[7-8] - GM (Iron Deficiency)

Question 1

What are the three broad categories of nutrients we get from plant-based food?

Answer 1

1. MACRONUTRIENTS - contain energy/calories (e.g., carbohydrates, lipid oils, proteins, fibre for bowel health and satiation)
2. MICRONUTRIENTS - vitamins and trace minerals
3. PHYTONUTRIENTS - protective functions

Question 2

Explain the fundamental issue with many of the plants we use as a source of food in terms of nutrition

Answer 2

Although plant foods can contain almost all of the mineral and vitamin nutrients that are essential for human nutrition, concentrations of many nutrients are low in the crops that form the majority of our diets

For example, zinc is low in many plant species, but EXTREMELY low in almost all wheat bean varieties

Calcium is more variable across plant species, but is also EXTREMELY low in beans and wheat

This is because crops are selected based on ease, energy and taste, but not necessarily on micronutrient content - thus, crops represent a small proportion of all plants

Question 3

What is the major downside of the priorities of the Green Revolution?

Answer 3

The Green Revolution (1960s-70s) increased high-yielding cereal production at the expense of nutrient-rich pulse/legume cultivation

These high yielding varieties of rice, wheat and maize were attractive to farmers, and cereal crops do provide calories and protein

However, they provide very few other nutrients, and half the global population now suffer from at least one nutritional deficiency (with women, children and poor people being disproportionately affected)

RICE is an especially prominent example (see other FC and notes)

Question 4

Explain how the world’s reliance on rice presents an issue for nutrition?

Answer 4

Rice is a poor source of many minerals, as grains are milled to prevent spoilage, which removes the nutrient-rich embryo and aleurone layers

Since half the world population eats rice daily, and many are entirely reliant on it, this means many people receive limited micronutrients

Question 5

Explain the concept of biofortification and the possible types

Answer 5

Fortification is the supplementation of additional nutrients, e.g., vitamins or minerals, into commonly consumed foods (e.g., cereals are fortified post-harvest during processing, many vegetable oil spreads are fortified with vitamins A and D)

BIOFORTIFICATION is the improvement of plant nutrient content PRE-harvest:
- AGRONOMIC biofortification via fertilizers (e.g., zinc in wheat in Turkey) - not possible for all minerals
- GENETIC biofortification (selective breeding) - limited due to existing diversity in varieties
- TRANSGENIC biofortification (genetic engineering)

Question 6

Explain how Biofortification may be able to mitigate Vitamin A deficiency

Answer 6

Background: 100 million children are VitA deficient, 500,000 become blind each year; improving dietary uptake of carotenes could reduce child mortality by 25%

GOLDEN RICE is a form of GM rice aiming to prevent Vitamin A deficiency

ß-carotene biosynthesis genes are introduced into rice grain endosperm tissue, causing increased ß-carotene to be produced, which is then converted to vitamin A in the body upon consumption

Initial research began in the 1990s and showed high ß-carotene yields; human feeding trials confirmed efficient conversion into VitA; field trials have taken place, but Golden Rice is still not available to most farmers and public due to strong political resistance

Question 7

Name the 5 highlighted examples of minerals which are lacking in many people’s diets, and the effects thereof

Answer 7

Iron -> 60% of pop; weakened immune function and impaired growth

Zinc -> 30% of pop; impairs development and immune system

Iodine -> 30% of pop; Goiter disease

Selenium -> 15% of pop; low fertility in men, cancer risk

Calcium -> 40-60% of women in UK/USA; osteomalacia, osteoporosis

Question 8

Explain the severity of global iron deficiency

Answer 8

It affects 4.5 billion people - more than any other condition

2 billion people are anaemic (48% of all pre-school-aged children)

It weakens the immune system, impairs growth and development and is exacerbated by malaria and worm infections

Question 9

What are the main strategies to reduce iron deficiencies?

Answer 9

1. Increased Fe uptake via supplements
2. Control of parasites (e.g., worms, malaria, schistosomiasis)
3. Improved or balanced diet
4. Fe BIOFORTIFICATION?

Note: 7.5X increase in iron per gram of dry weight of rice needed to reach recommended levels (currently 2µg Fe/g)

Question 10

What are the 4 (initially mentioned and highlighted) factors affecting the amount of iron available to the consumer?

Answer 10

Soil availability, assimilation efficiency into plant, sequestration into grain/food, assimilation efficiency and bioavailability

Question 11

What are the 4 possible strategies for transgenic biofortification of rice with iron?

Answer 11

Could introduce genes to increase:
- Mineral accumulation into roots
- Mineral transfer into edible tissues (e.g., grains)
- Mineral storage
- Mineral assimilation efficiency

Question 12

What is the Rhizosphere, and what major soil factor affects nutrient bioavailability to the plant?

Answer 12

The Rhizosphere is the root-soil interface, including the root surface, soil solution and soil particles

The soil solution is the pool of dissolved nutrient ions which are available for root uptake (but low concentration), while the soil particles are the main nutrient reserve in the rhizosphere

SOIL pH affects bioavailability ot the plant - but the optimum pH varies greatly for different minerals, meaning the optimum growth pH overall is around 5.5-6.5 (iron specifically is most available at pH 4.5, and decreases in alkaline conditions)

Question 13

Explain why iron bioavailability can be a problem despite the abundance of iron in the earth’s crust

Answer 13

Most forms of iron cannot be taken up by plants:
- In soil, Fe is present in various oxidation states
-> Fe(III) oxides are essentially insoluble in water
-> Fe(II) is soluble in water, and gives the divalent cation Fe2+, which is how Fe is mainly taken up by plants
-> HOWEVER, the solubility of Fe(II) depends strongly on pH - NOT bioavailable in alkaline soils (pH>7), leading to iron deficient plants

Question 14

What is the significance of root growth in terms of bioavailability of nutrients?

Answer 14

When roots take up minerals, a “nutrient depletion zone” quickly forms in the soil surrounding the root

For maximum nutrient uptake, roots must avoid depletion zones, by elongating and exploring new areas (e.g., vira formation of secondary roots and root hairs)

Question 15

Explain how plants attempt to increase nutrient bioavailability via control of soil conditions

Answer 15

Plants control soil pH to improve bioavailability:
-> A proton pump (H+-ATPase) releases protons from the root cell into the soil solution, acidifying it
-> Release of CO2 forms carbonic acid (H2CO3) which yields more H+ in the soil - this H+ displaces positively charged metal ions from negatively charged soil particles, allowing uptake
-> Thus, the plant both increases the solubility of key elements such as iron, AND releases H+ ions to displace and release positive metal ions

A pH indicator dye (bromocresol purple) clearly shows the acidification of soil surrounding grass roots

Question 16

Explain what is meant by “Strategy 1” Iron Uptake Method, and which plants use this

Answer 16

DICOT plants (e.g., peas, tomatoes, soybean, etc.) use Strategy 1:
-> Ferric [Fe(III)] chelate reductase (FRO) reduces oxidised iron(III) into soluble Fe2+, and secretes Chelates - small molecules which “pull” iron off the soil particles
-> Soluble Fe2+ then enters the root cell via the IRT1 transporter, assisted by the low pH induced by the H+-ATPase pump

Note: IRT1 (iron regulated transporter 1) is a high-affinity Fe2+ transporter for uptake into plant roots. Its importance is show by the poor growth of irt1-1 KO Arabidopsis mutants, which are rescued by addition of Fe

Question 17

Explain the principle of GM to increase mineral accumulation into roots (the first of the 4 broad strategies) in Strategy 1 plants, and evaluate its success

Answer 17

Aim: Overexpression of IRT1 to increase iron uptake into roots

Put IRT1 behind a strong 35S promoter
-> More iron does get into the LEAVES than in the WT
-> BUT plant height and number of tillers both decrease
-> Notably, no increase in iron in the GRAINS

Also, increasing Fe(III)-chelate reductase by expressing a FRO gene from yeast failed to increase rice content in each grain (although these plants did show better growth on low Fe soil)

Limitations of Manipulating Strategy 1 Genes:
- Overexpression of FRO or IRT1 did successfully increase iron transfer into the roots and up to the leaves of rice plants
- BUT iron content is NOT increased in the grains
-> This suggests a potential bottleneck in grain iron transfer, and implies that other mechanisms are needed to enhance this process

Question 18

Explain how root exudates can facilitate plant mineral uptake

Answer 18

Root cap cells and epidermal cells secrete:
1. High molecular weight compounds - mainly mucilage (a gelatninous material consisting of polysaccharides, which forms complexes with organic and inorganic soil particles and acts as a carbon source for symbiotic microorganisms, which reduce metal ions more efficiently than plants can)

2. Low molecular weight compounds:
   -> Organic acids (e.g., malate, citrate, carboxylate) which increase the solubility of metals by forming complexes with them
   -> Phytosiderophores - small molecular weight compounds that chelate metals and facilitate their uptake

Question 19

Explain what is meant by “Strategy 2” Iron Uptake, which plants use it, and how it differs from Strategy 1

Answer 19

Monocot plants (i.e., grasses such as rice, wheat and maize) use Strategy 2, though many also retain the Strategy 1 process of Fe2+ uptake via IRT1 transporter

In Strategy 2:
- Methionine is converted to Nicotinamine, then to Phytosiderophores (PS), which are secreted into the rhizosphere through the TOM1 transporter
- In the rhizosphere, PS form complexes with Fe(III), thus removing them from soil particles
- Rather than Fe(III) being reduced before uptake, Fe(III)-PS complexes are directly taken up into the cytoplasm, where Fe is removed and quickly reduced, and PS is recycled

Question 20

Explain the process of Phytosiderophore (PS) biosynthesis in Strategy 2 plants

Answer 20

1. Methionine is converted to S-Adenosylmethionine (SAM) by SAM Synthase
2. SAM is converted to Nicotinamine by Nicotinamine Synthase (NAS)
3. Nicotinamine is converted to Deoxymugineic acid (DMA), a phytosiderophore, via Nicotinamine aminotransferase (NAAT)

Question 21

Explain the feature that correlates with Fe deficiency tolerance in barley

Answer 21

Secretion of DMA (a phytosiderophore) is much higher in barley than rice, and correlates with greater tolerance of iron deficiency in barley

This suggests barley is more efficient at DMA biosynthesis, and that this gives it an advantage in tolerating Fe-deficiency

Question 22

Explain the SECOND route by which mineral uptake was targeted by GM, and evaluate how successful this was

Answer 22

Overexpression of Nicotinamine aminotransferase (NAAT) in rice (by isolating two barley genes, HvNAAT-A and HvNAAT-B, and introducing them into rice using Agrobacterium)

WT and NAAT rice plants were then grown in low iron conditions

NAAT plants did grow taller and had greater dry weight, HOWEVER there was, again, no significant increase in rice grain iron content

Question 23

Explain what the three transgenic experiments targeting mineral uptake suggest about transgenic biofortification of iron

Answer 23

All three experiments (targeting IRT1, FRO, and NAAT) failed to increase iron content in the actual rice GRAINS, despite increasing uptake into the roots

This suggests that accumulation of Fe into the plant is not a problem, but that sequestration of iron into the seed is the major bottleneck

Possible rate-limiting steps include:
-> Phloem transport from leaves
-> Unloading (from xylem and phloem) for grain filling
-> Grain sink strength

Question 24

Name the (mentioned) protein and molecule which are vital for transport of iron within the plant, and how these can potentially be targeted for transgenic biofortification

Answer 24

NICOTINAMINE (NA) is important for iron distribution in the phloem and movement to the seed, as iron is much more readily translocated when bound to NA

The protein YSL2 transports FE(II)-NA from the phloem into the seed

When aiming to increase Fe mobilisation in the phloem and into the seed (the second of the four broad strategies), there are two routes:
1. Increase internal NA content via overexpression of NA synthase
2. Increase FE-NA transport via overexpression of YSL2 transporter

Question 25

Explain the process of Nicotinamine biosynthesis in plants

Answer 25

Methionine is converted to S-Adenosylmethionine (SAM) by SAM Synthase

2. SAM is converted to nicotinamine by Nicotinamine Synthase (NAS)

Note: these are also the first two steps in the biosynthesis pathway of DMA (a phytosiderophore)

Question 26

Explain the transgenic experiments that aimed to increase iron transport into the seed, and evaluate how successful they were

Answer 26

1. Overexpression of NAS:
   -> The barley HvNAS1 gene was transformed in rice, either using a 35S promoter, or an Actin1 promoter
   -> This was successful! A 2-3 fold increase in iron content of the rice grains was achieved
2. Overexpression of YSL2:
   -> The rice OsYSL2 gene was placed behind the SUT1 promoter, thereby increasing its expression in vascular pathway/transfer cells
   -> This was successful! A 4-fold increase in rice grain content was achieved

Question 27

Name and describe the key iron storage molecule which was targeted to increase mineral storage (the 3rd of the 4 broad strategies)

Answer 27

FERRITIN
-> a widely conserved Fe storage protein in many organisms
-> capable of binding up to 4300 Fe atoms
-> plays an important role in preventing anaemia and Fe deficiency

Question 28

Explain the transgenic experiment which aimed to increase iron storage in rice

Answer 28

Expression of the Fe storage protein Ferritin in rice grains using a seed-specific glutenin promoter
(Can see it is expressed in endosperm and subaleurone but NOT in embryo)

-> This was successful! A 2-3 fold increase in iron content for both whole grain and milled rice

Question 29

Summarise the first three successful strategies for biofortification of iron, and explain whether they can be combined

Answer 29

1. NAS overexpression: 2-3 fold increase in grain Fe
2. YSL2 overexpression: 4-fold increase in grain Fe
3. Ferritin expression in seed: 2-3 fold increase in grain Fe

A DNA plasmid was constructed that allows introduction of 5 different iron-related transgenes into the genome simultaneously (2 versions of YSL2 for phloem and endosperm; 2 versions of Ferritin under endosperm different promoters; NAS1 under a constitutive promoter) as well as marker and reporter genes (kanamycin, hygromycin and -glucoronidase)

These “Fer-NAS-YSL2 Plants” showed a 6-fold increase in rice grain iron content, and field trials showed a 4.4-fold increase in milled grain Fe

Question 30

Summarise the challenges of general mineral assimilation from the diet

Answer 30

• Assimilation of minerals can vary greatly in plants due to the presence of Anti-Nutrients
• Most bioavailable plant sources of Fe can be as low as 5%
• In many cereals and legumes, chemical binding of Fe significantly reduces Fe absorption

As such, for many micronutrients (including iron), the total content in a food can be misleading, as the DIET BIOAVAILABILITY (the proportion of the nutrient that is digested and absorbed/assimilated) is much lower than total content.

Question 31

In what form is iron commonly found in seeds, and how does this affect mineral assimilation?

Answer 31

Iron is often found in a PHYTATE-BOUND form

Phytate (inositol hexaphosphate) is a negatively charged molecule which binds positive ions and interferes with their assimilation, making iron from plants much less readily assimilated than haem iron from meat

Question 32

How can mineral assimilation of iron from plants be improved transgenically?

Answer 32

PHYTASE ENZYME:

• Phytase is an enzyme which breaks down phytate-metal complexes to release free metals
• M14 3-3 Maize expressing the phytase enzyme released more permeable iron which was more efficiently taken up by intestinal cells than control plants

Question 33

Summarise the perspective on the future of GM iron biofortified rice provided at the end of the lecture

Answer 33

• Significant promise so far from field trials of Fer-NAS-YSL2 plants
• Other approaches also show promise - e.g., including other genes in multiple combinations (such as IRT1 + Phytase)
• Need more extensive field trials (to test for reduced yield performance) and human feeding trials
• The rice variety background may be important to the success of GM (e.g., expression of Fer-NAS-YSL2 was less successful in a tropical Japonica rice background)
• There is also a need to assess the risk of increasing accumulation of toxic metals (e.g., Cd)
• There may be the potential to enhance multiple nutrients at once (e.g., iron, zinc and Vitamin A)