Silencing of ABCC13 transporter in wheat reveals its involvement in grain development, phytic acid accumulation and lateral root formation
Journal of Experimental Botany
Silencing of ABCC13 transporter in wheat reveals its involvement in grain development, phytic acid accumulation and lateral root formation
Kaushal Kumar Bhati 1
Anshu Alok 1
Anil Kumar 1
Jagdeep Kaur 0
Siddharth Tiwari 1
Ajay Kumar Pandey 1
Editor: Greg Rebetzke
0 Department of Biotechnology, Panjab University , Chandigarh, Punjab , India
1 National Agri-Food Biotechnology Institute (Department of Biotechnology), C-127, Industrial Area , Phase VIII, S.A.S. Nagar, Mohali-160071, Punjab , India
Low phytic acid is a trait desired in cereal crops and can be achieved by manipulating the genes involved either in its biosynthesis or its transport in the vacuoles. Previously, we have demonstrated that the wheat TaABCC13 protein is a functional transporter, primarily involved in heavy metal tolerance, and a probable candidate gene to achieve low phytate wheat. In the current study, RNA silencing was used to knockdown the expression of TaABCC13 in order to evaluate its functional importance in wheat. Transgenic plants with significantly reduced TaABCC13 transcripts in either seeds or roots were selected for further studies. Homozygous RNAi lines K1B4 and K4G7 exhibited 34-22% reduction of the phytic acid content in the mature grains (T4 seeds). These transgenic lines were defective for spike development, as characterized by reduced grain filling and numbers of spikelets. The seeds of transgenic wheat had delayed germination, but the viability of the seedlings was unaffected. Interestingly, early emergence of lateral roots was observed in TaABCC13-silenced lines as compared to non-transgenic lines. In addition, these lines also had defects in metal uptake and development of lateral roots in the presence of cadmium stress. Our results suggest roles of TaABCC13 in lateral root initiation and enhanced sensitivity towards heavy metals. Taken together, these data demonstrate that wheat ABCC13 is functionally important for grain development and plays an important role during detoxification of heavy metals.
ABC-type transporters; cadmium stress; cadmium tolerance; lateral roots; phytic acid; Triticum aestivum
In plants, phytic acid (myo-inositol
1,2,3,4,5,6-hexakisphosphate; PA; IP6) is a major storage form of seed phosphate
that chelates important micronutrients (Ca, Zn, Mg, Fe, Mn,
etc.) and therefore reduces their bio-availability
and Andersson, 1988; Raboy, 2001, 2009)
. One strategy for
enhancing the bioavailability of micronutrients is by
lowering the PA content in cereal grains
biosynthesis takes place by sequential phosphorylation of the
primary substrate, myo-inositol, in the cytosol
and Bewley, 1984; Rasmussen et al., 2010)
. PA is subsequently
compartmentalized into vacuoles by multidrug
(Raboy, 2002; Regvar et al., 2011)
Previous studies suggest that lowering the seed PA level can be
achieved either by targeting genes involved in its biosynthesis
or its compartmentalization
(Raboy, 2001; Rasmussen et al.,
2010; Sparvoli and Cominelli, 2015)
Multiple genes involved in different steps of PA
biosynthesis have been targeted to achieve low phytate content
in diverse cereals
(Shi et al., 2005; Kim and Tai, 2011; Ali
et al., 2013a,b)
. The maize lpa1-1 and lpa2-1 lines were the
first mutants studied, and produced seeds that had 50% to
66% lower PA levels and high inorganic phosphate
et al., 2000)
. The lpa1-1 mutation was mapped to a locus
encoding an ATP-binding cassette transporter C (MRP4)
with a function in PA transportation and the lpa2-1
mutation was mapped to a locus encoding an inositol phosphate
kinase (IPK1) with a function in PA biosynthesis
(Shi et al.,
. Mutants similar to maize lpa1 with defects in
MRP (ABCC type) transporter activity have also been
recovered from Arabidopsis
(ABCC5; Nagy et al., 2009)
(ABCC1; Dorsch et al., 2003)
, soybean (ABCC1; Panzeri
et al., 2011), and rice
(MRP5; Xu et al., 2009)
observations have encouraged reverse genetic approaches in crop
plants to further validate the role of ABCC transporters in
lowering the PA content. For example, MRP4 in Zea mays,
MRP1 in Phaseolus vulgaris, and MRP5 in Oryza sativa have
been targeted to generate low phytate content in grain
et al., 2007; Li et al., 2014)
. Among all lpa mutants, the lpa1
mutations have been most predominantly examined, with
breeding and genetic engineering efforts in multiple plants
(Supplementary Table S1 at JXB online).
Initially, plant ABCC transporters were known for their
roles in heavy metal sequestration and transport of
glutathione conjugates, but subsequently they have been
examined for their role in multiple physiological responses
(Ishikawa et al., 1997; Gaedeke et al., 2001; Martinoia et al.,
. Correlations were then observed between ABCC
transporter-mediated lowering of PA in cereal grains and
their pleiotropic effects. For example, MRP5 knockout in
Arabidopsis plants not only results in a decrease of seed PA
content, but also makes the plant more sensitive in its response
(Gaedeke et al., 2001; Nagy et al., 2009)
Similarly, Arabidopsis ABCC1, ABCC2, and ABCC3 are
well-known active glutathione conjugate transporters. In
addition, ABCC1 is a potential folate and metal transporter
whereas ABCC2 and ABCC3 are involved in chlorophyll
(Lu et al., 1997, 1998; Tommasini et al.,
1998; Frelet-Barrand et al., 2008; Raichaudhuri et al., 2009;
Song et al., 2010)
. Similarly, maize ABCC4/MRP4 performs
multiple functions including flavonoid transportation and PA
compartmentalization, and ZmMRP4 mutants have altered
(Goodman et al., 2004; Shi et al., 2007;
Badone et al., 2012)
Hexaploid wheat is one of the important food crops that has
been improved for numerous traits through breeding efforts.
The grains of wheat and other cereals store several essential
inorganic nutrients and micronutrients compartmentalized in
(Regvar et al., 2011; Singh et al., 2013; Bhati
et al., 2014)
. Wheat mutants and landraces have been studied
for seed PA, iron bioavailability, and agronomic performance
(Guttieri et al., 2004; Salunke et al., 2012; Gupta et al., 2015)
Surprisingly, the pleiotropic effects observed in wheat due
to lowering of PA are more drastic compared to other crop
species with either lpa1/2/3 mutations
(Guttieri et al., 2004;
Gupta et al., 2015)
Unlike in other crops, transgenic-based approaches have
not been utilized and assessed to address the roles of
candidate genes to develop low phytate in wheat. Wheat genes
involved in PA biosynthesis or its possible transport,have been
(Bhati et al., 2014; Aggarwal et al., 2015)
It has also been demonstrated that expression of TaABCC13
in a heterologous system conferred tolerance to heavy metals
via the utilization of glutathione conjugates as substrates. In
the present work, the functional role of ABCC13 in wheat
was investigated by RNAi-mediated gene silencing by using
a constitutive promoter. Silencing of ABCC13 in transgenic
wheat reduced PA in mature grains, and it caused
developmental defects during grain filling. Furthermore, wheat
transgenic roots showed sensitivity for the presence of cadmium
(Cd) and phenotypic defects were observed. These results
along with previous information for TaABCC13 suggest its
important role in wheat.
Materials and methods
RNAi vector preparation and Agrobacterium-mediated
A monocot-specific RNAi vector pMCG161 (TAIR
stockCD3-459) was used for silencing of the wheat (Triticum aestivum)
(Zalewski et al., 2010; Gasparis et al., 2011)
vector confers selection of transgenic plants in the presence of
the herbicide BASTA, and the RNAi cassette is expressed
ubiquitously under the control of the CaMV 35S promoter. Primers were
designed to amplify and clone a 394-bp conserved sequence from
TaABCC13 that was confirmed by sequencing (Chr 4B, 4D, and
5A; Supplementary Fig. S1). This nucleotide fragment was used to
clone sense (AscI/AvrII) and anti-sense (SpeI/SgfI) strands
following a one-step cloning method. The primer sequences used are listed
in Supplementary Table S2. The cloning of sense and antisense
sequences on either side of a rice waxy intron was subsequently
confirmed using multiple restriction digestions and sequencing. Final
constructs with proper insertion of sense and antisense sequences
was transformed into Agrobacterium tumefaciens strain AGL-1.
Positive colonies were screened and a single Agrobacterium clone
harboring a confirmed TaABCC13:pMCG161 RNAi construct was
used for wheat transformation.
Wheat transformation experiments were performed using
Agrobacterium-mediated transformation with minor modifications
(Przetakiewicz et al., 2004)
, while the growth and culture media
were used as per
Patnaik and Khurana (2003)
and Gasparis et al.
(2011). Bread wheat (cv C306) was grown in an experimental field
of the National Agri-food Biotechnology Institute, Mohali, India.
Immature seeds were collected at 12–16 d after anthesis (DAA) and
sterilized aseptically using HgCl2 (0.01% w/v). These sterilized seeds
were used to rescue immature embryos using manual dissection
under a laminar flow hood. About 700 dissected embryos, with their
scutella facing up, were cultured on callus-induction medium
consisting of MS medium with 2 mg L–1 of 2,4-Dichlorophenoxyacetic
acid (2,4-D). After 48 h of incubation at 22 °C in the dark, explants
were infected with an Agrobacterium suspension (OD600 = 0.4)
carrying the TaABCC13:pMCG161 construct. The Agrobacterium
suspension in liquid MS medium with Silwet (0.001% v/v) was poured
drop by drop over the explants
(Gasparis et al., 2011)
3 d of co-cultivation, growing calli were rinsed 4–5 times with
autoclaved MilliQ water to remove excess bacterial suspension, and
sub-cultured for 4 weeks on callus-induction medium containing
cefotaxime (200 mg L–1). Healthy calli were transferred to selection
and regeneration medium supplemented with 1 mg L–1 Zeatin and
2 mg L–1 of the herbicide BASTA
(Gasparis et al., 2011)
. At least
four rounds of selection, each for 3–4 weeks, were performed for the
regeneration of putative transformants. The plantlets were rooted
in MS medium containing 2.5 mg L–1 BASTA. Healthy and
wellrooted plants were transferred to soil-pots for hardening. The shoots
regenerated from independent calli were considered as independent
integration events and assigned as T0 putative plants.
Transgenic integration and genetic analysis
The plants those survived after hardening were selected for gene
integration analysis through PCR. Total genomic DNA was isolated
from fully expanded leaves of putative transgenic and
non-transformed (control) plants using the DNeasy Plant Mini Kit–QIAGEN
following manufacturer’s protocol. Two sets of specific primers were
designed for amplification of the bar gene and the OCS1
terminator region of RNAi cassette
(Gasparis et al., 2011)
Table S2). Amplicon generated by PCR from T0 plants was
purified and sequenced for confirmation. An individual tiller from each
T0 plant was also screened by PCR to eliminate the possibility of
chimera. Subsequently, seeds developed from the PCR-positive T0
plants were subjected to segregation analysis on 2 mg L–1 BASTA
that was optimized for cv C306 (control wheat) in hydroponic media.
The T1 progenies derived from positive transformation events that
fitted a 3:1 Mendelian segregation ratio based on BASTA selection
and bar gene amplification were cultivated up to the T2 and T3
generations. Homozygous lines were selected using PCR in the T2 and
T3 generations. These homozygous TaABCC13 RNAi lines were
maintained up to the T4 generation with consistent BASTA
selection as well as by PCR-based screening. At the onset of flowering,
the spikes were tagged on the day of anthesis for each line for
subsequent observation on days post anthesis. The mature seeds from
wild-type C306 and the RNAi lines were collected at approximately
56 d post anthesis and stored in cool and dry conditions.
RNA extraction and confirmation of gene silencing using
Tissue samples from selected homozygous T4 lines were
snap-frozen in liquid nitrogen and stored at −80 °C until further analysis.
Total RNA was extracted using the RNeasy Plant MiniKit (Qiagen,
Valencia, CA, USA), as per the manufacturer’s instructions. Two
micrograms of DNA-free total RNA were used for cDNA
preparation. Reverse transcription reactions were performed using the
Transcriptor First-Strand cDNA Synthesis Kit RT-PCR (Roche,
USA) according to the manufacturer’s instructions. Primers used
in the expression study are noted in Supplementary Table S2.
Quantitative real-time PCR analysis was performed by following
SYBR Green (QuantiFastTM SYBR Green PCR kit, QIAGEN)
chemistry using the ABI PRISM 7500 Fast Realtime Platform
(Applied Biosystems). Target genes were amplified by a two-step
PCR reaction with an initial denaturation at 95 °C for 5 min
followed by 40 cycles of 95 °C for 30 s and annealing/extension at 60 °C
for 30 s. Relative quantification for fold-changes were calculated and
Ct values were normalized against wheat ARF (ADP-ribosylation
factor, AB050957.1) as defined previously by Bhati et al. (2014) and
Alok et al. (2015) using the 2–ΔΔCT method (Livak and Schmitteng,
2001). The specificity of the amplification was also assessed for each
gene by dissociation curve analysis. A unique peak on the
dissociation curve was confirmed for each gene.
Phytic acid, free phosphate and total protein estimation
Total phytate in seeds was estimated by a colorimetric method using
a K-PHYT kit (Megazyme, Inc, Bray, Ireland). Mature seeds from
T4 homozygous lines were ground into a fine powder and extracted
using 0.66 N HCl for 6 h with continuous stirring. Reactions were
processed as described previously
(Bhati et al., 2014)
phosphate in mature seeds of selected RNAi lines was measured by the
ascorbate and ammonium molybedate method (Ames, 1966). Total
protein was estimated by using the Bradford method (Bradford,
1976) with comparison against a BSA standard curve.
Elemental analysis and cadmium uptake assays
Sterilized seeds of homozygous RNAi lines and non-transformed
plants were germinated on Hoagland liquid media
supplemented with 50 µM CdCl2. Sample collection,
phenotypic observations, and Cd estimation were performed after 7
d. Root and leaf samples were heat-dried and microwave-digested
with HNO3 (SuraPureTM, Merck). Total Cd uptake was measured by
using Inductive Coupled Plasma-MS (ICP-MS) as described
(Bhati et al., 2014)
. In the figures the error bars indicate the
standard deviation across two independent experiments.
Homoeologous specific expression pattern of
TaABCC13 was mapped to the long arm of the 4B, 4D, and
5A chromosomes with a similarity score of 99% and 97%
(4B allele with respect to 4D and 5A). Fine mapping of these
homoeologous sequences in the wheat genome assembly
from the Ensembl plant server (http://plants.ensembl.org/
Triticum_aestivum /Info/Annotation) helped in
identification of their physical location on the respective chromosomes
(Supplementary Table S3). The wheat ABCC13 transporter
was conserved among orthologs from other cereals that share
phylogenetic proximity (Supplementary Fig. S2). TaABCC13
was differentially expressed during the developmental stages
of wheat grains
(Bhati et al., 2014)
. To check the
homoeologbased expression of TaABCC13, gene-specific primers were
used to determine the contribution from different wheat
genomes. Expression of each of the TaABCC13 homoeologs
at two developmental stages during grain filling suggested
preferentially high expression of transcripts arising from the
B genome (Supplementary Fig. S3). The expression of all
TaABCC13 homoeologous transcripts was higher at 14 DAA
than at 21 DAA. The transcript accumulation derived from
the B genome was ~13-fold higher at 14 DAA compared to
Selection and screening of transgenic wheat lines
To gain insights into the function of TaABCC13 in wheat,
a binary vector pMCG161 with a constitutive promoter was
designed to target a conserved region of the homoeologous
gene sequences (Fig. 1A and Supplementary Fig. S1). The
pMCG161 binary vector was previously shown to be an
efficient vector for gene silencing in monocots such as barley,
wheat, and maize
(Zalewski et al., 2010; Gasparis et al., 2011)
Nine independent putative transgenic events survived
during multiple rounds of selection on BASTA (Supplementary
Fig. S4), but only four of the putative transgenic plants
survived the hardening procedure. These four putative
transformants were subsequently confirmed for the presence of the
transgene by amplifying and sequencing the bar and OCS1
terminator sequences (Fig. 1B; Supplementary Fig. S5A,
B). Additionally, mosaicism of transgenic integration in the
wheat callus was also ruled out by screening each tiller by
amplification and sequencing of bar (Supplementary Fig.
S5C), and the flag leaves of T0–1 plants were found positive for
bar transcript expression (Fig. 1C). The T1 progenies from the
third event (K3) failed to survive due to reduced seed setting
and subsequent failure of seed germination. Eventually, three
independent transgenic events (K1, K2, and K4) showed
healthy growth and seed germination for further analysis.
The lines from these three independent events were
propagated to the T4 generation, which was analysed in detail. Two
transgenic plants from lines K1 (K1B4-2–5, K1A13-8-2), K2
(K2C4-6–8, K2C9-2–3), and K4 (K4G3-5-1, K4G7-10–3)
selected randomly for further study.
Silencing of TaABCC13 in wheat RNAi lines
The selected T3 or T4 transgenic plants developed from three
events (K1, K2, and K4) were subjected to qRT-PCR to assess
the level of gene silencing in varying tissues. Transcript
abundance of TaABCC13 was quantified in T4 seeds at 14 DAA
in the grains and flag leaf of the same tiller (Supplementary
Fig. S6). The fold-decrease in the TaABCC13 transcript
for the T4 generation seed is presented in Fig. 2A and
Supplementary Fig. S6. The silencing of TaABCC13 resulted
in 20–60% reduction in the transcript levels in the seed
tissue, while 20–70% of silencing was observed in the flag leaf
(Fig. 2A and Supplementary Fig. S6A, B). Maximum seed
tissue silencing was observed for lines K4G7-10–3,
K4G35-1, K1A13-8-2, and K1B4-2–5 in both T3 and T4
developing seeds. The representative silenced lines were further
used for detailed characterization and phenotypic studies.
As noted above, chromosome 4B is the major contributor
of the TaABCC13 transcripts (Supplementary Fig. S3), and
we wanted to know how the silencing construct affected the
expression of each of the homoeologs. To study the relative
expression of the transcripts of TaABCC13,
homoeologspecific primers were employed and expression was compared
to respective non-transgenic plants. The results confirmed
that the construct was able to reduce the expression of the
transcripts derived from the 4B, 4D, and 5A chromosomes
by 40–72% (Supplementary Fig. S6C). In addition, to check
whether other closely related ABCC genes (TaABCC3 and
TaABCC4) were affected by silencing of TaABCC13,
expression analysis was performed for them. No significant change
in the transcript of TaABCC3 and TaABCC4 was observed in
the non-transgenic and transgenic lines (Supplementary Fig.
S6D). This suggests that the silencing construct was specific
for TaABCC13 and probably does not affect the expression
of the other studied ABCC genes.
TaABCC13 silencing reduces grain total PA content
To examine the effect of silencing of TaABCC13 in seeds,
PA content was measured in the mature grains. Significant
differences in the accumulation of PA were observed among
the transgenic lines that ranged from 22% to 34% reduction
when compared to the non-transformed mature seeds. The
maximal reduction in seed PA was observed in line K1B4-2–5
(~34%) followed by K4G7-10–3, which had a ~22% reduction
in the PA level (Fig. 2B). No significant changes in total seed
phosphorus (P) was observed for the ABCC13:RNAi lines
as compared to C306 (Supplementary Fig. S7). However,
the lowering of PA content was accompanied by a increase
in free phosphate (Pi) for these transgenic lines (data not
shown). Silencing of TaABCC13 in the selected transgenic
lines resulted in a slight reduction in seed weight (Fig. 2C).
Although this slight reduction was observed consistently in
other lines, it was most apparent in the K1B4-2–5 and
K4G710–3 lines. The calculated protein content range for C306 was
11–12%. Although our transgenic lines varied in protein
content from 10.3–11.2%, it was within the usual range expected
for C306 (Fig. 2D).
Reduction in PA is often accompanied by remobilization or
a change in the content of micronutrients
(Ali et al., 2013a)
Transgenic wheat seeds showed increases in the accumulation
of Ca, but no significant changes were observed for
micronutrients such as Zn and Fe. The accumulation of Ca was
~1.7–1.8-fold greater than in the non-transformed control
seeds (Table 1). Results of Perl’s staining showed a decreased
density of iron at the crease region in the transgenic seed as
compared to C306 (Supplementary Fig. S8). Enhanced
colouration was also observed on the germinating coleoptile
region, suggesting a possible early remobilization of iron in
TaABCC13 affects grain filling, spike characteristics, and lateral root formation
Previously, it was shown that wheat ABC transporters affect
anther development and subsequently the process of grain
(Niu et al., 2013; Walter et al., 2015)
from multiple studies have also suggested that lowering PA
generally results in altered grain morphology
(Guttieri et al.,
2004; Li et al., 2014)
. To assess the contribution of ABCC13
to grain development, developing spikes after heading were
examined for phenotypic changes in C306 and transgenic
plants. An altered spikelet arrangement was noticed in the
developing spikes of transgenic lines. In these lines, the
outer glumes are more exposed, thus causing spikes with an
altered spikelet arrangement that is always accompanied by
a reduction in total spikelet counts, when compared to the
primary tiller of the control plants (Fig. 3A, B, E). These
observations were consistently observed for both the silenced
lines. The occurrence of head sterility was also observed in
these RNAi lines in T2 to T4 progenies (Fig. 3C). Therefore,
seed setting and grain filling were affected in transgenic
lines, but the head development was normal at the pedicel
region in both plants. Taking into account the pleiotropic
effects of silencing TaABCC13, a reduction in the number
of seeds recovered from each primary spike was generally
observed (Fig. 3F). These results suggest the possibility that
grain development directly involves ABCC13, or it could be
affected by PA levels.
A reduction in PA often correlates with the rate of
germination for cereal grains. The germination rate of transgenic
wheat (T4) with low PA levels was reduced by 5–7% when
compared to the non-transgenic wheat seeds. Interestingly,
a slow rate of germination was observed for low-PA seeds,
which was apparent at the early times points (24 and 96 h)
after imbibition. In addition, we also observed that the
germination rate was comparatively slow in transgenic
compared to non-transgenic seeds during the early hours
of germination. (Fig. 4). At a later stage of development
(10 d), no significant difference in the height of germinated
seedlings was noted between transgenic or non-transgenic
lines (Fig. 4). These data confirm that silencing of ABCC13
in wheat is related to lowering of total PA and to grain
Plant ABCC transporters are also known for their role
during root development under stress conditions
et al., 2001)
. The K1B4-2–5 and K4G7-10–3 transgenic lines
were selected to determine the effect of TaABCC13
silencing on root development. In general, TaABCC13 transcript
was reduced by 30–65% in the roots of the transgenic lines
(Fig. 5A), but there was no difference in the root length
compared to the non-transgenic plants. Interestingly, early
emergence of lateral roots was observed in transgenic
seedlings in contrast to non-transgenic plants (Fig. 5B). The
formation of lateral roots in the transgenic lines was higher as
compared to the non-transgenic, suggesting that TaABCC13
might be involved in controlling the emergence of lateral
roots in wheat.
Influence of cadmium toxicity on the wheat transgenic lines
Heterologous expression of TaABCC13 in yeast has
suggested a role in Cd tolerance that could utilize glutathione
conjugates as substrates
(Bhati et al., 2014, 2015)
. In order
to further characterize the role of TaABCC13 in heavy
metal detoxification, wheat RNAi transgenic seedlings were
exposed to Cd. As expected, the emergence of multiple
lateral roots was observed in control plants treated with Cd.
Transgenic lines showed phenotypic sensitivity when exposed
to Cd compared to non-transgenic (Fig. 6A). Interestingly,
the TaABCC13:RNAi lines did not develop lateral roots
when exposed to Cd (Fig. 6A). This phenotype was noted
in two different transgenic lines (K1B4-2–5 and K4G7-10–
3). In general, a decrease in the root length was observed in
Cd-exposed plants when compared to untreated seedlings
(Fig. 6B). The shoot biomass was significantly higher in C306
plants as compared to TaABCC13:RNAi seedlings exposed
to Cd (Supplementary Fig. S9). The TaABCC13 silencing
and altered root phenotype under Cd stress resulted into
differences in the root and shoot uptake of Cd (Fig. 6C, D).
This in planta evidence shows that Cd uptake is reduced and
sensitivity to Cd is decreased in TaABCC13:RNAi lines,
thus signifying the importance of ABC transporters for Cd
The present study provides insights into multiple
physiological roles of ABCC13 in wheat. Previous studies have
proposed high expression levels of TaABCC13 in immature
seeds and roots, thus advocating TaABCC13 as a
potential candidate gene regulating seed and root phenotypes in
(Bhati et al., 2015)
. The constitutive expression of
the RNAi cassette targeting TaABCC13 enabled the
functional validation of this gene not only in seeds but also in
the root tissue. TaABCC13 is evolutionarily conserved with
orthologs of other candidate genes from maize (ZmMRP4)
and Arabidopsis (AtMRP5) that were demonstrated to have
multiple functions in earlier studies
(Gaedeke et al., 2001;
Badone et al., 2012)
. In the present study, we observed that
ABCC13-silenced wheat transgenic lines had reduced PA and
altered heavy metal detoxification.
ABCC13 as a candidate to achieve low phytic acid in wheat
Multiple approaches have been considered to generate a
lowphytate trait in crop plants such as soybean, rice, and maize
(Shi et al., 2007; Kim and Tai, 2011; Ali et al., 2013a,b; Li
et al., 2014)
. However, such studies have not previously been
undertaken for important crops such as wheat where most of
the PA–along with micronutrients–accumulates in the
(Bohn et al., 2008; Bhati et al., 2014)
a transgenic approach to modify the PA in wheat is
important since genetic variation for this trait in this species is
very limited. Although the transcript arising from
chromosome 4B was highly expressed, to evaluate the functionality
of TaABCC13 a conserved region of the three
homoeologous genes was targeted (Supplementary Figs S1 and S6). In
general, we observed that this construct was able to reduce
relative expression levels of all the transcripts of TaABCC13
irrespective of the origin.
Recently, wheat genes involved in biosynthesis of PA have
been reported, and these could be potential candidates for
developing low-PA traits
(Bhati et al., 2014)
transporters are involved in the transport or
compartmentalization of the PA after its synthesis in grains. Loss-of-function
mutations for ABCC transporters have been characterized
with varying levels of reduction in seed phytate
et al., 2000; Pilu et al., 2003; Raboy, 2009; Panzeri et al.,
. Moreover, ABCC13 also shares close homology with
ZmMRP4 and AtMRP5 (90.5 and 73.5%, respectively), with
a similar exon–intron arrangement
(Bhati et al., 2014)
studies strongly support this ABCC transporter as a strong
candidate for the development of a lpa phenotype in
agronomically important crops.
Wheat RNAi lines targeting this ABCC transporter gene
(K1B4-2–5 and K4G7-10–3) showed 34–22% reduction in the
PA level with a concomitant increase in calcium. Depending
on the crop and the candidate genes targeted, lowering of
PA is often accompanied by an increase in micronutrients, as
demontsrated in other studies
(Shi et al., 2007; Ali et al., 2013a)
In our transgenic seeds, although no significant increase in the
iron content was observed, our preliminary screening suggested
its faster remobilization in other tissues. This phenomenon was
consistently observed in most of our RNAi seeds, reinforcing
the correlation between the rate of iron remobilization and
lowering of PA. Studies utilizing spectroscopy-based methods
could be further used to validate these redistribution patterns.
The only previous report for reduced PA in wheat was a line
with mutations in two independent lpa1-related loci that caused
a 37% reduction
(Guttieri et al., 2004)
. Similar, the lpa-1
mutation in barley and maize showed reduction of 50–95% and
(Raboy et al., 2000; Dorsch et al., 2003)
Reduction of PA due to a defective PA transport mechanism
causes significant pleiotropic effects that results in unacceptable
agronomic field performance in wheat, maize, and rice
et al., 2004; Badone et al., 2012; Li et al., 2014)
. Some of these
negative impacts were also observed in the current study in
TaABCC13:RNAi lines, including reduced seed weight, slightly
delayed germination, and slow coleoptile growth. However,
these defects were not as drastic as reported in the first wheat
lpa mutant and in the low field performance by the lpa1-7
mutation in ZmMRP4
(Guttieri et al., 2004; Badone et al., 2012)
Similar agronomical impacts of reduced PA have been observed
in other crop plants silenced for genes involved in either the
early or late phase of PA biosynthesis
(Raboy, 2009; Ali et al.,
2013a,b; Sparvoli and Cominelli, 2015)
. Thus these impacts
may vary from crop to crop, and with the choice of the
candidate gene. Phenotypic effects on the lpa crops could be reduced
by utilizing tissue-specific targeting of the PA biosynthetic
(Shi et al., 2007; Ali et al., 2013a)
. Such strategies could
also be designed to target ABCC13 in the aleurone to develop
an lpa trait in wheat, because the aleurone is the tissue where the
TaABCC13 transcript is abundant
(Bhati et al., 2014)
Role of TaABCC13 in grain development and physiological characteristics
The phenomenon of multi-functionality is common among
plant ABCC transporters
(Gaedeke et al., 2001; Nagy et al.,
2009; Walter et al., 2015)
. Functional studies of the wheat
ABCC transporter have established its roles in responses to
fungal pathogens, and in grain formation and maturation
et al., 2015)
. Subsequently, multiple wheat ABCC transporters
were reported that are highly expressed in developing grains
(Bhati et al., 2015)
. These reports have emphasized the need
to expand our knowledge of the role of ABCC transporters in
wheat. Our data confirm the importance of TaABCC13 and
generally support the emerging roles of ABCC transporters
in grain development and maturation. The early development
of wheat spikes is regulated by abscisic acid (ABA) and
gibberellic acid (GA) responses
(Thiel et al., 2008; Pearce et al.,
. Previously we have reported that the exogenous
application of GA significantly stimulated TaABCC6, TaABCC8,
and TaABCC13 transcript levels in wheat seeds
(Bhati et al.,
. Plant ABCC transporters are known to have a role in
the transport of derivatives in hormone biosynthesis to
further facilitate localized hormonal response
(Gaedeke et al.,
2001; Ko et al., 2014; Borghi et al., 2015)
. Inositol phosphate
biosynthesis pathways control signaling responses in
. In plants PA also acts as a signaling
molecule or a co-factor that controls the physiological response
to hormones or oxidative stress
(Lemtiri-Chlieh et al., 2003;
Tan et al., 2007; Doria et al., 2009; Sparvoli and Cominelli,
. The biosynthesis of wheat PA starts at the early stages
of seed development. Therefore, one could speculate that the
phenotypes observed during wheat spike development are
possibly effects of reduced PA signaling and/or perturbation
in the ABCC13-dependent transport of hormonal derivatives.
Plant roots may develop different phenotypes and anatomical
features when exposed to metal stress. Higher plants commonly
develop lateral roots in an attempt to block the radial transport
of heavy metals
(Hu et al., 2013; Sofo et al., 2013)
. In our
experiments, non-transgenic wheat lines also developed lateral roots
under Cd stress, reinforcing the conserved mechanism of
sensitivity in plants. Plant ABCC transporters may have direct or
indirect roles in regulation of plant development. Interestingly,
the untreated transgenic lines silenced for TaABCC13 showed
an early emergence of lateral roots, but they were inhibited in
the presence of Cd (Figs 5 and 6). Low PA mutants of maize
(zmmrp4) and Arabidopsis (atmrp5) also have altered root
phenotypes that include the early emergence of lateral roots
(Gaedeke et al., 2001; Nagy et al., 2009; Badone et al., 2012)
The early emergence of lateral roots on TaABCC13:RNAi
lines suggests a conserved role for TaABCC13 in wheat root
development. These observations coupled with previous
evidence suggest a correlation for the possible homeostatic role of
TaABCC13 in lateral root formation and metal stress.
It may be speculated that altered root development might
occur as a result of changes in auxin flux
(Gaedeke et al., 2001)
If this is the case, then we could propose that TaABCC13 may
function in the transport of auxin or a derivative; however, our
experiments do not directly test this hypothesis. In our study,
10-d-old C306 seedlings formed lateral roots when exposed to
Cd, whereas TaABCC13-silenced plants were not able to develop
lateral roots. This surprising reversal of lateral root development
on TaABCC13:RNAi seedlings under Cd stress suggests that
there is a complex interaction between the processes
regulating root development and responses to Cd stress. In plants, the
response activated by metals is mediated through the
biosynthesis of signalling molecules such as phytohormones. Multiple
reports have suggested that Cd interferes with the maintenance
of auxin homeostasis
(Xu et al., 2010; Elobeid et al., 2012; Hu
et al., 2013)
. Previous reports have also suggested that Cd alters
the expression of multiple genes responsible for auxin
biosynthesis and distribution, which leads to increased lateral root
(Hu et al., 2013, Yuan and Huang, 2015)
. Our data not only
validate the role of TaABCC13 during lateral root formation,
but also link with the possibility for metal–auxin homeostasis.
The current study provides in planta evidence for the
previously speculated role that TaABCC13 has in yeast.
Taken together, our data reinforce the importance of
targeting ABCC transporters to achieve low PA, and we further
demonstrate their roles in heavy metal detoxification. This
research lays the groundwork for further examination for the
functions of ABCC transporters in wheat, especially those
that are highly expressed during grain development.
Supplementary data are available at JXB online.
Table S1. List of ABCC transporters reported from
different plant systems together with the approaches used to
develop the low phytic acid trait.
The authors would like to thank the Executive Director of NABI for facilities
and support. This research was funded by the Department of Biotechnology
(DBT), Government of India (BT/PR5989/AGII/106/867/2012) to AKP and
ST. The authors would like to thank Dr Sebastian Gasparis, Department
of Functional Genomics, Instytut Hodowli i Aklimatyzacji Roslin, Poland
for his suggestions relating to the screening of wheat RNAi plants. We also
thank Dr Steven Whitham (Iowa State University, Ames, Iowa, USA) for
critically reading the manuscript. KKB was supported by a Senior Research
Fellowship from the DBT, AA was supported by a fellowship from the
NABI-CORE fund, and AK was supported by a fellowship from the DBT
grant. Technical assistance from Pankaj Pandey was greatly appreciated.
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