Genetic Engineering of Iron Accumulation in Crops

What is the iron problem? About 2 billion people suffer from iron deficiency anemia.

The causes are three-fold: dietary (insufficient iron uptake), demographic, and medical/social (infections or parasites like hookworm, malaria, or schistosomiasis that cause blood loss from the intestines. Loss of 5 ml of blood per day is enough to cause anemia. The remedies are greater iron intake (from food or iron supplements) and infection control.

Iron deficiency chlorosis in plants.Plant productivity is reduced because of Fe

deficiencyIron deficiency is very widespread on alkaline (pH 7.5 and above)and calcerous soils.Remedies are: acidify soils (this often does not work due to high calcium), apply iron chelate to the soil, apply a mixture of iron sulfate and sulfur, use foliar spray). These remedies are onlypractical in developed countries with “high-input” agriculture.

Iron deficiency is widespread in the world.

40% of all women are iron deficient. Healthy people have about 3.7 g of iron. 2.5 g is in hemoglobin. 1 g is stored in the liver as ferritin. Very little is

actually in the iron proteins (cytochromes etc) needed for cellular metabolism.

Iron deficiency causes anemia (low RBC count and RBCs with less hemoglobin). Iron is taken up in the small intestine by receptors after

exchange from its chelator.

Iron abundance and Bio-availabilityFood sources contain different levels of iron.

In animal tissues, iron is present as heme iron or ferritin (in liver). Levels differ markedly. Lamb kidneys: 10 mg/100g; lean beef: 4 mg/100g; chicken:

1 mg/100g;In plant tissues iron is found as phytoferritin in seeds and choroplasts, but

also in chelated form (citrate) or as Fe-Sulfur clusters in chloroplasts. Legume seeds and very green leafy vegetables are the best plant sources:

Seeds: peas and beans 1.2 mg/100 g; Leaves: spinach: 0.5 mg/100 g Little is known about the efficiency of iron absorption, but it is thought that

heme iron is 20 X more bio-available compared to non-heme iron.You need to eat 10-20 mg/day to absorb 1-2 mg/day.

Poor people rely heavily on cereal staples, which are low in iron and have low iron bioavailability. There is more iron in legume seeds (beans)

Bioavailability of Fe is diminished by antinutrients

Bioavailability is depressed by “antinutrients” such as phytic acids (in cereals and legumes) and polyphenols (tea and coffee ). Bioavailability can also be enhanced by S-amino acids and vitamin C.

Phytate (hexa-inositol phosphate complexed with cations) can be hydrolyzed by phytase (phosphatase) at the time a cereal porridge is prepared. Degrading phytate with a fungal phytase caused an increase of iron absorption of maize porridge from 1.8% to 8.9%, for wheat from 1% to 11.5 % and for rice from 1.7% to 5.3%.

Traditional maize porridge cooking pot in the Transkei. Phytate

Free iron is toxic in all cells.Iron and other transition metals are toxic if they are “free” in the cytoplasm (as hydrated ions). They generate reactive oxygen species (ROS) such as hydroxyl radicals.

Fe 2+ + H2O2 >>> Fe 3+ + OH_ + OH.

Iron is essential and its abundance in the cytosol must be tightly regulated. Iron must be stored and deposition in stores and remobilization depend on transporters.Transition metals must be chelated with organic acids, histidine or nicotianamine. In plants, transport across membranes is by transporters for these organic chelators. Transport is not very metal specific. Some iron chelate carriers will also carry zinc and other metals in lesser quantities.In humans, iron in supplements is readily absorbed only if it is present as glycinate or peptonate. Iron pills cause nausea and constipation. Some transition metals are toxic (cadmium, lead etc). Others, such as Cu are essential and yet they are very toxic.

Transport of iron in humans.Iron is absorbed by the enterocytes in the small intestine. Fe(II) and other divalent

cations are transported by the DMT1 receptor, but no receptor for Fe (III) is

known. The receptor for heme-iron is also unknown.

Once in the blood vessels, iron bound to carbonate is complexed by transferrin, a soluble protein in the blood plasma. The

complex binds to the transferrin receptor and is internalized into the liver by recycling endosomes. Acidification of the endosome causes iron to be released so that it can be

assembled in ferritin.Excessive iron absorption leads to

hemochromatosis or excessive iron accumulation (1 in 400 Caucasians).

We have no good way of eliminating iron.

Membrane

Transferrin receptor

Transferrin

Ferritin represents an iron storage in the cell that holds iron in a bioavailable and non-toxic form. 24 identical subunits of

apoferritin are assembled in a ball-like formation, each

monomer exhibiting a typical four-helix bundle fold. The ball

has 4500 iron atoms. Iron is thought to be taken up as Fe(II),

oxidized at the so called ferroxidase site within the helix

bundle, and accumulated as Fe(III) in a kind of crystalline

conformation.One ferritin ball has 8 pores through which the FE(III) is

mobilized and utilized,

Open pore Closed pore

Phytoferritin is a storage form of Fe in plants and is a good source of Fe for humans.

Two processes require large amounts of iron both as heme and in non-heme iron-sulfur clusters: photosynthesis and nitrogen fixation. Phytoferritin accumulates in plastids in leaves, and seeds (and smaller amounts in other organs). During senescence of leaves and nodules, Fe is mobilized and transported to the seeds.

Fe

Fe

FeSO4 Animal Ferritin Plant Ferritin

Hct = hematocrit

Plant and animal ferritin are equalin correcting iron deficiency in rats

Common bean accumulatesiron as phytoferritin in seeds.

Iron in the soil

Soils contain abundant supplies of iron,but most of it is insoluble. Iron is present as ferric ion Fe (III), as Fe(OH)2

+, Fe(OH)3 or Fe(OH)4

- . Solubility of Fe(III) is very low at neutral pH, and even lower at pH 8,typical of alkaline soils (30 % of soils worldwide).

Iron

Nitrogen

To increase iron availability, plants have three strategies:#1. Acidify the soil by releasing organic acids thereby increasing the solubility of ferric iron (malic acid and citric acid). This also promotes phosphate uptake. #2. Reduce ferric ion to the more soluble ferrous form#3. Secrete chelators (siderophores) that are then taken up again by the roots after they chelate iron.

Processes in iron homeostasis in plants

Iron needs to be “mobilized” (solubilized) in the soil. (How?)Uptake by root hairs across the plasma membrane (Carrier?)Loading into the xylem and transport as citrateTransport into leaves; 40 % of Fe is in thylakoids as heme iron or Fe sulfur clusters in photosynthetic electron carriers.Remobilization from leaves during senescence, loading into phloem. Chelator is nicotianamine. Transporter? Accumulation in seeds and transport into the vacuoles of the storage parenchyma cells (globoids in protein storage vacuoles) (Transporter?)Mobilization from seeds during germination.

Fe: electron scatterimage

Transmission EMimage

The xylem and the phloem are vascular or transporting tissues. The xylem conducts mostly waters and minerals from the soil to the leaves.

It consists of large vessels (cells without cytoplasm) surrounded by live cells. The phloem conducts organic substances (products of

photosynthesis or of macromolecule breakdown) all around the plant

(leaves to roots, leaves to seeds and flowers, etc). Larger sieve tubes are flanked by companion cells that load

the organic substances into the sieve tubes. Xylem and phloem

occur together in vascular bundles.

Xylem

Phloem

Two strategies for acquiring iron are found in different plants. Most plants use I, cereals use II

I: Fe(III) Iron reduction at the plasma membrane byFRO1, foollowed by uptake by IRT1

II: Synthesis of phyto-siderophores (PS) and excretion followed byre-uptake of chelated Fe(III)

Strategy II: Synthesis of the phytosiderophore mugineic acid by grasses

and transport into the plant.

(1) Barley is resistant to iron deficiency and grows well on alkaline soils, but rice is sensitive to iron deficiency. (2) Addition of mugeneicacid to barley roots in hydroponics stimulates iron uptake 30 X(3) Barley secretes much more mugineic acid than rice. How is mugineic acid synthesized? From 3 residues of S-Adenosyl-methionine.

Rice is sensitive toalkaline soils.

Mugineic acids are secreted siderophores that chelate iron and other heavy metals. Most plants make nicotianamine to chelate

and transport metals across internal membranes, but the synthesis of mugineic acids requires an additional step

Fe(III)

NAAT

Rice transformation with barley NAAT

genes.They used a genomic barley fragment with 2 different NAAT genes.

These genes had their own promoters

and were differentially expressed in shoots

and roots and regulated by iron

(strongly induced in roots by Fe deficiency)

Takahashi et al., Nature Biotechnology 19: 466-469, 2001

Increasing the synthesis of the siderophore mugineic acid in rice by introducing the NAAT

gene. DMA = desoxymugeneic acid

NAAT activity DMA secretion

Growth of transformed rice on alkalinesoil. Plants first established and thentransferred.

The maize Yellow stripe mutant (ys1) has yellow interveinal mesophyll and this looks like an Fe deficient plant. The mutants are unable to take up the Fe(III)-

siderophore (deoxymugineic acid) complex. Cloning of the gene YS-1 (using an Ac insertion mutant of maize) shows it to encode a protein with 14 trans-

membrane domains. (Curie et al, Nature 409:346-34, 2001; Walker lab). In maize, the gene is induced by iron starvation in the leaves, suggesting that it

has a function in transport within the plant. The gene restores growth of a mutant yeast that cannot grow on Fe (III).

Second question: how is Fe-mugineic acid taken up by the plant?

Uptake of radioactive 55Fe by mutant yeast strain unable to take up Fe and transformed

with maize YS1

The YS1 protein can transport mugineic acid (MA) complexed with Fe (III) or nicotianamine (NA) complexed with Fe(II). YS1 does not transport Zn.

Localization of barley HvYS1protein in barley roots underFe sufficient and deficientconditions.

control

14 members in rice; 8 members in ArabidopsisYSL2 encodes a plasma membrane protein of 674 AA with 14 membrane spanning domains.Iron deficiency induces expression in the leaves.YSL2 is expressed in the companion cells and other phloem cells, especially in iron deficient plants.The function of YSL2 is the lateral movement of iron and copper in the veins.Experiments with Xenopus oocytes show that the protein transports Fe(II) nicotianamine (NA), Mn (II) NA, but not Fe(III)siderophore (DMA).Kioke et al, Plant Journal 39: 415-424, 2004.

In Arabidopsis, YSL2 is expressed in many cell types indicating that it is also involved in iron transport generally.DiDonato et al, Plant Journal, 39:403-414, 2004

YSL (YS-like) is a large gene family in all plants.

Expression of YSL1 and YSL3 in Arabidopsis.

Promoter-GUS assays.

A-G: YSL1H-L: YSL3.

Note high expression in leaf veins (A and H), root veins (F) in oldest (senescing) leaves (G) and in stamens (J,K) (for storage in pollen?)

Central role of nicotianamine in iron transport in all plants.

In the phloem and xylem Fe has to be chelated by other molecules: citrate in the phloem and 11 kDa iron transport

protein (ITP) in the phloem.

Strategy I: Reduction of iron at the plasma membrane

In response to iron deficiency, all plants except the grasses, secrete protons in the soil medium with

their plasma membrane H+ATPase, induce Fe(III) chelate reductase and Fe(II) chelate transport

activity in the plasma membrane.

Proton secretion and Fe(III) chelate reductase can be measured non-invasively and used to screen for

mutants that lack the enzyme.

Visualization of acidification of the medium with bromocresol purple.

Bromocresol purple is purple at neutral pH and yellowish green atacid pH. In wild type plants, Fe deficiency induces acidification. In the frd3 mutant acidification is constitutive (with or without Fe)

Ferrozine Assay to Measure Root Fe(III)Chelate Reductase Activity Non-invasively. No color means no

enzyme!

Fe(III)EDTA + ferrozine Fe(II) ferrozine

colorless Purple complex

Ferrozine = 3-(2-PYRIDYL)-5,6-BIS(4-PHENYLSULFONIC ACID)-1, 2,4-TRIAZINE

Some frd mutants lack Fe(III)-chelate reductase.

FerroZine allows a quick visual screen for mutants in which Fe deficiency does not induce the enzyme (No purple color).

These mutants are termed frd. The mutants fail to take up radioactive 55Fe from the medium.The roots still secrete protons to acidify the medium and still

take up Zn and Mn.This shows that Fe(III) needs to be reduced before it can be

taken up.The frd mutation has been mapped and the gene identified.

In addition, because other organisms have similar reductases, homology cloning based on known sequences is possible.

Both approaches yielded the same gene: FRO2. FRO1 was also identified, but is not induced by iron deficiency.

Iron-chelate reductase from Arabidopsis.

The FRO2 protein has 725 amino acids with an FAD (flavin adenine

dinucleotide) binding site and 4 histidines that bind 2 heme groups. The

frd1-3 mutation is in the FAD binding site. High Fe-EDTA in the medium

overcomes the Fe deficiency phenotype of the mutant plants, possibly because

of low FRO1 levels that are constitutively present.

IRT1, an Fe(II) transporter, was identified by functional cloning of a plant cDNA library in a yeast mutant deficient in iron transport.

The gene restored iron transport in the yeast mutant.

The IRT1 gene also restores Mn transport in another yeast mutant, so IRT1 transports more than one

metal. IRT1 is a member of the ZIP family of metal transporters. It transports Fe, Zn, Mn and Cd.

Specific point mutations alter the metal specificity.

Fe, but no Zn

Mutational analysis of the iron transporter IRT1 Pink (E103A): eliminates zinc

Blue (D100A) :eliminates iron and manganeseGrey: knock out transport function - these residues are

essential for chelating the metal. It may be possible to make specific metal transporters

How is IRT1 regulated in Arabidopsis?Connolly et al, Plant Cell 17:1347-1357, 2002

Normal regulation of IRT. Panel A: Plants grown on Fe deficient medium induce the

IRT1 gene and the protein appears, but, Panel B, after shift to Fe sufficient medium the protein disappears rapidly.

Further analysis shows that this expression is only in the roots, but not in the shoots (not shown).

Induction by lack of Fe

When you express Irt gene with the 35S

promoter, the gene is expressed in both roots

and shoots and expression level is not dependent on the Fe nutrition status of the

medium. (35 S promoter is not Fe induced, of

course)

IRT1 protein in over-expressing plants grown with and without Fe. A surprising result: Fe in the medium suppresses protein

accumulation.In the immunoblot below, R and S refer to roots and shoots. In the transformant there is only protein in the root, not in the

shoot, and there is less protein in the + Fe, than the - Fe. Thus Fe suppresses protein accumulation by post-

translational regulation.

Transgenic

Does the protein stay around if you switch to + Fe?

Transfer of IRT transgenic to Fe sufficient medium suppresses mRNA levels and completely

eliminates the protein in 36 hours. IRT protein accumulation is regulated post-

translationally.

In addition, the transgenic plants contain no more Fe than the WT and they are more sensitive to Cd (which is toxic), suggesting that they take up more Cd. Life isn’t simple!

New frd screen: screen for expressors of FRO in the presence of Fe.

The frd3 mutant has constitutive activity of Fe(III) chelate reductase (even in the

presence of iron) and has high plant levels of Fe, Mn and Zn.

The genes for iron homeostasis are expressed, as is acidification of the

medium. This suggests that there may be a defect in iron sensing. Plants are unable to sense iron, so they activate their genes

for iron uptake.Why are the plants chlorotic? At the organ

or subcellular level, iron may be

mislocalized causing chlorosis.

The frd3 mutant has constitutive Fe uptake, but is chlorotic

Homology cloning using the yeast VIT transporter identifies plant VITs.

Yeast deletion mutant ccc1 is unable to grow, but plant VIT complements the yeast mutant.

Kim et al, 2006 Localization of iron in Arabidopsis seed requires the vacuolar membraneTransporter VIT1. Science 314: 1295-1298.

VIT-GFP localizes to the tonoplast.

NRAMP knockouts have a transient germination phenotype on normal

medium and are sensitive to low Fe

medium.

The EMBO Journal (2005) 24, 4041–4051 Lanquar et al. Mobilization of vacuolar iron by AtNRAMP3 and AtNRAMP4 is essential for seed germination on low iron.

NRAMP is a tonoplast protein and nramp3 nramp4 double knockout mutant fails to mobilize vacuolar Fe during seed

germination

Dry seeds

2-dayseedling

Dry seeds

2-dayseedling

EDX spectra of globoids

fe mutant Complement. with fer gene

Typical iron deficiency chlorosis intomato (left). Veins remain green.

fe mutant (below) is unable to turn on its iron uptake machinery (root acidifi-cation, FRO-1 gene) when the soil isdeficient in iron.

How is iron uptake

regulated?

Positional cloning shows the fe mutation to be encoded by fer, a helix-loop-helix

transcription factor. In situ hybridization showing localization of fer gene

expression in tomato roots:in the elongation zone (see

C) expression is in the epidermis, higher up (B) it is

in the vascular bundles.fer gene expression is

required in the roots, but not in the shoots (grafting

experiments)Ling et al, PNAS vol 99 13938 ff, 2002

Genetic engineering with phytoferritin:Expression of soybean phytoferritin in rice seeds with a seed storage protein (glutelin)

promoter A: iron content of seeds from individual plants

(transformants). B: iron content of 2nd generation seeds (T2 plants): control and

transformed.Goto et al. Nature Biotech. 17: 282-286 (1999)

Control Transform

Emb Endo Emb Endo

A

Iron supply down-regulates fer gene in tomato

A: PCRB: Immunoblot

100 molar Fe-EDTA is the normalconcentration in a nutrient medium.

Phytin, is the salt of phytic acid or inositol hexaphosphate. In seeds it is the storage form of phosphate and other minerals (metals). However, because animals have no intestinal phytase they cannot digest it. During germination, seeds make phytase and utilize the components of phytic acid. Phytic acid is an antinutrient because it prevents iron and zinc

absorption in the human body.

Phytate biosynthesis. D-Glc-6-P (1) is converted to

D-myo -inositol-3-phosphate (2) by the activity of MIPS (myo-Inositol 3-phosphate synthase).

D-myo -inositol-3-phosphate is further phosphorylated to yield myo -inositol hexakisphosphate (3) by several kinase steps.

Low phytate maize (LPM) has been developed by GM and by screening for mutants.

LPM contains only 40 % as much phytate as wild type. Iron absorption from tortillas made from both maize varieties

increases by about 50 % for the LPM (from 5.5% of intake to 8.2% of intake) in Fe deficient women.

Supplementation with small amounts of Fe-EDTA eliminates the difference between the two varieties.

There are no zero phytate mutants of maize (or other crops) as these seeds are not viable.

Conclusions

Many people are iron deficient because of high cereal diet (rice, corn) and intestinal parasites. Iron from animal sources I more bioavailable.Alkaline soils prevent iron uptake by plants.In the cell free iron is always toxic and needs to be chelatedPlants have different strategies to acquire iron. To be taken up iron must be reduced or chelated by a siderophore.Iron is stored in seeds as phytoferritin (good) or as phytate (bad).Genes regulating iron homeostasis include transporters and enzymes to synthesize chelators.Nicotianamine is an important iron chelator and a precursor to Siderophores.Regulation of iron acquisition occurs at all levels (transcriptional and post-translational).Transgenic approaches to create iron rich plants have not yet worked.

FRD3 is a member of the multidrug and toxin efflux family of proteins.

Rogers and Guerinot, Plant Cell: 14: 1787-1799, 2002

Complementation of frd3 with Frd3

This mutant is a good candidate for expression profiling to detectupregulated transcription factors that may be involved in the expression of Fe homeostasis genes.