Co-suppression of synthesis of major α-kafirin sub-class together with γ-kafirin-1 and...
Transcript of Co-suppression of synthesis of major α-kafirin sub-class together with γ-kafirin-1 and...
ORIGINAL PAPER
Co-suppression of synthesis of major a-kafirin sub-class togetherwith c-kafirin-1 and c-kafirin-2 required for substantiallyimproved protein digestibility in transgenic sorghum
Andile W. Grootboom • Nompumelelo L. Mkhonza • Zodwa Mbambo •
Martha M. O’Kennedy • Laura S. da Silva • Janet Taylor •
John R. N. Taylor • Rachel Chikwamba • Luke Mehlo
Received: 8 October 2013 / Revised: 4 December 2013 / Accepted: 18 December 2013 / Published online: 19 January 2014
� Springer-Verlag Berlin Heidelberg 2014
Abstract
Key message Co-suppressing major kafirin sub-classes
is fundamental to improved protein digestibility and
nutritional value of sorghum. The improvement is
linked to an irregularly invaginated phenotype of pro-
tein bodies.
Abstract The combined suppression of only two genes, ckafirin-1 (25 kDa) and c-kafirin-2 (50 kDa), significantly
increases sorghum kafirin in vitro digestibility. Co-sup-
pression of a third gene, a-kafirin A1 (25 kDa), in addition
to the two genes increases the digestibility further. The
high-digestibility trait has previously only been obtained
either through the co-suppression of six kafirin genes
(a-A1, 25 kDa; a-B1, 19 kDa; a-B2, 22 kDa; c-kaf1,
27 kDa; c-kaf 2, 50 kDa; and d-kaf 2, 18 kDa) or through
random chemical-induced mutations (for example, the high
protein digestibility mutant). We present further evidence
that suppressing just three of these genes alters kafirin
protein cross-linking and protein body microstructure to an
irregularly invaginated phenotype. The irregular invagin-
ations are consistent with high pepsin enzyme accessibility
and hence high digestibility. The approach we adopted
towards increasing sorghum protein digestibility appears to
be an effective tool in improving the status of sorghum as a
principal supplier of energy and protein in poor commu-
nities residing in marginal agro-ecological zones of Africa.
Keywords Sorghum � Biofortified � Transgenic � Protein
bodies � Protein digestibility � Gamma-kafirin
Introduction
Sorghum is a key C4 (carbon 4 photosynthesis) plant and is
endowed with many attractive attributes of C4s including
high photosynthetic efficiency, high water and nitrogen use
efficiency, and significant tolerance to biotic and abiotic
stresses (Ishimaru et al. 1997; Matsuoka et al. 2001).
Sorghum is a primary source of energy and nutrients for
many millions of people in marginal agricultural parts of
the semi-arid tropics of Africa, where other cereals have
inferior productivity (ICRISAT 2011; ICRISAT and FAO
1996). Unfortunately, sorghum has some nutritional limi-
tations; specifically, it is deficient in the essential amino
acid lysine and its protein has lower wet-cooked digest-
ibility when compared to other cereals. Comparative
studies have revealed the following in vitro digestibility
values for important cereals when cooked: 60 % white tan-
plant sorghum, 86 % wheat, 85 % maize, and 84 % rice
(Mertz et al. 1984). The question of what is the exact cause
of sorghum low protein digestibility has never been
answered satisfactorily because there are many factors that
play a significant role. Secondary metabolites such as
Communicated by L. Jouanin.
Electronic supplementary material The online version of thisarticle (doi:10.1007/s00299-013-1556-5) contains supplementarymaterial, which is available to authorized users.
A. W. Grootboom � N. L. Mkhonza � Z. Mbambo �M. M. O’Kennedy � R. Chikwamba � L. Mehlo (&)
CSIR/BioSciences, Meiring Naude Road, Brummeria,
Pretoria 0001, South Africa
e-mail: [email protected]
L. S. da Silva � J. Taylor � J. R. N. Taylor
Department of Food Science, University of Pretoria, Private Bag
X20, Hatfield 0028, South Africa
L. S. da Silva
Department of Biotechnology and Food Technology, Tshwane
University of Technology, Pretoria 0001, South Africa
123
Plant Cell Rep (2014) 33:521–537
DOI 10.1007/s00299-013-1556-5
tannins are rich in phenolic hydroxyl groups and therefore
form complex with proteins (Butler et al. 1984). This
makes them inaccessible to digestive enzymes. It is clear
that other factors besides tannins are influential because
low digestibility in sorghum proteins is still prevalent even
amongst tannin-free sorghums (Elkin et al. 1996). Much
evidence, however, suggests that the nature and arrange-
ment of kafirin storage proteins may be a major determi-
nant of protein digestibility. The a-, b- and c-kafirins make
up about 70–80 % of total endosperm protein (Hamaker
et al. 1995), and out of this combined value, the a-sub-class
alone comprises about 80 % of total kafirin endosperm
proteins (Shewry 2002). The a-kafirins are majorly located
at the centre of protein bodies, whereas the b- and c-kafi-
rins are concentrated at the periphery of the protein body
(Hamaker et al. 1995; Oria et al. 1995b; Shull et al. 1992;
Watterson et al. 1993).
The widely accepted theory suggests that poor digest-
ibility of the abundant and easily digestible (when isolated)
a-kafirins is a result of their interior location and the fact
they are surrounded by a kafirin protein complex of
disulphide-bonded c- and b-kafirins (Duodu et al. 2003;
Oria et al. 1995a; Rom et al. 1992). Important support for
this theory is the observation that a unique microstructure
of mutant sorghum protein bodies (derived from the
chemically induced opaque O-721 mutant), which are
characterized by irregularity and invagination, is consis-
tently correlated with the high protein digestibility of this
mutant (Oria et al. 2000). This probably is because of
increased protein body surface area which favours easy
accessibility to proteolytic enzymes. Significantly, in the
high protein digestibility mutant (Oria et al. 2000) and in
the original O-721 mutant (Sastry et al. 1986), the pro-
portions of the kafirin sub-classes appear to be normal, but
the c-kafirin sub-class is located at the base of the folds in
the invaginated protein bodies of the high-digestibility
mutant (Oria et al. 2000).
Efforts such as the African Biofortified Sorghum project
(ABS) aimed at improving the protein quality of sorghum
have been described (Lipkie et al. 2013; Zhao 2007). The
approach involved techniques such as the suppression of
the synthesis of the c-kafirin sub-class by the application of
RNAi technology (Shewry 2007). The transgenic lines
emanating from the ABS project had substantially
increased lysine content and improved in vitro digestibility
(Henley et al. 2010). A further examination of the
improved transgenic sorghum lines that were designed to
have various combinations of suppression of kafirin sub-
classes revealed that co-suppression of several kafirin sub-
classes was required to obtain high protein nutritional
quality in sorghum, but this seemed to result in a floury
type grain endosperm texture (Da Silva et al. 2011b). It was
further demonstrated that in transgenic sorghum designed
to have a wide range of kafirin sub-class suppression,
kafirin disulphide-bonded cross-linking was considerably
reduced and this seemed to improve protein digestibility
(Da Silva et al. 2011a). However, in both papers the work
was based on supposition as no direct evidence of specific
kafirin sub-class suppression was reported.
In the present study, we provide conclusive proof that
the combined suppression of two gamma-kafirins signifi-
cantly improves in vitro digestibility and that to obtain
further improvements in digestibility, a third gene (a-
kafirin) should also be co-suppressed. We further provide
evidence that the additional increase in protein digestibility
when the a-kafirin is suppressed probably results from
alterations in protein body ultrastructure.
Materials and methods
Transformation
Immature zygotic embryos isolated about 12 days post-
pollination from the sorghum public line P898012 (origi-
nally from Purdue University) were used for all transfor-
mations. P898012 is a purple plant, type II tannin sorghum.
Donor plants were grown in pots in the greenhouse in a soil
mix consisting of red soil, rough sand and compost in a
ratio of 1:1:1. Plants were watered three times per week
with a soluble fertilizer called Hortichem (Ocean Agri-
culture) with a nitrogen, phosphorus and potassium ratio of
3:1:5, respectively.
Two RNAi minimal cassette plasmid DNA constructs
designated as pABS042 and pABS044 were used for
transforming the sorghum embryos. These constructs were
obtained from Pioneer Hi-Bred International (Johnston,
Iowa, USA) and are outlined in a schematic way in Fig. 1.
The pABS042 construct was designed to co-suppress the
synthesis of the following sorghum kafirin grain storage
protein species: d-kafirin 2 (18 kDa), c-kafirin 1 (25 kDa)
and c-kafirin 2 (50 kDa). In addition to the kafirins, this
construct was also designed to suppress the synthesis of an
enzyme involved in lysine catabolism called lysine a-
ketoglutarate reductase (LKR). The pABS044 construct
was aimed at the combined suppression of all the kafirin
sub-classes in pABS042 combined with an additional
25 kDa a-kafirin-A1 sub-class (d-kafirin 2, c-kafirin 1, c-
kafirin 2, and a-kafirin-A1). Similar to pABS042, the
enzyme LKR was also targeted for suppression in
pABS044. Each of the constructs pABS042 and pABS044
was co-bombarded into sorghum immature embryos with a
minimal plasmid cassette encoding for mannose selection
as previously reported (O’Kennedy et al. 2004a). All
plasmid DNA preparations were carried out using the
Qiagen Maxiprep Kit (Qiagen) according to the
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123
manufacturer’s recommendations. Minimal transgene
expression cassettes were used for transformation follow-
ing restriction enzyme digestion of parent plasmids with
EcoRI to remove the backbone carrying the kanamycin
resistance gene (Fig. 1).
Explants were prepared and transformed essentially
according to previously described methods (Grootboom
et al. 2010). Briefly, immature zygotic embryos were pre-
cultured for 3–4 days in the dark on callus initiation
medium [CIM; medium J as previously described
(O’Kennedy et al. 2004b)] and then placed on CIM con-
taining 0.2 M D-sorbitol and 0.2 M D-mannitol as osmoti-
cum, for 3–4 h, as previously described (Vain et al. 1993).
Bombardment mixtures were prepared by DNA precipita-
tion on 0.6 lm gold particles with 2.5 M CaCl2 and 0.1 M
spermidine-free base (Vain et al. 1993). Following bom-
bardment with 40 ng linearized plasmid DNA, the embryos
were incubated on this medium for 16 h, followed by
7 days on osmoticum-free CIM. Selection and regeneration
of transgenic plants were carried out as previously descri-
bed (O’Kennedy et al. 2004b).
DNA extraction and Southern hybridization analysis
Genomic DNA was extracted from putative transgenic
sorghum leaf material using a mini-extraction procedure
(Dellaporta et al. 1983). The DNA was then resolved in a
0.8 % agarose gel, transferred to a nylon membrane and
probed with a DIG labelled DNA fragment spanning the
zein promoter and a-kafirin (44zk) and also the d- and c-
kafirin coding sequences (42cr). Southern hybridization
was carried out as previously described (O’Kennedy et al.
2004a). The 42cr probe was prepared using the following
primers: forward 50-GTTACGTGACCCGGACCGAA-30
and reverse 50-ACGCCGAAGATCGCCTGGTA-30. The
44zk probe was prepared using the following primers:
forward 50 GCTTGCTGCGATTGCCTGTT 30 as well as
reverse primer 50-CGGTGGGGATCGAGTGATTC-30.
Protein extraction from whole seeds
Ten mature dry T1 seeds from each transgenic event were
halved, and the halves were ground to a fine meal using a
mortar and pestle. As the T1 population was segregating, it
was expected that it would do so at a ratio of 3:1 (trans-
genic vs. non-transgenic) assuming that each of the genes
was heterogeneously integrated into one locus for each
genome. Each grain was sectioned longitudinally into half,
with one half retained for endosperm structure studies and
the other half for protein analysis. Endosperm texture,
defined here as the proportion of corneous endosperm
relative to floury endosperm in the grain, was determined
by viewing the longitudinal sections of half kernels under a
stereomicroscope and comparing them to sorghum
δ-kaf2 γ-kaf1 γ-kaf2 LKR
ADH1 Intron
δ-kaf2γ-kaf1γ-kaf2LKR Maize 19 kDaAlpha B1 zein promoter
EcoRI EcoRINdeI
δ-kaf2 γ-kaf1 γ-kaf2 LKR
ADH1 Intron
δ-kaf2γ-kaf1γ-kaf2LKR Maize 19 kDaAlpha B1 zein promoter
EcoRI EcoRINdeIα-kafA1 α-kafA1
a
b
Fig. 1 Plasmids used for transformation. a Plasmid pABS042.
b Plasmid pABS044. Both plasmids were digested with EcoRI and
gel purified to yield minimal transgene cassettes used for transfor-
mation. NdeI cuts once in each construct and the site is within the
maize 19-kDa Alpha B1 zein promoter. The NdeI site was used for
distinguishing independent transformation events through Southern
blot analysis. The main difference between pABS042 and pABS044
is that pABS044 is intended to silence the a-kaf-A1 subspecies in
addition to the kafirin subspecies indicated in pABS042. The two
plasmids were supplied by Pioneer Hi-Bred International
Plant Cell Rep (2014) 33:521–537 523
123
standards (ICC 2008). Use of SDS-PAGE analysis com-
bined with Western blot hybridization with cross-reacting
antibodies for each of the targeted proteins (d-kaf 2; c-kaf
1; c-kaf 2; a-kaf-A1) allowed the non-ambiguous identi-
fication of transgenic sorghum seeds and negative segre-
gants. To ensure that almost identical amounts of total
protein were analysed, 10 mg of each ground meal was
weighed separately and placed into 1.5 ml microfuge
tubes. Four hundred microlitres of a reducing total protein
extraction buffer (100 mM DTT; 2 % SDS; 60 mM Tris,
pH 6.8) was then added to each tube. The mixture was
vortexed briefly and the tubes were incubated at 100 �C for
8 min with intermittent vortexing. The mixture was cen-
trifuged at 14,000 rpm for 10 min at room temperature,
and the supernatant was collected and transferred into a
clean microfuge tube. Total protein concentration was
determined by the Bradford assay (Bradford 1976), using
bovine serum albumin (BSA) as the protein standard (Bio-
Rad). The BSA concentration range chosen for the standard
curve was 0, 1, 3, 5, 10, and 20 ng/ll protein.
RT-PCR assay for LKR
LKR suppression was only tested through reverse trans-
criptase PCR (RT-PCR) because currently there is no
monoclonal antibody specific for the LKR protein. Total
seed RNA was isolated at about 15 days post-anthesis
using Sigma-Aldrich (USA) total RNA extraction kit as per
manufacturer’s instructions. First-strand cDNA synthesis
was carried out from 1 lg total RNA using the manufac-
turer’s recommendations (cDNA synthesis kit, Roche-
South Africa). PCR methods were then carried out in 50 ll
reaction volumes using the following LKR-specific prim-
ers: forward 50-ACCGCATTCTGACAGGTCTTCTGA-30
and reverse 50-GGGCAATGGAGTTGTTGGAATTCT-30.Cycling conditions included an initial denaturation at
94 �C for 2 min, followed by 25 cycles consisting of
denaturation at 94 �C for 45 s, annealing at 64.6 �C for
30 s and extension at 72 �C for 45 s. The final extension
was for 3 min at 72 �C.
SDS-PAGE analysis
Ten micrograms of sample protein from each transgenic
line was resolved by SDS-PAGE. Electrophoresis was
performed using pre-cast 12 % Bis–Tris Bio-Rad Criterion
gel system (Bio-Rad). Briefly, protein extracts were pre-
mixed at a dilution of 4:1 with 49 sample loading buffer
containing dithiothreitol (DTT) (200 mM Tris–HCl, pH
6.8; 400 mM DTT; 8 % (w/v) SDS; 0.5 % (w/v) bromo-
phenol blue; 50 % glycerol). The samples were incubated
at 100 �C for 1 min before loading the gels. The voltage
was set at 120 V for 90 min and separation was monitored
using a pre-stained protein size marker (PageRulerTM
Prestained Protein Ladder, Fermentas). Visualization of
resolved protein bands was carried out after Coomassie
brilliant blue staining (0.2 % Coomassie stain, 45:45:10;
methanol:water:acetic acid) for 1 h, followed by destaining
in methanol:water and acetic acid combined in a ratio of,
45:45:10, respectively. Unless stated, the wild-type
P898012 or the null segregants of the transgenic plants
were used as reference controls.
Western blot analysis
Following separation and visualization of proteins by SDS-
PAGE, the semi-dry HoeferTM TE 77 transfer unit (Amer-
sham BioSciences) was used to transfer the resolved protein
bands onto the 0.45 lm pore size PVDF membranes (Sigma-
Aldrich). The PVDF membranes were pre-equilibrated in
99.9 % methanol for 5 min and re-suspended in Towbin’s
transfer buffer (25 mM Trizma-Base, pH 8.3; 192 mM
glycine; 20 % (v/v) methanol) for 5 min. The acrylamide gel
was also pre-equilibrated in Towbin’s transfer buffer for
15 min. The gel-membrane sandwich and current settings
were performed according to the manufacturer’s recom-
mendations (Bio-Rad). Membrane blocking was performed
for 1 h in blocking buffer (20 mM Tris, pH 7.5; 150 mM
NaCl; 0.05 % Tween 20; 3 % fat-free milk powder). Cross-
reacting antibodies directed against maize zein storage pro-
teins were used to probe their equivalent counterparts in
sorghum (25 kDa c kafirin-1, 50 kDa c-kafirin-2, 18 kDa d-
kafirin, and 25 kDa a-kafirin A1). Cross-reactivity was
previously demonstrated at Pioneer Hi-Bred International
(data not shown). All antibodies were used at a dilution of
1:10,000. Unbound antibodies were washed-off from the
membranes three times (10 min each wash) with TBST
buffer (20 mM Tris, pH 7.5; 150 mM NaCl; 0.05 % Tween
20) with constant shaking at 180–250 rpm. The membranes
were incubated for 1 h at room temperature with a 1:10,000
diluted secondary antibody (anti-rabbit conjugated horse-
radish peroxidase; Sigma-Aldrich). The membranes were
then washed three times for 10 min with TBST buffer. All
the steps were performed at room temperature. For protein
detection, the ECL PlusTM Western blotting system (Amer-
sham) was used according to the manufacturer’s instructions
to detect target antibody cross-reactions with targeted kafi-
rins. This was followed by autoradiography using the
‘‘8 9 10’’ BioMax MR Film (Kodak).
Transgenic trait: endosperm phenotype linkage and T1
Mendelian segregation
SDS-PAGE analysis of suppressed proteins, Western blot
analysis and seed endosperm texture of transgenic sorghum
were used to determine the correlation between the
524 Plant Cell Rep (2014) 33:521–537
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transgenic trait suppression using RNAi constructs and the
endosperm structure. Sectioned seeds were independently
scored for endosperm phenotype in two categories, i.e.
‘corneous’ vs. ‘floury’ endosperm texture. Ten seeds per
independent event, and from the same panicle, were inde-
pendently genotyped for the presence or absence of the
transgenic trait (suppression of target kafirin protein sub-
classes) as revealed by SDS-PAGE analysis. Western blot
analysis of the same protein extracts was performed to
confirm the identity of kafirin proteins suppressed as pre-
dicted from the constructs used. Known quantities of pro-
tein were loaded onto SDS-PAGE. A G:BOX gel
documentation system (Syngene) was used to capture
images of multiple replicates of the gels of each indepen-
dent transgenic line. In order to measure the extent of kafirin
suppression, intensities of specific bands (densitometry)
corresponding to gamma-kafirins were determined using
Gene Tools software (Syngene). Flouriness was determined
using a scheme devised by Rooney and Miller (1982) in
which one (1) represents corneous, three (3), intermediate
and five (5), floury endosperm. A statistical correlation
between the seed flouriness and extent of transgenic protein
suppression was then performed for seeds of both transgenic
events of pABS042 and pABS044. A Chi-square test, at a
5 % level, was also used to determine whether there was
any deviation from the expected T1 3:1 Mendelian segre-
gation ratio of transgenic to non-transgenic seeds. Whole T1
seeds were further screened for any possible abnormalities
as a result of the tissue culture stress by comparison to wild-
type seeds that had been subjected to the same tissue culture
and bombardment stress. Ten randomly chosen seeds per
event, from the same panicle, were weighed and compared
to wild-type P898012 seeds that had been regenerated on
the same medium, but lacking mannose selection.
Grain materials used for physical and chemical
characterization
Two groups of sorghums were analysed (Table 1): (1)
Transgenic sorghums and their null controls from cultivar
P898012 (a white type II tannin sorghum)—independent
transgenic events generated from constructs pABS042, and
pABS044) (Fig. 1), a null segregant P898012 control sample
(NS), and two parent types; and (2) Non-transgenic sorgh-
ums all white non-tannin tan-pant types—seven high protein
digestibility mutant lines (HPDM 1–7), either original lines
as described by Oria et al. (2000) or crosses between HPDM
and normal lines (all obtained from Texas A&M University);
five normal protein digestibility (NPD), either parent or
progeny lines (NPD 1–5); plus Macia, a popular improved
sorghum variety cultivated and used for food preparation in
southern Africa.
Table 1 Transgenic, high protein digestibility mutant and normal
sorghum lines studied
Sorghum lines Project
code
Sample
type
Source
Transgenic (type II tannin)
ABS042 (AGNM 42-2B) ABS042-1 H/CG Pretoria
(2007)a
ABS042 (AGNM 42-5C) ABS042-2 H/CG Pretoria
(2007)a
ABS042 (AGNM 42-6A) ABS042-2 H/CG Pretoria
(2007)a
ABS042 (AGNM 42-9B) ABS042-2 H/CG Pretoria
(2007)a
ABS044 (AGNM 44-1A) ABS044-1 H/CG Pretoria
(2007)a
ABS044 (AGNM 44-2G) ABS044-2 H/CG Pretoria
(2007)a
ABS044 (AGNM 44-3A) ABS044-3 H/CG Pretoria
(2007)a
Controls (type II tannin)
P898012 (null control tissue
culture)
P898012-
TC
H/CG Pretoria
(2007)a
P898012 (parent) P898012-
H/CG
H/CG Pretoria
(2007)a
P898012 (parent) P898012-
Bulk
Bulk
(WCG)
Johnston,
Iowa
(2007)b
High protein digestibility mutants (HPDM) (non-tannin)
HD parent PI851171 WES HPDM1 Bulk
(WG)
Weslaco,
Texas
(2006)c
HD parent PI850029 WES HPDM2 Bulk
(WG)
Weslaco,
Texas
(2006)c
HD parent PI851171 LUB HPDM3 Bulk
(WG)
Lubbock,
Texas
(2006)c
HD progeny 04CS11248-1
XTX436 WES
HPDM4 Bulk
(WG)
Weslaco,
Texas
(2006)c
HD progeny 04CS112278 X
851171/96GCP0124 WES
HPDM5 Bulk
(WG)
Weslaco,
Texas
(2006)c
HD progeny 04CS11186-1
X 850029/TX635 WES
HPDM6 Bulk
(WG)
Weslaco,
Texas
(2006)c
HD progeny 04CS11278 X
851171 and 96GCPO124
LUB
HPDM7 Bulk
(WG)
Lubbock,
Texas
(2006)c
Normal protein digestibility (NPD) (non-tannin)
LD parent 96GCPOB124
WES
NPD1 Bulk
(WG)
Weslaco,
Texas
(2006)c
LD parent BTX436 WES NPD2 Bulk
(WG)
Weslaco,
Texas
(2006)c
Plant Cell Rep (2014) 33:521–537 525
123
As indicated in Table 1, samples were received as
10–15 single half or crushed kernels, or a bulk samples
(3–15 kg), which were received as whole grains, or whole
crushed grains. For chemical analyses, whole or crushed
bulk samples were milled into flour using a hammer mill
fitted with a 500-lm opening screen. Small samples were
milled by hand using a mortar and pestle. All samples were
stored ±8 �C until use.
Physical characterization
Endosperm texture
To characterize endosperm texture, longitudinal sections of
20 half kernels were viewed under a stereomicroscope,
followed by comparison to sorghum standards (ICC 2008).
Due to the small sample size of the transgenic lines ana-
lysed, all half kernels obtained were viewed (±6 kernels).
Light micrographs were taken using a stereomicroscope
(Nikon Optiphot) fitted with a digital camera.
Transmission electron microscopy
For transmission electron microscopy (TEM), grains were
sectioned longitudinally using a sharp scalpel. The pericarp
was scraped from the top of the kernel directly opposite to the
germ, leaving the sub-pericarp and aleurone layers intact. In
brief, the preparation procedure involved taking small sec-
tions (1–2 mm thick) of cleaned peripheral endosperm using
a sharp scalpel. Specimens were fixed as described by Da
Silva et al. (2011b), in glutaraldehyde (2.5 %) in pH 7.4,
0.075 M phosphate buffer for 18 h before staining with
0.5 % aqueous osmium tetroxide for 2 h. The specimens
were then dehydrated in a graded aqueous acetone series
before infiltration with Quetol resin. Ultrathin sections were
cut with an ultra-microtome fitted with a diamond knife.
Sections were stained with aqueous uranyl acetate and then
further stained in Reynold’s lead citrate. Sections were
examined either with a Phillips EM301 or Phillips CM10
TEM (Eindhoven, the Netherlands).
Chemical characterization
Tannin content
Condensed tannin content was determined on all bulk
sorghum samples using the modified Vanillin HCl assay
(1 % conc. HCl in methanol extraction) according to
Maxson and Rooney (1972), with subtraction of sample
blanks. Tannin content was then expressed as catechin
equivalents. Total protein content (N 9 6.25) was deter-
mined by Dumas combustion, according to AACC standard
method 46–30 (AACC International 2000). Moisture con-
tent was determined by air oven drying using AACC
standard method 44–15A (AACC International 2000). For
small samples, 10 % moisture content was assumed. All
analyses were expressed on a dry weight basis.
Lysine determination
Lysine was determined on defatted samples using the Pico-
Tag method (Bidlingmeyer et al. 1984), with reversed-
phase high-performance liquid chromatography.
In vitro protein digestibility
In vitro protein digestibility (IVPD) was determined on whole
grain flour under wet-cooked conditions, using either 200 mg
or 20 mg flour scale using the pepsin digestibility method of
Hamaker et al. (1986), suitably modified for small-scale
assays. Briefly, the method involved incubating the flour with
pepsin at pH, 37 �C for 2 h. Protein digestibility was then
defined as the percentage N solubilised under the conditions of
the assay relative to flour total N. This was measured in terms
of insoluble residue by the above Dumas method.
Results
Suppression of c- and a-kafirin sub-classes
Several P898012 sorghum transgenic events were recov-
ered following particle bombardment transformation with
the constructs pABS042 designed to suppress the kafirin
Table 1 continued
Sorghum lines Project
code
Sample
type
Source
LD parent TX635 WES NPD3 Bulk
(WG)
Weslaco,
Texas
(2006)c
LD parent 96GCPOB124
LUB
NPD4 Bulk
(WG)
Lubbock,
Texas
(2006)c
LD progeny 04CS11199-1
X 850029/TX635 WES
NPD5 Bulk
(WG)
Weslaco,
Texas
(2006)c
Macia Macia Bulk
(WG)
Botswana
(2004)
H/CG half/crushed grain, WCG whole crushed grain, WG whole graina CSIR Biosciences, Pretoria, South Africab Pioneer Hi-Bred, Des Moines, Iowa, USAc Texas A&M University, College Station, Texas, USA
526 Plant Cell Rep (2014) 33:521–537
123
storage proteins d-kafirin 2, c-kafirin 1, c-kafirin 2 and the
catabolic enzyme lysine a-ketoglutarate reductase (LKR),
and pABS044 designed to suppress a-kafirin-A1 in addi-
tion to the suppression of the kafirin sub-classes in
pABS042. The pABS042 construct was similar in design to
that used to produce ABS 149 (Da Silva et al. 2011b).
However, this study looked at a similar construct individ-
ually and in combination with the suppression of the alpha
A1 kafirin sub-classes in events generated by particle
bombardment in a different sorghum type background.
Recovery of transgenic events was achieved at a transfor-
mation efficiency that varied between 2 and 5 %. Eight
independent transgenic events from each of the constructs
pABS042 and pABS044 were initially screened using
SDS-PAGE to ascertain the extent to which each construct
influenced the targeted kafirin sub-classes suppression. As
shown in Fig. 2, construct pABS042 was able to suppress
the c-kaf sub-classes (Fig. 2a), whereas pABS044 suc-
ceeded in suppressing both the c-kaf and the a-kaf sub-
classes (Fig. 2b), and this was confirmed further via
Western blot analysis in subsequent experiments. Further
verification of stable integration of each of the constructs
was carried out by Southern hybridization analysis of
genomic DNA extracted from each putative T0 transgenic
event following digestion with NdeI, a unique restriction
site within each construct. Independent events were
therefore distinguished from each other on the basis of the
occurrence of different integration patterns. Seeds from
transgenic progeny were also able to germinate on man-
nose-containing medium as previously described (O’Ken-
nedy et al. 2004a).
From the eight independent transgenic events containing
the pABS042 construct, four independent transgenic events
were chosen for further analysis. Three independent
transgenic events bearing the construct pABS044 were also
selected for further evaluation. These transgenic lines were
chosen on the basis that they exhibited complete suppres-
sion of the c-kafirin proteins (pABS042) and the combined
M W NS1 NS2 WpABS042 TRANSGENIC EVENTS
M W NS1 NS2 W pABS044 TRANSGENIC EVENTS
a
b
26 kDa
34 kDa
26 kDa
34 kDa
1 2 3 4 5 6 7 8
1 2 3 4 5 6 7 8
Fig. 2 SDS-PAGE analysis of kafirin storage proteins of transgenic
sorghum events with specific kafirin sub-classes targeted for
suppression. Protein profiles of eight independent events generated
from the pABS042 construct are shown in a, whereas eight
independent events generated from the pABS044 construct are also
shown in b. M 26 and 34 kDa bands of the protein molecular weight
marker, SM1841 from Fermentas Life Sciences. W protein profile of
wild-type (untransformed) P898012 sorghum seed total protein. NS1
and NS2 protein profiles of total seed protein of two independent null
segregants of transgenic events. The thick black arrow indicates the
relative apparent molecular weight position (about 26 kDa) of protein
bands identified as suppressed based on comparisons with the wild-
type and null segregant controls
Plant Cell Rep (2014) 33:521–537 527
123
complete suppression of c-kafirin and a-kafirin A1 protein
sub-class (pABS044) as confirmed by Western blot ana-
lysis (Fig. 3). In order to assess the impact of suppressing
either the c-kaf subspecies or the combined c-kaf and a-
kaf-A1 subspecies, it was important to choose only those
transgenic events exhibiting complete suppression. Our
observation of the two categories of independent events on
SDS-PAGE (pABS042; pABS044) indicated a range of
suppression encompassing complete-partial suppression
(compare for example, events 1–3 vs. events 6–8 in
Fig. 2b; events 1–3 exhibit complete suppression).
Even though the RT-PCR data for LKR suppression was
inconclusive, it appears that all 16 independent transgenic
events (8 of pABS042 and 8 of pABS044) had LKR
expression (supplementary data). Our inference was there-
fore that since LKR was not suppressed it is unlikely that it
(LKR) had much influence on lysine content, endosperm
microstructure and protein digestibility in transgenic events
of both pABS042 and pABS044. Western blot analysis of
the d-kaf 2 protein in both transgenic and non-transgenic
plants suggested that this protein was not expressed in the
sorghum line P898012. However, the d-kaf 2 gene is present
in the P898012 sorghum genome although not expressed.
This is the case with a limited number of other sorghum
varieties where allelic variations of kafirin genes are
observed (Laidlaw et al. 2010). Similar to LKR, no changes
in lysine, endosperm structure and protein digestibility in
transgenic P898012 events could therefore be directly
attributed to the suppression of d-kaf 2.
Physical and chemical characterization
To assess the effect of suppressing specific sub-classes of
kafirin proteins in sorghum, physical and chemical
M NS M NS M NS M NSEV1 EV2 EV3 EV4
NSNSNS EV2EV1 EV3
NS NS NS NS EV1 EV2 EV3 EV4
NS NS NSEV1 EV2 EV3e
pABS042 events
Typical pABS044 event
26 kDa 17 kDa 10 kDa
50 kDa
50 kDa 25 kDa: Not targeted in pABS042; But targeted in pABS044
a
b
c
d
Fig. 3 Western blot analysis of kafirin protein suppression in
transgenic P898012 sorghum plants generated from constructs
pABS042 and pABS044. a Gamma-kafirin-1 suppression in four
independent events of pABS042. Four replicates (clones) of each
independent event were used. b Gamma-kafirin-1 suppression in three
independent events of pABS044. Four replicates of each independent
event were used. c c-kafirin-2 suppression in four independent events
of pABS042. Each event was replicated four times (clones). d c-
kafirin-2 suppression in three independent events of pABS044. Each
independent event was replicated three times (clones). e Western
analysis of typical non-targeted 25 kDa alpha A1 kafirin protein in
independent events of pABS042 and targeted in a typical alpha A1
protein suppressed independent event of pABS044. As expected, the
alpha A1 protein was completely suppressed only in targeted
pABS044 events and not in pABS042. Total seed proteins of T1
transgenic sorghum seeds were used. EV1, EV2, etc. represent
independent events. NS is null segregant seed protein (control) and
was derived from each respective category of transgenic events
(pABS042 or pABS044)
528 Plant Cell Rep (2014) 33:521–537
123
analyses of the sorghum grains were carried out.
Transgenic sorghum lines (pABS042 and pABS044) all
had floury endosperm textures (Fig. 4a, b). Their normal
sorghum parent (P898012) had intermediate endosperm
texture (Fig. 4f). The HPDM also had floury endosperm
texture (Fig. 4c), but its parent had corneous endosperm
texture, as did Macia (Fig. 4d, e, respectively). Many of
the transgenic grains derived from the constructs
pABS042 and pABS044, and to a lesser extent their
normal parent, P898012, showed a distinct lumen (small
hole) in the centre of the endosperm (Fig. 4, black
arrows).
The floury endosperm texture was observed in trans-
genic seeds obtained from both constructs pABS042 and
pABS044 as explained in Fig. 4. There was variation
amongst transgenic events in the degree of endosperm
flouriness and the extent of gamma-kafirin suppression as
surmised from SDS-PAGE analysis. As such, 20 seeds
from each of the 8 independent transgenic events of
pABS042 and pABS044 were therefore sectioned longi-
tudinally and viewed under a stereomicroscope. The per-
centage of endosperm flouriness was estimated from
comparisons with sorghum standards (ICC 2008). A plot of
percentage flouriness and percentage gamma-kafirin
a b
dc
fe
Fig. 4 Longitudinal cross sections of representative sorghum grains
of the different types. a pABS042, b pABS044, c HPDM Progeny
04CS11248-1 XTX436 WES, d normal protein digestibility parent
BTX436 WES, e Macia, f P898012. Scale bar 2 mm. Black arrow
indicates lumen in centre of endosperm; black dashed arrow indicates
tannin staining, white arrow indicates dark pigmented testa layer
Plant Cell Rep (2014) 33:521–537 529
123
suppression indicated that there was a high probability that
the extent of floury endosperm texture in sorghum was
positively correlated with the extent of gamma-kafirin
suppression (Fig. 5).
All the P898012 lines were type II tannin sorghums
according to previously suggested classifications (Maxson
and Rooney 1972). The parent line showed typical tannin
staining of the corneous endosperm (Fig. 4f, black dashed
arrow). This is due to tannin leaching from the testa layer
and binding to the proteins in the dense peripheral endo-
sperm tissue. Tannin leaching was not visible in the
transgenic sorghum lines with floury endosperm. Never-
theless, a pigmented testa layer was visible between the
pericarp and endosperm layers of these grains (Fig. 4a, b,
white arrows). The tannin content of the parent, P898012,
was 2.77 mg catechin equiv./100 g flour, when extracted
with acidified methanol (Table 2). All the other sorghum
lines were classified as type I (non-tannin) sorghums, as
they contained low levels of phenols and no tannins (0.03
and 0.04 mg catechin equiv./100 g flour for HPDM and
NPD, respectively).
Considering grain protein content, the transgenic
sorghums had similar mean protein contents (12.7 % for
pABS042 and 14.3 % for pABS044) to their controls
(11.7 %) and the HPDM (12.8 %), and to the NPD
sorghums (13.2 %) (see Table 1 for notations, Table 2 for
data).
With regard to grain lysine content, the pABS042 con-
struct did not increase grain protein content of the P898012
line (Table 2). This is in agreement with previous findings
(Da Silva et al. 2011b) using a similar construct ABS049 to
transform TX430, a corneous endosperm sorghum. Due to
very limited sample size, only one pABS044 construct
sample was analysed for protein lysine content. Its protein
lysine content was high (3 %), so also was its protein
content (16 %) (Table 2). Hence, it is not clear whether
this gene construct caused increased protein lysine content.
In contrast, the protein lysine content of the HPDM (mean
2.1 %) was considerably higher than their normal digest-
ibility controls (1.4 %), as was previously reported (Wea-
ver et al. 1998) and thought to be as a result of
compensatory synthesis of lysine-rich non-kafirin proteins
(Guiragossian et al. 1978).
In vitro protein digestibility (IVPD)
The IVPD of the transgenic sorghum containing the
pABS042 gene construct (mean 39.2 %) was significantly
higher than its control (mean 28.5 %) (Table 2). It was
however not different from the mean IVPD of 41.1 % for a
non-tannin sorghum line TX430 transformed with the
similar pABS149 construct (Da Silva et al. 2011b) but was
higher than the 28.5 % cooked protein digestibility of the
TX-430 line with c-kafirin suppression (Kumar et al.
2012). This contrasted with the IVPD of cooked flours
from the transgenic sorghum lines with the pABS044 gene
construct (mean 53.7 %), which was considerably higher
than the control and also higher than the 35–39 % protein
digestibility of the TX430 line with the predicted 29 kDa
a-kafirin suppression (Kumar et al. 2012). Significantly,
the IVPD of pABS044 was similar to that of the HPDM
sorghums (mean 57.7 %). This is despite the fact that the
pABS044 sorghum contained tannins, which are well
known to reduce sorghum protein digestibility (Duodu
et al. 2003). As expected, the HPDM sorghums had sub-
stantially higher IVPD than their normal parent and normal
progeny. However, the IVPD of HPDM was similar to that
of Macia. The cooked IVPD values of the HPDM sorgh-
ums in this present study were much lower than those of
HPDM sorghums reported for decorticated grain (Tesso
et al. 2006; Weaver et al. 1998) (range 72.5–80.8 %),
where some factors inhibiting protein digestion would have
been removed (Duodu et al. 2003), but almost identical
(55.4 %) to that reported for whole grain (Taylor and
Taylor 2011).
To assess the possibility of a link between endosperm
texture and digestibility, protein body structure of trans-
genic sorghum obtained following suppression of the c-
and a-kafirin sub-classes was studied by TEM. A signifi-
cant finding was that the protein bodies from the pABS042
transgenic line (Fig. 6c) appeared to be identical in size
and shape to the protein bodies of its normal control,
y = 1.0195x - 0.0803
R2 = 0.9916
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100 120
% Floury endosperm
% g
amm
a ka
f b
and
su
pp
ress
ion
Fig. 5 Correlation chart for suppressed gamma-kafirin expression
with the floury endosperm phenotype for transgenic events. Twenty
seeds from each of the eight independent transgenic events of
pABS042 and pABS044 were cross sectioned and visualized under a
stereomicroscope and also analysed via SDS-PAGE and Western blot
analysis. When the extent of endosperm flouriness [as estimated from
sorghum standards (ICC 2008)] was plotted against the extent of
gamma-kafirin suppression as estimated from SDS-PAGE and
Western blot analysis, it appears that there was a positive correlation
between gamma-kafirin suppression and endosperm flouriness as
indicated by a co-efficient of 0.9916
530 Plant Cell Rep (2014) 33:521–537
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P898012 (Fig. 6a). This was in agreement with previous
observations (Da Silva et al. 2011b) where it was found
that the protein bodies of ABS 149 (with the same kafirin
suppression as pABS042) had protein bodies that were
normal in shape, packing density and size to that of the
parent. Similarly for TX430 (Kumar et al. 2012) with only
c-kafirin suppression, the protein bodies were normal in
appearance but those with the predicted 29-kDa a-kafirin
suppression had distorted protein bodies. pABS042 had
densely packed, individual spherical protein bodies,
2–3 lm in diameter. Some of the protein bodies exhibited
the internal concentric ring structure (black dashed arrow),
but were fewer in number than those observed in the pro-
tein bodies of normal sorghum (Fig. 6a) and in the litera-
ture (Shull et al. 1992). The more limited concentric ring
structures from the pABS042 construct compared to nor-
mal sorghum is probably related to the suppression of
c-kafirin sub-class, since the rings normally comprise high
concentrations of both b- and c-kafirins (Shull et al. 1992).
The protein body structure of pABS042 resembled that of
Table 2 Tannin, protein and lysine content and in vitro protein digestibility (IVPD) of wet-cooked whole grain flour of different transgenic and
high protein digestibility mutant sorghum compared to normal parents and Macia
Sorghum lines Tannin (mg CE/100 g flour) Protein (g/100 g flour db)
(N 9 6.25)
Lysine (g/100 g protein) IVPD (%) (wet cooked)
Transgenic (type II tannin)
pABS042-1 ND 10.57 ± 0.22 2.36 ± 0.12 *33.5 ± 8.3
pABS042-2 ND 9.07 ± 0.32 2.04 ± 0.31 *35.7 ± 6.0
pABS042-3 ND 13.21 ± 0.90 ND *41.7 ± 1.6
pABS042-4 ND 17.92 ± 0.39 ND *45.7 ± 9.3
Mean of ABS042 12.69a 2.20 39.2b
pABS044-1 ND 16.03 ± 0.01 3.06 ± 0.00 *55.4 ± 1.2
pABS044-2 ND 14.85 ± 0.29 ND *54.8 ± 0.8
pABS044-3 ND 12.12 ± 0.19 ND *50.9 ± 2.9
Mean of ABS044 14.33a 3.06 53.7c
Controls (type II tannin)
P898012-TC ND 10.19 ± 0.21 29.1 ± 9.3
P898012-H/CG ND 13.95 ± 3.81 30.7 ± 6.2
P898012-Bulk 2.77 ± 0.06 10.90 ± 0.51 2.05 ± 0.0.06 25.6 ± 1.5
Mean of controls 11.68a 2.05 28.5a
High protein digestibility mutant (HPDM) (non-tannin)
HPDM1 0.01 ± 0.02 10.36 ± 0.01 2.10 ± 0.12 60.8 ± 1.0
HPDM2 0.03 ± 0.03 13.21 ± 0.19 2.41 ± 0.31 55.2 ± 1.5
HPDM3 0.04 ± 0.03 14.61 ± 0.05 ND 61.4 ± 1.6
HPDM4 0.01 ± 0.01 11.90 ± 0.04 2.04 ± 0.18 51.9 ± 5.6
HPDM5 0.03 ± 0.02 10.83 ± 0.03 2.22 ± 0.06 58.1 ± 1.6
HPDM6 0.06 ± 0.03 13.37 ± 0.12 1.87 ± 0.06 53.1 ± 1.3
HPDM7 0.01 ± 0.01 15.02 ± 0.01 ND 63.1 ± 1.3
Mean of HPDM 0.03 12.76a 2.13 57.7c
Normal protein digestibility (NPD) (non-tannin)
NPD1 0.08 ± 0.04 13.44 ± 0.02 1.29 ± 0.11 33.3 ± 2.3
NPD2 0.03 ± 0.01 12.16 ± 0.05 1.25 ± 0.17 36.4 ± 1.7
NPD3 0.02 ± 0.02 14.51 ± 0.07 1.37 ± 0.11 33.1 ± 1.6
NPD4 0.02 ± 0.05 15.12 ± 0.17 ND 36.7 ± 2.3
NPD5 0.05 ± 0.05 13.46 ± 0.06 1.20 ± 0.06 32.6 ± 1.1
Macia 0.04 ± 0.03 10.61 ± 0.01 1.87 ± 0.23 59.8 ± 0.7
Mean NPD 0.04 13.22 1.40 38.7b
Values are the mean and standard deviation per cultivar; each analysis was repeated twice with two replicates per sample; except where indicated
(*), where two replicates were analysed once. Values with different letters indicate significant differences between the groups at the 95 % level
using Fisher’s least significant difference (LSD) procedure
ND not determined, CE catechin equivalents
Plant Cell Rep (2014) 33:521–537 531
123
parental line P898012 and as such also had low IVPD
(Table 2). In contrast, the pABS044 transgenic line had
modified peripheral endosperm protein body structure
(Fig. 6d). The protein bodies of pABS044 were densely
packed, 2–3 lm diameters and deeply folded (invaginated)
with dark-staining inclusions (black solid arrows). Signif-
icantly, the protein bodies of pABS044 were similar in
shape to those of the HPDM (Fig. 6b). However, the pro-
tein bodies of the HPDM were smaller (approx. 1 lm
diam.). The protein bodies of pABS044 and HDPM were
also similar in that the majority of inclusions seemed to
extend radially from the protein body periphery (black
solid arrows). In addition, the characteristic internal con-
centric ring structure common to normal protein bodies
was absent. The modified protein body structure of
pABS044 is similar to that of high lysine, high protein
digestibility mutants, where the protein bodies are descri-
bed as being highly invaginated (with deep folds) (Oria
et al. 2000).
Discussion
Previous studies have shown that even though sorghum and
maize share equivalent sequence homology of storage
proteins, sorghum contains higher levels of cross-linked
prolamin storage proteins with an overall bias towards
intermolecular disulphide cross-linking of kafirins (Ha-
maker and Bugusu 2003). The higher disulphide cross-
linking of sorghum kafirins than that of maize zeins may be
related to the fact that both the 19 and 22 kDa a-kafirin
sub-classes contain an additional cysteine residue com-
pared to the zein homologues (Belton et al. 2006). Further,
it has been proposed that disulphide bond formation in
kafirin as a result of heating in food processing also pro-
motes realignment of kafirin into b-sheet structures, and
that these conformational changes cause the lower prote-
olysis susceptibility of kafirin (Emmambux and Taylor
2009). This partly explains the persistence of encapsulated
sorghum proteins (rigid protein bodies) remaining
throughout most food preparations, thus having an impor-
tant bearing on sorghum protein digestibility (Hamaker and
Bugusu 2003). Previously, we reported that co-suppression
of six kafirin genes: a-kaf A1, 25 kDa; a-kaf B1, 19 kDa;
a-kaf B2, 22 kDa; c-kaf1, 27 kDa; c-kaf 2, 50 kDa; and d-
kaf 2, 18 kDa led to increased digestibility in sorghum and
that the digestibility is directly correlated with an alteration
of protein bodies to an invaginated, irregular phenotype
(Da Silva et al. 2011b). In the current research, we present
data indicating that, more specifically, co-suppression of
P P
C
a b
c d
P
Fig. 6 Transmission electron micrographs of protein bodies found in
peripheral endosperm of parent non-transgenic P898012 (a), HPDM
Progeny 04CS11248-1 TX436 WES (b), transgenic pABS042 (c) and
transgenic pABS044 (d) sorghum lines. Within the images, C
represents the cell wall, P protein body, S starch granule; Black
arrow indicates deep folds (invaginations) in protein body wall,
whereas black dashed arrow indicates dark-staining concentric ring
inclusion. Bar 1 lm
532 Plant Cell Rep (2014) 33:521–537
123
only two genes (c-kaf1 and c-kaf2) significantly increases
in vitro protein digestibility and that when an addition of
third gene, a-kaf A1, is also co-suppressed the in vitro
digestibility increases significantly further.
Many cereals including sorghum, maize and rice are
deficient in the essential amino acid lysine. Previous
research suggests that in cereals, for example maize,
increased lysine content could result from many factors.
These include low rate of lysine catabolism due to reduced
levels of two consecutive enzymes, lysine a-ketoglutarate
reductase (LKR) and saccharopine dehydrogenase (SDH),
which are linked on a single polypeptide encoded by a
single LKR/SDH gene; reduced prolamine synthesis;
increased non-zein protein content, and to some extent
through alterations in the regulation of enzymes involved
in the lysine biosynthesis (Azevedo et al. 2003). Since the
catabolism of lysine is carried out by the activities of the
two consecutive enzymes (LKR and SDH), as previously
reported (Fornazier et al. 2005), we made an assumption
that suppressing one of these enzymes, LKR, would pre-
serve any lysine accumulated in transgenic sorghum as a
result of kafirin subspecies suppression. This assumption is
further supported by evidence that in the Opaque-2 mutant
maize containing higher concentrations of soluble lysine in
seeds, there is a reduction in the lysine catabolic rate in the
endosperm, which therefore allows excess lysine to be
incorporated into storage proteins as well as to get accu-
mulated in soluble form (Arruda et al. 2000; Azevedo
2002; Gaziola et al. 1999). Unfortunately, the construct we
used to suppress LKR in both ABS042 and ABS044 did not
achieve the levels of suppression we expected (Supple-
mentary data). This is probably because LKR and SDH are
present in sorghum seeds in many multimeric forms with
distinct molecular masses (Fornazier et al. 2005). Future
experiments utilizing different sorghum genetic back-
grounds may serve to verify whether the LKR approach is
indeed unfeasible. The presence of the LKR suppression
sequence in both the constructs pABS042 and pABS044
(Fig. 1) also implies, though not proven, that the impact or
lack thereof LKR would be approximately similar for both
constructs.
Sorghum lines bearing the three suppressed genes pos-
sessed the typical irregular and invaginated phenotype
associated with high digestibility (Fig. 6d) which favours
easy accessibility of proteolytic enzymes as previously
described (Mehlo et al. 2013; Oria et al. 2000). The two
constructs we used, pABS042 and pABS044 (Fig. 1), were
crafted to differentiate between the effect of suppressing
only two c-kafirin sub-classes (c-kaf1, 27 kDa; c-kaf 2,
50 kDa in pABS042) or the combined suppression of the
two c-kafirin sub-classes, and an a-kafirin sub-class
(c-kaf1, 27 kDa; c-kaf 2, 50 kDa; a-kaf A1, 25 kDa in
pABS044). Analysis of four independent transgenic lines
derived from the pABS042, and three from pABS044,
together with suitable controls [including a high protein
digestible mutant) as shown in Table 1; Figs. 1, 3] con-
firmed the successful suppression of c- and a-kafirins
through the absence of relevant protein bands on SDS-
PAGE which was further confirmed by Western blotting
(Fig. 3). When transgenic seeds were sectioned, we
observed that suppression of c-kaf1 (27 kDa) and c-kaf 2
(50 kDa) alone, or the two c-kafirin sub-classes co-sup-
pressed with a-kaf A1 (25 kDa), resulted in floury endo-
sperm (Fig. 4). Since the two c-kafirin sub-classes (c-kaf1
and c-kaf 2) were common in both pABS042 and
pABS044, we ascribed the floury endosperm phenotype to
the suppression of c-kafirin. However, it appears that there
were two types of floury endosperm: one type resulted in a
lumen in the centre of the endosperm, whereas the other
had completely filled grains. The lumen was predominantly
associated with both pABS042 and pABS044 grains,
whereas some completely filled floury endosperm pheno-
type was associated only with pABS044 (combined c-
kafirin and a-kafirin suppression). The finding that there
was a direct relationship between suppression of c-kafirin
by the pABS042 construct alone and the proportion of
floury endosperm in the intermediate endosperm P8989012
line (Fig. 5) differs somewhat from the observed little or no
effect of the similar pABS149 construct on the corneous
endosperm phenotype of sorghum line TX430. The effect
in P890812 is presumably due to the suppression of
c-kafirin inhibiting the formation of inter-polypeptide
disulphide cross-linking involved in corneous endosperm
structure. This concept is supported by the fact that mod-
ifying genes can be used to increase the level of c-zein in
high-lysine maize to restore the hard endosperm charac-
teristic (Wu et al. 2010). Causes of a floury endosperm
have previously been described and include loosely pack-
aged granules with air spaces (Rooney and Miller 1982).
The floury endosperm particularly that of pABS044 was
similar to that of the HPDM sorghum endosperm (Fig. 4c).
The HPDM has been reported to have high in vitro
digestibility (Oria et al. 2000). The reported explanation
suggests that this is a consequence of the nature of protein
bodies, particularly the irregular, invaginated form which
favours high digestive enzyme accessibility.
Our next set of experiments therefore sought to unravel
the nature of protein bodies in both pABS042- and
pABS044-derived transgenic sorghum seeds and an
assessment of their digestibility. First, irregularly invagi-
nated protein bodies were observed only in the floury
endosperms of pABS044 (where the 27 kDa c-kaf 1;
50 kDa c-kaf 2 and the 25 kDa a-kaf A1 proteins were
targeted for suppression). The irregular and invaginated
protein bodies in seeds of pABS044 resembled those of the
HPDM seeds (compare Fig. 6b with d). We previously
Plant Cell Rep (2014) 33:521–537 533
123
reported a similar phenotype of protein bodies in seeds of
transgenic events in which six kafirin genes (a-A1, 25 kDa;
a-B1, 19 kDa; a-B2, 22 kDa; c-kaf1, 27 kDa; c-kaf 2,
50 kDa; and d-kaf 2, 18 kDa) were targeted for suppres-
sion and the events generated through Agrobacterium-
mediated transformation (Da Silva et al. 2011b). Protein
body structure of pABS42 was similar to those of the
parental low digestibility P898012 control (compare
Fig. 6a with c), though they were comparatively larger.
When assayed for digestibility, floury seeds of pABS044
had significant improvement in IVPD, comparable to that
of the HPDM (Table 2). However, those of pABS042 also
had significant improvement in digestibility over the
parental control even though this improvement was less
than that imparted by the pABS044 construct. When these
results are considered together, it appears that IVPD was
largely influenced by the protein body microstructure,
particularly irregular and invaginated protein bodies, and
not necessarily the floury trait. This is because the floury
trait was common for both constructs. We therefore con-
sider that the combined suppression of c- and a-kafirins
was the primary cause of a significant increase in protein
digestibility (since c-alone in pABS042 failed to achieve
the same levels of digestibility obtained for pABS044). The
fact that in this present study both the transgenic lines and
the HPDM sorghum had all floury endosperms, whereas
their parents did not suggest the suppression of particularly
the c-kafirin sub-class leads to alteration not only of protein
synthesis but also of the involvement of this kafirin sub-
class in cross-linking.
It is clear from the results that altered protein body
structure of the pABS044 transgenic line may be respon-
sible for its higher cooked IVPD compared to pABS042
and normal sorghum lines, as was proposed by Oria et al.
(2000) using high protein digestibility sorghum lines. Thus,
it would appear that the altered protein synthesis of
pABS044, namely the combined reduction in c-kaf 1
(27 kDa), c-kaf 2 (50 kDa) and a-kaf A1 (25 kDa), had a
major effect on the peripheral and internal protein body
structure. Suppression of the a-kaf A1 kafirin probably
adversely affects kafirin polymerization through disulphide
bonding, since c-kafirin and a-1 kafirin link together (El
Nour et al. 1998). This in turn may disrupt normal protein
body formation. It can be speculated that in the case of the
HPDM, where the proportions of the different kafirin sub-
classes are apparently normal (Oria et al. 2000), the
invaginated protein body structure could be due to differ-
ences in the sequence of kafirin sub-classes synthesis.
We were therefore interested in finding out whether the
two different phenotypes engendered by the constructs
pABS042 and pABS044, particularly the floury endo-
sperm, had any serious impact on seed density/weight. Our
results indicate that there was little variation in seed
weight, morphology and germination rates. The approach
we adopted in this study, i.e. suppression of only three
kafirin protein sub-classes (27 kDa c-kaf 1; 50 kDa c-kaf
2; and the 25 kDa a-kaf A1) as opposed to the suppression
of six kafirin protein sub-classes (Da Silva et al. 2011b),
appears to be an effective method for improving sorghum
protein digestibility. Up until recently, there has been
limited progress in improving sorghum protein digestibil-
ity. However, with the advent of recombinant DNA tech-
nology it is now a subject of intense research (Henley et al.
2010; Hoang 2008; Jung 2008; Zhao 2007). Other
researchers (Henley et al. 2010) directly compared the
improvement in protein digestibility and lysine of the
transgenic sorghums having suppressed kafirin sub-class
synthesis with the protein quality of other cereals, using the
international accepted measure of Protein Digestibility
Corrected Amino Acid Score (PDCAAS). They showed
that the PDCAAS of food (porridge) made from the
transgenic sorghums is similar to that of porridges made
from maize, pearl millet and wheat. Further, the levels of
protein digestibility obtained in this present research are
potentially higher than shown because the genotype we
used (P898012) is a type II tannin line that contains tannins
which naturally interfere with digestion.
Tannins are rich in phenolic hydroxyl groups. As such
they have high propensity to bind proteins through
hydrogen bonding, hydrophobic interactions, electrostatic
attraction (which does not occur in sorghum tannin-protein
system) and covalent bonding associated with oxidation
(no evidence in sorghum) (Leatham et al. 1980; Oh et al.
1980; Meek and Weiss 1979). The two major classes of
tannins, hydrolysable tannins and non-hydrolyzable tannins
(condensed), are widespread in plants, but sorghum con-
tains only one class—the condensed tannins, chemically
known as proanthocyanidins (Fraziera et al. 2010). Under
optimal conditions, sorghum tannins have the potential to
bind and precipitate proteins upwards of 12 times their
weight (Harris and Burns 1970). Sorghum grain contains
on average 10 % protein content. This implies that the
grain of high-tannin sorghum cultivars ([2 % tannin)
contains more than enough tannin to bind all the dietary
seed proteins, thus making the protein indigestible when
consumed (Neucere and Sumrell 1980). The most affected
proteins are those that are large, have an open structure,
contain no bound carbohydrate and are rich in proline
(Butler et al. 1984). To counteract the negative impact of
tannins on dietary protein digestibility, animals rely on the
use of several mechanisms. These include high pH gut
adaptations and detergency of insects and the production of
salivary tannin-binding proteins to protect valuable pro-
teins against precipitation by tannins (Martin et al. 1985;
Robins et al. 1991). The biological effects associated with
sorghum tannins are generally beneficial in the field
534 Plant Cell Rep (2014) 33:521–537
123
(against bird predation and fungal infection, and pre-har-
vest seed deterioration, etc.) and harmful in the diet
(binding dietary proteins). Many efforts are being explored
to deal with tannins in sorghum. These include the devel-
opment of lines in which tannins never polymerize to long
antinutritional forms (condensed tannins).
In subsequent phases of this project, the sorghum germ-
plasm developed in this project will be introgressed into non-
tannin sorghum lines. Further investigations are however
required to validate any additional nutritional benefits that
could arise from altered protein expression and accumulation
in sorghum grain following suppression of selected kafirin
sub-classes. In many poor countries, food security is a
complex interplay between availability, quantity and quality
in terms of nutritional composition. It has been argued pre-
viously that one nutritional trait, for example, ‘‘high protein
of sorghum’’ is not a miracle solution for the poor, mal-
nourished communities who farm marginal agro-ecological
zones in Africa (Mayer and Mayer 1974). Genetically
modified (GM) crops generally add another barrier to food
access because they are not readily accepted in developing
countries and the GMO regulations are strict. The transgenic
lines generated in this project are available free of charge to
communities in Africa. However, because Africa is the
centre of origin and diversity for sorghum, these lines still
have to go through rigorous assessments before they can be
grown in open fields.
We sincerely believe that our approach towards
improving sorghum nutrition is logical. Kafirins are the
major storage proteins in sorghum and are the most
abundant, yet they are deficient in many essential amino
acids like lysine, methionine and tryptophan. Previous
research (Kumar et al. 2012) involving molecular charac-
terization showed that downregulation of c-kafirin alone
was insufficient to change sorghum endosperm texture or
increase protein digestibility, but that a predicted 29-kDa
a-kafirin should perhaps be suppressed to alter protein
body morphology and protein digestibility. There is further
evidence which shows that sorghum protein digestibility,
endosperm texture and protein body morphology are highly
influenced by the dosage of high-digestibility mutant
alleles (Tesso et al. 2008). Also in relation to the effect of
kafirin cross-linking on sorghum protein digestibility,
Hoang (2008) overexpressed the reducing compound thi-
oredoxin in transgenic sorghum to produce mutants which
had improved digestibility. The c-kafirin sub-class there-
fore appears to play a major role in the low protein
digestibility of sorghum through its disulphide-bonded
cross-linking of the kafirin polypeptides (El Nour et al.
1998). A critical issue appears to be whether c-kafirin alone
is responsible, or whether additional suppression of other
kafirin sub-classes is necessary to improve protein digest-
ibility and nutrition. We have shown in this research that
the suppression of synthesis of three nutritionally chal-
lenged kafirin proteins leads to higher digestibility than that
imparted by the suppression of the two gamma-kafirins.
The approach we took in this research could be important
in efforts towards improving the nutrition of communities
subsisting on sorghum as a staple crop in marginal agri-
cultural ecologies of Africa.
Acknowledgments We are grateful to the Bill and Melinda Gates
Grand Challenges 9, Africa Biofortified Sorghum (ABS) Project
through a sub-grant from the Africa Harvest Biotechnology Founda-
tion International for funding. We would also like to thank Pioneer
Hi-Bred International for supplying the pABS042 and pABS044
constructs and antibodies, and Dr Dirk Hays of Texas A&M Uni-
versity for the high protein digestibility sorghums. Priscilla Dikiso,
Stella Manganye, Taola Shai and Moses Mokoena are thanked for
their skilled technical assistance.
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