Iron biofortification.

M.NARAYANAN
2019508202
GPB-513
Iron biofortification.

 Iron biofortification, the process of improving the bioavailability of iron in

food crops can be achieved via agronomic practices, conventional
breeding, and genetic engineering.
 Iron deficiency is a common health disorder affecting nearly 2 billion
people worldwide with other mineral and vitamin deficiency .
 Common effects of iron deficiency include anemia and impaired growth
development in pregnant women and preschool children .
Iron biofortification via agronomic practices

 Previously, several studies on fertilizer have been reported . positive

interactions between iron and zinc concentration in grains with
nitrogen, phosphorus, and potassium (NPK)
 The presence of nitrogen alone was reported to increase iron content in
brown rice by 15% and addition of potassium is able to further increase
the iron content in rice grain .
 Hence, combined application of both NPK fertilizer and iron fertilizer
could be a potential approach to increase iron bioavailability in rice .
Iron biofortification via conventional plant
breeding

 conventional plant breeding involves identifying and selection of parent

line, which contains desirable traits found in both parent plants.
 Parent lines are then crossed over for a few generations until daughter
plants with both desirable nutrient and agronomic traits are observed and
selected .
 For instance, iron bean is one of the successful products through
conventional plant breeding with high iron content, high bioavailability,
and high yield .
 In addition, the advancement of modern biotechnology techniques, such
as marker-assisted selection, improves the efficiency and precision in
identification of potential lines in daughter plants
Cont..
 An example of a product developed via plant breeding is high iron rice
variety (IR68144) with high yield, disease tolerant, good tolerant to
mineral deficient, and excellent seed vigor.
 The IR68144 rice variety was developed through crossing between semi
dwarf rice cultivar, IR8 and Taichung (Native)-1.
 This rice variety is able to produce 21 μg/g (2-fold) of iron concentration
in brown rice .
 In addition, IR68144 is able to retain most of the iron content
(approximate 80%) after polishing for 15 minutes compared to other .
 Iron uptake strategies in plants

Strategy I: reduction-based strategy

 Kim [20] suggested that ATPase are responsible for releasing protons into the
rhizosphere and reducing the pH of surrounding rhizosphere.
 The decrease in pH will increase the solubility of Fe3+ in the rhizosphere.
 In addition, NADPH-dependent Fe3+chelate reductase reduces Fe3+ into a more
soluble form of Fe2+ with the help of ferric reductase oxidase 2 (FRO2).
 Then, Fe2+ will be transported into the roots via ferric ion transporter controlled by
iron regulated transporter 1 (IRT1)
Strategy II: chelation-based strategy
 Grasses families such as maize, wheat, and rice are known as graminaceous plants. In
response to iron deficiency, these plants are able to increase iron uptake through
chelationbased strategy. Chelation-based strategy transports Fe3+ from rhizosphere
into the roots with the help of soluble siderophores.
 Mugineic acid (MA) family phytosiderophores are natural iron chelators and they have
a higher affinity toward Fe3
3. Iron uptake mechanism in rice

 Some graminaceous plants, in particular rice, can undergo combined

strategies of reductionbased strategy and chelation-based strategy for iron
uptake.
 Rice acquires Fe3+ via strategy I-like system and Fe2+ directly from the
surroundings via IRT1 or IRT2.
 However, there is no increase in Fe3+-chelate reductase levels detected in
the roots as compared to non graminaceous plants .
 Possible explanation is that adaptation of rice when grown in submerged
and anaerobic environment rich in Fe2+ compared to Fe3+ .
 Similarly to strategy II, MAs will be secreted into the rhizosphere to bind
with Fe3+ and the complexes will be transported into the root via YS-like
15 (YSL15).
 Between both strategies, rice is able to uptake iron from the surrounding
more efficiently through Fe3+-MA complexes as compared to direct Fe2+
uptake
Seven transgenic approaches to Fe
biofortification of rice
1. Enhancement of iron storage in rice via
ferritin genes

 Ferritin is an iron storage protein ubiquitously present in most organisms,

which is capable to store up to 4500 iron atoms in a complex and nontoxic
form .
 Thus, the first approach in iron biofortification is to enhance the
expression of ferritin by introducing soybean ferritin (SoyferH1 and
SoyferH2) genes into rice.
 In soybean, there are two types of ferritin proteins, known as SoyferH1
and SoyferH2, and both ferritin genes are controlled by endosperm
specific promoters [47].
 However, expression of multiple endosperm specific promoters (Oryza
sativa Globulin (OsGlb) and Oryza sativa Glutelin (OsGluB1) promoters)
did not produce a significant increase of iron concentration in rice grains
when compared to transgenic rice with ferritin genes expression driven by
single endosperm specific promoter .
2. Enhancement of iron transport in rice via
NAS genes

 The second approach involves enhancing iron transport in the plant via
overexpression of genes involves in biosynthesis of MA such as
nicotianamine synthase (NAS).
 NAS is able to catalyze the synthesis of nicotianamine (NA) from S-
adenosyl methionine .
 NA, a natural metal chelators for Fe(II) and Fe(III), are found in all higher
plants and involved with metal translocation and homeostasis in plants .
Rice comprises of three NAS genes, OsNAS1, OsNAS2, and OsNAS3.
cont..
 These genes are involved in long-distance transportation in plants and
each NAS gene is regulated at different parts of the plants in response to
iron deficiency .
 Overexpression of NAS gene enhances MA secretion into the
rhizosphere, and thus, increasing iron uptake into the plant via
chelationbased strategy .
 It has been demonstrated that over expression of rice OsNAS1, OsNAS2
and OsNAS3, OsNAS2 , OsNAS3 , and barley HvNAS1 genes are able to
increase the iron content by more than twofold in polished grain
3.Enhancement of iron influx into seeds via
OsYSL2 gene

 A total of 18 different YSL (yellow stripe-like) genes were identified by

Koike in rice.
 The rice YSL2 (OsYSL2) is the main focus in this approach as this gene
plays an important role as a metal-chelator transporter involved in
translocation and accumulation of iron in endosperm [73, 75].
 OsYSL2 was found to be highly expressed in leaves of iron-deficient rice
plants in contrast to other parts of the plant where no expression was
detected
Cont..
 Consistently, it was discovered that the iron influx into the rice endosperm
could be controlled through iron nicotianamine transporter OsYSL2 .
 Ishimaru successfully demonstrated that disruption of OsYSL2 gene in
rice decreased the iron content in both brown rice and polished grain by 18
and 39%, respectively with increased iron accumulation in roots as
compared to wild-type rice.
 Undoubtedly, the expression of OsYSL2 with OsSUT1 promoter is a
promising approach in iron biofortification of rice grains.
4.Enhancement of iron uptake and translocation via
IDS3 gene

 Introducing IDS3 gene from barley enables the synthesis and secretion of
different types of MAs from transgenic rice into the rhizosphere .
 In addition, formation of Fe(III)-MA complex has a better stability as
compared to Fe(III)-DMA complex when grown in a slightly acidic soil.
 This may enhance iron translocation in rice in combating iron deficiency
while increasing tolerance toward iron deficiency in rice plants
5: Enhanced Fe uptake from soil by overexpression of
the Fe transporter gene OsIRT1 or OsYSL15

 Lee et al. (2009a) produced transgenic rice that expressed the rice ferric
ion transporter gene OsIRT1 under the control of the Ubiquitin
promoter. This rice
 showed a 13% increase in Fe concentration in the brown seeds (Table 1),
while the Fe concentration in the leaves increased 1.7-fold. The authors
suggested that OsIRT1 could be used to enhance Fe levels in rice grains.
 Next, Lee et al. (2009b) reported that OsYSL15 over expression using the
OsActin1 promoter increased the concentration of Fe in brown seeds by
approximately 1.3-fold compared with non-transgenic rice
 In addition, Gómez- Galera et al. (2012) produced transgenic rice that
overex- pressed the barley Fe(III)-MA transporter gene HvYS1 under the
control of the CaMV35S promoter
 6: Enhanced Fe uptake and translocation by overexpression of
the Fe homeostasis-related transcription factor OsIRO2

 Ogo et al. (2006) identified a Fe-deficiency-inducible basic helix–loop–

helix (bHLH) transcription factor, OsIRO2, in rice.
 Ogo et al. (2007) intro- duced OsIRO2 under the control of the CaMV35S
pro- moter into rice plants. Rice that overexpressed OsIRO2 secreted more
DMA than non-transgenic rice, and exhib- ited enhanced Fe-deficiency
tolerance in calcareous soils
7: Enhanced Fe translocation from flag leaves to seeds
by knockdown of the vacuolar Fe transporter gene
OsVIT1 or OsVIT2
 Kim et al. (2006) have reported that the Arabidopsis vacuolar Fe
transporter, VIT1, is highly expressed in de- veloping seeds and transports
Fe and manganese into the vacuole.
 Zhang et al. (2012) reported that disruption of the rice VIT orthologues
(OsVIT1 and OsVIT2) in- creased the Fe concentrations by 1.4-fold in
brown seeds (Table 1) and decreased the Fe concentrations by 0.8- fold in
the source organ flag leaves.
 A possible explan- ation for these results is that the VIT genes are highly
expressed in rice flag leaves.
 Bashir et al. (2013) also re- ported that an OsVIT2-knockdown mutant
showed 1.3- fold and 1.8-fold increases in Fe concentrations in the brown
and polished rice seeds, respectively
Cont..
Combinational of multiple transgenes

 Multiple gene manipulation has been successfully carried out in rice.

 Wirth [23] has proven the synergism of three different genes expression
with the increased of iron content in rice by 6-fold through introducing
Arabidopsis thaliana NAS1 (AtNAS1), Phaseolus vulgaris ferritin
(Pvferritin), and Aspergillus fumigatus phytase (Afphytase) genes into
rice.
 The OsYSL15 or OsIRT1 genes are predominantly expressed in roots with
enhanced expression in response to iron deficiency [86].
 OsIRT1 gene encode for Fe2+ transporter involved in both strategy I and
II.Although overexpression of OsIRT1 alone could increase the iron
content in rice grain by 1.3-fold,
Conclusion

 Success of iron biofortification would results in improved nutritional value

of micronutrient-deficient affected areas in developing countries and as a
first step toward improving nutritional status worldwide.
 Transgenic approaches is manly used for improve the nutritional balance
for stable food cereals through traits specific stratigies.
 We generated transgenic rice by introducing multiple genes,including
ferritin under the control of endosperm-specific promoters, NAS
overexpression, OsSUT1 promoter-driven OsYSL2 expression, and the
barley IDS3 genome fragment, and showed increased concentrations of
bioavailable Fe.
 Increasing the expression of OsIRT1, OsYSL15, and OsIRO2, or
knockdown of OsVIT1 or OsVIT2 are the candidate approaches to improve
Fe bio- fortification of rice seeds.
 Further attempts are required to evaluate high-Fe rice varieties or other
target genes. A combination of these approaches will be beneficial to fu-
ture Fe biofortification work.
Challenges and future prospect

 In addition, there are possibilities of irreversibility effect on health and

environment due to the effects of GM crops on health and environment are
not fully understood and not sustainable in the long run
 To gain plant breeder acceptance, biofortified crops should contain visible
and favorable traits such as increased in yield, higher stress tolerant,
disease resistance, and other important agronomic traits
 Consumer acceptance on biofortified crops is not easy and achievable in a
short duration of time but it can be accomplished through thoroughly
planned strategies such as
 spreading knowledge among the people,
 raising awareness of micronutrient deficiency,
 creating new market opportunities,
 creating a demand on biofortified variety
REFERENCESS.
https://www.intechopen.com/books/rice-crop-current-developments/iron-
biofortification-of-rice-progress-and-prospects
 https://thericejournal.springeropen.com/articles/10.1186/1939-8433-6-40
 https://www.researchgate.net/publication/259385885_Iron_biofortification
_of_rice_using_different_transgenic_approaches
 https://www.nature.com/articles/srep00543
 https://www.irri.org/biofortification
 https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3567483/