Week 11: Food Quality; Biofortification

What are methods we can use to measure micronutrient deficiency

• hard, lots of issues in all & thus, lot of crtitque

• dietary intake (comparing what they eat vs reccomended diet)

• biochemical status (blood testing)

• functional/clinical outcomes (As many deficiencies are associated with disease)

Describe why measuring dietary intake allows measurement of micronutrient deficiency & note limitations

• can compare what individual is eating vs reccomended intake

limitations:

• people may not accurately recall what they ate (subjective)

• bioavailability is also a factor that affects micronutrient intake (Efficiency it absorbs into the gut)

• intensive to conduct as need to recruit lots of participants & survey etc.

• fails to consider demographical differences (different health burden, age, etc. may mean different reccomended intake)

Describe why measuring biochemical status allows measurement of micronutrient deficiency & note limitation

• blood testing to measure (e.g. ferratin - Fe or serum retinol - vitamin A)

• more objective

limitations:

• can be confounded by various states

• e.g. when sick ferratin levels spike, similar with serum retinol

Describe why observing functional/clinical outcomes allows measurement of micronutrient deficiency & note limitation

• many micronutrient deficiencies are assoicated with diseases - observing incidence can alert to various deficiencies

limitations

• counfounded by other causes of disease

List what these functional/clinical outcomes are due to:

• anaemia

• night blindness

• goitres

• neural tube defects

• Wernicke-Korsakoff syndrome

• Anaemia - low RBC count; associated with iron deficiency

• Night blindness - vitamin A deficiency

• Goitres - inflammation of thyroid - iodine deficiency

  • Often associated with increased consumption of pink himalyan salt (is not fortified with iodine unlike regular salt)

• Folic acid (especially important for pregant mothers for developing infant) -> neural tube defects (NTD)

• Thiamine (vitamine B1) -> Wrnicke-Korsakoff syndrome (fatal neurological disorder)

What is the best method to measure micronutrient deficiency?

Thus, there is no single best method

• biochemical status tends to be the most objective but also can be confounded

• need to carefully word statements to not overstate what our measurement tell us (e.g. insufficient consumption vs deficieny = dietary intake can only inform us of the former!)

Is micronutrient deficiency prevelant across the world? What is a study that illustrated this

• Consumption of 24hrs

• Observed distribution acorss population and modelled/projected this onto other countries

• Estimated that 68% of global population have dietary iodine inadequacy, 48% Vitamine A, 46% Zinc

What are measures to combat micronutrient deficiency

• supplementation (if deficient)

• food fortification (food manufacturers and farmers incorporate into food - add)

• biofortification (plant is fortified thus food is too)

What are limitations of supllementation & when is it applicable

• Take if reccomeneded (actually deficient)

• Feasbility is heavily dependent on consumer (behaviour)

  • Economic status (Affordability)

  • Acess to supplements (rural communities may not have)

  • Need to consistently take - requires a behavioural change by the consumer (problematic)

What are limitations/benefits of food fortification & what is it

• Food manufacturers and farmers incorporate micronutrients into food already consumed by people (e.g. wheat, rice, bread)

• Works well as there are foods the vast majority consume

• No change required by consumer; change is required by food manufacturers or bakers (problematic)

Describe food fortification in australia & methods (not highly important card)

• Fortification of certain micronutrients is mandated:

  • Thiamine fortficiation in 1991 into wheat flour

  • Later, in 2009 iodine and folic acid

• This enabled a 40% reduction in Wernickes-Korkakoff syndrome (thiamine)

• 14% national reduction in neural tube defects (folic acid); 74% in Aboriginal and Torres Strait Islander

  • Higher in Indigenous Australians as originally had higher deficiency incidence

  • Often micronutrient deficiencies disproportionately affect vulnerable communities

• Iodine - reduced 60% -> 9% in women of 'child-bearing age'

• Common methods:

  • Ferrous iron - blue powder appearance - common fortficant

    • Has a strong flavour + colour; not consumer appealing as alter flavour and texture

  • Iodine -> bread simply through iodised salt (instead of salt without iodine)

What is biofortification & what are the methods

• Fortification of certain micronutrients is mandated:

  • Thiamine fortficiation in 1991 into wheat flour

  • Later, in 2009 iodine and folic acid

• This enabled a 40% reduction in Wernickes-Korkakoff syndrome (thiamine)

• 14% national reduction in neural tube defects (folic acid); 74% in Aboriginal and Torres Strait Islander

  • Higher in Indigenous Australians as originally had higher deficiency incidence

  • Often micronutrient deficiencies disproportionately affect vulnerable communities

• Iodine - reduced 60% -> 9% in women of 'child-bearing age'

• Common methods:

  • Ferrous iron - blue powder appearance - common fortficant

    • Has a strong flavour + colour; not consumer appealing as alter flavour and texture

  • Iodine -> bread simply through iodised salt (instead of salt without iodine)

Why do biofortification & food fortification offer far reach across populations?

• Can deliver to vast majority as people generally consume the same food:

  • Rice - 16.5-20% of global calories; wheat - 15-19%; maize - 5% (more if consider animal feed (that we then eat))

  • Thus rice + wheat + maize = 50% global calories

  • Targetting these crops is highly effective

Compare impact across various measures to combat micronutrient deficiency

• $80 million dollars across each provides,

• Supplementation: vitamin A - 80 million women and children (1/5 population) in south asia - for 2 years (50 cents for 2 pills each effective for 6 months)

• Forfication: iron for 33% south asian population for 2 years (10 cents per person/ year)

• Biofortification (genetic): develop 6 nutrient dense crops

  • More economically effective/ viable

Describe prevelance of vitamin A deficiency

• 400 million people at risk

• Particularly in Asia and Africa; phillipines is severely affected

What is the other name for vitamin A

retinol

From what nutrient is vitamin A obtained

Beta-carotene

What is required for beta-carotene bioavailability in the gut?

Beta-carotene is highly hydrophobic thus needs to be consumed with fat/micelles for absorption

What cells absorb and metabolise beta-carotene & into what

enterocytes of small intestinal villi; convert into retinyl esters

What is a really good source of vitamine A & why

rich source of vitamin A is cod liver oil (already fatty and liver rich in vitamin A - retinyl ester form)

Describe the metabolic pathway of beta-caroten conversion into final useage sites

in enterocytes:

• beta-caroten converted by BCO1 into 2x retinal

• retinal converted by retinal reductase into retinol

• LRAT enzyme converts retinol into retinyl esters

retinyl esters enter lymph/blood and reach the liver

at the liver they are stored (80-90%) and when required converted back into retinol via hydrolases

retinol reenters the blood stream and reaches usage sites

in photoreceptors:

• via enzyme RDH5/ RPE655 (remember first & sight second)

• retinol converted into 11-cis-retinal

• this is combined with opsin, to form rhodopsin

in other cells

• via enzyme RDH10

• retinol converted into retinal

• then retinal → retinoic acid → RAR/RXR

What is the function of rhodopsin in photoreceptors?

Conformational shape change triggers electrical signal -> brain for sight

What is the function of retinoic esters in cells?

combined with proteins to transcribe genes important for retinol homeostasis

What are roles of vitamin A and what does deficiency cause?

• Roles of vitamin A:

  • Vision

  • Gene transcription

  • Immune function

  • Embryonic development

  • Bone metabolism

  • Haematopoiesis

  • Skin health

  • Antioxidant activity

• Vitamin A deficiency causes:

  • Increased heart disease risk

  • Vulnerability to infection

  • Higher risk of measles and diarrhoea

  • Night blindness & blindness

    • Implicated in 350,000 children going blind per year

  • Implicated in 670,000 child deaths (under 5 yrs old) per year

Why is rice a good target for vitamin A fortification?

Countries affected by VAD also have high rice consumption; thus, rice crop is a good target

Describe beta-carotene synthesis pathway in plants (only molecules)

GGPP → Phytoene → zeta-carotene → lycopene → beta-carotene

• grace pissed zack lost babe

Name enzymes required for each step in beta-carotene synthesis in plants

PSY: converts GGPP → Phytoene

PDS + Z-ISO: converts phytoene → zeta-carotene

ZDS + CRTISO: converts zeta-carotene → lycopene

LYCB: converts lycogene → beta-carotene

Why can rice not produce much beta-carotene for us?

• While rice plants have all genes necessary for enzymes, these are silenced in the endosperm/ rice grain (the part we consume); they are only expressed in vegeative tissue (As needed for photosynthesis)

• Thus, needed to activate enzyme expression in endosperm

What are the 3 genes added into golden rice 1 (GR1) & explain why & where from

• Gt1::NpPSY

  • Explaining gene name:

    • Part before :: is the promoter; part after :: is the gene

    • Gt1 promoter expressed specifically in the rice endosperm

      • Glutelin promoter

    • NpPSY: part capilised is the coding sequence

      • Np is the origin of gene - Np means daffodil

      • PSY - PSY enzyme

  • The PSY enzyme was thought as a rate limiting step, thus this gene was incorporated

• CaMV 35S::CRT1

  • CaMV 35S - constitutively active promoter

  • CRT1 - does the role of 4 enzymes (in typical pathway) - converts phytoene directly into lycopene

    • Simplifies process as now require less DNA to be incorporated

• Gt1::NpLCY

  • Endosperm promoter Gt1

  • Np = daffodil origin

  • LCY = enzyme gene

    • Thought was also rate limiting step thus added

Was GR1 successful?

• Produced 1.6 micrograms/g B-carotene

  • Crtiqued heavily by Greenpeace (an anti-GM originisation)

  • This is below the recommended intake - would need to consume 9kg to achieve this

Describe changes made to GR1 to produce golden rice 2

• Removed LCY gene which was found to actually not be rate limiting

  • This meant only had one T-DNA strand that needed to be inserted (simplified)

• Changed daffodil PSY into maize PSY gene which is more efficient

  • GR2 produces 31 microgram/g B-carotene (meeting the reccomended daily intake)

Compare T-DNA of GR1 & GR2

• Always need a terminator sequence (to stop polymerase)

• Tp - transit peptide gene required

  • B-carotene production usually occurs in the plastids

    • However, these enzymes are taken from soil bacterium thus don’t have a method to to enter the plastids

  • Thus needed for enzyme ability to enter plastids (organelles in plants) to carry out function

• Hygromycin in GR1 for selectable marker (hygromycin resistence gene)

• In GR2 pmi gene - pmi gene was used instead as a selectable marker,

  • Allows rice to metabolise mannose during tissue culture

	GR1	GR2
Number of T-DNA strands	2	1
enzymes incorporated	PSY, CRT1, LCY	Only PSY and CRT1
Origin of PSY	Daffodil (Np)	Maize
Selectable marker	Hygromycin resistence	Pmi (mannose metabolism)

Describe how patents was provided & how GR was distributed? + critique/ challenges

• Technology for production was protected by 70 patents across 32 organisations

• Would be expensive to pay the patent fee (for farmers) and hard to push for consumers, as this was intended as a humanitarian cause

• Parternered with Syngenta which obtained commercial rights without liscencing fee in any developing country

• Additionally, was able to get approval for seed collection, and re-use by the farmers (if making less than USD 10K in developing countries)

• Greenpeace attacked the parternship - as Syngenta was a private coorporation (may be corporate greed)

• Humanitarian board including ingo and peter to drive roll out to various countries

• Primarily given to international rice research institute (IRRI) in phillipines

Describe findings of GR1 field trials

• Conducted field trials for GR1 in 2004

  • Found there was no difference in yield or agronomic performance

    • This is crucial for farmer appeal

Describe relative amounts of beta-carotene across rice (wt, gr1, gr2)

Describe experimental evidence - feeding trials that affirmed GR improved vitamin A

• No evidence yet demonstrated that GR2 leads to increase vitamin A in humans

  • Attacked by greenpeace

• Experiment conducted with only 5 healthy people; it was difficult to conclude whether vitamin A improvements were due to golden rice or other dietary sources

  • To resolve this,

    • Grew golden rice in heavy water (water with an extra neutron in the hydrogen atom) (hydroponic growth)

      • This was very expensive

    • The heavy water incorporated into the beta-carotene -> retinol making them slighlty heavier than usual

    • In blood tests, the retinol with slightly heavier mass is linked to golden rice

    • Demonstrated promising results similar to that of supplements

• Replicated this study in China,

  • While the previous studies were only conducted on healthy adults, the target demongraphic for vitamin A deficiency is children

  • Demonstrated that GR provides 60% dietary intake

  • However, the study had to be retracted as later discovered researchers did not collect parental permission to feed the children GM rice

  • Despite being quite controversial, it demonstrates promising results

Describe challenges associated with GR & events

• In 2013 protesters uprooted all the field trial plants

  • Luckily other trials were there

  • However represents strong animosity toward GM

• IRRI released GR2 data showing it had a yield penalty (2016)

  • This was as the GR2 line had transgene intergration that disrupted the auxin gene (2017)

  • This greatly reduced root development

  • Luckily had progressed another line of GR2 that had the transgene insert into an intragenic (gene-less reigon) - GR2-R & transferred to GR2-E

• 2016: nobel lauretes signed a letter to request Greenpeace to stop protests particularly regarding golden rice

• 2018: FSANZ approved commercial sale of GR (not growth)

• 2021: Phillipines additionall approved a growing liscence however,

  • Greenpace and anti-GMO organisations filed a lawsuit to the court of appeals

  • Under the precuationary principle (which declares that if there is no scientific consensus, the government can ban technologies to protect environmental safety or human rigjts)

  • Can argue there is scientific consensus on safety

• 2026 - collated all field trials until the phillipine ban and published; demonstrated no agronomic penality in GR2-E event

  • Appeal to ban is pending

Why is Fe important in the body

• Iron is critical for function of several enzymes and proteins; particularly for haemoglobin in red blood cells

• It aids the storage of 2/3 of oxygen in the body & helps transport it

Is Fe deficiency common?

It represents one of the most common micronutrient deficiencies globally

How is Fe and Zn produced in monocots (wheat & rice)>

• Iron is not synthesised by plants, as it is a metal (atom)

• It is abundant in soil & intaken from there

• However, plants are not effective at intaking iron from soil

Why does wheat and rice not normally provide much Fe & Zn

• However, plants are not effective at intaking iron from soil

• Overall only 2-4 ppm Fe is present in rice grain, with most in the brain & germ

  • Bran and germ are removed when rice is polished to make white rice

• Additionally, iron is bound to phytate in the aleurone (outer part of rice; also part of brain)

  • Phytate is a bioavailability inhibitor

What is the target for Zn/Fe fortification in rice?

14ppm in endosperm

Describe Fe & Zn absorption in rice/wheat - process & controls

• In soil, iron is present in its insoluble form as ferric ions (Fe3+)

• Roots will secrete DMA (a metabolite) which chelates the insoluble (Ferric) form of iron

• The chealated complex can be absorbed into root cells

• There is an iron sensing gene network that tightly regulated iron uptake

  • This is because excessive iron can cause oxidative stress

  • Additionally, low iron reduces chlorophyll synthesis

  • The gene network provides an opportunity for modification!

Describe biosynthesis of DMA

• NAS enzyme produces Nicotianamine (NA) -> NAAT/DMAS enzyme produces DMA

Compare various Fe & Zn chelators & effects on bioavailability (of Fe in particular)

• NA increases iron bioavailability greater than DMA, vitamin C, and Epi

  • This was experimentally tested by growing intestinal cells & feeding them the iron + chelator complex and measuring ferratin production

  • Ferratin is a strong indicator for Fe bioavailability

What is the function of nicotianamine in Fe intake?

• Nicotianamine (NA) is also an iron & Zn chelator,

  • Increases greater Fe and Zn transport into the rice grain (within plant not soil)

  • Additionally increase NA -> some increase in DMA to increase uptake from soil itself

• Additionally, NA increases iron bioavailability greater than DMA, vitamin C, and Epi

What are the NA synthase genes in rice & which is most important

• These are OsNAS1, OsNAS2, OsNAS3 (note Os = rice; NAS = gene)

• Experimentally disocvered OsNAS2 was the most effective gene

Describe T-DNA for Fe & Zn fortified rice

• Constitutive promoter: 35S

• OsNAS2

• Additionally, 35S + nptII (neomycin resistence selective marker)

How were OsNAS2 rice plants grown & tested for increased Fe uptake?

• Placed in micronutrient-limiting conditions

  • Highly alkaline soil which promotes ferric ion formation in soil

  • This is the insoluble form of Fe, making it harder for plants to intake it

• WT plants grew poorly

• OsNAS2 plants demonstrated vigorous growth

  • This suggests increasing OsNAS2 expression, increases NA, which increases Fe

Describe field trials for OsNAS2 rice

• Partnered with global organisations to conduct field trials at IRRI (phillipines) and South America at the International Centre for Tropical Agriculture

• Here found there was no agronomic (yield and growth) impact on the GM plants

How was Fe & Zn content in OsNAS2 rice & wheat quantified?

• Utilised synchrotron x-ray fluroescence microscopy

  • Takes place in a giant circular building

  • The machine will shoot photons / x-ray

  • When these hit atoms they cause emission of an electron

  • The electron from outer shells will therefore jump to lower energy level (to replace the removed on)

  • This jumping form high to low, emits energy

  • This energy can be detected & corresponded to an atom

• very hard to do but snchotron allows!

Compare WT & OsNAS2 fe & Zn in rice?

• Found 35S:OsNAS2 rice lines was 4x Fe and 2x more Zn in the endosperm

• Not only this, but higher NA presence in endosperm also increases bioavailability during consumption

Describe final liscnecing & creation of OsNAS2 rice for distribution

• gave to IRRI (phillipines - international rice research insitutte)

• Added an extra apple ferratin gene, Mbfer

  • Mbfer attached to two promoters to express in different endosperm parts

  • Choice of apple vs soy genes:

    • Soy is a common allergen - precautionary

• Stacking OsNAS2 & Mbfer greately increased Fe in rice!

What is the target for Fe & Zn content in OsNAS2 wheat

• WT wheat already has 35-40ppm of Fe; biofortification presents a target of 59ppm

Why is wheat a useful crop to biofortify in AUs

Wheat is a crucial crop as in Australia it is grown more than rice

Describe T-DNA design for OsNAS2 wheat

• Ubi promoter (more suited to wheat) + OsNAS2

• 35S promoter + nptII (neomycin resistence marker); same as previous

How were single inert homozygous evetns isolated in OsNAS2 wheat trials

• Must ensure that the transgene insertion is single & stable (this is easier to regulate and track)

• However progeny may produce heterozygous offspring & gene will keep segregating out (lower and lower levels)

• For homozygous single insert events must look through several plants & check how progency are segregating

• Production of progeny through wheat breeding & growth takes 5-7 months (long long time)

• If under bright purple light for 22hrs a day & 2hrs dark = this period shortens to 3 months

Describe liscencing approvals & field trials for OsNAS2 wheat in AUs & phillipines

• In 2019, was graited dealing of intentional release by OGTR

• Grown across the wheat belt

• Different cultivars of wheat at different locations along this (to specifiy)

  • e.g. driver vs rainier climates

  • Need to consider agronomy to specific locations where typically grown

• Grown in Dookie campus for years!

  • Hand sown different wheat types

• is approved for growth in phillipines

compare WT wheat & OsNAS2 Fe and Zn content

• more of both present in the starchy endosperm of wheat!

Describe alternative approaches (3) for genetic Fe biofortification in wheat that would be more socially acceptable

• These are approaches that may help provide biofortified wheat to consumers faster, through the social pushback to GM crop

  • Currently only drought-resistence wheat is approved in australia; no rice yet approved for growth

  • GM crops such as canola and cotton are also approved (as not consuming the DNA; canola = only oil thus low risk); & feed corn (not human consumption)

• Intragenesis

  • Intead of using rice NAS can derive from wheat

  • This is plausible as wheat also has 34 (Several) NAS genes

  • Experimentally profiled 7 of these and had promising results of their effectivness

• Genome editing

  • CRISPR-Cas9

    • 3 categories of gene editing: SDN1, SDN2, and SDN3

      • SDN1 is cuting DNA & allowing natural repair mechanisms to create small mutations:

        • SDN1 is no longer regulated as a GM crop in Aus

        • (considered acceptable regulatory-wise as these mutations can naturally occur as well)

      • SDN2 and SDN3 require large templates (HR)

  • Targetted mutagenesis of negative regulators of Fe uptake / NAS2 (can knocout & increase it) - not GM

• Natural variation :

  • Conventional breeding approach

  • Must identify wheat varieties with higher grain NA and breed into crop

  • Challenging; limited success so far; because higher Fe uptake is associated with lower yield; agronomic penalities is not favourable for farmers (this is also vice-versa!)

  • Breeding for higher NAS might be more feasible