increases iron and zinc levels in rice, soybean, and sweet potato. [32] There are four important genes involved in the NAS pathway which I plan to overexpress in maize:
- HvNAS1
- OsNAS1
- OsNAS3
- OsIRO2.
Vitamin A
β-carotene is the biosynthetic pathway that is the precursor to Vitamin A. In the example of golden rice, genes involved in the β-carotene pathway were overexpressed, creating a transgenic strain of rice that contained 29 times more vitamin A than regular rice. [33]
Imitating golden rice, I believe that by overexpressing the crtB and crtI genes, the vitamin A concentration in the maize endosperm can be increased up to 34-times the regular amount (modeled after golden rice 2). The 8 genes involved in the β-carotene biosynthetic pathway are: y1, zds1, lcyE, crtRB3, lut1, crtRB1, zep1, and ccd1.
Contain no antinutrients.
Antinutrients are natural compounds in foods that interfere with the body’s absorption of nutrients, preventing the body from absorbing nutrients in food.
Maize contains the antinutrient phytic acid, which has a strong binding affinity for calcium, magnesium, iron, copper, and zinc, preventing their absorption. This means that no matter which nutrient biosynthetic pathways are genetically engineered in maize, if phytic acid is present in the endosperm, a very little amount of the vitamins will actually get absorbed. [34]
To prevent this from happening I want to down-regulate some of the essential genes in the phytic acid biosynthetic pathway. Attempts to do this have been previously made, but they have decreased germination rates and seed size. [35]
A newly discovered complex called Myo-inositol 3-phosphate synthase (MIPS) has been discovered as a precursor to the phytic acid biosynthetic pathway. By knocking out the genes involved in the early stages of the phytic acid biosynthetic pathway (which is the MIP pathway), I can prevent phytic acid from ever being produced. There are several genes that seem to be involved in the creation of MIP: MIPS, IMP, MIK, 2-PGK, IPK2, ITK, IPK1, MRP, AtMIPS1, AtMIPS2, and AtMIPS3. [36] [37]
The main three genes that work together to produce MIP are AtMIPS1, AtMIPS2, and AtMIPS3 [38] To achieve the MIP co-suppression, I will do a triple knockout of AtMIPS1, AtMIPS2, and AtMIPS3.
How will I actually overexpress and de-regulate all these complexes and genes?
I will be using two plasmids to create this transgenic maize strand. Plasmid 1 will be for CRISPR changes — Cas9, sgRNA, homologous regions — . Plasmid 2 will be for integration changes — shuttle vectors, integration sequences — .
Plasmid 1
Plasmid one will contain one cas9 enzyme that will make all the necessary cuts, but will contain a sgRNAs and homologous regions for each edit I am attempting to make.
Overexpression of the Rieske FeS subunit
Inspired by the work of Simkin et al. [39], a sgRNA will be created to target the Rieske FeS subunit. The full coding sequence length of the Rieske FeS subunit has been transcribed as the following: (5′caccATGGCTTCTTCTACTCTTTCTCCAG-3′) and (5′-CTAAGCCCACCATGGATCTTCACC-3′).
The sgRNA will be modified to contain the EcoRI restriction site which will make it possible to cut out three base pairs (CTT) of the target DNA. The three base pairs I am targeting to cut out are on the 2Fe-2S center of the Rieske subunit which is the main control center for the growth of the subunit. The absence of the three base pairs should result in a significant increase in protein production and growth of the subunit, resulting in a much higher photosynthetic efficiency rate and larger crop yields. This edit will be under the control of the 35S tobacco mosaic virus promoter. The homologous region of DNA will include the PSI Lhca1 protein which was shown to increase final crop yield by 40%, from the work of Simkin et al. [40] The plasmid will correspond to the NGG PAM site.
Overexpression of Abscisic Acid (ABA)
ABA will be under the control of the super promoter which will consist of the octopine synthase enhancer. Building off the work of Yue et. al [41], the plasmid will also include the manopine synthase promoter, a plant-specific promoter known for its strong expression rates.
Since the ABA biosynthetic pathway is already present in maize, the CRISPR-Cas9 system will be created to cut out a short 8-base pair sequence on the ABA complex which limits the production of the complex. By cutting out these 8 base pairs and inserting a gene with a high growth rate into that spot, the plant will require 40% less water. These 8 genes, as demonstrated by Lu et al.[42], are responsible for the formation of IAA which is a compound that limits the amount of ABA produced in cells. Getting rid of the gene involved in forming IAA will allow ABA to be mass-produced in cells. The sgRNA will contain the phloem pole pericycle (PPP) gene which should increase the production of ABA. The crRNA sequence of the sgRNA will be complementary to the DNA of the ABA complex and correspond to the NGG PAM site.
De-activate the Myo-inositol 3-phosphate synthase pathway
Modeling after the work of Fleet et. al [43], I will use Cas9 to deactivate the AtMIPS1, AtMIPS2, and AtMIPS3 genes involved in the creation of the MIP pathway. To prevent the creation of MIP, these three genes must be de-activated, or turned off. The cas9 will make a cut in any area of the gene sequence, allowing the homologous region of DNA to be inserted. The homologous DNA will include methylation, a molecule that is used to suppress genes by slowing transcription of the gene and turning off promoters. [44] Once the sequence of methylation is inserted into the gene, the DNA will repair the double-stranded break, smoothing methylation into the genome. Once the protein creation cycle begins, methylation should suppress the creation of the proteins for AtMIPS1, AtMIPS2, and AtMIPS3, effectively shutting off these three genes, preventing MIP from being created.
The work of Karmatar et. al [45] shows that if these three genes can successfully be deactivated, phytic acid should not be produced in maize, and even if it is, the production rate is 91% less than average.
Plasmid 2
Plasmid two will be a shuttle vector and include all the DNA that is being inserted into the maize genome, not DNA that I am changing in the genome like in plasmid one.
Plasmid two will include the following:
Overexpression of the Cry1F protein
Modeling after the MON810 strand of maize created by Monsanto [46], the Cry1F will be a synthetic replica of the Cry1F gene from the soil bacterium Bacillus thuringiensis (Bt). The expression of the Cry1F gene will be regulated by the cauliflower mosaic virus 35S promoter and hsp70 maize intron sequence which will drive the expression of the Cry1F gene. Introns are untranslated regions of a gene that are cut off when mRNA is produced, they sometimes contain short sequence motifs that regulate gene transcription. The hsp70 maize intron will help increase the transcription rates of the Cry1F protein inside cells. The overexpression of the Cry1F protein will result in resistance to the maize stalk borer and fall armyworm.
Overexpression of the ICARUS1 gene
The work of Lu et al [47] demonstrates how simple it is to design a shuttle vector to insert the ICARUS1 (ICA1) gene into the genome of a plant. The proteins coding for the ICA1 can be activated in the maize genome without the need for any molecular cuts. The MINSVGVVRK, EAENCNCLQV, and LHILSYS genes are the main genes involved in the formation of ICA1 [48]. Inserting the DNA of these three proteins via plasmid 2 into the maize genome will result in the formation of the ICA1 gene. The plasmid will be under the control of the cauliflower mosaic virus 35S promoter which will help increase the transcription rate of the ICA1 gene. If overexpression of the ICA1 gene can be achieved, the plant should be able to survive at temperatures up to 30°C higher than average. [49]
Activate β-Carotene biosynthetic pathway
The activation of the β-Carotene biosynthetic pathway is modeled closely after the work of Beyer and Protraykus, co-creators of golden rice [50].
Phytoene synthase, phytoene desaturase, ζ-carotene desaturase, and lycopene β-cyclase, four of the primary genes involved in the creation of Beta Carotene, will be enclosed in the plasmid and transfected into the maize genome. On top of these four genes, lcy, aph and nptII will also be added to the plasmid to increase the β-carotene production as much as possible.
The four genes will be under the control of the CaMV 35S promoter.
Overexpression of the Nicotianamine Synthase (NAS) pathway
The four genes (HvNAS1, OsNAS1, OsNAS3, OsIRO2) involved in the NAS biosynthetic pathway can be overexpressed with strong promoters to make more copies of these genes. Conveniently, maize already contains HvNAS1, OsNAS1, OsNAS3, and OsIRO2, so by putting the genes under the control of the maize ubiquitin promoter via a shuttle vector, the genes will be overexpressed. Nozoye et. al [51] demonstrate that iron fortification can be increased up to 3 fold by overexpressing the NAS pathway, by simply activating the key genes involved in the pathway.
The genes will be under the control of the maize ubiquitin promoter. The integration sequence will also include the HvNAS1 gene under the control of the CaMV 35S promoter which will help activate the vitamin D pathway as well as demonstrated by Suzuki et. al. [52]
Transfection
The transfection method I will use for both plasmids will be microparticle bombardment of plant cells using a biolistic (gene gun). The process of microparticle bombardment is shown below:
Once the plasmids are designed and created they will be transfected into the maize endosperm via the microparticle bombardment method shown above.
And that’s my idea!
If I were to start testing and experimenting with my idea in the lab and all the science matched up and it worked! — this strand of maize could provide millions more people with food, tackling the global crises of food shortage and malnutrition. My hope is that one day I will actually create a strand of maize similar to what I have just outlined and can then commercialize it across Africa and India, helping to provide millions more with food on their tables and a steady income.
My name is Rachel, I am a 15 y/o who’s main goal in life is to end poverty and hunger. I love being outside, spending time with my family, and learning about science and technology. You can email me at: runnerrachel.lee@gmail.com, or message me on LinkedIn. Don’t forget to sign up for my monthly newsletter here. Thanks so much for reading!
