Sara Miller
Bio509 Molecular Biology in Applications
Dr. Mourad
Fall 2010
Shi, Jinrui; Honcgyu Wang, Kathleen Schellin, Bailin Li, Marianna Faller, Johan M Stoop, Robert B Meeley,
David S Ertl, Jerry P Ranch and Kimberly Glassman. “Embryo-specific silencing of a transporter reduces
phytic acid content of maize and soybean seeds.” Nature Biotechnology 25(8):930-937. (5 August 2007)
Objective
Cereal grains and oilseeds contain large quantities of phytic acid which provides myo-inositol
and phosphorus required for seed germination and seedling establishment. Nutritionally adequate
phosphorus content of cereal grains and oilseeds is bound to phytic acid, and monogastric animals lack
sufficient phytase in their digestive tract to access the bound phosphorus. The undigested phytic acid
excreted in animal waste is considered a leading source of phosphorus pollution from agriculture. In
addition, phytic acid reduces the bioavailability of essential mineral cations Fe3+, Zn2+, Ca2+ as well as
interacts with basic amino acids, seed proteins and enzymes in the digestive tract to form complexes
that may reduce the amino acid availability, protein digestibility and the activity of digestive enzymes.
To compensate for monogastric animals inability to access the nutritional phosphorus in cereal grains
and oilseeds, feed has to be supplemented with Pi to meet the phosphorus requirement for optimal
animal growth.
In the past, breeding programs to produce plant mutants with less phytic acid and more
inorganic phosphate have had undesirable agronomic characteristics associated with the phytic acid
reducing mutations. Low phytic acid mutants (lpa) have been characterized to have reduced seed
weight, failure of seeds with strong lpa alleles to germinate, stunted vegetative growth, poor seed
germination and seedling establishment, and impaired seed development. The researchers of this study
indentified the gene disrupted in maize lpa1 mutants and showed that using transgenesis to specifically
reduce phytic acid levels in phytate-accumulating seeds can avoid undesirable agronomic characteristics
associated with known mutations. Using tissue-specific down regulation of an MRP (multidrug
resistance-associated protein) ABC (ATP-binding cassette) transporter enhanced the nutritional quality
of maize and soybean seeds. They show that maize lpa1 mutants are defective in a MRP ABC
transporter that is expressed most highly in embryos, but also in immature endosperm, germinating
seed and vegetative tissues. Silencing expression of this transporter in an embryo-specific manner
produced low-phytic acid, high inorganic phosphate transgenic maize seeds that germinate normally and
do not show any significant reduction in seed dry weight.
Experiments and Results
Cloning lpa1 gene. A collection of transposon-mutagenized F2 families derived from a variety of Mutator
(Mu)-active stocks were screened for high-Pi phenotype, and the high-Pi lines were crossed with
homozygous lpa1-1mutants to search for new allelic insertion mutations. Allelic mutations were
identified from lines producing high inorganic phosphate in F1 test crosses, and three independent lpa1
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mutants were identified in active Mu families and coined lpa1-mum1, lpa1-mum2, and lpa1-mum3
respectively. Initial efforts to clone the lpa1 gene from lpa1-mum1 mutants via PCR was unsuccessful,
but they enabled the identification of a Mu insertion in a gene encoding a transcriptional activator
protein (TAP) which was located ~1cM from the lpa1 locus and mapped to the terminal region of a BAC
clone named b167c.j1q from a library derived from chromosome 1. Fine mapping was done with
additional PCR markers and genetic analysis showed that marker m1 and i9 flanked the lpa1 locus.
Sequencing revealed 9 putative open reading frames one of which encoded an ABC transporter and
another encoded a protein with unknown function. Mu insertions were identified at independent sites
in exons of the ABC transporter gene in all three alleles which provides strong evidence that the ABC
transporter gene corresponds to the lpa1 locus.
Maize lpa1 gene encodes a MRP ABC transporter. Gene expression analysis was performed by massively
parallel signature sequencing. The MRP protein has 1,510 amino acid residues and contains two copies
of the modular structure consisting of an intergral transmembrane domain and a cytosolic ATP-binding
domain. Insertion of the Mu element interrupts the lpa1 gene expression, whileMRP4 transcripts were
undetectable in homozygous Mu-insertion mutant seeds by RT-PCR, but were present in the wild-type
control seeds. The lpa1-1 mutant carries an alanine-to-valine point mutation at amino acid position
1432. This amino acid is conserved in MRP proteins and is located in the second ATP-binding domain,
and it is very likely that this mutation accounts for the phenotype of the lpa1-1 allele.
Expression of the maize lpa1 gene. Northern Blot Hybridization analysis using total RNA from immature
seeds and seed parts revealed that lpa1 transcripts were detectable in the seeds 7 days after pollination
(DAP), while expression in the embryos was observed at 15 DAP which peaked at 21 DAP then declined
thereafter. Analysis of expressed sequence tags using massively parallel signature sequencing revealed
a broad expression pattern for lpa1 in roots, leaves, stalks, tassels, silks and developing seeds. The lpa1
gene was also expressed in germinating seeds and young seedlings while expression in vegetative
tissues was relatively low.
Impact of lpa1 gene mutation on germination and seed weight. Seeds homozygous for lpa1 knockout
alleles were indistinguishable in morphology and appearance from heterozygotes and wild-type seeds;
however, seed dry weights of lpa1 mutants as well as dry weights of embryos and ‘rest-of-seed’
fractions were less than those of the wild-type.
Production of transgenic low-phytic-acid, high-Pi maize plants. Maize transformants were produced by
using Agrobacterium-mediated transformation. The vector contains the plasmid backbone of super
binary vector pSB1 and a T-DNA region containing the selectable marker moPAT and a gene-silencing
cassette for suppressing the lpa1 expression. Ole and Glb maize embryo specific promoters were
chosen for gene-silencing constructs to ensure that the lpa1 expression would be suppressed in
embryos only where phytic acid is synthesized and accumulates in maize seeds. 21 independent
transformants were produced from the Ole::MRP4 construct, and 41 transformants were produced from
the Glb::MRP4 construct. 80% of the transformants exhibited the high-Pi phenotype, while reductions in
phytic acid levels to the tune of 68-87% were associated with the expression of Ole::MPR4. Phenotypes
of the Glb::MRP4 transformants were weaker than those of Ole::MRP4 with reductions in phytic acid
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levels ranging from 32-75%. The grain phenotype in progeny was stable in several independent
transformants tested with the levels of phytic acid reduced and Pi increased compared to selfed,
crossed, and back crossed seeds in each transgenic line. Similar to the lpa1 mutant, the transgenic seed
did not accumulate inositol phosphate intermediates.
Dry weights and germination rates of transgenic seeds. Using segregating ears for seed dry weight
analysis, Ole::MRP4 transformants were observed to have significant seed weight reduction while none
of the Glb::MRP4 transformants had a statistically significant reduction in seed dry weight. The
germination frequency of homozygous Glb::MRP4 transgenic seeds was 95-100% in filter-paper
germination tests and under field conditions the seeds from segregating ears produced glufosinateresistant and glufosinate-susceptible seedlings that segregated in the expected 1:1 ratio. Resistance to
glufosinate in transgenic seedlings is conferred by transformation with the selectable marker moPAT, a
maize optimized phosphinothricin acetyltransferase. This demonstrates that the Glb::MRP4 transgenic
and wild-type seeds have the same germination frequency. To determine the effect of transgene
expression on plant vegetative growth, seeds from segregating ears were planted in the field which
showed no obvious difference in seedling vigor, leaf number, plant heights, or tasselling or silking times
were observed between Glb::MRP4 transgenic plants and the wild-type.
Transgenic low-phytic acid and high-Pi soybean. The soybean transformation vector contained a
hygromycin B phosphotransferase gene under the control of the cauliflower mosaic virus 35S promoter
used for transgenic plant selection. Soybeans were used to determine whether the MRP gene can be
used to reduce phytic acid and increase Pi in other crops. The MPR homolog was identified in soybeans
and the expression was suppressed using a gene-silencing construct controlled by soybean Kunitz
trypsin inhibitor 3 (KTI3) promoter. Of the 23 independent soybean transgenics, 14 exhibited increased
inorganic phosphate levels. After selected transformants were analyzed quantitatively, results showed
that the transgenic seed typically showed a 15-30 fold increase in Pi and a 37-90% reduction in phytic
acid.
Conclusion
Cloning the maize lpa1 gene allowed for suppression of the lpa1 gene expression in an organspecific manner through a gene-silencing construct containing a maize embryo specific Glb promoter.
The resulting transgenic seeds germinate normally and have no significant reduction in seed weight
while exhibiting a low-phytic acid and high-Pi phenotype. Limiting the suppression of the lpa1 gene is
critical in grain because it is also expressed in vegetative tissues and disrupting its expression in the
mutant decreases vegetative growth. Although the Ole promoter produced the strongest phenotypes,
the seed dry weight was reduced and germination impaired in some transgenic lines. Reductions in
phytic acid levels in Glb::MRP4 transformants were not associated with significant decreases in seed dry
weights most likely because of the reduced activity of the Glb promoter relative to the Ole promoter.
Future research may uncover the mechanism of how MRP4 regulates phytic acid metabolism and
further research on how the MRP ABC transporter affects phytic acid transport and
compartmentalization may lead to more efficient methods of producing transgenic plants that are low in
phytic acid and high in inorganic phosphates that are commercially viable.
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