Genetic Engineering of Cereal Grains with Starch Consisting of ...

ISB NEWS REPORT APRIL 2013
Genetic Engineering of Cereal Grains with Starch
Consisting of More Than 99% Amylase
Kim H. Hebelstrup, Massimiliano Carciofi & Andreas Blennow
Numerous textbooks tell us that plant starches are a mix of two starch types: amylopectin and amylose. We recently
succeeded in engineering a cereal crop – a barley line – producing grain starch consisting of more than 99% amylose
1 . This amylose-only starch contains a high residual fraction that is resistant to enzymatic degradation, even when
gelatinized by cooking. The barley plants producing the grains had a moderate yield loss of 25% in comparison with
other barley plants of the same cultivar. We believe that the method can be applied to produce amylose-only starch
in other cereal crops including wheat and corn.
Background
Cereal grains usually consist of 50 – 80% starch. Biologically, starch acts as a storage form of energy and carbohydrate
biomass, to be transferred from one plant generation to the next. But for humans, cereal grains and their starch
content and other nutrients are a major agricultural source of food, food ingredients, and livestock feed.
Plant starches 2 are a mix of two types of glucan polymers: amylopectin and amylose. They both consist of polymers
of glucose, which are connected by α-1,4 glucosidic bonds. However, whereas amylose is mainly linear, amylopectin
is branched because 5% of the glucose units have additional α-1,6 glucosidic branches. Due to these structural
differences, amylose and amylopectin have different physio-chemical properties, such as amylose being much less
soluble in water and having a much higher gelatinization/melting temperature than amylopectin.
Natural plant starches usually contain 20 – 30% amylose and 70 – 80% amylopectin. Amylopectin and amylose
are organized in semi-crystalline granules, which results in the “powdery” appearance of purified starches. Some
crop varieties have been bred or engineered to contain either less or more amylose content of their starch. Among
those are the so-called waxy lines. These contain nearly 100% amylopectin and are therefore virtually amylose-free.
Several waxy varieties exist in different cereal crops. For example, sticky rices are often waxy variants. On the other
end, high amylose varieties such as amylose extender also exist, and have been identified in different cereal crops.
These varieties usually consist of starches with up to maximum 80% of amylose. These high- or low-amylose phenotypes
are often the result of a loss-of-function mutation in a gene that encodes an enzyme involved in biosynthesis
of the starch.
Cereal grain starch biosynthesis takes place in modified plastids called amyloplasts located in the cells of the
starchy grain endosperm. Starch is synthesized from sugars, which are loaded from the vascular plant tissue into
starchy endosperm cells of developing cereal grains. Amylopectin and amylose are both synthesized from ADPglucose,
which is either synthesized within or transported into the amyloplasts. This reaction is carried out by an
enzyme family of starch synthases that make the starch polymers by adding glucose units from ADP-glucose by
forming α-1,4 glucosidic bonds. But whereas amylose in cereal grains seems to be synthesized specifically by only
one member of this enzyme family, called Granular Bound Starch Synthase (GBSS), amylopectin is synthesized by
several members. In addition, the α-1,6 glucosidic branches are introduced only in amylopectin by another family
of enzymes, Starch Branching Enzymes (SBE). Hence, the waxy types have loss-of-function mutations in the gene
encoding grain GBSS, while most high-amylose varieties are the result of loss-of-function mutations in genes encoding
SBEs.
We hypothesized that a cereal with blocked activity of all starch branching enzymes would produce starch with
no branches and therefore consist of pure amylose. We tested this by silencing all three members of the SBE gene
family in barley (Hordeum vulgare var. Golden Promise). Barley is our choice of a model plant in genetic engineering
for small grain cereals, mainly for two different reasons: the plant is diploid and so allele segregation is simpler
than in polyploid plants such as wheats; and second, due to the cold-wet temperate climate, barley is a major crop in
Scandinavian countries.

ISB NEWS REPORT APRIL 2013
Gene silencing technology
We silenced expression of all three starch-branching enzyme genes by RNA interference. This was done by engineering
a DNA construct (Figure 1) containing the ubiquitin gene promoter from corn that activates expression of a
sense-loop-antisense RNA hairpin. The hairpin consisted of three consecutive parts of 300 basepairs, each targeting
one of the three members of the SBE gene family in barley (SBEI, SBEIIa and SBEIIb). Such hairpins are known
to induce degradation of mRNA with a matching nucleotide sequence. The DNA construct was transferred to the
genome of barley plants by Agrobacterium-mediated transformation. We identified 11 independent transgenic lines
showing insertion of the DNA construct into the genome, and confirmed a 50 – 90% reduction in the mRNA level of
all three members of the SBE gene family. Similarly we found an 80% reduction in starch branching enzyme activity
in the line with the highest reduction in mRNA levels.
Figure 1. DNA construct used to silence the gene family of starch branching enzymes (SBEI, SBEIIa and SBEIIb).
The promoter (pink) is the ubiquitin promoter from corn. The construct results in the expression of an RNA hairpin construct,
which promotes the breakdown of each of the three SBE mRNAs. Modified from Carciofi et al. 1
Properties of amylose-only starch and effects on yield
Amylose and amylopectin content was determined by combined size exclusion chromatography and iodine staining
(Figure 2). Amylopectin molecules are bigger than amylose molecules, and therefore amylopectin is eluted as an
early single peak, whereas amylose is eluted as a broader peak in later fractions of size exclusion chromatography. In
addition, amylopectin and amylose can be discriminated by staining with iodine. Complexes of iodine and amylose
are dark blue with an absorption maximum (λ-max) above 600 nm, whereas complexes of iodine and amylopectin
are brown with an absorption maximum at 540 nm. The size exclusion chromatography shows that normal Golden
Promise barley starch consists of a major amylopectin fraction, which is 70% of the total starch, and a much broader
fraction of amylose, corresponding to 30% of the total starch. In contrast, our genetically engineered line consisted of
two fractions of smaller amylose molecules corresponding to > 99% of the total starch and a fraction of amylopectin
corresponding to less than 1% of the total starch.
Figure 2 Combined size exclusion chromatography and iodine staining of normal barley
starch and amylose-only starch. Modified from Carciofi et al. 1

ISB NEWS REPORT APRIL 2013
This is the first time that an amylose-only plant starch type has been identified. We therefore studied the consequences
of eliminating amylopectin in grains. The amylose-only starch was organized in starch granules with
a different shape than normal barley starch granules (Figure 3). Normal barley starch consists of round granules
divided into two sizes—big A-granules and smaller B-granules. The amylose-only starch granules consisted of irregularly
shaped granules formed by the gluing of smaller round granules. Amylopectin gelatinizes between 60ºC
and 70ºC. No gelatinization of the amylose-only starch was observed at that temperature, confirming the absence of
amylopectin. The absence of amylopectin also resulted in complete loss of swelling when heated up to 100ºC, and
the solubility was reduced from 60% to 20% when boiled in water.
Figure 3. Scanning electron micrographs of normal barley starch and amylose-only starch. Scale bars represent
50 µm and 10 μm in the low and high magnification panels respectively. Arrows indicate A- and B-granules in
normal starch. Modified from Carciofi et al. 1
Such a drastic modification of endosperm starch could have a negative impact on grain development, yield,
and germination. But we found that the starch content of amylose-only grains was only 5% less than normal barley
grains. In a field trial, we found that the germination rate of grains from amylose-only plants were the same as in
normal grains. The yield (g grain / plant) was 25% lower in amylose-only plants, mainly due to a lower number of
spikes and only partly due to smaller grains.
Resistant starch
Digestibility of starch is important from a nutritional point of view. Rapidly digestible starch results in a quick glycemic
response, due to the formation of glucose after hydrolysis by salivary and pancreatic α-amylase. A fraction
of dietary starch escapes upper gut degradation and reaches the large intestine where it is fermented by gut bacteria.
This fraction is called resistant starch, and a definition by Englyst et al. 3 divides dietary starch in three fractions:
rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS). Amylose-content has a
direct positive correlation with RS content. We found that the amount of RDS in gelatinized (cooked) and retrograded
(cooled after cooking) amylose-only starch was 20% and 13%, respectively. In comparison, the amount of
RDS in the normal barley starch in gelatinized and retrograded starch was 41% and 42%, respectively. Similarly,
the RS fraction was 65% in gelatinized amylose-only starch and 68% in amylose-only retrograded starch, but only
29% both in gelatinized or retrograded normal barley starch.
Conclusions and perspectives
The generation of a high yielding amylose-only starch in barley is a proof-of-concept that it is possible to genetically
engineer cereal plants to produce starch granules without amylopectin. The mechanism of starch biosynthesis
is relatively similar among cereal grains, suggesting that amylose-only starch can also be produced in cereal crop
species other than barley. We used RNAi, but the concept may also be applied using other techniques, such as
mutagenesis breeding. However, it should be emphasized that by using the RNAi technique we present here, the
amylose-only trait segregates as a single locus dominant allele, because the silencing construct was designed to

ISB NEWS REPORT APRIL 2013
target three different genes simultaneously. In contrast, mutagenesis breeding will have to deal with segregation of
all SBE genes in several independent loci. Another aspect of the RNAi approach is that the amylose-only trait was
achieved even though not all SBE gene activity was silenced, and therefore we cannot exclude the possibility that
a full loss-of-function of all SBE gene activity would jeopardize grain filling or otherwise reduce yield much more
than in the barley amylose-only line presented here. A mutagenesis breeding approach may therefore have to include
the combination of alleles that do not represent full loss-of-function but only partial loss of expression levels.
The production of amylose-only starch in cereals may provide an opportunity to produce cereal starch for new
functionalities. Amylose, for example, is excellent for generating water-resistant films and bioplastics 2 . Amyloseonly
cereals could therefore provide production of starch-based bioplastics directly from extracted starch without
prior amylose extraction. We found that amylose-only starch is very resistant to enzymatic degradation, and that the
amount of readily digestible starch in raw as well as gelatinized (cooked) and retrograded amylose-only starch is
much reduced compared with normal starch. Such a reduction in amount of readily digestible starch is associated
with numerous dietary health promoting effects, suggesting that cereal amylose-only crops would be excellent for
dietary purposes.
In conclusion our amylose-only barley acts as a proof-of-concept demonstrating that amylose-only starch can be
produced in cereals. We see possible applications suggesting that cereals producing such amylose-only starch may
benefit both the producer by increased crop value due to specialized functionalities, and the consumer by improving
the nutritional value.
References
1. Massimiliano Carciofi, Andreas Blennow, Susanne L. Jensen, Shahnoor S. Shaik, Anette Henriksen, Alain Buléon, Preben B. Holm, Kim H. Hebelstrup (2012).
Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules. BMC Plant Biology, 12:223
2. Starch: Chemistry and Technology, 3rd Edition (2009). Edited by James BeMiller and Roy Whistler. Academic Press, Elsevier Inc.
3. Englyst HN, Kingman SM, Hudson GJ, Cummings JH (1996). Measurement of resistant starch in vitro and in vivo. British Journal of Nutrition, 75:749–755
Kim H. Hebelstrup, Massimiliano Carciofi & Andreas Blennow
Aarhus University, Denmark
University of Copenhagen, Denmark
Kim.Hebelstrup@agrsci.dk